Gribben, John G.

Abstract We performed immunophenotypic and IgVH mutation analyses on the chronic lymphocytic leukemia (CLL) cells of 307 patients for expression of CD38, ZAP-70, and IgVH mutation status to determine the relative strength of each in predicting the need for early treatment. For this we assessed the time from diagnosis to initial therapy for progressive and/or symptomatic disease. Patients confirmed to have remained untreated were censored for this endpoint. Expression of CD38 was defined as the proportion of CD38-stained CLL cells that had fluorescence intensity above a defined threshold. Similarly, cases were defined as ZAP-70+ when 20% or more of the ZAP-70-stained CLL cells had fluorescence intensity above a defined threshold, as described (NEJM351:893, 2004). CLL cells were classified as expressing mutated IgVH genes if they expressed IgVH with <98% homology with a known germline IgVH gene. The median percentage of cells expressing CD38 in our cohort was 11.5% (range 0.1% to 99.5%). In patients with multiple samples, the CD38 expression level appeared constant over time, with a median S.D. of 0.8%. We defined patient samples that had CD38 expression levels at or below the median as having low expression. There was a significant association between low expression of CD38 and lack of ZAP-70 or expression of a mutated IgVH (p < 0.0001 for each assessment by the Fisher exact test). Median level of CD38 among cases expressing mutated IgVH was 4.0%, compared to 27.4% among cases expressing unmutated IgVH (p < 0.0001 by the Wilcoxon rank sum test). Median level of CD38 among ZAP-70 negative cases was 5.3%, compared to 27.2% among ZAP-70+ cases (p < 0.0001 by the Wilcoxon rank sum test). Patients with low-level expression of CD38 had a median time to initiation of therapy of 8.2 years; whereas, patients with high-level expression of CD38 had a median time to initiation of therapy of 4.0 years (p<0.0001). Although high-level expression of CD38 was associated with a shorter time from diagnosis to initiation of therapy (p < 0.0001 in a Cox proportional hazards regression), the inclusion of IgVH mutation status (p < 0.0001) or expression of ZAP-70 (p < 0.0001) in these models reduced the contribution of CD38 status to a p-value of 0.051 (ZAP-70 model) or 0.052 (mutation status model). Although ZAP-70 expression and mutation status each contributed significantly to the hazard of initiating therapy in a Cox proportional hazards model, high-level expression of CD38 did not add significantly to the model that already addresses ZAP-70 and mutational status, p=0.425. As such, although knowledge of the CD38 expression level can be used as a predictor of the time from diagnosis to the initiation of therapy it provides only marginal value if either the IgVH mutational status or expression of ZAP-70 is known and no value if knowledge of both ZAP-70 expression and IgVH mutational status is available.

7514 Background: Relapse is expected in treated indolent lymphoma patients, and an unmet need exists to prolong remission with effective therapies. Lenalidomide + rituximab (R2) may improve the efficacy of next treatment by their unique mechanisms. Methods: The AUGMENT phase III study evaluated patients with R/R FL gr 1-3a (82%) and MZL (18%) after ≥ 1 prior systemic therapy (not rituximab refractory). Randomization was 1:1 to R2 (lenalidomide PO 20 mg/d, d1-21/28 X12 cycles [c] + rituximab [R] IV 375 mg/m2/wk, c1, d1, 8, 15, 22 and c2-5, d1) and R/placebo (same dosing schedule). The primary endpoint was PFS by 2007 IWG; secondary/exploratory analyses were time to next antilymphoma/chemotherapy treatment (TTNLT/TTNCT) and response to next treatment. Per regulatory guidance, PFS2 was defined as time from randomization to first PD or death from any cause after next antilymphoma treatment, or initiation of a third antilymphoma treatment. Results: Median PFS was superior for R2 over R/placebo (39.4 vs 14.1 mo; HR = 0.46; P < 0.0001). As of 22June2018, median TTNLT, TTNCT, and PFS2 were not reached for R2, and were significantly longer than R/placebo (HR = 0.54, 0.50, and 0.52, respectively). For 49/178 (28%) R2 and 80/180 (44%) R/placebo patients receiving next antilymphoma therapy, response was generally higher with R2 (57% ORR; 31% CR) than R/placebo (36% ORR; 16% CR; Table). Conclusions: These analyses suggest that R2 (vs R/placebo) prolonged time to subsequent treatment and is associated with longer PFS2, enabling greater response to next therapy. Although patient numbers were modest, it is hypothesized that patients who received R2 were generally more sensitive to subsequent therapy than those treated with R/placebo. Clinical trial information: NCT01938001. [Table: see text]

6521 Background: Genomic features including lack of IgVH mutations, del(11q), del(17p), and p53 mutations have been reported to predict clinical course and overall survival in CLL patients (pts). Bcl-2 family proteins and ZAP-70 have also been explored as predictors in CLL. Methods: We prospectively evaluated the prognostic significance of clinical features and laboratory variables on response and progression-free survival (PFS) following treatment with fludarabine (F, n=132) or fludarabine plus cyclophosphamide (FC, n=137) as part of the US Intergroup Trial E2997 for previously untreated CLL. Results: FC therapy had higher complete response (CR) (23.4% versus 4.6%), overall response (OR) (74.3% versus 59%), and median PFS (31.6 mos versus 19.2 mos) compared to F. CR and OR were not significantly different based on interphase cytogenetics, IgVH status, or p53 mutation. IgVH status or levels of ZAP-70, Bcl-2, Bax, Mcl-1, XIAP, Caspase-3, and Traf-1 proteins were not associated with clinical response or PFS. IgVH status and ZAP-70 levels were associated with time from diagnosis to treatment. Using a model including treatment arm, pts with del(17p) or (11q) had significantly shorter PFS (hazard ratios 3.54 and 2.05 respectively). In pts with a p53 mutation without del(17p) there was no enhancement of the model predicting poorer outcome, nor did IgVH status enter the model for predicting PFS. Conclusions: Combination chemotherapy is associated with a higher CR, OR, and PFS. Also, initiation of therapy for pts with Rai stages 0/1 resulted in a higher response rate. Del(17p) and del(11q) are highly predictive of shortened PFS with fludarabine-based chemotherapy. IgVH and p53 mutational status, as well as expression of ZAP-70, Bcl-2 family proteins, and CD38, did not predict response or PFS. ZAP-70 expression is associated with cytogenetic subsets predicted to have a worse overall prognosis, but did not identify pts who will do poorly with therapy. A combination of clinical staging and cytogenetics provide support for future risk-stratified treatment of CLL. Pts with a projected short response duration can be identified for future investigational strategies. No significant financial relationships to disclose.

Background: The combination of lenalidomide+rituximab (R2) recently showed superior efficacy vs R-placebo in patients (pts) with R/R iNHL (Leonard et al. J Clin Oncol 2019). Based on these AUGMENT study results, R2 was approved by the US FDA for treatment of adult pts with previously treated follicular lymphoma (FL) or marginal zone lymphoma (MZL). Advanced age at diagnosis is a risk factor in pts with iNHL. We performed post-hoc subgroup analyses by age from AUGMENT and data here focus on pts age ≥ 70 y. Methods: AUGMENT (NCT01938001) is a multicenter, double-blind, randomized phase III study of R2 vs R-placebo in pts with FL grade 1-3a or MZL previously treated with ≥ 1 systemic therapy with R/R disease but not refractory to rituximab. Pts were randomized 1:1 to R2 or R-placebo. R2 was oral lenalidomide 20 mg/d, d1-21/28 for 12c plus rituximab IV 375 mg/m2 weekly in c1 and d1 of c2-5. R-placebo was rituximab+placebo on the same schedule. The primary endpoint was progression-free survival (PFS) per 2007 IWG response criteria (without PET) as assessed by IRC (central review). Secondary endpoints included overall response rate (ORR), complete response (CR), time to next anti-lymphoma treatment (TTNLT) and safety. Post-hoc analyses were performed by dividing pts into age &lt; 70 y and ≥ 70 y subgroups, the latter group considered unfit for chemotherapy. Results: Of 358 pts randomized (R2, n = 178; R-placebo, n = 180), 267 pts were age &lt; 70 y (R2, n = 131; R-placebo, n = 136), and 91 pts were age ≥ 70 y (R2, n = 47; R-placebo, n = 44). Baseline characteristics including histology, disease status, and prior treatments are shown in the table and were similar across treatment arms in pts ≥ 70 y. At a median follow-up of 28.3 mo, the study met its primary endpoint of PFS, with a hazard ratio (HR) of 0.46 (95% CI, 0.34-0.62; P &lt; 0.0001) in the overall population. R2 had superior PFS vs R-placebo in both &lt; 70 and ≥ 70 y subgroups, with HR of 0.41 (95% CI, 0.29-0.59) and HR of 0.66 (95% CI, 0.37-1.18), respectively. In pts ≥ 70 y, median PFS with R2 vs R-placebo was 24.9 vs 14.3 mo; ORR/CR was 81%/26% vs 59%/16%; and TTNLT was not reached in either arm. Efficacy results for all pts and those &lt; 70 y are reported in the table; notably in pts receiving R2, mPFS was longer in pts &lt; 70 y vs ≥ 70 y (39.4 mo [95% CI, 22.9-NE] vs 24.9 mo [95% CI, 16.4-NE]). In pts ≥ 70 y, any-grade adverse events (AEs) with a ≥ 10 % difference between R2 vs R-placebo included neutropenia (63% vs 11%), constipation (33% vs 16%), cough (33% vs 16%), leukopenia (26% vs 2%), anemia (24% vs 9%), pyrexia (24% vs 9%), pruritus/pruritus generalized (24% vs 2%), muscle spasms (22% vs 11%), rash/rash maculopapular (22% vs 5%), headache (20% vs 9%), thrombocytopenia (17% vs 2%), dyspepsia (13% vs 2%), influenza (13% vs 2%), back pain (7% vs 18%), and nasopharyngitis (4% vs 16%). Also, tumor flare was reported in 9% vs 0% of pts, respectively. In pts ≥ 70 y, 75% of R2 pts vs 36% of R-placebo pts had ≥ 1 grade 3/4 AE, mainly due to neutropenia (50% vs 7%). All other grade 3/4 AEs occurred in &lt; 10% of pts ≥ 70 y in both treatment arms. One grade 5 AE occurred in pts ≥ 70 y (R-placebo arm). In the R2 arm, the median number of treatment cycles was 12 for both the &lt; 70 y vs ≥ 70 y subgroups; however, fewer older pts completed 12 cycles of lenalidomide (76% vs 57%), and more started lenalidomide at the lower dose of 10 mg (6% vs 35%) because of low creatinine clearance, respectively. In the R2 &lt; 70 y and ≥ 70 y subgroups, the average daily dose of lenalidomide was 17.9 mg/d (range, 5.6-20.0) and 14.4 mg/d (range 4.2-20.0), and median relative dose intensity was 95% and 86%, respectively. Conclusions: Similar to the results in the original population, R2 showed superior efficacy vs rituximab monotherapy (plus placebo) as measured by the primary end point of PFS and secondary end points of ORR and CR in pts with R/R FL grade 1-3a and MZL irrespective of age. The efficacy and safety profiles of R2 and R-placebo in pts ≥ 70 y were similar to those reported in the overall population. Older pts treated with R2 vs R-placebo had superior mPFS (24.9 vs 14.3 mo). They were more likely to start lenalidomide at a lower dose and had lower median dose intensity which may have contributed to their shorter mPFS vs younger pts receiving R2. These data show that R2 maintained efficacy improvements vs R-placebo in pts ≥ 70 y, despite higher unfit status and lower overall lenalidomide treatment/exposure. Thus, R2 is an effective and available treatment option for pts with iNHL, including those with advanced age. Disclosures Trněný: Amgen: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy; MorphoSys: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria. Leonard:Nordic Nanovector: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Bayer Corporation: Consultancy; Epizyme, Inc: Consultancy; Karyopharm Therapeutics: Consultancy; Sutro Biopharma: Consultancy; Merck: Consultancy; Gilead: Consultancy; BeiGene: Consultancy; ADC Therapeutics: Consultancy; Sandoz: Consultancy; Akcea Therapeutics: Consultancy; Miltenyi: Consultancy; MorphoSys: Consultancy. Nowakowski:F. Hoffmann-La Roche Ltd: Research Funding; NanoString: Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding. Izutsu:Chugai, Celgene, Daiichi Sankyo, Astra Zeneca, Eisai, Symbio, Ono, Bayer, Solasia, Zenyaku, Incyte, Novartis, Sanofi, HUYA Bioscience, MSD, Astellas Amgen, Abbvie, ARIAD, Takeda, Pfizer: Research Funding; Kyowa Kirin, Eisai, Takeda, MSD, Chugai, Nihon Medi-physics, Janssen, Ono, Abbvie, Dainihon Sumitomo, Bayer, Astra Zeneca, HUYA Japan, Novartis, Bristol-Byers Squibb, Mundi, Otsuka, Daiichi Sankyo, Astellas, Asahi Kasei: Honoraria; Celgene: Consultancy; Eisai, Symbio, Chugai, Zenyaku: Research Funding; Eisai, Chugai, Zenyaku: Honoraria. Fowler:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Thieblemont:Roche: Honoraria, Research Funding; Gilead: Honoraria; Novartis: Honoraria; Kyte: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Cellectis: Membership on an entity's Board of Directors or advisory committees. Zinzani:Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Honoraria, Speakers Bureau. Kalambakas:Celgene Corporation: Employment, Equity Ownership. Czuczman:Celgene Corporation: Employment, Equity Ownership. Fustier:Celgene Corporation: Employment, Equity Ownership. Wu:Celgene Corporation: Employment, Equity Ownership. Gribben:Janssen: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Background: Although diffuse large B cell lymphoma (DLBCL) can be cured using immuno-chemotherapy, 40% of patients experience relapse or refractory disease. Large-scale profiling studies have mainly focused on DLBCL at diagnosis, resolving different outcome groups based on gene expression (e.g. cell-of-origin (COO) or molecular high grade), MYC/BCL2 translocations (double-hit lymphoma) or gene mutations and copy number aberrations (Schmitz et al, NEJM 2018; Chapuy et al, NatureMedicine 2018). In comparison, longitudinal studies have been hindered by the limited availability of sequential biopsy samples. To date, the relapse-specific gene mutations identified are limited and inconsistent across studies. In our study, we have focussed attention on the changes in gene expression profile (GEP) accompanying DLBCL relapse. Methods: We retrospectively collected archival paired diagnostic/relapse formalin fixed paraffin embedded tumor biopsies from 38 de novo DLBCL patients collected from multiple UK sites treated with rituximab-based immuno-chemotherapy, where partial or complete remission was reported following treatment. COO classification was performed by the Lymph2Cx assay on NanoString to distinguish activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subtypes. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit was used to measure the expression levels of > 20,000 genes on the paired samples. Results: COO remained stable from diagnosis to relapse in 17 ABC-ABC pairs, 11 GCB-GCB pairs and 4 unclassified (UNC)-UNC pairs. Frank COO switching was observed in 6 cases (1 ABC-GCB, 2 ABC-UNC, 2 GCB-UNC, 1 UNC-ABC). Pairs with stable COO were taken forward for further analysis. Gene expression analysis using the limma R package identified 163 and 136 genes as differentially expressed (DE) (p <= 0.01 and absolute log2FC > 1) between the diagnostic and relapse biopsies in ABC and GCB tumors respectively, with only a one gene overlap. Gene Set Enrichment Analysis further suggested that ABC and GCB relapses are mediated via different mechanisms, with tumor growth and proliferation signatures enriched in ABC relapses, whilst adaptive immunity-related signatures accompanied GCB relapses. Next, we aimed to utilise our relapse-specific genes to identify outcome predictors at diagnosis using publicly available GEP datasets. In order to increase our discovery power and accuracy, a larger set of DE genes from the paired differential analysis (796 genes in ABC pairs and 387 from GCB pairs) were selected (p <= 0.05) and subsequently used in a training cohort (GEP from Reddy et al, Cell 2017). The Prediction Analysis for Microarrays R (PAMR) algorithm identified a 30-gene signature within DE genes from ABC pairs (Fig1.A), capable of separating the 249 ABC cases into 136 low and 113 high-risk cases with significantly inferior overall survival (Hazard Ratio (HR)=1.89, log-rank p=0.0017, measure of goodness-of-fit C-index=0.71; Fig1.B). No equivalent signature was found in the GCB cases using this approach. The prognostic significance of this 30-gene discriminator was successfully validated using a linear predictor in two independent GEP datasets: 1) a population-based cohort (Lenz et al, NEJM 2008) with 93 R-CHOP-treated ABC cases identifying 47 low and 46 high-risk cases (HR=1.92, p=0.046, C-index=0.77; Fig1.C) and 2) a clinical trial dataset (REMoDL-B, Davies et al, Lancet Oncol 2019) with 255 ABC cases identifying 110 low and 145 high-risk ABC cases (HR=1.95, p=0.0051, C-index=0.70; Fig1.D). Conclusions: Here we describe a 30-gene discriminator in ABC-DLBCL, derived from genes differentially expressed between diagnosis and relapse, that allowed the definition of clinically distinct high and low risk subgroups in ABC-DLBCLs at diagnosis. The clinical translation of such a tool may be useful to guide therapy for this unfavourable subgroup of ABC-DLBCLs. Validation of this signature is currently underway in additional datasets and further study is required to understand the contribution of these genes in DLBCL pathology. Disclosures Korfi: Roche: Consultancy. Burton:Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rule:TG Therapeutics: Consultancy, Honoraria; Napp: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Astra-Zeneca: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Crosbie:Janssen: Honoraria. Scott:Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Roche/Genentech: Research Funding. Rimsza:NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution]. Davies:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Bayer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Acerta Pharma: Honoraria, Research Funding; ADCT Therapeutics: Honoraria, Research Funding; BioInvent: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gribben:Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Okosun:Gilead Sciences: Honoraria, Research Funding. Johnson:Epizyme: Honoraria, Research Funding; Novartis: Honoraria; Kite: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Boehringer Ingelheim: Honoraria; Takeda: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Incyte: Honoraria. Fitzgibbon:Epizyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Speakers Bureau.

Abstract Background: Indolent non-Hodgkin lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL), is typically responsive to initial chemoimmunotherapy, but relapse is expected. Single-agent rituximab is FDA approved and frequently administered for patients with relapsed/refractory (R/R) low-grade or follicular CD20-positive B-cell NHL. Lenalidomide is an immunomodulatory agent with preclinical and clinical antilymphoma activity alone and in combination with rituximab. We compared the efficacy and safety of lenalidomide plus rituximab (R2) to rituximab (plus placebo) in patients with R/R iNHL. Methods: AUGMENT (NCT01938001) is a multicenter, double-blind, randomized phase III study of R2 vs rituximab/placebo (control) in patients with FL grade 1-3a or MZL who were previously treated with ≥ 1 prior systemic therapy with documented relapsed or refractory disease but not refractory to rituximab (refractory was defined as < partial response to rituximab or rituximab-chemotherapy OR disease progression < 6 months after last rituximab dose). Patients were stratified by prior rituximab treatment (yes vs no), time since last antilymphoma therapy (≤ 2 vs > 2 years), and histology (FL vs MZL), and then randomized 1:1 to R2 or control for up to 1 year. R2 patients received oral lenalidomide 20 mg/day (d), d1-21/28 for 12 cycles plus rituximab IV 375 mg/m2 weekly in cycle 1 and d1 of cycles 2-5. Control patients received rituximab and placebo on the same schedule. Dose modifications were pre-specified in the protocol to manage toxicities. The primary endpoint was progression-free survival (PFS) per 2007 IWG criteria without PET as assessed by Independent Review Committee (IRC; central review). Secondary endpoints included overall response rate (ORR), complete response (CR), duration of response (DOR), time-to-next antilymphoma treatment (TTNLT), overall survival (OS), and safety. Results: A total of 358 patients were randomized (n = 178 R2; n = 180 control), median age was 63 years (range, 26 - 88), 34% FLIPI score ≥ 3, and histologies of 82% FL/18% MZL. Median number of prior systemic treatments was 1 (range, 1 - 12); 84% received prior rituximab and 51% had prior antilymphoma therapy within 2 years of enrollment. At median follow-up of 28.3 months, the study met its primary endpoint of PFS with HR = 0.46 (95% CI, 0.34 - 0.62; P < 0.0001) (Figure 1). Median PFS was 39.4 months for R2 vs 14.1 months for control. IRC-assessed ORR for R2 was 78% vs 53% for control (P < 0.0001). CR was 34% for R2 vs 18% for control (P = 0.001). Median DOR was 36.6 and 21.7 months for R2 and control arms, respectively. TTNLT was improved for R2 vs control with HR = 0.54 (95% CI, 0.38 - 0.78; P = 0.0007). OS data were not mature with 16 deaths reported in the R2 arm vs 26 deaths in control (HR = 0.61 [95% CI, 0.33 - 1.13]). Selected all-grade treatment-emergent adverse events (TEAEs) of interest more common in the R2 vs control arm (≥ 10% difference) were infections (63% vs 49%), cutaneous reactions (32% vs 12%), constipation (26% vs 14%), thrombocytopenia (15% vs 4%), and tumor flare reaction (11% vs 1%). Grade 3/4 TEAEs were reported in 69% R2 and 32% control patients. More frequent grade 3/4 TEAEs in the R2 vs control arm were primarily attributable to increased neutropenia (50% vs 13%) and leukopenia (7% vs 2%). Grade 5 TEAEs were reported in 2 patients in each arm. TEAEs leading to discontinuation of lenalidomide occurred in 9% of patients vs 5% for rituximab + placebo. Neutropenia was the only TEAE leading to discontinuation of lenalidomide in > 1 patient (n = 5). Seventy-one percent of R2 patients completed all 12 cycles of planned treatment vs 61% of control. Disease progression was the leading reason for discontinuation of lenalidomide/placebo (n = 21 R2, n = 54 control). Conclusions: R2 demonstrated superior efficacy over rituximab monotherapy (plus placebo) as measured by the primary endpoint of progression-free survival as well as secondary endpoints of ORR, CR, DOR, and TTNLT in patients with R/R FL grade 1-3a and MZL. At this early analysis, fewer deaths have been observed in the R2 arm. Despite additional hematologic toxicity, greater efficacy of the R2 regimen (and fewer early progressions) allowed more patients to complete the planned therapy and delayed the need for subsequent treatment. R2 represents an important new treatment option in patients with previously treated FL/MZL, with meaningful advantages over single-agent rituximab. Disclosures Leonard: Celgene: Consultancy; Karyopharm: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; Juno: Consultancy; Bayer: Consultancy; AstraZeneca: Consultancy; Sutro: Consultancy; Biotest: Consultancy; United Therapeutics: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy; BMS: Consultancy. Trněný:F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria. Izutsu:Otsuka: Honoraria; Bristol- Myers Squibb: Honoraria; Nihon Medi-Physics: Honoraria; Novartis: Honoraria; Mundhi: Honoraria; HUYA Bioscience International: Research Funding; Kyowa Hakko Kirin: Honoraria; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Gilead Sciences: Honoraria; Eisai: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Astellas: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Zenyaku: Research Funding; Celltrion: Research Funding; MSD: Honoraria; Ono: Honoraria, Research Funding; Symbio: Research Funding; Celgene: Consultancy, Research Funding; Solasia: Research Funding; Sanofi: Research Funding; Meiji Seika: Honoraria; Shionogi: Honoraria; Asahi Kasei: Honoraria. Fowler:Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Zhu:Beijing Cancer Hospital (Peking University Cancer Hospital): Employment. Scheliga:INCA - Instituto Nacional Do Cancer, Brazil: Employment. Pinto:Servier: Consultancy; BMS: Honoraria, Research Funding; MSD: Honoraria; Roche: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Gilead: Honoraria. Scheinberg:Novartis: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding; Pfizer: Speakers Bureau. Flinn:Trillium: Research Funding; Takeda: Research Funding; Calithera: Research Funding; Incyte: Research Funding; Verastem: Research Funding; ArQule: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding; Forty Seven: Research Funding; Agios: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Portola: Research Funding; Verastem: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Forma: Research Funding; Merck: Research Funding; Novartis: Research Funding; Constellation: Research Funding; Curis: Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; Genentech: Research Funding; Gilead: Research Funding. Moreira:Instituto Português de Oncologia do Porto FG, EPE, Porto, Portugal: Employment. Liu:Celgene: Employment, Equity Ownership. Kalambakas:Celgene: Employment, Equity Ownership. Fustier:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Gribben:Cancer Research UK: Research Funding; Unum: Equity Ownership; Novartis: Honoraria; Abbvie: Honoraria; Wellcome Trust: Research Funding; Acerta Pharma: Honoraria, Research Funding; Kite: Honoraria; NIH: Research Funding; Roche: Honoraria; Janssen: Honoraria, Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; TG Therapeutics: Honoraria; Pharmacyclics: Honoraria.

The critical importance of specific proteins including Bcl-2, Mcl-1, XIAP, Bax, Caspase-3, Traf-1, and ZAP70 in predicting disease progression, prognosis, and response to therapy in CLL have been extensively studied through in vitro experiments. We now report molecular data obtained from a large prospective clinical trial involving 278 previously untreated patients with CLL. This ECOG-led intergroup trial involved participation from CALGB and SWOG, and currently provides quantitative information regarding protein levels on 217 cases assessed immediately prior to treatment. NCI criteria for therapy in CLL were met before patients were randomized to receive either Fludarabine or Fludarabine plus Cytoxan. On-study analyses included: interphase cytogenetics by Fluorescence In Situ Hybridization (FISH), p53 mutational analysis, immunoglobulin variable heavy chain (IgVH) gene rearrangement, and quantitative analysis of the aforementioned specific proteins. Peripheral blood mononuclear cells were obtained from each patient at registration before starting therapy. Lysates were prepared from the patient cells and separated by SDS-PAGE. Proteins were detected by immunoblot analysis using the multiple antigen detection method previously developed by our group. Detection was performed by an enhanced chemiluminescence method and quantified by densitometry. Expression levels were calculated relative to expression in the Jurkat T cell line (for ZAP70) or the RS11846 lymphoblastoid cell line (for apoptosis-related proteins). Using a two-sided Wilcoxon rank sum test, molecular data were compared with FISH and IgVH mutational status. Patients with either p53 mutations or del(17p) had significantly lower levels of XIAP (p=0.022). Patients with del(11q) had significantly higher Bcl-2/Bax ratios (p=0.052). Patients with trisomy 12 had lower levels of Bcl-2 (p=0.066); higher Mcl-1 (p=0.026); higher Bax (p=0.09); and higher Caspase-3 (p=0.019) levels. Interestingly, these specimens also had lower ratios of Bcl-2/Bax (p=0.0016). Patients with del(13q) had significantly higher levels of Bcl-2 (p=0.0059) lower levels of Caspase-3 (p=0.047), and higher Bcl-2/Bax ratios (p=0.0569). IgVH mutated patients had significantly lower levels of Caspase-3 using either a 98% cut-off (p=0.0008) or the 97% cut-off (p<0.0001). Finally, patients with higher ZAP70 protein expression had significantly higher levels of Caspase-3 (p=0.0099) and Traf-1 (p=0.016). In 163 patients evaluable for both response and the molecular studies, either elevation of Bcl-2 protein (p= 0.01) or an abnormality of p53 (either del 17p or a p53 mutation) (p=0.08) correlated significantly with attainment of a complete remission. Potential opportunities to predict response to therapy in CLL will hopefully identify those patients likely to benefit from therapy. Based upon our previous work, these molecular parameters can be modulated both in vitro and in vivo. Therefore, understanding the relationship of these important proteins to therapeutic response can facilitate strategic plans to circumvent drug resistance to chemotherapy and immunotherapy in CLL.

Several prognostic factors including un-mutated VH mutational status, select interphase cytogenetic abnormalities [del(11q22.3), del(17p13.1)] and p53 mutations have been associated with shorter interval time from diagnosis to symptomatic disease requiring treatment, shortened progression-free survival (PFS) and overall survival (OS). Limited prospective data exists relative to the relevance of these biologic markers on PFS following treatment with modern therapies for CLL. E2997 is a randomized phase III trial of 278 previously untreated, symptomatic CLL patients who received fludarabine and cyclophosphamide (FC) versus fludarabine (F). We analyzed the response rates and PFS of the patients for whom interphase cytogenetics, VH mutational status, and p53 mutational status studies have been completed. FC therapy had a higher complete response (CR) (23% versus 5%, p<0.002), overall response [OR] (73% versus 50%, p<0.002), and median PFS (33.5 months versus 15 months, p<.0001) as compared to F. The CR and OR was not different based upon interphase cytogenetics or VH mutational status for the entire group of patients or when analyzed by arm of treatment. However, the OR for pts with del(17) or p53 mutations was lower in both the FC arm (69%, versus 76% in patients with normal p53) and F arm (27%, versus 54% in normal p53). Using the Dohner hierarchical classification [del(17p)>del(11q)>del(6q)>tri12>normal> del(13q14)], the relationship of interphase cytogenetics, VH mutational status and p53 mutational status with PFS is summarized below: Median PFS on FC (months) Median PFS on F (months) NR = median not reached; p53 mut+ = mutation present del(17p) 11.9 8.2 del(11q) 30.6 12.8 del(6q) 26.9 26.7 tri 12 NR 20.3 normal NR 11.2 del(13q) NR 21.2 del(17p)/p53 mut+ 11.9 8.9 no del(17p)/p53 mut+ NR 17.8 VH<98% NR 21.2 VH>98% 21.4 13.4 Using the interphase cytogenetic groups, there was a significant difference in PFS for both the FC (p=0.04) and F (p=0.01) treatment arms. Similarly, the presence of a p53 mutation or deletion [del(17p)] predicted for shorter PFS for both FC (p=0.005) and F (p=0.02) therapies. PFS did not statistically differ among VH mutational groups for either arm. We next investigated a Cox proportional hazards analysis to assess the impact of multiple molecular features and treatment arm on PFS. Treatment with F (hazard ratio 3.14, p<0.0001), presence of del(17p) or p53 mutation (hazard ratio 3.13, p=0.0001), and presence of del(11q) (hazard ratio 2.067, p=0.019) were highly significant in the model. Finally, VH unmutated status and del(11q) were highly associated. In conclusion, our data demonstrate that high risk genomic features including p53 mutations, del(17p), and del(11q) are highly predictive of shortened PFS. Our data provide support for a risk-stratified therapy approach for the treatment of CLL.

Abstract Abstract 924 Background: Therapy-related myeloid neoplasia (t-MN), including myelodysplastic syndrome and acute myeloid leukemia, has been reported at a higher frequency with chlorambucil + fludarabine as compared to fludarabine alone, but has not been rigorously studied in the context of cyclophosphamide as an alkylator agent. The intergroup prospective randomized Phase 3 trial, E2997, compared FC with F alone as initial therapy for patients (pt) with chronic lymphocytic leukemia (CLL). FC therapy led to higher complete and overall response rates and longer progression-free survival at the initial analysis (Flinn et al JCO 2007). One rationale for combining F with C is that F can inhibit repair of DNA damage induced by C. FC did cause more myelosuppression that could lead to more long-term effects on myeloid hematopoietic function, including t-MN. Methods: E2997 enrolled 278 pts, 141 on FC and 137 on F. These cases were assessed for t-MN by required reporting of these events. Baseline genetic and molecular features of CLL were available through a companion lab correlative trial for 235 pts, 122 on FC and 113 on F. Results: With median follow-up of 6.4 yrs, there have been 13 (4.7%) reported cases of t-MN, 9 after FC and 4 after F. By cumulative incidence methodology, adjusting for the competing risk of death, the rates of t-MN at 7 yr are 8.2% after FC and 4.6% after F (p=0.18 by the Gray test). Median age at study entry of the t-MN pts was 60 (range 45–80) yrs vs 61 (range 33–86) yrs among those not reported to have t-MN. Median time from initial therapy to diagnosis of t-MN was 5 (range 0.7–8) yrs and did not differ between FC and F. Ten of 13 affected pts received 6 chemotherapy cycles. Additional therapy prior to occurrence of t-MN was given to only 2 of 9 FC pts, in contrast to 3 of 4 F pts. Thus, t-MN occurred in only 1 pt treated with F and no further therapy vs 7 who received FC and no further therapy. Ten of 12 pts with available cytogenetics at diagnosis of t-MN had an abnormality of chromosome 5 and/or 7, common to alkylating agent-induced t-MN, usually (n=8) with complex karyotype. Of the 9 pts with t-MN after FC, all 7 with available CLL data had lower risk IgVH mutated disease, in contrast to 1 of 4 with t-MN after F and 44% in the entire cohort. Conclusion: Our analysis suggests that a higher incidence of t-MN has occurred after FC than after F. t-MN after FC occurred most often without additional therapy and in IgVH mutated CLL which is associated with more favorable outcome. The increased incidence of t-MN after FC in this study, usually in the absence of additional treatment, suggests that FC is more leukemogenic than F. This finding emphasizes a need for longer follow-up of toxicity and survival before concluding that FC is preferable to F as the chemotherapy backbone for initial therapy of both low and high risk CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells. We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment. On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort. However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort. We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC). CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core. We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expressionthat could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1- High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years. We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups. We found that high-level expression of ROR1retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1- High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years. The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%). A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001). Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL. Table 1. Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohort Training Dataset Validation Dataset ROR1 MFI distribution (Median) 0~475 (35.5) 0~424(34.6) ROR1Low with Mutated IGHV 139 132 ROR1Low with Unmutated IGHV 64 84 ROR1High with Mutated IGHV 181 156 ROR1High with Unmutated IGHV 209 214 Total With Known IGHV Mutation Status 593 586 Disclosures Keating: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.