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Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: 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; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

Introduction: In 2017, a two-dose recombinant zoster vaccine (RZV) containing glycoprotein E (gE) and a novel adjuvant was approved to prevent herpes zoster and its complications in adults ≥50 years of age in the US. There are limited data on the immunogenicity and safety of RZV in patients with chronic lymphocytic leukemia (CLL). Methods: Between 8/15/2018 and 11/4/2019, RZV-naïve patients with CLL and monoclonal B-cell lymphocytosis (MBL) seen at Mayo Clinic, Rochester, MN were enrolled in this IRB-approved study. All participants received two doses of vaccine, at 0 and 2 months after enrollment. Cell-mediated immunity was measured by dual-color interferon γ (IFN- γ) and interleukin 2 (IL-2) FLUOROSpot in peripheral blood mononuclear cells (PBMC) after depletion of ≥50% leukemic B cells. Results were reported in spot forming cells (SFC)/106 PBMC in wells stimulated with gE overlapping peptides or varicella zoster virus (VZV) inactivated cell lysate. Response rate was defined as the proportion of participants who achieved a ≥2-fold increase in IFN- γ or IL-2 SFC/106 PBMC one month after the second dose of vaccine compared with pre-vaccination baseline levels. We also determined the geometric mean fold rise (GMFR) and geometric mean count (GMC) of SFC /106 PBMC at baseline and 90 days after the first dose of vaccine. Two cohorts were enrolled: Cohort 1 consisted of individuals with previously untreated MBL and CLL; and Cohort 2 consisted of CLL participants who were on Bruton tyrosine kinase inhibitor (BTKi) therapy. We compared the response data to historic age-and sex-matched healthy controls who received RZV. Chi-square or Fisher's exact tests measured differences in response rates and Kruskall-Wallis tested for differences in continuous variables. Among CLL participants, multivariable regression analysis was used to investigate the association between day 90 SFC and prior BTKi treatment, accounting for age at RZV, age at CLL diagnosis, sex, and day 0 SFC. Results: Sixty-one participants were enrolled. Paired samples were available for 41 participants (22 in Cohort 1 and 19 [18 ibrutinib and 1 acalabrutinib] in Cohort 2), and 68 age- and sex-matched healthy controls. Baseline characteristics of MBL/CLL participants are shown in Table 1. Compared to healthy controls, participants with MBL/CLL had a significantly lower IFN-γ VZV-specific response rate (41% vs. 63%, p=0.03) and IFN-γ gE-specific response (56% vs. 96%, p<0.001); although there were no differences in IL-2 response rates to VZV (51% vs 68%, p=0.09) or gE (95% vs. 100%, p=0.14, Table 2). There were differences in GMFR between participants with MBL/CLL and controls that reached statistical significance for IFN-γ and IL-2 gE-specific responses and borderline significance for IFN-γ VZV-specific responses (Table 2). VZV-specific response rates did not significantly differ between individuals with untreated MBL/CLL and those receiving BTKi (52% vs. 28%, p=0.12 for IFN- γ; (62% vs. 39%, p=0.15 for IL-2), nor did IL-2 gE-specific responses (96% vs. 95%, p=1.0). However, untreated MBL/CLL participants had significantly higher IFN- γ gE-specific response rate compared to BTKi-treated individuals (73% vs. 37%, p=0.02, Table 2). IFN- γ and IL-2 VZV- and gE-specific SFC GMC was significantly higher at Day 90 compared to Day 0 in all vaccinees, although the magnitude of response was significantly lower in MBL/CLL participants compared to healthy controls (Table 3). Among participants with MBL/CLL, age at diagnosis, age at receipt of RZV, and sex did not predict response to vaccination after accounting for prevaccination values. No serious adverse events were noted among MBL/CLL participants while on this study. Additional analyses including determination of humoral immunogenicity and long-term follow up to ascertain risk of herpes zoster infection are ongoing at this time. Conclusion: RZV was associated with lower cell mediated immunogenicity in MBL/CLL participants compared to healthy older adults. Among individuals with CLL, BTKi treatment was associated with decreased cell mediated immunogenicity of the vaccine. Given the inferior response rates, an additional dose of RZV may be considered to boost responses and should be studied in this patient population. Disclosures Johnson: GSK: Research Funding. Ding:Abbvie: Research Funding; Astra Zeneca: Research Funding; DTRM: Research Funding; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees. Kenderian:Humanigen: Consultancy, Patents & Royalties, Research Funding; Torque: Consultancy; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Mettaforge: Patents & Royalties; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Kay:Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levin:GSK: Research Funding. Kennedy:ICW Ventures: Research Funding. Weinberg:GSK: Research Funding. Parikh:GlaxoSmithKline: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; MorphoSys: Research Funding; Verastem Oncology: Honoraria; Pharmacyclics: Honoraria, Research Funding; Genentech: Honoraria; Ascentage Pharma: Research Funding.

Background: Clinical factors including previous history of AF, heart failure, hypertension, valvular heart disease, increased age and male gender increase the risk of AF in CLL patients (Shanafelt, Leukemia and Lymphoma 2017). Treatment with Bruton tyrosine kinase inhibitors (BTKi) such as ibrutinib has also been associated with an increased risk of AF in CLL. We evaluated the role of artificial intelligence electrocardiography (AI-ECG) in predicting ibrutinib-induced AF (and, for reference, AF unrelated to ibrutinib) in patients with CLL. Methods: We identified two cohorts of CLL patients using the Mayo Clinic CLL Database. Cohort 1 included patients evaluated within 12 months of CLL diagnosis who did not ever receive ibrutinib. Cohort 2 included patients who were treated with ibrutinib. The electrocardiographic signature of AF in sinus rhythm was detected by an AI-ECG algorithm previously developed using a convolutional neural network (Attia, Lancet 2019). The baseline AI-ECG AF score (positive defined as >0.10 on a scale of 0-1 which offers best balance between sensitivity and specificity per Attia et al.) was computed based on ECGs obtained within 10 years prior to CLL diagnosis (Cohort 1) or 10 years prior to initiation of ibrutinib therapy (Cohort 2). Patients with AF at baseline, missing data, or with ECGs previously used to train the AI algorithm were excluded. Reverse Kaplan Meier diagrams were plotted for both cohorts grouped by AI-ECG positivity. Cox proportional hazards were fitted to assess the predictive ability of AI-ECG in both cohorts. Results: After screening 2,739 patients and applying exclusion criteria (126 patients had baseline AF) a total of 1,149 patients with median 4 (interquartile range [IQR] 2-9) baseline ECGs were included in the analysis (Figure 1A). Cohort 1 included 951 patients with a median follow up of 3.0 (IQR 0.6-7.0) years and positive baseline AI-ECG in 546 (57%) patients. Cohort 2 included 198 patients with a median follow up of 1.6 (IQR 0.7-3.2) years and positive baseline AI-ECG in 91 (46%) patients. In Cohort 1, the median age was 67 years (IQR 58-72), 681 (72%) of patients were men, 68% had low/intermediate risk CLL-International Prognostic Index (IPI), and 32% had high/very high-risk CLL-IPI. In Cohort 2, the median age was 69 years (IQR 62-75), 139 (70%) of patients were men, 13% had low/intermediate risk CLL-IPI, and 87% had high/very high-risk CLL-IPI. AF occurred during follow up in 164 patients (17%) in Cohort 1 and 46 patients (23%) in Cohort 2. In both Cohorts 1 and 2, a positive baseline AI-ECG significantly increased the incidence of AF during follow up (log rank <0.001) (Figure 1B and C). Hazard ratios (for positive vs. negative AI-ECG) were 33.9 (95% confidence interval [CI] 15.0-76.6) for Cohort 1 and 14.8 (95% CI 5.3-41.3) for Cohort 2. Conclusion: The addition of AI to a standard 12-lead ECG obtained during normal sinus rhythm - an inexpensive and ubiquitous test - predicts the occurrence of future AF in patients with CLL. This holds true irrespective of BTKi -based therapy and has important implications for the management of CLL patients. Disclosures Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees. Kenderian:BMS: Research Funding; Gilead: Research Funding; Novartis: Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Juno: Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Torque: Consultancy. Wang:Novartis: Research Funding; Innocare: Research Funding; Incyte: Research Funding. Kay:Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Bristol Meyer Squib: 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. Kapoor:Cellectar: Consultancy; Janssen: Research Funding; Sanofi: Consultancy, Research Funding; Amgen: Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria; GlaxoSmithKline: Research Funding. Parikh:MorphoSys: Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria, Research Funding.

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: 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; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

INTRODUCTION T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive malignancy of mature T-cells. Although responses may been seen with initial alemtuzumab-based therapy, relapse is inevitable and relapsed/refractory disease carries a dismal prognosis. T-PLL has shown in vitro sensitivity to venetoclax, and short-lived clinical responses to venetoclax monotherapy (Smith, Blood Advances 2020; Boidol, Blood 2017) or when given in combination with pentostatin (Alfayez, Leukemia & Lymphoma 2020). Given the lack of standard treatment options, we reviewed the outcomes of patients (pts) with relapsed T-PLL on venetoclax-based therapy. METHODS Using an institutional clinical database of pts with T-PLL seen in the Division of Hematology at Mayo Clinic, Rochester, MN, all pts treated with venetoclax or venetoclax-based therapies in a relapsed/refractory setting were identified. Clinical, laboratory, and pathology data, as well as treatment dosing and toxicity were ascertained retrospectively from the medical records. Diagnostic criteria and response definitions were utilized as per the T-PLL International Study Group (Staber, Blood 2019). RESULTS Between 8/2017 and 5/2020, 9 pts with relapsed/refractory T-PLL treated with venetoclax were identified. The median age was 63 years (range 49-75), and one-third were male. Baseline characteristics are detailed in Table 1. Rearrangement of TCL1 family genes was present in all 8 pts evaluated with fluorescence in situ hybridization (FISH). Using conventional cytogenetics, a complex karyotype was present in 4/6 pts tested. The median prior lines of therapy was 3 (range 1-4), including alemtuzumab in 8 of 9 pts (intravenous 7/8); and two pts had undergone prior hematopoietic stem cell transplantation (1 allogeneic; 1 autologous) after achieving a complete remission. Additional individual pt details are included in Table 2. Prior to venetoclax therapy initiation, 2 pts had B-symptoms (2 night sweats; 1 fever) and 6 pts had an ECOG score ≥ 2. Lymphadenopathy and splenomegaly were present in 6 and 9 pts, respectively. Extranodal involvement included 3 pts with cutaneous involvement and 5 pts with effusions (4 ascites; 1 pleural). The target maximum dose of venetoclax (800 mg [n=4]; 400 mg [n=1]) was reached by 5 pts at a median of 12 days (range 7-40 days). The other 4 pts had disease progression after a median of 10 days (range 5-22 days) during the dose ramp-up, including 2 pts undergoing rapid dose escalation. Altogether, 6 of the 9 pts initiated venetoclax with a rapid dose escalation. Bendamustine was given with venetoclax in 5/9 pts; median number of cycles 1 (range 1-4) with variable starting dosing (range 60-100 mg/m2). The disease control rate was 56%; best response was partial remission (PR) in 4 (44%) pts and stable disease (SD) in 1 (11%) pt. The 1 pt with SD received venetoclax monotherapy; however, the overall response rate among pts who received the combination of venetoclax and bendamustine was 80% (4/5 pts). One pt with a partial remission met all criteria for complete remission but did not have a confirmatory bone marrow biopsy. Cutaneous disease improved in 2 of 3 pts (both with PR as best response), and effusions improved in 2 of 5 pts (1 PR and 1 SD as best response). Among those in PR, the median decrease in absolute lymphocyte count was 67x109/L and the median decrease in lactate dehydrogenase was 1,162 U/L (normal range 122-222 U/L). Median duration of treatment for all pts was 42 days (range 4-201 days). All pts ultimately died during follow-up with a median overall survival of 53 days (range 4-464 days); Figure 1. All pts experienced at least one adverse event and 8 of 9 pts had a grade ≥ 3 toxicity, most commonly edema (n=7) and neutropenia (n=6). Five pts required dose interruptions due to neutropenia (n=3), tumor lysis syndrome (n=1), and edema (n=1). Three pts had dose reductions, all from 800 mg dosing, due to hematologic toxicity (n=2) and nausea (n=1). No pts discontinued venetoclax as a result of adverse events. CONCLUSION Treatment of pts with relapsed/refractory T-PLL remains a significant unmet need. The 9 pts described herein represent the largest cohort of pts treated with venetoclax-based therapy in this setting to the current date. Despite initial reports of clinical efficacy, our results show that venetoclax (with or without bendamustine) has modest efficacy in the treatment of relapsed/refractory T-PLL after prior alemtuzumab exposure. Disclosures Parikh: TG Therapeutics: Research Funding; Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Merck: Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Ascentage Pharma: Research Funding; AstraZeneca: Honoraria, Research Funding. Shah:Dren Bio: Consultancy. Bennani:Verastem: Other: Advisory Board; Affimed: Research Funding; Purdue Pharma: Other: Advisory Board; Kite/Gilead: Research Funding. Wang:Incyte: Research Funding; Novartis: Research Funding; Innocare: Research Funding. Kenderian:MorphoSys: Research Funding; Lentigen: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties. Kay:MEI Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Cytomx: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: 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; Rigel: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: 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; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Ding:Octapharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Abbvie: Research Funding. OffLabel Disclosure: Venetoclax: off-label use in treatment of relapsed/refractory T-cell prolymphocytic leukemia

Introduction: Novel targeted therapies have revolutionized treatments for both upfront and relapsed CLL. However, effective therapies have not been established for progressive CLL after developing resistance to both BTK inhibitor and BCL-2 inhibitors. Similarly, Richter's transformation (RS) remains an area of unmet clinical need. Hypothesizing that progressive CLL and RS possess significant but differential molecular abnormalities, we aimed to dissect the initial genomic profiles in a clinical cohort of patients seen in our routine clinical practice using targeted DNA sequencing and transcriptome analysis. Methods: Twelve CLL patients who had lymph node biopsies for evaluation of clinical progression at Mayo Clinic were included in this study. RNA sequencing and targeted DNA sequencing on a focused oncology panel of 1,711 genes were performed on Illumina HiSeq through Tempus Labs, Inc. (Chicago, IL). Differentially expressed (DE) genes were identified using edgeR at the cutoff of FDR <=0.05 and fold-change >=2. Somatic mutations and copy number alternations (CNAs) were identified through Mutect2 and PatternCNV, respectively. CN loss was defined to have log2(ratio) <=-0.93 and CN gain to have log2(ratio) >= 0.585. Pathway analysis was done through the Enrichr package. Results: Among the 12 patients, 7 had confirmed RS (large cell lymphoma histology) and 5 had CLL based on pathological findings. The clinical and therapeutic history for each patient is illustrated in Fig 1A. FISH results from 11 of the patients showed chromosomal abnormalities including del(13q), del(11q), del(17p) and tri(12) (Fig 1A). Hierarchical clustering of expression profiles from the top 10,000 most variable genes in expression revealed two major clusters (Fig 1A). Cluster C1 contains five RS cases, of which case S207 did not receive ibrutinib, S256 was on high-dose steroid and anti-CD20 post-ibrutinib therapy for >1 year before biopsy, and case S025 was treated for < 6 months with ibrutinib. These three form a subgroup separate from the other two RS in C1 who had 8 and 42 months of continuous ibrutinib therapy pre-biopsy, respectively. C2 contains two RS and five CLL cases. Within C2, CLL and RS cases showed similar transcriptomic profiles. Over 90% of the DE genes showed an overall increase of expression in C1, particularly within the 3-sample subgroup (Fig 1B). These genes are enriched in pathways known to play key roles in CLL, including PI3K-AKT signaling, NOTCH signaling and RAS/MAPK signaling, while genes up-regulated in C2 are enriched in WNT signaling. The results suggest marked differences in gene expression profiles and indicate molecular heterogeneity among RS and CLL patients. In addition, targeted DNA sequencing data were analyzed to identify somatic mutations and CNAs. The analysis confirmed the FISH results of del(13q), del(11q), del(17p) or tri(12) in 8 of the cases (Fig 1A). The mutation profiles are similar between C1 and C2 for the recurrently mutated genes published in CLL. However, we identified recurrent CNAs that occurred preferentially in C1 (covering 9 genes) or C2 (48 genes) (Fig 1C). Manual inspections of the reads coverage revealed that 5 of the 9 genes are from chr1q21.1 to chr1q22 that showed clear CN gains largely in C1. Of the 48 genes, 6 are from chrXp11 that showed CN losses, and 27 are from chr17q12 to chr17q25 that showed CN gains. Therefore, the majority of the 57 genes are from large-scale CNAs. Notably, for the CNAs mostly occurring in C2, the affected genes are enriched in WNT signaling, Cell cycle/protein kinase activity and chromatin modifying enzymes. Collectively, the results support the variability of transcriptomic signatures and genetic lesions between RS and CLL. Conclusion: This study revealed a noticeable heterogeneity in gene expression and genetic variation between RS and CLL, albeit in a small cohort. The vast majority of the DE genes showed elevated expression in C1 containing only RS cases, and these genes are enriched in PI3K-AKT signaling, NOTCH signaling, and RAS/MAPK signaling. The pattern is more pronounced for a subgroup in C1 who were overall less exposed to ibrutinib. For somatic variation, there is a strong tendency of occurrence of recurrent CNAs towards C2 cohort, mainly involving gains of chr17q and deletions of chrXp11. Given these advances we are encouraged to further pursue the genetic abnormalities with defined roles in aggressive and transformed CLL. Disclosures Liu: Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Parikh:Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Verastem Oncology: Honoraria; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Merck: Research Funding; MorphoSys: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Wang:Incyte: Research Funding; Innocare: Research Funding; Novartis: Research Funding. Kenderian:Gilead: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Mettaforge: Patents & Royalties; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; Juno: Research Funding; Kite: Research Funding. Kay:Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Acerta Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: 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. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:Astra Zeneca: Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (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) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p<0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: 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; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: 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; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION Nearly 20% of patients (pts) are ≥ 80 years (yrs) of age at the time of CLL diagnosis. Increased medical comorbidities, as well as declining functional status and organ function, often accompany advanced age. However, clinical trials often enroll a disproportionally low number of people from this age demographic due to stringent eligibility parameters, which provides limited evidence to guide decisions. Here, we report clinical characteristics and outcomes of pts ≥ 80 yrs of age at the time of CLL diagnosis. METHODS Between 1/1995 and 3/2020, we identified previously untreated CLL pts from the Mayo Clinic CLL Database who were ≥ 80 yrs of age at the time of CLL diagnosis, and seen within 3 yrs of diagnosis. Baseline characteristics, treatments administered, and time to first treatment (TFT) were analyzed for all pts. Overall survival (OS) was measured from diagnosis date in all pts and separately from treatment date for treated pts. Time to next treatment (TTNT) was measured in treated pts from first treatment date to date of second treatment or last known treatment. TFT and TTNT were analyzed accounting for competing risk of death. Standardized mortality ratio (SMR) compared observed survival to survival in a Minnesota population. Cox multivariable regression models were used to determine which factors were associated with OS (treatment as a time-dependent covariate) and TTNT (accounting for competing risk of death). RESULTS Among the 213 pts identified, the median age was 83 yrs (range 80-97); baseline characteristics are summarized in Table 1. The median number of medical comorbidities among all pts was 4 (range 0-8). At least 1 comorbidity was present in 97% of pts, most commonly hypertension (57%), genitourinary (n=48%), rheumatological (46%), and dyslipidemia (n=43%). Median follow-up was 3.8 yrs. A total of 56 pts received treatment and the median TFT was 6.7 yrs. Over this 25 year interval, the treatment approaches included monoclonal antibody alone (n=17), chemotherapy alone (n=17), or chemoimmunotherapy (n=14), novel agents (n=3), and multi-agent Richter's transformation regimens (n=5). Chlorambucil-based treatments (chlorambucil n=13, chlorambucil, prednisone n=3) were the most frequently used chemotherapy regimens. The most common chemoimmunotherapy regimens included rituximab, cyclophosphamide, prednisone (RCP) with (n=3) or without vincristine (n=6), and chlorambucil with an anti-CD20 monoclonal antibody (n=4; obinutuzumab n=2, rituximab n=2). Among 17 pts who received monoclonal antibody alone, treatments included rituximab alone (n=11), rituximab plus methylprednisolone (n=2), obinutuzumab (n=2), and rituximab with alemtuzumab (n=2). Frontline novel agent treatments included ibrutinib alone (n=1), acalabrutinib alone (n=1), and ibrutinib, obinutuzumab, venetoclax (n=1). Five pts with Richter's transformation (diagnosed concurrent with CLL n=4) received multi-agent chemotherapy. Among the 56 treated pts, 22 pts received second line therapy after a median TTNT of 2.8 yrs from start of first therapy. The most common second line treatments were chlorambucil-based (n=6) and rituximab-based (n=6) regimens. Age, sex, absolute lymphocyte count, del17p, and unmutated IGHV were not predictive of TTNT. The median OS among all 213 pts was 4.6 yrs. SMR was 1.2 (p=0.008) when excluding pts with Richter's transformation. In univariable analyses, receipt of therapy (HR 3.92, 95%CI 2.11-7.28; p<0.001), age (per 5 year increase; HR 1.84, 95%CI 1.43-2.38; p<0.001) and unmutated IGHV (HR 2.03, 95%CI 1.12-3.67; p=0.02) were predictors of shorter OS. The OS among pts who received treatment (Figure 1) was similar when compared across time periods of treatment start year grouped as 1995-2001 (n=10), 2002-2013 (n=32), and 2014-2020 (n=14). Treatment categories by time period are shown in Table 2. CONCLUSIONS In this study of 213 pts ≥ 80 yrs of age at time of CLL diagnosis, the median time to first therapy was ~7 yrs. Although survival outcomes did not differ by era across the 25-year span of this study, pts treated with novel agents were underrepresented and are a patient population which warrants additional evaluation. The finding that pts with CLL in this cohort had a 20% higher risk of death compared to an age- and sex-matched population emphasize the need for ongoing efforts to improve clinical outcomes for CLL pts in this demographic category. Disclosures Kenderian: Torque: Consultancy; Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding. Wang:Incyte: Research Funding; Innocare: Research Funding; Novartis: Research Funding. Shanafelt:Genentech, Pharmacyclics LLC, an AbbVie Company, AbbVie, GlaxoSmithKline, and Merck: Research Funding; Mayo Clinic: Patents & Royalties: and other intellectual property. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: 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; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees. Ding:alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Parikh:Merck: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; TG Therapeutics: Research Funding.

Background: Cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) are known to differentially impact prognosis in patients with chronic lymphocytic leukemia (CLL). Deletion6q23 (del6q23) has been reported in ~2-3% of CLL patients at diagnosis; however, its prognostic significance remains indeterminate. Methods: We identified previously untreated CLL patients from the Mayo Clinic CLL Database seen between 1/1995 - 2/2020 who had FISH performed. The clinical characteristics, concomitant cytogenetic abnormalities, time to first therapy (TTFT), and overall survival (OS) were compared between patients with del6q23 to those without this abnormality. TTFT was analyzed from FISH date until treatment or last known untreated date, accounting for competing risk of death. OS was analyzed using Kaplan-Meier methods from FISH date. In a matched (CLL-IPI risk score and sex) analysis, Cox regression analysis was used to determine the association between TTFT/OS and presence of del6q23. The Mayo Clinic IRB approved this study. Results: A total of 3101 CLL patients were identified; their median age at diagnosis was 64 years and 66% were male. Of these patients, 42 (1.3%) had evidence of del6q23 by FISH. Compared to patients without del6q23, patients with del6q23 carried a more adverse clinical and cytogenetic profile at the time of diagnosis including advanced Rai stage (22% Rai III/IV vs. 9% Rai III/IV; P = 0.002), unmutated IGHV genes (91% vs. 43%; P < 0.001), and high/very high risk CLL-IPI (50% vs. 27%; P < 0.001). Del6q23 was the sole abnormality in 13 patients (31%). Of the remaining patients, del6q23 was concomitantly present with del13q in 8 patients (19%), and with either del11q or del17p in 21 patients (50%). Of the 42 patients with del6q23, 29 received first line therapy: chemoimmunotherapy in 16 patients, BTK inhibitor in 7 patients, and an anti-CD20 monoclonal antibody alone in 6 patients. The median follow-up of the entire cohort was 7.3 years. The median TTFT in patients with del6q23 was 0.7 years while the 5-year OS was 76%. Patients with del6q23 demonstrated significantly shorter TTFT and reduced OS when compared to patients without del6q23 (Figures 1A and 1B). The shorter TTFT was also evident for patients with del6q23 compared to patients without del6q23 divided according to the Dohner risk category (Figure 2A), although there was no difference in OS between these groups of patients (Figure 2B). Because del6q23 patients were enriched for other unfavorable prognostic markers, we identified four control patients without del6q23, matched by CLL-IPI risk score and sex, to each treatment-naïve patient with del6q23. Patients with del6q23 showed a borderline statistically significant shorter TTFT (median 0.7 years versus 3.9 years; P = 0.07) and a nonsignificant association with OS (median 10.9 years versus 10.3 years; P = 0.28). Conclusion: Del6q23 by FISH in previously untreated CLL represents an uncommon abnormality. When compared to patients without del6q23, patients with del6q23 tend to have more adverse clinical and cytogenetic profiles at baseline, which likely confer a worse prognosis as reflected by shorter TTFT compared to patients without this abnormality. Figure Disclosures Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding. Wang:Novartis: Research Funding; Innocare: Research Funding; Incyte: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Sunesis: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Kenderian:Juno: Research Funding; Gilead: Research Funding; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Tolero: Research Funding; BMS: Research Funding. Parikh:Ascentage Pharma: Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria.

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature-appearing malignant lymphocytes (CLL B-cells) in the blood, marrow, lymph nodes, and spleen. Despite improved outcome with the introduction of novel BCR and BCL-2 inhibitors, disease progression is still a therapeutic challenge from either differential responses or acquired drug resistance. Recent studies in CLL reported alterations of the epigenetic landscape as well as mutations of genes encoding key chromatin machineries. These aberrant chromatin structures may provide novel therapeutic targets for CLL. Here, we identify aberrant chromatin features in CLL B-cells as novel therapeutic targets. Methods: Histones were extracted by acid from B cells derived from 10 random selected CLL patients and 10 normal donors and histone modifications were checked by western blot. For ChIP-seq study, published H3K27me3 ChIP-seq data (GSE113336) were downloaded from and analyzed (Control samples, n= 6; CLL samples, n=16). Gene ontology analysis used the Panther Classification System. Cell Survival was determined by CellTiter 96® AQueous Assay (Promega). Results: While most histone modifications do not vary between CLL and controls, H3K27me3 and H3.3S31ph are increased and decreased respectively, albeit variably, in CLL B-cells (Fig1. A and B). Notably, the low level of H3.3S31ph was observed in a subset of samples (7 of 10 CLL samples tested). To further investigate the biology and role of H3K27me3 in CLL, we analyzed its genome-wide distribution by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our analysis showed that the genes with increased H3K27me3 occupancy were mostly enriched in tumor suppression pathways (e.g., negative regulation of PI3K-Akt pathway) or down-regulated genes in CLL such as genes involved in the pro-apoptotic pathway (FAS) (Fig1. C and D). These results suggested that high enrichment of H3K27me3 may regulate the expression of these genes, contributing to CLL survival. H3K27 methylation, an important suppressive histone modification that is associated with transcription repression, is catalyzed by Polycomb Repressive Complex 2 (PRC2). Therefore, inhibition of Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of PRC2, could be explored as a therapy approach in CLL. However, feedback activation of H3K27 acetylation (H3K27ac) can promote expression of pro-survival genes that confers EZH2 inhibitor (EZH2i) resistance, which limits its use in human malignancy. Thus, the epigenetic determinants that reliably overcome EZH2i resistance or sensitize cells to EZH2 inhibition have yet to be identified. As we observed that the CLL B-cells in a subset of CLL patients have low levels of H3.3S31ph, and a recent study showed the importance of H3.3S31ph for the enzymatic activity of p300 to acetylate H3 at lysine 27(Martire S et al., Nat Genet. 2019), we assessed the role of H3.3S31ph in the process of EZH2 inhibitor-mediated H3K27ac. Our results showed that inhibition of H3.3S31ph by CHK1 inhibitor MK-8776 abolished the activation of H3K27ac by EZH2i in MEC1 cells, which represents the patients who have CLL cells with relatively high level H3.3S31ph. However, we did not see a major increase of H3K27ac and H3.3S31ph in primary CLL B-cells with EZH2 inhibition (Fig. 1E), consistent with the relatively low expression of CHK1 protein in these cells (Fig. 1F). Because our data shows the requirement of H3.3S31ph in H3K27ac activation by EZH2 inhibition, we next tested if H3.3S31ph inhibition could overcome H3K27ac induced EZH2 inhibition resistance. We found that suppression of H3.3S31ph by CHK1 inhibitor MK-8776 sensitizes the CLL-like line MEC1 to EZH1/2 inhibition (Fig. 1 G). We then showed that an EZH2 inhibitor, Valemetostat, reduce the survival of the primary CLL B-cells (Fig. 1 H). These results suggest that the low level of H3.3S31ph may provide a therapeutic opportunity for CLL treatment with EZH inhibition. Conclusion: In summary, we have elucidated how epigenetic features in leukemic CLL B-cells (H3K27me3 and H3.3S31ph), can provide novel treatment targets for CLL (Fig. 1 I). Moreover, this study may provide a proof of concept to develop new treatment strategies based on epigenetic vulnerabilities in other hematological malignancies. Disclosures Parikh: GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Verastem Oncology: Honoraria; Merck: Research Funding. Kenderian:Kite: Research Funding; MorphoSys: Research Funding; Tolero: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; BMS: Research Funding; Gilead: Research Funding; Juno: Research Funding; Lentigen: Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Sunesis: Research Funding. Ding:Beigene: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding; DTRM: Research Funding; Abbvie: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; 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; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding.