Citations

Abstract Introduction: Given the availability of several active treatment (tx) options for CLL, providers frequently make tx recommendations based on their interpretation of objective data from consensus criteria and clinical trials. There are limited data describing patient (pt) experience, values, and insights when encountering multiple tx choices. The CLL Society, a pt-driven, physician-curated nonprofit organization focused on the unmet needs of the CLL community, sought to explore how CLL pts make tx decisions. Methods: A 64 question survey was distributed online and in paper to CLL pts from Oct-Dec 2017. The survey was developed by CLL experts. The research was IRB-approved. Chi square was used for statistical comparison; all other analyses are descriptive in nature. Results: 1147 pts from 48 states completed the survey. Median age was 65 (range 28-86), 46% male, 96% Caucasian. Who Influences Tx: Of the 79% of pts who have seen a CLL expert, defined as a provider focused on CLL at an academic/research center, 95% rated their provider as very/extremely influential. 83% of pts rated their own opinions as influential, and 59% rated the opinion of a general hematologist/oncologist (gen heme/onc), a provider who sees a variety of cancer types, as influential. Only 27% let their provider unilaterally decide on tx. Of these pts, 19% trusted their provider to select the best tx and 5% didn't have enough understanding to be involved in the decision. 64% were influenced by outside sources of information, such as family members, live CLL support groups, or online sources including pt experts and forums. Factors in Deciding Tx: Of the 67% pts surveyed who have been treated, 73% were offered > 1 tx choice by their provider. In deciding on a specific tx, 94% of respondents stated that it was very/extremely important to know there were tx options beyond their current regimen. The most important factors in selecting tx were response rate (91%), overall survival (88%), progression-free survival (86%), long-term side effects (82%), ability to achieve undetectable MRD (80%), cost/insurance coverage (66%), and location of tx (50%) with significant differences between subgroups (Table 1). Tx was impacted by cost or insurance issues for 16% (n=104). Of these 104 pts, cost/insurance issues resulted in tx delay for 34 and change in tx plan for 24 pts. 16 pts reported that an appeal led to coverage and 16 pts received financial assistance. Pts' willingness to accept potentially curable but high-risk tx was heavily influenced by availability of alternate options. For example, 18% of pts would be willing to consider cytotoxic chemotherapy if alternate options were available vs. 72% if no options were available. Similar patterns were seen with CAR-T tx (28% vs. 79%) and bone marrow transplantation (BMT) (15% vs. 66%) with significant differences between subgroups (Table 2). 82% were willing to take a life-long tx for long-term control without potential for cure. Willingness to accept long-term tx was significantly higher in treated vs. untreated pts (84% vs. 77%, p=.006) and men vs. women (84% vs. 78%, p=.008). Clinical Trial Education: Of those treated by a gen heme/onc, 31% received no education on clinical trials. Only 15% reported good understanding of trial opportunities vs. 52% treated by a CLL expert (p<.001). 9% of pts treated by gen heme/onc were offered clinical trial participation vs. 47% treated by a CLL expert (p<0.001). Pts who declined or would have declined participation cited reasons including preference for "proven" tx (38%), distance from trial site (29%), fear (20%), frequent imaging (20%), overall lack of understanding about trials (15%), and lack of information (14%). Summary: Most respondents want to be and are involved in tx decisions. While providers are influential in decision making, CLL pts also rely on outside sources. Gender, age, and tx status influence which factors drive decision making. Pts select tx based on response rate, survival, side effects, and ability to achieve deep response and want a plan in place for sequencing tx. The majority are willing to consider long-term tx without cure. Few want chemotherapy, CAR-T, or BMT unless they have no other options. A significant opportunity for improvement in education on clinical trial opportunities was identified. This description of pt experience, values, and preferences enriches the informed consent process and may lead to more tailored and patient-centered care. Disclosures Kennard: AbbVie, Gilead, Verastem: Consultancy. Furman:TG Therapeutics: Consultancy; Loxo Oncology: Consultancy; Sunesis: Consultancy; Verastem: Consultancy; Incyte: Consultancy, Other: DSMB; Genentech: Consultancy; Acerta: Consultancy, Research Funding; Gilead: Consultancy; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy. Pagel:Pharmacyclics, an AbbVie Company: Consultancy; Gilead: Consultancy. Davids:Sunesis: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Consultancy, Research Funding; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Merck: Consultancy; Merck: Consultancy; MEI Pharma: Consultancy, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Sunesis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Research Funding; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Verastem: Consultancy, Research Funding; BMS: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Surface Oncology: Research Funding; Verastem: Consultancy, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Roche: Consultancy. Nabhan:Cardinal Health: Employment, Equity Ownership. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees. Siddiqi:Juno Therapeutics: Other: Steering committee. Brander:Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Teva: Consultancy, Honoraria; Novartis: Consultancy, Other: DSMB; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Hill:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cheson:AbbVie, Roche/Genentech, Pharmacyclics, Acerta, TG Therapeutics: Consultancy. Choi:Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Rigel: Consultancy; Genentech: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau. Mato:AstraZeneca: Consultancy; Prime Oncology: Honoraria; Portola: Research Funding; Johnson & Johnson: Consultancy; Medscape: Honoraria; Celgene: Consultancy; TG Therapeutics: Consultancy, Research Funding; Acerta: Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Regeneron: Research Funding.

Abstract Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.

Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Abstract Introduction: Several prognostic models for overall survival (OS) in chronic lymphocytic leukemia (CLL) have been developed, but none were derived for patients (pts) with relapsed or refractory (R/R) CLL treated in the era of novel agents. We used a comprehensive approach to derive and validate a novel risk model for OS in 2,475 pts with R/R CLL who received therapy in randomized phase 3 trials evaluating ibrutinib (IBR), idelalisib (IDELA), and venetoclax (VEN) vs control arms, or the Mayo Clinic CLL Database (MCCD). Methods: In collaboration with Janssen, Gilead, Pharmacyclics, and Genentech/Roche, the analysis included 2,475 pts from 6 multicenter randomized phase 3 trials or the MCCD. Trials included were: IBR plus bendamustine-rituximab (BR) vs placebo plus BR (HELIOS: NCT01611090), IBR vs ofatumumab (RESONATE: NCT01578707), IDELA plus BR vs placebo plus BR (Study 115: NCT01569295); IDELA plus rituximab vs placebo plus rituximab (Study 116: NCT01539512); IDELA plus ofatumumab vs ofatumumab (Study 119: NCT01659021); VEN plus rituximab vs BR (MURANO: NCT02005471). All pts had R/R CLL and required treatment by iwCLL criteria. The model-building dataset (n=969) included pts from HELIOS and RESONATE, and was randomly assigned to the IBR/chemoimmunotherapy (CIT) training dataset (n=727) and IBR/CIT internal-validation dataset(n=242). Three independent external validation datasets included IDELA/CIT (n=897), VEN/CIT (n=389), and MCCD (n=220). We applied univariate and multivariate analyses (MVA) to 28 candidate clinical/laboratory and genetic prognostic factors to derive the risk model in the training dataset. The primary endpoint was OS. We assessed the need for separate models for targeted agents by interactions with treatment in univariate analyses. We proceeded to develop a single model for all pts with R/R CLL, as only one candidate factor (del(13q), interaction p value <0.1) demonstrated a marginal differential effect. We evaluated cutoffs for dichotomized covariates and for definition of risk groups after the final factors were selected. We fit a Cox regression on the training and internal/external validation datasets using the risk categories as the covariate to test the difference among the groups, and calculated the C-statistic as a measure of discrimination. Results: Of 11 selected factors with a significant univariate effect on OS (p<0.1), 6 were independently prognostic in the MVA of OS (p<0.05): Baseline β2-microglobubulin (B2M), lactate dehydrogenase (LDH), hemoglobin (HGB), IGHV mutational status, number of prior therapies, and time from initiation of last therapy. We excluded number of prior therapies from the model, reasoning that its optimal cutoff might change with advances in CLL therapies. IGHV did not remain significant (p=0.0592) in MVA of 5 remaining prognostic factors. Notably, del(17p) was not independently prognostic for OS. The final risk model consisted of 4 prognostic factors: B2M ≥5 mg/L, LDH elevated, HGB <110 mg/L for women or <120 mg/L for men, and time from initiation of last therapy <24 months. Because hazard ratios for each factor were similar (range, 0.55-0.68) with overlapping confidence intervals, 1 point was assigned to each factor. The resulting score separated pts into low (score 0-1), intermediate (score 2-3), and high risk (score 4) groups. Our risk model was prognostic for OS in the IBR/CIT training dataset (C-statistic 0.74 [95% CI 0.60-0.85]; log-rank p<0.0001) and was validated in the IBR/CIT internal validation (C-statistic 0.79 [95% CI 0.56-0.97]; log-rank p<0.0001), as well as in all 3 external validations: IDELA/CIT dataset (CS=0.71, 95% CI 0.59-0.81; log-rank p<0.001); VEN/CIT dataset (CS=0.76, 95% CI 0.66-0.85; log-rank p=0.01); MCCD (CS=0.61, 95% CI 0.56-0.66; log-rank p<0.001). Conclusions: We present the first validated risk model for OS in R/R CLL in the era of targeted therapies. Our model consists of 4 readily available factors (B2M ≥5 mg/L, LDH elevated, HGB <110 mg/L for women or <120 mg/L for men, and time from initiation of last therapy <24 months), identifies 3 prognostic groups with significantly different OS, and has been validated for pts treated with all FDA approved targeted therapies: IBR, IDELA, and VEN. This identifies a well-defined cohort of pts with R/R disease with an unmet clinical need who are suitable for prospective trials targeting these pts. An exploratory analysis regarding prediction of MRD is ongoing. Figure. Figure. Disclosures Darif: Jannsen: Employment. Londhe:Jannsen: Employment. Xing:Gilead Sciences, Inc.: Employment. Mun:Genentech: Employment, Equity Ownership. Kay:Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Mayo Clinic: Patents & Royalties: Physician Well-being Index, Medical Student Well-being Index, Well-being index; GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Furman:Loxo Oncology: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Gilead: Consultancy; Incyte: Consultancy, Other: DSMB; Acerta: Consultancy, Research Funding; Genentech: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Hillmen:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Novartis: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees. Sharman:Acerta: Consultancy, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Research Funding. Seymour:AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding. Jones:Celgene: Employment, Equity Ownership. Ferrante:Jannsen: Employment. Dreiling:Gilead: Employment. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Stark:Genentech: Employment. Reddy:Actinium Pharmaceuticals: Employment, Equity Ownership; Pharmacyclics: Employment. Howes:Janssen: Employment. James:Pharmacyclics: Employment. Bhargava:Gilead: Employment. Zelenetz:Novartis/Sandoz: Consultancy; Abbvie: Research Funding; Celgene: Consultancy; Amgen: Consultancy; Gilead: Consultancy, Research Funding; AstraZeneca: Consultancy; Genentech/Roche: Consultancy, Research Funding.

Abstract Introduction: Observation is the standard of care for asymptomatic early stage chronic lymphocytic leukemia (CLL) however these cases follow a heterogenous course. Recent studies show novel biomarkers can delineate indolent from aggressive early stage disease and current clinical trials are exploring the role of early intervention in high risk cases. Although several scoring systems have been established in CLL, most are designed for overall survival, do not circumscribe early stage disease, and require cumbersome calculations relying on extensive laboratory and clinical information. Aim: We propose a novel laboratory-based prognostic calculator to risk stratify time to first treatment (TTFT) in early stage CLL and guide candidate selection for early intervention. Methods: We included 1574 cases of early stage CLL in an international cohort from Italy, the United Kingdom and the United States using a training-validation model. Patient information was obtained from participating centers in accordance with the Declaration of Helsinki. The training cohort included 478 Rai 0 cases from a multicenter Italian cohort, all referred to a single center (Clinical and Experimental Onco-Hematology Unit of the Centro Riferimento Oncologico in Aviano, IT) for immunocytogenetic lab analyses. Considering TTFT as an endpoint, we evaluated 8 variables (age>65, WBC>32K, 17p-, 11q-, +12, IGHV status, CD49d+, gender) with univariate and multivariate Cox regression internally validated using bootstrapping procedures. FISH thresholds were 5% for 11q-, and +12 and 10% for 17p-. Cases were categorized according to the hierarchical model proposed by Dohner. IGHV status was considered unmutated at ≥98%. CD49d+ was set at >30%. WBC cutoff of >32K was established by maximally selected log rank analysis. Variables were weighted based on the proportion of their normalized hazard ratios rounded to the nearest whole integer. We used recursive partitioning for risk-category determination and Kaplan-Meier analysis to generate survival curves. We compared the concordance index (C-index) of our model with the CLL international prognostic index (CLL-IPI) for 381/478 cases in the training cohort with available beta-2-microglobulin data and for all validation cohorts. We used 3 independent single-center cohorts for external validation. Results: The training cohort had 478 cases of Rai 0 CLL with a median (95% CI) TTFT of 124 months (m) (104-183m). Five prognostic variables emerged with respect to TTFT, and each assigned a point value of 1 or 2 according to their respective normalized HR values as follows: 17p-, and UM IGHV (2 pts); 11q-, +12, and WBC>32K (1 pt). We identified three risk groups, based on point cut-offs of 0, 1-2, and 3-5 established by recursive partitioning analysis with a median (95% CI) TTFT of 216m (216-216m), 104m (93-140m) and 58m (44-68m) (p<0.0001, C-index 0.75) for the low, intermediate, and high-risk groups, respectively (figure 1). A comparison with the CLL-IPI was possible in 381 cases with available beta-2-microglobulin data. In this subset, the C-index was 0.75 compared to 0.68 when patient risk groups were split according to the CLL-IPI. The scoring system was then validated in 3 independent cohorts of early stage CLL: i) Gemelli Hospital in Rome, IT provided 144 Rai 0 cases. Median (95% CI) TTFT was 86m (80-94m, 95% CI). Median (95% CI) TTFT for the low, intermediate and high-risk groups was 239m (239-239m), 98m (92-132m) and 85m (60-109m) respectively (p=0.002 between low and intermediate groups, p=0.09 between intermediate and high groups; C-index 0.64 v 0.60 for CLL-IPI). ii) Cardiff University Hospital in Wales, UK provided 395 Binet A cases. Median (95% CI) TTFT was 74 m (67-81m) overall and NR, 111m (97-146m) and 70m (29-114m) for the low, intermediate and high-risk groups respectively (p<0.001 between low and intermediate groups, p=0.009 between intermediate and high groups; C-index 0.63 v 0.63 for CLL-IPI). iii) Mayo Clinic in Rochester, MN provided 557 Rai 0 cases. Median (95% CI) TTFT was 127m (96m-NR) overall and NR, 76m (64m-NR) and 36m (31-59m) for the low, intermediate and high-risk groups respectively (p<0.0001; C-index 0.72 v 0.68 for CLL-IPI). Conclusion: We present a novel laboratory-based scoring system for Rai 0/Binet A CLL to aid case selection in risk-adapted treatment for early disease. Further comparison to existing indices is needed to verify its utility in the clinical setting. Disclosures Zaja: Novartis: Honoraria, Research Funding; Takeda: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Sandoz: Honoraria. Fegan:Roche: Honoraria; Napp: Honoraria; Janssen: Honoraria; Gilead Sciences, Inc.: Honoraria; Abbvie: Honoraria. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents. Parikh:AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding; MorphoSys: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; Pharmacyclics: Honoraria, Research Funding. Kay:Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T cell (CART19) therapy and led to their FDA approval in diffuse large B cell lymphoma and in acute lymphoblastic leukemia. However, the complete response rate in chronic lymphocytic leukemia (CLL) after CART19 therapy is much lower, at approximately 20-30%, and the mechanism(s) for this relative lack of success is unclear. The dominant known mechanism(s) that prevent successful CART cell therapy in CLL have been limited to CART expansion and poor persistence. However, potential mechanisms are not limited to the CLL T-cell. Several immune defects have been identified in CLL that result from the complex bi-directional interaction between B-CLL cells and their microenvironment. In CLL the leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. There is growing evidence that these vesicles play an important role in intracellular communication by the delivery of growth factors, genetic material and microenvironmentally relevant molecules. Therefore, we aimed to investigate the role and interactions of EVs in the diminished or absent CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages (8 patients had early and 8 had advanced stage disease) and risk profile by FISH (8 patients had low risk and 8 patients had high risk disease, based on the presence of 17p deletion). Cytometry was used to determine size, number of particles per µl, Annexin V and CD19 expression. These variables were correlated to the Rai stage and risk category of the disease. To investigate the impact of EVs on CART cell functions, CART19 cells were stimulated with either CLL EVs alone or in combination with the CD19 positive cell line JeKo1. After coincubation different effector functions were analysed. Results: Two patterns of EVs in CLL patients were identified; a single versus two distinct EV size populations (small [EVssmall]; 50-240nm, median=110nm) and large [EVslarge]; 180-560nm, median = 360nm Fig 1.A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1.B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk) although some variation could be seen (Fig 1.C). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs, 1.5x10e5 particles, alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19 positive cell line JeKo1, their effector functions were reduced only in the presence of EVsCD19+, 50,000 particles, 2.5 x 10e3/ µl, but not EVsCD19- at the same concentration. This included a significant reduction in CART specific killing (Fig 1.D) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, i.e. there was no impairment of CART functions with large or small size EVCD19- in co-culture. Summary: We identify CD19 positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CART cell activity. Studies are ongoing to define the mechanism(s). Disclosures Parikh: Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; MorphoSys: Research Funding; Abbvie: Honoraria, Research Funding. Kay:Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Abstract Introduction: Richter transformation (RT) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Most studies on the management of RT were either small retrospective series or early phase non-randomized trials before the era of novel agents. The natural history, prognostication and optimal treatment of patients with RT remain undefined. Here we report the clinical characteristics and outcomes of a large series of RT from a single center. Methods: Biopsy-confirmed RT (limited to non-Hodgkin lymphoma) diagnosed from 4/1993 to 4/2018 were identified from the Mayo Clinic CLL database. Clinical characteristics, treatment information and follow-up data were abstracted by chart review. Overall survival (OS) was defined as time from RT diagnosis to death from any cause and analyzed using the Kaplan-Meier method. Statistical analysis was done in SAS 9.4. Results: A total of 204 patients with CLL who developed RT were identified. The median age at CLL diagnosis was 62 years (range 22-85), and 148 (73%) were male. The median time to transformation was 4.7 years (range 0-34.5). Prior to RT, 68 (33.3%) patients received no treatment for CLL, 109 (53.4%) received chemoimmunotherapy (CIT) only, and 27 (13.2%) received at least one novel agent (idelalisib, ibrutinib, or venetoclax) for CLL. The median lines of CLL therapy prior to RT was 2 (range 0-13). The median age at RT diagnosis was 69 years (range 30-88). Pathology of RT was DLBCL and high grade B-cell lymphoma in 193 (94.6%) and 11 (5.4%) patients, respectively. The median LDH was 306 IU/L (range 99-9000). 62/125 (49.6%) patients had bulky disease (≥ 5 cm), and the median PET SUVmax was 13.9 (range 2.9-30.0). 45/131 (34.4%) patients had del(17p) or TP53 mutation, 12 (9.2%) had del(11q), 21 (16.0%) had trisomy 12, 27 (20.6%) had del(13q), and 25 (19.1%) had normal FISH. The CLL and RT were clonally related in 12/21 (57.1%) patients. For the transformed lymphoma, cell of origin by Han's algorithm was germinal center B cell-like (GCB) and non-GCB in 31/100 (31.0%) and 69/100 (69.0%) patients, respectively. EBV was positive in 14/52 (26.9%) patients. The median Ki-67 was 80% (range 10-100). Myc and Bcl-2 were positive by IHC in 31/43 (72.1%) and 83/103 (80.6%) patients, respectively; 27/56 (48.2%) were double-expressors. MYC, BCL2, and BCL6 rearrangement was positive by FISH in 18/68 (26.5%), 10/34 (29.4%), and 4/31 (12.9%) patients, respectively; 8/66 (12.1%) were double/triple-hit. The most common first-line treatment (Table 1 notes) of RT was R-CHOP-like regimen (n=114, 65.5%). Other treatments included R-EPOCH-like (n=6, 3.4%), high-intensity chemotherapy (n=15, 8.6%), novel agents (eg, ibrutinib, venetoclax, pembrolizumab; n=19, 10.9%), other chemotherapy (n=12, 6.9%), and palliative therapy (n=8, 4.6%). Response to first-line treatment was CR in 57 (38.0%), PR in 33 (22.0%), SD in 18 (12.0%), and PD in 42 (28.0%) patients. The median OS of the entire cohort after RT diagnosis was 12.0 months. The median OS for patients who received no prior CLL treatment, CIT only or at least one novel agent for CLL were 65.5, 7.3, and 12.0 months, respectively (P<0.0001; Figure 1). Of note, in patients who received CIT only for CLL, ~10% and 60% received high-intensity and R-CHOP/R-EPOCH-like chemotherapy, respectively, as first-line RT therapy. In contrast, in patients who had prior novel agents for CLL, 56% and 26% were treated with novel agents and R-CHOP/R-EPOCH-like chemotherapy, respectively, as first-line RT therapy. Patients with or without del(17p)/TP53 mutation had a median OS of 8.3 and 12.8 months, respectively (P=0.046). Patients who were treated with high intensity chemotherapy, R-CHOP/R-EPOCH-like regimens, novel agents, and other therapies for RT had a median OS of 35.1, 14.4, 10.9 and 6.1 months, respectively (P=0.02; Figure 2). OS comparisons by CLL/RT clonal relationship, double expressor or double/triple-hit status are shown in Table 1, with no significant differences noted. Conclusions: Over two thirds of RT were the non-GCB subtype, and about half were Myc/Bcl-2 double expressors. Patients who developed RT without prior CLL therapies had a significantly better OS. In contrast, patients who had received prior CLL therapies had poor outcomes. Myc/Bcl-2 double expressor and MYC/BCL2/BCL6 double/triple hit status had no impact on OS. Disclosures Kenderian: Humanigen: Research Funding; Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Gilead: Honoraria; Pharmacyclics: Honoraria, Research Funding. Ding:Merck: Research Funding.

Abstract Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Introduction CLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance. Methods Purified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture. Results We observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture. Conclusions Here we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.