Transformation Of Chronic Lymphocytic Leukemia Research Articles

Guideline for the treatment of chronic lymphocytic leukaemia

Guideline for the treatment of chronic lymphocytic leukaemia

Unusual Illustration of Richter Transformation in Chronic Lymphocytic Leukemia on FDG PET/CT.

A man with history of chronic lymphocytic leukemia (CLL) in remission had multiple rapidly growing lumps in recent 2 months. FDG PET/CT demonstrated disseminated FDG-avid nodal and extranodal lesions including those in unusual locations such as the nasopharynx, salivary glands, lungs, gastric wall, peritoneum, bones, and muscles. Subsequent biopsy of the right axillary mass showed absolute lymphocytosis with focus of large B-cell lymphoma. Flow cytometric immunophenotyping demonstrated κ-restricted CLL and B-cell lymphoma, consistent with Richter transformation of CLL. FDG PET/CT images of this case illustrated the most severe and most disseminated nodal and extranodal involvements of Richter transformation in CLL.

CARD9 Forms an Alternative CBM Complex in Richter Syndrome.

Simple SummaryThe transformation process of chronic lymphocytic leukemia into an aggressive lymphoma, called Richter syndrome (RS), is incompletely understood, and therapeutic options are limited. Here, we report CARD9 to be expressed in a subset of RS tissue specimen and in the first and only available RS cell line, U-RT1. In U-RT1, CARD9 attaches to BCL10 and MALT1, and knockdown of CARD9 leads to a significant reduction in cell viability. We hypothesized that CARD9 plays an oncogenic role in RS through the activation of NF-κB signaling. Our findings may help to extend the current knowledge about the pathogenesis of RS and promote the development of targeted therapies for this aggressive disease.Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS.

Chronic lymphocytic leukemia (CLL) with Reed\u2013Sternberg-like cells vs Classic Hodgkin lymphoma transformation of CLL: does this distinction matter?

The distinction between chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with isolated Hodgkin/Reed–Sternberg cells (CLL-HRS; background milieu with a paucity of inflammatory cells) and overt transformation to classic Hodgkin lymphoma (CLL-HL; mixed inflammatory background) is incompletely understood. This retrospective study examined the clinicopathologic features of CLL-HRS (n = 15) and CLL-HL (n = 31) patients seen over the past three decades from a single institution. The phenotypic features of Reed–Sternberg cells in both groups were similar, including expression of CD30, CD15, and PAX5, as well as EBV status. However, a spectrum of background CLL/SLL infiltration amongst the HRS cells was noted on pathologic review, and four patients had both diagnoses, either concurrently or in succession. The median overall survival (OS) of patients with CLL-HRS was 17.5 months compared to 33.5 months for patients with CLL-HL (P = 0.24). Among patients with CLL-HRS, those who received Hodgkin-directed therapy had a significantly longer median OS (57 months) compared to those who received CLL-directed therapy (8.4 months, P = 0.02). Our clinical and pathologic findings suggest a biologic continuum between CLL-HRS and CLL-HL and indicate that CLL-HRS patients may benefit from Hodgkin-directed therapy.

Clonal Evolution of High-Risk Chronic Lymphocytic Leukemia: A Contemporary Perspective.

Clonal evolution represents the natural process through which cancer cells continuously search for phenotypic advantages that enable them to develop and expand within microenvironmental constraints. In chronic lymphocytic leukemia (CLL), clonal evolution underpins leukemic progression and therapeutic resistance, with differences in clonal evolutionary dynamics accounting for its characteristically diverse clinical course. The past few years have witnessed profound changes in our understanding of CLL clonal evolution, facilitated by a maturing definition of high-risk CLL and an increasing sophistication of next-generation sequencing technology. In this review, we offer a modern perspective on clonal evolution of high-risk CLL, highlighting recent discoveries, paradigm shifts and unresolved questions. We appraise recent advances in our understanding of the molecular basis of CLL clonal evolution, focusing on the genetic and non-genetic sources of intratumoral heterogeneity, as well as tumor-immune dynamics. We review the technological innovations, particularly in single-cell technology, which have fostered these advances and represent essential tools for future discoveries. In addition, we discuss clonal evolution within several contexts of particular relevance to contemporary clinical practice, including the settings of therapeutic resistance to CLL targeted therapy and immunotherapy, as well as Richter transformation of CLL to high-grade lymphoma.

Anti-CD37 Alpha-Amanitin Conjugated Antibodies As Therapeutic Weapons for Richter's Syndrome

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL.Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy.We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050).These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels.αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used).Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells.Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®.ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany

Real World Data on the Outcomes of Richter's Transformation of Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma in the Australian Population: An Australasian Lymphoma Alliance Study

Real World Data on the Outcomes of Richter's Transformation of Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma in the Australian Population: An Australasian Lymphoma Alliance Study

Radiation Therapy for Patients with Diffuse Large B Cell Richter's Syndrome of CLL

Introduction: The transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive B-cell lymphoma histology, known as Richter's syndrome (RS), is associated with rapid progression and remains a serious treatment challenge. Standard chemoimmunotherapy approaches often yield poor response rates, short remission durations, and limited overall survival. The role of radiation therapy (RT) in the management of RS has not been explored, and decision making regarding the use of RT in RS is extrapolated from the de novo diffuse large B cell lymphoma (DLBCL) literature. Herein, we report, to the best of our knowledge, the largest case series of patients with RS who received RT.Methods: We conducted a retrospective review of all patients Memorial Sloan Kettering Cancer Center diagnosed with DLBCL RS between April 2002 and August 2020 who received RT at any time following RS diagnosis. Baseline characteristics, information regarding CLL- and RS-directed therapy, RT characteristics including its therapeutic intent, and outcomes were collected. Progression free and overall survival (PFS, OS) were estimated using Kaplan Meier method. All other analyses were descriptive. Analyses were performed using STATA 17.0.Results: 34 RS patients who received RT were identified. Twenty-one received RT as part of a definitive therapeutic strategy while 13 received RT solely for palliation. Baseline characteristics for the entire cohort, as well as divided by RT therapeutic intent, are described in Table 1. Patients received a median of 1 (range 0-5) prior line of CLL-directed therapy prior to RS diagnosis; 24% had prior ibrutinib while 9% had prior venetoclax. Patients received a median of 2 (range 0-5) lines of RS-directed therapy prior to RT; chemoimmunotherapy was first-line RS-directed therapy for 82%. Figure 1 depicts a swimmer's plot for all patients from time of RS diagnosis and includes timing of RT and cellular therapy (CAR-T or allogeneic stem cell transplant, alloSCT).Heterogeneity in the parameters of radiation utilized reflected diverse clinical situations in this cohort. Across the cohort, most commonly irradiated areas included head and neck sites (n=11), abdomen (n=5), paraspinal (n=4) and pelvis/groin (n=4). Median delivered dose was 30.6 Gy (range: 18-54) and 25 Gy (range: 4.5-45) for the definitively and palliatively-treated patients, respectively. Four patients (3 palliative, 1 consolidative) did not complete prescribed course of RT.We subsequently focused on 21 patients treated with RT as part of a definitive therapeutic strategy. With a median follow-up time of 5.8 (range 0.2 - 18.5) years, median OS was estimated to be 6.8 years from RS diagnosis. Fourteen patients (67%) experienced DLBCL progression following RT.A subset of 12 patients received RT following chemoimmunotherapy without immediate cellular therapy in the front-line setting (58%; 7/12) or relapsed/refractory setting (42%; 5/12). Response at the time of RT was CR/clinical CR in 7 patients (58%), PR in 3 (25%), and PD in 1 (8%); response at the time of RT was unknown for 1 patient. In this subset, four (33%) experienced RS disease relapse with 2 occurring within the prior radiation field and 2 occurring distantly. Two patients received RT as an up-front therapy prior to any chemoimmunotherapy in the setting of localized disease. One of these two subsequently had a DLBCL relapse within the prior radiation field.RT was used as a bridging strategy to cellular therapy in 7 patients including alloSCT (n=4) and CAR-T (n=3). Four patients experienced RS relapse. Estimated median PFS was 1.1 years and median OS was 1.9 years from time of cellular therapy.Conclusion: In the largest single-institution series to describe the use of RT in RS, patients who received RT for the diagnosis of RS experienced favorable long-term survival when used as part of a definitive therapeutic strategy, including consolidation following chemoimmunotherapy and as bridging to cellular therapy. These data suggest a role for RT in the management of RS to be further explored through multicenter collaborations and prospective studies. [Display omitted] DisclosuresThompson: VJHemOnc: Honoraria; Curio Science: Honoraria; MJH Life Sciences: Honoraria. Mato: Acerta/AstraZeneca: Consultancy, Research Funding; LOXO: Consultancy, Research Funding; Nurix: Research Funding; Sunesis: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Genmab: Research Funding; AstraZeneca: Consultancy; DTRM BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Johnson and Johnson: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB, Research Funding; MSKCC: Current Employment. Roeker: AbbVie, AstraZeneca, Janssen, LOXO, Pharmacyclics, TG Therapeutics, Vaniam Group, Verastem: Consultancy; Pfizer: Consultancy, Research Funding; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy; Loxo Oncology: Consultancy; Abbot Laboratories: Current equity holder in publicly-traded company.

Richter Transformation in Chronic Lymphocytic Leukemia: Update in the Era of Novel Agents.

Simple SummaryRichter transformation is a significant and devastating complication of chronic lymphocytic leukemia. While its pathogenesis has been well-studied in terms of genetic and molecular changes and its diagnosis has been made easier by imaging and pathological techniques, its treatment is still an issue. Most patients are resistant to chemo-immunotherapy, and even novel agents do not seem to improve the prognosis in a significant way. Therefore, new combinations and novel drugs are currently being tested. In the current review, we summarize new data about the pathophysiology, biological, and clinical basis of Richter transformation, as well as the different treatments of this condition.Richter transformation (RT) is a poorly understood complication of chronic lymphocytic leukemia (CLL) with a dismal prognosis. It is associated with a switch in histopathology and biology, generally with a transformation of the original CLL clone to diffuse large B-cell lymphoma (DLBCL) or less frequently to Hodgkin’s variant of Richter transformation (HVRT). It occurs in 2–10% of CLL patients, with an incidence rate of 0.5–1% per year, and may develop in treatment-naïve patients, although it is more common following therapy. In recent years, there has been a deeper understanding of the molecular pathogenesis of RT that involves the inactivation of the TP53 tumor suppressor gene in 50–60% of cases and the activation of aberrations of NOTCH1 and MYC pathways in about 30% of cases. Compared to the preceding CLL, 80% of cases with DLBCL-RT and 30% of HVRT harbor the same IGHV-D-J rearrangements, indicating a clonal evolution of the disease, while the remaining cases represent de novo lymphomas that are clonally unrelated. Despite advances in understanding the molecular variations and the pathogenesis of the disease, there is still no significant improvement in patient outcomes. However, if no clinical trials were designed for patients with RT in the past, now there many studies for these patients that incorporate new drugs and novel combinations that are being explored. In this review, we summarize the new information accumulated on RT with special emphasis on results involving the novel therapy tested for this entity, which represents an unmet clinical need.

CXCR4 hyperactivation cooperates with TCL1 in CLL development and aggressiveness

Aberrant CXCR4 activity has been implicated in lymphoma pathogenesis, disease progression, and resistance to therapies. Using a mouse model with a gain-of-function CXCR4 mutation (CXCR4C1013G) that hyperactivates CXCR4 signaling, we identified CXCR4 as a crucial activator of multiple key oncogenic pathways. CXCR4 hyperactivation resulted in an expansion of transitional B1 lymphocytes, which represent the precursors of chronic lymphocytic leukemia (CLL). Indeed, CXCR4 hyperactivation led to a significant acceleration of disease onset and a more aggressive phenotype in the murine Eµ-TCL1 CLL model. Hyperactivated CXCR4 signaling cooperated with TCL1 to cause a distinct oncogenic transcriptional program in B cells, characterized by PLK1/FOXM1-associated pathways. In accordance, Eµ-TCL1;CXCR4C1013G B cells enriched a transcriptional signature from patients with Richter’s syndrome, an aggressive transformation of CLL. Notably, MYC activation in aggressive lymphoma was associated with increased CXCR4 expression. In line with this finding, additional hyperactive CXCR4 signaling in the Eµ-Myc mouse, a model of aggressive B-cell cancer, did not impact survival. In summary, we here identify CXCR4 hyperactivation as a co-driver of an aggressive lymphoma phenotype.

Abstract 1612: Tumor intrinsic changes determine immune-checkpoint response in Richter's transformation

Abstract Richter's transformation (RT) is a clinically challenging and aggressive transformation of CLL (Chronic lymphocytic leukemia) to lymphoma. Immune-checkpoint therapy has improved outcomes in selective lymphoma types. Nearly 40% of RT patients respond to anti-PD1, while the underlying CLL do not show any response, and 10% of CLL patients progress post-anti-PD1 treatment. Previous studies mostly focused on T-cell responses elicited by anti-PD1, however, intrinsic changes in CLL and RT B-cells induced by the treatment remains elusive. We hypothesized that pre-existing active transcriptional circuitry and distinct B-cell subsets in the CLL and RT B-cells may determine the response to anti-PD1 treatment. Peripheral blood mononuclear cells (PBMCs) from 3 patients who received pembrolizumab(anti-PD1) as a single agent. Samples were obtained at baseline and 3 months post-treatment initiation. Among these patients, 2 are progressors while 1 is a responder. We performed single-cell RNAseq (10X Genomics) on B-cells enriched from PBMCs derived from 3 samples (1 Responder: RS2, 2 progressors: CLL14 and CLL17) before and after treatment using chromium single cell 3' gene expression library. A total of ~10000 cells were captured with an average detection of 1000 genes/sample. To delineate the transcriptional changes upon the anti-PD1 treatment, we first integrated the single-cell data from 6 samples (before and after Pembro) and performed unsupervised clustering analysis using UMAP (Uniform Manifold Approximation and projection). We identified 8 different clusters based on highly expressed marker genes. Cluster 2(C2) was increased in the responder as compared to the progressors post-pem treatment. To determine the activated pathways in each cluster, we performed gene-set enrichment analysis (GSEA) using the Enrichr method. This analysis revealed upregulation of distinct pathways related to IFNG/MYC(C1), IFNG(C2), ER stress(C3), B-cell dependent T cell activation related pathway(C4), MYC targets/TNFα(C5,6,7) indicating intra-tumor transcriptional heterogeneity. To decipher the intratumor cell differentiation changes, we performed pseudotime trajectory analysis using Monocle 2 coupled with B cell developmental stage markers. We identified 7 different states, with distinct changes of state in responder sample RS2 (State 4,5, 6, and 7 were increased in RS2 post-treatment) while CLL samples(progressors) did not have distinct changes. Integration with B cell development revealed that aggressive B cells with CD38 expression were highly induced upon anti-PD1 in responder RS2 while regulatory (CD1d) and memory B cells (CD27) remained the same, suggesting PD1 induced intrinsic changes contributing to the disease response. We identified distinct transcriptional states in B cells within responders and progressors which may govern the intra-tumor and inter-tumor heterogeneity to the anti-PD1 response. Citation Format: Prajish Sundaram Iyer, Lu Yang, Yang Zhi Zhang, Hyo Jin Kim, Charla Secreto, Sutapa Sinha, Neil Kay, Stephen Ansell, Wei Ding, Lili Wang. Tumor intrinsic changes determine immune-checkpoint response in Richter's transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1612.

Clinical Outcome and Prognostic Factors of Patients with Richter's Syndrome: Retrospective Multicenter Study of the Spanish Chronic Lymphocytic Leukemia (CLL) Study Group (GELLC)

Richter syndrome (RS), the development of diffuse large B-cell lymphoma (DLBCL) in the context of CLL, is an uncommon complication with a dismal prognosis. Currently, data on biological and clinical features, and factors predicting the outcome of patients are scarce, and there is still no consensus on the best therapeutic approach for these patients (pts). The aim of this study was to analyze the presenting characteristics, treatment outcomes, and factors predicting for survival in a series of pts with RS from a large population-based cohort of the Spanish CLL Study Group (GELLC).Pts diagnosed with RS during 1988 - 2017 were retrospectively identified in eleven centers of the GELLC group. Only cases with confirmed biopsy-proven RS during follow-up were included. Clinical-biological features at diagnosis of RS, including cytogenetics, clonal relation with the pre-existing CLL, Epstein-Barr virus (EBV) status detected by in situ hybridization, cell of origin (COO) analyzed by immunohistochemistry, International Prognostic Index score (IPI) and the RS score (Tsimberidou AM et al, J Clin Oncol, 2006), as well as treatment and outcomes, were analyzed.One hundred and twenty-three pts with transformation of CLL to high-grade lymphoma were identified, including 108 classical DLBCL RS, 14 pts with Hodgkin lymphoma (HL), and one case of plasmablastic lymphoma. Median age of the whole series was 66 years (range, 31 to 92 years), and 64% were men. For the DLBCL RS, median time from the diagnosis of CLL to RS development was of 4.7 years (range, 0-24.8 years, with 9 simultaneous diagnosis of CLL and RS), and the median number of prior therapies for CLL/SLL was 1 (range, 0-7). The main clinical characteristics of the pts are shown in table 1. EBV was detected in 12/36 (33%) cases with classical RS and in 9/11 (82%) of patients with HL variant. According to COO the majority of RS tested (20 of 24, 83%) had a non-germinal center phenotype, whereas 19/23 (83%) cases were clonally related to the preceding CLL. Twelve out of 43 pts had 17p deletion or mutation of TP53 in the CLL clone at the time of RS diagnosis.Among the 108 patients with DLBCL RS, data on the treatment for RS was available in 99 patients. Twenty-one patients did not received treatment due to frailty, comorbidities, or poor performance status. Of the 78 patients that were treated for RS, 46 patients received chemoimmunotherapy, mainly R-CHOP (n=44), 29 patients chemotherapy (most commonly CHOP and purine analogue-based therapy), and others (n=3). The overall response rate for the 99 treated patients was 44% (chemotherapy, 24%; chemoimmunotherapy, 60%; P=0.003). A total of 13 patients underwent stem-cell transplantation (SCT), allogeneic SCT (n=7) or autologous SCT (n=6).After a median follow-up for surviving patients of 36 months (range 0.8-160 months), median overall survival (OS) from the time of transformation of CLL to high grade lymphoma was 7.3 months for the entire cohort, 5.7 months for DLBCL RS patients, and 30.8 months for patients with HL variant (P=0.07). Factors associated with shorter OS in the DLBCL RS series included, ECOG PS >1, Hb level < 10 g/dL, platelet count < 100x109/L, LDH > 1.5 UNL, β2-microglobulin > 3mg/dL, prior CLL therapies >1, p53 positive determined by IHQ, and 17p deletion or mutation of TP53 determined in the CLL clone. RS score was also predictive for OS (median OS of 59.5 months [0-1 points], 5.4 months [2 points], 2 months [3 points], and 3 months [4-5 points], p= 0.006); on the contrary, IPI was not discriminative for OS in this series. In addition, RS clonally related presented a median OS of 5.9 months in comparison with 74.8 months for the clonally unrelated RS (p= 0.071). Finally, patients treated with chemoimmunotherapy combinations presented a median OS of 35.4 months, and the 13 patients that could consolidated the response obtained with SCT (autologous or allogeneic SCT) obtained a median OS of 59.5 months.Although clinical outcome of patients with RS is generally poor, chemoimmunotherapy combinations are effective and obtain durable responses in a proportion of patients. RS score and TP53 disruption are predictive of survival. Finally, pts attaining a response should be considered for SCT. Further clinical development with novel promising treatment options is warranted. [Display omitted] DisclosuresDelgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau.

NOTCH1 Aberrant Activation Is a Common Nonmutational Early Event in Chronic Lymphocytic Leukemia Hematopoietic Stem-Cells

Chronic lymphocytic leukemia (CLL) is a B cell disorder characterized by constitutive NOTCH1 activation. NOTCH1 mutations are recurrently associated with CLL providing a new clonal marker to track CLL origin. To investigate CLL-initiating cells, we assessed NOTCH1 mutational status and signaling in hematopoietic stem (HSCs) and progenitor cells from CLL bone marrow. We collected a total of 29 bone marrow (BM) samples including 22 CLL patients and 7 healthy donors (HDs). BM cells were sorted into CD34+CD38- HSCs (purity 94.23% ± 3.04% ) and CD34+CD38+ progenitor fraction (98.12% ± 1.34%). While Sanger sequencing analysis of the NOTCH1 failed to detect alterations in CD34+/CD38- HSCs, AS-PCR and Droplet digital PCR (ddPCR) indicated the presence of small HSCs mutated clones in 57% of cases. Altogether, these data confirm that NOTCH1 mutation is an early event in CLL hematopoiesis. The analysis of CD34+/CD38+ progenitors detected the NOTCH1 mutation in the majority of samples (66%). The NOTCH1 mutational burden progressively increased along specific stages of HSC differentiation (6.4%± 4.7 in CD34+CD38- to 14.9%±11.3 in CD34+CD38+CD10+CD19+ cells, 22.7%±6.5 in CD34-CD38+CD10+CD19+ cells and 40.5%±4.3 in neoplastic CD5+CD19+ cells). This suggests that the NOTCH1 lesion is selected and expands during HSC differentiation toward a B neoplastic cell, thus strengthening the hypothesis that the genetic alteration is an initial event associated with the stepwise malignant transformation of CLL. Conversely, CD34+ cells (1x106) from CLL patients carrying the mutation, when transplanted into NOD/SCID/IL2Rgnull (NSG) mice generated a mature CLL phenotype (the median percentage of CD5+CD19+ cells in hCD45+ cells was 38.5% and 43.8% in BM and spleen respectively) lacking NOTCH1 mutation (as showed by AS-PCR assay performed on enriched hCD45 cells). This data is more in line with the hypothesis that this genetic abnormality is acquired at the mature B cell stage as an additional leukemogenic event to transform into clinical CLL. Thus, we analyzed the NOTCH1 signaling status in HSCs and progenitor cells of NOTCH1-mutated and unmutated CLL samples. The flow-cytometric analysis of the active NOTCH1-ICN protein level showed unmutated and mutated CLL has a significantly higher NOTCH1-ICN level than HDs samples in both CD34+/CD38- HSCs and CD34+CD38+ (73.4%±22.9 and 83%±16.4 vs 33.3%±14.8; 94.4%±7.3 and 92.8%±4.3 vs 47.9%±13.8, p<0.01 and p<0.001, respectively). c-MYC expression was found significantly higher in HSCs cells from NOTCH1 mutated and unmutated CLL samples compared to HD (3.5±0.7 and 2.6±0.08 vs 1.3±0.1). Western blot analysis of the expression levels of the NOTCH1-TM subunit revealed that HSCs from mutated and unmutated CLL patients always expressed the NOTCH1-TM protein, compared to HDs where NOTCH1-TM was either absent or expressed at lower levels. The present study indicated that the pool of CD34+ cells, including HSC and progenitor compartments, have NOTCH1 aberrantly expressed and activated in CLL patients compared to HDs demonstrating a common nonmutational NOTCH1 activation occurring early in CLL hematopoiesis. This selective pressure might contribute to the onset of specific NOTCH1 mutations in a DNA context that is prone to spontaneous microdeletion. These data represent a rationale for the use of therapies targeting the NOTCH1 signaling in CLL aimed to inhibit the survival of CLL-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

PRMT5 Initiates an Epigenetic Program That Promotes Progression of Chronic Lymphocytic Leukemia

Background: Epigenetic alterations that occur as a result of arginine methyltransferase activity play a critical role in normal biological functions, and increasingly their importance has been shown in cancer initiation and progression. In particular, protein arginine methyltransferase 5 (PRMT5) is an important epigenetic regulator that influences differentiation, cell cycle progression, and many other processes. Aberrant PRMT5 activity has been implicated in tumorigenesis, however its role in the biology of chronic lymphocytic leukemia (CLL) has not been established. Despite recent improvements in therapy, CLL remains an incurable disease. While newer therapies targeting the B cell receptor pathway have shown remarkable efficacy, complete responses are not frequent. In particular, transformation of CLL to aggressive lymphoma (Richter syndrome; RS) occurs in up to 15% of patients and confers a poor prognosis; however, the mechanisms by which RS occurs are poorly understood. A better understanding of CLL progression may facilitate the development of new targeted therapies. We hypothesized that PRMT5 dysregulation initiates an epigenetic program in CLL that contributes to disease progression and potentially transformation. Moreover, we provide evidence that inhibition of PRMT5 is a promising strategy in CLL.Methods: Eμ-PRMT5 transgenic mice were generated on the C57BL/6 background via pronuclear injection of human PRMT5 cDNA under the heavy chain promoter/enhancer. To investigate the effect of PRMT5 overexpression on CLL pathogenesis, Eμ-PRMT5 mice were crossed with Eμ-TCL1 mice, an aggressive, spontaneous model of CLL, and followed for disease onset, progression, and overall survival. Immunophenotypic analysis was performed using multicolor flow cytometry. Pharmacologic inhibition of PRMT5 was evaluated in freshly isolated CD19+-selected cells isolated from CLL patients. Kaplan-Meier curves were compared using a log-rank test.Results: We observed that Eμ-PRMT5/TCL1 had a shorter time to leukemia onset (defined as >20% CD45+/CD5+/CD19+ in peripheral blood) compared to the Eμ-TCL1 mice. The Eμ-PRMT5/TCL1 had significantly worse overall survival compared to the Eμ-TCL1 mice. In addition to more aggressive disease, Eμ-PRMT5/TCL1 mice developed focal lymphoid tumors and lymphadenopathy in contrast to Eμ-TCL1 mice. We generated an inducible knockout CLL-like cell line using CRISPR guide RNAs targeting PRMT5 . Genomic ablation of PRMT5 led to impaired protein expression and subsequent decrease in viable cells. We next assessed the cytotoxic effect of PRMT5 inhibition in CLL using the pharmacologic agents EPZ015666 (Chan-Penebre et al. 2015 Nat Chem Biol) and an in-house developed PRMT5 inhibitor. Both compounds inhibited growth of CD19+-selected cells isolated from CLL patients as well as CLL-like cell lines (Mec-1 and HG3). Pharmacologic PRMT5 inhibition in vivo was able to efficiently modulate symmetric dimethyl arginine motifs. Studies exploring the impact of in vivo PRMT5 inhibition on disease burden and overall survival in Eμ-PRMT5/TCL1 mice are currently ongoing.Conclusions: Our data indicate that PRMT5 facilitates CLL progression through altered epigenetic programming. PRMT5 overexpression in Eμ-TCL1 mice results in a more aggressive disease model with features of a focal lymphoid accumulation. Studies to further characterize the genomic and phenotypic features of this nodal disease are ongoing. Furthermore, our data support pharmacologic inhibition of PRMT5 as a promising therapeutic paradigm in CLL. DisclosuresByrd:Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics: Research Funding. Baiocchi:Prelude therapeutics: Research Funding; essanex: Research Funding; Theravectys: Consultancy; viracta: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia

AbstractThe transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor–associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase–related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.

Distinct immune signatures in chronic lymphocytic leukemia and Richter syndrome

Richter syndrome (RS) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. RS is known to be associated with a number of genetic alterations such as TP53 and NOTCH1 mutations. However, it is unclear what immune microenvironment changes are associated with RS. In this study, we analyzed expression of immune checkpoint molecules and infiltration of immune cells in nodal samples, and peripheral blood T-cell diversity in 33 CLL and 37 RS patients. Compared to CLL, RS nodal tissue had higher PD-L1 expression in histiocytes and dendritic cells (median 16.6% vs. 2.8%, P < 0.01) and PD1 expression in neoplastic B cells (median 26.0% vs. 6.2%, P < 0.01), and higher infiltration of FOXP3-positive T cells (median 1.7% vs. 0.4%, P < 0.01) and CD163-positive macrophages (median 23.4% vs. 9.1%, P < 0.01). In addition, peripheral blood T-cell receptor clonality was significantly lower in RS vs. CLL patients (median [25th–75th], 0.107 [0.070–0.209] vs. 0.233 [0.111–0.406], P = 0.046), suggesting that T cells in RS patients were significantly more diverse than in CLL patients. Collectively these data suggest that CLL and RS have distinct immune signatures. Better understanding of the immune microenvironment is essential to improve immunotherapy efficacy in CLL and RS.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1

AbstractRichter’s transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase–associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

ROR1 targeting with the antibody-drug conjugate VLS-101 is effective in Richter syndrome patient–derived xenograft mouse models

AbstractRichter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

Pleurisy in patients with chronic lymphoid leukemia

Aim . To establish the clinical and laboratory features of the pleurisy course in patients with chronic lymphocytic leukemia (CLL) living in the Amur Region. Materials and methods . We studied the case histories and outpatient records of 75 patients with CLL in stages B and C according to the J.Binet classification who were treated in the hematology department of the Amur Regional Clinical Hospital in 2012-2018. Results . Exudative pleurisy was found in 28 patients (37%). The most common cause of pleurisy in patients with CLL was lymphoid infiltration of the pleura (18 patients), less often – infectious pleurisy developed (10 patients). Based on the study of the peculiarities of the course of specific lymphoproliferative and infectious pleurisy, significant differences in the clinical course and laboratory data were established. With infectious pleurisy, “pleural” manifestations and clinical manifestations of pneumonia, the complication of which it was, prevailed; in laboratory examination of exudate, neutrophils prevailed. The prognosis of infectious pleurisy is favorable in most cases. The clinical picture of the tumor process prevailed in specific lymphoproliferative pleurisy, in the laboratory study of exudate ‒ tumor lymphocytes, the hemorrhagic nature of the fluid. Specific lymphoproliferative pleurisy occurs at the stage of malignant transformation of CLL, often being its first manifestation. Conclusion . The attachment of the specific lymphoproliferative pleurisy is an unfavorable prognostic factor for patients with CLL.

Central Nervous System (CNS) Involvement of Richter Transformation: A Single Center Experience

Central Nervous System (CNS) Involvement of Richter Transformation: A Single Center Experience