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CyBorD DARA as induction is well tolerated and induces deep responses when used in conjunction with ASCT for MM.Mechanism of action studies indicate that CyBorD DARA enhances macrophage activation, which may play a role in its clinical efficacy. Daratumumab (DARA) has shown impressive activity in combination with other agents for the treatment of multiple myeloma (MM). We conducted a phase 1b study to assess the safety and preliminary efficacy, as well as potential mechanisms of action, of DARA (16 mg/kg) in combination with a weekly schedule of subcutaneous bortezomib (1.3-1.5 mg/m2), cyclophosphamide (150-300 mg/m2), and dexamethasone (40 mg) (CyBorD DARA) as initial induction before autologous stem cell transplantation (ASCT). Eligible patients were ≤70 years of age with untreated MM requiring treatment and who lacked significant comorbidities. A total of 18 patients were enrolled. Their median age was 56 years (range, 32-66 years), and all patients had Eastern Cooperative Oncology Group performance status ≤1. The International Staging System stages were I, II, and III in 78%, 17%, and 6% of patients, respectively; 28% of patients had high-risk genetic features. There was no dose-limiting toxicity, and the incidence of grade 3 or 4 infection or neutropenia was

Introduction: DNA transposons are efficient, integrating, non-viral vector systems which have been applied to clinical scale CAR-T cell production, aiming to reduce cost and deliver larger genetic cargos relative to viral transduction. CAR-NK cells are a promising cell therapy platform, with a potential for 'off-the-shelf', allogeneic application. Inefficient delivery of plasmid DNA and substantial associated toxicity have limited the utility of DNA transposon systems in primary NK cell engineering. Here we describe an efficient process for the production of transposon engineered primary CAR-NK cells, and the in vitro activity of a CAR-NK cell product targeting human C-type lectin-like molecule-1 (CLL-1/ C-type lectin domain family 12 member A, CLEC12A) for acute myeloid leukemia (AML) produced using the Tc Buster (TcB) transposon system. Since CLL-1 is frequently expressed by leukemic stem cells (LSC) but absent on hematopoietic stem cells, it represents a rational antigenic target to enhance the innate activity of allogeneic NK cell therapies in AML. Methods: NK cells were isolated from 30ml of healthy donor peripheral blood by negative immunomagnetic selection (NK Cell Isolation Kit, Miltenyi Biotec), then activated and expanded using a good manufacturing practice (GMP) grade, 100 Gy irradiated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) in the presence of IL-2 and IL-21. On day 4, NK cells were electroporated with a nanoplasmid TcB transposon carrying a second generation CLL-1 CAR (CD28/CD3ζ or 41BB/CD3ζ) and hyperactive TcB transposase mRNA (Maxcyte ATx). Following transposition, the NK cells were stimulated with the feeder line for a second time. Purified CAR-NK cell populations were produced by immunomagnetic selection of CAR expressing cells using anti-biotin beads (Miltenyi Biotec) and biotinylated CLL-1 protein (ACRO Biosystems), which was also used to detect CAR expression by flow cytometry. For CRISPR/Cas9 gene knockout (KO), pooled sgRNAs targeting 3 sites within the target gene were complexed with Cas9 protein prior to co-electroporation with the TcB payload. CLL-1 CAR-NK cell function was evaluated in co-culture with AML cell lines or biobanked AML patient samples and analyzed by flow cytometry. Results: This approach (Fig 1A) produced efficient transposition with retained clinical-scale expansion capacity of primary CAR-NK cells with mean CAR expression of 37% (n=3, range 26-46%) at day 21, without selection (Fig 1B). By extrapolation, a mean 4,275-fold expansion was observed by day 21, increasing to 14,023-fold by day 25, sufficient to support many clinical doses of 1x10 7 cells/Kg (Fig 1C). Mean NK cell purity of the final product was 98%. A further increase in the proportion of CAR-NK cells was achieved by immunomagnetic selection, with mean CAR expression increasing to 89.5% (range 84-97%, n=3). A further feeder cell stimulation of these CLL-1 CAR selected cells produced a similar expansion trajectory, 3 days delayed relative to unselected cells (Fig 1C). CLL-1 CAR-NK cell function was confirmed by an enhanced ability to target CLL-1 positive AML cell lines and primary AML blasts relative to control electroporated NK cells (Fig 1 D,E). Increased elimination of a primary AML population enriched for LSC (CD34+, CD38-, CLL-1+) by CLL-1 CAR-NK cells was confirmed (mean 96% vs. 32%, p=0.0002, n=3 AML, n=2 NK donors) (Fig 1E). Preliminary data shows simultaneous KO of the NK cell checkpoint cytokine-inducible SH2 containing protein (CISH) using CRISPR/Cas9 while maintaining efficient transposition (Fig 1F). Conclusions: We describe a novel, non-viral approach to CAR-NK cell production using the TcB DNA transposon system, supported by a clinically validated, GMP grade, irradiated, EBV-LCL with clinical scale expansion capability. Purified CAR-NK cell populations were achieved by immunomagnetic sorting without requiring a selection marker or cytotoxic exposure. Preliminary data supports the ability to simultaneously perform CRISPR/Cas9 gene editing - in this case applied to KO of the CISH gene, an NK cell checkpoint with multiple established benefits, including enhanced in vivo NK cell persistence and improved metabolic health. Manufactured using this process, we also present the first reported pre-clinical activity of a CLL-1 CAR-NK cell therapy in AML, demonstrating enhanced targeting of populations enriched for LSC. Figure 1 Figure 1. Disclosures Gurney: ONK Therapeutics: Research Funding. O'Reilly: ONK Therapeutics: Research Funding. Corcoran: ONK Therapeutics: Current Employment. Brophy: ONK Therapeutics: Current Employment; Autolus: Ended employment in the past 24 months. Hardwicke: ONK Therapeutics: Current Employment; Novartis: Ended employment in the past 24 months. Hermanson: Bio-Techne: Current Employment. Szegezdi: ONK Therapeutics: Research Funding. O'Dwyer: Janssen: Consultancy; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

Introduction Cyclophosphamide (CTX) is a widely used anti-neoplastic, performing as an alkylating agent at high doses and immunomodulatory agent at low doses 1.. Combining CTX with monoclonal antibody (mAb) therapy has proven beneficial in potentiating relapsed and/or refractory multiple myeloma (RRMM) therapies, with daratumumab-directed MM cell death enhanced in the presence of CTX 2,3.. Elotuzumab (ELO), the second mAb approved for treating RRMM, promotes MM cell clearance by enhancing macrophage-mediated phagocytosis and CD16- and SLAMF7-directed NK cell cytotoxicity. ELO has been approved for use alongside dexamethasone and lenalidomide 4 or pomalidomide (POM) 5.. However, potential therapeutic benefits of ELO in combination with immunomodulatory drugs such as CTX and POM have yet to be examined. Our research investigates, the efficacy of combining low-dose CTX, alone or in combination with POM, and ELO in enhancing macrophage and NK cell infiltration and function in the MM tumour microenvironment. Materials and Methods Multiple myeloma cells (MM1S and H929) were treated with low-dose CTX and/or POM for 24hrs, washed to remove residual drug and resuspended in fresh media for tumour cell secretome (TCS) generation. Direct effects of CTX and/or POM on surface expression of checkpoint proteins (PD-1 and CD47) on MM cells was assessed by mean fluorescent intensity (MFI) flow cytometry. CD32/CD64 receptor expression on THP-1 macrophages, NKG2D, CD2, DNAM-1, CD96 and KIR2DL1 receptors on KHYG1 and primary NK cells, were measured using flow cytometry as a measure of activation. Migration of serum-starved, CFSE-labelled macrophages and NK cells towards CTX and/or POM TCS was assessed after 4hrs, with total number of migrated cells quantified using the Accuri flow cytometer. Immune cell function following indirect conditioning of macrophages/NK cells with MM cell TCS was measured by quantifying antibody-directed cellular phagocytosis (ADCP) or antibody-directed cellular cytotoxicity (ADCC), respectively. Conditioned immune cells were co-cultured with MM cells in a 2:1 effector to target ratio for 4hrs in the absence/presence of mAbs (ELO, nivolumab and anti-CD47), after which MM cell clearance was quantified by flow cytometry and presented as relative uptake (ADCP) and cytotoxicity (ADCC). One-way ANOVA statistical analysis was performed, followed by Tukey post hoc tests, with significance recognized at p Results Direct treatment of MM cells with CTX increased surface expression of immune evading checkpoint proteins PD-1 and CD47 (p Conclusions Low-dose CTX and POM potentiated the immunomodulatory effects of ELO, with NK-directed cytotoxicity of MM cells enhanced in the presence of this mAb. Our data therefore indicates that the inclusion of low-dose CTX and or POM in combination with ELO could be a novel immunotherapeutic strategy for treating RRMM. References 1. Swan et al., Hemasphere. 2020;4(2). 2. Pallasch et al., Cell. 2014; 156(3):590-602. 3. Naicker et al., Oncoimmunology. 2021; 10(1):1859263 4. Dimopoulos et al., Blood Cancer Journal. 2020 10:91 5. Hose et al., Journal of Cancer Research and Clinical Oncology. 2021; 147:205-212 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background and aims: Acute myeloid leukaemia (AML) is a relatively rare haematological malignancy which is the most common acute leukaemia in adults. Patients with AML often have a substantial comorbidity burden. Consequently, different scores are used in clinical practice to predict outcomes in patients with multiple comorbidities. The Charlson Comorbidity Index (CCI), calculated based on 19 different medical conditions, weighs the comorbidities to measure a patient's burden of disease. Previous publications have suggested that the CCI may be useful in determining survival in AML patients. However, the CCI is not in routine use in Ireland for assessing patients with AML. In this study we examined the usefulness of the CCI to predict early mortality in AML patients, drawing on data from the Extended Blood Cancer Registration (EBCR) in Ireland. Methods: The EBCR was undertaken by National Cancer Registry Ireland registrars trained by consultant haematologists and deployed in national centres. Data collection began in 2017 and continued to 2019; 141 AML patients underwent extended data registration. Comorbidities were identified by ICD-9 codes and chart review. Kaplan Meier curves and Cox regression analyses were used to determine the usefulness of the CCI to predict early mortality in AML patients. Results: Of the 141 AML patients, 82% were between 50 and 70 years of age and 84 had died by 31/12/2019 (median survival time = 289.0 days). The median survival time for patients in the lowest tertile of the CCI was 498.5 days, compared to 246.0 and 116.5 days for subjects in tertiles 2 and 3, respectively (Figure 1. Log rank P-value Conclusions: Although results demonstrate a strong relationship between the CCI and early mortality in AML patients, our findings suggest that the CCI provides little or no additional prognostic information beyond that which is obtained from age at AML diagnosis alone. This study highlights the importance of validating risk assessment tools in order to determine their potential usefulness in a clinical setting and emphasise the importance of weighing in the treatment decision making paradigm. The Blood Cancer Network Ireland (BCNI) thank the Science Foundation of Ireland (SFI) and the Irish Cancer Society (ICS) for funding (2015-2021). We also thank our colleagues in the National Cancer Registry Ireland for assistance, advice and guidance. Figure 1 Figure 1. Disclosures Quinn: Takeda: Honoraria. O'Dwyer: Bristol Myers Squibb: Research Funding; Janssen: Consultancy; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Szegezdi: ONK Therapeutics: Research Funding.

Introduction- The mainstream therapy for AML is cytarabine (AraC) and anthracycline-based intensive chemotherapy. Between 10-40% of patients however have a refractory disease. It is becoming increasingly accepted that the risk for refractory disease could be reduced by finding better ways of selecting the induction therapy. While multiple in vitro and in vivo experimental models exist for pre-clinical drug efficacy testing, they are either labour intensive, too time consuming or generate variable results, making them unsuitable for clinical application. Furthermore, very few, if any of these methods have been shown to replicate the clinical response of patients. The aim of this study was to develop a functional drug testing system that can rapidly and faithfully predict the clinical response of the patient and therefore be utilized to help the identification of patients suitable for AraC+daunorubicin (DnR) therapy. Methods- Primary samples were obtained through Blood Cancer Biobank Ireland. All patients were consented and the research project was approved by the local Research Ethics Committee of NUI, Galway. Mononuclear AML blasts were isolated from bone marrow (BM) aspirates of newly diagnosed patients by ficoll-paque gradient separation. HS-5 human bone marrow mesenchymal stromal cells (BMSC), healthy donor (HD)-derived BMSCs immortalised with hTERT (iMSC) and primary BMSCs (non-immortalised, pBMSC) from HDs and AML patients were used as feeder layers. Cytokine and chemokine secretion by BMSCs was determined using antibody arrays (R&D systems). Extracellular matrix (ECM) scaffold formation was determined by immunocytochemical detection of collagen I, III, IV, V, VI, laminin and fibronectin. AML blasts were cultured in direct contact on established BMSC feeder layer. BMSCs were identifiable by either GFP expression or green cell tracker labelling (CFSE) and the percentage of dying AML cells (negative for GFP/CFSE and positive for the viability dye, ToPro-3) was determined with FACS. Results- In order to choose the most suitable BMSC feeder layer, three main characteristics were studied: cytokine/chemokine secretion, formation of ECM-BMSC proteocellular scaffold and ability to support ex vivo AML blast survival were determined. HS-5 cells, iMSCs and primary, HD pBMSC secreted 66, 59 and 51 paracrine factors, respectively including angiopoietin 1, fibroblast growth factor, interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte colony stimulating factor (G-CSF), C-X-C motif chemokine 12 (CXCL12), CXCL10 and vascular endothelial growth factor. 51% of cytokines/chemokines were common between iMSCs and HD pBMSC, while the HS-5 secretome shared 43% commonality with HD pBMSC. iMSCs also showed an equal ability to establish an ECM-BMSC scaffold with HD pBMSC and they had a superior ability to support the ex vivo survival of AML blasts over HS-5 cells. To determine how closely iMSCs can replicate the effects of BMSCs of AML patients, AML blasts were cultured on matching BMSCs (BMSCs isolated from the same patient). When these AML-BMSC co-cultures were exposed to the BH3 mimetics ABT-199 and ABT-737 or AraC+DnR, iMSCs closely replicated the protective effect of the patients' own BMSCs. Using this optimized ex vivo co-culture model, we tested 16 AML patient samples by exposing iMSC-AML blast co-cultures to a clinically-relevant dosage of AraC+DnR. To quantify drug efficacy, the area under the curve (AUC) for each treatment timepoint (24 h and 48 h) was calculated. By applying a cut-off value of 35% efficacy, we found that the ex vivo test could predict the clinical response of the patient in 81.2% of cases (p-value= 0.002, Figure). Conclusions- The developed drug efficacy test can recapitulate the key features of the BM microenvironment and could predict the clinical response of patients with high accuracy. The advantages of this model over more complex pre-clinical AML models is its suitability to be developed into a laboratory diagnostic tool which could greatly advance the clinical decision on treatment choice. Figure. Figure. Disclosures Quinn: Janssen: Honoraria.

Introduction: Lenalidomide, bortezomib and dexamethasone (RVD) is considered a new standard of care regimen for patients with newly diagnosed multiple myeloma. A previous phase I/II study of RVD in front-line myeloma enrolled 66 patients and achieved a partial response rate or better of 100%, overall and a CR/nCR rate of 52% in the phase 2 portion of the study with encouraging tolerability, but high rates of peripheral neuropathy (PN), albeit mainly mild to moderate grade (Richardson et al, Blood 2010). Subcutaneous (SQ) administration of single agent bortezomib has been shown to be non-inferior to IV bortezomib and led to lower rates of PN, a common treatment-related toxicity (Moreau et al, Lancet Oncol 2011). Herein we present preliminary results of the RsqVD Study, a multi-center, open-label single arm phase II trial, incorporating SQ bortezomib with lenalidomide and dexamethasone and including patients who were considered either transplant eligible or ineligible. All patients subsequently received maintenance therapy with lenalidomide until progression, plus the addition of subcutaneous bortezomib twice monthly in high risk patients (ISS stage II or III and/or high risk cytogenetics features, t(4;14, t(14;16) and del17p). The primary endpoint was overall response rate (ORR) after 4 cycles of induction therapy (PR or better). Secondary endpoints include: rate and severity of PN, safety, time to progression, progression-free survival, duration of response and overall survival. Methods: Planned treatment was 4 cycles of lenalidomide 25 mg/day on days 1-14 and dexamethasone 20/mg/day on days 1, 2, 4, 5, 8, 9, 11 and 12 plus bortezomib 1.3 mg/m2as SQ injection on days 1, 4, 8 and 11 of a 21-day cycle. Thromboprophylaxis with aspirin 75 mg/day or higher was mandatory and HSV prophylaxis was as per institutional standard. Following 4 cycles, patients were planned to proceed with stem cell mobilization and autologous stem cell transplant (ASCT) or further induction therapy up to a total of 8 cycles. Following completion of ASCT or induction therapy, all patients were scheduled to receive lenalidomide maintenance in 28 - day cycle until progression, unacceptable toxicity or withdrawal of consent. Patients with high-risk features received SQ bortezomib on days 1 and 15 during maintenance phase. Response was investigator-assessed as per IMWG criteria. Sample size (n=42) was determined to provide 80% power to test an acceptable ORR of >70% versus an unacceptable ORR of Results: Between November 2014 and February 2016, 42 patients were enrolled across 8 sites in Ireland. Baseline demographic factors include: 64% males, 36% females; median age of 64 years (45-79 years); 41% ISS stage I, 59% ISS stage II/III. FISH analysis detected t(4;14) in 18% of patients (7/40), t(14;16) in 3% of patients (1/36) and del17p in 10% of patients (4/40). 64% (27/42) patients proceeded to stem cell mobilization and 60% (25/42) to ASCT. The median number of induction cycles completed was 4 (1 to 8 cycles). 40 of a total of 42 patients were considered evaluable for the primary endpoint of ORR. A preliminary analysis of ORR following 4 cycles of induction therapy indicates that 98% (39/40) of patients achieved partial response or better. PN of any grade has been reported by sites in 43% (18/42) of patients to date. Conclusion: RsqVD is a highly effective regimen in newly diagnosed multiple myeloma producing a very high ORR following initial induction therapy, with a lower overall rate of PN described by sites than expected. Full analyses of response and safety data for induction treatment and follow up will be presented, as well as preliminary evaluation of response to subsequent therapy. Disclosures O'Gorman: Janssen Cilag: Research Funding; Celgene: Research Funding. O'Dwyer:Celgene: Consultancy, Honoraria, Research Funding; Glycomimetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Quinn:Celgene: Honoraria; Janssen Cilag: Honoraria. Murphy:Celgene: Honoraria; Janssen Cilag: Honoraria. Crotty:BMS, Takeda, Novartis, Janssen, Roche: Honoraria. Hayden:Celgene: Honoraria; Janssen Cilag: Honoraria; Amgen: Honoraria. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Introduction: The Spectra Optia Apheresis System has been in use in our centre for 3 years for the collection of autologous peripheral blood stem cells (PBSC).We compared the Collection Efficiency (CE2) of the two Optias we have in use and also compared the CE2 of patients with Multiple Myeloma (MM) and Lymphomas, and those mobilised with and without Plerixafor. The Collection Efficiency (CE2) is calculated as follows: (Total CD34+ collected)/ (Whole Blood volume processed x Pre-CD34+ count). It is a crude estimation of the potential yield of a harvest and assumes that the CD34+ concentration is constant throughout a procedure. As the concentration of CD34+ cells in the peripheral blood will naturally drop off during a procedure, the CE2 can be expected to give a result that underestimates the yield. The pre-harvest FBC would normally be taken about 3 hours prior to commencement of the harvest, thus the peripheral CD34 may not fully reflect the count immediately before starting the harvest and this may also influence the accuracy of the CE2 result. Patients and Methods: Data on 143 peripheral blood autologous stem cell harvests from 87 patients collected using the Spectra Optia from mid-2011 to mid-2014 have been examined. Procedures without pre-harvest CD34+ counts or incomplete were excluded from the analysis. Procedures included in the analysis (138) are summarised in table 1. Median age of patients was 59 years (range 26-69). All patients were mobilised using disease specific standard protocols. Patients who fail to mobilise by the expected date were frequently given Plerixafor. A CD34+ dose of 5 x 106/kg was used as desirable target for reinfusion with enough for two reinfusion collected for MM patients to allow for repeat reinfusion if required. Statistical Analysis: Statistical analysis was performed using SPSS package. We have compared the CE2 achieved by each Optia machine, the CE2 results of the 2 main diseases MM and Lymphomas and to determine if any difference in Collection Efficiency is evident when patients are mobilised using Plerixafor. Results: 143 collections were included in the analysis. Fifty patients underwent one collection, 27 patients underwent two collections, and 10 patients required three collections to achieve the minimum CD34+ cell dose required. The mean CE2 for the entire cohort was 74.23% and mean CD34+ yield was 4.6 x 106/kg/collection. There were no statistically significant differences in pre-collection CD34+ count, collection efficiencies and final CD34+ yield between groups according to instruments used, administration of Plerixafor and diseases (Table 1). A significant correlation between the Preleukapheresis peripheral blood CD34+ cell count and the CD34+ cell yield of each leukapheresis was shown by a linear regression analysis (r2 = 0.867, p < 0.0001; Figure 1). Conclusions: For maximisation of PBSC yield, the timing for PBSC collection after these mobilizations is critical. Among criteria utilized to predict yield, the circulating peripheral blood CD34+ cell count has been a good predictor of PBSC yield mobilized with protocols used in our centre. In our data, pre-leukapheresis peripheral blood CD34+ cell count on the day of collection correlates with the final CD34+ yield. Interestingly, administration of Plerixafor in patients clinically at risk of poor mobilisation allows collection of comparable quality to patients who mobilised without Plerixafor. Abstract 3848. Table 1. Characterisation of collections, including device used, disease indication and use of Plerixafor. CD34_CE2 Periph_cd34 Actual yield Mean SD Number of procedures Mean SD Number of procedures Mean SD Number of procedures Optia 1 80.89% 85.37% 62 51.8 43.9 62 4.733 4.379 62 2 68.69% 17.38% 73 49.7 48.4 73 4.630 3.834 73 disease lymphoma 73.66% 28.94% 57 51.1 57.2 57 4.581 4.608 57 myeloma 75.39% 76.69% 73 52.7 36.5 73 5.008 3.651 73 other 65.45% 11.13% 5 15.4 12.0 5 .945 .677 5 Plerixafor no 73.79% 64.18% 107 55.1 47.6 107 5.078 4.208 107 yes 76.19% 35.77% 28 33.7 36.6 28 3.144 3.155 28 Total 74% 135 50.60 46.20 135 4.680 4.080 135 Fig. 1. Relation between pre-leukapheresis CD34+ count and actual leukapheresis yield. Fig. 1. Relation between pre-leukapheresis CD34+ count and actual leukapheresis yield. Disclosures No relevant conflicts of interest to declare.

Background Based on evidence from clinical trials there is a growing consensus that a three drug regimen involving at least one novel agent (proteasome inhibitor or immunomodulatory drug) should be used as initial induction in patients with newly diagnosed multiple myeloma (NDMM), usually for 3-4 cycles, prior to harvesting stem cells and proceeding to autologous stem cell transplantation (ASCT). Three drug regimens achieve deeper responses both before and after ASCT and this has been shown to translate into superior progression free survival (PFS). CR rates with the three drug regimens prior to ASCT are reported to be in the range of 20-30% with correspondingly high rates of very good partial response (VGPR) (50-60%). One such regimen is the combination of bortezomib with cyclophosphamide and dexamethasone (CyBorD). The published overall response rates (ORR) from phase II trials with CyBorD are approximately 90%, with at least 60% of patients achieving VGPR. At our center, CyBorD has been used as front line therapy since 2008. In this report we present our experience using this regimen as initial therapy in transplant eligible patients. Methods We retrospectively analysed clinical and laboratory records for 31 NDMM patients treated with CyBorD at our institution between 2008 and 2013, all of whom subsequently proceeded to ASCT. The standard protocol consisted of bortezomib 1.3 mg/m2 i.v. twice a week, cyclophosphamide orally at a dose of 300 mg/m2weekly, and dexamethasone orally of 40 mg daily given in 4 days long blocks weekly. Since November 2009 we have also used weekly CyBorD. Results The median age was 57 years (range from 45 to 65), including 24 males and 7 females. According to ISS, 45% patients were classified as stage I, 5% stage II, and 50% as stage III. The ORR was 90% post cycle I and increased to 96% post cycle IV. At least VGPR was observed in 13% of patients after cycle 1 and 56% after cycle IV. Transplantation had improved the responses and 79% had at least VGPR post transplant versus 63% prior, and 24% patients achieved CR post ASCT in comparison to 16% prior ASCT (Fig. 1). The therapy was well tolerated. Haematological adverse events were acceptable and no patient required significant dose reduction or experienced treatment delay due to haematological toxicity. Importantly, no patients developed > grade 3 peripheral neuropathy and no patients required dose reduction or discontinuation of bortezomib due to neurological complications. Stem cell mobilisation was performed using a combination of cyclophosphamide 1.5g/m2 and G-CSF. All patients mobilized successfully without requirement for plerixafor. The medium CD34+ yield after 2 collection days was 8.2 x106/kg (range 1.8 -19.4). The PFS at 2 years was 75% and 64% at three years of follow up. At 2 years post transplant, the patients achieving at least a VGPR had a longer median PFS in comparison to patients with PR (91% vs 76%, p=NS) (Fig. 2). The cost analysis has shown that on average, the drug only cost of 4 cycles of CyBorD was 18 000€. This compares to 38 000€ for 4 cycles of bortezomib, lenalidomide, dexamethasone (VRD). Conclusions Our data confirm the safety and efficacy of the CyBorD protocol and its applicability to routine clinical practice outside of large academic myeloma centres. CyBorD is an active, well tolerated and cost effective option for patients with NDMM, which could be an ideal backbone for the incorporation of new modalities, such as monoclonal antibodies in induction therapy. A randomized comparison with other triple drug regimens are required to fully establish its place in the treatment of newly diagnosed multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Abstract 3171 Autoimmune haemolytic anaemia (AIHA) is characterised by severe haemolysis due to development of auto antibodies directed against patient's own red cells. Rituximab is a chimeric monoclonal antibody that specifically depletes B cells by targeting CD20 on both immature and mature B lymphocytes We evaluated the use of Rituximab in patients with AIHA who are refractory to conventional Immunosupression in 5 centres in ireland. Methods: 24 cases of AIHA from five centres in the Republic of Ireland were identified who were treated with Rituximab at the standard dose of 375mg/m2 weekly for four weeks. All patients had received prior therapy with steroids and 50% of patients received two or more courses of Immunosupression. One patient had splenectomy prior to Rituximab. Five patients had an underlying lymphoproliferative disorder and two had a myeloproliferative neoplasm. Response was assessed at 1,3,6,12,36, 48 and 60 months. Medium duration of follow up was 34 months. Response was defined as normalisation of Haemoglobin concentration and haemolytic parameters reticulocyte count, bilirubin and lactate dehydrogenase (LDH). Overall response to Rituximab was 83.3% at 28 days post treatment. Four patients responded to Rituximab in combination with other Immunosupression such as Azathioprine or low dose steroids. Medium response duration was 11.5 months. Sustained response at 1 year, 2 years and 4 years post treatment were 60%, 55% and 30% respectively. Rituximab was well tolerated without significant complications in the majority of patients. One patient developed neutropenic sepsis. One patient received second course of Rituximab after relapse within a year and had sustained response of over two years after the second course. Conclusion: Rituximab is a safe and effective treatment for patients with AIHA who are resistant to steroids.The majority of patients will initially respond but a significant number will gradually relapse over time. Re-treatment with rituximab maybe considered on relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract 1914 Poster Board I-937 Introduction: Abnormal proliferation is associated with poor outcome in patients with diffuse large B cell lymphoma (DLBCL), with Ki-67 a well established prognostic marker. More recently gene expression profiling has shown high MYC expression, a marker of proliferation, to be associated with poor outcome in DLBCL. Thus, the cell cycle is an attractive therapeutic target in aggressive lymphoma. Cdc7, a serine/threonine kinase is essential for initiation of DNA replication and cell cycle regulation. Cdc7 phosphorylation of the replicative helicase Mcm2 is a key event in this process. Overexpression of Mcm2 has been linked to increased proliferation and poor outcome in a variety of cancers, including DLBCL. No data exists on the prognostic relevance of the phosphorylated form of Mcm2. Recently, overexpression of Cdc7 and its regulatory subunit Dbf4 has been demonstrated in multiple cancers but again, the prognostic relevance of this is unknown. Emerging data shows that inhibition of Cdc7 activity with a new class of pyrrolopyridinone kinase inhibitors has activity against a broad range of cancers, including lymphoma cell lines, even in cells lacking p53. We hypothesized that increased Cdc7 expression and/or Mcm2 phosphorylation may be associated with poor prognosis in diffuse large B cell lymphoma (DLBCL) and that if confirmed, Cdc7 inhibition could be a potential treatment strategy in DLBCL. Aim: The primary aim of this project was to explore the prognostic significance of Mcm2 phosphorylation and Cdc7 expression in archived diagnostic biopsy material from patients with DLBCL. Methods: All patients with a confirmed diagnosis of treated for DLBCL treated in Galway University Hospital between 2001 and 2009 and having suitable material were selected. Chart review was undertaken to document baseline demographics, International Prognostic Index (IPI), treatment, remission rate, progression free survival (PFS) and overall survival (OS). Two tissue cores (0.6mm diameter) from each specimen were arrayed onto a tissue microarray (TMA). Expression of phospho-Mcm2 and Cdc7 as well as Ki-67, p53, Mcm2, c-myc, standard germinal centre and non-germinal centre markers were evaluated by immunohistochemisty (IHC) on the TMAs. Expression of these markers was evaluated against clinical endpoints. PFS and OS curves were constructed using the method of Kaplan and Meier and evaluated using the log-rank test. The relationship between phospho-Mcm2, Cdc7 and the known prognostic factors: germinal center and non-germinal centre type, Ki-67, and c-myc were explored through Mann-Whitney rank test. Results: Seventy-eight patients including 43 males (median age 62) and 35 females (median age 67) were included. 10% of patients were in the low, 37% in the low-intermediate, 24% in the high —intermediate and 28% in the high IPI group. IPI score was significantly associated with survival (p10% was set as the cut off for high Cdc7. The cumulative OS at 50 months was 57% in the low Cdc7 group and 37% in the high Cdc7 group (p=0.26) for the whole cohort. For the patients with a low and low-intermediate IPI the cumulative OS at 50 months was 68% in the low Cdc7 group and 40% in the high Cdc7 group (p=0.1). For patients with a high and intermediate-high IPI, the cumulative OS at 50 months was 51% in low Cdc7 group and 42 % in high Cdc7 group (p=0.9). The PFS was not significantly different in the whole cohort as well as in IPI subgroups between patient with high and low Cdc7. Interestingly there was a significant correlation between Cdc7 and Ki67 expression (p Conclusion: Despite a relatively small sample size, our results suggest that increased activity of the Cdc7 kinase may be associated with a trend towards worse outcome. This is particularly apparent in low to low-intermediate IPI. Cdc7 could be also a potential therapeutic target in DLBCL. Further studies are needed to characterise prognostic and therapeutic significance of high expression of Cdc7 and pMcm2. Disclosures: No relevant conflicts of interest to declare.