ALBERT

Description: CLL develops from a small fraction of dividing monoclonal CD5+ B cells. The size and rate of growth of this proliferative fraction (PF) correlates inversely with time-to-first-treatment and directly with poor outcome prognostic markers. Furthermore, since the dividing cells upregulate DNA mutators such as AID and APOBEC family members, the PF has a greater propensity for acquiring new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy for patients with worst outcome category. The PF (CXCR4DimCD5Bright) differs by more than 1000 genes from the resting fraction (RF, CXCR4Bright CD5Dim); these genes relate to replication, migration, and regulation of gene expression. Some of these genes are also preferentially expressed in the PF of U-CLL cases. One such gene is Musashi 2 (MSI2). MSI2 regulates gene expression by binding consensus sequences of mRNA and blocking protein translation. High MSI2 expression is involved in proliferation of normal and malignant stem cells, tumorigenesis, and poor outcome. In CLL, high MSI2 mRNA expression has been identified in patients with worse prognosis. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we report studies of the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression. First, we evaluated MSI2 protein levels by flow cytometry in CD19+CD5- and CD19+CD5+ cells from healthy donors (HDs; n=25) and in CD19+CD5+ from CLL patients (n=55). Higher MSI2 expression was observed in CLL than HD B cells, whereas no differences were found in CD19+CD5+ and CD19+CD5- cells from HDs. Also, MSI2 protein levels were higher in U-CLL than M-CLL, and M-CLL B cells express more MSI2 than HDs. Finally, MSI2 protein levels correlated with CD38, a CLL poor prognosis marker, suggesting MSI2 associates with poor prognosis in CLL. Within the leukemic clone, we observed 25% more MSI2 in the PF than the Int (defined as CXCR4intCD5int) and 15% more in the Int than the RF (PF〉Int 〉RF). The PF contains 40% more MSI2 than the RF, suggesting the highest amounts of MSI2 protein are in dividing and recently-divided cells. Since CLL B cell proliferation occurs in the microenvironment of lymphoid organs, presumably delivered by external signals, we tested whether such signals could stimulate MSI2 expression. Results indicate that CD40L+IL4 and Toll-like 9 stimulation plus IL15 (TLR9+IL5) increase MSI2 synthesis in vitro 1.4 and 1.8 fold, respectively. The increases are associated with the appearance of phospho ERK and AKT. Also, inhibition of AKT signaling by a PI3K inhibitor decreases MSI2 levels, suggesting AKT is involved in MSI2 synthesis. In this regard, signals from the microenvironment inducing cell growth and proliferation promote MSI2 synthesis in B cells from CLL patients. In addition, cells entering the cell cycle (Ki-67+ cells, those incorporating the thymidine analogue EdU, and cells in S, G2 and M cell cycle phases) express higher MSI2 levels than quiescent cells. Furthermore, dividing cells contain higher MSI2 levels than non-dividing cells as determined by CFSE dilution. These results suggest that cells entering the cell cycle or recently dividing have greater MSI2 expression. Since high MSI2 levels associate with cell proliferation and its inhibition is said to promote apoptosis, we studied the effect of MSI2 downregulation in the CLL MEC1 cell line to determine if MSI2 is a potential therapeutic target for CLL. Our findings show that siRNAs decrease MSI2 mRNA (80%) and protein (40%) levels compared to negative controls. Downregulation of MSI2 in MEC1 led to cleaved caspase 3, TRAIL R1 and R2, FADD, TNFR1, P21, P27, phosho-p53, and decreased levels of inhibitors of apoptosis such as cIAP2 and survivin. Hence these data suggest downregulation of MSI2 in CLL cells could induce apoptosis. Thus, MSI2 levels are higher in B cells from poor outcome patients and also in the dividing/divided cells of the PF before and after stimulation. Also, MSI's downregulation induces apoptosis of CLL cell line. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention. Inhibiting its function and its role in cell proliferation will likely abort clonal evolution and disease progression, and make CLL an even more chronic and manageable condition. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: PI3Kδ signaling is critical for the proliferation, survival and homing/tissue retention of malignant B cells. Idelalisib is a first-in-class, highly selective, oral inhibitor of PI3Kδ recently approved for the treatment of relapsed CLL in combination with R. This report summarizes the long-term follow-up of the Phase 1 combination experience of idelalisib with anti-CD20 antibodies. Methods: This Phase 1 study evaluated idelalisib for relapsed/refractory CLL continuously given at 100 mg BID (4 of the pts receiving R) or 150 mg BID (all other pts) in combination with a total of 8 infusions of rituximab (R, 375 mg/m2 weekly x 8), or a total of 12 infusions of ofatumumab (O, 300mg initial dose either on Day 1 or Day 2 relative to the first dose of idelalisib, then 1,000 mg weekly x 7, then 1,000 mg every 4 wks x 4). Pts on treatment after 48 weeks were eligible to continue idelalisib on an extension study. Clinical response was evaluated according to published criteria (Hallek 2008; Cheson 2012). Results: 40 pts (12F/28M) with a median (range) age of 66 (43-87) years and a WHO performance status of 0 (24, 60%) or 1 (16, 40%) were enrolled. 19 pts received idelalisib in combination with R and 21 with O. Adverse disease characteristics (n, %) included Rai Stage III/IV (20, 50%), bulky lymphadenopathy (23, 58%), refractory disease (15, 38%), multiple prior therapies (median 2, range: 1-9). Almost all pts (39, 98%) had at least 1 prior therapy containing R, and 3 of the 21 pts (14%) receiving idelalisib + O had received prior O. 63% of the pts receiving idelalisib + R, and 48% of the pts receiving idelalisib + O were refractory to R. Prior therapies also included alkylating agents (31, 78%, [bendamustine: 20, 50%]) and purine analogs (31, 78%, [fludarabine: 28, 70%]). Data available from 39 pts showed that 11 (28%) pts had evidence of del(17p) and/or TP53 mutations and 30 (75%) had unmutated IGHV. As of 7/15/2014, the median (range) treatment duration was 18 (0-44) months. 23 (58%) pts have completed the primary study and enrolled into the extension study. Primary reasons for study discontinuation (as reported by investigators) included disease progression (14, 35%), adverse events (AEs) (12, 30%), investigator request (3, 8%), withdrawal of consent (n=1), BMT (n=1). There were a total of 8 deaths on study: 2 deaths occurred after disease progression, and 6 pts died because of AEs (all assessed as unrelated/unlikely related to idelalisib by investigators). A total of 4 pts (10%) were continuing idelalisib treatment on the extension study at time of analysis. Selected treatment-emergent AEs (any Grade/≥Gr 3, regardless of causality) included diarrhea/colitis (55%/23%), cough (40%/3%), pyrexia (40%/3%), dyspnea (30%/3%), fatigue (25%/0%) nausea (25%/0%), rash (20%/0%), pneumonia (20%/18%), and pneumonitis (8%/5%). Elevation of liver transaminases (TA, any Grade/≥Gr 3) was seen in 30%/10%. Re-exposure to idelalisib after resolution of TA elevation generally was successful; only 1 patient discontinued the study because of (recurrent) TA elevation. Other AEs leading to study discontinuation and reported as possibly/probably related to idelalisib included diarrhea/colitis (4, 10%), pyrexia (n=1), interstitial lung disease (n=1), pneumonia (n=1), rash (n=1), psoriasis (n=1). Secondary malignancies leading to discontinuation (all reported as unrelated) were breast cancer (n=1), recurrent colon cancer (n=1), AML (n=1). There was no obvious overall difference in the toxicity reported for pts receiving idelalisib with rituximab compared to those with ofatumumab. The ORR (N=40) was 83% (33/40), with 2 CRs (5%) reported. Median PFS (N=40) and duration of response (DOR) (n=33) were 24 months. Median (range) time to response was 1.9 (range 1.7-16.9) months. Median overall survival (OS) has not been reached with a KM estimate for OS of 80% at 24 months. For the 11 pts with del(17p) and/or TP53 mutations, the response rate was 73%, and the median PFS and DOR were 20 and 24 months, respectively. Conclusions: Combinations of idelalisib with anti-CD20 antibodies such as R or O represent non-cytotoxic regimens with acceptable safety profiles and considerable activity resulting in durable tumor control in pts with relapsed/refractory CLL, including those with high risk factors such as del(17p) or TP53 mutations. A Phase 3 trial evaluating the efficacy of idelalisib in combination with ofatumumab is ongoing (NCT01659021). Disclosures Furman: Gilead Sciences: Research Funding. Off Label Use: Zydelig is a kinase inhibitor indicated for the treatment of patients with: 1) Relapsed chronic lymphocytic leukemia (CLL), in combination with rituximab, in patients for whom rituximab alone would be considered appropriate therapy due to other co-morbidities; 2) Relapsed follicular B-cell non-Hodgkin lymphoma (FL) in patients who have received at least two prior systemic therapies; and 3) Relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies.. de Vos:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Boyd:Gilead Sciences: Research Funding. Fowler:Gilead Sciences: Research Funding. Leonard:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Kim:Gilead Sciences: Employment, Equity Ownership. Viggiano:Gilead Sciences: Employment, Equity Ownership. Jahn:Gilead Sciences: Employment, Equity Ownership. Coutre:Gilead Sciences: Research Funding.

Description: Targeted ultra-deep sequencing of chronic lymphocytic leukemia (CLL) cells has enabled the assessment of subclone development based on mutations in the IGHV-D-J signature sequence in the dominant CLL clone. We have utilized the Roche 454 FLX pyrosequencing system, which can generate long sequencing reads containing both the immunoglobulin variable region (IGHV-D-J) and part of the immunoglobulin μ constant region (IGHM) in a single sequence, to analyze the mutational characteristics of newly evolved subclones to determine if they derive from AID/APOBEC activity. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) is a family of cytidine deaminases that includes AID (activation-induced cytidine deaminase). AID is required for somatic hypermutation in germinal center B lymphocytes. CLL cells, like most non-germinal center B lymphocytes, generally do not express AID. However, AID expression in a small fraction of the CLL clone correlates with worse patient outcome. This observation has led to the hypothesis that abnormal AID expression promotes new off-target non-immunoglobulin mutations and DNA deletions and rearrangements leading to the development of more aggressive disease. CLL is not alone in this hypothesis, as AID is involved in the evolution of other leukemias/lymphomas and reportedly in other types of tumors such as breast and gastrointestinal cancers. Large scale cataloguing of somatic mutations by ultra-deep sequencing of a wide array of cancers has revealed an AID/APOBEC mutational signature in many cancers, including CLL (Alexandrov et al. 2013 Nature). Thus, AID/APOBEC family members may be involved in somatic mutations leading to the evolution of aggressive cancers. To test if AID/APOBEC proteins could be mutationally active in CLL, we analyzed the characteristics of new mutations found in IGHV-D-J-M in CLL cells that were activated in vivo after adoptive transfer into alymphoid NOD/Shi-scid,γcnull (NSG) mice. This CLL xenograft model activates a large fraction of CLL cells, which become AID+ and facilitates the detection of new subclones expressing mutated IGHV-D-Js by ultra-deep sequencing. Four unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (〉2% compared to germline) CLL (M-CLL) samples were each adoptively transferred into individual NSG mice. After expansion of CLL, the mice were sacrificed and the specific CLL clone IGHV-D-J-M was amplified from xenograft mouse spleen cDNA. Pre-transfer CLL cell cDNA was also amplified to establish a baseline comparison. Ultra-deep sequencing of these samples resulted in 2,318,800 sequence reads, which were subsequently trimmed according to the Roche 454 algorithm and further processed by custom R scripts. The sequence reads were aligned to the dominant CLL clone IGHV-D-J-M sequence to remove insertions, deletions, and poor quality (

Description: Introduction: Venetoclax (VEN) based therapy has become a standard of care in front line and relapsed-refractory (R/R) CLL based on favorable efficacy and toxicity. Whereas prospective data regarding activity of therapies following ibrutinib (IBR) or idelalisib (IDE) are available in the settings of progression (VEN, non-covalent BTKi) and intolerance (acalabrutinib), how best to manage patients (pts) who discontinue (dc) VEN remains a key unanswered question. With the increased use of VEN in early lines of therapy (LOT; CLL 14, MURANO), the activity of BTK inhibitors (BTKi) and cellular therapies following VEN becomes a critical issue. No prospective study has addressed this question, and currently reported VEN clinical trials have limited information about subsequent treatments. While recent data describe VEN resistance mechanisms (Guieze 2018, Blombery 2019), the impact of VEN resistance on efficacy of post VEN therapies is unknown. To address this gap, we conducted an international study to identify a large cohort of pts who dc VEN and have been subsequently treated. Methods: We conducted an IRB approved multicenter (31 US, EU, South American sites, in partnership with UK CLL Forum and CORE registry), retrospective cohort study of CLL pts who dc VEN for any reason. We examined demographics, dc reasons, responses, survival, adverse events (AEs) and activity of post VEN therapies. Primary endpoints were overall response rate (ORR) and progression free survival (PFS) for the post VEN treatments stratified by treatment type (BTKi, PI3Ki and cellular therapy: CAR-T or alloHSCT). ORR was defined by iwCLL criteria and PFS was defined from VEN dc to disease progression (PD), death, or last follow up for next treatment. Pts were further stratified by BTKi (resistant / intolerant) and PI3Ki exposure prior to VEN. PFS-2 was defined as time from VEN start to tumor progression on IBR or death from any cause. Results: 326 CLL pts who dc VEN in the front line (4%) and R/R settings (96%) were identified. The cohort was 69% male, 87% white, median (med) age 66 (38-91) at VEN start, 27% treated with VEN based combinations (n=88, med 6 cycles anti-CD20 abs). Pre VEN prognostic features: 82% IGHV unmutated (n tested=166), 47% del17p (n=306), 45% TP53 mut (n=217), 39% complex karyotype (n=273), 23% BTK mut (n=79), 18% NOTCH1 mut (n=103), 10% PLCγ2 mut (n=74). Pts received med 3 therapies (0-11) prior to VEN; 40% were BTKi naïve (n=130), 60% were BTKi exposed (196) and 81% were IDE naïve (n=263). Most common reasons for VEN dc were PD (38%), AE (20%), Richter's transformation (RT, 14%), 8% pt preference, and HSCT 5%. Of 326 pts who dc VEN, 188 (58%) were treated with a subsequent LOT, 61 are alive and untreated and 77 died prior to a subsequent LOT. Post VEN sequencing analyses focused on BTKi, PI3Ki and cellular therapy (CAR-T or alloHSCT) activities following VEN dc (Table1). ORR to BTKi was 84% (n=44) vs. 54% (n=30, p

Description: Although chronic lymphocytic leukemia (CLL) is an accumulative disorder of CD5+ B cells, a proliferative fraction exists and the extent of this proliferation correlates with clinical course. We previously demonstrated heterogeneous in vivo labeling kinetics of cells in 3 clonal fractions sorted based on reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and CD5 from CLL cases recruited on an in vivo labeling study. In that study, the CXCR4dimCD5br fraction contained more 2H-labeled DNA and hence recently divided cells (proliferative fraction) than the CXCR4brCD5dim(resting) fraction. Here, we have analyzed multiple CXCR4/CD5 based subclonal fractions and characterized their phenotypic differences and ability to respond to signals via the BCR and CXCR4 or a combination of the two. PBMCs from a cohort of 50 untreated IgM+ CLL cases were used for this study. Subclonal fractions marked based on differences in relative densities of CXCR4 and CD5 by CLL cells are referred to as CXCR4br, CXCR4int or CXCR4dim and CD5br, CD5int or CD5dim. Sub-populations adjacent to each other on flow cytometric plots differed at least 5 fold in the densities of both CXCR4 and CD5. When subcategorizing CLL clones based on these markers, 9 subfractions were identified. To permit comparisons between these fractions, each was defined as containing only 2-5% of the total clonal CD19+CD5+population while ensuring no overlap in CXCR4 or CD5 densities among each fraction. Interestingly, certain phenotypic and signaling features consistently clustered with distinct subfractions of the CLL clone. Subfraction analyses confirmed an enrichment of Ki-67+ cells in the CXCR4dimCD5br proliferative fraction of every CLL case. In addition, this subfraction showed the highest percentage of cells expressing smIgM and smIgD (p

Description: Abstract 315 Adoptive transfer of primary patient CLL cells into NOD/SCID/γcnull(NSG) mice results in engraftment and proliferation of CLL cells if autologous T cells are present. Formation of splenic follicles consisting of B cells interspersed and surrounded by T cells indicates engraftment. However, ultimately these CD20+ cells are lost several weeks later. We describe one of the mechanisms for this apparent loss: differentiation to plasma cells. Peripheral blood cells from 9 IgM+ CLL patients (6 U-CLL and 3 M-CLL) were adoptively transferred into NSG mice with enriched autologous CD3+ cells pre-activated with anti-CD3/28 beads. B and T cell engraftment and subset distributions were analyzed for 47 mice by immunohistochemistry (IHC) and flow cytometry (FC) at the time of sacrifice. The earliest and latest times of assessment were 12 and 124 days, respectively, after CLL cell injection. In some cases, CLL cells were labeled with CFSE to track cell division. At sacrifice, 3 engraftment patterns were observed. Pattern 1 (observed up to day 56) showed small follicles of CD20+ cells with low-moderate numbers of surrounding T cells. Intensely positive CD38 cells were inconspicuous. FC showed CD19+CD5+ cells with no increase in CD38 and variable CFSE dilution indicating lower levels of proliferation. Pattern 2 (observed throughout the study period) showed much higher T and B cell numbers. CD20+ cells were interspersed with and surrounded by principally CD4+ cells which were activated and functional as indicated by expression of Ki-67, PD-1, CD57, and T cell derived cytokines IFNγ and IL5 in plasma. Follicles contained CD20 and cytoplasmic Ig+ (cIg+) cells that double stained for IRF-4 and Blimp-1, transcription factors required for B cell differentiation. While Bcl-6 staining in these cells was minimal or absent, follicles from all 9 patients contained activation-induced deaminase (AID)+ cells. Cells with dim IgM expression localized to follicles; however, cells with intense IgM, IgA, or IgG were present both within, surrounding, and outside follicles matched by similar CD38 staining. Smaller populations of CD138+ cells surrounded follicles and were interspersed throughout non-follicular splenic areas. FC showed a novel CD19+CD5-CFSE-CD38++ population containing a CD138+ subset. Pattern 3 (observed in a limited subset of cases not before day 63) had minimal CD20+ cells by IHC, but noticeable populations of cIg+CD38+ and CD138+ cells interspersed amongst plentiful T cells. Such cells corresponded with cells with plasma cell morphology. Confirmation that differentiated cells were from the patient clone was achieved in 3 ways. First, in FACS sorted CD19+CD5+ and CD19+CD5-38++ cells from a subset of pattern 2 cases, RT-PCR revealed that all fractions contained both IGHC unswitched and switched clones identical to those found in the patients. Second, cases with pattern 3 engraftment generated CLL clonal switched and unswitched cDNA sequences. Finally, adoptive transfer of highly purified CD5+CD19+ patient cells generated IRF-4+Blimp-1+CD138+ cells. The generation of switched cells from all 9 patients indicated functional AID. In one U- CLL case, ultra-deep sequencing on pre-transfer and post-transfer human cells taken from mouse spleen revealed a significant number of new IGHVDJ mutations in spleen-derived cells. Such mutations targeted nucleotides typical for AID's action. In conclusion, CLL cells can diversify, switch, and differentiate in NSG mice in response to autologous T cell signals. The extent of this maturation is a function of T cell numbers and activity and the duration of the experiment. Differentiation without significant Bcl-6 expression suggests that follicles in NSG mice are not recapitulating classic germinal center reactions, possibly giving clues to the origin of CLL. Several features of poor prognosis disease were demonstrated (e.g., increased CD38 and AID expression with the development of clonally related switched transcripts) that might mirror clinical disease features. AID expressed by CLL cells is fully functional as indicated by de novo somatic hypermutation and class switch recombination. Both U-CLL and M-CLL clones respond in a similar manner in this model, suggesting the importance of T– B cell interactions in all types of CLL. Finally, the demonstration that cells can differentiate when appropriately induced may lead to novel therapeutic options for CLL. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most common lymphoid malignancies, accounting for ~11% of all hematologic neoplasms. Over the last 15 years, a series of phase 3 trials have established that chemoimmunotherapy (CIT) with fludarabine, cyclophosphamide, and rituximab (FCR) improves both progression free survival (PFS) and overall survival (OS) compared with chemotherapy alone. FCR is the gold standard for young fit patients with treatment naïve CLL. In parallel with the advances in CIT, a profound increase in the understanding of CLL B-cell biology led to new therapeutic approaches.1 Among these, ibrutinib (an irreversible inhibitor of Bruton's Tyrosine Kinase [BTK]) has had the largest impact on clinical practice to date. Initial trials of ibrutinib demonstrated robust and durable efficacy in patients with relapsed/refractory disease. Subsequent phase 3 trials showed improved PFS and OS with ibrutinib relative to chlorambucil in previously untreated, older CLL patients. Despite these advances, the efficacy of ibrutinib as a first-line treatment for younger CLL patients (i.e.

Description: Introduction: Venetoclax (VEN) is a selective, orally bioavailable BCL-2 inhibitor. This is a phase 1b, study of VEN plus rituximab (R) to determine safety, PK and preliminary efficacy in patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL). The recommended phase 2 dose for VEN plus R was previously reported to be 400mg/day. The aims of this analysis were to evaluate progression-free survival (PFS), overall survival (OS), and the durability of responses off all therapy in pts achieving CR. We also assessed minimal residual disease (MRD) status, which has previously been shown to be strongly predictive of PFS and OS after chemoimmunotherapy. Methods: Pts began once daily VEN (20 or 50 mg) to final cohort doses (200-600 mg/day) followed by R, given every 4 weeks for a total of 6 doses. VEN dosing was continuous. Responses were assessed by iwCLL criteria with CT scan and bone marrow (BM) biopsy after combination therapy (Month 7). MRD was assessed on BM aspirates in local laboratories using ≥4 color flow cytometry (minimum sensitivity of 0.01%). Results: 49 pts (48 CLL/1 SLL) were enrolled in 5 dose escalation cohorts (n=41; 200-600mg/day) and a safety expansion cohort (n=8; 400mg/day); results herein combine data from all cohorts. Median (range) age was 68 (50-88) years. The median (range) number of prior regimens was 2 (1-5). 45 (92%) received prior R and 14 (29%) had R-refractory disease; 29 (59%) received prior fludarabine and 9 (18%) had fludarabine-refractory disease. Of pts with available data, 9/46 (20%) had del(17p); 19/27 (70%) expressed unmutated IGHV. As of June 4, 2015, 12 pts have discontinued the study: 6 due to PD (5 were Richter's transformation), 3 due to AEs (neuropathy, TLS, and myelodysplasia [heavily pretreated and hypocellular marrow at study entry; pt achieved MRD-negative CR with incomplete marrow recovery, CRi, and proceeded to transplant]), and 3 withdrew consent (1 after achieving MRD-negative CR). The investigator-assessed ORR was 86% (42/49) with 20 (41%) CR/CRi, 1 (2%) nPR and 21 (43%) PR. 4 had SD, 2 had PD, and 1 died before assessment (fatal TLS). 9 with PR or SD at the 7 month assessment achieved CR after a median (range) additional time of VEN monotherapy of 6 (2-9) months. BM MRD was evaluated in 40 pts. MRD-negativity was achieved in 15/20 (75%) pts who achieved a CR/CRi and 26/49 (53%) overall. Disease has progressed in 5/42 (12%) responders; 89% are free from progression at 12-months. The median PFS has not been reached. At 12 and 24 months, actuarial PFS is 87% and 84%, respectively, with a median (range) follow-up for pts without events of 17.5 (0.03-32) months. 94% were alive at 12 months; the median OS has not been reached. Although the numbers are small, the ORR, CR rates, PFS, and OS were not significantly impacted by high-risk subgroups. 8 pts stopped VEN after achieving CR/CRi, 6 of whom were MRD-negative at the time. 2 withdrew from the study after achieving CR/CRi without evidence of progression; 6 remain in follow-up with a median (range) of 15 (4-24) months off VEN. The 2 MRD-positive pts had asymptomatic progression with rising lymphocytosis after 19 and 24 months off VEN; both are eligible for retreatment with VEN when clinically appropriate. Treatment-emergent AEs in 〉25% of pts were neutropenia (55%), diarrhea (53%), nausea (49%), upper respiratory tract infection (45%), fatigue and pyrexia (each 37%), cough (35%), and headache (33%). Grade 3/4 AEs in 〉10% were neutropenia (53%), thrombocytopenia (16%), anemia (14%), febrile neutropenia (12%), and leukopenia (10%). 1 treatment-emergent AE (TLS) led to death; no other fatal TLS events occurred after a protocol modification aimed at TLS risk management and prophylaxis. 2 deaths occurred after PD. Key efficacy data are summarized in the table. Conclusions: VEN plus R induces a high rate of deep and durable responses, independent of adverse prognostic factors, with a tolerable safety profile. 41% of pts achieved CR/CRi and 53% achieved BM MRD-negativity. Remission off all therapy has been maintained in pts achieving MRD-negative CR. The median PFS and OS have not yet been reached; 24-month PFS is estimated to be 84%. The high rate of MRD-negativity is an encouraging step towards prolonged PFS and durable elimination of CLL/SLL. VEN plus R versus bendamustine plus R is being evaluated in a phase 3 trial in pts with previously treated CLL (MURANO; NCT02005471). Table 1. Table 1. Disclosures Ma: Genentech, Pharmacyclics/Janssen and Gilead: Speakers Bureau; Genentech, Pharmacyclics/Janssen and Gilead: Consultancy; NCCN, AbbVie, Pharmacyclics, Novartis, Gilead, Celgene, and Xeme: Research Funding. Off Label Use: Venetoclax is an investigational drug that is not yet approved in this indication.. Seymour:Phebra: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kipps:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria. Barrientos:Gilead, Pharmacyclics, and AbbVie: Research Funding; Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Anderson:AbbVie and Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment. Choi:AbbVie: Consultancy, Other: Advisory Board, Research Funding; Gilead: Consultancy, Other: Advisory Board, Speakers Bureau. Tam:Roche: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Mason-Bright:AbbVie: Employment, Equity Ownership. Prine:AbbVie: Employment, Equity Ownership. Munasinghe:AbbVie: Employment, Equity Ownership. Zhu:AbbVie: Employment, Equity Ownership. Kim:AbbVie: Employment, Equity Ownership. Humerickhouse:AbbVie: Employment, Equity Ownership. Roberts:Walter and Eliza Hall Institute of Medical Research: Employment; Genentech: Research Funding; AbbVie: Research Funding; Servier: Research Funding.

Description: The recent generation of mice lacking functional SOCS3 in hepatocytes, macrophages, and neutrophils reveals SOCS3 to be an essential regulator of IL-6 signaling via mediation of gp130-related cellular complexes, as well as a negative regulator of G-CSF signaling in myeloid cells. Although SOCS3 would appear to be a critical physiologic regulator of inflammatory responses, its possible role in hematologic malignancies and the underlying mechanisms which regulate its expression in B cells remain to be clearly defined. We previously showed that CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice express high levels of SOCS3 in addition to overexpression of Bcl-2. Moreover, hematopoietic cell lines transduced to stably overexpress Bcl-2 exhibited marked induction of SOCS3 compared to controls, suggesting Bcl-2-associated pathways may play a role in the induction of SOCS3. In the current study, we describe SOCS3 overexpression limited to neoplastic follicular lymphoma (FL) cells in Bcl-2-associated human de novo FL and show that overexpression of SOCS3 is capable of stimulating cytokine-independent cellular proliferation of the BaF3 pro-B cell line. We measured SOCS3 protein levels by immunohistochemistry in paraffin-embedded biopsies from twelve patients diagnosed with de novo, untreated histologic grade I or II FL which harbored t(14;18) and Bcl-2 overexpression. In 9/12 de novo FL cases examined, immunostaining with two distinct antibodies to SOCS3 revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic FL cells and co-localized with Bcl-2 primarily in the nucleus of positive cells. In contrast, SOCS3 protein was not detected by immunostaining in germinal center follicular B cells from benign hyperplastic tonsil tissue. To further evaluate the role of SOCS3 in B cell biology, the IL-3-dependent BaF3 pro-B cell line was stably transduced with either a retroviral expression construct containing a 675bp human SOCS3 cDNA (BaF3SOCS3) or with vector only control (BaF3Δ). Whereas no SOCS3 protein was detected in control cells, high level expression of SOCS3 in transduced BaF3SOCS3 cells was confirmed by Western analysis using SOCS3 anti-sera. Furthermore, Bcl-2 protein was not detected in either BaF3SOCS3 or control cell lines. 2 x 105 BaF3SOCS3, BaF3Δ, and non-transduced BaF3 cell lines were initially grown in the presence 10% fetal bovine serum (FBS) and 5% WEHI 3B cell-conditioned medium as a source of IL-3. IL-3 was then removed by washing with DMEM/10% FBS. Cell viability was then measured by recording absorbance at 490nm using incorporation of the MTS tetrazolium compound. Interestingly, BaF3SOCS3 cells overexpressing SOCS3 did not undergo apoptosis but were able to proliferate in the absence of IL-3, with percent viable cells approaching 400% at 〉 96 hours, which represented the final time-point measured. In contrast, BaF3Δ and non-transduced BaF3 cells underwent apoptotic cell death between 8 and 36 hours in response to IL-3 withdrawal. Thus, SOCS3 overexpression confers IL-3-independent cell proliferation to the BaF3 cell line. These data indicate that unlike its negative regulatory effect on G-CSF signaling in myeloid cells, overexpression of SOCS3 in B cells may promote B cell proliferation rather than growth suppression and may play an important role in the pathogenesis of de novo FL in humans.