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Recent studies have suggested that rituximab has clinical activity and modulates antiapoptotic proteins associated with drug resistance in chronic lymphocytic leukemia (CLL). We performed a randomized phase 2 study to determine the efficacy, safety, and optimal administration schedule of rituximab with fludarabine in previously untreated CLL patients. Patients were randomized to receive either 6 monthly courses of fludarabine concurrently with rituximab followed 2 months later by 4 weekly doses of rituximab for consolidation therapy or sequential fludarabine alone followed 2 months later by rituximab consolidation therapy. A total of 104 patients were randomized to the concurrent (n = 51) and sequential (n = 53) regimens. During the induction portion of treatment, patients receiving the concurrent regimen experienced more grade 3 or 4 neutropenia (74% versus 41%) and grade 3 or 4 infusion-related toxicity (20% versus 0%) as compared with the sequential arm. The consolidation rituximab therapy was tolerated well in both arms. All other toxicities were similar in the 2 arms. The overall response rate with the concurrent regimen was 90% (47% complete response [CR], 43% partial response [PR]; 95% confidence interval [CI], 0.82-0.98) compared with 77% (28% CR, 49% PR; 95% CI, 0.66-0.99) with the sequential regimen. With a median follow-up time of 23 months, the median response duration and survival have not been reached for either regimen. Rituximab administered concurrently with fludarabine in previously untreated CLL patients demonstrates marked clinical efficacy and acceptable toxicity. Phase 3 studies using this combination approach for patients with CLL are warranted.

PURPOSE: This phase II pilot study determined the efficacy and safety of alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom) in patients with chronic lymphocytic leukemia (CLL), all of whom had previously received fludarabine and other chemotherapy regimens. PATIENTS AND METHODS: Twenty-four patients were treated with intravenous alemtuzumab at six centers in the United States. The target dose of 30 mg over 2 hours, three times weekly, was administered for up to 16 weeks. Responses were evaluated by an independent panel of experts using 1996 National Cancer Institute–sponsored Working Group criteria. Safety assessments included analysis of lymphocyte subpopulations. Antimicrobial prophylaxis was not mandatory. RESULTS: Eight patients (33%) achieved a major response (all partial remissions), with a median time to response of 3.9 months (range, 1.6 to 5.3 months). The median duration of response was 15.4 months (range, 4.6 to ≥ 38.0 months), the median time to disease progression was 19.6 months (range, 7.7 to ≥ 42.0 months), and the median survival time was 35.8 months (range, 8.8 to ≥ 47.1 months). Acute infusion-related events, mainly grades 1 and 2, were most common and most severe in the first week. Ten patients (eight nonresponders and two responders) experienced major infections on-study. Pneumocystis carinii pneumonia was reported in two patients on-study; neither had received prophylaxis. Median CD4+ and CD8+ counts decreased and then began to increase by the end of the study, with further recovery by 1-month follow-up. One of 53 samples obtained from 10 patients had a low titer of alemtuzumab antibodies. CONCLUSION: Alemtuzumab has significant activity in poor-prognosis, fludarabine-treated CLL patients. However, because of a relatively high incidence of opportunistic infections accompanying profound lymphopenia, future protocols should include mandatory prophylaxis.

In the treatment of hematological malignancies, the significance of achieving molecular complete remissions rather than a clinical complete remission has become increasingly apparent, as relapse is considered to be related to the presence and extent of residual, persistent malignant cells. Progress in the use of alemtuzumab (MabCampath™) to eradicate minimal residual disease in chronic lymphocytic leukemia (CLL) has positive implications for patients, particularly prior to stem cell transplantation. Another beneficial aspect of monoclonal antibody therapy under active investigation is the ability of alemtuzumab to repeatedly produce or maintain responses. Subcutaneous administration of alemtuzumab is now emerging as a key advancement in the treatment of CLL. A phase II trial of subcutaneous alemtuzumab has demonstrated an 87% response rate in 38 previously untreated patients, with a reduction in intravenous administration-related rigors, as well as the elimination of nausea, dyspnea, diarrhea, and hypotension, frequently seen following intravenous administration of alemtuzumab. This trial demonstrates the advantages for safety, cost, flexibility, and convenience offered by subcutaneous administration of alemtuzumab. Finally, encouraging results with the combination of alemtuzumab and fludarabine, which demonstrate eradication of malignant cells in patients who are resistant to either agent alone, open the way for such combinations to produce durable responses even in refractory disease.

This study investigated the efficacy, safety, and clinical benefit of alemtuzumab (Campath-1H) for patients with relapsed or refractory B-cell chronic lymphocytic leukemia exposed to alkylating agents and having failed fludarabine therapy. Ninety-three patients received alemtuzumab in 21 centers worldwide, with the aim to obtain an overall response rate of at least 20%. Dosage was increased gradually (target 30 mg, 3 times weekly, for a maximum of 12 weeks). Infection prophylaxis was mandatory, beginning on day 8, and continuing for a minimum of 2 months after treatment. Responses were assessed at weeks 4, 8, and 12, and patients were followed for 34 months. Overall objective response in the intent-to-treat population (n = 93) was 33% (CR 2%, PR 31%). Median time to response was 1.5 months (range, 0.4-3.7 months). Median time to progression was 4.7 months overall, 9.5 months for responders. At data cut-off, 27 patients (29%) were alive; overall median survival was 16 months (95% CI: 11.8-21.9) and 32 months for responders. Nineteen responders survived more than 21 months. Clinical benefit was observed both in responders and in patients with stable disease. The most common adverse events were related to infusion, generally grade 1 or 2 in severity, occurring mainly in the first week. Grade 3 or 4 infections were reported in 25 patients (26.9%). However, only 3 (9.7%) of 31 patients who responded to alemtuzumab treatment developed grade 3 or 4 infections on the study. Alemtuzumab induced significant responses in these patients with clinical benefit in the majority and with acceptable toxicity in a high-risk group.

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients often develop anemia due to the disease process and effects from disease therapy. Blood transfusion, the established treatment, has an immediate effect in improving patients' hemoglobin levels. However, this effect is transient and transfusion is associated with several risks, including infections and mild to life-threatening immunologic reactions. A newer option is recombinant human erythropoietin (epoetin); a biological treatment that leads to increased hemoglobin levels over an extended time without the risks of blood transfusion. Extensive evidence has shown that epoetin is effective in the treatment of cancer-associated anemia. An international expert panel met to develop treatment recommendations for the use of epoetin in MM and CLL patients. Based on the available data, it is recommended that treatment be initiated only after other possible causes of anemia are eliminated. Epoetin should be administered to any patient with hemoglobinor=10 g/dl. Patients with hemoglobin 10-12 g/dl should receive epoetin if they suffer from significant symptoms of anemia and/or have progressively decreasing hemoglobin values. Dosage should be initiated at 10 000 IU three times/week or 40 000 IU once/week and be titrated to maintain hemoglobin at 12 g/dl. Nonresponsive patients (1 g/dl increase over four weeks) may have their dose increased to 20 000 IU three times/week or 60 000 IU once/week, respectively. Epoetin treatment should be discontinued if there is no response to the increased dosage, or hemoglobin14 g/dl. Treatment should resume for patients who exceed 14 g/dl, at a reduced dosage, if their hemoglobin falls below 12 g/dl.

Featured Article: Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood 1975;46:219–34.3 Before publication of this article, clinicians treating chronic lymphocytic leukemia (CLL) experienced a high level of frustration because of the vast heterogeneity in the clinical courses of patients with CLL. After their initial diagnosis, some patients had an extremely aggressive course and would die in about 2 to 3 years despite therapy, whereas some CLL patients would live for years and sometimes die decades later from causes unrelated to CLL. CLL remained a relatively less investigated disease for more than 60 years of the 20th century. The publication of this article in 1975 provided an extremely practical, clinically usable, and reliable method of assigning CLL patients at the time of initial diagnosis into 3 broad prognostic groups: Those with evidence …

(mean of 3.7CNAs/case), with a higher frequency of complex genomes than at diagnosis (defined as 3 CNAs per case, 45% v 16%, P 0.001). In our total cohort, genome complexity was associated with a reduced overall survival (OS, 135 v 209mths, P 0.001). However, in our progressive cohort the reduced survival associated with genomic complexity was purely a reflection of underlying TP53/ATM loss or mutation. Only in our diagnostic cases was genomic complexity associated with reduced OS (155 v 321mths, P 0.02) independent of TP53 and ATM loss/mutation. Using a highly robust and systematic bioinformatic analysis we identify 37 novel recurrent ( 1% of all cases) minimally deleted (MDRs, n 32) and enhanced (MER, n 5) regions in our CLL cases ranging from 0.03-27.15Mb in size. In each instance, the MDR/MER was not defined by cases considered to have high levels of complexity ( 3 CNAs targeting the MDR/MER containing chromosome). These regions could be grouped as follows. (1) Of the 28 regions that contained genes, 16 were significantly associated (p 0.05) with the presence of loss/mutation targeting the ATM (n 2) or TP53 (n 14) loci. These regions are likely to be the result of genome instability, and could represent markers of TP53 pathway dysfunction; (2) Six of the remaining 12 regions occurred in complex genomes in cases that lacked a deletion or mutation of either TP53 or ATM, and included deletions of 6q, 8p, 8q, 9p, 19p and 19q. Genes within one or more of these regions may be contributing to genomic complexity in these cases. (3) The remaining eight regions, loss of 1q (two regions), 10q, 14q, 15q, 18q (x2) and gain of 3q, occurred independent of TP53/ATM loss or mutation and genomic complexity and may be important. Several of the novel regions are preliminarily associated with reduced survival, and within these regions are genes involved in cell cycle control, NF-Kappa-B signaling and chromatin modification. In conclusion, our data describe the detailed architecture of CNAs and aUPD events in CLL across the evolution of the disease. We show that the utility of established biomarkers, such as genome complexity, is dependent on the evolutionary stage of the disease. We use a systematic approach to identify a panel of novel regions of recurrent CNA that included genes implicated in carcinogenesis, where preliminary data suggests a prognostic utility. Currently, we are identifying causative genes within these regions, using next generation sequencing, and screening extended cohorts to confirm the prognostic relevance. It is hoped that several of the genes emerging from this study may represent novel biomarkers and therapeutic targets.

Background. Chronic lymphocytic leukemia (CLL) is a prototypic microenvironment-dependent B-cell malignancy, in which neoplastic B cells co-evolve with a supportive tissue microenvironment to promote leukemia cell survival, growth, and drug-resistance. Within the microenvironment, hematopoietic and non-hematopoietic stromal and tumor cells produce factors that recruit circulating monocytes to tumor sites and induce differentiation into macrophages. Mirroring the Th1/Th2 paradigm, cells of monocyte-macrophage lineage reprogram their functions in response to environmental signals, undergoing M1 (classical) or M2 (alternative) activation, which represent extremes of a broad continuum of functional states. Classical M1 cells (activated by IFNs and TLR) are involved as inducer and effectors cells in polarized Th1 responses and as effectors of resistance against intracellular parasites and tumors. In contrast, M2 cells (activated by IL4 and IL13) are poor at antigen presentation, suppress Th1 adaptive immunity, actively scavenge debris, contribute to the dampening of inflammation, promote angiogenesis and tissue remodeling, and support tumor progression. One way to distinguish the two types of macrophages is based on surface antigen expression: M1-like cells up-regulate Fcg receptors I, II, III such as CD16, CD32 and CD64, whereas M2-like macrophages display abundant levels of CD23, CD163, and scavenger receptors (e.g. MCR1). Understanding the microenvironment and the crosstalk between B-CLL cells and their tissue neighbors can give insight into disease biology and for therapy. Aim. To investigate if the CLL milieu, contained within serum, influences monocyte-to-macrophage differentiation, promoting an anti (M2)- or pro (M1)- inflammatory microenvironment. Methods. Monocytes from healthy donors were isolated using Monocytes Isolation Kit II (Miltenyi) and cultured in Ultra-Low Attachment plates with 10% normal human AB serum or 10% CLL-derived serum -/+ IL4 or IFNg for 3 days. Macrophages were stained for CD23, CD64, CD32, MRC1, CD14, CD16, and data were acquired with a BD LSRII flow cytometer and analyzed by FlowJo V7.2.4 software. Results. Normal monocytes were differentiated to macrophages in vitro in the presence of sera from 24 untreated CLL patients with different prognostic factors (genomic aberrations, % CD38 and IGHVmutational status). About 45% of the CLL sera (N=10; 6 M-CLL, 4 U-CLL) drove macrophage maturation toward an M2-like phenotype, as assessed by surface expression of CD23, CD64, CD32, CD36, MRC1, etc. These 10 sera induced higher CD23 expression after 3 days in culture compared to AB human serum, whereas the levels of M1-specific markers (CD64 and CD32) did not change relative to the control. Interestingly all of these 10 CLL sera came from patients bearing 13q14 Δ (N=5), 17p13 Δ (N=3) or a combination of these (13q14 Δ + 17p13 Δ; N=1) and 17p13 Δ + trisomy12; N=1)). On the contrary, no increase in CD23 expression was detected in presence of sera from patients with 11q22 Δ (N=1) alone or in combination with 13q14 Δ (13q14 + 11q22 Δ; N=5). Of note, treatment with a neutralizing mAb specific for IL-4 did not block the CLL serum induced up-regulation of CD23 (N=2). In a parallel set of studies, normal monocytes were incubated with each of the 24 CLL sera in combination with the M1 promoting cytokine, IFNg or the M2 promoting cytokine, IL4. In all cases IL4 induced CD23 up-regulation and an M2 phenotype. Paradoxically, IFNg, which normally induces an M1 phenotype, also induced an M2 phenotype (i.e., enhanced CD23 expression) when co-cultured with sera from a subset patients (N=8; 6 M-CLL and 2 U-CLL). Of note, the IFNg stimulatory effect on CD23 expression was observed with a different set of sera from those that directly stimulated CD23 expression. Furthermore, CD64 expression did change after incubation with IFNg + CLL serum in 6 of 8 cases, yielding another unusual (CD23+CD64+) macrophage phenotype. The 2 sera that did not yield such hybrids were from M-CLL patients. Conclusions. Sera from CLL patients contain two apparently novel activities that mature normal monocytes to M2-like macrophages. The first acts directly by an action that is apparently independent of IL-4 and associates with 13q14 Δ or 17p13 Δ abnormalities. The second acts indirectly through IFNg and leads to macrophages with a hybrid M2/M1 phenotype (CD23+CD64+), suggestive of a new type of macrophage. Disclosures No relevant conflicts of interest to declare.

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 ( AID mutational characteristics in new subclones were assessed using the SHMTool (http://scb.aecom.yu.edu/shmtool) algorithms to calculate mutation frequencies in IGHV-D-J relative to IGHM and at AID mutation hotspots and coldspots. Other APOBEC family members have a different mutation hotspot site, which we analyzed by custom R scripts. All CLL samples showed evidence of AID mutational activity: higher IGHV-D-J versus IGHM mutations in all cases, increased AID hotspot mutation frequency in five cases, and decreased AID coldspot mutation frequency in five cases. Increased APOBEC hotspot mutational activity was seen in four cases. This APOBEC mutational activity is consistent with increased APOBEC3 gene expression and the genomic somatic mutation pattern observed recently in CLL (Rebhandl et al. 2014 Leukemia). Thus, both U-CLL and M-CLL are capable of producing mutations characteristic of AID or APOBEC activity. These data are consistent with the hypothesis that AID/APOBEC may promote new mutations leading to the evolution of aggressive CLL. Disclosures No relevant conflicts of interest to declare.

Background: Although treatment with purine analogs is associated with a high response rate, hairy cell leukemia (HCL) remains incurable with a 30-40% relapse rate. For these patients (pts) and those intolerant to purine analogs, novel therapies are needed. The major finding that BRAFV600E mutations occur in 98% of HCL suggests BRAF as a promising therapeutic target. We therefore designed a phase II trial to (1) determine the clinical efficacy of the BRAF inhibitor vemurafenib in pts with relapsed/ refractory HCL and (2) identify biologic determinants of response and resistance to vemurafenib in HCL. Patients and Methods: Pts with BRAF mutant HCL who were refractory or resistant to purine analogs, or who had ≥2 relapses with an indication for treatment (ANC ≤1.0, HGB ≤10, or PLT ≤100K) were enrolled. Eligible pts received vemurafenib 960mg twice daily for 3 months. Bone marrow (BM) evaluations were performed after 3 months to assess response. Pts with partial (PR) or complete response (CR) with detectable minimal residual disease (MRD) were allowed to receive vemurafenib for up to 3 additional months. The primary endpoint of the study was overall response rate (ORR: CR + PR). Using a Simon’s mini-max two-stage design, if at least 4 of the 19 pts (≥20%) achieve ORR in the first stage, an additional 17 patients will be accrued to the second stage. If 11 or more pts achieve ORR out of the 36 pts, the study would be considered worthy of further investigation. Serial peripheral blood and/or BM samples were collected during the study for quantitative BRAF mutant allele burden, serum cytokine, multiparameter flow cytometry, and targeted next-generation sequencing analysis of a 350-gene panel to detect potential predictors of resistance and identify genes collaborating with BRAF mutations in HCL. Results: 22 pts have been enrolled and 20 pts received treatment. The median age was 61 years (range 44-77), and the median number of prior treatments was 3.5 (range 1-7). 20 pts are evaluable for toxicity and 17 patients for disease response with a median follow up of 10 months (range 2-19). The most common adverse events were rash (40%, Gr1-2), arthralgia (30%, Gr1-2), photosensitivity (20%, Gr1), and pruritis (15%, Gr1). Three pts developed squamous cell carcinoma (SCC), all of whom had previous history of SCC. All patients were able to complete the intended treatment (3 cycles: 11 pts, >3 cycles: 6 pts). All 17 evaluable pts achieved complete hematologic recovery with an overall response rate (ORR) of 100%. 6 pts achieved CR (4 MRD- and 2 MRD+) and 11 pts achieved PR with very minimal disease (Figure 1). Responses were rapid with reduction of circulating hairy cells within 24 hours and normalization of sCD25 within 2-3 weeks (Figure 2). This correlated with dephosphorylation of ERK at 1 month. Several additional inflammatory cytokines correlated with the leukemic cell burden identifying novel tumor markers in HCL including sTNF-R2, sIL-1R2, and sIL4R. Genetic analysis identified a median of 3 (range 0-5) somatic mutations co-existing with the BRAFV600E mutation in HCL including recurrent mutations in MLL2 and CREBBP (Figure 2). Several of these alterations were present as minor clones identifying a clonal architecture in HCL which was not previously appreciated. One pt was found to have de novo resistance to vemurafenib. Genetic analysis identified that this pt’s pretreatment HCL cells harbored a previously undescribed missense mutation in IRS1 (IRS1P1201S) in addition to BRAFV600E mutation. Given prior knowledge that IRS1 (Insulin Receptor Substrate 1) activates both MAP kinase and PI3K-AKT signaling, we compared the effects of expression of wildtype and mutant IRS1 cDNAs on signaling. This revealed robust activation of PI3K-AKT signaling induced by IRS1P1201S-mutant cells relative to wildtype. Conclusions: While longer follow-up is needed to assess the durability of the response, our results indicate that vemurafenib has potent antitumor activity in pts with relapsed/refractory BRAF mutant HCL. These data confirm the MAP kinase pathway as a rational promising therapeutic target in HCL and identify activation of signaling pathways parallel to the MAP kinase pathway as a first mechanism of RAF inhibitor resistance in a hematological malignancy. Subsequent studies will be required to prove that inhibition of B-raf may be the best initial therapy in HCL pts who require treatment. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Park: Genentech: Research Funding. Off Label Use: Vemurafenib in hairy cell leukemia. Rosen:Novartis: Consultancy.

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 When analyzing phosphorylation of Akt, Erk, p38MAPK and Syk by phosphoflow among all cases, the CXCR4brCD5br fraction exhibited the highest constitutive levels. Constitutive levels of phospho-Btk and - Syk were significantly higher in the CXCR4int CD5br subfraction in M-CLL than in U-CLL cases (p Together, these findings highlight the impact of the molecules expressed by the different subfractions on their ability to relay/inhibit signals elicited via the BCR or CXCR4. They also provide a general frame of reference to further understand functional post-replicative intraclonal heterogeneity of CLL clones and help define aggressive subfractions of the CLL clone as target populations for future therapies. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) cells interact with external stimuli via several receptors, including the B cell receptor (BCR) and cytokine/chemokine receptors. CLL cells also express varying levels of functional Toll-like receptors (TLR), including TLR7 and TLR9. In vivo, the interaction of CLL cells via BCR or TLR occurs within a supportive microenvironment that provides them with survival and proliferative signals. CLL clonal turnover, occurring as an outcome of these and other interactions might lead to the release of naked nuclear material into the microenvironment and permit an interaction with these remnants of dying/dead cells. This could lead to repeated low level T cell-independent activation, possibly resulting in disease progression. To this effect, concomitant engagement of BCR and TLR leads to differential Erk phosphorylation and apoptosis rescue in CLL cases segregated by IgV gene mutational status. Here, we have studied the impact of stromal cells on TLR-mediated activation of negatively selected CLL cells from 24 untreated cases. Marked increases in percentages of CD69+ CLL cells and density of HLA-DR expression were observed within 18 hrs after exposure to TLR7 or TLR9 agonists (Imiquimod or ODN2006, respectively). However, although CLL cells activated via their TLRs in the presence of irradiated stromal cells (HS-5) did not show further changes in CD69 and HLA-DR expression, they exhibited significantly elevated levels (p Ibrutinib covalently binds and inhibits Bruton tyrosine kinase, BTK, a molecule critical for BCR signaling. In addition, ibrutinib inhibits cell signaling occurring when CXCR4 binds its ligand, stromal derived factor-1/CXCL12. However, the effects of ibrutinib on concomitant TLR+ BCR-mediated signaling in CLL have not yet been reported. We evaluated the impact of ibrutinib over a dose range of 0.1 - 25mg/ml on TLR-mediated CLL cell apoptosis and proliferation in presence/absence of stromal help, as mentioned above. Pre-incubation of CLL cells with ibrutinib for 2 hrs significantly lowered TLR-mediated induction of Survivin and Mcl-1 within 36 hrs of culture. Exposure to ibrutinib also inhibited 3H-thymidine incorporation in non-stimulated (average: 22%; range: 7-43%) and TLR7- (average: 21%; range: 7-49%) or TLR9- (average: 43%; range: 11-98%) activated CLL cells in 14/14 cases. Ibrutinib treatment resulted in an increase in TLR7-induced apoptosis in 3 of 4 CLL cases and in TLR9-induced apoptosis in 2 of 4 CLL cases. Even when CLL cells from 6 patients were cocultured with stromal cells, ibrutinib reduced cell proliferation to 45-65% of that observed in untreated cells induced via TLR7 or TLR9 alone or when combined with an anti-BCR signal. These findings reiterate the pivotal role of the microenvironment in nurturing CLL cells activated via several types of receptors. Ibrutinib, known to be efficacious in inhibiting BCR signaling, can also abrogate events downstream of TLR7 and TLR9 signaling in CLL. Importantly, inhibition of TLR 7 and 9 signaling by ibrutinib can override the protective effects from stroma-derived signals. These anti-proliferative effects of ibrutinib may be especially relevant and important for curbing concomitant activation via the BCR, TLR, and chemokine receptor pathways that likely synergize and thereby contribute to the aggressiveness of the disease. Disclosures Barrientos: Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees. Chang:Pharmacyclics, Inc: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc: Research Funding.

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.

B-cell chronic lymphocytic leukemia (CLL) clones from different patients can express stereotyped BCR immunoglobulins (IGs) that share very similar IGHV-D-J rearrangements and HCDR3s, often with similar IGLV gene usage, suggesting that they were selected by common antigenic epitope(s). Patients bearing IGHV4-34/D5-18/JH6/VK2-30/JK2 rearrangements belong to one of such stereotyped subsets, subset 4. In addition, subset 4 BCRs are always IgG switched and exhibit IGHVsomatic mutations. The germline IGHV4-34 gene is inherently autoreactive and is often found in antibodies that bind to the surfaces of erythrocytes and B lymphocytes through the linear i blood group antigen, poly-N-acetyllactosamine (NAL). To test the reactivity of subset 4 monoclonal antibodies (mAbs) to auto and foreign antigens, we expressed a panel of CLL BCR IGs carrying IGHV4-34, 1-69, 3-21, and 4-39 as recombinant human IgGs and tested the binding of these CLL mAbs to HEp-2 cells by intracellular immunostaining. Three subset 4 mAbs (183, 240 and 342) showed only weak binding to permeabilized HEp-2 cells compared to other CLL mAbs which often showed strong binding to cytoplasmic structures. Furthermore, because apoptotic cells are a source of autoantigens in autoimmune conditions, the reactivity of CLL mAbs to non-permeabilized, irradiated Ramos B and Jurkat T cell line cells was also tested by flow cytometry. Surprisingly, the average binding percentage to apoptotic Ramos B cells of subset 4 mAbs 183, 240, and 342 was only 6, 5, and 4%, respectively. Moreover, all three subset 4 mAbs exhibited minimum binding to live or apoptotic Jurkat T cells (1-10% and 4-6% to each population, respectively). We next tested the reactivity of subset 4 mAbs to i/I carbohydrate epitopes, the known targets of many non-stereotyped IGHV4-34 mAbs, using an i/I focused microarray containing 60 i/I related oligosaccharides. Notably, the subset 4 mAbs showed no binding at a concentration of 10 μg/mL. Together, the above results suggest that CLL subset 4 mAbs have weak or no interaction to the known autoantigens recognized by other non-stereotyped IGVH4-34 IGs. Hence we hypothesized that the restricted IGHV-D-Jrearrangement, somatic mutations, and IgG switch of subset 4 were acquired by selection for other auto or foreign antigens. To test this possibility, we screened CLL mAbs against a library of over 8,000 full length human proteins, including an influenza A virus and some human viral gene encoded proteins. Each subset 4 mAb showed selective and strong binding to influenza A virus H3N2 Texas 1/77 at concentration of 50 μg/mL; this binding was 6-15 times higher than that of other non-subset 4 mAbs, which included two IGHV4-34, non-stereotyped CLL mAbs. Because the binding to virus is usually mediated via its glycoprotein hemagglutinin (HA), we tested reactivity of subset 4 mAbs to the HA of influenza A H3N2 Texas 1/77. Subset 4 mAbs had on average 2 fold (1.8-2.2) higher binding signals than the two non-stereotyped IGHV4-34mAbs. To explore if subset 4 mAbs recognize other influenza A virus strains, we tested the binding of a representative subset 4 mAb 183 to HAs from 11 strains that belong to 3 subtypes: H1 (4 strains), H2 (1 strain) and H3 (6 strains). Clear preferential binding to all 6 H3s and to some of H1s was found. mAb 183 did not bind to H2 (A/Japan/305/1957 H2N2). The average binding signal ratio to H3s, H1s and H2 was 2.4:1.6:1. Testing to another hemagglutinin subtype H7 (A/Anhui/1/13 H7N9) showed that mAb 183 did not interact with H7. Intrigued by the observation that CLL subset 4 BCR IGs are always the IgG isotype, we tested if isotype switching contributed to antigen recognition by expressing CLL 183 IGHV-D-Jas divalent human IgM (mAb183M) and checked its reactivity to virus A H3N2 Texas 1/77. Switching isotype from IgG to IgM led to a 48% loss in reactivity to intact virus and 74% loss to hemagglutinin H3 (A/Philippines/2/1982 H3N2). Taken together, our results indicate that unlike other IGHV4-34 bearing IGs, CLL subset 4 mAbs have only limited reactivity to autoantigens, including linear NAL epitopes. Instead, this subset has acquired specific recognition of subtypes of an infectious agent, influenza A (H3 and H1), apparently via its envelope protein hemagglutinin. The shift in antigen recognition appears to be dependent upon both the stereotyped IGVH-D-Jrearrangement and isotype switching to IgG. Disclosures No relevant conflicts of interest to declare.

Introduction: PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib, a selective oral inhibitor of PI3Kd, demonstrated considerable clinical activity as monotherapy in recurrent (Flinn, Blood 2014) or refractory iNHL subjects (Gopal, NEJM 2014). FDA granted accelerated approval for Idelalisib (ZYDELIG®) in patients who have received at least two prior systemic therapies with relapsed FL or SLL. This study evaluated Idelalisib in combination with rituximab, bendamustine, or both. We now present mature safety and response data with up to 4 years of follow up. Methods: Eligible patients had relapsed/refractory indolent NHL. Idelalisib (Z) was administered continuously with rituximab (R) (375 mg/m2 given weekly for 8 doses) (R/Z regimen), with bendamustine (B) (90 mg/m2 given on Days 1 and 2, for 6 cycles) (B/Z regimen), or in combination with R (375 mg/m2, on Day 1) and B (90 mg/m2 given on Days 1 and 2 of each cycle, for 6 cycles (BR/Z regimen). Initial subjects in the R/Z and B/Z groups (n=8 each), received Idelalisib 100 mg/dose BID. Thereafter, all patients received an Idelalisib dose of 150 mg/dose BID. Tumor response was evaluated according to standard criteria (Cheson 2007). The cutoff date for this analysis was June 2014, 26 months after the last patient enrolled. Results: Between April 2010 and May 2012, 79 subjects with iNHL were enrolled (including 59 with FL, 15 with SLL, and 5 with MZL). Median [range] age was 61 [37-84] years. At baseline patients had elevated beta-2 microglobulin (59%), stage IV disease (58%), bulky adenopathy (> 5cm) (48%), anemia (Hgb Frequent adverse events (all grade %/grade 3-4 %) included pyrexia (54/3), nausea (44/0), fatigue (43/4), diarrhea (39/15), rash (38/9), cough (35/0), pneumonia (22/19), pneumonitis (4/3), and febrile neutropenia (3/3). Laboratory abnormalities included lymphopenia (75/62), neutropenia (56/41), anemia (47/10), thrombocytopenia (42/8), and serum transaminase elevations (56/17). Drug was temporarily held for Grade 3/4 ALT/AST elevations, and 8/13 pts (62%) were re-treated without recurrence of ALT/AST elevation. 27% of pts have discontinued therapy due to adverse events. Of the 79 subjects enrolled, 64 had an objective response with an ORR of 81% (95% CI: 70.6-89.0). Complete responses were demonstrated in 26 patients (33%), and partial responses in 38 patients (48%). In addition, 7 patients had stable disease (9%), and 4 patients had progressive disease (5%) as best response on-study. Four patients were non-evaluable, as they did not have follow up CT scans. By treatment subgroup, the ORR were (n=24/32) 75% (95% CI: 57-89) for R/Z, (n=29/33) 88% (95% CI: 72-97) for B/Z, and (n=11/14) 79% (95% CI: 49-95) BR/Z. The CR rates were 25% (n=8/32), 36% (n=12/33), and 43% (n=6/14) respectively; stable disease was noted in 4/32 patients (13%), 3/33 patients (9%), and 0/14 patients in the three groups respectively. ORR/CR by iNHL subtype is: FL (81%/39%), SLL (73%/13%), and MZL (100%/20%). The median progression-free survival is 32.8 months. Median PFS for R/Z group is 29.7 months, B/Z group 32.8 months, and BR/Z group 37.1 months. The PFS at 24 months was 55%, 64%, and 71% for the R/Z, B/Z, and BR/Z groups respectively. The median duration of response has not yet been reached. Median DOR for the R/Z group is 28.6 months, for the B/Z, and BR/Z groups it is not yet reached. The DOR at 24 months was 65%, 67%, and 64% for the R/Z, B/Z, and BR/Z groups respectively. Figure 1: Median overall survival is not yet reached. Conclusions: Idelalisib in combination therapy was well tolerated, had an acceptable safety profile, and was highly effective in this recurrent iNHL population with an ORR of 81%, and CR rate of 33%. Responses are durable beyond 2 years, supporting further evaluation of these combination regimens. Phase 3 trials evaluating the efficacy of Idelalisib in combination with R or BR in iNHL are ongoing (NCT01732913, NCT01732929). Figure 1 Figure 1. Disclosures de Vos: 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.. Wagner-Johnston:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Fowler:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Boccia:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Boyd:Gilead Sciences: Research Funding. Furman:Gilead Sciences: Research Funding. Holes:Gilead Sciences: Employment, Equity Ownership. Kim:Gilead Sciences: Employment, Equity Ownership. Godfrey:Gilead Sciences: Employment, Equity Ownership. Leonard:Gilead Sciences: Research Funding.

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Patients receiving this treatment often develop lymphocytosis and concomitant reduced organomegaly. These actions of ibrutinib are believed due to egress of CLL cells from lymphoid compartments, although the mechanism(s) responsible for this are not clear. Overexpressed surface membrane chemokine receptor CXCR4 (smCXCR4) is a hallmark of CLL cells and is involved in CLL cell migration and interaction with protective niches. Surface expression of CXCR4 is regulated by phosphorylation at serine/theronine residues in the receptor’s cytoplasmic tail. Kinases including GRK and PIM are known to regulate phosphorylation and surface expression of CXCR4. The transcript and protein levels of BTK, GRK and PIM also correlate with smCXCR4 expression. Here we studied the effect of ibrutinib on CLL cell distribution using TCL1-192 cells, a clonal murine cell line that mimics aggressive CLL and involves active B-cell antigen receptor (BCR) signaling. Similar to patients, TCL1-192-bearing mice receiving ibrutinib developed almost immediately (≤ 1 hour) lymphocytosis that consisted of both non-divided and recently-divided cells. While TCL1-192 cells overexpress smCXCR4, ibrutinib promoted lymphocytosis was associated with a fall in smCXCR4 to a level similar to that on normal B cells from wild type C3H/B6 mice. Reduced levels of smCXCR4 were also observed at the later timepoints when mice still had the continued release of cells into circulation even though lymphocytosis was no longer evident. Despite this change in smCXCR4 expression, total CXCR4 was not changed after ibrutinib treatment. Importantly, cells obtained from ibrutinib treated animals that had reduced levels of smCXCR4 also failed to respond to their ligand CXCL12 in vitro, as measured by absence of smCXCR4 internalization or calcium mobilization. Furthermore, re-expression of smCXCR4 after withdrawal of CXCL12 was not observed in treated cells. We therefore next studied in vitro the mechanism whereby ibrutinib lowered smCXCR4 expression. Western blot analysis using cells treated with ibrutinib at the dose ≥0.1μM at 37⁰C for 2 hours in the presence of CXCL12 showed significantly reduced levels of phosphorylated CXCR4 at Ser339. Phosphorylation of Ser339 in the CXCR4 intracellular domain is known to be essential for normal receptor recycling, hyper-phosphorylated CXCR4 on Ser339 is associated with overexpressed smCXCR4 in CLL patient cells. Correlating with this, TCL1-192 cells treated with ibrutinib at 0.1μM not only de-phosphorylated CXCR4 on Ser339, but also significantly enhanced internalization of smCXCR4 after CXCL12 stimulation. We then examined the kinases responsible for the de-phosphorylation of CXCR4. Western blot analysis of CLL cells from animals treated with ibrutinib for 4 weeks showed minimal levels of total BTK protein. There was a direct positive correlation in between BTK protein expression and smCXCR4 expression. In addition to BTK, reduced levels of kinases known to regulate CXCR4 phosphorylation on Ser339 were also observed in cells collected from ibrutinib treated mice. Together, these results suggest blocking BTK, directly or indirectly causes de-phosphorylation of CXCR4, resulting in impaired smCXCR4 recycling and blocked CXCL12 signaling. The function of CXCR4 is dependent on its location on the cell membrane. Impaired CXCR4 recycling after ibrutinib treatment would lead to defective cell trafficking. Indeed, cell transfer studies involving TCL1-192 cells previously treated with ibrutinib indicated that persistent decrease in smCXCR4, which was ibrutinib-dependent, blocked the return of CLL cells to solid tissue niches. Finally, ibrutinib-fed mice eventually developed lower lymphocyte counts, reduced organomegaly, and prolonged survival. Ibrutinib-fed mice also had defective response to BCR stimulation, including abolished calcium mobilization, blocked cell proliferation and survival after anti-IgM antibody stimulation. Together, the data provide direct in vivo evidence of ibrutinib impairing CLL cell homing and retention and suggest a mechanism by which BTK targeted therapy affects not only BCR signaling but also CXCR4 phosphorylation and signaling, with the latter contributing to defective smCXCR4 expression. Disclosures Chang: Pharmacyclics, Inc: Employment, Equity Ownership. Chang:Pharmacyclics: Employment. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding.

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) is linked to clonal growth within pseudo-follicles, typically within lymph nodes, bone marrow and spleen, and occasionally lungs and skin. Both the clone’s antigen receptor and stromal milieu appear to influence its growth rate. An involvement of TLR signals seems probable based on atypically elevated TLR-9 expression within B-CLL cells and the likelihood that the specificity of B-CLL antigen receptors (BCR) facilitates the internalization of molecules from apoptotic cells and/or microbes that are physically linked to CpG DNA. Nevertheless, recent findings that a large subset of B-CLL undergoes in vitro apoptosis upon stimulation with CpG-rich oligodeoxynucleotides (ODN) raised questions about a central role for TLR-9 signaling. Using a CFSE-based model for examining in vitro B-CLL clonal expansion/viability and a cohort consisting of 19 IGHV mutated (M) and 19 unmutated (U) B-CLL, we report that TLR-9 signaling is uniformly stimulatory when accompanied by signals from IL-15. Importantly, this cytokine is known to be constitutively produced by stromal cells in normal bone marrow, lymph nodes, and spleen and in a constitutive/inducible manner within skin and lungs. We show that B-CLL display reproducible inter-clonal differences in the number of division cycles attained and/or lymphoblast survival that were not linked to IGHV mutation status, but were statistically linked to whether the patient leukemic population contained subclones with trisomy-12 (p=0.0003) or contained subclones with both an ATM anomaly (11q22 del and/or ATM mutation) and 13q14 del (p=0.009). When all B-CLL clones were assessed, in vitro high-division or high-viability status in response to ODN + IL-15 was not statistically linked to clinical progression as determined by time to first treatment (TFT). Nonetheless, in vitro high-division status showed a statistically-significant direct linkage to patient survival (OS) (p=0.019 for OS within B-CLL manifesting > 50% cells with > 2 divisions versus B-CLL with < 50% cells with > 2 divisions). Subdivision of the total cohort into U-CLL and M-CLL subsets revealed that the link of high division status with overall survival is most characteristic of U-CLL. Immunohistological evidence of IL-15-producing cells within or proximal to Ki-67-positive pseudo-follicles in B-CLL-infiltrated spleen is consistent with a role for ODN + IL-15 signaling in promoting in vivo leukemic cell growth. Taken together, the findings from this study support the concept that in vivo B-CLL clonal expansion is dependent upon leukemic B-CLL homing to tissue sites where IL-15 is typically sequestered along with intrinsic properties of the B-CLL clone, e.g. cytogenetic anomalies within members of a B-CLL clone that heighten leukemic cell growth and/or survival and an expression of U-BCRs specific for apoptotic cell debris that increase the opportunity for TLR-9 signaling. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: PI3Kδ signaling is critical for the proliferation and survival as well as for homing and tissue retention of malignant B cells. Idelalisib is a first-in-class, targeted, highly selective, oral inhibitor of PI3Kδ with considerable activity as monotherapy and in combination with R in patients with relapsed/refractory CLL, and was 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 chemo- and immuno-chemotherapies. METHODS: 74 subjects were sequentially enrolled into one of 5 regimens of idelalisib combined with either B (70 or 90 mg/m2 IV on D1,2 of cycles 1-6, N=18, enrolled Apr 2011-Nov 2011), BR (B: 70 mg/m2 IV on D1,2 of cycles 1-6; R: 375 mg/m2 IV on D1 of cycles 1-6, N=15, enrolled Apr 2011-Aug 2011), F (40 mg/m2 po D1-5 of cycles 1-6, N=12, enrolled Apr 2011-Aug 2011), Chl (10 mg/m2 po D1-7 x 3(min)-12(max) cycles, N=15, enrolled Mar 2012-Aug 2012), or ChlR (N=14, enrolled Mar 2012-Jul 2012). Idelalisib was administered at 100 (4 of the pts receiving B) or 150 mg po BID (all other patients) continuously. Patients on treatment after 48 weeks were eligible to continue idelalisib on an extension study. Clinical responses were evaluated according to published criteria (Hallek 2008; Cheson 2012). RESULTS: Of 74 subjects enrolled, 66% were male. Median age was 65 years, and median time since diagnosis was 8 years with a median of 3 (range 1-9) prior regimens. Overall, prior therapies included R (97%), F (78%), B (45%), and Chl (14%), and 55% of patients were refractory to last therapy. 65% of patients had Rai stage III/IV disease at enrollment. Adverse prognostic factors included del(17p) and/or TP53 mutation (30%), IGHV unmutated status (82%), NOTCH1 mutation (28%). As of 7/15/2014, the median idelalisib exposure for patients of all cohorts was 12.8 (range 1-48) months. 41 (55%) of patients completed the 48 weeks of the primary study, and 20 subjects (27%) were still on treatment on the extension study at the time of analysis. The most common reasons for discontinuation reported by investigators were adverse events (AEs) (15, 20%) or progressive disease (PD) (12, 16%). There were 13 deaths reported on study; 4 patients experienced PD before death. Selected treatment-emergent AEs (any Grade/≥Gr 3, regardless of causality) included diarrhea/colitis (51%/18%), pyrexia (47%/5%), fatigue (35%/7%), cough (35%/0%), nausea (31%/1%), pneumonia (23%/14%), rash (22%/7%), dyspnea (19%/3%), and pneumonitis (3%/3%). Elevation of liver transaminases (TA, any Grade/≥Gr 3) was seen in 37%/14%. Of those, only 1 patient discontinued the study because of (recurrent) TA elevation. Richter’s transformation as category of progressive disease was reported in 1 patient (3%). The ORR was 82% for all 74 patients with a CR rate of 10%. 4 of 74 patients (5%) were not evaluable because of missing follow-up assessments. Among 69 subjects with genetic data available, the ORR in the 22 subjects with either del(17p) and/or TP53 mutation was 68% vs. 87% among 47 subjects with neither aberration. The overall median PFS was not reached at the time of analysis (Figure 1); KM estimate of PFS was 57% [43-72%, 95% CI] at 24 months. Overall median time to response was 1.9 months, and overall median DOR was not yet reached. CONCLUSIONS: These preliminary results in patients with relapsed or refractory disease and multiple prior therapies suggest that idelalisib can be combined with B, BR, F, Chl, and ChlR with acceptable safety and that these combinations have substantial clinical activity. A Phase 3 study evaluating the combination of idelalisib with BR in relapsed CLL is ongoing (NCT01732926). Figure 1: Overall PFS Figure 1:. Overall PFS Disclosures Barrientos: 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.. Coutre:Gilead Sciences: Research Funding. de Vos:Gilead Sciences: Research Funding. Wagner-Johnston:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Boyd:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Leonard:Gilead Sciences: Research Funding. Kim:Gilead Sciences: Employment, Equity Ownership. Viggiano:Gilead Sciences: Employment, Equity Ownership. Jahn:Gilead Sciences: Employment, Equity Ownership. Furman:Gilead Sciences: Research Funding.

Background: Chronic lymphocytic leukemia (CLL) patients exhibit variable times from diagnosis to the need to initiate therapy. Previous studies have demonstrated that the rates of CLL cell proliferation (birth) and elimination (death plus recirculation to solid tissues) could be estimated using the incorporation of deuterium (2H), taken in the form of heavy water (2H2O), into the DNA of dividing cells. In a cross-sectional study of patients with early and late stage CLL, birth rates above 0.35% per day were associated with more aggressive disease. The NIH-sponsored CLL Research Consortium (CRC) conducted a prospective study on patients with early stage disease to assess the relationship between CLL-cell birth rate and the length of time to initiation of initial therapy. Methods: Eligible patients had untreated early stage (Rai 0, 1 or 2) disease, and were no more than 3 years from diagnosis. Additional clinical parameters included age at diagnosis, gender, mutational status of the immunoglobulin heavy chain variable region (IGHV), 70-kD zeta-associated protein (ZAP-70) (binary split at 20%) and CD38 (binary split at 30%) expression, and FISH cytogenetics by the Dohner hierarchy. Heavy water was administered daily for 6 weeks, and peripheral blood was obtained for kinetic measurements at the end of weeks 3, 6, 9, 12, and 16. CLL birth rates for blood samples were measured at the same facility (KineMed, Inc, Emeryville, CA) by quantifying deuterium incorporation into the deoxyribose moiety of DNA of tumor cells, using isotope ratio mass spectrometry (IRMS). Subjects were followed for time to administration of therapy; subjects still without treatment at time of analysis were censored, as were subjects who exited the study to receive treatment outside of the established criteria. To assess the impact of birth rate and other factors on time to first therapy, Kaplan-Meier curves were compared using the logrank test for univariable analyses; stepwise Cox proportional hazards regression models were used to build multivariable models. Results: Between July 2005 and February 2009, 119 subjects were enrolled through the CRC. 107 subjects were eligible. For 10 eligible patients, exit from the study prior to completion of protocol-mandated heavy water consumption (n=5) or technical issues (n=5) made it impossible to obtain estimates of birth rate. The 97 eligible subjects for whom birth rates were available form the basis of this report. 60% of subjects were male; median age 57 years (range 40-85). Median birth rate was 0.32%/day (range 0.07-1.31%/day). 43 subjects (44%) had birth rates higher than the previously reported threshold of 0.35%/day. With a median follow up of 4 years, 33 eligible subjects (34%) required treatment. IGHV status (31 subjects unmutated), elevated ZAP-70 (31 subjects), elevated CD38 (21 subjects), and hierarchical categorization of FISH anomalies (4 del(17p), 5 del(11q), 8 tri(12), 50 del(13q)) were examined for association with differential time to first therapy in univariable analyses. All were associated with earlier need for therapy (p 0.35%/day was also significantly associated with shorter time to first therapy (p < 0.001). In multivariable modeling, only birth rate and IGHV status met the criteria for inclusion (Wald p-value < 0.05). Hazard ratios were 3.51 (p = 0.002) for birth rate above 0.35%/day and 5.0 for unmutated IGHV status (p < 0.001). The association between elevated birth rate and shorter time to first treatment was observed within subjects with both unmutated and mutated IGHV (see Figure 1). Conclusion: An increased birth rate of CLL cells early in the disease course is a strong predictor of an earlier need for treatment in univariable analysis, as are unmutated IGHV, elevated ZAP-70 expression and elevated CD38 expression. However, only unmutated IGHV and an increased birth rate contribute significantly to the multivariable model. This adds a direct measure of B-cell kinetics to the array of biomarkers available to subjects with CLL. Figure 1 Time to treatment by birth rate and IGHV status Figure 1. Time to treatment by birth rate and IGHV status Disclosures Murphy: KineMed, Inc: Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Li:KineMed, Inc.: Employment, Equity Ownership. McConnel:KineMed, Inc.: Equity Ownership. Turner:KineMed, Inc.: Employment, Equity Ownership. Busch:KineMed, Inc.: Equity Ownership. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Chiorazzi:KineMed, Inc.: Stock options Other.

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 In summary, CLL patients demonstrate an altered HLA-DRlo/CD11b+/CD33+ population with significantly more g-MDSCs than m-MDSCs. CLL g-MDSCs are functional suppressors, whereas m-MDSCs are not, even though they exhibit increased levels of inhibitory molecules and can perform this function when induced from CD33+ cells. The latter suggest that that the poor suppressor activity of patient derived m-MDSCs is induced in vivo. Finally, different clinicobiological characteristics associate with the phenotype of MDSCs, especially an increase in suppressor molecules and need to treat. Disclosures No relevant conflicts of interest to declare.

B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.

PURPOSE: We sought to determine whether therapy with single-agent fludarabine compared with chlorambucil alone or the combination of both agents had an impact on the incidence and spectrum of infections among a series of previously untreated patients with B-cell chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: Five hundred fifty-four previously untreated CLL patients with intermediate/high-risk Rai-stage disease were enrolled onto an intergroup protocol. Patients were randomized to therapy with chlorambucil, fludarabine, or fludarabine plus chlorambucil. Data pertaining to infection were available on 518 patients. Differences in infections among treatment arms were tested with the Kruskal-Wallis, Wilcoxon, and χ2 tests. RESULTS: A total of 1,107 infections (241 major infections) occurred in 518 patients over the infection follow-up period (interval from study entry until either reinstitution of initial therapy, therapy with a second agent, or death). Patients treated with fludarabine plus chlorambucil had more infections than those receiving either single agent (P < .0001). Comparing the two single-agent arms, there were more infections on the fludarabine arm (P = .055) per month of follow-up. Fludarabine therapy was associated with more major infections and more herpesvirus infections compared with chlorambucil (P = .008 and P = .004, respectively). Rai stage and best response to therapy were not associated with infection. A low serum immunoglobulin G was associated with number of infections (P = .02). Age was associated with incidence of major infection in the combination arm (P = .004). CONCLUSION: Combination therapy with fludarabine plus chlorambucil resulted in significantly more infections than treatment with either single agent. Patients receiving single-agent fludarabine had more major infections and herpesvirus infections compared with chlorambucil-treated patients.

We thank Dr Foon for his letter, which allows us to clarify a few important points. The first question relates to the definition of (complete) response by the examination of peripheral blood counts and marrow. This point has been addressed by the final version of the manuscript[1][1]: With regard

The presence of hemolytic anemia (AIHA) and immune-mediated thrombocytopenia (ITP) poses a significant challenge in treating patients with chronic lymphocytic leukemia (CLL). Treatment with purine analogs has been known to induce autoimmune cytopenias, typically hemolytic anemia, but these occurrences are seen de novo as well. The pathophysiology of these autoimmune processes is believed to result from a defect or paucity of normal Tregulatory cells [1‐4]. Not only can the arrest of the autoimmune process be difficult, but its presence can preclude the use of purine analogs—arguably the most effective agents in the treatment of CLL itself. Although autoimmune cytopenias can present at any time in the disease course, they are more frequently seen in the later stages [5], making the requirement of treating both the CLL disease and a concomitant autoimmune process (‘complex autoimmune cytopenias’) a relatively common and difficult scenario. Purine analogs notwithstanding, overlap does exist between the treatments of immune cytopenias and CLL disease. Corticosteroids, and more recently rituximab and alkylating agents, have been shown to be effective in the treatment of AIHA and ITP; these agents are mainstays in the treatment of CLL itself. Recently, we published the results of a retrospective study using the combination of rituximab, cyclophosphamide, and dexamethasone (RCD) in 21 patients with CLL-associated immune cytopenias. The immune cytopenias in all of these patients responded, with a median duration of response of 22 months. Though this combination is effective in the treatment of CLL itself, we did not report on these results [6]. In this issue of Leukemia and Lymphoma, Bowen and colleagues report on 20 patients using the combination of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) in the treatment of progressive CLL complicated by autoimmune cytopenias (complex autoimmune cytopenias) [7]. The response rates achieved for both the immune phenomena as well as the progressive CLL are promising. A substantial number of complete responses (CRs) in each condition were achieved as well. The small sample size and the relatively short durations of response are obvious limitations to both the study and the therapy. Despite the limitations, the work of Bowen and colleagues provides an important evidence-based recommendation for treating autoimmune phenomena and CLL simultaneously. To our knowledge, little or no evidencebased guidance in the literature exists for dealing with the practical challenge posed by ‘complex autoimmune cytopenias’ faced by clinicians treating CLL. Additional combinations and newer agents will likely prove useful in these patients. Antibodies affecting the autoimmune phenomena are believed to be polyclonal, and not directly originating from the CLL clone. Nevertheless, newer therapies targeting B-cells in CLL will also likely decrease the activities of the aberrant polyclonal B-lymphocytes. The recent approval of ofatumumab, and the fast-coming addition of other monoclonal agents targeting B-cell antigens, broaden the potential options for new approaches in complex autoimmune cytopenias.

Fludarabine is an effective treatment for chronic lymphocytic leukemia that does not respond to initial treatment with chlorambucil. We compared the efficacy of fludarabine with that of chlorambucil in the primary treatment of chronic lymphocytic leukemia.Between 1990 and 1994, we randomly assigned 509 previously untreated patients with chronic lymphocytic leukemia to one of the following treatments: fludarabine (25 mg per square meter of body-surface area, administered intravenously daily for 5 days every 28 days), chlorambucil (40 mg per square meter, given orally every 28 days), or fludarabine (20 mg per square meter per day for 5 days every 28 days) plus chlorambucil (20 mg per square meter every 28 days). Patients with an additional response at each monthly evaluation continued to receive the assigned treatment for a maximum of 12 cycles.Assignment of patients to the fludarabine-plus-chlorambucil group was stopped when a planned interim analysis revealed excessive toxicity and a response rate that was not better than the rate with fludarabine alone. Among the other two groups, the response rate was significantly higher for fludarabine alone than for chlorambucil alone. Among 170 patients treated with fludarabine, 20 percent had a complete remission, and 43 percent had a partial remission. The corresponding values for 181 patients treated with chlorambucil were 4 percent and 33 percent (P0.001 for both comparisons). The median duration of remission and the median progression-free survival in the fludarabine group were 25 months and 20 months, respectively, whereas both values were 14 months in the chlorambucil group (P0.001 for both comparisons). The median overall survival among patients treated with fludarabine was 66 months, which was not significantly different from the overall survival in the other two groups (56 months with chlorambucil and 55 months with combined treatment). Severe infections and neutropenia were more frequent with fludarabine than with chlorambucil (P=0.08), although, overall, toxic effects were tolerable with the two single-drug regimens.When used as the initial treatment for chronic lymphocytic leukemia, fludarabine yields higher response rates and a longer duration of remission and progression-free survival than chlorambucil.

Response We are grateful to both Hanson and colleagues and Mulligan and his colleagues for welcoming our recent revision of the 1996 NCI CLL Guidelines and for raising several issues surrounding the diagnostic criteria of CLL and monoclonal B lymphocytosis (MBL). They particularly referred to the

Background : It is uncertain which people with chronic myelogenous leukemia (CML) in chronic phase should receive conventional treatment (interferon and/or chemotherapy) versus high-dose therapy and a bone marrow transplant. There are no randomized trials comparing these approaches and analyses of data from non-randomized studies are complex, contradictory without sufficient detail to allow subject-level treatment decisions. Objective : Determine appropriateness of high-dose therapy and bone marrow transplants in persons with CML in chronic phase with specific features. Develop a treatment algorithm. Panelists : nine leukemia experts from diverse geographic sites and practice settings. Evidence : Boolean MEDLINE searches of chronic myelogenous leukemia and chemotherapy and/or transplants . Consensus process : We used a modified Delphi-panel group judgment process. Age, prognostic score, disease duration, and type of conventional therapy and response were permuted to define 90 clinical settings. Each panelist rated appropriateness of high-dose therapy and a transplant versus conventional therapy on a 9-point ordinal scale (1, most inappropriate, 9, most appropriate) considering three types of donors: (1) HLA-identical siblings; (2) alternative donors (HLA-matched related or unrelated people other than an HLA-identical sibling); and (3) autotransplants. An appropriateness index was developed based on median rating and amount of disagreement. Relationship of appropriateness indices to permuted clinical variables was considered by analysis of variance and recursive partitioning. Preference between donor types was analyzed by comparing mean appropriateness indices of similar settings and a treatment algorithm developed. Conclusions : In people with CML in chronic phase and an HLA-identical sibling donor and in those with an alternative donor (but no HLA-identical sibling), a transplant was rated appropriate in those with a≤partial cytogenetic response to interferon and uncertain or inappropriate in all other settings. Autotransplants were rated uncertain or inappropriate in all settings. Most of the variance in appropriateness ratings between different clinical settings was accounted for by response to interferon: complete versus≤partial response. An HLA-identical sibling donor, when available, was always preferred to an alternative donor or autotransplant. In people without an HLA-identical sibling, an alternative donor was favored over an autotransplant at higher appropriateness indices and the converse at lower appropriateness indices.

Background: Despite considerable data, there is still controversy over which adults with acute myelogenous leukemia (AML) in 1st remission should receive high-dose therapy and a bone marrow transplant rather than conventional-dose chemotherapy. Analyses of data from randomized trials are complex, conclusions sometimes contradictory and results not sufficiently detailed to allow subject-level decisions.Objective: To determine appropriate use of high-dose therapy and bone marrow transplants in persons with AML in 1st remission with specific features. Develop a treatment algorithm.Panelists: Nine leukemia experts from diverse geographic sites and practice settings.Evidence: Boolean MEDLINE searches of acute myelogenous leukemia and chemotherapy and/or transplants.Consensus process: We used a modified Delphi-panel group judgment process. Age, WBC, cytogenetics and FAB-type were permuted to define 72 clinical settings. Each panelist rated appropriateness of high-dose therapy and a transplant versus conventional-dose chemotherapy on a nine-point ordinal scale (1, most inappropriate, 9, most appropriate) considering 3 types of donors: (1) HLA-identical siblings; (2) alternative donors (HLA-matched related or unrelated people other than an HLA-identical sibling); and (3) autotransplants. An appropriateness index was developed based on median rating and amount of disagreement. The relationship of appropriateness indices to the permuted clinical variables was considered by analysis of variance and recursive partitioning. Preference between donor types was analyzed by comparing mean appropriateness indices of comparable settings and a treatment algorithm developed. Conclusions: In people with an HLA-identical sibling, this type of transplant was rated appropriate in those with unfavorable cytogenetics and uncertain in all other settings. In people without an HLA-identical sibling, an alternative donor transplant was rated appropriate in those 30 years and unfavorable cytogenetics and inappropriate in all other settings. Autotransplants were rated appropriate in people with unfavorable cytogenetics and uncertain in all other settings. An HLA-identical sibling donor, when available, was always preferred to an alternative donor transplant or autotransplant. In people without an HLA-identical sibling, an autotransplant was almost always favored over an alternative donor transplant with the magnitude of preference inversely correlated with transplant appropriateness.

PURPOSE To analyze initial and long-term outcomes after treatment of patients with active hairy-cell leukemia (HCL) with a single cycle of cladribine (2-CdA). PATIENTS AND METHODS Forty-nine patients with active HCL were treated with 2-CdA by continuous intravenous infusion at 0.1 mg/kg/d for a total of 7 days at the Long Island Jewish Medical Center between September 1990 and August 1992. Here we report on all patients followed-up until April 1996. RESULTS At 3 months after treatment, complete response (CR) occurred in 37 patients (76%) and partial response (PR) occurred in 12 patients (24%), for an overall response rate of 100% (95% confidence interval, 94% to 100%). At a median follow-up of 55 months, the relapse-free survival is 80% and overall survival is 95%. Ten patients (20%) have relapsed. Of the 26 patients in whom lymphocyte phenotyping was performed, four were found to have a CD25-negative phenotype. All four of these patients had PRs only and all relapsed. Eight patients have been re-treated with 2-CdA, and all achieved at least a partial remission; two of these have already relapsed with remission durations of less than 1 year. Five second malignancies have occurred in four patients. CONCLUSION With a median follow-up of more than 4 years, 39 patients (80%) continue in remission. Only two deaths have occurred. A CD25-negative phenotype may predict for a poorer response to 2-CdA. Patients who relapse may be re-treated with 2-CdA, but subsequent remissions may be of shorter duration. There has not been a markedly increased incidence of second malignancies or late opportunistic infections.

Introduction PI3Kδ signaling is critical for the proliferation and survival as well as for homing and tissue retention of malignant B cells. Idelalisib is a first-in-class, targeted, highly selective, oral inhibitor of PI3Kδ that has shown considerable monotherapy activity in patients with heavily pretreated CLL. Methods This Phase 1 study evaluated idelalisib for relapsed/refractory CLL continuously given at 150 mg BID 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, 21 with O. Adverse disease characteristics (n, %) included Rai Stage III/IV (20, 50%), bulky lymphadenopathy (24, 60%), refractory disease (14, 35%), multiple prior therapies (median 2, range: 1-;9). Almost all patients (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 43% 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 May 2013, the median (range) treatment duration was 18 (0-33) months. 23 (58%) pts have completed the primary study and enrolled into the extension study. A total of 14 pts (35%) were continuing idelalisib treatment on the extension study. The most common reasons for discontinuation either from the primary or extension study were disease progression (10, 25%) and adverse events (AEs) (9, 23%). There were 6 deaths reported on study; 3 pts experienced PD before death. Selected treatment-emergent AEs (any Grade/≥Gr 3, regardless of causality) included diarrhea (53%/10%), cough (40%/3%), pyrexia (40%/3%), dyspnea (30%/3%), fatigue (25%/0%) nausea (25%/0%), rash (20%/0%), pneumonia (18%/15%), colitis (10%/10%) and pneumonitis (10%/7.5%). Elevation of liver transaminases (TA, any Grade/≥Gr 3) was seen in 30%/10%. Of those, only 1 pt discontinued the study because of (recurrent) TA elevation. The ORR (N=40) was 83% (33/40), with 3 CRs (8%), and a median (range) time to response of 1.9 (1.7-21.8) months. Median progression-free survival (PFS) for all patients (N=40) and duration of response (n=33) were 20 and 19 months, respectively. Median overall survival (OS) has not been reached. For the 11 pts with del(17p) and/or TP53 mutations, the response rate was 73% (8/11) and the median PFS and DOR were 19 months. Conclusions Combinations of idelalisib with therapeutic anti-CD20 antibodies such as rituximab or ofatumumab represent non-cytotoxic regimens with acceptable safety profiles and high activity resulting in durable tumor control in pts with heavily pretreated relapsed/refractory CLL. Phase 3 trials evaluating the efficacy of idelalisib in combination with R or O are ongoing (NCT01539512, NCT01659021). Disclosures: Furman: Gilead Sciences: Research Funding. Off Label Use: Idelalisib is a PI3K-delta inhibitor currently in phase III trials for multiple hematologic malignancies. De Vos:Gilead Sciences: Research Funding. Leonard: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. Rai:Gilead Sciences: Research Funding. Kim:Gilead Sciences: Employment. Holes:Gilead Sciences: Employment. Dansey:Gilead Sciences: Employment. Jahn:Gilead Sciences: Employment. Coutre:Gilead Sciences: Research Funding.

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.

Ultra-deep sequencing has revolutionized our ability to acquire large amounts of genetic data. We have applied this technology towards understanding the mutational process in B-cell chronic lymphocytic leukemia (CLL), which may be a key to understanding CLL pathogenesis. Acquisition of new cytogenetic aberrations and gene mutations in the CLL clone is associated with worse patient outcome. CLL is not unique in this aspect, as new somatic mutations and DNA rearrangements are also found during the evolution of other solid and liquid tumors. In many of these, activation-induced deaminase (AID), an enzyme normally expressed in germinal center B lymphocytes to induce IGHV-D-J mutations and isotype class switch recombination, is abnormally expressed. Its mutational activity, acting outside of the Ig loci, is implicated in the evolution to more aggressive disease. In CLL, the detection of leukemic cells expressing AID ex vivo correlates with significantly shorter patient survival. To test if AID mutational activity is functional in CLL cells and therefore could contribute to CLL evolution, we analyzed mutations in IGHV-D-J, the preferred substrate for AID. Because the rate of AID-induced mutation is low and only a small percentage of CLL cells express AID ex vivo, we used ultra-deep sequencing to analyze CLL cells that were activated under conditions that simulate the CLL microenvironment. Specifically, CLL cells were activated (1) in vitro by simulating the provision of T-cell help or (2) in vivo after adoptive transfer into alymphoid recipient mice, which requires the presence of T-cells for CLL cell growth. Each of these conditions induce AID in a large fraction of CLL cells. To analyze IGHV-D-J mutations, the specific CLL clone IGHV was amplified from cDNA obtained on day 0 or from the activated CLL samples using IGHV family-specific and IGHM primers to enable subsequent comparison of IGHV-D-J with IGHM mutation frequencies. Three unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were sequenced with the Roche 454 FLX system, resulting in a total of 1,367,522 sequence reads. After using the Roche 454 algorithm to trim sequence reads, they were prepared using custom R scripts that separated 5’ IGHV and 5’ IGHM primer sequences, aligned sequences to the CLL clone IGHV-D-J rearrangement, and removed poor quality ( To evaluate AID mutational characteristics in new subclones, SHMTool (http://scb.aecom.yu.edu/shmtool) was employed to calculate mutation frequencies in IGHV-D-J relative to the IGHM constant region, at AID mutation hotspot sites (GYW or WRC), at AID mutation coldspot sites (SYC or GRS), at C/G base pairs, and at error-prone DNA polymerase eta repair hotspot sites (WA or TW). To calculate statistical significance, we utilized a custom R script that used a bootstrap method to account for the large sample sizes provided by ultra-deep sequencing as well as to correct for differences in sequencing sample size. All samples showed an increase in IGHV-D-J versus IGHM mutations after T cell activation. Five of 6 cases showed an increase in AID hostpot mutation frequency. AID coldspot mutation frequency decreased in 3/6 CLL cases. Percent transition mutation at C/G sites was higher than random in 2/6 CLL cases, which correlated with low frequencies of DNA polymerase eta hotspot mutation. In the other 4/6 CLL cases, the lower percent transitions at C/G sites may reflect the contribution of error-prone DNA repair. In summary, we developed a method to analyze ultra-deep IGHV-D-J sequences that revealed AID mutational characteristics in both U-CLL and M-CLL cells after activation with T-cell help in vitro or in vivo. These data are consistent with the hypothesis that AID, perhaps along with error-prone DNA repair, creates new mutations leading to the evolution of aggressive CLL. Disclosures: Rai: Sanofi: Membership on an entity’s Board of Directors or advisory committees; GSK: Membership on an entity’s Board of Directors or advisory committees; Teva: Membership on an entity’s Board of Directors or advisory committees; Genentech: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

Introduction Chronic lymphocytic leukemia (CLL) develops in a stepwise fashion, starting in a restricted set of normal B lymphocytes that clonally expand, presumably due to antigen stimulation, reaching levels exceeding normal homeostasis but less than required for a diagnosis of CLL. Immunologic, genetic, and epidemiologic studies suggest these clonal expansions, termed monoclonal B lymphocytosis (MBL), are requisite precursors of CLL. Although ∼5% of normal subjects over age of 60 years old exhibit MBL, only ∼1% evolve to overt CLL each year. Hence, it is likely the genetic factors leading to the development of MBL from normal B cells are not sufficient to automatically lead to CLL and additional genetic lesions are needed for the final conversion to leukemia. To understand the development of MBL and its evolution to CLL, we investigated gene expression profiles of normal blood (N) B cells, MBL cells, and CLL cells using microarray technology. Methods RNA was purified from 31 N CD19+ B cells, 21 CD19+CD5+CD20dimIgL-restricted MBL cells, and 65 CD5+CD19+ CLL cells. Microarray assays were performed using Illumina Human HT12 BeadChips. Genes differentially expressed between the 3 populations were identified (MBL vs N and MBL vs CLL) and sets of significant genes (≥1.5 fold change and P Results Focusing on comparisons between MBL and N B cells, 1040 genes were higher and 868 lower in MBL than N B cells. Genes higher in MBL fall into different IPA categories including “PI3K/AKT Signaling” and “Inflammatory Disease”, thereby further underscoring a potential role of antigenic/inflammatory stimuli at the origin of MBL. In the “Inflammatory Disease” category, genes belonging to IFNα pathway were over-expressed in MBL. Eighteen of these IFN-associated genes overlapped with 74 genes in a type I IFN signature characteristic of systemic lupus erythematosus (SLE). Interestingly, 4 genes belonging to the “Post-Translational Modification” category that were more expressed in MBL - SPOP, SPK1, SRRM1, and ADAR (all P In contrast, the 683 genes more expressed in CLL than MBL B cells fall into the “Cell Death and Survival” and “Cancer” categories. Of note, several of the genes in the Wnt pathway over-expressed between MBL and N were lower in CLL B cells; however, since these comparisons were not paired (i.e., MBL clones that evolved to CLL were not studied), some of these differences may be spurious since we expect that not all MBL cases would evolve into CLL. Discussion Our studies implicate several pathways in the development and evolution of MBL. First, an interferon pathway, similar to that activated in SLE, may be operational in MBL, hinting such signaling is involved in the amplification of MBL clones and associating CLL precursors with autoimmunity, a well documented fact for overt CLL cells. Second, over-representation of genes involved in RNA processing suggests this function is also important in MBL. This again associates MBL with CLL and autoimmunity as splicing factor mutations have been identified in CLL and the small nuclear ribonucleoproteins involved in splicing are often autoantibody targets in SLE. Of note, mutations that affect SF3B1 and its splicing function have been recently described in a subset of CLL patients and implicated in its pathogenesis. Finally, our data strongly incriminate the Wnt/b-catenin pathway in MBL. Since both Wnt ligand and receptor genes are upregulated, autocrine as well as paracrine loops may be operative. It is interesting that the WNT effector, LEF1, which has been reported as upregulated in MBL previously, is an interferon-responsive gene, possibly linking those two pathways. Finally, if not an artifact of sample availability, it is curious that most of the Wnt pathway genes diminish in expression at the level of manifest CLL. Additional studies are needed to determine the functional relevance of our observations to MBL and CLL B-cell biology. Disclosures: No relevant conflicts of interest to declare.

Background PI3K-delta signaling is critical for proliferation, survival, homing and tissue retention of malignant B cells. Idelalisib is a selective, oral inhibitor of PI3Kδ that has been previously shown to be safe and tolerable and has demonstrated considerable activity as monotherapy or in combination with other agents in patients with relapsed/refractory (R/R) CLL. We provide an update on the safety and efficacy of a phase 1 study of idelalisib (IDELA) 150mg po BID administered continuously on a 28-day cycle in combination with two chemo-immunotherapy regimens: bendamustine/rituximab (IDELA+BR) and chlorambucil/rituximab (IDELA+ChR). Methods Twenty-nine adult subjects (15/14 IDELA+BR; IDELA+ChR) with R/R CLL requiring treatment were enrolled between April and Aug 2011 for the IDELA+BR cohort and Mar and Jul 2012 for the IDELA+ChR cohort. Median age 64 yrs (Min, Max: 41, 82), gender M/F (%) 65/35, WHO PS (%) 0/1/2 59/38/3, current Binet stage (%) A/B/C 21/28/41, and 62% of subjects had bulky adenopathy (presence of ≥1 node with diameter ≥5 cm). Median number of prior therapies was 3 (Min, Max: 1, 9). Thirty-five percent of subjects had refractory disease (not responding to a standard regimen or progressing within 6 month of the last course of a standard regimen) and 55% of subjects were refractory to rituximab. The treatment schedule was as follows: IDELA+BR (IDELA 150 mg BID po d1-28 until progression or withdrawal, rituximab 375 mg/m2 IV on d1 of cycles 1-6, bendamustine 70 or 90 mg/m2 intravenously on d1 and d2 of cycles 1-6). For the IDELA+RCh cohort (IDELA 150 mg BID po on d1-28 until progression or withdrawal, chlorambucil 10 mg/m2 po once a d1-7 of cycles 1-12, rituximab 375 mg/m2 IV d1 of cycles 1-6). Response was assessed by the investigators based on scheduled CT evaluations and clinical criteria following IWCLL 2008. Results The data cut-off date for this analysis was May 2013. Objective response rates for the 2 cohorts were 89.7% (95% CI (%): 72.6-97.8). Median time to response in both cohorts was 1.9 months. For IDELA+BR, the ORR was 86.7% (95% CI (%): 59.5-98.3) (CR 6.7%, PR 80%), neither median DOR nor median PFS have been reached and median exposure was 18.4 months (Min, Max: 1.2, 24.7). For IDELA+ChR, the ORR was 92.9% (95% CI (%): 66.1-99.8) (CR 14.3%, PR 78.6%), neither median DOR nor median PFS has been reached, and median exposure was 7.7 months (Min, Max: 1.9, 11.1). Commonly reported treatment-emergent AEs (≥20% of all subjects) and lab abnormalities of interest for IDELA+BR were (total(%)/≥grade 3 (%)): pyrexia (46.7/0) diarrhea (26.7/13.3), fatigue (20/0), neutropenia (86.7/60), thrombocytopenia (26.7/6.7), transaminase elevations (26.7/0), anemia (33.3/13.3). For IDELA+ChR, common TEAEs were: pyrexia (57.1/7.1), diarrhea (64.3/7.1), fatigue (42.9/21.4), neutropenia (64.3/42.9), thrombocytopenia (42.9/21.4), transaminase elevations (50.0/21.4), and anemia (42.9/14.3). Conclusion The combination of IDELA+BR or IDELA+ChR is tolerable and demonstrates strong activity with an ORR of 89.7%. Median PFS and DOR have not been reached in the IDELA+BR or the IDELA+ ChR cohorts. The ability to induce responses in this particularly difficult-to-treat patient population with refractory disease, the non-overlapping toxicity with these agents, and the ease of administration makes this an option in this patient population. These results support further studies with these chemo-immunotherapy regimens in patients with CLL. Disclosures: Barrientos: Gilead Sciences: Research Funding. Off Label Use: Idelalisib is a PI3K-delta inhibitor currently in phase III trials for multiple hematologic malignancies. Wagner-Johnston:Gilead Sciences: Research Funding. De Vos:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Leonard:Gilead Sciences: Research Funding. Dansey:Gilead Sciences: Employment. Kim:Gilead Sciences: Employment. Holes:Gilead Sciences: Employment. Adewoye:Gilead Sciences: Employment. Furman:Gilead Sciences: Research Funding.

Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model. After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses. There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%). T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells. For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%). An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition. The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures: Allen: Celgene Corporation: Honoraria.

Background Selective inhibition of Bruton's Tyrosine Kinase (BTK) is a novel approach to target diseases driven by BCR activation, such as B-cell lymphoma and chronic lymphocytic leukemia. Ibrutinib (PCI-32765) is a potent small molecule that binds covalently to a cysteine residue (Cys-481) in the BTK active site, resulting in inhibition of proliferation, migration and adhesion in CLL cells. Single agent ibrutinib orally-administered to relapsed/refractory (R/R) CLL patients was highly active as evidenced by an IWCLL overall response rate (ORR) of 71% and estimated 26 month PFS of 75% (Byrd et al, NEJM 2013). The chemoimmunotherapy regimen of bendamustine (B) and rituximab (R) (BR) is an active and well-established treatment regimen in R/R CLL patients and we report here the final data on the combination of ibrutinib with BR. Aims The primary objective of the study was to evaluate the safety and tolerability of ibrutinib in combination with BR administered to R/R CLL patients. The secondary objective was to estimate the efficacy of this novel combination. Methods 30 patients with R/R CLL/SLL and a median of 2 prior therapies (range 1-3) received up to 6 cycles of BR with a continuous fixed ibrutinib dose of 420 mg/day; ibrutinib dosing continued past 6 cycles until disease progression or other reason for discontinuation. (B was administered at 70 mg/m2 on D1 and D2 combined with R 375 mg/m2 on D1 for C1 and 500 mg/m2 on D1 for subsequent courses). Response was evaluated according to IWCLL criteria. Results The median age of the enrolled patients was 62 yrs (range 41-82) and the majority were male (83%). 53% of patients were Rai stage III/IV and 23% and 43% tested positive for del17p and del11q by FISH, respectively. Bulky disease (lymph nodes > 5 cm) was present in 53%. The observed safety profile for this combination therapy was generally consistent with the safety profile of BR and monotherapy ibrutinib. The most frequently reported treatment-emergent AEs were diarrhea (70%), nausea (66.7%), fatigue (46.7%), neutropenia (40%) and upper respiratory tract infection (36.7%). The most frequently reported treatment emergent AEs Grade 3 or higher in severity were neutropenia (40.0%), maculopapular rash and fatigue (10.0% each), and thrombocytopenia, febrile neutropenia, and cellulitis (6.7% each). Patients completed a median 6 cycles of BR (range 2-6); there have been no treatment discontinuations due to AEs and no deaths within 30 days after last dose. Of interest, the initial transient treatment-related lymphocytosis, seen in 78% of patients with ibrutinib monotherapy (Byrd et al, NEJM 2013), occurred less frequently (27%) in this combination approach. The study was completed on 12 November 2012. At the time of study completion 21 patients (70%) who were still receiving ibrutinib monotherapy after completion of BR rolled over to a long term extension study to continue treatment with ibrutinib. Nine patients (30%) discontinued the study treatment, 5 (17%) of whom proceeded to SCT and 4 (13%) of whom discontinued due to PD. With a median treatment duration of 16 months, the ORR was 93% (28/30 patients, including 5 CRs and 3 nPRs), and one additional patient achieved a PR with lymphocytosis. The estimated 12 month PFS was 90%. Moreover, responses appeared independent of high-risk features. Conclusions Ibrutinib orally-administered in combination with BR was tolerable and highly active in patients with R/R CLL/SLL. The high ORR and good tolerability compares very favorably with historical controls and is currently under further global investigation in a Ph3 trial (NCT01611090). Disclosures: Brown: Novartis: Consultancy; Genentech: Consultancy; Vertex: Consultancy; Pharmacyclics: Consultancy; Onyx: Consultancy; Emergent: Consultancy; Celgene: Consultancy; Sanofi: Consultancy, Research Funding. Off Label Use: The combination of bendamustine + rituximab and ibrutinib (a small molecule inhibitor of BTK) and the combinations of FCR+I to treat relapsed CLL patients is under investigation. Barrientos:AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead: Consultancy, Research Funding. Barr:Pharmacyclics: Research Funding. Flinn:Pharmacyclics: Research Funding. Burger:Pharmacyclics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Salman:Pharmacyclics: Employment, Equity Ownership. Clow:Pharmacyclics: Employment, Equity Ownership. James:Pharmacyclics: Employment, Equity Ownership. Graef:Pharmacyclics: Employment, Equity Ownership. Rai:Teva: Consultancy; Sanofi: Consultancy; GSK: Consultancy; Genentech: Consultancy; Celgene: Membership on an entity’s Board of Directors or advisory committees. O'Brien:Pharmacyclics: Research Funding.

Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones. B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied. Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures: No relevant conflicts of interest to declare.

TPS7133 Background: PI3K-delta is critical for the activation, proliferation and survival of B cells and plays a role in homing and retention of B cells in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that reduces proliferation, enhances apoptosis, and alters trafficking of malignant B cells in lymphoid tissues (Lannutti, 2011). Phase 1 trials demonstrated that idelalisib is highly active in heavily pretreated pts with CLL as a single agent or in combination with rituximab (R), bendamustine (B), or BR: pts experienced reductions in disease-associated chemokines, profound and rapid reductions in lymphadenopathy, and durable clinical benefit with an acceptable safety profile (Coutre et al, 2012; Sharman et al, 2011). Methods: Study will enroll 390 pts with previously treated CLL who have measurable lymphadenopathy, have received prior therapy containing a purine analog or B and an anti-CD20 monoclonal antibody, are not refractory to B, have experienced CLL progression within 36 months from the completion of the last prior therapy, and are currently sufficiently fit to receive cytotoxic therapy. Pts are randomized in a 1:1 ratio to Arm A or B. On Arm A, subjects receive idelalisib continuously at 150 mg BID + R at 375 mg/m2 (1st dose) and then 500 mg/m2 every 4 weeks for 6 cycles + B at 70 mg/m2 on Days 1 and 2 of each 4-week cycle for 6 cycles. On Arm B, subjects receive placebo instead of idelalisib. Stratification factors address IGHV mutational status, del(17p)/p53 mutation status, and refractory vs relapsed disease. The primary endpoint is PFS and key secondary endpoints include ORR, lymph node response rate, CR rate, and OS. This is an event-driven trial and primary endpoint evaluation will be based on independent central review. For the primary efficacy analysis, the difference in PFS between the treatment arms will be assessed in the ITT analysis set. The study was initiated in June 2012 and a data monitoring committee has begun regular review of data. Clinical trial information: NCT01569295.

8500 Background: PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that has shown considerable monotherapy activity in recurrent iNHL (Kahl, ICML 2011), as well as combination therapy (Fowler, ASCO 2012). Methods: This phase I study evaluated the activity of continuous (48 weeks) idelalisib (Id), 100/150 mg BID, in combination with rituximab (R) (375 mg/m2 weekly x 8 doses) (Id+R), with bendamustine (B) (90 mg/m2 x 2, for 6 cycles) (Id+B), or in combination with R (375 mg/m2 monthly x 6) and B (90 mg/m2 x 2), for 6 cycles (Id+BR). Investigators assessed response according to standard criteria (Cheson 2007). Patients who continued to benefit were able to enroll on an extension study. Results: Study enrolled 78 pts with relapsed/refractory iNHL, with 34 (44%) pts continuing on treatment in the ongoing extension protocol. The 3 cohorts included Id+R (N=30), Id+B (N=34), and Id+BR (N=14). Pts were 67% male, median age [range] of 62 [37E84] years, 41% with refractory disease, 88% stage III/IV, and 36% of FL with high FLIPI scores. The median [range] number of prior therapies was 3 [1E10]. The median [range] duration of treatment was 10.6 [0.5-29.2] months. Overall response rate (ORR) was 63/78 (81%), with 22/78 (28%) CR. The ORR/CR for Id+R was 77%/20%, Id+B was 85%/29%, and Id+BR was 79%/43%. At 20 months, the PFS was 66%. For responders, 73% were progression-free at 20 months. Most common adverse events included (total%/≥G3%) pyrexia (56/4), fatigue (45/4), nausea (41/0), rash (40/8), cough (37/0), diarrhea (36/8), chills (18/0), URI (18/1), and pneumonia (17/15). Lab abnormalities included (total%/≥G3%) ALT/AST elevations (56/17). Conclusions: Idelalisib-based combination therapy is highly active and well tolerated in patients with relapsed/refractory iNHL. These data support further clinical development. Phase III trials evaluating the efficacy of idelalisib in combination with R, or BR in iNHL are ongoing (NCT01732913, NCT01732926). Clinical trial information: NCT01732913, NCT01732929.

7017 Background: PI3K-delta signaling is critical for proliferation, survival, homing and tissue retention of malignant B cells. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that has shown considerable monotherapy activity in pts with heavily pretreated CLL. Methods: This phase I study evaluated idelalisib continuously given at 150 mg BID in combination with rituximab (R, 375 mg/m2 every wk x 8), bendamustine (B, 70 or 90 mg/m2 x 2, every 4 wks x 6) or BR (every 4 wks x 6) for relapsed/refractory CLL. Pts still 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: 52 pts (23F/29M) with a median (range) age of 64 (41-87) years were enrolled. Adverse disease characteristics included bulky lymphadenopathy (62%), refractory disease (50%), multiple prior therapies (median 3, range: 1-14) with 96% receiving prior R and 44% receiving prior B. As of 14 Jan 2013, the median (range) treatment duration was 18 (1-33) months. 31/52 (60%) pts enrolled into the extension study; of those, 24/52 (46%) pts are continuing idelalisib treatment on the extension study. The ORR was 81%, with 1 CR, and a median (range) time to response of 1.9 (1.5-8.3) months. The 2-year PFS and OS were 62% and 85%, respectively. At 2 years follow up, 71% of responses were still enduring. There was no difference in outcomes for pts with

Although chemoimmunotherapy has led to higher response rates in chronic lymphocytic leukemia (CLL), most patients relapse due to emergence of resistant subclones. Here, we analyzed changes in immune cell (IC) populations of 29 subjects with relapsed/refractory CLL enrolled in PCYC-1108, a Phase 1b, open-label, parallel-group, nonrandomized, multicenter study of ibrutinib (PCI-32765) in combination with BR (O'Brien et al; ASCO 2012). Mononuclear cells were collected and cryopreserved at 4 time points: 1: just prior to therapy, 2: cycle 1 day 15 (d15), 3: at start of cycle 3 (d 60) and; 4: at the end of study (d 180). Based on a decrease in absolute lymphocyte count (ALC), 27/29 patients responded to this combination therapy. We analyzed residual B, T and monocyte populations and prognostic markers, CD38 and CD49d in each patient and grouped the cases based on percent reduction of CD19+CD5+ (“CLL B cells”) at all time points while on therapy. 10/29 cases (∼34%) showed > 50% reduction (Grp1) whereas 41% (12/29) showed greater retention of CLL cells (Grp2), and 7/29 cases (∼24%) showed variable changes (Grp 3). Median reduction in ALC in Grp1 was 97.8% (range: 68.3-99.75) vs 87.7% (range: 77.3- 99.6) for Grp2. Grp 1 cases showed a decrease in CD23+ and CD38+ CLL B cells at d15; although CD23+ cells decreased further CD38+ cells increased in 8/10 Grp1 cases. CD49d+ CLL cells remained stable in 6/10 Grp 1 and 11/12 Grp 2 cases but increased in 4/10 Grp1 cases. A marked increase in CD3+ and CD14+ cells accompanied the decrease in CLL B cells in cases in Grp1. Grp2 followed a different trend: CD3+ cells decreased until d60 and eventually increased, albeit mildly whereas their percentages of CD14+ cells did not change throughout therapy. Such dynamic changes in IC populations prompted us to assess the presence of dividing CLL cells, in the face of reduced ALC. Surprisingly, although Grp1 showed decreased percentages of CLL cells, 3/10 cases developed increased numbers of Ki-67+ CLL cells, achieving 19-43% at d180. In Grp2, only 0.1-4.0% Ki-67+ CLL B cells were found. BCR-induced phosphorylation of BTK was analyzed by phosphoflow. Both Grp1 and Grp2 exhibited increased BTK phosphorylation at d15 which decreased precipitously at subsequent time points. Together, these studies indicate that combining ibrutinib with BR effectively reduced numbers of circulating CLL cells in >93% of cases, but relative proportions of CLL cells only in ∼1/3 relapse/refractory CLL cases. Of note, the proportion of circulating CD3+, CD14+, and Ki-67+ CLL B cells, increased considerably in Grp 1 cases, possibly representing compensatory proliferative changes due to increased availability of immune niches and/or release of cells from niches. A post-protocol correlation of clinical parameters with changes in circulating immune cell populations is pending. Citation Format: Rajendra N. Damle, Sonal Temburni, Prachi Aggarwal, Susan O'Brien, Jacqueline Barrientos, Jennifer R. Brown, Ian W. Flinn, Paul Barr, Jan Burger, Jonathan W. Friedberg, Kanti R. Rai, Betty Chang, Danelle James, Joseph Buggy, Nicholas Chiorazzi. Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2013-3531

Psychosocial interventions have been shown to improve quality of life (QOL) for many cancer patients. A pilot study was conducted to assess the feasibility of a psychoeducational intervention for cancer patients receiving chemotherapy. Eight patients receiving chemotherapy for colorectal carcinoma participated. The intervention is based on a modification of Interpersonal Therapy. It consisted of four sessions, administered biweekly, using a manual format, by a psychiatric nurse clinician over the telephone. The participants also completed a set of QOL measures by telephone to assess tolerance of the planned assessment. The patients received assistance with treatment-related side effects, reported improved ability to communicate with their physician, and gained an understanding of the stresses they discussed. The patients felt satisfied with the emotional support and medical information provided. A randomized trial is planned to test the intervention for patients participating in a cooperative trial sponsored by the National Cancer Institute.

The treatment of chronic lymphocytic leukemia includes the use of alkylating agents, steroids, and more recently nucleoside analogues. While prior studies have described potential mechanisms of 2-chlorodeoxyadenosine cytotoxicity including the accumulation of DNA strand breaks and induction of apoptosis or programmed cell death, the expression of p53 and its downstream target WAF1/CIP1 have not been examined. In this report we describe the induction of p53 and WAF1/CIP1 in the apoptotic chronic lymphocytic leukemia cells after exposure to 2-chlorodeoxyadenosine.