Unmutated Status Research Articles

Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia: A Retrospective, Single-Institution Analysis Based on Risk Features and Debulking Strategy with Anti-CD20 Monoclonal Antibody

Background: The treatment of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) has continued to evolve in recent years, offering the patients different therapeutic options. Venetoclax (ven) is a selective, small molecule inhibitor of B-cell receptor-2 (BCL-2) approved by the FDA for patients with newly diagnosed and relapsed/refractory CLL. Stratification based on tumor lysis syndrome (TLS) risk is recommended and may guide debulking strategies. Methods: We retrospectively analyzed 36 patients with relapsed/refractory (RR) CLL who received treatment with ven at the Moffitt Cancer Center between January 2016 and July 2019. Objective response to therapy was determined based on iwCLL. Progression free survival (PFS) and overall survival (OS) were evaluated via Kaplan-Meier method; overall response rate (ORR) and complete response (CR) via Fisher's exact test. Adverse events (AEs) were graded by CTCAEv5. Results: The median age was 58.5 years (28-82). Median follow up was 12.97 months. The vast majority of patients had high risk disease; Chromosomal analysis by Fluorescence In Situ Hybridization (FISH) reported Del17p and Del11q (+/- others, except Del17p) in 18 (50%) patients and 6 (16.7%) patients respectively. Twenty one (58.3%) patients had a TP53 mutation by next generation sequencing. Twenty five (80.6%) patients had unmutated IGHV status, and 24 (79.9%) patients had high or very high CLL-International prognostic Index (CLL-IPI) score. Twenty-four patients (66.7%) had been previously treated with Ibrutinib, 11 of those progressing on it. Further characteristics are described in table 1. Median OS was not reached. Median PFS was 23.93 months, figure 1a. Median duration of response was 25.97 months. The ORR (PR+CR) at the time of analysis was 63.9%, with CR rate of 33.3%, and PR rate of 30.6%. Nine patients (25%) had stable disease (SD), while four patients (11.1%) progressed on treatment. Minimal residual disease (MRD) was evaluated as part of the response assessment in 7 patients (19.4%) by flow cytometry (1 patient) and by clonoSEQ (6 patients), and 4 (11.1%) had achieved undetectable disease. Response rates were also based on risk stratification; OS and PFS were not affected by the presence of TP53 mutation (p=0.1145) as described in figure 1b, IGHV unmutated status, or NOTCH1 mutation. PFS was inferior in patients with high-very high CLL-IPI score (p=0.0414), figure 1c. The presence of bulky lymphadenopathy (≥5 cm) did not affect outcomes (p=0.5772), figure 1d. Reasons for treatment discontinuation were: progressive disease (PD) in 6 (16.7%), MD/patient preference in 5 (13.9%), Richter's transformation in 2 (5.6%), AEs in 1 (2.8%), allogeneic transplant in 1 (2.8%), and death while on treatment in 1 (2.8%) case. Twenty patients (55.6%) were considered to be at intermediate/high risk of developing TLS, of whom 15 (75%) received debulking therapy with rituximab (1), ofatumumab (11), or obinutuzumab (3) prior to ven. Based on the absolute lymphocyte count (ALC), the risk of TLS became low in 12 patients. The use of a debulking monoclonal antibody (MoAb) improved lymphocytosis, but did not impact PFS. Treatment was well tolerated, and was not dependent on the use of debulking MoAb; however, out of the 18 patients (50%) who underwent debulking therapy, 9 (25%) had grade 3/4 toxicities. Neutropenia was the most common grade 3/4 toxicity in all patients, which was seen in 7 (19.4%) patients, and diarrhea was the most common grade 3/4 non-hematologic toxicity, seen in 4 (11.1%) patients; grade 3/4 neutropenia was seen in 4 (57.1%) of those patients who received debulking therapy, while diarrhea was only seen in 1 (25%). No cases of TLS were observed. Ten patients (27.8%) were admitted for ramp up of ven as per physician preference, independent of TLS risk and comorbidities. Conclusion: Venetoclax is a very active agent for R/R CLL with an acceptable toxicity profile. Debulking strategy is a tolerable option for patients with high burden disease and may reduce the incidence of TLS and/or hospitalization. Nonetheless, based on this retrospective study, it does not seem to have a significant impact in outcomes, and it carries a higher risk for potential cumulative toxicity. Disclosures Chavez: Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees. Sandoval-Sus:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Shah:Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Bello:Celgene: Speakers Bureau. Sokol:EUSA: Consultancy. Nodzon:Pfizer: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy, Other: Speaker Fees; Abbvie: Other: Speaker Fees. Pinilla Ibarz:Bayer: Speakers Bureau; TG Therapeutics: Consultancy; Teva: Consultancy; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Sanofi: Speakers Bureau.

Next-Generation Optical Mapping Reveals Numerous Previously Unrecognizable Structural Variants in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in western countries. Although a number of genetic aberrations (complex karyotype, del(17p), del(11q), unmutated IGVH status, TP53 mutations) having predictive and prognostic significance in CLL was reported, there is still a lack of information about large structural variants, repetitive regions, copy number variations, and inversions in the CLL genome due to limitations of current methods (NGS, cytogenetics). Aim: Using novel next-generation optical mapping technology, we analyzed leukemic cells from peripheral blood of CLL patients at different stages of the disease. Methods and Patients: High-molecular weight DNA was isolated from peripheral blood (>90% CD5+/CD19+/CD20+/CD23+ B-cells) from two CLL patients (P1: 70-y-old male, diagnosis of CLL in 2018, Binet Stage B, unmutated IGVH, untreated; P2: 59-y-old male, diagnosis of CLL in 2005, Binet Stage A, unmutated IGVH, on ibrutinib (2nd line) since 4/2018). For optical mapping, DNA was labeled by DLS chemistry and visualized on Saphyr system (BioNano Genomics). The optical genome maps (480 Gbp, coverage 124X) were aligned to a human reference genome (GRCh38) and the detected structural variants (SVs) were compared against the database of healthy controls; only rare de novo variants were analyzed. In both patients, cytogenetic analysis (karyotype, FISH, and arrayCGH) and mutational analysis by NGS (TP53, ATM, BIRC3, NOTCH1, SF3B1, POT1 genes) were available. Results: For both CLL patients, the optical mapping method identified 97% of chromosomal abnormalities previously reported by standard cytogenetic methods such as deletions on 6q, 13q, 11q, 17p and trisomy 12. In both patients, same large deletions of 17.5 kbp in 9q21 and 7.1 kbp in 14q21 chromosome region were detected, undetectable by standard methods. Both patients also carried intra-chromosomal translocation t(13;13) located in 13q14 region. Additionally, P1 genome carried 2 kbp deletion in oncogene EGFR and 500 bp insertion affecting tumor suppressor TCF4, both genes previously associated with CLL. Moreover, numerous additional copy number variations and large somatic SVs, including on average tens of deletions, insertions, inversions, duplications, and translocations were detected in both patients. Conclusion: Our study revealed numerous novel genomic rearrangements in CLL genome, unrecognizable by standard methods. These data highlights a high potential of optical maps for refinement of genomic variability in CLL, mainly for detection of large structural variations and copy number variants. The study on larger patient cohort and longer follow-up of patients may identify genetic aberrations associated with the clinical course and therapy response. Support: MZ ČR VES16-32339A, MZ ČR - RVO (FNOl, 00098892), IGA UP_2019_014, IGA UP_2019_01 Disclosures Minarik: Celgene: Consultancy, Honoraria, Research Funding; Amgen, BMS, Janssen-Cilag, Takeda: Consultancy, Honoraria.

Rapid Dose Escalation of Venetoclax in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Previously Treated with B-Cell Receptor Inhibitor Therapy

Rapid Dose Escalation of Venetoclax in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Previously Treated with B-Cell Receptor Inhibitor Therapy

Involvement of RUNX1 Pathway Is a Common Event in the Leukemic Transformation of Chronic Myeloproliferative Neoplasms (MPNs)

Introduction. Runt-related transcription factor 1 (RUNX1) is a transcription factor critical for normal and leukemic hematopoiesis. Three main isoforms, RUNX1 i-1a, i-1b and i-1c, are produced through alternative splicing of RUNX1. Unlike i-1b and i-1c, i-1a has a Runt homology domain (RHD) but lacks the transactivation domain (TAD), and inhibits transcriptional activity of -1b by competing with higher affinity for target gene sequences. Overexpression of i-1a in cell lines inhibits terminal myeloid differentiation. The i-1a is expressed at high levels in patients (pts) with acute myeloid leukemia (AML). Mutations of RUNX1 are common in CMML and MDS, whereas incidence and clinical correlates of RUNX1 mutations in MPNs are poorly known. Aim: To analyze the spectrum and clinical correlates of RUNX1 genetic abnormalities (RUNX1-abn), and the expression of RUNX-1a isoform, in a large series of pts with MPNs. Methods. 686 MPN pts (50 PV, 165 ET, 154 overt-PMF, 143 prefibrotic-PMF, 74 PPV-MF and 100 PET-MF, diagnosed according to 2016 WHO) were included in the study; of these, 625 were analyzed in chronic phase (CP) and 61 at leukemic transformation (LT; of a total of 106 LTs, 58%), including 29 cases with paired samples at diagnosis and LT. Established methods were used for JAK2, MPL and CALR genotyping. A high molecular risk (HMR) category was defined according to Vannucchi A, [Leukemia 2013]. Ion Torrent NGS platform was used to genotype all RUNX1 coding sequence. In 21/29 paired samples, a long reads genome sequencing by Oxford Nanopore technology (ONT) was performed. Copy number variation (CNV) analysis was performed with NanoGLADIATOR package [Magi A, Genome Biol 2019]. Differential expression of RUNX1 mRNA isoform1a and 1b was analyzed by qRT-PCR. Nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimates and log-rank test were used as appropriate. Results. Somatic RUNX1 mutations were identified in 31 pts (4.5%): 14/625 in CP (2.2%) and 17/61 in LT (27.9%; P<.0001); 7/17 LT pts (41.1%) had two variants. 10/29 paired samples were RUNX1 mutated, in 8 of which mutations were acquired at LT. All mutations were heterozygous (median VAF 40%, range 0.4%-54%); 13 (34.2%) were frameshift, 5 (13.2%) nonsense and 20 (52.6%) missense. Most mutations clustered in RHD (26/38, 68.4%) and TAD (9/38, 23.7%). CNV involving RUNX1 was detected by ONT in 4/21 (19%) paired samples, including 2 gain and 2 loss, all acquired at LT; 2 pts had concurrent RUNX1 mutation. Overall, RUNX1abn, inclusive of mutations and CNV, occurred in 19/61 (31.1%) patients at LT. RUNX1-abn were more common in MPL (12.5%) and TN (7.5%) pts compared with JAK2 (4.3%) and CALR (2.0%) mutated (P=0.04), and were enriched in HMR category (61.3% vs 29.4% vs pts without RUNX1-abn; P=.001). We also analyzed CNV in a total of 47 selected genes included in the RUNX1 pathway (KEGG Orthology K08367); we found CNV in >1 of these genes in 16/21 (76%) pts at LT vs 2/16 (12.5%) of CP pts. After median follow-up of 4.9y (range 0.1-36.2y) 273 pts died (39.8%). 106 pts (15.4%) progressed to AML after median of 4.7y (0.1-36.2y) from diagnosis. Incidence of LT differed significantly among diseases: 3 pts in PV (5.9%), 17 in ET (10.1%), 39 in overt-PMF (23.5%), 12 in prefibrotic-PMF (8.3%), 11 in PPV-MF (14.1%) and 24 in PET-MF (22.6%). RUNX1-abn were detected, respectively, in 100%, 53.0%, 23.1%, 41.7%, 36.4% and 12.5% of cases at either CP and/or LT. Leukemia-free survival (LFS) was shorter in pts harboring RUNX1-abn in CP compared to RUNX1-WT pt (2.8y vs NR; P<.0001)(Fig 1A). Expression levels of i-1a and -1b were measured in 34 CP and 24 LT pts. Isoform-1a was significantly elevated in LT pts (mean 7.51±10.1) compared to CP (1.12±1.75; P=.009) (Fig 1B; lines represent the i-1a level rise from CP to LT in 4 paired samples). The i-1a expression levels were irrespective of RUNX1 mutated (mean 7.6±11.9) or unmutated (mean 9.2±6.3) status in LT pts (P=0.8). No difference of i-1b levels in CP (mean 0.9±0.7) versus LP (mean 1.6±1.4; P=0.08) was observed. The expression of i-1a was not dependent on gender, age, leukocyte or platelet count, hemoglobin and percentage of blast cells. Conclusions. These findings indicate that most MPN pts with LT have involvement of RUNX1 pathway through different mechanisms (mutations, CNV, altered isoform expression, genomic abnormalities of RUNX1 target genes), suggesting that a "RUNX1pathy" may represent a common event during leukemogenesis in MPNs. Figure 1 Disclosures Vannucchi: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: 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; Incyte: Membership on an entity's Board of Directors or advisory committees.

Lenalidomide and Rituximab Maintenance Therapy after Front-Line Induction Chemoimmunotherapy in Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma

Introduction: Chronic lymphocytic leukemia (CLL) is incurable with standard therapy. With first-line chemotherapy, some patients (pts) may achieve durable remissions of many months/years. Lenalidomide (LEN) has improved progression-free survival (PFS) when given as maintenance (MNT) therapy after front-line chemotherapy (CALGB10404, CLLM1). The combination of LEN + rituximab (LR) has activity in relapsed CLL, hypothesizing benefit as MNT therapy after first-line chemotherapy. Methods: Adult pts ≥18 years with previously untreated CLL received induction bendamustine (B) 90 mg/m2 IV days 1 & 2 and rituximab (R) IV day 1 (375 mg/m2 cycle 1, then 500 mg/m2 cycles 2-6) for 6 treatment cycles (as few as 4 cycles allowed). MNT therapy with LR was initiated within 12 weeks after cycle 6, day 1 of BR. Criteria to start LR MNT included: neutrophils ≥1000/microliter (uL), platelets ≥75 K/uL, and creatinine clearance ≥40 mL/min. LEN was administered in 28-day cycles for 24 cycles, initially 5-10 mg daily continuous dosing, later modified to 5-10 mg on days 1-21 of each 28-day cycle in 6/2018 due to neutropenia and second malignancy risk. LEN was reduced to 5 mg every other day for toxicities at 5 mg/day. R 375 mg/m2 IV was given every odd cycle (total of 12 doses). Patients discontinuing LEN for any reason were allowed to continue R MNT per protocol. The primary endpoint is PFS with LR MNT therapy, calculated from the first day of MNT therapy until progressive disease (PD), death, or start of a new therapy. Secondary endpoints are response rate and overall survival. Results: Thirty-four pts have enrolled beginning 11/2013, with follow-up through 6/2019. Median age is 64 years, with 8 pts ≥70 years; 8 women and 26 men. CLL FISH panel is available on all pts: 14 with 13q (as sole abnormality), 9 with 11q deletion, 6 with trisomy 12, 4 with normal FISH panel and 1 with 17p deletion. Heavy chain mutation analysis is available on 11 pts: 8 unmutated, 2 mutated, 1 indeterminate. Thirty-one pts completed 4 (n=2) or 6 cycles of induction BR; 3 pts are receiving induction BR. Twenty-four pts have received MNT LR; 7 did not receive LR for reasons of PD during induction (n=2), infection (n=1), pt preference (n=2), renal insufficiency (n=1), and new carcinoma (n=1). MNT LR was completed in 7 pts; 9 pts are still receiving LR. Fourteen subjects have discontinued protocol therapy, 3 during induction due to PD (n=2) and infection (n=1), and 8 during MNT. Toxicities that led to discontinuation of LR were recurrent infections in 7 pts, including 2 events of PJP pneumonia; 4 pts had recurrent neutropenia with infections; 1 pt had neutropenia without infections. Response is assessable in 31 patients using the International Working Group Consensus Criteria. Best responses to treatment were: partial response 65% (22/34), complete response (CR)/unconfirmed CR 24% (8/34). The median number of MNT cycles received is 16. The dose intensity of LEN across total cycles received (n=278): 5 mg every other day (52.5%), 5 mg/day (43.9%), and 10 mg/day (3.6%). The most common reason for dose reduction or dose holding was neutropenia. Most common Gr 3/4 toxicities (reported as events Gr3/Gr4) during MNT therapy were: neutropenia (20/20), leukopenia (19/4), febrile neutropenia (3/1), and infections (11/-). The majority of Gr3 infections were pneumonia/respiratory (n=5). One event of disseminated herpes zoster occurred. Second malignancies during MNT included: basal cell CA (n=1), squamous cell carcinoma (n=5), and colon cancer (n=1). No unexpected second malignancies were observed in pts receiving LR. Two-year PFS (defined from day 1 of MNT therapy) is 90% (95% confidence interval [CI] 0.78-1), and the median follow-up for 24 patient who started maintenance therapy is 1.79 years (95% CI 1.53-2.7). There have been no deaths. Conclusion: The combination of LR is effective in sustaining remissions after a BR induction in previously untreated CLL, but with frequent neutropenia and infections even at low doses of LEN. Most patients discontinuing MNT did so due to neutropenia and/or infections. A shorter planned interval of MNT LR (i.e., 6-12 months) may confer similar benefit to extended dosing that is more tolerable. Pts at high risk for short remissions after front-line chemotherapy (e.g., unmutated heavy chain status, 11q deletion and/or failure to achieve minimal residual disease after induction) may be the populations for which LR MNT therapy is most appropriate. Disclosures Chang: Genentech: Research Funding; Adaptive Biotechnologies: Research Funding; Celgene: Research Funding. OffLabel Disclosure: Lenalidomide administered as maintenance therapy for first treatment of CLL/SLL.

Efficacy of Phosphatidylinositol 3-Kinase (PI3K) Inhibitors in Patients with Relapsed and Refractory Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma

Introduction: Chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/ SLL) is the most common adult lymphoproliferative disorder in western countries and the B-cell receptor signaling pathway has been shown to be involved in the pathogenesis of CLL/ SLL. Phosphatidylinositol 3-kinase (PI3K) is a kinase protein in downstream signaling for multiple pathways in B cells, promoting B-cell survival, proliferation and metabolism. Two prominent PI3K inhibitors, idelalisib (PI3Kδ-selective inhibitor) and duvelisib (PI3Kδ/γ-combinatorial inhibitor), are currently being studied in the treatment of relapsed and refractory CLL/ SLL. The purpose of our study is to explore and consolidate the efficacy of PI3K inhibitors in patients with relapsed and refractory CLL/SLL. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 2019. Phase 3 RCTs utilizing PI3K inhibitors in patients with relapsed and refractory CLL/SLL were incorporated in the analysis. A generic inverse variance method was used to calculate the estimated pooled hazard ratio (HR) for progression-free survival (PFS) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q -statistic. Random effects model was applied. Results: Four phase 3 RCTs with a total of 1,216 patients with relapsed and refractory CLL/SLL were eligible for analysis. Studies compared ofatumumab vs idelalisib+ofatumumab, rituximab vs idelalisib+rituximab, bendamustine+ rituximab vs idelalisib+bendamustine+rituximab and ofatumumab vs duvelisib. The randomization ratio was 2:1 in the study by Jones et al. and 1:1 in other studies. The I2 statistic for heterogeneity was 82%, suggesting moderate heterogeneity among RCTs. The overall pooled HR for PFS was statistically significant at 0.30 (95% CI: 0.20- 0.47; P < 0.0001). The PFS benefit was observed across all ages and regardless of del 17p or TP53 status; age <65 (HR, 0.35; 95% CI: 0.27- 0.46; P < 0.0001), age ≥65 (HR, 0.32; 95% CI: 0.19- 0.54; P < 0.0001), either del 17p or TP53 cohort (HR, 0.33; 95% CI: 0.21- 0.52; P < 0.0001), and neither del 17p nor TP53 cohort (HR, 0.32; 95% CI: 0.19- 0.54; P < 0.0001). In the subset of patients with CLL treated with idelalisib, the pooled HR for PFS was statistically significant at 0.26 (95% CI: 0.18-0.37; P < 0.0001) and the PFS benefit was observed across all ages, and regardless of del17p or TP53 status and IGHV mutation status; age <65 (HR, 0.32; 95% CI: 0.24- 0.43; P < 0.0001), age ≥65 (HR, 0.26; 95% CI: 0.14- 0.47; P < 0.0001), either del17p or TP53 cohort (HR, 0.29; 95% CI: 0.15- 0.57; P = 0.0003), neither del17p nor TP53 cohort (HR, 0.26; 95% CI: 0.20- 0.35; P < 0.0001), IGHV mutated cohort (HR, 0.29; 95% CI: 0.17- 0.51; P < 0.0001), and IGHV unmutated cohort (HR, 0.25; 95% CI: 0.15- 0.40; P < 0.0001). Conclusions: Our study showed that PI3K inhibitors, idelalisib (PI3Kδ-selective inhibitor) and duvelisib (PI3Kδ/γ-combinatorial inhibitor), significantly improved PFS in patients with relapsed and refractory CLL/ SLL regardless of age and poor prognostic features such as del17p or TP53 and IGHV unmutated status, compared to control arm. The efficacy of these drugs must be balanced against the possible side effects. Disclosures No relevant conflicts of interest to declare.

Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) - 15-Month Safety, Response and MRD Evaluation: Third Interim Analysis from the Phase II Vision HO141 Trial

Venetoclax and Ibrutinib for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) - 15-Month Safety, Response and MRD Evaluation: Third Interim Analysis from the Phase II Vision HO141 Trial

A laboratory-based scoring system predicts early treatment in Rai 0 chronic lymphocytic leukemia.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x103/microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today’s chemofree era.

S105 GENETIC MARKERS AND OUTCOME IN THE CLL14 TRIAL OF THE GCLLSG COMPARING FRONT LINE OBINUTUZUMAB PLUS CHLORAMBUCIL OR VENETOCLAX IN PATIENTS WITH COMORBIDITY

Background:Genomic aberrations, IGHV mutation status and mutations in genes such as TP53 are established prognostic factors in CLL in the context of chemoimmunotherapy. Their role is less‐well established when using chemo‐free regimens such as obinutuzumab (GA‐101) plus venetoclax (Ven‐G).Aims:This study evaluates the prognostic impact of genetic risk factors on obinutuzumab+chlorambucil (G‐Clb) vs. obinutuzumab+Venetoclax (Ven‐G).Methods:We assessed the incidence and impact of genetic factors in the phase 3 CLL14 trial comparing G‐Clb vs.Ven‐G in patients with CIRS>6 or creatinine clearance < 70 ml/min. Genomic aberrations were assessed by FISH, IGHV by sequencing with a threshold of 98% homology and mutations via amplicon‐based targeted NGS for TP53, NOTCH1, SF3B1, ATM, MYD88, FBXW7, POT1, BIRC3, XPO1, NFKBIE, EGR2 and RPS15 for variants with allele fraction ≥10%. Of the intention to treat population (n = 432) FISH/IGHV/NGS was assessable in 418/408/421 cases. PFS and OS were estimated via Kaplan‐Meier method and compared by non‐stratified log‐rank test. Hazard ratios (HRs) were calculated via Cox proportional hazards regression modeling.Results:The incidence of genomic aberrations considering the hierarchical model were del(17p) 7%, del(11q) 18%, +(12q) 18% and del(13q) 35%. IGHV was unmutated in 61% of patients. The incidence of gene mutations was NOTCH1 23% (exon 34 and/or 3’UTR), SF3B1 16%, ATM 13%, TP53 10%, XPO1 6%, RPS15 5%, POT1 5%, BRAF 4%, BIRC3 4%, NFKBIE 4%, EGR2 4%, MYD88 2%, FBXW7 1%. High coincidence was found for del(17p) and TP53 mut in 77% of all del(17p) cases and 57% of all TP53 mut cases.None of the parameters impaired overall response rate (ORR) to Ven‐G at treatment completion. For G‐Clb, the ORR rate was lower in patients with del(17p) (36% vs. 73%), del(11q) (58% vs. 74%), TP53 mut (58% vs. 74%), ATM mut (56% vs. 75%) and BIRC3 mut (33% vs. 74%).At a median follow‐up of 29 months, there were 107 events for PFS and 37 for OS in the intention to treat population. Del(17p) was the only genomic abnormality with impact on PFS in G‐Clb (HR 4.6, p < 0.001) and Ven‐G (HR 4.4, p = 0.001; Fig 1a). Similarly, TP53 mutations affected PFS in both treatment arms (G‐Clb HR 2.7, p = 0.001; Ven‐G HR 3.1, p = 0.01). However, TP53 mut without del(17p) was not associated with shorter PFS. None of the other evaluated factors affected Ven‐G efficacy, while for G‐Clb del(11q) (HR 2.3, p = 0.002), BIRC3 (HR 4.0, p = 0.001), NOTCH1 (HR 1.8, p = 0.03) and unmutated IGHV (HR 3.4, p < 0.001) were adverse factors.In several genetic subgroups including del(17p), del(11q), TP53 mut, NOTCH1 mut, SF3B1 mut, ATM mut Ven‐G was superior to G‐Clb in PFS. Regarding IGHV, only patients with unmutated status had a significant PFS benefit from Ven‐G in comparison to G‐Clb (IGHVunm HR 0.2, p < 0.001; IGHVmut HR 0.6, p = 0.29; Fig.1b). Multivariable testing for interaction between treatment and IGHV mutational status was significant (p = 0.03) indicating unmutated IGHV as a predictive factor for increased benefit from Ven‐G. OS was lower with del(17p) in both treatment arms (G‐Clb: HR 11.0, p < 0.001; Ven‐G: HR 3.4, p = 0.03) and with TP53 mut, BRAF mut and IGHVunmut in the G‐Clb arm (HR 5.5, p = 0.002; HR 6.6, p < 0.01; HR 5.4, p = 0.03), while none of the other factors were significantly associated with OS.Summary/Conclusion:Prognostic value of genomic aberrations, IGHV and gene mutations were confirmed for G‐Clb, while with Ven‐G only del(17p) and TP53 mut were associated with short PFS and only del(17p) with short OS. Unmutated IGHV was identified as a predictive factor characterizing a group of patients with particular benefit from Ven‐G.image

PS1152 THE USE OF THE BCL‐2 INHIBITOR IN CLL PATIENTS WHO PROGRESSED AFTER B‐CELL‐RECEPTOR INHIBITORS: A RETROSPECTIVE MULTICENTER ITALIAN EXPERIENCE

Background:B‐cell receptor signaling inhibitors (BCRi), such as Ibrutinib (Ibr) and Idelalisib (Idela), have changed the treatment paradigm for chronic lymphocytic leukemia (CLL), and the BCL‐2 inhibitor Venetoclax (Ven) has shown promising efficacy across clinical studies in relapsed/refractory (R/R) CLL. To date, the exact timing on the use of new pathways inhibitors in the real life practice is not well established.Aims:In this multicenter retrospective real life study we evaluated the overall response rate (ORR), progression‐free survival (PFS), overall survival (OS) of CLL patients (pts) treated with Ven after one or two BCRi. Also the reasons for BCRi discontinuation ‐ adverse events (AE) vs progressive disease (PD) ‐ were evaluated.Methods:We report a multi‐center retrospective analysis that included 76 CLL pts from 18 different Italian centers treated with Ibr and/or Idela prior to Ven for progressive naïve CLL or with relapsed or refractory disease. Twenty‐four pts were treated with one BCRi, while 52 pts with both BCRi before Ven. Median age was 63 years (26–87); 78% pts had an unmutated IgVH status, 33% pts had 17p deletion and 32% pts had TP53 mutation. The sequence of the BCRi prior to Ven was the following: Ibr, Idela, Ven in 16 pts; Idela, Ibr, Ven in 8 pts; Ibr, Ven in 37 pts; Idela, Ven in 15 pts.Results:No statistically significant difference was found in terms of ORR to Ven in patients who had been exposed to one or two BCRi (p = 0.114). At the same time, we observed that pts who received only one BCRi prior to Ven showed a significantly better PFS and OS at 12 months: 76% vs. 35% (p = 0.011) and 88% vs. 57% (p = 0.015), respectively. BCRi treatment was stopped in 70% of pts because of PD and in 30% due to AE. When we considered the impact of the reasons of the first BCRi discontinuation ‐ AE vs PD ‐ on Ven response, we found that the ORR was significantly better in pts who stopped due to AE: 91% vs 49% (p = 0.03). PFS and OS were also significantly better in pts who discontinued treatment due to AE compared to PD, with a PFS of 84% vs 45% (p = 0.003) and an OS of 93% vs 62% (p = 0.028). When we considered the impact of discontinuation of both BCRi on Ven response, the PFS was similar in CLL patients who stopped BCRi due to AE (p = 0.31), while pts who experienced PD to both BCRi showed a worse PFS during Ven (p = 0.06).Summary/Conclusion:The number of BCRi received before BCL2i does not affect ORR to Ven. Single BCRi is better than double BCRi in terms of PFS and OS following Ven treatment, regardless of the BCRi used. Discontinuation of the first BCRi because of AE is associated with a better PFS and OS to Ven compared to pts who experienced PD. The discontinuation of double BCRi for AE did not impact on PFS to Ven. On the other hand, pts who stopped both BCRi for PD showed a tendency to a worse PFS on Ven, without reaching significance. Finally, these preliminary data seem to suggest the use of double BCRi before Ven for pts who discontinued due to AE; on the contrary, we suggest the use of Ven after single BCRi when the reason of discontinuation is disease progression.image

PB1894 INFECTIOUS RISK AND HYPOGAMMAGLOBULINEMIA IN A MONOCENTRIC REAL LIFE COHORT OF 211 CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS FOLLOWED FOR 25 YEARS

Background: Infections represent a major contributor to morbidity and mortality in chronic lymphocytic leukaemia (CLL). Hypogammaglobulinemia (HGG) is a common immune dysfunction in CLL and has been associated with an increased risk of infections. Aims: To analyze impact of HGG on infection free survival (IFS), infectious risk and major infections in a monocentric real life cohort of CLL patients followed for 25 years; to determine impact of IgA deficiency, combined antibody deficiency (CAD) and HGG late onset on infectious death risk. Methods: We retrospectively evaluated 211 unselected CLL patients, diagnosed from 01/1988 to 03/2018, median age 64 years (range 37-88). HGG was evaluated at diagnosis (n = 175), before first line therapy (n = 100) and 12 months after therapy (n = 92). Infection rate during course of disease and infectious morbidity have been collected. Results: During the course of disease 126 patients (60%) underwent at least one infectious event, regardless of severity. Of these, 51 (24%) reported at least one major infectious event (grade ≥3 CTCAE), mainly pneumonitis, skin and soft tissue infections. IgA levels at diagnosis were significantly lower in B and C Binet stage (p = 0.002); IgG (p < 0.001) and IgA levels (p = 0.03) were significantly reduced in patients who experienced infections within 12 months after diagnosis (Fig.1-A); 51 patients (24%) reported major infections and showed reduced IgG and IgA levels at diagnosis (p = 0.01 and p = 0.006, respectively). 25-year IFS for major infections was 67.2% for the entire cohort. Patients in C Binet stage showed significantly lower IFS in comparison with A and B stage (21.5% vs 73.5% and 73.3%, respectively. P < 0.001) (Fig.1-B). In multivariate analysis IGHV unmutated status was associated with lower IFS (p = 0.009). CAD was observed ad diagnosis in 60% of patients with severe infections versus 27% of patients who did not experience these events (p < 0.001). Furthermore, 20.4% of patients developed a late onset CAD with a significant reduction of IFS and an increase of serious infectious events. Overall survival (OS) was heavily reduced by early infectious events occurred during the first year from diagnosis, with higher mortality rate in patients with CAD at diagnosis or late onset; in both cases, risk of infectious death was 10 times higher compared to remaining cohort (p = 0.004).Summary/Conclusion: Our results confirmed the negative impact of HGG on infectious risk and major infections. IgA deficit and CAD were strong predictors for development of these events. Early identification of clinical profile of high risk infections (selective IgA deficit, CAD, late onset HGG) is useful in optimal management of CLL patients.

Outcomes in chronic lymphocytic leukemia (CLL) patients with NOTCH1 signaling pathway mutations.

7524 Background: NOTCH1 is one of the most frequently mutated genes in CLL and has emerged as a marker of poor prognosis. Additional mutations in the NOTCH1 signaling pathway, specifically MED12, FBXW7, and SPEN, have been identified in CLL but their clinical significance has yet to be fully determined. We evaluated the clinical outcome of time to first treatment (TTFT) to compare patients with mutations to those who are wild type for NOTCH1, MED12, FBXW7, and SPEN, hypothesizing that patients with these mutations will behave similarly to NOTCH1 mutated patients and have a shorter TTFT. Methods: We conducted a single center retrospective database review of 506 patients diagnosed with CLL from 1980 to 2018 who underwent whole exome profiling between 2015 to 2018 with a lymphoid specific 75-gene next generation sequencing (NGS) panel (Genoptix Inc). The TTFT was estimated using Kaplan-Meier methods, and the difference between groups was compared using the log-rank test. Multivariate analysis (MVA) was performed with Cox proportional hazards regression. Results: Of the 506 patients who underwent NGS testing, 121 (23.9%) had at least one mutation in the NOTCH1 signaling pathway. These patients were diagnosed at an older age (62.0 years vs. 60.0 years, p=0.04) and had higher rates of CD38 positivity (40.9% vs. 22.9%, p≤0.001), Trisomy 12 (36.0% vs. 15.8%, p≤0.001), and IGVH unmutated status (71.3% vs. 44.1%, p≤0.001). They also had a shorter TTFT with a median of 3.93 years compared to 5.02 years in patients without any mutation in the NOTCH1 signaling pathway (p=0.002). In MVA, IGVH unmutated status and CD38 positivity remained independently predictive for TTFT. Conclusions: We identified three mutations in genes associated with regulation of NOTCH1 signaling that appear to signify poor prognosis, predict for a shorter TTFT, and associate with similar baseline factors that NOTCH1 does such as Trisomy 12 and IGVH unmutated status. NOTCH1 mutated patients have poor response to chemoimmunotherapy and are associated with aggressive disease biology. Future research is needed to determine whether mutations in MED12, FBXW7, or SPEN may also predict for poor response to frontline chemoimmunotherapy or novel agents currently used in CLL.

Deep targeted sequencing of TP53 in chronic lymphocytic leukemia: clinical impact at diagnosis and at time of treatment.

In chronic lymphocytic leukemia, TP53 mutations and deletion of chromosome 17p are well-characterized biomarkers associated with poor progression-free and overall survival following chemoimmunotherapy. Patients harboring low burden TP53 mutations with variant allele frequencies of 0.3-15% have been shown to have similar dismal outcome as those with high burden mutations. We here describe a highly sensitive deep targeted next-generation sequencing assay allowing for the detection of TP53 mutations as low as 0.2% variant allele frequency. Within a consecutive, single center cohort of 290 newly diagnosed patients with chronic lymphocytic leukemia, deletion of chromosome 17p was the only TP53 aberration significantly associated with shorter overall survival and treatment-free survival. We were unable to demonstrate any impact of TP53 mutations, whether high burden (variant allele frequency >10%) or low burden (variant allele frequency ≤10%), in the absence of deletion of chromosome 17p. In addition, the impact of high burden TP53 aberration (deletion of chromosome 17p and/or TP53 mutation with variant allele frequency >10%) was only evident for patients with IGHV unmutated status; no impact of TP53 aberrations on outcome was seen for patients with IGHV mutated status. In 61 patients at time of treatment, the prognostic impact of TP53 mutations over 1% variant allele frequency could be confirmed. This study furthers the identification of a clinical significant limit of detection for robust TP53 mutation analysis in chronic lymphocytic leukemia. Multicenter studies are needed for validation of ultra-sensitive TP53 mutation assays in order to define and implement a technical as well as a clinical lower limit of detection.

Targeting IRAK4 Disrupts Inflammatory Pathways and Delays Tumor Development in Chronic Lymphocytic Leukemia

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediatedby the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Gene expression profiling of 423 CLL cases, assessed by the computational GSEA analysis tool, identified enrichment of gene sets related to cytokines and inflammation in the subgroup carrying mutations in MYD88. Accordingly, CLL cells from individuals with MYD88 mutations showed higher p65 NF-κB activity and secreted higher basal levels of CCL2, CCL3 and CCL4 cytokines compared with unmutated cases. To assess the effects of this pathway in CLL, we stimulated these cells with a TLR agonists mix, containing Pam3CSK4, HKLM, FSL1 and ODN2006, which was able to induce cytokines secretion, increase in NF-KB activity and STAT3 signaling as well as cells proliferation and migration. Here, we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. ND2158 was able to efficiently decrease all TLR effects seen with the mix like STAT3 signaling and the secretion of CCL2, CCL3, CCL4, TNFα, IL1β, IL10 and IL6 cytokines (assessed by Luminex). NF-κB signaling was also modulated by the drug, as observed by the downregulation of IκBα phosphorylation protein level, p65 translocation to the nucleus and decreased p65 DNA binding activity. ND2158 also impaired CLL proliferation (checked by carboxyfluoresceinsuccinimidyl ester staining) and migration towards CXCL12 in MYD88-mutated and -unmutated CLL cases.ND2158 also induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. To further validate this results, we checked the effect of ND2158 in splenocytes from leukemic Eμ-TCL1 mice and similar antitumor effects were seen.In addition to the direct impact on tumor cells, we analyzed whether ND2158 also affects other immune cells that also signal via MYD88/IRAK4. Monocytes, broadly known for their importance in tumor supporting, were also modulated by TLR agonists, and a reduction in activation markers and cytokines secretion was achieved after ND2158 treatment.In vivo studies in Eμ-TCL1 leukemic mice showed a significant reduction in tumor load in CLL-affected organs (peripheral blood, spleen, lymph node and peritoneal cavity) associated with reduced PD-L1 expression levels in mice treated with 100 mg/kg/day of the IRAK4 inhibitor. A decrease in spleen weight and size was also detected after the treatment. Corroborating the in vitro studies,ND2158 also modulated tumor-supporting monocytes. The effect of ND2158 was also assessed in CD8+ effector T cells, where a low proliferative (Ki-67+ percentage), exhausted phenotype (increase in TIGIT, CD244, CD160 and LAG3 markers) and less functional population (increase in the immune checkpoint protein PD-1) was found.Based on our in vitro data, it seems that ND2158 targets both MYD88 mutated and unmutated CLL cases to a similar degree. Since CLL progression in the TCL1 mouse model has not been reported to be driven by an activating MYD88 mutation, our in vivo results support the notion that IRAK4 inhibition might also be effective in CLL patients with an unmutated MYD88 status. To overcome the potential negative impact of IRAK4 inhibition on T cells, the combination treatment with drugs that improve T cell function and avoid their rapid exhaustion should be considered. As we observed increased expression of several inhibitory receptors on CD8+ T cells in ND2158-treated mice, including PD-1 and TIGIT, blocking these receptors with antibodies might be a successful strategy to overcome loss of T cell function and improve therapy outcome.In summary, our data show the importance of TLR signaling in CLL development and suggest IRAK4 as a novel treatment target for CLL that targets both the malignant cells and the tumor-supportive inflammatory milieu. DisclosuresNo relevant conflicts of interest to declare.

Outcome of Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) Treated with Ibrutinib within a Named Patient Program (NPP) in Italy. a Real-Life Retrospective Study

Outcome of Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) Treated with Ibrutinib within a Named Patient Program (NPP) in Italy. a Real-Life Retrospective Study

Identification of prognostic parameters in CLL with no abnormalities detected by chromosome banding and FISH analyses.

Chronic Lymphocytic Leukaemia (CLL) is a heterogeneous disease with a clinical course dependent on cytogenetic features. However, in 15-20% of cases both chromosome banding and fluorescence insitu hybridisation analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we performed a comprehensive analysis on 171 patients including genomic arrays (comparative genomic hybridisation and single nucleotide polymorphism), immunoglobulin heavy chain variable region genes (IGHV) status, flow cytometry and targeted sequencing. Genomic arrays detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV status was unmutated in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (15/171; 9%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of these, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, by integrating genomic and molecular data, we identified patients at higher risk for treatment need.

Cumulative experience and long term follow-up of pentostatin-based chemoimmunotherapy trials for patients with chronic lymphocytic leukemia

ABSTRACTBackground: 7 regimens of pentostatin based chemoimmunotherapy (CIT) for progressive previously untreated CLL primarily with long term follow-up to update both efficacy and toxicity.Research design and methods: Prognostic markers including assessment of IGVH and FISH status were done on all. Response rates and 95% binomial confidence intervals were calculated for each regimen and in the combined cohort. Overall survival and treatment-free survival were evaluated using Kaplan-Meier methods.Results: The initial CIT trial was pentostatin (2 mgs/m2), cyclophosphamide (600 mg/m2) and rituximab (PCR) but subsequent P based CIT trials with modifications in subsequent trials. The cohort (n = 288) included 52% with unmutated IGVH status and del17p (4.5%) and del11q (14.9%). Toxicity profiles were primarily hematologic and no patient has developed MDS or AML after a median follow-up of 6.4 years. The overall response rate across all trials was found to be over 90% with a 41% complete response rate. We validated that the CLL IPI model segregates progressive CLL patients into 4 risk groups associated with OS and TFS.Conclusions: The high overall and complete response levels in favorable genetic risk CLL along with favorable toxicity profiles provide rationale for consideration of a PC based strategy for previously untreated progressive CLL.

B-cell Receptor Signaling Regulates Metabolism in Chronic Lymphocytic Leukemia.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high β2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition.Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692-703. ©2017 AACR.

Lenalidomide maintenance after first-line therapy for high-risk chronic lymphocytic leukaemia (CLLM1): final results from a randomised, double-blind, phase 3 study

The combined use of genetic markers and detectable minimal residual disease identifies patients with chronic lymphocytic leukaemia with poor outcome after first-line chemoimmunotherapy. We aimed to assess lenalidomide maintenance therapy in these high-risk patients. In this randomised, double-blind, phase 3 study (CLLM1; CLL Maintenance 1 of the German CLL Study Group), patients older than 18 years and diagnosed with immunophenotypically confirmed chronic lymphocytic leukaemia with active disease, who responded to chemoimmunotherapy 2-5 months after completion of first-line therapy and who were assessed as having a high risk for an early progression with at least a partial response after four or more cycles of first-line chemoimmunotherapy, were eligible if they had high minimal residual disease levels or intermediate levels combined with an unmutated IGHV gene status or TP53 alterations. Patients were randomly assigned (2:1) to receive either lenalidomide (5 mg) or placebo. Randomisation was done with a fixed block size of three, and was stratified according to the minimal residual disease level achieved after first-line therapy. Maintenance was started with 5 mg daily, and was escalated to the target dose of 15 mg. If tolerated, medication was administered until disease progression. The primary endpoint was progression-free survival according to an independent review. The pre-planned interim analysis done by intention to treat was done after 20% of the calculated progression-free survival events. This study is registered with ClinicalTrials.gov, number NCT01556776; treatment in the lenalidomide group is still ongoing. Between July 5, 2012, and March 15, 2016, 468 previously untreated patients with chronic lymphocytic leukaemia were screened for the study; 379 (81%) were not eligible. Recruitment was closed prematurely due to poor accrual after 89 of 200 planned patients were randomly assigned: 60 (67%) enrolled patients were assigned to the lenalidomide group and 29 (33%) to the placebo group, of whom 56 (63%) received lenalidomide and 29 (33%) placebo, with a median of 11·0 (IQR 4·5-20·5) treatment cycles at data cutoff. After a median observation time of 17·9 months (IQR 9·1-28·1), the hazard ratio for progression-free survival assessed by an independent review was 0·168 (95% CI 0·074-0·379). Median progression-free survival was 13·3 months (95% CI 9·9-19·7) in the placebo group and not reached (95% CI 32·3-not evaluable) in the lenalidomide group. The most frequent adverse events were skin disorders (35 patients [63%] in the lenalidomide group vs eight patients [28%] in the placebo group), gastrointestinal disorders (34 [61%] vs eight [28%]), infections (30 [54%] vs 19 [66%]), haematological toxicity (28 [50%] vs five [17%]), and general disorders (28 [50%] vs nine [31%]). One fatal adverse event was reported in each of the treatment groups (one [2%] patient with fatal acute lymphocytic leukaemia in the lenalidomide group and one patient (3%) with fatal multifocal leukoencephalopathy in the placebo group). Lenalidomide is an efficacious maintenance therapy reducing the relative risk of progression in first-line patients with chronic lymphocytic leukaemia who do not achieve minimal residual disease negative disease state following chemoimmunotherapy approaches. The toxicity seems to be acceptable considering the poor prognosis of the eligible patients. The trial independently confirms the clinical significance of a novel, minimal residual disease-based algorithm to predict short progression-free survival, which might be incorporated in future clinical trials to identify candidates for additional maintenance treatment. Celgene Corporation.

Abstract 3547: B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia

Abstract Peripheral blood B cells in chronic lymphocytic leukemia (CLL) are quiescent, yet have an active RNA transcription and protein translation processes, suggesting that these malignant lymphocytes may be metabolically active. However, CLL metabolism and its relationship to disease biology has not been well-explored. In our previous investigation, we reported that compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in quiescent peripheral blood B lymphocytes from CLL patients (Vangapandu, H.V. et al, AACR 2014). In 45 patient samples tested, glycolysis (extracellular acidification rate, ECAR) was consistently low (1 to 15 mpH/min/5 x 105 lymphocytes), whereas, oxygen consumption rate (OCR) varied considerably (5 to 190 pMoles/min/5 x 105 lymphocytes). Among the prognostic factors, high OCR correlated strongly with ZAP 70 positivity, unmutated IGHV status, greater β2M levels and higher Rai stage. In contrast, glycolytic flux (ECAR) from same patient samples did not associate with prognostic factors. Further, OCR did not vary on the basis of frequently occurring cytogenetic abnormalities, 13q14, 17p, and 11q deletions or trisomy 12. Since, ZAP 70 and IGHV unmutated status are associated with augmented B-cell receptor (BCR) pathway signaling, we tested the impact on OCR after genetic ablation of B-cell receptor (CH2 region of IgM). A CRISPR-Cas9-mediated BCR knockout mitigated OCR in a malignant B cell line without impacting the rate of proliferation. A critical node in the BCR pathway is PI3 Kinase and PI3K isoforms, δ and γ are involved in B-cell malignancies. Consistent with BCR ablation results, knocking out PIK3CD (codes for catalytic subunit δ), dramatically reduced OCR and ECAR. Pharmacological inhibitors of the PI3K pathway, duvelisib (a PI3K δ/γ inhibitor in phase III clinical trials) or idelalisib (FDA approved PI3K δ inhibitor) also decreased OCR. Direct inhibition of AKT with MK-2206 showed similar results. Collectively, these data suggest that CLL cellular metabolism is associated with prognostic factors and linked to BCR signaling pathway. Since, the PI3K inhibitors used in our study are being tested clinically for patients with CLL, investigations on the impact on metabolomics during therapy and translation of results to combination strategies need to be explored. Note: This abstract was not presented at the meeting. Citation Format: Hima V. Vangapandu, Ondrej Havranek, William G. Wierda, Michael J. Keating, Richard Eric Davis, Christine M. Stellrecht, Varsha Gandhi. B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2017-3547