Chronic Lymphocytic Leukemia Subjects Research Articles

Time Limited Exposure to a ROR1 Targeting Bispecific T Cell Engager (NVG-111) Leads to Durable Responses in Subjects with Relapsed Refractory Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL)

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is absent or expressed at low levels in normal adult tissues but overexpressed in a range of malignancies including CLL and MCL. NVG-111 is a humanized first in class, tandem scFv, ROR1xCD3 bispecific T cell engager. NVG-111 mediates potent killing of ROR1 + tumors by engaging a unique epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder engineered for attenuated cytokine release. Methods: In this phase 1, first-in-human, dose-escalation study (ClinicalTrials.gov identifier: NCT04763083), NVG-111 was evaluated in relapsed/refractory CLL and MCL subjects with an ECOG PS of 0-2 who received ≥2 prior lines of treatment including a Bruton tyrosine kinase inhibitor (BTKi). Bayesian continual reassessment method with overdose control was implemented to guide escalation over a dose range of 0.3-45ug/day. Each subject received at least one dose of NVG-111, administered as a continuous intravenous infusion (cIV) over 21 days followed by 7 days off drug (=1 cycle). NVG-111 was administered in combination (N=8) with ibrutinib to subjects who had achieved a partial response to >1 year of ibrutinib therapy, or as monotherapy in subjects that progressed after covalent BTKi/B-cell lymphoma 2 inhibitor (BCL2i) (N=4). Primary objective was safety, with the secondary objectives being efficacy (overall response rates [ORR], minimal residual disease (MRD) measured by ERIC-compliant flow cytometry and duration of response [DoR]). Serum cytokine levels were determined using a human high sensitivity cytokine premixed magnetic Luminex assay and T cell function was longitudinally evaluated using cytometry by time of flight (CyTOF) analysis. Results: Between May 2021 and July 2023 12 subjects (10 males and 2 females, median age 60 years) completed a maximum of 6 cycles of treatment with NVG-111 (median=3 cycles [range 1-6 cycles]). Pharmacokinetic and pharmacodynamic data demonstrated systemic NVG-111 exposure and exposure-dependent NVG-111 targeting of ROR1 on circulating tumor cells. Adverse events (AEs) related to NVG-111 occurred in 10 subjects (83%), with the most common being nausea (58%), headaches (67%), and fatigue (33%), majority of which were grade 1 or 2. Grade 1(71%) or 2(29%) cytokine release syndrome (CRS) occurred in 58% of subjects during week 1 of cycle 1 except in one subject who had a second grade 1 CRS in cycle 2. Grade 3 dose limiting toxicities (DLTs) occurred in three subjects (25%) consisting of transient elevation of liver enzymes (ALT and AST) in one subject dosed at 3ug/day, immune effector cell-associated neurotoxicity syndrome-like symptoms (ICANS) in one subject dosed at 30ug/day and headache in one subject dosed at 45ug/day. All AEs were fully reversible with no late emergent grade 3 or greater toxicities. Evidence of T cell activation was observed in all 12 subjects with peak cytokine levels at the 30µg/day dose levels (mean±SEM): TNFα, 43±17pg/ml; IL6, 566±541pg/ml; IFNɣ, 18±12pg/ml; and IL10, 103±56pg/ml. Furthermore, T cell profiling showed an increase in cytotoxic CD8 + T cell activation marker expression during each NVG-111 treatment cycle without evidence of T cell exhaustion. Efficacy was evaluable in 11 subjects with objective clinical responses observed in 55% (6/11), including two subjects with clear evidence of single agent activity. Amongst these, three CLL subjects were rendered MRD negative in peripheral blood and one MCL subject achieved complete metabolic response by the Lugano criteria. Two subjects had stable disease but in one of these subjects there was a >50% reduction in peripheral blood lymphocytosis and a 40% reduction in lymphadenopathy. Despite time limited exposure to NVG-111, the restricted mean survival time for DoR is currently 13.6 months (SEM 3.07), the median is not yet calculable with response ongoing in four subjects. The median progression-free survival currently is 18.7 months (95% CI 2.6 - not calculable). Conclusion: These data provide clinical proof of concept for selective targeting of ROR1 with a TCE leading to objective evidence of antitumor activity with encouraging response durability even in CLL patients known to have defective T cell function. The safety profile was consistent with the mechanism of action. Further evaluation of this promising molecule is ongoing.

Comparison of Dietary Intake and Dietary Inflammatory Index in Patients with Chronic Lymphocytic Leukemia By Treatment Status to Cohabitating Controls

Chronic lymphocytic leukemia (CLL) is an incurable disease with a varied presentation ranging from indolent and asymptomatic to morbid and life-limiting. As tumor biology insufficiently explains this variability, we hypothesized that behavioral and environmental factors including diet and the gut microbiome, where diet is a key influence, may influence CLL pathogenesis and response to therapy. We performed a cross-sectional sample and survey collection study of the gut microbiome, blood, and diet in patients with CLL treated at the University of Nebraska Medical Center between 2021-2023. Eligible subjects included 1) untreated CLL with Rai stage 2-4 disease (untreated CLL), 2) CLL treated with a Bruton's tyrosine kinase inhibitor (BTKi), and 3) individuals without cancer cohabitating with enrolled CLL subjects (control). All subjects provided stool samples for microbial analysis, and subjects with CLL provided blood for serum cytokine analysis. All subjects completed the Diet History Questionnaire version 3.0 (DHQ-3). The DHQ-3 is a self-administered food frequency questionnaire, where participants complete 135 questions on food/beverages and 26 on dietary supplements consumed in the past month. A total and component healthy eating index (HEI) score was derived from the DHQ-3 and represents how well diet intake corresponds to the dietary guidelines recommended by the United States Department of Agriculture and Health and Human Services. The HEI is based on 13 individual components (Figure 1) and scored from 0-100, where higher scores indicate healthier diets. Inflammatory capacity of the diet was assessed by the Dietary Inflammatory Index (DII) (Shivappa et al, Public Health Nutrition, 2013), a measure of dietary components associated with serum markers of inflammation. The DII has a range of scores up to 7.98 suggestive of greater systemic inflammation (median DII score 0.23), and a minimum of -8.87, suggestive of consumption of anti-inflammatory foods. The DII was calculated and compared between each cohort. Statistical analysis was performed using ANOVA. A total of 58 subjects enrolled on the study, 42 with CLL and 16 cohabitating controls. A total of 27 subjects were included in the analysis after data cleaning. Fifteen subjects (56%) were treated with a BTKi, 5 (19%) had untreated Rai stage 2 disease, and 7 (26%) were cohabitating controls. Median ages (72 vs 67 vs 71 for BTKi, untreated, control, respectively) were similar. Median absolute lymphocyte count was 3.2 cells x10^3/µL (1.3 - 131 range) in the BTKi and 55 cells x10^3/µL (13-134 range) in the untreated cohort. There were 5/15 (33%) with 17p deletion and/or p53 mutation in the BTKi cohort and 2/5 (40%) in the untreated cohort. Median time from diagnosis to study entry was 30 months (range 3-151 months) in the untreated cohort, and median time on BTKi therapy was 30 months (range 2-92 months). Within the BTKi cohort, 33% received ibrutinib, 20% received zanubrutinib, and 47% received acalabrutinib. Overall dietary habits were largely similar between the 3 cohorts (Table 1). The mean total HEI score was higher in the untreated (66) and control (66) populations compared to the BTKi cohort (59) ( p = 0.23), although not significantly different. These scores are similar to the 2020 median score of 61 for Americans 60 years and above. Added sugar intake was highest in the untreated cohort (HEI component score of 9.6, 6.3, 8.6 untreated, BTKi, control p = 0.005, respectively) (Figure 1). After calculating the DII for each cohort, no statistically significant differences were observed (Table 1). The range of DII scores across subjects was -5.2 - 4.0, within the expected range for DII calculations. DII scores were 0.44, 0.2, 0.82 p = 0.89 for BTKI, untreated CLL, and control cohorts respectively. To our knowledge, this study is the first to analyze dietary patterns in well-defined homogenous CLL populations (BTKi vs Rai stage 2+ CLL cohort) and compare them to cohabitating individuals. Although overall dietary patterns and DII were similar across cohorts, slight differences in added sugar intake was observed between the cohorts, suggestive that subtle yet important dietary differences may be observed. These data provide a baseline to study personalize dietary approaches to patients with CLL, who increasingly ask clinicians for nutritional interventions as part of their comprehensive cancer care.

Mechanisms of MCL-1 Protein Stability Induced by MCL-1 Antagonists in B-Cell Malignancies.

Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i-induced Mcl-1 protein stability. Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein-protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling. Using both in vivo and in vitro analysis, we demonstrate that MCL-1i-induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors. We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.

CLL-050 Induction of Neutralizing Antibodies in Chronic Lymphocytic Leukemia Patients After SARS-CoV-2 mRNA Vaccination: A Monocentric Experience

Background: SARS-CoV-2 infection increases the clinical risk and mortality of patients with chronic lymphocytic leukemia (CLL). Covid-19 vaccinations have the potential to protect individuals from severe infection, but little is known about the antibody response profile in these patients after vaccination. We compared the anti-receptor binding domain (RBD) and neutralizing antibody responses of 27 patients with CLL to those of healthy donors after mRNA Covid-19 vaccination. Methods: Of the 27 patients enrolled in the study, 21 received the BNT162b2 mRNA vaccine and 6 received the Spikevax mRNA vaccine. Four patients were in clinical remission after treatment, 10 were treatment-naïve, and 13 were under treatment at the time of vaccination. Blood samples were taken before vaccination and after the second dose, with a median time between of 23 days. The levels of anti-RBD IgG, IgM, and IgA were evaluated by ELISA on recombinant RBD; the neutralizing antibody response was evaluated by spike protein inhibition assay (SPIA). We also performed a fluorescence-activated cell sorting study of peripheral blood mononuclear cell subpopulations, a disease burden assessment using blood indicators (lactate dehydrogenase, beta-2-microglobulin, lymphocytic count) and imaging, and an evaluation of IgG levels at the time of vaccination. Results: A comparison of anti-RBD antibody seroconversion (IgG, IgM, and IgA) and neutralizing antibodies present after vaccination between 27 patients with CLL and healthy control subjects indicated that CLL patients had a considerably lower response rate (50% vs. 100%) and lower antibody levels than the healthy controls. We found an inverse correlation between the spleen dimension and lymphonodal area. By stratifying patients according to the presence of neutralizing antibodies after vaccination, we found a larger prevalence of SPIA positives than SPIA negatives in treatment-naïve patients (60%); however, patients undergoing treatment had a much lower percentage of SPIA positives (23.1%). Conclusions: Only around half of vaccinated CLL patients acquire detectable anti-RBD and neutralizing antibodies, according to our findings. Furthermore, we discovered a substantial difference in the rates of detectable anti-SARS-CoV-2 antibodies between patients who were treatment-naïve/in clinical remission and those on CLL-directed treatment. The persistence and burden of disease represent a surrogate of vaccine failure, probably due to the persistence of immune dysfunction.

A sensitive and robust bioanalytical assay for pharmacokinetic analysis of ROR1xCD3 bispecific T cell engager (NVG-111) in a first-in-human study.

e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on hematological and solid tumors. NVG-111, a first in class humanized tandem scFv ROR1xCD3 bispecific antibody elicits potent killing of ROR1+ tumor cells in vitro and in vivo. This bispecific T-cell engager (TCE) is being evaluated in a first in human, Phase I trial in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The predicted therapeutic dose and steady state serum concentrations of NVG-111 were estimated by allometric scaling using relevant doses from a murine PK study. To assess free drug levels in patients following 21 days of continuous infusion of NVG-111, a bespoke, sensitive pharmacokinetics (PK) assay with high levels of specificity and sensitivity was developed. Methods: Anti-idiotype (anti-ID) antibodies directed to anti-ROR1 (αROR1-ID) and anti-CD3 (αCD3-ID) were generated by mouse immunization or by phage display from customized libraries. A proof-of-concept sandwich ELISA assay was developed using αCD3-ID to capture NVG-111 and detection by biotinylated hROR1-streptavidin-HRP. Gyrolab and Quanterix Simoa high sensitivity ELISA platforms were used to detect NVG-111 by αCD3-ID capture and αROR1-ID detection. The mesoscale discovery electrochemiluminescence assay (MSD-ECLA) was developed using a reversed format; NVG-111 capture with αROR1-ID and detection with αCD3-ID. Results: Allometric scaling predicted a theoretically relevant therapeutic dose and steady state serum concentration of 1ng/mL NVG-111 in humans, which was just at the level of sensitivity of a conventional ELISA under non-matrix conditions. Transferring the format to Quanterix Simoa had limited success due to high background levels in all configurations evaluated. The Gyrolab platform increased sensitivity to 75pg/mL, but suboptimal individual human sera matrix selectivity limited assay validity. Assessment of MSD-ECLA provided the best signal/noise, enhanced human disease and healthy sera selectivity, and a dynamic sensitivity range of 250pg/mL to 32ng/mL, which enabled the development of a GCLP qualified PK assay. The MSD-ECLA assay was employed to measure NVG-111 concentrations in CLL or MCL subjects dosed with 0.3-30µg/day NVG-111. MSD-ECLA detected drug in patients receiving NVG-111, with a range of steady-state serum concentrations (Cavg.ss) of 168-610pg/mL. This was in-line with the predicted drug levels from the single species allometric scaling, albeit with observed levels being marginally lower than expected. Conclusions: Development, custom optimization and validation of a highly sensitive MSD-ECLA PK assay has enabled GCLP-compliant measurement of circulating NVG-111 in CLL or MCL patients treated with at least 10µg/day cIV NVG-111.

Exposure-Response of Entospletinib (ENTO) for the Treatment of Relapsed or Refractory Chronic Lymphocytic Leukemia

Background: Entospletinib (ENTO) is a selective spleen tyrosine kinase (SYK) inhibitor that has demonstrated evidence of efficacy and tolerability in chronic lymphocytic leukemia (CLL). The relationship between ENTO plasma exposures and clinical efficacy and safety were evaluated in relapsed/refractory (R/R) CLL subjects, following administration of 100, 200, and 400 mg twice daily (BID) in a Phase 2 Study (NCT01799889; study -0102).Methods: ENTO pharmacokinetics (PK) and plasma exposures (Cmax, AUCtau, and Ctau) were characterized in 60 R/R CLL subjects using a population PK model developed from data collected in phase 1/2 clinical studies. ENTO plasma samples were collected on day 1, and day 8 (pre-dose,1.5 hours, and 4 hours post-dose), day 15, and day 22 (pre- and 1.5 hours post-dose) of cycle 1 and on day 1 (pre- and 1.5 hours post-dose) of subsequent 28 day cycles. Data for efficacy and safety endpoints were collected every 8 weeks (for cycles 1-6) followed by every 12 weeks (for cycles 7-18). Efficacy endpoints, including best overall response rate (BOR), progression-free survival (PFS), sum of products of the greatest perpendicular diameters (SPD) of index lesions, maximum change from baseline in lymphocytes, platelets, and hemoglobin were evaluated against ENTO plasma exposure (AUCtau and Ctau). Safety endpoints, including adverse events (all grades) of skin rash, infection, diarrhea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin (total), and neutrophils (maximum change from baseline) versus ENTO plasma exposure (AUCtau and Cmax) were evaluated.Results: A dose proportional increase in ENTO exposures was observed across 100, 200, and 400 mg BID regimen. A greater reduction in SPD and lymphocytes and an increase in platelets were associated with higher ENTO exposures. No exposure-response relationship was observed for PFS at Week 24 (i.e., there was an overlap in survival curves across quartiles of ENTO exposures) or hemoglobin. No association between ENTO exposures vs incidence of skin rash, infection, diarrhea, AST, ALT and neutrophils was observed. There was a slight trend toward elevated total bilirubin with higher ENTO exposures.Conclusions:In R/R CLL subjects, following ENTO 100, 200, and 400 mg BID regimen, higher exposures corresponding to 400 mg BID are associated with greater efficacy (reduction in SPD lymphocytes and an increase in platelets). No clinically relevant relationship between ENTO exposures and safety, except total bilirubin, was observed. Overall, the analyses support ENTO 400 mg BID regimen as a starting dose for future phase 2 trials in CLL patients. [Display omitted] DisclosuresGupta:Gilead Sciences, Inc.: Employment, Equity Ownership. Meng:Gilead Sciences, Inc.: Employment, Equity Ownership. Sharma:Gilead Sciences, Inc.: Employment, Equity Ownership. Ye:Gilead Sciences, Inc: Employment, Equity Ownership. Silverman:Gilead Sciences, Inc.: Employment, Equity Ownership. Abella:Gilead Sciences, Inc.: Employment, Equity Ownership. Mathias:Gilead Sciences, Inc.: Employment, Equity Ownership.

Do Statins Enhance the Anti-Cancer Activity of Venetoclax?

▪Introduction: Venetoclax is an oral, small-molecule BCL-2 inhibitor capable of restoring apoptosis in susceptible cancers. It is approved in the US for treatment of relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) with 17p deletion and in Europe for patients who are unsuitable for or who have failed B-cell receptor inhibitor (BCRi) therapy. It is also being evaluated in the broader CLL population and other hematologic malignancies. These diseases mostly afflict older patients, many of whom are treated with statins for high cholesterol. Aside from their cholesterol-lowering utility, statins have shown potential to induce apoptosis in various cancer cell lines, with evidence of synergism when combined with BCL-2 inhibition. On this background, post-hoc analyses of clinical trials in patients with CLL and multiple myeloma (MM) were undertaken to determine if statins have the potential to enhance the activity of venetoclax.Methods: Data from CLL subjects were pooled from 3 single-arm, open-label Phase 1 or 2 trials of venetoclax monotherapy in subjects with R/R disease: Study 1 (NCT01889186) enrolled subjects exclusively with 17p(del) CLL; Study 2 (NCT01328626) was a first-in-human, dose-escalation study in subjects with CLL/SLL and NHL; Study 3 (NCT02141282) enrolled CLL subjects previously treated with ibrutinib or idelalisib. MM data were from 2 single-arm, open-label Phase 1 studies in patients with R/R disease: Study 4 (NCT01794507) evaluated venetoclax in combination with bortezomib and dexamethasone (VenVd); Study 5 (NCT01794520) evaluated treatment with venetoclax monotherapy (Ven) or venetoclax plus dexamethasone (Vendex). Previously, it was reported that single-agent activity of venetoclax is almost entirely restricted to t(11;14) positive MM; therefore, analyses of response among subjects who received Ven or Vendex were limited to this subgroup. The following CLL endpoints were analyzed based on investigator assessment: overall remission rate (ORR), the composite complete remission (CR) rate (CR + CR with incomplete marrow recovery), progression free survival (PFS), and overall survival (OS). Odds ratios for ORR and CR were compared via Fisher exact test. PFS and OS were analyzed by Kaplan-Meier methodology. Multivariate analyses were conducted using Cox proportional hazard model (OS and PFS) and logistic regression model (ORR and CR) with implementation of a stepwise model selection procedure. For the MM studies, descriptive statistics were prepared based on investigator assessment for ORR, partial response (PR), and very good partial response (VGPR) or better. Exposure-response analyses have previously shown that higher exposures to venetoclax in CLL patients are associated with greater likelihood of CR and longer PFS; therefore, we also investigated the potential for statins to increase venetoclax exposures utilizing steady state pharmacokinetic sampling from Study 2.Results: In CLL studies, 22% of patients received a statin; statin users were more often male (79% vs. 67%), ≥65 years of age (73% vs. 54%), and enrolled at US sites (69% vs 46%). While the proportion of CLL subjects with any response (ORR) was similar for subjects with and without statin use (76% vs 75%), more statin users achieved CR, with cumulative CR rates of 26.7% vs. 15.2% in the pooled analysis. The unadjusted odds ratio (95% CI) for CR was 2.03 (1.099, 3.741; P=0.03) in favor of statin users, and the corresponding multivariable-adjusted odds ratio was 2.676 (1.338, 5.351; P<0.01). PFS and OS results were consistent with CR rates, with respective multivariable-adjusted hazard ratios of 0.61 (0.383, 0.956; P=0.03) and 0.50 (0.247, 1.007; P=0.05). The pharmacokinetic analysis showed no difference in venetoclax exposures between patients taking a statin versus those who did not. There were no unexpected safety observations in either group. The trend for improved response among statin-treated subjects was also present for the broad MM subject population treated with VenVd in Study 4 and t(11;14) positive MM subjects treated with Ven or Vendex in Study 5.Conclusions: Statins were associated with enhanced response to venetoclax in retrospective analyses of CLL and MM clinical trials. Given the post-hoc nature of these findings and lack of randomization, further evaluation in a prospective, randomized clinical trial is required to draw definitive conclusions. [Display omitted] DisclosuresRoberts:AbbVie: Employment, Equity Ownership. Xu:AbbVie: Employment, Equity Ownership. Jia:AbbVie: Employment, Equity Ownership. Agarwal:AbbVie: Employment, Equity Ownership. Salem:AbbVie: Employment, Equity Ownership. Bellin:Abbvie: Employment, Equity Ownership. Lam:AbbVie: Employment, Equity Ownership.

Exposure-Response of Idelalisib Administered in Combination with Bendamustine and Rituximab for the Treatment of Relapsed Chronic Lymphocytic Leukemi

Background: Idelalisib (IDELA) is a potent and selective PI3Kδ inhibitor approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) (in combination with rituximab) and relapsed indolent non-Hodgkin lymphoma (iNHL) subtypes, follicular lymphoma, and small-cell lymphocytic lymphoma. The relationship between plasma exposures of IDELA and clinical efficacy and safety were evaluated in relapsed/refractory CLL subjects following administration of IDELA 150 mg twice daily (BID) in combination with bendamustine (B) and rituximab (R; Study GS-US-312-0115).Methods: IDELA peak and trough plasma samples were collected every 4 weeks (up to 24 weeks) starting at baseline. Data for safety and efficacy endpoints were collected every 2 and 12 weeks, respectively. IDELA plasma exposures (Cmax, AUCtau, and Ctau) were generated using a previously developed population pharmacokinetic (PK) model (Jin F, et al. CCP . 77: 89-98, 2016). Efficacy and safety data from the phase 3, randomized, double-blind, placebo-controlled study (GS-US-312-0115) were included in the PK/pharmacodynamics (PD) analyses. Efficacy endpoints, including best overall response rate (BOR), duration of response (DOR), progression-free survival (PFS), sum of products of the greatest perpendicular diameters (SPD) of index lesions, and lymph node response rate (LNR), were evaluated against IDELA exposures (AUCtau and Ctau). Safety endpoints, including Grade 3 or 4 adverse events of neutropenia, skin rash, infection, pneumonia, pneumonitis, diarrhea, colitis, and aspartate aminotransferase (AST) or alanine aminotransferase (ALT) elevation, were evaluated versus IDELA exposures (AUCtau and Cmax). The relationship between average daily dose of IDELA versus exposures (AUCtau and Cmax) was also evaluated. Statistical analysis (such as Kaplan-Meier survival analysis, Cox proportional hazard model, or logistic regression) was conducted for selected efficacy and safety endpoints.Results: Median IDELA exposures were similar in subjects who achieved an objective response (complete response or partial response) and subjects who did not respond (stable disease or progressive disease) and across the 4 response categories (p=0.68; Table 1). As IDELA exposure increased over quartiles, there was no clear trend of prolonged DOR (p=0.50), PFS (p=0.69), or change in SPD. No relationship between LNR (responder versus non-responder) and IDELA exposure was observed. No association was observed between IDELA exposure vs incidence of neutropenia (p=0.35), diarrhea (p=0.78), pneumonia (p³0.12), colitis (p=0.62), AST or ALT elevation (p=0.45), skin rash, and pneumonitis. An increase in the incidence of Grade 3 or 4 infections with higher IDELA exposures was observed (p=0.021). Overall, IDELA in combination with BR was well tolerated, as indicated by the comparable average daily doses (~300 mg) administered across quartiles of IDELA exposure.Conclusions: No exposure-response relationships were observed for efficacy or safety (except infections) endpoints at IDELA 150 mg BID. These results are consistent with the previously observed IDELA exposure-response relationship in subjects with CLL (in combination with rituximab or ofatumumab) and iNHL. Overall, this analysis supports IDELA 150 mg BID in combination with BR for the treatment of relapsed CLL in patients who are fit for immunochemotherapy. [Display omitted] DisclosuresDaryani:Gilead Sciences, Inc.: Employment, Equity Ownership. Sharma:Gilead Sciences, Inc.: Employment, Equity Ownership. Xing:Gilead: Employment, Equity Ownership. Silverman:Gilead Sciences, Inc.: Employment, Equity Ownership. Adewoye:Gilead Sciences, Inc.: Employment, Equity Ownership. Mathias:Gilead Sciences, Inc.: Employment, Equity Ownership.

Abstract IA13: Treatment of COVID-19 pulmonary failure by targeting BTK

Abstract Pulmonary failure is the main cause of mortality related to COVID-19 infection. Up to 80% of patients hospitalized for COVID-19 infection require supplemental oxygenation, of whom 30-40% may require mechanical ventilation. SARS-CoV-2 binds via the ACE2-receptor that is highly expressed on alveolar type II (ATII) cells in the lung. ATII cells constitute 5-15% of the lung epithelium. While alveolar type I cells are highly adapted for gas exchange, alveolar type II cells have a specialized role in innate immune response. ATII cells express Toll receptors (TLRs) and can trigger inflammatory cytokines and chemoattractants in response to pathogens that recruit and activate other immune cells, including macrophages and neutrophils. We and others previously showed that BTK, and its upstream activator HCK, were involved in TLR-mediated signaling. Both BTK and HCK are triggered by MYD88, a TLR-adaptor protein that signals for all Toll receptors except TLR3 in response to viral and bacterial pathogens, including coronaviruses. ATII cells express TLRs, as do alveolar macrophages that coordinate inflammatory responses with ATII cells. As components of TLR/MYD88 signaling, BTK and HCK can drive inflammatory cytokine production through ERK1/2. The potential for BTK inhibitors to abrogate lung injury and death was demonstrated in an experimental model wherein mice challenged with a lethal intranasal inoculum of a mouse-adapted strain of H1N1 influenza virus were protected against lung injury. Control mice developed respiratory failure, along with histologic and CT findings consistent with lung injury in sharp contrast to the mice that received ibrutinib. Control mice also lost weight and died, whereas those treated with ibrutinib recovered their weight after a brief loss and all survived. Mice treated with ibrutinib also showed decreased inflammatory cell infiltration as well as proinflammatory cytokines in lung tissues that included proinflammatory and chemoattractant cytokines such as IL-1β, IL-6, KC/CXCL1, TNFα, and MCP-1 observed in SARS-Cov-1 and SARS-CoV-2 patients. In a series of Waldenstrom’s macroglobulinemia (WM) patients who were on full-dose ibrutinib (420 mg/day) and contracted COVID-19, none experienced dyspnea. In this series, one WM patient who contracted COVID-19 on reduced-dose ibrutinib (140 mg/day) experienced dyspnea and became hypoxic when ibrutinib was stopped and required mechanical ventilation. He was started on full-dose ibrutinib and showed rapid improvement, was extubated the next day, and did not require any oxygen supplementation two days later. Consistent with his rapid course of improvement on ibrutinib, his C-reactive protein level showed a marked decrease. In a subsequent and larger experience, Roschewski and colleagues administered acalabrutinib to 19 COVID-19 patients, 11 on supplemental oxygenation and 8 on mechanical ventilation. Eight of 11 (73%) and 2/8 (25%) on supplemental oxygenation and mechanical ventilation were discharged on room air, respectively. Ex vivo analysis of peripheral blood samples showed attenuation of activated monocyte BTK expression and IL-6 production following acalabrutinib. Serially collected blood samples from patients with chronic lymphocytic leukemia (CLL), WM, and chronic graft-versus-host disease (cGVHD) on ibrutinib monotherapy also showed marked reductions in proinflammatory and chemoattractant cytokines that greatly overlapped with those reported elevated in the plasma of SARS-Cov-1 and SARS-COV-2 patients, and in ACE2+ cells from lung tissue of SARS-CoV-1 patients. In the iLLUMINATE randomized study, CLL subjects treated with ibrutinib immediately prior to infusion with obinutuzumab also showed significantly decreased levels of inflammatory cytokines associated with infusion related reactions (a cytokine release syndrome). These findings are consistent with a shift from an M1 to M2 polarized macrophage response following ibrutinib and are supported by preclinical and clinical studies showing dependence of macrophage lineage commitment on BTK function. BTK-inhibitors may therefore provide protection against lung injury and even improve pulmonary function in hypoxic patients with COVID-19. Randomized clinical trials to examine the benefit of BTK-inhibitors in COVID-19 patients in pulmonary distress and/or accompanying corollary studies to identify response biomarkers have been initiated: NCT04439006, NCT04382586, NCT04346199, NCT04380688. Selected

Presence of serum antinuclear antibodies correlating unfavorable overall survival in patients with chronic lymphocytic leukemia.

Background:Serum antinuclear antibodies (ANAs) are positive in some patients with chronic lymphocytic leukemia (CLL), but the prognostic value of ANAs remains unknown. The aim of this study was to evaluate the role of ANAs as a prognostic factor in CLL.Methods:This study retrospectively analyzed clinical data from 216 newly diagnosed CLL subjects with ANAs test from 2007 to 2017. Multivariate Cox regression analyses were used to screen the independent prognostic factors related to time to first treatment (TTFT), progression free survival (PFS) and overall survival (OS). Receiver operator characteristic curves and area under the curve (AUC) were utilized to assess the predictive accuracy of ANAs together with other independent factors for OS.Results:The incidence of ANAs abnormality at diagnosis was 13.9%. ANAs positivity and TP53 disruption were independent prognostic indicators for OS. The AUC of positive ANAs together with TP53 disruption was 0.766 (95% confidence interval [CI]: 0.697–0.826), which was significantly larger than that of either TP53 disruption (AUC: 0.706, 95% CI: 0.634–0.772, P = 0.034) or positive ANAs (AUC: 0.595, 95% CI: 0.520–0.668, P < 0.001) in OS prediction. Besides, serum positive ANAs as one additional parameter to CLL-international prognostic index (IPI) obtained superior AUCs in predicting CLL OS than CLL-IPI alone.Conclusion:This study identified ANAs as an independent prognostic factor for CLL, and further investigations are needed to validate this finding.

NDRG2 mRNA levels and miR-28-5p and miR-650 activity in chronic lymphocytic leukemia

BackgroundNDRG2 is identified as a tumor suppressor gene in many tumors, and functions in cell proliferation, differentiation and apoptosis. Recent data indicate that NDRG2 expression is up-regulated by TP53. Moreover, proposed mechanisms of NDRG2 inactivation include epigenetic silencing of the NDRG2 promoter and down-regulation by microRNAs (miRNAs). However, few studies have ever been done on the role of NDRG2 and the NDRG2-regulating miRNAs interference in chronic lymphocytic leukemia (CLL).MethodsNDRG2 and microRNAs mRNA levels in CLL subjects were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The dual-luciferase reporter assay was performed to determine NDRG2-related miRNAs. Low expression of mature exogenous miRNAs in CLL cells was established by transient transfection. NDRG2 protein levels in CLL cells were detected by western blot. In addition, flow cytometry was conducted to examine the apoptosis of CLL cells.ResultsLower expression of NDRG2 was found in the B-cells from 102 CLL patients compared the 40 normal subjects (P < 0.001). Patients with advanced Binet stage (P = 0.001), high lactate dehydrogenase (LDH) level (P = 0.036), un-mutated immunoglobulin heavy chain variable region gene (IGHV) (P = 0.004) and those with p53 aberrations (P < 0.001) had a markedly lower levels of NDRG2 mRNA. This decrease was associated with briefer time-to-treatment (P = 0.001) and poorer survival (P < 0.001). High expression of miR-28-5p and miR-650 was associated with Binet B/C stage (P = 0.044) and IGHV un-mutated (P = 0.011), as well as Binet B/C stage (P = 0.013) and p53 aberrations (P = 0.037), respectively. Inhibition of miR-28-5p or miR-650 could induce more apoptosis in CLL cells with germline TP53.ConclusionsNDRG2 mRNA levels might be a useful prognostic variable for patients of CLL and up-regulating NDRG2 transcription may be a therapy approach in CLL without p53 aberrations.

Cholesterol Homeostasis in Isolated Lymphocytes: a Differential Correlation Between Male Control and Chronic Lymphocytic Leukemia Subjects

Background:This study was performed to investigate any association between cellular cholesterol homeostasis and chronic lymphocytic leukemia (CLL). CLL is characterized primarily by an abnormal accumulation of neoplastic B cells in the blood, bone marrow, lymph nodes and spleen.Methods:Men aged >50 years participated in this study. Enzyme-based plasma lipid profile estimations, peripheral blood lymphocyte isolation, lysate preparations, SDS-PAGE, western blotting, dil-LDL uptake and ultracentrifugation were employed.Results:Our study demonstrated hypocholesterolemia in lymphocytic leukemia in addition to hyper-expression of LDLRs in leukemic lymphocytes. Breakdown of intracellular cholesterol homeostasis and failure to maintain the feedback mechanism normally processed by the transcription factor SREBP-2 in the cytoplasm was apparent. The presence of cholesterol in the nucleus was noted in leukemic lymphocytes. A comparison of cholesterol homeostasis between healthy controls and CLL subjects showed that cholesterol may contribute to lymphocytic leukemia. While plasma cholesterol levels decreased (p < 0.0005), hyper-expression of LDLR (p=0.0001), SREBP-2 (transcription factor of LDLR) (p=0.0001) and PBR (nuclear cholesterol channel protein) (p=0.016) was observed in lymphocytes isolated from CLL subjects in association with a significant increase in intracellular cholesterol in the nuclear (p=0.036) and cytoplasmic (p=0.004) compartments.Conclusion:This study provided insights into cholesterol homeostasis in CLL subjects regarding LDLR, SREBP-2 and PBR. Cholesterol may enter the nucleus through highly expressed PBR and may be involved in development of leukemia by influencing cell cycle mechanisms in the lymphocytes of CLL subjects.

Night shift work and chronic lymphocytic leukemia in the MCC-Spain case-control study.

Chronic lymphocytic leukemia (CLL) has few known modifiable risk factors. Recently, circadian disruption has been proposed as a potential contributor to lymphoid neoplasms' etiology. Serum melatonin levels have been found to be significantly lower in CLL subjects compared with healthy controls, and also, CLL prognosis has been related to alterations in the circadian molecular signaling. We performed the first investigation of an association between night shift work and CLL in 321 incident CLL cases and 1728 population-based controls in five areas of Spain. Participants were interviewed face-to-face by trained interviewers to collect information on sociodemographic factors, familial, medical and occupational history, including work shifts and other lifestyle factors. We used logistic regression models adjusted for potential confounders to estimate odds ratios (OR) and 95% confidence intervals (CI). Seventy-nine cases (25%) and 339 controls (20%) had performed night work. Overall, working in night shifts was not associated with CLL (OR = 1.06; 95% CI = 0.78-1.45, compared with day work). However, long-term night shift (>20 years) was positively associated with CLL (OR(tertile 3 vs . day-work) = 1.77; 95% = 1.14-2.74), although no linear trend was observed (P trend = 0.18). This association was observed among those with rotating (OR(tertile 3 vs . day-work) = 2.29; 95% CI = 1.33-3.92; P trend = 0.07), but not permanent night shifts (OR(tertile 3 vs . day-work) = 1.16; 95% CI = 0.60-2.25; P trend = 0.86). The association between CLL and long-term rotating night shift warrants further investigation.

C–C motif ligand 11 reduction in CLL patients serum after vitamin D supplementation

Background: Vitamin D (VD) deficiency results in a worse prognosis in patients with chronic lymphocytic leukemia (CLL) and may affect the production of cytokines. Nonetheless, there is the lack of studies dealing with VD supplementation and its impact on chemokines in CLL patients.Aim: The primary endpoint of our interventional study was to evaluate the effect of cholecalciferol supplementation on serum chemokines levels in CLL patients.Materials and methods: Eighteen subjects with CLL were enrolled for the study. Six-month-long cholecalciferol supplementation was performed in CLL patients with serum 25-OH-D3 levels below 30 ng/ml. Cytokines levels were assessed at the beginning of the study and after 6 months. Baseline measurements of cytokines were compared to those in apparently healthy controls.Results: Increased levels of CCL2, CCL3, CCL4, CXCL8, CXCL10, TNFα, bFGF, G-CSF, and VEGF were found in CLL patients in comparison with the healthy controls. In the course of the VD supplementation a decrease in serum levels of chemokines CCL11, CCL3, and cytokine PDGF-BB was observed. The decrease of CCL11 was found in CLL patients on VD supplementation solely, whereas the decrease of CCL3 and PDGF-BB was observed in CLL subjects on both chemotherapy and VD supplementation.Conclusion: The VD supplementation may exert beneficial effect on chemokines levels in CLL patients with VD deficiency.

Phase 2 Trial of Entospletinib (GS-9973), a Selective Syk Inhibitor, in Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL)

Abstract Background: Spleen tyrosine kinase (Syk) is a mediator of B-cell receptor signaling in normal and transformed B-cells. GS-9973 is an orally bioavailable, selective inhibitor of Syk. Methods: This Phase 2 trial enrolled 41 patients with CLL and 15 patients with SLL treated with GS-9973 800 mg BID. Tumor imaging occurred at weeks 8, 16, 24 and then every 12. Response was independently evaluated according to Hallek 2008 as modified by Cheson 2012 for patients with CLL and Cheson 2007 for patients with SLL. Primary endpoint for the study was PFS at 24 weeks. Results: The median ages of CLL and SLL patients were 73 (range 51-89) and 70 (range 57-84), respectively. 68% of CLL subjects and 60% of SLL subjects were male. Ten patients had 17p deletions/TP53 mutations and 17 had SF3B1 or NOTCH1 mutation, or 11q22.3 deletion. The median number of prior regimens for CLL was 2 (range 1-8) and for SLL was 2 (range 1-10). Prior therapies included anti-CD20 antibodies (98%), alkylating agents (86%, [bendamustine 63%]) and fludarabine (66%). 12 CLL and 6 SLL patients are still on treatment; the median duration of treatment for all CLL and SLL patients was 36 weeks The most common treatment emergent AEs (any Grade/≥Gr 3, independent of causality) were fatigue (70%/7%), nausea (54%/2%), diarrhea(48%/0%), cough(34%/0%), dizziness (32%/2%), headache (29%/0%), pyrexia (29%/0%), decreased appetite (27%/2%), upper respiratory tract infection(27%/0%), constipation (23%/0%). Common laboratory abnormalities were increased AST (30%/5%), increased ALT (43%/4%), increased total bilirubin (41%/16%), anemia (50%/7%) and neutropenia (54%/29%). Forty-nine patients were treated for at least 8 weeks and 54 patients had ≥ 1 efficacy assessment, two patients discontinued prior to the first response assessment, one due to AE and one withdrew consent. Per investigator assessment, 51 out of 52 (98%) patients evaluable for SPD experienced reduced tumor bulk; 38 (73%) achieved a decrease of ≥ 50%. The ORR was 62.5% (95% CI: 48.6%, 75.1%), with 35 patients achieving a PR and no subject achieving a CR. Thirteen patients (23.2%) had stable disease. The primary end point of 24 weeks PFS was 72.3% (95% CI: 57.1%, 83.0%). Median PFS was 20.5 months (95% CI: 7.7 months, not reached). There were 24 patients (42.9%) with events, 22 (39%) with disease progression and two deaths (4%) attributed to septic pneumonia and pseudomonal infection which was unrelated to entospletinib by investigator assessment. Among the 35 responding patients, median DOR was 21.3 months (95% CI: 13.2 months, not reached). Results of an independent response assessment are pending and will be presented. Entospletinib was well tolerated and demonstrated substantial activity in patients with CLL, and SLL including those with poor prognostic features. Entospletinib activity seems comparable to that reported by other approved BCR pathway inhibitors with the median PFS reported for Idela of 15.8 months (Blood. 2014;123(22):3390-3397) and Ibrutinib reported 42.6% overall response rate and a PFS of 70-80% @ 12 months (ibrutinib PI) Current studies plans include studying Entospletinib in combination therapy. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Sharman: Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Research Funding; Calistoga: Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Research Funding; TG Therapeutics, Inc.: Research Funding; Janssen: Research Funding. Kolibaba:Janssen: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; TG Therapeutics: Research Funding; GSK: Research Funding; Celgene: Research Funding; Acerta: Research Funding; Seattle Genetics, Inc.: Research Funding; Takeda Pharmaceuticals International Co.: Research Funding; Gilead: Consultancy, Research Funding. Abella:Gilead: Employment. Di Paolo:Gilead Sciences: Employment, Equity Ownership. Eng:Gilead: Employment. Hu:gilead: Employment. He:Gilead Sciences: Employment. Reddy:gilead: Employment. Yasenchak:Seattle Genetics, Inc.: Research Funding.

Outcomes of Human Leukocyte Antigen–Matched Sibling Donor Hematopoietic Cell Transplantation in Chronic Lymphocytic Leukemia: Myeloablative Versus Reduced-Intensity Conditioning Regimens

Allogeneic hematopoietic cell transplantation (HCT) can cure some chronic lymphocytic leukemia (CLL) subjects. This study compared outcomes of myeloablative (MA) and reduced-intensity conditioning (RIC) transplants from HLA-matched sibling donors (MSD) for CLL. From 1995 to 2007, information regarding 297 CLL subjects was reported to the Center of International Blood and Marrow Transplant Research; of these, 163 underwent MA and 134 underwent RIC MSD HCT. The MA subjects underwent transplantation less often after 2000 and less commonly received antithymocyte globulin (4% versus 13%, P = .004) or prior antibody therapy (14% versus 53%; P < .001). RIC was associated with a greater likelihood of platelet recovery and less grade 2 to 4 acute graft-versus-host disease compared with MA conditioning. One- and 5-year treatment-related mortality (TRM) were 24% (95% confidence intervals [CI], 16% to 33%) versus 37% (95% CI, 30% to 45%; P = .023), and 40% (95% CI, 29% to 51%) versus 54% (95% CI, 46% to 62%; P = .036), respectively, and the relapse/progression rates at 1 and 5 years were 21% (95% CI, 14% to 29%) versus 10% (95% CI, 6% to 15%; P = .020), and 35% (95% CI, 26% to 46%) versus 17% (95% CI, 12% to 24%; P = .003), respectively. MA conditioning was associated with better progression-free (PFS) (relative risk, .60; 95% CI, .37 to .97; P = .038) and 3-year survival in transplantations before 2001, but for subsequent years, RIC was associated with better PFS and survival (relative risk, 1.49 [95% CI, .92 to 2.42]; P = .10; and relative risk, 1.86 [95% CI, 1.11 to 3.13]; P = .019). Pretransplantation disease status was the most important predictor of relapse (P = .003) and PFS (P = .0007) for both forms of transplantation conditioning. MA and RIC MSD transplantations are effective for CLL. Future strategies to decrease TRM and reduce relapses are warranted.

Treatment With The Potent PI3K-δ,γ Inhibitor IPI-145 Is Associated With Rapid Decreases In Specific Cytokines, Chemokines and Matrix Metalloproteinases In The Serum Of Patients With Chronic Lymphocytic Leukemia and Indolent Non-Hodgkin Lymphoma

Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node, relaying signals from cell surface receptors to downstream mediators such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. IPI-145 is a potent, oral PI3K-δ,γ inhibitor that has shown clinical activity in the Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies (ClinicalTrials.gov NCT01476657). Cytokines, chemokines, and matrix metalloproteinases (MMPs) play key roles in the homing, migration and activation of normal immune cells, and can have similar effects on malignant leukocytes. To further explore the biological effects of IPI-145, a panel of cytokines, chemokines, and MMPs were evaluated at several time points in the serum of patients enrolled in the IPI-145-02 trial.Serum was collected from consenting subjects at baseline, Cycle 1 Day 8 (C1D8), and Cycle 2 Day 1 (C2D1). Serum was frozen and stored at -80°C prior to analysis. Serum proteins were analyzed using Luminex xMAP(®) technology in which analytes are captured on uniquely labeled fluorescent beads, and the amount of analyte is quantified using an LED CCD camera contained in Millipore's MagPix(®) instrument. Multiplex panels of cytokines, chemokines and MMPs covering 72 analytes were evaluated in 30 chronic lymphocytic leukemia (CLL) and 19 indolent non-Hodgkin lymphoma (iNHL) subjects. Each sample was tested in duplicate, and duplicate measurements were averaged. Measurements were excluded from further analysis if duplicate readings exhibited a coefficient of variation of greater than 20% or if all values for a specific analyte and subject were below the limit of detection.Each analyte was evaluated for evidence of a consistent change (reduction or increase) in serum levels at C1D8 and/or C2D1 compared to baseline. When data were compared between CLL subjects treated with IPI-145 at 25 mg twice daily (BID) and those treated at 75 mg BID, no clear differences in serum analyte levels were observed, although the population subgroups were relatively small. For the purposes of this analysis, all doses were pooled together (n=1 at 8 mg BID, 2 at 15 mg BID, 15 at 25 mg BID, and 13 at 75 mg BID). Likewise, the iNHL dose groups were also pooled (n=1 at 15 mg BID, 12 at 25 mg BID, 1 at 50 mg BID, and 5 at 75 mg BID). In CLL subjects, 9 of 72 analytes decreased after IPI-145 treatment compared to baseline, whereas none increased significantly. Analytes that decreased after IPI-145 treatment include CXCL13, CCL3, CCL4, IL-10, TNFα, IL-12p40, MMP-9, CCL17 and CCL22. Median serum levels of these analytes decreased by C1D8, ranging from 16% to 59% of baseline. In iNHL subjects, median serum levels of 7 analytes decreased by C1D8 (ranging from 32% to 70% of baseline), whereas none increased significantly. Of the 7 analytes that decreased in iNHL subjects, 5 also decreased in CLL subjects (CXCL13, MMP-9, TNFα, CCL17 and CCL22) and 2 were distinct (CCL1 and MMP-12). Interestingly, many of the analytes that decreased with IPI-145 treatment are involved in the communication between malignant B-cells and the microenvironment. CCL3, CCL4, CCL17 and CCL22 are expressed by malignant B-cells and may play a role in recruiting T-cells to interact with the malignant B-cells. CXCL13 is secreted by stromal cells and recruits malignant B-cells to the lymph nodes. In addition, IL-10 is produced by many normal immune cell types as well as by neoplastic B-cells. IL-10 is known to be an autocrine growth factor for B-cell lymphoma cell lines.These pharmacodynamic data provide further evidence for biological activity of IPI-145 in patients with CLL and iNHL and suggest both similarities and differences in how these two malignancies respond to IPI-145. The association of many of these pharmacodynamic factors with the tumor microenvironment suggests a mechanistic basis for the clinical observation of lymphocytosis and nodal reduction with IPI-145 treatment of CLL subjects. Cytokine, chemokine and MMP levels from patients in IPI-145-02 are being evaluated further for associations with multiple clinical parameters to determine if there is evidence for biomarkers predictive of efficacy and tolerability. Disclosures:Douglas:Infinity Pharmaceuticals, Inc.: Employment. Allison:Infinity Pharmaceuticals, Inc.: Employment. Ted:Infinity Pharmaceuticals, Inc.: Employment. Allen:Infinity Pharmaceuticals, Inc.: Employment. Kahl:Infinity Pharmaceuticals, Inc.: Consultancy, Research Funding. Horwitz:Celgene, Allos, Seattle Genetics, Bristol-Myers Squibb, Genzyme, Kyowa, Janssen, Johnson & Johnson, Millenium: Consultancy; Celgene, Allos, Seattle Genetics, Kyowa, Infinty, Millenium: Research Funding. Flinn:Infinity Pharmaceuticals, Inc.: Consultancy, Research Funding. Kelly:Infinity Pharmaceuticals, Inc.: Employment. Stern:Infinity Pharmaceuticals, Inc.: Employment.

BMS-936564 (Anti-CXCR4 Antibody) Induces Specific Leukemia Cell Mobilization and Objective Clinical Responses In CLL Patients Treated Under a Phase I Clinical Trial

Expression of CXCR4 receptor (CD184) has been reported in different malignancies including Chronic Lymphocytic Leukemia (CLL) and the CXCR4-CXCL12 axis has been described to play a very important role in cancer development. Moreover, this pathway can be inhibited using CXCR4 antagonists.Previously, we have shown that BMS-936564, an anti-CXCR4 IgG4 antibody, can block in vitro the CXCR4/CXCL12 pathway in CLL overcoming stromal cell protection by inducing apoptosis, inhibiting F-actin polymerization, and migration of cells. These findings provide the rationale for an ongoing phase I clinical study in which CLL subjects received four weekly infusions of BMS-936564 antibody followed by 5 monthly cycles of bendamustine, rituximab and BMS-936564 antibody (BRB chemoimmunotherapy). Eleven (n=11) subjects with relapse / refractory disease have been enrolled and treated in this study. Here, we present correlative studies performed in three of those CLL subjects with samples collected at different time points (day 1, 2, 8, 15 and 22) during the monotherapy cycle of BMS-936564.All three subjects showed leukocytosis that was detected as early as in 4 hours following the initial BMS-936564 infusion (median increase of 64.6% above base line; range: 59.7% - 112.7%). Leukocytosis was present during the entire four weeks of monotherapy with BMS-936564. Leukocytosis in these three patients was due primarily to increase in absolute counts of CLL cells (Median increase of 129.6%; range: 95.3% - 324.8%). Interestingly, there was no evidence of increase in the absolute number of normal lymphocytes. Only one of the three patients showed an increase in neutrophil counts after infusion of BMS-936564.We observed changes in the level of CXCR4 expression after infusion with BMS-936564. All subjects showed CXCR4 down-regulation in peripheral CLL cells with a median percentage decrease in the level of expression of 106.7% (Range: 25.1% - 350.7%). We did not observe changes in CXCR4 expression in normal B cells.In contrast to our in vitro studies where we observed that all tested CLL samples underwent apoptosis after treatment with BMS-936564, only one subject showed significant increase in the percentage of apoptosis after infusion of BMS-936564 in vivo. The levels of apoptosis detected were relatively low (<18%) and this suggests a potential mechanism of rapid clearance of apoptotic cells.Importantly, associated with the mobilization of CLL cells, all three patients showed significant decrease in the lymphadenopathy size with a median decrease of 59.83% (Range: 55.6-72%). Lymphadenopathy response was assessed using stringent IWCLL response assessment criteria by an independent radiologist using CT scan measurements.All three subjects tolerated the infusions well with no evidence of side effects or toxicities. After the initial monotherapy treatment phase with BMS-936564 antibody, all three subjects received BRB chemoimmunotherapy combination and had a dramatic decrease in lymphocytosis during the first week after initiation of combination therapy. These subjects are currently finishing treatment under protocol and response assessments will be presented during the meeting.In conclusion, we found that BMS-936564 infusion in subjects with CLL induces specific leukemia cell mobilization associated with an objective decrease in the lymphadenopathy bulk and phenotypic changes associated with CXCR4 down-regulation in peripheral leukemia cells. These findings strongly suggest a CXCR4-CXCL12 dependent mobilization process of CLL cells from lymphatic tissues. Together, our study provides for the first time evidence at in vivo level, which supports inhibition of the CXCR4-CXCL12 pathway mediated by BMS-936564 in CLL patients. These findings suggest a compartmental biological distribution and recirculation of CLL cells opening the possibility to explore targeted therapies against minimal residual disease that traditionally has been refractory to conventional chemoimmunotherapy treatment and contributes to the incurable characteristics of this disease. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Kipps:Bristol-Myers Squibb: Research Funding. Cardarelli:Bristol-Myers Squibb: Employment.

Update On The Safety and Efficacy Of The Pan Class I PI3K Inhibitor SAR245408 (XL147) In Chronic Lymphocytic Leukemia and Non-Hodgkin's Lymphoma Patients

BackgroundConstitutive activation of phosphatidylinositol 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) pathway by various mechanisms has been implicated in the pathogenesis of chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). There is mounting evidence suggesting that along with PI3Kγ, PI3Kα may be involved in CLL/NHL. SAR245408 is a potent and selective inhibitor of all α, γ and δ class I PI3K isoforms. It has been shown to inhibit PI3K signaling and impact tumor growth in preclinical tumor models. The impact of SAR245408 on safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor effect was evaluated in patients with relapsed/refractory CLL and NHL from the Sanofi sponsored phase 1 single-agent study (NCT00486135) [Brown et al. ASH Annual Meeting Abstracts 2011. 118 (21): 2683]. MethodsSAR245408 was administered orally, once daily, with continuous dosing in monthly cycles. A total of 25 patients were enrolled at the maximum tolerated dose identified in solid tumor patients as part of the expansion cohort in relapsed/refractory lymphoproliferative malignancies. Plasma cytokines and chemokines were evaluated using the Myriad RBM Human Discovery MAP250+ panel (> 250 analytes) and ELISA assays. ResultsAmong the 25 patients (pts), 40% (n=10) had refractory CLL and 60% (n=15) had various relapsed/refractory lymphomas (R/RL), including follicular lymphoma (FL) (n=5), diffuse large B-cell lymphoma (DLBCL) (n=4), Waldenstrom's macroglobulinemia (WM) (n=3), Hodgkin lymphoma (n=2) and B-cell prolymphocytic leukemia (n=1). The median age was 65 years (range 28–83), and 56% were female. Eighty percent of pts had stage III-IV disease and 48% had bulky disease. Five pts were categorized as having refractory disease and the median number of prior regimens was 1 (range 1-7) for CLL pts and 3 (range 0-9) for R/RL pts. For all 25 patients, the median starting absolute lymphocyte count was 1.15 x 103/μL (range 0.3–37.2), the median starting hemoglobin was 11.4 g/dL (range 9.1–15) and the median platelet count was 162 x 103/μL (range 60–431). The median number of cycles administered in CLL pts was 10 (range 5-24) and 5 (range 1-26) in R/RL pts. Six CLL pts experienced an increase in absolute lymphocyte counts within 2-4 weeks of treatment, as has been seen with other PI3K inhibitors. According to modified International Workshop on Chronic Lymphocytic Leukemia (IWCLL) response criteria, 4 CLL pts had partial response (PR), 1 had nodal PR with increased lymphocytosis, and 5 had stable disease (SD); the progression free survival (PFS) in the responding pts was 22, 21.2, 15.6 and 15.4 months while in the SD pts was 12.5, 9.2, 7.4, 5.6, 4.6 and 3.6 months. According to the modified International Working Group response criteria, 3 PRs were reported in R/RL pts [1 WM, 1 DLBCL (transformed) and one FL with PFS of 23.7, 18.4 and PFS 4.8 months respectively]. One hundred percent of CLL and 80% of R/RL pts reported grade 3 or higher AEs, with the most common (≥ 10% of patients) including neutropenia, diarrhea, anemia and hypotension. SAR245408 induced a reduction in levels of chemokines involved in lymphocyte trafficking in CLL subjects (n=8), including CXCL13, CCL3, CCL22 and CCL19 (64, 58, 52 and 54% reduction, respectively, p<0.05) similar to what was reported with PI3K δ specific inhibitors. In addition, a reduction of tumor necrosis factor receptor 2 (TNFR2) and interleukin-2 receptor alpha (IL-2Rα) levels was observed (63% reduction each, p<0.01). ConclusionsThe recommended phase 2 dose of SAR245408 in solid tumor patients was confirmed as safe and tolerable in patients with CLL and R/RL. Single agent SAR245408 demonstrates clinical activity in patients with relapsed or refractory CLL and promising pharmacodynamic effects on chemokine levels involved in lymphocyte trafficking. Disclosures:Brown:Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding; Avila: Consultancy; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy. Off Label Use: The abstract shows scientific information on SAR245408 which is an investigational product developed by Sanofi. This investigational product is not approved by any health authority for any indication. Egile:Sanofi: Employment. Ruiz-Soto:Sanofi: Employment. Awan:Lymphoma Research Foundation: Research Funding; Spectrum Pharmaceuticals Inc.: Speakers Bureau.

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.