Chronic Lymphocytic Leukemia Tumor Research Articles

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Shelter in place: Live CLL cells inside the bone marrow fibroblasts and its implication in drug resistance

Introduction: Cell-in-cell (CIC) describes a century old observation where an intact cell can be seen inside another whole cell. It has been associated with human cancers, especially hematopoietic malignancies. However, most observations have been published as case reports. In the absence of systematic studies of case cohorts, understanding of the pathological significance of live CIC remains challenging. Methods: In this investigation, we studied primary CLL samples using an ex vivo co-culture model system which mimics the CLL tumor microenvironment (TME). In these TME models, CLL cells were cocultured with bone marrow fibroblasts (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4 and upregulate Ki67 expression. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. In this study, cells were observed and analyzed with confocal microscopy and 3D reconstruction. Results: We observed that CLL cells were actively internalized by BMF in 41 CLL cases studied. The internalized CLL cells were alive and mobile within the BMF’s cytoplasm. Moreover, the tumor cells were seen leaving their BMF host and re-entering into other BMFs. The number of internalized CLL cells were quantified and correlated with patients’ clinical and pathological parameters. No correlations were found between cell internalization and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that treated patients had higher level of CIC than those not treated. We hypothesized that live CIC may contribute to drug resistance and internalization may be an important mechanism for tumor cells to evade harmful drug insult and survive as residual disease. To test the hypothesis, we exposed CLL cells in the TME models to BTK inhibitors including ibrutinib and pirtobrutinib. Our results showed that the drug exposure drove CLL cells into BMF. Additionally, we observed that increased CIC was associated with higher cellular proliferation capacity. Importantly, chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, CXCL12 ligand promoted cell internalization, while a CXCR4 antagonist partially prevented CIC from occurring. The research was funded by: NCI Keywords: Chronic Lymphocytic Leukemia (CLL), Microenvironment, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Molecular characterization of Richter syndrome identifies de novo diffuse large B-cell lymphomas with poor prognosis

Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). We characterize 58 primary human RS samples by genome-wide DNA methylation and whole-transcriptome profiling. Our comprehensive approach determines RS DNA methylation profile and unravels a CLL epigenetic imprint, allowing CLL-RS clonal relationship assessment without the need of the initial CLL tumor DNA. DNA methylation- and transcriptomic-based classifiers were developed, and testing on landmark DLBCL datasets identifies a poor-prognosis, activated B-cell-like DLBCL subset in 111/1772 samples. The classification robustly identifies phenotypes very similar to RS with a specific genomic profile, accounting for 4.3-8.3% of de novo DLBCLs. In this work, RS multi-omics characterization determines oncogenic mechanisms, establishes a surrogate marker for CLL-RS clonal relationship, and provides a clinically relevant classifier for a subset of primary “RS-type DLBCL” with unfavorable prognosis.

Practice Gaps and Barriers in Optimal Care Among Healthcare Professionals Treating Patients with CLL and MCL with BTK Inhibitors: Results of a 2-Phase Qualitative/Quantitative Study

Background: Multiple BTK inhibitors (BTKis) are approved for treating patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), with further approvals expected in the near future, increasing the clinical complexity of decision-making in patient care. This study sought to quantify knowledge and practice gaps among oncology healthcare professionals (HCPs) in the use of BTKis to treat patients with CLL and MCL, with the goal of informing the design of evidence-based education interventions aimed at addressing these gaps. Methods: From November 2021 - January 2022, 488 eligible HCPs (including physicians, physician assistants, nurse practitioners, and pharmacists) participated in a 2-phase study focused on knowledge and use of BTKis for CLL and MCL. Participants were required to be active practitioners who treated patients with CLL and/or MCL. In the exploratory qualitative phase of this study, 30 US participants completed a 45-minute telephone interview focusing on factors that influence clinical reasoning in selecting and providing treatment for CLL and MCL. Interviews were transcribed, and thematic analysis was performed. The quantitative phase of this study consisted of an online survey composed of multiple-choice questions and semantic differential rating scales. Respondents' answers to each question in the quantitative survey were compared with optimal answers as identified by treatment guidelines and specialists in CLL and MCL patient care. Results: Of responding HCPs, 59% were physicians, 64% had been in practice >10 years, 73% practiced in a nonacademic setting, and 73% cared for ≤10 patients with CLL and/or MCL per week. A group of 7 core practice gaps were identified through combined analysis of data from the qualitative and quantitative surveys; these included optimal therapy selection in the first- and later-line settings for CLL and MCL, awareness of mechanisms of resistance to approved BTKi therapy, and knowledge of investigational therapies and appropriate clinical trial referral. For example, of 270 responding HCPs, 47% were unable to select preferred first-line therapy (per expert recommendations) for a patient with CLL and high tumor burden and renal insufficiency, with 9% recommending a chemoimmunotherapy regimen. Likewise, a significant percentage of surveyed HCPs lacked confidence in their approach to therapy selection for patients with relapsed/refractory CLL or MCL, with optimal sequencing identified as a primary concern. Many HCPs were unfamiliar with BTKi mechanisms of action. Gaps in knowledge were particularly evident with novel BTKis, including pirtobrutinib, with 46% of online survey respondents uncertain about the noncovalent mechanism of this agent and only 33% aware that pirtobrutinib has shown activity in patients with CLL and disease progression on ibrutinib due to the appearance of a BTK C481 mutation. In total, 60% of respondents would not consider recommending a newly approved agent if they were not familiar with the class or mechanism of action of this agent. Conclusions: Many HCPs lack confidence and knowledge in applying optimal care for patients with CLL and MCL, particularly with regard to the use of BTKis, both in first- and later-line therapy. A lack of familiarity with promising novel BTKis under clinical investigation may lead to missed opportunities to enroll eligible patients on clinical trials and potentially delay integration of these agents into clinical practice as new indications emerge. Results of this study provide evidence to support the design of clinical tools, educational programs, and performance improvement interventions. Detailed findings will be presented, including group-specific analyses and investigation of the causalities of each of the identified practice gaps.

Abstract IA25: Human genetic studies: CLL and transformation

Abstract Across cancers, histologic transformation remains a major challenge due to its association with rapid tumor progression and resistance. This is well-exemplified by Richter syndrome (RS) – the occurrence of an aggressive lymphoma of dismal prognosis in patients with chronic lymphocytic leukemia (CLL). RS is now a major obstacle to long-term CLL cure since the targeted agents developed for CLL have failed to either prevent or treat RS. In short, RS is becoming a frequent mode of progression and escape through therapeutic resistance. Thus far, definitive evolutionary studies and understanding of the genomic basis of transformation have yet to be completed. Major challenges towards this goal include the very fact that patient biopsy specimens contain an admixture of both CLL and RS tumor cells within the same tumor microenvironment, thereby limiting reliable investigations of RS cells. For CLL, large-scale sequencing studies have successfully demonstrated the diverse spectrum of recurrent gene mutations associated with CLL and the high level of genetic heterogeneity amongst patient samples, consistent with the high degree of clinical variability that is characteristic of this malignancy. We will review new studies to integrate multiple data layers in a cohort of >1000 patients, providing us with a ‘CLL map.’ Recently, we have been developing highly novel analytics that allow us to discern, within a sample that is admixed with both CLL and RS cells, the contributions of CLL vs that of RS. In doing so, we are able to identify new drivers and molecular subtypes of RS, and profile the trajectory from CLL to RS. Such studies are paradigm shifting for the clinic in relationship to both detection, diagnosis and prognosis of RS, and advances our efforts to attain a molecular definition of RS. Moreover, the methodologies, conclusions and detailed studies presented in our work are highly relevant to other hematologic malignancies and solid tumors. Citation Format: Catherine J Wu. Human genetic studies: CLL and transformation [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr IA25.

P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image:Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.

ROR-1 Expression in the Diagnosis and Monitoring of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Background. In view of similar morphological and phenotypic characteristics of some B-cell lymphoproliferative diseases and despite the known phenotype of tumor cells, a search is currently underway for new diagnostic markers, the expression of which remains stable during chronic lymphocytic leukemia (CLL) treatment and can be used for both diagnosis and assessment of residual tumor population. One of such markers is ROR-1. Aim. To assess the expression and feasibility of the ROR-1 marker using В-lymphocytes in minimal residual disease (MRD) dynamics and monitoring in CLL. Materials & Methods. Hematological and immunophenotypic analyses were performed in 110 CLL patients (50 of them with newly diagnosed disease and 60 on therapy). In addition to that, 20 patients with reactive lymphocytosis and 32 donors were examined. The ROR-1 expression in В-lym-phocytes were measured with FACS Canto II flow cytometer using the following monoclonal antibody panel: CD45, CD19, CD20, and ROR-1. Results. The analysis showed that ROR-1 is essentially not expressed in normal and reactive В-lymphocytes and is detected in 100 % of CLL tumor cells both at disease onset and on therapy. The ROR-1 expression does not change during CLL treatment and can be used not only for CLL diagnosis but also for detection of MRD. Bone marrow aspirates (п = 64) and peripheral blood samples (п = 6) were analysed for MRD assessment by two methods: according to the standardized protocol, recommended by ERIC (European Research Initiative on CLL) in 2007, with FACS Canto II flow cytometer (BD Biosciences) and using DuraClone RE CLB Tube (Beckman Coulter) with Navious flow cytometer (Beckman Coulter).

BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies.Methods: Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h).Results: T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME.Conclusion: Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. [Display omitted] DisclosuresLunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

Effects of the Gamma Secretase Inhibitors (GSI) Dapt (GSI-IX) and LSN424354 on Gene Expression and Shedding of Bcma from Multiple Myeloma and Chronic Lymphocytic Leukemia Tumor Cells

Introduction: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have high levels of solubilized (s) BCMA in the blood, and this protein is responsible for the immune deficiency of MM. sBCMA may also interfere with the efficacy of anti-BCMA-CAR-transduced T cells or other anti-BCMA antibody-based immunotherapy for multiple myeloma patients. Recently, γ-secretase has been identified as the enzyme that leads to the shedding of BCMA from B-cells. Gamma secretase (GS) is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 acts as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 among MM pts with progressive disease (PD) and complete remission (CR). We also determined the effects of the GS inhibitors (i) DAPT (GSI-IX) and LSN424354 (Eli Lilly, Indianapolis, IN) on solubilized BCMA levels from cultured human MM and CLL tumor cells.Study design: Bone marrow (BM) mononuclear cells (MCs) and serum were collected from monoclonal gammopathy of undetermined significance (MGUS) individuals, MM patients and healthy subjects after obtaining IRB approval. BMMCs from MM patients were treated with or without DAPT (GSI-IX) in a concentration dependent fashion for 24 hours. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY) following the manufacturer's instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the left superficial gluteal muscle of SCID mice. Cells were cultured and treated with the LSN424354 in a concentration dependent fashion. Supernatant BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay.Results and Discussion: qPCR showed PEN-2 gene expression was only slightly increased among MM patients with progressive disease (PD) compared to those in complete response or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared with those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we examined the effect of the DAPT (GSI-IX) on primary human MM tumor cells. Supernatants from primary human MM cells cultured for 24 hours in DAPT showed markedly reduced BCMA in a concentration dependent fashion (range,0.2 nM-2µM). Using qPCR, DAPT markedly reduced PEN-2 gene expression in a concentration dependent fashion. On the other hand, this GSI did not effect BCMA gene expression. DAPT reduces shedding of BCMA from B-cells but not its gene expression. We also determined BCMA levels in supernatants from cultured tumor cells derived from the human MM LAGκ-1A xenograft following 5 days of tissue culture with LSN424354 at concentrations ranging from 100 pM to 10 nM. Supernatant sBCMA levels were reduced > 90% at the highest concentrations (10nM). Similarly, freshly obtained CLL tumor cells cultured with LSN424354 at concentrations ranging from 100 pM to 10 nM also showed a marked reduction in supernatant BCMA levels at the higher concentrations.Conclusion: Gamma secretase sheds BCMA off B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression among MM pts with PD compared with those in CR or MGUS individuals. The GSIs DAPT and LSN424354 reduce shedding of BCMA from malignant cells from MM and CLL patients. Since GSIs reduce solubilized BCMA levels, it is possible that treatment with these agents can reverse the immune dysfunction caused by this solubilized protein, and also improve the efficacy of anti-BCMA immunotherapies for treating patients with multiple myeloma and CLL. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Evidence for Non-Cancer-Specific T Cell Exhaustion in the Tcl1 Mouse Model for Chronic Lymphocytic Leukemia.

The reinvigoration of anti-cancer immunity by immune checkpoint therapies has greatly improved cancer treatment. In chronic lymphocytic leukemia (CLL), patients as well as in the Tcl1 mouse model for CLL, PD1-expressing, exhausted T cells significantly expand alongside CLL development; nevertheless, PD1 inhibition has no clinical benefit. Hence, exhausted T cells are either not activatable by simple PD1 blocking in CLL and/or only an insufficient number of exhausted T cells are CLL-specific. In this study, we examined the latter hypothesis by exploiting the Tcl1 transgenic CLL mouse model in combination with TCR transgene expression specific for a non-cancer antigen. Following CLL tumor development, increased PD1 levels were detected on non-CLL specific T cells that seem dependent on the presence of (tumor-) antigen-specific T cells. Transcriptome analysis confirmed a similar exhaustion phenotype of non-CLL specific and endogenous PD1pos T cells. Our results indicate that in the CLL mouse model, a substantial fraction of non-CLL specific T cells becomes exhausted during disease progression in a bystander effect. These findings have important implications for the general efficacy assessment of immune checkpoint therapies in CLL.

Derived from a Platform of Innovative Therapeutic Molecular Clusters (TMC), Ag5 Is a Highly Potent and Differentiated Molecule That Promises to be Efficacious in Hard-to-Treat Hematological Cancers

[Background on medical need in hematology]Even though significant progress has been made in a number of hematological malignancies in recent years, an unmet medical need still remains in indications such as multiple myeloma (MM), chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML) due to refractory disease, severe adverse events or the failure to achieve sustained minimal residual disease. As a consequence, novel therapies are required to prevent relapse, be safer in administration and to target residual disease. We present here for the first time a novel and innovative therapeutic approach with the potential to treat patients suffering from hard-to-treat hematological malignancies based on the tumor´s pathophysiology and reactive oxygen species (ROS) phenotype. [Background on Ag5]TMC are created by combining the atoms of certain transition metals under specific conditions to form novel molecules with entirely distinct properties from "traditional" metal. For example, silver can be used to create clusters of different and defined sizes, based on the number of atoms making up the final drug candidate. More specifically, Ag5 contains five silver atoms arranged in a specific conformation, is the first TMC derived from this novel platform and has entirely different physiological properties than the three silver atom containing Ag3. Ag5 is a water-soluble and heat stable molecule, it is orally bioavailable and a freely diffusible pan-tumor therapeutic. It selectively kills those cells with high ROS concentrations by oxidizing their antioxidant systems and subsequently drives these cells to programmed cell death. In consequence, Ag5 will preferentially kill cancer cells which typically have higher ROS levels, but will spare normal cells which display lower ROS due to their functional REDOX homeostasis. More specifically, Ag5 efficiently catalyzes the oxidation of thiol groups of thioredoxins and peroxiredoxins and thereby drives sensitive cells above a threshold to irreversible protein misfolding, protein degradation and programmed cell death. [Background on Ag5 experiments]We characterized Ag5 efficacy using a wide range ofin vitroassays and found potent Ag5 efficacy against a number of MM, CLL and AML cell lines with an IC50 in the low nM range. Ag5 sensitivity of all cell lines was correlated with ROS levels, more specifically superoxide, as measured by dihydroethidium (DHE) or MitoSOX. Furthermore, we were able to demonstrate that Ag5 treatment resulted in a concentration dependent cell cycle arrest in G1 phase, mitochondrial swelling and induction of apoptosis. Treatment of primary CLL tumor samples resulted in low nM efficacy. Finally, we could demonstrate that Ag5 was not only safely administered without any side effects in mouse and rat studies, but was equally effective as the stand-of-care bortezomib in a multiple myelomain vivomodel. [Conclusion and clinical significance]In summary, Ag5 is a novel and innovative therapeutic candidate that was shown to be safe and effective in preclinical studies, and has the promise to address the unmet medical need in hard-to-treat hematological malignancies. Keywords: Ag5, Therapeutic molecular cluster (TMC), Redox, ROS, catalysis, Ag3, AML, MM, CLL Disclosures Porto González: Arjuna:Research Funding.Carneiro:Arjuna:Research Funding.Lopez-Quintela:Arjuna:Current equity holder in private company.Treder:Arjuna:Current Employment.Dominguez:Arjuna:Current equity holder in private company.

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

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

Inferring clonal heterogeneity in cancer using SNP arrays and whole genome sequencing.

MOTIVATION Clonal heterogeneity is common in many types of cancer, including chronic lymphocytic leukemia (CLL). Previous research suggests that the presence of multiple distinct cancer clones is associated with clinical outcome. Detection of clonal heterogeneity from high throughput data, such as sequencing or single nucleotide polymorphism (SNP) array data, is important for gaining a better understanding of cancer and may improve prediction of clinical outcome or response to treatment. Here, we present a new method, CloneSeeker, for inferring clinical heterogeneity from sequencing data, SNP array data, or both. RESULTS We generated simulated SNP array and sequencing data and applied CloneSeeker along with two other methods. We demonstrate that CloneSeeker is more accurate than existing algorithms at determining the number of clones, distribution of cancer cells among clones, and mutation and/or copy numbers belonging to each clone. Next, we applied CloneSeeker to SNP array data from samples of 258 previously untreated CLL patients to gain a better understanding of the characteristics of CLL tumors and to elucidate the relationship between clonal heterogeneity and clinical outcome. We found that a significant majority of CLL patients appear to have multiple clones distinguished by copy number alterations alone. We also found that the presence of multiple clones corresponded with significantly worse survival among CLL patients. These findings may prove useful for improving the accuracy of prognosis and design of treatment strategies. AVAILABILITY AND IMPLEMENTATION Code available on R-Forge: https://r-forge.r-project.org/projects/CloneSeeker/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

PB1876 GENETIC MARKERS OF THE PRIMARY AND SECOND TUMORS RISK DEVELOPMENT IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Malignant tumors in patients with chronic lymphocytic leukemia (CLL) occur with equal frequency in both previously untreated patients and those receiving high‐dose therapy. This is due not only to the presence of CLL‐associated, but with drug‐induced immunodeficiency, and, probably, with modifications in the immune response genes.Aims:Identify the relationship of the several immune response genes mutational status with the development of primary and second tumors in patients with CLL.Methods:Surveyed 202 patients in the beginning of CLL at the age of 32 to 87 years (median‐63 years), 112 men (55.4%), 90 women (44.6%). Genotyping of 20 polymorphic regions of the 14 immune response genes (TLR2, TLR3, TLR4, TLR6, TLR9, IL1β, IL2, IL4, IL6, IL10, IL17A, CD14, TNFα, FCGR2A) in genomic DNA obtained from peripheral blood leukocytes was carried out by the PCR‐ASP. Intergenic interactions analysis was performed using the MDR program version 3.0.2, http://www.mybiosoftware.com/mdr‐2‐0‐multifactor‐dimensionality‐reduction.html.Results:Patients were divided into 3 groups. The first group consisted of 6 patients who had other malignancies before onset CLL. The appearance of CLL was preceded in 2 cases by the breast cancer and one: cancer of the testicle, ovary, skin and fungal mycosis from 36 to 252 months (median‐135.5 months). The second ‐ consisted of 190 CLL patients with no previous or second malignant tumors history. The third included 6 patients, who developed the second solid tumors in CLL remission after FCR treatment. Within 13‐94 months (median‐53.5 months) from the onset of CLL, they had skin cancer in 2 cases, colon cancer in 2 patients, melanoma and breast cancer in one case. In all selected groups, age and gender differences were not found. All possible combinations of the key immune response genes polymorphic variants were analyzed. It was found that the first group of patients is characterized by 2 statistically significant models: single‐ FCGR2A (A519C) (p = 0.0001) and two‐locus TLR4 (G896A)/FCGR2A (A519C) (p &lt; 0.0001). Patients who had a second tumors after onset CLL also distinguished two models: single‐locus with the participation of the IL17A gene (G197A), p = 0.00006 and three‐locus model TLR6 (C745T)/TLR9 (A2848G)/IL1β (T511C) (p &lt; 0.0001). It is very likely that the triggering factor for the second malignant tumors occurrence in these patients was high‐dose chemotherapy of CLL, which aggravated the existing CLL‐mediated immunodeficiency. As a result of the intergenic interactions evaluation, it was noted that in patients with a previous CLL cancer history, the risk factors for the development of primary tumors were 3 combined genotypes: FCGR2A‐519AA/TLR4‐896GG; FCGR2A‐519AA/TLR9‐2848AA/TLR4‐896GG; FCGR2A‐519AA/TLR9‐2848AG/TLR4‐896AG. For patients with CLL and second tumors detected in remission of leukemia, the most prognostically important were the combinations of genotypes: IL1β‐511CC/TLR9‐2848AA/TLR6‐745TT; IL1β‐511CC/TLR9‐2848AG/TLR6‐745TT; IL1β‐511TT/TLR9‐2848AG/TLR6‐745TT.Summary/Conclusion:Studies have confirmed the involvement of immune mechanisms in the carcinogenesis. Analysis of the polymorphic status genes FCGR2A, TLR6, TLR9 (A2848G), IL1β (T511C), IL17A, which are involved in the development and progression of various human tumors, will allow to select among patients with CLL a group with an increased risk of the development of previous and second malignant neoplasms.

S897 VALIDATION OF A NOVEL MECHANISM OF ACTION FOR TREATMENT OF CCR7+ HEMATOLOGICAL MALIGNANCIES WITH FIRST‐IN‐CLASS ANTIBODY CAP‐100

Background:The homeostatic CC‐chemokine receptor 7 (CCR7) controls trafficking of immune cells to the lymph node (LN). Accordingly, CCR7 is highly expressed in hematological malignancies with nodal involvement including chronic lymphocytic leukemia (CLL), several B‐cell non‐Hodgkin lymphomas (NHL) such as mantle cell lymphoma (MCL), and various mature T‐cell neoplasia like T‐cell prolymphocytic leukemia (T‐PLL) and Sézary syndrome (SS). Upon engagement by its ligands, the chemokines CCL19 and CCL21, CCR7 directs tumor cells to LN. In this protective microenvironment, CCR7 contributes to tumor cell survival and proliferation. Indeed, both high CCR7 surface expression levels and high CCR7‐triggered migratory responses correlate with lymphadenopathy, adverse prognostic factors, and shorter patient survival. Therefore, strategies targeting CCR7 could provide a novel therapeutic approach for CCR7+ hematological malignancies.Aims:In this study we aimed to validate CCR7 as a novel target for antibody‐based therapies in CCR7‐expressing blood cancers.Methods:We have generated CAP‐100, the first humanized IgG1 monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand‐mediated signaling, and evaluated the antibody in various in‐vitro and in‐vivo preclinical models.Results:CAP‐100 potently inhibited in vitro migration of primary CLL, MCL, T‐PLL and SS tumor cells. Furthermore, in vivo pre‐clinical studies confirmed that CAP‐100 effectively blocked entry of CCR7‐expressing cells to LNs. CAP‐100 also abrogated survival elicited by CCR7 in CLL, and showed potent antibody‐dependent cell‐mediated cytotoxicity (ADCC) against CLL or CCR7+ T‐lymphomas cells. This Fc‐mediated cell killing activity clearly outperformed anti‐CD20 mAb rituximab and anti‐CD52 mAb alemtuzumab, standards of care in B‐cell and T‐cell lymphomas respectively. In all cases, CAP‐100 activity was independent of prognostic markers for high risk disease. Finally, when given as monotherapy in disseminated B‐NHL and CLL xenograft tumors in SCID mice, CAP‐100 inhibited tumor growth and extended survival significantly. In addition, CAP‐100 strongly inhibited the infiltration into as well as the survival of tumor cells in the LN.Summary/Conclusion:Our results demonstrate that CAP‐100, the first‐in‐class anti‐CCR7 mAb, is a potent antagonist with biological activity in several CCR7+ hematological malignancies, including relapsed/refractory disease. Moreover, these results highlight the relevance of the CCR7‐CCL19/CCL21 pathway as a therapeutic target in these diseases. CAP‐100's unique ability to block migration of tumor cells to the LN (where also inhibits tumor cell survival), in combination with its potent cell killing activity, represents a novel and differentiating mechanism of action. This provides the biological rationale for use of CAP‐100, either as monotherapy or in combination with novel agents. Clinical trials in CLL and CCR7‐expressing NHL will be initiated soon.

Leukemic B Cell CTLA-4 Suppresses Costimulation of T Cells.

The clinical benefit of CTLA-4 blockade on T cells is known, yet the impact of its expression on cancer cells remains unaddressed. We define an immunosuppressive role for tumor-expressed CTLA-4 using chronic lymphocytic leukemia (CLL) as a disease model. CLL cells, among other cancer cells, are CTLA-4+ Coculture with activated human T cells induced surface CTLA-4 on primary human CLL B cells. CTLA-4 on CLL-derived human cell lines decreased CD80 expression on cocultured CD80+ cells, with restoration upon CTLA-4 blockade. Coculture of CTLA-4+ CLL cells with CD80-GFP+ cell lines revealed transfer of CD80-GFP into CLL tumor cells, similar to CTLA-4+ T cells able to trans-endocytose CD80. Coculture of T cells with CTLA-4+ CLL cells decreased IL-2 production. Using a human CTLA-4 knock-in mouse lacking FcγR function, antitumor efficacy was observed by blocking murine CTLA-4 on tumor cells in isolation of the T cell effect and Fc-mediated depletion. These data implicate tumor CTLA-4 in cancer cell-mediated immunosuppression in vitro and as having a functional role in tumor cells in vivo.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

Inferring clonal heterogeneity in cancer using SNP arrays and whole genome sequencing.

Clonal heterogeneity is common in many types of cancer, including chronic lymphocytic leukemia (CLL). Previous research suggests that the presence of multiple distinct cancer clones is associated with clinical outcome. Detection of clonal heterogeneity from high throughput data, such as sequencing or single nucleotide polymorphism (SNP) array data, is important for gaining a better understanding of cancer and may improve prediction of clinical outcome or response to treatment. Here, we present a new method, CloneSeeker, for inferring clinical heterogeneity from sequencing data, SNP array data, or both. We generated simulated SNP array and sequencing data and applied CloneSeeker along with two other methods. We demonstrate that CloneSeeker is more accurate than existing algorithms at determining the number of clones, distribution of cancer cells among clones, and mutation and/or copy numbers belonging to each clone. Next, we applied CloneSeeker to SNP array data from samples of 258 previously untreated CLL patients to gain a better understanding of the characteristics of CLL tumors and to elucidate the relationship between clonal heterogeneity and clinical outcome. We found that a significant majority of CLL patients appear to have multiple clones distinguished by copy number alterations alone. We also found that the presence of multiple clones corresponded with significantly worse survival among CLL patients. These findings may prove useful for improving the accuracy of prognosis and design of treatment strategies. Code available on R-Forge: https://r-forge.r-project.org/projects/CloneSeeker/. Supplementary data are available at Bioinformatics online.

Combination Therapy with BTK Inhibitor Plus Anti-PD-1 Antibody Results in a Hyperprogressor Phenotype in a Mouse Model of CLL

Chronic lymphocytic leukemia (CLL) is often driven by aberrant activation of the B-cell receptor signaling pathway. We and others have shown in preclinical animal models as well as human clinical trials that the BTK inhibitor, ibrutinib, is effective in reducing CLL tumor burden and improving survival rates in both humans and mice. Preclinical studies also suggest that BTK inhibitors likely act not only through their direct effect on CLL tumor cells but also by alleviating immunosuppression in the tumor microenvironment through BTK inhibition in myeloid-derived suppressor cells. Additionally, we have previously demonstrated that CLL cells are susceptible to cytotoxic killing by T cells targeting the aberrantly expressed TCL1 oncoprotein (Weng et al. Blood 2012). Therefore, we hypothesized that the combination of BTK-pathway inhibition in conjunction with activation of antigen-specific T-cells by immune checkpoint blockade will be a synergistic therapeutic strategy. Here, we examined the effect of acalabrutinib (previously known as ACP-196), a selective BTK inhibitor with limited effect on other kinases, alone or in combination with immune checkpoint blockade in a mouse model of CLL.Eµ-TCL1 mice, which overexpress TCL1 in B-cells, were used as a model of CLL. We generated cohorts of Eµ-TCL1 mice and treated them with anti-PD-1 antibody (n = 18), acalabrutinib (n = 20), and acalabrutinib+anti-PD-1 antibody (n= 19). Treatment cohorts had a significant reduction in CD5+CD19+ tumor cells in peripheral blood and spleens as compared to vehicle-treated mice (n =16) (p <0.05). However, the most pronounced anti-tumor effects were observed in mice treated with acalabrutinib alone. In fact, MRI imaging of splenic volume and flow cytometry of CD5+CD19+ cells during the treatment phase revealed that acalabrutinib monotherapy was superior to the combinatorial therapy. Even more surprising was the fact that while anti-PD-1 and acalabrutinib monotherapies provided an improvement in survival compared to vehicle treatment (p = 0.05 and p < 0.0001, respectively), the combination of these two agents actually diminished overall survival (p = 0.77). Shockingly, histopathological analyses of tumors from these acalabrutinib+anti-PD-1 treated mice revealed a significant increase in lymph node involvement and tumors with a high mitotic index. Consistent with this, we observed that treatment of CD5+CD19+ tumor cells from Eµ-TCL1 transgenic mice in vitro with anti-PD-1 antibody plus acalabrutinib markedly increased the proliferative index as measured by EdU incorporation assay compared to acalabrutinib treated group.To understand the molecular events responsible for the observed acalabrutinib+anti-PD-1 hyperproliferative phenotypes, we are examining complementary pathways that may be aberrantly activated/repressed by this combination. Currently, we have identified expression changes in both the NFkb and PKC signaling pathways when these agents are used in combination, and are working to understand how this combination may impact tumor progression. We are also assessing the possibility that anti-PD-1 interferes with the anti-tumorigenic effect of acalabrutinib by enhancing the BCR signaling pathway in CLL tumor cells.In summary, our results demonstrate that the selective BTK inhibitor, acalabrutinib greatly enhances the survival of Eµ-TCL1 mice compared to either vehicle or ibrutinib alone. Surprisingly, we found that the combination of anti-PD-1 antibody + acalabrutinib therapy is actually detrimental in this CLL model and results in a hyperprogressor phenotype. Therefore, results from ongoing clinical trials evaluating combination strategies of anti-PD-1 antibody therapy plus BTK inhibitor need to be analyzed carefully to ensure that this combination is not leading to a similar hyperprogressor phenotype in patients. The results provided here offer some initial insights into the potential mechanisms of the “hyperprogressive” phenotypes following anti-PD-1 treatment and may highlight pathways that could be useful in blocking these deleterious effects. DisclosuresNo relevant conflicts of interest to declare.

Ibrutinib-Based Therapy Improves Anti-Tumor T Cell Killing Function Allowing Effective Pairing with Anti-PD-L1 Immunotherapy Compared to Traditional FCR Chemoimmunotherapy; Implications for Therapy and Correlative Immune Functional Data from the Phase III E1912 Trial

The targeted therapy ibrutinib inhibits B cell receptor signaling (BTK inhibitor) and has yielded high response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL). However, it is widely believed that the addition of immune therapies to targeted drugs will be required to activate anti-tumor immunity and work towards curative therapy. Identifying effective combinations of targeted drugs and/or standard chemotherapy with immunotherapy is a priority research area and particularly relevant for CLL, as patients' T cells have been shown to exhibit profound tolerance/exhaustion and notably, no activity was reported in a recent trial of anti-PD-1 immunotherapy for relapsed disease.Ibrutinib has shown beneficial immunomodulatory activity in CLL by inhibiting IL-2-inducible T cell kinase (ITK) as well BTK that is associated with increased effector CD4+and CD8+ T cell numbers and decreased expression of inhibitory checkpoint receptors such as PD-1 on patient T cells. Here we have performed comparative immune bioassays from a randomized phase III trial comparing ibrutinib-based therapy to traditional FCR chemoimmunotherapy to assess the effects of treatments on anti-tumor T cell function.Viable peripheral blood mononuclear cell samples were collected serially (baseline, 6 months and 12 months) from CLL patients on the randomized phase III E1912 trial of ibrutinib and rituximab versus FCR for previously untreated disease to allow longitudinal batched immune analysis.Cytotoxicity assays revealed that highly purified CD3+ T cells from the FCR treated patients at 6 and 12-month time-points did not change their activated killing function against autologous baseline CD19+ CLL tumor B cells (acting as target antigen-presenting cells pulsed with superantigen, sAg) compared to pre-treatment/baseline exhausted T cells (n=22). In contrast, patients treated with ibrutinib-based therapy had a significant increase in activated anti-tumor T cell killing function (P<.01, n=22) at both 6-month (66% increase) and 12-month (89% increase) time-points.Flow cytometric analysis of circulating immune subsets revealed that the percentage of PD-1 and PD-L1 positive cells among CD8+ and CD4+ T cells (particularly effector compartments) were reduced with ibrutinib-based therapy, whereas only a partial reduction was detected following FCR treatment. However, patients' T cells from both treatment arms responded normally to T cell receptor engagement by upregulating these checkpoint molecules. This led us to explore ex vivo treatment of highly purified CD3+ T cells and CD19+ CLL B cells from both treatment arms with anti-PD-L1 or anti-PD-1 immunotherapy prior to cytotoxicity assays. Our functional data revealed that the T cells from both FCR time-points (6 and 12-months) were not sensitive to either anti-PD-L1 (n=14) or anti-PD-1 (n=14) treatment. In contrast, ibrutinib-based treatment sensitized anti-tumor T function (23% increase in killing) following anti-PD-L1 treatment (n=14) at the 6-month time-point only (P<.01) but not with anti-PD-1.To investigate the mechanism underlying these effector function differences, we compared the ability of highly purified CD3+ T cells from each treatment arm (n=45) time-point to form F-actin immunological synapses with baseline autologous CLL tumor B cells. Quantitative confocal image analysis revealed that ibrutinib-based therapy significantly (P<.01) enhanced polarization of F-actin, tyrosine-phosphorylated proteins and granzyme B at immune synapses with tumor cells at both 6 and 12-month time-points, whereas FCR treated patient T cells failed to mobilize these lytic synapse molecules. Importantly, our assays have revealed that T cells from both FCR treatment time-points formed “non-polarized” immune synapses with tumor cells, in keeping with cytotoxic dysfunction and insensitivity to additional checkpoint immunotherapy. In contrast, our functional correlative bioassays have revealed that ibrutinib-based therapy can reactivate exhausted cytolytic T cell function and suggest to us, a potential therapeutic window for anti-PD-L1 immunotherapy at the earlier 6-month time-point.We believe this data supports the concept of incorporating functional bioassays to immune-monitoring assays associated to clinical trials that should aid knowledge-led design of future combination immunotherapy. DisclosuresKay: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; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Infinity 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; 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; 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. Shanafelt:Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding; MedImmune: Research Funding.