Download ARVO 2016 Annual Meeting Abstracts 206 Eye Cancer Biology and

ARVO 2016 Annual Meeting Abstracts
206 Eye Cancer Biology and Therapeutics
Monday, May 02, 2016 8:30 AM–10:15 AM
608 Paper Session
Program #/Board # Range: 1364–1369
Organizing Section: Anatomy and Pathology/Oncology
Program Number: 1364
Presentation Time: 8:30 AM–8:45 AM
Proteomic analysis of uveal melanoma (UM) secretome reveals
novel biological insights and potential biomarkers
Sarah E. Coupland1, 3, Sam Prendergast1, 3, Martina Angi3,
Deborah Simpson2, Robert Beynon2, Helen Kalirai1, 3. 1Molecular
and Clinical Cancer Medicine, University of Liverpool, Liverpool,
United Kingdom; 2Centre for Proteomics, University of Liverpool,
Liverpool, United Kingdom; 3LOORG, University of Liverpool,
Liverpool, United Kingdom.
Purpose: Proteins secreted or shed from cancer cells, the “cancer
secretome”, represent an important class of bioactive molecules
thought to play a causal role in cancer progression. We investigated
the UM secretome as a source of biomarkers of early metastatic
disease and its capacity to provide novel insights into tumour biology.
Methods: Secretomes of short-term UM cultures established
from patient tumours with a high- (HR; n=10) or low- (LR; n=4)
metastatic risk were analysed using nanoLC-MS/MS-based label-free
quantitative proteomics. They were compared with the secretomes
of normal cultured choroidal melanocytes (NCM; n=5). Protein
identification was performed using Proteome Discover v3.1 and
protein quantification was undertaken using Progenesis®v2.0.
Bioinformatics analyses applied SecretomeP.v2.0, SignalP.v4.1,
Exocarta.v5 and Ingenuity® Pathway Analysis (IPA).
Results: Initial analysis produced a dataset of 1843 proteins, which
were common to all three analysed subgroups (HR, LR, NCM).
Of these, 758 proteins with ≥3 unique peptides were identified.
These 758 proteins could be subdivided into: 25% classically/
non-classically secreted, 46% exosomal proteins, and 29% other.
Taking only the 538 proteins into account that were secreted by
classical, non-classical or exosomal mechanisms, IPA suggested that
they have biological involvement in cell proliferation, growth and
movement. Further, IPA highlighted top canonical signalling pathway
involvement of these proteins, particularly associated with hepatic
fibrosis/hepatic stellate cell activation and the mTORC1-S6K axis.
Finally, CCN Family Member 3 and Neuropilin 2, both previously
shown in cancer to promote metastasis, were highly upregulated in
HR vs LR UM.
Conclusions: UM secretome analysis identified cancer-associated
proteins likely to represent important biomarkers of disease and/or
play a functional role in disease progression. Exosomes are a likely
mode of local and distal communication in UM.
Commercial Relationships: Sarah E. Coupland, None;
Sam Prendergast, None; Martina Angi, None; Deborah Simpson,
None; Robert Beynon, None; Helen Kalirai, None
Program Number: 1365
Presentation Time: 8:45 AM–9:00 AM
Host Microenvironment Shapes Uveal Melanoma Tumor
Properties
Matthew W. McEwen1, Qing Zhang2, Bradley Gao1, Hua Yang2,
Zachary Goldsmith1, Hans E. Grossniklaus2, Matthew W. Wilson1, 3,
Vanessa M. Morales1, 4. 1Ophthalmology, University of Tennessee
Health Science Center, Memphis, TN; 2Ophthalmology, Emory
University, Atlanta, GA; 3Surgery, St. Jude Children’s Research
Hospital, Memphis, TN; 4Microbiology, Immunology and
Biochemistry, University of Tennessee Health Science Center,
Memphis, TN.
Purpose: Approximately 50% of patients diagnosed with uveal
melanoma (UM) will develop liver metastases. Metastasis may be
present at the time of initial diagnosis in the form of micrometastases.
The pathways by which these UM micrometastases transform
into clinically detectable metastases are not well understood. By
comparing UM cells cultured in vitro with those recovered from an in
vivo orthotopic xenograft, our study tested the hypothesis that tumormicroenvironment interactions in the liver affect UM cell properties.
Methods: Athymic nude mice were inoculated intraocularly with Mel
270, a primary UM cell line known to metastasize. Livers and spleens
were recovered and used to prepare ex vivo single cell suspensions.
Mel 270 and its corresponding metastatic cell line OMM 2.5 were
used as in vitro controls. Quantitative PCR (qPCR) was performed
to determine relative gene expression among the tumor cells using
HPRT1 controlled delta delta Ct analysis. Cell culture supernatants
were examined using multiplex assay to quantify levels of secreted
proteins from tumor cells.
Results: qPCR analysis of genes associated with cytoskeletal
signaling, angiogenesis, and cell death and survival revealed a
liver-specific (compared to spleen) upregulation of pro-angiogenic
molecules, including VEGFA (79.2-fold) and the transcription factor
HIF1A (72.2-fold). Our work also revealed an in vivo, liver-specific
blockade of the tumor suppressor TP53 (141.2-fold) in UM tumor
cells by HDM2 (102.7-fold) and HDMX (96.0-fold) sequestration.
Multiplex analysis of human secreted proteins revealed a high
concentration of IFN-α2 (344.09 pg/mL) specific to the liver sample.
VEGF concentration was elevated in both liver and spleen samples
(377.17 pg/mL and 191.27 pg/mL, respectively).
Conclusions: Our data show a host-dependent, liver-specific
upregulation of several UM tumor genes and cytokines. This is
consistent with our hypothesis that interaction with the host liver
microenvironment plays a role in shaping UM tumor properties.
Further study is needed to characterize the host tissue mediators.
Commercial Relationships: Matthew W. McEwen, None;
Qing Zhang, None; Bradley Gao, None; Hua Yang, None;
Zachary Goldsmith, None; Hans E. Grossniklaus, None;
Matthew W. Wilson, None; Vanessa M. Morales, None
Support: Research to Prevent Blindness, SJCRH Endowment, West
Cancer Center, NIH Medical Student Research Fellowship Program
at UTHSC
Program Number: 1366
Presentation Time: 9:00 AM–9:15 AM
Deciphering the molecular basis of periocular infiltrative basal
cell carcinoma
John Bladen1, Michèle Beaconsfield2, Edel O’Toole1,
Michael Philpott1. 1Centre for Cell Biology and Cutaneous Research,
Blizard Institute, London, United Kingdom; 2Moorfields Eye
Hospital, London, United Kingdom.
Purpose: Basal cell carcinoma (BCC) is the commonest cancer
worldwide and constitutes the vast majority of eyelid tumours.
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ARVO 2016 Annual Meeting Abstracts
Infiltrative BCC (iBCC) is a particularly aggressive subtype, which
invades local tissue and adjacent structures in an uncompromising
manner. Little is known about the genetics of iBCC and the
differences that render it more aggressive compared to the more
benign nodular (nBCC) subtype
Methods: Fresh frozen tissue was collected from 20 BCC patients;
10 iBCC and 10 nBCC subtypes. Laser capture microdissection and
nucleic acid extraction was performed. Whole exome sequencing of
20 BCC tumour and blood-matched controls were undertaken along
with RNA sequencing of 3 iBCC and 3 nBCC tumour and stromamatched controls. Differential expression (DE) from normal eyelid
was deemed significant if P<0.01, Log2FC >1 or <-1. Quantitative
RT-PCR and protein immunohistochemistry was performed for
validation.
Results: For iBCC and nBCC, the average tumour mutational
burden was 1533 and 2073, and UV signature was 88% and 85%,
respectively. PTCH1 mutations were present in 80% of IBCC and
60% of nBCC. Novel iBCC driver included EPHA3 in 3 out of
10 patients. DE revealed 288 and 276 genes for iBCC and nBCC
compared to normal, respectively. When both subtypes were
compared, 128 genes were differentially expressed, with the majority
upregulated in iBCC including LAMB3 and EPHB4. Shared genes
included VCAN and GPC3. Immunohistochemistry confirmed
Hedgehog (Hh) pathway expression, but to a much greater extent
in iBCC including the surrounding non-tumour stromal tissue. Two
key novel activated pathways were detected; axonal guidance and
extracellular matrix (ECM) receptor interaction pathways.
Conclusions: Despite a reduced mutational burden in iBCC, the
presence of significant driver mutations may explain its aggressive
nature. Furthermore, the hyper-expression of the Hh pathway
compared to nBCC including surrounding non-tumour stromal tissue
may aid its local migration. Discovery of novel axonal guidance and
ECM receptor interaction pathways could also play a role in this
behaviour. Regardless of trends, the extent of tumour heterogeneity
demands personalised genetic mapping of the tumour to direct
developing novel treatment modalities such as inhibitors of EPHA3,
VCAN, Gli1/2 and EPHB4.
Commercial Relationships: John Bladen; Michèle Beaconsfield,
None; Edel O’Toole, None; Michael Philpott, None
Support: Fight For Sight UK
Program Number: 1367
Presentation Time: 9:15 AM–9:30 AM
Successful treatment of recurrent ocular surface squamous
neoplasia (OSSN) with topical cidofovir
Matthew H. Ip1, 2, Robert George3, William Rawlinson1, 3,
Minas T. Coroneo2, 1. 1Faculty of Medicine, University of New South
Wales, Sydney, NSW, Australia; 2Department of Ophthalmology,
Prince of Wales Hospital, Sydney, NSW, Australia; 3South Eastern
Area Laboratory Services, Prince of Wales Hospital, Sydney, NSW,
Australia.
Purpose: Despite the success of topical medical treatments,
particularly with Interferon alpha-2b in treatment of OSSN, there is
a failure rate of ~2%. A previous study2 showed OSSN responds to
cidofovir (CDV), although our data showed no herpes viruses present
in OSSN1. We have previously demonstrated that 6.5% of OSSN
specimens are HPV-positive1, and considered the possibility that
patients unresponsive to interferon may be HPV-positive and respond
to antiviral therapy with CDV.
Methods: A single center retrospective observational case series
was performed to evaluate the efficacy of topical CDV for treatment
of recurrent OSSN in 7 eyes of 7 patients. Each patient had been
previously diagnosed with OSSN utilizing slit-lamp examination
confirmed with histopathology, and had failed other interventions
including Interferon alpha-2b. Each patient was treated with 2.5mg/ml
TDS topical CDV for a period of 4-6 weeks. Patients were then
reviewed clinically with slit-lamp examination and photography to
look for recurrence.
Results: Topical CDV was effective in 6 out of 7 eyes treated, with
improved clinical appearance. After 1-month of CDV treatment,
each of the 6 eyes demonstrated a marked reduction in mass size.
Furthermore, no residual scar tissue was visualized (Figure 1 and 2).
The mean follow-up with no clinical relapse is currently 6.4 months,
with a range of follow-up of 1-12 months. In 1/7 cases we detected
high risk HPV in a biopsy specimen using a hybrid capture assay.
A further 5/7 were negative for high risk HPV using PCR, and 1/7
remains untested.
Conclusions: Topical CDV appears to be effective for treatmentrefractive OSSN in the short-medium term. The efficacy of CDV
raises the possibility of a viral etiology of OSSN in cases resistant to
treatment with Interferon Alfa-2b.
1. Woods M et al. Cornea 2013;54:8069-8078.
2. Sherman MD et al. Am J Ophthal 2002;134:432-3.
Figure 1. The initial slit-lamp photograph of the right nasal
conjunctiva of one patient.
Figure 2. A comparative clinical photograph of the right nasal
conjunctiva 12 months after biopsy and topical cidofovir
administration for 6 weeks.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Matthew H. Ip, None; Robert George,
None; William Rawlinson, None; Minas T. Coroneo, None
Program Number: 1368
Presentation Time: 9:30 AM–9:45 AM
PDGF-PDGFR Signaling Sustain Angiogenesis in an Autocrine
and Paracrine Fashion in Retinoblastoma
Matthew W. Wilson1, 2, Zachary Goldsmith1, William Coppess1,
Bradley Gao1, Matthew McEwen1, Andrew Irvine1,
Rachel C. Brennan3, 1, Vanessa M. Morales1. 1Ophthalmology, Univ
of Tennessee Health Sci Ctr, Memphis, TN; 2Surgery and Pathology,
St Jude Children’s Research Hospital, Memphis, TN; 3Oncology,
St Jude Children’s Research Hospital, Memphis, TN.
Purpose: Purpose: The presence of vitreous seeding is considered
a poor prognostic factor for ocular survival in Retinoblastoma (Rb)
patients. External beam radiotherapy and chemotherapy have failed to
increase ocular survival rates beyond 70% for eyes with vitreous seeding
at the time of diagnosis, in part due to the presence of dormant tumor
cells. Platelet derived growth factor is highly abundant in the vitreous
microenvironment, especially in patients suffering from proliferative
retinal disorders. We hypothesize reduction of the PDGF-PDGFR
signaling may control vitreous seeds in Rb.
Methods: Methods: We used the Rb cell lines Y79 and Weri-1
as they represent the metastatic and non-metastatic forms of the
disease. Cells were cultured with or without PDGF-AB as primary
stimulus and evaluated for the expression of angiogenesis-related
genes, production of angiogenic factors, and morphological
changes by qPCR, Multiplex assays and confocal microscopy. Next,
we evaluated how PDGF regulates Rb tumor proliferation and
angiogenesis via autocrine and paracrine signaling by modulation
of the PDGFR-b by imatinib mesylate (IM) and a neutralization
antibody against the PDGF-BB isoform.
Results: Results: We found high expression of the PDGFA and
PDGFB isoforms in Rb cells by qPCR analysis. Western blot,
Multiplex and flow cytometry analyses revealed a reduction in the
PDGF-AB/BB isoforms (p=0.04), FLT-3L (p=0.02), and VEGF
(p=0.08) after concomitant incubation of IM and recombinant human
PDGF-AB (rhPDGF-AB), when compared to untreated and rhPDGFAB stimulation. We observe striking differences in cellular migration
both in 2D and 3D culture models when Rb cells were treated with
IM. Additional studies utilizing the neutralizing antibody increased
the magnitude of the angiogenic reduction.
Conclusions: Conclusions: Our work suggests PDGF-PDGFR
signaling sustain angiogenesis in an autocrine and paracrine fashion.
These studies are a step in the pursuit of our goal of development
of identifying targets of immunotherapy that will control the factors
sustaining Rb.
Commercial Relationships: Matthew W. Wilson, None;
Zachary Goldsmith; William Coppess, None; Bradley Gao,
None; Matthew McEwen, None; Andrew Irvine, None;
Rachel C. Brennan, None; Vanessa M. Morales, None
Support: Research to Prevent Blindness and St Jude Endowed Chair
in Pediatric Oophthlamology
Purpose: HER2 (ERBB2), a member of the epidermal growth factor
family, is overexpressed in variety of malignancies including breast,
ovarian, gastric, colorectal, pancreatic and endometrial cancers.
Overexpression and gene amplification of HER2 is associated with
aggressive malignancies accompanied by chemoresistance and poor
prognosis. HER2 has been successfully targeted in breast cancer
therapy by humanized anti-HER2 antibody trastuzumab (Herceptin)
and an antibody drug conjugate trastuzumab-DM1 (Kadcyla).
In this study, we tested the hypothesis that HER2 is expressed in
retinoblastoma (RB), a childhood malignancy of the retina, and can
be targeted by anti-HER2 antibody based therapeutics.
Methods: To test the hypothesis that HER2 is expressed in
retinoblastoma, we compared HER2 expression in four RB cell lines
(Y79, WERI-RB27, RB143 and RB116) vs. breast cancer cell lines
(BT474 and MDA-MB231), RB patient samples and breast cancer
tumor arrays, as well as normal human ocular tissues by a variety of
methods, including immunocytochemistry, flow cytometry, western
immunoblot and RT-PCR. We utilized PCR primers that would detect all
HER2 variants, as well as a variety of antibodies against HER2-specific
epitopes. Once HER2 expression was confirmed, we tested the cytotoxic
effect of trastuzumab and trastuzumab conjugates (T-vc-MMAE [DAR4
and DAR8]) on RB cells in vitro using an MTS assay.
Results: HER2 expression was established in RB by flow cytometry,
RT-PCR, immunohistochemistry of RB cell lines and an RB tumor
tissue array. HER2 was not immunoreactive in normal ocular
tissues. Western immunoblot analysis suggested a possible HER2
truncation in RB. This truncation spared the trastuzumab binding site
and allowed us to test the cytotoxic effects of trastuzumab and its
conjugates (T-vc-MMAE [DAR4 and DAR8]) on RB cells in vitro.
Our data suggested that while trastuzumab itself was not cytotoxic in
vitro, T-vc-MMAE was very effective in killing RB cells, where the
DAR8 ADC (IC50=0.03-0.04 μM) was more potent than the DAR4
ADC (IC50=0.1-0.3 μM).
Conclusions: For the first time, we have reported on the expression
of HER2 in retinoblastoma and the potential for anti-HER2 based
therapy. Our discovery of HER2 expression in RB may lead
to innovative and targeted drug treatment options with a wider
therapeutic index and fewer systemic side effects, designed to spare
the eye and preserve vision in RB patients.
Commercial Relationships: Gail M. Seigel, None; Sharad Sharma,
None; Abigail Hackam, None; Dhavalkumar Shah, None
Support: GMS is supported by the Cornell Center on the
Microenvironment & Metastasis through Award Number
U54CA143876 from the National Cancer Institute, as well as a grant
from the SUNY Brain Network of Excellence. ASH is supported
by the Karl Kirchgessner Foundation. Institutional support to
Bascom Palmer Eye Institute was provided by a Research to Prevent
Blindness Unrestricted Grant and an NEI Center Core Grant P30
EY014801. SS is supported by a Postdoctoral Fellowship grant
from Roche Inc. to DKS. This work was in part supported by NIH
grant GM114179 to DKS, and funding from the Center for Protein
Therapeutics at the State University of New York at Buffalo.
Program Number: 1369
Presentation Time: 9:45 AM–10:00 AM
HER2: A novel therapeutic target for retinoblastoma
Gail M. Seigel1, 2, Sharad Sharma3, Abigail Hackam4,
Dhavalkumar Shah3, 2. 1Ctr for Hearing & Deafness, University
at Buffalo, Buffalo, NY; 2SUNY Eye Institute, Buffalo, NY;
3
Pharmaceutical Sciences, University at Buffalo, Buffalo, NY;
4
Bascom Palmer Eye Institute, University of Miami, Miami, FL.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.