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HSANZ POSTERS
P173
Philadelphia Positive ALL in Pregnancy - Successful Outcome with
Antenatal Chemotherapy Followed by Single-cord Haematopoetic Stem
Cell Transplant
Dustin Hall, Julian Cooney, Stephanie P’Ng
Royal Perth Hospital, Western Australia
Aim
We present the case of a 34 year old female who was diagnosed with Philadelphia
positive B Acute Lymphoblastic Leukaemia at 22 weeks gestation. Challenges
including limited clinical evidence regarding safety of therapeutic agents during
pregnancy and long-term disease control make regime selection difficult.
Results
The patient received 3 cycles of hyperfractionated Cyclophosphamide, Doxorubicin,
Vincristine and Prednisolone (Arm A HyperCVAD without intrathecal methotrexate,
reduced dose steroid) chemotherapy at 3 weekly intervals, combined with Imatinib
600mg daily. This achieved normalisation of peripheral blood counts and a bone
marrow BCR-abl/ABL% of 0.308.
At 34 weeks gestation the patient was induced and gave birth to a healthy boy via a
normal vaginal delivery.
Four weeks post-partum the patient received her first Arm B of HyperCVAD, and
subsequently completed six cycles (four A and two B arms) in combination with
imatinib therapy Four months post-partum the patient successfully underwent an
unrelated single-cord haematopoetic stem cell transplant with a cyclophosphamide
plus total body irradiation conditioning regime.
Now five months post transplant the patient is well with a peripheral blood and bone
marrow BCR-abl/ABL% of 0.000. At age nine months, her son is well with no
apparent complications of his mother’s antenatal therapy.
Conclusion
This challenging case has demonstrated a successful outcome with imatinib and
chemotherapy during the second and third trimesters of pregnancy for Philadelphia
positive B Acute Lymphoblastic Leukaemia, with subsequent allograft.
Keywords
Pregnancy, Leukaemia, Chemotherapy
Conflict of interest No conflict of interest to disclose
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HSANZ POSTERS
P174
Lenalidomide Synergises with Azacitidine to Enhance Effector Cytokine
Production by T Cells in Acute Myeloid Leukaemia Patients
Chindu Govindaraj1, Peter Tan2, Andrew Spencer2, Andrew Wei2 and Magdalena
Plebanski1
1
Department of Immunology, Central Clinical School, Monash University,
Melbourne, VIC, Australia. 2Department of Clinical Haematology, The Alfred
Hospital, Monash University, Melbourne, VIC, Australia
Background
Immunomodulatory drugs (iMiDs), Lenalidomide (Len) and Azacitidine (Aza) are
effective treatments for haematological malignancies including Acute Myeloid
Leukaemia (AML). The combined effect of the above drugs on the remission immune
system, particularly on T cells have not been previously investigated.
Aim
To investigate the combined effect of Len and Aza on T cell subsets in acute myeloid
leukaemia patients in complete remission after chemotherapy.
Methods
AML patients were treated with Aza subcutaneously on days 1-5 for each 28-day cycle
combined with Len orally on days 5-25, as a maintenance therapy. Four patients were
treated with Aza alone as a control cohort. Peripheral blood samples were obtained at
the end of each cycle and the levels and function of T cells were compared to initial pretreatment samples. Flow cytometry was performed to determine levels of T cells.
PBMCs were stimulated with PMA/Ionomycin followed by incubation with Brefeldin A to
determine effector cytokine production by T cells.
Results
No significant differences in CD3+ cell numbers were observed between patients and
healthy donors. CD4+ T cells were significantly lower, whilst CD8 T cells were higher in
AML patients when compared to healthy donors. CD4 and CD8 T cells from patients
with AML in remission produced lower levels of effector cytokines like IL-2. Upon
treatment, patients receiving Aza alone had significantly higher increases in intracellular
IL-2 in both CD4 and CD8 T cells at the end of cycle 3. In patients who received Aza
and Len, significantly higher levels of IL-2 production were noted at the end of cycle 1.
Conclusion
This study suggests that T cells remain abnormal in AML patients even when in
remission. Our results suggest beneficial effects from Azacitidine and lenalidomide on
effector T cell function that occur within 1 month of therapy. The correlation between
these immunological changes and clinical outcome is ongoing.
Keywords
IL2, Azacytidine, Lenalidomide
Conflict of interest No
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HSANZ POSTERS
P175
Clinical Outcomes of Allogeneic Stem Cell Transplantation for non-Hodgkin
Lymphoma with Combined MYC and BCL2 Translocations
Tasman Armytage1, Barbara Withers1, Keith Fay1,2, Luke Coyle1, Ian Kerridge1,
Chris Arthur1, Naomi MacKinlay1, William Stevenson1, Chris Ward1, Matthew
Greenwood1
1
Department of Haematology, Royal North Shore Hospital, Sydney, Australia
2
Department of Haematology, St. Vincent’s Hospital, Sydney, Australia
Aim
B-cell non-Hodgkin lymphoma with concurrent translocations of MYC and BCL2 has
recently been identified as a distinct clinical entity characterised by aggressive
clinical course and poor response to therapy. We determined the clinical outcomes
of patients with MYC+/BCL2+ ‘double hit’ lymphoma who received allogeneic
transplantation following chemotherapy.
Methods
All patients who were referred to our institution between 2005 and 2012 and
diagnosed with B-cell non-Hodgkin lymphoma with concurrent MYC+/BCL2+
translocations and undergone allogeneic transplantation were and analysed by
medical and database records.
Results
Of 16 patients treated for ‘double hit’ non-Hodgkin lymphoma between 2006 and
2012, 7 patients underwent allogeneic transplantation with mean age 53 years (3665), with 6/7 (86%) attaining CR prior to transplant. 4/7 (57%) patients received
reduced intensity conditioning, and 3/7 (43%) had matched unrelated donors. Acute
and chronic graft-vs-host disease rates were 6/7 (86%) and 5/7 (71%) respectively.
DFS and OS for those undergoing allogeneic transplantation was 86% with a
median post-transplant follow-up of 456 days (60-1999). OS was 33% for
untransplanted patients with a median follow-up of 133 days.
Conclusion
MYC+/BCL2+ ‘double hit’ lymphoma is a heterogeneous disease entity with
generally poor treatment response and rapid progression. This retrospective
analysis of a series of patients treated with chemotherapy followed by allogeneic
stem cell transplant demonstrates the utility of transplantation for long–term disease
free survival.
Keywords Double hit lymphoma, allogeneic stem cell transplantation, MYC
Conflict of interest No
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HSANZ POSTERS
P176
Analysis of Patients with Relapse of Acute Myeloid Leukaemia after
Allogeneic Stem Cell Transplantation: Post-relapse Graft-versus-Host
Disease and Prior T cell Depletion Are Associated with Better Outcome
Andrew Lim1, Kate Fielding1, Cameron Curley2, Kate Mason1, Marnie Collins3, Jeff
Szer1, Glen Kennedy 2, David Ritchie1
1
Royal Melbourne Hospital, Parkville, Vic, Australia. 2Royal Brisbane and Women’s
Hospital, Herston, Qld, Australia. 3Centre for Biostatistics and Clinical Trials, East
Melbourne, Vic, Australia
Aim
To describe the management and outcome of salvage therapies in patients with
relapse of acute myeloid leukaemia (AML) after allogeneic haematopoietic stem cell
transplantation (alloHSCT), and to evaluate factors that predict improved event-free
survival (EFS) from time of relapse. An event was defined as failure to achieve
further remission, a further relapse after achieving remission, or non-relapse death.
Results
Between the years 2000 and 2011, 135 patients transplanted for AML (median age
47, 45% female, RMH n=63, RBWH n=75) relapsed at a median of 130 days postalloHSCT. 88 patients received salvage: 40 had donor cell-based salvage (second
alloHSCT n=31, donor lymphocyte infusion n=9), while 48 did not (interferon n=6,
immunosuppression withdrawal n=5, fludarabine plus cytarabine n=20, other
therapies n=17). Outcome data were available for 81 patients (median age 45, RMH
n=46, RBWH n=35, median follow-up 215 days), with 2-year EFS of 15%. From
univariate analysis, improved EFS was predicted by T cell depletion in the original
allo-HSCT, remission duration greater than 6 months post-alloHSCT, donor cellbased salvage, and post-salvage graft-versus-host disease (GVHD). From
multivariate analysis, GVHD post-salvage (HR 0.26, 95%CI 0.14-0.48, p < 0.001)
and T cell depletion (HR 0.19, 95%CI 0.06-0.64, p=0.007) remained predictive for
superior EFS.
Conclusion
The presence of post-salvage GVHD, and prior in vivo T cell depletion, are
associated with improved outcome from salvage therapy in patients with relapse of
AML after alloHSCT. These findings underline that in this situation, salvage should
incorporate therapies directed at inducing a graft-versus-leukaemia effect.
Keywords
allogeneic transplantation, acute myeloid leukaemia, relapse
Conflict of interest
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No conflict of interest to disclose.
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HSANZ POSTERS
P177
Outcomes Compared between Reduced Intensity and Myeloablative
Conditioning for Allogeneic Transplant in Acute Myeloid Leukaemia
Barbara Withers, Kelly Wong, Chris Arthur, Keith Fay, William Stevenson, Ian
Kerridge, Matthew Greenwood
Department of Haematology, Royal North Shore Hospital, Sydney, Australia
Aim
RIC transplant is increasingly offered to older, poorer performance status patients,
as a potentially curative therapy for acute myeloid leukemia (AML). Data suggests
similar overall survival for patients offered MAC vs RIC transplantation in AML. We
reviewed the outcomes of patients undergoing RIC vs MAC transplant for AML at
our centre.
Results
Between 2000 and 2012, 51 patients underwent allogeneic transplant for AML (49%
MAC, 51% RIC). 51% of patients were female, and 25.5% aged >60yrs (18-67y).
Donor source was matched sibling in 42 pts, matched and mismatched unrelated in
8, and umbilical cord blood in 1. Most patients were in CR1 (60.8%).
Busulfan/Cyclophosphamide conditioning was the most common MAC (41.2%), and
Fludarabine/ Melphalan the most common RIC (33.3%). Acute graft versus host
disease (GVHD) occurred in 43.1% of pts, and chronic GVHD in 47.1% of pts. Acute
GVHD was seen in 40% MAC pts, and 46.2% of RIC pts (p=NS). Chronic GVHD
was noted in 48% MAC pts, and 46.2% of RIC pts (p=NS). Relapse rates in MAC vs
RIC were 32% vs 19.2% (p=NS). Overall 5 year survival and disease-free survival
for the cohort was 57%, and 50% respectively. There was no significant difference in
survival outcomes between the MAC vs RIC group.
Conclusion
The outcomes and survival of AML patients offered different conditioning regimes
for allogeneic transplant were not significantly different. RIC broadens access to
potentially curative therapy, with equivalent outcomes to MAC in our centre.
Keywords Allogeneic transplant, AML, Reduced intensity conditioning
Conflict of interest No conflict of interest to disclose
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HSANZ POSTERS
P178
Outcome of Acute Myeloid Leukaemia in the Era of Molecular Diagnostics –
A Single Institution Experience
Nagendra prasad Sungala, Silvia Ling, Michael Harvey, David Rosenfeld, Lindsay
Dunlop, Penelope Motum, Anne-Marie Watson, Nicholas Viaala, Danny Hsu,
Samantha Day, Lyelin Ho
Department of Haematology, Liverpool Hospital, Sydney
Aim
In 2008, molecular testing was not available in this region of NSW was not readily
available. Hence molecular testing was established,
1. To improve prognosis of local AML patients at Liverpool Hospital.
2. To set up molecular testing that can be used in diagnostic setting
Results
There were 59 patients with AML between June 2008 and December 2010. On
univariate analysis, age (P<0.001), chemotherapy (P<0.001), LDH (P=0.002), blast
percentage (P=0.076), and cytogenetics (P<0.001), were statistically significant.
On the multivariate analysis, only age (P=0.047), chemotherapy (P<0.001), and
cytogenetics (P<0.001), were statistically significant. However, there was an
interaction between age and chemotherapy (P=0.005), indicating that older patients
were less likely to receive chemotherapy and more likely to receive best supportive
care. There were 8 patients in the good cytogenetic risk group, 29 in the
intermediate group and 21 in the poor risk group. 18 among the 29 patients in the
intermediate group had normal karyotype. Among the patients with normal
karyotype, 10 patients were negative for both FLT3-ITD and NPM1 genetic
mutations. 5 patients had both FLT3-ITD and NPM1 mutations. 2 patients had FLT3ITD mutation and were negative for NPM1. Only one patient had NPM1 genetic
mutation without FLT3-ITD. Patients with NPM1 mutation had trend towards better
survival than those without NPM1 mutation, and patients with FLT3-ITD genetic
mutations show trend towards poor survival than those without FLT3-ITD mutations,
but not statistically significant. This was probably due to small numbers.
Conclusion
This study confirms the importance genetic mutation testing in AML and ideally
would be performed in all new patients. In transplant eligible patients, these
molecular markers will help select patients in the standard and intermediate risk
group who would benefit from allogeneic stem cell transplantation. In transplant
ineligible patients, the molecular markers may help to choose the right category of
patients who will benefit from intensive chemotherapy.
Keywords
AML NPM1 FLT3-ITD Conflict of interest No
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176
HSANZ POSTERS
P179
Abbreviated HypeCVAD Chemotherapy Combined with Long Term
Imatinib Can Result in Prolonged Survival in de-novo Ph Positive Acute
Lymphoblastic Leukaemia
R Wooldridge, P Tsang, G Seeley, T Cochrane
Department of Haematology, Gold Coast Hospital, Qld, Australia
Introduction
Philadelphia positive acute lymphoblastic leukaemia (Ph+ALL) is the most frequent
genetic abnormality seen with ALL and the incidence rises with age. The dismal
outcome of Ph+ALL has been tempered by incorporating tyrosine kinase inhibitors
into standard chemotherapy. The Hyper CVAD regimen consists of 8 cycles of
alternating doxorubicin, vincristine, dexamethasone, cyclophosphamide with
methotrexate and cytarabine. This regimen has successfully been combined with
tyrosine kinase inhibitors in Ph+ ALL, producing long term survivors in the absence
of allogeneic transplant. Unfortunately due to advanced age, toxicity development or
co-morbidity a significant proportion of patients is unable to complete a full course of
HyperCVAD – Imatinib based chemotherapy
Aim
The aim of this report was to ascertain whether abbreviated HyperCVAD
chemotherapy in combination with Imatinib could result in prolonged survival.
Methods
This was a retrospective, single centre report from Gold Coast Hospital from 20092012. All data were obtained from the medical record.
Results
We describe 2 patients with Ph+ALL who have achieved prolonged survival with
abbreviated HyperCVAD chemotherapy in combination with Imatinib. The first
patient, a 62yo female, received only 3 cycles of HyperCVAD due to severe sepsis.
She remains alive and in complete molecular remission on imatinib 26 months post
diagnosis. The second patient, a 65yo male, received only 4 cycles of HyperCVAD
due to decline in performance status and liver impairment. He is maintained on
600mg of Imatinib and is in a complete haematological remission with a 4 log
reduction in transcript level at 14 months post diagnosis.
Conclusion
Prolonged survival is possible in patients with Ph+ ALL who are unable to complete
a full course of standard chemotherapy, provided long term Imatinib is maintained.
Keywords
Imatinib, Ph+ALL, HyperCVAD
Conflict of interest No
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HSANZ POSTERS
P180
Immunophenotype Negative AML
R Wooldridge1, M Bryson1, T Cochrane1, B Willimas2, C McCarthy3, M Self3,
J Wellwood1
1
Department of Haematology, Gold Coast Hospital, 2 Royal Brisbane Hospital,
3
Pathology Queensland
We report a case of a 66 year old male with acute leukaemia with
immunophenotype negative blasts. The case was associated with a diagnostic delay
and is noteworthy due to poor response to therapy. He presented with a 1 month
history of lethargy and dyspnoea. Testing revealed anaemia and significant
thrombocytopenia. His medical history was significant for obesity and a clipped
cerebral aneurysm.
Marrow showed marked megaloblastic dyserythropoesis, megakaryocytic dysplasia
and blasts (20%) with cytoplasmic blebbing and no Auer rods. Flow cytometry was
negative for any clonally restricted population with no significant blast population
identified. Parvo virus PCR was negative as was peripheral blood serology. The
Trephine showed erythroid left shift and increased numbers of immature cells.
Immunohistochemistry showed CD 117 uptake in the blasts, with minor
subpopulations expressing CD34 and FVIII. Molecular markers were negative, and
Cytogenetics demonstrated a complex karyotype with amplification of MLL in 12.5%
of cells and hemizygous deletion of TP53 in 6% of cells detected by FISH. The
consensus diagnosis was AML with myelodysplasia related change.
Standard Induction therapy for AML was completed uneventfully with post Induction
marrow showing morphological remission, with blasts less than 5%. MRD analysis
by flow cytometry was non-contributory however FISH showed low level persistence
of the MLL amplification (4%).
Post consolidation marrow showed morphologic relapse with 35% blasts showing
the same morphological appearance as the diagnostic marrow. Flow cytometry was
again negative with only a small population of normal myeloblasts. FISH identified
increased MLL amplification (21%) consistent with early relapse. The patient was
not considered fit for transplant and a palliative course was undertaken.
This case highlights the importance of morphological diagnosis of acute leukaemia,
in an era of increasing use of ancillary testing to define and risk stratify cases.
Cytogenetics was abnormal in both diagnosis and MRD assessment however flow
cytometry was not and based on this case we may postulate that immunophenotype
negative AML could be seen as a poor prognostic factor.
Keywords
Immunophenotype, AML, Cytogenetics
Conflict of interest No
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HSANZ POSTERS
P181
MLL-AF9 Mouse Models Identify Key Molecular Targets for the
Treatment of This Acute Leukaemia (AL)
A Baker, I Verbrugge, K Stanley, B Martin, M Ghisi, E Gerace, J Shortt, R Johnstone
Cancer of Immunology, Gene Regulatory Laboratory, Peter MacCallum Cancer
Centre, Melbourne
The mixed lineage leukaemia (MLL) mutation accounts for 60-80% of all infant acute
leukaemias and is classified as aggressive tumours that require high amounts of
poly-chemotherapy and still result in a poor prognosis outcome. Genetically,
paediatric and adult acute leukaemic patients are characterized by distinct genetic
rearrangements of the MLL gene, located at the 11q23 chromosomal band (Alonso,
Longo et al. 2008). MLL has been found in 73 different translocations and 64 partner
genes have been cloned (Meyer, Kowarz et al. 2009). Only 6 the frequent partner
proteins constitute 85% of all clinical cases of MLL, one of which is AF9 (Wang, Wu
et al. 2011). Fusion partner proteins (FPs) are generally transcriptional activators
that induce ectopic expression of target genes in hematopoietic precursor cells.
MLL-FP’s are capable of leukemic transformation and dysregulation of multiple
genes, often through the aberrant recruitment of epigenetic modifying enzymes such
as histone deacetylases and methyltransferases.
Using retroviral gene transduction of hematopoietic stem cells we will express MLLFP AF9 and aim to produce mice that develop AML driven by this rearrangement.
These mice will be utilized to study disease onset and progression and to determine
the oncogenic potential of the MLL-FP. Moreover, the AML cells from these mouse
models will be used to test the efficacy of epigenetic modifying agents that inhibit
both histone deacetylases and histone methyltransferases. Furthermore, as MLLFP’s recruit a transcriptional elongation complex with CDK9 as the key enzymatic
component, CDK9 inhibitors will be tested to determine its oncogenic potential within
the MLL complex.
These studies will contribute to a better understanding of the biology and the
tumorigenic process driving MLL-AF9 associated acute leukaemias and to the
identification of more targeted therapies for this aggressive class of leukaemias.
Keywords
Mixed lineage leukaemia (MLL), HDAC, CDK9
Conflict of interest This research was supported by MERCK. The company had
no role in analysing the data or preparing the abstract.
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HSANZ POSTERS
P182
A Genome-Wide Analysis of the Transcriptional Changes in t(8;21) AML Blasts
following Treatment with Panobinostat
Jessica Salmon, Michael Bots, Ben Martin, Kym Stanley, Inge Verbrugge, Ricky
Johnstone
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
Gene transcription is tightly controlled by the activity of chromatin modifiers such as
histone acetyltransferases (HATs) and histone deacetylases (HDACs).
Inappropriate gene expression due to altered function of HATs or HDACs has been
detected in several haematological malignancies including acute myeloid leukemia
(AML).
The t(8;21) translocation is one of the most common karyotypic abnormalities found
in AML and results in a transcriptionally active fusion protein, AML1-ETO, which
aberrantly recruits HDACs to AML1/ RUNX1 target promoters. This results in the
deacetylation of histones, inappropriate repression of target genes and an
accumulation of myeloid blasts in the blood, bone marrow and spleen. HDAC
inhibitors (HDACi) are chemotherapeutic agents that have been shown to induce
histone hyperacetylation, alter gene expression and mediate diverse anti-tumour
effects such as induction of apoptosis, cell-cycle arrest, differentiation and
senescence. We have examined the therapeutic effect of panobinostat, a multiHDACi, using a clinically relevant mouse model of t(8;21) AML. Treatment of mice
bearing AML1-ETO-driven tumours with panobinostat results in a significant
decrease in tumour burden and a clear survival advantage.
Gene expression changes in tumour cells treated with panobinostat in vivo were
analysed by RNA-sequencing. These results showed a signature consistent with cell
cycle arrest. To further investigate the effects of HDACi on the regulation of cell cycle
progression and myeloid differentiation we treated AML1-ETO AML cells in vitro with
panobinostat and analysed alterations in potential downstream target genes by qRTPCR. Together, these analyses revealed a restored expression of myeloid lineageassociated transcription factors Scl, GATA2 and CEBPα and an increase in the cellcycle inhibitors CDKN1a and CDKN2a.
These studies provide evidence for the rational use of HDACi as a differentiation
therapy for t(8;21) AML. Inhibition of HDAC activity does not cause tumour cell
apoptosis but rather induces cell cycle arrest, myeloid differentiation and a decrease
in tumour burden through the reactivation of critical target genes.
Keywords
AML, HDAC-inhibitors, RNA-sequencing
Conflict of interest This research was supported by Novartis. The company had
no role in analysing the data or preparing the abstract.
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HSANZ POSTERS
P183
Inhibition of Human Haematological Malignant Cell Lines by Capsaicin
is not TRPV1-Mediated
Sofia Omari, Dominic Geraghty, Dale Kunde and Murray Adams
School of Human Life Sciences, University of Tasmania, Tasmania, Australia
Aim
Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel
activated by a variety of endogenous and exogenous stimuli, including the major
active component of ‘hot chilli peppers’, capsaicin. Recent evidence suggests that
capsaicin induces apoptosis and inhibits cell proliferation, although this has not
been extensively investigated in haematological malignancies. The aims of this
study were to: 1) investigate whether capsaicin kills human haematological
malignant cells, and if so, 2) whether this action was TRPV1-mediated.
Methods
THP-1 (acute monocytic leukaemia), U266B1 (myeloma) and U937 (histiocytic
lymphoma) cells were exposed to increasing concentrations of capsaicin (8-1000
µM) in the presence and absence of TRPV1, and cannabinoid 1 and 2 receptor
(CB1, CB2; 0.1-100 µM) antagonists. Cell metabolic activity (indicative of viability)
was measured after 24hrs using the alamarBlue® method (resazurin reduction
assay).
Results
Capsaicin reduced viable THP-1, U266B1 and U937 cell numbers in a
concentration-dependant manner. A biphasic effect was observed on THP-1 cells
[EC50 and IC50(95% CI) = 32.9(19.9-54.3) and 219(144-246) µM]. SB452533 and
AM251 (100 µM) suppressed the capsaicin-induced increase in THP-1 cell activity
(P<0.001). U266B1 cells were more resistant to capsaicin than THP-1 and U937
cells. Cell activity was significantly inhibited by capsaicin in U937 compared to U266
cells (IC50: 197 vs. 431 µM, respectively, P<0.008). AM251 and SB452533 appeared
to act as partial agonists and displayed a synergistic effect with capsaicin in U937
cells.
Conclusion
THP-1, U266B1 and U937 cells responded differently to capsaicin. TRPV1, CB1 and
CB2 antagonists did not affect capsaicin-induced changes in U266B1 cell activity
although CB1 and CB2 receptors appeared to mediate an increase in cell activity in
THP-1. We conclude that capsaicin inhibits the viability of haematological malignant
cells through a non-TRPV1-dependent mechanism.
Keywords: Capsaicin, TRPV1, Haematological malignancy.
Conflict of interest: No.
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HSANZ POSTERS
P184
Identification and Analysis of Oncogenic Pathways in Deletion 20q
Acute Myeloid Leukaemia
Matthew Ku1,2, Nisha Narayan1,2, Ruth Mackinnon3, Meg Wall3, Lynda Campbell1,3,
Harshal Nandurkar1,2
1
Department of Medicine, University of Melbourne, Melbourne, Australia
2
Immunology Research Centre, St Vincent’s Hospital, Melbourne, Australia
3
Victorian Cancer Cytogenetics Services, Melbourne, Australia
Aim
The oncogene and tumor suppressor gene (TSG) identified in patients with deletion
20q acute myeloid leukaemia [del(20q)AML] are tested in the laboratory to confirm
their oncogenic potential. We are analysing the oncogene Haemopoietic cell kinase
(HCK) in causing AML, on the background of the loss of the TSG Protein tyrosine
phosphatase receptor type T (PTPRT).
Method
Haemopoietic stem cells (HSC) are isolated from the bone marrows of wild type and
PTPRT-null mice by FACS sorting for Lineage negative, C-kit and Sca-1 positive
cells (LKS+). Isolated LKS+ HSC will be transduced by either the retroviral construct
of HCK or the vector control. These cells are then used in in vitro assays such as
methylcellulose assay and STAT3 antibody assay to assess features of malignancy.
These cells will also be used for in vivo reconstitution where they are transplanted
into recipient mice and AML formation is assessed. Kaplan-Meier curves will be
used to assess the survival times of these recipient mice.
Result
1.HCK mRNA expression increased in del(20q)AML
2.PTPRT mRNA expression is reduced in del(20q)AML
3.HCK amplification and PTPRT loss confer higher methylcellulose colony numbers
4.HCK causes STAT3 hyperphosphorylation in the PTPRT-null HSC
5.Direct interaction between HCK and PTPRT raising the possibility that they are
substrates for each other
Conclusion
Our preliminary data reveal a possible novel oncogenic cascade: HCK amplification
and PTPRT loss may cooperate to cause del(20q)AML directly, or by aberrant
transcriptional activity of hyperphospho-STAT3.
Keywords – Leukaemogenesis, oncogene, TSG
Conflict of interest - No conflict of interest to disclose.
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HSANZ POSTERS
P185
A Case of Mixed Phenotype Acute Leukaemia with Burkitt-like
Morphology and Expression of MPO in Conjunction with Typical B Cell
Antigens
Yasmin Harvey, Tennille Pelly, Tee Beng Keng, Helen Wordsworth
Department of Haematology, Sullivan Nicolaides Pathology
Aim
To describe a case of Mixed Phenotype Acute Leukaemia with unusual
morphologic, immunophenotypic and genetic features.
Results
We report the case of a 65 year old lady with mixed phenotype acute leukaemia.
The blast cell morphology was typical for Burkitt lymphoma/ leukaemia. The
immunophenotype was however, diagnostic of a mixed phenotype acute leukaemia
(MPAL) with expression of immature and B cell markers in conjunction with
myeloperoxidase (MPO) as the sole myeloid marker. FISH for translocations
involving the MYC gene was negative. The combination of Burkitt-like morphology
and B/Myeloid markers on immunophenotyping has previously been reported in only
a small number of cases.
Conclusion
This case highlights the utility of a multimodality approach to the diagnosis and
classification of acute leukaemia and that analysis of blast cell morphology can be
misleading.
Keywords
Mixed phenotype leukaemia
Conflict of interest
No
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HSANZ POSTERS
P186
Jacking Up Jak2: Resolving an IP Issue with High Resolution Melting
Chor Ee Tan1, Robyn Mardell1, Neil Van De Water2, Kenneth Romeril3, Julia
Philips3, Serena Rooker1,*
*Correspondence; 1Molecular Diagnostics, Wellington Hospital, New Zealand;
2
Labplus, Auckland Hospital, New Zealand; 3Haematology Department, Wellington
Hospital, New Zealand
Aim
The purpose of this study was to develop an in-house High Resolution Melting
(HRM) analysis assay to detect the Jak2 V617F mutation (c.1849G>T), a molecular
marker for myeloproliferative disorders. This method is a sensitive, specific and cost
effective alternative to commercially available real-time PCR kits. Prior to this study,
we used a commercial kit supplied by TIB MolBio. TIB MolBio were disadvantaged
when the patent for the Jak2 gene was imposed upon them by the exclusive
licensee, Ipsogen.
Method
We amplified an area of ~150 bp surrounding this mutation using the HRM Master
kit (Roche) on the LightCycler 480. We compared the results of 60 patients (N=25
positive, N=35 negative) who had been previously tested. Unlike the commercial
method that uses the melting profile of hybridisation probes, HRM detects single
nucleotide polymorphisms (SNP) by calculating the differences between melt
curves. A limitation of this method is the difficulty of HRM to detect homozygous
mutants. To address this, we analysed specimens in duplicate, spiking one with 1
µL of wildtype DNA control.
Results
The results of our in-house assay were concordant with those of the commercial kit.
25 patients carrying the mutation had a normalised & temperature-shifted difference
plot above the wildtype baseline. A further 35 patients who were wildtype had a
melting curve close to the wildtype baseline. One patient carrying a homozygous
mutation was identified through the spiked reaction. By spiking the reaction, we
resolved the atypical difference plot of a homozygous mutation by making the
reaction heterozygous.
Conclusion
We have implemented a HRM assay for routine diagnostic testing of the Jak2
V617F mutation which is as sensitive and specific as commercially available
methods. This assay will also detect rare variants in this region implicated in
haematological disorders. This assay provides a 96% reagent cost saving per test
for our district health board.
Keywords
Jak2, HRM, Myeloproliferative disorder
Conflict of interest No conflict of interest to disclose
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184
HSANZ POSTERS
P187
The Long and Winding Road: the Path to Standardisation of Quantitative BCRABL using Cepheid GeneXpert BCR-ABL Monitor
Neil Granter 1, Susan Dooley 1, Esther Leit1, Sharon Louise Way1, Anoop Enjeti 2
1
Genetics Department, Hunter Area Pathology Service, John Hunter Hospital, New
Lambton, NSW, Australia
2
Haematology Department, Calvary Mater Hospital, Waratah, NSW, Australia
Aim
To introduce an automated quantitative BCR-ABL testing in our laboratory and
participate in the International Standardization Program for quantitative PCR testing.
Method
In March 2010 trials of the GeneXpert BCR-ABL monitor kit from Diagnostic
Technology were commenced. This is an automated assay that performs RNA
extraction, cDNA production, nested real-time PCR and signal detection in a single
cartridge on whole blood samples.
In order to participate in the International Standardization program, it was necessary
for a minimum of 60 samples to be tested in parallel between our laboratory and
IMVS. These samples all had to be BCR-ABL positive and within the range 10%
BCR-ABL to 0.01%BCR-ABL. An initial batch of 30 samples was tested for
standardization in January and a second batch in May 2011.
Results
The standardization of the conversion factor was completed in April 2011.
A further 30 samples were sent in May 2011 to validate the correction factor.
The laboratory conversion factor was validated in June 2011. The Value obtained
was 0.43
qBCR-ABL testing as a service from HAPS laboratory was started in September
2011 and a total of 231 patient samples have been tested to end of MAY 2012. 5
tests needed to be repeated and a valid result was obtained when repeated.
Conclusion
The GeneXpert BCR-ABL monitor assay is automated, easy to perform with a rapid
turn-around time of QPCR results for the clinicians.
So although the journey was long and arduous the end result made it all worthwhile.
Keywords bcr abl, quantitative pcr, chronic myeloid leukemia
Conflict of interest No
185
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HSANZ POSTERS
P188
Audit of Genexpert Quantitative bcr-abl Assay
Asma Ashraf1, Neil Granter2, Susan Dooley2, Esther Leit2, Sharon Louise Way2,
Sam Yuen1, Philip Rowlings1, Anoop Enjeti1
1
Haematology Department, Calvary Mater Hospital, Waratah, NSW, Australia
2
Genetics Department, Hunter Area Pathology Service, John Hunter Hospital, New
Lambton, NSW, Australia
Aim
To assess the turn around time and error rate for an automated quantitative bcr-abl
assay using GeneExpert.
Method
A retrospective analysis of quantitative PCR for bcr-abl using an automated method
GeneExpert was undertaken. It included total 215 samples from 31st August 2011
until 29th May 2012. Turn-around time of reporting and error rates were evaluated.
Results
A total of 215 samples in were evaluated. The average turn-around was 2.45 days
and the range was 0-7days. There were a total of 4 errors in this period. These are
described as follows:
Error 1 and 2.High white cell at collection ( >80.0 x 10^9/L total white cell count)
caused a fail in the test. Test was repeated with a smaller volume of sample and
was successful.
Error 3. Possible operator error (new operator performing the test) caused a fail in
the test. Blood was recollected and test repeated.
Error 4. Test failed as sample processed 48hrs after sample was received. Delay in
testing as sample received late on friday afternoon. There is a protocol in place for
sample collection to be avoided on Fridays.
Conclusion
GeneExpert provides a rapid automated method of measuring quantitative bcr-abl
for Chronic Myeloid Leukemia patients. Although the sample is processed to give a
result on the GeneExpert instrument in 2 to 4 hours, a practical turn around time of
<3 days from the time of sample collection to the time of report issue is achievable
as demonstrated by our results. The error rate is low with 4 errors occurring in a 9
month period.
Keywords
bcr abl, quantitative pcr, chronic myeloid leukemia
Conflict of interest
II:186
No
186
HSANZ POSTERS
P189
Dendritic Cells from Myelodysplastic Patients Have Reduced Expression of
Co-stimulatory Antigens But Are Able to Stimulate Autologous T-cells
Glenda Mary Davison1, Nicolas Novitzky2, 3, Rygana Abdulla 2
1
Cape Peninsula University of Technology, Cape Town, South Africa. 2 University of
Cape Town, Cape Town, South Africa. 3 National Health Laboratory Service, South
Africa
Aim
Research has implied that the immune system plays a role in the pathogenesis of MDS
and is a possible cause of the cytopaenias and increased apoptosis observed in these
patients. Dendritic cells play a role in the immune response and as they are part of the
MDS clone, their ability to stimulate T-cells is important. The aim of this study was to
examine the interaction of mature dendritic cells with allogeneic and autologous T-cells
in a cohort of MDS patients.
Methods
Dendritic cells (MoDC) were generated in 6 MDS patients and 9 controls by culturing
peripheral blood monocytes with GM-CSF and IL-4. The immature MoDc were activated
using LPS and TNFα and analyzed for expression of co-stimulatory and activation
antigens. Thereafter, they were co-cultured with autologous and allogeneic T-cells. In
order to evaluate the T-cell response, the percentage change in expression of the
activation antigen CD69 was analyzed on CD3+, CD4+ and CD8+ T-cells.
Results
MDS MoDC had reduced expression of HLA-DR (p=0.006), CD11c (p=0.0004), CD80
(p=0.03) and CD86 (p=0.003), while resting T-cells from MDS patients, prior to culture
with the MoDC had higher expression of the activation antigen CD69 on CD3+
(p=0.009), CD4+ (p=0.05) and CD8+ (p=0.05) subsets. The % change in CD69
expression was significantly increased for both control and MDS T-cells after co-culture
with allogeneic dendritic cells, however this change was lower in the MDS group
especially in the CD3 (p=0.05) and CD8 (p=0.02) subsets. Nevertheless, despite the
increased CD69 expression prior to culture, MDS MoDC significantly up-regulated CD69
expression on autologous CD3+, CD4+ and CD8+ T-cells to values that were
statistically higher than control cells.
Conclusion
This study is in agreement that T-cells in MDS are able to respond normally to dendritic
cells and are therefore probably not part of the malignant clone. It further implies that
although myeloid dendritic cells are part of the malignant clone they are able to interact
with the T-cells and influence the immune response. In conclusion, the abnormal
activation of the immune system observed in MDS patients is complex and could be due
to other factors present in the malignant micro-environment.
Keywords
Myelodysplasia, dendritic cells, T-cells
Conflict of interest This research was supported by the National Health laboratory
Service, South Africa. The company had no role in analysing or preparing the abstract.
187
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HSANZ POSTERS
P190
Long Term Outcomes of Seven Day Consecutive 5’Azacitidine (Vidaza®)
Usage in a Regional Hospital
Caroline McNamara, Ian Irving, Edward Morris, Emily Wenta, Hock Choong Lai
Department of Haematology and Bone Marrow Transplantation, The Townsville
Hospital, Douglas, Qld, Australia
Aim
To evaluate the indications, tolerability and efficacy of seven day consecutive
5’azacitidine in a regional hospital.
Method
A retrospective chart review of all patients treated with 5’azacitidine at The
Townsville Hospital (TTH) between March 2009 and June 2012. Overall survival
was calculated using Kaplan-Meier estimates.
Results
5’azacitidine was prescribed to 25 patients, with a median age of 64 years (range:
24-77yrs) and 76% (19/25) were male. Indications included: intermediate-2 and high
risk myelodysplastic syndrome (MDS) (12 patients), acute myeloid leukaemia (AML)
with 20-30% blasts (5 pts), relapsed AML post autologous or allogeneic peripheral
blood stem cell transplant (5 pts), MDS post therapy for AML (2 pts) and acute
leukaemia of ambiguous lineage with less than 30% blasts (1 pt).
Patients with relapsed AML received a median of 5 months (4-6 months) of
5’azacitidine with all patients progressing and dying. Of the remaining 20 patients,
complete response (CR) was observed in 1 patient (5%), partial response (PR) in 8
patients (40%) and 3 patients (15%) had haematologic improvement. The median
duration of therapy for this group was 12 months (2-34 months). Only 1 patient
discontinued 5’azacitidine due to toxicity and 4 patients have died. The progressive
median overall survival for this group is 61%.
Conclusions
5’Azacitidine is well tolerated and effective for high risk MDS and AML with less than
30% blasts. In the relapsed AML setting, 5’azacitidine responses are not durable but
may provide a bridge to more definitive therapy. 5’Azacitidine administered on a 7day consecutive schedule is deliverable in a regional setting.
Keywords 5’azacitidine myelodysplasia leukaemia
Conflict of interest No conflict of interest to disclose.
II:188
188
HSANZ POSTERS
P191
Myelofibrosis Associated Pulmonary Hypertension with Resolution on
Treatment with Hydroxyurea. A Case Study and Review of the Literature
Helen McDougall, Ali Bazargan
St Vincent’s Hospital, Melbourne, Vic, Australia
We present a case of a previously well 59 year old woman who presented in right
heart failure with massive splenomegaly, anaemia and marked thrombocytosis. She
was found to have Myelofibrosis (IPSS: 3) and Pulmonary Hypertension (right
ventricular systolic pressure 37mmHg). She was commenced on Hydroxyurea,
which led to marked improvement of her symptoms as well as resolution of her
Pulmonary Hypertension.
The pathogenesis Myelofibrosis and Pulmonary Hypertension are not completely
understood and are both areas of ongoing development. There are several reported
cases of association between these conditions but few have reported improvement
in Pulmonary Hypertension with systemic treatment. Those that have reported some
response have generally been associated with marked thrombocytosis suggesting a
link between platelets and pulmonary hypertension either due to the development of
pulmonary microthrombi or by vasoconstriction or vascular remodelling due to
platelet derived factors such as Thromboxane A2 and Platelet Derived Growth
Factor.
Other proposed mechanisms are the development of pulmonary extramedullary
haematopoiesis, or pulmonary vascular remodelling effects from bone marrow
derived cytokines.
Due to the absence of pulmonary biopsy or Tc99m sulphur colloid scan we cannot
definitely say the cause of Pulmonary Hypertension in our patient. However we can
demonstrate improvement in this poor prognosis condition with early detection and
treatment, emphasizing the importance of suspecting Myelofibrosis in those with
new onset pulmonary hypertension and echocardiogram in those with Myelofibrosis.
Keywords
Myelofibrosis, Pulmonary Hypertension, Hydroxyurea
Conflict of interest No conflict of interest to disclose
189
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HSANZ POSTERS
P192
An Audit of Anagrelide Use and Outcome at the Gold Coast Hospital
Jason Restall, Tara Cochrane
Gold Coast Hospital, Southport, Queensland, Australia
Background
The phosphodiesterase inhibitor Anagrelide was approved by the US FDA in 1988
for use in myeloproliferative neoplasms. It selectively inhibits megakaryocytic
differentiation and proliferation via the thrombopoietin-mediated intracellular
signalling pathway. The aim of Anagrelide therapy is a reduction in platelets by
>50% or to an absolute count of <600x10^9/L in order to reduce the risk of
thrombocytosis-associated microvascular events. Long term studies have
demonstrated a 75% treatment efficacy with a reported adverse event rate between
8%-28% and an associated 10% discontinuation of therapy.
Patients and Methods
Pharmacy dispensing records from the Gold Coast Hospital identified 34 patients
who had been prescribed Anagrelide between 2009 and 2011. These patient
records were audited for patient characteristics and treatment outcomes.
Results
The audited group was 59% male, with an average age of 71. Primary diagnoses
were ET (14), PRV (11), PMF (5), MPN NOS (2) and CMML (2). 62% were
asymptomatic, 20% reported fatigue and 9% had a prior history of thrombosis. They
were treated for a median of 22 months (range 2 to 42) months with doses ranging
from 0.5mg daily to 3.5mg daily. Five (15%) were on concurrent hydroxyurea
treatment. The median pre-Anagrelide platelet count was 816x10^9/L which reduced
to a median 374x10^9/L on therapy. Six (18%) failed to achieve aforementioned
treatment goals. During therapy 4 (12%) patients reported new symptoms of
congestive heart failure and 1 reported abdominal pain. At the time of audit 22
patients remained on Anagrelide, 7 patients were deceased (average age 82), and 5
were discontinued (3 converted to Ruxolitinib, 1 pancytopenia, and 1 anagreliderelated cardiomyopathy).
Conclusion
This audit demonstrates similarity between published Anagrelide trials and Gold
Coast Hospital population outcomes.
Keywords Anagrelide, Audit, Thrombocytosis.
Conflict of Interest: No conflict of interest to disclose.
II:190
190
HSANZ POSTERS
P193
Treatment of Chronic Myelomonocytic Leukemia with Azacitidine
E Wong1, M Kenealy2, JF Seymour1,3, D Westerman1,3, K Herbert1, M Dickinson1,3
1
Peter MacCallum Cancer Centre, East Melbourne, Victoria; 2 Cabrini Medical
Centre, Malvern, Victoria; 3 University of Melbourne, Victoria
Aim
There are limited published data on the outcomes of patients with CMML treated
with hypomethylating agents including azacitidine. The aim of this study was to
retrospectively analyse the efficacy of azacitidine in patients with CMML at our
institutions.
Methods
Patients were identified from the pharmacy database. Azacitidine was administered
subcutaneously at a dose of 75mg/m2 daily for 7 days in 28 day cycles. Patients
who received at least 1 cycle of azacitidine were included in the analysis. Response
was assessed using the modified IWG criteria (2006). Kaplan-Meier surival analyses
were used.
Results
Eleven patients were treated between 2008 and 2012. The median age was 65
years (range 42-80). Four were classified as having CMML-1 and 7 with CMML-2.
Four patients had a white cell count >13x109/L at baseline. Three patients (27%)
had poor-risk cytogenetics. Five patients received concurrent therapy with either
thalidomide or lenalidomide. After a median of 8 cycles (range 2-29) and a follow-up
time of 16.1 months (range 2.8-38.1), 6 patients (55%) had a response of
hematologic improvement (HI) or better (one CR, 3 marrow CR, 1 PR, 1 HI). Four
patients had stable disease and one had progressive disease. In patients who
responded, the median time to response and duration of response were 4.1months
(range 1.6-8.2) and 7.0months (range 2.3-13.4) respectively. Response rates were
similar between patients with CMML-1 or 2. The response rate appeared higher and
duration of response longer in patients with a lower white cell count (<13) at
baseline (71% vs 25%, p=0.24; 3.8vs8.2months). Seven patients discontinued
treatment; 2 due to transformation to AML. The median overall survival was
17.2months. The most common toxicities included local skin reactions and nausea.
Fourteen hospitalisations were recorded in 8 patients, the most common cause
being infection.
Conclusion
Azacitidine had efficacy in our cohort of patients wtih CMML with response rates
comparable to that seen in MDS. Patients with lower white cell counts appeared
more likely to benefit.
Keywords
Chronic Myelomonocytic Leukemia; Azacitidine;
Conflict of interest No conflict of interest to disclose
191
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HSANZ POSTERS
P194
A Case of JAK2 Positive Chronic Neutrophilic Leukaemia (CNL)
Associated with Plasma Cell Myeloma
Marija Nedeljkovic1, Surender Juneja1, Peter Hughes2, Simon He1
1
Diagnostic Haematology, The Royal Melbourne Hospital
2
Nephrology, The Royal Melbourne Hospital
Chronic neutrophilic leukaemia is a rare myeloproliferative neoplasm characterised
by hepatosplenomegaly and granulocytic proliferation mainly comprised of mature
neutrophils. In up to 20% of reported cases there is an association with an
underlying neoplasm, most commonly myeloma. There have been as yet no
reported cases of clonality in the granulocytic lineage in such cases. This has led to
speculation that the granulocytic proliferation in these cases may be secondary to
cytokine release by the clone of plasma cells.
We report a case of JAK2 V617F mutation positive myeloproliferative neoplasm,
consistent with CNL, associated with IgG plasma cell myeloma.
The patient is an 81yo man presenting with proteinuria and worsening renal
impairment (creatinine 0.253mmol/L) in the context of previous nephrectomy for
renal cell carcinoma. Further investigation revealed an IgG kappa paraprotein of
16g/L. Renal biopsy was not performed due to the risk of complications with a
solitary kidney. No lytic lesions were detected on skeletal survey. There was mild
anaemia (Hb109g/L), which may have been related to the degree of renal
impairment. In addition, the patient was noted to have moderate splenomegaly,
moderate thrombocytopenia (82x109/L) and a raised peripheral blood white cell
count (26.8x109/L) with a neutrophilia (24.7x109/L) but no left shift, basophilia or
excess of blasts. Testing for BCR-ABL was negative, but a JAK2 Exon 14 mutation
was detected. Bone marrow biopsy revealed a markedly hypercellular marrow with
granulocytic hyperplasia and minimal fibrosis. There was an infiltrate of plasma cells
accounting for approximately 15-20% of cellularity, which demonstrated kappa light
chain restriction on immunohistochemistry. Thus a diagnosis of chronic neutrophilic
leukaemia associated with plasma cell myeloma was made.
To date, this is the only reported case of CNL associated with myeloma with
convincing evidence of clonality (JAK2 positivity) for the myeloid proliferation.
However, the possibility of co-existing dual pathology cannot be excluded.
Keywords
CNL, myeloma
Conflict of interest
II:192
No
192
HSANZ POSTERS
P195
Acute Kidney Injury Requiring Dialysis Following Carmustine and
Etoposide During Autologous Stem Cell Transplant
Jane Li, Amit Khot, Kate Burbury
Department of Haematology, Peter MacCallum Cancer Centre, Melbourne, Victoria,
Australia
The Stanford BCNU protocol (carmustine, etoposide and cyclophosphamide) is a
high-dose conditioning regimen widely used prior to autologous stem cell
transplantation. While acute renal failure requiring renal replacement therapy is a
known but rare complication of autologous stem cell transplantation, acute
nephrotoxicity following carmustine and etoposide has not yet been reported. We
present the first case of carmustine-induced acute kidney injury in the setting of
autologous stem cell transplantation and perform a review of the literature. The
renal failure was associated with a sharp rise in serum creatinine, oliguria and trace
proteinuria. Urgent haemodialysis was required however the renal failure resolved
after seven days. Although a rare complication, the severity mandates close
monitoring of renal function as early recognition and treatment may limit long-term
sequelae.
Keywords
carmustine, acute kidney injury, autologous stem cell transplant
Conflict of interest
No conflict of interest to disclose
193
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HSANZ POSTERS
P196
Autologous Stem Cell Transplantation in Myeloma: the Geelong
Hospital Experience, 2004-2012
Hannah Rose1, Adam Friebe2, John Hounsell3, Philip Campbell1
1. Department of Haematology, Geelong Hospital, Geelong, Victoria. 2. St. John of
God Pathcare, Geelong, Victoria. 3. Warrnambool Base Hospital, Warrnambool,
Victoria, Australia
Aim
To evaluate outcomes of patients undergoing HDT and ASCT for myeloma at
Geelong Hospital since the inception of the transplant program.
Methods
A retrospective audit was performed, collating clinical and laboratory data from
patient medical records and pathology systems. Response assessment used IMWG
criteria.
Results
40 consecutive patients underwent ASCT between May 2004 and February 2012,
median age was 59 years (range 47-70 years), 29/40 (73%) patients were male.
7/40 (18%) patients had advanced (ISS III) disease and 11/40 (28%) patients had
one or more high-risk abnormalities on cytogenetic studies. 32/40 (80%) patients
received novel agents prior to ASCT, with 12/40 (30%) patients treated on a trial.
Response to induction treatment was CR in 5/40 (13%), VGPR in 12/40 (30%), PR
in 20/40 (50%), SD in 2/40 (5%) and PD in 1/40 (2%). Response post ASCT has
been evaluated in 37 patients: 11/37 (30%) achieved CR, 12/37 (32%) VGPR,
12/37(32%) PR and 2/37 (5%) showed SD. 7/40 (18%) patients reverted to an
MGUS state post ASCT. Median TTP was 12.5 months (range 0.4 -78.3 mths) for
the entire cohort, with a median OS from transplant of 26.1 months (0.5 -88 mths)
after a median follow up of 24.2 months (0.5 -84.5 mths). Patients reverting to
MGUS post transplant had longer median PFS compared to the remainder of the
cohort (29.8 months vs 9.2 months) (p 0.002). 5 patients with t4;14 on cytogenetic
studies all demonstrated disease progression within 12 months of ASCT despite 4/5
achieving ≥VGPR post ASCT. 14/40 (35%) patients have died at time of analysis,
with causes of death PD (11), TRM (1), other malignancy (1) and unknown (1).
Conclusion
Patient outcomes are comparable to those in larger published series, and support
the use of ASCT in the selected patient population. Patients who revert to MGUS
post ASCT show improved PFS compared to those who do not, while patients with
t4;14 relapse early despite initial good response post transplant.
Keywords: myeloma, transplant, response
Conflict of interest: No conflict of interest to disclose
II:194
194
HSANZ POSTERS
P197
Fifteen Year Update on the Safety and Feasibility of Autologous
Peripheral Blood Stem Cell Transplant in a Regional Centre – The
Townsville Hospital
Caroline McNamara,1 Georgina Hodges,² Andrew McCutchan,1 Joanne Kanakis,1
Edward Morris,1 Hock Choong Lai,1 Ian Irving1
1
Department of Haematology and Bone Marrow Transplantation, The Townsville
Hospital, Douglas, Qld, Australia. ²Pathology Queensland, The Royal Brisbane and
Women’s Hospital, Herston, Qld, Australia
Aim
To evaluate the safety and outcomes of autologous peripheral blood stem cell
transplants (PBSCT) performed at The Townsville Hospital.
Method
A retrospective chart review on all patients who have undergone an autologous
PBSCT at The Townsville Hospital between July 1998 and June 2012. Overall
survival was calculated using Kaplan-Meier estimates.
Results
Two hundred and fifty-one (251) patients have undergone 271 autologous PBSCT
at The Townsville Hospital from July 1998 until June 2012. Ten year overall survival
for acute myeloid leukaemia, Hodgkin lymphoma, non Hodgkin lymphoma and
multiple myeloma was 57%, 54%, 50% and 30% respectively. Overall transplant
related mortality (TRM) was 3% (9/270) and in the last five years TRM was 1%
(1/97). Specifically in the last two years (July 2010 until June 2012), there have
been 50 autologous PBSCT in 49 patients with no deaths secondary to TRM.
Conclusions
The data demonstrate that having designated cancer centres acting as “regional
hubs” is a safe and feasible way of delivering highly specialised and best practice
care to rural and regional residents. This approach helps combat deficiencies in
cancer services in rural and regional Australia.
Keywords autologous, transplant, regional
Conflict of interest No conflict of interest to disclose.
195
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HSANZ POSTERS
P198
A Single Institutional Experience of Plerixafor (Mozobil®) HPC-A
Mobilisation and Historical Comparison with Ancestim (Stemgen®)
Caroline McNamara, Andrew McCutchan, Ian Irving, Hock Choong Lai, Joanne
Kanakis, Jane La, Edward Morris
Department of Haematology and Bone Marrow Transplantation, The Townsville
Hospital, Douglas, Qld, Australia
Aim
To review a single centre experience of haemopoietic progenitor cells - apheresis
(HPC-A) mobilisation with plerixafor and ancestim.
Method
Retrospective chart review of all patients who received plerixafor or ancestim at The
Townsville Hospital between May 2004 and June 2012.
Results
Before November 2010, patients failing HPC-A mobilisation were given repeat
mobilisation using combination chemotherapy, G-CSF and ancestim (20mcg/kg/day
SC). Prophylaxis was essential with phenergan, ranitidine and salbutamol; patients
required prolonged observation following administration. There were 17 patients (10
NHL, 5 MM, 2 germ cell tumour); 13/17 (76%) achieved ≥2x106 CD34+ cells/kg with
a median total yield of 4.82x106 (0-15.52) CD34+ cells/kg. The majority of patients
developed local urticarial reactions. Three patients (18%) receiving ancestim,
developed cardiac arrhythmias requiring admission to the Coronary Care Unit.
Since November 2010, 11 patients (9 NHL, 1 MM, 1 AML) failing HPC-A
mobilisation (10 chemotherapy, 2 G-CSF alone) have received plerixafor
(0.24mg/kg/day SC) as “immediate rescue”. 9 of 12 (75%) collections harvested
≥2x106 CD34+ cells/kg with an average of 2 doses; median total yield was 2.57x106
(0.09-3.84) CD34+ cells/kg. Two patients (18%) developed self limiting diarrhoea
with no grade 3/4 events. Five patients have proceeded to HPCT. Median time to
neutrophil and platelet engraftment was 13 and 28 days respectively. No
unexpected toxicity was observed.
Conclusions
Plerixafor allows “immediate rescue” for patients failing HPC-A mobilisation with
minimal toxicity.
Keywords mobilisation, plerixafor, stem cells
Conflict of interest No conflicts of interest to disclose.
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196
HSANZ POSTERS
P199
Neutrophil Contamination in Autologous Peripheral Blood Stem Cell
Products: Impact on Post-Transplant Outcome
Wei Xia, Lijun Bai, Kelly Wong, Cassandra Reid, David Collinls, Ian Kerridge, Chris
Ward, Matthew Greenwood
Cellular Therapeutic Laboratory, Department of Haematology, Royal North Shore
Hospital, Sydney, NSW, Australia
Aim
To evaluate whether neutrophil contamination of HPC-A product could impact
transplant related outcomes at a single centre.
Methods
126 patients undergoing PBSC mobilization/collection were identified during 20092012. The instrument modes of apheresis included Cobe Spectra manual, Cobe
Spectra automated and Optia. The patient characteristics, collected HPC-A volume,
neutrophil% in HPC-A, infused neutrophil dose, and transplant outcomes are
evaluated.
Results
54%, 38% and 8% of patients received manual, auto and Optia mode collection.
Numbers of neutrophil infused positively correlated with collected HPC-A volume
and neutrophil % in HPC-A (p<0.001, R2=0.262 and 0.255). Neutrophil
contamination significantly increased if collected HPC-A volume>300mL and
neutrophil% in HPC-A>50% (p<0.001). Excepting limited using of Optia, manual
mode, comparing auto mode, significantly caused larger collected HPC-A volume
(mL) (308+14.8 vs 123+10.23, p<0.001); and more neutrophil% in HPC-A (40+2.33
vs 14+2.66, p<0.001). Other pre-harvest factors, e.g. gender, disease, G-CSF using
and body weight, didn’t significantly affect neutrophil number in HPC-A product.
Following infusion of HPC-A, infused neutrophils >4x108/kg in HPC-A significantly
delayed neutrophil engraftment vs HPC-A containing neutrophils <4x108/kg,
(13d+0.79 vs 12d+0.24, p=0.037). Infused neutrophil number significantly correlated
to neutrophil engraftment time (R2=0.1, p<0.05); but didn’t impact platelet
engraftment, TRM and survivals.
Conclusions
1. Neutrophil contamination significantly increased if collected HPC-A volume
>300mL
2. Infused neutrophils >4x108/kg in HPC-A is associated with delayed neutrophil
engraftment, but doesn’t impact other post-transplant outcomes
3. Automated mode of apheresis collected smaller volume of HPC-A, less
neutrophils in HPC-A product than manual mode
Keywords: neutrophil contamination, autologous HSCT, apheresis
Conflict of interest: no
197
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HSANZ POSTERS
P200
Use of ‘Rainy Day’ Autologous Haematopoietic Stem Cells: A Single
Institution Experience over 10 Years
Lucy Fox, Scott Ragg, Ray Lowenthal, Elizabeth Tegg, Anna Johnston
Royal Hobart Hospital, Tasmania, Australia
Aim
High dose therapy and autologous haematopoietic stem cell (AHSC) transplantation
is an important treatment for a variety of haematologic malignancies. AHSCs can be
harvested during remission or consolidation treatment for subsequent
transplantation if required (‘rainy day’ collection). Although the practice is
widespread, evidence base is minimal. This study investigated the eventual
transplantation of ‘rainy day’ collections, the delay between collection and
transplantation and the outcome of patients collected with rainy day intent.
Methods
This was a retrospective audit of practice at the Royal Hobart Hospital between
1/1/2001 and 31/12/2010. A ‘rainy day’ harvest was defined as collection of AHSCs
where transplantation was not anticipated in the next 6 months.
Results
During the audit period, a total of 532 collections were performed in 474 patients for
haematological indications. 342 (71%) patients had rainy day AHSCs collected.
Disease indications for ‘rainy day’ collections included NHL (N=205, 60%), MM
(45,13%), HL (38, 11%), AML (20, 6%), CML (5, 1%), ALL (6, 2%), other
myeloproliferative neoplasms (21, 6%), CLL (3, 1%). The overall rate of
transplantation of ‘rainy day’ products was 14%. 27% of collections for MM resulted
in transplant, compared to 16% of collections for HL and 14% of collections for NHL.
Only 5% of collections for AML resulted in transplantation and there were no
transplants from ‘rainy day’ collections for CML. The median delay from collection to
transplantation was 18 months (range 6.3 to 112). 81% of patients transplanted
engrafted within specified limits. Of the 296 patients who had rainy day AHSC
collections that have not been transplanted, 255 (85%) are still alive
Conclusions
‘Rainy day’ AHSC collection is resource intensive. We found high rates of eventual
usage for certain indications (MM, HL) but only 5% of collections for AML resulted in
transplant and there were no transplants for CML. Thus far 14% of ‘rainy day’
collections for NHL have resulted in transplant. Findings from this study may help to
inform guidelines for ‘rainy day’ AHSC collection.
Keywords
high dose therapy, autologous stem cell transplant, rainy day
Conflict of interest None to report
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HSANZ POSTERS
P201
Clinical Aspects of Poor Peripheral Blood Stem Cell (PBSC) Mobilisation –
Experiences at RNSH
Lijun Bai, Barbara Withers, Cassandra Reid, Wei Xia, Kelly Wong, Ian Kerridge,
Matthew Greenwood
Cellular Therapeutic Laboratory, Department of Haematology, Royal North Shore
Hospital, Sydney, Australia
Aim
Reliable engraftment following autologous haematopoietic stem cell transplantation
(ASCT) requires a minimum CD34 dose of 2x106/kg recipient body weight. Failure
to mobilize sufficient HPC occurs in 10-25% of ASCT candidates and is often cited
as a reason for not proceeding with ASCT. We report the results of a retrospective
analysis of HPC mobilization in ASCT candidates in our institution between 2002
and 2012, focusing on the characteristics of poor mobilisers.
Results
562 patients (median age 57yrs, male 63%) underwent initial HPC mobilisation.
Poor mobilization (defined as either (1) failure to mobilise sufficient peripheral blood
CD34<15/µl prior to apheresis, or (2) the collection of CD34<2x10^6/kg body
weight.) occurred in 98 patients (17%); median age 59yrs with 62% males. There is
no significant difference from those who mobilized successfully (P=0.565 & 0.324
respectively). Of poor mobilisers the diagnosis was MM in 22 cases (22.4%); NHL in
54 cases (55%), Hodgkin’s lymphoma in 2 cases (2%); leukemia in 17 cases
(17.4%), and other diseases in 5 cases (5.1%), which is similar frequency of each
diagnosis in those success mobilisation groups. Patients diagnosed with Diffuse
large B-Cell lymphoma (DLBCL) had a higher frequency of failed mobilisation (32%)
compared to other cases of NHL (19.8%, P=0.007). The mobilization regimen
consisted of G-CSF alone and chemotherapy + G-CSF, being 41.8% and 58.2% in
those who failed to mobilize. The median number of cycles of chemotherapy before
mobilisation was 4 (1 ~ 9). All patients had a second mobilisation attempt with GCSF, chem+G or plerixafor. Only30 (30.6%) successfully mobilized sufficient HPC
for ASCT. 28 patients (28.6%) went on to successful bone marrow harvest. 11% of
poor mobilizers underwent ASCT. These patients had equivalent outcomes to
successful first mobilisers.
Conclusion
We describe the frequency and outcomes of poor mobilizers in our institution.
Identification of those likely to fail mobilisation – either before mobilisation
commences or at the point of HPC apheresis may assist with patient selection,
optimise resource allocation, assist with scheduling of apheresis and enable the
development of a predictive algorithm for the pre-emptive use of plerixafor.
Keywords
poor, mobilization, clinical
Conflict of interest No
199
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HSANZ POSTERS
P202
Outcomes of Autologous Transplantation for Primary Refractory
Hodgkin Lymphoma
MM Spanevello, K Morris, GA Kennedy
Dept of Haematology, Royal Brisbane and Women’s Hospital, Brisbane Qld,
Australia
Background
Autologous stem cell transplantation (ASCT) for Hodgkin lymphoma (HL) is widely
used and effective for relapsing disease, but in patients with primary refractory and
early relapsing (< 12 month) disease the benefit is less certain and historically poor.
To attempt to overcome this, we have since 2009 adopted individualised
conditioning regimens based on protocols published by MSKCC / Cleveland Clinic:
cyclophosphamide-carmustine-etoposide (CBV) for previously irradiated patients
and cyclophosphamide-etoposide + total lymphoid irradiation (CV-TLI) for
unirradiated patients.
Aim and Method
Consecutive patients receiving ASCT for primary refractory Hodgkin lymphoma from
2001 to 2011 were identified from an institutional transplant database, with analysis
of patient and disease characteristics, treatment, and survival determined by
retrospective chart review.
Results
12 patients were identified. All patients had nodular sclerosing HL at early
unfavourable (n = 7) or advanced stage (n = 5), and received ABVD as first-line
therapy. All patients except one received ESHAP as their salvage therapy prior to
ASCT. Prior to transplantation, 7 patients achieved a complete response, 3 a partial
response, and 2 patients had stable disease. Transplant conditioning was CBV or
CV-TLI in 6 patients (all since 2009), BEAM in 4 (all except one prior to 2009), and
BuMelThiotepa in 2 (both prior to 2009). At a median follow up of survivors of 20
months (range 2.4-49 months), 3 patients (25%) have progressed, and 2 of these
(17%) have died from disease. Factors analysed as predictors of progression
included initial disease characteristics (age, stage, Hasenclever prognostic score),
characteristics at the time of salvage therapy (presence of B symptoms, bulk, or
extranodal disease), response to salvage, conditioning regimen and date of
transplant (before / after 2009). None were found to be statistically significant.
Conclusions
Autologous stem cell transplant can be effective even in patients with primary
refractory Hodgkin lymphoma. Further follow up of our data is required to determine
factors influencing outcome and the benefit of alternative conditioning regimens.
Keywords
Hodgkin Lymphoma, Autologous Transplant, Primary Refractory
Conflict of interest No conflict of interest to disclose
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HSANZ POSTERS
P203
Case study: T cell Lymphoma With Isolated Bone Marrow Involvement
Amanda Ormerod1, Teresa Leung1, Phillip Campbell1, Adam Friebe2, Geoff Davey 2
1. Department of Haematology, Barwon Health, Geelong,Victoria Australia
2. St John of God Pathcare, Geelong, Victoria, Australia
Background
Cases of T cell lymphoma with bone marrow involvement only have not been widely
published1,2,3. Here we describe a previously well 73 year old female presenting
with severe pancytopenia, with the rare finding of T cell lymphoma with involvement
limited to the bone marrow
Results
Repeated bone marrow biopsies noted an absence of normal haematopoesis. An
abnormal infiltration by T lymphocytes was noted, which was confirmed by flow
cytometry. Clonality was suspected but unable to be diagnostically proven. T cell
gene rearrangement studies were positive. Commenced on a 21 day cycle of
CHOP chemotherapy with GCSF support
Conclusion
Currently completed cycle 6 CHOP with interval marrow demonstrating partial
recovery of normal haematopoesis. This has been associated with count recovery
and transfusional independance. The patient remains well and in remission, with
ECOG 0.
Keywords
T cell lymphoma, bone marrow
Conflict of interest
No conflict of interest to disclose
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HSANZ POSTERS
P204
Prognostic Value of Secondary Chromosomal Abnormalities in
Follicular Lymphoma
Nathanael Lucas1, Richard Parfitt2, Robert Hill 3,Takayoshi Ikeda 4, Alwyn D’Souza 1
1
Wellington Blood and Cancer Centre, Wellington Hospital, Wellington, New
Zealand, 2 Central and Southern Regional Genetic Services Laboratory , Wellington
Hospital, Wellington, New Zealand, 3 Department of Anatomic Pathology, Wellington
Hospital, Wellington, New Zealand, 4 Department of Biostatistical Services,
University of Otago School of Medicine, Wellington, New Zealand
Aim
Follicular lymphoma (FL) is a slowly progressive malignancy which is characterised
by the t(14;18) translocation and numerous secondary genetic alterations. We
performed an audit to determine common secondary chromosomal abnormalities in
follicular lymphoma (FL) and whether these had prognostic value independent of the
follicular lymphoma international prognostic index (FLIPI).
Results
We analysed the total number of chromosomal abnormalities in each sample. The
most common secondary chromosomal abnormalities found were +7, +X,
+der(18)t(14;18), +12, +8, +2 , del(1)(p36), -13, +5, -17 and add(3q). Of these only
the total number of chromosomal abnormalities per sample (p= 0.036) and
del(1)(p36) (p = 0.001) demonstrated decreased overall survival on univariate
analysis. When the total number of chromosomal abnormalities per sample and the
del(1)(p36) were added to the FLIPI as extra parameters they added no prognostic
value.
Conclusion
We confirmed that the FLIPI remains the most powerful prognostic tool in FL. This
may be because the del(1)(p36) and number of chromosomal abnormalities are
generally associated with a higher FLIPI score and merely highlight patients who
have already been identified as being in a poorer prognostic group.
Keywords
Follicular lymphoma, cytogenetics
Conflict of interest No
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202
HSANZ POSTERS
P205
Case Report: Atraumatic Splenic Rupture in a HIV Patient with
Undiagnosed Hodgkin Lymphoma
Swe Myo Htet, Nicholas Murphy
Clinical Haematology, Northern Hospital, Epping, Victoria, Australia
Introduction
Hodgkin Lymphoma is a known malignancy associated with HIV infection. It can
present with atraumatic splenic rupture.
Case report
A 37 year old man with known HIV infection presented with spontaneous peri
splenic haematoma. HIV was diagnosed 3 years prior after presenting with
lymphadenopathy, excisional lymph node biopsy showing reactive hyperplasia.
Lymphadenopathy resolved after initiating highly active antiretroviral therapy
(HAART).
In the four months prior to presentation, the patient was admitted on three occasions
with fevers and night sweats, all of which responded to antibiotic therapy. Blood
cultures on each occasion were negative. CT of the abdomen noted mild
splenomegaly and retroperitoneal lymphadenopathy (1.5 cm diameter).
After presenting with left sided abdominal pain and hypotension, a CT scan noted
peri-splenic haematoma, and he underwent embolization of the splenic artery. The
patient improved haemodynamically, however had ongoing fevers with progressive
pancytopenia. Bone marrow biopsy was performed. The patient went into
haemorrhagic shock and cardiac arrest on the evening of bone marrow biopsy
secondary to splenic rupture. He underwent emergency splenectomy.
Perioperatively, the patient received 14 units of packed cells, 2 bags of fresh frozen
plasma (FFP) and 2 bags of poled platelets. Postoperatively, he became
progressively hypotensive, hypothermic and had worsening metabolic acidosis. He
had massive blood transfusion associated coagulopathy, and died from
haemorrhagic complications. Spleen and bone marrow biopsy showed classic
Hodgkin lymphoma.
Conclusion
We describe a case of undiagnosed Hodgkin lymphoma in HIV positive patient who
presented with atraumatic splenic rupture, progressive cytopenias and who died
after splenic rupture and transfusion associated coagulopathy.
Keywords
HIV spleen lymphoma
Conflict of interest No
203
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HSANZ POSTERS
P206
Audit of Western Health Intermediate Grade Lymphoma Management
D Carradice, W Renwick
Western Hospital Footscray, Western Health, Melbourne
Aim
The DHS document “Patient Management Framework (PMF)-Haematological
tumour stream: intermediate grade non-Hodgkin’s lymphoma” details strategies and
protocols to maximize patient experience and clinical outcome through the
lymphoma treatment journey. We assessed Western Health performance and
practice relating to key KPIs from the PMF document, for patients with Diffuse Large
B cell Lymphoma (DLBCL) and Hodgkin lymphoma.
Results
Data on 50 patients from with an initial diagnosis of DLBCL/Hodgkin lymphoma
made from 1/1/2008 to 31/12/2010 were retrieved from the BioGrid database
(www.biogrid.com.au) and analysed for key KPIs relating to initial referral to Western
Health, lymphoma diagnosis and treatment planning.
7 patients were excluded from analysis (relapsed or diagnosis/treatment outside
Western Health). Diagnoses were Hodgkin lymphoma in 11/43 and DLBCL in 32/43.
Average duration of symptoms prior to referral was 99 days, some patients
experienced symptoms for an extended period prior to referral (range 0-700 days).
Average “Waiting time” was 17 days for outpatient appointment (KPI=14 days),
23/43 patients (54%) presented to the Emergency department. Referrals were
mainly to surgical units (13/43) or Oncology/Haematology (12/43). Diagnostic
procedures were excisional lymph node biopsy (26/43) and radiological core biopsy
(17/43). First consultation with Oncology/Haematology was often after tissue biopsy
had been performed (24/43 (56%), KPI=0%) and ancillary testing on the tissue (flow
cytometry (21/43), cytogenetics (0/43), molecular studies (1/43), KPI=100% for
each) were variably requested. The majority of patients were not discussed in an
MDM or “team meeting” prior to treatment commencement (only 4/43, KPI=100%).
PET was only performed in 16/34 (47%) patients with stage I/II disease (KPI 100%).
Time from Oncology/Haematology consultation to commencement of treatment
(mean 32 days) was within an acceptable timeframe
Conclusion
The clinical pathway for patients with intermediate grade lymphoma from initial
presentation, through diagnosis, staging and treatment planning was variable within
our institution. Efficiency in the process and improvement in patient experience
could be achieved by development of a defined lymphoma clinical pathway and
encouraging early consultation with Haematology. Relevant stakeholders are
identified for involvement in this process
Keywords
Lymphoma, Audit, Clinical pathway
Conflict of interest No
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HSANZ POSTERS
P207
Persistent Lymphadenopathy After Appropriate Treatment. The Role of
Re-biopsy
R Wooldridge, J Wellwood, T Cochrane, M Bryson
Department of Haematology, Gold Coast Hospital, Qld, Australia
A 47 year old female presented with right hip pain, CT scans revealed a large lytic
lesion, without evidence of disease elsewhere. A diagnosis of Peripheral T cell
lymphoma - not otherwise specified (PTCL-NOS) was made following biopsy based
on immunohistochemistry and the detection of a monoclonal population with TCR
rearrangement studies. Treatment with CHOPx6 was completed, restaging
demonstrating persistent disease in the marrow and new mesenteric
lymphadenopathy. Salvage therapy with ICE was commenced, during which
recurrent fevers upon neutrophil recovery were noted. Restaging showed stable low
volume disease, and a break in therapy was decide upon, during which she
developed a subcutaneous lesion on the forehead approximately 4cm in diameter,
biopsy confirming recurrent PTCL-NOS. GEM-P was commenced which rapidly
resolved this lesion, however previous lymphadenopathy persisted and treatment
was again complicated by fevers. Referral for a Clinical trial was made and biopsy of
the mesenteric nodes was done revealing Mycobacterium Avian Complex (MAC). A
repeat bone marrow and culture showed disseminated MAC along with low-level
PTCL. She completed and responded well to anti-tuberculous therapy however in
early 2011, hypercalcaemia and renal impairment developed, investigation showed
persistent low-level stable PTCL and cultures were again positive for MAC.
Currently she is receiving second line therapy for MAC without lymphoma
progression. The degree of T cell dysfunction as a consequence of PTCL and
chemotherapy is highlighted in this case not only due to MAC but also notably since
2010, this patient has had treatment for invasive aspergillus and disseminated
varicella zoster.
It is common practice to repeat lymph node biopsies at relapse to exclude other
pathologies, but this case highlights that even in histologically confirmed lymphoma,
co-existent and treatable pathologies occur and biopsies of more than one site may
be required when the clinical picture is not typical. This may result in fewer adverse
events related to prolonged chemotherapy.
Keywords
Lymphadenopathy, Peripheral T cell Lymphoma
Conflict of interest No
205
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HSANZ POSTERS
P208
Intrapleural Rituximab for the Treatment of Malignant Pleural Effusion
Due to B-cell Lymphomas
Man Fai Law1, Hay Nun Chan2, Ho Kei Lai2, Chung Yin Ha2, Celia Ng2, Yiu Ming
Yeung2, Sze Fai Yip2
1
Department of Medicine and Therapeutics, Prince of Wales Hospital, the Chinese
University of Hong Kong 2Department of Medicine, Tuen Mun Hospital, Hong Kong
Background
Pleural effusion due to lymphoma involvement can be massive and is sometimes
resistant to chemotherapy treatment. Rituximab is proven effective for the treatment
of B cell lymphoma when given intravenously. The intrapleural route of
administration was not explored. We report our experience of the intrapleural
rituximab in controlling malignant pleural effusion due to B-cell lymphoma.
Method
Patients with malignant pleural effusion due to B-cell lymphoma were identified.
They were given systemic chemotherapy and pleural tapping. Those with persistent
pleural effusion were recruited and given intrapleural rituximab (50-100mg in 50ml
saline as a bolus) and the chest tube was clamped for two hours.
Results
Two patients were given intrapleural rituximab. The first patient suffered from stage
IV mantle cell lymphoma. He was given two cycles of standard R-CHOP (rituximab,
cyclophosphamide, doxorubicin, vincristine and prednisolone) chemotherapy and
pleural tapping but the pleural effusion persisted. At the third cycle of chemotherapy,
rituximab (100mg in 50ml saline) was added and given intrapleurally. Repeat chest
X-ray showed resolving pleural effusion. After the fourth cycle of R-CHOP, the
patient had lymphoma progression with central nervous system involvement.
Further salvage chemotherapy was given. The lymphoma did not respond to
salvage chemotherapy. Despite this, there was no recurrence of the pleural effusion.
The second patient suffered from marginal zone B-cell lymphoma. Two cycles of RCHOP chemotherapy were given but there was persistent massive effusion.
Rituximab (50mg in 50ml saline) was then given intrapleurally together with the first
day of intravenous chemotherapy at the third cycle of therapy. Two more doses of
intrapleural rituximab (100mg each) were given three weeks apart in subsequent
cycles and the effusion subsided. Both patients tolerated the intrapleural rituximab.
Conclusion
Intrapleural rituximab has an adjuvant role in the control of malignant pleural
effusion due to CD20+ B-cell lymphoma and it was well tolerated. It is worthwhile
exploring further this novel route of administration.
Keywords: Intrapleural rituximab, malignant pleural effusion, B-cell lymphoma
Conflict of interest: No
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HSANZ POSTERS
P209
Lymphoplasmacytic Lymphoma Presenting with Oculomotor Nerve
Palsies and Associated Myasthenia Gravis
Omar Mansour1, Sandeep Bhuta2, Jason Restall1, Jeremy Wellwood1, Tara
Cochrane1
1
Department of Haematology, Gold Coast Hospital, Queensland Australia
2
Department of Radiology, Gold Coast Hospital, Queensland Australia
Background
Lymphoplasmacytic lymphoma (LPL) is a slowly progressive, clonal disorder of
mature B cells, with features of plasma cell differentiation and IgM paraproteinemia.
LPL commonly presents in the lymph nodes and bone marrow and may show
features of hyperviscosity. Neurological and orbital involvement is exceedingly rare.
Case Presentation
A 50 year-old male presented with a two week history of right upper eye-lid
weakness and fatigability, diplopia on left medial gaze, and photophobia.
Neurological examination revealed a right third nerve palsy, bilateral partial sixth
nerve palsies and right-sided ptosis. CT scan demonstrated widespread
lymphadenopathy above and below the diaphragm but no thymoma. MRI orbits
revealed bilateral extensive infiltration of the extra-ocular muscles and loss of the
ocular retrobulbar fat. Inguinal node biopsy confirmed a histological diagnosis of
LPL with 30% infiltration of bone marrow. Further investigations demonstrated an
IgM kappa paraprotein band of 16g/L, small lymphocytes in the CSF which were
clonally restricted by flow cytometry, and positive anti-acetylcholine-receptor
antibodies. The working diagnosis was LPL with CSF involvement and
secondary/co-existent myasthenia gravis. The patient was treated with
pyridostigmine (10mg QID) and R-CHOP chemotherapy with IT methotrexate.
Following 3 cycles of chemotherapy there has been a reduction in IgM kappa
protein levels to 6g/L, a reduction in the lymphadenopathy and there is less extra
ocular muscle infiltration. However the CSF remains positive in the absence of any
neurological symptoms. High dose methotrexate (3g/m2) will be added to the
subsequent 3 cycles of R-CHOP chemotherapy in an effort to eradicate the CNS
disease.
Conclusion
LPL can present with CNS involvement and may exhibit unusual signs not usually
associated with lymphoma, such as incidental oculomotor nerve palsies. Although
extremely rare, cases of myasthenia gravis associated with lymphoma have been
reported. It is likely that this patient has a dual diagnosis of both ocular myasthenia
and infiltrative ocular lymphoma.
Keywords
Lymphoplasmacytic lymphoma; myasthenia gravis, ocular palsies
Conflict of interest No
207
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HSANZ POSTERS
P210
Nodular Sclerosis Classical Hodgkin Lymphoma Type Post Transplant
Lymphoproliferative Disorder. A Case 10 Years Post Renal
Transplantation Treated with AVD
Dejan Radeski1, Jeremy Parry2, Julian Cooney 1
1
Department of Haematology, Royal Perth Hospital. 2Department of Anatomical
Pathology, Royal Perth Hospital, Perth Western Australia
Aim
We present a case of classical Hodgkin lymphoma post transplant
lymphoproliferative disorder treated with AVD chemotherapy.
Results
A 28 year old female of subcontinental origin had a live related donor renal
transplant performed for chronic renal impairment secondary to vesicoureteric reflux
in 2002. Post transplant she remained on long term immunosuppresion with
Tacrolimus, Azathioprine and Prednisolone. The patient had a 6 month history of
slowly enlarging left cervical lymphadenopathy without B symptoms. Excisional
lymph node biopsy demonstrated nodular sclerosis classical Hodgkin lymphoma.
The malignant cells demonstrated expression of CD30 as well as nuclear
expression of EBV encoded RNA (EBER) and cytoplasmic and membranous
expression of latent membrane protein 1 (LMP1). CD3, CD15, CD20, CD45 or EMA
were not expressed. The positive EBER and LMP1 demonstrates the presence of
EBV expression and favours that the Hodgkin lymphoma arised in the post
transplant setting. Staging investigations demonstrated Stage IIA disease with the
presence of left supraclavicular and left superior mediastinal lymphadenopathy in
conjunction with disease in the left cervical chain. Her Hasenclever score was 2
(Haemoglobin <105 g/L and Albumin <40 g/L). Complicating her therapy was
moderate renal impairment (GFR = 41 ml/min/1.73m2), reduced DLCO (46%) and
ongoing requirement for immunosuppression including Tacrolimus. As a
consequence of her renal and lung function Bleomycin was ommitted. AVD
chemotherapy was commenced with dose adjustment for the patient’s renal function
and ongoing Tacrolimus use.
Conclusion
Classical Hodgkin Lymphoma Post Transplant Lymphoproliferative Disorder is a
rare malignancy. We present the first document case treated with AVD
chemotherapy.
Keywords
Hodgkin Lymphoma PTLD
Conflict of interest
II:208
The authors have no conflict of interest to disclose.
208
HSANZ POSTERS
P211
Experience with Mantle Cell Lymphoma at Sir Charles Gairdner Hospital
Marianne Elder1, Duncan Purtill1, Julie Crawford1,2, Rebecca Howman1,2, Brad
Augustson1,2, Gavin Cull1,2, David Joske1,2,3
1
Sir Charles Gairdner Hospital, Perth, Western Australia
2
Pathwest, Perth, Western Australia
3
University of Western Australia
Aim
Mantle cell lymphoma is an aggressive form of non-Hodgkin lymphoma which is
held to be incurable with conventional chemotherapy. We audited all 39 patients
who were diagnosed with mantle cell lymphoma at our centre from October 1999
until November 2010.
Results
The median age at diagnosis was 59 years (range 46-84). 63% were men. Of 19
with available data, ECOG performance score was 0 (n=8) or 1 (n=12). Splenic
involvement was documented in 16 of 25 with available data, and 20 of 21 had
evidence of lymphoma cells in the peripheral blood. Median LDH at diagnosis was
207U/L (range: 111-506). MIPI score was calculated for 17 patients and was 1-3
(n=5), 4 (n=6), 5 (n=3) or 6-8 (n=3). Of 25 patients with available data, 3 declined or
did not require induction chemotherapy, 12 received CHOP chemotherapy, 8
received hyperCVAD, one received both regimens and one received fludarabine
and cyclophosphamide. 17 (71%) of these patients received rituximab with
chemotherapy. Thirteen (54%) went on to high dose chemotherapy and autologous
stem cell transplant, and one eventually received an allogeneic stem cell transplant
from his HLA-matched brother. Remission was achieved in 16 of 22 evaluable
patients; of these, 7 went on to relapse. Of the 6 who did not achieve remission, 2
had received no induction chemotherapy, 2 had received CHOP, 1 hyperCVAD and
one had received both regimens. A total of 14 of 29 patients with available data
were alive at last follow-up. Median follow-up of these patients was 33 months
(range: 6-125 months). The probability of survival at 3 years was 64+/-10%
(Kaplan-Meier method), and median survival was 44 months (95% confidence
interval: 31-57 months). For the 16 patients who achieved remission with induction
therapy, the probability of progression-free survival at 3 years from diagnosis was
69+/-15%. Of the 6 patients who remain alive >4 years after diagnosis, all had a
MIPI<6, 5 had received CHOP induction therapy and all had received rituximab.
Conclusion
From these preliminary data, it appears that durable remission is feasible after
chemotherapy for mantle cell lymphoma, and that low MIPI score, rather than type
of chemotherapy administered, is associated with long-term survival. Data
collection continues for those cases with missing information.
Keywords
Mantle cell lymphoma, NHL, treatment Conflict of interest No
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HSANZ POSTERS
P212
A Single Institution Experience of Angioimmunoblastic T-cell Lymphoma:
Presenting Features, Management and Outcome
Valentine Ho1, 2, Stephen Opat1, 2, Andrew Spencer2, 3, Jake Shortt1,2
Department of Clinical Haematology, Monash Medical Centre, 2 Monash University,
Faculty of Medicine, Dentistry & Health Sciences, 3Department of Malignant
Haematology & Stem Cell Transplant, Alfred Hospital; Melbourne
1
Angioimmunoblastic T-cell Lymphoma (AITL) is a rare lymphoma with poor clinical
outcomes and lack of standard, effective treatment.
Aim
To describe presenting clinical features, therapeutic responses and survival in a
cohort of patients diagnosed with AITL at Southern Health Hospitals.
Methods
The Southern Health Pathology database was searched for all biopsy-proven AITL
cases accrued between 1997 and 2012. Cases were cross-referenced against
medical records to capture standard measures of prognosis and response.
Results
Ten cases were identified, including 8 with confirmed AITL and 2 with
angioimmunoblastic features but not otherwise characterised. Median age of at
diagnosis was 58 (41-73; M/F 7:1). Baseline blood tests were available in 7/8
confirmed cases and demonstrated anaemia in 3/7 and elevated LDH in 6/7. AnnArbor stage was advanced (3-4) in 7/8. 6/8 patients had baseline
hepatosplenomegaly, with 3/8 having a prior diagnosis of chronic liver disease.
One patient was managed palliatively. The remainder received CHOP-based
therapy (2/7 CHOEP, 5/7 CHOP). All responded with 5 in CR and 2 in PR after initial
therapy. Median time to progression was 31 weeks (range 21-75). Responses to
salvage therapy were poor with a median overall survival of only 55 weeks. Novel
therapies utilised at relapse included lenalidomide (1 patient, no response),
cyclosporin (1 patient, SD for 2 months) and brentuximab (1 patient, PR/SD for 6
months). The only long term-survivor received an autograft in first relapse and
remains well with 8 years follow-up.
Conclusions
Although limited by small numbers, our data highlight the need for better
consolidation and remission maintenance strategies in AITL. Despite good initial
responses, early chemoresistant relapses resulted in premature deaths. The only
favourable long-term outcome was observed with autologous stem cell transplant.
Further clinic trials investigating the role of stem cell transplant or novel agents in
first remission are required.
Keywords: Angioimmunoblastic T-cell lymphoma Conflict of interest No
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HSANZ POSTERS
P213
Administration of High Dose Methotrexate and Cytarabine in a Patient
with Primary Central Nervous System Lymphoma (PCNSL) and Renal
Dysfunction
Karim Ibrahim1, John Moore2, Grace Gifford2, John Ray3, Jacob Sevastos4
Pharmacy Department, St Vincent’s Hospital, Sydney, NSW, Australia
2
Haematology Department, St Vincent’s Hospital, Sydney, NSW, Australia
3
Clinical Pharmacology, St Vincent’s Hospital, Sydney, NSW, Australia
4
Nephrology Department, St Vincent’s Hospital, Sydney, NSW, Australia
1
Aim
To describe the safe administration of intravenous high-dose methotrexate (HDMTX) and cytarabine for a patient with primary central nervous system lymphoma
(PCNSL) and renal impairment.
Result
A 64-year-old male with a creatinine clearance (CrCl) of 20mL/min secondary to
lupus nephritis is diagnosed with multifocal, EBV-driven PCNSL. Systemic
chemotherapy with HD-MTX (500mg/m2 over 15mins, then 3000mg/ m2 over 3hrs)
on day 1 and Cytarabine 2g/m2 twice daily on days 2 and 3 are the current PCNSL
treatment (Ferreri et al, 2009). Methotrexate is predominantly renally excreted, and
high doses when CrCl<50mL/min is not recommended. Cytarabine is metabolised to
uracil arabinoside (Ara-U), a metabolite responsible for neurotoxicity. Ara-U is
predominantly renally excreted, with increased neurotoxicity when CrCl<60ml/min.
In discussion with the patient, HD-MTX was administered with dosage determination
by an area under the curve (AUC) method, the target AUC of 1000 to 1100 µmol/L.h
based on literature review. Methotrexate at 500mg/m2 was initially trialled; with 20%
of the dose administered over 15mins, then the remaining 80% over 3hrs. Serum
Methotrexate levels were collected at 0, 6, 8, 12 and 24hrs from the end of the
infusion, which yielded an AUC of 414µmol/L.h. Intravenous Leucovorin was
administered 6 hourly at 15mg/m2 until serum Methotrexate was undetectable. The
Methotrexate dose was subsequently increased to 1000mg/m2; this yielded an AUC
of 1080µmol/L.h. Daily haemodialysis was undertaken after dose reduced
cytarabine (1g/m2/d, 2 days) to remove Ara-U to minimize neurotoxicity.
Our patient tolerated the first 2 cycles of dose adjusted HD-MTX/Ara-C with no
major toxicities, and a brain MRI demonstrated radiological response. Our patient
had 2 further cycles of this regimen. An end of treatment MRI is scheduled for July.
Keywords
methotrexate, cytarabine, area under the curve
Conflict of interest No conflict of interest to disclose
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HSANZ POSTERS
P214
Successful Stem cell Mobilisation with Plerixafor in a Patient with
Multiple Meyloma and Dialysis-Dependant Renal Failure
John Moore1, Annabel Horne1, Grace Gifford1, Karim Ibrahim2
1
Haematology Department, St Vincent’s Hospital, Sydney, NSW, Australia
1
Pharmacy Department, St Vincent’s Hospital, Sydney, NSW, Australia
Aim
To report on the use of plerixafor in a patient with mutiple myeloma and dialysisdependant renal failure.
Result
A 43-year-old man with multiple meyloma and dialysis-dependent renal failure was
evaluated for an autologous stem cell transplant (ASCT). Following Stem cell
mobilisation with our standard regimen of cyclophosphamide and 9 doses of
granulocyte colony-stumulating factor (G-CSF) 10mcg/kg/day the patient’s pre
apheresis CD34+ count was inadequte at 2.18 cells/µL.
Plerixafor was prescribed to achieve stem cell mobilisation. There is no dose
recommendation for plerixafor in patients with CrCl < 20mL/min or those on dialysis.
In this patient we used 0.16mg/kg/day dose, which is the dose recommended for
patients with CrCl 20-50mL/min. The first plerixafor dose was given subcutaneously
post-dialysis 8 hours before apheresis and the second dose was given the next day
9 hours prior to second apheresis session. The pre-apheresis CD34+ count was
11.99 and 8.82 cells/µL with a total White cell count of 22.2 and 17.3 x 10^9/L after
the first and second doses respectively. The patient underwent stem cell collection
via the Spectra Optia cell separator with a total yield of 2.4 x 10 6 cells/kg. There
were no observed toxicities with plerixafor. The patient underwent ASCT with
reduced dose of Melphalan 140mg/m2. 6 weeks after stem cell collection. Neutrophil
engraftment occurred at day +11, the patient was discharged at day +16.
Conclusion
Whilst information regarding dosing and safety of plerixafor remains limited, this
case report illustrates that it can be safely and effectively used to mobilise adequate
stem cells in patients with end stage renal failure. This report may be useful for other
clinicians who are considerding the use of plerixafor in this setting
Keywords
plerixafor, haemodialysis, renal dysfunction
Conflict of interest
II:212
No conflict of interest to disclose
212
HSANZ POSTERS
P215
Subcutaneous Velcade: A Feasible Option?
Rebecca Powell, Manjula Deo
Waitemata District Health Board, New Zealand
Background
Velcade is given to patients with Multiple Myeloma and has traditionally been given
intravenously. Velcade is a proteasome inhibitor that returns to normal the cells self
destruct programme. It has been approved for use in the United States of America
(USA) since 2003. In 2011 Pharmac approved its use for Myeloma in New Zealand.
An international trial involving 222 patients found that Velcade administered
subcutaneously compared with intravenously had less side effects but was still
efficacious (Moreau, et al, 2011). Our centre opted to trial this change in
administration technique in the hope of reducing side effects and patients treatment
times whilst still being effective against myeloma.
Methods
The change of administration technique was explained and patients were given the
option of changing from intravenous (IV) to subcutaneous. Days of treatment were
day 1 and 8 until recently when our protocol changed to days 1, 8, 15 and 22. The
main side effects we monitored were; peripheral neuropathy, bone marrow
suppression, nausea and vomiting and in this study localised reactions.
Results
Three patients temporarily opted to have IV, only 1 continues with this route and 1
has stopped Velcade altogether.
• Nausea was experienced with 8 out of 91 injections, majority being mild
nausea, only 1 patient vomited
• 34 patients experienced peripheral neuropathy and 43 did not (yes/no
answers, no scale used)
• 19 injections produced a localised reaction at the injection site to varying
degrees (statistically our study had a rate of 12.6% vs 6%)
• Two of these patients had thrombocytopenia of less than 75,000 mm3
Conclusion
Treatment times were halved and most patients tolerated the side effects without
having to be swapped to intravenous dosing. Our consultants are seeing
comparable disease responses.
Moreau, P., Pylypenko, H., Grosicki, S., Karamanesht, L., Leleu, X., Grishunina, M., Rekhtman, G.,
Masliak, Z., Robak, T., Shubina, A., Arnulf, B., Kropff, M., Cavet, J., Esseltine, D., Feng, H., Girgis, S.,
Velde, H., Deraedt, W., & Harousseau, J. (2011). Subcutaneous versus intravenous administration of
bortezomib in patients with relasped multiple myeloma: a randomised, phase 3, non-inferiority study.
Lancet Oncology Journal, 12, 431-40.
Keywords: Myeloma, Velcade, Subcutaneous. Conflict of interest: No
213
II:213
HSANZ POSTERS
P216
Slan Dendritic Cells and Tumour-Induced Clonal T cell Anergy in
Multiple Myeloma
Hayley Suen1, Ross Brown1, Narelle Woodland2, Shihong Yang1, Phillip Fromm3,
James Favaloro1, P Joy Ho1, John Gibson1, Derek Hart3, Douglas Joshua1
1
Institute of Haematology, Royal Prince Alfred Hospital, Sydney, NSW, Australia;
2
School of Medical and Molecular Biosciences, UTS, Sydney, NSW, Australia; and
3
DC Biology & Therapeutics, ANZAC Research Institute, Sydney, NSW, Australia.
Aim
6-Sulfo-LacNAc dendritic cells (SlanDC) are major producers of IL-12, which has the
potential to correct the tumour-induced immune defect observed in patients with
multiple myeloma (MM). We aimed to determine the number and function of
SlanDCs in patients with MM and whether SlanDC stimulation could overcome
clonal T cell anergy induced by MM tumour cells.
Method
Peripheral blood (PB) samples were collected from patients (57) and age-matched
normal controls (20) attending the haematology clinic at the Royal Prince Alfred
Hospital. Enumeration, endocytosis by Dextran uptake and CD80 expression of
SlanDCs were determined using flow cytometry. T cells from MM patients (4) were
stimulated with flow sorted SlanDCs (>90% purity) or mononuclear cells (MNC) from
healthy donors and cultured for 4 days with IL-2 to determine the capacity of
SlanDCs to allostimulate and reverse clonal T cell anergy in MM patients.
Results
SlanDCs were reduced in the PB of MM patients, compared to monoclonal
gammopathy of undetermined significance (MGUS) patients (t=3.10; p=0.0007) and
normals (t=3.32; p=0.003). This reduction was not seen in 10 year survivors of MM
(n=15), suggesting SlanDCs have prognostic implications. Endocytosis and CD80
expression in MM SlanDCs was normal. SlanDC stimulation alone was not sufficient
in overcoming anergy of the clonal T cells in MM patients. However, SlanDCs
demonstrated a moderate allo-stimulatory capacity to other T cell subsets and were
more efficient at stimulating T cell proliferation than the crude MNC population.
Conclusion
With the exception of 10 year survivors, SlanDCs in MM patients are decreased but
may be prognostically significant. Their endocytic function and antigen presentation
potential were normal and the cells were superior to MNC in stimulating MM T cells.
The data suggests SlanDCs are functional in MM and are potentially candidates for
immunotherapy programs and for overcoming MM-associated T cell anergy.
Keywords Slan dendritic cells, Multiple myeloma, T cell anergy
Conflict of interest No
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214
HSANZ POSTERS
P217
The Routine Detection of Cytogenetic Abnormalities in Plasma Cell
Myeloma: Local Experience over a Five-Year Period
Adam Friebe1, Adrian Zordan2, Lynda Campbell2, Meaghan Wall2
1
St John of God Pathology, Geelong, Victoria, Australia; 2Victorian Cancer
Cytogenetics Service (VCCS), Fitzroy, Victoria, Australia
Aim
Best practice guidelines recommend cytogenetic assessment for risk-stratification in
plasma cell myeloma (PCM). This study aimed to measure the local demand for
conventional karyotyping and FISH testing for PCM, to evaluate the quality of local
testing procedures and to identify the key factors that limit the acquisition of
prognostic information by this testing in a routine setting.
Methods
1347 newly diagnosed PCM cases referred to the VCCS between March 2007 and
March 2012 were identified by database interrogation. Cytogenetic results were
analysed with regards to the annual frequency of testing and the rates of
abnormality detection and testing failure. All statistical analyses were descriptive.
Results
Testing demand increased over the review period, although the proportion of
patients evaluated with FISH remained static at 30-40%. Testing failure rates were
low. Detection rates of specific abnormalities among abnormal metaphases and
FISH tests performed were consistent with rates in the literature. However, the
overall proportion of patients with an abnormality identified remained low (34-43%
annually). Major limitations to detecting cytogenetic abnormalities were identified as
a low yield of abnormal metaphases by conventional karyotyping (28%), low uptake
of FISH testing and low percentage of PCM cells by bone marrow aspiration.
Conclusion
Local demand for cytogenetic evaluation in PCM is increasing. Although local
technical methods and scientific analysis have been validated, a low proportion of
patients have an abnormality identified. In addressing the limitations to abnormality
detection identified in this study, the yield of prognostic information could be
improved through better-performed marrow aspiration, expeditious delivery to the
laboratory and wider application of FISH testing.
Keywords
Myeloma, Cytogenetics, FISH
Conflict of interest
No conflict of interest to disclose.
215
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HSANZ POSTERS
P218
A Comparison of Response Rates of Two Romidepsin Dose Strategies
When Combined With Bortezomib, and Dexamethasone in Relapsed or
Refractory Multiple Myeloma
Amit S Khot1, H Miles Prince1-4, Hang Quach2-4, John F. Seymour1,2, David S
Ritchie1,2, Sam Ruell1, Henry Januszewicz1, Simon J Harrison1,2
1
Haematology Department, Peter MacCallum Cancer Centre, East Melbourne,
Australia; 2University of Melbourne, Melbourne, Australia; 3Faculty of Medicine,
Monash University, Victoria, Australia; 4Haematology Service, Monash Medical
Centre, Melbourne, Australia
Aim
There is a strong rationale for combining a proteasome inhibitor with a HDAC
inhibitor to target multiple pathways to overcome drug resistance in myeloma. The
current analysis was carried out to compare the response rate (RR) and toxicity of 2
schedules of romidepsin (Rom) in combination with bortezomib (Bor) and
dexamethasone (Dex) in patients with relapsed / refractory myeloma (RRM).
Methods
Patients with RRM and measurable disease needing treatment, were treated with
Rom10 mg/m2 D1,8,15 at a 28-day (C1) or D1,8 (C2) at a 21-day interval in
combination with Bor 1.3 mg/m2 D1,4,8,11 and Dex 20 mg D1,2,4,5,8,9,11,12 in a
phase 1/2 trial. Responses were defined in study criteria. Fisher’s test was used to
analyse the difference between the RR and toxicity in the 2 groups.
Results
25 patients were enrolled in the phase 1/2 study (C1) in which the maximum
tolerated dose (MTD) of romidepsin was determined to be 10 mg/m2. 13 patients
were treated in C2. The median age (58 yrs vs 59yrs), performance status, stage
and median number of prior therapies (2 vs 2) were similar across cohorts. The
median number of cycles of treatment was 5 (range 1-8) in C1 and 4 (range 2-8) in
C2. The overall RR was 72% (8% CR, 52% PR, 12% MR) in C1 and 61% (8% CR,
38% PR, 15% MR) in C2 (p=0.71). There was no significant difference between the
two groups regarding grade > 3 toxicity (88% vs 69%, p=0.2) and also regarding
haematologic and non-haematologic toxicities including peripheral neuropathy.
Conclusion
Similar ORR and toxicity profile is seen when romidepsin is used in either a 28-day
or 21-day regimen along with bortezomib and dexamethasone in patients with RRM.
The shorter schedule may be preferable; this needs to be confirmed with additional
patient enrolment and longer-term follow up.
Keywords
Myeloma, Bortezomib, Romidepsin
Conflict of interest:
HMP, HQ, JFS, DSR, and SJH receive research funding
from Celgene. SJH and HMP are members of advisory boards for Janssen Cilag.
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216
HSANZ POSTERS
P219
Dendritic Cell Subsets in Myeloma Have Altered Distribution and
Activation with Implications for Disease Behaviour
Christian Bryant1,2, Phillip Fromm1,2, Ross Brown2, James Favaloro2, Hayley Suen2,
John Gibson2, Phoebe Joy Ho2, Douglas Joshua2, Derek Hart1
1
Dendritic Cell Biology and Therapeutics Group, ANZAC Research Institute,
Sydney. 2Institute of Haematology, Royal Prince Alfred Hospital, Sydney
Aim
Despite a growing understanding of the importance of dendritic cells (DC) in multiple
myeloma (MM) our knowledge of their subset distribution, number and activation
state in the bone marrow (BM) is incomplete. Understanding DC biology within the
MM BM niche may contribute to strategies for disease monitoring and the
development of therapeutics.
Methods
We analysed DC in peripheral blood (PB) and BM specimens from patients with MM
(n=25), MGUS (n=8), smouldering myeloma (n=8) and age-matched controls (n=9)
using flow cytometry. Panels included lineage markers, HLA-DR, CD1c, CD141 and
CD304. CD80, CD86 and PDL-1 expression was measured on fresh specimens.
Results
Plasmacytoid DC (pDC) and CD141+ DC were significantly reduced in the PB of
patients with MM but not SM or MGUS compared to healthy controls. In contrast,
they were relatively enriched in the BM of MM patients. pDC increased from a mean
of 12.57+/- 2.08% in the PB, to 49.2 +/- 7.7% of DC in the BM (p<0.05) and CD141+
DC rose from 3.1 +/- 0.7% in the PB to 13.9+/- 5.8% in the BM. In addition, CD80
and CD86 expression was low on DC in the BM in MM and lower in the BM than in
the PB. This relationship was less evident in patients with SM and MGUS.
Conclusion
We describe differential DC compartmentalization between the PB and BM in
patients with MM. Our novel finding of reduced co-stimulator molecule expression
on DC in the BM in MM may be explained by the microenvironment suppressing DC
maturation. The different DC activation in MM and MGUS suggests a role in disease
progression. These data may be useful in monitoring disease and in selecting a
potential therapeutic DC target.
Keywords
Dendritic cell, multiple myeloma, immunobiology
Conflict of interest No
217
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HSANZ POSTERS
P220
Early Application of High Cut-Off Haemodialysis for De-Novo Myeloma
Nephropathy is Associated With Dialysis-Independency and Renal
Recovery
Alhossain Khalafallah1,2,3, Muhajir Mohamed1,2, Sarah Love4, Sie Wuong Loi1, Rose
Mace1, Ramy Khalil2, Miriam Girgs2, Rajesh Raj1, Mathew Mathew1
1
Launceston General Hospital, Launceston, Tasmania, Australia; 2School of
Medicine, University of Tasmania, Australia; 3School of Human Life Sciences,
University of Tasmania, Tasmania, Australia; 4Faculty of Health and life Sciences,
Coventry University, Coventry, West Midlands, UK
Background
Multiple myeloma (MM) is a haematological malignancy associated with kidney
injury resulting from cast nephropathy, which can be caused by monoclonal free
light chains (FLC). It has been demonstrated that early reduction of FLCs can lead
to a higher proportion of patients recovering renal function with a better outcome,
especially if extended high cut-off haemodialysis (HCO-HD) combined with
chemotherapy is used.
Patients and Methods
In this study, four cases with MM nephropathy were treated with HCO-HD and
chemotherapy at the Launceston General Hospital. All of the patients were
presented with acute renal failure and high serum FLCs. Three patients had de novo
MM and one patient had relapsed light chain myeloma disease.
Results
Our data showed that all patients had a significant decrease in serum FLC through
HCO-HD, proving the effectiveness of HCO-HD in managing light chain MM.
Patients who restored renal function and achieved low-level FLC early on in the
treatment had a better chance of becoming dialysis-independent. The patient with
relapsed myeloma remained dialysis dependant.
Conclusion
In summary, our study suggests that if myeloma nephropathy is associated with
light-chain MM, HCO-HD should be initiated as early as possible. At the same time a
specific MM treatment should be initiated to gain control of the disease and salvage
the kidneys to achieve dialysis-independency. Further randomized trials to confirm
our results are warranted.
Key Words: Multiple myeloma, renal failure, High cut-off haemodialysis,
chemotherapy, outcome.
Conflict of interest No
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218
HSANZ POSTERS
P221
Whole Body Magnetic Resonance Imaging and Sestamibi Technetium99m Bone Marrow Scan in Prediction of Multiple Myeloma Disease
Progression and Outcome
Alhossain A. Khalafallah1,2, Andrew Snarski3, Robert Heng1, Ryan Hughes1,
Shamsunnaher Renu1, Jameen Arm1, Richard Dutchke3, Iain Robertson2
1
Launceston General Hospital; 2School of Human Life Sciences, UTAS, Australia;
3
Northern Nuclear Medicine Tasmania, Launceston, Australia
Background and Aim
Bone disease occurs in about 90% of multiple myeloma (MM) patients. There are
few available radio-nuclear imaging techniques for the diagnosis of bone disease in
MM. There are limited data comparing the new diagnostic modalities of MM. This
prospective study was conducted to compare whole body Magnetic Resonance
Imaging (WB-MRI) and Sestamibi Technetium-99m-MIBI Bone Marrow Scan (MIBI)
in assessing bone disease and prognosis in MM.
Subjects and Methods
Sixty two consecutive patients with confirmed WHO-criteria of MM underwent
simultaneous WB-MRI and MIBI scans at the Launceston General Hospital from
January 2010 to January 2011, and their survival status was determined in January
2011. The median age was 62 years (range 37-88) with a male to female ratio of
33:29.
Results
Both MRI and MIBI scans showed bone involvement of MM. In vertebrae and long
bones, MRI scan detected more disease compared to MIBI scan (p<0.001) but there
was less difference in the skull (p=0.09). In the rib-cage, the MIBI scan detected
more lytic lesions of the ribs compared to MRI scan (p<0.001). Thirteen of the 62
patients died during the 24 months follow-up. Analysis of the association between
MM bone disease and mortality rate showed that increased disease detected by
both scans was associated with increased mortality risk (p<0.025). In thoracic and
lumbar vertebrae (p=0.013), and all bone groups (P<0.04), the mean MIBI scan
results provided a better prediction of disease progression and mortality than MRI
scan over the 24 month follow-up period (p=0.012).
Conclusions
Although WB-MRI detected more bone disease than MIBI scan, MIBI scan was able
to predict overall disease outcome and mortality better than MRI scan. Further
studies to confirm our preliminary findings and to define optimum use of these
imaging techniques are warranted.
Keywords Multiple myeloma; MRI scan; Sestamibi bone marrow scan; Outcome,
Mortality.
Conflict of interest No
219
II:219
HSANZ POSTERS
P222
A Randomised Phase 2 Study of Elotuzumab with Lenalidomide and
Low-dose Dexamethasone (Elo/Len/Dex) in Patients With
Relapsed/refractory Multiple Myeloma (RR MM)
1
2,3
3,4
3,5
Philippe Moreau, Paul G Richardson, Andrzej J Jakubowiak, Sundar Jagannath, Marc
6
7
3,8
9
10
11
S Raab, Thierry Facon, Ravi Vij, Donna E Reece, Darrell White, Lotfi Benboubker,
12
13
14
14
15
Jeffrey Zonder, Jean-Francois Rossi, Claire Tsao, Teresa Parli, Glenn Kroog, Anil K
14
3,16
on behalf of the 1703 Study Investigators
Singhal, Sagar Lonial,
1
2
3
Hematology Department, University Hospital, Nantes, France; Dana-Farber Cancer Institute, Boston, MA, USA; Multiple
4
5
Myeloma Research Consortium, Norwalk, CT, USA; University of Chicago, Chicago, IL, USA; Mount Sinai Medical
6
7
Center, New York, NY, USA; Universitaetsklinikum Heidelberg, Heidelberg, Germany; Hopital Claude Huriez, Service
8
9
des Maladies du Sang, Lille, France; Washington University School of Medicine, St. Louis, MO, USA; Princess Margaret
10
11
Hospital, Toronto, Ontario, Canada; Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada; CHU
12
13
Tours-Hopital Bretonneau, Tours, France; Karmanos Cancer Institute, Detroit, MI, USA; CHU de Montpellier-Hopital
14
15
Saint-Eloi, Montpellier, France; Abbott Biotherapeutics Corporation, Redwood City, CA, USA; Bristol-Myers Squibb,
16
Princeton, NJ, USA; Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA.
Background
Elotuzumab is a monoclonal antibody targeting CS1, a cell surface glycoprotein highly
expressed on MM cells. A phase 1 trial of Elo/Len/Dex showed an 82% objective
response rate (ORR) in RR MM (Lonial et al. J Clin Oncol 2012;30(16):1953–9).
Methods
In this phase 2 study, previously-treated patients (pts) with MM were randomised
to Elo 10 or 20 mg/kg IV (days 1, 8, 15, and 22 for two cycles and days 1 and 15 for
subsequent cycles) plus Len 25 mg PO (days 1–21) and Dex 40 mg PO weekly.
Treatment continued until disease progression or unacceptable toxicity. Prophylaxis
for infusion reactions (IRs) was administered prior to each Elo infusion. The primary
objective was to assess efficacy (ORR ≥partial response [PR]).
Results
Among 73 pts (median age 63 years; range, 39–82), 55% had received ≥2 prior
therapies. ORR was 84% for all pts including 53% ≥very good PR. ORR was 92% in
the 10 mg/kg group (n=36), 76% in the 20 mg/kg group (n=37), and 80% in pts with
high-risk cytogenetics. Median time to best response was 2.5 months (range, 0.7–
21.4). Median PFS was not yet reached for the 10 mg/kg group at 17.2 months median
follow up, and was 18.6 months in the 20 mg/kg group. The most common grade 3/4
toxicities were neutropaenia (16%), lymphopaenia (16%), and thrombocytopenia (16%).
IRs were reported in 12% of pts (all grades); 1 pt (1.3%) had grade 3 IR (rash).
Conclusion
Elo/Len/Dex was generally well tolerated and resulted in a high ORR. Median PFS
was not yet reached for the 10 mg/kg group at 17.2 months median follow-up. Phase 3
trials of 10 mg/kg Elo/Len/Dex are ongoing in newly-diagnosed MM and RR MM.
Keywords: elotuzumab, multiple myeloma, phase 2.
Conflict of interest: This research was supported by Abbott Biotherapeutics Corp.
and Bristol-Myers Squibb. Editorial support and graphic services were provided by
StemScientific and funded by both sponsor companies.
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220
HSANZ POSTERS
P223
The Anti-kappa Myeloma Antigen Antibody MDX-1097 Synergises with
Novel Therapeutics to More Effectively Kill Multiple Myeloma Cells
Andrew R Cuddihy1, Tiffany Khong1, Parisa Asvadi2, Rosanne Dunn2, Andrew
Spencer1
1
Division of Blood Cancers, Australian Centre for Blood Diseases, Monash
University, Melbourne, VIC Australia
2
Immune Systems Therapeutics Ltd. Ultimo, NSW Australia
Aim
Multiple Myeloma (MM) is an incurable disease resulting from the clonal proliferation
of malignant B-cells in the bone marrow, with an average survival duration of 3.5
years. To improve survival rates, combinations of established and novel anti-MM
treatments are being assessed. This study examined whether the anti-kappa
myeloma antigen (KMA) antibody MDX-1097, currently being assessed as a single
agent in a phase II clinical trial, could be combined with immunomodulatory drugs
(IMiDs) such as lenalidomide or pomalidomide or histone deacetylase inhibitors
(HDACi) such as panobinostat or vorinostat, to more effectively kill MM cells in vitro.
Results
In vitro IMID-exposed normal PBMCs were 1.5-fold more effective at killing MDX1097 bound JJN3 cells via antibody-dependent cell cytotoxicity (ADCC) compared
to control PBMCs. Similarly, PBMCs from IMiD-treated patients were 1.8 fold more
effective in killing MDX-1097 bound JJN3 cells compared to PBMCs obtained from
the same patient prior to IMID treatment. Treating JJN3 cells with IMiDs resulted in
a 2-fold increase in KMA expression and enhanced ADCC by 1.7 fold in the
presence of normal control PBMCs compared to untreated JJN3 cells. A further
modest increment in ADCC was observed when IMiD-exposed PBMCs were mixed
with IMiD-treated JJN3 cells. Exposure of JJ3 cells to either HDACi caused a 1.5
fold increase in KMA expression levels. Work is underway to determine whether
HDACi-treated JJN3 cells are more susceptible to MDX-1097 mediated ADCC.
Conclusion
These data provide a strong rationale for the further evaluation and clinical
development of MDX-1097 as a therapeutic for MM in combination with other novel
anti-MM agents.
Keywords
Multiple Myeloma, Monoclonal Antibodies, Immunomodulatory drugs
Conflict of interest No
221
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HSANZ POSTERS
P224
High Response Rates with CyBorD as Induction Therapy for Transplant
Eligible Multiple Myeloma
K Romeril, A D’Souza, H Buyck, J Phillips, K Nelson
Wellington Blood and Cancer Centre, Wellington Hospital, New Zealand
Aim
We studied the triplet combination of cyclophosphamide, bortezomib and
dexamethasone (CyBorD) in a 28 day cycle as induction treatment for transplant
eligible de novo multiple myeloma patients. The primary end point was to assess
response after four cycles of therapy and document any toxicity. Twenty-five
patients were analysed after completion of four cycles (16 weeks) of therapy.
Patients received cyclophosphamide 300mg/m2 po, and bortezomib 1.5mg/m2
intravenously on days 1, 8, 15 and 22. Dexamethasone was given at 14mg po on
days 1 – 4, 9 – 12, and 17 – 20 in cycle 1, then 14mg weekly on subsequent cycles
to reduce toxicity. Six patients (24%) had a high risk genetic signature, 17p deletion
in four, t(4;14) in two.
Results
The patients included 16 males and 9 females with a median age of 57. All patients
completed four cycles of therapy and were evaluable for response by modified
EBMT criteria. The overall response (≥ PR) was 88%. 48% had a CR/NCR and 64%
had VGPR or better. Two of the four 17p deletion cases had non-responsive
disease and experienced early death. Fifteen patients have now been auto-grafted
without incidence and some have been evaluated and improved their initial
response.
Conclusion
Weekly bortezomib dosing with a low dose dexamethasone approach has become
the standard of care at our institution and produces a rapid and deep response. The
CR rate is superior to most currently used induction regimes in myeloma. The
combination however does not always overcome high-risk disease and in particular
17p deletion.
Keywords
Myeloma induction Bortezomib
Conflict of interest No
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HSANZ POSTERS
P225
Evidence for Increased Sphingosine Kinase Activity in Acute
Lymphoblastic Leukaemia
Craig Wallington-Beddoe1, Kenneth Bradstock2,Linda Bendall1
1
Westmead Institute for Cancer Research, Westmead Millennium Institute, The
University of Sydney, NSW Australia; 2Haematology Department, Westmead
Hospital, Sydney, NSW Australia.
Aim
Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and
survival, produced by the sphingosine kinases, SK1 and SK2. These enzymes are
under investigation as oncogenic targets, however, little is known about the
interaction of SKs with intracellular signalling pathways. Here we investigate
intracellular interactions of SKs by analysing gene regulation in ALL cells.
Results
Published gene signatures for activation of SK1 or SK2 are not available, so we
treated ALL cell lines with the SK1 or SK2 specific inhibitors SK1-I and ABC294640
respectively, and analysed gene expression by microarray. Although a signature for
SK1 was not obtained, two independent methods of analysis of the microarray data
generated gene signatures that segregated control and drug treated cell lines using
Hierarchical Clustering and Qlucore software. The SK2 signatures were used to
interrogate a large publicly available gene expression dataset obtained from
paediatric ALL patients at the time of diagnosis (GSE28497). This revealed that SK2
activity signatures were more highly expressed in ALL samples (p=0.000007 and
p=0.045 for the smaller and larger signatures respectively) than normal B cell
progenitors. SK1 or SK2 genes were not over expressed in this dataset, except for a
small fraction of BCR/ABL-driven cases. However, increased SK1 protein was found
in ALL cell lines and in patient samples. The SK2 signatures included decreased
expression of the NF-κB inhibitor TRIB3 and the sphingolipid/cholesterol transporter
ABCA1 as validated by RT-QPCR. The former being a component of the NF-κB
proliferative pathway, commonly upregulated in ALL whilst the latter is a cholesterol
and sphingolipid transporter, the function of which may relate to the movement of
S1P produced by SK2 across intra- and extracellular membranes.
Conclusion
This is the first report to suggest that sphingosine kinase 2 activity is increased in
ALL, providing support for targeting SK2 as a therapeutic strategy.
Keywords
Leukaemia, sphingosine kinase, gene expression.
Conflict of interest
No.
223
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HSANZ POSTERS
P226
Molecular Analysis of Two Novel HBA2 Promoter Point Mutations
Causing Down Regulation of HBA2 Transcripts
Talal Qadah1,2,3, Jill Finlayson1,2, Maxine Dennis1, Reza Ghassemifar1,2
1
Department of Haematology, PathWest Laboratory Medicine, QEII Medical Centre,
Nedlands, Western Australia, 2School of Pathology and Laboratory Medicine,
University of Western Australia, Nedlands, Western Australia, and 3Department of
Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz
University, Jeddah, Saudi Arabia.
Aim
The HBA2.c-59C>T and HBA2.c-91G>A point mutations were identified during
routine clinical investigations for thalassemia screening. The aim of this study was to
analyse their molecular effect on HBA2 transcription levels.
Methods
The two HBA2.c-59C>T and HBA2.c-91G>A point mutations were created using our
previously designed HBA2-WT (wild type) expression constructs and site directed
mutagenesis protocol. Human bladder carcinoma cell line (5637) cells were
transfected with either the wild type or the two mutated constructs followed by HBA2
gene transcription analysis using Real-Time PCR.
Results
The analysis showed that HBA2c.-59C>T and HBA2.c-91G>A caused down
regulation of the HBA2 transcripts by 53.7 % and 36.2 %, respectively, compared to
the HBA2-WT.
Conclusion
We have provided experimental evidence that these point mutations reduce the
HBA2 promoter efficiency with a subsequent reduction in HBA2 transcript levels
thus resulting in α-thalassemia phenotype. Such experimental evidence is essential
to assist laboratory diagnosis and genetic counselling.
Keywords: HBA2 promoter region, Point mutation, Real Time PCR
Conflict of interest: No conflict of interest to disclose.
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224
HSANZ POSTERS
P227
Enhancement of Antigen Presenting Ability in the Leukemic
Plasmacytoid Dendritic Cell Line (PMDC05) by Lentiviral Vectormediated Transduction of CD80 Gene
Akie Yamahira1, Miwako Narita1, Kayoko Ishii1, Minami Iwabuchi1, Naoya Satoh1,
Takayoshi Uchiyama1, Tomoyo Taniguchi1, Shigeo Hashimoto2, Noriyuki Kasahara3,
Emmanuelle Faure3, Masuhiro Takahashi1
1
Laboratory of Hematology and Oncology, Graduate School of Health Sciences,
Niigata University, Niigata, Japan
2
Division of Hematology, Nagaoka Red Cross Hospital, Nagaoka, Niigata, Japan
3
Depts. of Medicine and Molecular & Medical Pharmacology, CURE Vector Core &
JCCC Vector Shared Resource Facility University of California, Los Angeles
Aim
PMDC05, a leukemic plasmacytoid dendritic cell (pDC) line which was established
from CD4+CD56+ leukemia in our laboratory, showed a capacity of generating
antigen-specific cytotoxic T lymphocytes (CTLs). In order to enhance an antigen
presenting ability of PMDC05, PMDC05 was transduced with CD80 gene by
lentiviral vector, which was named as PMDC11. We investigated whether PMDC11
could enhance allogeneic T cell response.
Results
PMDC11 enhanced the expression of antigen presentation-associated molecules
and displayed strong antigen presenting ability even without any stimulation
compared with PMDC05. By culturing with stimulators such as calcium ionophore,
PMDC11 gained much more potent antigen presenting ability. Accordantly, IFN-γ
secretion of allogeneic T cells cultured with PMDC11 was increased in PMDC11.
Conclusion
Our data suggested PMDC11 could be applied as antigen presenting cells more
efficiently in adoptive cellular immunotherapy for tumors and severe infections
compared with PMDC05.
Keywords
PMDC11, CD80 gene transduction, lentiviral vector
Conflict of interest
No conflict of interest to declare.
225
II:225
HSANZ POSTERS
P228
Induction of TNF-α Expression in NKT Cells by Sphingosine-1phosphate and α-galactosylceramide
Shiori Ito1, Rie Kondo1, Soichiro Iwaki1, Kazuya Iwabuchi2, Ryunosuke Ohkawa3,
Yutaka Yatomi3, Satoshi Fuji1
1
Department of Molecular and Cellular Pathobiology and Therapeutics, Nagoya City
University Graduate School of Pharmaceutical Sciences, 2Department of
Immunology, Kitasato University School of Medicine, 3 Department of Clinical
Laboratory Medicine, The University of Tokyo
Aim
Natural killer T (NKT) cell is a T lymphocyte with NK markers that recognizes
glycolipid antigens and produces Th1/Th2 cytokines. NKT cells contribute to insulin
resistance and atherosclerosis via cytokine secretion. Sphingosine-1-phosphate
(S1P) is a breakdown product of sphingolipid metabolism that has various
bioactivities. We tried to determine the effects of S1P on the production of
proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas.
Materials and methods
Established NKT cell hybridomas were used for the experiments. NKT cell
hybridomas were stimulated with S1P or glycolipid ligand, α-galactosylceramide
(αGalCer). TNF-α mRNA and protein levels were measured by qPCR and ELISA.
Plasma S1P levels from 120 patients with uncomplicated hypertension and
dyslipidemia were determined by HPLC. Plasma TNF-α levels were measured by
ELISA.
Results
S1P receptors S1P1, S1P2 and S1P4 were expressed in NKT cell hybridomas. Both
S1P and αGalCer increased TNF-α mRNA expression and protein production. S1P
enhanced the induction of TNF-α by αGalCer. The increase of TNF-α mRNA
expression by S1P was reduced by S1P receptor inhibitors and MAP kinase
inhibitor. Plasma S1P levels in patients were correlated with their body mass index.
Conclusion
S1P can regulate the production of TNF-α in NKT cells. Overexpression of TNF-α by
elevated S1P in obesity may induce insulin resistance. S1P receptors on resident
NKT cells may play important roles for development of diet induced insulin
resistance and atherothrombosis.
Keywords
NKT cell, sphingosine-1-phosphate, TNF-α
Conflict of interest
II:226
No
226
HSANZ POSTERS
P229
The Advantages of Novel Dual PI3K/mTOR Inhibitors Over Everolimus
in Pre-B ALL is Restricted to Cases Where Everolimus Administration
Results in AKT Activation
Jacky Wong1, Ken Bradstock2, Linda Bendall1
1
Westmead Institute for Cancer Research, Westmead Millennium Institute, 2Dept.
Haematology, Westmead Hospital, Westmead, NSW, Australia
Aim
To investigate the efficacy of the dual PI3K/mTOR inhibitors BEZ235 and BGT226 in
comparison to everolimus in a NOD/SCID xenograft model of human ALL.
Methods
NOD/SCID mice were engrafted with human ALL xenografts and treatment
commenced once 1% ALL was detected in the blood. Mice were given either
vehicle, 40mg/kg of BEZ235 daily, 10mg/kg BGT226 5 times per week or 5mg/kg
everolimus three times per week. Signalling pathways were examined by western
blot.
Results
The dual PI3K/mTOR inhibitors BEZ235 and BGT226 significantly extended survival
in all 6 xenografts tested compared to control vehicle treated animals. However,
they only produced superior results to everolimus in two xenografts (2032 and 0407)
and were clearly inferior in another (1345). In xenograft 0407 and 2023, exposure to
everolimus increased pAKT (Ser473) levels, demonstrating activation of AKT,
reducing the effects of mTOR inhibition. This AKT activation was prevented by
simultaneous inhibition of PI3K when BEZ235 and BGT226 were used, and was not
observed in most xenografts where dual PI3K/mTOR inhibition was not superior to
mTOR inhibition alone. The level of basal AKT activation differed between
xenografts, however, this did not correlate with in vivo responses to single mTOR or
dual PI3K/mTOR inhibition.
Conclusion
In the majority of ALL samples examined, the survival advantage provided by
everolimus was not further increased by dual inhibition of PI-3K/mTOR. Everolimusinduced phosphorylation of AKT prospectively identified samples where a further
survival benefit was achieved with dual PI-3K/mTOR inhibition.
Keywords
ALL, PI3K/mTOR, BEZ235
Conflict of interest No
227
II:227
HSANZ POSTERS
P230
CD45 Expression Versus Right-angle Light Scatter (CD45/SSC) ‘Cartography’
in Haematological Malignancies. Part 1: Acute Leukaemia
Suneet Sandhu1, Surender Juneja1, 2, Neil Came1, 2
1
The Royal Melbourne Hospital (RMH), Parkville, Victoria, Australia
2
University of Melbourne, Parkville, Victoria, Australia
Aim
CD45 directed cell gating has been utilised for assessing normal and leukaemic
bone marrow in clinical cytometry for nearly two decades although explicit
descriptions of the use of this method for orientation and interpretation of ‘down
stream’ flow cytometric data are relatively few. The geometric increase in
informational content of data generated by modern instruments requires a standard
reference point to guide the most efficient use and interpretation of this information.
CD45/SSC characteristics can provide definitive clues to the lineage of acute
leukaemia even before analysis by specific antibody markers. It can also be very
helpful when assessing residual disease post treatment. The purpose of the first part
of this brief review is to reinforce the importance of the CD45/SSC plot when triaging
acute leukaemia by flow cytometry.
Results
Six cases of newly diagnosed acute leukaemia presenting to RMH in 2011/12 were
chosen for their illustrative value in demonstrating how the CD45/SCC histogram
related back to the morphological characteristics and thus alluded to the final
diagnosis before further detailed immunophenotyping was reviewed. A pictorial ‘test
and teach’ approach was chosen with the CD45/SSC plots matched to respective
photomicrographs of the following acute leukaemic subytpes: Precursor Blymphoblastic, myeloid without maturation, promyelocytic, monoblastic, monocytic
and pure erythroid. A brief explanation of the CD45/SSC characteristics of each
disorder will be provided, referring back to a normal reference CD45/SSC ‘map’.
Conclusion
Early reference to this standard ‘map’ is strongly recommended in the systematic
approach to diagnostic flow cytometry. Knowledge of CD45 and light scatter
characteristics of normal and abnormal haematopoietic cell populations permits
rational predictions about morphological subtypes and helps with further
characterisation of haematological malignancies.
Keywords
II:228
CD45, immunophenotyping, diagnosis
228
Conflict of interest
No
HSANZ POSTERS
P231
CD45 Expression Versus Right-angle Light Scatter (CD45/SSC) ‘Cartography’
in Haematological Malignancies. Part 2: Lymphoma
Sumita Ratnasingam1, Surender Juneja1, 2, Neil Came1, 2
1
The Royal Melbourne Hospital (RMH), Parkville, Victoria, Australia
2
University of Melbourne, Parkville, Victoria, Australia
Aim
CD45 directed cell gating was originally utilised for assessing lymphocytes in clinical
cytometry in order to minimise non-lymphocyte contaminants in blood. However,
CD45/SSC also provides a standard reference point to guide the efficient use and
interpretation of increasingly complex data sets generated by modern instruments.
The purpose of this brief review is to reinforce the early contribution of the
CD45/SSC plot towards the diagnosis of mature lymphoproliferative disorders (LPD)
by flow cytometry.
Results
Ten cases of newly diagnosed lymphoma and an acute infectious mononucleosis
presenting to RMH in 2011/12 were chosen for their illustrative value in
demonstrating how the CD45/SCC histogram relates back to the morphological
characteristics and thus alludes to the final diagnosis before further detailed
immunophenotyping was reviewed. A pictorial ‘test and teach’ approach was chosen
with the CD45/SSC plots matched to respective photomicrographs of the following
lymphoma subtypes: Diffuse large B-cell, grade III follicular, Burkitt, pleomorphic
variant mantle cell, hairy cell leukaemia, and overlayed plots of chronic lymphocytic
leukaemia, mantle cell, follicular and marginal zone lymphoma highlighting
additionally the relative similarity of these latter four cases on the reference plot. A
brief explanation of the CD45/SSC characteristics of each disorder will be provided,
referring back to a normal CD45/SSC ‘map’.
Conclusion
CD45/SSC cartography can provide predictive value as to the subtype in the
diagnostic algorithm of LPD and thus guide the optimum use of complex antibody
panels targeted towards mature and/or precursor lymphoid neoplasms.
Keywords
CD45, immunophenotyping, diagnosis
229
Conflict of interest
No
II:229
HSANZ POSTERS
P232
Direct Effect of Dasatinib on Proliferation and Cytotoxicity of NK Cells in vitro
Study
Takayoshi Uchiyama, Naoya Sato, Miwako Narita, Akie Yamahira, Minami Iwabuchi,
Masuhiro Takahashi
Laboratory of Hematology and Oncology, Graduate School of Health Sciences,
Niigata University, Niigata, Japan
Aim
Lymphocytosis predominantly due to NK cell proliferation has been reported in
nearly a half of CML patients who were being treated with dasatinib. Besides,
dasatinib-treated patients with lymphocytosis have a better prognosis than patients
without lymphocytosis. The aim of this study was to elucidate the direct effects of
dasatinib on the proliferation of lymphocyte subset.
Method
Peripheral blood mononuclear cells (PB-MNCs) isolated from healthy volunteers
were cultured in RPMI1640 (10% FBS) supplemented with IL-2 (50U/ml) and
zoledronate (1µM) for γδT cell culture or IL-2 (500U/ml) for LAK culture. Surface
antigens of the cultured PB-MNCs were analyzed with FACSCalibur.
Cytotoxicity assay was also performed using PB-MNCs cultured in the way
mentioned above as the effector cells and K562 cells transfected with EGFP as the
target cells.
The statistical relevance of differences in the percentages and numbers of
lymphocyte subpopulations in the cultured cells were evaluated using paired
Student’s t-test. The value of P0.05 was considered to be statistically significant.
Results
In both fashions of culture, the addition of dasatinib had a direct effect on NK cell
increase both in percentage and the absolute number. The increase was observed
in the range of dasatinib concentration from 2 to 25nM and was dose-dependent
manner. Lytic ability of cultured cells against EGFP-K562 cells was much higher in
the cells cultured with dasatinib than those without it.
Conclusion
This study suggests that dasatinib-associated lymphocytosis is due to the direct
effect of dasatinib on NK cell expansion in some patients. The dasatinib-induced
expansion of NK cells in CML patients might trigger NK cell cytotoxicity in vivo and
give patients better prognosis.
Keywords
dasatinib, NK cells, proliferation, cytotoxicity
Conflict of interest No conflict of interest to disclosed
II:230
230
HSANZ POSTERS
P234
The Effect of Excessive EDTA on the Morphology of Bone Marrow Smears
Szu-Hee Lee, Carrie van der Weyden, Eleni Mayson, Shrinivas Desai
Department of Haematology, St George Hospital, South Eastern Area Laboratory
Services, Kogarah, New South Wales, Australia
Aim
To document the changes in bone marrow (BM) morphology due to excessive
EDTA, which can arise from underfilling of the collecting tube or inadequate mixing
of the specimen.
Methods
Subjects with negative staging investigations for lymphoma and normal BM
morphology in bedside smears were studied. Bedside smears and smears made
from BM stored in excessive EDTA for two hours at room temperature were stained
with May–Grunwald Giemsa, and cell morphology was evaluated microscopically.
Neutrophil and megakaryocyte size were measured using a calibrated eyepiece
graticule.
Results
Excessive EDTA resulted in nuclear and cytoplasmic contraction with respective
membrane damage, pyknotic nuclei and smudged cells. Specific changes that
mimicked dysplasia were hypolobated neutrophils, small neutrophils and
micromegakaryocytes. In erythroblasts, nuclear contour irregularities were induced,
but in general, artefactual changes did not mimic dyserythropoiesis.
Conclusion
The storage of BM aspirates in excessive EDTA results in morphological changes
that can mimic or obscure dysplasia, thus highlighting the importance of collection of
aspirates in an appropriate concentration of EDTA.
Keywords
EDTA, bone marrow, myelodysplasia.
Conflict of interest No conflict of interest to disclose.
231
II:231
HSANZ POSTERS
P235
A Case of Progressive Mutlifocal Leukoencephalopathy and Outcome
After Treatment with High Dose Cytarabine
R Wooldridge, J Wellwood, M Bryson, G Seeley
Department of Haematology, Gold Coast Hospital, Qld, Australia
A 77 year old male presented to GCH in December 2010 with subacute onset of
weakness and numbness of his left leg, weakness of left arm, a facial drop and
dysphagia. He was 7 months post treatment for low grade Non Hodgkin Lymphoma
(Fludarabine, Cyclophosphamide and Rituximab), and in Remission. A CT scan
showed an area of white matter hypoattenuation in the right parietotemporal region.
Original diagnosis of stroke was made and appropriate stroke management
instituted. He represented in Jan 2011 with worsening symptoms, CT and MRI
showed multiple white matter lesions with no enhancement with gadolinium and no
mass effect. Biopsy of a lesion showed active demyelinating process involving the
cerebral white matter with viral inclusions, consistent with Progressive Multifocal
Leukoencephalopathy. PCR was positive for JC viral DNA and flow cytometry was
negative for a monoclonal B cell population. Infectious diseases Consultation was
sought and a literature review found use of High Dose Cytarabine in non-HIV
patients had shown stabilization of disease for a period of around 3 years. This
treatment was offered to the patient, who accepted. The patient initially became
unresponsive, suggesting inflammation of the brain. He recovered to pre-treatment
status within 1 week, a PEG tube was placed for feeding and he was transferred to
a subacute facility awaiting NH placement. A repeat MRI at 6 weeks post treatment
showed progressive disease, and the patient passed away 7 months post diagnosis.
Keywords
Progressive Multifocal Leukoencephalopathy, Rituximab,
Conflict of interest No
II:232
232
HSANZ POSTERS
P236
Case Report of Refractory HHV8 Driven Haemophagocytic
Lymphohistiocytosis in an HIV Seropositive Patient
Charmaine Wong, Matthew Greenwood
Royal North Shore Hospital, Sydney, NSW, Australia
Haemophagocytic Lymphohistiocytosis (HLH) is a rare, life threatening condition
that can be associated with both HIV and HIV associated Multicentric Castleman’s
Disease (MCD). Management is directed at treatment of the underlying precipitant
while stabilizing the unregulated NK response that characterizes the immune
dysregulation in HLH. A 45 year old recently diagnosed HIV positive male presented
with refractory HLH secondary to HHV8 infection in the absence of morphologically
defined MCD. The patient presented with fevers, lymphadenopathy, splenomegaly,
pancytopenia and hyperferritinemia 3 months after commencing HAART. His HIV
viral load was 125 copies/ml, and CD4 count 380. Bone marrow biopsy revealed
prominent macrophages with haemophagocytosis and an occasional HHV8 positive
cell. Splenic biopsy revealed expanded and congested red pulp with numerous
HHV8 positive cells. There were increased lambda positive cells but no diagnostic
features of MCD. Peripheral blood HHV8 DNA was positive. NK cell function was
reduced, and Perforin activity was reduced. Treatment consisted of HLH2004
protocol with Rituxmab. Rituximab was associated with evidence of tumor lysis
syndrome. Despite an initial response to HLH-2004 and rituximab, the patient
deteriorated in week 3 with recurrent fevers, pancytopenia and progressive
splenomegaly. Repeat BM biopsy demonstrated persistent haemophagocytosis.
Foscarnet and high dose IVIg was added with good clinical effect, with repeat HHV8
PCR showing undetectable levels. HHV8 reactivation in HIV is associated with
immune reconstitution syndrome (IRS), MCD, Kaposi’s Sarcoma, Primary Effusion
Lymphoma and Plasmablastic Lymphoma. HIV MCD is associated with high levels
of HHV8 viremia and has been associated rarely with the development of HLH. NK
dysfunction and low perforin activity suggest a primary role of HHV8 reactivation in
the development of HLH. This case of refractory HLH and subsequent response to
antiviral therapy suggests that the HLH2004 protocol induced immunosuppression
in the setting of HIV may have resulted in prolonged HHV8 viremia. There is no
standard approach to the management of HHV8 associated HLH and this case
suggests the possible utility of antiviral therapy in this disease.
Keywords
Disease
Haemophagocytic Lymphohistiocytosis, HIV, Multicentric Castlemans
Conflict of interest
No conflict of interest
233
II:233
HSANZ POSTERS
P237
Oral Ribavirin is Effective and Well-tolerated for the Treatment of
Respiratory Syncitial Virus and Parainfluenza III Virus Respiratory Tract
Infections in Allogenic Haemopoetic Stem Cell Transplant Recipients
John Casey, Kirk Morris, Manjunath Narayana, Midori Nakagaki, Glen Kennedy
Haematology and Bone Marrow Transplant Unit, Royal Brisbane and Women’s
Hospital, Brisbane, Qld, Australia
Background and Aim
The prognosis for patients with respiratory syncytial virus (RSV) and parainfluenzaIII (PIV-III) respiratory tract infection post allogenic haemopoetic stem cell transplant
(alloSCT) is historically poor. Use of ribavirin by the oral route has not been widely
studied in this patient population. We examined the effectiveness and tolerability of
oral ribavirin in alloSCT recipients infected with RSV and PIV-III.
Methods
Patients with viral respiratory tract infection from November 2009 until March 2012
that were treated with oral ribavirin were identified from an institutional database.
Outcomes were subsequently identified in a retrospective chart audit. Ribavirin dose
was initially 10mg/kg given four times a day and titrated to up to 60mg/kg given four
times a day based on response.
Results
14 patients were identified, 13 were infected with RSV and 1 with PIV-III. 12 patients
were diagnosed during their acute transplant admission and 2 others more than 12
months post-transplant. 9 presented initially with upper respiratory tract symptoms, 1
with lower respiratory tract infection symptoms and 3 with fever. All patients
developed changes on plain chest X-ray consistent with viral pneumonitis. 10 of 14
patients survived the acute infective episode, with 4 deaths attributed to RSV
complications (2 progressive RSV, 1 post-RSV pneumonitis despite clearing the
virus and 1 cerebrovascular accident while intubated). 1 additional patient infected
with RSV cleared the virus but later that admission died from HHV-6 encephalitis.
Radiologic changes on chest X-ray (infiltrates, bronchial wall thickening) often
persisted despite even if the virus was cleared. Treatment was well tolerated with no
patient experiencing adverse effects that could be attributed to ribavirin.
Conclusion
Oral ribavirin appears to be a safe and effective intervention which may assist with
viral clearance and reduction of mortality and morbidity in allo-SCT group.
Keywords
Ribavirin, Respiratory syncytial virus, parainfluenza virus
Conflict of interest No
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234
HSANZ POSTERS
P238
Hepatitis Screening and Monitoring During Rituximab Containing
Regimes at a Single Institution
Jennifer Brotchie, Duncan Carradice, William Renwick, Nicholas Murphy
Western Health, Melbourne, Victoria, Australia
Aim
To analyse current institutional practice with respect to hepatitis B screening prior to
commencement of Rituximab-containing chemotherapy regime, and to describe the
monitoring and management of patients with positive screening results.
Method
We used a retrospective analysis to identify all patients receiving Rituximab with
chemotherapy during the period 1/7/2006 to 12/4/2012.
169 being patients identified from whom we then reviewed results (Hepatitis
serology and biochemistry) and management plans through our electronic records
system and hard-copy files.
Results
71/169 (42%) had documented hepatitis B testing with hepatitis B surface antigen
(HepBsAg) tested. Only 26/71 (36%) had core antibody (HepBcAb) testing. 54/71
had testing within 3 months of chemotherapy.
3/71 (4%) had evidence of chronic hepatitis B infection. 8/71 (11%) had evidence of
prior hepatitis B infection.
All 3 HepBsAg positive patients were treated with anti-viral therapy from the
commencement of chemotherapy. None had evidence of clinical reactivation of
hepatitis B. Strategies employed for management of patients HepBcAb positive only
included prophylactic anti-virals or monitoring. There was no reactivations in the 3
patients on prophylactic anti-virals. Of the 3 patients managed by monitoring
alone, 1 developed laboratory evidence of hepatitis B reactivation. She was
managed with commencement of anti-virals which achieved some control of her
reactivation with undetectable DNA load but continued seropositivity. This patient
was managed with R-CHOP.
No patient developed overt jaundice or acute liver decompensation.
Conclusion
Current practice with regards to hepatits B screening prior to commencement of
Rituximab-containing regimes is suboptimal with varying strategies for management
of patients HepBcAb positive implimented.
Patient care could be improved by implication of an evidence-based policy including
universal hepatitis B screening prior first chemotherapy treatment
Keywords
Rituximab, Hepatitis B, Screening
Conflict of interest No
235
II:235
HSANZ POSTERS
P239
Evaluation of CD64 as a Marker of Early Sepsis and Predictor of
Recovery in Neutropenic Patients Post-Myeloablative Chemotherapy,
and its Correlation with Laboratory Markers of Sepsis and Absolute
Neutrophil Count
Rachel Cooke, Stephen Valentine, Carole Smith, Ray Dauer
Austin Pathology, Melbourne, Victoria, Australia
Aim
Sepsis invariably occurs in patients with prolonged, severe neutropenia (<0.5
x109/L) and, in patients receiving myeloablative chemotherapy, it is critical to
commence antibiotics at the first sign of sepsis. CD64 is a high affinity receptor to
IgG that is up-regulated on polymorphonuclear (PMN) cells in response to IFNγ and
growth-colony stimulating factor (GCSF). Quantitative PMN CD64 expression by
flow cytometry has been shown to be a sensitive and specific marker of sepsis in
emergency and post-operative settings. This study aims to show that PMN CD64
can be a valid and early marker of sepsis in patients with severe neutropenia.
Method
20 patients were recruited from the haematology unit who were receiving
myeloablative chemotherapy as treatment for various haematological malignancies.
Daily PMN CD64 index measurements were correlated with clinical and laboratory
evidence of sepsis. A PMN CD64 index cut-off of 2.0 for a positive result was used.
Result
Positive PMN CD64 results were achieved with severe neutropenia that were
comparable with measurements taken at a normal/elevated neutrophil count. False
negative results occurred when the number of events within the PMN gate on flow
cytometry was less than 500-700. After excluding results obtained when there was
no clearly definable population to gate on flow cytometry, PMN CD64 was a
sensitive but not specific marker for sepsis (sensitivity 97%, specificity 49%, PPV
57%, NPV 96%). 70% of the false positive results were obtained in the days prior to
sepsis, and in some cases these occurred in the context of developing mucositis or
viral pneumonitis (before an increasing trend in CRP).
Conclusion
Provided there is a clearly definable population of PMNs to gate, PMN CD64 is a
sensitive marker of sepsis when patients were severely neutropenic. PMN CD64
may be up-regulated with mucosal/bronchial tissue damage (in the context of severe
neutropenia) prior to an acute phase response and thus may serve as an early
warning of ensuing sepsis. An alternative explanation is that patients with certain
haematological diseases constitutively have higher levels of IFNγ.
Keywords: Sepsis, neutrophil, neutropenia
Conflict of interest: No
II:236
236
HSANZ POSTERS
P240
Cytomegalovirus Disease in Acute Lymphoblastic Leukaemia Treated With
HyperCVAD Chemotherapy
Tharma Balakrishnan1, Richard Yiu2
1
Department of Internal Medicine, Singapore General Hospital, Singapore
2
Department of Haematology, Singapore General Hospital, Singapore
Background
Cytomegalovirus (CMV) disease is a common viral infection in bone marrow
transplant patients with 19% in seronegative and as high as 63% in seropositive
recipients. Therefore routine CMV surveillance is recommended in bone marrow
transplant setting. However there are no data on the prevalence of cytomegalovirus
infection in patients treated with intensive hyperCVAD chemotherapy for acute
lymphoblastic leukaemia (ALL). The role of routine CMV screening in the setting of
intensive chemotherapy is unclear.
Aim
To determine the prevalence of CMV disease in ALL treated with hyperCVAD
chemotherapy.
Results
Total 140 patients with ALL treated with hyperCVAD, were included in this analysis
with median follow up duration of 7 months. The median age of this cohort was
36.9years (range 14 to 67years). There were 74 males and 66 females. Total 9
(6.4%) patients had CMV antigenaemia and previous CMV exposure with positive
CMV IgG noted in 49 (35%) patients. CMV disease was noted in 6 patients (4.3%).
Of these 1 patient had pneumonitis, 1 had meningoencephalitis, 1 had hepatitis and
3 others had viremia with positive antigenaemia. Death occured in 1 patient with
CMV meningoencephalitis.
Conclusion
Our analysis shows that the prevalence of Cytomegalovirus Disease among patients
with diagnosis of Acute Lymphoblastic Disease treated with hyperCVAD
chemotherapy is relatively low. Therefore routine CMV surveillance testing is not
necessary.
Keywords
Acute Lymphoblastic Leukaemia, Cytomegalovirus Disease,
HyperCVAD Chemotherapy
Conflict of interest No
237
II:237
HSANZ POSTERS
P241
Expansion of Adenovirus Specific T Cells Suitable for Clinical Use
Rebecca Brown1, Renee Simms1, Jane Burgess1, Leighton Clancy1,2, David
Gottlieb1,2,3
1
Westmead Millennium Institute, Sydney, Australia, 2Sydney Cellular Therapies
Laboratory, Westmead Hospital, Sydney, Australia. 3University of Sydney, Australia
Introduction
Adenovirus (AdV) infection causes significant morbidity in paediatric patients
following HSCT. Treatment options are limited, cidofovir is not always effective and
is associated with significant toxicity. Control of infection correlates with the
emergence of T cells specific for the AdV hexon protein. Recently clinical grade AdV
hexon peptides have become available to expand T cells for adoptive transfer.
Aim
To compare a clinically acceptable method for the ex vivo expansion of AdV specific
T cells using dendritic cells (DC) matured with TNF-α alone or a cytokine cocktail.
Methods
Monocyte derived DC were matured with either 200U/ml of TNF-α or a cocktail
including 1000U/ml TNF-α, 1µg/ml PGE-2, 1900U/ml IL-1β and 1000U/ml IL-6. DC
were pulsed with an AdV hexon protein peptide mix and co-cultured with autologous
PBMC to stimulate expansion of AdV CTL. Cultures were restimulated after 7 days
and supplemented with IL-2 every 2-3 days for 14 days.
Results
We compared the expansion of AdV specific T cells in 3 donors using DC matured
with TNF-α alone or a cytokine cocktail reported to increase expansion and improve
CD8 responses. There was a mean 9 fold increase (range 5-11 fold) in cell number
using TNF-α treated DC and 7 fold increase (range 4-10 fold) with cocktail treated
DC. All cultures consisted primarily of T cells (mean 94.5%) with a higher proportion
of CD4 (mean 59.8%) compared to CD8 (mean 15.2%) T cells. The proportion of
CD8 T cells was higher in all cultures expanded with cocktail treated DC. The
proportion of T cells in starting donor PBMC that produced interferon-γ in response
to AdV hexon ranged from 0.1 to 0.3%. This increased to 25.3% (13-41%) of T cells
when expanded with TNF-α treated DC and 20% (18-24%) with cocktail matured
DC. For 2 of 3 donors a higher proportion of CD8 T cells were specific for AdV when
expanded with cocktail treated DC compared to TNF-α DC (13.6%±13.8 and
4.8%±2.7 respectively).
Conclusion
This study has provided an acceptable method for the ex vivo expansion of AdV
specific T cells. Using the cytokine cocktail to mature DC proved to be effective at
inducing a higher number of AdV specific CD8 T cells.
Keywords: Adoptive transfer, Adenovirus, Infection Conflict of interest: No
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238
HSANZ POSTERS
P242
Pathway Use in the Emergency Department Reduces the Time to
Antibiotic Administration for Infected Immunocompormised
Haematology Patients
Natalia Gavrilova1, Lois Surgenor1,2, Polly Grainger3, , Ruth Spearing1,2
1
Department of Haematology, Christchurch Hospital, New Zealand
2
University of Otago at Christchurch, New Zealand
3
Emergency Department, Christchurch Hospital, New Zealand
Background
Sepsis in imumunocompromised patients, especially those with neutropenia, is
potentially life-threatening. In non-immunocopromised patients with severe sepsis,
the giving of antibiotics in <1hr leads to a significantly lower mortality. For the last
four years the Emergency Department (ED) at Christchurch Hospital has been using
an Immunosuppressed Patient’s Clinical Pathway (Pathway) to expedite the
management of immunocompromised patients.
Aim
This study assesses the use of the Pathway for haematology immunocompromised
patients with fever or other features of infection.
Method
Systematic file review was undertaken for admissions of haematology patients with
problems related to infections and neutropenia/severe immunocompromised state
presenting through the ED from November 2010-2012.
Results
Of the 80 admissions identified, 70% utilised the Pathway. Time to antibiotics was
significantly shorter for those on the Pathway compared with those were not (p
<.05), and importantly, Pathway use was significantly associated with receiving
antibiotics within an hour (p <.01). There was also a trend for those on the Pathway
to spend less time in the ED (p <.10). Unlike other studies, time of day or day of
presentation to the ED was not significantly associated with time to antibiotics.
Conclusion
Being on the Immunocompromised Clinical Pathway means that patients are
significantly more likely to receive antibiotics within the time recommended by the
National Chemotherapy Advisory Group (UK). Further research is required to
understand the patient, clinician and system factors involved in non-use of the
Pathway, and in turn how a higher rate of compliance can be achieved. This study is
limited by a small sample and thus larger studies are needed to enable examination
of the other contextual factors that may explain use of the Pathway.
Keywords
Immunocompromised; Neutropenic Fever
Conflict of interest No
239
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HSANZ POSTERS
P243
Generation of T-cells Expressing a CD19-Specific Chimeric Antigen
Receptor Using the PiggyBac Transposon/Transposase Gene
Modification System
Saumya Ramanayake, Ian Bilmon, David Gottlieb, Kenneth Micklethwaite
Westmead Millennium Institute, University of Sydney, Westmead, Australia
Aim
To optimise the electroporation and culture conditions for the generation of T-cells
expressing CD19-specific chimeric antigen receptors (CARs) using the non-viral
PiggyBac transposon/transposase system of gene modification.
Methods
4x106 peripheral blood mononuclear cells (PBMC) from donors undergoing
venesection for haemochromatosis were electroporated in the presence of 5ug each
of the PiggyBac transposase plasmid and a corresponding transposon plasmid
encoding a CAR specific for CD19. Electroporation was carried out at a range of
voltages (2000-2400v) and pulse widths (2x15ms and 1x20ms) using the Neon
electroporation system (Invitrogen). Transduced cells were rested for 24 hours and
then stimulated on day+1 with CD19+ targets at effector:stimulator ratios ranging
from 1:10 to 10:1. CAR T-cells were re-stimulated on day+8 and harvested on
day+15. Cells were enumerated by trypan blue exclusion and viability, CD3 and
CAR expression were analysed by flow cytometry.
Results
Electroporation of PBMC with a single 20ms pulse at 2400v produced optimal
transduction efficiency with mean percentage CAR expression on day+1 of 25.6% of
CD3+ cells (n=6, range 12.2%-47.6%) and a mean percentage recovery of 31%
(range 18.0%-44.0%). Stimulation with the CD19+ Nalm-6 cell line at an E:S ratio of
4:1 or PBMC at an E:S ratio of 1:1 produced optimal CAR T-cell expansion.
Stimulation with Nalm-6 cells led to enrichment of mean CAR expression over a 14
day culture period from starting expression of 30.4% to 78.2% (n=4, range 62.5%80.3%) with 12.3-fold overall cell expansion (range 3.7-20.8). Stimulation with
PBMC produced enrichment of mean CAR expression to 64.75% (range 56.4%77.9%) and 45.5-fold expansion (range 19.7-61.3).
Conclusion
Genetic modification using the non-viral PiggyBac system followed by stimulation
with autologous PBMC provides a simple and effective means of generating T-cells
expressing a CD19-specific CAR. This work lays the foundations for generating
clinical grade T-cell products for phase I trials in patients with relapsed B-cell
malignancies.
Keywords
Genetic modification, chimeric antigen receptors, immunotherapy
Conflict of interest No conflict of interest to disclose
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240
HSANZ POSTERS
P244
Detecting Folate Receptor Autoantibodies and the Implications They
Might Have in Relation to Stroke Patients
Lisa Holmes 1,2, Quintin Hughes1,2, Ross Baker1,2
1
The Thrombosis and Haemophilia Centre, Murdoch University, Murdoch, Western
Australia
2
Department of Haematology, Royal Perth Hospital, Perth, Western Australia
Background
Folate deficiency causes increased levels of homocysteine that has been
associated with thrombotic events such as stroke. Interestingly folate
supplementation is reported to decrease the risk of stroke by up to 18%. Recent
studies have described autoantibodies directed against a key transporter of folate,
Folate Receptor alpha (FRα) that block folate uptake causing folate deficiency within
the target tissues.
Aim
To investigate if there is an association between the presence of FRα
autoantibodies and stroke
Methods and Results
An Enzyme linked Immunosorbant assay (ELISA) has been developed to detect
FRα autoantibodies present in patient serum and plasma. A normal FRα
autoantibody range was established using 26 control subjects. Based on the values
obtained cut-off values were established to describe low and high positive antibody
titres. A low positive is defined as an optical absorbance (OA) of >2 standard
deviations above the mean normal value, with a high positive defined as > 5
standard deviations. The Thrombophilia in Stroke Study (TISS) cohort (Royal Perth
Hospital) consisting of 219 first ever ischemic stroke patients, and 205 matched
control subjects is currently undergoing screening using the ELISA. The cohort has
extensive vascular risk factor data including folate and homocysteine levels, c677t
Methylene Tetrahydrofolate Reductase (MTHFR) polymorphism status and data
relating to autoimmune conditions including Antiphospholid syndrome.
Conclusion
A valuable ELISA tool for detecting FRα autoantiboides has been developed and a
normal population range has been established. Ongoing screening of a well
characterised stroke cohort, should provide valuable information to determine if the
presence of these autoantibodies constitute a risk for stroke.
Keywords
Folate Receptor alpha, Autoantibodies, Stroke
Conflict of interest No
241
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HSANZ POSTERS
P245
Effects of Daily Iron Supplementation in 2-5 Year Old Children:
Systematic Review and Meta-Analysis
Jane Thompson1, Beverly-Ann Biggs2, Sant-Rayn Pasricha2
1
School of Medicine, Faculty of Health Sciences, The University of Adelaide
2
Department of Medicine, Royal Melbourne Hospital, Faculty of Medicine, Dentistry
and Health Sciences, University of Melbourne
Aim
Causes and epidemiology of anaemia differ in 2-5 year olds compared with their
younger counterparts. To develop evidence to support clinical practice and public
health, we summarized evidence for benefit and safety of daily iron supplementation
with regard to haematological, growth and cognitive outcomes in preschool children.
Methods
We performed electronic searches of MEDLINE, CENTRAL, SCOPUS and WHO
regional databases for randomised controlled trials comparing daily oral iron
supplementation with control in 2-5 year old (preschool) children. Random effects
meta-analysis was used to synthesise pre-defined outcomes reported by ≥2 studies.
Results
Of 9169 references identified, 15 studies met inclusion criteria, none of which were
considered at low risk of bias. Children receiving iron had a mean endpoint
hemoglobin (Hb) 6.97g/L (p<0.00001, I2=82%) greater than controls, while mean
endpoint ferritin was 11.64µg/L (p<0.0001, I2=48%) greater. Subgroup analysis
showed that iron supplementation resulted in a greater improvement in Hb in
anemic subjects compared with non-anemic subjects. A total iron intake of >2500mg
showed no additional advantage over an intake of <2500mg for improving Hb but
did improve ferritin more. No trials reported effects of iron on iron deficiency or iron
deficiency anemia and only one reported on anemia (finding no benefit from iron).
Limited evidence suggested that iron supplementation produced a small
improvement in cognitive development. Iron had no effect on physical growth.
Conclusion
In 2-5 year olds, daily iron supplementation increases Hb and ferritin. Our analysis
highlights a concerning lack of data on the effect of iron supplementation on
clinically important outcomes including anaemia and cognitive development. Given
the public health burden imposed by iron deficiency, especially in the developing
world, further studies in this important age group are needed.
Keywords
Iron, anaemia, supplementation, haemoglobin, preschoolers
Conflict of interest
II:242
No conflict of interest to disclose
242
HSANZ POSTERS
P246
Clinical Symptoms of Hemolysis Are Predictive of Disease Burden and
Mortality in Asian Patients with Paroxysmal Nocturnal Hemoglobinuria
(PNH)
Jong Wook Lee,1 Jun Ho Jang,2 Jin Seok Kim, 3 Sung-Soo Yoon,4 Je-Hwan Lee,5
Yeo-Kyeoung Kim,6 Deog-Yeon Jo,7 Jooseop Chung,8 Sang Kyun Sohn9
1
2
The Catholic University of Korea, Seoul, South Korea; Sungkyunkwan University School of
3
Medicine, Seoul, South Korea; Yonsei University College of Medicine, Seoul, South Korea;
4
5
Seoul National University, Seoul, South Korea; University of Ulsan College of Medicine,
6
Seoul, South Korea; Chonnam National University Hwasun Hospital, Hwasun, South Korea;
7
8
Chungnam National University Hospital, Daejeon, South Korea; Pusan National University
9
Hospital, Pusan, South Korea; Kyungpook National University Hospital, Daegu, South
Korea
Aim
This study aims to describe disease manifestations and burden in Asian PNH
patients.
Method
Retrospective analysis of 301 patients from the national South Korean PNH registry.
Result
At diagnosis, the median lactate dehydrogenase (LDH) level was 4.1-fold above
upper limit of normal. Corticosteroids and immunosuppressive therapies were in use
by 77% and 22% of patients, respectively. Thromboembolism (TE) was detected in
18% of patients and was a strong risk factor for mortality (odds ratio [OR] 8.42, 95%
confidence interval [CI] 4.15-17.08, P<0.0001). Impaired renal function was reported
in 16.9% of patients and was a significant risk factor for TE (OR 3.78, 95% CI 1.937.42, P<0.001) and mortality (OR 3.41, 95% CI 1.66-7.02, P=0.001). 56% of
patients reported pain, with 39% requiring medical intervention. Abdominal pain
(47%) was a strong marker for TE (69% of patients with TE had abdominal pain)
and a significant risk factor for TE (OR 2.94, 95% CI 1.57-5.51, P<0.001) and
mortality (OR 2.104, 95% CI 1.087-4.08, P=0.026). Symptoms of pulmonary
hypertension were prominent (43%), with dyspnea (OR 2.52, 95% CI 1.382-4.58,
P=0.002) and chest pain (OR 2.82, 95% CI 1.33-5.95, P=0.009) identified as
significant risk factors for TE.
Conclusion
These data demonstrate that Asian PNH patients frequently suffer disabling
symptoms during the course of PNH disease. Abdominal pain, dyspnea, and chest
pain were identified as risk factors for TE, and TE was found to be a risk factor for
mortality. Complications and early mortality continued despite medical intervention.
Keywords: paroxysmal nocturnal hemoglobinuria, hemolysis, thrombosis
Conflict of interest: Editorial support for the abstract was supported by Alexion
Pharmaceuticals. The company had no role in collecting or analyzing the data.
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HSANZ POSTERS
P247
Lactate Dehydrogenase (LDH) ≥1.5× Above Normal: A Sensitive and
Specific Marker of Patients at Risk of Clinical Complications and
Mortality Associated with Paroxysmal Nocturnal Hemoglobinuria (PNH)
Jong Wook Lee,1 Jun Ho Jang,2 Jin Seok Kim, 3 Sung-Soo Yoon,4 Je-Hwan Lee,5
Yeo-Kyeoung Kim,6 Deog-Yeon Jo,7 Jooseop Chung,8 Sang Kyun Sohn9
1
2
The Catholic University of Korea, Seoul, South Korea; Sungkyunkwan University School of
3
Medicine, Seoul, South Korea; Yonsei University College of Medicine, Seoul, South Korea;
4
5
Seoul National University, Seoul, South Korea; University of Ulsan College of Medicine,
6
Seoul, South Korea; Chonnam National University Hwasun Hospital, Hwasun, South Korea;
7
8
Chungnam National University Hospital, Daejeon, South Korea; Pusan National University
9
Hospital, Pusan, South Korea; Kyungpook National University Hospital, Daegu, South
Korea
Aim
Elevated LDH is a marker for uncontrolled complement activation and hemolysis in
PNH. The study aim is to evaluate whether LDH ≥1.5× upper limit of normal (ULN) at
diagnosis is a risk factor for thromboembolism (TE) and mortality.
Method
Retrospective analysis of 301 patients from the national South Korean PNH registry.
224 patients who had reported LDH level at diagnosis were analyzed.
Result
Patients with LDH ≥1.5×ULN at diagnosis had a 4.8-fold greater mortality rate compared
with an age- and gender-matched general population (P<0.001). A multivariate analysis
that controlled for age, gender, and the presence of bone marrow disorder at diagnosis
confirmed that LDH ≥1.5×ULN was an independent risk factor for mortality (odds ratio
[OR] 10.57, 95% confidence interval [CI] 1.36-81.93, P=0.024). Patients with LDH
<1.5×ULN had a similar mortality rate as the general population (P=0.824). A threshold
of LDH ≥1.5×ULN detected 93% of patient deaths and 96% of patients with TE. Early
mortality was not predicted by higher LDH thresholds of ≥3×ULN (OR 1.8, 95% CI 0.784.09, P=0.162) or ≥5×ULN (OR 2.0, 95% CI 0.91-4.32, P=0.082), and these thresholds
only detected 67% and 47% of patients, respectively, who experienced a TE.
Conclusion
These data demonstrate that chronic, uncontrolled complement activation indicated by
LDH ≥1.5×ULN at PNH diagnosis is a strong and independent risk factor for clinical
complications and mortality in PNH patients. Thus, physicians should consider LDH
≥1.5×ULN as a strong indicator of risk for clinical complications and mortality that
warrants early intervention.
Keywords: paroxysmal nocturnal hemoglobinuria, lactate dehydrogenase, thrombosis
Conflict of interest: Editorial support for the abstract was supported by Alexion
Pharmaceuticals. The company had no role in collecting or analyzing the data.
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244
HSANZ POSTERS
P248
Aplastic Anaemia: Establishment of a New International Registry
Simon Wilkins1, Zoe McQuilten1,2,3, Louise Phillips 1, Xavier Badoux4, Ashish Bajel5,
Christina Brown6, Catherine Cole7, Gillian Corbett13, Gavin Cull8, Devendra Hiwase9,
John Gibson6, Anna Johnston10, Tony Mills11, Stephen Opat3, Fernando Roncolato4,
Jeff Szer5, Merrole Cole-Sinclair12, Erica Wood1,2,3,5, Frank Firkin12
1
Monash University, VIC; 2Australian Red Cross Blood Service; 3Monash Medical
Centre, VIC; 4St George Hospital, NSW; 5Royal Melbourne Hospital, VIC; 6Royal
Prince Alfred Hospital, NSW; 7Princess Margaret Paediatric Hospital, WA; 8Sir
Charles Gardiner Hospital, WA; 9Royal Adelaide Hospital, SA; 10Royal Hobart
Hospital, TAS; 11Princess Alexandra Hospital, QLD; 12St Vincent’s Hospital, VIC, All
in Australia; 13Waikato Hospital, New Zealand.
Aim
Aplastic anaemia (AA) is a rare disease with significant morbidity & mortality and
substantial transfusion requirements. Information regarding the natural history of AA
is scarce. Currently there are few local data on AA incidence, therapies or clinical
outcomes. The relative rarity of AA makes both accrual of data and material to
support scientific studies, and the establishment of high quality randomised
prospective trials, challenging. The existing literature may be biased towards
reporting good outcomes and/or serious or unusual events. Establishment of a
registry for all Australian and New Zealand (NZ) patients with AA would provide an
important community resource to address these issues.
Results
A new registry to determine incidence and natural history of adult and paediatric AA,
explore factors influencing clinical outcomes, inform patient management and
inspire further research has been developed. Over 50 sites in Australia and NZ have
agreed to participate and ethics applications are underway. Patients are identified
and registered by treating clinicians. Data are recorded via specifically designed
web-based forms, recording details of presentation, management and outcomes.
Sharing information with participating clinicians and hospitals is a high priority.
Regular reports regarding accrual and outcomes, including analyses of national,
state and local incidence and management will be provided.
Conclusion
This new registry for AA in Australia and NZ will provide a means to improve clinical
practice, inform future prospective clinical trials, and will deliver important insights
into the disease.
Keywords aplastic anaemia, registry, international
Conflict of interest The registry is partly supported by Alexion Pharmaceuticals
Australasia and Genzyme Inc. Neither company had any role in analysing the data
or preparing the abstract.
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HSANZ POSTERS
P249
Daily Iron Supplementation for Improving Growth and Development in
Children Aged 4-23 Months: A Systematic Review and Meta-Analysis
Emily Hayes, Kongolo Kalumba, Sant-Rayn Pasricha
Monash Medical Centre, Southern Health, Clayton, VIC, Australia
Aim
Daily iron supplementation has been suggested to affect physical and cognitive
development in children. To better inform public health policy and clinical practice
we sought to determine the potential risks and benefits of iron supplementation in
young children aged 4-23 months.
Methods
Electronic databases were searched for randomised controlled trials comparing daily
oral iron to control in 4-23 month old children. Eligibility was screened independently
by two authors with data extracted by one author. Random effects meta-analysis
was used to synthesise pre-defined outcomes reported in at least two studies.
Results
9169 studies were identified in the search, of which 76 were screened for eligibility,
52 met our inclusion criteria, and 41 had useable data for meta-analysis. We found
that children receiving iron experienced less weight gain (standardised mean
difference (SMD) -1.25 95% CI -2.2 to -0.3, p=0.01, I2=97%) and linear growth (SMD
-0.88 [-1.68 to -0.08], p=0.03, I2=96%) during the intervention than did controls. Iron
did not improve Z-scores for weight-for-age (MD -0.01 [-0.09 to 0.07], p=0.79,
I2=24%), length-for-age (MD -0.03 [-0.09 to 0.03], p = 0.36, I2 = 0%) or weight-forlength (MD 0.03 [-0.06 to 0.12], p=0.50, I2=46%). Developmentally, iron
supplemented children had similar final Bayley’s indices of mental (MD 1.68 [-0.65
to 4.01], p=0.16, I2=69%, 6 studies) and psychomotor (MD 1.08 [-1.36 to 3.51],
p=0.39, I2=69%, 6 studies) development compared with controls, and iron did not
benefit cognitive development even where only anaemic children were recruited.
Conclusion
Our findings suggest that daily iron supplementation to children 4-23 months of age
does not clearly improve physical or cognitive development; iron supplementation
may even be detrimental to physical growth. Further trials in this age group
evaluating growth and developmental outcomes with iron are urgently needed.
Keywords: Iron, anaemia, children, systematic review
Conflict of interest: No conflicts of interest to disclose
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246
HSANZ POSTERS
P250
Intravenous Iron Therapy is Associated with Improved Maternal Quality
of Life, Less Postnatal Depression and Longer Breastfeeding after
Treatment of Iron Deficiency Anaemia in Pregnancy
Alhossain Khalafallah1,2,3, Amanda Dennis1,2, Kath Ogden2, Iain Robertson1,3 Ruth
Charlton1,2, Jackie Bellette1,2, Jessica Shady1,2, Nep Blesingk1,2, Madeleine Ball3
1
Launceston General Hospital; 2School of Human Life Sciences, UTAS, Australia
Background and Aims
To date there are no data available regarding the impact of intravenous versus oral
iron on the wellbeing and health-related quality of life (HRQoL) in particular
postnatal depression and duration of breast- feeding during and after pregnancy.
Design and Interventions
We conducted a prospective randomised controlled open label trial of intravenous
versus oral iron therapy for pregnancy-related iron deficiency anaemia between
March 2007 and January 2009 at the Launceston General Hospital, Tasmania,
Australia. The follow up study was conducted between June and October 2010
using a modified version of the SF-36 questionnaire together with the original
prospective HRQoL data collected during 2nd and 3rd trimesters of pregnancy as well
as 6-8 weeks post delivery.
Patients and Methods
126 women completed the follow up HRQoL study. The participants were followed
up post-delivery for a median period of 32 months (range, 26-42) with a well-being
and health-related QoL questionnaire using a SF-36 QoL survey and child growth
charts as set by the Australasian Paediatric Endocrine Group (APEG).
Results
Patients who received intravenous iron demonstrated significantly higher Hb and
serum ferritin levels (p<0.001). There were strong associations between iron status
and a number of the HRQoL scales with improved general health (P=0.021),
improved physical energy (P=0.016), less psychological downheartedness
(P=0.005), less clinical depression (P=0.003), and overall improved mental
component scale (P<0.001). The duration of breastfeeding was longer (P=0.046) in
women who received intravenous iron. The babies born in both groups recorded
similarly on APEG growth chart assessments.
Conclusions
Our data suggest that HRQoL is improved in anaemic pregnant women by repletion
of their iron stores during pregnancy. About 80% of the intravenous iron polymaltose
group showed a maintained normal ferritin until delivery with long-term benefits and
a minimal effect on their babies. Further studies are warranted.
Key words Quality of life, iron deficiency, oral iron, intravenous iron, pregnancy.
Conflict of interest No
247
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HSANZ POSTERS
P251
Identification of a Novel High Reticulocyte ENU Murine Strain as a
Model of Human Ankyrin Related Spherocytosis
Thu V Tran1, Katrina Kildey1,2, Robert Tunningley3, Robert L Flower1,2, Melinda M
Dean1
1
Research and Development, Australian Red Cross Blood Service, Brisbane QLD
Australia, 2Queensland University of Technology, Brisbane QLD Australia,
3
Australian Phenomics Facility, Canberra ACT Australia
Aim
The study of immune responses in animal models has been paramount in guiding
current understanding of human immune function. N-ethyl-N-nitrosourea (ENU)directed mutagenesis was utilised to identify mutations with relevance to
haematological disease states and blood transfusion.
Methods
33 ENU-induced mutant mouse lines were screened for abnormalities in blood
physiology. Evidence of a changed blood picture was assessed via differential cell
count (RBC, platelet, neutrophil lymphocyte, monocyte, eosinophil, basophil, and
reticulocytes) and measurement of key haematological parameters (haemoglobin,
haematocrit, mean cell volume). Blood physiology was compared between affected
and unaffected mice (Unpaired T test, 95% CI).
Results
An ENU-mutant phenotype with significantly elevated reticulocyte count was
identified (P<0.0001). The high reticulocyte count was compounded with
significantly reduced RBC count (P<0.0001), haemoglobin (P<0.0001), haematocrit
(P<0.0001), and mean cell volume (P<0.0001). Sequencing revealed the phenotype
was the result of a novel splice variant of ankyrin-1 (ANK-1), specifically G→A
substitution at first intronic base for exon 35. ANK-1 provides the link between RBC
membrane structural proteins (spectrin and band 3) and the inner surface of the lipid
bilayer. A number of human ANK-1 mutations have been reported which are
associated with hereditary spherocytosis. The phenotype of this novel murine ANK1 ENU-pedigree reflects the human clinical scenario. The ANK-1 strain is viable and
heterozygous mice exhibit a milder form of spherocytosis with significantly elevated
reticulocyte count (P<0.01), and reduced mean cell volume (P<0.05).
Conclusions
This novel ENU-induced ANK-1 mutation models human hereditary spherocytosis
and is a valuable tool for further characterisation of this blood disease that may
require transfusion support.
Keywords: Spherocytosis, Reticulocyte, Ankyrin -1, ENU mutagenesis
Conflict of interest: No conflict of interest to disclose
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248
HSANZ POSTERS
P252
Identification of Haemoglobin Variants Using Nanolitre Blood Samples,
Microwave-Mediated Chemical Cleavage and Mass Spectrometry
Reinhard Boysen1, Asif Alam1,2, Agron Mataj1, Yuanzhong Yang1, Donald Bowden2,
Milton Hearn1
1. Centre for Green Chemistry, Monash University, Melbourne, VIC, Australia.
2. Clinical Genetics Laboratory, Southern Health, Melbourne, VIC, Australia.
Aim
In clinical diagnosis, mass spectrometry is an emerging technique for the
characterization of disease-specific proteins, protein variants of clinical relevance or
post-translational modified proteins as biomarkers of a disease. The emphasis is
frequently placed on the identification of proteins at the peptide level relying almost
exclusively on expensive, resource-intensive and time-consuming enzymatic proteolysis procedures for sample preparation and is lacking tools for the identification of
protein variants/isoforms. This investigation assessed an alternative approach to
obtain “signature” peptides of haemoglobin A and its structural variants in blood
combining microwave-assisted chemical cleavage and mass spectrometric analysis.
Methods
A sensitive, rapid analytical method has been developed for the characterization of
human haemoglobin disorders with very small volumes (<1 µL) of blood. As an
alternative to conventional enzymatic digestion, a site-specific chemical cleavage
method has been established using 0.05% formic acid under microwave-irradiation
conditions for short time intervals, for example, less than 10 min. Peptide analysis
was performed by MALDI TOF MS and capillary liquid chromatographic ESI MS/MS.
Results
The cleavage of the haemoglobin chains with formic acid occurred at either side or
at both sides (C- and N-terminal) of aspartic acid residues, but preferentially Nterminally. The method has been applied to blood samples from haemoglobin S
carrier heterozygotes and haemoglobin S thalassaemia compound heterozygotes
with a reduced expression level of haemoglobin S. Both MALDI TOF MS and ESI
MS/MS analysis allowed the identification of the haemoglobin S “signature” peptide.
Conclusion
This fast and cost effective method of sample preparation is compatible with MS
techniques and is expected to significantly contribute to the further development of
rapid, robust, reproducible and sensitive analytical methods in proteomics and
biomedical diagnostics where protein variant characterization is a crucial factor for
biomarker discovery and disease identification.
Keywords Chemical proteolysis, haemoglobin variant identification, mass
spectrometry
Conflict of interest None.
249
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HSANZ POSTERS
P253
Catastrophic Acute Refractory Immune Thrombocytopenia (ITP) with
Intracerebellar Haemorrhage Successfully Salvaged with
Thrombopoietin (TPO) Mimetics – A Case Report and Review of the
Literature
Michael Gilbertson, Andrew W. Roberts, Jeff Szer, Annabel Tuckfield, Simon He
Department of Clinical Haematology & BMT Service, The Royal Melbourne Hospital
VIC, Australia
Aim
We present the case of a 43-year-old previously well woman who first presented to
our institution in January 2012 with severe thrombocytopenia (3x109/L) and
evidence of iron deficiency anaemia as an illustration of the potential life-saving
properties of TPO mimetic agents.
Results
A clinical diagnosis of acute ITP was made and subsequently confirmed by bone
marrow biopsy. Despite two weeks of prednisolone at 1mg/kg/day and two doses of
intravenous immunoglobulin (IVIg) at 1g/kg/day, marked thrombocytopenia (<10
x109/L) persisted. 19 days after the initial diagnosis, she developed acute
spontaneous onset of headache, confusion and nystagmus. A CT brain revealed a
large right posterior cranial fossa haemorrhage with mass effect. Platelet count was
6x109/L. Her conscious state rapidly deteriorated requiring intubation. Despite
aggressive support with multiple platelet transfusions, IVIg, high-dose
dexamethasone and activated factor VIIa, the patient had ongoing intracerebellar
bleeding and further neurological decline requiring urgent decompression. Postoperatively, off-label use of romiplostim, a TPO mimetic, was commenced at
5mcg/kg/week by subcutaneous injection as a salvage measure resulting in
progressive stable platelet counts and reduction of platelet support. She eventually
underwent an urgent splenectomy one week later with only transient response. She
continued to respond to weekly romiplostim at 10mcg/kg/week with low-dose of
prednisolone and has made a successful neurological recovery.
Conclusion
This case highlights the potential use of TPO mimetics in acute refractory ITP. We
review the current literature and summarise the management of this benign but lifethreatening condition in the era of TPO mimetics.
Keywords
ITP, refractory, bleeding, romiplostim
Conflict of interest
II:250
No conflict of interest to disclose
250
HSANZ POSTERS
P254
Experience with Rituximab Treatment on Paediatric Patients with
Refractory Immune Thrombocytopenia (ITP)
Nalini Pati, Catriona Buchanan, Anthea Greenway, Chris Barnes
Department of haematology, The Royal Children’s Hospital, Melbourne, Australia
Aim
Retrospective review of children with refractory ITP treated with rituximab.
Results
Immune thrombocytopenic purpura (ITP) that is unresponsive to conventional
treatment is uncommon. In this situation, additional therapeutic options are limited
and management is challenging. Several recent reports in children suggest a
potential role for rituximab in the treatment of ITP that is unresponsive to
conventional treatment. In paediatric population with refractory ITP data on the use
of rituximab are extremely limited. We share the experience of five children with the
age group ranging from 10weeks to 15years with severe life-threatening bleeding
complications as a result of refractory ITP and successfully managed with rituximab.
All of them received weekly infusions for total of 4 doses except one who received
three monthly infusions subsequently. Complete response was seen within first
week of starting the treatment and sustained in all but one patient who required
three monthly doses to maintain remission. There were no complications seen both
immediately and up to 16moths following rituximab treatment. Majority developed
lymphopenia and reduced immunoglobulin level, but shown signs of recovery over a
follow up period of 8-10 months while maintaining remission.
Conclusion
We conclude rituximab is a very effective modality of treatment in refractory ITP who
develop life threatening bleeding complications, however further prospective studies
focussing on long term outcomes are essential in children with refractory ITP.
Keywords ITP, Paediatric, Rituximab.
Conflict of interest No conflict of interest to disclose.
251
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HSANZ POSTERS
P255
Successful Treatment of Refractory Immune Thrombocytopenia with
Rituximab in a 10-week Old Infant
Nalini Pati, Charmaine Gray, Kongolo Kalumba, Anna Peterson, Tom G Connell
Department of Haematology, The Royal Children’s Hospital, Melbourne, Australia
Immune thromobycytopenia purpura (ITP) that is unresponsive to conventional
treatment is uncommon. In this situation, additional therapeutic options are limited
and management is challenging. Several recent reports in children suggest a
potential role for rituximab, a monoclonal anti-CD20 antibody, in the treatment of ITP
that is unresponsive to conventional treatment. In infants with refractory ITP data on
the use of rituximab are extremely limited. We describe the case of a 10 week-old
infant who developed severe life-threatening (Intracranial haemorrhage) as a result
of refractory ITP that was successfully managed with rituximab. Although he
developed lymphopenia and low in immunoglobulin level, both shown signs of
recovery over the next 8-10 months of follow up period while maintaining remission.
Keywords: ITP, rituximab, infancy
Conflicts of interest: Nothing to disclose.
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252
HSANZ POSTERS
P256
The Synergistic Effect of Taurine and Caffeine on Platelet Function and
Hemostatic Activity
Abishek B Santhakumar, Indu Singh
School of Medical Science, Gold Coast Campus, Griffith University, Parklands Drive
Qld, Australia
Aim
Several studies have emphasized the deleterious effects, of high concentrations of
Taurine and Caffeine present in the energy drinks, on cardiac health. The purpose
of this study is to examine the underlying controversies in the use of energy drinks.
Increased platelet activation is one of the known predictor of thrombotic and cardiac
disorders. The aim of this study was to evaluate the in vitro synergistic effects of
lower concentrations of the Taurine and Caffeine on platelet activity and
haemostatic function.
Method
Blood from 12 healthy volunteers was analysed after in-vitro treatment with either
Taurine (500uM) or Caffeine (700uM) or both together to examine synergistic effect.
Platelet function was tested using platelet aggregometry. Coagulation screen, lipid
profile and inflammation marker were also tested.
Results
Taurine and Caffeine (T+C) synergistically inhibited platelet aggregation. Taurine
(p=0.0446), T+C (p=0.0117) raised prothrombin time (PT) compared to the baseline
while Caffeine decreased (p=0.0263) PT and increased (p=0.0387) CRP. There was
no effect on the lipid profile.
Conclusion
These data support our hypothesis that synergistic effect of Taurine and Caffeine in
lower concentrations may be instrumental in reducing platelet activity and hence
these drinks could be more beneficial and effective with lower concentrations of
Taurine and Caffeine.
Keywords:
Taurine, Caffeine & Platelet aggregation
Conflict of interest: There is no conflict of interest to disclose.
253
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HSANZ POSTERS
P257
Successful Use of Eltrombopag Without Splenectomy in Refractory HIVrelated Immune Reconstitution Thrombocytopenia
Pasquale Fedele1, Lai-Yang Lee2, Tony Korman3, Brodie Smith4, Ian Woolley5, Hang
Quach6
1,6
Department of Haematology, Monash Medical Centre, Clayton, Victoria, Australia
4
Pharmacy Department, Monash Medical Centre, Victoria, Australia
2,3,5
Department of Infectious Disease, Monash Medical Centre, Victoria, Australia.
Aim
We report a case of refractory immune thrombocytopenic purpura (ITP) occurring in
the setting of immune reconstitution following the commencement of anti-retroviral
therapy for HIV, successfully treated with eltrombopag.
Results
A 47 year old man, recently diagnosed with human immunodeficiency virus (HIV)
and commenced on combination antiretroviral therapy (cART), developed severe
thrombocytopenia (platelet nadir 10x109/L) in the setting of immune reconstitution
with a concurrent rise in CD4 count from 123x106/L to >400x106/L. Investigations
including full blood examination, blood film and bone marrow aspiration, trephine
and flow cytometry were all consistent with a diagnosis of ITP. While initially
responsive to prednisolone and intravenous immunoglobulin (IVIG), ITP relapsed on
weaning of steroid dose and was subsequently refractory to azathioprine,
hydroxychloroquine and change to alternative antiretroviral regimen. One week
following the commencement of eltrombopag 50mg PO daily, platelet count rose to
87x109/L. Prednisolone was weaned and ceased after eight weeks. Six months
later platelet count is stable between 90-126 x 109/L, HIV viral load is undetectable
and CD4 count is >400x106/L.
Conclusion
While thrombocytopenia in the setting of HIV is well documented, particularly in the
period prior to highly active antiretroviral therapy (HAART), to our knowledge, this
case represents the second report of ITP presenting as an immune reconstitution
syndrome in HIV, and furthermore is the first documentation of efficacy of
eltrombopag in this setting. This case highlights the therapeutic challenges
associated with immunosuppression and splenectomy in HIV-related IRIS
associated ITP with increased risk of opportunistic infections, as well as the lack of
current treatment guidelines and the need for further studies in this area.
Keywords
ITP, HIV, Eltrombopag
Conflict of interest No conflict of interest to disclose
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254
HSANZ POSTERS
P258
Testing for Antiplatelet Antibodies: Retrospective Audit
Ferenc Szabo, Laura Taverna, Khin Chann
Haematology Unit, Royal Darwin Hospital, NT
Aim
The value of testing for antiplatelet antibodies in patients suspected of immune
thrombocytopenia remains controversial. MAIPA was considered useful in
discriminating between immune and non-immune thrombocytopenia, but more
recently an international consensus panel recommended against the routine testing
for antibodies to specific platelet glycoproteins because platelet-associated IgG
(PaIgG) is elevated in both immune and non-immune thrombocytopenia.
Material and methods
Retrospective review of antiplatelet antibody test requests at the Royal Darwin
Hospital over a 5 years period. The primary data was extracted from the laboratory
information system and patient notes were reviewed. We looked at clinical diagnosis
on request form, requesting ward, platelet count, test results and their influence on
clinical management.
Results
We have identified 54 patients with a median age of 26 years (2 days-78 y). There
were 44 female and 10 male. 4 tests were cancelled after consultation with
haematologist, 5 patients had been tested twice and 8 specimens were not
processed due to collection error (and not recollected by clinicians). As expected,
most requests for antiplatelet antibodies came from Obstetric & Gynaecology
department (n=33) with 15 requests from antenatal clinic.
The platelet count at the time of request was > 100 x 109/L in 28 patients (100 – 365
x 109/L), between 50 and 100 x 109/L in 18 patients and < 50 x 109/L in 9 patients.
Out of the latter 9 patients, the cause of thrombocytopenia was identified as: HIV
and sepsis related pancytopenia (n=1), need for HLA and not antiplatelet antibody
test (n=1), alcoholic liver cirrhosis and hepatitis C on Interferon (n=1), clinically
evident ITP (n=1), progressive CLL (n=1), TTP (n=1), hypothyroidism (n=1) and SLE
(n=1). Below normal platelet count in pregnant women were mainly gestational
thrombocytopenia and the diagnosis evident clinically.
Test results did not influence clinical decisions in any of tested cases.
Conclusion
We found little evidence for testing for antiplatelet antibodies as in most cases the
cause of thrombocytopenia could be established clinically. Results did not influence
decision making in our cohort.
Keywords
antiplatelet antibody immune
Conflict of interest No
255
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HSANZ POSTERS
P259
Use of Romiplostim in Patients with Chronic Thrombocytopenic Purpura
(cITP) During Peri-Operative Period
William Alexander
Illawarra Private Cancer Care Centre, NSW, Australia
Aim
In patients with cITP, the platelet count tends to be quite variable and, in the
majority of cases, specific therapy is not warranted on a regular basis. However,
patients with low platelet count (<30nL) or with bleeding complications would require
therapy such as prednisolone, IVIG infusions, splenectomy, and/or
immunosuppression. Romiplostim, a thrombopoietin agonist, has also proven to be
useful in improving platelet counts.
cITP can be associated with bleeding complications peri-operatively. As such, a
higher platelet count is warranted (>100nL), particularly for invasive surgeries where
risk of bleeding is high.
Seven patients were treated with Romiplostim short-term to improve platelet counts
peri-operatively, ensuring adequate haemostasis was achieved and hence reducing
the risk of bleeding. The seven patients had all failed to achieve an adequate
response to IVIG and steroid use.
Results
None of the patients treated with this protocol have run into any bleeding
complications peri-operatively, and they all achieved satisfactory platelet increment.
None of the patients experienced any significant rebound thrombocytopenia or other
adverse events as a result of Romiplostim administration.
Conclusion
The following protocol has been suggested to aid clinicians managing cITP perioperatively:
Romiplostim 3µg/kg subcutaneously on day 15 pre-op.
Repeat FBC on day 10 pre-op.
Increase Romiplostim dosage by 1µg/kg and administer on day 7 pre-op.
Repeat FBC Day 3 pre-op and if platelet count <90nL, repeat Romiplostim dosage.
Repeat FBC and, if platelet count is satisfactory, surgery day 0.
If necessary, repeat Romiplostim administration day 7 post-op.
Keywords
Romiplostim; ITP; peri-operative
Conflict of interest No conflicts of interest.
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256
HSANZ POSTERS
P260
Long Term Outcome of Splenectomised Patients with Idiopathic
Thrombocytopenic Purpura
Danielle Oh1, Ian Simpson2, Huyen Tran1
1
Department of Haematology, Monash Medical Centre, Southern Health, Melbourne
2
Department of Pathology, Monash Medical Centre, Southern Health, Melbourne
Aim
Splenectomy is an effective treatment for patients with refractory and relapsing
Idiopathic Thrombocytopenic Purpura (ITP). The aim was to evaluate the outcome
of splenectomised ITP patients at a tertiary haematology centre.
Method
All splenectomised ITP patients between1987 to 2010 were retrospectively
analysed. Attention was paid to additional treatments and response at pre-defined
time periods post splenectomy. Complete response (CR), Partial response (PR) and
Minimal response (MR) were defined as platelet ≥ 100x109/L, platelet 50 – 100
x109/L and platelet 30 -50 x109/L respectively. Overall response (OR) was defined
as CR + PR whereas No response (NR) was defined as platelet <30 x109/L.
Infection complications and mortality outcomes were also studied.
Results
50 patients underwent splenectomy for ITP. Prior to splenectomy 17 (34%) had
primary refractory ITP, 5 (10%) had relapsing disease with multiple relapses and 25
(50%) had chronic ITP. The median age at diagnosis and at splenectomy was 34
years (range 2 – 80) and 37 years (range 4 – 81) respectively. Median time to
splenectomy from diagnosis was 67 weeks (range 2 - 1016) with the median time to
last follow-up platelet count post splenectomy being 483.6 weeks (range 4.2 - 1300).
38/44 (86%) patients had two or more lines of treatment prior to splenectomy. At 1
year post splenectomy, 35/41 (85%) patients achieved OR. 33 (80%) achieved CR,
2 (5%) PR, 2 (5%) MR and 4 (10%) NR, platelet count was not available for 9
patients. At 5 year post splenectomy, 28/32 (88%) achieved OR. All 28 patients
were in CR with no one in PR. 3 (9%) achieved MR and 1 (3%) NR. Data was not
available for 9 patients. Among those patients who ever relapsed post splenectomy
(13/46; 28%), the median number of treatment received was 3 (range 1-9). Overall
there were 5 deaths, all with refractory ITP, 2 died from infection, 1 from
gastrointestinal bleed, 1 from metastatic adenocarcinoma and 1 from unknown
cause.
Conclusion
Our study supports the safety and efficacy of splenectomy in ITP patients with 88%
of patients remaining in OR in 418.5 person-years of follow-up.
Keywords splenectomy, idiopathic thrombocytopenic purpura, long term follow-up
Conflict of interest No conflict of interest to disclose
257
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HSANZ POSTERS
P261
Elevated Lactate Dehydrogenase (LDH) Is Associated with Occurrence of
Thromboembolism (TE) and Mortality Within 12 Months of LDH Assessment at
Diagnosis in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)
Jun Ho Jang,1 Jong Wook Lee,2 Jin Seok Kim, 3 Sung-Soo Yoon,4 Je-Hwan Lee,5
Yeo-Kyeoung Kim,6 Deog-Yeon Jo,7 Sang Kyun Sohn,8 Jooseop Chung9
1
Sungkyunkwan University School of Medicine, Seoul, South Korea; 2The Catholic
University of Korea, Seoul, South Korea; 3Yonsei University College of Medicine,
Seoul, South Korea; 4Seoul National University Hospital, Seoul, South Korea;
5
University of Ulsan College of Medicine, Seoul, South Korea; 6Chonnam National
University Hwasun Hospital, Hwasun, South Korea; 7Chungnam National University
Hospital, Daejeon, South Korea; 8Kyungpook National University Hospital, Daegu,
South Korea; 9Pusan National University Hospital, Pusan, South Korea
Aim
TE is associated with morbidities and mortalities in PNH and is the result of hemolysis
and blood cell activation. The aim of this study was to evaluate the association of LDH
≥1.5× the upper limit of normal (ULN), a measure of hemolysis, and granulocyte clone
size with the risk of TE and mortality.
Method
Retrospective analysis of 301 patients from the national South Korean PNH registry.
LDH levels that were assessed at diagnosis were analyzed.
Result
A significant association was found between LDH and the occurrence of a TE within 12
months of diagnosis. The odds of experiencing a TE were 4.4 times greater if LDH was
≥1.5×ULN compared with <1.5×ULN (P=0.020). Importantly, 22 patients with LDH
≥1.5×ULN had a TE within 12 months of diagnosis, and 82% (18/22) of the patients
experienced the TE within the first 6 months. Furthermore, 5% (7/150) of patients with
LDH ≥1.5×ULN died within 12 months following LDH assessment at diagnosis, reflecting
a significantly higher mortality compared with 0% (0/51) of patients with LDH <1.5×ULN
(P=0.004). A multivariate analysis of all patients showed that there was no significant
association between clone size and risk of TE (P=0.292) or early mortality (P=0.247).
Conclusion
Elevated LDH is a significant risk factor for increased occurrence of TE and mortality
within 12 months of LDH assessment at diagnosis, while granulocyte clone size is not.
The relatively high incidence of TE within 12 months of diagnosis in patients with LDH
≥1.5×ULN, with most events occurring within the first 6 months, highlights the medical
need and urgency for early therapeutic intervention in PNH patients with elevated LDH.
Keywords: paroxysmal nocturnal hemoglobinuria, lactate dehydrogenase, thrombosis
Conflict of interest: Editorial support for the abstract was supported by Alexion
Pharmaceuticals. The company had no role in collecting or analyzing the data.
II:258
258
HSANZ POSTERS
P262
Multiplex Ligation-Dependent Probe Amplification Assay for Mutation
Detection of the Human Globin Gene Clusters in Victoria, Australia
Asif Alam, Jeremy Wells, Ruoxin Li; Anastasia Adrahtas, Mioara Gavrila, Penny
Matthews, Kerryn. Weekes, Donald Keith Bowden, Sant-Rayn Pasricha
Clinical Genetics Laboratory, Monash Medical Centre, Clayton, Victoria, Australia
Aim
Over the past several years, the number of samples for molecular screening for
haemoglobin disorders has dramatically increased for the multiethnic Victorian
population. Mutations from the bulk of the samples can be diagnosed with relative
ease with routine methods used in our laboratory. But the number of cases where
routine molecular screening by GAP PCR, RFLP and Sanger sequencing fail to
detect mutations are increasing. The aim of this study was to apply the Multiplexed
Ligation Dependent Probe Amplification (MLPA) Assay to investigate all these
unresolved cases and test the methods ability to detect known common and rarer
mutations of the globin gene clusters.
Methods
MLPA analysis was performed according to the manufacturer’s instruction. The
commercial kit, SALSA® MLPA® probemix for the alpha globin gene complex
including the upstream HS 40 regulatory region is designed to detect copy number
changes of 24 different sequences with one probe for detection of the Constant
Spring mutation. The SALSA® MLPA® probemix P102-B1 HBB MLPA kit contains 28
probes for the beta-like globin genes including the upstream regulatory sequences
and one probe for HbS. Electrophoresis was performed by ABI 3130xl genetic
analyser. The raw data analysis was performed using a spreadsheet designed to
use the peak heights of the probes of controls and samples to generate histograms
for each sample from which interpretation is made by observing the dosage
changes.
Results
A cohort of samples where no mutations had been detected by routine methods
employed in our laboratory were investigated along with some common mutations to
test the applicability of MLPA technique. Out of 121 cases, we were able to
establish the presence of deletions or duplications in 63 cases. We identified
fourteen known deletions and the break points for another thirteen mutations
detected by MLPA are currently being determined by GAP-PCR.
Conclusion
The data presented here show that MLPA is a powerful tool for the referral
laboratories. Both known and unknown mutations detected by this method aided in
solving a significant number of difficult cases.
Keywords
MLPA, globin gene complex, deletions, insertions
Conflict of interest None
259
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HSANZ POSTERS
P263
Quantification of Alpha and Beta Globins by Mass Spectrometry for the
Detection of Haemoglobin Expression Disorders
Asif Alam1,2, Reinhard Boysen1, Donald Bowden1,2, Milton Hearn1
1. Centre for Green Chemistry, Monash University, Melbourne, VIC, Australia
2. Clinical Genetics Laboratory, Southern Health, Melbourne, VIC, Australia.
Aim
Tandem mass spectrometry based techniques offer a relatively cost-effective, highthroughput method for multiple mutation analysis and quantification of clinically
significant proteins from proteolytically derived samples. This investigation aimed at
establishing an absolute quantification (AQUA) method for α- and β-globin chains of
adult haemoglobin in blood samples at the peptide level using liquid
chromatography/tandem mass spectrometry (LC MS/MS).
Methods
A rapid AQUA method has been developed for detection of haemoglobin disorders
in very small volumes of whole EDTA-treated human blood. The selection of two
internal standards for each globin chain was based on globin chain homology
mapping. Wild-type and corresponding stable isotope-labelled peptides containing
13
C/15N-labelled amino acids were custom-synthesised. These synthetic peptides
were used as internal standards/calibrators in the proteolysis of whole blood and in
the quantification of the wild-type peptides using fast LC and optimised multiple
reaction monitoring (MRM) with electrospray triple quadrupole (ESI-QQQ) MS/MS.
The method was developed using an adult haemoglobin A standard, optimized for
blood samples containing normal haemoglobin and applied to samples with aberrant
haemoglobins, i.e. HbE.
Results
The MRM transitions for the qualifier and quantifier precursor/product ions for each
wild-type/internal standard peptide pair have been validated. The developed AQUA
method was successfully applied to the absolute quantification of adult Hb A globin
chains in samples containing both, normal and aberrant haemoglobins.
Conclusion
The AQUA method allows the rapid quantitative analysis of Hb A globin chains and
is suitable for clinical diagnostics. The method can also be adopted for identifying
silent carrier states or for detecting the presence of excess alpha globin in patients
with triplicated alpha chains.
Keywords Absolute quantification, haemoglobin, mass spectrometry
Conflict of interest None
II:260
260
HSANZ POSTERS
P264
Application of MLPA Assay to Characterize α-globin Gene
Rearrangements and Copy Number Variations Caused by Deletions or
Duplications
Asif Alam1, Sudha Raghunath2, Anastasia. Adrahtas1, Ruxion Li1, Jeremy Wells1,
Kerryn Weekes 1, Donald Keith Bowden1,2, Sant-Rayn Pasricha1,2
1
Clinical Genetics Laboratory & 2 Medical Therapy Unit, Monash Medical Centre,
Southern Health, Vic, Australia.
Aim
MLPA (multiplex ligation-dependent probe amplification) is a flexible and robust
technique that enables detection of deletions and duplications for more than 52
genomic loci in one assay. This detection of quantitative changes in genomic DNA is
often incorporated routinely as an additional test to complete mutation screening for
selected cases or as a primary screening technique for monogenic diseases.
The two alpha globin genes are embedded withing two highly homologous
duplication units. Unequal homologous recombination within these different
homologous regions result in a variety of deletions and duplication. The aim of this
study was to apply MLPA techniques and to validate the dosage changes observed
for the probes for -α3.7 deletional variants, hybrid variants and triple alpha variants.
Method
The commercial kit, SALSA® MLPA® probemix for alpha globin gene (P140-B3 HBA
ver 22) was used for MLPA assay. Deletional variants -α3.7 were detected by Apa
restriction enzyme digestion target alpha globin gene amplification product. A gap
PCR was also employed to confirm presence of triple α. Direct sequencing was
performed to check for presence of other mutations. The dosage changes for
different probes for samples with hybrid alpha globin gene were investigated as part
of this study. Finally, a large cohort of beta thalassaemia heterozygotes where an
increased copy number of alpha globin gene was suspected were investigated using
MLPA assay to identify zygosity of the duplicated alpha globin gene.
Results
This study systematically documents copy number variations (CNVs) by observing
dosage changes calculated for each probe, indicating the presence of each
mutation investigated. This study further documents the CNVs of alpha globin gene
from a large cohort of beta thalassaemia carriers to clarify their genotype-phenotype
correlation.
Conclusion
MLPA is a powerful screening tool to complement other routine methods used by
molecular screening laboratories.
Keywords
MLPA, Triple alpha, hybrid alpha globin gene
Conflict of interest None
261
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HSANZ POSTERS
P265
Trends in Alpha Globin Gene Genetic Testing in Victoria
Jeremy Wells, Kerryn Weekes, Asif Alam, Anastasia Adrahtas, Ruoxin Li,
Carly Wishart, Donald K Bowden, Sant-Rayn Pasricha
Clinical Genetics Laboratory, Monash Medical Centre, Clayton VIC Australia
Aim
Haemoglobinopathies are the most common genetic disorders worldwide. The
Clinical Genetics Laboratory at Monash Medical Centre is a state-funded reference
centre for haemoglobinopathy genetic testing. Ongoing migration to Australia and
increased awareness regarding Thalassaemia may be expected to increase the
number of tests requested. Testing aims is to identify couples at risk of having
children with severe thalassaemias and haemoglobinopathies to facilitate prenatal
testing. We sought to study the change in patterns of testing over time performed at
our laboratory and thus, the state of Victoria.
Results
Our custom multiplex PCR test detects the most common alpha-globin gene
deletions and has consistently comprised the largest volume of tests, followed by
Restriction Fragment Length Polymorphism (RFLP) testing and sequencing. A
breakdown of alpha-globin gene deletions detected over time is as follows:
3.7†
4.2†
2001 (255)
45% (114) 5% (12)
2002-2006 (1609)
54% (960) 4% (71)
2007-2001 (4505)
62% (2967)5% (229)
† One gene deletion
SEA‡
MED‡
31% (79) 2% (4)
30% (426)2% (34)
23% (952)1% (55)
‡ Two gene deletion
THAI‡ 20.5‡ FIL‡
9% (24) <1% (1) 1% (3)
<1% (6) <1% (4) 2% (13)
<1% (17)<1% (14) 2% (54)
††Non-deletional
Hph1†† Nco1††
5% (14) 2% (4)
6% (106) 2% (19)
6% (236) 1% (36)
raw no. in brackets
The volume of tests has increased for all test types, from ~3800 p.a. 2002-2006 to
~7200 p.a. 2007-2011. However, the number of prenatal tests has remained stable
(32, 25, 38, 29, 24, 31, 31, 41, 39, 32, 32, 30; 2001-2011 p.a. respectively).
Conclusion
We have observed an increase in sample and testing numbers over the last decade.
This increase in workload may be due to an increase in antenatal screening for
haemoglobinopathy. It may also be attributable to an increasing migrant population.
From 2009-2010 the largest proportion of population growth in Victoria was due to
international immigration (1.1%), compared with 0.67% natural and 0.05% interstate
growth. However, testing has resulted in increased detection of single gene alpha
deletions which do not pose as serious a risk of haemoglobinopathy in offspring
(HbH disease) suggested by the stable prenatal testing workload over time.
Keywords
thalassaemia, haemoglobinopathy, genetic testing, mutations.
Conflict of interest No conflict of interest to disclose.
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262
HSANZ POSTERS
P266
Complications of HbH Disease in an Adult Population
Claire Sheeran1, Roger Peverill3, Kerryn Weekes2, Giovanni Romanelli3, Donald K
Bowden1,2, Sant-Rayn Pasricha1,2
1
Medical Therapy Unit, 2Clinical Genetics Laboratory, Monash Medical Centre
Clayton. 3MonashHEART and Monash Cardiovascular Research Centre, Southern
Clinical School, Monash University, Melbourne, Victoria, Australia
Aim
HbH disease is a common thalassaemia syndrome; the natural history of this
condition in adulthood is not well described. In an adult Australian multiethnic cohort
we evaluated iron overload, abdominal organomegaly, transfusion dependence,
bone and cardiac outcomes, and assessed correlation between genotype and
phenotype in HbH disease.
Methods
Data were collected from medical records of patients attending the Thalassaemia
service at Monash Medical Centre, Clayton. The study population comprised of
patients with HbH disease attending service for both regular transfusions and
outpatient reviews (if transfusion independent).
Results
There were 32 patients. The mean age of patients was 42 years; there were 68%
females. 68% had a deletional genotype and 32% had a non-deletional genotype;
32.3% had received long term transfusions. Mean Hb was 98.5g/L [95% CI 94.9102.1]; mean MCV was 66fL [61-71]; geometric mean ferritin was 404.3ng/mL
[281.0-581.8] (in non transfused patients, 272.3 [171.2-432.9]); mean HbH% was
15.8% [11.0-20.5] and mean spleen size was 650.3cc [486.4-814.3]. All patients had
normal left ventricular ejection fractions (mean 65.3%). 28% of patients had
osteoporosis. Mean total body bone t score was -0.63, mean lumbar bone t score
was -1.4, and mean femoral bone t score was -0.80. There were significant
differences between patients with deletional and non-deletional HbH in terms of:
HbH% (8.5% vs 29.1%, p<0.0001), MCV (60.8fL vs 75.8fL, p=0.0229), spleen size
(471.9cc vs 993cc, p=0.0041) and femoral t score (-1.17 vs -0.23, p=0.0371).
Conclusion
With aging, patients with HbH disease are at risk of osteoporosis, splenomegaly and
iron loading. Our data suggest patients with HbH disease require lifelong clinical
monitoring.
Keywords
HbH Disease, thalassaemia, genetics
Conflict of interest No conflict of interest to declare
263
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HSANZ POSTERS
P267
Comparison of the BioRad Variant High-Pressure Liquid
Chromatography (HPLC) and Sebia Capillarys 2 Flex Piercing Capillary
Electrophoresis in Haemoglobin Analysis
Claudine Ho, Ross Brown, Konstantinos Zarkos
Institute of Haematology, Royal Prince Alfred Hospital, New South Wales
Aim
Numerous methods are available for the evaluation of haemoglobinopathies. Here
we compare a new instrument, the Sebia Capillarys 2 Flex Piercing capillary
electrophoresis (Sebia), with the BioRad Variant II high pressure liquid
chromatography (BioRad) in haemoglobin analysis.
Method
This was a prospective study of 132 whole blood samples. Hb A2 and Hb F values
of both methods were correlated in samples with and without variant haemoglobins.
The mean Hb A2, F and S in patients with Hb S were compared. Within-run and
between-run precisions were tested with the Sebia. The BioRad chromatogram and
Sebia electrophoresis patterns were examined for clear haemoglobin separation.
The haemoglobin variants studied included 5 common (Hbs S, C, E, Lepore and H)
and 2 rare (Hbs J and Hasharon).
Results
Hb A2 measurements showed excellent agreement between the two methods in
patients who did not have a structural variant (mean, 2.9%; SD, 0.6% by BioRad;
mean, 2.9%; SD 0.7% by Sebia; r=0.953). The agreement of Hb F was also very
good (mean, 0.6%; SD, 0.8% by BioRad; mean, 0.3%; SD, 0.8% by Sebia; r=0.986).
In patients with Hb S, good correlation was found with Hb F and Hb S. The mean
Hb A2 was significantly higher in the BioRad (mean, 4.1%; SD 1.1%) than by Sebia
(mean, 3.5%; SD, 0.9%).Results of within-run and between-run precisions were
good for Hb A2. Hb F showed poor precision when the Hb F was less than 0.5% but
had a more favourable CV when the high Hb F control was used. The Sebia
electrophoresis patterns were easier to read and had less HbA2 comigration issues
than with HPLC.
Conclusion
The performance of the Sebia Capillarys in the evaluation of haemoglobinopathies
compares well with the existing HPLC method.
Keywords
Capillary electrophoresis; haemoglobinopathy; high-pressure liquid
chromatography
Conflict of interest No
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P268
In vitro Analysis of the HBB:c.129delT Beta Thalassaemia Mutation
(Haemoglobin Yala)
Luke Forster1,2, Vip Viprakasit3, Worrawut Chinchang3, Suchada Riolueang3, Talal
Qadah1,2,4, Jill Finlayson1,2, Reza Ghassemifar1,2
1
Department of Haematology, PathWest Laboratory Medicine, QEII Medical Centre,
2
Nedlands, Western Australia, School of Pathology and Laboratory Medicine, University of
3
Western Australia, Nedlands, WA, Australia, Division of Hematology/Oncology, Department
of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand and
4
Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences and King
Abdulaziz University, Jeddah, Saudi Arabia
Aim
Hb Yala (HBB:c.129delT) was described in a Thai patient who presented with
compound heterozygous HbE/Beta thalassaemia. The mutation creates a frameshift
and a premature termination codon (PTC) at the new codon 60 and is expected to
result in a β0-thalassaemia phenotype, however the clinical presentation of this
patient was surprisingly mild and experimental analysis was undertaken to
definitively characterise the mechanism for β thalassaemia in this patient.
Method
The HBB:c.129delT mutant was generated by site directed mutagenesis in our
expression vector containing the HBB-WT gene. Following successful transfection
into the 5637 cell line, the cytoplasmic fraction was isolated and levels of the beta
globin transcript were assayed by quantitative PCR at timepoints: untreated, 0, 1, 2
and 3 hours.
Results
In intact cells, the mutant mRNA level was 76% of the level expressed by the WT
vector. In the isolated cytoplasmic fraction, the rate of decay of mutant mRNA was
greater than that for the WT. (mRNA levels were 100%, 11%, 4%, 1% and 0% at
defined time points for the mutant mRNA compared with 100%, 46%, 30%, 12% and
4% for the WT mRNA) The degradation rate for the endogenously produced
GAPDH was the same for both samples.
Conclusion
We have demonstrated enhanced degradation of the mRNA from the
HBB:c.129delT beta globin mutation. This is likely to be due to nonsense-mediated
decay, a protective mechanism in cells which prevents the translation of genes
carrying premature termination codons. As suspected this would result in the
phenotype of β0 thalassaemia.
Keywords: Nonsense Mediated Decay, Premature Termination Codon, β-Globin
Conflict of interest: No
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