Reduction In Absolute Lymphocyte Count Research Articles

OP03 Efficacy and safety of the oral selective sphingosine-1-phosphate-1 receptor modulator VTX002 in moderately to severely active Ulcerative Colitis: results from a randomised, double-blind, placebo-controlled, phase 2 trial

Abstract Background VTX002 is a novel oral selective sphingosine-1-phosphate-1 (S1P1) receptor modulator in development for the treatment of ulcerative colitis (UC). The efficacy and safety of VTX002 in patients with ulcerative colitis (UC) was assessed in a phase 2, multicentre, randomised, double-blind, placebo-controlled study (NCT05156125). Methods Adults (N=213) with moderately to severely active UC (modified Mayo score [MMS] 4-9) and inadequate response/intolerance to conventional and/or advanced (approved biologics or Janus kinase inhibitor) therapies were randomised 1:1:1 to once daily treatment with placebo (n=70), 30 mg (n=73) or 60 mg (n=70) VTX002 tablets for 13 weeks. Patients underwent a 6–8-day dose titration up to the assigned treatment dose. The primary endpoint was clinical remission (MMS stool frequency subscore ≤ 1, rectal bleeding subscore=0, and endoscopic subscore [ES] ≤ 1) at week 13. A protocol amendment limiting eligibility to patients with baseline MMS 5-9 resulted in 213 and 209 patients in the safety and efficacy populations, respectively. Key secondary endpoints were endoscopic improvement, symptomatic remission, histologic remission, and endoscopic improvement-histologic remission. Endoscopic remission and histologic-endoscopic mucosal improvement (HEMI) were also assessed (see Table 1 for outcome definitions). Adverse events (AEs) and laboratory abnormalities were assessed for safety. Results The primary endpoint of clinical remission at week 13 was achieved (27.9% of patients who received VTX002 60 mg vs 11.4% of patients who received placebo, ∆=16.5%, p=0.0184). Both doses of VTX002 achieved nominal statistical significance for all key secondary endpoints (Table 1). Significantly greater proportions of patients who received VTX002 60 mg achieved endoscopic remission and HEMI compared to patients who received placebo (29.4% VTX002 60 mg vs 7.1% placebo, ∆=22.3%, p=0.0012, and 32.4% VTX002 60 mg vs 10.0% placebo, ∆=22.4%, p=0.0026, respectively). A dose-dependent decrease of 67.7% and 53.9% in mean absolute lymphocyte count (ALC) from baseline was observed at week 8 for patients who received VTX002 60 mg and 30 mg, respectively. Most AEs were mild to moderate (Table 2). No AEs of bradycardia, atrioventricular block, serious infections, macular oedema, or deaths were reported. Conclusion The S1P1 receptor modulator VTX002 was well-tolerated and statistically superior to placebo for induction of clinical, endoscopic, histologic, HEMI, and symptomatic remission/response at week 13. These positive findings, including a dose-dependent pharmacodynamic reduction in ALC, support phase 3 development of VTX002 for treatment of UC.

P748 Pharmacokinetics, relative bioavailability, and dose proportionality of a tablet formulation of the sphingosine 1-phosphate-1 receptor modulator VTX002 (formerly OPL-002)

Abstract Background VTX002 is an oral selective sphingosine 1-phosphate-1 receptor (S1P1R) modulator in clinical development for treatment of ulcerative colitis (UC). Single and multiple doses of a spray-dried dispersion (SDD) oral suspension of VTX002 were well-tolerated up to 45 mg in a phase 1 study in healthy participants and were accompanied by dose proportional reductions in lymphocyte counts, an expected pharmacodynamic outcome of treatment.1 A separate phase 1 study compared the pharmacokinetics (PK) and pharmacodynamics of the SDD oral suspension and a tablet formulation of VTX002. Methods Healthy adult participants (N=12) were randomised (4 per dose group) in an open-label 3-way crossover study to receive single oral doses of 5 mg or 20 mg VTX002 tablets, or 20 mg VTX002 SDD suspension followed by at least a 1-week washout between each dose as shown in Fig. 1. Study assessments included: VTX002 plasma concentrations, PK parameters, change from baseline in absolute lymphocyte count (ALC), and adverse events (AE). Results A total of 10 participants completed the study. Mean plasma VTX002 concentrations were greatest with a single oral dose of 20 mg VTX002 SDD suspension (Fig. 2). The geometric mean peak (Cmax) and total (AUCinf) exposure to VTX002 were lower with the 5 mg (dose normalized to 20 mg tablet) and 20 mg VTX002 tablets compared to the 20 mg VTX002 SDD suspension (Table 1). Geometric mean ratios for the tablet and SDD formulations of VTX002 are shown in Table 2. Maximum percent (SD) reduction in ALC from baseline was greatest for the 20 mg VTX002 SDD suspension and was observed at 6 hours post-dose both for the SDD suspension and the 20 mg VTX002 tablet (59.9% [11.0%] and 51.6% [9.4%], respectively). The maximum percent reduction in ALC from baseline for the 5 mg VTX002 tablet was 22.4% (9.2%) which occurred 2 hours post-dose. A total of 23 AEs were reported; of which 19 were considered mild, and 4 were moderate. Conclusion All doses and formulations of VTX002 were well-tolerated and associated with ALC reductions. Data from this relative bioavailability study was used to convert the results from the prior phase 1 multiple ascending dose study performed with VTX002 SDD suspension to AUC equivalent tablet doses and enable tablet dose selection for phase 2. A phase 2 study of VTX002 tablets for treatment of UC is underway (ClinicalTrials.gov Identifier:NCT05156125). 1Luo A, et al. 2020 Gastroenterology. 2020;158(6):S1188.

Predictive role of absolute lymphocyte count in daratumumab-treated patients with relapsed/ refractory multiple myeloma.

Daratumumab has shown an encouraging antitumor effect in patients with multiple myeloma (MM), and was known to alter the immune properties by off-targeting immunosuppressive cells. Here, we aimed to evaluate the change in absolute lymphocyte count (ALC) as a surrogate marker for predicting survival outcomes of patients treated with daratumumab. Between 2018 and 2021, the medical records of patients with relapsed/refractory MM (RRMM) treated with daratumumab monotherapy at 10 centers in South Korea were reviewed. We collected the ALC data at pre-infusion (D0), day 2 after the first infusion (D2), and prior to the third cycle of daratumumab therapy (D56). Fifty patients who were administered at least two cycles of daratumumab were included. Overall response rate was 54.0% after two cycles of daratumumab treatment. On D2, almost all patients experienced a marked reduction in ALC. However, an increase in ALC on D56 (ALCD56) was observed in patients with non-progressive disease, whereas failure of ALC recovery was noted in those with progressive disease. Patients with ALCD56 > 700/μL (n = 39, 78.0%) had prolonged progression- free survival (PFS) and overall survival (OS) than those with ALCD56 ≤ 700/μL (median PFS: 5.8 months vs. 2.6 months, p = 0.025; median OS: 24.1 months vs. 6.1 months, p = 0.004). In addition, ALCD56 >700/μL was a significant favorable prognostic factor for PFS (hazard ratio [HR], 0.22; p = 0.003) and OS (HR, 0.23; p = 0.012). Increase in ALC during daratumumab treatment was significantly associated with prolonged survival outcomes in patients with RRMM. The ALC value can predict clinical outcomes in patients treated with daratumumab.

Absolute Lymphocyte Count and Outcomes of Multiple Myeloma Patients Treated with Idecabtagene Vicleucel: The U.S. Myeloma CAR T Consortium Real World Experience

Introduction: Chimeric Antigen Cell (CAR) T-cell therapy with idecabtagene vicleucel (ide-cel) is FDA approved for relapsed refractory multiple myeloma (MM). Investigating factors that may predict response, toxicity and survival is important to guide therapy and inform prognosis. Low pre-lymphodepletion (LD) absolute lymphocyte count (ALC) was found to be associated with worse outcomes in a small cohort of patients (pts) treated with ide-cel (Liu et al. Transplant Cell Ther 2022). We evaluated the impact of pre-apheresis (pre-A) and pre-LD peripheral blood ALC on post-ide-cel outcomes in a large patient cohort. Methods: A total of 215 myeloma pts were treated with ide-cel at 11 U.S. academic medical centers. We studied the effect of ALC including pre-A, pre-LD, absolute and percent reduction in ALC between apheresis and LD on progression free survival (PFS), overall survival (OS), overall hematologic response rate (ORR which included VGPR, CR and sCR) and cytokine release syndrome (CRS) incidence. Associations of ALC parameters with PFS and OS were evaluated using Kaplan-Meier (KM) method and Cox proportional hazard model. Patient demographics and clinical characteristics between ALC groups were compared using Chi square test and Wilcoxon rank test. Results: The median patient age was 64 years (range, 36-83) and 60 % were male. The median prior lines of therapy was 6 (IQR, 5-9), 84.2% had a prior autologous hematopoietic cell transplant (AHCT), 44.7% had extramedullary disease, and 77.1% received bridging therapy (BT). CRS was seen in 81.1% of the pts, mostly grade 1. The median pre-A and pre-LD ALC's were 0.65 x109/L (range, 0.06-5.3) and 0.55 (range, 0-5.0), respectively. The pre-A ALC was significantly higher than pre-LD ALC (mean difference and 95% CI: 0.10 (0.04, 0.40), p=0.002). The median follow-up time after CAR-T infusion was 5.4 (IQR, 2.1-8.3) months. Pre-A and pre-LD ALC's were not significantly associated with PFS or OS, while pts with a high absolute reduction in ALC between apheresis and LD (≥0.27 or top quartile) had a significantly worse six-month PFS (48.4 vs 63.5%, p= 0.04) and OS (76.0 vs 87.7%, p= 0.03). Similarly, patients with a high percent reduction between pre-A and pre-LD ALC (≥37.5% or top quartile) had a worse six-month PFS (41.9 vs 65%, p <0.001) and OS (70.5 vs 89%, P=0.001). PFS and OS differences remained statistically significant after adjusting for age, race, CRS grade, ECOG-PS, extramedullary disease and prior AHCT. Kaplan-Meier estimated PFS and OS curves for absolute ALC reduction groups and percent ALC reduction groups are displayed in Figure 1. The one-month and three-month ORR, and CRS grade were not significantly different between pre-A (≥1 or <1), pre-LD (≥1 or <1), absolute reduction and percent reduction ALC groups. ECOG-PS ≥2 (p= 0.004), ISS III (p= 0.015), and implementation of BT (p <0.01) were found to be associated with high ALC reduction or percent reduction. There was no difference in ALC between pts who received BT vs not. However, more patients in the high ALC reduction (>=0.27: 90.4% vs 72.6%, p=0.008) and high ALC percent reduction (>=37.5: 96.2% vs 70.5%) groups received BT. Moreover, pts who received BT had a worse PFS (six-month PFS: 53.2% vs 85.3%, p <0.001) and OS (six-month OS: 81.9% vs 95.8%, p= 0.02) likely indicating aggressive disease biology. The specific types of BT were not available in our dataset. Conclusion: Pre-apheresis ALC did not impact survival in this large cohort of myeloma patients treated with ide-cel. Reduction in ALC between apheresis and lymphodepletion is a potentially predictive tool of survival outcomes and may be associated with more aggressive disease biology and the need for more aggressive bridging therapy. Investigating the association between specific types of bridging therapy, ALC reduction and outcomes may guide clinical decision making in this heavily pretreated myeloma patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1 Study with the Novel B-Cell Lymphoma 2 (Bcl-2) Inhibitor Bgb-11417 As Monotherapy or in Combination with Zanubrutinib (ZANU) in Patients (Pts) with CLL/SLL: Preliminary Data

Background/introduction: The effectiveness of Bcl-2 inhibitors as a treatment for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was established by the approval of venetoclax in pts with CLL/SLL across all lines of therapy. However, the related adverse events (AEs) and emergence of BCL2 mutations, resulting in resistance, can limit the utility of venetoclax. BGB-11417 is a highly selective Bcl-2 inhibitor with potency >10 times that of venetoclax in biochemical assays. BGB-11417 monotherapy is tolerable, with no maximum tolerated dose (MTD) reached after dose escalation through all planned doses to 640 mg once daily (QD) in pts with non-Hodgkin lymphoma (EHA 2022. Abstract P687). The combination of Bcl-2 and Bruton tyrosine kinase (BTK) inhibitors is tolerable with synergistic activity in CLL and mantle cell lymphoma (MCL) (J Clin Oncol 2019;37:2722-9; N Engl J Med 2019;380:2095-103; EHA 2020. Abstract S158; N Engl J Med 2018;378:1211-23). ZANU, a next-generation BTK inhibitor, has shown favorable activity and safety in pts with CLL/SLL (EHA 2021. Abstract LB1900) and Waldenström macroglobulinemia (Blood. 2020;136(18):2038-2050). BGB-11417-101 is an ongoing first-in-human phase 1/1b dose-escalation/expansion study (NCT04277637). Pts with various B-cell malignancies were enrolled; data from CLL/SLL cohorts are presented here. Methods: In separate monotherapy and combination therapy cohorts, pts received escalating doses of BGB-11417 (40, 80, 160, 320, or 640 mg QD) with a ramp-up to the intended target dose to minimize risk of tumor lysis syndrome (TLS). In combination therapy cohorts, pts received ZANU (320 mg QD or 160 mg twice daily) beginning 8-12 weeks before BGB-11417. Dose-limiting toxicity for each cohort was evaluated by a Bayesian logistic regression model during dose ramp-up through day 21 at the intended dose. AEs were reported per Common Terminology Criteria for AEs v5.0. Minimal residual disease (MRD) was assessed by a European Research Initiative on CLL flow cytometry assay. Results: As of May 15 2022, 50 pts with CLL received treatment: 6 monotherapy (all relapsed/refractory [R/R]) and 44 combination (22 R/R; 22 treatment naïve [TN]). The monotherapy CLL cohort received BGB-11417 doses up to 160 mg. Based on emerging safety data from other cohorts, pts in combination cohorts with R/R CLL received BGB-11417 up to 640 mg and pts with TN CLL received up to 320 mg (data include 8 pts in ZANU pretreatment not yet dosed with BGB-11417). MTD has not yet been reached for any CLL cohort, with dose escalation ongoing. Median follow-up was 11.5 mo (range 8.5-18.3) for monotherapy and 5.8 mo (range 0.2-10.5) for combination. Treatment-emergent AEs (TEAEs) across all doses are listed in the Table. With monotherapy, cytopenias were the most common TEAEs (≥50%), with 33% grade ≥3. With combination, contusion, neutropenia, and low-grade gastrointestinal toxicity were the most common TEAEs (≥22.7%); neutropenia was the most common grade ≥3 TEAE (11.4%) with 5 pts. No pts discontinued monotherapy treatment, and 1 pt discontinued combination treatment (disease progression; Richter transformation). Only 1 high-risk pt with CLL on monotherapy had laboratory TLS that resolved with no intervention (overall laboratory TLS ≤2%). No clinical TLS was reported. Diarrhea was mostly grade 1 and grade ≥3 was not seen. Although efficacy data are early, most pts with CLL/SLL had notable reductions in absolute lymphocyte count (ALC) with responses seen at doses as low as 1 mg (Figure), consistent with improved potency of BGB-11417 vs venetoclax. Four responses (66%, partial response [PR] or better) and 32 responses (72.7%, PR with lymphocytosis or better) were observed with mono- and combination therapy, respectively. MRD data are early: among 4 MRD evaluable pts at 160 mg, 3 pts (2 monotherapy; 1 combination) had a peripheral blood CLL count <10-4 at 24 weeks after BGB-11417 initiation. Conclusion: These preliminary data show BGB-11417, alone or in combination with ZANU, was well-tolerated in most patients. Grade ≥3 neutropenia was uncommon and manageable. Efficacy is supported by the rapid reduction in ALC during ramp-up, and early response data are promising. TLS rates are low; the prophylactic measures and ramp-up schedule seem to adequately mitigate TLS across all dose levels tested. Mature MRD data are forthcoming, and venetoclax-treated CLL/SLL cohorts will soon be open for enrollment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lymphopenia in Multiple Sclerosis patients treated with Ocrelizumab is associated with an effect on CD8 T cells

BackgroundIn the phase III, OPERA I and OPERA II, clinical trial lymphopenia was reported in 20.7% of relapsing–remitting multiple sclerosis (RRMS) patients taking Ocrelizumab (OCR). ObjectiveThe objective of this study was to investigate the effect of OCR on lymphocyte subtypes in MS patients with and without lymphopenia. MethodsRetrospective study comparing lymphocyte subtypes in OCR-treated MS patients with low (G1) and normal (G2) absolute lymphocyte count (ALC) at the six-month follow-up (cut-off: 1000 × 106/L). Mann Whitney U test was used to compare ALC, CD19, CD4 T, CD8 T and NK cell counts at baseline and at the six-month follow up between the two groups. A linear mixed model was applied to compare changes in ALC, and subset counts and proportions between patients with and without lymphopenia. We performed the same analyses in a subpopulation of naïve to treatment patients to exclude the possible influence of the previous disease modifying therapy (DMT) in the different kinetics observed between the two groups. Results: One hundred sixty-seven patients were included (G1, n = 34; G2, n = 133). At the six-month follow-up, compared with baseline, in the whole population we observed a significant reduction in ALC (p<0.0001), CD19 (p<0.0001) and CD8 T (p<0.0288) lymphocytes. We also found and increase in CD4/CD8 ratio after six months of treatment (p = 0.0098). G1 showed a lower ALC than G2 at baseline. At six months, mean ALC was 896.41 ± 156.25 × 106/L in G1 and 1909.9 ± 629.07 × 106/L in G2. CD4 and CD8 T cell mean counts were lower (p < 0.0001) in G1 than G2. At the linear mixed model analysis, we found a more pronounced increase in CD8 T percentage in G2 than G1 (p = 0.008). In the naïve to treatment group fifty patients were included. CD4 and CD8 T cell mean counts at six months were lower (p = 0.0074 and p = 0.0032, respectively) in G1 than G2. At the linear mixed model analysis, we found a more pronounced decrease of CD8 T cell count in G1 than G2 (p = 0.0103). Furthermore, we found an increase in CD8 T percentage in G2 whereas a profound decrease of CD8 T percentage was observed in G1 (p = 0.0052). After adjusting for confounders, significantly positive correlations were noted between ALC and both CD4 and CD8 T cell counts. Negative correlation was observed between ALC and CD4/CD8 ratio driven by low CD8 T cell counts. ConclusionOCR decreases ALC. Among T cells, the treatment predominantly impacts CD8 cells. However, CD8 T cell decrease was more pronounced in patients with lymphopenia. Further studies are needed to establish the relationship between the effect of OCR on ALC and CD8 T cells and its potential implication in the early clinical response and risk for viral infections.

Model-informed assessment of ethnic sensitivity and dosage justification for Asian populations in the global clinical development and use of cladribine tablets.

Cladribine tablets have been approved in many countries for the treatment of patients with various forms of relapsing multiple sclerosis (MS). Cladribine has a unique pharmacokinetic/pharmacodynamic (PK/PD) profile with a short elimination half‐life (~ 1 day) relative to a prolonged PD effect on specific immune cells (most notably a reversible reduction in B and T lymphocyte counts). This results in a short dosing schedule (up to 20 days over 2 years of treatment) to sustain efficacy for at least another 2 years. Global clinical studies were conducted primarily in White patients, in part due to the distinctly higher prevalence of MS in White patients. Given the very low prevalence in Asian countries, MS is considered as a rare disease there. In spite of the limited participation of Asian patients, to demonstrate favorable benefit/risk profile in the treatment of MS demanded application of a Totality of Evidence approach to assess ethnic sensitivity for informing regulatory filings in Asian countries and supporting clinical use of cladribine in Asian patients. Population PD modeling and simulation of treatment‐related reduction in absolute lymphocyte count, as a mechanism‐related biomarker of drug effect, confirmed consistent PDs in Asian and non‐Asian patients with MS, supporting absence of ethnic sensitivity and a common dosage across populations. Through this example, we demonstrate the value of holistic integration of all available data using a model‐informed drug development (MIDD) framework and a Totality of Evidence mindset to evaluate ethnic sensitivity in support of Asia‐inclusive development and use of the drug across populations.

TCL-194: Change in Lymphocyte Count as a Predictor of Response to Mogamulizumab in Patients with Mycosis Fungoides or Sézary Syndrome

Context Mogamulizumab is an anti-CCR4 antibody for patients with relapsed/refractory mycosis fungoides (MF) or Sezary syndrome (SS). The mechanism of action of mogamulizumab involves depletion of CCR4+ T cells and potential to induce lymphopenia. Objective This post hoc analysis of the phase 3 MAVORIC study evaluated the shift in lymphocyte populations during therapy and correlated changes from baseline to highest grade of lymphopenia with clinical outcomes. Design MAVORIC patients received mogamulizumab (1.0 mg/kg IV weekly for 4 weeks, then biweekly; n=186) or vorinostat (400 mg/d PO; n=186). Patients were stratified by baseline blood involvement (B0, B1, B2). Changes in cell types (absolute counts of CD8+CD3+/-, CD4+CD26-, CD4+CD7-, and CD4/CD8 ratio) were assessed. Response was determined using global composite response, as well as separately within skin. Results Grade 1–4 lymphopenia occurred in 80% (138/174) of mogamulizumab patients. Shifts from grade 0 at baseline to 1 during therapy occurred in 6% of patients, grade 0 to 2–3 in 47%, and grade 0 to 4 in 6%. Infections/infestations after a reduction in absolute lymphocyte count were reported in 18 (11.3%), 53 (33.3%), 22 (13.8%), or 5 (3.2%) of patients with grade 1, 2, 3, or ≥4 lymphopenia, respectively. Blood involvement (B1=detectable Sezary cells Conclusions Mogamulizumab induced early and sustained lymphopenia. No increase in the incidence of infections with severity of lymphopenia was observed. There was no correlation with the depth of lymphopenia and clinical response, but patients with advanced disease and circulating Sezary cells had higher grade lymphopenia, reflecting mogamulizumab’s effect on clearing CCR4-expressing Sezary cells from blood. Depth of lymphopenia in mogamulizumab-treated patients did not correlate with clinical response, but early change in CD4+/CD8+ may be a biomarker for eventual skin response. Supported by Kyowa Kirin, Inc.

Abstract 13352: Decreased Absolute Lymphocyte Count and Increased Neutrophil Lymphocyte Ratio With Immune Checkpoint Inhibitors-associated Myocarditis

Introduction: Myocarditis due to immune checkpoint inhibitor (ICI) therapy is a potentially fatal immune-related adverse event (irAE). Hypothesis: Limited data have suggested an association between baseline and on-treatment absolute lymphocyte count (ALC) and neutrophil lymphocyte ratio (NLR) and the development of other irAEs; there are no data characterizing the role of ALC and NLR in ICI-associated myocarditis. Methods: This was a case control study of 55 patients with ICI myocarditis and 55 controls without any post-ICI irAEs. We leveraged clinical testing where patients underwent routine serial blood counts before and with each ICI cycle to compare the baseline and change in ALC and NLR between cases and controls. The association between the change in these parameters with clinical variables and major adverse cardiac events (MACE) was also tested. Results: In those who developed myocarditis, there was a decrease in ALC from baseline (1.6 K/μL, IQR 1.1-1.9) to admission (1.1 K/μL, IQR 0.7-1.3, p&lt;0.001, Panel A). Similarly, among those who developed myocarditis, there was an increase in NLR from baseline (3.5, IQR 2.3-5.4) to admission (6.6, IQR 4.5-14.1, p&lt;0.001, Panel B). There was no change in patients treated with an ICI who did not develop myocarditis. Among those who developed myocarditis, a greater decrease in ALC or increase in NLR was associated with a higher heart rate and a lower blood pressure at admission. In follow-up, there were 20 events; larger decreases in ALC or increases in NLR were associated with MACE (Panel C and D). Conclusions: A reduction in ALC and an increase in NLR was seen with ICI myocarditis. A greater decrease in ALC or increase in NLR were associated with subsequent MACE.

Decreased Absolute Lymphocyte Count and Increased Neutrophil/Lymphocyte Ratio With Immune Checkpoint Inhibitor-Associated Myocarditis.

BackgroundMyocarditis attributable to immune checkpoint inhibitor (ICI) therapy is a potentially fatal immune‐related adverse event. Limited data have suggested an association between baseline and on‐treatment absolute lymphocyte count (ALC) and neutrophil/lymphocyte ratio (NLR) and the development of other immune‐related adverse events; there are no data characterizing the role of ALC and NLR in ICI‐associated myocarditis.Methods and ResultsThis was a case control study of 55 patients with ICI myocarditis and 55 controls without any post‐ICI immune‐related adverse events. We leveraged clinical testing, where patients underwent routine serial blood counts before and with each ICI cycle to compare the baseline and change in ALC and NLR between cases and controls. The association between the change in these parameters with clinical variables and major adverse cardiac events was also tested. In cases, there was a statistically significant decrease in ALC with myocarditis from baseline (1.6 thousands per cubic milliliter (K/μL); interquartile range, 1.1–1.9 K/μL) to admission (1.1 K/μL; interquartile range, 0.7–1.3 K/μL; P<0.001). Similarly, there was an increase in NLR from baseline (3.5; interquartile range, 2.3–5.4) to admission (6.6; interquartile range, 4.5–14.1; P<0.001). There was no statistically significant change in controls. In follow‐up, there were 20 events; larger decreases in ALC (44.6% versus 18.2%; P<0.001) or increases in NLR (156.5% versus 65.1%; P=0.019) were associated with major adverse cardiac events.ConclusionsA reduction in ALC and an increase in NLR was seen with ICI myocarditis. A greater decrease in ALC or increase in NLR was associated with subsequent major adverse cardiac events.

Temporal profile of lymphocyte counts and relationship with infections with fingolimod therapy in paediatric patients with multiple sclerosis: Results from the PARADIGMS study

Background: Reduction in absolute lymphocyte count (ALC) is expected with fingolimod treatment. Objective: To evaluate the effect of fingolimod 0.5 mg versus intramuscular interferon β-1a (30 μg) on ALC and its relationship with infections in paediatric-onset multiple sclerosis (POMS) up to 4 years. Methods: We assessed ALC at baseline, monthly till 3 months, and every 3 months (core phase) and with variable periodicity (extension phase) of Phase 3 PARADIGMS study (N = 215). Incidence rates (IRs) of infection-related adverse events (infAEs)/100 patient-years were analysed by on-study nadir ALC. Results: With fingolimod, ALC rapidly reduced to 29.9%–34.4% of baseline values within 2 weeks and remained stable thereafter; no relevant changes observed with interferon. IRs of infAEs were 67.6 with fingolimod and 61.8 with interferon; IR ratios with respect to interferon, overall: 1.09, by nadir ALC 0.2–0.4 × 109/L: 1.13 and >0.4 × 109/L: 0.91. Three patients had a single episode of ALC <0.2 × 109/L (core phase). No opportunistic infections were observed and infection risk did not increase during the extension phase. Conclusion: In paediatric patients, the overall incidence of infections was comparable between fingolimod and interferon. No association was observed between nadir ALC and infections in POMS, although sample size may have been too small to rule an association.

Chemoradiation-Induced Lymphopenia in Glioblastoma Patients and the Influence of Total Radiation Exposure Time

There is a clear relationship between the adaptive immune response and solid tumor control. However, many of our effective cancer therapies are delivered at the cost of treatment-induced lymphopenia. It has been shown that patients with glioblastoma (GBM) treated with standard post-operative chemoradiation develop a significant, lasting treatment-induced lymphopenia. Others have shown in this patient population that severe CD4-postive T-cell depletion (<200cells/mm3) significantly reduces overall survival. We hypothesized that dosimetric and treatment planning parameters, including the cumulative time a patient is exposed to radiation (beam-on-time, BoT) and total monitor units (MU) delivered, increase lymphocyte toxicity and degree of lymphopenia. Here, we report a single institutional retrospective analysis of 128 patients with pathologically-proven WHO grade IV GBM treated with surgical resection followed by the standard 6000cGy of IMRT in 30 fractions with concurrent and adjuvant temozolomide from 2007-2016. In our cohort, the mean pre-treatment absolute lymphocyte count (ALC) was 1,528 cells/uL. Over the 6-week course of radiation therapy, patient’s ALC dropped by 47% (mean 811 cells/uL) immediately after completion of treatment and remained reduced by 38% (mean 950 cells/uL) and 36% (mean 980 cells/uL) 1- and 2-months post treatment, respectively. Furthermore 20.3% (26/128) developed severe lymphopenia (ALC < 500 cells/uL) immediately after treatment. The mean BoT was 42.3 minutes (13.6 – 116.1 minutes) and mean MU delivered per patient was 15787 MU (7904 – 39805 MU). Both BoT and MU did not correlate with post-treatment ALC or absolute reduction in ALC at the end of treatment (R2 < 0.01). The general neuroanatomic location of each tumor was included in the analysis and was also found not influence the degree of lymphopenia (p > 0.1), while pre-treatment ALC was indeed correlated with post-treatment ALC (p<0.001). These data suggest that the complexity of an IMRT plan that may increase a patient’s BoT and/or total MU delivered does not increase a patient’s risk of treatment-induced lymphopenia. Instead, our data confirmed that a low pre-treatment ALC is a risk factor for more severe post-treatment lymphopenia.

Mitochondrial Apoptotic Priming Is Associated with Clinical Response to the Bcl-2 Antagonist ABT-199 in Chronic Lymphocytic Leukemia

BACKGROUNDBH3 profiling is a functional assay that assesses the degree to which cells are primed to die via mitochondrial apoptosis. We previously used this technique to show that pre-treatment CLL cells from patients who responded to frontline chemoimmunotherapy had higher levels of apoptotic priming than cells from refractory patients (Davids et al., Blood, 2012). BH3 profiling also detects dependence on individual anti-apoptotic Bcl-2 family proteins. Recently, ABT-199, a selective, potent, small molecule antagonist of the anti-apoptotic protein Bcl-2, has been found to be highly active in patients with relapsed or refractory CLL, even those with high-risk markers such as del(17p) (Seymour et al., EHA, 2014). Unlike its Bcl-XL/Bcl-2-targeting predecessor navitoclax (ABT-263), ABT-199 spares platelets, which rely on Bcl-XL for survival. We hypothesized that baseline levels of apoptotic priming would be associated with the depth of initial clinical response to ABT-199 in CLL patients. METHODSPeripheral blood samples were obtained just prior to initial dosing on the phase I, first-in-human study of ABT-199 in relapsed/refractory CLL. Viability was determined by Annexin V–FITC and propidium iodine by FACS. BH3 profiling was performed by exposing gently permeabilized CLL cells to a panel of BH3-domain peptides, fixing the cells, and quantifying apoptotic priming by assessing cytochrome C release by FACS. Platelets from 3 healthy donors were also analyzed. Clinical response was assessed by comparing baseline values to first-re-staging, as follows: peripheral blood: % reduction in absolute lymphocyte count, lymph node: % reduction in the sum of the product of the diameters of the 6 target lesions, and bone marrow: % reduction in morphologic bone marrow intertrabecular space involvement. Linear regression was used to assess clinical responses as continuous variables, and non-parametric Mann-Whitney testing was used for intergroup comparisons. Two-tailed p values were used in all cases. RESULTSEx vivo treatment of platelets with navitoclax induced substantial apoptotic priming, whereas minimal baseline priming was unchanged after ex vivo treatment with ABT-199. Baseline blood samples from 14 relapsed/refractory CLL patients on the ABT-199 phase I study were analyzed for viability after ex vivo treatment with ABT-199 and for pre-treatment levels of apoptotic priming. With ex vivo treatment, ABT-199 induced dose-dependent apoptosis, with a median EC50 of 23 nM in CLL cells (n=14); however, the ABT-199 EC50 was not associated with clinical response in blood, lymph node, or bone marrow. In contrast, samples with higher levels of apoptotic priming as measured by BIM BH3 peptide had a significantly deeper response in the bone marrow (Figure 1, n=9, p=0.01) and blood (n=13, p = 0.05), with a trend toward better response in lymph node (n= 14, p = 0.19). Reflecting the observation in the clinic that ABT-199 has an equivalent response rate irrespective of del(17p) status, we found no difference in apoptotic priming in patients with (n=5) or without (n=9) del(17p). Interestingly, although patients who were fludarabine-refractory at study entry (n=8) were highly primed at baseline, they had lower levels of apoptotic priming (median 92% depolarization) than those who were not refractory (n=6) (median 98%) (p=0.04). To provide further validation of the on target effect of ABT-199 on Bcl-2 on CLL mitochondria, we also studied low concentration ABT-199 used like a BH3 peptide in the BH3 profiling assay. We found an excellent correlation between the amount of mitochondrial depolarization caused by ABT-199 and BAD BH3 peptide (Figure 2, n=13, p = 0.0001). CONCLUSIONSBH3 profiling provides valuable insights into the mechanism of action of the promising Bcl-2 antagonist ABT-199. In contrast to traditional EC50 assays, the pre-treatment level of apoptotic priming predicts initial clinical response to ABT-199 in CLL. An analysis of the association between apoptotic priming and progression free survival is ongoing. With additional validation, BH3 profiling may eventually guide optimal patient selection for Bcl-2 directed therapy, and potentially for other therapies in CLL. [Display omitted] DisclosuresDavids:Genentech: Consultancy; Infinity Pharmaceuticals: Consultancy. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Letai:Abbvie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

Streptozotocin-associated lymphopenia in cynomolgus monkeys

Streptozotocin (STZ) is used to induce diabetes in experimental animals. It has a variety of adverse effects, ranging from nausea, emesis, and weight loss to liver damage, renal failure, and metabolic acidosis. STZ also has effects on the immune system, being associated with lymphopenia in rodents, the mechanism of which is not fully understood. We present data on a significant STZ-associated reduction in lymphocyte count in nonhuman primates. We report a significant reduction in absolute lymphocyte count; in 2 monkeys, the lymphopenia persisted for >100 d. However, a significant increase in absolute monocyte count was noted. Furthermore, an increase in serum monocyte chemoattractant protein-1 (MCP-1) was observed. The reduction in lymphocyte numbers may contribute to immunomodulation that may be beneficial to a subsequent islet graft, and may reduce the need for immunosuppressive therapy. The increase in monocytes and MCP-1, however, may be detrimental to the islet graft. Studies are warranted to explore the mechanism by which STZ has its effect.

Safety, pharmacokinetics, pharmacodynamics, and bioavailability of GSK2018682, a sphingosine-1-phosphate receptor modulator, in healthy volunteers.

The tolerability, pharmacokinetics, and pharmacodynamics of single (SD) and repeat (RD) doses of GSK2018682, a selective S1P1 receptor modulator, were evaluated in healthy volunteers. The bioavailability (BA) of different formulations and effects of food were also evaluated. SD of up to 24 mg and RD of up to 6 mg/day for 28 days were reasonably tolerated, despite higher incidences of gastrointestinal and cardiovascular adverse events compared to placebo. There was a linear relationship between dose and systemic exposure with a dose-independent half-life (t1/2 ) between 44.9 and 63.3 hours. GSK2018682 induced acute, transient and non-symptomatic decreases in heart rate and blood pressure. Dose-dependent reduction in absolute lymphocyte count (ALC), and all tested subsets, was observed to various degrees, up to a nadir of over 70% reduction from baseline. There was no difference in major pharmacokinetic parameters among three formulations of GSK2018682 and between fasted and fed subjects. However, there was a reduction in the extent of bradycardia following dosing in the fed state. Additionally, exercise induced robust increase in heart rate in subjects who had bradycardia following RD of GSK2018682 up to 6 mg, suggesting possible physiological methods of reducing the extent of S1P mediated bradycardia and subsequent AV-block.

Abstract 3531: Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR).

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

Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia

Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia

Effects of Hydroxyurea (HU) On Lymphocyte Subsets and the Immune Response to Pneumococcal, Measles, Mumps and Rubella Vaccination in the Pediatric Hydroxyurea Phase III Clinical Trial - BABY HUG - (ClinicalTrials.gov Identifier: NCT00006400)

AbstractAbstract 243▪▪This icon denotes a clinically relevant abstractSusceptibility to encapsulated bacteria, particularly Streptococcus pneumonia, is well known in sickle cell disease (SCD). Hydroxyurea (HU) is commonly used in adults and children with SCD, but, little is known about the effects of HU on immune function in SCD and recommendations for immunization are lacking. As HU reversibly inhibits ribonucleotide reductase, causing cell cycle arrest at the G1-S interface, we postulated that HU might delay the transition from naïve to memory T cells, resulting in a delay in immunologic maturation, with deleterious effects on vaccine responses; therefore, T cell subsets, including conversion of naïve to memory T cells and antibody (Ab) responses to pneumococcal (Pnu), measles, mumps and rubella (MMR) vaccinations were studied during the BABY HUG Trial of HU for infants and toddlers with SCD. Methods:Blood was collected for measurement of: T cell subsets at entry (9–18 mos of age), age 24 mo and exit after 2 yrs of treatment; Pnu Ab levels were measured (by EIA after preabsorption with C-polysaccharide) at entry, before and 2–8 wks after 23-valent polysaccharide (PS) PCV23 vaccine at age 24 mo, and exit; and MMR antibodies at entry, 1–10 weeks after MMR at age 12 mo, age 24 mo and exit. Results:Of the 193 subjects in BABY HUG, T cell subsets were available for 91 HU and 88 placebo (PL) subjects. At entry, there were no significant differences in absolute lymphocyte count (ALC), or % or absolute T cell subsets between the HU and PL groups. At age 24 mo, both groups had an age-related decline in ALC, with significant differences between the HU and PL groups in ALC (5740 vs. 7323/mm3; p=0.003), absolute CD4 cells (1912 vs. 2247/mm3; p=0.022) and memory CD4 cells (392 vs. 487/mm3; p=0.003). The same pattern was seen at exit for ALC (4836 vs. 5764/mm3; p=0.015), absolute CD4 cells (1510 vs. 1747/mm3; p=0.043) and memory CD4 cells (360 vs. 444/mm3; p=0.003).IgG Ab levels to Pnu antigens were measured at entry [after 3 doses of PS/protein conjugate (PC) PCV7 containing Pnu PS type 26 (6B), but not 51 (7F)], before and after immunization with PS PCV23 vaccine at 24 mo, and at exit, with no significant differences between the HU and PL groups in mean Ab levels at any time point for either serotype (Fig 1&2). [Display omitted] For those immunized with MMR prior to study treatment, there were no differences in % immune for measles, mumps, or rubella between the HU and PL groups at any time point. For 40 HU and 38 PL subjects immunized after starting study treatment, there was a smaller % of HU-treated children with protective response to measles vaccine 1 – 10 wks after immunization [HU 71.4% (10/14) vs. PL 100% (24/24); p=0.014]. Response to mumps [HU 78.6% (11/14) vs. PL 91.7% (22/24)] and rubella [HU 66.7% (10/15) vs. PL 92% (23/25)] vaccines showed similar trends, but no statistical significance. The HU vs. PL difference in failure of response to at least one vaccine virus was of similar significance (p = 0.021) to that of measles alone. By age 24 mo, there were no significant differences in response to MMR (89.7 to 100%). Mean Ab levels to measles and rubella were also significantly lower in the HU group 1–10 wks after immunization (p=0.005 and p=0.011, respectively). Conclusions:HU given to infants and young children with SCD causes a reduction in ALC, CD4 T cell and memory T cell counts. Additional monitoring of HU-treated children with SCD for these and related T cell parameters is warranted until the biological effects on immunity are known with certainty.Response to Pnu vaccine was not affected by HU therapy, suggesting that there is no increased risk for Pnu sepsis in infants and children with SCD treated with HU, consistent with the clinical results in the BABY HUG trial.Although the sample was small, a delay in achieving protective measles Ab levels was seen in the HU group, and possibly for mumps and rubella. This delay cannot be estimated precisely, due to study design, but Ab levels to all 3 viruses were similar at exit, indicating that effective immunization can be achieved despite HU use. For endemic or local epidemics of measles, mumps, rubella or other pathogens requiring immunization, adherence to recommended accelerated immunization schedules will be especially important for children with SCD treated with HU. Disclosures:Off Label Use: Off-label use of hydroxyurea in infants and children. Casella:Adventrx: Consultancy, Research Funding.

High-Resolution Sequencing Identifies NOXA1 De-Methylation As a Novel Strategy to Overcome Bortezomib Resistance in Mantle Cell Lymphoma

Abstract 557Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with frequent relapses after initial response to standard chemotherapy. Bortezomib is the first proteasome inhibitor with promising clinical efficacy in relapsed MCL, where it achieved durable responses in 30%–50% of patients. The mechanisms contributing to Bortezomib resistance remain poorly understood. Our group and others have identified aberrantly methylated genes in MCL controlling critical cellular processes like cell cycle, transcription and regulation of gene expression. We therefore hypothesized that a subset of aberrantly methylated genes may be contributing to Bortezomib resistance.Using a massively parallel sequencing approach, HELP-tagging, we analyzed genomic methylation in MCL patients treated in a Phase II trial at the NIH. This high-resolution technique covers about 1.8 million CpGs in the human genome including gene promoters, gene bodies and CpG shores and is more sensitive and yields greater coverage of the genome as compared to array-based platforms. Unsupervised analysis of genome-wide promoter methylation of Bortezomib sensitive patients (having more than 50% reduction in absolute lymphocyte count after one cycle of Bortezomib) as compared to resistant patients revealed that methylation patterns could independently distinguish between these two groups. Similarly, three-dimensional Principal Component Analysis also clustered Bortezomib resistant patients distinctly from Bortezomib sensitive patients, confirming genomic differences in methylation between these patients. We further analyzed the differentially methylated genes using pathway analysis software, gene ontology and gene set enrichment analysis. We found that majority of differentially methylated genes were relevant to cancer biology involving metabolic pathways, regulation of transcription, cell cycle and programmed cell death. Comparing methylation profiles of patient samples taken before and 96 hours after Bortezomib treatment showed a striking genome-wide hypomethylation following Bortezomib. Western blotting of MCL cell lines showed a significant reduction in DNMT1 levels following Bortezomib treatment, which may help us understand the post-treatment hypomethylation seen in patients.NOXA1 is a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of Bortezomib. The NOXA1 gene promoter was differentially methylated in Bortezomib-sensitive MCL patients. NOXA1 was also significantly hypomethylated to a greater extent 96 hours following Bortezomib treatment in Bortezomib sensitive patients. NOXA1 hypomethylation was associated with induction of NOXA1 mRNA and protein expression in sensitive patients alone. These findings suggest a possible role for dynamic NOXA1 methylation and expression as biomarkers for Bortezomib sensitivity.Treatment of MCL cell lines Z138 and MINO with demethylating agents Decitabine also induced NOXA1 hypomethylation and increased NOXA1 protein expression. Low dose (0.1-0.5μM) Decitabine pretreatment potentiated low (IC25) dose Bortezomib causing more than 80–90% cell kill in MINO and Z138 cells. The combination of Decitabine and Bortezomib led to increased NOXA1 hypomethylation and protein expression as compared to the individual drugs. Depletion of NOXA1 by specific shRNA rescued 40–50% of the cells from the cytotoxicity of this combination. Tumor progression and chemoresistance are commonly associated with the overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1). ABT-263 is a small molecular inhibitor of the Bcl-2 family of proteins, excepting Mcl-1, which can be neutralized by an increase in NOXA1. Decitabine treatment also potently enhanced the cytotoxicity of ABT-263 in MCL cell lines.Our data suggest that inducing hypomethylation of NOXA1 promoter can be a novel therapeutic strategy for overcoming Bortezomib resistance in MCL, and that genomic methylation patterns and specifically NOXA1 hypermethylation may be useful biomarkers for Bortezomib response. More broadly, these data demonstrate that high-resolution genomic methylation profiling can identify genes and signatures with important prognostic and therapeutic implications. Disclosures:No relevant conflicts of interest to declare.

Epigallocatechin-3-Gallate (EGCG) Modulates Cytokine Production When Leukemic CLL B-Cells and Marrow Stromal Cells Are Co-Cultured: Correlations with Clinical Activity in a Phase II Trial,

Abstract 3882 PURPOSE:Nurturing of leukemic B-cells by the microenvironment appears to be an important mechanism of chemoresistance. Recently, several groups have shown that immune cytokines such as MIP-1a (CCL3) and MIP-1b (CCL4) are upregulated when CLL B-cells interact with stroma or nurse-like cells. It appears that stroma may nurture CLL B-cells by providing critical growth factors and signals enhancing survival. Disruption of these signals could be a strategy to overcome stromal cell mediated chemoresistance. METHODS:We cultured CLL B-cells alone or with human stromal cells (HS-5) and evaluated levels of immune cytokines in the media using multiplex cytokine arrays (Invitrogen). To explore the effect of different drugs on cytokine release, CLL B-cells were treated with sub-lethal doses of fludarabine (F) and chlorambucil (C) in co-culture with the stromal cell line HS-5 cells. Based on our previous observation that pharmacologically achievable doses of EGCG are able to overcome stromal rescue of CLL B-cells exposed to purine nucleoside analogues and alkylating agents (ASH 2010), we examined the effects of EGCG on stromal cell mediated cytokine release in co-cultures. In addition, plasma samples collected prior to and after 1 month of treatment from Rai stage 0-II CLL patients participating in a phase II trial of EGCG were used to explore whether there was a clinical correlation of the in vitro effects of EGCG. RESULTS:Co-culture of CLL B-cells with HS-5 cells resulted in upregulation of multiple cytokines. In addition to the previously reported upregulation in MIP-1a and MIP-1b, increases in IL-1RA (5 fold) and MCP-1 (4 fold) were also observed. Upregulation of these cytokines in co-culture was largely contact dependent.When CLL B-cells and HS5 cells were co-cultured with sub-lethal doses of F, modest reductions in the upregulation of IL-1RA (12%) and MCP-1 (18%) were observed compared to co-culture without drug exposure. Similar experiments using sub-lethal doses of C resulted in more substantial reductions in IL-1RA (68%) and MCP-1 (48%) upregulation. Interestingly, the effect of sub-lethal doses of EGCG was similar to C with respect to reducing upregulation of IL-1RA (69%) and greater than both F and C with respect to upregulation of MCP-1 (76%; Figures 1A and B). [Display omitted] To explore the potential clinical correlations of these in vitro observations, we assessed the effects of EGCG on plasma IL-1RA and MCP-1 levels in 42 CLL patients participating in a phase II trial evaluating the efficacy of EGCG (2000 mg BID). Of these 42 patients, 13 (31%) were Rai stage 0 and 29 (69%) Rai stage I-II at enrollment. As previously reported (Shanafelt, ASCO 2010), clinical activity was observed with 13 (31%) patients experiencing a sustained ≥20% reduction in absolute lymphocyte count (ALC) and 20 of 29 (69%) patients with palpable adenopathy experiencing at least a 50% reduction in the sum of nodal products with treatment. Median baseline plasma MCP-1 levels prior to treatment were 687 pg/ml and median IL-1RA levels were 468 pg/ml. At the cohort level, no change in median MCP-1 (p=0.21) or IL1-RA (p=0.95) levels were observed after 1 month of EGCG treatment. We next explored whether changes in the MCP-1 or IL1-RA levels of individual patients correlated with response. Although no correlation between reductions these cytokines and declines in ALC were observed, a significant association between reductions of both MCP-1 (Spearman coefficient: 0.51; p=0.02) and IL1-RA (Spearman co-efficient: 0.60; p=0.006) and reductions in lymphadenopathy was found suggesting EGCG effects on these cytokines may relate to clinical efficacy. CONCLUSIONS:Co-culture of CLL B-cells with marrow stromal cells resulted in contact dependent up-regulation of multiple immune cytokines including IL-1Ra and MCP-1 which we speculate may partially facilitate stromal cell mediated chemoresistance. Several drugs, including EGCG appear to modulate this up-regulation. An association between changes in these cytokine levels and reductions in lymphadenopathy in a phase II trial of EGCG suggests that interrupting signaling by these cytokines may be a potential therapeutic target. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees. Shanafelt:Polyphenon E International: Research Funding.