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Introduction: Patients (pts) with CLL may be at particular risk of severe COVID-19 given advanced age and immune dysregulation. Two large series with limited follow-up have reported outcomes for pts with CLL and COVID-19 (Scarfò, et al. Leukemia 2020; Mato, et al. Blood 2020). To provide maximal clarity on outcomes for pts with CLL and COVID-19, we partnered in a worldwide effort to describe the clinical experience and validate predictors of survival, including potential treatment effects. Methods: This international collaboration represents a partnership between investigators at 141 centers. Data are presented in two cohorts. Cohort 1 (Co1) includes pts captured through efforts by European Research Initiative on CLL (ERIC), Italian CAMPUS CLL Program, and Grupo Español de Leucemia Linfática Crónica. The validation cohort, Cohort 2 (Co2), includes pts from US (66%), UK (23%), EU (7%), and other countries (4%). There is no overlap in cases between cohorts. CLL pts were included if COVID-19 was diagnosed by PCR detection of SARS-CoV-2 and they required inpatient hospitalization. Data were collected retrospectively 2/2020 - 5/2020 using standardized case report forms. Baseline characteristics, preexisting comorbidities (including cumulative illness rating scale (CIRS) score ≥6 vs. The primary endpoint of this study was to estimate the case fatality rate (CFR), defined as the proportion of pts who died among all pts hospitalized with COVID-19. Chi-squared test was used to compare frequencies; univariable and multivariable analyses utilized Cox regression. Predictors of inferior OS in both Co1 and Co2 were included in multivariable analyses. Kaplan-Meier method was used to estimate overall survival (OS) from time of COVID-19 diagnosis (dx). Results: 411 hospitalized, COVID-19 positive CLL pts were analyzed (Co1 n=281, Co2 n=130). Table 1 describes baseline characteristics. At COVID-19 dx, median age was 72 in Co1 (range 37-94) and 68 in Co2 (range 41-98); 31% (Co1) and 45% (Co2) had CIRS ≥6. In Co1, 48% were treatment-naïve and 26% were receiving CLL-directed therapy at COVID-19 dx (66% BTKi ± anti-CD20, 19% Venetoclax ± anti-CD20, 9.6% chemo/chemoimmunotherapy (CIT), 1.4% PI3Ki, 4% other). In Co2, 36% were never treated and 49% were receiving CLL-directed therapy (65% BTKi ± anti-CD20, 19% Venetoclax ± anti-CD20, 9.4% multi-novel agent combinations, 1.6% CIT, 1.6% PI3Ki, 1.6% anti-CD20 monotherapy, 1.6% other). Most pts receiving CLL-directed therapy had it held at COVID-19 diagnosis (93% in Co1 and 81% in Co2). Frequency of most COVID-19 symptoms/laboratory abnormalities were similar in the two cohorts including fever (88% in both), lymphocytosis (ALC ≥30 x 109/L; 27% vs. 21%), and lymphocytopenia (ALC < 1.0 x 109/L; 18% vs. 28%), while others varied between Co1 and Co2 (p Median follow-up was 24 days (range 2-86) in Co1 and 17 days (1-43) in Co2. CFRs were similar in Co1 and Co2, 30% and 34% (p=0.45). 54% and 43% were discharged while 16% and 23% remained admitted at last follow-up in Co1 and Co2, respectively. The proportion of pts requiring supplemental oxygen was similar (89% vs. 92%) while rate of ICU admission was higher in Co2 (20% vs. 48%, p Conclusions : In the largest cancer dx-specific cohort reported, pts with CLL hospitalized for COVID-19 had a CFR of 30-34%. Advanced patient age at COVID-19 diagnosis was an independent predictor of OS in two large cohorts. This CFR will serve as a benchmark for mortality for future outcomes studies, including therapeutic interventions for COVID-19 in this population. The effect of CLL treatment on OS was inconsistent across cohorts; COVID-19 may be severe regardless of treatment status. While there were no significant differences in distribution of current lines of therapy between cohorts, prior chemo exposure was more common in Co1 vs. Co2, which may account for difference in OS. Extended follow-up will be presented. Disclosures Roeker: American Society of Hematology: Research Funding; Abbott Laboratories: Other: spouse with minority ownership interest ; AbbVie: Other: spouse with minority ownership interest . Scarfo:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau. 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There is a Blood Commentary on this article in this issue., Key Points Both watch-and-wait and treated CLL patients have high mortality rates when admitted for COVID-19. Receiving a BTKi for CLL at COVID-19 diagnosis severe enough to require hospitalization did not influence case fatality rate in this study., Given advanced age, comorbidities, and immune dysfunction, chronic lymphocytic leukemia (CLL) patients may be at particularly high risk of infection and poor outcomes related to coronavirus disease 2019 (COVID-19). Robust analysis of outcomes for CLL patients, particularly examining effects of baseline characteristics and CLL-directed therapy, is critical to optimally manage CLL patients through this evolving pandemic. CLL patients diagnosed with symptomatic COVID-19 across 43 international centers (n = 198) were included. Hospital admission occurred in 90%. Median age at COVID-19 diagnosis was 70.5 years. Median Cumulative Illness Rating Scale score was 8 (range, 4-32). Thirty-nine percent were treatment naive (“watch and wait”), while 61% had received ≥1 CLL-directed therapy (median, 2; range, 1-8). Ninety patients (45%) were receiving active CLL therapy at COVID-19 diagnosis, most commonly Bruton tyrosine kinase inhibitors (BTKi’s; n = 68/90 [76%]). At a median follow-up of 16 days, the overall case fatality rate was 33%, though 25% remain admitted. Watch-and-wait and treated cohorts had similar rates of admission (89% vs 90%), intensive care unit admission (35% vs 36%), intubation (33% vs 25%), and mortality (37% vs 32%). CLL-directed treatment with BTKi’s at COVID-19 diagnosis did not impact survival (case fatality rate, 34% vs 35%), though the BTKi was held during the COVID-19 course for most patients. These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Future epidemiologic studies are needed to assess severe acute respiratory syndrome coronavirus 2 infection risk, these data should be validated independently, and randomized studies of BTKi’s in COVID-19 are needed to provide definitive evidence of benefit., Visual Abstract

Introduction: Ibrutinib (Ibr) is the first approved irreversible BTK inhibitor. Cardiovascular (CV) adverse events, include hypertension (HT) and atrial fibrillation (AF). Ventricular tachyarrhythmias and sudden death have been reported possibly due to off target kinase inhibition. Several randomised Phase III trials have reported low numbers of cardiac deaths, sudden/unexplained deaths, and/or ventricular arrhythmias with Ibr including 9 deaths in RESONATE-2 Trial, 11 unexplained or unwitnessed deaths in Alliance Trial A041202 (med age 71; Ibr (n=7 [4%]), Ibr+ritux (IR; 4 [2%]), one death in the IR arm of ECOG1912 Trial (mean age 56.7) and 3 unexplained plus 5 cardiac deaths with BR + Ibr in the HELIOS trial (med age 64). Methods: FLAIR, a phase III, multicentre, randomised, controlled, open, trial recruiting 771 pts with untreated CLL requiring therapy. Pts were ≤75 years, considered fit for FCR and were randomized to IR or FCR. Pts with inadequately controlled symptomatic cardiac failure or unstable angina were excluded. We conducted ad hoc evaluations of the potential association of sudden or cardiac deaths with prior medical history or therapies for cardiac disorders (AF, Ischaemic heart disease, MI or angina) and HT via univariate analyses. We used Fisher's Exact test and estimated relative risk (RR) and associated confidence intervals under a normal approximation. Results: In FLAIR, 384 and 378 pts were treated with IR and FCR, respectively, median 52.7 months FU and treatment durations of 47.2 and 4.7 months. Median age 62. 84/384 (22%) IR pts had pre-existing HT and 17/384 (4%) a prior history of a cardiac disorder. 74/378 (20%) FCR pts had HT and 18/378 (5%) a cardiac disorder. To date 10 pts had a sudden or cardiac death: 2 (0.5%) FCR and 8 (2%) IR - median 33 mo (range: 13-48) from initiating IR to death. 9/10 pts were male aged 54 to 73 years at randomisation. 7/ 8 IR pts experiencing a sudden or cardiac death, had a prior history of HT and/or cardiac disorder (RR 23.6 vs pts with no HT/CV history, 95%CI [2.9-195]; Fisher's Exact P=0.0003; Table 1). Hypertensive cardiomyopathy and/or coronary artery disease was found at post mortem of all 3 IR pts having a PM. The risk of sudden or cardiac death with IR was 0.3% (1/291) in pts without pre-existing risk. The number of pts taking anti-hypertensives at trial entry (some taking more than one class) was: ACE inhibitors (ACEi) 47 IR, 37 FCR; Angiotensin II receptor blockers (ARB's) 15 IR, 17 FCR; Ca channel blockers 30 IR, 34 FCR; B-blockers 24 IR, 20 FCR; Diuretics 13 IR, 16 FCR; and alpha blockers 7 IR, 5 FCR. Overall, 291/384 (76%) IR and 296/378 (78%) FCR were taking no HT or cardiac medications at trial entry. The use of ACEi at study entry was associated with an increased risk of sudden or cardiac death with IR: 7/47 (15%) pts on ACEi at trial entry had a sudden or cardiac death compared to 1/336 (0.3%) not taking ACEi (RR 50.2, 95%CI [6.3-399]; P < 0.0001; Table 1). 2/7 pts had discontinued ACEi and switched to ARB's due to cough sometime prior to death. In contrast 0/37 FCR pts with a history of HTN/CV taking ACEi experienced a sudden or cardiac death. In the IR arm, none of the 46 pts receiving cardiac medication but not ACEi had a sudden or cardiac death suggesting that the risk was not simply a prior history of HT or cardiac disorder. 3/11 (27%) IR pts taking an ACEi plus a beta blocker at study entry had a sudden or cardiac death vs 4/32 (12.5%) pts on ACEi without a beta blocker. Factors potentially confounding the interpretation: 1) analyses did not take into account severity of underlying HT/CV risk, as it is possible that pts on ACEi had more severe underlying CV disease than others; and 2) analyses did not adjust for changes in HT/CV medications during the course of the study. However, the lack of any such signal in pts with a prior cardiovascular/HT history on therapies other than ACEi suggests a potential association with the medication itself. Conclusion: Sudden or cardiac deaths seen on IR in FLAIR were observed predominantly among male pts with a prior history of HT or cardiac disease. The prior use of ACEi was correlated with the risk of sudden or cardiac death in IR pts. In contrast, pts treated with IR who were not on ACEi (regardless of medical history) had a very low rate of cardiac or sudden death. Whilst these findings need confirming in other Ibr trials we believe that caution should be taken when concurrently administering ibrutinib and ACE inhibitors in pts with a medical history of HT or CV disease. Figure 1 Figure 1. Disclosures Munir: F. Hoffmann-La Roche: Consultancy; Alexion: Honoraria. Bloor: Novartis: Honoraria; Kite, a Gilead Company: Honoraria. Broom: AbbVie: Honoraria; AstraZeneca: Honoraria; Janssen-Cilag Ltd: Honoraria; Takeda UK Ltd: Honoraria; Celgene Ltd: Honoraria; Gilead: Honoraria. Furtado: Abbvie: Other: Conference support. Morley: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference support; AbbVie; Takeda: Other: Conference support; Janssen: Honoraria; Kite: Honoraria. Cwynarski: Atara: Consultancy; BMS/Celgene: Other; Celgene: Consultancy; Gilead: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Janssen: Consultancy, Other; Kite, a Gilead Company: Consultancy, Speakers Bureau; Roche: Consultancy, Other, Speakers Bureau; Takeda: Consultancy, Other, Speakers Bureau. Gatto: Roche: Consultancy. Paneesha: AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Roche: Honoraria; Celgene: Honoraria. Fox: F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees. Howard: Roche: Current Employment. Cairns: Takeda: Research Funding; Amgen: Research Funding; Merck Sharpe and Dohme: Research Funding; Celgene / BMS: Other: travel support, Research Funding. Patten: NOVARTIS: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria; JANSSEN: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; ROCHE: Research Funding. Hillmen: BeiGene: Honoraria; SOBI: Honoraria; AstraZeneca: Honoraria; Gilead: Research Funding; Roche: Research Funding; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding.

Introduction: The most effective chemoimmunotherapy (CIT) in previously untreated CLL is the combination of fludarabine, cyclophosphamide and rituximab (FCR). Ibrutinib (I), the first irreversible inhibitor of Bruton's tyrosine kinase approved for CLL, has improved outcomes in numerous clinical trials compared to different CIT. Methods: FLAIR (ISRCTN01844152) is an ongoing, phase III, multicentre, randomised, controlled, open, parallel group trial for previously untreated CLL requiring therapy according to the IWCLL 2008 guidelines. Patients over 75 years or with >20% 17p-deleted cells were excluded. Participants were randomised on a 1:1 basis to receive 6 cycles of FCR (oral fludarabine 24mg/m 2/day for 5 days, oral cyclophosphamide 150mg/m 2/day for 5 days with IV rituximab [375 mg/m 2 on day 1/2 of cycle 1; 500 mg/m 2 on day 1 of cycles 2-6]) every 28-days or IR (Ibrutinib [420mg/day] plus rituximab [6 doses as for FCR]) given for up to 6 years with stratification by disease stage, age, gender and centre. The primary endpoint was to assess whether IR was superior to FCR in terms of investigator-assessed PFS. Secondary endpoints included overall survival,; attainment of undetectable MRD; response to therapy; safety and toxicity; health-related quality of life and cost-effectiveness. A formal interim analysis was planned when 191 events were observed in both arms or 109 events in the FCR arm alone with a p-value of 0.005 leading to reporting of the trial. Here we report the results of this planned interim analysis. Results: A total of 771 patients were randomised (385 to FCR and 386 to IR) from 113 UK Centres between 9/19/2014 and 7/19/2018. The data was locked on 5/24/2021. 73.3% were male, median age was 62 years (33.6% >65yo) and 45.1% were Binet Stage C. IGHV data was available for 728 (94.4%) patients with 53.2% IGHV unmutated (≥98% homology to germline), 40.5% IGHV mutated and 6.3% Subset 2. Hierarchical FISH testing revealed 0.4% 17p del, 15.4% 11q del, 12.3% trisomy 12, 29.7% normal and 35% 13q del; with 7.1% failed. The arms were well-balanced for disease variables with no significance differences. Median follow-up was 52.7 months. IR had a superior PFS compared to FCR (Median PFS not reached for IR versus 67 months for FCR; HR: 0.44; p Conclusion: Ibrutinib plus rituximab resulted in a superior PFS compared to FCR. There was no difference in overall survival, most likely due to effective second-line targeted therapy in patients progressing after FCR. Figure 1 Figure 1. Disclosures Hillmen: Janssen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; AbbVie: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Research Funding; Gilead: Research Funding; SOBI: Honoraria; BeiGene: Honoraria; AstraZeneca: Honoraria. Bloor: Novartis: Honoraria; Kite, a Gilead Company: Honoraria. Broom: AbbVie: Honoraria; AstraZeneca: Honoraria; Janssen-Cilag Ltd: Honoraria; Takeda UK Ltd: Honoraria; Celgene Ltd: Honoraria; Gilead: Honoraria. Furtado: Abbvie: Other: Conference support. Morley: Kite: Honoraria; Janssen: Honoraria; AbbVie; Takeda: Other: Conference support; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference support. Cwynarski: Adienne, Takeda, Roche, Autolus, KITE, Gilead, Celgene, Atara, Janssenen: Other. Paneesha: Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Bristol Myers Squibb: Honoraria; AbbVie: Honoraria. Howard: Roche: Current Employment. Cairns: Merck Sharpe and Dohme: Research Funding; Amgen: Research Funding; Takeda: Research Funding; Celgene / BMS: Other: travel support, Research Funding. Patten: NOVARTIS: Honoraria; ROCHE: Research Funding; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding. Munir: F. Hoffmann-La Roche: Consultancy; Alexion: Honoraria.

Introduction: CNS relapse of DLBCL is associated with poor prognosis. Estimated incidence varies between 1.9 and 8.4%1. The CNS-International prognostic index (IPI)2 help risk stratify and estimate the 2-year risk of CNS relapse in DLBCL patients treated with R-CHOP chemotherapy. CNS prophylaxis is indicated in patients with a high risk of CNS relapse (a score of ≥4 equated to a 10.2% risk). High-risk DLBCL patients outside the CNS-IPI system include double/triple-hit (MYC/BCL-2/BCL-6 translocations) lymphoma, HIV lymphoma, testicular lymphoma, primary cutaneous lymphoma-leg type, stage IE breast lymphoma3. IT methotrexate or cytarabine administered during the course of systemic chemotherapy has been the most widely employed method of CNS prophylaxis but there is paucity of data validating its efficacy. Aim: The primary aim of the study was to evaluate the CNS relapse rates in DLBCL patients who received CNS prophylaxis. Patients and Methods: This was a single-centre retrospective observational study conducted in a district general hospital. Data was extracted from the regional (Dorset Cancer Network) DLBCL database and laboratory reports for CSF analysis at the time of the first intrathecal chemotherapy. Medical records of patients with DLBCL who received CNS prophylaxis were evaluated for the following patient-related and disease-related demographics: age at diagnosis, gender, stage, systemic treatment, CNS prophylaxis, treatment response, remission duration, systemic relapse rates, CNS relapse rates and survival. CNS-IPI scores were retrospectively calculated and additional indications evaluated for patients who received CNS prophylaxis. Results: Between 2013 and 2018, 178 patients were diagnosed with DLBCL. All patients were treated with RCHOP chemo-immunotherapy. CNS prophylaxis was administered in 47 (26%) patients. Median age was 69 years (range 20-86 years) and 62% were males. All 47 patients (100%) received IT methotrexate as CNS prophylaxis, with 43 (91%) receiving all of the planned 4 doses of IT methotrexate 12.5 mg each. A CNS-IPI score of ³4 was present in 31 (66%) patients, and a score of 2-3 in 9 (19%) patients. Additional risk factors identified included testicular lymphoma in 3 patients, breast lymphoma in 2 patients and oropharyngeal lymphoma in 2 patients. Ten (21%) patients received their treatment at the outset with courses 1-4 of R-CHOP. Of the 47 patients who received CNS prophylaxis, 5 (10%) relapsed; all had isolated CNS lymphoma at relapse. Median time to CNS relapse was 25 months (range 12-36 months) from initial diagnosis of DLBCL. Median survival after CNS relapse was 5 months (range 2-9 months). Of the remaining 141 patients, 2 patients relapsed with isolated CNS lymphoma. Conclusion: Although the overall incidence was low (4%), CNS relapse was observed in 10% of high-risk patients all of whom received CNS prophylaxis with IT methotrexate. The efficacy of CNS prophylaxis with IT chemotherapy remains unproven. There is no randomised study to show that IT prophylaxis alone is effective. Current British guidelines recommend high-dose intravenous methotrexate over IT methotrexate if patient's physiological fitness and renal function are acceptable4. The median age in our cohort was 69 years which makes it challenging to deliver dose-intensive systemic therapy concurrently with intravenous high-dose methotrexate. The role of CNS prophylaxis in high-risk patients including its efficacy and safety in older patients need further evaluation in prospective randomised studies. References Eyre T et al.Efficacy of central nervous system prophylaxis with stand-alone intrathecal chemotherapy in diffuse large B-cell lymphoma patients treated with anthracycline-based chemotherapy in the rituximab era: a systematic review. Hematologica. 2019;105(7):1914-1924.Norbert Schmitz et al.CNS International prognostic Index: A risk model for CNS relapse in patients with diffuse large B-cell lymphoma treated with R-CHOPJ Clin Oncol 2016; 34:3150-3156.Andrew D Zelenetz et al.National Comprehensive Cancer Network (NCCN) Guidelines: B-Cell Lymphomas.Version 2.2020.Pamela McKay et al.The prevention of central nervous system relapse in diffuse large B-cell lymphoma: a British Society for Haematology good practice paper. Onlinelibrary.wiley.com. 2020. Available from: https://onlinelibrary.wiley.com/doi/epdf/10.1111/bjh.16866 Disclosures Hall: Janssen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Karyopharm:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Takeda:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Celgene:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings.Killick:Celgene:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Jazz Pharmaceuticals:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Novartis:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Gilead:Honoraria, Other: Support for attending education meetings.McCarthy:Janssen:Honoraria;Abbvie:Membership on an entity's Board of Directors or advisory committees.Walewska:AbbVie:Other: sponsored for educational meetings, Speakers Bureau;Janssen:Other: sponsored for educational meetings, Speakers Bureau;Gilead:Speakers Bureau;Astra Zeneca:Membership on an entity's Board of Directors or advisory committees.Chacko:Astellas:Honoraria;Daiichi-Sankyo:Honoraria;Novartis:Honoraria, Other: Travel Grants;Gilead:Other: Travel grants;Jazz Pharmaceuticals:Other: Travel grants;Celgene:Other: Travel grants.

To the editor: Ibrutinib, approved by the US Food and Drug Administration (FDA), is an inhibitor of Bruton tyrosine kinase (BTK).[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Ibrutinib use is associated with atrial fibrillation (AF), with an incidence of 5% to 6% after 18 months on therapy[4][4][⇓][5]-[6

High-level BCL2 expression is seen in most patients with chronic lymphocytic leukemia (CLL) in the absence of BCL2 chromosomal translocation. A single nucleotide polymorphism (SNP; −938C>A) within an inhibitory region of the BCL2 promoter has been reported to regulate BCL2 protein expression and to be associated with adverse prognostic features in CLL. We screened 276 patients with CLL for this SNP and 100 patients by quantitative Western blot for BCL2 expression. In contrast to the previous report, we found no association with BCL2 protein levels or with any clinical or laboratory parameters. BCL2 protein levels remained constant in 10 individual patients at different time points. A total of 19 patients with the lowest levels of BCL2 protein expression were biologically and clinically heterogeneous; 5 patients exhibited high-level BCL2 RNA expression and 4 were fludarabine resistant. BCL2 protein levels in CLL reflect a complex interplay of transcriptional and posttranscriptional controls, but do not appear to be associated with the −938C>A promoter SNP.

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse–polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5′ or 3′ of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)–PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Whilst the spectrum and clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies is well established, their incidence, biological and clinical importance in splenic marginal zone lymphoma (SMZL) remains uncertain. Accordingly we screened 175 well-characterised SMZL cases for mutations in 768 genes (Haloplex Target Enrichment System) with a known or postulated role in B-cell physiology, B-cell malignancies in general and SMZL pathophysiology in particular. After sequencing (HiSeq 2000) we achieved a mean depth across our gene panel of 297-fold (range 128-702x), with more than 85% of all bases covered at >50-fold. After comprehensive filtering, 1,374 single nucleotide variants and insertions/deletions were identified. 221 genes were recurrently mutated at a gene frequency of 2-16% [n=2-28]. Sanger sequencing confirmed 86/86 selected variants in our recurrent genes, and showed 99% concordance between our Haloplex and Sanger sequencing of NOTCH2 exon 34, which was performed in all patients. Comprehensive validation of both germ-line Haloplex [n=18 patients] and Sanger sequencing established the sensitivity and specificity of our approach, and confirmed the biological importance and somatic origin of the genes described herein. To identify biologically relevant genes, we employed MutSigCV analysis, an algorithm that identifies significantly mutated genes by accounting for background mutation rate, DNA replication time and the gene size. 18 mutated genes were identified with TP53 [n=28], KLF2 [n=21], MYD88 [n=12], NOTCH2 [n=17], TNFAIP3 [n=13] and CCND3 [n=15] being the most significant; genes that encode components of pathways important in the regulation and differentiation of mature B-cells were also identified, including CREBBP [n=9], MAP3K6 [n=5], KDM2B [n=7], SETD1B [n=6], TRAF3 [n=9], ARID1A [n=10], BIRC3 [n=3], BCL10 [n=5], BTG1 [n=3], ATM [n=10], NFKBIE [n=4] and DDI1 [n=4]. Then, we searched for significant pairwise gene correlations and mutually exclusive relationships between our mutated genes demonstrating the following: (1) independent events, such as MYD88, where a mutation is invariably observed as an isolated event; (2) cancer drivers that have a similar proportion of co-occurring and mutually-exclusive relationships, such as NOTCH2, TP53, TNFAIP3 and CREBBP, and (3) genes such as KLF2, CCND3 and ARID1A that have proportionally more co-occurring relationships, thus suggesting a synergistic function to promote tumorigenesis. Finally, we studied clonal evolution, by differentiating between early, clonal events, and later, subclonal mutations (ABSOLUTE algorithm), and we were able to classify clonal or subclonal mutation in 6/18 of our MutSigCV genes. Paradigmatically, we observed that all the CREBBPmutations were fully clonal. Amongst our most novel findings was KLF2, or Krüppel-like factor 2, mutations that were distributed across the entire protein, with a cluster in the C2H2 domain and were all somatically acquired. All mutations tested were clonal, significantly associated with del(7q) (P=0.001), IGHV1-2*04 gene usage (P Genes associated with reduced time to first treatment (TTFT) included KLF2 (HR 1.93, 95%CI 1.16-3.32, p=0.01), and NOTCH2 (HR 2.13, 95%CI1.26-3.58, p=0.003). TP53 mutations were associated with shorter event-free (EFS) and overall survival (OS) (HR 2.17, 95%CI 1-4.74, p=0.05 and HR 2.16, 95%CI 1.05-4.42, p=0.032, respectively). Using multivariate Cox proportional hazard analysis, which included base-line clinical varaibles, both NOTCH2 (HR 2.12, 95%CI 1.02-4.4, p=0.044) and 100% germline IGHV gene identify (HR 2.19, 95%CI 1.05-4.55, p=0.036) were independent risk factors for TTFT. Furthermore, the presence of TP53 mutation was an independent risk factor for OS (HR 2.16, 95%CI 1.05-4.43, p=0.032). In summary, we have defined the mutational landscape in a large cohort of patients with SMZL and identified novel recurrent mutations, especially involving KLF2. Importantly we show for the first time, that gene mutations and immunogenetic features have independant prognostic significance. Disclosures Anagnostopoulos: Gilead Sciences: Research Funding. Fazi:Rhizen Pharmaceuticals SA: Research Funding. Ghia:Merck: Consultancy; GSK, Roche Italia: Consultancy; Gilead, Pharmacyclics, Boehringer Ingelheim, Celgene, Roche Italia: Membership on an entity's Board of Directors or advisory committees.

SF3B1 is a critical component of the RNA splicing machinery that achieves successful transcription and provides the functional diversity of protein species through alternative splicing. Recent studies have identified SF3B1 mutations in several tumours. In chronic lymphocytic leukemia (CLL) these mutations are associated with altered splicing, reduced survival and resistance to treatment. We therefore hypothesised that inhibition of the spliceosome may be an effective therapeutic strategy in SF3B1 mutated individuals. Initially, we utilized Spliceostatin A (SSA), an agent known to inhibit SF3B1 and alter gene splicing in various cell types. We showed that in CLL cells (SF3B1 WT and mutated samples), SSA treatment resulted in a dose-dependent increase in the un-spliced mRNA of DNAJB1 and RIOK3, two genes known to be regulated by SF3B1. We then assessed the ability of SSA to kill CLL cells with an SF3B1 mutant (n=12) or wild-type (n=28) genotype over a 24 hour period. CLL cells, regardless of SF3B1 mutational status, underwent apoptosis in a dose- (Figure A) and time-dependent manner (data not shown), with a mean IC50 of 5.1 nM. Interestingly, SSA induced more apoptosis in IGHV unmutated-CLL (U-CLL) compared to mutated-CLL samples (M-CLL) (p=0.037) and was active in 17p-deleted CLL (n=7), an aggressive disease sub-type that exhibits pronounced resistance to standard treatments. Normal B and T cells were significantly more resistant to SSA treatment than CLL samples purified from age-matched normal donors (B-cells p=0.006, T-cells p=0.001) and CLL derived T cells (p=0.003). SSA-induced apoptosis proceeded via a caspase-dependent mechanism (inhibited by ZVAD.FMK) coincident with elevations in Noxa. No changes in Bcl-2 or Bcl-xL were observed. Given the known ability of Noxa to interact with, and destabilise, Mcl-1, we next assessed the ability of SSA to modulate Mcl-1, and observed a substantial decrease in protein expression (Figure B, p=0.028). SSA treatment also altered the splicing of MCL-1; reducing the proportion of full length MCL-1L and increasing the amount of MCL-1s transcripts produced. The key role of Mcl-1 in regulating SSA-induced apoptosis was also observed in Ramos cells where over-expression of Mcl-1L, significantly protected them from SSA-induced apoptosis (p=0.001). Furthermore, in CLL cells exposed to CD40L and IL-4, factors known to induce Mcl-1 expression, SSA-induced apoptosis was significantly reduced (p=0.008). Taken together, these data implicate aberrant Mcl-1 splicing and Noxa upregulation in the apoptosis induced after SSA exposure. CD40L and IL-4 are proposed to be key factors preventing the apoptosis of CLL cells in the proliferative niche. Therefore, to explore possible strategies for overcoming this resistance, we investigated combining SSA with the Bcl-2/Bcl-xL inhibitor ABT-263 and showed that dual exposure to both inhibitors significantly increased apoptosis overcoming the protective effects of IL-4/CD40L (p=0.004; Figure C). In conclusion, our data shows that SSA induces apoptosis at nanomolar concentrations in CLL cells, independently of SF3B1 mutational status, through effects on Mcl-1. In combination with ABT-263, we show that SSA is particular active and can overcome the protective effects of CD40L and IL-4, suggesting promising clinical utility. Figure Legend. (A) Annexin V /PI analysis of CLL cells treated with SSA, (B) CLL cells treated with SSA for 24 h followed by immunoblotting for various proteins. (C) Drug combination studies with ABT-263 and SSA at a 1:1 ratio at 10 nM or 20 nM following IL-4/CD40L treatment. Figure Legend. (A) Annexin V /PI analysis of CLL cells treated with SSA, (B) CLL cells treated with SSA for 24 h followed by immunoblotting for various proteins. (C) Drug combination studies with ABT-263 and SSA at a 1:1 ratio at 10 nM or 20 nM following IL-4/CD40L treatment. Disclosures No relevant conflicts of interest to declare.

Background The JAK2 V617F mutation is present in almost 95% of patients with Polycythemia Vera (PV). Prior to the discovery of this mutation, red cell mass and plasma volume measurement to establish the presence of true erythrocytosis was required first, before the diagnosis could be confirmed using other criteria. The revised WHO criteria no longer mandate this. Besides, access to this test is restricted in our region, due to non-availability of radio-isotopes. We previously showed the utility of early JAK2 mutation screening when used with history, physical examination, oxygen saturation, complete blood count, blood film examination, splenomegaly, serum erythropoietin, phlebotomy trial, bone marrow biopsy and, when available, red cell mass studies. Patients Between 2002 and 2006 we screened 231 consecutive patients referred to our institution with raised hemoglobin and hematocrit for JAK2 mutation. PV was diagnosed in 27 patients. Polycythemia of unknown cause (idiopathic) was identified in 40 patients after excluding patients with transient polycythemia and secondary polycythemia due to hypoxia, sleep apnoea, excessive smoking, alcohol abuse, renal disease, androgens or inherited causes. All PV patients were assessed at 3-6 monthly intervals from diagnosis until death or to present day. All idiopathic polycythemia patients were assessed at 6-12 monthly intervals until discharge, death or to present day. Patients with idiopathic polycythemia were discharged from follow-up when their hematocrit returned to normal range without any interventions for at least 12 months. All patients received thromboprophylaxis with aspirin or vitamin K antagonists as appropriate unless contra-indicated. Results Of 27 PV patients, 15 were males and 13 females. The median age at diagnosis was 69 years (range 21-88 years). Leucocytosis was present at diagnosis in 14 and thrombocytosis in 15 patients. Patients were treated with phlebotomy to a target hematocrit Conclusion Disease progression occurred in 11% (3/27) and thrombosis in 33% (9/27) of patients with PV. Idiopathic polycythemia was associated with thrombosis in 37% (15/40) of patients. Our findings indicate that thrombosis occurs with equal or higher frequency in idiopathic group when compared to PV group. These patients require a stricter target hematocrit control to Disclosures: Chacko: Amgen : Membership on an entity’s Board of Directors or advisory committees; GSK: Membership on an entity’s Board of Directors or advisory committees. Killick:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.

Most cases of CLL express BCL2 constitutively at levels comparable to those seen in follicular lymphoma with t(14;18)(q32;q21) and BCL2 is considered to play a pivotal role in the suppression of apoptosis in this disease. The BCL2 antagonist ABT-263 has recently entered phase I/IIa clinical trials for potential treatment of CLL. ABT-263 and ABT-737 are rationally designed BH3 mimetics that are thought to act by displacing pro-apoptotic BIM from BCL2 and thus activating the apoptotic cascade. While being resistant towards some commonly used apoptotic stimuli like etoposide or TRAIL, we found that all primary CLL cells are highly and rapidly sensitive to ABT-737, with an average EC50 in 60 patients of only 6.7 nM and after only 4 h of treatment. ABT-737 induced BAX/BAK oligomerization followed by rapid and extensive cytochrome c release, caspase activation and chromatin condensation. Incubating unfractionated samples in a whole blood assay did not significantly alter sensitivity. Notably, sensitivity of CLL cells to ABT-737 was independent of the patient’s stage of disease, p53 mutation or function, cytogenetics, or IGVH mutational status. Furthermore, ABT-737-induced apoptosis was observed irrespective of previous treatment or in vivo resistance to fludarabine, suggesting that ABT-737 might induce apoptosis even in patients with highly progressive and otherwise resistant disease. We also analyzed expression levels of BCL2 and MCL1 in CLL patients by western blot, since MCL1 is considered to be the most important factor for resistance towards ABT-737. Interestingly, sensitivity to ABT-737 was independent of BCL2 and MCL1 levels in CLL, indicating that expression of MCL1 was not sufficient to render CLL resistant to ABT-737. In addition, we further induced expression of MCL1 by either IL4 or interferon γ treatment, which resulted only in a minor delay of apoptosis that was easily overcome by treatment with slightly higher concentrations of ABT-737. Hence, our data suggest that in contrast to previous reports obtained in a variety of cell lines, MCL1 upregulation is not sufficient to induce resistance to ABT-737 in primary CLL cells. Next, we investigated whether exposure to ABT-737 resulted in a displacement of BH3-only proteins from BCL2. Surprisingly, we could not detect a significant displacement of BIM from BCL2. In contrast, we found that the multidomain proapoptotic protein BAK is directly bound to BCL2 in CLL and this interaction is abrogated by ABT-737, indicating that ABT-737 can directly displace BAK but not BIM from BCL2. Taken together, our results indicate that targeting BCL2 with ABT-263 or ABT-737 may be a highly promising strategy for treatment of CLL.

Abnormalities of the ATM and p53 genes can be detected by interphase FISH, mutational analysis or functional assays. The latter measure up-regulation of p53 or activation of downstream targets following induction of dsDNA breaks. To assess functional integrity of the TP53/ATM pathway we have exposed CLL cells with defined abnormalities of both ATM and TP53 to etoposide alone and to etoposide + an MDM2 inhibitor, nutlin 3 that up-regulates p53/p21 expression in cells with wt p53. Leukemic cells from 98 patients previously screened by FISH for TP53 and ATM loss, del 13q14 and +12 were incubated with etoposide alone. 68 cases were functionally normal; of these, 8 had ATM loss and 6 had TP53 loss. 9 patients had high basal p53 expression and did not up-regulate p53/p21 (type A dysfunction); 7 of these had TP53 loss, none had ATM loss. 20 patients with low basal p53 expression failed to up-regulate p53/p21 (type B dysfunction); of these, 14 had ATM loss and 5 had TP53 loss. 1 case showed a normal p53 response but did not show any p21 up-regulation (type C dysfunction) consistent with a p21 polymorphism. 46 cases were selected for analysis using etoposide/nutlin based on their ATM and TP53 loss and mutational status; see table for summary. 20 of these 46 were cases showing type B dysfunction. Exposure of the 20 type B dysfunctional cases to etoposide/nutlin resulted in normal up-regulation of p53/p21 in cases with ATM loss but not in those with TP53 loss. The expression levels of p53/p21 following exposure to etoposide and nutlin led us to redefine the criteria for identifying the type A and B groups (see table). The assay detected type A or B dysfunction in all 17 cases with 11q/17p loss with associated ATM/TP53 mutation respectively. However, no dysfunction was detected in all 11 patients with either ATM or TP53 loss and no mutation of the remaining allele. In contrast 3/6 patients with TP53 and 3/3 with ATM mutations without associated chromosomal loss were functionally abnormal, suggesting a dominant negative effect of the mutant clone. TP53 loss clone size by FISH was >48% in cases with TP53 mutations and Category No of Patients Function No Loss No Mutation 6 All Normal p53 Loss no mutation 5 All normal Loss and mutation 8 All type A No Loss with mutation 6 Normal, 3 type A ATM Loss no mutation 6 All normal Loss and mutation 9 All type B No Loss with mutation 3 All type B

Background: The doses and timing necessary for effective cytoreduction with monoclonal antibodies remain largely unknown. The human IgG1 CD52 MAb alemtuzumab is normally given intravenously (IV) three times per week at a standard dose of 30mg, following dose escalation. However, comparable results may be observed with fewer side effects using subcutaneously (SC) administered MAb, although the pharmacokinetics of SC administered MAb are slower than IV and therapy may have to be administered on a thrice weekly schedule for up to 18 weeks to achieve maximal efficacy. Efficacy with alemtuzumab has been shown to relate directly to serum levels of the MAb. We wished to assess whether more rapid cytoreduction with acceptable toxicities might be attained with daily alemtuzumab dosing without dose escalation. Patients and methods: Patients with CLL (either as first-line therapy or at first or subsequent relapse) received SC alemtuzumab for 15 consecutive days at a dose of 30mg daily and thereafter three times per week for up to 8 weeks. To minimize adverse skin and possible systemic reactions, dexamethasone (12mg) was given along with acetaminophen (1g) for the first two injections only. Results: 7 patients (aged 55– 73, 6 male 1 female) have been treated comprising 3 patients with Binet stage A progressive and 4 stage C. Use of steroid premedication for the first two doses virtually abolished all acute toxicities with only one patient showing any significant cutaneous reaction. CMV PCR was performed weekly. 4 patients showed low-level CMV (up to 7000 viral copies/ml) reactivation in the absence of any symptoms. All were treated successfully with oral valganciclovir 900mg bd whilst continuing with alemtuzumab without interruption. All 7 patients showed rapid reductions of peripheral blood lymphocytes as shown in the figure. Rates of drop in lymphocye count were much more rapid with daily than with conventional thrice weekly dosing schedules. Despite the more rapid reductions in lymphocytes, no patient developed tumor lysis. All patients showed significant reduction in bone marrow infiltration after the 15 days of daily therapy although a further 8 weeks of therapy was necessary to induce CR. Conclusions; these data indicate that daily SC dosing with alemtuzumab results in rapid cytoreduction without increased adverse events. Prolonged daily dosing may result in enhanced efficacy of alemtuzumab in CLL. Figure Figure

Bortezomib (PS-341/Velcade™) is a reversible inhibitor of the proteasome that has shown promising activity in clinical trials in several malignancies including multiple myeloma, mantle cell lymphoma and follicular lymphoma, including those with refractory disease. However, results have been less encouraging in chronic lymphocytic leukemia (CLL) and we have, therefore, sought to determine the barriers to effective therapy with bortezomib in this disease. Patients with CLL were eligible but were required to have received no therapy in the six months prior to the study. In a panel of 26 patients with CLL, both purified mononuclear cells and whole blood were tested for their apoptotic response to bortezomib (1–100 nM) up to 24 h by flow cytometry and western blotting. In all cases, purified CLL cells were sensitive to bortezomib-induced apoptosis in a concentration and time-dependent fashion, irrespective of stage of disease, resistance to prior therapy, IGHV mutational status or the presence of TP53 mutations. Apoptosis was induced at low (>10 nM) nanomolar concentrations of bortezomib by activation of the intrinsic apoptotic pathway. Bortezomib-induced apoptosis correlated with levels of ubiquitination, Bax activation, and caspase cleavage. Apoptosis of CLL cells was obtained at drug levels readily obtained in vivo using currently-used dosing protocols. However, in vitro, it was necessary to maintain these concentrations for 16–24 hours to obtain maximal apoptosis. Apoptosis measured in a whole blood apoptosis assay was markedly less than in isolated lymphocytes at comparable time points and concentrations. Activity of bortezomib in purified cells was not diminished by addition of exogenous plasma but was abrogated by addition of autologous red blood cells (RBC), suggesting preferential active uptake of the drug by these cells. These data were confirmed in animal models showing preferential distribution of bortezomib to the RBC fraction. RBC uptake may therefore account for the low serum levels of bortezomib attained in vivo during terminal half-life and thus the lack of activity against cells in the peripheral blood. Together with pharmacokinetic and in vivo data, these studies suggest that different dosing schedules of bortezomib other than bolus injections may be more effective in patients with CLL.

The B-cell lymphoma/leukaemia 11A (BCL11A) gene was first identified in the rare t(2;14)(p16;q32.3) translocation involving the IGH locus in aggressive B-cell chronic lymphocytic leukaemia (CLL) and, together with REL, is a candidate for the 2p16 chromosomal amplifications in B-cell malignancies. BCL11A encodes a Kruppel zinc finger transcription factor essential for pre-B-cell development and thymocyte maturation. Alternative RNA splicing generates at least three BCL11A transcripts encoding proteins that share a common N-terminus with the BCL11AXL transcript being the most abundant BCL11A transcript in normal tissues. We have produced a murine monoclonal antibody (BCL11A/123, isotype IgG1) specific for the BCL11AXL protein and, using this reagent, provide the first description of BCL11AXL protein distribution in normal and malignant human tissues. Initial studies using another antibody to the common N-terminus indicated the BCL11AXL protein to be the dominant isoform. In lymph node, tonsil and spleen, BCL11AXL expression was detected in B-cell nuclei in the mantle zone, germinal centers, interfollicular areas, splenic marginal zones and in tonsillar epithelium, but not in plasma cells or T cells. The differential expression of BCL11AXL in B-cell populations suggests its downregulation might be a requirement for plasma cell differentiation and is consistent with reports that BCL11A is transcriptionally repressed by Blimp-1. A subpopulation of cells, mostly B-cells, in the thymic medulla and scattered cells in the cortex were BCL11AXL+. Labeling of a very small number of CD3+ T-cells in the cortex and epithelial cells around the cortical periphery was also observed. Labeling of B-cell follicles in rat spleen demonstrated the BCL11A/123 epitope to be conserved across species. Another important finding was the high level expression of BCL11AXL in CD74+CD68+CD123+ plasmacytoid dendritic cells (pDCs). Publicly available microarray expression data are consistent with these findings, showing BCL11A transcripts to be present at high levels in peripheral blood B cells, in tonsil, lymph node and fetal brain with the highest expression in pDCs. BCL11AXL therefore represents an additional marker for the identification of pDCs and may be important in the differentiation and/or functional activity of this cell type. BCL11AXL was commonly detected in B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia and a subgroup of Hodgkin’s lymphomas, with the exception of multiple myeloma. Tumor cells in a small number of T-cell lymphomas (3/29) also expressed BCL11AXL. In a tissue microarray, comprising 107 cases of de novo DLBCL at diagnosis, BCL11AXL expression correlated with that of its interaction partner BCL6 and showed a trend towards increased overall survival. The study of this molecule should provide additional invaluable information concerning the possible role of BCL11A in lymphomagenesis.

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We proposed that semi-autologous myeloma hybrid cells, formed by fusing malignant plasma cells with the human lymphoblastoid cell line, HMy2, would generate immortalised cell lines with enhanced immunogenicity for T cell responses, which could be used as potential therapeutic vaccines. The specific goals of the project were: to generate stable hybrid cell lines in vitro, by fusion of HMy2 with ex vivo tumour cells from patients with MM; to characterise the hybrid cells with respect to growth in culture, stability, and surface marker phenotype; and to investigate the ability of the hybrid cells to stimulate T cell immune responses in vitro as compared with parental tumour cells. The parent HMy2 cell line was fused with ex vivo myeloma plasma cells (purified from the marrow using CD138+ magnetic beads) from 5 patients, and chemically selected in HAT medium (to which HMy2 is sensitive), to form stable cell lines that grew continuously in culture. Efforts of growing myeloma cell lines from all of the patients were unsuccessful. The hybrid status of 4 of the 5 fused cell lines was confirmed by microsatellite testing using polymorphic loci on chromosomes X, 16q, 7q, 13q, 5q, 2p, 11p, 12p. Furthermore, the microsatellite analysis was checked after 3 months of continuous culture (approximately 40 passages), and all of the hybrids remained genetically stable over this period. Hybrid cells were analysed for expression of immunologically relevant accessory molecules using flow cytometry, in comparison with parent tumour cells and HMy2. The hybrids retained CD138 expression, although expression of CD56 was reduced in hybrids from two of three CD56+ myeloma cases. CD54 (ICAM-1) and CD50 (ICAM-3) were uniformly expressed in all hybrids. CD58 (LFA-3) was expressed more strongly in the hybrids than in original tumour cells, but CD11a (LFA-1) was weakly expressed or absent, in line with expression on HMy2 cells. All of the hybrids expressed CD40, CD80 (B7-1) and CD86 (B7-2), although CD80 was weakly or not expressed by myeloma plasma cells. Both MHC class I and II were expressed in all the hybrids, suggesting the potential for antigen presentation to both CD8+ and CD4+ T cells, in contrast to the majority of myeloma plasma cells, which failed to express MHC class II. The hybrid cells therefore expressed important markers to facilitate effective antitumour responses, which were absent on the parent tumour cells. The hybrid cells stimulated allogeneic T cell proliferative (3H thymidine uptake) responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, which failed to induce T cell proliferation above background levels. In addition, T cells from patients from whom hybrid cell lines had been derived responded to stimulation with both autologous and allogeneic hybrid cell lines, whilst failing to proliferate in response to autologous or allogeneic myeloma plasma cells. These data indicate that myeloma patients are immunocompetent, suggesting that active immunisation with semi-autologous hybrid cell lines represents a valid approach to immunotherapy in these patients. We have shown the generation of myeloma hybrid cell lines that grow continuously in tissue culture and have increased immunogenicity for T cell responses in vitro. This approach represents a novel strategy for the development of therapeutic vaccines for the treatment of multiple myeloma.