Transformation Of Chronic Lymphocytic Leukemia Research Articles

Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter’s syndrome

Richter’s syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Mouse models of CLL: In vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

A multicenter study of venetoclax-based treatment for patients with Richter transformation of chronic lymphocytic leukemia

AbstractPatients with chronic lymphocytic leukemia (CLL) who develop Richter transformation (RT) have a poor prognosis when treated with chemoimmunotherapy regimens used for de novo diffuse large B-cell lymphoma. Venetoclax, a BCL2 inhibitor, has single-agent efficacy in patients with RT and is potentially synergistic with chemoimmunotherapy. In this multicenter, retrospective study, we evaluated 62 patients with RT who received venetoclax-based treatment outside of a clinical trial, in combination with a Bruton tyrosine kinase inhibitor (BTKi; n=28), rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) (n=13), or intensive chemoimmunotherapy other than R-CHOP (n=21). The best overall and complete response rates were 36%/25%, 54%/46%, and 52%/38%, respectively. The median progression-free and overall survival estimates for the same treatment groups were 4.9/14.3 months, 14.9 months/not reached, and 3.3/9 months, respectively. CLL with del(17p) was associated with a lower complete response rate in the total cohort (odds ratio [OR] 0.15; 95% confidence interval [CI] 0.04-0.6; p=0.01) and venetoclax-naïve subgroup (OR 0.13; 95%CI 0.02-0.66; p=0.01). TP53 mutated CLL was associated with a lower complete response rate (OR 0.15; 95%CI 0.03-0.74; p=0.02) and shorter progression-free survival (hazard ratio 3.1; 95%CI 1.21-7.95; p=0.02) only in venetoclax-naïve subgroup. No other clinical or baseline characteristics, including prior venetoclax treatment for CLL, showed statistically significant association with outcomes. Grade 3-4 neutropenia and thrombocytopenia events were most frequent with intensive chemoimmunotherapy + venetoclax; grade 3-4 infection rates were similar across treatment groups. In this difficult-to-treat RT patient population, venetoclax-based combination regimens achieved high response rates, with durable remission and survival observed in a subset of patients.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Prognostication in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western countries. CLL is a highly heterogeneous disease: some patients may never require therapy and others relapse several times after different therapeutic strategies. Therefore, in CLL, prognostic markers are essential to capture high-risk patients for different clinical endpoints including early treatment requirement, early progression after BTK or BCL2 inhibitors and Richter transformation. In early stage CLL, different biological and clinical biomarkers have been identified to predict time to treatment requirement that could be used to identify the most appropriate population for early intervention clinical trial. However, at the moment, the standard of care for early stage CLL remains watch & wait since no survival benefit has been identified in clinical trials with chemoimmunotherapy and with BTK inhibitors. In patients requiring treatment TP53 disruptions identify high-risk patients who benefit the most from long-term continuous therapy with BTKi. On the opposite side of the spectrum, IGHV mutated patients devoid of TP53 disruption benefit the most from fixed-duration therapy with venetoclax-obinutuzumab. In between, the highly heterogenous subgroup of patients with IGHV unmutated genes represents the group in which further efforts are needed to identify additional prognostic biomarkers aimed at selecting patients who can benefit from fixed-duration and patients who can benefit from long term BTKi therapy. In the context of the aggressive transformation of CLL, namely Richter syndrome, the clonal relationship to the CLL counterpart represents the strongest prognostic biomarker. Clonally related Richter syndrome still represents an unmet clinical need which requires further efforts to identify new therapeutic strategies.

An extranodal Richter’s syndrome presenting with cardiac diffuse large B-cell lymphoma: a case report

BackgroundRichter’s syndrome (RS) defines the transformation of chronic lymphocytic leukemia into high-grade lymphoma, which usually involves lymph nodes and bone marrow. Extranodal involvement of the heart is an extremely rare condition. Patients with heart involvement tended to have a low response to chemotherapy and relative poor prognosis. The transformation process of RS is often insidious and nonspecific making it challenging to diagnose.Case presentationA 64-year-old woman wih a history of chronic lymphocytic leukemia (CLL) presented with intermittent chest pain and was diagnosed with non-ST-elevation myocardial infarction (NSTEMI). However, the contrast enhanced echocardiography revealed a large irregular mass, measuring about 75.4 mm × 37.5 mm, located on the lateral and posterior wall of the right ventricle. Biopsy of the cardiac mass and the results revealed diffuse large B-cell lymphoma.ConclusionsWe present a case of a 64-year-old woman with aggressive diffuse large B-cell lymphoma involving the heart. This case could provide some insights in the diagnosis of cardiac lymphoma.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Real-World Tisagenlecleucel Outcomes in Richter-Transformed Chronic Lymphocytic Leukemia: A Center for International Blood & Marrow Transplant Research (CIBMTR) Analysis

Background: Richter transformation (RT) is a rare but serious transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most commonly large B-cell lymphoma (LBCL). There is currently no standard therapy for RT lymphoma and median overall survival (OS) is estimated to be only 8-12 months. Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR)-T cell therapy approved for the treatment of r/r LBCL in patients who have received ≥2 lines of prior therapy. Here, we describe the efficacy and safety of tisagenlecleucel in patients with RT in a real-world setting with a median follow-up (infusion to data cutoff) of 31 months. Methods: Data were collected as part of a noninterventional, prospective, longitudinal study using the CIBMTR registry. All patients were treated in the United States, Canada, or Israel. Efficacy outcomes analyzed included overall response rate (ORR), progression-free survival (PFS), and OS. Safety outcomes included the incidence and severity of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Results: As of May 4, 2023, 48 patients with RT received a single tisagenlecleucel infusion. The median age at infusion was 68 years (range, 39-83). Fifty-eight percent of patients were ≥65 years old. The median number of all prior lines of therapy was 3 (range, 1-8); 42% of patients received ibrutinib as a prior therapy. No information confirming clonal relation of LBCL to CLL in evaluated patients was available. Fifty percent of patients had primary refractory disease. Prior to infusion, 46% of patients were reported to have elevated lactate dehydrogenase (LDH). Two patients who received tisagenlecleucel infusion, and were alive at data cutoff, had not yet reached 100 days post-infusion and were therefore not evaluable for efficacy and safety. Among the 46 patients evaluated for efficacy and safety, the ORR (complete response [CR] + partial response) was 63% (95% CI, 47.5-76.8) and CR rate was 50% (n=23). In total, 23 patients (23/46, 50%) experienced disease relapse, 17 within 6 months of infusion (Figure). Twelve- and 24-month PFS were 43% and 39%, respectively; 12- and 24-month OS rates were 51% and 44%, respectively. Among 23 patients with a best overall response of CR, 57% remained in remission at data cutoff. The most common adverse events of any grade were CRS (78%), hypogammaglobinemia (46%), and ICANS (35%). Grade 3/4 CRS was reported in 11% of patients. No patient experienced grade 5 CRS. CRS was managed with tocilizumab in 81% of cases. Grade 3 ICANS was reported in 9% of patients. No grade 4/5 ICANS events were observed. Two patients went on to receive a stem cell transplant following tisagenlecleucel infusion. In total, 25 deaths were reported. The most common cause of death was progressive disease (13/48, 27%). Twelve patients (12/48, 25%) died in the absence of disease progression. Causes of non-relapse-related mortality included infection (including COVID-19, n=5), respiratory failure (n=2), malignant neoplasm (n=2), prior malignancy (n=1), multiorgan dysfunction (n=1), and severe veno-occlusive disease (n=1). Conclusions: In the largest cohort of patients with RT receiving treatment in a real-world setting, tisagenlecleucel resulted in a high overall response rate and durable survival. The incidences of CRS and ICANS were similar to other real-world reports of tisagenlecleucel for patients with LBCL. Overall, these findings demonstrate that tisagenlecleucel could be considered a potential treatment option for patients with RT who have a high unmet medical need.

Co-Expression of C-MYC/BCL2 Is Associated with Inferior Survival Outcomes in Relapsed/Refractory Diffuse Large B-Cell Lymphoma after Autologous Stem Cell Transplantation - a Nationwide Retrospective Analysis in Singapore

Introduction Co-expression of C-MYC and BCL2 in diffuse large b-cell lymphomas (DLBCL), also termed double-expressor lymphomas (DEL), has been shown to be associated with poorer outcomes after standard R-CHOP induction therapy. Approximately 70% of DEL will relapse within 5 years. The subsequent traditional approach with potential for cure has been high dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT). Whilst factors such as remission status after front-line treatment and early relapse have been associated with poorer outcomes after ASCT, data are limited regarding significance of DEL status on survival outcomes post-ASCT. We retrospectively studied the prognostic impact of DEL status on outcomes in patients with relapsed or refractory (R/R) DLBCL who underwent ASCT. Methods This was a retrospective, nationwide study of adults patients, >18 years old, with R/R DLBCL who underwent autologous hematopoietic stem cell transplantation (ASCT) between 2010 and 2022 at the only 2 transplant centers in Singapore - Singapore General Hospital and National University Cancer Institute. Patients with double/triple hit lymphoma, defined as concurrent rearrangements of MYC and BCL2 and/or BCL6 by fluorescence in situ hybridization (FISH) analysis, were not included in this study. Patients with transformed indolent B-cell NHL (non hodgkin lymphoma), primary mediastinal B-cell lymphoma, primary CNS (central nervous system) lymphoma, or Richter transformation of chronic lymphocytic leukemia were excluded. Only patients that had available MYC and BCL-2 immunohistochemistry (IHC) were included in this study. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method. Cox proportional hazards regression models were used to assess associations between covariates of interest and OS or PFS. Results A total of 72 patients were included, 44 (61.1%) non-double expressor DLBCL (non-DEL) and 28 (38.9%) double expressor DLBCL (DEL). Patient demographics and disease characteristics at relapse were similar in both groups aside from a higher incidence of CNS involvement in the non-DEL group as compared to DEL group, 34.1% vs 10.7% (Table 1). Both groups achieved a >90% response (CR and PR) after second-line salvage chemotherapy as well as a >90% response post-transplant (CR and PR). Patients with DEL however were more likely to relapse post-transplant, 69.7% vs 40.9% (p = 0.032) and were also associated with increased odds of all-cause mortality, 60.7% vs 36.4% (p=0.043). This significance was maintained on multivariable analysis, HR 4.22 95% CI 1.20 - 14.84 (p=0.025) and HR 4.32 95% CI 1.10 - 17.15 (p=0.038) respectively. The median follow up for survivors were 42 months (range, 26 to 64 months) in the DEL patients and 60 months (range, 46 to 88 months) in the non-DEL patients. The 5-year PFS in patients with DEL was inferior to non-DEL patients, 23.3% vs 49.6% respectively, p=0.022. DEL patients were also associated with inferior 5-year OS, 40.2% vs 65.7%, p=0.041. In multivariable model, DEL status remained significantly associated with inferior PFS and OS. We also observed that patients with CNS involvement at relapse had inferior PFS and OS. Male gender and a less than complete metabolic response (CR) status prior to transplant, assessed on PET-CT, was also associated with inferior OS. Conclusion Co-expression of C-MYC/BCL2 was associated with inferior outcomes after ASCT in patients with R/R DLBCL. While ASCT has been the standard curative approach in the R/R setting, considerations for novel targeting therapies to achieve a deeper response prior to transplant or the upfront use of chimeric antigen receptor T-cell therapy at first relapse may be further explored in this high-risk subset of patients.

PD-1 Expression in Lymphoma Cells Mediates Cellular Proliferation By Engaging Phosphatase SHP-1/SHP-2

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression 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) as determined by IHC. 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 tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. 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, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

CXCR4 Loss Reduces the Local Growth of Richter Syndrome Murine and Patient-Derived Xenograft Models and Impairs BCR and VLA-4 Activation

Richter Syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma and is considered the most important unmet clinical need in CLL because of the lack of effective treatments. An important feature of RS cells is the frequent acquisition of genetic lesions in cell cycle regulators, such as CDKN2A/CDKN2B and TP53, resulting in increased proliferative capacity of the transformed cells and reduced dependence on proliferative microenvironmental signals. However, interactions with the tumor microenvironment are still required to promote the expansion of the transformed cells, as evidenced by the delayed growth of murine TCL1-derived or patient-derived xenograft (PDX) RS models following BCR disruption or macrophage depletion (Chakraborty S et al. Blood 2021, Martines C et al. Blood 2022). To further define the relevance of individual microenvironmental signals in supporting the growth of RS cells in vivo, in this study we evaluated the effects of CRISPR/Cas9-mediated knockout of the chemokine receptor CXCR4, which plays a central role in regulating the homing and interaction with the tumor microenvironment of non-transformed CLL cells. CRISPR/Cas9 editing of CXCR4 was done in the murine TCL1-derived RS model TC1-355 TKO and in the RS-PDX model RS9737, which are both characterized by biallelic disruption of TP53, CDKN2A and CDKN2B. The edited cells, comprising a mixed population with more than 80% disrupted CXCR4 alleles, were then transplanted in the peritoneal cavity and subcutaneous tissue of recipient mice (n=3 C57BL/6 mice for TCL1-355 TKO, n=8 NSG mice for RS9737). Analysis of the mutant allele frequency several weeks after injection revealed a significant reduction in the proportion of cells with mutated CXCR4 in the spleens of the injected mice and to a lesser extent in the peritoneal cavity and subcutaneous tumors, suggesting that disruption of CXCR4 signaling affects not only the migration and homing of the malignant B cells but also their local growth. To further evaluate the latter possibility, we transplanted CXCR4 knockout and CXCR4 wild type RS9737 cells in separate flanks of NSG mice (n=8) and after 4 weeks investigated the percentage of proliferating cells in the tumors by in vivo BrdU incorporation assay. A significantly lower percentage of proliferating cells was observed in the CXCR4 knockout compared to CXCR4 wild type tumors (22.3 ± 2.1 vs 37 ± 1.4, respectively, p<0.001), further suggesting that loss of CXCR4 affects the proliferation of the tumor cells. Since CXCL12 stimulation had no effect on the in vitro proliferation rate of the RS cells, we next investigated whether the reduced in vivo proliferation could be an indirect effect. For this purpose, we investigated whether CXCR4 knockout would affect integrin VLA4 or BCR signaling, as both BCR and CXCR4 can mediate inside-out VLA4 activation and there are data suggesting a potential crosstalk between the BCR and CXCR4 pathways (Maity PC et al. Front Immunol. 2018). Analysis of BCR signaling in the TCL1-355 TKO model showed a significant reduction in anti-IgM induced activation of AKT, ERK and reduced calcium mobilization in the CXCR4 knockout cells despite increased surface IgM expression. However, no effect of CXCR4 knockout on BCR signaling was observed in RS9737 cells, whereas CXCL12-induced VLA4 activation and VCAM1 adhesion were impaired in both models by CXCR4 knockout. To investigate whether impaired VLA4 activation could be responsible for the reduced in vivo proliferation rate of CXCR4 knockout RS cells, we performed BrdU incorporation analysis of RS9737 CXCR4 knockout and CXCR4 wild type cells co-cultured with the human stromal cell line HS5 + CXCL12. Co-culture with HS5 + CXCL12 resulted in a significantly increased proliferation of RS9737 CXCR4 wild type cells compared to the unstimulated control condition (58.5% ± 0.9% vs 50.1% ± 4.3%, respectively, p=0.031), whereas no such difference was observed with RS9737 CXCR4 knockout cells (50.0% ± 1.5% vs 49.1% ± 0.5%, respectively, p=n.s.). Together, these data show that CXCR4 disruption results in negative selection and reduced growth of murine and PDX RS models in vivo and affects BCR and VLA-4 signaling, suggesting that the CXCR4 pathway could represent a therapeutic vulnerability in Richter Syndrome.

A Prospective, Phase-II Study to Evaluate the Efficacy and Safety of Obinutuzumab, Ibrutinib, and Venetoclax (GIVeRS) in Patients with Richter's Syndrome

Background: Richter's syndrome (RS) is an aggressive histologic transformation of chronic lymphocytic leukemia (CLL), most commonly to diffuse large B-cell lymphoma (DLBCL). RS is characterized by a rapid clinical course, low responses to therapy, and poor long-term survival. Chemoimmunothreapy for RS achieves overall response rates of 40% to 60%, and median progression-free survival (PFS) and overall survival (OS) of 3 to 10 months and 6 to 21 months, respectively. More recently, single targeted agents of either ibrutinib or venetocolax have been shown some clinical activity in RS. Furthermore, ibrutinib combined with venetoclax with or without anti-CD20 antibodies has a synergistic activity in CLL, resulting in deep and prolong responses along with a good safety profile. Methods: This is a prospective phase 2, open-label, non-randomized, single-arm, multi-center study aimed to assess the efficacy and safety of ibrutinib, venetoclax, and obinutuzumab in treatment-naïve or relapsed/refractory patients with biopsy-confirmed RS to DLBCL (NCT04939363). The experimental regimen consisted of 12 months of fixed-duration treatment with obinutuzumab, ibrutinib, and venetoclax. Obinutuzumab was administered at a dose of 100 mg intravenously on day 1, 900 mg on day 2, and then 1000 mg on day 8 and 15 of cycle 1. On day 1 of the subsequent five cycles, obinutuzumab at a dose of 1000 mg was administered. Ibrutinib was administered at a dose of 560mg orally daily initiated together with the first obinutuzumab infusion on day 1 of cycle 1 and was continued throughout the 12 treatment 28-day cycles. Venetoclax was given at a dose of 400 mg orally daily for 12 treatment cycles after an accelerated ramp-up phase from day 15 in cycle 1. The primary endpoint was investigator-assessed overall response rate (ORR) defined as the proportion of patients who achieve complete metabolic response (CR) and partial metabolic response (PR) determined by PET-CT imaging at 6 months per Lugano 2014 criteria. Other key endpoints included; investigator-assessed ORR at 3 and 12 months, PFS, Duration of response (DOR), OS, and safety. Results: From August 2021 until the data cut-off on July 20, 2023, a total of 10 patients with RS were enrolled. Median age was 76.0 (range, 62‒88) years and 70% were male. Six patients were treatment-naïve, 4 had relapsed/refractory RS, 50% had extra-nodal disease, 70% had elevated LDH, 70% were double or triple expressors, and the median number of prior therapies among the R/R patients was 1 (Table 1). Median follow-up was 255 days (95% CI, 158.7-351.3), median treatment duration was 160 days (range: 29-362) and treatment is still ongoing in 3 patients. At 3 and 6 months, the ORRs were 70.0% (7/10) and 22.2% (2/9), respectively, and CR rates (CRRs) were 40.0% (4/10) and 11.1% (1/9), respectively. At the data cutoff, 6/10 (60.0%) patients progressed and 5/10 (50.0%) died. The median PFS was 162 days (95% CI, 66.9-257.1), median DOR 272 days, and median OS was 228 days (95% CI: 184.2-271.8). The causes of death included RS in 2 patients and one case each of Covid-19, secondary malignancy, and general deterioration. Most common related treatment-emergent AEs (TEAEs) of all grades were neutropenia 40%, thrombocytopenia 50%, diarrhea 40%, and skin rash 50%, 2 patients developed Covid-19 who recovered during the protocol. None of the patients developed tumor lysis syndromes. Conclusions: In patients with RS triplet treatment with ibrutinib, venetoclax, and obinutuzmab was well tolerated, achieved high rates of early metabolic response (ORR-70% and CRR-40%) at 3 months. Given the high early response rate but subsequent progression, this triplet therapy may serve as a bridge to consolidation with cellular therapy.

Multicenter Study to Assess the Outcomes and Prognosis of Richter's Transformation in the Era of Novel Agents and Cell Therapy

INTRODUCTION: Richter's transformation (RT) is the transformation of chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL) into an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). RT occurs in 2-10% of CLL patients, usually between 2-5 years after CLL diagnosis. The prognosis of RT is poor and the historical median overall survival (OS) after diagnosis is about 8-14 months. However, with the development of novel therapeutic agents including Bruton tyrosine kinase inhibitors (BTKi) and anti-CD19 CAR T-cell therapy, the natural history and prognosis of RT have evolved. To date there is limited data on the prognosis and management of RT in the era of novel therapies. To better understand the prognosis, impact of new therapeutics, and survival for RT in the modern era we performed a multicenter retrospective study to assess the outcomes of RT. METHODS: We performed a multicenter retrospective study including 4 US centers evaluating patients who were diagnosed with RT between 2012-2022 with the current analysis limited to those patients with large B-cell lymphoma transformation. Statistical analyses include descriptive statistics, t-tests for normally distributed continuous variables, non-parametric tests for skewed variables, and chi-square for categorical variables. Overall survival (OS) was calculated using Kaplan-Meier estimates. RESULTS: We analyzed 78 patients with DLBCL transformation from underlying CLL. The median age at RT diagnosis was 66 years (43-84 years). Most patients had advanced stage III-IV disease 92% (n=72). Extranodal disease was present in 68% (n=53). Among 31 patients with 17p deletion or p53 mutational data available at the time of CLL diagnosis, one of these findings was present in 18 patients. Patients had 2 median lines (range 0-7) of treatment for CLL prior to RT diagnosis. 49% (n=38) had BTKi exposure and 28% (n=22) had Venetoclax exposure before RT diagnosis. 24% (n=19) had no prior treatment for CLL before RT. The median time from CLL to RT diagnosis was 42.5 (0-332) months. Median lines of therapy for RT were 2 (range 1-8). The most common frontline regimens for RT included R-CHOP/ R-CHOP-like backbone in 58% (n=45) or high intensity chemoimmunotherapy regimens (R-EPOCH/CVAD) in 14% (n=11). Overall response rate (ORR) to frontline treatment was 56% (complete response (CR)=32/partial response (PR)=12). Of the patients who received 1 st line treatment and achieved a CR/PR (n=44), the 1-year duration of response (DOR) was 50% and the median DOR was 14 months. Fifty-one patients either relapsed or had refractory/progressive disease after frontline treatment and 48 patients received further treatment. Among these 48 patients, 29% (n=14) received a covalent or a non-covalent BTKi in the 2 nd or higher line setting, with an ORR of 43% (CR=4, PR=2). 23% (n=11) received venetoclax with/without other agents in the 2 nd or higher line setting, with an ORR of 36% (CR=3, PR=1). In total, 17 patients received CD19 based CAR T-cell therapy as part of treatment for RT, all in a second or later line of treatment. The ORR and CR rate to this therapy was 82% (n=14). Of these 14 responding patients, the 1-year DOR was 57% and the median DOR was not reached. 12 patients underwent allogeneic transplant (alloHCT), 11/12 as a consolidative treatment option. At first disease assessment after allo-HCT, the ORR was 92% (CR=11). Of the 11 responders, the 1-year DOR was 73% and the median DOR was 42 months. For the entire cohort (n=78), the 1-year OS was 62% and the median OS was 31 months (95% CI 22-NR), which suggest improvement compared to historical reports. CONCLUSION Although RT has a poor prognosis, with the use of novel therapies such as BTKi and CAR T-cell therapy, the overall survival of these patients has improved in the current era. The overall response to chemoimmunotherapy in the first line setting remains poor. Despite this, advances in the management of RT with incorporation of targeted and cellular therapies has improved clinical outcomes of RT. Incorporating these agents either alone or in combination with chemoimmunotherapy in the earlier setting may further improve outcomes in patients with this aggressive disease.

Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation

Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT

Response-adapted, time-limited venetoclax, umbralisib, and ublituximab for relapsed/refractory chronic lymphocytic leukemia

AbstractMany patients with chronic lymphocytic leukemia (CLL) will develop treatment resistance to Bruton tyrosine kinase (BTK) inhibitors. Phosphatidylinositol-3-kinase (PI3K) inhibitors, including umbralisib, have significant clinical activity in relapsed/refractory CLL, but prolonged exposure is associated with potential toxicities. Owing to the synergistic antitumor effects of combined PI3K and BCL-2 inhibition, we sought to explore the feasibility of response-adapted, time-limited therapy to optimize disease control while mitigating the risks of prolonged treatment. We conducted a phase 1/2 clinical trial to determine the safety and efficacy of venetoclax in combination with umbralisib and the anti-CD20 monoclonal antibody, ublituximab, (U2-VeN) in patients with relapsed/refractory CLL (N = 46) and Richter transformation (N = 5). After 12 cycles, treatment was stopped for patients with CLL who achieved undetectable minimal residual disease (uMRD). Adverse events of special interest included diarrhea in 50% of patients (11% grade 3/4), and aspartate aminotransferase and/or alanine aminotransferase elevation in 15 patients (33%), with 3 (7%) grade 3/4. There were no cases of tumor lysis syndrome related to venetoclax, with outpatient initiation in 96% of patients. The intent-to-treat overall response rate for CLL was 98% with best response of 100% in evaluable patients (42% complete responses). The end-of-treatment rate of uMRD at 10−4 in bone marrow was 77% (30/39), including a 71% uMRD rate among 14 patients refractory to prior BTK inhibitor. Time-limited venetoclax and U2 is safe and highly effective combination therapy for patients with relapsed/refractory CLL including those who have been previously treated with covalent BTK inhibitors. This trial was registered on www.clinicaltrials.gov as #NCT03379051.

Factors associated with long-term outcomes of CD19 CAR T-cell therapy for relapsed/refractory CLL

AbstractHigh response rates have been reported after CD19-targeted chimeric antigen receptor–modified (CD19 CAR) T-cell therapy for relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL), yet the factors associated with duration of response in this setting are poorly characterized. We analyzed long-term outcomes in 47 patients with R/R CLL and/or Richter transformation treated on our phase 1/2 clinical trial of CD19 CAR T-cell therapy with an updated median follow-up of 79.6 months. Median progression-free survival (PFS) was 8.9 months, and the 6-year PFS was 17.8%. Maximum standardized uptake value (hazard ratio [HR], 1.15; 95% confidence interval [CI], 1.07-1.23; P < .001) and bulky disease (≥5 cm; HR, 2.12; 95% CI, 1.06-4.26; P = .034) before lymphodepletion were associated with shorter PFS. Day +28 complete response by positron emission tomography–computed tomography (HR, 0.13; 95% CI, 0.04-0.40; P < .001), day +28 measurable residual disease (MRD) negativity by multiparameter flow cytometry (HR, 0.08; 95% CI, 0.03-0.22; P < .001), day +28 MRD negativity by next-generation sequencing (HR, 0.21; 95% CI, 0.08-0.51; P < .001), higher peak CD8+ CAR T-cell expansion (HR, 0.49; 95% CI; 0.36-0.68; P < .001), higher peak CD4+ CAR T-cell expansion (HR, 0.47; 95% CI; 0.33-0.69; P < .001), and longer CAR T-cell persistence (HR, 0.56; 95% CI, 0.44-0.72; P < .001) were associated with longer PFS. The 6-year duration of response and overall survival were 26.4% and 31.2%, respectively. CD19 CAR T-cell therapy achieved durable responses with curative potential in a subset of patients with R/R CLL. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

518 Testicular Mass: A Rare Presentation of Richter’s Syndrome

Abstract A 72-year-old male patient who presented with a painless right sided scrotal swelling for few months. History of stage A Chronic Lymphocytic Leukemia (CLL) since 2015- under surveillance by hematology team. On examination; 10 X 10 cm hard non-tender right-sided scrotal mass, normal left testicle, no peripheral lymphadenopathy or organomegaly. WBC count = 17.5 × 109 /L with neutrophils of 4.39 × 109 /L &amp; lymphocytes of 11.16 × 109 /L. Testicular tumour marker; within normal CT chest, abdomen, and pelvis (CAP); some borderline enlarged left para-aortic and aorto-caval lymph nodes and unremarkable abdominal viscera. Right inguinal orchiectomy Histopathology and immunohistochemistry profile were consistent with a high-grade Richter’s transformation to diffuse large B cell lymphoma. MDT plan with hematologist: 6 cycles of R-CHOP and IV as well as intrathecal Methotraxate for CNS prophylaxis. Patient was given the option testicular radiotherapy vs orchidectomy and he opted for orchidectomy. After more than 24 months follow up, the patient had no relapse. Richter’s syndrome (RS) is the transformation of CLL to an aggressive form of lymphoma mostly represented by diffuse large B cell lymphoma (DLBCL) in 2 -10% of CLL patients. RS is clinically characterized by development of B symptoms (fever, night sweats and weight loss), rapid clinical deterioration and a rapid increase in the size of lymphadenopathy and /or splenomegaly. RS commonly arises in lymph nodes or bone marrow and disseminates to other organs. Presenting as a testicular mass, as in this case, is quite rare and our patient represents the 4th case reported in the literature.

Plasmablastic transformation of chronic lymphocytic leukemia: a review of literature and report on 2 cases.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most common leukemia in adults in Western countries. Transformation of CLL/SLL to plasmablastic lymphoma (PBL) is exceedingly rare and often has an extremely poor response to treatment. A thorough molecular workup may help in determining clonality-relatedness and prognosis. We describe two cases of CLL/SLL that transformed into PBL, with an extensive molecular workup in one case, and a review of the literature.

GLOFITAMAB MONOTHERAPY INDUCES DURABLE COMPLETE REMISSIONS AND HAS A MANAGEABLE SAFETY PROFILE IN PATIENTS WITH RICHTER’S TRANSFORMATION

Introduction: Richter's transformation (RT) is characterized by transformation of chronic lymphocytic leukemia to an aggressive lymphoma, most commonly CD20+ large B-cell lymphoma (LBCL). Prognosis for patients (pts) with RT is poor and no standard of care exists; there remains a major unmet medical need. Glofitamab is a T-cell engaging bispecific antibody (Ab) with a novel 2:1 (CD20:CD3) format. In a Phase I/II study (NCT03075696), fixed-duration glofitamab monotherapy demonstrated durable responses and a manageable safety profile in pts with relapsed/refractory LBCL (Dickinson et al. 2022). We report the efficacy and safety of glofitamab monotherapy in pts with RT after a median follow-up of 40.6 months (range: 0.3–53.9). Methods: All pts had RT and had received ≥1 prior regimen including ≥1 anti-(a) CD20 Ab. Pts received obinutuzumab pretreatment (1000 or 2000mg) 7 days before the first glofitamab dose and intravenous glofitamab at a fixed dose (0.6, 16, or 25mg) or with step-up dosing (SUD) in Cycle 1 (target dose 16 or 30mg) every 3 weeks for up to 12 cycles. Responses were assessed using Lugano 2014 criteria. Cytokine release syndrome (CRS) events were graded by ASTCT criteria. Results: As of 10 October 2022, 11 pts had received glofitamab at a fixed dose (0.6–25 mg, n = 5) or with SUD (2.5/10/16 mg, n = 3; 2.5/10/30 mg, n = 3). Median age was 71 years (range: 48–76); six pts were aged >70 years. Overall, 91.0% of pts had Ann Arbor stage III–IV disease and 45.5% had an IPI score ≥3. Median number of prior therapies was 3 (range: 1–4) and 54.6% of pts had ≥3 prior therapies. Most pts were refractory to a prior aCD20 Ab-containing regimen (90.9%) and all to their most recent regimen (100%); 54.5% of pts were refractory to their initial therapy. Investigator-assessed overall response rate and complete response (CR) rate were 63.6% and 45.5%, respectively. Median time to CR was 3 months (95% CI: 2.5–not estimable [NE]). Complete responses were durable and most (4/5; 80%) had been ongoing for ≥24.9 months at data cut (Figure). CRS occurred in 72.7% of pts, was primarily associated with the initial doses, and was mostly Grade (Gr) 1 (27.3%) or Gr 2 (27.3%); Gr 3 (9.1%, n = 1) or Gr 4 (9.1%, n = 1) events were uncommon. Glofitamab-related neurologic adverse events (AEs) potentially consistent with immune effector cell-associated neurotoxicity syndrome occurred in 5 pts (Gr 3, n = 1 [syncope]; Gr 1, n = 4). No glofitamab-related Gr 5 (fatal) AEs or glofitamab-related AEs leading to discontinuation were reported. The research was funded by: NCT03075696 is sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing assistance, under the direction of all authors, was provided by Dikeledi Matlebjane, MSc of Ashfield MedComms, an Inizio company, and was funded by F. Hoffmann-La Roche Ltd. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, ongoing trials Conflicts of interests pertinent to the abstract C. Carlo-Stella Employment or leadership position: Humanitas University Consultant or advisory role: Sanofi, ADC Therapeutics, Karyopharm Tx, Celgene/BMS, Roche, Merck Sharp & Dohme, Scenic Biotech, Novartis Honoraria: Celgene/BMS, Incyte, Roche, Janssen Oncology, Merck Sharp & Dohme, Astra-Zeneca, Gilead Research funding: Sanofi, ADC Therapeutics, Roche Other remuneration: Travel, accommodation, expenses - Takeda, Janssen Oncology M. Hutchings Consultant or advisory role: Takeda, Roche, Genmab, Janssen, Abbvie Research funding: Celgene, Genmab, Roche, Takeda, Novartis, Janssen, Merck, Abbvie, AstraZeneca E. Mulvihill Employment or leadership position: F. Hoffmann-La Roche Stock ownership: F. Hoffmann-La Roche Other remuneration: Travel, accommodation, expenses - F. Hoffmann-La Roche J. Relf Employment or leadership position: Roche Products Limited Stock ownership: Roche, F-Star Therapeutics and Harpoon Therapeutics B. Byrne Employment or leadership position: Roche Products Ltd, Welwyn Garden City, United Kingdom Stock ownership: Roche Products Ltd, Welwyn Garden City, United Kingdom L. Lundberg Employment or leadership position: F. Hoffmann-La Roche Stock ownership: F. Hoffmann-La Roche Other remuneration: Patent with F. Hoffmann-La Roche M. Dickinson Consultant or advisory role: Novartis, BMS, Gilead, Roche, Janssen, Abbvie, Genmab Honoraria: Roche, Amgen, MSD, Janssen, BMS, Novartis, Gilead, Abbvie Research funding: Novartis, Roche, Takeda, Celgene, MSD, Abbvie, Lilly Other remuneration: Travel, accommodation, expenses - Roche

Zanubrutinib, Alone and in Combination With Tislelizumab, for the Treatment of Richter Transformation of Chronic Lymphocytic Leukemia.

Zanubrutinib, Alone and in Combination With Tislelizumab, for the Treatment of Richter Transformation of Chronic Lymphocytic Leukemia.