Sinha, Sutapa

ABSTRACT Richter transformation of Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) into classic Hodgkin lymphoma (CHL-RT) is rare and remains incompletely understood. Two histologic subtypes are recognized: type 1 (CLL/SLL with scattered Hodgkin/Reed-Sternberg (HRS) cells) and type 2 (HRS cells within a polymorphous inflammatory background). In this multi-institutional study of 77 patients with CHL-RT (27 type 1 and 50 type 2), we characterized immune evasion markers, PD-L1 / PD-L2 copy number alterations, tumor microenvironment, and performed targeted next-generation sequencing on 37 CLL/SLL samples. HRS cells in CHL-RT displayed immune-evasion phenotypes similar to de novo CHL, though PD-L1 expression was lower in type 1 cases. PD-L1/PD-L2 gain/polysomy were frequent (83.3%). CLL/SLL with CHL-RT harbored increased mutations in XPO1, FBXW7, BIRC3, TRAF3 , and HLA-A versus reference CLL/SLL. Similar mutational profiles, demographics, and survival outcomes support a biological continuum between type 1 and type 2 CHL-RT, with distinct genetic features in CLL/SLL predisposing to CHL transformation. STATEMENT OF SIGNIFICANCE Classic Hodgkin lymphoma-type Richter transformation arises from convergent Hodgkin-like immune evasion on a genetically primed CLL/SLL background. Through integrated genomic and tumor microenvironment analyses in a large multicenter cohort, we establish this as a biologically coherent entity and provide rationale for PD-L1/PD-L2-directed therapeutic strategies.

Abstract Bruton tyrosine kinase (BTK) is central to chronic lymphocytic leukemia (CLL) biology, yet the regulatory programs that depend on BTK remain poorly defined. Here, we used acute NX-5948 (Bexobrutideg)-mediated BTK degradation to define BTK-dependent regulatory circuitry in CLL. Integrating early transcriptional and chromatin responses with chromatin-state annotation, target-gene linkage, and transcription factor assignment, we resolved a chromatin-informed framework linking BTK loss to downstream gene-regulatory output. This framework is separated into two directional programs: a dominant BTK-maintained network that collapsed after BTK degradation and a smaller BTK-released network that emerged upon BTK loss. BTK-responsive elements were concentrated in promoter- and enhancer-linked chromatin compartments, consistent with a model in which BTK-dependent transcriptional output is mediated through specific active regulatory elements. Functional validation in a CLL-like cell model provided orthogonal support for the NFAT-linked component of the BTK-maintained regulatory circuitry. BTK-dependent regulon activity was associated with clinical outcome, and projection of bulk-derived programs into longitudinal single-cell RNA-seq resolved malignant CLL cells into distinct regulatory states with maintained-like and released-like features. Together, this work defines BTK-dependent regulatory programs in CLL through a chromatin-informed framework linking BTK activity to gene-regulatory output. These findings lay the foundation for understanding how BTK-dependent circuitry contributes to malignant cell-state diversity, clinical outcome, and remodeling during therapy.

Abstract Introduction: A subset of patients with chronic lymphocytic leukemia (CLL) experiences disease progression from CLL to Richter transformation (RT), characterized by the occurrence of a high-grade lymphoma. The outcomes of patients with RT are typically dismal and identifying better therapeutic strategies remains critically important for these patients. To discern the biologic underpinnings driving this transformation, we studied immune changes and genetic evolution in samples from CLL patients who developed RT. Methods: We identified 18 patients with CLL who developed RT; and performed single cell (sc) RNA-sequencing(seq) on sorted B and T cells from peripheral blood mononuclear cells (PBMCs) at the time of RT (n=18) and at the earlier time of CLL diagnosis (n=11). The median time between the CLL sample and the one closest to RT was 4.3 years (range, 0.2-7.3); and patients received a median of 1 CLL directed treatment (range, 0 – 7). A total of 749,906 cells and 36,601 genes were captured during scRNAseq and 720,580 cells and 21,892 genes were retained for downstream analysis, respectively. Analysis was performed using the immunopipe (https://github.com/pwwang/immunopipe) that wraps around Seurat (v5.1.0) and identified enriched pathways using Enrichr service. The sc-T cell receptor (TCR)-repertoire(R)-seq data was analyzed and visualized using scplotter (). The whole exome sequencing (WES) analysis with tumor B-cells from same patients of CLL & RT stages were done using the Agilent sureselect Human all exon v5 + UTR capture kit. Reads from paired end sequencing were processed with the sarek pipeline. Results: Analysis of 720,580 cells from 11 CLL and 18 RT samples showed 16 distinct cell types- 3 B and 13 T cell subtypes. We observed an increasing trend of CD8 TEM [mean 0.548 vs. 0.384, p = 0.12 (Wilcoxon test)] but a decreasing trend of CD4 TCM (mean 0.236 vs. 0.337, p = 0.08) and B-memory (mean 0.0085 vs. 0.0195, p = 0.51) subsets in RT compared to their CLL stage. When comparing transcriptional changes, we found that among the top 18 highly variable genes, the expression of FP236383.3 (lncRNA) and TCL1A was significantly higher (p<0.0001) in the B cell subsets of patients with RT vs. CLL stage. Additionally, in T cell subsets—primarily dnT, γδT, and CD8/CD4 naïve cells—the expression of FP236383.3, NKG7, GNLY, and CCL5 was significantly higher (p<0.0001), while the expression of LDHB, GZMK, and IL32 was significantly lower (p<0.0001) in RT vs. CLL stage samples. Comparison of specific gene set enrichment analysis showed significantly increased expression of GZMB in gdT (p<0.001) and decreased expression of GZMA, GZMK, TIGIT and TCF7 in dnT and CD8 Naïve cells (p<0.001) for RT vs. CLL stage. The top 10 differentially expressed pathways in RT included oxidative phosphorylation, allograft rejection, IFN-α/-γ responses, TNF-α signaling via NF-κβ, complement, Myc targets V1, mTORC1, hypoxia, and p53 signaling across various B and T cell subsets compared to the CLL stage. Initial analysis of TCR-R data showed that patients with RT stage have lower number of unique clones (p<0.5) and reduced clonal diversity (p<0.5), and a higher number of large and hyperexpanded clones compared to their CLL stage. Additionally, WES data indicated that the most frequently mutated gene is TP53 in tumor B cells of both CLL and RT stages. However, a higher percentage of RT-stage samples carry mutations in MGA, ATM, and IRF2BP2 genes compared to their CLL-stage. WES data also showed a trend toward differing frequencies of copy number amplification at 19q12 (p=0.10) and 4q13.1 (p=0.52) between RT and CLL stages. Conclusions: The main findings of our study are as follows:The transcriptional landscape showed significant changes in multiple genes and pathway enrichment related to cell growth, differentiation and immune response in RT vs. CLL stage.A decreased fraction of CD4 TCM cells and reduced TCR clonal diversity were observed in samples at the RT stage and may indicate a poor prognosis for the patients at this stage. WES data of tumor B cells also indicated possible genetic alterations in the RT vs. CLL stage. Data from in-depth analyses on understanding of the dynamics of T cell response and additional experiments on these patients are expected to provide valuable insights into tumor genetic heterogeneity, transcriptional and immune regulation in blood-based immune subsets.

Abstract Despite the advent of novel targeted therapies with high overall response rates, chronic lymphocytic leukemia (CLL) is still largely incurable due to the development of resistance. The bone marrow microenvironment (BMME) plays an important role in the survival and drug resistance in CLL. BM stromal cells (BMSCs) are considered a key component of BMME. Our long-term goal is to gain further insights of BMSCs-CLL crosstalk influencing both survival and drug resistance in CLL. To understand the global biologic changes that occur in leukemic CLL cells when in contact with BMSCs, we assessed RNA-seq in paired CLL cells isolated from untreated blood and BM and in untreated patients’ CLL cells cultured alone or with BMSCs. CLL cells from blood/BM and cultured alone/with BMSCs were also examined by Immunoprecipitation (IP), Western blot (WB), untargeted metabolomics (LCMS+GCMS), and preclinical drug sensitivity assays. We detected only 13 upregulated genes that overlap between CLL cells from BM and cocultured with BMSCs (p&lt0.05, fold change &gt1.5). When we analyzed the expression of these 13 genes in CLL cells from a cohort of 162 untreated patients by RNA-seq, we found associations between 4 genes (PNP, C16orf54, MOB3A, CDK2AP2) and clinical outcomes including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine pathway, had the most significant association with OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine pathway metabolites adenosine, adenine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis showed variable baseline PNP protein expression with 2 cohorts: low /no (L) and high (H) PNP using CLL blood cells. An induction in PNP protein levels were noticed in CLL cells expressing only LPNP when cocultured. Given the association of HPNP with adverse clinical outcomes, we tested for L vs. HPNP cohorts drug sensitivity to known BTK inhibitors(is) or Bcl2is. Treatment of CLL cells from untreated patients with Bcl2is venetoclax, S55746, and LP-118 (Bcl2-Bclxli, Newave) showed increased sensitivity to the Bcl2/Bclxlis in HPNP but not in LPNP cells. In addition, BTKis showed no difference in drug sensitivity associated with CLL PNP levels. To explain this discrepancy, WB and IP analyses were conducted in L vs. HPNP leukemic cells for apoptotic profiles, where we found increased phosphorylated-Bcl2(S70) and an increased association of Bcl2 with Bax in LPNP-expressing CLL cells compared to HPNP. These findings support increased resistance to apoptosis afforded by inhibiting Bcl2 for LPNP vs. HPNP-expressing CLL cells. Our future goals are to continue to understand the prognostic significance of the 4 genes with adverse clinical outcomes and to investigate how best to exploit the peculiar, enhanced sensitivity to Bcl2/Bclxlis found in the HPNP CLL cells. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, Brianna M. Finke, Heather C. Darby, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Yi Chen, Fenlai Tan, Stephen P. Anthony, Yu Chen, Bing Dai, Yue Shen, Neil E. Kay. CLL drug resistance- A multifaceted mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4423.

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia among adults in the US. Despite significant improvement of current novel agent treatments, CLL remains incurable with many eventually experiencing disease relapse. Therefore, it remains of high interest to expand therapeutic options. Transcriptional chemical inducers of proximity (TCIPs) are a new class of anticancer drugs designed and developed by Stanford teams. These agents recruit cancer drivers or transcription factors to activate proapoptotic genes' expression and kill cancer cells. Current TCIPs are produced by covalently linking small molecules that bind B-cell lymphoma 6 (BCL6) to those that bind to transcriptional activators, such as bromodomain-containing protein 4 (BRD4) (BRD4-BCL6, TCIP1) or cyclin-dependent kinases 9 (CDK9) (CDK9-BCL6, TCIP2). In collaboration with the Stanford teams, we have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients and found heterogeneous but evident drug responsiveness with a panel of TCIPs. Our current work has shown that TCIP2 demonstrated remarkable killing ability of CLL B cells with IC50 of 9nM in 72-hours (h), while the IC50 of TCIP1 was 560nM. TCIPs showed time and dose-dependent drug-associated killing in primary CLL B cells over a 24-72h interval using a flow cytometry-based apoptosis assay. After 24, 48, and 72h exposure of CLL B cells to TCIPs, the IC50 was 1054, 820 and 555nM for TCIP1, and 9, 8, and 9nM for TCIP2, respectively. TCIP2 is designed and produced by covalently linking BI-3812 (BCL6 inhibitor) as a BCL6 binder and SNS-032 (CDK9 inhibitor) as a CDK9 binder with a rigid linker. Comparing TCIP2 to its negative controls (TCIP compounds with only one active binder, either BCL6 or CDK9), TCIP2 exhibited significant cytotoxicity in primary CLL B cells. TCIP2 also showed a significant increase in potency, compared to SNS-032 and BI-3812 alone at 72h. Normal human peripheral blood mononuclear cells (PBMCs) were 6-fold less sensitive to TCIP2 compared to CLL PBMCs with high percentage of CLL B cells. Immunoblotting data showed induction of BCL6 protein level after 20nM TCIP1 treatment in CLL B cells. In samples from relapsed/refractory (RR) CLL patients, TCIPs maintained good cytotoxicity with IC50 of 1805nM for TCIP1 and 84nM for TCIP2 in 24h using a cell viability assay. Our study indicates that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in primary CLL B cells. This notable killing effect may be due to its unique gain-of-function drug mechanism where BCL6 is induced. This is the first study of TCIPs ability to target CLL B cells from both untreated and RR patients. Further studies to more fully evaluate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the potential role of TCIPs as a novel therapeutic approach in CLL. Citation Format: Weiguo Han, Aisharya Bhattacharya, Basel Karim, Wenzhi Ji, Roman C. Sarott, Meredith Nix, Sai Gourisankar, Andrey Krokhotin, Sutapa Sinha, Zhiquan Wang, Tait D. Shanafelt, Sameer A. Parikh, Nathanael S. Gray, Gerald R. Crabtree, Neil E. Kay. Rewiring cancer drivers to induce apoptosis in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5633.

Chronic lymphocytic leukemia (CLL), one of the most common types of leukemia among adults in the US, is characterized by the accumulation of CD5+CD19+ monoclonal B cells. Current clinical treatment approaches for progressive CLL patients focus on novel agents targeting Bruton Tyrosine Kinase (BTK) and Bcl-2. Although clinical trials combining novel agent have shown significant efficacy with high overall response and, in some cases, achievement of high rates of undetectable measurable residual disease (MRD), CLL remains incurable with a majority of patients eventually experiencing relapse of their disease. Therefore, it remains of high interest to expand therapeutic options. Transcriptional chemical inducers of proximity (TCIPs) are small molecules designed and developed by Dr. Gerald R. Crabtree at Stanford University. TCIPs are a new class of anticancer drugs that recruit endogenous cancer drivers or a downstream transcription factor to the promoters of cell death genes and activate their expression, resulting in apoptosis of cancer cells. Current TCIPs are produced by covalently linking small molecules that bind B-cell lymphoma 6 (BCL6) to those that bind to transcriptional activators, such as Bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinases 9 (CDK9). The Stanford team has demonstrated TCIPs generate remarkable killing in diffuse large B cell lymphoma (DLBCL) cell lines, including chemotherapy-resistant, TP53-mutant lines, with a half maximal effective concentration (EC50) of 1-10nM at 72hours (h) and exhibited cell-specific and tissue-specific effects (Gourisankar et al, 2023). We have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients. Testing two types of TCIPs with a flow cytometry-based apoptosis assay, we found time and dose-dependent drug-associated killing in primary CLL B cells collected from untreated CLL patients over a 24-72h interval. The IC50 for BRD4-BCL6 TCIP JWZ-7-133 at 72h was 560nM while CDK9-BCL6 TCIP BAK-04-232 at 72h was 10nM. After 24, 48, and 72h exposure of CLL B cells to TCIP JWZ-7-133, the IC50 was 1050, 820 and 560nM, respectively. We also found that TCIP JWZ-7-133 exhibited a similar ability to inhibit leukemic cell viability with an IC50 of 1070nM using a cell viability assay, compared to a flow cytometry-based cell death assay (24h IC50 1050nM). CDK9-BCL6 TCIPs are designed and produced by covalently linking BI-3812 (binding to BCL6) and SNS-032 (binding to CDK9) with a rigid linker. BI-3812 is a potent and efficacious BCL6 inhibitor, inhibiting the BTB domain of BCL6, and SNS-032 is a potent and selective inhibitor of CDK9. When CLL B cells were treated with only BI-3812 or SNS-032 or TCIP BAK-04-232 as single agents for 72h, we found that TCIP BAK-04-232 had superior and significant killing effects compared to the single agents, with an IC50 of 10nM. Specifically this killing activity was more than 10-fold compared to SNS-032 alone (IC50 over 100nM), and more than 1,000-fold compared to BI-3812 alone (IC50 over 10,000nM). There remains an urgent to identify new categories of potent drug, inhibitor, or small molecules to expand potential clinical treatment options for patients with CLL. The results presented here indicate that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in CLL B cells at nanomolar concentrations, and this remarkable killing effect may be due to its unique gain-of-function drug mechanism. TCIPs provide a completely new drug direction and strategy for the design of anticancer drugs: to rewire cancer drivers to activate apoptosis in cancer cells. This study is the first of a kind demonstration of TCIPs in robustly targeting CLL B cells. Further studies to fully validate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the role of TCIPs as a novel therapeutic approach in CLL.

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant lymphocytes in the lymph nodes, spleen, blood, and bone marrow. Although current clinical treatment approaches targeting Bruton Tyrosine Kinase (BTK) and Bcl-2, either as monotherapy or in combination are highly effective with high overall response, complete responses, and achievement of undetectable measurable residual disease (MRD), most patients eventually experience disease relapse. In this study, we utilized our unique mesenchymal stromal cells (MSCs) system, which mimics the in vivo stromal environment in CLL, to explore the combination interaction effect of the first-in-class BTK inhibitor, ibrutinib, with novel tumor microenvironment (TME)-directed drugs in CLL. Methods: Since 1999, patients with CLL seen at Mayo Clinic in Rochester, MN have been offered participation in a longitudinal study of CLL B-cell Biology (CLL Tissue Bank). This Tissue Bank is seamlessly integrated into the Mayo Clinic CLL Database where the clinical characteristics of all patients are prospectively maintained. Using this resource, we identified previously untreated CLL patients (n=10) for our experiments. Individuals with high risk CLL (defined as those with both unmutated IGHV gene mutations and either del17p- or del11q- by FISH) (n=5) and with low risk CLL (defined as those with both mutated IGHV genes and no del17p- or del11q- by FISH) (n=5) were studied. Annexin-V/PI apoptotic cell death assays were employed to evaluate the effects of TME-directed drugs including everolimus (mTOR inhibitor), MI-2 (MALT1 inhibitor), ruxolitinib (JAK1/2 inhibitor), S63845 (MCL-1 inhibitor), and venetoclax (Bcl-2 inhibitor) either as single agent, or in combination with ibrutinib on CLL B cells in direct contact with MSCs. Dose response curves and IC50 levels were generated for single agent killing effect in CLL. The combination interaction effects were determined by Combination Index (CI) values generated by CalcuSyn software indicating synergistic (CI < 0.9), additive (CI between 0.9 - 1.1), or antagonistic (CI > 1.1) interactions. Among drug combinations with synergistic CI values, the following indicated different levels: below 0.1 very strong, 0.1 - 0.3 strong, 0.3 - 0.7 good, and 0.7 - 0.9 moderate synergism. Results: All tested drugs exhibited different IC50 levels in CLL cells co-cultured with MSCs: MI-2 IC50 of 3.5uM, ruxolitinib IC50 of 18.5uM, S63845 IC50 of 0.115uM, venetoclax IC50 of 0.006uM, and both everolimus and ibrutinib did not reach IC50 levels at maximum tested doses. Of interest we observed more killing in high versus low-risk cohort when treated with everolimus (19uM vs. undetectable), MI-2 (2.5 vs. 4uM) and S63845 (0.09 vs. 0.14uM) as single agents (IC50, high vs. low, respectively), while ruxolitinib and venetoclax showed no differences. Combination interaction effect results showed highly synergistic interactions for all patients (good to strong synergism) when ibrutinib was combined with S63845 or venetoclax, moderate to strong synergism for ibrutinib and everolimus, moderate synergism to additive levels for ibrutinib and MI-2, and moderate synergism to antagonism for ibrutinib and ruxolitinib. We also observed more synergism in the low-risk cohort when treated with ibrutinib and MI-2/ruxolitinib combinations, but no difference with ibrutinib and everolimus/S63845/venetoclax combinations between low and high-risk cohort. Conclusions: In summary, we evaluated the pre-clinical combination effects of ibrutinib when used together with other novel agents on CLL B cells in the presence of TME, where patients were assigned to low or high-risk cohorts. Our study showed that ibrutinib exhibits highly synergistic effects with venetoclax, S63845 and everolimus in both cohorts. The observed difference in effectiveness between high and low-risk cohorts when treated with ibrutinib plus MI-2 or ruxolitinib suggests these combinations would be beneficial for a subset of patients with high risk CLL. These preclinical data offer preliminary insights that support the evaluation of therapeutic combinations in patient with high-risk CLL: a population with significant unmet medical needs. Funding Statement: This study was funded and supported by Pharmacyclics LLC, an AbbVie company.

Abstract Purpose: Although the B-cell receptor (BCR) signal plays a critical role in chronic lymphocytic leukemia (CLL) cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined. Experimental Design: Patients with CLL were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism. Results: We detected that compared with normal B cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its noncanonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR signal in CLL cells via LYN/Bruton’s tyrosine kinase axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR inhibitors or venetoclax (a BCL2 inhibitor), induced apoptosis synergistically in CLL cells. Conclusions: These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some patients with CLL.

7050 Background: The management of Richter transformation (RT) of CLL represents an unmet clinical need. We previously reported modest efficacy of pembrolizumab monotherapy in an initial cohort of patients with RT (Ding, et al, Blood 2017). Here we report updated results of this trial, including an expansion cohort and a combination therapy cohort. Methods: Patients with biopsy proven RT to diffuse large B-cell lymphoma were enrolled and treated with single agent pembrolizumab (200 mg IV Q3W). Patients with progressive disease (PD) or stable disease (SD) after 3 months of pembrolizumab monotherapy were allowed to add a B-cell receptor (BCR) kinase inhibitor. The primary end point was overall response rate (ORR). Results: A total of 26 patients with RT were enrolled (9 in initial cohort and 17 in expansion cohort). The median age was 68.5 years. Ten (38%) patients had del(17p) or TP53 mutation. The median number of prior lines of therapy was 3 (range 1-10). Fifteen (58%) patients had received ibrutinib previously (14 of whom had PD on ibrutinib). With pembrolizumab monotherapy (n=26), the ORR was 23.1%, with 2 (7.7%) complete response (CR), 4 (15.4%) partial response (PR), and 7 (26.9%) SD. Of the 6 patients with confirmed CR or PR, responses occurred after a median of 2 cycles of monotherapy. The median duration of response (DOR) was 3.2 months (95% CI 2.8-NA). The median progression-free survival (PFS) was 2.6 months (95% CI 1.6-3.6). Sixteen patients went on to receive a BCR kinase inhibitor (ibrutinib [n=15], idelalisib [n=1]) in combination with pembrolizumab, after SD or PD on pembrolizumab monotherapy. The ORR to combination therapy was 62.5%, with 4 (25.0%) CR and 6 (37.5%) PR. Of the 10 patients with confirmed CR or PR to combination therapy, responses occurred after a median of 4 cycles of therapy. The median DOR was 4.5 months (95% CI 1.6-NA). The median PFS was 7.6 months (95% CI 2.3-13.3). After a median follow-up of 50.4 (95% CI 34.0-56.0) months, the median overall survival (OS) for all 26 patients with RT was 11.6 months (95% CI 7.5-20.1), and the 2-year OS rate was 27%. Treatment-related adverse events (TRAEs) of any grade occurred in 24 (92%) patients, including anemia (54%), thrombocytopenia (58%), neutropenia (54%), leukopenia (35%), lymphopenia (23%), dyspnea (23%), diarrhea (19%), and nausea (23%). Treatment-related grade 3 or above AEs occurred in 21 (81%) patients, including neutropenia (38%), leukopenia (31%), thrombocytopenia (27%), anemia (15%), febrile neutropenia (12%), and lung infection (12%). Conclusions: This expanded analysis confirmed that pembrolizumab has modest single agent activity in RT. Combination therapy with pembrolizumab and a BCR kinase inhibitor is associated with increased efficacy (ORR >60%), similar to other reports. These results support further investigation of immune checkpoint inhibitor-based combination therapy in RT. Clinical trial information: NCT02332980 .