ALBERT

Description: Abstract 975 We recently described a xenograft model of chronic lymphocytic leukemia (CLL) using NOD/SCID/γcnull (NSG) mice. Adoptive transfer of primary patient PBMCs into these mice results in engraftment and proliferation of CLL cells if autologous activated T cells are present. To date, such CLL-derived T cell proliferation has been achieved by co-transfer of third party antigen presenting cells (APCs). Unfortunately, in this setting, mice succumb to graft-versus-host disease, due to complex interactions between CLL cells, alloAPCs, T cells, and the xenogeneic host. We hypothesized that alternative strategies of autologous T cell activation might refine the model, ultimately providing longer CLL cell engraftment and animal survival. We describe 3 approaches to achieving engraftment and activation of CLL-derived T cells that support B cell growth in vivo. First, we activated CLL CD3+ cells isolated from PBMCs of 4 patients with anti-CD3/28 beads for 72 hours in vitro. Cells were then mixed with CFSE-labeled PBMCs from the same patient at varying ratios (1:50 to 1:1000 CD3+ cells:CLL PBMCs) and injected intraorbitally (io) into a total of 17 mice. CD4, CD8 and CD19 cell engraftment, identified by a human CD45 lymphocyte gate (hCD45), and proliferation, assessed by CFSE dilution of labeled cells, were monitored weekly. All mice demonstrated detectable CD3+ and CD5+CD19+ cells from week (wk) 1. The percent (%) CD3+ cells, as a proportion of hCD45, increased weekly in all mice receiving anti-CD3/28-activated cells. While overall % CD5+CD19+ cells decreased weekly, the % proliferating increased and strongly correlated with increasing % of T cells (r2=0.7799, p

Description: Chronic Lymphocytic Leukemia (CLL) is an incurable disease in which most of the tumor cells in the blood are arrested in G0/G1 stages of the cell cycle with only a minimal number displaying proliferative activity. In this regard, our group has found by gene expression profiling (GEP) that the proliferative fraction (PF) of CLL cells is enriched in the intraclonal subset marked by CXCR4dim CD5brite expression. Indeed, this subset differs by more than 1000 genes from the CXCR4brite CD5dim resting fraction (RF). The genes over-expressed in the PF relate to replication and migration as well as regulation of gene expression. One of these genes is Musashi 2 (MSI2). Of note, MSI2 is expressed at the highest levels in IGHV unmutated CLL (U-CLL) clones and their PFs. Normally, MSI2 binds mRNA and blocks translation of proteins, playing an important role in post-transcriptional regulation. In addition, MSI2 has been linked to proliferation of normal and malignant stem cells, tumorigenesis, and poor prognosis. 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 have begun to study the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression. Fist, we studied MSI2 protein expression by flow cytometry in CD19+ B cells from healthy donors (HD) and CD5+CD19+ cells from CLL patients, observing an up-regulation in CLL compared to HD. Also, we documented higher MSI2 expression in U-CLL compared to IGHV-mutated (M-CLL) CLL as well as HD. Within the leukemic clone, we observed that MSI2 expression was highest in the PF, lower in the intermediate (INT) fraction (defined as CXCR4int CD5int), and much lower in the RF (PF〉INT〉RF). The PF expressed 40% more MSI2 than the RF, suggesting that the highest amounts of MSI2 protein is in dividing and recently-divided cells of the clone. 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. After stimulating CLL cells with TLR9 agonist + IL15 + IL2 in vitro MSI2 protein was up-regulated form 0.3 to 2.5 fold. In addition, the increase in MSI2 protein was associated with an enhancement in Ki-67+ cells and in phosphorylation of MAPK/ERK and AKT signaling components, measured by flow cytometry. These results suggest that signals from the microenvironment that induce cell growth and proliferation lead to MSI2 synthesis in CLL B cells. In order to study a possible association between MSI2 expression and cell division, we labeled CLL PBMCs with a dilutable cell tracer, CFSE, and then stimulated them in vitro with TLR9 agonist + IL15 + IL2. These studies indicated that MSI2 protein synthesis was increased in the activated cells and that MSI2 protein levels increased with each cell division. However, it was also clear that this increase was not directly associated to the extent of cell replication as CLL B cells from only 10% of the patients underwent 4 cycles of cell division. Since we observed an increase in MSI2 and Ki-67 expression after stimulation in all patients' clones but did not detect replication of CLL cells in all patients, we studied the effects of in vitro stimulation on cell cycle entry and completion and how this related to MSI2 expression. Experiments using propidium iodide to evaluate DNA content of PBMCs showed that in vitro stimulation increased the percentage of cells in S phase (5-25%) compared to control cells without activation (

Description: Introduction: Autoimmune hemolytic anemia (AIHA) and immune mediated thrombocytopenia (ITP) are frequent complications of chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) that may evolve independently or occur at any stage of disease progression. Ibrutinib (Imbruvica®), a first-in-class BTK inhibitor, is a once-daily single-agent approved by the US FDA for CLL patients (pts) who had received ≥1 prior therapy, and for CLL pts with deletion 17p. Comprehensive efficacy and safety results from the interim analysis of the phase III RESONATE (PCYC-1112) study have previously been reported, demonstrating that improved progression-free survival (PFS) and overall survival were seen with ibrutinib (ibr) as compared to ofatumumab (ofa) in pts with previously treated CLL/SLL. Interim analysis data from the Phase III RESONATETM study are presented for pts with autoimmune complications, including that of a case report in which recurrent AIHA/ITP episodes resolved following initiation of ibr. Methods: History of AIHA and ITP along with status on study entry and resolution date when applicable were collected from 386 enrolled pts in both arms (ibr n=195; ofa n=191) who received study treatment. Ibr was administered at 420 mg once daily until PD or unacceptable toxicity. Ofa was administered at 300 mg followed by 2000 mg dose for up to 12 doses. Treatment emergent adverse events of ITP and AIHA are summarized for treated pts based on randomized arm as of the interim analysis. Pts with uncontrolled AIHA or ITP, defined as declining counts in the 4 weeks prior to randomization or requirement for steroids 〉20 mg/daily were excluded per study eligibility criteria. In addition, detailed medical history was reviewed for RESONATE patient MXC, as this patient was diagnosed with rapidly progressing CLL at its inception complicated by recurrent AIHA/ITP episodes over a 10-year course of CLL treatment. Results: In the RESONATE trial, median age was 67 years with 40% ≥70 years, and median number of prior therapies was 3 (ibr) vs 2 (ofa). In all treated pts (ibr n=195; ofa n=191), 29 (15%) pts in ibr arm had a history of AIHA with 20 (10%) ongoing at study entry, compared to 30 (16%) pts in the ofa arm with only 9 (5%) ongoing at study entry. 18 (9%) pts in ibr arm had a history of ITP with 12 (6%) ongoing at study entry, compared to 20 (10%) pts in ofa arm with 10 (5%) ongoing at study entry. Nine ibr and 8 ofa pts reported both AIHA and ITP at baseline, including patient MXC. No pts on the ibr arm developed treatment-emergent AIHA or idiopathic thrombocytopenic purpura. Two pts on the ofa arm developed AIHA, 1 of which was Grade 3/4. Two pts on the ofa arm developed idiopathic thrombocytopenic purpura, both of which were Grade 3/4. Case history showed that patient MXC underwent first-line fludarabine followed by alemtuzumab as consolidation treatment for rapidly progressing CLL diagnosed in 2004. Patient had unmutated IGHV status and deletion 17p. In 2005, the patient underwent autologous peripheral blood stem cell transplant. First AIHA episode was noted in 2007, followed by ITP in 2008 despite prior steroid and IVIG treatment. Recurrent episodes of AIHA (n=6) and ITP (n=3) transpired over the course of CLL, not always related to simultaneous disease progression. After several treatment failures during the 3rd AIHA episode and 2nd ITP episode, splenectomy was performed to obtain temporary clinical control of autoimmune events, and low dose steroids were successfully administered. In March 2013, patient was randomized to ibr as part of the RESONATE trial, steroid use was discontinued, and no further episodes of AIHA/ITP have been observed since ibr initiation. The patient showed Coombs test negativity after only a few weeks following ibr treatment, and no CLL progression has been observed to date. Conclusions: Efficacy and safety of ibrutinib has been evaluated in CLL/SLL pts including pts with ongoing AIHA/ ITP, both frequently noted complications of this disease. Data from the Phase III RESONATE study suggest that these CLL disease-related autoimmune complications did not limit ibrutinib treatment. This is supported by the lack of AIHA and ITP adverse events on the ibrutinib arm despite 19% having a history of these complications and further exemplified by a case report from the RESONATE study, where sequential episodes of severe AIHA/ITP ceased following ibrutinib initiation in the setting of disease control. Disclosures Montillo: Janssen: Honoraria. O'Brien:Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding. Hillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding. Dearden:Roche, GSK, Gilead, Janssen, Napp: Honoraria. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Barrientos:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mulligan:Roche, Abbvie : Consultancy, Honoraria. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Cymbalista:Janssen, Roche, GSK, Gilead, Mundipharma: Honoraria. Plascencia:Pharmacyclics: Employment. Chang:Pharmacyclics: Employment. Hsu:Pharmacyclics: Employment. James:Pharmacyclics: Employment. Byrd:Pharmacyclics: Research Funding.

Description: Immunoglobulin heavy variable (IGHV) gene replacement is a RAG-mediated recombination event that modifies a rearranged IGHV-IGHD-IGHJ sequence by replacing the original IGHV with another IGHV. As the excision of the original IGHV occurs at a cryptic heptamer near the end of FR3 of the original IGHV, a trace (“footprint”) remains embedded in the V-D (“N1”) junction of the heavy complementarity-determining region 3 (HCDR3) sequence of the new rearrangement. Since IGHV-replaced antibody sequences are over-represented in several autoantibody-associated syndromes, it is assumed that the process is designed to eliminate unwanted antibody specificities, particularly those which are autoreactive. Because CLL antibodies/B cell receptors (BCRs) are often autoreactive, we sought to determine if IGHV replacement plays a significant role in the formation of these antibodies. To this end, we analyzed IGHV-IGHD-IGHJ rearrangements in a CLL sample set of 6,121 sequences and in another set of 3,326 sequences from normal B lymphocytes culled from the GenBank Database. Sequences without positional information for their IGHV match were eliminated, resulting in 6023 CLL and 3202 normal sequences. From among these, we looked for the presence of IGHV footprints in the N1 and N2 junctional regions. Sequences were processed by IMGT to determine the particular IGHV used as well as junctional N1, N2 sequences. The resulting junctional regions were analyzed using the Java-based VH Replacement Footprint Analyzer (VHRFA) program [Huang, L et al (2013) PLOS ONE] to identify footprints (≥5-mer) in the N1 and N2 regions. Various CLL subgroups based on ZAP-70 expression, CD38 expression, IGHV mutation status, BCR stereotypy, and genomic aberrations were also examined for differences in the presence of footprints. Comparison of CLL with normal sequences in the N1 regions revealed a slight yet significant difference in footprint frequency (12.62% versus 11.06%, respectively; P = 0.03). Comparison of IGHV-mutated (M) versus unmutated (U) subgroups of CLL also disclosed a significant difference in frequencies (M: 13.58%, U: 11.51%, P = 0.02). Using footprints in the N2 regions as an internal control, we found the frequency of ≥5-mer replacement footprint motifs to be significantly higher in N1 over N2 (P = 1.1E-7), supporting the validity of this approach. Other comparisons led to a significant enrichment of footprint frequencies, such as 13.71% for the ZAP-70-negative group (P = 0.029), 13.36% for the CD38-negative group (P = 0.013), and 14.70% for the 13q-deletion group (P = 0.016) compared with the normal group. Note, an overall comparison of stereotyped versus non-stereotyped cases revealed highly statistically significant differences in N1 footprint frequencies (9.4% for the 2084 cases having defined subsets vs 14.32% for the 3,939 cases without defined subsets; P = 2.7E-8). Focusing on stereotyped cases that exhibited evidence for IGHV replacement, we found significantly different footprint frequencies in the N1 region among different stereotyped subsets, even when restricting the comparison to subsets utilizing the same IGHV and belonging to the same mutational category (U-CLL or M-CLL). Relevant examples include differential frequencies among U-CLL, IGHV1-69-expressing subsets #3, #5, #6, #7, and #59 (0 - 15.5%; P = 0.01) and M-CLL, IGHV4-34-expressing subsets #4, #16, #29, and #201 (0 - 40.9%; P = 0.005). In summary, CLL had an elevated IGHV replacement rate compared to normal controls. In addition, M-CLL, ZAP-70-neg, CD38-neg and 13q del CLL subgroups were also elevated above normal controls. The most dramatic differences in IGHV replacement frequency were found between CLL non-stereotyped and stereotyped CLL cases, with an intriguing difference within stereotyped subgroups exhibiting the same IGHV where IGHV replacement varied by as much as 40-fold. Because higher levels of IGHV replacement are found in ZAP-70-neg, CD38-neg, 13q del CLL, and non-stereotyped cases that in general are less aggressive, the presence of IGHV replacement may pinpoint CLL clones whose precursor B cells had edited their BCRs away from autoreactivity and therefore antigen-drive. This conclusion suggests a differential role for autoantigen drive in certain stereotyped subsets: CLL subsets not exhibiting IGHV replacement appear to be more susceptible to antigen drive than those that do. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The role of various prognostic factors in CLL/SLL is not yet fully understood, including the implications of new genetic markers associated with high risk. Ibrutinib (Imbruvica®), a first-in-class Bruton’s tyrosine kinase inhibitor, is a once-daily single agent approved by the US FDA for CLL patients (pts) who have received ≥ 1 prior therapy, and for pts with 17p deletion CLL. We report updated efficacy results for the phase 3 RESONATETM(PCYC-1112) study of ibrutinib (ibr) vs ofatumumab (ofa), relative to genetic features and prior treatment exposure, and provide updated adverse event (AE) data. Methods: Pts received 420 mg oral ibr daily until disease progression or unacceptable toxicity or IV ofa for up to 24 weeks. Primary endpoint was IRC-assessed PFS with secondary endpoints OS and IRC ORR (Byrd et al, NEJM 2014). At interim analysis, with median follow-up of 9.4 months, the Independent Data Monitoring Committee recommended pts in the ofa arm be provided access to ibr. Additional endpoints including investigator-assessed efficacy, investigation of potential predictive biomarkers, and safety with longer follow-up are reported. Results: The 391 pts were randomized to ibr (n=195) or ofa (n=196). Median age was 67 years, with 40% ≥70 years, 57% Rai stage III/IV disease. Median number of prior therapies was 3 (ibr) vs 2 (ofa) with 23% having 1, 28% with 2, and 49% ≥ 3 prior therapies. Approximately 32% had del(17p) and 32% (122/381) del(11q). 72 of 300 pts had complex cytogenetics (≥3 abnormalities) and 68% (181/266) unmutated IGHV. With outcome analyses pending, preliminary analysis revealed that 19% of pts had NOTCH1, 23% SF3B1, 36% TP53, and 1% MYD88 gene mutations at baseline. With median follow-up of 16 months, investigator-assessed PFS was significantly longer for ibr vs ofa (median NR vs 8.1 months, [HR 0.106, 95%CI 0.073-0.153, P

Description: Abstract 2699 Introduction: PI3Kδ is expressed in cells of hematopoietic origin where it regulates survival and proliferation of normal and malignant B-cells. CAL-101 (GS-1101) is an orally bioavailable, small-molecule inhibitor that selectively targets PI3Kδ. A prior Phase 1 study established 150 mg/dose BID as an appropriate single-agent starting dose for GS-1101 and demonstrated that GS-1101 monotherapy is associated with substantial clinical activity in patients with hematologic malignancies. Methods and Patients: This Phase 1 study has evaluated repeated 28-day cycles of GS-1101 in combination with rituximab and/or bendamustine in patients with previously treated iNHL. GS-1101 (G) was administered starting on Day 1 of Cycle 1 with rituximab (R) (375 mg/m2 given weekly for 8 doses, GR regimen), with bendamustine (B) (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles, GB regimen), or in combination with R (375 mg/m2, on Day 1 of each cycle for 6 cycles) and B (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles, GRB regimen). Initial cohorts of patients received GS-1101 at a dose of 100 mg/dose BID in the GR or GB regimens. Thereafter, all patients received GS-1101 at a dose of 150 mg/dose BID in the GR, GB, or GRB regimens. Chemokine/cytokine plasma levels were assessed at baseline and on Day 28 of therapy using multiplexed bead suspension arrays. Tumor response was evaluated according to standard criteria (Cheson et al. 2007). Results: At data cutoff, the study had enrolled 37 patients with iNHL. Patient characteristics, histological subtyping, safety, and efficacy results are depicted in the table. The majority of patients were 〉60 years of age and had undergone extensive prior therapy. Grade ≥3 adverse events were generally consistent with those expected with each of the single agents. All evaluable patients had reductions in lymphadenopathy, resulting in an overall response rate (ORR) of 〉65% for all regimens. In 3 patients among the 24 patients receiving either GB or GRB, 3 complete responses were observed. Lymph node shrinkage was rapid; responses generally occurred within ≤2 cycles. Disease-associated chemokines/cytokines were commonly elevated at baseline and were reduced by GS-1101 treatment; in 20 evaluable patients, mean (±SEM) values declined from 302 (±68) to 62 (±14) pg/mL for CCL17 (p=0.001), from 2512 (±332) to 879 (± 88) pg/mL) for CCL22 (p=0.0001), from 517 (± 88) to 107 (± 34) pg/mL for CXCL13 (p=0.0001), and from 33 (± 5.4) to 18 (± 1.8) pg/mL for TNFα (p=0.007). Conclusions: A favorable safety profile and lack of overlapping toxicities allows the oral PI3Kδ inhibitor, CAL-101 (GS-1101), to be delivered at the full single-agent starting dose when coadministered with chemoimmunotherapies in heavily pretreated patients with iNHL. GS-1101-based combination therapy with rituximab and/or bendamustine offers major and rapid reductions in lymphadenopathy. The data from this trial will be used to design Phase 3 combination studies of GS-1101 in patients with iNHL. Disclosures: de Vos: Gilead Sciences Inc: Consultancy. Flinn:Gilead Sciences Inc: Research Funding. Coutre:Gilead Sciences Inc: Consultancy. Leonard:Gilead Sciences Inc: Consultancy. Fowler:Gilead Sciences Inc: Consultancy. Sharman:calistoga: Honoraria; Pharmacyclics: Honoraria; Genentech: Honoraria; Rigel: Research Funding; Portola: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy. Holes:Gilead: Employment. Lannutti:Gilead: Employment. Johnson:Gilead Sciences Inc: Employment. Jahn:gILEAD: Employment. Miller:Gilead: Employment.

Description: Abstract 291 Lenalidomide (L) is an immunomodulatory agent with therapeutic activity in CLL; rituximab (R), a CD20 mAb, has limited activity as monotherapy in CLL. In preclinical models, L resulted in NK cell expansion and synergized with R. Therefore, a phase II, 2-stage study was designed to evaluate this combination. Treatment-naïve patients (pts) were eligible if they had an indication for therapy, normal kidney function, and no history of deep vein thrombosis or pulmonary embolic events. Pts started L at 2.5 mg per day (D) and could escalate to 5 mg/D on D8 and again on C3D1 to a maximum of 10 mg/D if tolerated. Pts received L 21 of 35 D for cycle (C) 1, then for 21/28 D for C2-7 (7 cycles total). R was started at the end of C1 at 50 mg/m2 D29, 325 mg/m2 D31, 375 mg/m2 D33, C1, then 375 mg/m2 weekly × 4 for C2 and on D1 for C3-7. Pts received allopurinol 300 mg daily, and following a protocol amendment, aspirin at 81 mg daily. The study has closed to accrual with 69 pts enrolled at four CRC clinical sites. 40 pts enrolled into arm A (age under 65). 29 in arm B (age 65 or older). 57 now have final response data. The median age on arm A was 57 years (range 45–64) and arm B 70 years (range 65–80). Advanced Rai stage (III-IV) was present at baseline more frequently in arm B pts (48%) compared to arm A pts (23%) (p= 0.04). 73% of pts on arm A had an excellent performance status (ECOG 0/5) compared to only 54% of pts on arm B. Arm A pts frequently had aggressive leukemia features including unmutated IgVH genes and/or elevated CLL expression of ZAP-70 in 70%, these features were present in 52% of those on arm B. 4 pts on arm A and 2 on arm B had del(17p) by FISH whereas 5 pts in arm A and 4 in arm B had del(11q). A significantly lower proportion of pts on arm B (16/27) were able to complete the 7 cycles of therapy compared to (35/39) of eligible pts on arm A (p=0.013) mainly due to toxicity. Similarly, older patients were limited in dose escalation or maintenance of the maximum L dose (10 mg). 27/39 pts on arm A achieved a median L dose of 10 mg while only 13/27 pts on arm B tolerated the 10 mg L dose. The most frequent and severe adverse events (AEs) are summarized in Table 1. Infusion and tumor flare reactions (TFR) were observed more frequently during cycle 1 than in subsequent cycles. Neutropenia was frequently grade (G) 3/4 in severity. Severe (G3/4) AEs were reported in 72% of arm A pts and 82% of pts on arm B (p =.55), with non-heme G3/4 toxicities reported in 37% of arm A and 52% of arm B (p =.31).Table 1Frequent and Severe AEsArm AArm BGradeI/IIIII/IVI/IIIII/IVTFR32–161Neutropenia1119115Anemia153141Thrombocytopenia211131Fatigue25–142AST/ALT elevation183113Hypophosphatemia19271Hypocalcemia21–15–Infusion Reaction20–53Respiratory Infection17–5–Pneumonia–123Rash142121Neutropenic Fever–2–2PE/DVT–––2 Responses are summarized in Table 2. The overall response rate (ORR) to therapy for arm A was 94%, 90% CI (83%, 99%) with 20% (10%, 34%) achieving a complete remission (CR) and 17% a nodular PR (nPR). The ORR for arm B was 77% with 90% CI (58%, 91%) with 9% (2%, 26%) achieving a CR. Arm A patients had median follow-up of 17 months (from D 1 of treatment) with an estimated median progression free survival of 19 months. Arm B had a median follow-up of 7 months, with an estimated 85% remaining progression free at 7 months.Table 2ResponsesArm A (Age 〈 65) N = 35Arm B (Age 〉= 65) N = 22NCR N (%)nPR N (%)PR N (%)ORR N (%)NCR N (%)PR N (%)ORR N (%)All patients357 (20)6 (17)20 (57)33 (94)222 (9)15 (68)17 (77)IgVH unmutated224 (18)2 (9)15 (68)21 (96)131 (8)10 (77)11 (85)IgVH mutated133 (23)4 (31)5 (38)12 (92)91 (11)5 (56)6 (67)Median L dose 10 mg247 (29)5 (21)12 (50)24 (100)82 (25)5 (63)7 (88)Rai stage III/IV92 (22)1 (11)5 (56)8 (89)111 (9)6 (55)7 (64)17p del3002 (67)2 (67)100011q del31 (33)02 (67)3 (100)403 (75)3 (75)TFR present285 (18)5 (18)16 (57)26 (93)14011 (79)11 (79) A defined course of seven cycles of L and R administered for the initial treatment of CLL was associated with a high response rate (ORR 88%) with 16% CRs and 11% nPRs. Older patients were more likely to have advanced Rai stage at baseline and were less likely to escalate or maintain the maximum L dose and/or complete the 7 cycles of therapy, factors that may have contributed to a lower CR, and ORR rate in arm B. An additional 11 patients will have final response assessments by October 2011. Disclosures: James: Celgene: Honoraria, Research Funding. Off Label Use: Lenalidomide for Chronic lymphoyctic leukemia. Brown:Calistoga: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Research Funding. Rai:Celgene: Membership on an entity's Board of Directors or advisory committees. Neuberg:Celgene: Research Funding. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Description: Chronic lymphocytic leukemia (CLL) is a disease commonly associated with an immune disturbance. These alterations of the immune system have been generally considered due to the direct effects of CLL cells. In the last years, myeloid derived suppressor cells (MDSCs) have been found to be expanded in several cancers and to play a major role in helping tumor cells escape from immune surveillance. MDSCs represent a heterogeneous population of HLA-DRlo/CD11b+/CD33+ cells that are subdivided into monocyte-like (CD14+, m-) or granulocyte-like (CD15+, g-) subsets. Here we have investigated the extent that patients with CLL have expansions of MDSCs, their types and functions, and how these correlate with clinical and laboratory characteristics. Using flow cytometry on cryopreserved PBMCs, we did not observe differences in the percentages of HLA-DRlo/CD11b+/CD33+ cells between 17 untreated CLL patients and 10 healthy controls (HC) (3.3% vs. 3.1%). However, the distribution between m-MDSCs and g-MDSCs was dramatically different, with CLL patients exhibiting significantly higher levels of g-MDSCs (79.7% vs. 4.1%, p

Description: Abstract 1787 Introduction: PI3Kδ is expressed in cells of hematopoietic origin where it regulates survival and proliferation of normal and malignant B-cells. CAL-101 (GS-1101) is an orally bioavailable, small-molecule inhibitor that selectively targets PI3Kδ. A prior Phase 1 study established 150 mg/dose BID as an appropriate single-agent starting dose for GS-1101 and demonstrated that GS-1101 monotherapy is associated with substantial clinical activity in patients with hematologic malignancies. Methods and Patients: This Phase 1 study has evaluated repeated 28-day cycles of GS-1101 in combination with rituximab and/or bendamustine in patients with previously treated CLL. GS-1101 (G) was administered starting on Day 1 of Cycle 1 with rituximab (R) (375 mg/m2 given weekly for 8 doses, GR regimen), with bendamustine (B) (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles, GB regimen), or in combination with R (375 mg/m2, on Day 1 of each cycle for 6 cycles) and B (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles, GRB regimen). Initial cohorts of patients received GS-1101 at a dose of 100 mg/dose BID in the GR or GB regimens. Thereafter, all patients received GS-1101 at a dose of 150 mg/dose BID in the GR, GB, or GRB regimens. Chemokine/cytokine plasma levels were assessed at baseline and on Day 28 of therapy using multiplexed bead suspension arrays. Tumor response was evaluated according to standard criteria (Hallek et al, 2008). Results: At data cutoff, the study had enrolled 27 patients with CLL. Patient characteristics and safety and efficacy results are depicted in the table. The majority of patients were 〉60 years of age, had bulky adenopathy, and had undergone extensive prior therapy. Grade ≥3 adverse events were generally consistent with those expected with each of the single agents. During combination therapy, the substantial majority of patients had marked reductions in lymphadenopathy, resulting in a ≥50% decrease in lymph node area in 〉75% of patients on all regimens. Lymph node shrinkage was rapid, generally occurring within ≤2 cycles. The lymphocyte mobilization that is expected with PI3Kδ inhibition was observed in some patients but was not as prominent as had previously been seen with single-agent GS-1101 therapy. More than 80% of patients receiving each regimen met criteria for CLL response. Disease-associated chemokines/cytokines were commonly elevated at baseline and were reduced by GS-1101 treatment; in 20 evaluable patients, mean (±SEM) values declined from 116 (±36) to 33 (±5.5) pg/mL for CCL3 (p=0.022), from 217 (±83) to 55 (± 28) pg/mL) for CCL4 (p=0.052), from 220 (± 57) to 46 (± 6.6) pg/mL for CXCL13 (p=0.004), and from 100 (± 20) to 30 (± 3.6) pg/mL for TNFα (p=0.001). Conclusions: A favorable safety profile and lack of overlapping toxicities allows the oral PI3Kδ inhibitor, CAL-101 (GS-1101), to be delivered at the full single-agent starting dose when coadministered with chemoimmunotherapies in heavily pretreated patients with CLL. GS-1101-based combination therapy with rituximab and/or bendamustine offers major and rapid reductions in lymphadenopathy. Patients with CLL are currently being enrolled in this study to receive GS-1101 with fludarabine or ofatumumab; data for all regimens will be presented. The data from this trial will be used to design Phase 3 combination studies of GS-1101 in patients with CLL. Disclosures: Sharman: calistoga: Honoraria; Pharmacyclics: Honoraria; Genentech: Honoraria; Rigel: Research Funding; Portola: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy. de Vos:Gilead: Consultancy. Leonard:Gilead: Consultancy. Coutre:Gilead: Consultancy. Flinn:Gilead: Research Funding. Fowler:Gilead: Consultancy. Holes:Gilead: Employment. Lannutti:Gilead: Employment. Johnson:gILEAD: Employment. Jahn:gILEAD: Employment. Miller:Gilead: Employment.

Description: Abstract 3690 Mantle cell lymphoma (MCL), a less common non-Hodgkin's lymphoma (NHL), often has a poor prognosis and a median survival time of 3–5 years. Historically, MCLs were believed to originate from mature but naive B cells; this notion has now changed based on the demonstration of somatically mutated IgHV sequences in the lymphoma cells from a subset of cases. Indirect evidence suggesting that the B-cell receptor (BCR) pathway may be at the base of the observed activation in the disease exists; however, that extent that this activation results from Toll-like receptor (TLR), B-cell antigen receptor (BCR), or a combination of signaling from both has not been adequately addressed. In this study, the responsiveness of purified primary B cells isolated from peripheral blood (PB) and/or bone marrow (BM) of MCL patients in the leukemic phase of the disease to triggering via the BCR or via TLR-9 alone or in context with selected chemokines – CCL17, CCL22, or CXCL12 - was assessed using various early and late cell signaling readouts. Phosphoflow analysis revealed that within 5 minutes of stimulation both PB and BM B cells significantly increased levels of pAkt and pNFkB in response to BCR crosslinking by an anti-IgM monoclonal antibody (mAb). When PB B cells were cultured for 3 days in the presence of various stimuli to evaluate their proliferative response (uptake of 3H-thymidine), anti-BCR triggering stimulated 2 to 5.5 fold increases in DNA synthesis, whereas the TLR-9 agonist ODN2006 elicited 55 to 235 fold increases. In addition, conditions simulating T-cell help (anti-CD40 mAb + IL-4 in the presence of CD32-transfected fibroblasts) stimulated significant (40–65 fold) proliferative responses in MCL B cells. Simultaneously, a significant increase in HLA-DR (anti-BCR: 49%; ODN2006: 61%; T-cell help: 20%) and Bcl-2 expression (anti-BCR: 21%; ODN2006: 36%; T-cell help: 25%) was induced by these stimuli. Furthermore, B cells from the BM of the same cases differed in their proliferative responses based on the agonist. Thus, in response to BCR triggering, B cells from BM proliferated to a greater extent compared with PB B cells, whereas in response to TLR-9 stimulation PB B cells proliferated to a greater extent than those from BM. In independent experiments, B cells were incubated with various stimuli including those simulating T-cell help and chemokines for 3 days. Cells were harvested and extracts prepared from viable cells to determine telomerase activity using the telomere repeat amplification protocol (TRAP). Anti-BCR stimulation and anti-TLR-9 stimulation independently increased telomerase activity 1.7 and 1.9 fold, respectively, whereas in combination with CCL17 and CCL22, anti-TLR-9 stimulation further increased telomerase activity to 2.28 and 2.36 fold, respectively. In summary, these findings suggest an important role for commonly encountered microenvironmental influences interacting with TLR9 and to a lesser extent the BCR in promoting the aggressiveness of MCL. They also suggest that responses to these stimuli differ between MCL cells residing in the BM and those circulating in the blood. Finally, the data suggest that ligands for CCR4 may play an enhancing role for signals transduced by the BCR and TLR-9 in this disease. If documented in a larger number of cases, treatment regimens that target these signaling pathways might be of therapeutic value. Disclosures: No relevant conflicts of interest to declare.