Download ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group

ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
105 Retinoblastoma: Basic and clinical
Sunday, May 03, 2015 8:30 AM–10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 65–86/A0097–A0118
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Biochemistry/Molecular Biology, Clinical/
Epidemiologic Research, Genetics, Immunology/Microbiology,
Physiology/Pharmacology
Program Number: 65 Poster Board Number: A0097
Presentation Time: 8:30 AM–10:15 AM
The cone-rod homeobox transcription factor (CRX) mRNA as a
molecular marker in metastatic retinoblastoma
Viviana E. Laurent1, Ana V. Torbidoni1, Claudia Sampor1, Daniela
Ottaviani1, Mariano R. Gabri3, Jorge Rossi4, María T. García de
Dávila2, Cristina Alonso1, Daniel F. Alonso3, Guillermo L. Chantada1.
1
Hematology-Oncology, Hospital de Pediatría Prof. Dr. J.P. Garrahan,
Ciudad Autónoma de Buenos Aires, Argentina; 2Pathology Service,
Hospital de Pediatría Prof. Dr. J.P. Garrahan, Ciudad Autónoma de
buenos Aires, Argentina; 3Molecular Oncology Laboratory, Quilmes
National University, Bernal, Argentina; 4Immunology Service,
Hospital de Pediatría Prof. Dr. J.P. Garrahan, Ciudad Autónoma de
buenos Aires, Argentina.
Purpose: Disseminated retinoblastoma is still the major cause of
mortality for this tumor worldwide. Research on the dissemination of
this neoplasm has been hindered by the rarity of extraocular cases in
developed countries. However, it would be of interest for developing
countries. Using a prospective evaluation of a diagnostic clinical test,
we evaluated the cone-rod homeobox transcription factor (CRX) as a
new lineage-specific molecular marker for metastatic retinoblastoma
and its usefulness as a tool for improving diagnostic accuracy and
assessing the response to treatment and the patterns of disease
dissemination in different clinical scenarios by the evaluation of
minimal dissemination (MD) in extra-ocular sites.
Methods: To validate CRX mRNA as a marker, we evaluated its
expression in 17 retinoblastoma primary tumors, two cell lines,
and 47 specimens from other malignancies as negative controls.
Seventeen consecutive patients with metastatic retinoblastoma (9 at
diagnosis, 8 at relapse) were included. CRX mRNA was evaluated by
retrotranscription followed by real-time polymerase chain reaction
(RT-qPCR) in bone marrow (BM), peripheral blood (PB), and
cerebrospinal fluid (CSF) at diagnosis, after induction chemotherapy,
and during follow-up after autologous BM transplantation.
Results: With a sensitivity of 1 in 107 cells, CRX mRNA was
expressed in all tumors and cell lines studied but it was negative
in all control samples. BM metastatic cells showed expression of
CRX in all nine children presenting with metastasis. After induction
chemotherapy, no MD was evident in any of the eight responding
children. In the CSF of children who had a metastatic relapse, CRX
mRNA detection was positive in all 11 cases studied. MD in the CSF
heralded a clinical relapse in three cases. No concomitant MD was
evident in the BM in any case.
Conclusions: CRX mRNA is a novel marker for retinoblastoma
at extraocular sites. In patients with BM metastasis, there is
quick, complete and sustained molecular response after induction
chemotherapy. In all patients with secondary metastasis, CSF relapse
occurs independently from the BM suggesting a sanctuary site.
Commercial Relationships: Viviana E. Laurent, None; Ana V.
Torbidoni, None; Claudia Sampor, None; Daniela Ottaviani,
None; Mariano R. Gabri, None; Jorge Rossi, None; María T.
García de Dávila, None; Cristina Alonso, None; Daniel F. Alonso,
None; Guillermo L. Chantada, None
Program Number: 66 Poster Board Number: A0098
Presentation Time: 8:30 AM–10:15 AM
Adherence Capability of Cultivated Retinoblastoma Cells
Narges Fazili6, Sahar Balagholi2, 1, Mozhgan Rezaeikanavi6,
3
, Somayeh Asadi4, 1, Seyed Bagher Hosseini5. 1Ocular Tissue
Engineering Research Center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran, Tehran, Iran (the Islamic Republic of); 2Iran
Blood Transfusion Organization, Tehran, Iran (the Islamic Republic
of); 3Ophthalmic Research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Iran (the Islamic Republic of); 4K.N.Toosi
of Technology, Tehran, Iran (the Islamic Republic of); 5Central Eye
Bank of Iran, Tehran, Iran (the Islamic Republic of); 6Ocular Tissue
Engineering Research Center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran (the Islamic Republic of).
Purpose: To investigate adherence capability of cultivated
retinoblastoma (RB) tumorspheres with prolonged cultivation.
Methods: RB cells from two Iranian patients were cultivated
in DMEM supplemented with 15% FBS for four weeks in
each passage. Fresh medium was added on a weekly basis and
immunocytochemistry for synaptophysin was performed. All the
experiments were done in duplicate. Cell attachment capability was
studied during three consecutive passages.
Results: A biphasic population of cultivated cells was observed
during the first week in each passage; composed of RB tumorspheres
and a single-cell suspension overlying a layer of fibroblastic cells
that had adhered to the bottom of flask. Early adherence of RB
tumorspheres to the bottom of flask, while surrounded by fibroblasts,
was observed in the second week and increased by the fourth week
(figure).
Conclusions: Compared to previous data, this study demonstrated
the adherence capability of RB tumorspheres to the underlying
surface with prolonged cultivation; which after 4 weeks seems to be
independent of the fibroblasts.
Figure: Cultivated retinoblastoma (RB) cells. A-B: Note the presence
of RB tumorspheres (circle 1), single-cell suspension (circle 2) in the
first week C-F: Cultivated RB cells in the second week: the circled
area shows adhered RB tumorspheres; merged FITC synaptophysine
expression and DAPI in cultivated RB cells (C), adhered RB
tumorsphere (D), DAPI of nucleus of RB and fibroblast cells (E),
merged FITC synaptophysine expression and DAPI in cultivated RB
cells (F).
Commercial Relationships: Narges Fazili, None; Sahar Balagholi,
None; Mozhgan Rezaeikanavi, None; Somayeh Asadi, None;
Seyed Bagher Hosseini, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 67 Poster Board Number: A0099
Presentation Time: 8:30 AM–10:15 AM
The Effects of Modulation of MMP-2 and MMP-9 in
Angiogenesis and Invasive Potential in Retinoblastoma
Anderson H. Webb1, Nabil Saleh1, Bradley T. Gao1, Ryan P. Lee1,
Justin B. Lendermon1, Matthew W. Wilson1, Vanessa M. Morales1,
2 1
. Ophthalmology, University of Tennessee Health Science Center,
Memphis, TN; 2Microbiology, Immunology and Biochemistry,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: Retinoblastoma (Rb) is the most common primary
intraocular tumor in children. Effective local treatment exists, but
occasionally Rb can metastasize. Metastases are attributed to extraretina invasion of the ocular coats and the optic nerve. In this study
we investigate modulation of Matrix Metalloproteinases (MMP),
responsible for degradation of the extracellular matrix and invasion,
as a potential adjuvant therapeutic target for Rb.
Methods: Three different Rb cell lines, Rb-Y79, Rb-Weri and Rb355, were analyzed by gene expression, protein levels, and secretion
of MMP-2 and MMP-9 by RT-PCR, Zymmography, and ELISA.
Flow cytometry examined the levels of ICAM, VEGF, and CD31 as
surrogates of adhesion, invasion, and angiogenesis. We evaluated
the effect of pharmacological inhibition of MMP-2 (ARP100, Santa
Cruz Biotechnology, Dallas, TX) and MMP-9 (AG-L-66085, Santa
Cruz Biotechnology) by magnetic levitation (Nano3D Biosciences,
Inc, Houston, TX) to determine their effects on angiogenesis and
invasion.
Results: Our results show the three different Rb cell lines express
different levels of MMP-2 and MMP-9 mRNA. MMP-9 expression
increased upon cell activation by using Phorbol 12-myristate
13-acetate (PMA) and was reduced upon use of the MMP-2 and
-9 inhibitors. Magnetic levitation analysis showed reduction in Rb
tumor masses in vitro by pharmacological inhibition of MMP-2 and
MMP-9.
Conclusions: Inhibition of MMP-2 and MMP-9, both markers of
invasion, decreased expression of CD31 and VEGF, both markers of
angiogenesis. MMP2 and MMP9 are potential candidates for targeted
therapy.
Commercial Relationships: Anderson H. Webb, None; Nabil
Saleh, None; Bradley T. Gao, None; Ryan P. Lee, None; Justin
B. Lendermon, None; Matthew W. Wilson, None; Vanessa M.
Morales, None
Program Number: 68 Poster Board Number: A0100
Presentation Time: 8:30 AM–10:15 AM
HLA class I expression in cell lines derived from conjunctival
melanoma and retinoblastoma using allele-specific monoclonal
antibodies and flow cytometry
Rind Smesseim1, Jinfeng Cao1, T H. Van Essen1, Arend Mulder1,
Elsbeth van Zeeburg2, Bruce R. Ksander3, Martine J. Jager1.
1
Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands; 2The Rotterdam Ophthalmic Institute and the Rotterdam
Eye Hospital, Rotterdam, Netherlands; 3Schepens Eye Research
Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical
School, Boston, MA.
Purpose: The expression of human leukocyte antigen (HLA) on
tumor cells may influence the immunological recognition and
response. Malignant cells often change their HLA class I antigens to
escape immunosurveillance. To examine whether immunotherapy
is possible in conjunctival melanoma and retinoblastoma we
investigated the HLA class I surface expression by using allelespecific antibodies in flow cytometry. We furthermore analyzed the
effect of interferon gamma (IFN-γ) stimulation on HLA expression.
Methods: Three conjunctival melanoma (CRMM1, CRMM2
and CM2005.1) and three retinoblastoma (Rb116, Rb125 and
Rb143) cell lines were HLA gene-typed at the Department for
Immunohematology and Blood Transfusion (IHB), The Netherlands.
Cells were grown in culture in the presence or absence of
recombinant IFN-γ (100 and 200 U/ml) for 48 hours and prepared
for flow cytometry. Based on the HLA-typing, HLA allele-specific
monoclonal antibodies against class I were selected. Flow cytometry
was performed and cellular surface HLA-expression measured. For
Western blot analysis, a lysate was made to determine the expression
of MHC class I.
Results: We analyzed the HLA allele-specific expression on three
conjunctival melanoma and three retinoblastoma cell lines by flow
cytometry. In general, a lower expression of antigen specific and
allele-specific HLA class I expression was observed in both types
of malignancies. In one retinoblastoma cell line (Rb143) no class
I expression was observed without IFN-γ stimulation but it was
restored after incubation with IFN-γ. Two of the three conjunctival
melanoma (CRMM1 and CRMM2) cell lines showed loss of
expression of specific HLA class I alleles (respectively HLA-A2 and
HLA-B44) which did not recover after IFN-γ stimulation.
Conclusions: We were able to determine HLA expression on three
new retinoblastoma cell lines and three conjunctival cell lines,
and found a defect in expression of particular HLA specific alleles
which were not restored by IFN-γ stimulation. This loss of antigen
expression may help ocular tumors to escape the immune response
and complicate the development of immunotherapy.
Commercial Relationships: Rind Smesseim, None; Jinfeng Cao,
None; T H. Van Essen, None; Arend Mulder, None; Elsbeth van
Zeeburg, None; Bruce R. Ksander, None; Martine J. Jager, None
Program Number: 69 Poster Board Number: A0101
Presentation Time: 8:30 AM–10:15 AM
Gonadotropin releasing hormone receptor is expressed in
retinoblastomas and a retinoblastoma cell line
Sultan Aldrees, Pablo Zoroquiain, Mohammed F. Qutub, Sarah
Alghamdi, Taylor Nayman, Miguel N. Burnier. Mcgill University,
Montreal, QC, Canada.
Purpose: Despite the fact that retinoblastoma treatment has
dramatically increased the survival and vision preservation in these
patients, it is still important to pursue new therapeutic targets to
minimize the side-effects of current therapy. Gonadotropin releasing
hormone (GnRH) has been shown to exert a direct antiproliferative
effect on many types of reproductive tissue cancers, such as breast
cancer, skin melanoma, and glioblastoma. The aim of this study is to
describe the presence of GnRH receptor (GnRHR) in retinoblastoma
in order to identify a new possible therapeutic target for this disease.
Methods: Protein expression of GnRHR was studied by
immunohistochemistry in 32 eyes with retinoblastoma and in the Y79
retinoblastoma cell line. Expression was scored according to intensity
(1–3) and distribution (1–4), which were multiplied to generate an
immunoreactive score (IRS). Low expression was considered an IRS
score of 1 to 4, moderate 5 to 8, and high 9 to 12. GnRHR mRNA
expression in Y79 cells were analyzed using reverse transcription
polymerase chain reaction (RT-PCR). The Student’s t-test was used
to compare GnRHIRS cases with or without each morphological high
risk features.
Results: GnRHR was expressed in all retinoblastoma cases and in the
Y79 cell line. There was no expression in normal ocular structures.
High, moderate, and low expression according to IRS score was
evident in 16%, 36%, and 48% of cases. There were no differences
in GnRH IRS with respect to uni- versus multifocal tumors, type of
growth (mixed/endophytic), rosette formation, choroidal invasion,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
extraocular extension or extension to the sclera, or optic nerve
invasion. In Y79 cells, RT-PCR showed amplification of GnRHR
mRNA.
Conclusions: GnRHR is expressed in differing degrees in
retinoblastomas, but did not correlate with prognostic factors of this
particular tumor. Therefore, GnRHR may be a novel therapeutic
target for the treatment of retinoblastoma. Further studies to analyze
the response of the Y79 cell line to agonist and antagonist drugs are
required to confirm the functionality of this receptor.
Commercial Relationships: Sultan Aldrees, None; Pablo
Zoroquiain, None; Mohammed F. Qutub, None; Sarah Alghamdi,
None; Taylor Nayman, None; Miguel N. Burnier, None
Program Number: 70 Poster Board Number: A0102
Presentation Time: 8:30 AM–10:15 AM
Identification Of Differentially Expressed Protein Targets In
HPV Infected Retinoblastoma Using 2D-DIGE Coupled Mass
Spectrometry Approach
Jasmine Naru1, 2, Ritu Aggarwal2, Ashok K. Mohanty3, Usha Singh4,
Deepak Bansal5, Manoj K. Jena3, Surender Singh3, Nandita Kakkar6,
Navneet Agnihotri1. 1Biochemistry, Panjab University, Chandigarh,
India; 2Immunopathology, Post Graduate Institute of Medical
Education and Research, Chandigarh, India; 3Anima Biotechnology
Center, National Dairy Research Institute, Karnal, India;
4
ophthalmology, Post Graduate Institute of Medical Education and
Research, Chandigarh, India; 5Pediatrics, Post Graduate Institute of
Medical Education and Research, Chandigarh, India; 6histopathology,
Post Graduate Institute of Medical Education and Research,
Chandigarh, India.
Purpose: In India, population based cancer registries have reported
retinoblastoma (RB) among the top five childhood cancers. There are
reports on association of RB with Human Papilloma Virus (HPV)
and abrogation of pRB function. But no reports are available on the
proteomic profile of HPV positive and negative RB. We hypothesize
that HPV may play a role in development of RB with several proteins
differentially expressed in patients and controls that may act as
potential candidates for therapy.
Methods: Fresh RB tumor and normal retinal tissues (n=42each)
were recruited. Screening of 21 different HPV genotypes was done
using HPV genoarray kit. Proteomic analysis was performed on four
samples each from HPV positive, HPV negative RB and controls.
2D-DIGE coupled MALDI- TOF/TOF mass spectrometry was
employed. Dye swapping was done. Image analysis was done using
Decyder 2D software. Differentially expressed spots were identified
and picked. Using PCA, the two tumor groups could be delineated
from normal tissue based on protein expression pattern. mRNA
expression of the identified proteins was verified and some selected
proteins were validated by western blot as well.
Results: Disease was bilateral in 33% cases. Of the 39 eyes with nonfamilial RB, 25.6% tested positive for HR HPV16. Among the three
groups, 102 protein spots were differentially regulated (p<0.05,±1.5
folds) constituted by 39 unique proteins determined by MALDI-TOF/
TOF. 12 proteins were up regulated in HPV positive cases vis-a-vis
HPV negative. Patient group exhibited upregulation of 7 proteins
compared to controls. Highly deregulated proteins were GFAP,
RBP3, CRABP1, SAG and TF. Significant mRNA levels (p<0.05) of
RBP3,GFAP,CRABP1,PDIA3,ATP5B,PITPNA were observed in RB
compared to normal. Gene ontology revealed majority of proteins to
be associated with metabolic processes (26%) and catalytic activity
(38%). Pathway analysis identified to be the proteins involved in
acute phase signalling in tumor(p=1.42-08). Whereas CTTNB1 and
TP53 signalling pathways were more significantly regulated in HPV
positive than HPV negative RB.
Conclusions: The study provides a dynamic protein profile
of retinoblastoma (HPV positive and negative) and highlights
significantly relevant protein targets like GFAP, RBP3 and CRABP1.
Their prospects of being used as potential candidates in therapy needs
to be further explored.
Commercial Relationships: Jasmine Naru, None; Ritu Aggarwal,
None; Ashok K. Mohanty, None; Usha Singh, None; Deepak
Bansal, None; Manoj K. Jena, None; Surender Singh, None;
Nandita Kakkar, None; Navneet Agnihotri, None
Program Number: 71 Poster Board Number: A0103
Presentation Time: 8:30 AM–10:15 AM
Interferon-gamma is required for protecting against intraocular
tumor growth after peripheral immunization but not for the
generation of tumor-specific cytotoxic T-lymphocytes.
Ann J. Ligocki, Jerry Y. Niederkorn. Ophthalmology, UT
Southwestern Medical Center, Dallas, TX.
Purpose: To determine the effect of interferon-gamma loss on ocular
tumor growth after peripheral immunization.
Methods: C57BL/6 or interferon-gamma knock-out mice (IFN-γ KO)
were immunized with the syngeneic Ad5E1 tumor (adenovirus type
5 transformed mouse embryo cells) in the anterior chamber (AC),
subcutaneously (SC), or in a protective model of SC immunization
prior to an AC challenge. Ocular tumor growth was measured by
percent AC occupied with tumor. Spleens were isolated from mice
and tested for tumor-specific cytotoxic T-lymphocytes (CTLs) using a
51
Cr release assay.
Results: AC immunization of tumor cells into C57BL/6 mice resulted
in ocular tumor growth followed by necrotizing immune rejection of
the tumor. However, when the same AC immunization was performed
in IFN-γ KO mice, the tumor grew progressively in the eyes of all
of the mice. SC immunization with the same tumor cells into both
C57BL/6 and IFN-γ KO mice resulted in peripheral tumor rejection.
Furthermore, both C57BL/6 and IFN-γ KO mice generated tumorspecific CTLs post SC immunization. In a protective immunization
model, mice were immunized peripherally with tumor cells prior to
an AC challenge. This SC immunization provided protection against
ocular tumor growth in 79% of wild-type C57BL/6 mice (N=15/19).
Conversely, 100% of the IFN-γ KO mice (N=18/18) developed
progressive ocular tumors. Interestingly, both the C57BL/6 and
IFN-γ KO mice still generated tumor-specific lysis CTL responses
in the periphery, yet only the wild-type C57BL/6 mice rejected their
intraocular tumors.
Conclusions: IFN-γ is critical for immune rejection in this intraocular
tumor model but is not required for the peripheral rejection of these
tumors. Despite the generation of peripheral tumor-specific CTL
responses in both C57BL/6 and IFN-γ KO mice, only an IFN-γ
replete environment produces a protective immune response within
the eye after a peripheral immunization. This suggests that IFN-γ is
required for the protective CTLs to either enter or function within the
eye.
Commercial Relationships: Ann J. Ligocki, None; Jerry Y.
Niederkorn, None
Support: R01 EY05631-28A1
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 72 Poster Board Number: A0104
Presentation Time: 8:30 AM–10:15 AM
Effects of pretreatment with the radioprotector ortho-phosphoL-tyrosine (pTyr) on Rb+/- mice after radiation exposure –
Implication for the treatment of retinoblastoma patients with
radiotherapy
Alexander Tschulakow1, Stephan Huber2, Monika Rittgarn1, HansPeter Rodemann3, Ulrich Schraermeyer1, 4, Sylvie Julien1, 4. 1Section
of Experimental Vitreoretinal Surgery, Center for Ophthalmology,
Tuebingen, Germany; 2Laboratory of Experimental Radiooncology,
Division of Radiooncology, University of Tuebingen, Tuebingen,
Germany; 3Division of Radiobiology & Molecular Environmental
Research, Department of Radiation Oncology, University Hospital,
Tuebingen, Germany; 4STZ OcuTox, Preclinical Drug Assessment,
Hechingen, Germany.
Purpose: Retinoblastoma (Rb) is the most frequent ocular tumor
in children and if let untreated, can cause death. Like the most head
and neck tumors it is sensitive to radiotherapy (RT). However, the
therapy has its risks like damage of healthy tissues recurrence and
development of treatment-induced secondary tumors.
The aim of this study is to investigate the ability of the radioprotector
pTyr to prevent RT-induced secondary tumors and other side-effects
observed after RT.
Methods: B6;129-Rb1tm3Tyj/J mice having a mutation in one of the
Rb -gen allele were used. Although these mice do not develop a Rb,
this model was chosen because Rb-patients having a similar mutation
have a higher risk of developing secondary tumors induced by RT.
One group of mice was treated with intraperitoneal injections of pTyr
16 hours before each irradiation. Another group was only irradiated.
Both groups were irradiated over a period of 3 weeks 3 times a
week with a dosage of 5 Gy per exposure (Fig.1). All animals were
investigated using SLO/OCT and histologically 1, 3, 6 and 9 months
after irradiation (Ir) . Radiation-induced tumor induction as well as
normal tissue radiation toxicity were evaluated as function of pTyrtreatment.
Results: The first visible effect of the Ir was the graying of the hair
coat in the area of Ir. This effect was reduced in the pTyr treated
mice. The results of the OCT- analysis showed that 3 and 6 months
after Ir the thickness of the retina of the mice was significantly
lower in the untreated group (n=12) compared to the pTyr treated
one (n=12) (p<0.05 3 months and p<0.001 6 months after Ir). The
histological analysis of the retina showed a significant photoreceptor
loss in the untreated group vs. the pTyr treated one (p<0.001 3
months and p<0.0001 6 months after Ir) .
Conclusions: Our results show, that the application of pTyr before
irradiation significantly reduces the negative effects of radiation on
the hair coat and retina. The results of the analysis 9 months after Ir,
the appearance of secondary tumors as well as the potential benefit of
the pretreatment with pTyr are currently under investigation.
fig. 1: (a) linear accelarator Linac-G (Phillips), (b) the mouse fixation
units, (c) 6 Rb+/- mice fixed for radiation exposure, (d) scheme of the
assembly of the
experiment.
Commercial Relationships: Alexander Tschulakow, None;
Stephan Huber, None; Monika Rittgarn, None; Hans-Peter
Rodemann, None; Ulrich Schraermeyer, None; Sylvie Julien,
None
Support: DKKS, DKS 2012.08
Program Number: 73 Poster Board Number: A0105
Presentation Time: 8:30 AM–10:15 AM
Invasiveness and metastasis of retinoblastoma in an orthotopic
zebrafish tumor model
Xiaoyun Chen, Wei Xiao, Yizhi Liu. State Key Laboratory of
Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen
University, Guangzhou, China.
Purpose: Retinoblastoma is a highly invasive malignant tumor that
often invades the brain and metastasizes to distal organs through
the blood stream. Invasiveness and metastasis of retinoblastoma can
occur at the early stage of tumor development. However, an optimal
preclinical model to study retinoblastoma invasiveness and metastasis
in relation to drug treatment has not been developed. In this study,
we developed an orthotopic zebrafish model in which retinoblastoma
invasion and metastasis can be monitored at a single cell level.
Methods: Human and mouse retinoblastoma cell lines were
labeled with fluorescent DiI dye before injection. At 48 hours
after fertilization, wild type and fli1: EGFP-transgenic zebrafish
embryos which exhibit green fluorescent signals in blood vessels
were anesthetized and injected about 100-200 retinoblastoma cells
into the vitreous cavities using the Pneumatic PicoPump under
the stereomicroscope. Zebrafish embryos were examined every 2
days under a fluorescent microscope to monitor tumor cell growth,
invasion, metastasis, and the interactions between retinoblastoma
cells and surrounding microvasculatures. Further, to prove this
method can be used to evaluate the efficacy of new therapies,
sunitinib was added directly to the aquaria water after tumor cells
implantation to attain a final concentration of 1 μM, and zebrafish
embryos were examined with a fluorescent microscopy after 4 days.
Results: After 2 days implantation, dissemination of tumor cells from
the primary sites could be detected using fluorescent microscopy.
Total numbers of metastatic foci was progressively increased and
reached the maximal level at day 6 after tumor implantation. Tumor
cells could disseminate to the heads, the lateral healthy eyes and
the tails of zebrafish. Additionally, the primary tumor masses could
stably maintain in the vitreous cavity of zebrafish for 2 days after
implantation, but progressively decreased after 4 days. We also found
that most tumor cells formed clusters around the hyaloid vessels
attached to lens at day 2 after tumor cell implantation. Finally,
treatment with retinoblastoma-bearing zebrafish embryos with 1 μM
of sunitinib could significantly attenuate retinoblastoma invasion and
metastasis.
Conclusions: Thus, this orthotopic retinoblastoma model in zebrafish
offers a new and unique opportunity to study the early events of
tumor invasion, metastasis and drug responses.
Commercial Relationships: Xiaoyun Chen, None; Wei Xiao,
None; Yizhi Liu, None
Program Number: 74 Poster Board Number: A0106
Presentation Time: 8:30 AM–10:15 AM
Kif14 overexpression accelerates tumor development in the TAgRB transgenic model of retinoblastoma
Michael O’Hare1, 3, Shadmand Mehdi1, Timothy W. Corson1, 2.
1
Ophthalmology, Glick Eye Institute, Indianapolis, IN; 2Biochemistry
and Molecular Biology, Indiana University School of Medicine,
Indianapolis, IN; 3Biomedical Science, University of Ulster,
Coleraine, United Kingdom.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Purpose: The mitotic kinesin KIF14 is a molecular motor that
plays a pivotal role in the final stages of cytokinesis. In most
cases of retinoblastoma the KIF14 locus at 1q32.1 is gained as an
important event after mutation of the RB1 gene. Moreover, KIF14
is overexpressed in retinoblastoma, strongly suggesting its role as
an oncogene. Despite this, KIF14’s effects on retinoblastoma in
vivo have not previously been analyzed. We aimed to determine
Kif14’s role in promoting retinal tumor formation using a novel
Kif14 overexpressing, TAg-RB retinoblastoma mouse model.
Understanding the effects of Kif14 overexpression in vivo will allow
for a greater understanding of the biology of post RB1 loss events and
how they contribute to retinoblastoma progression.
Methods: By crossing transgenic mice constitutively overexpressing
Kif14 into the SV40 T-antigen expressing model of retinoblastoma
(TAg-RB) we generated Kif14; TAg-RB double transgenic mice.
The Micron III rodent imaging system was used to obtain fundus
photographs as well as optical coherence tomography (OCT) images.
Double transgenics and TAg-RB littermates were imaged in both eyes
over a time-course to document tumor development.
Results: Compared to the TAg-RB single transgenic mice, the
Kif14; TAg-RB double transgenic mice showed greatly accelerated
formation of tumor-like clusters of hyper-reflective cells in the
inner nuclear layer of the retina from as early as 3-4 weeks of age,
as visualized by OCT. Pale intraretinal tumors were first visible by
funduscopy in the periphery of the retina by week 6 in the Kif14;
TAg-RB mice compared to week 8 in the TAg-RB mice.
Conclusions: The over-expression of the Kif14 oncogene in the
TAg-RB model of retinoblastoma leads to accelerated onset of
tumor formation, providing strong evidence that Kif14 promotes
retinoblastoma formation in vivo. Further investigation will include
quantitative immunohistochemical analysis of these mice to
complement the OCT findings and further validate the importance of
Kif14 for the genesis of retinoblastoma.
Commercial Relationships: Michael O’Hare, None; Shadmand
Mehdi, None; Timothy W. Corson, None
Support: American Cancer Society Institutional Research Grant,
Research to Prevent Blindness, NIH NCATS KL2TR001106
Program Number: 75 Poster Board Number: A0107
Presentation Time: 8:30 AM–10:15 AM
Retinal Toxicity of Intravitreal Melphalan in Albino Rabbit
Shai M. Bar-Sela1, 2, Shiri Zayit-Soudry3, 4, Amir Massarweh5, Anat
Loewenstein1, 2, Ido Perlman5. 1Ophthalmology, Tel Aviv Medical
Center, Tel Aviv, Israel; 2Sackler Faculty of Medicine, Tel Aviv
University, Tel Aviv, Israel; 3Ophthalmology, Rambam Medical
Center, Haifa, Israel; 4Ruth & Bruce Rappaport Faculty of Medicine,
Technion-Israel Inst of Tech, Haifa, Israel; 5Physiology & Biophysics,
Medicine, Technion-Israel Inst of Tech, Haifa, Israel.
Purpose: Intravitreal melphalan injections at doses of 8-30mg have
been successfully used for treating retinoblastoma with vitreous
seeds, but there is insufficient data regarding their safety. This study
was designed to evaluate the toxicity of intravitreal melphalan in a
rabbit model.
Methods: Eighteen albino rabbits were treated with a single
intravitreal melphalan injection (0.1ml) to the right eye (experimental
eye): group 1: 5mg, n=4; group 2: 15mg, n=4; group 3: 30mg, n=5;
group 4: 60mg, n=5. The left eye of each rabbit was injected with
0.1ml saline (control eye). Indirect ophthalmoscopic examination,
electroretinography (ERG) and visual evoked potentials (VEP)
testing were performed at baseline and periodically during
4-week follow-up. After 4 weeks the retinas were prepared for
histological examination and glial fibrillary acidic protein (GFAP)
immunocytochemistry. Analysis of variance for repeated measures
was used for statistical analysis of the electrophysiological
parameters.
Results: Indirect ophthalmoscopy after injections revealed sclerotic
retinal vessels and retinal whitening in the experimental eyes of
groups 2, 3 and 4, but not in rabbits of group 1, injected with the
lowest melphalan dose. Mean (±SD) dark-adapted (DA) ERG b-wave
Vmax ratios (experimental eye/control eye) in groups 1-4, measured
at 4-weeks after injection were 1.12±0.08, 0.77±0.20, 0.42±0.11 and
0±0, respectively, and light adapted (LA) b-wave amplitude ratios
were 1.19±0.18, 0.77±0.27, 0.43±0.24 and 0±0, respectively. Thus,
increasing melphalan dosage significantly predicted reduced DA
b-wave Vmax ratios (p<0.013) and reduced LA b-wave amplitude
ratios (p<0.026), indicating dose-dependent functional retinal damage
induced by melphalan. However, similar flash VEP responses were
found in experimental and control eyes of all groups, suggesting
no melphalan induced functional damage to the optic nerve.
Morphological studies supported the ERG findings demonstrating
retinal structural damage and GFAP expression in Müller cells, at a
magnitude that paralleled the ERG deficit.
Conclusions: Intravitreal melphalan dose of 5μg in rabbits,
approximately equivalent to 10μg in human, appears to be safe
to the retina. However, rabbit doses of 15μg and higher, roughly
corresponding to human doses of 30μg and higher, are toxic, and
their utilization for treating retinoblastoma should be executed with
caution, particularly if visual potential exists.
Commercial Relationships: Shai M. Bar-Sela, None; Shiri ZayitSoudry, None; Amir Massarweh, None; Anat Loewenstein, None;
Ido Perlman, None
Program Number: 76 Poster Board Number: A0108
Presentation Time: 8:30 AM–10:15 AM
Ocular Toxicity of Intravitreal Melphalan for Retinoblastoma
Stephen J. Smith1, Brian Smith2. 1Kellogg Eye Center, Univeristy of
Michigan, Ann Arbor, MI; 2Hematology/Oncology, University of
Rochester, Rochester, NY.
Purpose: To describe the ocular side effects in patients receiving
melphalan intravitreal injection therapy (IViT) for retinoblastoma.
Methods: PUBMED (1946-present), SCOPUS (all years), Science
Citation Index (1900 – present) and Conference Proceedings Citation
Index - Science (1990 – present) electronic databases were searched
to identify all published reports of therapeutic intravitreal injections
for retinoblastoma in humans.
Results: Eleven studies with original melphalan IViT ocular side
effect data were included in this systematic review. In these combined
reports, a total of 1311 intravitreal injections were given to 327
eyes of 317 patients. Melphalan IViT doses ranged from 8 to 50
micrograms. Ocular side effects occurred in 50 patients, with 37
patients experiencing side effects likely secondary to melphalan
toxicity. The proportion of patients experiencing drug related
ocular side effects following melphalan IViT regiments was 0.117
(37/317). Some patients experienced more than one side effect. Drug
related side effects in patients receiving doses ranging from 8 to 40
micrograms included salt and pepper retinopathy, ERG reduction,
iris atrophy, chorioretinal atrophy, and peripheral lens opacity. Four
patients received a single dose of 50 micrograms of melphalan and
developed retinal necrosis and gliosis, choroidal congestion, optic
nerve atrophy, cataract, and retinal neovascularization.
Conclusions: Ocular toxicity following melphalan IViT occurs in
a dose dependent fashion, and can be severe. There appears to be
significant variability in assessing and reporting ocular side effects,
potentially underestimating drug related toxicity in these patients.
Care must be taken in the dosing of intravitreal melphalan treatments
to avoid potentially irreversible vision loss.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Stephen J. Smith, None; Brian Smith,
None
Program Number: 77 Poster Board Number: A0109
Presentation Time: 8:30 AM–10:15 AM
Toxic Effects of Melphalan on Retinal Pigment Epithelial Cells
Sabine Aisenbrey1, 2, Ulrike Hagemann1, Merle Schrader1, 2, Kai
Januschowski1, Sven Schnichels1, Daniela Suesskind1. 1Department
of Ophthalmology, Eberhard Karls University Tuebingen, Tuebingen,
Germany; 2University Eye Hospital, University of Oldenburg,
Oldenburg, Germany.
Purpose: The cytostatic drug Melphalan is widely used in the
treatment of retinoblastoma. Special emphasis lies on superselective
intraarterial and intravitreal chemotherapy with great success
regarding tumor control. Recently, clinical evidence of retinal
pigment epithelial (RPE) atrophy and vascular complications after
intraarterial and intravitreal application of the drug have been
reported in single cases. Facing this background we investigated
cellular toxic effects of Melphalan on RPE in a cell culture model.
Methods: ARPE19 cells were used after reaching 90% confluence.
The effects of Melphalan (4, 10, 20, 50, 100, 166, 200 mg/ml) on cell
morphology via phase contrast microscopy, proliferation using BrdU
assay, cell viability via MTS assay, cell mass estimation using Crystal
violet staining and apoptosis via Caspase 3/7 activity assay were
examined in triplicate. Measurements were carried out after 24h of
incubation time. Staurosporin and the Melphalan diluent were applied
as positive and negative controls, respectively.
Results: Morphologically increasing number and size of gaps were
detectable in the cell layer with increasing Melphalan concentrations.
In parallel proliferation of ARPE19 as well as their cell mass
were decreasing. Cell viability was influenced by Melphalan
concentrations of 50mg/ml and higher. After 24h apoptosis reached a
maximum value with the 100mg/ml Melphalan concentration.
Conclusions: In a cell culture model using ARPE19 cells we
could observe a decrease in proliferative activity, cell amount, and
cell viability of RPE cells as well as an increase in apoptosis after
24h Melphalan incubation suggesting a direct toxic effect of the
chemotherapeutic remedy. A direct toxic effect of Melphalan in
vivo after intraarterial or intravitreal application on the RPE may be
probable and may explain the clinical and angiographic alterations.
Additional cytostatic drugs currently used in retinoblastoma treatment
have to be investigated in this context regarding tumor control versus
toxicity.
Commercial Relationships: Sabine Aisenbrey, None; Ulrike
Hagemann, None; Merle Schrader, None; Kai Januschowski,
None; Sven Schnichels, None; Daniela Suesskind, None
Program Number: 78 Poster Board Number: A0110
Presentation Time: 8:30 AM–10:15 AM
Diagnosing pathological prognostic factors in retinoblastoma:
correlation between traditional microscopy and digital slides
Christina Mastromonaco, Patrick T. Logan, Pablo Zoroquiain, Sarah
Alghamdi, Matthew Balazsi, Miguel N. Burnier. Pathology, Henry C.
Witelson Ocular Pathology Laboratoy, Montreal, QC, Canada.
Purpose: Digital pathology is a tool that converts a microscope
slide into a digital image using a scanner that can be viewed on a
computer screen rather than on a microscope. Whole slide imaging
(WSI) possess the unique feature of having a global view of the
entire eye in order to visualize the relationships between the tumor
and ocular structures. The aim of the present study was to determine
the diagnostic accuracy, using WSI generated by a scanner, of
high-risk prognostic factors and morphological characteristics of
retinoblastomas.
Methods: Forty-seven enucleated eyes with retinoblastoma from the
Henry C. Witelson Ocular Pathology Laboratory, Montreal, Quebec,
were stained with hematoxylin and eosin. Whole slide images at 40×
magnification were reviewed by a pathologist using the Virtuoso
image analyzer, and the following prognostic factors were evaluated:
muticentricity, type of growth, choroidal, anterior chamber, and
optic nerve invasion, rosette formation, necrosis, and Azzopardi
effect. These results were compared with results obtained from the
same pathologist after reviewing the slides in a random order using
a regular microscope as the gold standard. McNemar’s test (MT),
percentage of agreement (POA), and sensibility (S) and specificity
(Sp) were evaluated between WSI and conventional microscopy.
Results: There were no differences with respect to the determination
of multicentricity, type of growth, rosette formation (Homer Wright,
Flexner-Wintersteiner, and fleurettes), choroidal invasion, invasion
of anterior chamber structures, extraocular extension, extension to
the sclera (including vortex vessels), optic nerve invasion (head or
prelaminar, laminar, or postlamellar invasion), necrosis, or Azzopardi
effect between WSI analysis and light microscopy (MT, P = 1.0; POA
= 100%; S = 100%; and Sp = 100%).
Conclusions: To the best of our knowledge, this is the first report
using digital pathology (WSI) to evaluate prognostic factors in eyes
containing retinoblastomas. Using WSI, the pathologist was able
to detect high-risk morphological features in retinoblastoma. WSI
is an important tool now in particular for ophthalmic pathologists
examining enucleation and exenteration specimens. WSI should be
considered as a viable alternative to traditional microscopy in ocular
pathology.
Commercial Relationships: Christina Mastromonaco, None;
Patrick T. Logan, None; Pablo Zoroquiain, None; Sarah
Alghamdi, None; Matthew Balazsi, None; Miguel N. Burnier,
None
Program Number: 79 Poster Board Number: A0111
Presentation Time: 8:30 AM–10:15 AM
PAR-1 and Maspin expression in Retinoblastoma and their
correlation with histopathological prognostic features
nadine marques1, Ana Beatriz T. Dias2, Sarah Alghamdi2, Cristina
Fonseca3, Tânia Borges4, Miguel N. Burnier2. 1ophthalmology,
hospital garcia de orta, Lisbon, Portugal; 2Ocular pathology, Mcgill,
Montreal, QC, Canada; 3Ophthalmology, Centro hospitalar de
Coimbra, Coimbra, Portugal; 4Ophthalmology, Centro hospitalar do
Porto, Porto, Portugal.
Purpose: Maspin is a tumor suppressor protein expressed in normal
mammary and other epithelial cells and is reduced or absent in
breast, ovarian carcinomas, and gliomas. Protease activated receptor
1 (PAR-1) is related to tumor growth and metastatic potential.
Maspin expression, when co-expressed with PAR-1, can counteract
its malignant potential, as the maspin gene is downstream of PAR-1
signaling. Our purpose was to evaluate, for the first time, maspin and
PAR-1 expression in retinoblastomas and in normal retinas, and to
correlate them with histopathological prognostic features.
Methods: Maspin and PAR-1 expression were evaluated in 40
retinoblastoma eyes. Nine normal human eyes from the Eye Bank of
Canada were used as controls. Positive controls were skin for maspin
and pancreas for PAR-1. Maspin and PAR-1 immunostains were
evaluated using a score considering extent of tumor/structure staining
(0=none, 1=<50% and 2=>50%) and intensity (0=none, 1=intensity <
positive controls, 2=intensity > positive controls). A total final score
ranging between 0 and 4 was established (final score = proportion
x intensity of staining). Invasive phenotype was considered when
invasion of the optic nerve or choroid were present. Fisher exact and
Student’s t-test analyses was performed to compare variables.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Results: A maspin score of 0 (neither nuclear nor cytoplasmic
expression) correlated with invasive phenotype (P=0.03). No
difference in invasive phenotype was found for isolated PAR-1 or
PAR-1 without maspin expression in the same tumor. In normal eyes,
no nuclear or cytoplasmic maspin staining in the retina was found.
Both retinal plexiform layers showed PAR-1 expression in 6 of the
same samples (4 with score 2 and 2 with score 4). No differences in
PAR-1 score between normal retinas and retinoblastoma were found.
Conclusions: Absence of maspin expression is associated with
more aggressive biological behavior in this study. PAR-1 expression
was found only in plexiform layers of normal retinas and does
not differ according to retinoblastoma expression score. Maspin
is not expressed in the normal retina. The identification of maspin
suggests that it may be a novel therapeutic target for aggressive
retinoblastomas.
Commercial Relationships: nadine marques, None; Ana Beatriz
T. Dias, None; Sarah Alghamdi, None; Cristina Fonseca, None;
Tânia Borges, None; Miguel N. Burnier, None
Program Number: 80 Poster Board Number: A0112
Presentation Time: 8:30 AM–10:15 AM
Rates of Pineal Cysts Detected by MRI Associated with
Successful Intravenous Chemotherapy Treatment of Bilateral
Retinoblastoma
Felix Y. Chau, Kai B. Kang, Michael P. Blair, Michael Shapiro.
Ophthalmology & Visual Sciences, University of Illinois at Chicago UIC, Chicago, IL.
Purpose: Retinoblastoma (RB) treatments include enucleation,
radiotherapy, cryotherapy, laser photocoagulation and chemotherapy.
Pinealoblastomas are known to potentially occur in germline RB,
most often with bilateral RB (“trilateral RB”), and may regress to
pineal cysts with systemic chemotherapy. The purpose of this study
is to evaluate rates of pinealoblastomas and pineal cysts detected on
magnetic resonance imaging (MRI) in bilateral RB patients treated
with intravenous chemotherapy (vincristine, etoposide, carboplatin VEC) with focal consolidation or enucleation as primary therapy.
Methods: Retrospective review of digital fundus images and
medical records of RB patients who presented to the University of
Illinois at Chicago, Illinois Eye and Ear Infirmary Retina Clinic from
November 1, 2004 to November 1, 2014.
Results: 34 patients received treatment over the study period. 18
patients had unilateral RB, and 16 had bilateral RB. The mean age
at diagnosis was 16 months. No pinealoblastomas were detected by
MRI in any RB patient. Pineal cysts detected by MRI occurred in
none (0%) of the 18 unilateral patients and 2 of 16 (12.5%) bilateral
patients. All bilateral RB patients received 6 cycles of VEC. One
bilateral RB patient (International Classification of Retinoblastoma
– ICRB Group B or C in both eyes) was diagnosed at age 12 months
and developed pineal enhancement at age 18 months (within the last
month of 6 rounds of VEC) and a pineal cyst at age 21 months, 3
months after completing VEC. The other bilateral RB patient (ICRB
Group E OD [enucleated], Group C or D OS) was diagnosed at age 7
months and had a pineal cyst detected at age 26 months, 16 months
after completing VEC.
Conclusions: The rate of pinealoblastoma detected on MRI was 0%
in bilateral RB patients receiving VEC. Pineal cysts were detected by
MRI in 12.5% of bilateral RB patients receiving VEC. These findings
may confirm the successful prevention of pinealoblastoma growth by
systemic intravenous vincristine, etoposide, and carboplatin treatment
in patients with bilateral retinoblastoma.
Commercial Relationships: Felix Y. Chau, None; Kai B. Kang,
None; Michael P. Blair, None; Michael Shapiro, None
Support: Research to Prevent Blindness; K12 EY021475.
Program Number: 81 Poster Board Number: A0113
Presentation Time: 8:30 AM–10:15 AM
Identification of RB1 gene germline mutations in Mexican
patients with sporadic unilateral retinoblastoma
Dalia C. Guadarrama Vallejo1, Juan C. Zenteno1, 2, Arturo Flores
Cuevas1. 1Genetics- retina, Instituto de Oftalmologia Conde de
Valenciana, Mexico city, Mexico; 2Biochemistry, Faculty of
Medicine, National Autonomous University of Mexico, Mexico, city,
Mexico.
Purpose: Purpose: to determinate the percentage of cases with
sporadic unilateral retinoblastoma carrying RB1 gene germline
mutations in a sample of Mexican patients.
Methods: Methods: an observational, cross-sectional and descriptive
study was performed; patients with sporadic (non-familial) unilateral
retinoblastoma evaluated between March 2005 and December 2011
in a reference center in Mexico City were selected. Inclusion criteria
were both genders, older than 12 months, and unilateral tumor.
Patients with a family history of fibrosarcoma, lymphoma, leukemia,
or melanoma were excluded. Patients developing contralateral
retinoblastoma during the study were eliminated.
Genomic DNA was obtained from peripheral blood leukocytes in
each patient; PCR amplification and direct nucleotide sequencing
of the 27 exons and exon/intron junctions of the RB1 gene was
performed. Possible gross gene deletions or duplications were
investigated by means of MLPA (Multiplex Ligation-Dependent
Probe Analysis). In addition, promoter sequencing and DNA
methylation analyses were performed. Potential pathogenicity of
novel mutations was analyzed with PolyPhen software.
Results: Results: Twenty (9 female and 11 male) Mexican patients
with sporadic unilateral retinoblastoma were included; average age
at diagnosis was 26.3 months. Germline mutations were identified
in two patients (10%): a mutation in exon 17 predicting a nonsense
mutation at residue 533 and a mutation in exon 20 predicting a
p.R661W missense mutation. Parental DNA’s of these two cases were
negative for the mutations, indicating de novo origin. No mutations in
exonic or promoter regions or methylation of the RB1 promoter were
demonstrated in the remaining 18 patients.
Conclusions: Conclusion: This is the first study in Mexican
population using a number of molecular diagnostic tests to identify
RB1 germline defects in unilateral retinoblastoma. Our results
contrast with figures from other populations showing up to 20% of
RB1 mutations in sporadic unilateral retinoblastoma. Our results
are of extreme importance for genetic counselling in this group of
retinoblastoma patients and their families.
Commercial Relationships: Dalia C. Guadarrama Vallejo, None;
Juan C. Zenteno, None; Arturo Flores Cuevas, None
Program Number: 82 Poster Board Number: A0114
Presentation Time: 8:30 AM–10:15 AM
Retinoblastoma in South Africa – A 20-year retrospective study at
two tertiary academic hospitals in Johannesburg
Saadiah Goolam, Nicky D. Welsh, Ismail Mayet. Ophthalmology,
University of Witwatersrand, Johannesburg, South Africa.
Purpose: To characterise retinoblastoma in the South African
population through a 20-year review of patient records at two tertiary
academic hospitals in Johannesburg
Methods: Retrospective clinical case series analysis of medical
records of patients with retinoblastoma presenting to Charlotte
Maxeke Johannesburg Academic Hospital and Chris Hani
Baragwanath Academic Hospital between 01 January 1992 and 31
December 2011
Results: The total number of cases identified was 282, with 245
meeting the study inclusion criteria. Retinoblastoma comprised 6.9%
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
of total pediatric oncology presentations. 65.3% were unilateral,
34.3% bilateral and 0.4% trilateral. The overall male to female
ratio was 1.08. Mean age at presentation overall was 32.6 months
(median 28.0 months), unilateral 39.4 months (median 33.0 months)
and bilateral 19.7 months (median 17.0 months). The mean delay to
presentation overall was 7.0 months (median 4.0 months), unilateral
8.5 months (median 5.0 months) and bilateral 4.4 months (median 3.0
months). The most frequent presenting symptoms were leukocoria
(37.1%) and proptosis (34.7%). Distribution of disease stage at
presentation (International Retinoblastoma Staging System) was
1.6% with Stage 0, 24.1% with Stage I, 27.8% Stage II, 16.3% Stage
III and 25.3% Stage IV. 26.5% of patients defaulted care. The fiveyear survival rate using the Kaplan-Meier survival curve was 57.7%
in the overall study population, and according to disease stage at
presentation: 95.3% - Stage I, 84.8% - Stage II, 49.7% - Stage III and
5.7% - Stage IV
Conclusions: This study showed that delay in presentation of
retinoblastoma cases remains a significant barrier to effective
treatment in this African setting. Intervention to streamline referrals
is indicated and may include outreach programs and education of
referring hospitals
Commercial Relationships: Saadiah Goolam, None; Nicky D.
Welsh, None; Ismail Mayet, None
Program Number: 83 Poster Board Number: A0115
Presentation Time: 8:30 AM–10:15 AM
Development and Delivery of Candidate Retinoblastoma
Therapeutics
Eleanor M. Pritchard1, 2, Rachel Brennan5, Lyra Griffiths2,
Elizabeth Stewart2, Cori Bradley2, Burgess B. Freeman3, William
Caufield3, Michael A. Dyer2, 4, R K. Guy1. 1Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN;
2
Developmental Neurobiology, St. Jude Children’s Research Hospital,
Memphis, TN; 3Preclinical Pharmacokinetics Shared Resource,, St.
Jude Children’s Research Hospital, Memphis, TN; 4Department of
Ophthalmology, University of Tennessee Health Sciences Center,
Memphis, TN; 5Oncology, St. Jude Children’s Research Hospital,
Memphis, TN.
Purpose: While mortality is low with aggressive multimodal therapy,
partial or full loss of vision occurs in approximately 50% of patients
with advanced bilateral retinoblastoma. There is an urgent need to
develop targeted therapies that preserve vision and reduce late effects
of current treatment modalities, which include facial malformations
and increased incidence of secondary malignancies. Candidate
therapeutics should ideally (1) completely clear disease to prevent
progression and metastasis, (2) be well tolerated locally to preserve
vision, and (3) act quickly to overcome rapid clearance from the eye.
Phase I and II clinical trials for retinoblastoma are difficult due to the
young age of the patient population and relative rarity of the disease.
Therefore robust preclinical testing of new therapies is critical.
Methods: To meet this need, we conducted library screening of
approximately 300 compounds using focused libraries from St.
Jude Children’s Research Hospital (SJCRH) chemical collection
to determine in vitro potency in retinoblastoma cell lines (RB355,
Weri, and Y79) and a normal human fibroblast control cell line (BJ).
Further in vitro testing was conducted to prioritize “hits” (compounds
active in retinoblastoma cells, but not BJ cells). For lead compounds,
we determined speed of effect with washout studies and cidality
with outgrowth studies. Synergy testing was performed to eliminate
candidates with antagonistic interactions with current standard of
care retinoblastoma drugs and prioritize any with strong agonism. We
characterized pharmacokinetics (PK) to compare intraocular exposure
for different routes of delivery (local and systemic) for high priority
candidates.
Results: Screening identified several candidates to advance to the
preclinical testing phase, including histone deacetylase (HDAC)
inhibitors and FDA-approved oncology drugs that might be
repurposed for ocular use with local delivery methods. Consistent
with treatment goals and the unique constraints of reaching an
intraocular target, we prioritized HDAC inhibitors with rapid, cidal,
potent and selective effects on retinoblastoma cells in vitro.
Conclusions: In vitro and PK testing suggest local delivery of HDAC
inhibitors represents a promising potential targeted therapy for
retinoblastoma. Next steps will include toxicity and efficacy testing in
preclinical animal models of retinoblastoma.
Commercial Relationships: Eleanor M. Pritchard, None;
Rachel Brennan, None; Lyra Griffiths, None; Elizabeth Stewart,
None; Cori Bradley, None; Burgess B. Freeman, None; William
Caufield, None; Michael A. Dyer, None; R K. Guy, None
Support: Knights Templar Eye Foundation (KTEF) Pediatric
Ophthalmology Career Starter Grant
Program Number: 84 Poster Board Number: A0116
Presentation Time: 8:30 AM–10:15 AM
Safety and efficacy of digoxin as a potential candidate agent for
retinoblastoma treatment
URSULA A. WINTER6, Emiliano Buitrago6, Hebe Mena1, Soledad
Negrotto1, Maria J. del Sole2, Hakim Djaballah3, Juan O Croxatto4,
Guillermo L. Chantada7, David H. Abramson5, Paula Schaiquevich6.
1
Experimental Thrombosis Laboratory, Institute of Experimental
Medicine (IMEX), National Academy of Medicine-CONICET,
Capital Federal, Argentina; 2Pharmacology Laboratory, CIVETANCONICET, Faculty of Veterinary, National University of the Centre
of Buenos Aires, Tandil, Argentina; 3HTS Core Facility, Memorial
Sloan-Kettering Cancer Center, New York, NY; 4Argentinean
Ophthalmic Foundation Jorge Malbrán, Buenos Aires, Argentina;
5
Ophthalmic Oncology Service, Memorial Sloan-Kettering Cancer
Center, New York, NY; 6Clinical Pharmacokinetic Unit, Hospital
de Pediatria JP Garrahan, Buenos Aires, Argentina; 7Service of
Hematology-Oncology, Hospital de Pediatria JP Garrahan, Buenos
Aires, Argentina.
Purpose: Despite recent advances of local routes for chemotherapy
delivery there have been few agents incorporated in the
chemotherapy armamentarium for retinoblastoma treatment with
almost no new schedules for drug administration. This study assessed
the anti-tumor and antiangiogenic effect of digoxin in vitro under
conventional and protracted schedules and the ocular and systemic
toxicity of repeated intravitreal injections in rabbits.
Methods: Two retinoblastoma (Y79, WERI-RB1) and three
endothelial cell types (HMEC, HUVEC, EPC) were exposed
to increasing concentrations of digoxin after a single (1 day) or
protracted (7 days) treatment fashion. Cytotoxicity was assessed with
a vital dye and induction of apoptosis and the cell-cycle status were
evaluated by flow cytometry. A cohort of 4 New Zealand rabbits (1.82.2 kg) received 4 bi-weekly doses of 1μg of intra-vitreal digoxin
and the same volume (0.1 ml) of vehicle into the fellow eye. Animal
controls included clinical, hematological and ocular evaluations
(fundoscopy and electroretinograms). After 2 weeks of the last
dose rabbits were euthanized and samples were obtained for retinal
histology.
Results: Digoxin was cytotoxic in retinoblastoma and endothelial
cells after a single exposure. Single and protracted treatments of all
five cell types did not show a significant difference in terms of the
IC50 (p>0.05). Both treatment schedules with digoxin at the IC50
induced apoptosis and cell cycle arrest at G0. Retinal toxicity was
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
evident after the third intravitreal dose of digoxin with statistical
changes of the ERG components against the control eye and
considerable but local histologic damage of the retinas.
Conclusions: Digoxin showed cytotoxic effects in retinoblastoma
cell lines while exerting an antiangiogenic activity in vitro at similar
concentrations. A protracted treatment was assessed in retinoblastoma
cells without an advantage in terms of the dose for antitumor
effect with respect to single dose. Despite promising antitumor and
antiangiogenic activity in vitro four bi-weekly injections of digoxin
lead to significant but local toxicity to the retina of rabbits. Thus, a
counterbalance between efficacy and toxicity should be taken into
account if translated into the clinics for retinoblastoma treatment.
Commercial Relationships: URSULA A. WINTER, None;
Emiliano Buitrago, None; Hebe Mena, None; Soledad Negrotto,
None; Maria J. del Sole, None; Hakim Djaballah, None; Juan
O Croxatto, None; Guillermo L. Chantada, None; David H.
Abramson, None; Paula Schaiquevich, None
Support: This work was supported by 2013 ARVO/Merck
Collaborative Research Fellowship; Agencia Nacional de Promoción
Científica-FONCYT (PICT Bicentenario, 2010-2271); Fund for
Ophthalmic Knowledge (GLC, ACF), New York, NY, USA and
Fundación Natalie D Flexer de Ayuda al Niño con Cáncer (GLC and
ACF), Buenos Aires, Argentina.
Program Number: 85 Poster Board Number: A0117
Presentation Time: 8:30 AM–10:15 AM
Orbital Retinoblastoma: Enucleation vs. Ophthalmic Artery
Chemosurgery for Advanced Intraocular Retinoblastoma
Nicolas A. Yannuzzi1, Jasmine H. Francis1, Brian P. Marr1, Irina
Belinsky1, Ira Dunkel1, Y. Pierre Gobin2, David H. Abramson1.
1
Ophthalmology, Memorial Sloan-Kettering Cancer Center, New
York, NY; 2Interventional Radiology, Weill Cornell Medical College,
New York, NY.
Purpose: To determine the incidence of orbital recurrence following
enucleation and ophthalmic artery chemosurgery (OAC) as primary
treatments for advanced stage retinoblastoma.
Methods: Single center retrospective study of 73 patients (74
eyes) of Reese-Ellsworth group V, or International Classification of
Retinoblastoma group D or E primarily treated with enucleation and
76 patients (90 eyes) primarily treated with OAC. Endpoints analyzed
were the development of orbital disease, incidence of metastasis, and
death from metastatic retinoblastoma.
Results: There were 6 orbital recurrences (incidence 8.1%) in the in
the primary enucleation group and 1 orbital recurrence (incidence
1.1%) in the primary OAC group during median follow up times
of 32.2 months (range 0.1-97.1) and 36.8 months (range 3.0-104.3)
respectively. The 24-month Kaplan Meier estimate for orbital
recurrence free survival was significantly worse for the enucleation
group 92.6% (95% C.I. 83.2-96.8) than for the OAC group 98.5%
(95% C.I. 89.9-99.7), Log-Rank p-value = 0.02. The enucleation
group had 6 cases of metastatic disease and 2 deaths representing
8.2% and 2.7% of patients respectively. In the OAC group, there
were 3 (3.9%) cases of metastatic disease and 0 deaths. Kaplan Meier
analysis of metastasis free survival and overall survival yielded no
statistically significant differences between the two treatment groups.
Analysis of a large number of features of the two groups suggested
no difference except more rubeotic eyes in the enucleated group
(24%) than in the OAC group (6%).
Conclusions: In this single institution retrospective study of
advanced intraocular retinoblastoma there was significantly more
orbital retinoblastoma in the primarily enucleated group. OAC for
advanced intraocular retinoblastoma does not increase the chance
of orbital recurrence or metastatic disease compared to primary
enucleation.
Commercial Relationships: Nicolas A. Yannuzzi, None; Jasmine
H. Francis, None; Brian P. Marr, None; Irina Belinsky, None; Ira
Dunkel, None; Y. Pierre Gobin, None; David H. Abramson, None
Program Number: 86 Poster Board Number: A0118
Presentation Time: 8:30 AM–10:15 AM
Rates and Features of Neovascularization Associated
with Successful Intravenous Chemotherapy Treatment of
Retinoblastoma
Kai B. Kang, Michael Shapiro, Michael P. Blair, Felix Y. Chau.
Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary,
Chicago, IL.
Purpose: Retinoblastoma (RB) treatments include enucleation,
radiotherapy, cryotherapy, laser photocoagulation and chemotherapy.
Certain features of RB may be safely monitored without further
treatment. The purpose of this study is to evaluate rates and describe
features of retinal neovascularization associated with successful
intravenous chemotherapy and focal consolidation of tumors with
laser or cryotherapy.
Methods: Retrospective review of digital fundus images and
medical records of RB patients who presented to the University of
Illinois at Chicago, Illinois Eye and Ear Infirmary Retina Clinic from
November 1, 2004 to November 1, 2014. Only surviving eyes that
received treatment for RB with a minimum length of follow up of one
year were included.
Results: 50 eyes of 34 patients received treatment over the study
period. 18 patients had unilateral RB, and 16 had bilateral RB. The
mean age at diagnosis was 16 months. Of the 50 eyes, 22 were
enucleated upon diagnosis. Most (86%) of the enucleated eyes were
classified as International Classification of Retinoblastoma (ICRB)
Group E. Twenty-five eyes received intravenous chemotherapy
consisting of six cycles of carboplatin, etoposide and vincristine
as the primary treatment. Twenty-two (88%) eyes (with 2 in ICRB
group A, 6 in Group B, 10 in Group C and 4 in Group D) showed
no tumor progression over the study period. Neovascularization was
identified in 3 eyes (14% of 22 eyes) in 3 patients. These eyes were
monitored closely without further treatment. The three eyes showed
no tumor growth over respective periods of 9.1 years, 3.2 years, and
1.2 years. All 3 were associated with calcified regression, and 2 with
preretinal fibrosis and subretinal fluid (one with inferior detachment
successfully treated with scleral buckle).
Conclusions: The rate of retinal neovascularization in eyes with
successful intravenous chemotherapy was approximately 14%.
Neovascularization associated with retinoblastoma may be safely
monitored without the need for further treatment if there are no signs
of tumor progression.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
A) Fundus photos illustrating regressed tumor with calcified remnants
after chemotherapy and laser with neovascularization and fibrosis
from 2005 to 2009
Purpose: Melanin is an abundant endogenous chromophore in the
iris, ciliary body and choroid of the eye, altered in many diseases
such as macular degeneration and eye tumors. Here we attempt
to evaluate melanin distribution in intact eyes using a novel
photoacoustic imaging tool.
Methods: Following St. Michael’s Hospital Research Ethics Board
approval, we imaged whole eyes by photoacoustic imaging (MSOT
inVision128, iThera Medical Inc., Germany), constructed with a
Nd:YAG laser (1064/532 nm) with 8 ns pulse length and 10 Hz pulse
frequency coupled to optical parameter oscillator. Five minipig eyes
and three human eyes (Human Eye Biobank for Research) fixed in
10% formalin were embedded in 1.5% agar and placed in a clear
plastic bag with distilled water in the imaging chamber. Images were
taken in 300mm steps with wavelengths from 680-980 nm in 5 nm
steps, and 4 averages per scan. Spectral unmixing was performed
using a linear regression algorithm with spectra for deoxygenated and
oxygenated hemoglobin and for melanin (Fig. 1A), providing images
of entire eye sections. Known concentrations of synthetic melanin
in 1.5% agar were used to determine the relationship between
concentration of melanin and signal intensity.
Results: In all human eyes, melanin was detected by photoacoustic
imaging in the iris, ciliary body and choroid (Fig. 2A, melanin in
green). In all Yucatan minipig eyes, in addition to the iris, ciliary
body, and choroid, melanin was observed in optic nerve head (Fig.
2B, melanin in green). Histological examination of the eyes was
used to confirm the location of melanin rich eye tissues. A linear
relationship was found between the concentration of synthetic
melanin and photoacoustic signal intensity (R2 = 0.99, Fig. 1B).
Conclusions: Melanin distribution was reliably detected by
photoacoustic imaging in whole intact human and minipig eyes.
The linear relation between melanin and photoacoustic signal
intensity supports the potential use for melanin quantification in eye
specimens. Photoacoustic imaging may be useful for qualitative and
quantitative assessment of the enucleated eye with uveal melanoma,
including extraocular extension, and other eye diseases.
B) Fundus photographs of the same eye illustrating continued
neovascularization with fibrosis and adjacent calcified remnants
without further tumor growth from 2011 to 2014
Commercial Relationships: Kai B. Kang, None; Michael Shapiro,
None; Michael P. Blair, None; Felix Y. Chau, None
149 New challenges in anatomy
Sunday, May 03, 2015 3:15 PM–5:00 PM
1AB Mile High Blrm Paper Session
Program #/Board # Range: 896–901
Organizing Section: Anatomy and Pathology/Oncology
Figure 1: Absorption spectra of chromophores used in spectral
unmixing (A) and photoacoustic signal at known concentrations of
synthetic melanin (B).
Program Number: 896
Presentation Time: 3:15 PM–3:30 PM
Melanin Distribution in Intact Human and Minipig Eyes
Detected by Photoacoustic Imaging
Shireen Khattak1, Neeru Gupta1, 2, Clinton Hupple3, Yeni H. Yucel1,
2 1
. Keenan Research Centre for Biomedical Science, St. Michael’s
Hospital, Toronto, ON, Canada; 2Ophthalmology & Vision Sciences,
Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, ON, Canada; 3iThera Medical GmbH, Munich, Germany.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
state in aniridic eyes due to elevated Wnt signaling, which may lead
to AFS following surgery.
Commercial Relationships: Yichen Wang, None; Yan Wang,
None; Christopher Riemann, None; Melinda K. Duncan, None
Support: NIH Grant EY015279
Figure 2: Photoacoustic images of human eye (A) and minipig eye
(B) with melanin signal in green.
Commercial Relationships: Shireen Khattak, None; Neeru Gupta,
None; Clinton Hupple, iThera Medical (E); Yeni H. Yucel, None
Support: Canadian Foundation for Innovation, Dorothy Pitts Fund,
Nancy and Thor Eaton Fund, Henry Farrugia Fund
Program Number: 897
Presentation Time: 3:30 PM–3:45 PM
Molecular Mechanisms of Aniridia Fibrosis Syndrome (AFS)
Yichen Wang1, Yan Wang1, Christopher Riemann2, Melinda K.
Duncan1. 1Biological Sciences, University of Delaware, Newark, DE;
2
Cincinnati Eye Institute, Cincinnati, OH.
Purpose: Congenital aniridia (CI) is defined as iris hypoplasia at
birth, and often results from PAX6 mutations/deletions. Surgical
interventions for common CI sequala such as cataract, glaucoma,
and keratopathy may be complicated by AFS which can lead to
devastating fibrotic complications. While little is known about the
pathogenesis of AFS, previous studies showed that Pax6 negatively
regulates Wnt signaling, the chronic upregulation of which can drive
fibrosis. Thus, this work tests the hypothesis that haploinsufficiency
of Pax6 leads to the upregulation of Wnt signaling, which may
contribute to a pro-fibrotic state, resulting in AFS following surgeries.
Methods: Immunofluorescence (IF) staining was used to characterize
human AFS samples by detecting the expression of fibrotic markers.
Penetrating central corneal wounding was performed in Pax6+/tm1Pgr
and wildtype (WT) mice to study fibrotic responses at day 5/9 postsurgery, followed by IF staining to detect the expression of fibrotic
markers. To study the signaling pathways, the activation state of Wnt
and TGF-β signaling was measured by the levels of β-catenin and
pSMAD3 respectively.
Results: In human AFS samples, abundant cells expressing
myofibroblast markers were found, confirming that AFS is a classic
fibrotic disease. Further, both β-catenin and pSMAD3 was detected,
indicating the activation of Wnt and TGF-β signaling. In unoperated
Pax6+/tm1Pgr mice, elevated levels of β-catenin were observed in
pockets of spontaneous fibrosis, suggesting the chronic elevation
of Wnt signaling. After surgery, Pax6+/tm1Pgr mice displayed severe
fibrosis at the injury site. They also developed distal fibrosis at the iris
root, which was absent in WT mice. Higher levels of β-catenin and
pSMAD3 were found in Pax6+/tm1Pgr mice at both the injury site and
iris root compared to WT.
Conclusions: AFS is a classic fibrotic disease. Chronic upregulation
of Wnt signaling was confirmed in unoperated Pax6+/tm1Pgr mice.
Compared to WT, Pax6 mutant mice exhibited increased fibrosis
along with higher levels of β-catenin and pSMAD3 post-surgery.
These data suggest that Pax6 haploinsufficiency leads to a pro-fibrotic
Program Number: 898
Presentation Time: 3:45 PM–4:00 PM
Analysis of the volumetric relationship among human ocular,
orbital and visual cortical anatomy
Michael P. Masters1, Emiliano Bruner2, Sarah Queer1, Sarah
Traynor3, Jess Senjem3. 1Montana Tech, Butte, MT; 2Centro Nacional
de Investigación sobre la Evolución Humana, Burgos, Spain;
3
University of Wisconsin, Madison, WI.
Purpose: Recent research on the visual system has examined the
volumetric relationship among the eye, orbit, and visual cortex in
humans. Some studies have also suggested that light levels may drive
eye size, which was hypothesized to in turn dictate orbital and visual
cortical size, as a product of adapting or acclimating to differences
in available daylight at disparate latitudes. However, further research
is necessary in order to establish how these variables are related, and
to what extent ocular volume influences orbital and visual cortical
volume in humans.
Methods: Relationships among these anatomical components were
investigated using MRIs from a large sample of 83 individuals, which
also included each subject’s height, age, sex, and uncorrected visual
acuity scores. Frontal and occipital gyri volumes were calculated
using two different cortical parcellation tools, Brain Parser 56 ROI
and FreeSurfer 5.3.0, in order to provide a better understanding
of how the eye and orbit vary in relation to the visual cortex, and
in association with cerebral gyri of the frontal cortex that are not
directly related to vision.
Results: Results indicated that ocular and orbital volumes were
weakly correlated, but that eye volume explains only 14.7% of the
variance in orbital volume. Ocular and orbital volumes were also
found to be equally, and in most cases, more highly correlated with
five frontal lobe gyri than with occipital lobe gyri associated with V1,
V2, and V3 of the visual cortex. Additionally, after accounting for
age, sex, visual acuity, and body size differences, the relationships
between eye and visual cortical volumes were no longer statistically
significant for all variables using Brain Parser 56 ROI, and for all
except the lingual gyrus using FreeSurfer 5.3.0. The relationship
between orbital and visual cortical volumes remained significant
for a number of occipital lobe gyri even after accounting for these
cofactors, however it was again found to be equally, and often more
highly correlated with the frontal lobe gyri than with occipital lobe
gyri involved in visual processing.
Conclusions: These results indicate that eye volume explains only a
small amount of variation in orbital and visual cortical volume, and
that the eye and orbit are generally more structurally associated with
the frontal lobes than they are functionally associated with the visual
cortex of the occipital lobes.
Commercial Relationships: Michael P. Masters, None; Emiliano
Bruner, None; Sarah Queer, None; Sarah Traynor, None; Jess
Senjem, None
Support: Montana INBRE - National Institute of General Medical
Sciences of the National Institutes of Health, award number 8 P20
GM103474-12
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 899
Presentation Time: 4:00 PM–4:15 PM
Cell−ECM interactions during formation of the zebrafish hyaloid
vasculature
Andrea Hartsock, Victoria Arnold, Jeffrey Gross. Biological Sciences,
University of Texas, Austin, Austin, TX.
Purpose: Vasculature formation requires an orchestrated series of
morphogenetic changes to generate an integrated vessel system.
Previous work by our laboratory demonstrated there are three stages
of hyaloid development in zebrafish: Stage I- arrival of hyaloid cells
at the lens and formation of the hyaloid loop, Stage II- formation
of a branched hyaloid network, and Stage III- refinement of the
hyaloid network (Hartsock et al., 2014). The lens is not required for
recruitment of hyaloid precursor cells, but is required for Stage II and
III development and maturation. The lens is surrounded by the lens
capsule, an ECM-rich basement membrane. It is not known how the
ECM of the lens capsule contributes to hyaloid formation. Here, we
test the hypothesis that distinct cell-ECM interactions play an integral
role in building the hyaloid.
Methods: Fixed sample and in vivo time lapse imaging of fli1a:GFP
(a hyaloid marker) were performed in a variety of zebrafish lines
possessing mutations in ECM components or their cellular interaction
partners: lamining1 (lamc1), fibronectin1b (fn1b), integrin a5 (itga5),
and integrin b1 (itgb1). All images were processed as maximum
projections via ImageJ.
Results: in vivo time-lapse imaging revealed that mutations in all
cell-ECM components resulted in Stage II and III hyaloid defects.
Unique defects were identified in each mutant. For example, Stage II
hyaloid network branching was disrupted in lamc1 and fn1b mutants;
fn1b mutants possessed clumps of vascular precursor cells on the
lens that did not organize into mature vessels. itga5 mutants also
possessed Stage II defects, with vessels that were reduced in branch
number and thickness. Stage III, refinement of the vascular network
appeared normal in itga5 and itgb1 mutants, but not in fn1b or lamc1
mutants.
Conclusions: Analysis of hyaloid formation in cell-ECM component
mutants revealed requirements for these proteins during hyaloid
development. Mutations in ECM components (fn1b, lamc1) were
more severe, likely affecting multiple cellular interacting partners.
Conversely, hyaloid defects in mutants affecting the interacting
partners (itga5, itgb1) were more limited, suggesting specific roles
for these in distinct phases of hyaloid development. Further analyses
of downstream regulators of these cell-ECM pathways will generate
a comprehensive understanding of the cellular underpinnings of
hyaloid morphogenesis during embryonic eye development.
Commercial Relationships: Andrea Hartsock, None; Victoria
Arnold, None; Jeffrey Gross, None
Support: F32 EY023910
Program Number: 900
Presentation Time: 4:15 PM–4:30 PM
Identification of a novel cause of ocular coloboma
Sonya Widen1, Prajakta Desai2, Mika Asai-Coakwell2, Matthew
Benson2, Curtis French2, Ordan J. Lehmann2, Andrew Waskiewicz1.
1
Biological Sciences, University of Alberta, Edmonton, AB, Canada;
2
Medical Genetics, University of Alberta, Edmonton, AB, Canada.
Purpose: Ocular coloboma results from the incomplete fusion of
the optic fissure and is a major cause of pediatric vision loss. We
investigated a microphthalmia, anophthalmia and coloboma (MAC)
cohort to advance understanding of these disorders’ genetic etiology.
Methods: Exomic next generation sequencing (NGS) was performed
in a large coloboma pedigree and the mutated gene was subsequently
screened in 150 MAC DNA samples. Luciferase reporter assays,
western blots and in silico ANOLEA modeling were used to
investigate the pathogenicity of the identified mutations. Zebrafish
morpholino (MO) inhibition was used to investigate gene function
together with in situ hybridization (ISH), and analysis of transgenic
GFP reporter lines.
Results: NGS identified a BMP3 mutation in affected individuals
of the MAC pedigree. The identified variant, A470P, alters a residue
that is invariant across vertebrates. Four additional variants were
identified in the larger cohort (A188D, K345N, S393F, F450Y), all
of which were absent from controls. Consistent with these variants
being disease causing, western immunoblots and luciferase reporter
assays demonstrate significantly altered activity relative to wildtype
BMP3. Supporting bmp3’s role in ocular development, antisense
MO inhibition induces zebrafish coloboma and lens defects (85%,
N=46). Furthermore, we show that bmp3 is expressed in a neural
crest subpopulation (periocular mesenchyme, POM) with a critical
role in eye development. Although POM migration to the eye in
bmp3 morphants is unaffected, analysis of BMPRE:GFP (BMP
Responsive Element) transgenics demonstrates retinal BMP signaling
is profoundly altered (90%, N=10).
Conclusions: We have identified a novel gene involved in MAC
disorders, BMP3. While the precise regulation of ocular BMPs is
required for optic fissure closure, BMP3’s role in eye development
and disease is unstudied. Our research extends the role of BMPs
in eye development and implicates BMP signaling in POM in this
process. These data are compatible with a model in which POM
bmp3 regulates retinal BMP signaling, identifying a mechanism for
bmp3 in eye development and MAC disorders.
Commercial Relationships: Sonya Widen, None; Prajakta Desai,
None; Mika Asai-Coakwell, None; Matthew Benson, None; Curtis
French, None; Ordan J. Lehmann, None; Andrew Waskiewicz,
None
Program Number: 901
Presentation Time: 4:30 PM–4:45 PM
Hypothermic treatment induces the expression of cold sensing
proteins CIRP and RBM3 in the retina, both in vitro and in vivo
Alfredo Martinez1, Manuel Rey-Funes2, Daniela S. Contartese2,
Verónica B. Dorfman3, Federico Rolón2, Anibal Sarotto2, Fabián
Loidl2, 4, Ignacio Larrayoz1. 1Oncology, CIBIR, Logroño, Spain;
2
Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis”,
University of Buenos Aires, Buenos Aires, Argentina; 3Centro de
Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico
(CEBBAD), Universidad Maimónides, Buenos Aires, Argentina;
4
Facultad de Medicina, Universidad Católica de Cuyo, San Juan,
Argentina.
Purpose: Hypothermia has been described as a very effective
intervention to prevent or treat brain and retinal damage. Under cold
conditions, there is a general reduction in protein expression, but
there are 2 proteins whose expression is upregulated by hypothermia:
CIRP and RBM3. Both are RNA-binding proteins that nowadays
are considered cold sensors, thus providing a molecular mechanism
of action for the advantages of hypothermia. These proteins have
never been characterized in the retina and here we offer a preliminary
description of their behaviour in this organ, both in vitro and in vivo.
Methods: Retinal cell lines R28 (rat neural retina) and mRPE
(monkey retinal pigment epithelium) were exposed to different
temperatures in a time-dependent manner and the expression of
RBM3 and CIRP was measured through quantitative real time PCR
(qRT-PCR) and Western blotting. In addition, Sprague-Dawley
albino rats of different ages (newborns and adults) were exposed
to a cold environment (8 0C) for different periods of time. Retinas
were either snap frozen for molecular analysis (qRT-PCR and
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Western blotting) or fixed in paraformaldehyde for histological and
immunohistochemical analysis with specific antibodies against CIRP
and RBM3. Data were analyzed with ANOVA tests.
Results: In both cell lines, there was a time-dependent significant
increase of RBM3 and CIRP expression after exposure to a cold
environment. Maximum expression was reached after 6 h at 32
0C. Immunoreactivity (IR) for RBM3 and CIRP was negligible in
retinas not exposed to hypothermia. On the other hand, retinas of
both neonate and adult rats that had been exposed to hypothermia
presented high levels of IR for both proteins with different
colocalization patterns (Fig. 1).
Conclusions: As happens in the central nervous system, cells in the
retina can sense cold exposure through elevation of RBM3 and CIRP
expression. These proteins are expressed in several cell types and can
be responsible for the beneficial effects of hypothermia through the
binding of specific molecules of mRNA.
Figure 1. Retinas of newborn rats that were exposed to room
temperature (CTL) or to hypothermia (HYP), stained with specific
antibodies against RBM3 (green) and CIRP (red). Nuclear contrast
was accomplished with DAPI (blue).
Commercial Relationships: Alfredo Martinez, None; Manuel
Rey-Funes, None; Daniela S. Contartese, None; Verónica B.
Dorfman, None; Federico Rolón, None; Anibal Sarotto, None;
Fabián Loidl, None; Ignacio Larrayoz, None
Support: U.S. Department of Defense, Vision Research ProgramHypothesis Development Award (MR130239).
204 Tumors - Inside and around the eye, I
Monday, May 04, 2015 8:30 AM–10:15 AM
1AB Mile High Blrm Paper Session
Program #/Board # Range: 1287–1293
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Anatomy/Pathology
Program Number: 1287
Presentation Time: 8:30 AM–8:45 AM
Mitogen-activated protein kinase (MAPK) inhibitors in
conjunctival melanoma cell lines
Jinfeng Cao1, Robert M. Verdijk2, Aart G. Jochemsen3, Martine J.
Jager1. 1Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands; 2Pathology, Erasmus Medical Center, Rotterdam,
Netherlands; 3Molecular Cell Biology, Leiden University Medical
Center, Leiden, Netherlands.
Purpose: Current therapies of conjunctival melanoma (CM)
mainly focus on radical excision of the tumor, and are frequently
supplemented with cryotherapy or local therapy. As activating
mutations of BRAF and NRAS have been discovered in 47% of CMs,
we studied the antitumor effect of mitogen-activated protein kinase
(MAPK) inhibitors in CM cell lines.
Methods: Sanger sequencing was used to detect the mutation status
of three human CM cell lines (CRMM1, CRMM2 and CM2005.1).
The MEK inhibitor MEK162 (ARRY-162) and BRAF inhibitors
Vemurafenib (PLX4032) and Dabrafenib (GSK2118436) were chosen
to treat CM cells. Cell viability was determined with WST-1 and
in-cell western assay. The phosphorylation status of ERK protein was
evaluated using western blot.
Results: The BRAF T1799A mutation was identified in CRMM1
and CM2005.1, and the NRAS A182T mutation in CRMM2. The
growth of all three CM cell lines was inhibited by MEK162 with a
low concentration (IC50 < 45 nM). CRMM1 and CM2005.1 were
sensitive to Vemurafenib (IC50 < 200 nM) and Dabrafenib (IC50
< 20 nM) in a dose-dependent manner, while CRMM2 was not.
Consistent with the result of cell viability, a concentration-dependent
decrease of ERK phosphorylation was detected in all three CM
cell lines after MEK162 treatment, and in CRMM1 and CM2005.1
after Vemurafenib and Dabrafenib treatment. Paradoxically, the
phosphorylated ERK of CRMM2 was activated by both BRAF
inhibitors.
Conclusions: Growth inhibition of CM cell lines can be achieved by
the appropriate MEK or BRAF inhibitors. Our findings demonstrate
that MAPK inhibitors might be potential therapeutic drugs against
conjunctival melanoma and its metastasis.
Commercial Relationships: Jinfeng Cao, None; Robert M.
Verdijk, None; Aart G. Jochemsen, None; Martine J. Jager, None
Support: China Scholarship Council
Program Number: 1288
Presentation Time: 8:45 AM–9:00 AM
Role of L1 Cell Adhesion Molecule in Adhesion-Mediated
Proliferation and Chemoresistance of Retinoblastoma
Dong Hyun Jo1, 2, Jin Hyoung Kim1, 4, Young Hoon Kim3, YoungLai Cho5, Young Suk Yu6, Jeong-Ki Min7, Jeong Hun Kim1, 2. 1Fight
against Angiogenesis-Related Blindness (FARB) Laboratory,
Clinical Research Institute, Seoul National University Hospital,
Seoul, Korea (the Republic of); 2Department of Biomedical
Sciences, College of Medicine, Seoul National University, Seoul,
Korea (the Republic of); 3Department of Pathology, College of
Medicine, Seoul National University, Seoul, Korea (the Republic
of); 4Tumor Microenvironment Research Center, Global Core
Research Center, Seoul National University, Seoul, Korea (the
Republic of); 5Center for Nanosafety Metrology, Korea Research
Institute of Standards and Science, Daejeon, Korea (the Republic of);
6
Department of Ophthalmology, College of Medicine, Seoul National
University, Seoul, Korea (the Republic of); 7Research Center for
Integrated Cellulomics, Korea Research Institute of Bioscience and
Biotechnology, Daejeon, Korea (the Republic of).
Purpose: Although retinoblastoma is the most common intraocular
malignancy in children, there have been limited studies on which
drives distinct proliferation patterns and responses to chemotherapy
in retinoblastoma. In this study, we investigated the role of L1 cell
adhesion molecule (L1CAM) in adhesion-mediated tumor behavior
and chemoresistance of retinoblastoma.
Methods: We evaluated the expression of L1CAM in retinoblastoma
tissues from 30 patients by immunohistochemistry and in cell lines
by Western blotting. To study the role of L1CAM in proliferation and
chemoresistance in retinoblastoma, we utilized two retinoblastoma
cell lines, Y79 and SNUOT-Rb1, with knockdown and
overexpression of L1CAM, respectively. Then, the effects of L1CAM
expression on proliferation, cell-cell adhesion, and chemoresistance
of retinoblastoma cells were investigated. We injected naïve and
stable cell lines into the vitreous cavity of BALB/c nude mice (n =
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
6) to demonstrate the relation between L1CAM and in vivo tumor
formation.
Results: We observed varying degrees of L1CAM positivity in
86.6% (26/30) of retinoblastoma tissues. Interestingly, there was
an inverse relation between the degree of Flexner-Wintersteiner
rosette formation and that of L1CAM positivity. In vitro studies
using stable cell lines demonstrated that L1CAM was associated
with cell-cell adhesion and further adhesion-mediated proliferation
of retinoblastoma cells. In addition, L1CAM was related with
chemoresistance to carboplatin, one of the most widely utilized drug
in the treatment of retinoblastoma. In line with in vitro results, in vivo
tumor formation in rodent eyes was also more prominent with cell
lines with higher expression of L1CAM.
Conclusions: Our results show that L1CAM is expressed in
retinoblastoma and plays an important role in adhesion-mediated
proliferation and chemoresistance of tumor cells. We suggest that
profound understanding of the roles of L1CAM may open up another
therapeutic approach against retinoblastoma.
Representative photographs of L1CAM expression in retinoblastoma
tissues. Scale bar, 20 μm.
Expression of L1CAM protein among different cells. HRMEC,
human retinal microvascular endothelial cells; Rb1, SNUOT-Rb1
cells.
Commercial Relationships: Dong Hyun Jo, None; Jin Hyoung
Kim, None; Young Hoon Kim, None; Young-Lai Cho, None;
Young Suk Yu, None; Jeong-Ki Min, None; Jeong Hun Kim, None
Program Number: 1289
Presentation Time: 9:00 AM–9:15 AM
Diagnosis of Vitreoretinal Lymphoma: The Use of Cytology, Gene
Rearrangement, and IL-10/IL-6 Ratio
Jacob Pe’er1, Shahar Frenkel1, Inna Kalickman2, Yoav Sherman3, Bela
Maly3, Dina Ben Yehuda4, and Vivian Barak2
Departments of 1Ophthalmology, 2Oncology, 3Pathology, and
4
Hematology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
.
Jacob Pe’er. Ophthalmology, Hadassah-Hebrew Univ Med Ctr,
Jerusalem, Israel.
Purpose: Purpose: To compare three methods of diagnosis of
suspected vitreoretinal lymphoma: cytology, IgH gene rearrangement,
and IL-10/IL-6 ratio.
.
Methods: Methods: Diluted vitreous fluid obtained in diagnostic
vitrectomy from patients with suspected vitreoretinal lymphoma
were delivered immediately after the surgery to the cytopathology
laboratory; to the hematology laboratory for PCR studies, and to the
cancer-markers laboratory for ELISA test for IL-10/IL-6 ratio.
.
Results: Results: Forty-seven specimens were evaluated using the
three methods: 21 of them (44.7%) were positive for lymphoma
in cytology examination, 13 (27.6%) were positive in Ig-H gene
rearrangement evaluation, and 25 (53.2%) were positive by IL10/IL-6 ratio that was higher than 1.0. Nine of 21 specimens
(42.8%) with positive cytology were positive also for Ig-H gene
rearrangement and 18 of the 21 (85.7%) were positive also for IL-10/
IL-6 ratio. Nine specimens were positive for lymphoma via all three
methods. Seven specimens in which the IL-10/IL-6 ratio was positive
for lymphoma were negative in cytology, 4 of them developed CNS
lymphoma, and 3 that were positive for Ig-H gene rearrangement
were negative in cytology.
Conclusions: Conclusions: In our series, the examination of the IL10/IL-6 ratio was much more sensitive and much more comparable
to cytology than examination for Ig-H gene rearrangement which
showed low sensitivity.
Commercial Relationships: Jacob Pe’er, None
Program Number: 1290
Presentation Time: 9:15 AM–9:30 AM
The Classification of Vitreous Seeds in Retinoblastoma: Response
to Intravitreal Melphalan
Jasmine H. Francis1, 2, David H. Abramson1, 2, Marie-Claire
Gaillard3, Brian P. Marr1, 2, Maya Beck-Popovic4, Francis L. Munier3.
1
Ophthalmic Oncology, Memorial Sloan Kettering Cancer Center,
New York, NY; 2Weill-Cornel Medical Center, New York, NY; 3JulesGonin Eye Hospital, Lausanne, Switzerland; 4University Hospital
CHUV, Lausanne, Switzerland.
Purpose: Vitreous seeds in retinoblastoma have clinical
heterogeneity and we have previously proposed a classification
to distinguish between them. This study evaluates the clinical
characteristics of the three vitreous seed classes: dust (class
1), spheres (class 2) and clouds (class 3) and their responses to
intravitreal melphalan.
Methods: Bi-institutional cohort study of 87 patient eyes that
received 475 intravitreal injections of melphalan (median dose 30mg)
given weekly, a median of 5 times (range 1-12). At presentation,
the vitreous seeds were classified into three groups: dust, spheres
and clouds. Indirect ophthalmoscopy, fundus photography,
ultrasonography and ultrasonic biomicroscopy were used to evaluate
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
clinical response to weekly intravitreal melphalan injections and time
to regression of vitreous seeds. Kaplan-Meier estimates of time to
regression, ocular, patient and event-free survival were calculated and
Log-rank test of curve comparison.
Results: There was a significant difference in time to regression for
the three seed classes (p<0.0001): the median time to regression was
0.6 months, 1.7 months and 7.7 months for dust, spheres and clouds,
respectively. Dust received significantly fewer injections, a lower
median and cumulative dose of melphalan; while clouds received
significantly more injections, a higher median and cumulative dose
of melphalan. Kaplan-Meier estimates for two-year ocular, patient,
and event-free survival (related to target seeds) was not significantly
different between the three seed groups: overall, they were 90.4%
(95% confidence interval (CI) 79.7-95.6), 100%, 98.5% (95%CI 9099.7), respectively.
Conclusions: The three vitreous seed classes have a significantly
different time to regression in response to intravitreal melphalan. The
vitreous seed classification can help to predict time to regression,
number, median dose and cumulative dose of intravitreal melphalan
injections required.
Commercial Relationships: Jasmine H. Francis, None; David H.
Abramson, None; Marie-Claire Gaillard, None; Brian P. Marr,
None; Maya Beck-Popovic, None; Francis L. Munier, None
Support: The Fund for Ophthalmic Knowledge and Perry’s Promise
Fund
Program Number: 1291
Presentation Time: 9:30 AM–9:45 AM
Evaluating the in vivo efficacy of a novel first in class drug for the
treatment of primary uveal melanoma
Patrick T. Logan, Sultan Aldrees, Mohammed F. Qutub, Natalia Vila,
Vasco Bravo-Filho, Miguel N. Burnier. Henry C Witelson Lab, Ocular
Pathology, McGill University, Montreal, QC, Canada.
Purpose: The standard for treating uveal melanoma is plaque
radiotherapy; however, tumors that are too large or close to the optic
nerve cannot be treated using this method. Moreover, as radiotherapy
is a non-specific treatment, it can cause serious side-effects, such
as retinopathy, cataract, and glaucoma. Thus, there is a need for a
specific uveal melanoma treatment with limited side-effects. Herein,
we tested the efficacy of a nanoparticle with a conjugated photoactive
dye (collectively, AU-011) in a uveal melanoma rabbit model.
Methods: One million 92.1 uveal melanoma cells were injected
into the suprachoroidal space of twenty albino New Zealand
rabbits. Tumor growth was assessed weekly by ultrasound and
fundoscopy. After the presence of a tumor was confirmed clinically,
treatment commenced. Treatment consisted of intravitreal injection
of AU-011; 20 hours after injection, tumors were lasered using an
ophthalmic laser (690 nm, 50 J/cm2) to activate the photoactive dye.
This treatment was repeated weekly for 3 weeks, and animals were
sacrificed 1 week after the last treatment. Post-mortem, tumors were
examined on gross pathology and by histopathology.
Results: Twelve of the 20 animals developed intraocular tumors.
Two animals with tumors died unexpectedly due to cyclosporine
complications and did not receive treatment; these animals acted
as controls. For the other 10 treated animals, a noticeable tumor
response was observed, which was characterized by three elements:
1) induction of extensive tumor necrosis; 2) change in the growth
pattern (“sleeve-like pattern”); and 3) sparing of the adjacent retina.
On ultrasound, tumor growth cessation or shrinkage was generally
observed. In 4 of these treated animals, histopathology revealed that
no tumor was present (complete response); instead, the intraocular
masses noted by ultrasound and fundus were comprised of only
inflammatory cells and fibrosis. Histopathology of all treated animals
showed widespread necrosis (>80% in most cases) that was not seen
in controls or in previous animal models (<20% necrosis).
Conclusions: In this pilot study, we show that AU-011 is efficacious
for treating uveal melanoma tumors in an orthotopic xenograft
rabbit model. In addition to the widespread necrosis noted in treated
tumors, several animals experienced complete responses. Future
studies investigating different AU-011 dosing regimens are currently
underway.
Commercial Relationships: Patrick T. Logan, Aura Biosciences
(C); Sultan Aldrees, None; Mohammed F. Qutub, None; Natalia
Vila, None; Vasco Bravo-Filho, None; Miguel N. Burnier, Aura
Biosciences (C)
Program Number: 1292
Presentation Time: 9:45 AM–10:00 AM
Intravitreal Aflibercept as Rescue Therapy for Post-Radiation
Cystoid Macular Edema
Mohammed A. Khan1, 2, Arman Mashayekhi1, Jerry A. Shields1,
Carol L. Shields1. 1Ocular Oncology Service, Wills Eye Hospital,
Philadelphia, PA; 2Retina Service, Wills Eye Hospital, Philadelphia,
PA.
Purpose: To investigate the safety and efficacy of intravitreal
aflibercept as rescue therapy for persistent post-radiation cystoid
macular edema (CME) following prior treatment with intravitreal
bevacizumab.
Methods: Retrospective, observational, consecutive case series
of patients who received intravitreal aflibercept (2mg/0.05mL) for
persistent post-radiation CME by the Oncology Service, Wills Eye
Hospital (Philadelphia, PA). Primary outcomes were change in
central macular thickness (CMT) by optical coherence tomography
(OCT) and visual acuity after initiation of intravitreal aflibercept
therapy.
Results: Five eyes of five patients with persistent CME following
plaque radiotherapy for choroidal malignant melanoma were
included. Mean tumor basal diameter was 10.4 mm (range 6 - 16
mm) and mean tumor thickness of 3.56 mm (range 2.2 – 4.7 mm).
Mean radiation dose to the macula was 6158 cGy (mean rate to
macula 65.5 cGy/hour). OCT evident CME occurred at a mean of
22.8 months (range 8 – 43 months) post plaque radiotherapy. All
patients were prior treated with intravitreal bevacizumab (mean 11.6
injections per patient, range 6 – 22 injections). Following treatment
with three monthly doses of intravitreal aflibercept, logMar visual
acuity improved from mean 0.47 (standard deviation 0.08, Snellen
equivalent 20/60) to mean 0.30 (standard deviation 0.24, Snellen
equivalent 20/40) (p=0.19). CMT reduced significantly from mean
478.8 microns (standard deviation 123.3) to mean 292.4 microns
(standard deviation 46.7)(p=0.01). No patient experienced worsening
of CMT or visual acuity while treated with intravitreal aflibercept.
No alternative therapies were necessary. No patient experienced an
adverse event. At mean final follow-up of 4.2 months (range 3-8
months), mean CMT and logMar visual acuity remained stable at
294.6 microns and 0.35 (Snellen equivalent 20/44), respectively.
Conclusions: Intravitreal aflibercept can be an effective rescue
therapy for persistent post-radiation CME in patients with poor
response to bevacizumab, with reduction in central macular thickness
and improvement in visual acuity.
Commercial Relationships: Mohammed A. Khan, None; Arman
Mashayekhi, None; Jerry A. Shields, None; Carol L. Shields, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 1293
Presentation Time: 10:00 AM–10:15 AM
Second primary cancers in uveal melanoma survivors and the
role of radiotherapy: a long-term population-based study
Inês Laíns1, 2, Carla Bartosch3, Vera Mondim4, Deeba Husain1, Joan
W. Miller1. 1Department of Ophthalmology, Massachusetts Eye and
Ear Infirmary, Harvard Medical School, Boston, MA; 2Faculty of
Medicine, University of Coimbra, Coimbra, Portugal; 3Instituto
Português de Oncologia do Porto, Porto, Portugal; 4Centro Hospitalar
de Lisboa Central, Lisboa, Portugal.
Purpose: The long term survival in uveal melanoma (UM) patients
is about 50% and these patients may be at higher risk of developing
second primary cancers (SPC). In other malignancies, the risk of
SPC has been linked with radiotherapy (RT). Despite this knowledge,
no population-based studies have been done on UM survivors after
radiation became standard of care. The aim of this study was to
characterize the long-term SPC risk in subjects previously diagnosed
with UM and to determine if RT is an independent risk factor.
Methods: This is a retrospective cohort study. Using the
Surveillance, Epidemiology and End Results (SEER) 9 database from
the United States of America, we identified patients with a diagnosis
of UM as their first malignancy between 1973 to 2011. The second
cancer events were defined as those diagnosed at least two months
after the UM. Poisson regression was used to model standardized
incidence ratios (SIR) and excess absolute risks (EAR) of SPC,
compared with a reference SEER population matched for sex, 5-year
age group, race, site and calendar year. Multivariate Cox regression
model was used to evaluate the effect of RT in SPC risk.
Results: Of the 3,736 UM patients identified, 15.3% developed
a SPC during a median follow-up of 139 months (range: 12-463
months). This represented a 10% higher risk compared to the general
reference population, mainly due to a significantly increased risk
of salivary gland tumors (SIR=4.27, p<0.05, EAR=1.22), skin
melanomas (SIR=2.94, p<0.05, EAR=10.18) and kidney tumors
(SIR=2.05, p<0.05, EAR=3.67). The occurrence of second UM was
also increased (SIR=16.95, p<0.05, EAR=3.26), which may represent
a recurrence of melanoma or true second primary malignancies. RT
was performed in 38.3% of the patients. The multivariate analysis
revealed that this treatment was not an independent risk factor for
SPC (HR=1.27, 95%CI: 0.97-1.65, p=0.08), after adjusting for age at
diagnosis, sex, race and surgical treatment.
Conclusions: Uveal melanoma survivors presented a 10% higher risk
of SPC as compared to the general population. RT does not seem to
be an independent risk factor for second primary cancers.
Commercial Relationships: Inês Laíns, None; Carla Bartosch,
None; Vera Mondim, None; Deeba Husain, None; Joan W. Miller,
None
229 Tumors - Retinoblastoma
Monday, May 04, 2015 11:00 AM–12:45 PM
1AB Mile High Blrm Paper Session
Program #/Board # Range: 1660–1665
Organizing Section: Anatomy and Pathology/Oncology
Program Number: 1660
Presentation Time: 11:00 AM–11:15 AM
Generation of in vitro intraocular tumor models for pre-clinical
drug screening using nanotechnology
Justin B. Lendermon1, Nabil Saleh1, Anderson H. Webb1, Bradley T.
Gao1, Ryan P. Lee1, Matthew W. Wilson1, Jena J. Steinle1, Vanessa
M. Morales1, 2. 1Ophthalmology, University of Tennessee Health
Science Center, Memphis, TN; 2Microbiology, Immunology and
Biochemistry, University of Tennessee Health Science Center,
Memphis, TN.
Purpose: Three dimensional (3D) tumor cell cultures more
closely resemble tumors in vivo, thus providing a better model for
pre-clinical drug screening. This technology, Nano3D (Nano3D
Biosciences, Inc., Houston, TX), was recently developed by
Souza and colleagues to study glioblastomas. We adopted Souza’s
techniques to generate 3D cell cultures of retinoblastoma and uveal
melanoma for pre-clinical drug screening.
Methods: We used two retinoblastoma cell lines, Y79 and Weri,
and three uveal melanoma cell lines, OMM1, 92.1 and Mel 270.
First, we determined the minimum number of tumor cells to obtain
significant data, maximizing their potential. Cells were co-cultured
with nanoshuttle particles, made of iron oxide, to allow the formation
of tumor spheroids upon contact with magnet in the dock station with
capacity to record, at 37°C. Cells were dissociated and analyzed for
cell viability and different surface markers. Then, we generated, in
vitro, an extracellular matrix-like microenvironment to analyze tumor
migration potential and disruption of extracellular matrix, in a similar
fashion to the classical “scratch wound assay”. Third, we optimized
tumor cell number and media volume to analyze cell growth by
spheroid size.
Results: Comparison of tumor growth for functional analyses in
flat 2D surfaces versus the 3D nanotechnology system revealed
significant differences. First, we found a more homogeneous
phenotype in 2D flat surfaces than in the 3D. Second, we observed
slight differences in adhesion molecules, specifically in UM, as they
are adherent in 2D surfaces and not in the 3D.
Conclusions: The use of 3D cell culture is a powerful tool with
potential applications for preclinical drug screening in intraocular
tumors.
Commercial Relationships: Justin B. Lendermon, None; Nabil
Saleh, None; Anderson H. Webb, None; Bradley T. Gao, None;
Ryan P. Lee, None; Matthew W. Wilson, None; Jena J. Steinle,
None; Vanessa M. Morales, None
Program Number: 1661
Presentation Time: 11:15 AM–11:30 AM
Discovery of novel cell cycle regulatory and signal transduction
modules driving Retinoblastoma using a correlative multi-omics
approach
Arkasubhra Ghosh1, 2, Ashwin C. Mallipatna1, Nilanjan Guha3,
Deepak S A3, Syed Lateef3, Vishnu Babu1, Seetaramanjaneyulu
Gundimeda3, Arunkumar Padmanabhan3, Rohit Shetty1, 2. 1GROW
Research Laboratory, Narayana Nethralaya Foundation, Bangalore,
India; 2Singapore Eye Research Institute, Singapore, Singapore;
3
Agilent Technologies, Bangalore, India.
Purpose: Applying a multi-omics methodology to molecular analysis
of primary intraocular retinoblastoma tumor samples covering global
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
transcriptomic and metabolomics assays for elucidating functional
pathways driving this cancer.
Methods: All samples were collected after informed written consent
and approval of Institutional Ethics Committee. We obtained total
RNA from tumors of 9 patients (5 male, 4 female; age range 4-30
months) who underwent enucleation of the affected eyes. Additional
tumor, aqueous humor, vitreous humor and tear samples were also
collected from the same patients for metabolomics analyses. Control
retina, vitreous and aqueous humor was extracted from pediatric
donor eyes. Global gene expression and miRNA microarrays
were performed simultaneously with the tumor RNA, followed by
pathway analysis. Metabolites were extracted using monophasic
solvent extraction of aqueous and vitreous humor samples followed
by analysis on Accurate Mass QTOF mass spectrometer on reverse
phase C18 and HILIC columns.
Results: Differential expression analysis carried out using moderated
t-test with Benjamini Hochberg multiple testing correction revealed
1404 significantly regulated genes (p≤0.005 and fold change≥10)
as compared to normal pediatric retina. Analysis of miRNA arrays
revealed 18 previously unreported, deregulated miRNAs (p≤0.005
and fold change≥10). Using these data sets in concert, pathway
analysis of the miRNAs and their possible target differentially
expressed genes revealed novel networks. The results show that cell
cycle, mTOR, PI3-AKT, retinal function and Rap1 signaling modules
are most significantly deregulated. We further validated 20 genes
selected from these deregulated pathways by quantitative PCR. IHC
staining of 33 independent patient tumor cohort further validated
E2F and Rap1 up regulation. Analysis of the differentially expressed
metabolites in vitreous and aqueous humor revealed enrichment of
several additional pathways.
Conclusions: The study illustrates an integrated method of
discovering new biological insights that are made accessible by
correlating data from different large scale techniques in the same
patient sample, thereby reducing intra-cohort bias. In particular,
E2F, Rap1, CDK and cyclin dependent signaling pathways were up
regulated while retinal function and visual cycle related pathways
were down regulated in Rb patients.
Commercial Relationships: Arkasubhra Ghosh, None; Ashwin
C. Mallipatna, None; Nilanjan Guha, None; Deepak S A, None;
Syed Lateef, None; Vishnu Babu, None; Seetaramanjaneyulu
Gundimeda, None; Arunkumar Padmanabhan, None; Rohit
Shetty, None
Program Number: 1662
Presentation Time: 11:30 AM–11:45 AM
Targeting Notch signaling as a novel therapy for Retinoblastoma
Laura Asnaghi1, Arushi Tripathy1, Charles Eberhart1, 2.
1
Neuropathology, Johns Hopkins University, Baltimore, MD;
2
Ophthalmology, Johns Hopkins University, Baltimore, MD.
Purpose: Retinoblastoma, a malignant tumor of the retina, is the
most common intraocular cancer of childhood. Our goal was to study
the role of Notch signaling in promoting growth and proliferation of
retinoblastoma cells to determine if inhibiting this pathway might
represent a novel approach to treatment.
Methods: WERI Rb1 and Y79 retinoblastoma lines were used as
model. Expression of Notch pathway components was measured
using qPCR and Western blot. Downregulation of the Jag2 ligand
was achieved using short hairpin RNA (shRNA). Cell growth,
proliferation, and invasion of these cell lines, upon downregulation
of Jag2, were determined respectively using the colorimetric CCK8
(Cell Counting kit8) assay, bromodeoxyuridine incorporation and
transwell invasion assays.
Results: We found that Notch pathway components, including
Jag1-2 ligands, Notch receptors and Hes1, Hey1-2 target genes, were
expressed at various levels in WERI Rb1 and Y79 retinoblastoma
lines. Notch ligand Jag2 RNA and protein were more highly
expressed as compared to Jag1 in the retinoblastoma lines. Notch1
receptor was found to be more abundantly expressed than Notch2 and
Notch3 receptors in these lines. The cleaved, active form of Notch1
was detected under standard culture conditions, further supporting
a potential role for the pathway in retinoblastoma. Because Jag2
was highly expressed, we performed loss-of-function studies using
shRNA. Downregulation of Jag2 significantly suppressed cell
growth and proliferation by 40 to 50% in both lines, as determined
respectively by CCK8 and bromodeoxyuridine assays, and partially
reduced invasion, as found by transwell invasion assay.
Conclusions: Our findings indicate that Notch pathway components
are expressed in WERI Rb1 and Y79 retinoblastoma lines, with the
presence of cleaved Notch1 receptor and downstream targets further
supporting pathway activity. Jag2 ligand appeared to be highly
expressed and promoted retinoblastoma growth. These data suggest
that Notch inhibitors represent a new potential therapeutic strategy in
retinoblastoma.
Commercial Relationships: Laura Asnaghi, None; Arushi
Tripathy, None; Charles Eberhart, None
Support: Knights Templar Eye Foundation Grant for Career
Development to Laura Asnaghi
Program Number: 1663
Presentation Time: 11:45 AM–12:00 PM
Aqueous seeding: fall of the ultimate intraocular retinoblastoma
sanctuary by a new in situ chemotherapy technique
Francis L. Munier1, Marie-Claire Gaillard1, Sarah Decembrini1,
Maja Beck-Popovic2. 1Ophthalmology Department, Jules-Gonin Eye
Hospital, Lausanne, Switzerland; 2Pediatric Hematology Oncology
Unit, University Hospital CHUV, Lausanne, Switzerland.
Purpose: The presence of primary or secondary aqueous seeding in
retinoblastoma (rb) represents a universal indication for enucleation.
All previous attempts at conservative treatment have been associated
with 100% failure rate. Here we present a novel technique of in situ
chemotherapy specifically developped to eradicate aqueous seeding.
Methods: Retrospective review of two patients presenting with
primary (patient#1) and secondary (patient# 2) aqueous seeding
respectively. Combined injections of melphalan were given in the
vitreous (200 μg/mL) and in both posterior (PC) and anterior (AC)
chambers (15 μg/mL). The intra-cameral procedure consisted of 5
successive steps: 1) long needle passage (34G) across the peripheral
clear cornea, 2) aqueous aspiration of of both AC and PC volumes, 3)
melphalan injection of 1/3 of the paracentesis volume in AC, 4) transiridal injection into the PC of the last 2/3 and AC retrofilling, 5) triple
freeze and thaw cryo-application at the entry site. Per-operative intravenous acetazolamide is administred to suppress aqueous secretion.
Results: Patient#1 was diagnosed with unilateral group E anterior
diffuse rb at 11 years of age. Following first line intra-arterial
chemotherapy, she received 10 intravitreal and 12 intra-cameral
injections. Complete regression was achieved with an event-free
follow-up of 27 months. The patient is binocular with visual acuity of
20/20 OU.
Patient#2 was diagnosed with bilateral rb, group D OD and group E
OS at 6 months of age. Following first line systemic chemotherapy
and intra-arterial injections in OS, he developed secondary anterior
seeding in OD at 25 months of age. He received 4 intravitreal and 5
intra-cameral injections. Complete regression was obtained with an
event-free follow-up of 7 months since the last injection. The patient
has good fixation without nystagmus.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Conclusions: This is the first study to report globe salvage in case
of anterior chamber seeding using a new intra-cameral injection
technique. Moreover, both eyes retained normal visual function
including binocularity in patient#1. We would like to stress that
this approach is safe and efficient provided that 1) careful 35mHz
ultrasonic biomicroscopy have excluded invasion of the ciliary body,
iris and Schlemm’s canal, 2) the injection is performed in the PC
with retofilling of the AC, 3) pharmacologic suppression of aqueous
secretion is observed.
Commercial Relationships: Francis L. Munier, None; MarieClaire Gaillard, None; Sarah Decembrini, None; Maja BeckPopovic, None
Program Number: 1664
Presentation Time: 12:00 PM–12:15 PM
Intra-arterial chemotherapy rescue for recurrent or persistent
subretinal seeds in retinoblastoma: Tumor control and globe
salvage in 30 eyes
Prashanth G. Iyer2, Emil A. Say1, Murat Hasanreisoglu1, Sara Lally1,
Pascal Jabbour3, Carol L. Shields1. 1Ocular Oncology Service,
Wills Eye Hospital, Philadelphia, PA; 2Sydney Kimmel Medical
College, Philadelphia, PA; 3Thomas Jefferson University Hospital,
Philadelphia, PA.
Purpose: To describe the efficacy of intra-arterial chemotherapy
(IAC) for control of persistent or recurrent subretinal seeds in
retinoblastoma.
Methods: In this retrospective non-comparative interventional case
series, there were thirty eyes in 29 patients with retinoblastoma who
have failed previous treatment with systemic chemotherapy, plaque
radiation, or focal therapy and persistent or recurrent subretinal
seeds. Superselective ophthalmic artery chemotherapy infusion under
fluoroscopic guidance with melphalan (3, 5, or 7.5 mg) and additional
topotecan (1 mg) and/or carboplatin (30 mg) was administered as
necessary. The main outcome measures were tumor control and globe
salvage.
Results: The mean patient age was 19 months. Secondary IAC
was given to patients with recurrent or persistent subretinal seeds
who failed previous treatment regimens (n=30 eyes). Each eye
received a mean of 3 IAC per eye (median, 2; range, 1-7). After IAC
with a mean follow-up of 14 months, 27 of the 30 eyes (90%) had
subretinal seed regression after completion of IAC cycles. Longterm control with global salvage was maintained in 19 eyes (63%),
with enucleation being required for recurrent subretinal seeds in 3
eyes (27%) and for recurrent subretinal and vitreous seeds in 3 eyes
(27%).
Conclusions: IAC is effective as a secondary agent in the
management of subretinal seeds in retinoblastoma achieving
complete regression of subretinal seeds in 90% of eyes.
Commercial Relationships: Prashanth G. Iyer, None; Emil A.
Say, None; Murat Hasanreisoglu, None; Sara Lally, None; Pascal
Jabbour, None; Carol L. Shields, None
Support: Eye Tumor Research Foundation
Program Number: 1665
Presentation Time: 12:15 PM–12:30 PM
Management of retinal detachment after first-line intra-arterial
chemotherapy for advanced retinoblastoma
Jelena Potic1, 2, Jean-Antoine C. Pournaras1, Marie-Claire Gaillard1,
Thomas Wolfensberger1, Maya Beck-Popovic3, Francis L. Munier1.
1
Jules-Gonin Eye Hospital, Lausanne, Switzerland; 2Univ Eye Hosp,
Clinical Center of Serbia, Belgrade, Serbia; 3Paediatric Oncology and
Haematology, CHUV, Lausanne, Switzerland.
Purpose: First line intra-arterial chemotherapy was proposed
recently as a new conservative treatment for retinoblastoma patients.
The aim of this retrospective study is to analyze the occurrence
of rhegmatogenous retinal detachment in patients with advanced
retinoblastoma treated with first line intra-arterial chemotherapy and
their surgical outcome.
Methods: All consecutive patients with advanced retinoblastoma
treated with first line intra-arterial chemotherapy at the Jules-Gonin
Eye Hospital (November 2008 to October 2014) were included.
Indications for retinal detachment surgery were either new or
persistent detachment. Scleral buckle surgery with anterior chamber
ponction was performed without drainage of the sub-retinal fluid.
Results: 30 eyes from 30 patients responded to the inclusion criteria,
including 3 eyes from bilaterally affected patients: Group C (n=1),
Group D (n=23), Group D/E (n=2), Group E (n=4). Average age
at the first intra-arterial injection was 28.7 months. Cumulative
injected dose of Melphalan was 11.44 mg, with a mean number of 2.7
injections. Before treatment, 11 patients (37%) had retinal detachment
at presentation, and 19 (63%) patients had none. After chemotherapy,
16/19 (84.2%) patients developed retinal detachment (1 localized, 6
subtotal, 9 total). All the 11 patients presenting retinal detachment at
the first examination regressed spontaneously after treatment (100%).
Mean time between the intra-arterial chemotherapy and beginning of
retinal detachment was 41 days. Average observation period before
surgery was 4.1 months. In 10 patients, scleral buckle was indicated,
but only 9 underwent surgery. In 1 patient with shallow retinal
detachment, it was decided to continue with clinical follow-up only.
Regression of retinal detachment was seen in 8/9 cases (89%), with
complete reapplication in 6/9 patients (67%). One patient showed no
change (11%).
Conclusions: First line intra-arterial chemotherapy for advanced
retinoblastoma provides good tumor control, but in this series
secondary retinal detachment occurred in 16/30 cases (50%). Retinal
reapplication was obtained with scleral buckling in more than two
thirds of them. The early treatment of this complication is important
for salvage of the globe and visual function.
Commercial Relationships: Jelena Potic, None; Jean-Antoine
C. Pournaras, None; Marie-Claire Gaillard, None; Thomas
Wolfensberger, None; Maya Beck-Popovic, None; Francis L.
Munier, None
275 Myopia
Monday, May 04, 2015 3:45 PM–5:30 PM
Exhibit Hall Poster Session
Program #/Board # Range: 2148–2182/B0001–B0035
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Clinical/Epidemiologic Research
Program Number: 2148 Poster Board Number: B0001
Presentation Time: 3:45 PM–5:30 PM
The effect of combination of white and monochromatic light on
eye growth of normal chicks
Rachel Ka-man Chun1, Danyang Wang1, 2, King Kit Li1, Thomas
C Lam1, Quan Liu2, Chi Ho To1, 2. 1Laboratory of Experimental
Optometry, Centre for Myopia Research, School of Optometry, The
Hong Kong Polytechnic University, Hong Kong, Hong Kong; 2State
Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,
Sun Yat-sen University, Guangzhou, China.
Purpose: To examine the effect of combination of white and
monochromatic light on eye growth of normal chicks
Methods: White Leghorn chicks aged 4 days (n = 8 in each group,
three groups in total) were raised in a cabinet of 80 x 80 x 125 cm
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
under three different lighting. They were white (585 nm), white
(585 nm) and red (630 nm), white (585 nm) and blue (450 nm).
The average luminances in these three environments were around
260 lux. The ratio of those light combination was 50:50. The ocular
parameters and refractive errors were measured before and after
14 days of exposure. The ocular parameters were measured by
high frequency A-scan ultrasonography while the refractive errors
were examined by streak retinoscopy. Percentage changes in the
ocular parameters and refractive errors relative to the baseline
were calculated and compared among different groups by two-way
ANOVA and Fisher’s least significant difference (LSD) post hoc test.
Results: After 14 days of exposure, the percentage increase in
anterior chamber depth (ACD), lens thickness, vitreous chamber
depth (VCD), retinal thickness and axial length were found to be
significantly greater in chicks raised under white and red light when
compared with those under white light only (p < 0.001). Besides,
more myopic shift was shown in the chicks under white and red light
(p < 0.05). However, the choroid did not demonstrate the thinning
as expected but significant thickening was found (mean choroidal
thickness ± SEM; white vs. white and red; 264.3 ± 4.43 mm vs. 280.8
± 5.18 mm).
Chicks raised in white and blue light had a smaller increase in ACD,
lens thickness, VCD and axial length when compared with chicks
under white light only (p< 0.001). Significant thinning of the retina
was found (percentage change of the retinal thickness ± SEM; white
vs. white and blue; -6.1 ± 1.2% vs -11.6 ± 0.7%, p = 0.001). Changes
in refractive errors and choroidal thickness were comparable to the
chicks under white light only.
Conclusions: Combination of the white and monochromatic light
provided two peak wavelengths across the spectrum. It significantly
affected the eye growth of normal chicks. Greater elongation of
eyeball was found in the chicks under white and red light than those
under white light only whereas ocular growth was slower in the
chicks under white and blue light. Monochromatic light seems to play
a role in modulating the ocular growth in chicks.
Commercial Relationships: Rachel Ka-man Chun, None;
Danyang Wang, None; King Kit Li, None; Thomas C Lam, None;
Quan Liu, None; Chi Ho To, None
Support: This work was supported by research grants GU986,
GU839, GUA32 and GYK89 from The Hong Kong Polytechnic
University and the Henry G. Leong Endowed Professorship in
Elderly Vision Health.
Program Number: 2149 Poster Board Number: B0002
Presentation Time: 3:45 PM–5:30 PM
Effects of 430nm monochromatic light on defocus-induced
myopia in guinea pigs
Yi-Feng Qian1, rui liu2, jinhui dai2. 1Department of Ophthalmology,
First Affiliated Hospital of Soochow University, Suzhou, China;
2
Department of Opthalmology, Eye & ENT Hospital of Fudan
University, Shanghai, China.
Purpose: To investigate the effects of 430nm monochromatic light
on defocus-induced myopia in guinea pigs.
Methods: Eighteen 2-week-old pigmented guinea pigs were
randomly assigned to two groups based on the mode of illumination:
short-wavelength light (SL) for 8 weeks and broad-band white light
(BL) for 8 weeks. All animals of the two groups were worn -5D
lenses on right eye. Biometric and refractive measurements were then
performed every 2 weeks. The illuminative parameters of all groups
were identical and the light quantum number was 3×10-4μmolcm-2s-1.
Results: After the beginning of the experiment, the right eyes of the
two groups decreased in refraction. At the end of the experiment,
relative myopia of the right eye was about 2.72D in the SL and about
3.03D in the BL when compared with the fellow eye. But a relative
hyperopia, about 1.2D, was induced in the SL compared with the BL
group in the end. From 4 to 8 week, there was significant difference
in radius of corneal curvature between the two eyes of the SL group.
But, there was no significant difference in corneal curvature between
the two eyes of the BL group. At the end of the experiment, there was
significant difference in radius of corneal curvature between the right
eyes of the two groups. The difference was not significant in vitreous
length of right eye between the two groups from beginning to the
end of experiment. There was no significant difference in vitreous
length between the two eyes of the SL group in the end. But finally,
significant difference existed in vitreous length between the two eyes
of the BL group. There were no significant inter-group or intra-group
differences in length of anterior segment and Lens thickness.
Conclusions: 430nm monochromatic light could interfere with
the development of defocus-induced myopia in guinea pigs. The
effect of the monochromatic light may be achieved by influencing
the developments of vitreous chamber and corneal curvature. The
recognition of defocus under the monochromatic light may be
achieved by the function from only one type of cone.
Commercial Relationships: Yi-Feng Qian, None; rui liu, None;
jinhui dai, None
Support: This work was supported by Grants 81400429,
81100689,81271040 from the National Natural Science Foundation
of China
Program Number: 2150 Poster Board Number: B0003
Presentation Time: 3:45 PM–5:30 PM
Antagonistic effects of atropine and timolol on the color and
luminance emmetropization mechanisms
Laura A. Goldberg, Frances J. Rucker. New England College of
Optometry, Boston, MA.
Purpose: The role of the autonomic nervous system in the color
and luminance emmetropization mechanisms is unknown. This
study analyzed the response to the non-selective, parasympathetic
antagonist, atropine, and the sympatholytic, beta-adrenergic
antagonist, timolol, in chicks subjected to illumination conditions
that selectively stimulate the color and luminance emmetropization
mechanisms.
Methods: Chicks were binocularly exposed eight hours each day,
for four days, to one of three illumination conditions: 2Hz sinusoidal
luminance flicker (LUM), 2Hz sinusoidal color flicker (B/Y), or
steady light. Mean illuminance was 680 lux. Eyes received daily
injections of either 20μl atropine (18nmol) (N=8), 2 drops of 0.5%
timolol (N=8), 20μl phosphate-buffered saline (N=8), or no injection
(N=8). Measurements of the axial dimensions of ocular components
and refraction were performed using A-scan ultrasonography,
photorefraction and a Hardinger Refractometer. In each illumination
condition, the saline effect was subtracted from the drug effect [Drug
(ΔX-ΔN) – Saline (ΔX-ΔN)].
Results: LUM flicker demonstrated opposite effects on eye growth
and refraction with atropine and timolol treatment. Atropine caused
a reduction in eye growth (-0.08 ± 0.02 mm, p=0.01) and a reduction
in vitreous chamber depth (-0.10 ± 0.02 mm, p=0.004), evoking a
hyperopic shift in refraction (3.40 ± 1.77 D), despite an antagonistic
increase in lens thickness (0.14 ± 0.05 mm, p=0.004). In contrast,
timolol elicited a myopic shift in refraction (-4.07 ± 0.92 D, p=0.001),
due to an increase in eye length (0.045 ± 0.030 mm). Color flicker
induced choroidal compensation for eye growth, preventing refractive
shifts with atropine and timolol. With atropine, hyperopia was not
observed, because a reduction in eye length (-0.05 ± 0.02 mm,
p=0.01) was compensated for by choroidal thinning (-0.05 ± 0.02
mm, p=0.03). With timolol, myopia did not occur because a reduction
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
in eye length (-0.05 ± 0.018 mm, p=0.02) was also compensated for
by choroidal thinning (-0.052 ± 0.015 mm, p=0.01).
Conclusions: The opposing growth and refractive effects of atropine
and timolol with luminance flicker, and the compensatory choroidal
compensation with color flicker, suggest a precise balancing
mechanism between the parasympathetic and sympathetic nervous
system, and the visual environment, in achieving emmetropization.
Commercial Relationships: Laura A. Goldberg, None; Frances J.
Rucker, None
Support: NEI T35 Student Research Fellowship and Beta Sigma
Kappa Student Research Grant
Program Number: 2151 Poster Board Number: B0004
Presentation Time: 3:45 PM–5:30 PM
Antagonistic Effect of Ciliary and Superior Cervical Ganglion
Sections on the Color and Luminance Emmetropization
Mechanisms
Frances J. Rucker1, Falk Schroedl2. 1Biomedical Science, New
England Coll of Optometry, Boston, MA; 2Paracelsus Medical
University, Salzburg, Austria.
Purpose: Longitudinal chromatic aberration produces changes in
retinal color and luminance contrast that guides emmetropization.
An eye exposed to luminance contrast becomes hyperopic, like an
atropine treated eye, an eye exposed to color contrast becomes more
myopic. This experiment investigates the role of the autonomic
nervous system in the control of emmetropization.
Methods: One to two week old, white leghorn chicks, underwent
unilateral lesion of the ciliary ganglion (CGX; N=16) or superior
cervical ganglion (SCGX; N=16). Animals were allowed to recover
for one week, and were then placed in cages illuminated with
sinusoidally modulated light (2 Hz: 80% contrast) that changed in
luminance (LUM) contrast or COLOR (red to green) contrast (mean
illumination 680 lux). Animals were kept in these illumination
conditions for three days (9am-5pm), and otherwise in the dark.
Changes in ocular components after the recovery period, and after
exposure to the illuminants, were measured with OCT (Lenstar) and
refraction with a Hartinger Refractometer. Changes in the lesioned
eye were compared with the unlesioned fellow eye.
Results: After recovery: CGX produced an eye with relative
hyperopia (2.01 ± 0.63D; p=0.006) and thinning of the anterior
chamber (25 ± 11 mm; p=0.037). SCGX produced an enlarged eye
(114 ± 26 mm; p<0.001) with longer vitreous chamber depth (154 ±
22 mm; p<0.001). With subsequent exposure to flicker: With CGX,
LUM prevented significant eye growth (47 ± 30 mm) but the choroid
thinned slightly (-17 ± 17 mm; p = 0.03) increasing vitreous depth (79
± 17 mm; p < 0.01) without refractive shift (-1.1 ± 1.45 D; p = 0.44).
The lens thickened (36 ± 8 mm; p = 0.002) and anterior chamber
thinned (-41 ± 12 mm; p = 0.009). COLOR increased eye growth (101
± 34 mm; p<0.05) but the choroid thickened (41 ± 19 mm), preventing
significant vitreal (59 ± 33 mm) and refractive change (0.38 ± 1.0 D).
With SCGX, LUM prevented vitreal growth (-13 ± 19 mm), and the
choroid thickened slightly (16 ± 6 mm; p = 0.03), without refractive
shift (0.3 ± 0.6 D). The lens thinned (32 ± 9 mm). COLOR increased
vitreal growth (40 ± 14 mm; p=0.02) without refractive shift (0.8 ±
1.0 D).
Conclusions: The results indicate common neural pathways for CGX
and LUM flicker, slowing growth and mostly affecting the anterior
eye, and for SCGX and color flicker, increasing growth and affecting
the posterior eye.
Commercial Relationships: Frances J. Rucker, None; Falk
Schroedl, None
Support: Research Promotion Fund of the Paracelsus University
S13/05/007-SCH: F. Rucker and F. Schroedl. Neural pathways for
Emmetropization
Program Number: 2152 Poster Board Number: B0005
Presentation Time: 3:45 PM–5:30 PM
Scotopic and Photopic Lighting Prevents Lens-induced Myopia
in Mice
Erica Landis1, Han na Park2, Megan Prunty2, 3, Curran Sidhu2,
P M. Iuvone2, 4, Machelle T. Pardue2, 3. 1Neuroscience, Emory
University, Atlanta, GA; 2Ophthalmology, Emory University,
Atlanta, GA; 3Rehab Center for Excellence, Atlanta VA, Atlanta, GA;
4
Pharmacology, Emory University, Atlanta, GA.
Purpose: The goal of this study was to determine the effects of
different ambient illumination levels on dopaminergic signaling in
the retina and on the susceptibility of the mouse eye to lens-induced
myopia. Previous studies have shown photopic lighting to protect
against myopia in both controlled animal studies and correlational
human population studies. Photopic lighting was tested as well as
scotopic lighting since rods may be needed for emmetropization
(Park et al IOVS 2014).
Methods: Male C57BL/6J mice were exposed to photopic (15,000
lux, n=38), mesopic (50 lux, n=36), or scotopic (0.005 lux, n=38)
lighting during the light phase of a 12:12 hr light cycle, starting at
postnatal day 23 (P23). At P28, half the mice received head-mounted
monocular lens defocus (-10D). Retinas were enucleated at P36 and
analyzed for dopamine and DOPAC levels via HPLC. At each time
point, the refractive error, corneal curvature, and ocular parameters
of the mice were measured. The DOPAC/dopamine ratios were
calculated as a measure of dopamine turnover.
Results: After two weeks of exposure to photopic, mesopic, or
scotopic light there was no myopic shift (OD minus OS) in the
refractive development of the control animals. Mice with lens
defocus under mesopic light had significantly larger myopic shifts
(normalized to P28, -4.741±0.608; p<0.005) by P34 compared to
mice exposed to photopic (-2.604±0.544) or scotopic (-1.807±0.608).
Additionally, in a subset of mice, the difference in DOPAC/dopamine
ratio between the lens defocused and opposite eyes was significantly
increased in mice exposed to scotopic light levels (0.022±0.007);
decreased in mice exposed to mesopic light levels (-0.020±0.003;
p<0.001); and showed no significant change in mice exposed to
photopic light (0.002 ±0.003). No significant differences were found
in corneal curvatures or axial lengths.
Conclusions: While photopic and scotopic light levels were
protective against lens induced myopia with increases in dopamine
turnover, mesopic light levels increased the development of lensinduced myopia with a decrease in dopamine turnover. This implies
that high and low intensities of light may prevent myopia, while
intermediate intensities, similar to indoor lighting, promote myopia.
Commercial Relationships: Erica Landis, None; Han na Park,
None; Megan Prunty, None; Curran Sidhu, None; P M. Iuvone,
None; Machelle T. Pardue, None
Support: NEI Grant EY016435, NEI Grant EY004864, NEI Core
Grant P30EY006360, Research to Prevent Blindness
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 2153 Poster Board Number: B0006
Presentation Time: 3:45 PM–5:30 PM
Retinal-specific Dopamine Knock-out Mice are Myopic
Michael A. Bergen3, 4, Han na Park1, Ranjay Chakraborty1, 4, Erica
G. Landis5, 4, Curran Sidhu1, P M. Iuvone1, 2, Machelle T. Pardue4, 1.
1
Ophthalmology, Emory University School of Medicine, Atlanta, GA;
2
Pharmacology, Emory University School of Medicine, Atlanta, GA;
3
Biology, Emory University, Atlanta, GA; 4Rehab R&D Center of
Excellence, Atlanta VA Medical Center, Decatur, GA; 5Neuroscience,
Emory University, Atlanta, GA.
Purpose: Dopamine has been implicated as a stop signal for
refractive eye growth based on pharmacological studies in chickens,
mammals, and primates. More recently, dopamine receptor knockout mice have been used to elucidate dopaminergic mechanisms of
refractive development (Huang et al. IOVS 2014). In this study, a
Cre-mediated, retinal-specific tyrosine hydroxylase knockout (KO)
mouse was studied to determine the effect of eliminating retinal
dopamine on refractive development and susceptibility to form
deprivation (FD) myopia.
Methods: KO mice were on a C57BL/6J background and were
homozygous for both the Chx10 Cre-recombinase and floxed tyrosine
hydroxylase alleles. Mice were randomly assigned to two groups, one
undergoing normal refractive development and the other undergoing
FD. Refractive development of KO mice and age-matched C57BL/6J
wild-type (WT) mice was measured every 2 weeks from post-natal
day 28 (P28) to P112. Under the FD paradigm, mice received a headmounted diffuser goggle at P28 over their right eye (OD) and were
measured weekly until P77. Measurements of refractive error, corneal
curvature, and ocular biometrics were obtained using an automated
photorefractor, a keratometer, and a spectral-domain optical
coherence tomography system, respectively.
Results: During normal refractive development, KO mice were
significantly more myopic (at P70, KO 2.74 ± 2.18 D, n=19; WT 7.08
± 1.47 D, n=9; p < 0.01) and had significantly shorter axial lengths (at
P56, KO 3.18 ± 0.01 mm; WT 3.23 ± 0.04 mm; p < 0.05) than their
WT counterparts. KO mice also had significantly steeper corneas than
WT mice (at P56, KO 1.42 ± 0.03 mm; WT 1.44 ± .03 mm; p < 0.05).
Both WT and KO form-deprived mice showed similar magnitudes of
myopic shift (difference of right and left eyes) (at P42, KO -2.29 ±
3.59 D, n=8; WT -3.85 ± 1.35 D; n=7).
Conclusions: Our results support the hypothesis that dopamine is
a stop signal for refractive eye growth. KO mice showed greater
variability in FD myopic shifts than WT mice, which may indicate
varying levels of retinal dopamine depletion in this model. Future
work will correlate dopamine and DOPAC levels with refractive error
and ocular parameters to comprehensively examine how dopamine
concentration affects refractive development
Commercial Relationships: Michael A. Bergen, None; Han na
Park, None; Ranjay Chakraborty, None; Erica G. Landis, None;
Curran Sidhu, None; P M. Iuvone, None; Machelle T. Pardue,
None
Support: NEI Grant EY016435, NIH Core grant P30EY006360,
Research to Prevent Blindness Grant
Program Number: 2154 Poster Board Number: B0007
Presentation Time: 3:45 PM–5:30 PM
Apomorphine attenuates form-deprivation myopia (FDM) by a
dopamine D2R-independent mechanism
Xiangtian Zhou, Furong Huang, Jiangfan Chen, Jia Qu. School
of Ophthalmology and Optometry, Wenzhou Medical College,
Wenzhou, China.
Purpose: The dopamine agonist apomorphine (APO) can profoundly
attenuate form-deprivation myopia (FDM) development in animals.
Here, we determine whether APO acts at dopamine D2 receptors
(D2R) to exert its effect on myopia development using D2R knockout
(KO) mice.
Methods: Wild-type (WT) littermates and D2R KO were subjected
to FDM at postnatal days 28-56. Both groups were intraperitoneally
injected daily with either APO (5 mg/kg/day) or vehicle for 4 weeks
(starting from postnatal day 28). Their body weight, refraction,
corneal radius of curvature and ocular axial components were
measured at the end of 4-week treatment.
Results: Consistent with our recent report, D2R KO attenuated
FDM development compared to WT littermates. As expected, APO
treatment attenuated myopia development compared with vehicle
treatment in WT mice. Importantly, APO treatment in D2R KO mice
further attenuated myopia development compared with the vehicle
treatment in D2R KO mice. In parallel with refractory changes, D2R
KO alone or APO alone also attenuated FDM-induced elongation
of vitreous chamber depth and axial length compared to their
corresponding controls. Moreover, combined treatment of D2R KO
an APO treatment attenuated FDM-induced elongation of vitreous
chamber depth and axial length compared to the D2R KO treated
with vehicle.
Conclusions: The inhibition of APO on FDM development was still
effective in absence of D2Rs, suggesting that APO attenuates myopia
development by a D2R-independent mechanism.
Commercial Relationships: Xiangtian Zhou, None; Furong
Huang, None; Jiangfan Chen, None; Jia Qu, None
Support: 973 project:2001CB504602
Program Number: 2155 Poster Board Number: B0008
Presentation Time: 3:45 PM–5:30 PM
Effectiveness of Low-dose Atropine on the Marmoset Eye
Alexandra Benavente-Perez1, Ann Nour1, Eric R. Ritchey2, David
Troilo1. 1Biological Sciences, SUNY College of Optometry, New
York, NY; 2Johnson & Johnson Vision Care, Inc, Jacksonville, FL.
Purpose: Low dose atropine (0.5%, 0.1% and 0.01%) has been
shown to reduce myopia progression in humans by up to 24.5%
(0.01%) to 40% (0.5%). The purpose of this study was to develop
reliable measures to evaluate the effects of low doses of atropine on
marmoset eyes for studies of myopia control.
Methods: Nine age-matched young adult marmosets were divided
into three treatment groups of low-dose atropine (0.1%, 0.01% or
0.005%). Subjects received one drop of atropine in one eye. The
contralateral eye served as control. Pupil diameter (PD) under fixed
background illumination (254lux) and pupillary light reflex (PLR)
to ophthalmoscope illumination were monitored at baseline and
after atropine every 5mins for the first 30mins, every hour for the
first 9hrs, and daily for 7 days. The accommodative response to 1D
of imposed hyperopic defocus was measured with a Shin-Nippon
autorefractor in one marmoset from each group for 5 days following
the atropine drop.
Results: The interocular PD difference, normalized to baseline,
reached maximum at 4hrs (0.1%), 3hrs (0.01%) and 2hrs (0.005%)
after instillation (+1.96±0.26mm,+1.46±0.28mm and +1.45±0.49mm
respectively). PDs returned to baseline levels 6 (0.1%) or 7 days
(0.01% and 0.005%) after instillation. The PLR was blocked 15 to
25mins after 0.1% instillation and recovered after 2 days, but was
always present for the 0.01% and 0.005% dosages. Accommodation
was reduced 3hrs after instillation of 0.1% and recovered after 4
days (baseline accommodation: 1.11±0.56D; 3hrs post: 0.05±1.08D,
p=0.03; 4days post: 1.45±1.20D, p>0.05). Lower doses of atropine
did not block the accommodative response (p>0.05).
Conclusions: A single drop of atropine had measurable effects on
pupil function lasting several days and showed a dose response. Only
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
the highest dose tested affected accommodation. While the effects of
extended treatment have yet to be examined, these results will help
guide further investigation of the effects of low-dose atropine for
myopia control using the marmoset model.
Commercial Relationships: Alexandra Benavente-Perez,
2Johnson & Johnson Vision Care, Inc (F); Ann Nour, Johnson &
Johnson Vision Care, Inc (F); Eric R. Ritchey, Johnson & Johnson
Vision Care, Inc (E); David Troilo, Johnson & Johnson Vision Care,
Inc (C), Johnson & Johnson Vision Care, Inc (F)
Support: Johnson & Johnson Vision Care, Inc
Program Number: 2156 Poster Board Number: B0009
Presentation Time: 3:45 PM–5:30 PM
The control effects of Fenofibrate on the axial length elongation
in lens-induced myopia chicken model
Panfeng Wang1, 2, Thomas C Lam2, Chi Ho To2. 1Zhongshan
Ophthalmic Center, Sun Yat-sen University, Guangzhou, China;
2
Laboratory of Experimental Optometry,Centre for Myopia Research,
School of Optometry, The Hong Kong Polytechnic University, Hong
Kong, Hong Kong.
Purpose: Apolipoprotein A1 (ApoA1), the major component of high
density lipoprotein, was suggested to be down-regulated in the retina
of lens-induced myopia (LIM) animal model. The growth of axial
length was reduced in LIM chicken possibly through an up-regulation
of the ApoA1. Fenofibrate, a peroxisome proliferator-activated
receptor α (PPARα) agonist, is the first line therapy to regulate lipid
metabolism by increasing the synthesis of ApoA1. The current study
investigated the efficacy of fenofibrate on the growth of eye axial in
lens-induced myopia chicken model.
Methods: At 4-day old, male chicken was divided into different
groups after gender determination by PCR method. In the LIM group,
negative powered lenses (-10D) were worn on the right eyes and the
left eyes were kept untreated as controls. In the treatment groups,
20uM, 100uM, 200uM fenofibrate delivered at an injection volume
of 10ul, were injected into the bottom of the vitreous chamber of
the right eye, and vehicle solutions were injected into the left eyes
as control on day 5. Then, lenses (-10D) were worn on both eyes
of treated chicken. An extra group of chicken without lens received
200uM fenofibrate intravitreal injection at the right eyes, and the left
eyes were kept untreated as normal control. A high-frequency A-scan
ultrasound system was used to measure the ocular parameters of all
the chicken before the treatment and on day 8. Chicken with body
weight growth abnormality or vitreous hemorrhage were ruled out.
The changes of ocular parameters among different treated eyes were
statistically analyzed by t-test.
Results: Fenofibrate dose dependently suppressed the growth of
ocular axial length, and statistical difference was recorded at the
concentration of 200uM. On day 8, LIM chicken (n=15) treated
by fenofibrate (200uM) had shorter axial length than LIM chicken
(n=13) by 32.7% (P= 5.15078E-07). The fenofibrate showed no
effect on the growth of ocular axial length of normal eyes. There was
no different between fenofibrate treated eyes and LIM eyes at the
choroid recovery changes after the negative lenses were taken off on
day 8.
Conclusions: 200uM of Fenofibrate is protective against LIM
development. The results not only demonstrate the therapeutic effects
of fenofibrate on myopia, but they also support the possible role of
PPARα-dependent mechanism in the development of myopia.
Commercial Relationships: Panfeng Wang, None; Thomas C
Lam, None; Chi Ho To, None
Support: XJ2012061
Program Number: 2157 Poster Board Number: B0010
Presentation Time: 3:45 PM–5:30 PM
Effect of oral administration of nicotinic acid on ocular growth of
lens-induced myopic chicks
Hu XIAO, Panfeng Wang, King Kit Li, Rachel Ka-man Chun,
Thomas C Lam, Chi Ho To. School of Optometry, The Hong Kong
Polytechnic University, Hong Kong, Hong Kong.
Purpose: To explore the effect of oral administration of nicotinic
acid on eye growth of normal and myopic chicks. The nicotinic acid
is the normal drug used in human to raising the HDL in blood. The
Apolipoprotein A1 is the potential in protect myopic eye growth in
previous studies.
Methods: White Leghorn chicks aged at 4 days (n = 48 in total) were
randomly allocated into 4 groups. Chicks in group A and B were
orally administered a single dose of nicotinic acid daily (150mg/
ml, 1ml per chick) while the chicks in group C and D were received
saline orally as control (1ml per chick). The oral administration last
for 11 consecutive days. After 7 days of oral administration, -10D
lenses were attached to both eyes of the chicks in group A and C
while the chicks in group B and D worn plano lenses for 4 days. The
refractive errors and ocular dimension components were examined
using streak retinoscopy and high resolution A-scan ultrasonography
before and after 11 days of the oral administration respectively. The
changes of refractive errors and vitreous chamber depth between 1
days and 11 days of oral administration were gained. T-test was used
to analysis the difference.
Results: After 4 days of lens wear, chicks with -10D lenses became
significantly more myopic than the chicks with plano lenses (A and
B group: P= 0.0274; C and D group: P= 0.0013; t-test). In the groups
with -10D lenses (group A and C), the changes in vitreous chamber
depth (VCD) in chicks treated with nicotinic acid (mean ± SEM;
0.568 ± 0.146mm) were significantly less than that of the salinetreated chicks (0.778 ± 0.197 mm, p = 0.007, t-test). The change in
refractive errors in chicks treated with nicotinic acid (mean ± SEM;
-8.35 ± 1.11D) were significantly less than that of the saline-treated
chicks (-10.81± 0.75D, p = 2.20E-06, t-test). There was no significant
difference in VCD between the groups wearing plano lenses (group
B: nicotinic acid vs. group D: saline; 0.398 ± 0.202mm, vs. 0.514±
0.151mm, p = 0.125, t-test). But there was significant difference in
refractive errors between the groups wearing plano lenses (group B:
nicotinic acid vs. group D: saline; -3.25 ± 0.30D, vs. -4.54 ± 0.35D, p
= 2.20E-09, t-test).
Conclusions: The nicotinic acid intake could retard the elongation of
VCD in lens-induced myopic chicks. Its effect on the normal ocular
growth is however not apparent.
Commercial Relationships: Hu XIAO, None; Panfeng Wang,
None; King Kit Li, None; Rachel Ka-man Chun, None; Thomas C
Lam, None; Chi Ho To, None
Support: PolyU research grants: GYK89, GU986; RGC GRF:
BQ29N
Program Number: 2158 Poster Board Number: B0011
Presentation Time: 3:45 PM–5:30 PM
Form-deprived highly myopic chick eyes have lower than normal
corneal stiffness than emmetropic eyes
Byung Soo Kang1, Li Ke Wang2, Yong-Ping Zheng2, Chea-su Kee1.
1
School of Optometry, The Hong Kong Polytechnic University,
Hong Kong, Hong Kong; 2Interdisciplinary Division of Biomedical
Engineering, The Hong Kong Polytechnic University, Hong Kong,
Hong Kong.
Purpose: This study aimed to determine whether corneal stiffness
differed between normal and highly myopic eyes in the chick model
of myopia
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Methods: Starting from day 5 post-hatching, the right eyes of 21
chicks (Gallus Gallus Domesticus) were covered with a translucent
occluder for 7 days to induce form-deprived myopia. At the end
of the treatment period, spherical equivalent (SE) refractive error
was measured under anesthesia (1.5% Isoflurane) by Hartinger
coincidence refractometry, and in-situ corneal stiffness (CS) was
measured using a custom-made air-jet optical coherent tomography
(OCT) system. Specifically, triplicate CS measurements were
obtained, with corneal deformation (in mm) assessed in response
to 5 cycles of increasing/decreasing air pressure (in N). A custom
algorithm calculated the slope of CS load-deformation curve (N/mm)
and the correlation between these parameters
Results: Compared to fellow untreated eyes, form-deprived eyes
developed significant myopia (mean±SEM: SE= -20.65 ± 1.41D
vs. -1.22 ± 0.26D; paired t-test, p<0.001) and exhibited reduced
corneal stiffness (mean±SEM: CS= 0.0203 ± 0.0008mm vs. 0.0239
± 0.0009mm; paired t-test, p<0.05). When data from both eyes were
used for correlation analyses, both SE (r=+0.462, p<0.002) and J45
astigmatic component (r=+0.351, p<0.05) were moderately correlated
with corneal stiffness. Expressing the corneal stiffness as percentage
of interocular difference in CS [100 %*( treated eye – fellow eye)
/ fellow eye], we found that in the 17 birds that showed a reduction
in corneal stiffness in the treated eyes (range= -0.35% ~ -46.65%),
eleven (64.7 %) of them had at least 15% reduction in corneal
stiffness (mean= -24.46%, one-sample t-test, T= -4.57, p<0.01)
Conclusions: Form deprivation induced high myopia and reduced
corneal stiffness. The correlation between SE and CS indicates that
the changes occurring in the biomechanical properties of the cornea
may be quantitatively related to those occurring in the sclera
Commercial Relationships: Byung Soo Kang, None; Li Ke Wang,
None; Yong-Ping Zheng, None; Chea-su Kee, None
Program Number: 2159 Poster Board Number: B0012
Presentation Time: 3:45 PM–5:30 PM
Regional Differences in Gene Expression with Imposed Defocus
in Chick RPE
Yan Zhang, Emily Eng, Christine F. Wildsoet. School of Optometry,
Univ of California, Berkeley, Berkeley, CA.
Purpose: We previously reported bidirectional gene regulation of
members of the Bone Morphogenetic Protein (BMP) family (2, 4, &
7) in chick retinal pigment epithelium (RPE) in response to as little as
2 h of imposed optical defocus. This study investigated whether there
were also regional differences in the effects of imposed defocus on
the expression of these genes in chick RPE.
Methods: 19-day old White-Leghorn chicks wore monocular +10
or -10 D lenses for 2 h. At the end of the treatment period, RPE
was isolated and divided into 3 circular zones using punches of 3
and 6 mm radius: central 3 mm zone (T3 or F3; T: treated eyes, F:
fellow controls), middle 3-6 mm zone (T6 or F6), and peripheral 6-9
mm circular zone (T9 or F9). RPE RNA was purified and reverse
transcribed to cDNA. qPCR was performed to examine the gene
expression of BMP2, 4, and 7. Expression levels were compared
between lens treated and fellow control eyes. Paired Student’s t test
was used for statistical analysis.
Results: The +10 D lens treatment induced up-regulation of both
BMP2 and BMP4 gene expression in the central and middle zones
(T3 & T6 regions compared to F3 & F6). For BMP2, gene expression
was highly up-regulated, by 129- and 28-fold (T3/F3, n = 3, p =
0.09; T6/F6, n = 4, p = 0.05). The equivalent values for BMP4 gene
expression were 13- and 10-fold (T3/F3, n = 3, p < 0.05; T6/F6, n =
4, p < 0.05). BMP7 expression was 3-fold up-regulated for the T3/
F3 comparison only. The most peripheral region (T9/F9) did not
show differential gene expression between treated and fellows for
any of the genes. As expected, the -10 D lens induced down- instead
of up-regulation; significant changes were recorded for both BMP2
and BMP4, for both T3/F3 and T6/F6 comparisons. BMP2 was
down-regulated by 32- and 12-fold for T3/F3 and T6/F6 comparison,
respectively (n = 4), but not for the T9/F9 comparison. Likewise,
BMP4 gene expression was down-regulated 12- and 3.6-fold in
central (T3/F3) and middle (T6/F6) regions respectively. BMP7 gene
expression did not change for any of these three regions.
Conclusions: This study provides evidence of regional variations in
the response of chick RPE to the same defocus, i.e. as imposed by
single vision lenses, generally decreasing with increasing eccentricity.
The significant up- or down-regulation of BMP gene expression in
central RPE points to a critical role of the central retina/RPE in early
stage of eye growth regulation.
Commercial Relationships: Yan Zhang, None; Emily Eng, None;
Christine F. Wildsoet, None
Support: NIH grants R01EY012392 (CFW), K08EY023609 (YZ),
K12EY017269 (YZ), T35EY007139 (EE)
Program Number: 2160 Poster Board Number: B0013
Presentation Time: 3:45 PM–5:30 PM
Retinal profile asymmetries in myopes and emmetropes
Christopher A. Clark, Ann E. Elsner, Bryan Haggerty, Joel A. Papay.
School of Optometry, University of Indiana, Bloomington, IN.
Purpose: Previous work has shown that peripheral refractive error
asymmetries exist between different locations in the retina and those
asymmetries are greater in myopes than emmetropes. This could be
due to a number of factors including physical restriction from the
optic nerve and differences in optical alignment between the optical/
visual axis. The purpose of this study is to investigate the source of
asymmetry.
Methods: Fifty-six subjects (refractive error +1.50 to -11.15)
had a battery of tests performed including axial length, corneal
topography, anterior chamber depth, peripheral refraction, peripheral
partial coherence interferometry, and SD OCT for retinal thickness.
Turning point location (TPL) was classified as the retinal location in
degrees where the retinal profile was at a minimum. Angle alpha was
measured as the distance from the apex of the corneal topography to
the visual axis. Statistics including repeated measures ANOVA were
performed with SPSS (IBM, Endicott, NY.)
Results: The TPL was greatest in high myopes with an average
displacement of three degrees temporal from the fovea compared
to approximately zero degrees for emmetropes (P = 0.045.) No
correlation was found in this study between angle alpha and either
the TPL or retinal profile asymmetry along the horizontal axis.
Asymmetry did increase with refractive error (P = 0.008.) Visual
inspection of the SD OCT images showed greater optic nerve head
tilt in subjects with higher asymmetries regardless of refractive error.
Conclusions: Retinal profile asymmetries increase with refractive
error which is consistent with previously reported data. These
asymmetries appear to be largely due to physical constriction by the
optic nerve rather than to do optical effects such as angle alpha.
Commercial Relationships: Christopher A. Clark, None; Ann E.
Elsner, None; Bryan Haggerty, None; Joel A. Papay, None
Support: NIH Grant K23EY022064
Program Number: 2161 Poster Board Number: B0014
Presentation Time: 3:45 PM–5:30 PM
Effects of 6-hydroxydopamine on refractive development and
form-deprivation myopia in C57BL/6 mice
Shi-Jun Weng, Xiao-Hua Wu, Yun-Yun LI, Kang-Wei Qian, Xiong-Li
Yang, Yong-Mei Zhong. Institute of Neurobiology, Fudan University,
Shanghai, China.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Purpose: In various species, reduced retinal dopamine (DA) levels
are thought to mediate the development of myopia. However, recent
evidence shows that in the C57BL/6 mouse, retinal DA levels are
unaltered when form-deprivation myopia is developed. Here, to
further explore the role of retinal DA in mouse eye growth, we
examined whether refractive development could be disturbed by
destroying retinal DA pathway in this mouse strain.
Methods: 6-hydroxydopamine (6-OHDA, 50 μg) was intravitreally
applied to the right eye using a micro-injector, and the left eye serves
as the control. Refractive errors were measured using an automated
eccentric infrared photorefractor, in animals raised in normal visual
environment, or in those with the injected eye wearing an occluder
for 4 weeks to induce form-deprivation myopia. The levels of retinal
DA and its primary metabolite 3,4-dihydroxyphenylacetic acid
(DOPAC) were assessed by HPLC analysis.
Results: Administration of 6-OHDA significantly reduced retinal
DA levels by 40-80%, and the effect lasted for at least 31 days.
With normal visual experience, the 6-OHDA-injected eyes became
markedly myopic relative to their fellow eyes (~6D of interocular
difference). Furthermore, in injected eyes, form-deprivation did not
induce further myopic shifts, nor did it cause further reduction in
retinal DA and DOPAC levels.
Conclusions: An intact retinal dopaminergic system, including
healthy dopaminergic amacrine cells and complete retinal DA
stores, is essential for both normal refractive development and the
generation of form-deprivation myopia in the C57BL/6 mouse. In this
mouse strain, refractive development could be interfered by reducing
retinal DA levels dramatically, even though the generation of formdeprivation myopia is not associated with retinal DA levels.
Commercial Relationships: Shi-Jun Weng, None; Xiao-Hua Wu,
None; Yun-Yun LI, None; Kang-Wei Qian, None; Xiong-Li Yang,
None; Yong-Mei Zhong, None
Support: Ministry of Science and Technology of China
(2011CB504602); the National Natural Science Foundation of China
(31171055, 31121061, 31100796); ARVO/Pfizer Collaborative
Research Fellowship to Shi-Jun Weng
Program Number: 2162 Poster Board Number: B0015
Presentation Time: 3:45 PM–5:30 PM
Identification of apolipoprotein A-I as a novel retinoic acid
binding protein
Jody A. Summers1, Angelica Harper1, Hanke Van-Der-Wel2,
Marcela Hermann3, Christopher M. West2. 1Cell Biology, University
of Oklahoma Health Science Center, Oklahoma City, OK;
2
Biochemistry, University of Oklahoma Health Science Center,
Oklahoma City, OK; 3Medical Biochemistry, Medical University of
Vienna, Vienna, Austria.
Purpose: All-trans-retinoic acid (atRA) may be an important
molecular signal in the postnatal control of eye size. Retinoids are
closely associated with atRA binding proteins, which are important
in regulating their transport, metabolism and biological activity. We
and others (Mertz and Wallman, Exp. Eye Res. 2000) have previously
identified a protein with an apparent Mr of 27 kD (p27) that
represents the major secreted atRA binding protein in chick choroids.
The goal of the current study was to identify p27 as we hypothesize
that p27 may play a role in the regulation of atRA activity during
visually guided ocular growth.
Methods: atRA binding proteins were initially identified from
conditioned medium of chick ocular tissues by photoaffinity 3H-atRA
labeling, SDS-PAGE and autoradiography. 3H-atRA binding proteins
were purified using a combination of anion exchange (Q-sepharose),
gel filtration (Superdex 200) columns and SDS-PAGE. Unlabeled
samples were processed in parallel and peak fractions of unlabeled
protein, corresponding to the elution position and mass of 3H-atRAlabelled protein were identified by mass spectrometry. The identify of
p27 was confirmed using immunoprecipitation of 3H-atRA-labelled
p27 from conditioned medium using anti-chick apolipoprotein A-I
antibodies.
Results: Following photoaffinity labeling of choroid and sclera
conditioned medium, radiolabeled proteins migrating at 60 kD
and 27 kD were detected by autoradiography. These proteins coeluted from Q-sepharose and Superdex 200. Mass spectrometry
analyses identified the 60 kD protein as serum albumin and the 27
kD protein as apolipoprotein A-I. Following immunoprecipitation
of 3H-atRA labeled proteins from sclera conditioned medium with
anti-chick apolipoprotein A-I, a single 27 kD band was detected on
autoradiograms.
Conclusions: Apolipoprotein A-I is the 27 kD 3H-atRA-binding
protein present in chick choroid and sclera conditioned medium. The
expression of this protein may play a role in the regulation of atRA
signaling in the choroid and sclera in postnatal ocular growth.
Commercial Relationships: Jody A. Summers, None; Angelica
Harper, None; Hanke Van-Der-Wel, None; Marcela Hermann,
None; Christopher M. West, None
Support: NIH Grant EY09391
Program Number: 2163 Poster Board Number: B0016
Presentation Time: 3:45 PM–5:30 PM
Transcriptome Analysis Indicates Adaptive Responses to
Physiological Stress in Recovery from FDM
Loretta Giummarra1, Nina Riddell1, Nathan Hall2, 3, Melanie
Murphy1, Sheila G. Crewther1. 1Psychological Science, La Trobe
University, Melbourne, VIC, Australia; 2Life Sciences Computation
Centre (LSCC), Victorial Life Sciences Computation Centre
(VLSCI), Melbourne, VIC, Australia; 3La Trobe University,
Melbourne, VIC, Australia.
Purpose: Form deprivation myopia (FDM) is associated with
dramatic increases in ocular volume, axial length, thinning of the
retina and choroid and hyperosmotic stress. Thus this study aimed to
assess the associated gene pathway changes using high throughput
RNA-sequencing and comprehensive bioinformatic analysis. Given
our previous ultrastructural and elemental microanalyses it was
hypothesized that profile changes would involve energy metabolism,
ionic solute changes and evidence of oxidative stress
Methods: Twelve male hatchling chicks were monocularly occluded
from days 4-11 after which chicks were given T=0hr, T=6hr or
T=24hr of normal vision. Four chicks were used as aged-matched
unoccluded controls. Biometrics were measured prior to tissue
collection. RNA was isolated from choroid/retina/RPE tissue and
prepared for sequencing on the Illumina HiSeq™ 1500. Raw reads
were mapped onto the chicken genome and counts determined for
each gene. Differential expression analysis was undertaken with
voom/EdgeR with an FDR of 0.05. Gene Set Enrichment Analysis
(GSEA) software was used to determine whether a priori defined
set of genes were significantly altered (FDR<0.25) during the
induction and recovery of FDM. Curated gene sets were obtained
from BioCarta, KEGG, and the Pathway Interaction Database and the
Reactome database.
Results: FD Chicks were ~20D myopic. Refractive normalization
began with removal of occlusion. GSEA analysis revealed an overall
suppression in genes associated with metabolism and ion homeostasis
at T=0hr. GSEA during the recovery period revealed an increase in
expression of genes associated with glucose metabolism, potassium
transport and hypoxia. These changes were positively correlated with
reduction in refraction.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Conclusions: Increased axial growth during FD is accompanied by
suppression of gene pathways associated with retinal metabolism
and refractive normalization. Removal of FD and reintroduction of
the normal visual environment with constantly changing luminance
levels requires upregulation of metabolic pathways and normalization
of ion distribution profiles across the eye. These results confirm our
previous work and build our understanding of the importance of
osmoadaptive pathways that use energy metabolism, ion transport, to
reduce hypoxia and restore osmotic homeostasis.
Commercial Relationships: Loretta Giummarra, None; Nina
Riddell, None; Nathan Hall, None; Melanie Murphy, None; Sheila
G. Crewther, None
Program Number: 2164 Poster Board Number: B0017
Presentation Time: 3:45 PM–5:30 PM
RNAseq gene expression analysis highlights the correlation
of changes in metabolic and structural pathways with axial
elongation during refractive compensation.
Nina Riddell1, Loretta Giummarra1, Nathan Hall4, 2, Melanie
Murphy1, David P. Crewther3, Sheila G. Crewther1. 1Psychological
Science, La Trobe University, Bundoora, VIC, Australia; 2La Trobe
University, Melbourne, VIC, Australia; 3Swinburne University,
Melbourne, VIC, Australia; 4Life Sciences Computation Centre
(LSCC), VLSCI, Melbourne, VIC, Australia.
Purpose: High throughput transcriptome studies in animal models
of refractive error have primarily analysed data at the single-gene
level. Results from these studies are disparate and a comprehensive
framework for understanding the biological cascades underlying
ocular growth regulation remains elusive. Thus, this study aimed to
identify characteristic biological features of refractive compensation
to myopic and hyperopic defocus in chick by correlating axial length
across lens-groups during defocus induction with expression of genes
in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
Methods: Chicks were raised with ±10D lenses, or no lens.
Following biometric measurements at 1, 2, and 3 days, 3-4 chicks per
lens-group were euthanized and RNA extracted from the retina/RPE/
choroid. Libraries were sequenced on the Illumina HiSeq1500, raw
reads mapped to the chick genome, and counts determined for each
gene. Counts/million were imported into GSEA and expression of
KEGG pathways correlated with axial length phenotype across lensgroups at each time-point (FDR cut-off <.25).
Results: Refractive and axial length change was rapid during the
first day of defocus for both lens-types and slower over subsequent
days (particularly for plus lenses). Consistent with the initially rapid
change in ocular morphology, expression of structural pathways
including focal adhesion, tight junction, and vascular smooth muscle
contraction was positively correlated with axial length at 1 day.
Fatty acid metabolic and signalling pathways were also correlated
with axial length at this time. Although no structural pathways were
identified following 2 and 3 days of lens-wear when morphological
changes had slowed, metabolic pathways (such as oxidative
phosphorylation) were implicated at both time-points.
Conclusions: This study is the first to correlate ocular axial length
changes with pathway enrichment across a period of refractive
compensation to lenses. Results suggest that changes in structural
pathway expression are linked to periods of rapid axial growth
change. Perturbed metabolism was characteristic of all stages of
compensation, with implication of oxidative phosphorylation and
related pathways suggesting that growth changes elicit a shift in
energy homeostasis that may alter redox state and vulnerability to
later development of ocular pathologies.
Commercial Relationships: Nina Riddell, None; Loretta
Giummarra, None; Nathan Hall, None; Melanie Murphy, None;
David P. Crewther, None; Sheila G. Crewther, None
Program Number: 2165 Poster Board Number: B0018
Presentation Time: 3:45 PM–5:30 PM
Anti-Diuretic Hormone in the Regulation of Ocular Volume in
Compensation to Defocus
Melanie Murphy1, Loretta Giummarra1, Nina Riddell1, David P.
Crewther2, Vinh Nguyen1, Sheila G. Crewther1. 1Psychological
Science, La Trobe University, Melbourne, VIC, Australia; 2Centre for
Human Psychopharmacology, Swinburne University of Technology,
Melbourne, VIC, Australia.
Purpose: The hormone Arginine Vasopressin (AVP) is a
vasoconstrictor and anti-diuretic that is commonly associated with
stress. Our previous results show that AVP causes a myopic shift
in refractive compensation (RC) to +10D defocus (ARVO, 2013).
Further, environmental stress in the form of asymmetric flicker
impacts ocular growth (Crewther et al, 2006). Thus the current
experiment aimed to investigate whether AVP plays a role in RC to
defocus, and whether flicker affects this process.
Methods: Experiment 1: RNA was extracted from the retina/RPE/
choroid of chicks with + or -10D, or no defocus on days 5-7 posthatching (n = 3 per lens group, per day) and prepared for sequencing
on the Illumina HiSeq1500. Raw reads were mapped onto the chick
genome and counts determined for each gene. Counts per million
were imported into Pathway studio and GSEA conducted using the
Mann-Whitney U-test algorithm (p<.05).
Experiment 2: Chicks (n=360) were raised from day 5-9, with or
without asymmetric flicker in the 12 hr day cycle, with + or -10D
defocus (or non lens), following intravitreal injection of 5ml of either
PBS, AVP or the AVP receptor antagonist ([des-Gly9-β-Mercapto-β,
β cyclopentamethylenepropionyl1, O-Et-Tyr2,Val4,Arg8]Vasopressin) (in PBS) into the experimental eye. Fellow eyes
were injected with PBS. Retinoscopy and A-scan ultrasonography
was performed on day 9. Tissue was collected and prepared for
immunohistochemistry to examine AQP-4 and Kir4.1 expression.
Results: Experiment 1: RNAseq revealed sign-dependent changes in
AVP-related pathways over 3 days of rearing with defocus.
Experiment 2: Flicker alone induced a myopic shift in both lens
conditions. Flicker+AVP reduced hyperopia, axial elongation
and anterior chamber depth in +10D lenses. Flicker+AVP
antagonist reduced RC and ocular growth to -10D lenses.
Immunohistochemistry showed altered AQP-4 and Kir4.1 staining
across flicker conditions.
Conclusions: Results indicate that changes in AVP-related gene
expression occur concomitantly with changes in ocular volume
during the induction of RC. Further, AVP and its antagonist also
differentially interfered with the typical pattern of compensation
to lenswear. Physiological stress induced by flickering light further
influenced this. These results implicate stress-induced changes in the
rate of transretinal fluid movement in the development of refractive
error.
Commercial Relationships: Melanie Murphy, None; Loretta
Giummarra, None; Nina Riddell, None; David P. Crewther, None;
Vinh Nguyen, None; Sheila G. Crewther, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 2166 Poster Board Number: B0019
Presentation Time: 3:45 PM–5:30 PM
Hyperosmotic Stress and Osmo-Gene Adaptation During Early
Induction of Refractive Errors.
Sheila G. Crewther1, Nina Riddell1, Alan Marshall1, Loretta
Giummarra1, Melanie Murphy1, Melinda J. Goodyear1, David P.
Crewther2. 1Psychological Science, La Trobe University, Melbourne,
VIC, Australia; 2CHP, Swinburne University of Technology,
Melbourne, VIC, Australia.
Purpose: Why is myopia a common risk factor for most sight
threatening disorders? Our earlier biometric, ultrastructural
and elemental analyses of the chick form deprivation model
have provided evidence of severe physiological, oxidative and
hyperosmotic stress. More recently prolonged hyperosmotic stress
has been shown to lead to chronic inflammation in a number of
diseases (Brocker etal 2013). We hypothesized that perturbation
of axial growth during induction of refractive errors would also be
accompanied by hyperosmosis and osmoadaptative gene changes,
that should be demonstratable with elemental microanalysis (EDX)
and RNA seq respectively.
Methods: Chicks were raised with ±10D lenses, or no lens.
Following biometric measurements at 1, 2, and 3 days, 8 chicks per
lens group were euthanized. RNA was extracted from the retina/RPE/
choroid of 4. Four were used for scanning electron-microscopy and
EDX. Libraries were sequenced on the Illumina HiSeq1500. Counts
per million were imported into GSEA and expression of KEGG and
Reactome pathways during myopia/hyperopia induction compared to
age-matched no lens chicks (FDR cut-off <.25).
Results: Refractive compensation (RC) to -10D defocus continued
for 72hrs whereas RC to +10D was in near completion after 24hours.
EDX shows sodium and chloride ion distributions were greatly
upregulated in outer retina by -10D over the 72hrs but only at the
retino-vitreal border in +10D at 72hrs. Potassium profiles in RC
to +10D remained upregulated across the retina for 72 hrs with
concurrent up-regulation of reactome potassium channel pathways at
72hrs in RNAseq data. Consistent with altered osmotic and oxidative
stress, implicated pathways during refractive compensation included
those related to synthesis of small molecule osmolytes, structural
remodelling, inflammation, and metabolism.
Conclusions: The EDX results demonstrate that RC to optical
defocus is accompanied by hyperosmotic shifts in ion distribution
profiles across the entire posterior eye, while concurrent changes
in gene expression profiles were seen in metabolic and ion solute
processes. These pathways have previously been associated with
osmoadaptation and more severe disease states such as ARM and
diabetes. The findings suggest the need for further experimental
considerations of hyperosmotic changes as risk factors for severe
visual impairments and for development of therapeutics.
Commercial Relationships: Sheila G. Crewther, None; Nina
Riddell, None; Alan Marshall, None; Loretta Giummarra, None;
Melanie Murphy, None; Melinda J. Goodyear, None; David P.
Crewther, None
Program Number: 2167 Poster Board Number: B0020
Presentation Time: 3:45 PM–5:30 PM
Differences in the sensitivity to myopia-inducing stimuli of young
guinea pigs sourced from different colonies
Mariana Garcia2, David Hammond1, Christine F. Wildsoet2. 1Deakin
University, Geelong, VIC, Australia; 2Vision Science, University of
California, Berkeley, Berkeley, CA.
Purpose: To characterize the responses of guinea pigs sourced from
different breeding colonies to myopia-inducing stimuli using negative
lens and form deprivation paradigms.
Methods: English Short Hair guinea pig breeders were obtained from
a commercial vendor (Elm Hill Labs, Chelmsford, MA – designated
“Elm Hill” guinea pigs) and from a University-based breeding colony
(University of Auckland, NZ – designated “NZ” guinea pigs). Elm
Hill guinea pig pups were fitted with either negative lenses (-10, -5,
or 0 D) or diffusers at 10 days of age. NZ guinea pigs were fitted
with diffusers at 7 days of age. Both sets of animals were treated for
4 weeks. Ocular axial lengths were measured twice a week using
high frequency A-scan ultrasonography, cycloplegic refractions were
measured on treatment days 0, 14, and 28, and behavioral visual
acuity measured on treatment day 28.
Results: Elm Hill guinea pigs fitted with lenses exhibited minimal
interocular differences in axial length and refractive error after
28 days of treatment; likewise, form deprivation (FD) failed to
significantly affect the rate of ocular elongation or to induce a myopic
shift in refractive error. Overall changes in interocular difference
in axial length (treated minus control) were 0.03±0.1 mm for -10 D
lenses, -0.20±0.43 mm for -5D lenses, -0.01±0.21 mm for 0 D lenses,
and 0.07±0.16 mm for FD. Conversely, the NZ guinea pigs exhibited
a 0.17±0.12 mm increase in interocular differences in axial length
after 28 days of FD treatment.
Conclusions: A systematic study of the ocular growth responses of
young guinea pigs to myopia-inducing stimuli revealed significant
strain-related differences. These results point to genetically
determined differences in the sensitivity of emmetropization
mechanisms to visual manipulation, even within the same breed.
Finally, this works suggests that research groups wishing to work
with a guinea pig myopia model should carefully consider the source
of their animals.
Commercial Relationships: Mariana Garcia, None; David
Hammond, None; Christine F. Wildsoet, None
Support: EY012392
Program Number: 2168 Poster Board Number: B0021
Presentation Time: 3:45 PM–5:30 PM
Compensatory eye growth responded to the imposed defocus is
influenced by spatial content in chick
Man Pan Chin, Zhe Chuang Li, Allen Ming Yan Cheong, Ho Lung
Henry Chan. School of Optometry, The Hong Kong Polytechnic
University, Hong Kong, Hong Kong.
Purpose: Emmetopization is a visually guided eye growth, and the
compensatory eye growth is depending on the imposed defocus.
This study hypothesized if different spatial contents can affect the
compensatory eye growth responded to imposed defocus.
Methods: The right eyes of White Leghorn chicks from 10 to 12 days
old were glued with a cone-shaped lens system using Velco. Animals
were divided into six groups (n=8 to 12) for various levels of defocus
magnitude, including plano, -15D and -25D (lenses were attached
at the proximal end of the cone), with two different spatial stimulus
patterns at the other end of the cone: 1) high spatial frequency: 0.88
cycle/deg (0.4mm white/black checkers) with 100% of contrast, and
2) a lower spatial frequency: 0.28 cycle/deg (1.25mm white/black
checkers) with 100% of contrast. Axial ocular dimensions, including
anterior chamber depth, lens thickness, vitreous chamber depth
(VCD) and axial length, were obtained using A-scan ultrasound.
Measurement was carried out prior to fitting the lens system and on
the fourth day after treatment. Analysis of variance (ANOVA) was
used for statistical analysis.
Results: After 4 days of wearing the cones, the VCD of the right eye
increased with the imposed defocuses. Under the same magnitude
of defocus, chick eyes with low spatial stimulus had consistently
longer VCD than those with high spatial stimulus (p<0.05). Both
defocus and spatial stimulus showed significant main effect on VCD
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
percentage change (ANOVA, p<0.005). Significant group differences
of VCD percentage change were observed in Group -15D (low
vs high, 10.4±2.2 vs 7.25±3.35, p<0.05) and -25D (9.53±3.82 vs
4.93±4.79, p<0.05), but not in plano group. Similar trend was also
observed in axial length, but neither in anterior chamber depth nor
lens thickness.
Conclusions: Our results confirm our hypothesis that spatial
content in emmetropization can affect the compensatory eye growth
responded to same magnitude of imposed defocus. Effects of
different optical defocus on compensatory eye growth significantly
interacted with the spatial frequency of the visual stimulus. Further
studies are important to understand the mechanism of defocus and
spatial interaction in emmetropization.
Commercial Relationships: Man Pan Chin, None; Zhe Chuang
Li, None; Allen Ming Yan Cheong, None; Ho Lung Henry Chan,
None
Support: General Research Funds (PolyU5605/13M), Health and
Medical Research Fund (01121876) and PolyU Internal Grants
(G-YBBS, G-UA2E, Z-0GF, G-YM70)
Program Number: 2169 Poster Board Number: B0022
Presentation Time: 3:45 PM–5:30 PM
Effects of the relative strength of the more positive-powered
component in dual focus lenses on emmetropization in macaques
Baskar Arumugam1, 2, Li-Fang Hung1, 2, Chi-ho To3, Brien A. Holden2,
Earl L. Smith1, 2. 1College of Optometry, University of Houston,
Houston, TX; 2Vision CRC, Sydney, NSW, Australia; 3Hong Kong
Polytechnic University, Kowloon, Hong Kong.
Purpose: Dual focus lenses that impose relative myopic defocus
over a large part of the visual field can slow myopia progression in
children. Our aim was to determine how the relative surface area
devoted to the more positive-powered lens component influenced the
ability of dual focus lenses to alter refractive development.
Methods: Beginning at 3 weeks of age, infant rhesus monkeys
were reared with Fresnel lenses that had central 2mm zones of zero
power and concentric annular zones that had alternating powers of
+3.0D or 0D. The relative spatial widths of the annular zones were
varied from 1:1 (i.e., equal widths) to 1:4.5 (+3D:0D) between
treatment groups (n≥6 per group). The monkeys wore the treatment
lenses over both eyes continuously until 151±4.2 days. Comparison
data were obtained from monkeys reared with full field +3D lenses
over both eyes (FF+3D, n=6) and from 34 control monkeys reared
with unrestricted vision. Refractive status, corneal power and axial
dimensions were assessed every 2 weeks throughout the lens rearing
period.
Results: All of the dual focus lens designs produced relative
hyperopia. At the end of the treatment period, the median refractive
errors for the monkeys reared with dual focus lenses that had width
ratios of 1:1, 1:2, 1:3 and 1:4.5 were +5.25D, +5.19D, +4.31D, and
+4.28D, respectively, which were similar to the refractive errors
exhibited by animals reared with FF+3D lenses (+4.63D; p=0.22 to
0.94), but significantly more hyperopic than those found in agematched control monkeys (+2.50 D; p=0.0002 to 0.004). The average
vitreous chamber depths for the dual-lens-reared animals were also
not significantly different from those found in FF +3D lens-reared
monkeys (OD:+3D/pl 1:1; 9.31±0.34mm, 1:2; 9.44±0.60mm, 1:3;
9.74±0.38mm, 1:4.5; 9.55±0.25mm vs 9.58±0.32mm, respectively,
p=0.18 to 0.87). In addition, there were no significant differences
in either the median refractive errors (p=0.08 to 1.0) or the average
vitreous chamber depths (p=0.06 to 0.71) between the dual focus lens
groups.
Conclusions: The results demonstrate that even when the more
positive-powered zones make up only about 1/5th of a dual-focus
lens’ surface area, refractive development is dominated by relative
myopic defocus. Overall, the results emphasize that myopic defocus
distributed across the visual field evokes strong signals that can slow
eye growth in primates.
Commercial Relationships: Baskar Arumugam, None; Li-Fang
Hung, None; Chi-ho To, Inventor (P); Brien A. Holden, Zeiss (P);
Earl L. Smith, Zeiss (P)
Support: National Eye Institutes EY03611 and EY07551 and funds
from Vision CRC, Sydney, Australia
Program Number: 2170 Poster Board Number: B0023
Presentation Time: 3:45 PM–5:30 PM
Two-zone bifocal lenses with peripheral negative additions
control lens-induced hyperopia in young chicks
Huamao Miao1, Christine F. Wildsoet2. 1Department of
Ophthalmology, Eye and ENT Hospital of Fudan University,
Shanghai, China; 2Center for Eye Disease & Development, School of
Optometry, University of California Berkeley, Berkeley, CA.
Purpose: It has been proven that bifocal lenses designed with relative
positive additions can slow ocular elongation and thus myopia
progression. This study addressed a related question of whether
similar designed lenses with relative negative additions can control
hyperopia using young chicks as an animal model.
Methods: To induce hyperopia, chickens wore monocular +5
diopter (D) single vision lenses (SVLs) from 7 days of age for 3
days; the lenses were then switched for either +10 D SVLs or 2-zone
concentric bifocal lenses (BFLs), which were worn for 5 days. BFLs
had a central zone power of +10 D and one of 3 peripheral zone
powers (plano, +5 or +8 D, corresponding to additions of -10, -5 or
-2 D, respectively). For all BFL designs, both 2.5 and 4.5 mm central
zone diameters (CZDs) were tested. Central refractive errors and
ocular axial parameters were measured using static retinoscopy and
high frequency A-scan ultrasonography.
Results: At the last time point, the control group (i.e., wearing +10
D SVLs) was most hyperopic (+9.37 D), with the group wearing
2.5 mm CZD BFLs with the highest (-10 D) addition being least
hyperopic (+4.28 D). For both the 2.5 and 4.5 mm CZDs, there
were trends towards decreasing induced hyperopia with increasing
negative add power, with this dose effect being significant for 2.5
mm CZD lenses. Induced changes in vitreous chamber depth and
optical axial length (relative shortening) as well as choroid thickening
followed trends consistent with induced refractive errors.
Conclusions: Our study explored the possible application of BFLs
as a treatment to control hyperopia. Our data provide evidence that
2-zone concentric BFLs incorporating peripheral negative additions
could restrain lens-induced hyperopia progression in young chicks,
and treatment effects increasing with both add power and larger
peripheral zones. Further studies are warranted to examine whether
mammals and primates show similar beneficial effects.
Commercial Relationships: Huamao Miao, None; Christine F.
Wildsoet, None
Support: NIH/NEI Grants R01 EY012392 (CFW) & exchange
program fund for doctoral student, Graduate Medical Education,
Fudan University (HMM)
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 2171 Poster Board Number: B0024
Presentation Time: 3:45 PM–5:30 PM
Evaluation on the changes of 90 degree visual field defects
accompanied peripheral retinal degenerative lesions in high
myopic eye——the roles in following-up and prophylaxis in
retinal detachment
Yining Shi1, Yanming Chen2, 3, Ji Liu3. 1Department of Ophthalmology,
Shaanxi Provincial People’s Hospital, Xian, China; 2Department
of Ophthalmology, China Medical University, Shenyang, China;
3
Department of Ophthalmology and Visual Science, Yale University
School of Medicine, New Haven, CT.
Purpose: To observe the relationship between peripheral retinal
degenerative changes in high myopic eyes and 90 degree visual fields
defects, and the influence with the refractive and aging factors. We
performed a retrospective, observational clinical study to provide a
safer prophylactic treatment of retinal detachment (RD).
Methods: 360 cases of high myopia were tested by LVP, OCTOPUS
101 perimeter. and compared with the 161 fellow eyes of high
myopia with RD, 118 eyes of high myopia with RD, 41 fellow eyes
of low myopia with RD, 54 eyes of low myopia with RD, and 108
normal eyes.High risk RD eyes were treated by defined equatorial
pan-retinal photocoagulation, at meantime detected the visual field
changes and posterior vitreous detachment(PVD) developing, posttreated complications.
Results: The 90 degree mean light sensitivity (MS) of high myopia
eyes was (21.34±5.40)dB (Fig. 1). The MS in high myopia was
declined with aging and severity of myopia, the linear regression
formula was R=32.981-0.161ages+0.468 diopters.There was definite
co-relationship with 2 kinds of visual defect and the degenerations:
1. The peripheral absolute defects, related with lattice degeneration;
2. The peripheral MS declining, with white-without-pressure.
Following 32 to 74 months after the defined equatorial pan-retinal
photocoagulation,there were complete PVD formation observed
under split lamp with 90°Volk pre-set lens (Fig. 2), and there were no
significant changes between the MSs pre- and post treatment.
Conclusions: There are linear relations with MS and aging, myopic
degree. The MS may indicate the peripheral degenerative lesions and
its degrees, played a roles in following-up and prophylaxis in retinal
detachment.The defined equatorial pan-retinal photocoagulation is a
safer, effective intervene for prophylaxis of retinal detachment and
forming iatrogenous PVD.
Commercial Relationships: Yining Shi, None; Yanming Chen,
None; Ji Liu, None
Program Number: 2172 Poster Board Number: B0025
Presentation Time: 3:45 PM–5:30 PM
Identification of Elements of the BMP2 Signaling Pathway in
Cultured Chick Scleral Fibroblast
Emilia A. Zin, Yan Zhang, Christine F. Wildsoet. University of
California, Berkeley, Berkeley, CA.
Purpose: Previous studies have indicated the role of Bone
Morphogenetic Protein 2 (BMP2) in eye growth regulation. This
study investigated the role of BMP2 in scleral remodeling using a
chick scleral fibroblast culture model to look for evidence of BMP2
signaling.
Methods: Primary chick scleral fibroblast (CSF) were cultured
on 24-well plates or chamber slides in DMEM/F-12 medium with
10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2
incubator. CSF total RNA was collected and purified using RNeasy
Mini Kit and then subjected to cDNA synthesis and real-time PCR
semi-quantification. Gene expression was examined for BMP2,
BMP receptors (BMPR1A, 1B, -2), SMAD1, -5, and -9, and the
localization of relevant proteins, BMP2, BMPR1A, BMPR2,
SMAD1, -4, and -5, phosphorylated SMAD1 (p-SMAD1), and
p-SMAD1/5 in CSF and 293T cell lines was investigated using
immunocytochemistry. Protein expression was also validated with
Simon automated western blot.
Results: Cultured CSF showed detectible expression of BMP2,
BMP receptors, and SMAD 1, 5, 9 genes and immunohistochemistry
confirmed the expression of BMPR1A, BMPR2, SMAD1, -4, and
-5 proteins in CSF as well as 293T cells. Western blot analyses
confirmed expression of the p-SMAD1/5 protein in both CSF and
293T cells, while SMAD1 and p-SMAD1 proteins were only detected
in 293T cells.
Conclusions: That cultured chick scleral fibroblasts express many
of the components of the BMP2 signaling pathway, at both gene and
protein levels, points to their likely involvement in scleral remodeling
and ocular growth. These results provide a foundation for future
in vivo studies into the role of BMP2 in ocular (scleral) growth
regulation.
Commercial Relationships: Emilia A. Zin, None; Yan Zhang,
None; Christine F. Wildsoet, None
Support: NIH grant R01EY012392, NIH grant K08EY023609, NIH
grant K12EY017269
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 2173 Poster Board Number: B0026
Presentation Time: 3:45 PM–5:30 PM
Growth and completion of emmetropization in the normally
developing chick eye
Zheng Shao1, 2, Marsha Kisilak1, Elizabeth L. Irving2, Melanie C.
Campbell1, 2. 1Physics and Astronomy, University of Waterloo,
Waterloo, ON, Canada; 2School of Optometry and Vision Science,
University of Waterloo, Waterloo, ON, Canada.
Purpose: Normal emmetropization has long been assumed
to result in zero refractive error, but recently this has been
questioned. In normally growing chick eyes, we are interested in
objectively determining when emmetropization is complete. We
are also interested in whether growth during and following normal
emmetropization differs.
Methods: From literature values of chick eye parameters, functions
were fit to MOR (mean ocular refraction or spherical equivalent) and
optical axial length (OAL; anterior cornea to anterior retina) vs. age.
Dioptric length (K’) and eye power (F) were derived up to day 75
using our previously reported method to calculate eye power. Pupil
size data were also used to calculate the angular and linear retinal
blurs (EB and LRB) due to defocus.
Results: Eye power and K’ decrease exponentially with age at
slightly different rates until power and K’ reach almost equal values
about day 35. Subsequently, power and K’ decrease almost identically
with age. This gives an initial rapid exponential decrease in MOR,
which reaches a relatively stable value of 1.0 D of hyperopia beyond
day 35. The completion of emmetropization is defined as the first
time point beyond which MOR remains relatively stable, estimated as
between 30 and 35 days. EB and LRB decrease almost exponentially
until day 35. After emmetropization is complete, MOR changes little,
EB remains almost constant while LRB increases slowly from about
day 45 to the end of available measurements on day 75, in agreement
with predictions of an almost uniformly expanding eye model. The
radius of the blur on the retina is larger than cone spacing prior to
completion of emmetropization, and approaches cone spacing as
emmetropization is completed.
Conclusions: Concurrent variations in eye power and length
combine to produce the smaller, more rapid changes in MOR during
emmetropization. The time point at which emmetropization is
complete can be defined as the first time point after which MOR and
angular retinal blur are stable. Emmetropization appears to be driven
by an active reduction of EB to a value close to cone resolution.
After emmetropization is complete, the subsequent change in retinal
blur is consistent with a slow, almost uniform ocular expansion.
However, after the age when normal emmetropization is complete,
an emmetropization response to additional imposed defocus blur has
been observed.
Commercial Relationships: Zheng Shao, None; Marsha Kisilak,
None; Elizabeth L. Irving, None; Melanie C. Campbell, None
Support: NSERC Canada 35321
Program Number: 2174 Poster Board Number: B0027
Presentation Time: 3:45 PM–5:30 PM
Adaptive optics measurements of cone density in chick eyes
during lens-induced myopia
Marsha Kisilak, Laura Emptage, Ian Andrews, Melanie C. Campbell.
University of Waterloo, Waterloo, ON, Canada.
Purpose: In vivo measurements of cones in the chick eye, an animal
model of myopia, are desirable as a marker of retinal changes
during axial length increases. In vivo images allow longitudinal
measurements of the angular cone spacing in the growing chick eye
and during lens-induced myopia. We can then compare measured
densities with models of retinal changes during eye growth and
myopia development.
Methods: Four Ross Ross chicks were acquired on the day of
hatching. Axial length was measured using A scan ultrasound and
aberrations and defocus were measured in a custom built HartmannShack aberrometer. Eyes were imaged in an adaptive optics corrected
scanning laser ophthalmoscope modified for small animal use (2.5
mm diameter pupil). After this, the right eyes were goggled with
-15D lenses. Measurements were repeated on days 7 and 14. All
measurements were taken close to the optical axis and the anatomical
position of the area centralis. Angular cone densities were measured
directly. Linear cone spacings on the retina were calculated from
published schematic eye models modified for measured eye lengths
and cone packing properties were assessed. Paired t-tests were
performed to compare between days and between treated and control
eyes.
Results: By day 14 goggled eyes were on average 15D myopic.
Cones were successfully imaged on all days. The angular density of
cones was not significantly different between control and goggled
eyes (p > 0.2) on any day. As seen in previous control birds, angular
density was not significantly different between days 0 and 7 (p =
0.1) in control eyes, after which it significantly increased (p < 0.02).
Goggled eyes showed no significant change in angular density with
growth. The calculated linear distance between cones increased
significantly from 6.4 microns on day 0 to 7.0 microns on day 14 in
control eyes and did not differ from goggled values of 6.2 microns
on day 0 (before goggling) and 8.3 microns on day 14. On average,
cones were 38% hexagonally packed across all days and for both
control and treated eyes.
Conclusions: Average cone spacings in control eyes on day 14
were within 10% of some literature values. Results for control eyes,
showing initial uniform expansion followed by either cone migration
or optic pole elongation are consistent with our previous data. Eyes
with lens-induced myopia expand uniformly relative to control eyes.
Commercial Relationships: Marsha Kisilak, None; Laura
Emptage, None; Ian Andrews, None; Melanie C. Campbell, None
Support: NSERC Canada 35321
Program Number: 2175 Poster Board Number: B0028
Presentation Time: 3:45 PM–5:30 PM
Peripheral Wavefront Aberration in Myopia with and without
Orthokeratology Lenses
Young Sik Yoo1, Kyung-Sun Na1, Choun-Ki Joo1, Geunyoung Yoon2.
1
The Catholic University of Korea, Seoul, Korea (the Republic of);
2
University of Rochester, Rochester, NY.
Purpose: Peripheral refractive error degrades the quality of retinal
images and has been hypothesized as a potential factor to stimulate
the development of refractive error. Various contact lens designs
based on the hypothesis have shown the efficacy of controlling
myopia progression. The aim of the study was to evaluate the impact
of orthokeratology lens (OK lens) on the peripheral wavefront
aberration in myopic eyes.
Methods: We conducted a cross-sectional study to evaluate the
effect of OK lens on the peripheral aberration profile of myopic
subjects in adolescents. Study subjects were divided into two groups;
one was OK lens group and the other was myopic patients who
did not experience the OK lens correction. A custom-developed
Shack-Hartmann aberrometer was used to measure ocular wavefront
aberrations along different horizontal retinal eccentricities with ten
degree step across the central 30 degrees of visual field. The study
subjects maintain their natural foveal fixation while the aberrometer
is rotated around the eye for the off-axis measurements. Wavefront
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
refraction for each retinal eccentricity was quantified over 4 and 6
mm pupils.
Results: The mean refractive error was -1.92D ± 0.83 in the OK
lens group and -5.84D ± 2.47 in the naked eye group, respectively.
Hyperopic defocus at the peripheral visual field in the myopic eyes
increases with increasing amounts of forveal refractive error. These
effects varied with the degree of the corrected power after a treatment
of OK lens. Significant difference (p < 0.05) in the value of defocus
was found at the peripheral retinal eccentricity in the OK lens group
compared to the naked eye group. In the analysis of high order
aberration, values of on-axis and off-axis showed a different tendency
along with the symmetry of each aberration.
Conclusions: OK lens treatment is found to be effective in reducing
the degree of peripheral hyperopic defocus in myopic eyes.
Commercial Relationships: Young Sik Yoo, None; Kyung-Sun Na,
None; Choun-Ki Joo, None; Geunyoung Yoon, None
Program Number: 2176 Poster Board Number: B0029
Presentation Time: 3:45 PM–5:30 PM
Hyperoxic Myopia: A Prospective Study of Twelve Divers with
Six Hours of Exposure to 1.35 ATM PO2 for Five Consecutive
Days
Jonathan W. Brugger1, 2, Anita Gupta1, Barbara Shykoff2, John
Florian2. 1Ophthalmology, New York Eye & Ear, New York, NY;
2
Navy Experimental Diving Unit, Panama City Beach, FL.
Purpose: Hyperoxic myopia is a phenomenon associated with
prolonged exposure to an increased partial pressure of oxygen (PO2)
resulting in a myopic shift of refractive error. This has been described
in patients undergoing hyperbaric oxygen therapy and in divers
exposed to high PO2. The mechanism of action for hyperoxic myopia
is not understood. This prospective study collected ocular data in
healthy divers exposed to 1.35 ATM PO2 at the Navy Experimental
Diving Unit to better characterize hyperoxic myopia PO2 thresholds
and the mechanism of action.
Methods: Twelve healthy healthy U.S. Navy Divers participated in
five consecutive days of exposure to 100% Oxygen via surfacedsupplied, open-circuit MK20 breathing apparatuses at the bottom of
a 15-foot pool (PO2 of 1.35 ATM) for 6 hours. Prior to diving, and
three days after the last dive, subjects had an ocular examination
consisting of visual acuity (VA), autorefraction, intraocular pressure
(IOP), biometry, and corneal topography. Before and after every dive,
subjects had VA, and autorefraction. IOP was measured on the first,
third, and fifth day.
Results: Two of the twelve divers had subjective symptoms of blurry
vision 2-3 days after the last dive. The first diver had a myopic shift
of -0.50 diopters OS via autorefraction and VA change from 20/16 to
20/20-2. The other diver had a myopic shift of approximately -0.25
diopters OU via autorefraction with a VA shift from 20/30-1 to 20/100
OD and 20/20-1 to 20/40 OS. Both subjects had no significant changes
in IOP, topography, and biometry measurements and both had
spontaneous resolution of their myopia over two to three weeks with
no residual symptoms.
Conclusions: Two healthy divers exposed to an increased PO2
(1.35ATM for 30 hours in 5 days) developed symptomatic myopia
with no changes in corneal topography and biometry (axial length,
lens thickness, aqueous depth). With no appreciable changes in eye
structure, a change in refractive index of the lenticular crystalline lens
is likely responsible for the myopic shift. Hyperoxic myopia is a risk
for those conducting intense diving with a PO2 between 1.3-1.6 ATM
and warrants additional studies to better define risk factors, recovery
time, mechanism of action, and PO2 thresholds.
Commercial Relationships: Jonathan W. Brugger, None; Anita
Gupta, None; Barbara Shykoff, None; John Florian, None
Program Number: 2177 Poster Board Number: B0030
Presentation Time: 3:45 PM–5:30 PM
Malondialdehyde in high myopia.
Amparo Navea1, Francisco Bosch-Morell2, 1, Salvador Mérida
Donoso2. 1Oftalmología Médica, FISABIO, Valencia, Spain; 2Instituto
de Ciencias Biomédicas, Universidad CEU Cardenal Herrera,
Valencia, Spain.
Purpose: Malonyldialdehyde (MDA), a secondary product of lipid
peroxidation is widely used as an indicator of lipid peroxidation.
Lipids and lipid-soluble compounds are essential constituents of the
cells and tissues that comprise the eye. Simultaneously, lipids are
also crucial targets of the attack by reactive oxygen species such as
oxygen free radicals. The role of lipid peroxidation, a process under
which oxidants such as free radicals attack lipids containing carboncarbon double bond(s), especially polyunsaturated fatty acids, has
been described in several ocular pathologies in the past decades.
The aim of this work is to establish, if any, the relationship between
myopia and oxidative damage in subretinal fluid of myopic patients
with retinal detachment.
Methods: Protein content and MDA was evaluated in subretinal
fluid of 71 myopic and no myopic patients with retinal detachment.
Samples were collected in three different groups attending to myopia
degree of the subjects: group 1, non-myopic, group 2, low myopia
(patients with less than 6 dioptries) and group 3, high myopia
(patients with more than 6 dioptries).
Results: Similar data were obtained for groups 1 and 2 (group 1: 0,20
± 0,09 mM MDA and 9,24 ± 4,54 mg protein/ml; group 2: 0,22 ± 0,06
mM MDA and 9,26 ± 4,29 mg protein/ml). However high myopia
patients displayed statistically significant higher values (p<0,001)
of both components: MDA (0,39 ± 0,10 mM) and proteins (17,47 ±
4,55 mg/ml). One of the most remarkable result was the high positive
correlation obtained (r=0,87) when representing individual data pairs
of MDA and myopia degree of myopic patients.
Conclusions: These results ratify the direct contribution of oxidative
stress in retinal detachment. They also suggest that myopia may play
a role (qualitative and quantitative), that deteriorate the natural course
of ocular diseases that involve oxidative stress.
Commercial Relationships: Amparo Navea, None; Francisco
Bosch-Morell, None; Salvador Mérida Donoso, None
Support: CEU-SANTANDER PRCEU-UCH 13/17
Program Number: 2178 Poster Board Number: B0031
Presentation Time: 3:45 PM–5:30 PM
Collagen crosslinking using genipin diminishes cyclic softening of
tree shrew sclera during lens-induced myopia development
Alexander Levy2, Sarah M. Baldivia2, Rafael Grytz1. 1Ophthalmology,
University of Alabama at Birmingham, Birmingham, AL;
2
Biomedical Engineering, University of Alabama at Birmingham,
Birmingham, AL.
Purpose: To assess the effect of exogenous crosslinking using
genipin on the cyclic softening response of the remodeling tree shrew
sclera during monocular -5 diopter (D) lens wear.
Methods: Cyclic tensile tests were performed on 2-mm wide scleral
strips, first at physiological loads (50 cycles, 0-3.3 g, 30 sec/cycle)
and subsequently after 10 minutes rest at supra-physiological loads
(50 cycles, 0-33.3 g, 60 sec/cycle) conditions. Two scleral strips
were obtained from each eye of two juvenile tree shrews exposed to
4 days of monocular -5 D lens wear to induce axial elongation and
myopia. The scleral strips of the control eye were mechanically tested
immediately after enucleation or after 24 hours incubation at 37°C
in PBS. The scleral strips of the lens treated eye were tested after 24
hours incubation in PBS or PBS supplemented with genipin at a low
cytotoxicity concentration (1mM). Cyclic softening was defined as
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
the incremental strain increase from one to the next cycle. This value
was averaged over both animals and cycles 5 to 50. Cycles that led to
tissue failure were excluded.
Results: At both loading conditions (physiological / supraphysiological loads), the average incremental strain increase (%
per cycle) was nearly identical in the fresh (0.03 /0.2) and PBS
incubated tissue of the control eye (0.03/0.26). The cyclic softening
was approximately four times higher in the sclera of the myopic
eye (0.14/0.81) and two orders of magnitude lower after genipin
crosslinking (0.001/0.004).
Conclusions: Results indicate that cyclic tensile loading leads to
continued softening of the juvenile tree shrew sclera. The softening
rate increases during lens-induced myopia and is diminished after
genipin crosslinking. This finding suggests that axial elongation in
myopia may be due to a remodeling mechanism that increases the
cyclic softening response of the sclera, which can be inhibited by
scleral crosslinking using genipin.
Cyclic tensile tests at supra-physiological loads of three scleral strips
of one animal showing the increased cyclic softening leading to tissue
failure in the treated sclera versus the control and the diminished
cyclic softening after genipin crosslinking.
Commercial Relationships: Alexander Levy, None; Sarah M.
Baldivia, None; Rafael Grytz, None
Support: NIH grant EY003909 (P30); EyeSight Foundation of
Alabama; Research to Prevent Blindness
Program Number: 2179 Poster Board Number: B0032
Presentation Time: 3:45 PM–5:30 PM
Immunolesioning of glucagonergic amacrine cells in the chick
retina
Diane Nava1, 2, Tatiana Lupashina2, Bhavna Antony1, Li Zhang3,
Michael D. Abramoff3, Christine F. Wildsoet1, 2. 1Vision Science
Graduate Group, UC Berkeley, Berkeley, CA; 2Center for Eye
Disease and Development, Berkeley, CA; 3Department of Electrical
and Computer Engineering, University of Iowa, Iowa City, IA.
Purpose: Neurotoxins have been used in myopia research to ablate
inner retinal cells in order to study their contributions to eye growth
and refractive error regulation, although those used in past studies
have been relatively unselective. The purpose of this study is to
investigate immunolesioning as a potential tool to selectively ablate
glucagon amacrine cells (GACs) in the chick and to compare its
selectivity to previous methods.
Methods: A Saporin immunotoxin conjugated to a primary antiglucagon antibody was injected intravitreally in the left eyes of 7-day
old chicks as a 10uL solution in one of 5 concentrations (0.125, 0.25,
0.5, 0.75 or 1 uM).
TUNEL staining (Roche) was used to determine the distribution of
apoptotic cells and immunohistochemistry on vertical sections used
to assess changes in the glucagonergic cell population.
Optical coherence tomography imaging was used to investigate
changes in the retina and choroid, and flash electroretinograms
(ERGs) were recorded to assess changes in retinal function 4, 6 and 9
days after injection.
Results: The maximum loss of GACs was seen with the 1 uM
concentration and for concentrations lower than 1 uM, the central
retina seems to be more affected than peripheral retina, where GAC
immunoreactivity in the IPL/INL boundary was more apparent than
in the center.
The 1uM concentration significantly attenuated the photopic
negative response of the flash ERG at both 4 and 7 days (p=0.0236
& p=0.0393), with no significant effect on b-wave and a-wave
amplitudes. The peak of the flash ERG at approximately 200 ms was
also significantly attenuated at 4 days after injection (p=0.03), but not
at later time points.
With the 1 uM concentration, total retinal thickness was not
significantly reduced in injected eyes at any time point, while
choroidal thickness underlying the area centralis was significantly
increased compared to values for the contralateral eyes at both 6 and
9 days post injection (p=0.0475 & p=0.04097 respectively).
Conclusions: The above immunotoxin conjugate, injected
intravitreally, appears to more selectively lesion GACs in the
chick retina than previously tested neurotoxins, as evidenced by
histological as well as retinal structural and functional data, and thus
represents a suitable tool for investigating the role of GACs in eye
growth regulation. The finding of choroidal thickening in lesioned
eyes is a novel finding that warrants further investigation.
Commercial Relationships: Diane Nava, None; Tatiana
Lupashina, None; Bhavna Antony, None; Li Zhang, None;
Michael D. Abramoff, University of Iowa (P); Christine F.
Wildsoet, None
Support: NIGMS 3R25GM090110-04S1
Program Number: 2180 Poster Board Number: B0033
Presentation Time: 3:45 PM–5:30 PM
Ciliary Muscle Cell Changes During Guinea Pig
Emmetropization
Andrew D. Pucker1, Ashely R. Carpenter2, Hugh J. Morris1, Andrew
J. Fischer3, Kirk M. McHugh2, Donald O. Mutti1. 1Optometry,
Ohio State University, Columbus, OH; 2Center for Molecular and
Human Genetics, Nationwide Children’s Hospital, Columbus, OH;
3
Department of Neuroscience, Ohio State University, Columbus, OH.
Purpose: To establish normal morphological parameters and to
characterize ciliary muscle (CM) cell changes with age during guinea
pig emmetropization.
Methods: Three pigmented guinea pig eyes were collected at three
different ages (n = 9 eyes). Mean refractive error was determined
with retinoscopy by two trained examiners. Eyes were then
enucleated, hemisected, and fixed with paraformaldehyde. Temporal
eye segments were then embedded in OCT compound and 30 mm
serial sections were collected; the two most temporal slides of each
eye were then labeled with anti-α-smooth muscle actin antibodies
(smooth muscle) and Draq5 nuclear stain. Sections were then
visualized with a fluorescent microscope (Leica Microsystems) and
analyzed with Stereo Investigator (MBF Bioscience) to determine
the mean CM cross-sectional area, nuclei number, and cell crosssectional area.
Results: Guinea pigs displayed emmetropization as refractive error
decreased from +9.08 ± 2.75 D at 1-day-old to +2.91 ± 0.88 D at
90-days-old. The mean CM length and CM cross-sectional area both
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
significantly increased with age, from 0.42 ± 0.035 mm to 0.90 ±
0.095 mm (p = 0.003) and from 0.037 ± 0.005 mm2 to 0.057 ± 0.015
mm2 (p = 0.011), respectively. The mean cross-sectional area covered
by each CM cell did not change (p = 0.82), which is consistent with
the marginal increase in the mean CM cell number from 72.0 ± 7.4
cells to 105.5 ± 19.2 cells per section (p = 0.059; all by JonckheereTerpstra test).
Conclusions: Guinea pig CM undergoes morphologic changes during
development in the first 90 days of life characterized by significant
increases in cross-sectional area and length while the mean area
occupied by each cell does not significantly change. These data
suggest that the CM grows via cell proliferation rather than through
hypertrophy. These normative data will be useful when contrasted
with potential CM changes during myopia induction experiments.
Commercial Relationships: Andrew D. Pucker, None; Ashely
R. Carpenter, None; Hugh J. Morris, None; Andrew J. Fischer,
None; Kirk M. McHugh, None; Donald O. Mutti, None
Support: NIH/NEI: K08EY023264
Program Number: 2181 Poster Board Number: B0034
Presentation Time: 3:45 PM–5:30 PM
Eye size and shape in newborn children and its relation to axial
length and refraction at three years
Laurence S. Lim1, 2, Sharon Chua2, Pei Ting T. [email protected],
Shirong Cai2, Yap Seng Chong2, Kenneth Kwek3, Marielle Fortier3,
Cheryl Ngo2, Anqi Qiu2, Seang-Mei Saw2. 1Ophthalmology, Singapore
National Eye Center, Singapore, Singapore; 2National University
of Singapore, Singapore, Singapore; 3KK Women’s and Children’s
Hospital, Singapore, Singapore.
Purpose: Eye shape has been postulated to be a risk factor for
refractive error. The purpose of this study is to determine if eye shape
and size at birth are associated with refractive error and eye size 3
years later.
Methods: A subset of 173 full-term newborn infants from the
Growing Up in Singapore Towards healthy Outcomes (GUSTO)
birth cohort underwent magnetic resonance imaging (MRI) to
measure the axial length (AL), height, width, volume and surface
area of the internal eye at birth. Eye shape was assessed by an index
of oblateness, calculated as 1–(AL/width) or 1–(AL/height). Oblate
eyes had oblateness >+0.01, spherical eyes had oblateness between
+0.01 and -0.01, and prolate eyes had oblateness <-0.01. Cycloplegic
autorefraction and optical biometry (IOLMaster) were performed 3
years later.
Results: In total, 346 eyes of 173 children were analysed. The
majority were male (94 children, 54%) and of Malay (43%) or
Chinese (43%) origin. Most eyes were prolate at birth. At three years,
the mean AL was 21.74±0.68mm (range 19.77-23.84), representing
a mean increase from birth of 4.47±0.94mm (1.71–7.20). The mean
spherical equivalent refraction(SER) was 0.91±0.80D (-2.40 to
+3.47) and only a small proportion of eyes was myopic (8 eyes,
3.6%). After multivariate adjustment, eyes with longer AL at birth
had smaller increases in AL at 3 years (p<0.001). Eyes with larger
baseline volumes and surface areas had smaller increases in AL at
3 years (p<0.001 for both). Eyes which were more oblate at birth
had greater increases in AL at 3 years (p<0.001). Using width to
calculate oblateness, prolate eyes had smaller increases in AL at 3
years compared to oblate eyes (p<0.001), and, using height, prolate
and spherical eyes had smaller increases in AL at 3 years compared to
oblate eyes (p<0.001 for both). There were no significant associations
between eye size and shape at birth and SER, corneal curvature or
myopia at 3 years.
Conclusions: Eyes that are longer, larger and have prolate or
spherical shapes at birth exhibit smaller increases in AL over the
first 3 years of life. Eye size and shape at birth influence subsequent
eye growth but not the development of refractive error, suggesting
adequate compensatory mechanisms to maintain emmetropia for at
least the first 3 years of life.
Commercial Relationships: Laurence S. Lim, None; Sharon
Chua, None; Pei Ting T. [email protected], None; Shirong Cai,
None; Yap Seng Chong, None; Kenneth Kwek, None; Marielle
Fortier, None; Cheryl Ngo, None; Anqi Qiu, None; Seang-Mei
Saw, None
Support: This work is supported by the Translational Clinical
Research (TCR) Flagship Program on Developmental Pathways
to Metabolic Disease funded by the National Research Foundation
(NRF) and administered by the National Medical Research
Council (NMRC), Singapore- NMRC/TCR/004-NUS/2008.
Additional funding is provided by the Young Investigator Award
at the National University of Singapore (NUSYIA FY10 P07) and
Singapore Ministry of Education Academic Research Fund Tier 2
(MOE2012-T2-2-130).
Program Number: 2182 Poster Board Number: B0035
Presentation Time: 3:45 PM–5:30 PM
Normative ocular biometric values for the adult mouse, rat,
rabbit, dog, pig, non-human primate, and human
Joshua S. Eaton1, Andrea D. Rodrigues2, Craig B. Struble3, Sara M.
Thomasy4, Christopher J. Murphy4, 1. 1Ocular Services on Demand,
Madison, WI; 2Non-Clinical Development - Toxicology, Allergan,
Irvine, CA; 3Covance Laboratories, Madison, WI; 4Department of
Surgical and Radiological Sciences, University of California - Davis,
Davis, CA.
Purpose: Confidence in knowledge of biometric dimensions of
the ocular chambers and structures is essential in pharmacokinetic/
dynamic modeling and preclinical development of therapeutic
compounds and devices. Furthermore, knowledge of comparative
biometric values between animal models and humans can impact
dosing methods, concentration delivered, and techniques employed
in nonclinical studies. Consideration of comparative differences also
improves accuracy in prediction of human safety risk. Our objective
was to summarize available normative data for the human and six
common animal models - mouse, rat, rabbit, dog, pig, and non-human
primate (NHP).
Methods: References citing normative ocular biometric values in
all seven species were collected by searching online publication
databases, textbooks, and journals available to the authors. Biometric
parameters researched included: axial globe length (AGL), anterior
chamber depth (ACD), axial lens diameter (ALD), vitreous chamber
depth (VCD), and vitreous chamber volume (VCV). Available data
were assembled and evaluated. For scarce or unavailable values,
additional data was generated to establish normative values. All data
are presented as mean ± SD.
Results: Reported values were obtained using both in vivo and ex
vivo techniques as well as calculation and/or estimation. Parameters
with values of higher intraspecies variability (>25% coefficient of
variation) included: ACD in the mouse (0.50 ± 0.17); ALD in the rat
and rabbit (3.55 ± 1.08 and 6.18 ± 1.88 mm, respectively); VCD in
the mouse and NHP (0.61 ± 0.16 and 8.72 ± 3.58 mm, respectively);
and VCV in the mouse, rat, dog, and NHP (0.01 ± 0.01, 0.03 ± 0.02,
2.63 ± 0.81, and 2.20 ± 0.89 ml, respectively). Relative to other
laboratory species, fewer values were reported for AGL in the rat;
ACD in the rat, rabbit, pig, and NHP; VCD in the rabbit, pig, and
NHP; and VCV in the rat, dog, and NHP.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Conclusions: Reliable normative ocular biometric values are critical
for use of animal models in eye research and preclinical ocular
drug and device development. This review summarizes published
data, highlighting parameters of greater variability and identifying
data gaps for species commonly used in the development of new
therapeutics.
Commercial Relationships: Joshua S. Eaton, None; Andrea D.
Rodrigues, None; Craig B. Struble, None; Sara M. Thomasy,
None; Christopher J. Murphy, None
302 Glial cell pathology in blinding disease - Minisymposium
Tuesday, May 05, 2015 8:30 AM–10:15 AM
1AB Mile High Blrm Minisymposium
Program #/Board # Range: 2561–2567
Organizing Section: Anatomy and Pathology/Oncology
Program Number: 2561
Presentation Time: 8:30 AM–8:45 AM
Why Glial Cells Matter to Retinal Function?
Vijay P. Sarthy. Northwestern University, Chicago, IL.
Presentation Description: Retinal health and function are supported
by several types of glial cells: Müller cells, astrocytes, microglia
and oligodendrocytes. This presentation will focus on our current
knowledge of the structural, nutritional and trophic roles of
retinal glia. It will describe novel roles for Müller cells in retinal
regeneration following injury and in the cone-visual cycle, and
potential roles of astrocytes in retinal ganglion cell function.
Commercial Relationships: Vijay P. Sarthy, None
Support: EY019325
Program Number: 2562
Presentation Time: 8:45 AM–9:00 AM
Retinal glial cells in development and aging
Tailoi Chan-Ling. University of Sydney, Sydney, NSW, Australia.
Presentation Description: The retina is an unistratified avascular
neuroepithelium early in human embryonic development. Retinal glia
including Muller cells and astrocytes play pivotal roles in the normal
development of the retina. This presentation will review the complex
interactions between astrocytes, pericytes, blood vessels and neurons
and how these complex interactions regulate retinal vascularization
during development and disease. Changes in astrocytes including the
decrease in the ratio of astrocytes servicing neurons likely affect the
ability of astrocytes to service the functions of neurons with aging
and thus contribute to disease pathogenesis.
Commercial Relationships: Tailoi Chan-Ling, None
Support: National Health and Medical Research Council of
Australia. Rebecca Cooper Medical Research Foundation
Program Number: 2563
Presentation Time: 9:00 AM–9:15 AM
Glial cell pathology in glaucoma
Yeni H. Yucel. 1University ofToronto, Toronto, ON, Canada; 2Keenan
Research Centre for Biomedical Science, St. Michael’s Hospital,
Toronto, ON, Canada.
Presentation Description:
In addition to neuron loss, glial cell pathology is implicated in
glaucoma damage. Glial cell pathology in optic nerve and central
visual pathways will be reviewed, in addition to relevant potential
strategies to prevent vision loss in glaucoma.
Commercial Relationships: Yeni H. Yucel, None
Support: Canadian Institutes of Health Reseacrh, Canada Foudation
for Innovation, Glaucoma Research Society of Canada,
Program Number: 2564
Presentation Time: 9:15 AM–9:30 AM
Role of retinal microglia in retinal degeneration
Wai T. Wong. NEI, Bethesda, MD.
Presentation Description: Microglia, the resident immune cell
in the retina, are prominently involved in the pathology of retinal
degenerations. However, whether microglia contribute or are simply
reactive to photoreceptor degeneration has been unclear. This
presentation describes how retinal microglia are altered during the
course of retinal degeneration and the mechanisms by which they
contribute to the progression of degeneration.
Commercial Relationships: Wai T. Wong, None
Support: NEI Intramural Research Program
Program Number: 2565
Presentation Time: 9:30 AM–9:45 AM
Astrocyte and Muller cell pathology in diabetic retinopathy
Elia J. Duh. Johns Hopkins School of Medicine, Baltimore, MD.
Presentation Description: Diabetic retinopathy (DR) is one of
the leading causes of blindness in industrialized countries. Long
regarded as a microvascular disease, it is increasingly evident that
other retinal cell types are involved in the pathogenesis of DR. There
is increasing awareness of the potential role of glial cells - Muller
cells and astrocytes. Emerging evidence supports the concept that
glial elements may contribute significantly to the progression of DR.
This talk will discuss current knowledge of pathologic changes in
astrocytes and Muller cells in diabetic retinopathy as well as their
possible role in progression of the disease.
Commercial Relationships: Elia J. Duh, None
Support: NIH Grant EY022383, EY022683
Program Number: 2566
Presentation Time: 9:45 AM–10:00 AM
Lessons from glial cell tumors of the eye
Charles Eberhart. 1Wilmer Eye Institute, Baltimore, MD; 2Pathology,
Johns Hopkins University, Baltimore, MD.
Presentation Description: Ocular glia can proliferate and form
both neoplastic and reactive mass lesions. The most common
ocular gliomas occur in the optic nerve, and are generally low
grade pilocytic astrocytoma. Many of these are associated with
neurofibromatosis type 1 (NF1), and activation of the BRAF/MEK
and AKT/mTOR pathways has been documented in NF1 deficient
lesions. It has also recently been shown that small duplications of
the BRAF gene leading to a constitutively active fusion protein are
common in sporadic optic nerve gliomas not associated with NF1
mutation. The activation of BRAF in both syndromic and sporadic
tumors leads to induction of p16 and oncogene induced senescence,
which most likely explains the growth arrest and even regression
often seen clinically in optic nerve low grade gliomas. Unfortunately,
directly targeting mutant BRAF can cause paradoxical tumor
growth, thus more sophisticated pharmacological approaches will
be necessary to block this molecular arm in aggressive optic nerve
gliomas. The mTOR pathway is also active in the majority of both
NF1 associated and sporadic optic nerve gliomas, and clinical trials
are underway in children. Finally, the Notch pathway can drive ocular
glioma formation in mice, and is active in many human tumors.
Intraocular gliomas are rare, and include retinal astrocytic neoplasms
in tuberous sclerosis patients similar to giant cell astrocytomas seen
in the brain. A number of “reactive retinal astrocytic tumors” have
also been reported, and while their biology is not entirely clear,
genetic studies suggest that they are not clonal neoplasms. The
clinical, pathological and molecular features of the above tumors will
be discussed.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Charles Eberhart, None
Program Number: 2567
Presentation Time: 10:00 AM–10:15 AM
Harnessing glial cells to understand and treat diseases of the
retina
Deniz Dalkara. Institut de la Vision INSERM, Paris, France.
Presentation Description: Müller glial cells are the principal glial
cells of the retina. They provide architectural support and form
the limits of the neurosensory retina at the outer and inner limiting
membranes. They are involved in virtually every aspect of retinal
homeostasis and maintenance. They are thus a main player in both
acquired and inherited retinal disease and a target for therapeutic
intervention. Müller glia respond to retinal disease and injury in ways
that can be protective or detrimental to retinal function. Here, I will
speak about viral tools to direct gene expression to Müller cells. Such
viral tools allow us to both investigate Müller glial response to retinal
disease and harness glial cells in gene therapeutic intervention to
slow down or reverse retinal disease.
Commercial Relationships: Deniz Dalkara, INSERM (P)
Support: Association Francaise contre les Myopathies (AFM);
Grant number: 14853, LABEX LIFESENSES [reference ANR-10LABX-65]
were identified as multiple, small, amorphic calcified material within
the dermis that was positive for calcium with Von Kossa stain. The
spherical calcified deposits were surrounded by foreign body giant
cells and lymphoplasmocytic chronic inflammation (image 1). On
the other hand, the biopsy findings in elderly patients (3 cases)
were characterized by a single, large, well-demarcated amorphous
calcified material that also stained positive with Von Kossa, and was
surrounded by fibrous tissue without chronic inflammation or foreign
body reaction (image 2)
Conclusions: SCN of the eyelid is a rare condition, but should
be considered in any patient presenting with a painless white to
yellowish
colored nodule of the ocular adnexa. Clinician should be aware that
this entity could be associated particularly in elderly patients, with
systemic conditions. We have found 2 different histopathological
patterns according to patients’ age that are associated with SCN of the
eyelid and have not been described yet.
345 Tumors - Inside and around the eye, II
Tuesday, May 05, 2015 11:00 AM–12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 3407–3435/C0218–C0246
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Cornea, Eye Movements/Strabismus/
Amblyopia/Neuro-Ophthalmology, Immunology/Microbiology,
Multidisciplinary Ophthalmic Imaging, Physiology/Pharmacology
Program Number: 3407 Poster Board Number: C0218
Presentation Time: 11:00 AM–12:45 PM
Subepidermal calcified nodule of the eyelid
Saeed AlWadani2, Charles Eberhart3, Hind ALKatan1, Maria
J. Suarez B3, Jonathan Kass3. 1KKESH, Riyadh, Saudi Arabia;
2
Ophthalmology department, King Saud University, Riyadh, Saudi
Arabia; 3johns Hopkins Hospital, Baltimore, MD.
Purpose: Subepidermal calcified nodule of the eyelid (SCN) is
considered one of the types of Calcinosis cutis.The purpose of this
study was to study the clinical features and histopathological findings
in patient diagnosis with SCN of the eyelid
Methods: There were 13 patients identified who had been diagnosed
with SCN of the eyelid. A chart review was conducted on all patients
to identify any salient past medical history, trauma or concurrent
systemic disease. Histopathological analysis was performed for
each case using hematoxylin-eosin stains. In selected cases, Von
Kossa stain was also used to identify and characterize the nature of
calcium deposition, as well as immunostains including CD3, CD20
for chronic inflammation and CD68 to recognize granulomatous
inflammation.
Results: We have found 13 cases of SCN diagnosis in our
ophthalmic pathology archive. Clinical information is summarized
in table 1.Two of these patients presented with systemic disease
association including hypertension and gout. The other 11 patients
were otherwise healthy and most of them presented as white, firm,
nodules. All cases from our review received surgical excision of
the lesion as treatment under local anesthsia. Microscopically, we
described 2 different histopathological patterns according to the age
of patients. In younger patients (10 cases, histopathological features
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
pattern and it may be confused with other tumors, thus in order
to make the diagnosis of this adenoma, resection is needed along
with histopathologic analysis and the identification of specific
immunohistochemical markers.
Commercial Relationships: Laura Andrea Torrado, None; Juan
Carlos Serna-Ojeda, None; Enrique Ariza-Camacho, None;
Alberto Collado- Solorzano, None; Blanca C. Flores- Sanchez,
None; Abelardo Rodriguez- Reyes, None; Emiliano Fulda-Graue,
None
Commercial Relationships: Saeed AlWadani, None; Charles
Eberhart, None; Hind ALKatan, None; Maria J. Suarez B, None;
Jonathan Kass, None
Support: Non
Program Number: 3408 Poster Board Number: C0219
Presentation Time: 11:00 AM–12:45 PM
Adenoma of the nonpigmented ciliary body and iris epithelium in
Mexican mestizo patients
Laura Andrea Torrado1, Juan Carlos Serna-Ojeda1, Enrique ArizaCamacho1, Alberto Collado- Solorzano1, Blanca C. Flores- Sanchez2,
Abelardo Rodriguez- Reyes2, Emiliano Fulda-Graue1. 1Instituto de
Oftalmologia, Mexico, Mexico; 2APEC, Mexico, Mexico.
Purpose: Describe the clinical characteristics, ultrasound findings,
and histopathologic and immunohistochemical results from mexican
mestizo patients with adenoma of the nonpigmented ciliary body and
iris epithelium
Methods: Retrospective analysis of the interventional case series of
four patients with final diagnosis of adenoma of the nonpigmented
ciliary body and iris epithelium.
Results: Median age at presentation was 50 years (range 15 - 75
years). Half of the patients presented with decreased visual acuity
and the other half with changes in iris pigmentation. Ultrasound
revealed a solid mass of homogeneous density and high internal
reflectivity and the biomicroscopy showed the anatomical origin.
In all the patients an excisional biopsy with partial lamellar
sclerouvectomy was performed for accurate diagnosis. One patient
required a 23-gauge vitrectomy at the same surgical time because
of retinal detachment. Histopathology reported sheets of cells with
clear cytoplasm surrounded by basement membrane and tubular
differentiation. Inmunohitochemistry was positive por vimentin,
S-100, epithelial membrane antigen (EMA) and neuron specific
enolase (NSE).
Conclusions: The adenoma of the nonpigmented ciliary body
is a benign rare tumor with few reports in literature, being this
the first latin american case series of this entity. Because of its
origin, the clinical presentation is varied without an established
Program Number: 3409 Poster Board Number: C0220
Presentation Time: 11:00 AM–12:45 PM
Topographic distribution of ocular vascular lesions: a 20-year
study
Tânia Borges1, 2, Taylor Nayman2, Ana Beatriz T. Dias2, Sultan
Aldrees2, Beatriz Nugent da Cunha2, Miguel N. Burnier2. 1Centro
Hospitalar do Porto, Porto, Portugal; 2Henry C. Witelson Ocular
Pathology Laboratory, Montreal, QC, Canada.
Purpose: The clinical diagnosis of ocular vascular lesions is
challenging for ophthalmologists due to similarities between the
different categories of these lesions and between them and other nonvascular lesions. The purpose of this study is to describe the clinical
and pathological characteristics and the frequency of different ocular
vascular lesions in order to assist with differential diagnoses.
Methods: We reviewed 15,512 cases diagnosed at the Henry C.
Witelson Ocular Pathology Laboratory at McGill University during a
20-year period. Clinical and pathological data of all ocular vascular
lesions diagnosed during this period were retrieved. Descriptive
analysis of various lesions based on site (eyelid, conjunctiva, and
orbit), frequency, gender, and age was performed.
Results: Of the 15,512 specimens reviewed, 115 (0.74%) cases were
ocular vascular lesions. Female patients represented approximately
half the study population (55.17%). Most patients presented
in the 41–60 year age group (42.61%). The most common site
of involvement was the eyelid (52.17%), followed by the orbit
(27.83%) and conjunctiva (20%). The most common eyelid lesion
was capillary hemangioma (36.67%). However, the most common
orbit and conjunctiva lesions were cavernous hemangioma (81.25%)
and vascular hamartoma (52.17%), respectively. Four malignant
lesions were found: Kaposi’s sarcoma (n = 2) and epithelioid
hemangioendothelioma (n = 2). Three of these lesions were in the
eyelid and one case was conjunctival. One case was misdiagnosed
clinically as a benign lesion. Of the 101 cases with clinical diagnoses,
28 cases (27.72%) were misdiagnosed clinically as non-vascular
lesions. Encountered lesions include: cavernous hemangioma
(38.26%), vascular hamartoma (25.22%), capillary hemangioma
(22.61%), lymphangioma (6.96%), vascular ectasia with thrombosis
(1.74%), epithelioid hemangioma (0.87%), and intravascular
papillary endothelial hyperplasia (0.87%).
Conclusions: The location of ocular vascular lesions is an important
feature for differential diagnoses. There was a lack of a correlation
between clinical and histopathological diagnosis in one third of
cases. The eyelid is the most common site of ocular vascular lesions.
Capillary hemangioma is the most common eyelid vascular lesion;
however, cavernous hemangioma is the most frequent lesion of all
studied sites. Malignant ocular vascular lesions are exceedingly rare.
Commercial Relationships: Tânia Borges, None; Taylor Nayman,
None; Ana Beatriz T. Dias, None; Sultan Aldrees, None; Beatriz
Nugent da Cunha, None; Miguel N. Burnier, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 3410 Poster Board Number: C0221
Presentation Time: 11:00 AM–12:45 PM
The Occurrence of Lens Tumors in Humans and Other Species
and a Perceived Opportunity for Further Study
Krishna R. Surapaneni1, Paul O. Phelps1, Bradley Thuro1, Richard
R. Dubielzig2, Daniel M. Albert1. 1Department of Ophthalmology and
Visual Sciences, University of Wisconsin – Madison, Madison, WI;
2
Department of Pathobiological Sciences, University of Wisconsin –
Madison, Madison, WI.
Purpose: The purpose of this census was to determine in which
species and under what conditions lens tumor occurs using human
and veterinary pathological material available to us.
Methods: Material in two major archived collections at the
University of Wisconsin medical and veterinary schools were
studied for occurrence of lens tumors. A database of material from
1920 to 2014 was included, which was approximately 17500 human
eye pathology cases, and 45000 veterinary cases. In addition,
cases presented in every major eye pathology society (VerhoeffZimmerman, Eastern Ophthalmic Pathology Society, and Armed
Forces Institute of Pathology Alumni Society) were reviewed. Finally,
a careful search of the literature was carried out using variations of
the terms “Lens, Crystalline”[Mesh]) AND “Neoplasms”[Majr].
Results: The database search revealed that lens tumors occurred
under natural conditions in cats and rabbits. Five percent of feline
neoplasms (257/5048) in the veterinary school database were
designated a Feline Ocular Post-traumatic Sarcoma (FOPTS), a
tumor demonstrated to be of lens epithelial origin. Three similar
tumors seen in chronically disease rabbit eyes are suspected because
or remarkable similarity to FOPTS. There are three reports of these
FOPTS-like tumors rabbits in the literature. All cat and rabbit
sarcomas had a history of either ocular trauma or protracted uveitis.
Literature search also revealed cases where lens tumors were induced
in zebrafish, rainbow trout, hamsters, and mice, by carcinogenic
agents (methylcholanthrene, thioacetamide), oncogenic viruses
(SV40, HPV-16), and genetic manipulation (bumper, p53). No
evidence of lens tumors was found in human pathologic material or
extensive literature search.
Conclusions: Following lens capsule rupture a malignant lens tumor
can occur in other species, but not in humans. We hypothesize that
a genetic mechanism exists which prevents lens tumors in man. We
have begun a search for candidate genes in other species involved in
tumor formation for subsequent comparison in humans.
Commercial Relationships: Krishna R. Surapaneni, None; Paul
O. Phelps, None; Bradley Thuro, None; Richard R. Dubielzig,
None; Daniel M. Albert, None
Support: Department of Ophthalmology and Visual Sciences CORE
Grant (UW – Madison), and Financial Support from Ocular Services
on Demand (OSOD)
Program Number: 3411 Poster Board Number: C0222
Presentation Time: 11:00 AM–12:45 PM
Clinical relevance of EMT in eyelid sebaceous gland carcinoma:
an immunohistochemical and molecular study
Mansi Bhardwaj1, Seema Sen1, Anjana Sharma2, Neelam Pushker3,
Seema Kashyap1, Mandeep S. Bajaj3, Kunzang Chosdol4, Sameer
Bakhshi5. 1Ocular Pathology, Dr.R.P.Centre for Ophthalmic
Sciences, All India Institue of Medical Sciences, New Delhi,
India; 2Ocular microbiology, Dr.R.P.Centre, AIIMS, New Delhi,
India; 3Ophthalmology, Dr.R.P.Centre, AIIMS, New Delhi, India;
4
Biochemistry, AIIMS, New Delhi, India; 5Medical Oncology, IRCH,
AIIMS, New Delhi, India.
Purpose: Sebaceous gland carcinoma (SGC) is the most common
malignant eyelid tumor with a high rate of incidence in Asia.
Invasion and metastasis in several carcinomas is promoted by
‘Epithelial Mesenchymal Transition’ (EMT) where epithelial cells
acquire a mesenchymal phenotype.It is mediated by transcription
factors like Slug,ZEB1 & ZEB2 which causes loss of cell-cell
adhesion molecule E-cadherin.The present study was designed
to determine the status of EMT markers Slug,ZEB1,ZEB2 and
E-cadherin and to correlate with high risk features of eyelid
SGC.
Methods: Prospective analysis of 30 eyelid SGC patients was
undertaken. Clinicopathological features were noted and H&E
stained sections were reviewed to confirm the diagnosis.AJCC
staging (2009) was done and patients were followed up for 37
months(Mean: 22.5±9.08).Immunohistochemistry(IHC) was
performed using antibodies against ZEB1,ZEB2,Slug and E-cadherin.
mRNA analysis by quantitative Real Time PCR was performed in
all the tumour samples and adjoining normal skin tissues.Results
were correlated with high risk features and follow up data.Statistical
analysis was performed using Fisher Exact and Spearman’s Rank
Correlation Tests.
Results: Mean age of patients was 58.9±13.2yrs (male:female
ratio1.1:1).IHC and mRNA analysis revealed ZEB2,ZEB1 and
Slug overexpression in 67%(20/30),40%(12/30) and 10%(3/30)
cases respectively.E-cadherin loss was seen in 63%(19/30) cases.
IHC expression of all 4 EMT markers significantly correlated with
mRNA levels.ZEB2 overexpression was significantly associated with
high risk features like poor histological differentiation (P=0.017) &
pagetoid spread (P=0.023),whereas E-cadherin loss was significantly
associated with large tumour size(P=0.023) & pagetoid spread
(P=0.018).Both ZEB2 overexpression and E-cadherin loss were
present in 88%(7/8) cases with recurrence.ZEB2 overexpression
was also seen in 75%(6/8) cases with lymph node metastasis.There
was a significant inverse correlation between ZEB2 and E-cadherin
expression (P=0.038).Other EMT markers ZEB1 and Slug did not
correlate with any clinicopathological high risk features.
Conclusions: EMT is an important phenomenon in eyelid SGC
which could be responsible for its aggressive behaviour.Both ZEB2
and E-cadherin are important biomarkers for detection of high risk
SGC cases.Further validation on a larger series of cases,with longer
follow up is however necessary.
Commercial Relationships: Mansi Bhardwaj, None; Seema Sen,
None; Anjana Sharma, None; Neelam Pushker, None; Seema
Kashyap, None; Mandeep S. Bajaj, None; Kunzang Chosdol,
None; Sameer Bakhshi, None
Program Number: 3412 Poster Board Number: C0223
Presentation Time: 11:00 AM–12:45 PM
Ocular Ultrastructural Features of Gaucher Disease
Mones S. Abu-Asab, Christopher Ardeljan, Chi-Chao Chan. Lab of
Immunol/Sect of Immunopath, National Eye Institute, Bethesda, MD.
Purpose: Gaucher disease is a lysosomal storage disorder
resulting from mutations in the enzyme glucocerebrosidase
(aka b-glucosidase). It leads to the massive accumulation of
glucocerebroside (glucosylceramide) within phagocytic cells
throughout the body, particularly white blood cells such as
macrophages. Lysosomes turn into Gaucher bodies, and cells with
Gaucher bodies turn into Gaucher cells. In addition to histology,
we have undertaken an ultrastructural approach to examine a case
of Gaucher disease in order to uncover the state of affected ocular
tissues and their cellular organelles. Currently, there is no adequate
ultrastructural survey of Gaucher in the eye.
Methods: A postmortem ultrastructural examination and
immunohistochemistry was performed on ciliary body (OD & OS),
retina (OD & OS), choroid (OD & OS), sclera (OD & OS), corneal
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
limbus (OS), and cornea (OD). The patient was a 61 year old woman
with type I Gaucher since the age of 14. The tissues were prepared
for transmission electron microscopy.
Results: By light microscopy, Gaucher body inclusions (GBIs)
were seen in ciliary body, choroid, sclera, and some infiltration in
the retina. There were lipid-laden foamy histiocytes throughout the
choroid, many CD68+ macrophages, and a few of CD45RO+ T cells,
CD3+ T cells, and CD20+ B cells. EM showed GBIs present in ciliary
pigmentary epithelium, while ciliary non-pigmentary epithelium
appeared vacuolated probably due to the remains of storage vacuoles.
Axons of the ciliary body had abnormal and degenerate myelin
sheaths and numerous membranous myelin inclusions; and Schwann
cells contained cholesterol inclusions. Choroid was full of Gaucher
cells filled with GBIs. GBIs were found mainly along the vessels in
sclera. In the retina, some neuronal cells contained fine homogenous
granular materials that had replaced normal cytoplasmic organelles
mainly in the GCL and INL. No GBIs were seen in the cornea,
corneal limbus, neural retina, or RPE. In the cornea, there were
melanosome laden macrophages, but no GBIs.
Conclusions: Gaucher disease affected the ocular tissues and GBIs
were evident in the ciliary body, choroid, and sclera. Furthermore,
there were other pathological transformations such as degenerate
axons in ciliary body, large inclusions in retinal ganglion cell layer,
melanosome-laden macrophages in the cornea, and condensed
Bruch’s membrane with loss of elastin fibrils.
Commercial Relationships: Mones S. Abu-Asab, None;
Christopher Ardeljan, None; Chi-Chao Chan, None
Program Number: 3413 Poster Board Number: C0224
Presentation Time: 11:00 AM–12:45 PM
An immunohistochemical study for the tumorigenesis of feline
ocular post-traumatic sarcoma
Hiroki Takahashi1, Shunichiro Ueda1, Jun Matsubayashi2, Leandro
B. Teixeira3, Richard R. Dubielzig3, Hiroshi Goto1. 1Ophthalmology,
Tokyo Medical University, Shinjyuku-ku, Japan; 2Anatomic
Pathology, Tokyo Medical University, Tokyo, Japan; 3Pathological
Sciences, University of Wisconsin, Madison, WI.
Purpose: Feline ocular post-traumatic sarcomas (FOPTS) are
malignant intraocular neoplasms that are frequently associated with
a history of ocular trauma and chronic inflammation. However, the
tumorigenesis of FOPTS is unknown. In this study, we evaluated the
possible association between the epithelial mesenchymal transition
(EMT) of lens epithelial cells (LEC) and the tumorigenesis of
FOPTS.
Methods: The database of the Comparative Ocular Pathology
Laboratory of Wisconsin (COPLOW) was searched and records
of FOPTS spindle cell variant were examined. FOPTS spindle
cell variant were divided into 4 categories: Spindle cell metaplasia
(No tumor but LECs have proliferated), Early FOPTS (LECs were
extending beyond the lens capsule), Full FOPTS (Fully developed
tumors which have spread entirely around the eye), and Extensive
FOPTS (Fully developed tumors extend beyond the sclera). 17
cases from each category were examined immunohistochemical
phenotypes. Immunohistochemistry were performed to detect
expression of Pancytokeratin (P-CK: AE1/AE3), Vimentin, α-SMA,
S100A4, and Ki-67. We also compared the number of P-CK and
α-SMA positive case of the inside and outside the lens capsule.
Results: In all cases, tumor cells were positive for Vimentin and
S100A4. The number of P-CK positive cases gradually decreased
with tumor progression. On the other hand, the number of α-SMA
positive cases gradually increased with tumor progression. Ki-67
labeling index also gradually increased with tumor progression. In
cases of early FOPTS intracapsular LECs were more frequently
P-CK positive than extracapsular LECs and extracapsular LECs were
more frequently α-SMA positive than intracapsular LECs. However,
there was no significant difference between the number of P-CK and
α-SMA positive cells of the inside and outside the lens capsule in
Full FOPTS.
Conclusions: In FOPTS, EMT phenomenon was enhanced with
the category progression. Our immunohistochemical findings were
suggested that lens epithelial cells might associate with tumorigenesis
of FOPTS.
Commercial Relationships: Hiroki Takahashi, None; Shunichiro
Ueda, None; Jun Matsubayashi, None; Leandro B. Teixeira, None;
Richard R. Dubielzig, None; Hiroshi Goto, None
Program Number: 3414 Poster Board Number: C0225
Presentation Time: 11:00 AM–12:45 PM
A case series of canine pleomorphic iridociliary adenocarcinomas
Gillian C. Shaw, Leandro B. Teixeira, Richard R. Dubielzig.
COPLOW & Pathobiological Sciences, University of Wisconsin,
Madison, WI.
Purpose: To describe a case series of pleomorphic iridociliary
adenocarcinomas in dogs.
Methods: The COPLOW database was mined for cases of canine
pleomorphic iridociliary adenocarcinomas. Cases were reviewed
and described, and additional clinical data were collected and
summarized. Immunohistochemical staining was performed on a
subset of cases.
Results: There are 22 cases of canine pleomorphic iridociliary
adenocarcinomas in the COPLOW collection representing 1.5%
of all canine iridociliary epithelial tumors. The average age at
enucleation was 10.9 years and there were 13 males and 8 females.
There were 7 Labrador retrievers, 4 golden retrievers, 2 shih tzus,
3 mixed breeds and a single dog from the following breeds: border
collie, Shetland sheepdog, boxer, vizsla, Maltese, dachshund.
Elevated intraocular pressure, uveitis, corneal disease and hyphema
were common presenting complaints. Seven cases (31.8%) had
a history of chemical ciliary body ablation with an intravitreal
gentamycin injection and three (13.6%) had known or suspected
ocular trauma. All had a history of ocular abnormalities for months
to years. Histologically, the tumors are composed of cuboidal to
polygonal cells forming cords, trabeculae and sheets with variably
prominent PAS-positive basement membranes and exhibit extensive
invasion of intraocular structures and/or sclera. Of the tumors stained
immunohistochemically, the neoplastic cells stain positively for
vimentin and variably positive for pancytokeratin.
Conclusions: These canine neoplasms represent the most malignant
of tumors arising from the anterior uvea and share histologic
and immunohistochemical features with human pleomorphic
adenocarcinomas of the ciliary body. The development of these
tumors in canine globes is associated with a history of chemical
ciliary body ablation, trauma and long standing ocular disease.
Commercial Relationships: Gillian C. Shaw, None; Leandro B.
Teixeira, None; Richard R. Dubielzig, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 3415 Poster Board Number: C0226
Presentation Time: 11:00 AM–12:45 PM
Mantle Cell Lymphoma of the Ocular Adnexa
Roxana Saucedo Urdapilleta2, Abelardo A. Rodriguez-Reyes2,
Hector A. Rodriguez-Martinez1, Carmen Lome-Maldonado3, Ivette
Hernandez-Ayuso2, Rosario Gulias-Cañizo2, Dolores Ríos y VallesValles2. 1Experimental Medicine, Hospital General de México,
Mexico city, Mexico; 2Ophthalmic Pathology, Asociación para Evitar
la Ceguera en México I.A.P. Dr. Luis Sánchez Bulnes, Mexico
city, Mexico; 3Pathology, Instituto Nacional de Ciencias Médicas y
Nutrición, Salvador Zubirán, Mexico city, Mexico.
Purpose: Mantle cell lymphoma (MCL) is a rare type of lymphoma.
Reports of MCL in the ocular adnexa have been published in only
some studies with a limited number of cases. We reviewed the
frequency of MCL of the ocular adnexal region in a large Mexican
referral center.
Methods: Retrospective review of clinical records, histopathology
and immunohistochemistry of medical cases (all genders and ages)
diagnose with MCL of ocular adnexal from 1958-2013. a. Files from
the Ophthalmic Pathology Service from the “Asociación para Evitar
la Ceguera en México I.A.P. Dr. Luis Sánchez Bulnes”. b. Cases with
diagnosis of Mantle cell lymphoma. c. Clinical data recorded for
each patient (included year of diagnosis, gender, age, symptoms and
clinical findings). d. Evaluation of clinical pictures, ocular ultrasound,
computed tomography and magnetic resonance imaging. e. Followup.
Results: Twelve patients with MCL of the ocular adnexa were
identified comprising 8% (12/155) of all lymphomas in the ocular
region. There were 6 male patients and 6 female patients with an age
range from 32 to 79 years old (median 66 years). Forty two percent
had bilateral involvement. The orbit (83%) and the lacrimal gland
(42%) were the most commonly affected sites. MCL of the adnexal
region was the first manifestation of systemic disease. Fifty percent
presented in stage II, one stage IV. Microscopically all the cases had a
diffuse architectural pattern and expressed CD20 and cycline D1.
Conclusions: Twelve of one hundred and fifty five patients were
diagnosed with MCL. Commonly ocular adnexa MCL presents in
elderly males, however our patients did no present predominance
of gender. The orbit and lacrimal gland were frequently involved.
Less than 50% of our patients had bilateral orbital involvement. In
the current series the end clinical stage was not as common as other
series in the literature. Inmmunohistochemistry is mandatory to
establish the definitive diagnosis.
Commercial Relationships: Roxana Saucedo Urdapilleta, None;
Abelardo A. Rodriguez-Reyes, None; Hector A. RodriguezMartinez, None; Carmen Lome-Maldonado, None; Ivette
Hernandez-Ayuso, None; Rosario Gulias-Cañizo, None; Dolores
Ríos y Valles-Valles, None
Program Number: 3416 Poster Board Number: C0227
Presentation Time: 11:00 AM–12:45 PM
Lymphoproliferative Disease of the Ocular Adnexa: Clinical
Features and Subtypes
Nitika Arora1, Catherine E. Cuite2. 1Internal Medicine, University of
Illinois College of medicine, Peoria, IL; 2Oculoplastics, Illinois Eye
Center, Peoria, IL.
Purpose: To study the clinical features of patients presenting with
ocular adnexal Lymphoproliferative disease (OALD) and their
relationship with different histologic subtypes.
Methods: Data was collected retrospectively for 36 cases of OALD
that were biopsied by one surgeon between 2000 and 2014. All
patients were classified according to the World Health Organization
modification of the Revised European American Classification.
Descriptive statistics were calculated for demographics, histologic
subtype of tumor, clinical stage at presentation, tumor location,
presenting symptoms and tumor related mortality. Tumor location
was reported as orbital, lacrimal or conjunctival. Presenting
symptoms included swelling, double vision, prominent globe and
tearing. Relationships between the histologic subtype and other
variables were explored using two sample t test and Fisher’s exact
test.
Results: The mean age of the patients was 68.6 ± 13.4 years, and
47% were males. Fifty percent of patients had mucosa associated
lymphoid tissue (MALT) followed by follicular type in 27.8%.
The rest were small cell, large cell and mantle cell lymphomas.
On comparing MALT and follicular type, there was no significant
difference in the age of the presentation, gender, location of the tumor
or clinical features of presentation (p>0.05 for all).
Eighty percent of follicular lymphomas are secondary as compared
to 11% in case of follicular (p=0.023). 69.4% of all the tumors were
primary.
Conclusions: The diagnosis of orbital lymphomas is challenging
because these present with few specific features. According to
our study, clinical presentation, age and gender are not significant
determinants of histologic subtype.
Although majority of tumors were primary, follicular Lymphoma is
more likely to be a secondary tumor. Follicular Lymphoma may have
increased tumor-related death but our small cohort could not detect
statistical significance of this finding.
Commercial Relationships: Nitika Arora, None; Catherine E.
Cuite, None
Program Number: 3417 Poster Board Number: C0228
Presentation Time: 11:00 AM–12:45 PM
Incidence of retinal detachment in primary vitreoretinal
lymphoma (VRL)
Irina Belinsky1, Song Eun Lee2, William M. Schiff3, Brian P. Marr1.
1
Ophthalmic Oncology, Memorial Sloan-Kettering Cancer Center,
New York, NY; 2Ophthalmology, Harkness Eye Institute, Columbia
University, New York, NY; 3Ophthalmology, Manhattan Eye Ear and
Throat Hospital, New York, NY.
Purpose: Primary vitreoretinal lymphoma (VRL) is rare but
potentially fatal, presenting a diagnostic and therapeutic challenge
by masquerading as infectious or inflammatory conditions. We have
observed an increased incidence of retinal detachment in our cohort
of patients with VRL. To characterize this observation further, we
studied this population with attention to demographics, clinical
course, and treatment.
Methods: Retrospective review of 26 consecutive cases at a large
Ocular Oncology Center.
Results: Between 2006 and 2014, a total of 26 patients (44 eyes)
were identified with primary vitreoretinal lymphoma, of whom 81%
were female and 19% male, 92% were Caucasian, and the mean age
at presentation was 62 years. The mean follow up period was 32
months. 81% (n=21) had biopsy proven VRL and 29% (n=5) were
diagnosed clinically. 69% (n=18) had bilateral disease and 31% (n=8)
unilateral. 27% (n=7) of patients had a history of retinal detachment
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
(RD), 86% (n=6) unilateral and 14% (n=1) bilateral and one patient
developed a recurrent RD in one eye, totaling 9 retinal detachments
in this cohort. 33% (n=3) of RD’s occurred prior to the diagnosis of
VRL, 56% (n=5) of RD’s occurred at presentation, and 11% (n=1)
occurred during the course of the disease. 89% (n=8) received some
form of surgical treatment, with no recurrences except one to date.
58% (n=15) of patients had associated primary central nervous
system lymphoma (PCNSL) and 12% (n=3) died as a result of their
lymphoma.
Conclusions: We observe a high incidence of retinal detachment
in patients with primary vitreoretinal lymphoma. The etiology of
this association is unclear. Retinal detachments may be caused
by some effect of lymphoma on the vitreous, the retina, or the
interface. Furthermore, these patients receive numerous diagnostic
and therapeutic interventions. Further studies with more patients
and longer follow up are needed. We propose that this observation
is clinically relevant and warrants careful surveillance for retinal
detachment during follow up and treatment as well as patient
counseling.
Commercial Relationships: Irina Belinsky, None; Song Eun Lee,
None; William M. Schiff, None; Brian P. Marr, None
Program Number: 3418 Poster Board Number: C0229
Presentation Time: 11:00 AM–12:45 PM
A large series of blind painful eyes: potential causes and
associated histopathological features
Cristina Fonseca1, 2, Norah Alsaif2, Mohammed F. Qutub2, Silvin
Bakalian2, Vasco Bravo-Filho2, Miguel N. Burnier2. 1Opthalmology,
Centro Hospitalar e Universitario de Coimbra, Coimbra, Portugal;
2
Henry C. Witelson Ocular Pathology Laboratory, Montreal, QC,
Canada.
Purpose: A blind painful eye is the end stage of several ocular
conditions, including inflammatory, vascular, and glaucomatous
changes. The aim of this project is to describe the morphological
findings of blind painful eyes and to evaluate the most frequent
histopathological features of this condition.
Methods: One hundred and seventy-two cases of enucleated or
eviscerated blind painful eyes were reviewed from the Henry C.
Witelson Ocular Pathology Laboratory, McGill University over
a 21 year period (1993–2014). A review of the histopathological
features to establish criteria for the diagnosis of blind painful eyes
was performed. The underlying causes of the blind painful eyes were
determined clinically and histopathologically.
Results: Of the 172 blind painful eyes, 95 (55.2%) were eviscerated
and 77 (44.7%) were enucleated. Phthisical eyes were diagnosed
by the presence of disorganization with osseous metaplasia and
calcification in shrunken eyes (<16 mm in diameter), and represented
18% of all cases. Mean patient age was 55.6 years (1–88) with
equal sex distribution. Fifty-four histopathological features were
identified, the most common being retinal gliosis (70.9%), chronic
keratitis (69.1%), and non-granulomatous chronic uveitis (50%).
Other findings included total retinal detachment (34.3%), subretinal
hemorrhage (32.5%), anterior synechia (29.6%), optic nerve atrophy
(25.5%), subepithelial pannus (25%), bone metaplasia (16.8%),
and rubeosis iridis (8.1%), among others. The histopathological
changes were further classified according to clinicopathologic
features: over 90% of cases had findings consistent with retinal
vascular diseases, retinal detachment (83%), uveitis (79%), keratitis
(72%), glaucomatous changes (37%), cataract (21%), and corneal
decompensation (18%).
Conclusions: To the best of our knowledge, this is the first study
of blind painful eyes describing the histopathological features and
the underlying ocular disease. The clinical criteria for the diagnosis
of a blind painful eye includes retinal detachment and gliosis,
disorganization of ocular structures, and glaucomatous atrophic
changes. A wide spectrum of ocular conditions can lead to a blind
painful eye. Pathological evaluation of blind painful eyes may
help ophthalmologists formulate an accurate clinicopathological
correlation of the baseline ocular disease.
Commercial Relationships: Cristina Fonseca, None; Norah Alsaif,
None; Mohammed F. Qutub, None; Silvin Bakalian, None; Vasco
Bravo-Filho, None; Miguel N. Burnier, None
Program Number: 3419 Poster Board Number: C0230
Presentation Time: 11:00 AM–12:45 PM
Genetic alterations in conjunctival melanoma and relation to
clinical outcome
Nihal Kenawy1, Sarah E. Coupland1, Bertil E. Damato2, Heinrich
Heimann3, Sarah L. Lake1. 1University of Liverpool, Liverpool,
United Kingdom; 2Ocular Oncology Service, University of
California, San Fransisco, CA; 3Liverpool Ocular Oncology Centre,
Royal Liverpool University Hospital, Liverpool, United Kingdom.
Purpose: Despite improved understanding of the molecular changes
in conjunctival melanoma (CoM), the underlying aetiology of this
tumor remains unclear. In this study, we determined gene copy
number variations (CNV) in CoM aiming to identify disease-specific
genetic biomarkers to facilitate prognostication, as has been achieved
in uveal melanoma.
Methods: Ninety two patients with primary CoM seen between
2005 and 2014 were recruited in eight international ocular oncology
centres. Clinical and histological data were collected. DNA was
extracted from paraffin-embedded samples. Fifty-nine samples
yielded sufficient DNA for Affymetrix 6.0 Single Nucleotide
Polymorphism (SNP) microarray testing. SNP data were analysed by
Partek Genomic suite. Patients who developed regional lymph nodes
and/or systemic metastases were compared to a sex, age-matched
cohort of low risk clinical and pathological criteria for dissemination
and with similar follow up duration.
Results: Over 40% of the samples showed amplifications of histone
gene cluster (6p22.2), ETV1 (7p21.2), FOXQ1 (6p25.3 - 6p25.2) and
SOX4 (6p22.3) and deletions in ASNS (7q21.3), CHEK2P2 (15q11.1),
RET (10q11.21) and BAGE (21p11.1). CNVs in the two groups of
metastatic CoM and the low risk comparative group were consistent.
None of the chromosomes identified with gains or losses showed
loss of heterozygosity or isodisomy. Based on the current median
follow-up time of 2.5 years, no statistically significant correlation has
been detected between any of the genetic alterations and features of
poor outcome, i.e. caruncular involvement, epithelioid cell type or
metastatic spread.
Conclusions: This is the largest cohort of CoM samples analysed by
SNP genotyping to date and describes CNVs not previously reported.
Longer follow-up is required to facilitate our understanding of CoM
and identification of patients at high metastatic risk.
Commercial Relationships: Nihal Kenawy, None; Sarah E.
Coupland, None; Bertil E. Damato, None; Heinrich Heimann,
None; Sarah L. Lake, None
Support: Eye Tumour Research Fund A0722/CF
Program Number: 3420 Poster Board Number: C0231
Presentation Time: 11:00 AM–12:45 PM
HSP90 expression is a useful tool for the differential diagnosis of
ocular surface squamous neoplasia
Ana Beatriz T. Dias, Pablo Zoroquiain, Dominique F. souza, Dana
Faingold, Patrick T. Logan, Miguel N. Burnier. McGill University,
Montreal, QC, Canada.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Purpose: HSP90 is a chaperone protein that stabilizes and activates
client proteins involved in malignant transformation. HSP90 is
overexpressed in many malignant tumors, including squamous cell
carcinomas of the head and neck. The aim of this study is to evaluate
HSP90 expression in ocular surface squamous neoplasia (OSSN) and
to assess its diagnostic value for the different lesions of the OSSN
spectrum.
Methods: Seventy conjunctival squamous lesions, including 19
papillomas (Pa), 22 conjunctival intraepithelial neoplasia (CIN) I,
11 CIN II, 6 CIN III, and 12 squamous carcinoma (sqCA), were
evaluated using automated immunohistochemical staining against
HSP-90. As controls, 15 normal conjunctiva specimens were used.
The staining was scored by evaluating staining intensity (0–3)
and extent (0–4) and these values were multiplied to generate an
immunoreactive score (IRS; 0–12). This process was performed for
nuclear (nIRS) and cytoplasmic (cIRS) HSP90 staining. Also, intraepithelium staining was evaluated as a percentage of total thickness
(PTE).
Results: All OSSN and 90% of controls were positive for HSP90;
however, mean benign and malignant OSSN IRS were significantly
higher than normal conjunctiva (P<0.0001). With respect to OSSN,
cIRS had a higher HSP90 IRS in high grade compared to low grade
lesions (Pa and CIN I; P<0.001). nIRS was significantly different
between high grade lesions (CIN II vs CIN III-sqCA; P=0.0162). A
cIRS >6 correlated with a high grade lesion (sensitivity [S] 58.3%,
specificity [Sp] 97.4%), while a cIRS <4 correlated with a low
grade lesion (S 43.6%, Sp 100%). An nIRS >6 in high grade lesions
correlated with CIN III-sqCA (S 55.6%, Sp 81.8%), while an nIRS <4
correlated with CINII (S 45.5%, Sp 94.4%). PTE staining of <73%
differentiated between CIN III and sqCA with an S of 91.7% and Sp
of 100% for sqCA. nIRS, cIRS, and cIRS + nIRS did not correlate
with the depth of infiltration or sqCA differentiation degree.
Conclusions: To the best of our knowledge, this is the first
study using HSP90 as a marker to differentiate between benign,
premalignant, and malignant lesions of the conjunctiva. The
expression of HSP90 is particularly useful to differentiate low from
high grade CIN and invasive sqCA of the conjunctiva. HSP90 may
play an important role in stabilizing and activating client proteins
involved in this malignant transformation.
Commercial Relationships: Ana Beatriz T. Dias, None; Pablo
Zoroquiain, None; Dominique F. souza, None; Dana Faingold,
None; Patrick T. Logan, None; Miguel N. Burnier, None
Program Number: 3421 Poster Board Number: C0232
Presentation Time: 11:00 AM–12:45 PM
Expression of nestin in conjunctival melanoma
Marie-Sophie Hanet, Henning Thomasen, Henrike Westekemper,
Klaus-Peter Steuhl, Daniel Meller. Ophthalmology, University of
Duisburg-Essen, Essen, Germany.
Purpose: Conjunctival melanoma is a rare malignant tumor, of
which the biology remains largely unknown. Nestin, an intermediate
filament protein described as neural stem cell marker, has been
reported in cutaneous melanocytic tumors with potential implication
for their diagnosis and staging. We analyzed the immunological
properties of conjunctival melanomas and hypothesized that nestin
could also have clinical interest as a biomarker in conjunctival
melanomas.
Methods: Samples of tumoral tissue from five patients with a
primary conjunctival melanoma and four cell lines (UKE29, UKE17,
CR1 and CR2) derived from conjunctival melanoma were analyzed.
Ten samples of limbal (n=5) or fornical (n=5) conjunctiva obtained
from healthy subjects were used as controls. Expression of nestin and
other pluripotency markers (Sox-2, Oct-4, Nanog, c-Myc, ABCG2,
p63, and c-KIT) was examined using real-time polymerase chain
reaction. Expression of nestin was additionally examined using
immunohistochemical staining.
Results: Expression of nestin was significantly higher in melanoma
tissue than in controls (p<0.008 vs. limbal and p<0.02 vs. fornical
conjunctiva), which was consistent with the results of immunological
staining. The expression of other markers of pluripotency was not
statistically different between melanomas and normal tissues from
either limbal or fornical conjunctiva. In all cell lines the expression of
nestin was inferior to the melanoma tissues. The expression of other
markers of pluripotency was generally lower in cell lines compared
to melanoma tissues, except for ABCG2 in UKE29 and p63 in
UKE17. Compared to control tissues, nestin expression was higher
in the cell line UKE17 than in limbal controls, and higher than both
limbal and fornical controls in the cell line UKE29. The expression of
other pluripotency markers was inferior in cell lines compared to all
controls, apart from c-Myc in the cell line CR1 and ABCG2 in UKE
29.
Conclusions: Nestin is significantly overexpressed in conjunctival
melanoma, whereas the expression shows a decrease in cell lines
consistent with the general pattern of expression of other pluripotency
markers in those cell lines. This observation supports the hypothesis
that nestin can potentially serve as diagnostic adjunct in conjunctival
melanoma.
Commercial Relationships: Marie-Sophie Hanet, None; Henning
Thomasen, None; Henrike Westekemper, None; Klaus-Peter
Steuhl, None; Daniel Meller, None
Program Number: 3422 Poster Board Number: C0233
Presentation Time: 11:00 AM–12:45 PM
Clinicopathologic Characterization of Amyloid Deposition in
Ocular Surface and Adnexa
Maria J. Suarez B1, Roxana Rivera-Michlig2, Fausto Rodriguez1.
1
Ophthalmic Pathology, Johns Hopkins University, Baltimore, MD;
2
Ophthalmology, Wilmer Eye Institute/Johns Hopkins, Baltimore,
MD.
Purpose: To describe the histopathological features of amyloid
deposition seen in the ocular surface and/or adnexa biopsy specimens
and further characterize the type of amyloid with proteomic analysis.
Methods: This is a retrospective study in which the medical records
from patients that were diagnosed with primary and secondary ocular
and orbital amyloid deposition at our institution were retrieved
between 1991-2014. The demographic data, clinical findings and
pathology reports were also reviewed. Mass spectrometry-based
proteomic analysis was performed in one case using formalin-fixed
paraffin-embedded tissue.
Results: The study included 9 patients (5 females, 4 males). The
mean age was 59.1 years (range 39 – 88 years). Eight cases presented
as unilateral lesions in otherwise healthy patients and one case was
bilateral, in a patient with a previous history of multiple myeloma
confirmed by electrophoresis. Four cases involved the conjunctiva,
three cases with lesions in the eyelid and two cases presented as
orbital masses, one of them with ptosis. Congo red stain was positive
in eight cases; one case was unequivocal but moderately positive for
Thioflavine T. Proteomic analysis performed in one of the orbital
masses demonstrated lambda light chain-derived peptides (but not
kappa). Systematic clinical evaluation in this patient was performed,
and no evidence of a systemic plasma cell dyscrasia was identified.
Conclusions: Our study describes the clinicopathologic features of
amyloid deposition in the ocular surface and adnexa in patients with
no evidence of predisposing disease, as well as secondary amyloid
orbital deposition seen in one patient with a preexisting plasma cell
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
dyscrasia (multiple myeloma). Proteomic analysis may be of value in
biopsies from these patients and deserves further study.
Commercial Relationships: Maria J. Suarez B, None; Roxana
Rivera-Michlig, None; Fausto Rodriguez, None
Program Number: 3423 Poster Board Number: C0234
Presentation Time: 11:00 AM–12:45 PM
Secreted Ly6/urokinase-type plasminogen activator receptor
related protein-1 (Slurp1) expression by strain, age, gender and
ocular surface health
Sudha Swamynathan, Emili E. Delp, Stephen A. Harvey, Leela Raju,
Shivalingappa K. Swamynathan. Ophthalmology, University of
Pittsburgh, Pittsburgh, PA.
Purpose: Previously, we demonstrated that Slurp1 serves an
immunomodulatory function in the ocular surface. Although Slurp1
transcripts are the 11th most abundant in the mouse cornea, it is not
known if this high expression is reflected at the protein level. Also,
several tear proteomic studies have not identified SLURP1, resulting
in ambiguity about the baseline SLURP1 concentration in human
tears. Here, we examine the mouse Slurp1 expression in different
strains, age-groups, genders, and ocular surface health conditions,
and quantify SLURP1 expression in human tears collected from
healthy or inflamed ocular surfaces.
Methods: Early embryonic (E) and postnatal (PN) mouse corneal
Slurp1 expression was quantified using QPCR. The influence of
mouse genetic background, age, and gender on Slurp1 expression
was examined by QPCR and immunofluorescent staining in Balb/C,
FVBN, C57Bl/6, and DBA/2J strains at PN10, PN20 and PN56.
Human tears were collected from 34 adults (18-80 years) with
absorbent wicks using an IRB-approved protocol, and the SLURP1
levels quantified by ELISA.
Results: Slurp1 expression, undetectable in E13, E16, and PN1
mouse corneas and barely detectable in PN10, increased rapidly
in post-eyelid opening stages levelling off around PN20. Slurp1
expression did not differ significantly between different strains,
or genders. Human tear SLURP1 levels did not vary significantly
between genders and age groups. However, tears from inflamed
human ocular surface contained significantly decreased amounts of
SLURP1 (0.35 ng/100ng tear protein) compared with those from
healthy individuals (0.68 ng/100ng tear protein).
Conclusions: These data provide the first detailed description of
the influence of age, gender, and genetic background on Slurp1
expression in the mouse cornea, establish the baseline for SLURP1
concentration in human tears, and demonstrate that the tears from
inflamed ocular surface contain decreased amounts of SLURP1.
Commercial Relationships: Sudha Swamynathan, University of
Pittsburgh (P); Emili E. Delp, None; Stephen A. Harvey, None;
Leela Raju, None; Shivalingappa K. Swamynathan, University of
Pittsburgh (P)
Support: NIH Grant R01EY022898
Program Number: 3424 Poster Board Number: C0235
Presentation Time: 11:00 AM–12:45 PM
Orbital Invasion From Ocular Surface Squamous Neoplasia
Benjamin P. Erickson1, Anat Galor2, Thomas Johnson1, Wendy W.
Lee1, Carol L. Karp2. 1Oculoplastics, Bascom Palmer Eye Institute,
Miami, FL; 2Cornea, Bascom Palmer Eye Institute, Maim, FL.
Purpose: Orbital invasion from ocular surface squamous neoplasia
(OSSN) is a rare and potentially devastating complication. The
majority of studies arise from Sub-Saharan Africa and the Middle
East, where HIV and poor access to care are significant factors. The
North American literature primarily consists of isolated reports. We
present our experience with 7 cases of OSSN invading the orbit.
Methods: Retrospective review of 500 consecutive patients with
biopsy-confirmed OSSN and radiologic and/or histologic evidence of
orbital invasion.
Results: Seven patients with orbital extension of OSSN were
identified. Average age was 59.7±15.8 years old; 5 patients were
male and 2 female. 5/7 were black and the remainder Hispanic
(1/7) or non-Hispanic Caucasian (1/7). 4/7 were current or former
smokers. 3/7 patients (age 48±10 years old) were HIV positive. None
had a known history of hematogenous malignancy, head & neck
radiation, immunosuppressive medication use, skin cancer, or HPV.
Five of the lesions were primary, while the remaining 2 represented
recurrence of previously treated OSSN. Lesions occupied 6.8±5.0
clock hours, with 5 of 7 cases involving the perilimbal conjunctiva.
The one case with multiple recurrences had only a single clock hour
of superficial involvement. Orbital invasion was clinically detected
in the form of significant motility restriction in 4/7 patients, diplopia
in 2/7, profound vision loss (light projection or worse) in 2/7, and
epiphora in 2/7. Only the patient with multiple recurrences lacked
clear external stigmata of invasion, which was identified by computed
tomography (CT). Scans revealed involvement of the extraconal
fat in 6/7, intraconal fat in 2/7, lacrimal drainage system in 2/7, and
optic nerve in 1/7. Four of the 7 patients underwent total, and 2/7 lid
sparing, exenteration; the remaining patient (age 88) refused surgery
and died of unrelated causes. 2/7 underwent negative sentinel node
biopsy. One patient each was treated with adjuvant chemotherapy and
radiation. A single patient had disease specific mortality.
Conclusions: OSSN is statistically a disease of older Caucasian
men with history of sun exposure, but the majority of patients in our
study were black with delayed primary diagnosis and/or underlying
immunosuppression. Orbital invasion appears rare in cases of
appropriately treated but recurrent OSSN, but associated external
findings can be minimal and close surveillance is essential.
Commercial Relationships: Benjamin P. Erickson, None; Anat
Galor, None; Thomas Johnson, None; Wendy W. Lee, None; Carol
L. Karp, None
Program Number: 3425 Poster Board Number: C0236
Presentation Time: 11:00 AM–12:45 PM
Fibrosis, Gene Expression, and Orbital Inflammatory Disease
Stephen R. Planck1, 2, Dongseok Choi1, Christina A. Harrington4,
David J. Wilson1, Hans E. Grossniklaus3, Patrick Stauffer1, James T.
Rosenbaum1, 2. 1Casey Eye Institute, Oregon Health & Science Univ,
Portland, OR; 2Devers Eye Institute, Legacy Health System, Portland,
OR; 3Ophthalmology, Emory University, Atlanta, GA; 4Integrated
Genomics Laboratory, Oregon Health & Science Univ, Portland, OR.
Purpose: Fibrosis is an important association with inflammation that
affects the orbital tissue. To clarify the pathogenesis of fibrosis in
orbital diseases, we analyzed the gene expression in orbital biopsies
and compared our results to those reported for idiopathic pulmonary
fibrosis.
Methods: We collected 153 biopsies including 68 from the lacrimal
gland and 85 from orbital fat. Diagnoses included healthy controls
(n=27), nonspecific orbital inflammation (NSOI) (n=64), thyroid
eye disease (TED) (n=33), sarcoidosis (n=20), and granulomatosis
with polyangiitis (GPA) (n=9). Fibrosis was scored on a zero to three
scale by two expert, ophthalmic pathologists. Gene expression was
quantified using Affymetrix U133 plus 2.0 microarray.
Results: Within orbital fat, fibrosis was greatest among subjects with
GPA (2.75±0.46) and significantly increased in tissue from subjects
with GPA, NSOI, or sarcoid (p<0.01), but not for TED, compared
to controls (1.13±0.69). For the lacrimal gland, the average fibrosis
score among healthy controls (1.36±0.48) did not differ statistically
from any of the 4 disease groups. 73 probe sets identified transcripts
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
(~54 genes) correlating with fibrosis in orbital fat (false discovery
rate < 0.05 and fold-difference >1.5). Transcripts with increased
expression included fibronectin, lumican, thrombospondin, and
collagen types I and VIII. Many of these transcripts were also
increased in pulmonary fibrosis.
Conclusions: A pathologist’s recognition of fibrosis in orbital tissue
correlates well with increased expression of transcripts considered
essential in fibrosis. Furthermore, overlapping genes have been
detected in pulmonary fibrosis. The study supports the accuracy of
histological scoring of fibrosis and conversely, the results help to
validate gene expression analysis from formalin-fixed tissue as an
accurate methodology to understand pathophysiology.
Commercial Relationships: Stephen R. Planck, None; Dongseok
Choi, None; Christina A. Harrington, None; David J. Wilson,
None; Hans E. Grossniklaus, None; Patrick Stauffer, None; James
T. Rosenbaum, Genentech (C)
Support: NIH Grants EY020249, EY010572 and RR024140;
Research to Prevent Blindness
Program Number: 3426 Poster Board Number: C0237
Presentation Time: 11:00 AM–12:45 PM
A Novel Murine Model of Orbital Inflammation
N. Grace Lee1, Lindsay L. Wong2, Dhanesh Amarnani2, Suzanne K.
Freitag1, Diane Bielenberg3, Patricia A. D’Amore2, Leo A. Kim1,
2 1
. Ophthalmology, Harvard Medical School - MEEI, Boston, MA;
2
Schepens Eye Research Institute, Boston, MA; 3Boston Children’s
Hospital, Boston, MA.
Purpose: Orbital inflammation from Graves’ disease or nonspecific
orbital inflammation can cause devastating compressive optic
neuropathy. Current therapeutic options which primarily consist of
steroids and decompression surgery may not be sufficient to resolve
the optic neuropathy. Progress in this area is hindered by the lack of
animal models of acute orbital inflammation. We have developed an
animal protocol to induce inflammation in the murine orbit with the
use of a skin sensitizer, oxazolone.
Methods: Eight-week-old female BALB/c mice were sensitized with
a topical application of 2% oxazolone solution in olive oil/acetone
(2:1 vol/vol) to the shaved abdomen. Five days after sensitization
(day 0), the right eye was challenged with a 10uL orbital injection of
2% oxazolone solution. The left eye, serving as a control, received
a sham injection of the vehicle alone (olive oil/acetone). Mice then
underwent daily magnetic resonance imaging (MRI) before being
euthanized at various time points. Their exenterated orbits were
examined histologically.
Results: Twenty-four hours following the orbital challenge, mice
were observed to demonstrate mild proptosis and dermatitis. MRI on
day 1 confirmed the findings of exophthalmos as well as retrobulbar
inflammation and periorbital edema. On day 3, there was relative
reduction of edema and proptosis compared to day 1. Histopathologic
examination of the mouse orbit from day 1 to day 5 corroborated the
MRI findings of periocular and intraocular inflammation consisting
of neutrophils with a transition to chronic inflammation with
lymphocytes by day 3 and early fibrosis on day 5.
Conclusions: We present a novel animal model of orbital
inflammation utilizing a type 4 hypersensitivity reaction. This model
should allow a better understanding of why the orbit is preferentially
affected in certain systemic disorders and may provide a way to
evaluate potential alternatives to steroid and surgical treatment.
A. Sub-Tenon’s injection of 2% oxazolone in the right orbit and
vehicle in the left on Day 0.
B. Dermatitis of the right upper and lower eyelids, right facial edema,
and right orbital inflammation in contrast to the normal-appearing left
side on Day 1.
C. MRI reveals asymmetric retrobulbar and facial edema right greater
than left on day 1.
D. Exenterated right orbit on day 1 shows retrobulbar edema and an
aggregate of neutrophils, suggesting acute inflammatory response.
Commercial Relationships: N. Grace Lee, None; Lindsay L.
Wong, None; Dhanesh Amarnani, None; Suzanne K. Freitag,
None; Diane Bielenberg, None; Patricia A. D’Amore, AGTC (C),
Eleven Biotherapeutics (S); Leo A. Kim, None
Support: American Thyroid Association Grant, Lions Eye
Foundation Research Award
Program Number: 3427 Poster Board Number: C0238
Presentation Time: 11:00 AM–12:45 PM
Differences in the expression of immunohistochemical markers in
solid and fibrosing basal cell carcinoma
Viola Graham1, 2, Ute Kaiser1, 2, Frank G. Holz1, Martina C. Herwig1,
2
, Karin U. Loeffler1, 2. 1Department of Ophthalmology, University of
Bonn, Bonn, Germany; 2Department of Ophthalmology, Division of
Ophthalmic Pathology, University of Bonn, Bonn, Germany.
Purpose: Basal cell carcinoma (BCC) is the most frequent cancer of
the ocular adnexae. Semimalignant, it can be subdivided into a solid
type and a more aggressive and invasive fibrosing type. In this study,
different molecules related to infiltrative growth were investigated to
further help our understanding of these different growth patterns.
Methods: 20 formalin-fixed paraffin-embedded specimens of basal
cell carcinoma of the eyelid were investigated and subdivided
into two groups. Group I consisted of 10 solid BCC, group II of
10 fibrosing BCC (mean age 71 versus 75,1 yrs). There were 4
male and 6 female patients in both groups. Immunohistochemistry
was performed for matrix metalloproteinases (MMP) 1, 2 and 9
(Santa Cruz), Ki67 (Dako), p53 (Dako) and FHOD1 (Novusbio).
Immunoreactivity was evaluated by light microscopy and graded
semiquantitatively by two readers using a score from 0 to 3.
Statistical analysis was performed with SPSS (IBM SPSS Statistics
20; Armonk, NY). The probability for the alpha error was set to p <
0.05.
Results: As could be expected, MMP 1, 2 and 9 were present in a
membrane-bound fashion, whereas Ki67 and p53 were located in
the nucleus. MMP1, MMP2 and p53 showed a stronger expression
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
in fibrosing BCC compared to solid BCC. This was only statistically
significant for MMP1 (p=0,024) with a mean staining reaction of
0,95 for solid BCC and 1,7 for fibrosing BCC. For MMP2 (p=0,105)
and p53 (p=0,233), there was only a trend for a more intense staining
in fibrosing BCC (MMP2: 2,45; p53: 1,9) compared to solid BCC
( MMP2: 2,05; p53:1,4). MMP9 and FHOD1 did not as yet yield
reliable results. Regarding Ki67, there was no significant difference
between both groups.
Conclusions: Molecules associated with infiltrative growth such as
MMP1 showed a higher staining intensity in fibrosing BCC and may
explain - among other factors - their invasive behaviour. Adressing
those as therapeutic target might perhaps help in developing new
adjuvant treatment options. To confirm our observations, studies with
a larger number of basal cell carcinomas are warranted and may also
address this topic on a genetic level.
Commercial Relationships: Viola Graham, None; Ute Kaiser,
None; Frank G. Holz, None; Martina C. Herwig, None; Karin U.
Loeffler, None
Program Number: 3428 Poster Board Number: C0239
Presentation Time: 11:00 AM–12:45 PM
Macrophages in basal cell carcinoma
Karin U. Loeffler1, Ute Kaiser1, Frank G. Holz2, Martina C. Herwig1.
1
Division of Ophthalmic Pathology, University Eye Department,
Bonn, Germany; 2Ophthalmology, University of Bonn, Bonn,
Germany.
Purpose: Basal cell carcinoma (BCC) is a locally invasive tumor
which can be subdivided in a fairly benign solid variant and a more
aggressive fibrosing type. Since in oncology there is increasing
evidence for the importance of macrophages (Ms) interacting
between tumor cells and their surroundings, thereby affecting not
only the infiltrative potential but also the patients’ prognosis, we
wanted to compare these two BCC variants regarding the presence
and localisation of tumor-associated Ms.
Methods: We studied 15 specimens of solid BCC (BI) and 15 of
fibrosing BCC (BII). Mean age in both groups was similar (73
vs.75 years). Fibrosing BCC was located almost exclusively in
the lower eyelid while solid BCC was located more diversely
around the eye. H&E sections were evaluated for the presence of
associated inflammation (score + to +++). Immunohistochemistry
was performed with antibodies against CD68 (Dako;1:100), CD163
(EnzoLifeSciences;1:100) and Ki67 (Dako;1:100). Positive reacting
cells were visualized either using AEC or immunofluorescence and
counted in a standard fashion and/or by means of Automatic Nuclei
Counter plug-in for ImageJ. Evaluation was performed by at least 2
different readers, and IBM SPSS 20 was used for statistical analysis.
Results: In all specimens, an inflammatory component was observed
which - as judged by H&E - was significantly more pronounced
in BII compared to BI (p<0.001). Ms were also more frequent in
BII (p<0,05), however, no significant difference in M subtype was
found between both groups. In both BI and BII, Ms were much more
frequent in the surrounding stroma compared to the tumor itself
(p<0.001). Ms within the tumor were more frequent in BII compared
to BI (p<0,05), and in both groups those Ms always belonged to the
M1(CD68+CD163-) category. Ki67 index was similar in both groups.
Conclusions: Fibrosing BCC, more likely to be located at the lower
eyelid, is associated with a significantly more intense inflammatory
reaction in the surrounding tissue. From our data, however, there
was no significant difference in the subtype of Ms when comparing
both groups. Thus, in BCC, macrophage polarization does not seem
to be particularly relevant regarding infiltrative growth, and tumorassociated chemokines attracting/stimulating inflammatory cells other
than Ms might also be important in tissue destruction and thereby
invasive potential. It remains unclear whether localisation of the
tumor plays a major role in this.
Commercial Relationships: Karin U. Loeffler, None; Ute Kaiser,
None; Frank G. Holz, None; Martina C. Herwig, None
Program Number: 3429 Poster Board Number: C0240
Presentation Time: 11:00 AM–12:45 PM
Clinical and histopathological characteristics of periocular basal
cell carcinoma in a low UV geographic region
Beatriz Nugent da Cunha1, Dominique F. de Souza2, Nadine
Marques2, Patrick T. Logan2, Jordan Discepola2. 1Federal University
of São Paulo, São Paulo, Brazil; 2Ophthalmology, McGill University,
Montreal, QC, Canada.
Purpose: Basal cell carcinoma (BCC) accounts for more than 90% of
all eyelid malignancies. Risk factors for BCC include age, ultraviolet
light [UV] exposure, genetic background, and fair skin. The
objective of this study is to investigate the frequency of periocular
BCC subtypes, in particular sclerosing type, in a low UV exposure
area (Quebec, Canada) and to compare these results to published
data from a high UV exposure area (Australian Mohs Nationwide
Database).
Methods: A total of 387 excised periocular BCCs from January
1998 to June 2014 from the Henry C. Witelson Ocular Pathology
Laboratory, Montreal, Quebec were evaluated. Demographic
data were retrieved, including gender, age, tumor localization,
prior clinical diagnosis, histological subtype, and ulceration. For
comparisons with a high UV geographic location, we obtained
published data (ocular location and histopathological subtype) from
the Australian Mohs Database, which includes 1,295 periocular BCC
cases.
Results: The average age of our patients was 67.6 ± 15.3 years
(range: 8–101). Tumors were more frequent in women (57.1%).
Clinical misdiagnosis occurred in 15.2% of the cases, 70% of which
were diagnosed as benign lesions. BCC location frequency in
descending order was the lower eyelid (58%), upper eyelid (12%),
and medial canthus (8.5%). Histopathological subtypes identified
were as follows: nodular (79.6%), sclerosing type (7.5%), squamous
differentiation (6.5%), sebaceous differentiation (2.8%), and mixed
types (3.6%). Twenty tumors were ulcerated (5.2%). The Australian
Mohs Database published the following results: 615 (47.5%) BCCs
were located in the lower eyelid; 627 (48.3%) in the medial canthus;
and 51 (3.9%) in the upper eyelid. The most common subtype in this
geographic region was nodular (39.5%) followed by sclerosing type
in 34.8% of cases.
Conclusions: There are significant differences in periocular BCC
location and subtype between low and high UV geographic locations.
In Quebec, the most common ocular location is the lower eyelid,
whereas in Australia it is the medial canthus. Sclerosing type was
significantly higher in Australia compared to Quebec. The higher
UV exposure in Australia relative to Quebec is the likely factor
explaining the much higher frequency of this most aggressive type of
periocular BCC in the Australian population.
Commercial Relationships: Beatriz Nugent da Cunha, None;
Dominique F. de Souza, None; Nadine Marques, None; Patrick T.
Logan, None; Jordan Discepola, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 3430 Poster Board Number: C0241
Presentation Time: 11:00 AM–12:45 PM
Multispecialty approach to management of sebaceous carcinoma
of the eyelids
Seanna R. Grob1, 2, N. Grace Lee1, 3, Francis Creighton4, Frederick
Jakobiec1, 5, Kevin Emerick4, Suzanne K. Freitag3, 1. 1Ophthalmology,
Massachusetts Eye and Ear Infirmary, Boston, MA; 2Ophthalmology,
Harvard Medical School, Boston, MA; 3Oculoplastics and
Reconstructive Surgery, Massachusetts Eye and Ear Infirmary,
Boston, MA; 4Otolaryngology, Massachusetts Eye and Ear Infirmary,
Boston, MA; 5Ophthalmic Pathology, Massachusetts Eye and Ear
Infirmary, Boston, MA.
Purpose: To report a multispecialty approach to the management of
periocular sebaceous carcinoma and the clinical outcomes of patients
who have been treated with this approach.
Methods: Retrospective review of 6 patients with periocular
sebaceous carcinoma managed by the Oculoplastics Service at
Massachusetts Eye and Ear Infirmary (MEEI) was conducted. Data
collected included duration of symptoms, previous ocular diagnoses,
eyelids affected, method of excision and reconstruction, results of
sentinel lymph node biopsy and metastatic work up, recurrence rate,
and duration of follow up.
Results: Six patients were identified (mean 70.5 years, 4 female). All
were of a Caucasian background. Four patients had a family history
of any cancer; only one patient had a prior history of skin cancer –
basal cell carcinoma. Prior to evaluation at MEEI, four patients were
diagnosed with blepharitis. The mean duration of symptoms prior to
biopsy proven diagnosis and evaluation at MEEI was 28.5 months.
The location of the sebaceous carcinoma was most commonly
in the upper lids (4 patients). After diagnosis was confirmed by
histopathologic analysis, all patients were evaluated by the head and
neck oncology service at MEEI for discussion of sentinel lymph node
biopsy, medical oncology for staging and metastatic work up, and
radiation oncology for possible adjuvant therapy. An average of 2.2
total sentinel lymph nodes were removed per case. The parotid and
cervical level 2 sentinel lymph node basins were the most common
sites (each 50% of cases). Surgical margins were evaluated by frozen
section and confirmed with rapid permanent section within 24 hours.
All patients had negative lymph node biopsies and metastatic work
ups on initial presentation. Only one patient has had recurrence since
initiation of care at MEEI, but this patient had extensive eyelid and
ocular surface disease on initial presentation.
Conclusions: The optimal management of sebaceous carcinoma of
the ocular surface and adnexa requires an integrated multidisciplinary
approach involving an ophthalmic plastic surgeon, a head and
neck oncologist, a medical and radiation oncologist, experienced
pathologists and radiologists, and at times, an anterior segment
surgeon specializing in ocular surface tumors.
Commercial Relationships: Seanna R. Grob, None; N. Grace Lee,
None; Francis Creighton, None; Frederick Jakobiec, None; Kevin
Emerick, None; Suzanne K. Freitag, None
Program Number: 3431 Poster Board Number: C0242
Presentation Time: 11:00 AM–12:45 PM
Visual Outcomes in Non-Neurofibromatosis-Related Optic
Pathway Gliomas
Michael J. Wan, Gena H. Heidary. Ophthalmology, Boston Children’s
Hospital and Harvard University, Boston, MA.
Purpose: Optic pathway gliomas (OPGs) are low-grade tumors
of childhood that have a high survival rate but can cause
significant visual impairment. While commonly associated with
Neurofibromatosis Type 1 (NF1), non-NF1-related OPGs have
been reported to cause more visual disability, but long-term data are
limited. The purpose of this study was to report the long-term visual
outcomes of a cohort of pediatric patients with non-NF1-related
OPGs.
Methods: This was a retrospective cohort study at a tertiary care
pediatric hospital and cancer institute. The study included all patients
with non-NF1-related OPGs seen between 1995-2013. The clinical
features at presentation, tumor location, type of treatment, evidence
of progression during treatment or recurrence after treatment, and the
results of all visual assessments were recorded. The primary outcome
was visual acuity at final follow-up.
Results: There were a total of 61 patients with non-NF1-related
OPGs. The most common presenting features were vision loss (26%),
nystagmus (26%), neurological findings (23%), strabismus (13%)
and headache (12%). The median age of onset was 2.6 years old
and 52 cases (85%) presented with bilateral involvement. Fifty-four
patients (89%) received treatment; the most common treatment
was chemotherapy and surgery (44%), followed by chemotherapy
alone (39%) and surgery alone (5%). Fifteen patients (28%) showed
progression during treatment requiring a change in therapy and 29
patients (54%) had evidence of recurrence after completion of initial
therapy. Of the 54 patients who received treatment, 18 (33%) did not
have evidence of progression or recurrence. The median follow-up
interval was 5.1 years after presentation. In the worse eye at final
follow-up, 18 patients (30%) were ≥ 20/30, 9 patients (15%) were
between 20/40-20/80, and 34 patients (56%) were ≤ 20/100. In the
better eye at final follow-up, 35 patients (57%) were ≥ 20/30, 11
patients (18%) were between 20/40-20/80, and 15 patients (25%)
were ≤ 20/100.
Conclusions: In this cohort of pediatric patients with non-NF1related OPGs, the long-term visual outcomes were poor, with severe
visual deficits in over half of the patients at last follow-up. Even with
treatment, the majority of patients had evidence of either progression
or recurrence. Frequent visual assessments for patients with nonNF1-related OPSs, both during treatment and afterward, are crucial to
minimize long-term visual impairment.
Commercial Relationships: Michael J. Wan, None; Gena H.
Heidary, None
Program Number: 3432 Poster Board Number: C0243
Presentation Time: 11:00 AM–12:45 PM
The influence of race and gender on the risk of hair loss
secondary to methotrexate.
Sarah Escott1, Julia Malalis1, Yosuke Harada1, David Mai2, Debra A.
Goldstein1. 1Ophthalmology, Northwestern University, Chicago, IL;
2
University of Illinois, Peoria, IL.
Purpose: To determine if there is a difference in terms of selfreported race and gender in the incidence of hair loss with
methotrexate (MTX).
Methods: A retrospective review of all patients at a University based
practice seen between August 2012 and September 2014 with a
history of MTX treatment for uveitis was performed. Data collected
included self-reported race, diagnosis, age at start of MTX therapy,
self-reported hair loss, and both dose and duration of MTX at time
of hair loss. To qualify for inclusion patients had a minimum of 6
months follow up after reported hair loss and cessation of MTX
therapy.
Results: Complete records of 97 uveitis patients who met the above
criteria were reviewed. There were 25 males and 72 females. Of
these, 28 patients self-reported African American race, 60 reported
Caucasian, and the remaining 9 reported Asian, Hispanic, or Arabic
ethnicity. Hair loss was reported in 9.2% of patients. Average age
at the time of hair loss was 38.6 years (range 9-59 years). The
incidence of hair loss was similar in women (10%) and men (8%);
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
but was twice as common in African American (AA) patients (14%)
compared with Caucasian patients (6.6%). All AA patients reporting
hair loss were female, although there were only 3 AA males included
in the analysis. Hair loss developed on weekly doses of 10-25mg
MTX after an average exposure time of 24.8 months (range 7 months
– 8 years). Reversal of hair loss occurred in all cases following
cessation of treatment.
Conclusions: These findings suggest hair loss is twice as common in
African Americans compared to Caucasians exposed to MTX. There
was no effect of gender on risk of hair loss. Alopecia may develop
on any dose of the drug, and is reversible on cessation of therapy.
This information may be useful to clinicians when considering this
medication for treatment, and in counseling patients on risk profile.
Commercial Relationships: Sarah Escott, None; Julia Malalis,
None; Yosuke Harada, None; David Mai, None; Debra A.
Goldstein, None
Program Number: 3433 Poster Board Number: C0244
Presentation Time: 11:00 AM–12:45 PM
The effect of tumor formation and treatment on retinal
dysfunction in mouse models of Neurofibromatosis type 1 (NF1)associated optic glioma
Joseph Toonen, Aparna Kaul, Scott Gianino, David Gutmann.
Neurology, Washington University in St. Louis, Saint Louis, MO.
Purpose: Neurofibromatosis type 1 (NF1) is an autosomal
dominant cancer predisposition syndrome, affecting 1 in 2,500
people worldwide. Approximately 15-20% of children with NF1
develop low-grade gliomas of the optic pathway (OPG), leading to
visual imparment in 30-50% of affected children. While bi-allelic
inactivation of the NF1 gene is the most common cause of OPG, a
small group of patients with OPGs have additional genetic alterations
resulting in greater tumor volume and proliferation. Our aim was to
determine the impact of optic glioma formation on retinal dysfunction
utilizing genetically engineered mouse (GEM) models. Additionally,
we tested the outcome of tumor treatment on retinal function.
Methods: Mice used in the study were Nf1 flox/mut; GFAP-Cre (FMC)
Nf1flox/mut; f-BRAF; GFAP-Cre mice (FMBC) and Nf1flox/mut; Pten flox/
wt
; GFAP-Cre (FMPC) mice. Mice were perfused at 3 months of age
and processed for paraffin embedding. Immunohistochemistry was
performed on paraffin sections using antibodies Brn3a and SMI-32.
TUNEL staining was also performed. 3 month old FMC mice were
treated for 4 weeks with PD901, BKM120, or vehicle controls via
oral gavage (n=6).
Results: FMC, FMBC, and FMPC retinae had a 6-fold (±5.6%
–6.9%) increase in RGC death and a 31-45% reduction in RGCs
compared to controls. While all of the Nf1-OPG mouse strains had
decreased RNFL thickness, FMPC mice had 3-fold decreased RNFL
thickness in comparison with 2-fold reductions observed in FMC
and FMBC mice. We found an inverse correlation (R2 = 0.803)
between RGC death and RNFL thickness among all 3 mouse models.
Following treatment with either BKM120 or PD901 which each
reduced FMC-OPG volume to wild-type levels, we found 1.9-fold
and 4.9 -fold decreases in TUNEL+ cells in BKM120-treated and
PD901-treated mice, respectively. Similarly, BKM120-treated and
PD901-treated mice retained 40% (± 14%-57%) and 50% (± 34%60%) more RGCs than controls. Lastly, RNFL thickness following
BKM120 and PD901 treatment was increased by 1.6-fold and 2.3fold, respectively, compared to vehicle-treated FMC mice.
Conclusions: The results from this study show that a larger, more
proliferative OPG leads to increased retinal dysfunction. Likewise,
using biological targets that inhibit tumor growth reversed these
effects. These findings have implications for assessing visual
outcome in children with NF1-OPG.
Commercial Relationships: Joseph Toonen, None; Aparna Kaul,
None; Scott Gianino, None; David Gutmann, None
Support: NIH Grant NS007205
Program Number: 3434 Poster Board Number: C0245
Presentation Time: 11:00 AM–12:45 PM
The utility and technique for infraorbital and supraorbital nerve
biopsies
Valerie Chen, Hee Joon Kim, Brent Hayek, Hans E. Grossniklaus,
Ted H. Wojno. Ophthalmology, Emory University, Atlanta, GA.
Purpose: Enlargement of the infraorbital and supraorbital nerves
can most commonly indicate perineural invasion of a malignancy
or benign conditions such as idiopathic orbital pseudotumor. The
purpose of this study is to review the role of supraorbital and
infraorbital nerve biopsies in patients presenting with radiographic
enlargement of these nerves and to elucidate the surgical technique
involved in obtaining these biopsies.
Methods: A 5-year chart review (2009-2014) was performed at The
Emory Clinics. Patients with radiographic confirmation of enlarged
supraorbital and/or infraorbital nerves that underwent a biopsy were
included in the review. Charts were reviewed for the following data:
patient demographics and history, clinical symptoms and findings,
radiographic findings, surgical method, and treatment.
Results: A total of 6 patients met the inclusion criteria. Five patients
(83%) were female and 1 (17%) was male with the average age being
72.3, ranging from 36-90 years. Five of the 6 patients had a history of
a cutaneous malignancy. All 6 patients presented with either diplopia
and/or dysesthesias on the affected side. Clinical examination
confirmed decreased V1 and/or V2 sensation for 5 of the 6 patients.
Imaging revealed enlargement of V1, V2, and/or V3 for all of the
patients.
Supraorbital nerve biopsies were performed for 2 patients via a
sub-brow incision onto the superior orbital rim with reflection of
the periosteum that revealed the nerve. One confirmed squamous
cell carcinoma and one confirmed mucoepidermoid carcinoma.
The remaining 4 patients underwent infraorbital nerve biopsies
via a transconjunctival fornix-based orbitotomy with subperiosteal
dissection along the orbital floor followed by unroofing of the
infraorbital canal. The biopsy confirmed squamous cell carcinoma for
the 3 patients with a history of cutaneous squamous cell carcinoma.
One patient confirmed idiopathic orbital inflammation. Five of the 6
patients had initiation of treatment in less than a month. One patient
was lost to follow-up.
Conclusions: For patients presenting with enlarged supraorbital and/
or infraorbital nerves, biopsies of these nerves can rapidly confirm
the underlying condition that can facilitate early treatment. A subbrow approach offers a direct access to the supraorbital nerve while a
transconjunctival fornix-based anterior orbitotomy with unroofing of
the infraorbital canal allow access to the infraorbital nerve.
Commercial Relationships: Valerie Chen, None; Hee Joon Kim,
None; Brent Hayek, None; Hans E. Grossniklaus, None; Ted H.
Wojno, None
Program Number: 3435 Poster Board Number: C0246
Presentation Time: 11:00 AM–12:45 PM
Prevalence of intraocular tumors detected by ultrasonography in
eyes with opaque media
Saranya C. Balasubramaniam, Sophie J. Bakri. Mayo Clinic,
Richmond, VA.
Purpose: To study the prevalence of intraocular tumors detected by
screening ultrasonography in eyes with opaque media.
Methods: A retrospective review of ultrasounds done in 119 eyes
that had opaque media and a diagnosis of blindness or phthisis
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
between January 1, 1994 and December 31, 2013. Patients were
excluded if ultrasound was ordered in eyes with clear media or when
ultrasound was used for acute etiologies of vision loss including
endophthalmitis, retinal detachment, or acute vitreous hemorrhage.
Data were extracted on visual acuity, intraocular pressure, presence
or absence of ocular pain, etiology of opaque media, number of
ultrasounds received during study time period, and ultrasound
findings. Follow up was defined as the time range for which an eye
was followed from initial documentation of opaque media to last visit
with opaque media. In addition, ultrasounds obtained for screening
prior to evisceration or enucleation was noted along with pathology
findings.
Results: A total of 173 ultrasounds corresponding to 119 eyes
were reviewed. No intraocular tumors were detected. Mean age of
patients was 59 years. Visual acuity was hand motions or worse
in 89 eyes (74.8%), elevated intraocular pressure was found in 23
eyes (19.3%) and ocular pain was noted in 30 eyes (25.4%). 50
eyes had less than twelve months of follow up. The remaining 69
eyes with opaque media (58%) had at least one year follow up. The
mean follow was 65 months (median 56 months; range 12-129). Of
these, 2 eyes (2.9%) had an annual ultrasound, 43 eyes (62%), had
an ultrasound done every 13 months- 5 years, and 19 eyes (27.5%)
had an ultrasound every 61 months to 10 years. In addition, 16 eyes
with opaque media for at least 6 years only received a screening
ultrasonography at presentation (11 eyes had 6-8 years follow up;
5 eyes had more than 8 years of follow up). 6 eyes had screening
ultrasonography prior to evisceration or enucleation with pathology
clear of intraocular tumors.
Conclusions: No prior studies have been done to examine the utility
of interval screening ultrasonography in blind eyes with opaque
media. In this series of eyes with opaque media, no intraocular
tumors were detected by screening ultrasonography. Further long
term study is necessary to determine the utility of consecutive
ultrasonography in eyes with opaque media and the appropriate
interval of screening ultrasonography.
Commercial Relationships: Saranya C. Balasubramaniam, None;
Sophie J. Bakri, None
Support: Research to Prevent Blindness
427 Tumors - Uveal melanoma
Wednesday, May 06, 2015 11:00 AM–12:45 PM
1AB Mile High Blrm Paper Session
Program #/Board # Range: 4331–4337
Organizing Section: Anatomy and Pathology/Oncology
Program Number: 4331
Presentation Time: 11:00 AM–11:15 AM
HIF-1α upregulates ANGPTL4 to promote angiogenesis in Uveal
Melanoma
Ke Hu1, 2, Savalan Babapoor-Farrokhran1, Murilo W. Rodrigues1,
Brooks Puchner1, Monika Deshpande1, Laura Asnaghi1, Charles
Eberhart1, Silvia Montaner1, Akrit Sodhi1. 1Wilmer Eye Institute,
Johns Hopkins School of Medicine., Baltimore, MD; 2The First
Affiliated Hospital of Chongqing Medical University, Chongqing,
China.
Purpose: The transcriptional response promoted by hypoxiainducible factor (HIF)-1α has been associated with angiogenesis and
metastatic spread in uveal melanoma (UM). Expression of one HIF-1
target gene, vascular endothelial growth factor (VEGF) correlates
with tumor vascularity in UM, as well as other tumors. However,
treatment of patients with therapies targeting VEGF has not proven
sufficient alone to prevent UM growth or spread. Here we set out to
evaluate the potential role of a novel HIF-regulated gene product,
angiopoietin-like 4 (ANGPTL4), in the promotion of angiogenesis in
UM.
Methods: UM cell lines were examined for expression of HIF1α, VEGF, and ANGPTL4. Expression of HIF-1α, VEGF, and
ANGPTL4 were further assessed by immunohistochemical analysis
in UM tissue and quantitated using a UM tissue array. Expression of
ANGPTL4 was also examined in vitreous biopsies from patients with
uveal melanoma. The role of ANGPTL4 in angiogenesis in UM was
assessed using endothelial cell (EC) tubule formation (TF) assays.
Inhibition of HIF-1α, VEGF, and ANGPTL4 was performed using
RNAi.
Results: Expression of HIF-1α was detected in all UM cell lines
tested. Hypoxic stabilization of HIF-1α resulted in the promotion
of TF in treated ECs in vitro; this affect was inhibited by blocking
HIF-1α translation, but only partially inhibited by blocking VEGF
expression, implicating additional HIF-regulated genes in the
promotion of angiogenesis in UM. We demonstrate that HIF-1α
stabilization results in ANGPTL4 mRNA and protein expression
in UM cell lines. These results were corroborated in tissue samples
from patients with UM. Using a UM tissue array, we further observed
expression of HIF-1α in a majority of tumors in the array. Expression
of either ANGPTL4 or VEGF was detected in almost all (99%) of the
tumors in the UM tissue array. ANGPTL4 expression was increased
more than 50 fold in vitreous biopsies from UM patients at levels
that were sufficient to promote angiogenesis in vitro. RNAi targeting
ANGPTL4 inhibited the promotion of TF induced by UM cell lines;
this effect was additive to RNAi targeting VEGF.
Conclusions: We propose that therapies targeting both VEGF and
ANGPTL4 will be necessary to effectively inhibit tumor-induced
angiogenesis in patients with UM.
Commercial Relationships: Ke Hu, None; Savalan BabapoorFarrokhran, None; Murilo W. Rodrigues, None; Brooks Puchner,
None; Monika Deshpande, None; Laura Asnaghi, None; Charles
Eberhart, None; Silvia Montaner, None; Akrit Sodhi, None
Support: The K08 grant: K08EY021189, The RPB Career
Development Award: 109622
Program Number: 4332
Presentation Time: 11:15 AM–11:30 AM
Modulation of Cytoskeletal Associated Proteins Regulates Cell
Death and Survival in Uveal Melanoma
Matthew W. Wilson1, 2, Ryan P. Lee1, Sumana R. Chintalapudi1,
Bradley T. Gao1, Justin B. Lendermon1, Nabil Saleh1, Anderson H.
Webb1, Hans E. Grossniklaus4, Vanessa M. Morales1, 3. 1Ophthal/
Hamilton Eye Int, Univ of Tennessee Health Sci Ctr, Memphis,
TN; 2Surgery, St Jude Children’s Research Hospital, Memphis,
TN; 3Microbiology, Immunology and Biochemistry, University of
Tennessee Health Science Center, Memphis, TN; 4Emory Eye Center,
Eye Univeristy, Atlanta, GA.
Purpose: Purpose: Approximately 50% of Uveal Melanoma (UM)
patients will be diagnosed with liver metastases within 5-years
of diagnosis. Micro-metastases are present in the liver as early as
2-years before diagnosis of the intraocular malignancy. Recent
work from our laboratory suggests that inhibiting the upregulation
of cytoskeletal signaling could offer potential therapeutic targets
for metastasis control. In this study we elucidate the mechanism(s)
involved.
Methods: Methods: We performed gene transcription, flow
cytometry as well as cellular and molecular biology analyses after
pharmacological modulation of paxillin Y118 using the paxillin
inhibitor 6-B345TTQ. The effects of paxillin inhibition (pY118) were
measured on pAKT, Protein Kinase C-delta (PKC-δ), pro-apoptotic
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
molecules expression, actin polymerization and cellular proliferation
in UM cell lines and immunohistochemical (IHC) analyses in
normal and with UM. We performed cutting edge Nano3D (Nano 3D
Biosciences Inc, Houston, TX) technology to investigate changes in
cellular migration and proliferation after paxillin inhibition.
Results: Results: We observed an increase in the alpha-4 subunit
of VLA-4 and paxillin both in the UM cell lines by flow cytometry
analysis (p<0.05) and in UM eyes compared to normal eyes by IHC.
Targeting of paxillin through inhibition of pY118 reduced cellular
proliferation in primary, not metastatic, UM. Mechanistically, we
observed a significant reduction in pAKT signaling in metastatic
UM (p<0.05) by Western blot analysis and phospho-PKC-δ in the
metastatic UM cell lines by flow cytometry. IHC analysis in human
eyes revealed differences in PKC-δ expression in normal and UM
eyes.
Conclusions: Conclusions: Our in vitro results identified paxillin as
a potential therapeutic target to arrest UM cellular proliferation and
migration. Inhibition of paxillin preferentially affected primary UM
cells compared to metastatic cell lines through pAKT and PKC-δ and
may represent a potential targeted therapy for micrometastases.
Commercial Relationships: Matthew W. Wilson, None; Ryan P.
Lee, None; Sumana R. Chintalapudi, None; Bradley T. Gao, None;
Justin B. Lendermon, None; Nabil Saleh, None; Anderson H.
Webb, None; Hans E. Grossniklaus, None; Vanessa M. Morales,
None
Support: Unrestricted Grant from Research to Prevent Blindness,
Inc. New York, New York
Program Number: 4333
Presentation Time: 11:30 AM–11:45 AM
FNAB of Uveal Melanoma : Outcomes and Complications
Arun D. Singh1, Carlos A. Medina Mendez2, Nakul Singh2, Mary
E. Aronow3, Hassan A. Aziz1, Charles V. Biscotti4. 1Ophthalmic
Oncology, Cole Eye Institute, Cleveland, OH; 2Case Western Reserve
University, Cleveland, OH; 3WIlmer Eye Institute, Baltimore, MD;
4
Anatomy PAthology, Cleveland Clinic, Cleveland, OH.
Purpose: To report outcomes and complications of diagnostic fineneedle aspiration biopsy (FNAB) of uveal melanoma.
Methods: Prospective interventional series of 150 consecutive
patients with uveal melanoma treated at the Cleveland Clinic Cole
Eye Institute between May 2009 and August 2012. The FNAB
approach (trans corneal [TCO], trans scleral [TSC], and trans vitreal
[TSV] were primarily determined by the location of the tumor. The
FNAB was performed using 25 Gauge needle using previously
published technique. All aspirated material was flushed into in
Cytolyt® solution for ThinPrep® processing. The diagnosis of
uveal melanoma was based upon characteristic cellular features. The
cytological reporting was divided into 4 conventional categories:
1. Unsatisfactory for interpretation, 2. Negative for melanoma,
3. Atypical cells (not diagnostic or consistent with melanoma)
and 4. Positive for melanoma. Patients were evaluated 1-4 weeks
postoperatively, then every 3 months for the first year, followed by
every 6 months thereafter. Data were analyzed using STATA version
11.
Results: FNAB was obtained via TCO (8), TSC (71), and TVT
(64) approach and impression smear in 7 cases. Diagnostic yield
was 92% (Positive for melanoma 122, Atypical cells : consistent
with melanoma 9). False negative results were oserved in 8%
(Unsatisfactory 7, Negative for melanoma 3, Atypical cells : not
diagnostic 2). Diagnostic yield was significantly correlated to biopsy
approach (TCO 100%, TSC 96%, 86%; p = 0.029, Fisher’s exact
test) and tumor size (basal diameter >5.0 mm; height >2.5 mm).
Visual acuity (<20/40 and > 20/400) at baseline and at 3 months
was recorded in 68%,13% and 57%, 22% respectively). Visually
significant complications such as persistent hemorrhage (sub retinal
hemorrhage or vitreous) requiring surgical intervention (1%) and
rhegmatogenous retinal detachment (1%) were rare. Endophthalmitis,
hypotony, and detectable needle tract seeding were not observed.
Conclusions: FNAB for uveal melanoma with 25-gauge needle is a
safe procedure that can yield diagnostic samples in more than 90%
of cases. Possibility of negative diagnostic FNAB yield should be
considered when counseling patients with small tumors.
Commercial Relationships: Arun D. Singh, None; Carlos A.
Medina Mendez, None; Nakul Singh, None; Mary E. Aronow,
None; Hassan A. Aziz, None; Charles V. Biscotti, None
Support: Falk Trust; Supported in part by the Cole Eye Institute,
Research to Prevent Blindness Unrestricted Grant.
Program Number: 4334
Presentation Time: 11:45 AM–12:00 PM
Tumor diameter contributes prognostic information that
enhances the accuracy of gene expression profile molecular
classification in uveal melanoma
Scott Walter1, Daniel L. Chao2, Joyce C. Schiffman1, William J.
Feuer1, J. William Harbour1, 3. 1Department of Ophthalmology,
Bascom Palmer Eye Institute, University of Miami, Miami, FL;
2
Department of Ophthalmology, UCSF School of Medicine, San
Francisco, CA; 3Sylvester Comprehensive Cancer Center, University
of Miami Miller School of Medicine, Miami, FL.
Purpose: Molecular classification using gene expression profiling
(GEP) is currently the most accurate single predictor of mortality
from uveal melanoma. The purpose of this study was to determine
whether clinical or cytopathologic features may provide independent
prognostic value in assessing mortality risk.
Methods: 336 consecutive cases of uveal melanoma were analyzed
using Kaplan-Meier survival analysis and Cox regression. The
primary outcome measure was 5-year all cause mortality. Dependent
variables included GEP molecular classification (class 1 vs. 2),
patient age and sex, tumor thickness and basal diameter, ciliary body
involvement and cell type.
Results: After controlling for GEP molecular classification,
no clinicopathologic features provided independent prognostic
information except basal tumor diameter (p=0.002, Cox regression).
For both class 1 and class 2 tumors, the best dichotomous cutoff
for basal tumor diameter was <12mm (small) vs. ≥12mm (large).
The risk of mortality relative to small class 1 tumors was 8.6 for
large class 1 tumors, 9.1 for small class 2 tumors, and 78.1 for large
class 2 tumors (all p<0.05). Similar results were demonstrated for
melanoma-specific mortality. The model-based predicted 5-year
survival was 98% for small class 1 tumors, 85-87% for large class 1
and small class 2 tumors, and 16% for large class 2 tumors.
Conclusions: For both class 1 and class 2 uveal melanomas, basal
tumor diameter was the only clinicopathologic factor that provided
additional prognostic information that enhanced the accuracy of GEP
molecular classification. Basal tumor diameter, in combination with
GEP, may be a useful metric for risk stratification, especially for
tumors less than 12mm in diameter.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
However, because receiving both Class 1 and Class 2 test results
are possible in the setting of a non-melanoma malignancy as we
have demonstrated, we recommend that cytopathology and/or other
melanoma-specific testing, (e.g. fluorescence in-situ hybradization
for monosomy 3, GNAQ mutation analysis) also be performed.
DecisionDx-UM GEP testing alone should be interpreted with
caution in choroidal lesions which are not melanomas.
Commercial Relationships: Michael A. Klufas, None; Sujit Itty,
None; Colin A. McCannel, None; Ben J. Glasgow, None; Christian
Moreno, None; Tara A. McCannel, None
Figure 1: Reverse Kaplan-Meier plot of all cause mortality over time
for patients with class 1 (A) and class 2 (B) uveal melanoma by gene
expression profiling. Results are stratified according to basal tumor
diameter <12mm (blue line) or ≥12mm (green line). Vertical steps
represent mortality events. Vertical notches represent patients alive at
last follow-up.
Commercial Relationships: Scott Walter, None; Daniel L. Chao,
None; Joyce C. Schiffman, None; William J. Feuer, None; J.
William Harbour, Castle Biosciences (C), Castle Biosciences (P)
Support: R01 CA125970, Melanoma Research Alliance,
Melanoma Research Foundation, and Dr. Mark J. Daily Fund (to
JWH), and NIH Core Grant P30EY014801, Research to Prevent
Blindness Unrestricted Grant, and Department of Defense Grant
#W81XWH-09-1-0675 (to Bascom Palmer Eye Institute). Funding
organizations had no role in the design or conduct of this research.
Program Number: 4335
Presentation Time: 12:00 PM–12:15 PM
Variable Results for Uveal Melanoma Specific Gene Expression
Profile Prognostic Test in Choroidal Metastasis
Michael A. Klufas, Sujit Itty, Colin A. McCannel, Ben J. Glasgow,
Christian Moreno, Tara A. McCannel. Ophthalmology, Jules Stein
Eye Institute/UCLA, Los Angeles, CA.
Purpose: DecisionDx-UM (Castle Bioscience, Phoenix, AZ) gene
expression profiling (GEP) of uveal melanoma tissue obtained by fine
needle aspiration biopsy is a frequently used prognostic test for the
identification of patients at high risk for uveal melanoma metastasis.
Benign melanocytic lesions are believed to provide a “Class 1”,
or low-risk for metastasis designation. However, cytopathology
is not typically obtained to confirm the actual lesion diagnosis. At
some centers, performing GEP testing on indeterminate choroidal
lesions may be used to direct a decision for treatment. We report our
experience with uveal melanoma specific GEP testing on a series of
choroidal metastatic tumors confirmed by cytopathology.
Methods: Retrospective review of all cytopathology and
DecisionDx-UM GEP reports between 2012 to 2014 from
intraoperative fine needle aspiration biopsy (FNAB) of choroidal
tumors undergoing brachytherapy at our center. Patient medical
records including ancillary testing (fundus photography, fluorescein
angiography, B-scan ultrasonography) were also reviewed.
Results: Four patients (four eyes) were identified to have
cytopathology consistent with a non-melanoma primary. All patients
presented with a unilateral, single choroidal lesion. 50% of the
patients were male. Mean patient age was 74.75 years (median 72.5
years, range 67-87 years). Two patients (50%) had a history of treated
systemic cancer with no previous evidence of metastatic disease (one
prostate, one lung). For the two patients with no history of systemic
cancer, further systemic work-up revealed primary lung cancer in
both cases. A GEP result was provided for each patient, three were
Class 1A, one was Class 2.
Conclusions: DecisionDx-UM GEP may be a helpful test to
provide molecular prognostication in patients with uveal melanoma.
Program Number: 4336
Presentation Time: 12:15 PM–12:30 PM
Correlation of Iris Color with Gene Expression Profile in 146
consecutive Fine Needle Biopsies of Uveal Melanoma
Peter G. Hovland1, Marc Zafferani2. 1Ophthalmology, Colorado
Retina Associates, Denver, CO; 2College of Osteopathic Medicine,
Rocky Vista University, Parker, CO.
Purpose: It has been previously demonstrated that light iris color
is a risk factor for the development of uveal melanoma. This study
is estimates the correlation of iris color to gene expression profile
(GEP), which is an independent measure of likelihood of metastasis.
Methods: A retropective chart review of a consecutive series of
patients treated for uveal melanoma was performed. Patients were
selected for study if they had been treated for uveal melanoma
with either brachytherapy or enucleation, and if they had GEP
classification (Castle BioSciences) into Class 1A, Class1B, or
Class 2. Iris color classification was derived from drivers license
information as administered by the Department of Motor Vehicles.
Iris color was classified into one of four groups: Brown, Blue, Hazel,
or Green. Complete data was available for 146 patients. Differences
in GEP distributions in the separate classes of iris color was analyzed
with students t-test and chi-squared analysis.
Results: The population of patients was predominantly Caucasian
(98%). The incidence of iris colors in the 146 patients with uveal
melanoma was: Blue 66/146 (45%), Hazel 26/146 (18%), Green
14/146 (10%), Brown 40/146 (27%). Blue iris color had the
following percentage distribution of GEP results: Class 1A 58%,
Class 1B 14%, Class 2 29%. Brown iris color had the following
percentage distribution of GEP results: Class 1A 35%, Class 1B 33%,
Class 2 33%. Differences in the distributions of GEP class in different
iris colors was found to be not statistically significant.
Conclusions: This study is consistent with previous studies which
show light iris color is a risk factor for uveal melanoma in Caucasians
in that light iris color is predominant in this patient group, with brown
iris color represented in a minority of 25%. The separate classes of
iris color, however, do not appear to be associated with significant
differences in GEP result. This study supports the hypothesis that iris
color does not confer clinically significant information regarding risk
of developing metastatic disease in patients with uveal melanoma.
The limitations of this study are it’s relatively small size, and the
dependence on the unknown relaibility of iris color determination as
present in the database of the department of motor vehicles.
Commercial Relationships: Peter G. Hovland, None; Marc
Zafferani, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 4337
Presentation Time: 12:30 PM–12:45 PM
Novel combinatorial treatment option for metastatic uveal
melanoma
Shahar Frenkel1, Dudi Shneor1, 2, Alik Honigman2, Jacob Pe’er1.
1
Ophthalmology, Hadassah-Hebrew Univ Med Ctr, Mevaseret Zion,
Israel; 2Biochemistry and Molecular Biology, IMRIC, The Hebrew
University-Hadassah Medical School, Jerusalem, Israel.
Purpose: To date, chemotherapy for metastatic uveal melanoma
(mUM) is limited to dacarbazine (DTIC) and fotemustine. We tested
the effect of the common chemotherapeutic drug doxorubicin (DOX)
on cell mortality in order to expand the chemotherapeutic arsenal for
mUM.
Methods: We examined the effect of both DITC and DOX in five
different uveal melanoma cell lines – originating from metastases
(OMM1, OMM2.3 and OMM2.5) and from primary tumors (92.1
and MEL270) and performed dose response tests using both drugs.
Based on our previous results, we hypothesized that combining DOX
and knockdown of CREB will increase cellular death. To test our
hypothesis, we infected cells with replicative competent retroviruses
(RCR) expressing shRNA against CREB to create a continuous
infective knockdown of CREB.
Results: Both chemotherapeutic drugs induced cell death in a dose
dependent manner. Knockdown of CREB in these cells increased the
effect of DOX on cell mortality.
Conclusions: Treatment with DOX is at least as efficient and in some
cases even more efficient than DTIC in inducing UM cell mortality
in vitro. Moreover, the ability of combining CREB knockdown and
DOX treatment to achieve the same amount of cell death in lower
concentrations of DOX may result in fewer side effects from DOX.
This combination is a possible new treatment for metastatic uveal
melanoma.
Commercial Relationships: Shahar Frenkel, None; Dudi Shneor,
None; Alik Honigman, None; Jacob Pe’er, None
Support: Israel Science Foundation (ISF) for physician-researcher
444 Orbit, whole globe, components
Wednesday, May 06, 2015 11:00 AM–12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 4719–4732/D0164–D0177
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Clinical/Epidemiologic Research,
Eye Movements/Strabismus/Amblyopia/Neuro-Ophthalmology,
Glaucoma
Program Number: 4719 Poster Board Number: D0164
Presentation Time: 11:00 AM–12:45 PM
Isolation Of Several Choroidal Cell Types And Retinal Pigmented
Epithelial Cells From A Single Rabbit Eye
Stephanie Proulx2, 1, Rémi Parenteau Bareil2, Olivier RochetteDrouin2, Solange Landreville2, 1. 1Ophthalmology, Laval University,
Quebec, QC, Canada; 2Centre de recherche du Centre hospitalier
universitaire (CHU) de Québec, axe médecine régénératrice, Quebec,
QC, Canada.
Purpose: Melanocytes, fibroblasts and endothelial vascular (EV)
cells from the choroid as well as retinal pigment epithelial (RPE)
cells are required to generate functional tissue-engineered choroidal
substitutes. The purpose of this study was to develop an isolation
protocol that enables the establishment of pure cultures of choroidal
and RPE cells.
Methods: Enucleated rabbit eyes were cut in a half just below the
ora serrata. The retina and the optic nerve were then carefully
removed. A 20 min dispase treatment (2.5%) and/or gentle pipetting
were used to separate small sheets of RPE that were then put in
culture. After a complete removal of the RPE, the choroid was peeled
out of the sclera and incubated overnight in collagenase H (0,125U).
The resulting cell suspension was centrifuged and incubated 10
min in trypsin-EDTA (0,05%-0,01%). The cell suspension was then
filtered with a cell strainer. Dynabeads and anti-CD31 antibody
were used to sort choroidal EV cells from the cell suspension. The
residual mixture was equally plated in two selective growth media:
a non-restrictive medium allowing fibroblast proliferation and a
melanocyte growth medium containing G418 selection agent for
eradication of contaminating fibroblasts. Cells were seeded onto glass
coverslips and purity was assessed by phase contrast microscopy and
immunofluorescence (K8/18, CD31, HMB45, vimentin).
Results: The method described herein allowed for the isolation of
four ocular cell types. RPE cells expressing K8/18 were successfully
isolated. EV cells positive for CD31 were expended for at least three
passages without any loss of phenotype. Pure cultures of HMB45positive melanocytes were obtained after a two-week culture period
in G418 selective medium. Finally, cultures of fibroblasts positive for
vimentin and negative for the other markers were established after
the third passage. At low passages (P1), less than 1% of melanocytes
were present in the fibroblast cultures. Melanocyte contamination
was absent at higher passages (P3 and over).
Conclusions: Our results show the feasibility of obtaining pure
cultures of four ocular cell types required to develop functional
tissue-engineered choroidal substitutes. These substitutes may be
used as allogeneic sub-retinal grafts in preclinical studies for the
treatment of retinal diseases.
Commercial Relationships: Stephanie Proulx, None; Rémi
Parenteau Bareil, None; Olivier Rochette-Drouin, None; Solange
Landreville, None
Support: Foundation Fighting Blindness, FRQ-S, CFI
Program Number: 4720 Poster Board Number: D0165
Presentation Time: 11:00 AM–12:45 PM
Choroidal mast cells in retinal pathology: a potential target for
intervention
Francine F. Behar-Cohen1, 2, elodie bousquet2, Min Zhao2, Brigitte
Goldenberg2, Lorena Vieira2, Marie-Christine Naud2, Ciara Bergin1,
Yvonne De Kozak2. 1Jules-Gonin Eye Hospital, Lausanne, Paris,
France; 2INSERM U1138, Centre de Recherche des Coreliers, Paris,
France.
Purpose: We previously showed that mast cells play a role in the
initiation of acute ocular inflammation but the exact consequences
of mast cell degranulation on ocular pathology have not been
fully characterized. The aim of this study was to analyse in depth
the kinetics of events following local and acute ocular mast cells
degranulation.
Methods: Adult female Lewis rats (6–8 weeks old, Janvier, Le
Genest-Saint-Isle, France) were used in this study. Animals were
housed in a 12-hrs light and 12-hrs dark cycle and fed water and
dried ration ad libitum. Experimental procedures were submitted
and approved by the ethic committee of Paris Descartes University
(number: Ce5/2012/122).
We induced mast cells depletion of their inflammatory mediators by
the local unilateral sub-conjunctival injection of 48/80 drug. Clinical
examination (slit-lamp, SD-OCT) were preformed and animals
were sacrificed at different time points for pathology and cytokines
analysis.
Results: Initial degranulation of mast cells was observed in the
choroid 15 min after the injection of 48/80, with degranulation
continuing to increase up to 3-6 hrs after the injection. The signs
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
of anterior segment clinical inflammation paralleled mast cell
degranulation. Posterior segment imaging using optical coherence
tomography showed increased choroidal thickness and serous retinal
detachments, which were confirmed by histological analysis. The
infiltration of polymorphonuclear cells was associated with increased
ocular media levels of TNF-a at 1 and 3 hrs, followed by CXCL1, IL6, IL-5, CCL-2 at 6 and 24 hrs, and IL-1b at 24 hrs. Analysis of levels
of VEGF and IL-18 at all time intervals showed an opposite evolution
of VEGF as compared to IL-18 levels.
Conclusions: These findings suggest that the local degranulation
of ocular mast cells provokes acute ocular inflammation, increased
choroidal vascular permeability and serous retinal detachments.
The involvement of mast cells in retinal diseases, particularly when
associated with serous detachments, should be investigated and the
pharmacological inhibition of mast cells degranulation considered as
a potential intervention target.
Commercial Relationships: Francine F. Behar-Cohen, None;
elodie bousquet, None; Min Zhao, None; Brigitte Goldenberg,
None; Lorena Vieira, None; Marie-Christine Naud, None; Ciara
Bergin, None; Yvonne De Kozak, None
Program Number: 4721 Poster Board Number: D0166
Presentation Time: 11:00 AM–12:45 PM
N-cadherin regulates morphogenesis and secretion of the ciliary
body in the mouse eye
Yi Zhou1, 2, Christopher P. Tanzie1, 2, Ting Xie1, 2. 1Xie Lab, Stowers
Inst for Medical Research, Kansas City, MO; 2Anatomy and Cell
Biology, University of Kansas Medical Center, Kansas City, KS.
Purpose: High intraocular pressure (IOP) is often a risk factor for
glaucoma. The ciliary body (CB) is an apically adherent epithelial
structure responsible for aqueous humor secretion and thus IOP
modulation. However, the regulation of its development and secretion
remains poorly understood. This study reveals an important role of
N-cadherin in the regulation of CB morphogenesis and a surprising
role in the regulation of CB secretion.
Methods: Trp1-cre was used to conditionally delete N-cadherin
(Ncad) in both CB epithelial layers. Mutant phenotypes
were characterized by standard procedures, including
immunohistochemistry, mRNA in-situ hybridization, BrdU
labeling, TUNEL labeling and Western blotting. Additionally, coimmunoprecipitation was performed to verify protein interactions. To
further support our hypothesis, injection of Par3 shRNA lentiviruses
into developing CBs was utilized to demonstrate the role of the cell
polarity in CB morphogenesis.
Results: Ncad mutant CBs show defective morphogenesis. Those
mutant CBs maintain normal expression of the CB markers, Msx1,
Otx1, Pax6 and Notch2, suggesting that N-cadherin is dispensable
for CB specification. Our BrdU labeling results show that cell
proliferation is drastically reduced in the Ncad mutant CBs. In
addition, the cellular localization of the Par3/Par6/aPKC polarity
complex is compromised in the Ncad mutant CBs, and shRNAmediated knockdown of Par3 in the developing CB also disrupts
its morphogenesis, suggesting that one of the N-cadherin functions
in the regulation of CB morphogenesis is to maintain the epithelial
polarity. Finally, secretion of Collagen IX is also reduced in the
Ncad mutant CBs. Our co-IP results show that N-cadherin physically
interacts with gap junction protein Connexin43, which is essential for
aqueous humor secretion, indicating that N-cadherin regulates CB
secretion by modulating the function of Connexin43.
Conclusions: Our findings in this study have revealed two important
roles of N-cadherin in the CB: regulation of CB morphogenesis and
secretion. N-cadherin regulates CB morphogenesis at least in part by
stabilizing the epithelial polarity and maintaining cell proliferation.
N-cadherin also regulates aqueous humor secretion by directly
modulating the function of the gap junction protein Connexin43.
Therefore, this study has provided important insights into how CB
morphogenesis and secretion are regulated at the molecular level.
Commercial Relationships: Yi Zhou, None; Christopher P.
Tanzie, None; Ting Xie, None
Program Number: 4722 Poster Board Number: D0167
Presentation Time: 11:00 AM–12:45 PM
COMPARISON OF BRUCH MEMBRAN OPENING
BETWEEN HEALTHY SUBJECTS, GLAUCOMATOUS EYES
ET CENTRAL VEIN OCCLUSION (CRVO) EYES
Arnaud GEORGE. Ophtalmologie, Clinique Sainte Jeanne D’arc,
Saint-Brieuc, France.
Purpose: To determine if there is any correlation between Bruch
Membran Opening Area and CRVO, and if a small BMO area is a
risk factor for CRVO.
To our knowledge, this is the first study using the Heidelberg
Spectralis’ new algorithm.
Methods: We compare the Bruch Membran Opening Area, as
measured by the new algorithm of the Spectralis (R) Optical
Coherence Tomography in 20 patients with Central Retinal Vein
Occlusion, their fellow eyes, 20 glaucomatous eyes and 20 normal
eyes, comparable in age, gender and sex.
Results: The first results show no correlation between BMO area and
CRVO, as hypothesized by Hayreh.
Conclusions: In this small study, we found no correlation between
BMO and CRVO.
We need further studies and larger samples to confirm these results.
BMO area sample
BMO area sample
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Arnaud GEORGE, None
Program Number: 4723 Poster Board Number: D0168
Presentation Time: 11:00 AM–12:45 PM
Age related changes in the Korean pediatric human orbit on CT
Choi Woo seok, Hee-Bae Ahn, Sang wook Jin, Woo Jin Jung, Yoon
Hyung Kwon, Won Yeol Ryu, Hyun Wook Shin. Ophthalmology,
DongA medical center, Busan, Korea (the Republic of).
Purpose: The purpose of this study was to determine the
relationships among orbital dimensions, globe diameter and age.
Methods: A retrospective analysis of 136 CT scans of subjects
with no orbital or globe disease was performed and 10 lengh
measurements and 3 angle measurement of various aspects of the
orbit was obtained.
Results: 136 CT scans of 136 korean subjects without identifiable
globe and orbital disease were included in this study. 29 subjects ≥
17 years of age were considered mature adults and grouped together,
while the remaining 107 subjects were grouped according to age. All
orbital length measurements except globe protrusion increased most
rapidly over the first 12 to 24 months. Vertical and horizontal orbital
length was reached to 97-98% of their adults value at age 8 years and
highly correlated to linear orbital measurement. Central orbital axis
angle and intraorbital angle in neonate group were found to be greater
than their adults group and decreased rapidly over the first 12 months.
Orbital protrusion continued to increase through the neonate until
adulthood.
Conclusions: The growth of the korean human orbit increased most
rapidly over the first 12 to 24 months and reached to 96-98% of their
adults value at age 8 years. With this attempt to define normal agerelated measured orbital value and orbital changes, this is helpful to
treat the pediatric orbital disease and abnormalies.
Commercial Relationships: Choi Woo seok, None; Hee-Bae Ahn,
None; Sang wook Jin, None; Woo Jin Jung, None; Yoon Hyung
Kwon, None; Won Yeol Ryu, None; Hyun Wook Shin, None
Program Number: 4724 Poster Board Number: D0169
Presentation Time: 11:00 AM–12:45 PM
Changes in Angle of Optic Nerve and Angle of Ocular Orbit with
Increasing Age in Japanese Children
Hideyuki Tsukitome1, Yoshikazu Hatsukawa2, Tomoko Morimitsu2,
Teiji Yagasaki3, Mineo Kondo1. 1Department of Ophthalmology, Mie
University School of Medicine, Tsu, Japan; 2Osaka Medical Center
and Research Institute for Maternal and Child Health, Sakai, Japan;
3
Yagasaki Eye Clinic, Ichinomiya-shi, Japan.
Purpose: To study the changes in the opening angle of the optic
nerve and angle of the ocular orbit with increasing age in normal
Japanese children.
Methods: We studied 147 normal children whose ages ranged from
6 months to 18 years who had undergone computed tomography(CT)
as a diagnostic procedure. Measurements were done on the axial
CT images that included the entire optic nerves of both eyes. The
opening angle of the optic nerve was defined as the angle formed
by the intersection of a line running through the left optic nerve and
a vertical line passing through the center of the nose. The opening
angle of the orbit was defined as the angle formed by the intersection
of a line running tangentially along the deep lateral wall of left
orbit and a vertical line passing through the center of the nose.
The relationships between the age and these opening angles were
analyzed by regression analysis.
Results: The correlation between the age and the opening angle of
the optic nerve was not significant. The opening angle of the orbit
decreased relatively rapidly until about 2-3 years of age, and then
it stabilized. The decrease in the opening angle of the orbit with
increasing age was significant(P<0.001). The relationship between
these two parameters was best fit by a logarithmic regression curve.
Conclusions: Because the Opening angle of the orbit decreased
significantly with increasing age, this factor must be considered when
diagnosing and treating strabismus in children.
Commercial Relationships: Hideyuki Tsukitome, None;
Yoshikazu Hatsukawa, None; Tomoko Morimitsu, None; Teiji
Yagasaki, None; Mineo Kondo, None
Program Number: 4725 Poster Board Number: D0170
Presentation Time: 11:00 AM–12:45 PM
Gene expression profiling of orbital adipose tissue in thyroid
orbitopathy
Jwu Jin Khong1, 2, Lynn Wang3, Gordon Smyth3, Alan A. McNab2,
Thomas Hardy2, Dinesh Selva4, Shiwani Sharma6, Kathryn P.
Burdon5, Ebeling Peter1, 7, Jamie E. Craig6. 1Department of
Medicine, University of Melbourne, Melbourne, VIC, Australia;
2
Orbital, Plastics and Lacrimal Unit, The Royal Victorian Eye and
Ear Hospital, Melbourne, VIC, Australia; 3Bioinformatics Division,
Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC,
Australia; 4Ophthalmology and Visual Sciences, South Australian
Institute of Ophthalmology, Adelaide, SA, Australia; 5Menzies
Institute for Medical Research, Hobart, TAS, Australia; 6Department
of Ophthalmology, Flinders University, Adelaide, SA, Australia;
7
Department of Medicine, Monash University, Melbourne, VIC,
Australia.
Purpose: Thyroid orbitopathy(TO) results from autoimmunity
against orbital tissue. The underlying molecular mechanisms
remain incompletely understood. We aim to determine differentially
expressed genes that may be involved in stimulating orbital
inflammation and fat expansion in TO using microarray gene
expression profiling in a case control study.
Methods: Human orbital adipose samples were obtained during
orbital decompression and upper lid surgeries in patients with
TO. Patients with TO were subclassified as active(n=12) or
inactive(n=21). Normal controls(n=21) were patients without
autoimmune thyroid disease with adipose tissue harvested from
corresponding anatomical locations at the time of unrelated orbital
and lid surgeries.
RNA was extracted then hybridized to Illumina humanHT12
v4 microarray. Expression signals were analyzed using R. Top
differentially expressed genes were compared between active and
inactive TO, and between active TO and normal controls ranked
by fold change, level of expression and adjusted false discovery
rate<0.05. Top ranked and some lower ranked genes of interest
were validated by real time PCR in additional 8 active, 13 inactive
TO and 11 normal controls. Gene set enrichment analysis(GSEA)
was performed. Molecular pathways were analysed using DAVID
bioinformatics.
Results: 721 probes representing 626 annotated genes were
differerentially expressed in active compared to inactive TO.
Defensins(DEFA1, DEFA1B, DEFA3) were overexpressed by
3.05-4.14 fold. TIMD4 was over-expressed by 4.3 fold. CD247,
CD3A, CAMP, SLAM family member 6, GZMA, NKG7 were
upregulated by 1.75-1.95 fold. Several markers for adipogenesis were
overexpressed by 1.91-2.6 fold including SCD, FADS and ELOVL6.
Increased expression of FADS1 and SCD were confirmed in active
TO versus normal controls.
GSEA revealed a significant proportion of genes upregulated in gene
sets tyrosine kinase protein CSK pathway, cytotoxic T lymphocyte, T
cell apoptosis, glycolysis, IL-12, T helper, T cell receptor activation,
caspase cascade, TOB1, cell to cell adhesion signaling and strathmin
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
pathways. Molecular pathway analysis correlated well with GSEA
analysis.
Conclusions: Active TO is marked by up-regulation of genes
involved in cellular, innate immune and inflammatory response
and enhanced orbital adipogenesis.TIMD4, DEFA1, DEFA1B and
DEFA3 may be involved in stimulating immune mediated orbital
inflammation.
Commercial Relationships: Jwu Jin Khong, None; Lynn Wang,
None; Gordon Smyth, None; Alan A. McNab, None; Thomas
Hardy, None; Dinesh Selva, None; Shiwani Sharma, None;
Kathryn P. Burdon, None; Ebeling Peter, None; Jamie E. Craig,
None
Support: Ophthalmic Research Institute of Australia new
investigator grant
Program Number: 4726 Poster Board Number: D0171
Presentation Time: 11:00 AM–12:45 PM
Serum Total IgG and IgG4 Levels in Thyroid Eye Disease
Aileen Sy, Rona Silkiss. Ophthalmology, California Pacific Medical
Center, San Francisco, CA.
Purpose: IgG4-related disease (IgG4-RD) has recently been
recognized as an etiology of previously described cases of orbital
inflammatory disease or pseudotumor. The most common orbital
inflammatory disease is thyroid eye disease (TED). The authors
sought to evaluate total IgG and IgG4 levels in a TED population to
determine if a subset of this population demonstrated latent IgG4-RD.
Additionally, the relationship between total IgG, IgG4 and TSI levels
was evaluated.
Methods: TED patients evaluated in the practice of one author
between December 2013 and July 2014 were reviewed. Patients with
total and IgG subclass and thyroid function tests were included in the
study.
Results: Of 19 patients studied, three were found to have elevated
serum IgG4 levels by current criteria. All three patients presented
with proptosis, extraocular muscle enlargement on MRI and elevated
TSI, but no other systemic findings. One patient had elevated IgG4
and IgG4: total IgG ratio, positive by IgG4-RD criteria. The IgG4
levels of the remaining two patients were not positive by IgG4-RD
criteria, but were positive by IgG4: total IgG ratio. In all patients,
elevated IgG4 levels were not reflected in the total IgG levels.
Additionally, elevated TSI levels were not reflected in levels of total
IgG or any particular subclass of IgG.
Conclusions: We measured the serum IgG4 and IgG4: total IgG
levels in patients with TED. Measurement of these levels in TED
patients specifically has not been previously reported. Total IgG
levels were not an adequate proxy for either IgG4 or TSI elevation.
The results of this study suggest there may be a subpopulation of
TED patients with elevated IgG4 without other systemic findings
to suggest a type of IgG4-RD. This subpopulation of TED may be
responsive to rituximab selectively. Further immunologic evaluation
of TED patients may provide insight into the pathogenesis of the
disease and aid in the selection of novel biologic therapy.
Commercial Relationships: Aileen Sy, None; Rona Silkiss, None
Program Number: 4727 Poster Board Number: D0172
Presentation Time: 11:00 AM–12:45 PM
Galanin receptor detection in the human eye: first results
Falk Schroedl1, 2, Alexandra Kaser-Eichberger1, Andrea Trost1,
Barbara Bogner1, Christian Runge1, Karolina Motloch1, Daniela
Bruckner1, Clemens Strohmaier1, Barbara Kofler3, Herbert A.
Reitsamer1. 1Ophthalmology and Optometry, Paracelsus Medical
University Salzburg, Salzburg, Austria; 2Anatomy, Paracelsus
Medical University Salzburg, Salzburg, Austria; 3Dept. of Pediatrics,
Laura-Bassi Centre of Expertise, THERAPEP, Paraelsus Medical
University Salzburg, Salzburg, Austria.
Purpose: The neuropeptide galanin (GAL) is widely distributed
within intrinsic and extrinsic sources supplying the eye. It is
involved in regulation of the vascular tone, thus important for
ocular homeostasis. Since the presence/distribution of its receptors
is unknown, we here screen for the presence of the various GAL
receptors in the human eye.
Methods: Meeting the Helsinki-Declaration, human eyes
(n=6; 45 -83 years of age, of both sex, post mortem time 1019 hrs) were obtained from the cornea bank and prepared for
immunohistochemistry against GAL receptors 1 to 3 (GALR1GALR3). Over-expressing cell assays served as positive controls and
confocal laser-scanning microscopy was used for documentation.
Results: In the cornea, GALR1-GALR3 were detected in basal
layers of the epithelium, stroma, endothelium, as well as in adjacent
conjunctiva. In the iris, GALR1-GALR3 were detected in iris
sphincter, dilator and iris vessels. In the ciliary body, GALR1GALR3 were detected in ciliary muscle, with highest signal for
GALR3, and in ciliary body epithelium (GALR1 >>GALR3>
GALR2), while ciliary body vessels were positive for GALR3
only. In the retina, GALR1 was present in fibers of the IPL/NFL,
many cells of the INL and only few cells of the ONL. GALR3 and
GALR2 were present in few neurons of the INL, while GALR2 was
also found surrounding retinal vessels. In the choroid, GALR1-3
were detectable in nerve fibers surrounding vessels and in intrinsic
choroidal neurons.
Conclusions: This is the first report of the various GALRs in the
human eye. While the presence of GALR in cornea is enigmatic, the
detection of GALR1-3 in ocular vessels (iris, choroid) highlights
the role of GAL in vessel dynamics. High GALR3 presence in
ciliary body vessels might indicate importance for aqueous humor
production, whereas retinal GALR distribution might contribute to
signal transduction.
Commercial Relationships: Falk Schroedl, None; Alexandra
Kaser-Eichberger, None; Andrea Trost, None; Barbara Bogner,
None; Christian Runge, None; Karolina Motloch, None; Daniela
Bruckner, None; Clemens Strohmaier, None; Barbara Kofler,
None; Herbert A. Reitsamer, None
Support: Research Promotion Fund of the Paracelsus Medical
University PMU-FFF (E-11713/068-SRO), The Fuchs Foundation,
Adele Rabensteiner Foundation, Lotte Schwarz Endowment
Program Number: 4728 Poster Board Number: D0173
Presentation Time: 11:00 AM–12:45 PM
Optical clearing of the human eye using the See Deep Brain
(SeeDB) technique
Antonio Bergua1, Bettina Hohberger1, Winfried Neuhuber2.
1
Department of Ophthalmology, University of Erlangen-Nuremberg,
Erlangen, Germany; 2Department of Anatomy, University of
Erlangen-Nuremberg, Erlangen, Germany.
Purpose: Previous studies showed clearing of the brain using a
water-based optical clearing method denominated See Deep Brain
(SeeDB). However, although the eye belongs to the central nervous
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
system, this histological technique was not used until now. Applying
this new clearing technique could open new possibilities for the
histological visualization of the normal anatomy or pathological
changes of the eye maintaining original architecture. We applied this
optical clearing method for visualization of intraocular structures.
Methods: Four eyes of cornea-donors (2 male, 2 female: 73 to
84 years) obtained from the Cornea Bank of the Department of
Ophthalmology, were used. After enucleation and extraction of the
corneoscleral button, bulbi were fixed with 4% paraformaldehyde
in PBS and treated with increasing concentrations of aqueous
fructose solution with 0.5% α-thioglycerol. After the optical clearing
procedure, transscleral microphotographs of the choroid were taken
using confocal microscopy.
Results: Complete transparency of the sclera was obtained in the
SeeDB treated enucleated human eyes. Transscleral visualization
of the choroid is possible. The transparency of the choroid is only
partial, because the SeeDB method does not act on melanocytes of
the uvea. Microscopical observation and photographic documentation
of vessels and other choroidal tissues is also allowed without
additional processing of the specimens.
Conclusions: The SeeDB method allows visualization of intraocular
structures in fixed enucleated human eyes through a completely
translucent sclera. This innovative imaging technique could facilitate
comprehensive qualitative and quantitative studies of the whole
human eye, preserving its 3D relations. Of special interest, supra- and
intrachoroidal ganglionic plexus could be visualized transsclerally.
Also choroidal vessels and melanocytes are now accessible for direct
visualization without opening the fixed, enucleated eyes. Finally,
clinical-pathological correlations of some intraocular diseases – e.g.
choroidal or retinal tumors will be possible in intact eyes.
Commercial Relationships: Antonio Bergua, None; Bettina
Hohberger, None; Winfried Neuhuber, None
Program Number: 4729 Poster Board Number: D0174
Presentation Time: 11:00 AM–12:45 PM
Surgical Considerations in Vascularized Composite
Allotransplantation of the Eye
Nikisha Richards4, Edward Davidson1, Eric Wang3, Jenny Y.
Yu4, Juan Fernandez-Miranda2, Kia M. Washington1. 1Plastic
Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA;
2
Neurosurgery, University of Pittsburgh Medical Center, Pittsburgh,
PA; 3Otolaryngology, University of Pittsburgh Medical Center,
Pittsburgh, PA; 4Ophthalmology, University of Pittsburgh Medical
Center, Pittsburgh, PA.
Purpose: The concept of eye transplantation is not entirely novel.
In 1885, Chibret unsuccessfully transplanted a rabbit eye into a
blind girl and in 1977, the National Eye Institute called for a limited
and thoughtful laboratory effort in eye transplantation. Methods for
reconstruction or replacement of a non-functioning eye have to date
traditionally been limited to prostheses with the limiting factors of
whole eye transplantation including: failure by ganglion cell axon
survival, insuring adequate circulation of blood to the transplanted
eye and immune rejection of foreign tissue. We propose the surgical
considerations in patient selection, implantation and surgical
procedure in this prospective experimental model.
Methods: The critical technique prerequisite for successful
transplantation surgery was the development of techniques for
suturing blood vessels. Vascular anastomosis techniques have
evolved tremendously most recently with vascularized composite
allotransplantation (VCA). Anastomosis of donor and recipient
ophthalmic arteries is well within the capabilities of a microsurgeon.
Through cadaveric studies, we propose the anatomic considerations
for the use of the angular or internal maxillary artery (IMAX) as ideal
recipient vessels, a solution to the challenge of an anastomosis to
the ophthalmic artery due to limited exposure and need to escape the
zone of injury or pathology.
Results: Given the larger caliber of the IMAX, it is preferred over
the angular artery in some instances. We have successfully duplicated
a surgical approach to the IMAX for recipient grafting: bicoronal
incision followed by temporalis muscle turndown and extended
lateral orbitotomy.
Conclusions: Given the highly specialized function of the eye and
its unique anatomical components, VCA of the eye is an appealing
novel method for restoration, replacement and reconstruction of
the non-functioning eye. With the advent of image guidance and
evolution of endoscopic techniques, this is certainly within the realm
of practicality and this trial helps to bring eye transplantation closer
to clinical reality.
Commercial Relationships: Nikisha Richards, None; Edward
Davidson, None; Eric Wang, None; Jenny Y. Yu, None; Juan
Fernandez-Miranda, None; Kia M. Washington, None
Program Number: 4730 Poster Board Number: D0175
Presentation Time: 11:00 AM–12:45 PM
Suture fixation for Monocanalicular stenting
Milap Mehta1, 2, Nitasha Gupta2. 1Eye and Vision, Northshore
University, Glenview, IL; 2Surgery, Division of Ophthalmology,
University of Chicago, Chicago, IL.
Purpose: Epiphora may result from multiple etiologies including
increased lacrimation, decreased tear outflow, and eyelid malposition.
Monocanalicular stenting using the mini-Monoka has been used to
treat punctal stenosis but often results in premature stent extrusion
or migration. We sought to test a simple technique to reduce stent
extrusion using an externalized fixation suture.Our study was a
retrospective chart review of 3 patients undergoing a modified
surgical technique.
Methods: Our exclusion criteria included patients with canalicular
stenosis, nasolacrimal duct obstruction, dry eyes or significant
ectropion as a cause for epiphora. Probing and irrigation were
performed on each patient to demonstrate canalicular and
nasolacrimal duct patency. Tear break-up time and fluoroscein
staining were used to evaluate for dry eyes. Three patients (4 puncta)
fulfilled our exclusion criteria and demonstrated only punctal
stenosis. One patient had bilateral punctal stenosis. Our cohort
included 2 male and 1 female patients with an average age of 52
years. Each patient underwent punctal dilation, limited punctoplasty
with a single vertical blade incision using a # 11 blade, insertion of
mini-Monoka® stenting, and suture fixation. A double-armed 6-0
chromic suture or double-armed 6-0 vicryl suture was first looped
around the stent lip. Each arm was then passed from the punctal
lumen through the eyelid skin and externalized. The ends of the
sutures were tied 3 millimeters from the eyelid margin to prevent
corneal irritation. The sutures were allowed to reabsorb and the
patients were followed at 1 week, 1 month and 3 month intervals. Our
end points were extrusion or migration of the stent prior to surgical
removal. Each stent was removed after 3 months.
Results: There were no cases of premature migration or extrusion.
All 3 patients underwent uncomplicated stent removal after 3 months.
One punctum (25%) developed a conjunctival pyogenic granuloma
which was treated conservatively. All 3 patients (100%) developed
symptomatic relief and all 4 puncta (100%) maintained anatomic
patency at 3 month follow-up with a repeat probe and irrigation.
Conclusions: The mini-Monoka® stent is an excellent option for
the treatment of epiphora from punctal stenosis. Suture fixation with
a double-armed 6-0 chromic or 6-0 vicryl suture reduces the risk of
stent migration and extrusion with limited complications.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Milap Mehta, None; Nitasha Gupta,
None
Program Number: 4731 Poster Board Number: D0176
Presentation Time: 11:00 AM–12:45 PM
Immunopathology of conjunctival biopsies in patients with
punctal stenosis
Amit Reddy1, Meredith Baker1, Amanda Maltry1, Nasreen A. Syed1, 2,
Richard C. Allen1, 3. 1Ophthalmology and Visual Sciences, University
of Iowa Hospitals and Clinics, Iowa City, IA; 2Pathology, University
of Iowa Hospitals and Clinics, Iowa City, IA; 3Otolaryngology - Head
and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa
City, IA.
Purpose: Numerous etiologic processes and pharmacologic agents
have been associated with punctal stenosis, yet until two recent
papers examining punctal histopathology in patients with punctal
stenosis, little was known about the underlying pathologic processes.
These two papers reported that nearly all punctal specimens displayed
signs of chronic inflammation. The purpose of this study is to expand
upon these previous findings and examine the utility of conjunctival
biopsy in patients with punctal stenosis without known risk factors.
Methods: A retrospective chart review was performed of patients
who presented to the University of Iowa Hospital and Clinics
between August 2009 and October 2014 with idiopathic epiphora,
were clinically diagnosed with punctal stenosis, and underwent
conjunctival biopsy for histopathologic and direct immunofluorescent
(DIF) examination. Patients with known systemic or pharmacologic
etiologies were excluded.
Results: 12 patients met inclusion criteria. Conjunctival biopsies
from all 12 patients – 18 specimens – underwent histological
examination. All 18 specimens showed a stromal lymphocytic
infiltrate. Conjunctival biopsies from nine of the 12 patients – 12
specimens – were also evaluated by DIF. Three patients (33.3%)
had depositions of shaggy and/or duplicative fibrinogen along the
basement membrane zone.
Conclusions: Conjunctival specimens of all patients displayed
stromal lymphocytic infiltrate, confirming the previously reported
findings in punctoplasty specimens. While these previous studies
were performed on punctal specimens, conjunctival tissue tends
to be easier to access and provides greater amounts of tissue. That
conjunctival biopsies displayed similar findings to those in the
previous punctal specimens suggests that conjunctival biopsies may
be useful in studying these patients.
Three of nine patients showed abnormal fibrinogen morphologies that
are consistent with lichen planus (LP). None of these patients had
signs or symptoms of LP elsewhere in the body. These cases suggest
that isolated LP may be a common and underdiagnosed cause of
punctal stenosis in patients without other risk factors. Thus, patients
meeting these criteria may benefit from prompt histopathologic and
DIF examination.
Commercial Relationships: Amit Reddy, None; Meredith Baker,
None; Amanda Maltry, None; Nasreen A. Syed, None; Richard C.
Allen, None
Program Number: 4732 Poster Board Number: D0177
Presentation Time: 11:00 AM–12:45 PM
Feline Neovascular Vitreoretinopathy: A Histological Study of a
Newly Described Cause of Feline Glaucoma
Richard R. Dubielzig1, Billie Beckwith-Cohen1, Alison Hoffman2.
1
Pathobiol Sciences, Univ of Wisconsin-Madison, Madison, WI; 2Eye
Care for Animals, Pasadina, CA.
Purpose: To characterize the histopathology of twenty-one feline
globes enucleated because of intractable glaucoma.
Methods: The submission forms of twenty-one feline globes
diagnosed with neovascular vitreoretinopathy were examined
retrospectively. Case histories and follow-up information were
reviewed when available. Globes were examined grossly and
histologically. Histological sections were stained with hematoxylin
and eosin and selected special and immunohistochemical stains were
used in individual cases.
Results: The median±SD age of cats diagnosed with feline
neovascular vitreoretinopathy was 6±14 months. Eighteen cats
were domestic, 2 were Persian and one was a British shorthair.
All cats (21/21) had clinical or histological signs of glaucoma and
buphthalmos. Intraocular pressure measurements were available for
twelve cats and had a median±SD of 47±11mmHg. Histologically in
21/21 globes the peripheral retina was avascular and gliotic and there
were epiretinal vascular profiles extending from the peripapillary
retina into the vitreous. Other common histological abnormalities
included: anterior segment dysgenesis (20/21), retinal detachment
(20/21), lymphoplasmacytic anterior uveitis (18/21), preiridal
fibrovascular membranes (21/21), peripheral anterior synechia
(19/21), and ectropion uvea in 16/18 eyes where the pupillary
margin was sampled. Keratitis was present in 14/21 globes, likely
secondary to exposure. The status of the fellow eye was known in
seventeen cats: the fellow eye was variably affected in ten. Physical
examination results were available for thirteen cats and were
otherwise unremarkable.
Conclusions: Feline neovascular vitreoretinopathy is a rare disease
of kittens and young cats. Either one or both eyes may be involved
and cats often present with multiple ocular abnormalities. Etiology
remains unknown.
Commercial Relationships: Richard R. Dubielzig, None; Billie
Beckwith-Cohen, None; Alison Hoffman, None
505 Tumors - Melanomas
Thursday, May 07, 2015 8:30 AM–10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 5307–5349/A0156–A0198
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Biochemistry/Molecular Biology,
Genetics, Physiology/Pharmacology
Program Number: 5307 Poster Board Number: A0156
Presentation Time: 8:30 AM–10:15 AM
A novel classification of canine uveal melanoma: the importance
of melanocytoid-type of uveal melanoma in dogs
Miguel N. Burnier1, Silvin Bakalian1, Erin Mayo Goldberg2, Eduardo
Perlmann3, Paulo S M. Barros3, Nancy E. Mayo4. 1Ophthalmology,
McGill University, Montreal, QC, Canada; 2Henry C. Witelson
Ocular Pathology Laboratory, McGill University, Montreal, QC,
Canada; 3Surgery/School of Vet Medicine, University of Sao Paulo,
Sao Paulo, Brazil; 4McGill University, Montreal, QC, Canada.
Purpose: Canine uveal melanoma is a pigmented uveal tumor that
rarely metastasizes. They are erroneously referred in the literature
as melanocytomas, which is a benign melanocytic tumor. In contrast
to human melanoma, the histopathological features of canine uveal
melanomas exhibit classical and melanocytoma-type cells that
display malignant features. The aim of this study is to describe these
intraocular melanocytic lesions in dogs, propose a new classification,
and compare these features with human uveal melanoma.
Methods: Sixty-seven enucleated canine eyes with the clinical
presentation of an intraocular pigmented tumor were evaluated. Three
cases were excluded from the study (one squamous cell carcinoma
and two melanocytomas). In total, 64 malignant melanomas were
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
classified into two groups (melanocytoid or classic) according to the
presence or absence of large melanocytes with abundant cytoplasm
and medium sized nuclei (M cells), respectively. Histopathological
characteristics were compared between the two groups using Chisquare and t-tests. Multivariate discriminant analysis was conducted
to identify features that discriminated the two groups.
Results: Among the 64 tumors, 28 were classic and 36 were
melanocytoid type. Classic melanomas were larger than melanocytoid
type (18.0 mm vs. 13.6 mm; P=0.0033). Four variables (tumor
size, presence of histiocytes, degree of pigmentation, and mitotic
activity) were identified as discriminating between these two types,
with an accuracy of 68% for classic and 100% for melanocytoid.
Melanocytoid-type tumors had a smaller average size, abundant
histiocytes, high degree of pigmentation, and the majority low mitotic
activity (0–1 mitoses).
Conclusions: To the best of our knowledge, this is the largest series
of canine uveal melanomas.
Canine uveal melanoma should be further classified into classic
and melanoytoid-type. The melanocytoid-type melanomas possess
fewer malignant features and are characterized by the presence of
M cells. This classification suggests that melanocytoid-type tumors
have a better prognosis than the classic type. This study reclassifies
previous tumors that were called “melanocytomas” as malignant
“melanocytoid-type” tumors. Thus, the term melanocytoma used
ubiquitously is a misnomer in dogs as it is suggestive of a benign
lesion when these lesions have an infiltrative pattern.
Commercial Relationships: Miguel N. Burnier, None; Silvin
Bakalian, None; Erin Mayo Goldberg, None; Eduardo Perlmann,
None; Paulo S M. Barros, None; Nancy E. Mayo, None
Program Number: 5308 Poster Board Number: A0157
Presentation Time: 8:30 AM–10:15 AM
Inferring an evolutionary tree of uveal melanoma
Nakul Singh1, Arun D. Singh3, Winston Hide2. 1School of Medicine,
Case Western Reserve University, Solon, OH; 2Department of
Neuroscience, University of Sheffield, Sheffield, United Kingdom;
3
Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Lack of repeated access to pathologic samples over time
in a given patient limits study of tumor evolution. Herein, we apply
tools developed within the field of evolutionary biology for the study
of evolution of species to the genomes of primary uveal melanoma.
Methods: Primary uveal melanoma genomic DNA was assayed on
the Illumina Human660WQuad v1.0 DNA Analysis Bead Chip. Raw
signal intensity data were quantile normalized, which were then used
to estimate copy number aberration with the Genome Alteration
Print algorithm. Distance between samples was calculated as the
Manhattan distance between the copy number profiles of the tumors.
From the distance matrix, a phylogenetic network (evolutionary
relationship inference) was estimated using the SplitsTree package.
Each copy number segment was tested for an association with tumor
clade by means of a Fisher’s exact test, using a Benjamini-Hochberg
corrected p value of 0.05.
Results: Of the 57 tumors, 1(1.7%) was discarded because of
a failed assay, and 8 (13.8%) revealed to be mixtures of several
cell populations that could not be resolved by the GAP algorithm.
Three clades of tumor, each following a distinct evolutionary path,
were identified. The clades contained 29 (59.2%), 16 (32.7%),
and 3 (6.1%) samples each, and were labeled Clade A, B and C,
respectively. These clades were associated with metastatic status (p
value 0.04 by Fisher’s exact test). From a normal diploid melanocyte,
a few tumors (Clade C) lose a large portion of chromosome 6q. A few
tumors within this group subsequently lose almost all of chromosome
arm 1p. Clade C tumors do not develop any mutations on 8q. In an
alternate path, the vast majority of tumors (Clade A and Clade B)
gain a copy of the telomeric half of 8q. A majority of these tumors
(Clade A) then subsequently lose a copy of chromosome 3, as well as
the rest of 8q. The other tumors (Clade B) gain copies of 6p, as well
as regions on 11p and 22q.
Conclusions: Applying an evolutionary framework to uveal
melanoma genome reveals that there are distinct subtypes of uveal
melanoma, and these subtypes resemble each other at the beginning
of their development, but diverge soon thereafter. Our data also
suggests that there is little overlap in the subtypes of uveal melanoma
after divergence that are not likely to crossover or transform from one
major clade to another major clade.
Commercial Relationships: Nakul Singh, None; Arun D. Singh,
None; Winston Hide, None
Program Number: 5309 Poster Board Number: A0158
Presentation Time: 8:30 AM–10:15 AM
Generation of Luc2-eGFP-UM Cell Lines for Pre-Clinical Studies
on Metastatic Uveal Melanoma
Ryan P. Lee1, Michelle Sims2, Bradley T. Gao1, Hans E.
Grossniklaus3, Lawrence M. Pfeffer2, Matthew W. Wilson1,
Vanessa M. Morales1, 4. 1Ophthalmology, University of Tennessee
Health Science Center, Memphis, TN; 2Pathology, University of
Tennessee Health Science Center, Memphis, TN; 3Ophthalmology,
Emory University, Atlanta, GA; 4Microbiology, Immunology and
Biochemistry, University of Tennessee Health Science Center,
Memphis, TN.
Purpose: Metastatic Uveal Melanoma (UM) has no available
treatment. UM is the most common primary intraocular malignancy
in adults. Almost half of cases metastasize to the liver, resulting in a
very poor 2-year survival, but little is known about how UM invades
new tissue. Of particular interest is the ability of UM to remain
dormant as sub-clinical micro-metastasis. The goal of our study was
to generate UM cell lines transduced with Luciferase bioluminescent
protein (Luc2) and a fluorescent reporter gene (eGFP) to investigate
in vivo (1) how UM migrates to, seeds, and invades the liver and
(2) the kinetics of the transition from micro-metastasis to detectable
metastasis.
Methods: The UM cell lines, Mel 270, 92.1, and OMM1, were
transduced with a lentiviral construct (pLenti-UBC-Luc2-EGFP).
Transduced cell lines were screened using IVIS® Xenogen (Caliper
Life Sciences, MA) for bioluminescence and confocal microscopy for
fluorescence. Validation of the transduced cell lines was performed
using flow cytometry to analyze (a) cell surface expression of
ICAM-1 (CD54), ICAM-2 (CD102), VCAM-1 (CD106), ALCAM
(CD166), EpCAM (CD326), and VLA-4 (CD49d), (b) cell death by
Annexin V and Propidium Iodide (PI), and (c) proliferative capacity
by Ki-67. Cell line authentication was performed by Genetica DNA
Laboratories, Inc. (Cincinnati, OH) by comparison to published
material.
Results: All three UM cell lines exhibited significant
bioluminescence and green fluorescence in vitro 48 hours after
transduction, which was sustained through time, evidencing stable
transduction. Comparing transduced to non-transduced control cells,
we found no difference in (a) the cohort of surface molecules, (b)
the live/dead cell ratio by Annexin V and PI, and (c) proliferative
capacity by Ki-67. Authentication analysis showed cell line properties
were consistent with published characterization data.
Conclusions: We have generated a valuable tool for the in vivo study
of metastatic UM, allowing for extremely sensitive tracking of cancer
cells in animal models. Using this non-invasive tool, we are currently
performing in vivo analyses of the kinetics of UM metastasis.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Ryan P. Lee, None; Michelle Sims,
None; Bradley T. Gao, None; Hans E. Grossniklaus, None;
Lawrence M. Pfeffer, None; Matthew W. Wilson, None; Vanessa
M. Morales, None
Support: Fight for Sight - Summer Fellowship
Program Number: 5310 Poster Board Number: A0159
Presentation Time: 8:30 AM–10:15 AM
Phenotypic heterogeneity of uveal melanoma cell lines
Michael J. Young1, Natalia Vila2, Vasco Bravo2, Petr Y. Baranov1,
Burke Lieppman1, Alexis Palazzolo1, Miguel N. Burnier2. 1Schepens
Eye Research Institute, Massachusetts Eye and Ear, Boston, MA;
2
Ophthalmology, McGill University, Montreal, QC, Canada.
Purpose: Uveal melanoma (UM) is the most common primary
intraocular tumor in adults, with 2,500 new cases diagnosed every
year in United States. The “cancer stem cell” paradigm has been
explored in skin melanoma and has led to better understanding of
disease manifestation, progression and metastasis. The expression
of stem cell markers (ABCB5, CD133, Sox2, Pax6, nestin) by small
subpopulation of uveal melanoma cells in vitro and in vivo has also
been previously described, although the possibility of using this
“stem” subpopulation for drug discovery has not been addressed.
The goal of this study was to identify new targets for drug therapy,
which would be focused on suppressing proliferative potential of
dedifferentiated melanoma cells.
Methods: Immunocytochemistry, flow cytometry and RT-PCRT
analysis was performed for 3 adherent UM cell lines: MKT-BR,
OCM1, 92.1. The expression of stemness (Oct4, Nanog, Sox2,
Klf4, cMyc, NMyc, LMyc, Lin28, hTERT), proliferative (FGFR,
HGFR, Ki67, EpoR), eye field and melanocyte (Lhx2, Mitf, Nestin,
TRP1, TRPM1), known skin melanoma markers (ABCA5, ABCB5,
ABCG2) as well as several cell surface markers, expressed through
neural crest (CD73, CD38, PSA-NCAM, PTK7, A2B5, CD133,
HLA-ABC).
For proliferation assays we have plated cells at 10,000 cells/well
in 96 well plate, cultured them for 24 hours before drug treatment
and assessed the population change at 48 hours after stimulation
using CyQuant. The dose-response curves (7-point dilution) were
calculated for several small molecules and growth factors.
Results: We have observed the uniform expression of early
development markers (Sox2, Klf4), proliferative marker Ki67 and
melanoma markers (ABCA5, ABCB5, ABCG2) within population.
We have also confirmed the expression of growth factor receptors.
Cultured cell lines (92.1, MKT-BR and OCM1) contain a small
subpopulation of Lhx2, CD38 (2%, 3%, 8%), CD24 (2%, 11%,
5%), PSA-NCAM (2%, 3%, 4%) and CD133 (15%, 6% and 3%,
respectively) – positive cells.
Proliferation studies showed the cytostatic effect of Iodoacetic acid,
Rapamycin, PI3K inhibitor, Akt inhibitor, DAPT, AMPK inhibitor
and Valproic acid. Wnt-3a, EGF and FGF stimulated the proliferation.
Conclusions: We have identified several immature markers that
are heterogeneously expressed in 3 uveal melanoma cell lines. This
identified subpopulation may be considered as a target for drug
therapy.
Commercial Relationships: Michael J. Young, None; Natalia
Vila, None; Vasco Bravo, None; Petr Y. Baranov, None; Burke
Lieppman, None; Alexis Palazzolo, None; Miguel N. Burnier,
None
Program Number: 5311 Poster Board Number: A0160
Presentation Time: 8:30 AM–10:15 AM
Identification and proteomic analysis of exosomes derived from
human uveal melanoma cultures
Manuel Bande1, Maria Santiago-Varela1, Maria Jose BlancoTeijeiro1, Carmela Capeans1, Antonio Pineiro1, Maria Pardo2. 1Ocular
oncology Unit. Ophthalmology. Complexo Hospitalario Santiago de
Compostela, Santiago de Compostela, Spain; 2Grupo obesidomica,
Laboratorio de Endocrinologia molecular, Complexo Hospitalario
Santiago de Compostela (SERGAS), Santiago de Compostela, Spain.
Purpose: Exosomes are particles derived from cell membranes with
sizes ranging between 20-120 nm. They are also attributed a role
of intercellular messengers since the interior contains a variety of
proteins and microRNAs. This fact makes them object of study as
oncogenic biomarkers with prognostic value. The objective was to
determine whether uveal melanoma cells (UM) release exosomes and
to analyze their proteome.
Methods: The secretome from a primary human UM cell culture
established previously by our group (UM-A), and from a spontaneous
cell line arisen from this culture, characterized by more invasive
capability, were collected. Exosomes were isolated (ExoQuick-TC)
and analyzed by 2-dimensional Differential In Gel Electrophoresis
(DIGE) to quantitatively compare the differences in the protein
content of exosomes from both cultures. Significant protein
differences among exosomes (SameSpots, TOTALLAB) were
identified by mass spectrometry analysis (MALDI-TOF/TOF).
Results: 118 significant differences (p<0.001) between the two
samples of exosomes were detected. From 67 proteins identified,
proteins such as the mitogen activated protein kinase 3 (ERK1),
and transcription regulators such as zinc finger protein 224 or
LAMTOR3, were elevated in exosomes secreted by the more
invasive cell line; angiogenesis inhibitors such as PEGF were found
in the less invasive primary culture.
Conclusions: We show for the first time the identification and
characterization of exosomes released by UM cells with different
invasion potential. Our results indicate that the proteomic exosome
profiles vary depending on the aggressiveness of the cell line.
Certain proteins from UM exosomes could be considered as potential
biomarkers or treatment targets in the future.
Commercial Relationships: Manuel Bande, None; Maria
Santiago-Varela, None; Maria Jose Blanco-Teijeiro, None;
Carmela Capeans, None; Antonio Pineiro, None; Maria Pardo,
None
Support: Grant Instituto de Salud Carlos III PI11/00972
Program Number: 5312 Poster Board Number: A0161
Presentation Time: 8:30 AM–10:15 AM
Epigenetic Drugs Inhibit Uveal Melanoma Cell Proliferation and
Cell Cycle Progression
Weiwei Chen1, Jiao Wang1, Dan-Ning Hu2, Dongsheng Yan1. 1school
of opthalmology and optometry, Wenzhou medical university,
Wenzhou, China; 2The New York Eye and Ear Infirmary, New York,
NY.
Purpose: Emerging evidence indicates that epigenetic drugs, such
as DNA hypomethylating agents and histone deacetylase (HDAC)
inhibitors have substantial efficacy in treating some cancers. Their
effects on uveal melanoma, however, are largely unknown. To deal
with this question, we determined the effects of four epigenetic drugs
on uveal melanoma cell proliferation and apoptosis. The drugs used
include two hypomethylating agents and two HDAC inhibitors.
Methods: The uveal melanoma cell lines M23 and SP6.5 were
cultured and treated, respectively, with 5-azacytidine and decitabine,
two hypomethylating agents approved by the FDA for the treatment
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
of myelodysplastic syndrome, SAHA and romidepsin, two HDAC
inhibitors approved by the FDA for cancer therapy in treating
T-cell lymphomas. The MTS assay measured uveal melanoma
cell proliferation. Flow cytometry analyzed cell cycle progression.
Caspase 3/7 assays examined cell apoptosis.
Results: Uveal melanoma cell proliferation was dramatically
inhibited after treatment with each of these four epigenetic drugs.
5-azacytidine, SAHA and romidepsin induced G1-arrest, while
decitabine blocked the G2 phase. All of these epigenetic drugs did
had no obvious effects on cell apoptosis.
Conclusions: Our results imply that these epigenetic drugs may be
developed as potential agents in uveal melanoma treatment.
Commercial Relationships: Weiwei Chen, None; Jiao Wang,
None; Dan-Ning Hu, None; Dongsheng Yan, None
Support: National Natural Science Foundation of China(81077682
& 81272286)
Program Number: 5313 Poster Board Number: A0162
Presentation Time: 8:30 AM–10:15 AM
Oncogenic GNAQ orchestrates multiple signaling pathways via
ARF6
Dallas S. Shi1, 3, Jae Hyuk Yoo2, Weiquan Zhu4, Dean Li1, 2. 1Human
Genetics, University of Utah, Salt Lake City, UT; 2Oncological
Sciences, University of Utah, Salt Lake City, UT; 3MD/PhD Program,
University of Utah, Salt Lake City, UT; 4Molecular Medicine,
University of Utah, Salt Lake City, UT.
Purpose: Activating mutations in GNAQ and GNA11, occur in
over 80% of uveal melanomas. However, the molecular mechanisms
governing this stimulation remain unknown. Using biochemical
tools, cellular assays, a small molecule inhibitor, and animal models
of uveal melanoma, we tested the hypothesis that the small GTPase,
ARF6, is necessary and sufficient for oncogenic GNAQ signaling.
Methods: For in vitro experiments, Mel 92.1 and Mel 202 cells were
maintained in RPMI 1640 with 10% FBS. Cells were transfected
with siRNA against ARF6 or control siRNA using Lipofectamine
RNAiMAX (Invitrogen). Transfected cells were then assessed for
proliferation using CyQUANT (Invitrogen), anchorage-independent
colony growth using CytoSelect 96-Well Cell Transformation Assay
(Cell Biolabs), cell invasion using BD BioCoat Tumor Invasion
Assay System (BD Bioscience), and ARF6/RhoA/Rac1/PLC levels
using pull-down kits (Cells Biolabs, Millipore, Invitrogen).
For in vivo experiments, nude mice (Jackson) were anesthetized
with ketamine/xylazine. Then, 105 cells transfected with either ARF6
siRNA or control siRNA were injected into the posterior chamber.
In a subset of mice, non-transfected cells were injected and the mice
were subsequently treated with either 30mg/kg of NAV-2729 or
DMSO control. All mice were euthanized after 5 weeks, and eyes
were collected, fixed, embedded, sectioned, stained with H&E, and
examined histologically for primary tumors by a pathologist who was
blinded to the treatment regimen.
Results: Compared to controls, uveal melanoma cells with ARF6
knockdown exhibited decreased proliferation, decreased anchorageindependent colony growth, decreased cell invasion, and decreased
downstream signaling to PLC-PKC and Rac1/Rho-MAPK pathways
(n=3, p<0.01). In vivo, mice with uveal melanoma xenografts using
ARF6 knocked down cells exhibited lower tumor incidence and
smaller tumor size compared to mice treated with control xenograft
cells (n=12, p<0.05). Mice with uveal melanoma xenografts also had
decreased tumors when treated with a novel ARF6 inhibitor, NAV2729 (n=9, p<0.05).
Conclusions: In this study, we demonstrate that an activated GNAQARF6 complex orchestrates the activity of distinct tumorigenic
pathways in uveal melanoma. This suggest that targeting ARF6
may inhibit all of the currently known GNAQ-mediated oncogenic
signaling pathways and presents a new strategy for treating uveal
melanoma.
Arf6 in oncogenic GNAQ signaling.
Commercial Relationships: Dallas S. Shi, None; Jae Hyuk Yoo,
None; Weiquan Zhu, None; Dean Li, Navigen LLC (C)
Support: NH Grants RO1CA163970, RO1NS080893,
U54HL112311, RO1HL077671, ROLHL084516, and RO1AR064788
Program Number: 5314 Poster Board Number: A0163
Presentation Time: 8:30 AM–10:15 AM
Effects of ranibizumab and amfenac on the functional abilities of
uveal melanoma cells
Vasco Bravo-Filho1, Patrick T. Logan1, Sultan Aldrees1, Natalia
Vila1, Ayman Oweida2, Miguel N. Burnier1. 1Ophthalmology, McGill
University, Montreal, QC, Canada; 2McGill University, Montreal,
QC, Canada.
Purpose: Uveal Melanoma (UM) is the most common primary
intraocular tumor in adults and even with recent progress in
treating the primary tumor, mortality rate is still high. Also, some
tumors are too large at presentation and do not qualify for radiation
therapy, which is the standard treatment. Therefore, there is a need
for alternative treatment options. Our purpose was to evaluate
the effects of ranibizumab in association with amfenac in human
uveal melanoma cell lines. Moreover, we tested the ability of these
compounds to sensitize uveal melanoma cells to radiation therapy.
Methods: Proliferation and migration of the 92.1 uveal melanoma
cell line were assessed after pretreatment with ranibizumab (125
microg/ml) or amfenac (150 nM), and the combination of both
compounds. In addition, proliferation rates were assessed after
treatment with ranibizumab and amfenac and subsequent radiation
exposure. After treatment with ranibizumab and amfenac, cells
were exposed to various doses of radiation: 0, 4, and 8 Gy. An MTT
assay was used to assess proliferation rates 48 hours after radiation
exposure, and these values were compared to control cells (without
radiation and without treatment, but with radiation).
Results: Cells treated with ranibizumab and amfenac had lower
proliferation rates compared to controls (P=0.016), and to the
group treated only with ranibizumab (P=0.033). Migration was
only statistically lower in the group treated with amfenac relative to
the control (P=0.014). Treatment with ranibizumab, amfenac, and
the combination of both prior to the 8 Gy radiation dose led to a
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
marked reduction in proliferation rates (P=0.009; P=0.01; P=0.034;
respectively). There was no statistical difference between the three
treatment groups with the 8 Gy dose.
Conclusions: The combination of ranibizumab and amfenac
decreased the proliferation rate of uveal melanoma cells; however,
only amfenac monotherapy significantly decreased cell migration.
Other studies as an animal model, would help us to clarify if using
this association we could avoid tumor growth. The radiosensitivity
of the 92.1 UM cell line was increased by the administration
of ranibizumab, amfenac, and combination therapy. Further
investigation is warranted to determine if this treatment is a
viable pretreatment strategy to render large tumors amenable to
radiotherapy.
Commercial Relationships: Vasco Bravo-Filho, None; Patrick T.
Logan, None; Sultan Aldrees, None; Natalia Vila, None; Ayman
Oweida, None; Miguel N. Burnier, None
Program Number: 5315 Poster Board Number: A0164
Presentation Time: 8:30 AM–10:15 AM
Influence of VEGF isoforms and VEGF inhibition on uveal
melanoma cell lines – implications for therapeutic VEGF
inhibition
Alexa K. Klettner1, Michaela Dithmer1, Anna M. Kirsch1, Lidia
Graefenstein1, Sarah E. Coupland2, Johann Roider1. 1Ophthalmology,
Univ of Kiel, Univ Medical Center, Kiel, Germany; 2University of
Liverpool, Liverpool, United Kingdom.
Purpose: The influence of VEGF and its potential inhibitory impact
on uveal melanoma (UM) cell growth is controversial. In this study,
we investigated the effect of the VEGF isoforms VEGF165 (proangiogenic) and VEGF165b (anti-angiogenic) as well as of VEGFantagonist bevacizumab on UM cells.
Methods: Five UM cell lines were used, derived from both primary
tumors (92.1, Mel 270) and metastases (OMM2.5, OMM2.3,
OMM1). Secretion of VEGF-A was evaluated in ELISA. Influence
of 10 ng/ml and 100 ng/ml VEGF165 and VEGF165b, respectively,
on UM cells was assessed. Proliferation and toxicity was assessed
by WST assay, cell migration using a scratch assay. Cell death was
induced by hydrogen peroxide. The expression and phosphorylation
of the mitogen activated kinase, Erk1/2, and the expression of the
apoptosis-related proteins, Bax and Bcl2, were evaluated in Western
blots.
Results: All evaluated cell lines secreted VEGF-A. Application of
VEGF165 resulted in a significant reduction in cell proliferation in
all lines, while VEGF165b only displayed minimal (but significant)
reducing effects on the proliferation of OMM1 and OMM2.3. The
application of bevacizumab (250mg/ml) had no effect. VEGF165 and
VEGF165b were able to accelerate wound healing in OMM1 cells;
however, they displayed a slight decelerating effect in OMM2.3 cells,
with VEGF165 additionally having some effect on both OMM2.5 and
Mel270 cells. Bevacizumab again displayed no effect in either cell
line. In Mel270 cells, VEGF165 and VEGF165b reduced the Bax/
Bcl2 ratio.
The susceptibility of UM cells to hydrogen peroxide-induced
cell death varied. In all cell lines, VEGF165 protected cells from
hydrogen peroxide-induced cell death; however bevacizumab was
also protective. We found that in Mel 270 cell line, bevacizumab
increased the pErk/Erk ratio.
Conclusions: The inconsistent effect of VEGF and VEGF inhibition
found in the literature can be supported by these results. VEGF
was able to protect UM cells from oxidative stress-induced cell
death, but, contrary to expectations, proangiogenic VEGF inhibited
the proliferation rate of UM cells, while the effect of VEGF and
VEGF165b on wound healing was cell line dependent. Bevacizumab,
on the other hand, protected UM cells from oxidative stress-induced
cell death. These data indicate that a therapy targeting VEGF only in
UM is not likely to be beneficial to patients.
Commercial Relationships: Alexa K. Klettner, Novartis Pharma
(C), Novartis Pharma (R); Michaela Dithmer, None; Anna M.
Kirsch, None; Lidia Graefenstein, None; Sarah E. Coupland,
None; Johann Roider, None
Program Number: 5316 Poster Board Number: A0165
Presentation Time: 8:30 AM–10:15 AM
Chrysin reduces cell viability and induces apoptosis in cultured
human uveal melanoma cells
Codrin E. Iacob. Pathology, The New York Eye &Ear Infirmary of
Mount Sinai, New York, NY.
Purpose: Chrysin is a natural flavnoid and has been reported to
inhibit proliferation and induce apoptosis in various tumor cells.
However, the effect of chrysin on uveal melanoma cells has not been
reported. The present study investigated the effects of chrysin on the
viability of cultured human uveal melanoma cells and compared with
normal ocular cells.
Methods: Cell viability of two immortal cell lines of uveal
melanoma (SP6.5 and M17) treated by chrysin (0, 10, 30 and 100
μM) was studied using the MTT test. Normal uveal melanocytes and
retinal pigment epithelial cells (ARPE19) were used as the controls.
Cell apoptosis was determined by annexin V-FITC staining. All
studies were conducted in triplicate.
Results: Chrysin showed a dose-dependent inhibitory effect on the
cell viability in two uveal melanoma cell lines treated. Cell viability
in cells treated with 30-100 μM chrysin was significantly (P < 0.05)
lower than that in cells not treated with chrysin. The ID50 in SP6.5
and M17 cell lines were 28.3 and 35.8 μM, respectively. Annexin
V-FITC staining showed that chrysin (30-100 μM) could induce
apoptosis of uveal melanoma cells. Chrysin at 10-100 μM did not
change the cell viability in normal uveal melanocytes or retinal
pigment epithelial cells.
Conclusions: Chrysin reduced the cell viability and caused apoptosis
of uveal melanoma cells at 30-100 μM, but did not influence the cell
viability of normal uveal melanocytes and retinal pigment epithelial
cells at identical dosages. These findings suggest that chrysin has a
specific cytotoxic effect on uveal melanoma cells.
Commercial Relationships: Codrin E. Iacob, None
Program Number: 5317 Poster Board Number: A0166
Presentation Time: 8:30 AM–10:15 AM
MicroRNA-135b Inhibits Uveal Melanoma Cell Proliferation and
Migration
Xiaoyan Chen1, Jiao Wang1, Lihua Wang1, Dan-Ning Hu2, Dongsheng
Yan1. 1Sch of Ophthal & Optometry, Wenzhou Medical College,
Wenzhou, China; 2The New York Eye and Ear Infirmary, Tissue
Culture Center, New York Medical College, NY.
Purpose: MicroRNAs (miRNAs) can act as either oncogenes
or tumor suppressors in tumorigenesis. Evidence indicates that
miRNAs play important roles in uveal melanoma cell proliferation
and migration. The role of miR-135b in uveal melanoma, however,
remains unclear. Here, we investigated the function of miR-135b in
uveal melanoma cells.
Methods: Realtime RT-PCR was performed to detect the expression
of miR-135b in uveal melanoma specimens as well as normal
controls. Transfection of miR-135b into uveal melanoma cells was
carried out by Lipofectamine RNAiMAX reagent. The proliferation
of uveal melanoma cells was measured by MTS assay. Cell cycle
was analyzed by flow cytometry. Cell migration was examined by
transwell migration assay.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Results: miR-135b was decreased in uveal melanoma specimens
as compared with normal controls. Transfection of miR-135b into
uveal melanoma cells dramatically inhibited cell proliferation and
blocked cell cycle at G1 phase. Furthermore, miR-135b suppressed
cell migration.
Conclusions: Our results demonstrated that miR-135b may act as a
tumor suppressor in uveal melanoma cell proliferation and migration.
Commercial Relationships: Xiaoyan Chen, None; Jiao Wang,
None; Lihua Wang, None; Dan-Ning Hu, None; Dongsheng Yan,
None
Support: National Natural Science Foundation of China(81077682
& 81272286)
Program Number: 5318 Poster Board Number: A0167
Presentation Time: 8:30 AM–10:15 AM
The Importance of Sox-10 Expression in Uveal Melanoma
Sarah Alghamdi, Ana Beatriz T. Dias, Mohammed F. Qutub, Julia
Caminal, Josep M. Marti, Miguel N. Burnier. Henry C. Witelson
Ocular Pathology Lab, McGill University, Montreal, QC, Canada.
Purpose: An immunohistochemical panel of S100 protein, Melan A
and HMB-45 is commonly used to confirm the diagnosis of malignant
melanoma due to the lack of adequate specificity and sensitivity of
a single marker. Sox10 is a neural crest transcription factor crucial
for specification of Schwann cells and melanocytes. Sox-10 has been
shown to be a sensitive marker of cutaneous melanoma, including
spindle and desmoplastic subtypes, which are known to be negative
for melanocytic markers such as Melan A and HMB-45. This study
aimed to evaluate Sox-10 expression in uveal melanoma.
Methods: Thirty-nine uveal melanoma cases over a period of 35
years (1980–2014) were retrieved from the Henry C. Witelson Ocular
Pathology Laboratory. Formalin-fixed, paraffin-embedded blocks of
enucleated eyes with uveal melanoma were cut and stained using an
anti-Sox-10 mouse monoclonal antibody. The staining was scored
based on the extent of the nuclear expression: diffuse when staining
was seen in more than 50% of cells, and focal when it was seen in
less than 50% of cells.
Results: Of the 39 uveal melanomas studied, 12 were epithelioid
(31%), 13 were spindle (33%), and 14 were mixed (36%) subtypes.
The mean age of diagnosis was 69 with no gender predilection.
Thirty-five showed diffuse nuclear positivity for Sox-10 (90%), two
showed focal nuclear staining (5%), while two were negative (5%).
Positivity for Sox-10 was also noted in the inner and outer nuclear
layers of the retina in 78% of enucleated eyes. Overall, 17% of cases
showed Sox-10 nuclear staining in retina pigmented epithelium. The
uninvolved ciliary body, choroid, and iris showed focal positivity in
48%, 44%, and 11%, respectively. Five cases expressed Sox-10 in
Schwann cells of the scleral nerves (13%).
Conclusions: To the best of our knowledge, Sox-10 expression in
uveal melanoma has never been investigated. Sox-10 expression was
a sensitive, easily recognizable marker for uveal melanoma; however,
it was also positive in normal ocular structures. This study suggests
that Sox-10 can be incorporated in the panel for diagnosing uveal
melanoma tumors. Furthermore, the observation of distinct, diffuse
nuclear Sox-10 expression in retinal inner and outer nuclear layers, is
a finding that warrants further investigation.
Commercial Relationships: Sarah Alghamdi, None; Ana Beatriz
T. Dias, None; Mohammed F. Qutub, None; Julia Caminal, None;
Josep M. Marti, None; Miguel N. Burnier, None
Program Number: 5319 Poster Board Number: A0168
Presentation Time: 8:30 AM–10:15 AM
Programmed cell death ligand 1 is highly expressed in uveal
melanoma
Pablo Zoroquiain1, 2, Dominique F. de Souza1, Joao Mansure1,
Mohammed F. Qutub1, Juliana Portela Passos1. 1Pathology, McGill
University, Montreal, QC, Canada; 2Pathology, Pontificia Universidad
catolica de Chile, Santiago, Chile.
Purpose: Programmed cell death-1/ ligand (PD1/PDL1) is a pathway
that negatively regulates T-cell activity. A targeted therapy blocking
PD1/PDL1 has shown unprecedented tumor response in metastatic
cancers. PDL1 expression in tumors has been associated with therapy
response; however, this association is controversial due to the lack
of a validated commercially available antibody. Despite advances
in uveal melanoma (UM) treatment, 40% of patients will develop
metastases from which 90% will die. The aim of this study was to
analyze PDL1 expression and its prognostic value in UM.
Methods: Placenta sections and 92.1 UM cells were used as a
positive control to validate a newly PDL1 antibody (E1L3N clone).
Negative controls were established by silencing the PDL1 gene with
siRNA in UM cell lines. After validation, 40 eyes with UM and
clinical information were analyzed. The retinal pigmented epithelium
present in each case was used as an internal positive control.
Immunohistochemical expression of PDL1 was scored based on
extent:(0-3) and intensity (1= less than internal control; 2= higher or
equal to internal control). A final score(IRS)was obtained multiplying
both values. Low expression was considered an IRS 0-3 and high 4-6.
Chi-square, and survival analysis using log-rank test were used to
determine statistical significance.
Results: Placenta showed staining limited to the trophoblast. UM
92.1 cells showed positive PDL1 staining. After gene silencing, there
was a 9-fold decrease in PDL1 mRNA, which corresponded with a
significant decrease in IRS (P<0.0001). In UM samples, 4 cases were
excluded due to absence of staining in the inner positive controls.
PDL1 expression was seen in 88.6% of the cases, with 38.9% of
cases showing high expression. Lower PDL1 IRS was observed in
epithelioid cells compared to spindle and mixed cell type (P=0.03).
No differences were seen in tumor size, vascular loops, lymphocytic
infiltration, and metastases.
Conclusions: The commercially available anti PDL1 monoclonal
antibody (E1L3N) shows high specificity. To the best of our
knowledge, this is the first report showing positivity for PDL1 in UM
cases. In our series, PDL1 is expressed in most cases. These results
support the evaluation of anti-PD/PDL1 therapy in UM. Further
studied are needed to determine the importance of this marker for
predicting response to treatments targeting this pathway.
Commercial Relationships: Pablo Zoroquiain, None; Dominique
F. de Souza, None; Joao Mansure, None; Mohammed F. Qutub,
None; Juliana Portela Passos, None
Program Number: 5320 Poster Board Number: A0169
Presentation Time: 8:30 AM–10:15 AM
Deferoxamine inhibits the growth of uveal melanoma cells in
vitro
Thanos D. Papakostas1, Fotini Nicolaou2, Ahmad Al-Moujahed1,
John B. Miller1, Haijiang Lin1, Evangelos S. Gragoudas1, Demetrios
Vavvas1. 1Ophthalmology, Massachusetts Eye & Ear Infirmary,
Boston, MA; 2Surgery, Massachusetts General Hospital, Boston, MA.
Purpose: To evaluate the effect of deferoxamine, an iron chelator
used in clinical practice, on the growth of uveal melanoma cell lines.
Methods: Two different uveal melanoma cell lines (MEL 270 and
MEL285) were treated with various doses of deferoxamine (0.05
- 0.2 mg/ml). Cell growth was assessed by 3-(4,5-dimethylthiazol-
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was
conducted by flow cytometry; additionally, expression of cell-cycle
control proteins and cell growth transcription factors, was determined
by Western blot.
Results: Treatment with deferoxamine inhibited cell growth in a
dose dependent manner and caused cell cycle arrest in G1 phase after
1 and 2 days of treatment and in S phase after 4 days of treatment.
There was a significant increase in the population of apoptotic
cells, as determined by flow cytometry. Deferoxamine decreased
phosphorylation of ribosomal S6 kinase and decreased expression
of cyclin D1 and cyclin dependent kinase 2. Furthermore, it resulted
in activation of pro-apoptotic molecule Bcl2 and pro-senescence
molecule p21.
Conclusions: Deferoxamine inhibits proliferation of uveal melanoma
cells in vitro via cell cycle arrest and pro-apoptotic mechanisms.
These effects were seen at doses encountered in clinical use of
Deferoxamine. In vivo animal studies are needed to further examine
its potential for treatment of patients suffering from uveal melanoma.
Commercial Relationships: Thanos D. Papakostas, None; Fotini
Nicolaou, None; Ahmad Al-Moujahed, None; John B. Miller,
None; Haijiang Lin, None; Evangelos S. Gragoudas, QLT (P);
Demetrios Vavvas, Genentech (C), Kala Pharmaceuticals (P), Roche
(C)
Program Number: 5321 Poster Board Number: A0170
Presentation Time: 8:30 AM–10:15 AM
The effects of polarized macrophages on melanoma growth
characteristics in vitro and in vivo
Marta M. Kilian1, Karin U. Loeffler1, Christiane Pfarrer2, Christian
Kurts3, Frank G. Holz1, Martina C. Herwig1. 1Ophthalmology,
University of Bonn, Bonn, Germany; 2Anatomy, University of
Veterinary Medicine Hannover, Hannover, Germany; 3Experimental
Immunology, University of Bonn, Bonn, Germany.
Purpose: To investigate the influence of different tumor
microenvironments on melanoma growth characteristics in vitro
and in vivo. The effects of age as well as of M1- and M2- polarized
macrophages on tumor growth characteristics shall be verified.
Methods: Murine macrophages were polarized in vitro into
M1- and M2- macrophages. The result was verified by a multiple
cytokine ELISA and immunocytology. The murine melanoma cell
line HCmel12 was incubated with the supernatant of M1- or M2polarized macrophages, respectively. In vitro proliferation of M1- and
M2- conditioned HCmel12 cells was examined by a BrdU assay. In
vivo, 26 CX3CR1+/GFP mice (M1 young n=7, M1 old n=6, M2 young
n=7, M2 old n=6; young 8-12 wk, old >10 m) received an intravitreal
injection of M1- or M2- conditioned HCmel12 cells. Enucleated eyes
were processed for histological (H&E) and immunohistochemical
(CD31, collagen 4, laminin, GFP) evaluation of tumor size and
different tumor growth characteristics.
Results: In vitro polarization of macrophages was effective and was
confirmed by a multiple cytokine ELISA and by immunocytology.
Intraocular tumor growth was determined in all mice by H&E
stains. By immunohistochemistry, M2- conditioned tumors showed
a higher mean vascular density (CD31 positive vessels), more
frequent collagen 4- positive structures and an increased infiltration
by immune cells, particularly lymphocytes and macrophages, in
comparison to M1- conditioned intraocular tumors. Tumor size
did not differ between M1- and M2- conditioned tumors. Age
did not have any impact on tumor size or on other tumor growth
characteristics.
Conclusions: After in vitro M1- or M2- polarization of macrophages
and subsequent M1- or M2- conditioning of HCmel12 melanoma
cells, tumor growth characteristics revealed a more aggressive
phenotype in M2- conditioned tumors since a high mean vascular
density, infiltration of inflammatory cells and extracellular matrix
structures are described as poor prognostic factors in uveal melanoma
in humans. This animal study indicates that a high mean vascular
density and increased collagen 4 expression might be attributed to a
M2- characteristic local proangiogenic tumor microenvironment and
supports the concept that M2 macrophages have a modulating effect
on angiogenesis in melanoma.
Commercial Relationships: Marta M. Kilian, None; Karin U.
Loeffler, None; Christiane Pfarrer, None; Christian Kurts, None;
Frank G. Holz, None; Martina C. Herwig, None
Support: BONFOR Research Grant, intramural research fund of the
University of Bonn
Program Number: 5322 Poster Board Number: A0171
Presentation Time: 8:30 AM–10:15 AM
Role of Chemokine Receptor 7 in Uveal and Conjunctival
Melanoma
Ludwig M. Heindl, Nasrin Refaian, Claus Cursiefen, Simona L.
Schlereth. Department of Ophthalmology, University of Cologne,
Cologne, Germany.
Purpose: Conjunctival (CM) and uveal melanoma (UM) show a
different metastatic behavior. While CM spreads mainly via the
lymphogen routes to the draining lymph nodes, UM spreads primarily
via the hematogen routes into the liver. In this study, we investigated
the role of chemokine receptor 7 (CCR7) in relation to the metastatic
behavior of UM and CM.
Methods: Different human cell lines of uveal (Mel202, Mel270,
OM431 and Mel290) and conjunctival melanoma (CRMM1,
CRMM2 and CM2005) were investigated for their expression levels
of CCR7 by using real time PCR and immunocytochemistry.
Results: CM cell lines clearly expressed CCR7 histologically in all
cell lines. The highest signal was detectable in CRMM2. UM cell
lines expressed CCR7 very weakly. In some UM cell lines, nearly no
signal was detectable.
Real time PCR showed the highest CCR7 expression in CM2005
(p<0.01 compared to Mel270 and p<0.05 compared to CRMM2).
From the tested UM cell lines OM431 showed the highest CCR7
expression level.
Conclusions: Metastatic behavior of CM cells to the draining lymph
nodes might be influenced by CCR7 expression, whereas the tested
UM cells express very little CCR7.
Commercial Relationships: Ludwig M. Heindl, None; Nasrin
Refaian, None; Claus Cursiefen, None; Simona L. Schlereth, None
Support: German Research Foundation (HE 6743/2-1, CU 47/61, CU 47/4-1, Priority Research Project SFB 643: B10), GEROK
program by University of Cologne
Program Number: 5323 Poster Board Number: A0172
Presentation Time: 8:30 AM–10:15 AM
Loss of BAP1 expression is associated with increased angiogenesis
in uveal melanoma
Gülçin Gezgin1, Inge H. Bronkhorst1, T H. Van Essen1, Sake van
Pelt1, Robert M. Verdijk2, Pieter A. van der Velden1, Gregorius P.
Luyten1, Thorbald van Hall3, Sjoerd H. van der Burg3, Martine J.
Jager1. 1Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands; 2Pathology, Erasmus University Medical Center,
Rotterdam, Netherlands; 3Clinical Oncology, Leiden University
Medical Center, Leiden, Netherlands.
Purpose: High risk uveal melanoma (UM) is associated with an
inflammatory infiltrate and show an increased amount of vessel
growth. UM with monosomy 3 have an increased inflammatory
infiltrate in comparison to UM with disomy 3. Although monosomy
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
3 is associated with loss of BAP1, there are monosomy 3 cases
that still have BAP1 expression. We wondered whether loss of
BAP1 is associated with higher inflammatory infiltrate and more
vessel growth. Therefore, the aim of this study was to analyse the
association of BAP1 with inflammatory infiltrate, vessel density and
pro-angiogenic factors in uveal melanoma.
Methods: Fifty-four eyes with uveal melanoma obtained by
enucleation between 2001 and 2007 were included and were analysed
for a variety of infiltrating cells, vessel density and pro-angiogenic
factors by immunostaining and RNA sequencing with Illumina.
These parameters were compared with BAP1 immunohistochemistry
staining.
Results: Immunohistochemical staining of BAP1 was negative in
30 tumors, whereas 24 tumors stained BAP1 positive. Negative
BAP1 staining showed a significant increase of HCA2, HC10,
HLA-DR, CD68, CD163, CD4, FOXP3, CD8 and CD3 compared
to BAP1 positive tumors (p=0.003, p=0.000, p=0.035, p=0.000,
p=0.000, p=0.003, p=0.000, p=0.003, p=0.002). Also, a significantly
increased CD34 staining was observed in tumors with negative BAP1
compared to BAP1 positive tumors (p=0.005), indicating that these
tumors contain a higher vessel density. The immunostained BAP1
tumors were also compared with gene expression of infiltrating
cells. Again, negative BAP1 tumors showed a significant increased
gene expression of CD3, CD4, CD8, HLA-A, HLA-B, CD68 and
CD163L1 compared to positive BAP1 tumors (p=0.015, p=0.042,
p=0.016, p=0.000, p=0.000, p=0.002 and p=0.001). Gene expression
of pro-angiogenic factors, such as HIF1A, PECAM1, CDH1, vWF,
PDGFRB, MMP14, MMP25 and MMP9 were significantly increased
in BAP1 negative tumors compared to BAP1 positive tumors
(p=0.000, p=0.004, p=0.000, p=0.013, p=0.004, p=0.002, p=0.024
and p=0.025). However, some other factors, such as VHL and
VEGFB, showed the opposite (p=0.003 and p=0.001).
Conclusions: Loss of BAP1 expression in UM is not only associated
with an increased inflammatory infiltrate, but also with an increase
in vessel density and pro-angiogenic factors, suggesting that BAP1
associated leukocyte influx may stimulate angiogenesis.
Commercial Relationships: Gülçin Gezgin, None; Inge H.
Bronkhorst, None; T H. Van Essen, None; Sake van Pelt, None;
Robert M. Verdijk, None; Pieter A. van der Velden, None;
Gregorius P. Luyten, None; Thorbald van Hall, None; Sjoerd H.
van der Burg, None; Martine J. Jager, None
Support: ANVVB, SNOO, Stichting Blinden-Penning, LSBS
Program Number: 5324 Poster Board Number: A0173
Presentation Time: 8:30 AM–10:15 AM
Effect of Anti-angiogenic Agents Targeting Integrins ανβ3 on
human uveal melanoma and vascular endothelia
Hua Yang1, Hans E. Grossniklaus1, Wayne Harris2, Qing Zhang1,
Edmund K. Waller2, Ravi Chakra3, Zhi-Ren Liu3. 1Ophthalmology,
Emory University Eye Center, Atlanta, GA; 2Hematology and
Medical Oncology, Emory University, Atlanta, GA; 3Biology,
Georgia State University, Atlanta, GA.
Purpose: Angiogenesis is a prerequisite for the growth and
metastasis of solid tumors. Targeting the proliferation of tumor
neovasculature will form an effective anti-cancer therapy. ProAgio
is a noval anti-angiogenesis agent that targets Integrins ανβ3 of
endothelial cells. In this study, we investigate the effect of the noval
anti-angiogenesis agent on human uveal melanoma, mouse melanoma
and human/mouse vascular endothelia cells, and screen the sensitivity
of tumor cells for the further in vivo experiment.
Methods: Primary human uveal melanoma cells Mel290, Mel270,
OMM3, 02-1486, mouse melanoma Queens, mouse vascular
endothelium sVEC and human vascular endothelium HUVEC
were subcultured in 6-well-plates and treated with 5μm ProAgio or
equal volume of PBS as control for 24 hours after more than 90%
confluence. Harvested cells were stained with Caspase 3, CXCR4
and VEGFR2. The stained cells were acquired by FACScanto and
analyzed by FlowJo software.
Results: Comparing with the PBS control, ProAgio resulted in
the elevated level of caspase 3 in vascular endothelia HUVEC and
sVEC, the unchanged level of caspase 3 in human uveal melanoma
and mouse melanoma cells. ProAgio decreased the level of CXCR4
in human uveal melanoma OMM3 and mouse vascular endothelium
sVEC, also reduced the level of VEGFR2 in human uveal melanoma
OMM3 and 02-1486, and vascular endothelia HUVEC, sVEC.
Conclusions: ProAgio effectively caused the apoptosis in human
and mouse vascular endothelia, not human uveal melanoma in vitro,
suppressed the expression of VEGFR2 related with integrins ανβ3
and the migration indicator CXCR4 in vascular endothelia and some
types of human uveal melanoma cells. We are able to select human
uveal melanoma cells whose expressions of VEGFR2 and CXCR4
can be inhibited by ProAgio for the animal model, in purpose to
know the effect of ProAgio on tumor growth and metastasis in vivo.
Commercial Relationships: Hua Yang, None; Hans E.
Grossniklaus, None; Wayne Harris, None; Qing Zhang, None;
Edmund K. Waller, None; Ravi Chakra, None; Zhi-Ren Liu, None
Support: NIH Grant R01 CA12644, R01 EY13165 and P30
EY03630, and RPB, Inc.
Program Number: 5325 Poster Board Number: A0174
Presentation Time: 8:30 AM–10:15 AM
The Effects of Gold Nanoparticles on the Choroidal Melanoma
Cells: An In Vitro Study
Hamid Ahmadieh1, Somayeh Asadi4, 3, Sahar Balagholi4, 2, Mozhgan
Rezaeikanavi4, 1, Mostafa Olfat3, Narges Fazili4, Mehdi Vaez-zadeh3.
1
Ophthalmology, Ophthal Rsch Ctr, Shahid Beheshti Univ Med Sci,
Tehran, Iran (the Islamic Republic of); 2Iranian Blood Transfusion
Organizatio, Tehran, Iran (the Islamic Republic of); 3K.N.Toosi
University of Technology, Tehran, Iran (the Islamic Republic of);
4
Ocular Tissue Engineering Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran (the Islamic Republic
of).
Purpose: To study the effects of different sizes and concentrations
of gold nanoparticles (GNPs) on the cell viability in choroidal
melanoma.
Methods: GNPs were synthesized following the Fern’s method.
Two different diameters of 20 and 40 nm were prepared by making
changes in the citrate volume. The uveal melanoma (UM) tissue was
obtained after enucleation of an eye. UM cells were cultivated in
DMEM with 20% FBS. In the seventh passage, cells were grown on
two 24-well plates in which the first five wells were incubated with
different concentrations of GNPs and the sixth well saved as control
in both plates. The first five wells in the first and second plates had
the concentrations of 200, 150, 100, 50 and 25 mg for NPs with the
diameter of 20nm and 600, 400, 200, 100 and 50 mg for NPs with
the diameter of 40nm, respectively. The MTT assay was done after
incubating the cells for 48 hours at 37°C.
Results: The larger diameter of GNPs correlated with a higher
reduction in cell viability. At a fixed size of GNPs, cell viability
decreased with increasing GNPs concentration. The concentrations
of 600 and 400 mg for GNPs with the diameter of 40 nm obviously
affected the cell viability . For 20 nm diameter, nearly similar
behavior for all concentrations of GNPs was observed although the
concentration of 200 mg showed a more obvious decrease in the cell
viability of the choroidal melanoma cells ( Fig. 1).
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Conclusions: Higher concentrations have more cytotoxic effects
on the choroidal melanoma cells in GNPs with a larger diameter
compared with those with a smaller diameter. The concentrations
lower than 200 mg show better biocompatibility for both sizes
of GNPs. Further in vivo cytotoxicity studies are required before
high-concentration GNPs can be used for the treatment of choroidal
melanoma .
Figure 1: Cytotoxicity of gold nanoparticles with different
concentrations and sizes following incubation with uveal melanoma
cells after 48 hours.
Commercial Relationships: Hamid Ahmadieh, None; Somayeh
Asadi, None; Sahar Balagholi, None; Mozhgan Rezaeikanavi,
None; Mostafa Olfat, None; Narges Fazili, None; Mehdi Vaezzadeh, None
Program Number: 5326 Poster Board Number: A0175
Presentation Time: 8:30 AM–10:15 AM
Characterizing the role of the p53 effector PERP in cell
migration: Insights from uveal melanoma cells
Raheela Awais, Lyndsay Davies, Luminita Paraoan. Eye and Vision
Science, University of Liverpool, Liverpool, United Kingdom.
Purpose: PERP (p53 apoptosis-effector related to PMP-22) is a
tetra-span membrane protein specifically expressed during p53mediated apoptosis. PERP also plays a pivotal role in cell-cell
adhesion by enabling desmosome function. Previously, we identified
PERP as an important molecular determinant of apoptosis in primary
uveal melanoma (UM) tumours. We demonstrated that PERP is
significantly down-regulated in the highly metastatic (monosomy
3)-type compared with the less aggressive (disomy 3) tumours.
We hypothesize that down-regulation of PERP results in enhanced
migratory and invasive properties of UM tumour cells due to
decreased cell- cell adhesion in the absence of functional PERP.
Methods: PERP expression was characterized in UM cell lines
OCM1, MEL202, MEL285 and MEL 290 using qPCR and western
blotting. The effect of PERP expression on the migration of GFPPERP transfected UM cells was assessed using an in vitro scratch
assay and time-lapse microscopy, with non-transfected (NT) and
GFP-only transfected cells as controls. The kinetic behaviour of
cells was analyzed in real time with the Essen Bioscience Cell
Player Migration and Kinetic Cell Invasion assay and data analyzed
using IncuCyte software. Experiments were done in triplicates and
statistical analysis was performed using the Student’s T-test.
Results: Endogenous PERP expression was significantly reduced
both at transcriptional and protein level compared to ARPE19 cells.
Migration of NT and GFP-only transfected OCM1 cells led to the
reduction in the gap created by the scratch. However, GFP-PERP
transfected cells migrated significantly less than NT (p=0.005) or
GFP-only cells (p=0.02). Kinetic quantification in four UM cell lines
also demonstrated a reduction in cell migration capacity following
GFP-PERP expression, with highest (71% reduction) in MEL202 and
the lowest (13.5% reduction) in MEL285 compared to NT control.
Conclusions: The results are consistent with the hypothesis that
reduced levels of PERP lead to enhanced migratory and invasive
properties of UM tumour cells resulting in metastasis. Further studies
will examine whether PERP’s role in cell adhesion in UM is via
direct association with the focal adhesion assembly or by indirect
regulation of focal adhesion components.
Acknowledgements – The work has been supported by The Humane
Research Trust, UK.
Commercial Relationships: Raheela Awais, None; Lyndsay
Davies, None; Luminita Paraoan, None
Program Number: 5327 Poster Board Number: A0176
Presentation Time: 8:30 AM–10:15 AM
Fine-Needle Aspiration Biopsy of Solitary Amelanotic Choroidal
Tumors in 200 Cases
Chloe T. Khoo, Wasim A. Samara, Adel A. Set, Arman Mashayekhi,
Emil A. Say, Jerry A. Shields, Carol L. Shields. Ocular Oncology
Service, Wills Eye Hospital, Philadelphia, PA.
Purpose: To analyze outcomes of fine-needle aspiration biopsy
(FNAB) of indeterminate solitary amelanotic choroidal tumors.
Methods: Retrospective case series of 200 eyes with solitary
amelanotic choroidal tumors that underwent FNAB.
Results: Of 200 consecutive solitary amelanotic choroidal tumors
that underwent FNAB for diagnosis, sufficient sample for a
conclusive diagnosis was obtained in 170 (85%). The most frequent
diagnoses were choroidal metastasis (81/200, 41%), choroidal
melanoma (74/200, 37%), non-specific inflammatory cells (8/200,
4%) and atypical lymphoid cells (3/200, 2%). There were 33 (33/81,
41%) patients with FNAB-proven choroidal metastasis without a
known history of primary cancer and further evaluation revealed
primary lung carcinoma in 21 (21/33, 64%) and 17 (17/21, 81%)
of these patients were smokers. Further, the majority (16/17, 94%)
of patients who smoked and did not have prior history of cancer
were found to have smoking-related primary cancers (lung = 14/17,
82%; esophagus = 1/17, 6%; kidney = 1/17, 6%) following FNAB.
Subgroup analysis showed patients with tumors cytopathologically
proven to be choroidal metastasis (versus choroidal melanoma) were
more likely to be older (64 vs 59, p = 0.021), have prior history of
lung carcinoma (19% vs 0%, p = 0.001), better presenting visual
acuity (20/40 vs 20/60, p = 0.024), and have smaller (thickness 4.5 vs
5.4 mm, p = 0.028) tumors. Prior history of breast, kidney, and other
systemic cancers, as well as smoking history were not statistically
different between both groups.
Conclusions: FNAB is a reliable diagnostic modality for
indeterminate solitary amelanotic choroidal tumors. Older patients
with prior history of lung carcinoma and smaller solitary amelanotic
tumors were more likely to have choroidal metastasis rather than
melanoma on cytopathology.
Commercial Relationships: Chloe T. Khoo, None; Wasim A.
Samara, None; Adel A. Set, None; Arman Mashayekhi, None;
Emil A. Say, None; Jerry A. Shields, None; Carol L. Shields, None
Program Number: 5328 Poster Board Number: A0177
Presentation Time: 8:30 AM–10:15 AM
Rates of Sample Acquisition in Uveal Melanoma Fine Needle
Aspiration Biopsies Using Transscleral and Transvitreal
Techniques
Amy C. Schefler1, 2, Maru E. Bretana1, Patricia Chevez-Barrios2, Bin
Teh2, Neda Nikpoor3, Daniel Gologorsky3. 1Ophthalmology, Retina
Consultants of Houston, Houston, TX; 2Ophthalmology, Houston
Methodist Hospital, Houston, TX; 3Ophthalmology, Bascom Palmer
Eye Institute, Miami, FL.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Purpose: The purpose of this study was to compare the rate of
successful sample acquisition during uveal melanoma fine needle
aspiration biopsies using transscleral versus transvitreal techniques.
Methods: This was a single-center, retrospective review of
patients who chose to undergo fine needle aspiration biopsies for
cytopathologic and genomic analyses of uveal melanoma concurrent
or before definitive treatment with Iodine-125 plaque or biopsy in
2014. Only patients with 3 months or more of follow-up and adequate
data for analysis were included. Clinical variables were reviewed
including: patient demographic data (age, sex, right/left eye); clinical
tumor data (size of tumor, presence of subretinal fluid, location of
tumor); biopsy technique details (transscleral versus transvitreal,
number of passes needed, adequacy of sample obtained, GEP class
and discriminant value).
Results: There were 30 patients who met the inclusion criteria.
Ten were females and 20 were males. Seventeen of the eyes in the
study were right eyes, and 13 were left eyes. The mean age of the
patients was 61 (range, 19-82). Seventeen underwent biopsies with
a transvitreal approach and 13 underwent biopsies via a transscleral
approach. Of the 30 biopsies, only two patients who underwent
transvitreal biopsies had an inadequate sample (93% success rate).
Sixteen were Gene Expression Profiling Class 1A; Three patients
were Class 1B; and 11 were Class 2. Mean discriminant value was
0.86 for the gene expression profiling sample and all patients except
one in the study had a discriminant value > 0.1. The vast majority of
the time, only one biopsy pass was needed. No patients developed
a retinal detachment and transient vitreous hemorrhage occurred in
nearly all patients. There were no cases of extraocular extension.
Various techniques were used to ensure biopsy success including
adequacy checks with the ophthalmic pathologist present in the
operating room for the procedure, use of the 27 g vitreous cutter for
lesions < 1.5 mm, direct visualization with a retinal viewing system,
and others.
Conclusions: Very high success rates can be achieved with both
transscleral and transvitreal approaches to fine needle aspiration
biopsy for uveal melanoma using the technical approaches described
in this study.
Commercial Relationships: Amy C. Schefler, None; Maru E.
Bretana, None; Patricia Chevez-Barrios, None; Bin Teh, None;
Neda Nikpoor, None; Daniel Gologorsky, None
Program Number: 5329 Poster Board Number: A0178
Presentation Time: 8:30 AM–10:15 AM
Comparison of Gene Expression Profiling and Chromosome 3
Analysis by Fluorescent In Situ Hybridization in Fine Needle
Aspiration Biopsy Specimens of Uveal Melanoma
Elizabeth Richter, Sujit Itty, Tara A. McCannel. Retina, Jules Stein
Eye Institute, Los Angeles, CA.
Purpose: To assess the concordance between results of DecisionDxUM uveal melanoma specific Gene Expression Profiling (GEP)
(Castle Biosciences, Phoenix, AZ) and Fluorescent in Situ
Hybridization (FISH) for chromosome 3 analysis in uveal melanomas
undergoing intraoperative fine needle aspiration biopsy (FNAB) for
metastatic prognostication during brachytherapy.
Methods: Consecutive patients diagnosed with uveal melanoma
who underwent intraoperative transscleral or transvitreal fine needle
aspiration biopsy with 30-gauge needle prior to placement of
iodine-125 radioactive plaque between November 2012 and January
2014, were retrospectively reviewed. Patient demographics and
tumor characteristics were obtained. The results of biopsy specimens,
analyzed by cytopathology, FISH for monosomy 3 and 6p gain, and
for GEP analysis with the DecisionDx-UM assay, were reviewed.
Results: A total of 99 intraocular tumors underwent brachytherapy
with intraoperative biopsy, including 90 choroidal melanomas, 6 iris
melanomas, and 3 uveal metastatic lesions. FISH and GEP results
were both available in 44 (44%) patients all with iris or choroidal
melanoma. Of these 44, FISH and GEP results were discordant in
7 tumors (15.9%). Six tumors were classified as “Class 1” (four
1A, two 1B), or low risk-for metastasis designation, by GEP but
monosomy 3 by FISH; and one tumor was found to be “Class 2”
by GEP and disomy 3 by FISH analysis. There was no significant
difference with regard to tumor height (p=0.94), patient age (p=0.95),
or ciliary body involvement (p=0.97) between discordant and
concordant cases. No patients with discordant tumors had confirmed
metastatic disease with limited follow up (mean 8.4 months, range
1- 15.5 months).
Conclusions: Discordance between GEP and Chromosome 3 status
by FISH occurred in our series at a rate of 15.9%, which is similar to
previously published reports. No patient or tumor characteristics were
identified as risk factors for discordance. The metastatic prognosis of
uveal melanoma patients with discordant results is unclear from our
study due to limited follow up.
Commercial Relationships: Elizabeth Richter, None; Sujit Itty,
None; Tara A. McCannel, None
Program Number: 5330 Poster Board Number: A0179
Presentation Time: 8:30 AM–10:15 AM
Genetic status of chromosome 3 in posterior uveal melanoma in
relation to the development of second primary malignancies
Mette Bagger2, 1, Karin Wadt2, Morten T. Andersen2, Steffen
Heegaard3, 1, Mette K. Andersen2, Jens Kiilgaard1. 1Dept of
Ophthalmology, Rigshospitalet/ Glostrup hospital, Copenhagen,
Denmark; 2Dept of Clinical Genetics, Rigshospitalet, Copenhagen,
Denmark; 3Inst. of Eye Pathology, Copenhagen University,
Copenhagen, Denmark.
Purpose: To evaluate if the genetic profile of chromosome 3 in
posterior uveal melanoma is indicative of the development of second
primary malignancies after treatment for posterior uveal melanoma.
Methods: Charts from a retrospective consecutive cohort of 153
Patients with posterior uveal melanoma treated from 2009 through
2012 were reviewed. Information on genetic analysis of chromosome
3 and synchronous cancers including histopathological descriptions
were collected.
Results: A total number of 28 patients (18.3 %) presented at least
one second primary malignancy (excluding non-melanoma skin
cancers and carcinoma in situ). In 15 patients (9.8 %) a second
primary malignancy was diagnosed after treatment of posterior
uveal melanoma during a median follow-up time of 3.2 years (range:
0.2-5.7, interquartile range: 2.2-4.3). All secondary cancers were
histologically verified. Breast cancer, Prostate cancer, lung cancer
and cutaneous melanoma were the most common. Cause of death
included metastatic spread of uveal melanoma (30 ptt.), metastatic
spread of other primary cancers (7 ptt.) and other causes (7 ptt.). The
group of patients who developed a second primary malignancy after
treatment demonstrated a significantly (p=0.03) higher frequency of
chromosome 3 aberrations compared to patients who did not show
any signs of new primary malignant lesions during follow-up (Figure
1). Furthermore, an increased risk of second primary malignancies
was observed in patients with gain of chromosome 3 (p=0.0006).
Conclusions: Our data demonstrates the importance of surveillance
for second primary malignancies in patients treated for posterior
uveal melanoma. Furthermore, the determination of chromosome
3 status in posterior uveal melanoma could identify a subgroup of
patients with an increased risk of second primary malignancies.
However this finding needs to be reproduced in a larger dataset.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
incorporated GEP class and largest basal diameter was associated
with strong independent significance of each of the factors.
Conclusions: Although the currently available GEP test for
prognostic testing of uveal melanoma samples has asserted that
recognized clinical prognostic factors for metastasis do not modify
the metastatic risk of a patient with a GEP class 2 tumor, our single
center study that included a substantial proportion of smaller tumors
showed that both GEP class and LBD of the tumor are independent
prognostic factors for metastasis and metastatic death in multivariate
analysis.
Commercial Relationships: Zelia M. Correa, None; James J.
Augsburger, None
Support: Research to Prevent Blindness
Figure 1 Chromosome 3 status of posterior uveal melanoma in
patients who developed a second primary malignancy during followup compared to the rest of the cohort (2).
Commercial Relationships: Mette Bagger, None; Karin Wadt,
None; Morten T. Andersen, None; Steffen Heegaard, None; Mette
K. Andersen, None; Jens Kiilgaard, None
Support: Grant information: This study was funded by research
grants from Fight for Sight Denmark, The Research Fund of
Rigshospitalet and The Danish Medical Research Grant/The
Højmosegård Grant
Program Number: 5331 Poster Board Number: A0180
Presentation Time: 8:30 AM–10:15 AM
Independent Prognostic Significance of Gene Expression Profile
Class and Largest Basal Diameter of Posterior Uveal Melanomas
Zelia M. Correa, James J. Augsburger. Ophthalmology, University of
Cincinnati, Cincinnati, OH.
Purpose: To determine whether any conventional clinical prognostic
factors for metastasis from uveal melanoma retain prognostic
significance in multivariate models that includes gene expression
profile (GEP) class of the tumor cells.
Methods: Prospective IRB-approved clinical study of GEP
testing and other conventional prognostic factors for metastasis
and metastatic death in 299 patients with primary posterior uveal
melanoma evaluated by fine-needle aspiration biopsy (FNAB) of the
tumor at the time of or shortly prior to initial treatment with GEP
testing of some of the aspirated tumor cells. Univariate prognostic
significance of all evaluated potential prognostic variables (patient
age, largest basal diameter of tumor, thickness of tumor, intraocular
location of tumor, cytopathological class of tumor cells, and GEP
class) was performed by comparison of Kaplan-Meier event
rate curves and univariate Cox proportional hazards modeling.
Multivariate prognostic significance of combinations of significant
prognostic factors identified by univariate analysis was performed
using step-up and step-down Cox proportional hazards modeling.
Results: GEP class of the tumor cells was the strongest prognostic
factor for metastatic death in this series. However, largest basal
diameter of the tumor, thickness of the tumor and intraocular tumor
location also proved to be significant individual prognostic factors
in this study. On multivariate analysis, a two-term model that
Program Number: 5332 Poster Board Number: A0181
Presentation Time: 8:30 AM–10:15 AM
Uveal melanoma: clinical features and intraocular radiation
treatment response based on the gene expression profiling assay
Euiyong Kweon, Namchun Cho, Min Ahn, Dongwook Lee, Incheon
You, Youra Kim, Woojin Kim. Chonbuk National University Medical
School, Jeonju, Korea (the Republic of).
Purpose: To characterize the clinical features and intraocular
radiation treatment response of class 1 and class 2 uveal melanomas
based on gene expression profiling assay.
Methods: The charts of one hundred thirty patients who diagnosed
uveal melanoma by tumor biopsy from a single institution
were reviewed. The medical records of the ninety patients who
underwent intraocular radiation treatment were analyzed for clinical
characteristics and radiation response based upon molecular pattern
of uveal melanomas.
Results: Fifty-eight patients (64.4%) had class 1 tumors and thirtytwo patients (35.6%) had class 2 tumors. The mean age of patients at
the time of diagnosis was significantly greater in patients with class 2
tumors (68.8±15.3 years) as compared to patients with class 1 tumors
(60.1±15.2 years) (P=0.047). There was no difference in fundus
findings and fluorescein angiographic features (presence of orange
pigment, drusen, and subretinal fluid) (P=0.197, 0.285, 0.334) and
statistically borderline difference in the rate of enucleation between
2 classes during follow-up periods (P=0.052). Mean pretreatment
ultrasound thickness was significantly greater for class 2 patients
(6.61±3.0 mm) as compared to class 1 patients (4.80±2.4 mm)
(P=0.003). Moreover, there was significant difference between class
1 and class 2 tumors based on the extent of change in ultrasound
thickness at approximately 6, 12 months after intraocular radiation
treatment (P=0.018).
Conclusions: Age and ultrasound tumor thickness were pretreatment
findings to predict molecular pattern, but there was a large variability
in most clinical parameters (gender, tumor location, chorioretinal
findings, metastasis and adjunctive treatments) in both classes in
determination of molecular characteristics of the uveal melanomas.
This study demonstrated that class 2 uveal melanomas are generally
greater in tumor thickness at diagnosis and had significantly greater
decrease in thickness after intraocular radiation treatment than class 1
uveal melanomas.
Commercial Relationships: Euiyong Kweon, None; Namchun
Cho, None; Min Ahn, None; Dongwook Lee, None; Incheon You,
None; Youra Kim, None; Woojin Kim, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 5333 Poster Board Number: A0182
Presentation Time: 8:30 AM–10:15 AM
Validation of the Liverpool Uveal Melanoma Prognosticator
Online for predicting survival of patients with choroidal
melanoma
Sarah W. DeParis, Bertil E. Damato. Ophthalmology, University of
California San Francisco, San Francisco, CA.
Purpose: Almost 50% of patients with choroidal melanoma
develop metastatic disease despite successful eradication of the
primary tumor. Accurate prognostication for patients with choroidal
melanoma enhances care planning and improves patient quality
of life, even when the prognosis is found to be poor. However,
univariate prognostication based on anatomic, histologic or genetic
predictors is not accurate enough to be relevant to individual patients.
Further, the accuracy of various prognostic models is diminished by
missing data.
The Liverpool Uveal Melanoma Prognosticator Online (LUMPO)
performs multivariate analysis of genetic, histologic and anatomical
data, taking the patient’s age and sex into account and compensating
for any missing data, providing prognostications that are accurate
enough to be relevant to individual patients. This tool has been
validated in the United Kingdom, but it remains to be seen whether
it is generalizable to other patient populations. In this study, we seek
to validate LUMPO in a group of patients who received care for
choroidal melanoma at the University of California, San Francisco.
Methods: A retrospective chart review was performed for all
patients who were treated for choroidal melanoma at the University
of California, San Francisco between 2002 and 2007. Tumor
characteristics were recorded including largest basal tumor diameter
and thickness, extraocular extension, and ciliary body involvement.
In patients who underwent enucleation, histopathological data was
gathered, including the presence of epithelioid cells, vascular loops,
and mitotic count. Survival data nonspecific to cause of death were
gathered from the UCSF Cancer Center tumor database. The data
were analyzed using the method previously outlined by Eleuteri et al.
Results: A total of 409 patients were compared to the established
database of patients in the United Kingdom. Patient characteristics
including age at diagnosis, race, gender, and laterality were found to
be similar across both patient groups. Time from diagnosis to death
was as well similar between the two groups. LUMPO accurately
predicted survival in our cohort of patients, even when information
was missing including histopathological and genetic data.
Conclusions: These results confirm that LUMPO is a valuable
method of prognostication for choroidal melanoma and is
generalizable to patients in the United States.
Commercial Relationships: Sarah W. DeParis, None; Bertil E.
Damato, None
Program Number: 5334 Poster Board Number: A0183
Presentation Time: 8:30 AM–10:15 AM
Sirtuin 2 expression in uveal melanoma correlates with metastasis
in an animal model
Juliana Portela Passos, Ana Beatriz T. Dias, Pablo Zoroquiain,
Christina Mastromonaco, Sarah Alghamdi. Henry C. Witelson Ocular
Pathology Laboratory, McGill University, Montreal, ON, Canada.
Purpose: Sirtuins (Sirt) are a family of seven enzymes that are
involved in the cell cycle. Previous studies have shown that Sirt2 acts
both as a tumor suppressor and as an oncogene. In uveal melanoma,
we have shown that Sirt2 is preferentially expressed in malignant, but
not in normal, uveal melanocytes. The purpose of this study was to
analyze Sirt2 expression in primary tumors and metastases from an
animal model of uveal melanoma.
Methods: The rabbit model of uveal melanoma with human uveal
melanoma cells injected into the suprachoroidal space used in this
study has been previously described.
Seventeen formalin-fixed, paraffin-embedded uveal melanoma eyes
from the rabbits were immunostained for Sirt2 and were analyzed.
In addition, three lung metastases were all stained and scored.
Immunostaining was scored based on the intensity and distribution
of the immunoreaction. Intensity was classified as 0, 1, 2, or 3,
which corresponds to negative, weak, moderate, or strong staining,
respectively. Extent was classified as 0 for negative, 1 for less than
50% positive cells, 2 for cells with positivity between 50% to 80%, or
3 for cells with >80% positivity. A total immunoreactive score (IRS)
was then generated by multiplying intensity by extent.
Results: Sirt2 was expressed in 14 of 17 primary uveal melanoma
tumors analyzed with an average IRS of 1.71. Three of these animals
also had metastases, which had a mean IRS of 2.33. The difference
in Sirt2 staining was not statistically different between the primary
tumors and metastases (P>0.05). We also compared the IRS in
primary tumors from rabbits that did not have metastases (n = 14;
IRS = 1.93) to those animals that had metastases (n = 3; IRS = 0.67;
P = 0.19).
Conclusions: Primary tumors that generated metastases had a higher
expression of Sirt2 than tumors that did not metastasize. Moreover,
metastastic uveal melanoma showed higher expression of Sirt2 than
primary tumors. These results strongly suggest that Sirt2 acts as an
oncogene in this uveal melanoma animal model. Although our data
did not reach statistical significance, this may be a consequence of
the limited number of metastases investigated. Future studies are
warranted to confirm these findings in human ocular tumors and
metastases.
Commercial Relationships: Juliana Portela Passos, None;
Ana Beatriz T. Dias, None; Pablo Zoroquiain, None; Christina
Mastromonaco, None; Sarah Alghamdi, None
Program Number: 5335 Poster Board Number: A0184
Presentation Time: 8:30 AM–10:15 AM
Analysis of DNA hydroxymethylation in uveal melanoma
Cindy Weidmann1, 2, Christine Yao1, 2, Jade Pomerleau1, 2, Solange
Landreville1, 2. 1Ophthalmology, Université Laval, Quebec City, QC,
Canada; 2Axe médecine régénératrice, Centre de recherche du CHU
de Québec, Quebec City, QC, Canada.
Purpose: Epigenetic regulation of cancer involves posttranslational
modifications of histones and DNA methylation as a means of
controlling gene expression without modifying the DNA sequence.
These dynamic mechanisms allow tumor cells to adapt faster to their
changing microenvironment. The enzymatic conversion of cytosine
(C) to 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5hmC) silences the methylation and reactivates gene transcription.
Hypoxia plays a role in the etiology of uveal melanoma (UM)
according to increased expression of HIF1A by tissue microarray
immunostaining. Our general hypothesis is that hypoxia modulates
DNA methylation in UM. The objectives of our study are to
compare the overall level of hydroxymethylation between choroidal
melanocytes and UM cells, as well as to evaluate the influence of
hypoxia on this epigenetic mark.
Methods: Skin (N=1) and eye (N=6) melanoma tissue sections
were used for immunofluorescence analyses using 5-mC and
5-hmC antibodies to study the overall levels of methylation and
hydroxymethylation in situ. Melanocytes from the choroid (N=3) and
UM cells (N=6) were then exposed to oxygen levels of 21% and 1%,
and DNA was isolated to perform immunofluorescence analyses and
slot blot using the same antibodies. Levels of 5-mC and 5-hmC were
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
quantified by measuring the densitometric mean (normalization with
DAPI or an anti-ssDNA antibody; Mann-Whitney test).
Results: In situ, there was no staining for 5-hmC in UM tumors while
neural cells of the retina were positive. The 5-mC staining was fainter
or absent in tumor cells compared to retina cells. These results were
similar to the positive control (skin). In vitro, both methylation and
hydroxymethylation levels were decreased in UM cells compared to
normal melanocytes (P < 0.0001). Hypoxic conditions also impaired
the overall levels of 5-mC and 5-hmC in all cell types (P < 0.0005
and P < 0.05, respectively).
Conclusions: Overall levels of methylation and hydroxymethylation
were reduced in the UM genome, and hypoxia accentuated the loss of
these epigenetic marks. Unlike genetic mutations, alterations of DNA
methylation are reversible, and represent potential therapeutic targets
for restoring the epigenetic balance in tumor cells.
Commercial Relationships: Cindy Weidmann, None; Christine
Yao, None; Jade Pomerleau, None; Solange Landreville, None
Support: Doctoral Research Training Award from Université
Laval, Fondation du CHU de Québec, Fondation de l’Université
Laval, Fondation des maladies de l’oeil, Fondation “Ophtalmologie,
recherche et développement”, Canada Foundation for Innovation,
Vision Health Research Network Uveal Melanoma Quebec Database
and Eye Tissues Bank (FRQS), ThéCell Network (FRQS)
Program Number: 5336 Poster Board Number: A0185
Presentation Time: 8:30 AM–10:15 AM
The effects of acetylsalicylic acid as an anti-tumor agent in a
metastatic ocular melanoma cell line
Dominique Fausto de Souza, Sultan Aldrees, Mohammed F. Qutub,
Sarah Alghamdi, Ana Beatriz T. Dias, Miguel N. Burnier. The Henry
C. Witelson Ocular Pathology Laboratory, McGill University,
Montreal, QC, Canada.
Purpose: Acetylsalicylic acid (ASA) was shown to inhibit
proliferation, angiogenesis and induce apoptosis in colorectal,
ovarian and endometrial cancer cells. Moreover, ASA has been
shown to abrogate various processes that contribute to tumor growth
and progression, via both COX-2 dependent and independent
mechanisms. Currently, ASA is being evaluated in clinical trials as
an adjuvant therapy to treat multiple cancer types. The goal of the
current study is to evaluate the effects of ASA on metastatic uveal
melanoma (UM) cells.
Methods: OMM1.5, a well characterized UM cell line derived from
a metastatic liver nodule, was used in the current study. OMM1.5
cells were treated with 5 mM ASA for 48 hours and compared to
their untreated counterparts for their ability to secrete a panel of
10 proangiogenic cytokines using a sandwich ELISA-based array.
Additionally, the functional characteristics of treated and untreated
cells were compared using proliferation, invasion, and migration
assays.
Results: Treatment with ASA caused a significant decrease in
angiogenin and PIGF secretion, and an increase in HB-EGF
secretion (P<0.05). No significant change was seen for the other
seven proangiogenic cytokines, which included ANG-2, EGF, bFGF,
HGF, Leptin, PDGF-BB, and VEGF. Moreover, treatment with ASA
significantly inhibited the proliferation and invasion capabilities of
OMM1.5 cells (P<0.05 for both).
Conclusions: These results suggest that ASA may be effective
as an adjuvant therapy to reduce the proliferation and invasion
of metastatic UM. Future studies are needed to determine the
mechanisms underlying how ASA simultaneously increase and
decreases some proangiogenic cytokines in metastatic UM cell lines.
Commercial Relationships: Dominique Fausto de Souza, None;
Sultan Aldrees, None; Mohammed F. Qutub, None; Sarah
Alghamdi, None; Ana Beatriz T. Dias, None; Miguel N. Burnier,
None
Program Number: 5337 Poster Board Number: A0186
Presentation Time: 8:30 AM–10:15 AM
Bromodomain (BRD) inhibition as a novel strategy to inhibit the
Microphthalmia-associated transcription factor (MITF) axis in
uveal melanoma (UM)
Vassiliki Poulaki1, 2, Nicholas Mitsiades3, Warren Fiskus3.
1
Ophthalmology, Veterans Affairs Boston Healthcare System, Jamaica
Plain/Boston, MA; 2School of Medicine, Boston University, Boston,
MA; 3Medicine, Baylor College of Medicine, Houston, TX.
Purpose: UM is universally lethal when metastatic, creating an
unmet need for novel, effective, targeted systemic therapies. MITF
is a critical oncogenic transcription factor in UM, yet no targeted
therapies are available to inhibit it. BRDs are epigenetic readers
that recognize acetylated lysine on chromatin proteins and regulate
gene transcription. Lysine 27-specific acetylation of Histone 3
(H3K27ac) is associated with enhancer sites on chromatin and active
transcription.
Methods: We performed Chromatin ImmunoprecipitationSequencing (ChIP-Seq) analysis for BRD4, H3K27ac, MITF and
RNA Polymerase II (RNAP2) in UM cells. We also examined the
transcriptomic and proteomic effects of BRD4 siRNA and of the
BRD inhibitor JQ1 alone and in combination with the PKC inhibitors
bisindolylmaleimide I (BIM) or AEB071 against a panel of five UM
cell lines in vitro and in a UM xenograft model.
Results: Integrated ChIP-Seq analysis demonstrated strong
genome-wide co-localization of BRD4, H3K27ac, MITF and
RNAP2, suggesting that they cooperate to drive MITF-dependent
transcription. Treatment of UM cells with JQ1 abolished the
recruitment of BRD4 to the c-Myc and MITF genes, and suppressed
the expression of c-Myc and MITF mRNAs and proteins, as well
as of MITF target genes, in multiple UM cell lines. These effects
were also encountered upon treatment with BRD4 siRNA. Global
gene expression analysis revealed that JQ1 induces a transcriptional
signature that is associated with decreased MITF activity, decreased
metastatic potential and improved patient survival when applied to
publicly available UM patient datasets. JQ1 increased expression
of p21, p27, BCL2L11 and cleaved PARP proteins in UM cells.
These effects were enhanced by the combination of JQ1 with
BIM or AEB071, which led to synergistic loss of cell viability (by
isobologram analysis; CI < 1.0). JQ1 significantly inhibited the
growth of UM xenografts in SCID/Beige mice (p < 0.01).
Conclusions: BRD4 is a central regulator of MITF-driven oncogenic
signaling in UM. BRD inhibition silences c-myc and MITF
expression and function, inhibits proliferation and induces apoptosis,
holding promise for the treatment of UM, either as monotherapy or in
combination with PKC inhibitors.
JQ1 significantly inhibited the growth of UM 92.1 xenografts in
SCID/Beige mice, as evidenced by bioluminescence (A) and tumor
size (B and C).
Commercial Relationships: Vassiliki Poulaki, None; Nicholas
Mitsiades, None; Warren Fiskus, None
Support: Conquer Cancer Foundation of the American Society of
Clinical Oncology (ASCO) Career Development Award
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 5338 Poster Board Number: A0187
Presentation Time: 8:30 AM–10:15 AM
Modulation of MMP-2 and MMP-9 as Potential Adjuvant
Therapy to Limit the Growth of Metastatic Uveal Melanoma
Nabil Saleh1, Anderson H. Webb1, Bradley T. Gao1, Ryan P. Lee1,
Justin B. Lendermon1, Matthew W. Wilson1, Vanessa M. Morales1,
2 1
. Ophthalmology, University of Tennessee Health Science Center,
Memphis, TN; 2Microbiology, Immunology and Biochemistry,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: Our study focused on the progression and invasion of uveal
melanoma (UM), the most common intraocular malignancy in adults.
Degradation of the extracellular matrix is a critical step during tumor
progression as it correlates with the ability of the microenvironment
to restrict tumor growth. Matrix metalloproteinases (MMPs) are a
family of zinc ion-dependent endopeptidases, which digest different
substrates present in the tissue. Regulation of MMPs during the
interplay of the tumor and the surrounding microenvironment is still
unclear.
Methods: We cultured three lines of UM cells (Mel 270,
OMM1, and 92.1). Each cell lines were exposed to the common
phorbol esters Phorbol 12-myristate 13-acetate (PMA) and
12-O-Tetradecanoylphorbol-13-Acetate (TPA) at varying
concentrations, and to pharmacological inhibitors of MMP-2 and
MMP-9 (ARP100 and AG-L-66085, Santa Cruz Biotechnology,
Dallas, TX). Genomic studies and protein analyses were conducted to
determine the expression of MMP-2 and MMP-9. To study effect of
drug on tumor (spheroid) size, Nano3D BioPrinting assays (Nano3D
Biosciences, Inc, Houston, TX) were performed.
Results: Our results show MMP-2 and MMP-9 mRNA expression
in UM cell lines. The expression levels are higher in the metastatic
UM cell line OMM1. Gene expression was reduced upon use of
inhibitors, even when phorbol esters were used. Tumor shrinkage was
observed when MMP-9 inhibitors were used in the metastatic UM
cell line as measured by the Nano3D BioPrinting assay.
Conclusions: In this study, we investigated the changes in uveal
melanoma progression in vitro after pharmacological inhibition
of MMP-2 and MMP-9. Both inhibitors reduced expression of the
expected targeted genes, however only the MMP-9 slowed the
reduction in spheroid size. Our studies revealed pharmacological
inhibition of MMP-9 reduced the size of the tumor spheroids of
metastatic UM cell line in vitro.
Commercial Relationships: Nabil Saleh, None; Anderson H.
Webb, None; Bradley T. Gao, None; Ryan P. Lee, None; Justin
B. Lendermon, None; Matthew W. Wilson, None; Vanessa M.
Morales, None
Program Number: 5339 Poster Board Number: A0188
Presentation Time: 8:30 AM–10:15 AM
Protein Candidate Biomarkers of Uveal Melanoma Metastasis
John W. Crabb1, 2, Bo Hu2, Jack S. Crabb1, Arun D. Singh1. 1Cole
Eye Institute, Cleveland Clinic, Cleveland, OH; 2Lerner Research
Institute, Cleveland Clinic, Cleveland, OH.
Purpose: Proteomic analysis of metastasized and non-metastasized
uveal melanoma primary tumors was pursued for insights into
mechanisms and biomarkers of uveal melanoma metastasis.
Methods: Fifteen uveal melanoma specimens collected from
enucleated eyes at the Cole Eye Institute, Cleveland Clinic were
subjected to global quantitative proteomic analysis using LC MS/
MS iTRAQ technology. Bruch’s membrane choroid complex protein
extracts from nine normal human postmortem eyes were pooled
and utilized as a reference control specimen. Tumor and control
protein was extracted in SDS and digested with trypsin. Peptides
were labeled with iTRAQ tags, fractionated by cation exchange
chromatography, and analyzed by LC MS/MS. Protein identification
utilized the Mascot search engine and the human Uni-Prot/SwissProtein database with the false discovery rate £ 1%. iTRAQ tags were
quantified by the weighted average method (Matrix Science, 2.4.1).
Differentially expressed proteins exhibited group differences with
p £ 0.05 (t-test) in a sample set composed of five metastasized and
five non-metastasized ocular tumors. Logistic regression modeling
and principal component analysis was used to test the quantitative
capability of differentially expressed proteins to classify the
metastatic status of five independent ocular tumor specimens
Results: Over 1700 proteins were quantified with two or more
peptides from 15 uveal melanoma primary tumors. Thirty-six
differentially expressed proteins were identified from quantitative
comparison of proteins in 5 metastatic and 5 non-metastatic ocular
tumors. Among differentially expressed proteins, the levels of
collagen alpha-3 (VI) and heat shock protein beta-1 correctly
classified metastasis in five independent tumor samples. The
metastatic status of one independent tumor specimen was also
classified correctly by 13 other differentially expressed proteins and
by principal component analysis.
Conclusions: While additional uveal melanoma specimens must
be studied to confirm the identification of differentially expressed
proteins and to establish their discriminatory capabilities between
metastatic and non-metastatic tumors, the current results suggest
collagen alpha-3 (VI) and heat shock protein beta-1 as candidate
biomarkers of metastasis.
Commercial Relationships: John W. Crabb, None; Bo Hu, None;
Jack S. Crabb, None; Arun D. Singh, None
Support: Supported in part by NIH grants EY021840, EY022134,
Research to Prevent Blindness (RPB) Unrestricted Grant to the Cole
Eye Institute, RPB Senior Investigator Award, and the Cleveland
Clinic.
Program Number: 5340 Poster Board Number: A0189
Presentation Time: 8:30 AM–10:15 AM
Insights into Genetic Alterations of Liver Metastases from Uveal
Melanoma
Conni Hanke1, Andrew Dodson2, Sarah Lake1, Helen Kalirai1,
Sarah E. Coupland1. 1Department of Molecular and Clinical Cancer
Medicine, University of Liverpool, Liverpool, United Kingdom;
2
Institute of Cancer Research, London, United Kingdom.
Purpose: The liver is the main organ affected by metastatic uveal
melanoma (MUM), but current treatments of metastases rarely
prolong life and most patients die within a year of diagnosis. In this
study, the genomic aberrations present in MUMs were identified and
compared with those found in matched primary UMs (PUMs) to shed
light into the molecular characteristics of these tumors.
Methods: DNA was extracted from 13 liver MUMs and six matched
PUMs. The Affymetrix SNP Array 6.0 platform was used to detect
copy number variations (CNVs), and data analysis was performed
using the Partek Genomic Suite™ software. During analysis the
samples were divided into different groups: (i) MUMs only, (ii)
PUMs only, and (iii) matched PUM/MUM pairs.
Results: Across the whole genome a broad spectrum of CNVs,
mainly amplifications (87%), was observed, which confirmed the
main chromosomal changes, i.e. deletions on chromosome (chr.) 3
and amplifications on chr. 8q. In total > 17000 genes with CNVs were
detected for MUMs and > 20000 for PUMs. A group comparison of
the MUMs with the PUMs found that the similarities between these
groups varied, e.g., 94% of the amplifications on chr. 8q, but only
34% of the amplifications on chr. 9 were shared. Within the MUMs
the most common CNVs were gene amplifications on chr. 8q, 20,
17, and 19 and gene deletions on chr. 3. Amplifications of the genes
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
GNAQ and GNA11 (commonly mutated in PUM), were observed
in 100% of PUMs but only 58% of MUMs; the BAP1 gene was
deleted in 42% of MUMs and 40% of PUMs. CNVs of the SF3B1
and EIF1AX genes were not identified in MUMs, but the former
was amplified in 60% and the latter deleted in 20% of PUMs. Other
interesting genes like PTP4A3 or WISP1 were amplified in 100% of
both, MUMs and PUMs. For the PUM/MUM pairs between > 4500
and > 20000 genes with CNVs were identified, and between 30-55%
of commonly altered genes were found. However, the comparison
also revealed that the more similar the CNVs on chr. 3 and chr. 8q in
these pairs, the fewer CNVs were present on other chromosomes.
Conclusions: The genomic characterization of MUMs revealed a
broad spectrum of CNVs with a high prevalence of amplifications
on chr. 8q. Our data underline the importance of chr. 8q in UM
metastatic disease. By validating the biological importance of specific
gene amplifications in MUMs, novel therapeutic targets are likely to
be identified.
Commercial Relationships: Conni Hanke, None; Andrew
Dodson, None; Sarah Lake, None; Helen Kalirai, None; Sarah E.
Coupland, None
Support: Liverpool RLBUHT Charitable Funds to cover salary and
bench fees
Program Number: 5341 Poster Board Number: A0190
Presentation Time: 8:30 AM–10:15 AM
MIRNA PROFILING IN VITREOUS HUMOR, VITREAL
EXOSOMES AND SERUM FROM UVEAL MELANOMA
PATIENTS: PATHOLOGICAL AND DIAGNOSTIC
IMPLICATIONS
Michele Reibaldi, Teresio Avitabile, Andrea Russo, Antonio Longo,
Mario D. Toro, Caterina Gagliano, marco ragusa, Santo Stella,
Vincenza Bonfiglio, Cesare Mariotti. Catania University, Catania,
Italy.
Purpose: Uveal melanoma (UM) is the second most common form
of melanoma, as it represents approximately 5–6 % of all melanoma
diagnoses. Actual therapeutic impact on patients survival is debatable
at best, given that up to 50% of patients succumb to their disease.
Although several methods are available, accurate diagnosis is not
always easily feasible due to potential accidents (e.g., intraocular
hemorrhage). Accordingly, there is a great need for improved,
minimally invasive diagnostic methods for UM. Based on the
assumption that the profile of circulating miRNAs is often altered
in human cancers, we sought to verify whether UM patients show
different serum or vitreous humor (VH) miRNAs profiles respect to
healthy controls.
Methods: By using TaqMan Low Density Arrays, we analysed 754
miRNAs from VH, vitreal exosomes, and serum of 6 UM patients
and 6 healthy donors.
Results: Our data demonstrate that VH miRNAs profile from UM
patients is unique and only partially overlaps with that from serum of
the same patients.Moreover, 90% of miRNAs are shared between VH
and vitreal exosomes. Interestingly, also the alterations of miRNAs
expression profiles in VH and vitreal exosomes of UM patients
overlap in a statistically significant manner. This could suggest that
miRNAs profile alterations in VH result from the dysregulation of
the exosomal molecular cargo. We reported 32 miRNAs differentially
expressed in UM patients in at least two different types of samples
analyzed. Comparable modifications were detected in an independent
cohort of twelve ocular melanoma patients. Most alterations were
common to VH and vitreal exosomes. Interestingly, miR-146a was
upregulated in the serum of UM patients. Upregulation of miR-21
and miR-146a was also detected in formalin-fixed and paraffinembedded UM, suggesting that VH and serum alterations in UM
patients could be the consequence of molecular mutations arising
in tumoral cells. Computational functional analysis on miR-146a
(the only miRNA dysregulated according to all biological matrices)
showed an overrepresentation of biological functions related to
cancer and immunity.
Conclusions: Our findings suggest the possibility to screen the
blood of UM patients to identify diagnostic miRNAs released by the
affected eye: based on this, miR-146a could be considered a potential
non invasive blood marker of UM.
Commercial Relationships: Michele Reibaldi, None; Teresio
Avitabile, None; Andrea Russo, None; Antonio Longo, None;
Mario D. Toro, None; Caterina Gagliano, None; marco ragusa,
None; Santo Stella, None; Vincenza Bonfiglio, None; Cesare
Mariotti, None
Program Number: 5342 Poster Board Number: A0191
Presentation Time: 8:30 AM–10:15 AM
Ultrasonography versus Transillumination to Localize Choroidal
Melanomas in Preparation for Proton Beam Irradiation
Jonathan Lu1, 2, Ellen Redenbo2, Susanna S. Park2, 1. 1UC
Davis School of Medicine, Sacramento, CA; 2Department of
Ophthalmology & Vision Science, University of California Davis Eye
Center, Sacramento, CA.
Purpose: Proton beam therapy is a highly effective, globe saving
local treatment for ocular melanoma, but efficacy of treatment is
dependent on proper localization and placement of tantalum rings
in preparation for irradiation. The ring placement currently relies
on tumor localization intraoperatively using transillumination of the
globe. The goal of this study was to evaluate the relative accuracy of
this localization method compared to ultrasonography, and to identify
tumor characteristics that would affect the relative accuracy of these
two methods.
Methods: This study is a retrospective chart review of all eyes
with ocular melanoma from 2006 to 2014 who had tantalum
rings placed in preparation for proton beam therapy where the
placement of the rings were localized using both transillumination
and ultrasonography. All tantalum rings were placed under general
anesthesia by the same surgeon. Each eye had four rings placed at
the edges of the tumor. Transillumination through the globe was
performed intraoperatively to determine the distance between the
rings to tumor edge. The distance between the ring and tumor edge
was reconfirmed by ultrasonography postoperatively among the
identified patients.
Results: Among 53 patients with ocular melanoma treated with
proton beam irradiation, we identified 18 patients (18 eyes) who
had tumor localization performed using both ultrasonography and
transillumination. The mean age was 63 years (range 22 to 89 years)
and 44% were female. Among 71 rings placed around 18 tumors, 30
rings (42%) had tumor to ring distance of 1mm or greater difference
between the two methods (range 0 to 4 mm difference, mean 1.1 mm
+ 0.3 mm). The tumor feature associated with a greater difference
in tumor localization between the two imaging techniques was
increasing tumor height (p value = 0.04). Tumor pigmentation (p
value = 0.77) and tumor location (p value = 0.16) did not significantly
affect the difference in tumor localization using the two imaging
methods in this study.
Conclusions: Tumor localization with tantalum rings in preparation
for proton irradiation for treatment of choroidal melanoma may have
some limitation in accuracy when using transillumination method
alone. Postoperative ultrasonography may be a useful supplemental
method of reconfirming the accuracy of tumor localization especially
in tumors of increasing thickness.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Commercial Relationships: Jonathan Lu, None; Ellen Redenbo,
None; Susanna S. Park, None
Support: UC Davis Medical Student Research Fellowship
Program Number: 5343 Poster Board Number: A0192
Presentation Time: 8:30 AM–10:15 AM
Irradiation enhances chromosomal aberrations in uveal
melanoma
Mehmet Dogrusoz1, Wilma G. M. Kroes2, Sjoerd V. Duinen3,
Carien L. Creutzberg4, Mieke Versluis1, Jaco C. Bleeker1, Marina
Marinkovic1, Gregorius P. Luyten1, Martine J. Jager1. 1Department
of Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands; 2Department of Clinical Genetics, Leiden University
Medical Center, Leiden, Netherlands; 3Department of Pathology,
Leiden University Medical Center, Leiden, Netherlands; 4Department
of Clinical Oncology, Leiden University Medical Center, Leiden,
Netherlands.
Purpose: We wondered whether uveal melanoma (UM) would
show different chromosomal aberrations after previous irradiation.
Chromosome analysis of UM was performed by karyotyping and
fluorescence in situ hybridization (FISH). We determined the
spectrum of chromosomal aberrations in a retrospective cohort
of enucleated UM, some of which had previously undergone
brachytherapy or proton beam irradiation. Furthermore, we analyzed
which tumor features determined success or failure of karyotyping
and FISH.
Methods: Material from 327 UM-containing enucleated eyes was
submitted for karyotyping, while FISH analysis for chromosome 3
was performed in 240 samples. 36 UM had previously undergone
irradiation. The outcome of karyotyping was studied, and the
success rate of both techniques was evaluated and compared to
clinicopathologic characteristics of the tumor and to prior irradiation
treatment.
Results: Karyotypes showed that aberrations occur in all
chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16 and Y being
altered in at least 15% of the tumors. Aberrations were more common
in previously irradiated tumors, compared to primarily enucleated
cases, and reached significance for chromosomes 5 and 13 (p=0.004
and p=0.04, respectively, Table 1).
Karyotyping was successful in 79% of primarily enucleated tumors,
but only in 25% of previously irradiated tumors (p<0.001). In
primarily enucleated cases, both a large tumor prominence (p=0.004)
and a high mitotic count (p=0.007) were associated with successful
karyotyping. In previously irradiated tumors, this was only true
for a high mitotic count (p=0.03). FISH analysis for monosomy 3
failed significantly more often in irradiated tumors as well (p=0.04),
and was more often successful when primarily enucleated tumors
contained epithelioid cells (p=0.002); in previously irradiated cases,
FISH analysis was more often successful in tumors with a large
diameter (p=0.015) and prominence (p=0.001), and a high mitotic
count (p=0.019).
Conclusions: Karyotyping demonstrated that all chromosomes in
UM can be aberrant, with an increased frequency after irradiation.
Prior irradiation and histopathologic features characteristic of
low tumor aggressiveness were associated with both unsuccessful
karyotyping and FISH. The frequent lack of success in irradiated
tumors emphasizes the shortcomings of these techniques and shows
the relevance of taking biopsies for chromosomal testing prior to
irradiation treatment.
Commercial Relationships: Mehmet Dogrusoz, None; Wilma G.
M. Kroes, None; Sjoerd V. Duinen, None; Carien L. Creutzberg,
None; Mieke Versluis, None; Jaco C. Bleeker, None; Marina
Marinkovic, None; Gregorius P. Luyten, None; Martine J. Jager,
None
Program Number: 5344 Poster Board Number: A0193
Presentation Time: 8:30 AM–10:15 AM
Diffusion-Weighted Magnetic Resonance Imaging And
Ultrasound Evaluation Of Choroidal Melanomas After Proton
Beam Therapy
Livio Giulio Marco Franco1, Teresio Avitabile1, Michele Reibaldi1,
Antonio Longo1, Vincenza Bonfiglio1, Seby Flavio Gulisano1,
Pietro Valerio Foti2, marco ragusa3, Rosario Caltabiano4, Andrea
Russo1. 1Institute of Ophthalmology, University of Catania, Catania,
Italy; 2Department of Radiology, University of Catania, Catania,
Italy; 3Department Gian Filippo Ingrassia, Unità di BioMedicina
Molecolare Genomica e dei Sistemi Complessi, Genetica, Biologia
Computazionale, University of Catania, Catania, Italy; 4Department
Gian Filippo Ingrassia, Unità di Anatomia Patologica, University of
Catania, Catania, Italy.
Purpose: To compare the ultrasound and magnetic resonance
imaging parameters of ocular melanoma and to assess their variation
after proton-beam therapy.
Methods: Fifteen choroidal melanoma patients treated with protonbeam therapy were enrolled in the study. All patients underwent
ophthalmologic evaluations, ultrasound, conventional magnetic
resonance (MR) imaging and diffusion-weighted MR imaging before
the start of therapy and 3 and 6 months after therapy. Basal diameters,
thickness, internal reflectivity, tumor volumes and apparent diffusion
coefficient (ADC) values of ocular melanomas were measured at
each examination. Correlations between internal reflectivity and ADC
were investigated.
Results: No significant changes were seen in tumor diameters
and tumor height as assessed by B-scan and A-scan respectively.
Significant increase in mean tumor internal reflectivity was detected
at 6 months (baseline: 35 % ± 11; 6 months: 48 % ± 8, Tukey-Kramer
p=0.005). By MRI, compared to baseline (mean 547 ± 262 mm3), a
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
significant reduction in volume was seen at 6 months (Tukey-Kramer
p= 0.045) (mean volume 339 ± 170 mm3, mean reduction 38%).
A significant increase in ADC (baseline 1002 ± 109 mm2/sec) was
detected both at 3 and 6 months after proton-therapy (respectively
1454 ± 90 and 1833±261, mm2/sec, both p<0.001).
Conclusions: By MRI, in particular by ADC assessment, it is
possible to detect early variations in melanoma treated by proton
beam therapy. This examination could be used together with
ultrasound in the follow-up of this treatment.
Commercial Relationships: Livio Giulio Marco Franco, None;
Teresio Avitabile, None; Michele Reibaldi, None; Antonio
Longo, None; Vincenza Bonfiglio, None; Seby Flavio Gulisano,
None; Pietro Valerio Foti, None; marco ragusa, None; Rosario
Caltabiano, None; Andrea Russo, None
Program Number: 5345 Poster Board Number: A0194
Presentation Time: 8:30 AM–10:15 AM
Diffuse Intraocular Proliferation of uveal melanoma following
radiotherapy
Amir Mohsenin1, Vivek A. Rudrapatna2, George J. Harocopos3,
Sander R. Dubovy1, J William Harbour1. 1Department of
Ophthalmology, Bascom Palmer Eye Institute, Miami, FL; 2Graduate
School of Biological Sciences, Icahn School of Medicine at Mount
Sinai, New York, NY; 3Department of Ophthalmology, Washington
University School of Medicine, St. Louis, MO.
Purpose: To describe the presentation and management of diffuse
intraocular proliferation of uveal melanoma following radiotherapy
Methods: A retrospective chart review was conducted identifying
patients with uveal melanoma treated with radiotherapy who later
developed vitreous and retinal invasion by melanoma cells.
Results: 5 patients were identified. All patients had a collarbutton tumor configuration. 1 patient had a biopsy performed
at time of radiotherapy. 1 patient was managed with vitrectomy
alone. 2 patients that were initially treated with vitrectomy and
intravitreal methotrexate required enucleation. Enucleated specimens
demonstrated retinal and vitreous invasion by melanoma cells.
Conclusions: Intraocular proliferation of uveal melanoma is a
rare and challenging sequela after radiotherapy that may warrant
aggressive intervention.
Commercial Relationships: Amir Mohsenin, None; Vivek A.
Rudrapatna, None; George J. Harocopos, None; Sander R.
Dubovy, None; J William Harbour, None
Support: NIH Center Core Grant P30EY014801, Research to
Prevent Blindness Unrestricted Grant
Program Number: 5346 Poster Board Number: A0195
Presentation Time: 8:30 AM–10:15 AM
Uveal Melanoma Regression After Brachytherapy: Relationship
with Monosomy Chromosome 3
Hassan A. Aziz1, Sachin Salvi2, Nakul Singh3, Suhail Dar3, brandy
hayden1, Arun D. Singh1. 1Cole Eye Institute, Miami, FL; 2Royal
Hallamshire Hospital, Sheffield, United Kingdom; 3Case Western
Reserve University School of Medicine, Cleveland, OH.
Purpose: To explore relationship between regression rate of ciliary/
choroidal melanoma after plaque radiotherapy and percentage
composition with chromosome 3 monosomy tumor cells.
Methods: One hundred fifty consecutive patients underwent fine
needle aspiration biopsy at the time of plaque radiation therapy
to sample tumor cells for prognostication. Fluorescence in situ
hybridization (FISH) was performed to evaluate the percentage
of tumor cells with chromosome 3 monosomy (200 cell count).
Regression rate was calculated as percent change in tumor height
(measured by ultrasonography). Relationship between regression rate
and tumor location, initial tumor height, and percentage chromosome
3 monosomy was assessed by univariate linear regression at months
3, 6, and 12 following plaque radiation therapy (R version 3.1.0).
Results: Of the 150 patients, 75 patients were excluded because
treatment (enucleation and resection, 48) or location of the tumor
(irido ciliary, 16 ) or lack of cytological confirmation (11) of the
diagnosis. At time of diagnosis the mean tumor height was was 5.15
mm (1.90-13.00mm). Percent chromosome 3 monosomy ranged
from 0-20% (35) to 81-100 (10%). The percentage of tumor height
at months 3, 6, and 12 did not statistically correlate with tumor
location (ciliray or choroidal), initial tumor height, and percentage
chromosome 3 monosomy (Figure 1).
Conclusions: Regression rate in height of uveal melanoma following
brachytherapy does not correlate with the percentage composition of
tumor cells with chromosome 3 monosomy.
Commercial Relationships: Hassan A. Aziz, None; Sachin Salvi,
None; Nakul Singh, None; Suhail Dar, None; brandy hayden,
None; Arun D. Singh, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 5347 Poster Board Number: A0196
Presentation Time: 8:30 AM–10:15 AM
Sorafenib in metastatic uveal melanoma : A multicentric
prospective phase II study
Frederic Mouriaux1, Vincent Servois2, Sophie Piperno-Neumann2,
Thierry Lesimple3, Antoine Thyss4, Thomas Jouary5, Eve-Marie
Neidhart-Berard6, Nicolas Penel7, Corinne Delcambre8, Françoise
Joly8. 1Ophthalmology, CHU, Rennes, France; 2Institut Curie, Paris,
France; 3Centre Eugène Marquis, Rennes, France; 4Centre Antoine
Lacassagne, Nice, France; 5Dermatology, CHU, Bordeaux, France;
6
Centre Antoine Berard, Lyon, France; 7Centre Oscar Lambret, Lille,
France; 8Centre François Baclesse, Caen, France.
Purpose: Uveal melanoma is the most common primary ocular
malignancy in adults, accounting for 70 % of all eye malignancy.
Despite adequate local treatment, about 30-50 % of patients develop
metastasis and the survival rate is 50% after 10 years and the median
survival time is 5-7 months after development of metastasis. There is
no consensual and effective treatment in metastatic uveal melanoma.
Preclinical data suggest that Sorafenib, a multikinase inhibitor of
cell proliferation and angiogenesis may be a potential treatment of
metastatic uveal melanoma. The primary objective of this study was
the non-progression rate at 6 months with 800 mg/day of Sorafenib in
patient with metastatic uveal melanoma.
Methods: Multicenter, non-comparative, non-randomized, phase II
study was conducted. The primary objective was to determine the
non-progression rate at 6 months for patients receiving Sorafenib at
dose of 400 mg twice per day orally according the RECIST criteria.
Quality of Life and adverse events were evaluated. Secondary
efficacy endpoints included progression-free survival, overall
survival, quality of life and adverse events
Results: 32 patients were enrolled in this study and 29 patients were
evaluable. Non-progression at 6 months was observed in 11 patients
(38%). Non-progression at 12 months was observed in 4 patients
(13%). The overall survival was 10 months. Fatigue was a frequent
adverse event and limited the quality of life.
Conclusions: Sorafenib at dose of 400 mg twice per day orally
showed non-progression rate at 6 months in some patients with
metastatic uveal melanoma
Commercial Relationships: Frederic Mouriaux, None; Vincent
Servois, None; Sophie Piperno-Neumann, None; Thierry
Lesimple, None; Antoine Thyss, None; Thomas Jouary, None;
Eve-Marie Neidhart-Berard, None; Nicolas Penel, None; Corinne
Delcambre, None; Françoise Joly, None
Clinical Trial: N° EudraCT : 2010-022527-29
Program Number: 5348 Poster Board Number: A0197
Presentation Time: 8:30 AM–10:15 AM
Unsuspected and misdiagnosed ciliary body and choroidal
melanoma after enucleation and evisceration: review of cases in
the Ottawa-Gatineau region from 1996-2012
Solin Saleh1, 2, Seymour Brownstein1, 2, André Jastrzebski1, 2, David
Jordan1, Steven Gilberg1, Brian Leonard1, Bernard Hurley1.
1
Ophthalmology, University of Ottawa, Ottawa, ON, Canada;
2
Pathology, University of Ottawa, Ottawa, ON, Canada.
Purpose: There is little in the recent literature on the topic of
clinically unsuspected and misdiagnosed ciliary body and choroidal
melanomas with a calculation of the frequency of these events for a
set geographical area. We present 2 cases unexpectedly diagnosed
with a melanoma of the ciliary body and choroid after enucleation
and evisceration, respectively, and 2 cases of clinically misdiagnosed
ciliary body and choroidal melanoma. As the only ophthalmic
pathology laboratory in our region serving a population of 1,236,300,
we determined the rate of these outcomes over a period of 16 years.
Methods: We carried out a retrospective single centred, population
based case review in the Ottawa-Gatineau region. We reviewed
3,320 total cases, of which 98 were ciliary body and choroidal
melanomas. Four cases were included in the study group. We
calculated the frequency of clinically unsuspected melanoma of the
ciliary body and choroid in enucleated and eviscerated eyes and of
clinically misdiagnosed melanoma of the ciliary body and choroid in
enucleated eyes in our region. We also determined the rate of uveal
melanoma undergoing enucleation in our region by calculating the
annual population and the mean number of cases within this area over
a 16-year-span.
Results: Out of 98 diagnoses of ciliary body and choroidal
melanoma, we identified 2 cases where the diagnosis was clinically
unsuspected. We also identified 2 cases clinically misdiagnosed as a
ciliary body or choroidal melanoma, with one of these being a nonneoplastic lesion. In our region, 2.0% of ciliary body and choroidal
melanomas were clinically unsuspected and 2.0% of clinically
diagnosed ciliary body and choroidal melanomas were misdiagnosed.
We also calculated the rate of uveal melanoma undergoing
enucleation in our region of 5.4 cases/1,000,000 population/year.
Conclusions: We present the first and only single centred, population
based data on the rates of unsuspected and misdiagnosed ciliary body
and choroidal melanoma in the current era of modern diagnostic
imaging. Unsuspected melanoma of the ciliary body or choroid is
a rare occurrence, with a rate significantly lower than previously
published. The rate of clinically misdiagnosed ciliary body or
choroidal melanoma at our centre is within the range of recent reports
of this encounter.
Commercial Relationships: Solin Saleh, None; Seymour
Brownstein, None; André Jastrzebski, None; David Jordan, None;
Steven Gilberg, None; Brian Leonard, None; Bernard Hurley,
None
Program Number: 5349 Poster Board Number: A0198
Presentation Time: 8:30 AM–10:15 AM
First description of a melanoma-associated paraneoplastic
syndrome with retinopathy and cochleopathy
Armin Mohi1, Mahdy Ranjbar1, Salvatore Grisanti1, Henning
Frenzel2, Martin Rudolf1. 1Department of Ophthalmology, University
of Luebeck, Luebeck, Germany; 2Department of ENT, University of
Luebeck, Luebeck, Germany.
Purpose: Melanoma-associated retinopathy (MAR) is a known
paraneoplastic syndrome in some patients with cutaneous
malignant melanoma. It is characterized by the presence of serum
auto-antibodies (IgG) against retinal proteins, affecting retinal
photoreceptor function and leading to retinal damage. While a
reaction of melanoma-associated auto-antibodies against various
neuronal proteins is already known, an association with symptoms of
other sensory organs has not been published yet. In this case-report
we are able to demonstrate, that a symptomatic cochleopathy due to
paraneoplastic serum auto-antibodies can be present in patients with
cutaneous malignant melanoma and MAR.
Methods: A 60 year old women with cutaneous malignant melanoma
and visual affection was presented in our hospital for further
diagnostics and treatment. She reported flickering photopsias and
night-blindness. We were able to detect a reduction of visual acuity,
a distinct loss of the visual field and reduced b-waves in the ERG.
MAR-auto-antibodies were detected in an independent referencecentre. An advanced staging examination revealed a metastatic
recurrence of the melanoma. Noteworthy was an early development
of a tinnitus. We initiated an immune-suppressive therapy with
prednisolone, performed weekly clinical examinations and took
blood samples in a 14 days interval. The blood samples were used
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
for indirect-immune-fluorescence on macaque retina and cochlea
sections, to detect auto-antibodies.
Results: We were able to observe a quick improvement of BCVA
and field of view after one month of prednisolone treatment, while
the b-waves in the ERG were still reduced. The tinnitus remained
unchanged. A cerebral MRI showed no affection of the auditory
pathway as an explanation for the tinnitus. The indirect-immunefluorescence showed a specific pattern, staining the cell-bodies of
photoreceptors and hair-cells. Under therapy this pattern showed
a reduction of intensity. Controls with immune sera of healthy
individuals showed no staining at all.
Conclusions: To our knowledge, we demonstrated for the first time
the presence of specific auto-antibodies against hair cells of the
cochlea which was associated with MAR. Together with the tinnitus
it is the first description of symptomatic melanoma-associated
cochleopathy (MAC).
Commercial Relationships: Armin Mohi, None; Mahdy Ranjbar,
None; Salvatore Grisanti, None; Henning Frenzel, None; Martin
Rudolf, None
527 New insights in myopia
Thursday, May 07, 2015 12:00 PM–1:45 PM
2C/3C Mile High Blrm Paper Session
Program #/Board # Range: 5841–5847
Organizing Section: Anatomy and Pathology/Oncology
Contributing Section(s): Biochemistry/Molecular Biology
Program Number: 5841
Presentation Time: 12:00 PM–12:15 PM
Rates of change of ocular properties during emmetropization in
the developing chick eye vary linearly with retinal blur
Melanie C. Campbell1, 2, Zheng Shao1, 2. 1Physics & Astronomy,
University of Waterloo, Waterloo, ON, Canada; 2School of Optometry
and Vision Science, University of Waterloo, Waterloo, ON, Canada.
Purpose: Emmetropization is an active process involving retinal
feedback. We examine changes in ocular properties during
emmetropization in the normal chick eye to determine which changes
are proportional to the retinal blur due to defocus.
Methods: Mean ocular refraction (spherical equivalent, MOR),
dioptric length (Kʹ), eye power, pupil radius and cornea and lens
powers were calculated from literature values of ocular components
(including our results). They and their rates of change were then fitted
versus MOR, rate of change in optical axial length (OAL-cornea to
retina) and calculated retinal blur due to defocus. The relationships
of corneal radius, corneal and lens powers to other parameters and to
angular (EB) and linear retinal blur (LRB) were examined.
Results: Because of its exponential decrease with age during
emmetropization, the rate of change of MOR (D/day) varies linearly
with MOR. However, it also varies approximately linearly with
retinal blur (both EB and LRB; p<0.001) before emmetropization is
complete (up to day 30). During emmetropization, the rate of increase
of OAL decreases, as a linear function of decreasing retinal blur (EB
p=0.004 and LRB p<0.02). This relationship breaks down around
the time that emmetropization is complete (~day 30). Similarly,
during emmetropization, the rate of increase in corneal radius varies
linearly with retinal blur (EB p=0.002 and LRB p<0.01). The rate of
change in lens power is almost proportional to the rate of change of
retinal blur (p<0.0001) before the completion of emmetropization. As
expected from the above results, the rate of change of corneal radius
(p<0.0001) and lens power (p<0.0001) are proportional to the rate of
change of OAL during emmetropization.
Conclusions: In chick, emmetropization occurs with a rate of ocular
elongation proportional to the size of the hyperopic defocus blur
on the retina until about day 30. When retinal blur approaches cone
spacing, it no longer decreases, MOR is stable and many relationships
above no longer apply during slower eye growth to day 75. Thus
emmetropization appears to be driven by the angular defocus blur
directly causing proportional changes in OAL, corneal radius and
lens power until blur reduces sufficiently and emmetropization is
complete. Many of these results are consistent with findings during
emmetropization in children but here we show a direct link to retinal
blur.
Commercial Relationships: Melanie C. Campbell, None; Zheng
Shao, None
Support: NSERC Canada 35321
Program Number: 5842
Presentation Time: 12:15 PM–12:30 PM
The Effect of Discontinuous Wear of 2-zone Concentric Bifocal
Spectacle Lenses on Refractive Error Development & Eye
Growth in Young Chicks
Nevin W. El-Nimri, Christine F. Wildsoet. Vision Science, UC
Berkeley, Berkeley, CA.
Purpose: To characterize in young chicks the myopia control effects
of two-zone, distance or near center concentric multifocal lens
designs worn on an alternate day schedule interleaved with standard
single vision corrective lenses.
Methods: Monocular -10 Diopter (D) single vision (SV) lenses were
worn by ten-day-old chicks for 4 days to induce myopia, after which
the SV lenses were replaced by one of 2 multifocal lens designs (-10
D center/ -5 D periphery, MFDC) or -5 D center/ -10 D periphery
(MFDP)) every other day for a total of 6 days. A control group wore
-10 D SV lenses every day. A minimum of 4 birds was included in
each treatment group. Refractive error and axial ocular dimensions
were monitored every three days with retinoscopy and high frequency
A-scan ultrasonography respectively.
Results: The MFDC lens had greater myopia control effects than
MFNC lenses although both groups recorded less myopia than the
SV group and intergroup differences in on-axial dimensions were
consistent with refractive errors. Mean baseline interocular optical
axial length differences (± SD) were 0.29 ± 0.14 mm (SV), 0.26 ±
0.14 mm (MFNC), and 0.32 ± 0.17 mm (MFDC). After 6 days of lens
wear, equivalent values were 0.46 ± 0.27 mm (SV), 0.39 ± 0.19 mm
(MFNC), and 0.28 ± 0.17 mm (MFDC). Mean interocular refractive
error differences (± SD) on day 6 were -12.18 D ± 1.77 (SV), -11.01
± 1.14 D (MFNC), and -7.60 ± 0.97 D (MFDC) compared to baseline
values of -12.32 ± 0.95 D (SV), -12.17 ± 1.56 D (MFNC), and -12.48
± 0.41 D (MFDC).
Conclusions: The results demonstrate that discontinuous wear of
multifocal lenses, e.g., every other day, interleaved with standard
single vision correction of refractive errors, may be sufficient to
control the progression of myopia.
Commercial Relationships: Nevin W. El-Nimri, None; Christine
F. Wildsoet, None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Program Number: 5843
Presentation Time: 12:30 PM–12:45 PM
Melanopsin knock-out mice have abnormal refractive
development and increased susceptibility to form-deprivation
myopia
Ranjay Chakraborty1, 3, Duk C. Lee1, 3, Erica G. Landis1, 3, Michael A.
Bergen1, 3, Han na Park1, Curran Sidhu1, Samer Hattar5, P M. Iuvone1,
2
, Richard A. Stone4, Machelle T. Pardue3, 1. 1Ophthalmology, Emory
University School of Medicine, Atlanta, GA; 2Pharmacology, Emory
University, Atlanta, GA; 3Rehab R&D Center of Excellence, Atlanta
VA Medical Center, Decatur, GA; 4Ophthalmology, University of
Pennsylvania School of Medicine, Philadelphia, PA; 5Departments
of Biology and Neuroscience, Johns Hopkins University, Baltimore,
MD.
Purpose: Melanopsin, a photopigment in intrinsically photosensitive
retinal ganglion cells (ipRGCs), is involved in regulating visual
signaling, retinal dopaminergic cell activity, and circadian activity.
Here, we examined the contribution of melanopsin to normal
refractive development and to visual form-deprivation (FD) in a
mutant mouse lacking melanopsin (Opn4-/-).
Methods: Refractive development of Opn4-/- and age-matched wildtype (WT) mice, both on mixed C57BL/6 and 129S1/Sv background,
were measured every 2 weeks from 4 to 16 weeks of age. Weekly
measurements were performed on a separate cohort of mice that
underwent monocular FD in the right eye from 4 weeks of age using
head-mounted diffuser goggles. Refraction, corneal curvature, and
ocular biometry were obtained using photorefraction, keratometry
and 1310 nm spectral-domain optical coherence tomography.
Results: Under normal visual conditions, Opn4-/- mice (n=10) were
significantly more myopic than WT mice (n=10) at early ages (mean
refractive error at 4 weeks, Opn4-/-: -3.56 ± 0.35 D; WT: +0.94 ± 0.35
D, p<0.05), and they became relatively more hyperopic than the WT
mice by the end of 16 weeks (Opn4-/-: +6.53 ± 0.25 D, WT: +4.52 ±
0.64 D, p<0.05). The axial length of Opn4-/- mice (mean at 12 weeks,
3.29 ± 0.01 mm) was significantly shorter (p<0.001) than that of WT
animals (3.35 ± 0.01 mm). After 3 weeks of FD, goggled Opn4-/- mice
(n=6) showed a significant myopic shift (difference between the right
and left eyes) of -4.08 ± 0.89 D compared to non-goggled controls
(n=6, + 0.43 ± 0.52 D, p<0.05). No significant refractive shift was
observed in WT mice goggled for 3 weeks (-1.65 ± 0.65 D). Also,
Opn4-/- mice showed a significant increase (p<0.05) in axial length
with FD at 3 weeks (difference of right and left eyes) of 0.03 ± 0.01
mm in comparison with WT animals (-0.01 ± 0.004 mm). There were
no significant differences in corneal curvatures of Opn4-/- and WT
mice under either normal or FD conditions.
Conclusions: Our findings suggest that melanopsin signaling
pathways contribute to normal refractive development, and the
development of FD myopia in mice. Future studies are needed
to determine other mechanisms underlying abnormal ocular
development in Opn4-/- mice, and the extent to which these findings
reflect an interaction of refractive development and circadian biology.
Commercial Relationships: Ranjay Chakraborty, None; Duk C.
Lee, None; Erica G. Landis, None; Michael A. Bergen, None; Han
na Park, None; Curran Sidhu, None; Samer Hattar, None; P M.
Iuvone, None; Richard A. Stone, None; Machelle T. Pardue, None
Support: NEI Grant EY022342, NEI Grant EY004864, NEI Grant
EY016435, NIH Core grant P30EY006360, Research to Prevent
Blindness Grant and Mackall Foundation Trust
Program Number: 5844
Presentation Time: 12:45 PM–1:00 PM
Expression of soluble Bmp antagonists from the zebrafish RPE
leads to eye enlargement and myopia
Ross F. Collery, Brian A. Link. Cell Biology, Neurobiology and
Anatomy, Medical College of Wisconsin, Milwaukee, WI.
Purpose: Bmp signaling is involved in early eye development, but
negatively regulates growth in the adult eye. bugeye zebrafish lacking
the large endocytic receptor Lrp2, which is expressed in the RPE
and binds Bmp4, show grossly enlarged eyes and high myopia. Here
we investigate the effects of expressing antagonists to Bmp (Nog3,
Grem2) as well as a soluble domain of Lrp2 found to bind Bmp4, and
analyze the effects on eye size and refractive state.
Methods: An RPE-specific promoter was used with the bipartite
Gal4/UAS system to express Nog3, Grem2, and the first LDLA1
domain of Lrp2. Zebrafish expressing transgenes and control siblings
were imaged using a Bioptigen SD-OCT system at 2 months of age,
measuring eye axial length, retinal radius and lens diameters. Using
these metrics, eye sizes were normalized and degrees of refractive
errors were calculated.
Results: Zebrafish expressing Nog3 from the RPE showed enlarged
eyes and increased myopia compared to their control siblings. lrp2-/zebrafish show enlarged and myopic eyes relative to wild-types, and
this phenotype is exacerbated with RPE-driven Nog3 expression.
Similarly, expression of Grem2 from the RPE also increased eye
size and degree of myopia, but only in lrp2-/- zebrafish. Finally we
demonstrated that Bmp4 binds in vivo to the LDLA1 extracellular
domain of Lrp2, and found that expression of soluble Lrp2 (LDLA1)eGFP from the RPE also increased eye size and degree of myopia.
Conclusions: Bmp antagonists exert their effect by binding
extracellular ligands and prevent them from activating their cognate
receptors, thus modulating signaling. We propose that overexpression
of Bmp antagonists in the RPE leads to a large, myopic eye
phenotype in part by inhibiting Bmp signaling. In ongoing
experiments, we will investigate whether Lrp2 is endogenously
cleaved to generate a soluble LDLA1-containing ectodomain, and
examine whether such putative processing alters its effects on Bmp
signaling during emmetropization and with myopia.
Commercial Relationships: Ross F. Collery, None; Brian A. Link,
None
Support: NIH/NEI RO1EY016060; P30EY001931
Program Number: 5845
Presentation Time: 1:00 PM–1:15 PM
Increased retinaldehyde dehydrogenase activity during recovery
from induced myopia
Angelica Harper1, Gennadiy P. Moiseyev2, Jody A. Summers1. 1Cell
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK; 2Physiology, University of Oklahoma Health Sciences
Center, Oklahoma City, OK.
Purpose: Previous studies have identified significant increases in
retinaldehyde dehydrogenase 2 (RALDH2) steady state mRNA
in the chick choroid during recovery from myopic defocus. Thus,
RALDH2 may be responsible for the increase in choroidal all-transretinoic acid (atRA) synthesis observed during recovery from myopic
defocus. The present study examines RALDH activity and RALDH2
protein expression in control and recovering chick ocular tissues
to further elucidate the link between RALDH2, atRA, and visually
guided ocular growth.
Methods: Myopia was induced in chicks by form deprivation for 10
days, followed by up to 15 days of recovery. Retina/RPE, choroid,
and/or sclera were isolated from control (not treated) and recovering
(treated) eyes at various points during recovery. RALDH catalytic
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
activity was measured in tissue homogenates using an in vitro atRA
synthesis assay together with HPLC quantification of synthesized
atRA. RALDH2 protein expression in control and recovering
homogenates was compared by western blotting.
Results: RALDH activity was increased in recovering choroids as
compared to controls by 66.5% (p > 0.01, paired t-test) at day 3 of
recovery and by 49.7% (p > 0.05, paired t-test) at day 7 (n = 6). When
RALDH activity in control and day 4 recovering retina/RPE, choroid,
and sclera was compared, RALDH activity could only be detected in
the choroid (n = 9). Western blot analyses indicated that RALDH2
protein expression was highest in the choroid and minimal in retina/
RPE and sclera. Choroidal RALDH2 expression was 281.1% greater
(p > 0.05, paired t-test) in recovering eyes as compared with controls
(n = 3).
Conclusions: These results demonstrate that the choroid is the
major ocular tissue exhibiting RALDH activity, which is responsible
for increased RALDH activity during recovery. These increases
correspond to increases in choroidal RALDH2 protein expression,
suggesting that increased RALDH activity in the recovering choroid
is due to increased RALDH2 protein expression. These results
suggest that RALDH2 may be useful as a therapeutic target for the
treatment of myopia.
Commercial Relationships: Angelica Harper, None; Gennadiy P.
Moiseyev, None; Jody A. Summers, None
Support: NEI Grant R01 EY09391
Program Number: 5846
Presentation Time: 1:15 PM–1:30 PM
RNA sequencing of sclera from form-deprived guinea pigs
identifies multiple signalling pathways underlying myopia
Nethrajeith Srinivasalu1, 2, Sally A. McFadden2, Callan Medcalf2,
Gayle Philip3, Michael Zhang1, Paul N. Baird1. 1Centre for Eye
Research Australia, University of Melbourne, Royal Victorian Eye
and Ear Hospital., Melbourne, VIC, Australia; 2School of Psychology,
University of Newcastle., Callaghan, NSW, Australia; 3Victorian Life
Sciences Computation Initiative, Melbourne, VIC, Australia.
Purpose: Remodelling of the posterior sclera accompanies
myopia, and in young guinea pig eyes, early changes occur in the
peri-papillary zone (PPZ) around the optic nerve. We examined
the differential expression of genes in the PPZ sclera to identify
significant pathways associated with myopia.
Methods: One eye of guinea pigs (n=3) was form-deprived for 2
weeks (day 6-20) using a translucent diffuser to induce myopia.
The other eye was untreated and served as a matched control. At the
end of 2 weeks, refractive error was measured in cyclopleged eyes
using a Nidek auto-refractor. Posterior scleral punches (4 mm) were
collected from the PPZ from myopic and control eyes. Total RNA
was extracted using an RNeasy® Fibrous tissue mini kit. RNA was
sequenced by a commercial provider using an Illumina HiSeq 2000
platform. RNA-seq data were analysed using three different statistical
programs (CuffDiff, edgeR and Voom) and the most differentially
expressed genes (p value < 10-3) were used to identify significant
pathways.
Results: Form-deprivation induced relative myopia in all animals.
The mean difference between myopic and control eyes was
-3.76±0.6 D at the end of the treatment period. The number of
genes differentially expressed between myopic and control sclera
were 26,088, 14,301, and 14,298 using CuffDiff, edgeR and Voom
analysis tools, respectively. Differences in gene expression (p<10-3)
were found for 850 (CuffDiff), 794 (edgeR) and 270 (Voom) genes
using the different analysis programs. Pathway analysis conducted on
significant genes identified more than 50 different pathways, of which
four have previously been reported as associated with myopia.
Conclusions: Number of differentially expressed genes were
identified in the posterior myopic sclera compared to non-myopic
eyes, which varied between analysis packages. Several signalling
pathways were consistently identified across the different analysis
programs with some of these having previously been identified as
associated with myopia in humans and animal models. These findings
provide insights into potential pathways involved in active scleral
remodelling. Further targeted investigation of these genes may
provide potential avenues for development of treatment therapies.
Commercial Relationships: Nethrajeith Srinivasalu, None; Sally
A. McFadden, None; Callan Medcalf, None; Gayle Philip, None;
Michael Zhang, None; Paul N. Baird, None
Support: NHMRC Senior Research Fellowship 1028444, Coal
Port Authority and Hunter Medical Research Institute, Melbourne
International Fee Remission Scholarship
Program Number: 5847
Presentation Time: 1:30 PM–1:45 PM
Eye Elongation and Retinal Degeneration in the IRBP Knockout
Mouse
Natecia Williams, Shannon Getz, Curran Sidhu, Jeffrey H. Boatright,
Machelle T. Pardue, P M. Iuvone, J M. Nickerson. Ophthalmology,
Emory University, Atlanta, GA.
Purpose: Interphotoreceptor retinoid-binding protein (IRBP) is
abundant in the subretinal space and binds retinoids and lipophilic
molecules. Despite initial expression at embryonic day 12 (E12),
the role of IRBP in eye development is unknown. Congenic IRBPdeficient mice (KO) show eye elongation starting at postnatal day 8
(P8), abnormal pruning indicated by reduced TUNEL-positive cell
death in the inner nuclear layer by P12, increased TUNEL-positive
cell death of photoreceptors at P23-P27, and profound myopia by
P30 followed by slow retinal degeneration (RD) (Wisard et al. IOVS.
2011; 52:5804-11). Based on experimental myopia (Stone et al.
PNAS 1989; 86:704-6), we propose KO mice will show a decrease in
dopamine (DA) and the DA metabolite, 3,4-dihydroxyphenylacetic
acid (DOPAC) and number of retinal dopaminergic cells (DA-cells)
coinciding with abnormal eye elongation, abnormal pruning of inner
retinal cells, and RD.
Methods: Using immunohistochemistry for tyrosine hydroxylase
(TH) on retinal flat mounts, we counted the DA-cells in retinas
of C57BL/6J (WT) and KO mice at P12-P30. HPLC was used to
analyze the amounts of DA and DOPAC at P12-P30. t-tests for agematched mice determined P-values.
Results: Although no difference was observed at P12, DOPAC levels
in KO retinas increased by 64% at P23 (N=9 WT, 8 KO, P < 0.001)
and 73% by P30 compared to WT mice (N=9 WT, 7 KO, P < 0.01).
Similarly, KO retinal DA levels increased by 25% at P23 (N=9 WT,
8 KO, P < 0.01) and 21% at P30 compared to WT mice (N=9 WT, 7
KO, P < 0.05). The number of retinal TH+ cells was 28% greater in
KO retinas compared to WT at P30 (N=4 WT, 4 KO, P<0.05).
Conclusions: Despite development of myopia, KO mice have
increased levels of DA and DOPAC contrary to the expectation that
high levels of DA should slow eye growth. The data would suggest
that myopia observed in the KO mice is atypical to refractive myopia,
independent of dopaminergic activity and visual input. The excess
of TH+ cells in the KO retina may result from insufficient pruning
of retinal DA-cells in the development of the KO inner retina. In
addition, this high dopaminergic activity seems to be characteristic of
KO retinas after P23, the age in which we begin to see a rise in cell
death in the KO retina, which may suggest a role for DA or DA-cells
in this RD.
Commercial Relationships: Natecia Williams, None; Shannon
Getz, None; Curran Sidhu, None; Jeffrey H. Boatright, None;
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology
Machelle T. Pardue, None; P M. Iuvone, None; J M. Nickerson,
None
Support: NIH Grant R01EY021592, NIH Grant R01EY016470,
NIH Grant T32EY007092, NIH Grant P30EY006360, NIH Grant
R01EY004864
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].