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 171 II:171 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 II:172 172 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 173 II:173 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 II:174 No conflict of interest to disclose. 174 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 175 II:175 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 II:176 176 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 177 II:177 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 II:178 178 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. 179 II:179 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. II:180 180 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. 181 II:181 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. II:182 182 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 183 II:183 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 II:184 184 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 II:185 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 II:187 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 II:189 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 II:191 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 II:193 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 II:195 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. II:196 196 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 II:197 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 II:198 198 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 II:199 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 II:200 200 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 201 II:201 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 II:202 202 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 II:203 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 II:204 204 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 II:205 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 II:206 206 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 II:207 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 II:209 209 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 II:210 210 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 211 II:211 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 II:214 214 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 II:215 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. II:216 216 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 II:217 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 II:218 218 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. II:220 220 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 II:221 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 II:222 222 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 II:223 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. II:224 224 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 II:234 234 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 II:238 238 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 II:239 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 II:240 240 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 II:241 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. 243 II:243 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. II:244 244 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. 245 II:245 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 II:246 246 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 II:247 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 II:248 248 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 II:249 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 II:251 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. II:252 252 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 II:253 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 II:254 254 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 II:255 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. II:256 256 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 II:257 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 II:259 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 II:261 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. II:262 262 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 II:263 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 II:264 264 HSANZ POSTERS 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 265 II:265 HSANZ POSTERS Notes: II:266 266
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