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Background: Tisagenlecleucel is approved in the United States and Europe for adults with relapsed/refractory follicular lymphoma (r/r FL) after ≥2 lines of prior therapy. The primary analysis of the Phase II ELARA trial (median follow-up: 17 months) reported high response rates and a favorable safety profile in heavily pretreated patients with r/r FL. Here we report longer-term efficacy, safety, pharmacokinetic, and exploratory biomarker analyses after a median follow-up of more than 3 years.

Introduction:Multiple myeloma (MM) is an incurable cancer, but a subset of patients can achieve long-term remission and overall survival (OS). We aimed to study the clinical and biological factors associated with short OS defined as less than 3 years.

Introduction:

BACKGROUND

Mollee: Amgen: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Reynolds:Novartis AG: Current equity holder in publicly-traded company. Janowski:Janssen: Membership on an entity's Board of Directors or advisory committees; BMS/ Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZenica: Consultancy. Quach:Amgen, Celgene, karyopharm, GSK, Janssen Cilag, Sanofi.: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Honoraria; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Consultancy; Amgen, sanofi, celgene, Karyopharm, GSK: Research Funding. Campbell:Amgen, Novartis, Roche, Janssen, Celgene (BMS): Research Funding; AstraZeneca, Janssen, Roche, Amgen, CSL Behring, Novartis: Consultancy. Gibbs:Janssen, BMS/Celgene, Amgen, Takeda, Pfizer, Caelum, Abbvie and Eidos: Membership on an entity's Board of Directors or advisory committees. D'Rozario:Abbvie: Membership on an entity's Board of Directors or advisory committees; BMS/ Celgene: Membership on an entity's Board of Directors or advisory committees. Wallington-Beddoe:Amgen: Membership on an entity's Board of Directors or advisory committees. Weber:Amgen: Membership on an entity's Board of Directors or advisory committees. Spencer:Celgene, Janssen and Takeda: Speakers Bureau; AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria.Daratumumab as initial treatment of myeloma

Introduction

Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/ Juno/ BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eusa: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Consultancy, Honoraria. Mollee: Janssen, Pfizer: Research Funding; Amgen, BMS, Janssen, Caelum, EUSA, Pfizer, SkylineDx, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: No personal fees received. Quach: GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria. Wood: Amgen, Celgene, Gilead, Janssen, Novartis, Sanofi, Takeda: Research Funding; Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda.: Other: The Australian and New Zealand Myeloma and Related Diseases Registry (MRDR) has received funding from Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda. . McQuilten: Amgen, Celgene, Gilead, Janssen, Novartis, Sanofi, Takeda: Research Funding; Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda.: Other: The Australian and New Zealand Myeloma and Related Diseases Registry (MRDR) has received funding from Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda. .

Kalff: Janssen: Honoraria; Amgen: Other: travel to preceptorship; Celgene: Honoraria; Takeda: Honoraria. Reynolds:Novartis: Equity Ownership, Other: former employee of Novartis AG and holds stock in the company. . Quach:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy; Sanofi Genzyme: Research Funding. Ho:Amgen: Honoraria; Celgene: Other: Travel to meeting; Janssen: Honoraria; Novartis: Honoraria; Takeda: Honoraria, Other: travel to meeting. Mollee:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Estell:Janssen: Membership on an entity's Board of Directors or advisory committees.

Ho: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel to meeting; Celgene: Other: Travel to meeting ; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Spencer:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria. Mollee:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bryant:Celgene: Consultancy, Honoraria. Trotman:PCYC: Research Funding; Beigene: Research Funding; Celgene: Other: Unremunerated member of Ad Board, Research Funding; F. Hoffman-La Roche: Other: Travel to meeting, Unremunerated member of Ad Board, Research Funding; Janssen: Other: Unremunerated member of Ad Board, Research Funding; Takeda: Other: Unremunerated member of Ad Board. Gibbs:Amgen: Honoraria. Joshua:Amgen: Honoraria.

Whilst the addition of dexamethasone to upfront therapy with immunomodulatory (IMiD®) agents in myeloma (MM) is important to mediate rapid reduction in disease burden, preliminary findings suggest that the NK stimulatory effects of IMiD® compounds are best harnessed without the co-administration of dexamethasone. This may be especially effective in the setting of minimal disease burden (i.e. maintenance) when some inherent immune recovery has occurred, however this has yet to be confirmed in a prospective clinical trial.

Talaulikar: Takeda: Research Funding; Novartis: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau. Ho:Celgene: Other: Travel to meeting ; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel to meeting; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Simpson:Roche: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Acerta: Research Funding; Merck: Honoraria, Research Funding; BeiGene: Research Funding; Sanofi: Research Funding; Bristol-Myers Squibb: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Novartis: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; MSD: Honoraria; Abbvie: Honoraria, Research Funding; Amgen: Research Funding, TRAVEL, ACCOMMODATIONS, EXPENSES; Janssen: Honoraria, Research Funding. Tam:AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Beigene: Honoraria. Castillo:Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding.

BACKGROUND & METHODS

Introduction and aims:Bing Neel syndrome (BNS) is a rare complication of Waldenström Macroglobulinaemia (WM)/ Lymphoplasmacytic Lymphoma (LPL). It is a clinicopathological entity characterised by central nervous system involvement with malignant cells. It presents with diverse symptoms and can be difficult to recognise. However, it is a treatable condition amenable to systemic and intrathecal treatment. We present the Australasian experience of this rare entity.

Many acute myeloid leukemia (AML) patients achieve a complete remission (CR) with chemotherapy, but relapse is common. Removal of residual disease remains the greatest challenge. The potential of the immune system to control chemotherapy resistant malignant cells is evident in the success of allogeneic transplantation (aTx), and early results with T cell checkpoint inhibition. However, these non-specific interventions lead to considerable morbidity.

Introduction:The global JULIET trial (NCT02445248) evaluates the efficacy and safety of CTL019, a single infusion of genetically modified autologous chimeric antigen receptor-expressing T cells targeting CD19+ cells, in adult r/r DLBCL pts. Primary analyses showed a best overall response rate of 53%, which included patients with 40% complete response (CR) and 14% partial response (PR). A serious adverse event rate of 51% was observed within 8 weeks of infusion, decreasing to 23% after 8 weeks. This analysis evaluates patient reported QoL assessed before and after CTL019 infusion.

Autologous hematopoietic stem cell (HSC) gene therapy could be an effective treatment for transfusion-dependent β-thalassemia (TDT) without some of the limitations and risks associated with allogeneic HSC transplantation. LentiGlobin Drug Product (DP) contains autologous HSC (CD34+ cells) transduced ex vivowith the betibeglogene darolentivec (BB305) lentiviral vector encoding a human β-globin gene with the anti-sickling T87Q mutation (HbAT87Q). The Northstar Study(HGB-204; NCT01745120) is an international, multi-center phase 1/2 clinical trial examining the safety and efficacy of LentiGlobin investigational gene therapy in patients with TDT. As of the most recent data cut (June 2, 2017), all patients have 1 to 3 years of follow-up post DP infusion.

Hsu: DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Bryant:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Fromm:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Papadimitrious:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Orellana:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Suen:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Yang:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Weatherburn:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gasiorowski:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Iland:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Brown:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Joshua:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Ho:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gibson:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Clark:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hart:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.

Many acute myeloid leukemia (AML) patients achieve a complete remission (CR) with chemotherapy but relapse is common. Removal of residual disease remains the greatest challenge. Allogeneic transplantation (alloHCT) addresses this through an immune-mediated graft versus leukemia effect (GVL), but has high morbidity and mortality. Therapeutic dendritic cell (DC) vaccination has the potential to provide immune control with limited toxicity. Previous trials using monocyte-derived DC (Mo-DC) have demonstrated modest clinical effects. This is understandable as Mo-DC have demonstrably poor migration in vivoand relatively inferior antigen processing and presentation compared to blood dendritic cells (BDC). We have developed a more practical, functionally superior vaccine composed of natural blood DC (BDC). This is achieved using the human-mouse chimeric monoclonal antibody CMRF-56 to enrich BDC from patient peripheral blood after a short incubation.We assessed the potential for preparing a CMRF-56+ BDC vaccine from AML patients in CR.

Ho: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aspen: Other: Conference Grant. Hiwase:Celgene Corporation: Research Funding. Viiala:Novartis: Honoraria. Zor:Novartis: Employment. Gervasio:Novartis: Employment. Bowden:Novartis: Research Funding.

Abstract 3950

No relevant conflicts of interest to declare.

Over 40% of patients with the most common lymphoid malignancy worldwide, DLBL, are over the age of 70. Although R-CHOP is inarguably the mainstay of therapy for DLBL patients, a significant number of elderly patients do not tolerate the regimen due to underlying frailty and/or co-morbidities. Most elderly patients with significant co-morbidities have limited treatment options and are not offered anthracycline-containing chemotherapy due to concerns regarding toxicity. Here we describe our single center experience with CEEP, a lower intensity regimen for elderly patients with newly diagnosed or relapsed DLBL whom are deemed inappropriate for CHOP-based chemotherapy.All patients >70 years old (median 78.5, range 71 – 85) with histologically proven DLBL treated with CEEP ± Rituximab (R) at Royal Prince Alfred Hospital from 2000 to 2010 were retrospectively reviewed. Modified CEEP, Cyclophosphamide 300mg/m2 Day 1 (D1) and D15, Epirubicin 50mg/m2 D1 and D15, Etoposide 100mg/m2 D1 and D15, and Prednisolone 50mg D1-D5 (reduced dose from original CEEP protocol) was administered every 2 weeks. Rituximab 375mg/m2 (when approved for use in Australia) was administered every 28 days. As per institutional protocol, all patients received Bactrim prophylaxis for Pneumocystis. Baseline characteristics, Charlson Comorbidity Index, Revised International Prognostic Index (RIPI), the number of CEEP cycles, treatment response and toxicity from treatment were identified and reviewed.A total of 22 patients were identified, 10 were male. 15 received CEEP as initial therapy, and 7 for relapsed disease. 23% (n=5) had an ECOG score ≥ 2. 55% (n=12) had RIPI ≥ 3. All patients had a Charlson Comorbidity Index ≥ 2, with 23% (n=5) ≥ 5, which was considered sufficient to preclude conventional CHOP-based chemotherapy. Median cardiac ejection fraction was 62% (range 55 – 85%). 73% (n=16) received Rituximab and 50% (n=11) received primary GCSF prophylaxis. The median number of CEEP ± R cycles was 6 (range 2 – 9 cycles). 5% (n=1) required dose reduction and 9% (n=2) required delays in treatment due to haematological toxicity.Median follow-up was 10.0 months (range 1 – 92.7 months). At completion of therapy, complete responses (CR) were demonstrated in 10 patients (45%), with partial responses (PR) seen in 32% (n=7). 18% (n=4) demonstrated progressive disease (PD) despite therapy. Of the 7 patients with relapsed disease prior to CEEP ± R, CR was seen in 2 cases, both of whom had previous exposure to R-CVP (cyclophosphamide, vincristine, prednisolone) chemotherapy. At most recent follow up, 32% (n=7) have remained in CR with a median follow up period of 28.1 months (range 13 – 92.7 months), 36% (n=8) had disease progression, 9% (n=2) demonstrated stable residual disease, while 23% (n=5) have died. Of the 5 deaths, 3 were attributed to progressive DLBL. The other deaths were a result of complications following further salvage chemotherapy.Grade 3 – 4 haematological toxicity was observed in 72% (n=16) of patients. Febrile neutropenia occurred in 41% (n=9). Overall, 50% (n=11) required at least one re-admission to hospital. Non-haematological grade 3 – 4 toxicity was detected in 2 patients, one of whom suffered unstable angina in the setting of anaemia, the other an acute cerebrovascular event in the setting of new atrial flutter post-chemotherapy.Although limited by a small sample size, our retrospective single center experience demonstrates that CEEP ± R chemotherapy can be administered to elderly patients with significant co-morbidities. Our cohort was all aged >70, with medical co-morbidities leading to the unsuitability of conventional CHOP-based therapy. Whilst an overall response rate of 77% (CR + PR) was observed, on prolonged follow up, 32% of patients remained in CR. Significant haematological toxicity (72%) and infectious complications (41%) were observed, however no deaths were directly attributed to the chemotherapy. Future prospective studies are required to further evaluate the safety and efficacy of R-CEEP in the elderly.No relevant conflicts of interest to declare.

Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Abstract 1655

Abstract 4957

No relevant conflicts of interest to declare.

Abstract 983

No relevant conflicts of interest to declare.

Abstract 1820

Trogocytosis is the transfer of cell membrane material from one cell to another during short term cell-cell contact. Recent studies have suggested that the transfer of cell membrane patches across the immunological synapse is at least partly responsible for immune tolerance, tumor escape and the production of adaptive T regulatory cells (Tregs). We sought to identify the extent of tumor-related trogocytosis in patients with multiple myeloma and have developed an in vitro model for further study. Trogocytosis in patients with multiple myeloma was demonstrated by flow cytometry, immunohistochemistry, confocal microscopy, lack of mRNA expression and by the failure to upregulate costimulatory molecules in vitro after stimulation by IL2 and huCD40LT. Of the costimulatory molecules CD80, CD86, B7-H1, B7-H3 and PD-L2, only CD80 and CD86 showed significant transfer to T cells. Increased CD80 expression was found on the T cells of 9% of patients and CD86 on 13% of patients (n= 95). Both CD4 and CD8 memory (CD45RO+) cells were involved but not naïve T cells (CD45RA+). HLA-G expression was found on less than 1% of T cells in 69/70 different myeloma blood samples. Following biotinylation of plasma cells (CD38++) using an in vitro culture model, trogocytosis was demonstrated in up to 36% of T cells. The presence of trogocytosis on CD3- TCRαβ-mutant Jurkat cells (J.RT3-T3.3) and on normal T cells with HLA incompatibility infers that trogocytosis is independent of immune recognition and TCR engagement. In vitro stimulation with IL-2 and huCD40LT and mRNA studies showed that T cells acquire CD80 and CD86 cell surface antigen but unlike B cells do not produce CD80 and CD86 mRNA (n=5) and cannot be stimulated to express CD80 and CD86 (n=6). Trogocytosis was not evident in age-matched control lymphocytes but could be induced in normal lymphocytes in vitro after exposure to malignant plasma cells. Trogocytosis is common in patients with multiple myeloma and involves the transfer of costimulatory molecules and other cell membrane proteins from malignant plasma cells to T cells. Tumor-induced trogocytosis may be a common cause of the failure of cytotoxic T cells and provide a mechanism of tumor escape.

Trogocytosis is the transfer of cell membrane material from one cell to another during short term cell-cell contact. Recent studies have suggested that the transfer of cell membrane patches across the immunological synapse is at least partly responsible for immune tolerance, tumor escape and the production of adaptive T regulatory cells (Tregs). We sought to identify the extent of tumor-related trogocytosis in patients with multiple myeloma and have developed an in vitro model for further study. Trogocytosis in patients with multiple myeloma was demonstrated by flow cytometry, immunohistochemistry, confocal microscopy, lack of mRNA expression and by the failure to upregulate costimulatory molecules in vitro after stimulation by IL2 and huCD40LT. Of the costimulatory molecules CD80, CD86, B7-H1, B7-H3 and PD-L2, only CD80 and CD86 showed significant transfer to T cells. Increased CD80 expression was found on the T cells of 9% of patients and CD86 on 13% of patients (n= 95). Both CD4 and CD8 memory (CD45RO+) cells were involved but not naïve T cells (CD45RA+). HLA-G expression was found on less than 1% of T cells in 69/70 different myeloma blood samples. Following biotinylation of plasma cells (CD38++) using an in vitro culture model, trogocytosis was demonstrated in up to 36% of T cells. The presence of trogocytosis on CD3- TCRαβ-mutant Jurkat cells (J.RT3-T3.3) and on normal T cells with HLA incompatibility infers that trogocytosis is independent of immune recognition and TCR engagement. In vitro stimulation with IL-2 and huCD40LT and mRNA studies showed that T cells acquire CD80 and CD86 cell surface antigen but unlike B cells do not produce CD80 and CD86 mRNA (n=5) and cannot be stimulated to express CD80 and CD86 (n=6). Trogocytosis was not evident in age-matched control lymphocytes but could be induced in normal lymphocytes in vitro after exposure to malignant plasma cells. Trogocytosis is common in patients with multiple myeloma and involves the transfer of costimulatory molecules and other cell membrane proteins from malignant plasma cells to T cells. Tumor-induced trogocytosis may be a common cause of the failure of cytotoxic T cells and provide a mechanism of tumor escape.

Bortezomib is the first proteasome inhibitor in clinical use. It is particularly effective in myeloma compared with other cancers; as a single agent, its response rate in relapsed myeloma is about 40%. Nevertheless, a large proportion of patients are primarily resistant and acquired bortezomib resistance is also an emerging issue. The mechanism of bortezomib resistance in myeloma remains elusive. We have previously shown that bortezomib sensitivity correlates inversely with the levels of XBP-1mRNA in vitro and in myeloma patients. XBP-1 is a major regulator of the unfolded protein response (UPR), which is essential for immunoglobulin folding and assembly, and plasma cell development. Previous studies suggested that immunoglobulin production sensitises myeloma cell lines to proteasome inhibitors (S Meister, et al., 2007, Cancer Res 67:1783). We hypothesize that dependence on XBP-1and the UPR mediate sensitivity to bortezomib. The goal was to develop bortezomib resistant myeloma cell lines, study their phenotype, characterise the mechanism of resistance and, in particular, study the changes in the UPR. Bortezomib resistant sublines (9 in total) were derived from 4 myeloma cell lines (KMS-11, H929, U266 and OPM2), by long term exposure to bortezomib. The fold resistance varies from 3 to 12 and is stable for at least 4 passages without bortezomib. During the evolution of resistance, the myelomas all adopted adherent growth, independent of the morphology of the parent cell lines. When adherent versions of the parent cell lines were derived without exposure to bortezomib, they were, surprisingly, resistant to bortezomib. All bortezomib-resistant myeloma sublines downregulated XBP-1mRNA levels and the reduction was correlated with the fold resistance. Moreover, splicing of XBP-1mRNA to the active form was also reduced. The adherent versions of the parent cell lines that showed de novo resistance to bortezomib also had lower levels of XBP-1. Bortezomib normally induces XBP-1 expression and splicing acutely within 12–24 hours (AH Lee, et al., 2003. PNAS 100: 9946) but the resistant sublines were less responsive than the sensitive parents in this respect. Protein levels of other components of the UPR were evaluated by immunoblotting; BiP, phosphorylated-eIF2α, ATF6α and p58IPK were found to be unchanged in the resistant sublines. Cytoplasmic and secreted immunoglobulins were assayed by immunoblotting and ELISA respectively but no differences between the parent and resistant sublines were observed. In a small cohort of relapsed/refractory patients whose bone marrow biopsy was collected prior to bortezomib treatment, the quantity of intracytoplasmic light chain in the myeloma cells, as detected by flow cytometry, did not predict clinical response. The bortezomib-resistant myeloma sublines showed significant cross-resistance to doxorubicin and, to a lesser extent, vincristine, but none to melphalan. The cross-resistance is associated with adherent growth, as it was also observed in the adherent versions of the parent cell lines which were never exposed to bortezomib. Moreover, functional analysis of the multi-drug transporter activity, by drug accumulation assay, showed no difference between parent and resistant sub-lines. These results suggest that resistance to bortezomib is associated with downregulation of the IRE-XBP-1 pathway, but not necessarily other components of the UPR, consistent with observations by others concerning BiP (DT Rutkowski, et al., 2006. Plos Biology 4, e374). The association between adherence, bortezomib resistance and low XBP-1requires further study.

Bortezomib is the first proteasome inhibitor in clinical use. It is particularly effective in myeloma compared with other cancers; as a single agent, its response rate in relapsed myeloma is about 40%. Nevertheless, a large proportion of patients are primarily resistant and acquired bortezomib resistance is also an emerging issue. The mechanism of bortezomib resistance in myeloma remains elusive. We have previously shown that bortezomib sensitivity correlates inversely with the levels of XBP-1 mRNA in vitro and in myeloma patients. XBP-1 is a major regulator of the unfolded protein response (UPR), which is essential for immunoglobulin folding and assembly, and plasma cell development. Previous studies suggested that immunoglobulin production sensitises myeloma cell lines to proteasome inhibitors (S Meister, et al., 2007, Cancer Res 67:1783). We hypothesize that dependence on XBP-1 and the UPR mediate sensitivity to bortezomib. The goal was to develop bortezomib resistant myeloma cell lines, study their phenotype, characterise the mechanism of resistance and, in particular, study the changes in the UPR. Bortezomib resistant sublines (9 in total) were derived from 4 myeloma cell lines (KMS-11, H929, U266 and OPM2), by long term exposure to bortezomib. The fold resistance varies from 3 to 12 and is stable for at least 4 passages without bortezomib. During the evolution of resistance, the myelomas all adopted adherent growth, independent of the morphology of the parent cell lines. When adherent versions of the parent cell lines were derived without exposure to bortezomib, they were, surprisingly, resistant to bortezomib. All bortezomib-resistant myeloma sublines downregulated XBP-1 mRNA levels and the reduction was correlated with the fold resistance. Moreover, splicing of XBP-1 mRNA to the active form was also reduced. The adherent versions of the parent cell lines that showed de novo resistance to bortezomib also had lower levels of XBP-1. Bortezomib normally induces XBP-1 expression and splicing acutely within 12–24 hours (AH Lee, et al., 2003. PNAS 100: 9946) but the resistant sublines were less responsive than the sensitive parents in this respect. Protein levels of other components of the UPR were evaluated by immunoblotting; BiP, phosphorylated-eIF2α, ATF6α and p58IPK were found to be unchanged in the resistant sublines. Cytoplasmic and secreted immunoglobulins were assayed by immunoblotting and ELISA respectively but no differences between the parent and resistant sublines were observed. In a small cohort of relapsed/refractory patients whose bone marrow biopsy was collected prior to bortezomib treatment, the quantity of intracytoplasmic light chain in the myeloma cells, as detected by flow cytometry, did not predict clinical response. The bortezomib-resistant myeloma sublines showed significant cross-resistance to doxorubicin and, to a lesser extent, vincristine, but none to melphalan. The cross-resistance is associated with adherent growth, as it was also observed in the adherent versions of the parent cell lines which were never exposed to bortezomib. Moreover, functional analysis of the multi-drug transporter activity, by drug accumulation assay, showed no difference between parent and resistant sub-lines. These results suggest that resistance to bortezomib is associated with downregulation of the IRE-XBP-1 pathway, but not necessarily other components of the UPR, consistent with observations by others concerning BiP (DT Rutkowski, et al., 2006. Plos Biology 4, e374). The association between adherence, bortezomib resistance and low XBP-1 requires further study.

There is increasing evidence that the Cancer Stem Cell (CSC), a small percentage of cancer cells that have the characteristic to self-renew, proliferate, differentiate and replenish the bulk of the tumor, may be the leading cause of cancer relapse after chemotherapy. A distinct subpopulation within cancer cells known as the side population (SP), distinguished via Hoechst 33342 staining, has been repeatedly reported to be enriched with cells possessing CSC characteristics. In this study, we first examine if SP can be identified in four established myeloma cell lines. Hoechst-33342 stained cells were analyzed through FACS LSRII using a blue and red dual-wavelength analysis after UV excitation to detect the important red-blue differential dye emission feature of the SP cells. An average of 26.5% of SP cells was found in RPMI8226, 21.4% in KMS-11, 5.4% in U266 and 10.1% in OPM-2 within the viable cells compartment in four separate experiments. In addition, 4.9% of SP cells were also identified in the plasma cell leukemia cell line ARH77. SP and non-SP cells of RPMI8226 and KMS-11 were then sorted and respective RNAs were extracted, amplified and labeled for subsequent Affymetrix microarray scanning. The gene-expression profiles of SP and non-SP cells from RPMI8226 and KMS-11 were compared before selecting statistically significant genes of interest. Two genes were found to be highly upregulated in SP cells. The first one was the Receptor for Hyaluronan-Mediated Motility (RHAMM) gene of 29.0 and 3.9 fold increase in RPMI8226 and KMS-11 respectively. Increased expression of RHAMM has been previously reported to correlate with human B cell malignancies. The second upregulated gene was the human inhibitor of apoptosis protein-family gene, Apollon, of 4.4 and 3.2 fold increase in RPMI8226 and KMS-11 respectively. Another gene - interferon gamma receptor 1 (IFNGR1) gene was found to be down-regulated in SP cells, of 4.3 and 1.2 fold decrease in RPMI8226 and KMS-11 that may provide an explanation of their escape from immune response via T cells. We then examine if SP cells that represent putative CSC exist in primary myeloma cells. Seven myeloma patients’ bone marrow samples were investigated and 0.1 to 5.2 %, of an average of 1.4% of SP cells were identified in the CD38++ plasma cells compartments in 6 out of 7 bone marrow samples. This study reports the existence of SP in both myeloma cell lines and primary myeloma cases. Further studies will be required to confirm the protein expression of selected genes selected from the gene-expression profiles of SP versus non-SP from myeloma cells for the insights into the characteristic and tumorigenicity of CSC in myeloma.

Previous studies have suggested that expanded T-cell clones are found in the blood of 59% of patients with multiple myeloma. These expanded T-cell clones are associated with prolonged overall survival and thus it has been suggested that they may have anti-tumor activity. We have previously reported similar T-cell clones exist in the peripheral blood of patients with Waldenstrom’s Macroglobulinemia (WM) by using flow cytometry to determine the T cell receptor (TCR) Vβ repertoire. Expanded T-cell clones were detected in 9 of 15 (60%) patient samples. Of the nine patients with TCR Vβ clones, four patients had multiple clones. The TCR Vβ clones were not identical, representing a variety of families across the TCR Vβ repertoire. We have previously found that while the TCRVβ+CD8+CD57 negative subset represents polyclonal populations, the CD57 positive subset represents either monoclonal or biclonal populations. By comparing the genetic profiling of these two subsets from a statistically significant gene list, two genes have been found to be highly upregulated in the CD57 negative polyclonal subset. These two genes are i.) SESN3, a member in the Sorting Nexin (SNX) protein family which is implicated in regulating membrane traffic capable of interaction with phosphatidylinositol-3-phosphate (10.4 fold, p=0.0241); ii.) Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled receptor) EBI2 (7.4 fold, p=0.0207): This finding is in contrast to previous report that EBI2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. For the CD57 positive clonal T cell expansions, consistent with our previous reports, CD28 expression was found to be down regulated by 2.6 fold. There are two genes found to be highly upregulated. They are i.) Granzyme B (4.3 fold, p=0.0337) also called Cytotoxic T-lymphocyte proteinase 2. This enzyme is necessary for target cell lysis in cell-mediated immune responses through caspase-dependent apoptosis; ii.) Granzyme H, also called Cytotoxic T-lymphocyte proteinase and probably necessary for target cell lysis in cell-mediated immune responses. In summary, we have shown that CD57 positive clonal T cell populations exist in some patients with WM. Importantly, microarray results have indicated some genes and proteins that may related to better patients survival as previously demonstrated in patients with Multiple Myeloma.

There is increasing evidence that the Cancer Stem Cell (CSC), a small percentage of cancer cells that have the characteristic to self-renew, proliferate, differentiate and replenish the bulk of the tumor, may be the leading cause of cancer relapse after chemotherapy. A distinct subpopulation within cancer cells known as the side population (SP), distinguished via Hoechst 33342 staining, has been repeatedly reported to be enriched with cells possessing CSC characteristics. In this study, we first examine if SP can be identified in four established myeloma cell lines. Hoechst-33342 stained cells were analyzed through FACS LSRII using a blue and red dual-wavelength analysis after UV excitation to detect the important red-blue differential dye emission feature of the SP cells. An average of 26.5% of SP cells was found in RPMI8226, 21.4% in KMS-11, 5.4% in U266 and 10.1% in OPM-2 within the viable cells compartment in four separate experiments. In addition, 4.9% of SP cells were also identified in the plasma cell leukemia cell line ARH77.

Previous studies have suggested that expanded T-cell clones are found in the blood of 59% of patients with multiple myeloma. These expanded T-cell clones are associated with prolonged overall survival and thus it has been suggested that they may have anti-tumor activity. We have previously reported similar T-cell clones exist in the peripheral blood of patients with Waldenstrom's Macroglobulinemia (WM) by using flow cytometry to determine the T cell receptor (TCR) Vβ repertoire. Expanded T-cell clones were detected in 9 of 15 (60%) patient samples. Of the nine patients with TCR Vβ clones, four patients had multiple clones. The TCR Vβ clones were not identical, representing a variety of families across the TCR Vβ repertoire.

The impact on patients' quality of life (QoL) is an important consideration in both decisions of individual patient care, and in allocating health care resources between competing treatments. This study aims to better inform those decisions by investigating the differential impact on patients' QoL of two equally safe and effective iron chelators: one a subcutaneous infusion, the other a dispersible tablet. The current standard of care in patients with chronic iron overload secondary to haematological conditions such as the thalassaemias, myelodysplastic syndrome and sickle cell disease is the subcutaneous administration of desferrioxamine (DFO), for 8 to 12 hours per day, 5 to 7 days a week. The burden of this treatment regimen in some cases leads not only to poor compliance, and hence a reduction in the extent of effective iron chelation, but also to a reduction in patients' QoL. Novartis Pharmaceuticals AG has developed a once daily oral iron chelator (deferasirox, DFX) shown to be as safe and effective as DFO in treating iron overload. To investigate the differential impact of DFO and DFX on patients' QoL, a community sample of 110 individuals were asked to participate in a time trade off (TTO) exercise to elicit community utility values, or preferences, for their different modes of administration. Following standard TTO methodology, participants were asked how they value ten years of full health compared with time in each of three health states for patients with thalassaemia:

Although thalidomide has been reported to increase T cell activity and NK cell cytotoxicity in the blood of patients with multiple myeloma (MM) it is not known whether thalidomide stimulates tumour-specific T cells or causes a general lymphocytosis. We investigated the immunodulatory effects of thalidomide by studying peripheral blood samples from 64 patients in the Australian ALLG MM6 trial, a multicentre randomised phase III study of low-dose thalidomide, prednisolone and Zometa versus prednisolone and Zometa used as post-autologous stem cell transplant (ASCT) maintenance therapy in patients with MM. We have previously demonstrated that TCR Vβ clonal expansions are present in approximately 50% of patients with MM and that their presence correlates with a good prognosis. Sequencing has confirmed that these cells are a clonal expansion of CD3+CD8+CD57+CD28−CD27− late-differentiated cytotoxic effector cells. Flow cytometric analysis of TCR Vβ expansions (24 families) was performed on blood samples from 64 patients enrolled in the MM6 trial both prior to and 12 months post transplant. Prior to transplant, 59% of the 64 patients had TCR Vβ expansions. After thalidomide, T cell expansions were found in a further 8/34 patients compared with 3/30 in the control, no thalidomide group. In the thalidomide group, 70% of the patients had an expansion of more than one T cell clone compared with 32% in the control group (chi2=28.8; p<0.01). In the thalidomide treated patients there was a total of 32 new clones compared with 22 in the patients who did not receive thalidomide. There was a trend for an increase in NK and NKT cells following thalidomide. Further patients will be studied and the prognostic significance will be evaluated but these preliminary studies provide further evidence that thalidomide has an immunomodulatory action in patients with MM and that this is due, at least in part, to a clonal expansion of late-differentiated cytotoxic effector cells.

The impact on patients’ quality of life (QoL) is an important consideration in both decisions of individual patient care, and in allocating health care resources between competing treatments. This study aims to better inform those decisions by investigating the differential impact on patients’ QoL of two equally safe and effective iron chelators: one a subcutaneous infusion, the other a dispersible tablet. The current standard of care in patients with chronic iron overload secondary to haematological conditions such as the thalassaemias, myelodysplastic syndrome and sickle cell disease is the subcutaneous administration of desferrioxamine (DFO), for 8 to 12 hours per day, 5 to 7 days a week. The burden of this treatment regimen in some cases leads not only to poor compliance, and hence a reduction in the extent of effective iron chelation, but also to a reduction in patients’ QoL. Novartis Pharmaceuticals AG has developed a once daily oral iron chelator (deferasirox, DFX) shown to be as safe and effective as DFO in treating iron overload. To investigate the differential impact of DFO and DFX on patients’ QoL, a community sample of 110 individuals were asked to participate in a time trade off (TTO) exercise to elicit community utility values, or preferences, for their different modes of administration. Following standard TTO methodology, participants were asked how they value ten years of full health compared with time in each of three health states for patients with thalassaemia: an anchor/base state that described a patient who has iron chelation without describing the treatment itself; the anchor state plus iron chelation via a subcutaneous infusion; the anchor state plus iron chelation via a once daily oral medication. To avoid ordering bias, respondents were randomised to respond to either scenario (2) or (3) first after the anchor state. Scenarios were not labelled in terms of the iron chelator included and differed only in their description of the mode of treatment administration. Maximum possible utility values are 1 for perfect health and 0 for death. Preliminary results based on the first 32 respondents showed an average utility value of 0.73 for the anchor state, 0.57 for the subcutaneously infused iron chelator and 0.82 for the once a day oral iron chelator. The difference of 0.25 in the utility value for the two treatments was statistically significant (Wilcoxon-signed rank test, p=0.002). Treatment with the subcutaneously infused agent was associated with a lower QoL value, being 0.16 below that for the anchor state (p=0.025). In contrast, treatment with a once daily oral iron chelator had a utility value that was 0.08 (p=0.002) higher than the anchor state. While data collection is ongoing (completion date late Sept), these preliminary results indicate that society associates oral administration of an iron chelator such as DFX with an improvement in the QoL of patients requiring such treatment. Assuming equal safety and efficacy, a once daily oral treatment may therefore be preferred to a burdensome subcutaneous infusion due to the benefits it confers on patients’ QoL.