Citations

Abstract Richter’s syndrome (RS) arising from chronic lymphocytic leukemia (CLL) is a striking example of an aggressive malignant histology that emerges from indolent cancer. RS is a major barrier to disease control in CLL and is associated with poor clinical outcomes and limited survival. The genetic basis of RS is poorly understood, and its relationship to the antecedent CLL remains incompletely characterized. To study RS, we performed whole-exome sequencing (WES) on samples collected from 52 patients with RS of diffuse large B cell lymphoma (DLBCL) histology. For this genomic characterization, samples from 42 patients were analyzed as ‘trios’ (matched germline, CLL and RS tissue DNA) and those from 10 as ‘duos’ (matched CLL and RS DNA). Beyond addressing contamination of CLL DNA in the germline sample, we developed methods for discriminating between the RS and CLL clones which often coexist in the same samples. The discovery cohort revealed that RS and CLL were clonally-related in 45/52 (87%) cases based on WES analysis, with a distinct RS clone emerging from a CLL subclone. The remaining 13% were determined to be clonally unrelated. RS clones presented ~3x higher rates of additional mutations than the ancestral CLL clones from which they developed. We identified novel RS somatic driver mutations (in IRF2BP2, SRSF1, B2M, DNMT3A and others), a high rate of copy number variations with recurrent deletions (e.g., del(17p) [TP53], del(13q14.3), del(7q36), and del(15q13.11) [MGA], del(9p21.3) [CDKN2A/B], del(16q12.2)), focal amplifications (amp(7q21.2) [CDK6], amp(8q24.2) [RECQL4, MYC], amp(13q31.2) [ERCC5], and frequent whole genome duplication. To further investigate RS and CLL clonal evolution, we performed single-cell RNA-sequencing on biopsies at the time of RS diagnosis in 5 individuals with clonally related transformation. Using our novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations, yielding cluster-specific copy number profiles that matched the WES results of individual subclones of the RS and CLL populations. This enabled mapping genetic clones to specific expression patterns. Finally, we devised and tested a methodology that uses cfDNA for early detection of emerging Richter’s disease and have successfully identified Richter‘s tumor DNA in the blood several months prior to the clinical diagnosis. Our study thus defines drivers, distinct molecular subtypes and evolutionary path to RS and suggests strategies for its improved detection. Citation Format: Erin M. Parry, Ignaty Leshchiner, Romain Guièze, Connor Johnson, Eugen Tausch, Sameer Parikh, Camilla Lemvigh, Conor Messer, Daniel Rosebrock, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew Davids, Raquel A. Jacobs, Kara Slowik, Julien Broseus, Shanye Yin, Shuqiang Li, Geoff Fell, Ziao Lin, Binyamin A. Knisbacher, Neil Ruthen, Dimitri Livitz, Christof Schneider, Jialin Ma, Julian Hess, Laura Z. Rassenti, Thomas J. Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E. Kay, Kenneth J. Livak, Brian P. Danysh, Chip Stewart, Donna Neuberg, Jennifer R. Brown, Laxmi Parida, Stephan Stilgenbauer, Gad Getz, Catherine J. Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4007.

Abstract MBL is a common pre-malignant condition characterized by circulating clonal B-cells with an absolute B-cell count <5x109/L and no lymphadenopathy, organomegaly, or cytopenias. MBL is the precursor to CLL. The incidence of cutaneous SCC in CLL is significantly higher compared to controls. The incidence of SCC in MBLs has not yet been determined. Study participants from the Mayo Clinic Biobank who had no prior history of hematologic malignancy, were 40 years or older, and were Olmsted County residents completed a health questionnaire and provided blood samples between 7/2009 and 12/2020. Stored peripheral blood mononuclear cells were screened for MBL using flow cytometry. We defined three MBL immunophenotypes: CLL-like MBL (CD5+, CD20dim), atypical MBL (CD5+, CD20+), and non-CLL-like MBL (CD5-, CD20+). MBL individuals were also classified by cell count into low-count MBL (LC-MBL) and high-count MBL (HC-MBL), with HC-MBL having a percent clonal B-cell count ≥85%. Data on newly diagnosed SCC was abstracted from the medical records, and prior history of skin cancer before sample collection was ascertained from patient questionnaires. Individuals were followed from sample date to the earliest of SCC, death, loss to follow-up, progression, or 12/31/2020. Cumulative SCC incidence was adjusted for competing risk of death. Cox regression was used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) adjusted for age and sex. A total of 5,470 participants were screened for MBL and included 949 (17%) with LC-MBL, 63 (1%) with HC-MBL, and 4,458 (81%) controls (negative for MBL). Individuals with HC-MBL (median age 75 years) or LC-MBL (median age 73 years) were significantly older (P<0.001) than controls (median age 66 years). There were 41 (65%) males among HC-MBL, 447 (47%) among LC-MBL, and 1,551 (35%) among controls. Prior skin cancer history was highest among HC-MBLs (N=17, 27%) or LC-MBLs (N=236, 25%) compared to controls (N=767, 17%). After a median follow-up of 18 months (range 0-138), 154 of the 5,470 individuals were identified to have incident SCC following MBL screening. At least one SCC was observed in 3 individuals with HC-MBL, 33 individuals with LC-MBL, and 118 controls. The 5- and 10-year cumulative incidence of SCC in individuals with MBLs was 7% and 16%, respectively; control estimates were 4% and 8%, respectively. However, after adjusting for age and sex, we observed no evidence of an association between MBL and risk of incident SCC (HR=0.95, CI=0.65-1.40, P=0.80), nor when we stratified individuals by age or by sex (all P>0.05). In the largest MBL screening cohort to date, individuals with MBL do not have an increased risk of incident SCC compared to controls. In contrast to individuals with CLL, these individuals with screening MBL do not need increased dermatologic examination for skin cancer, which is important given the high prevalence of MBL (18% of the population above age 40). Citation Format: Ahmed Nadeem Ansari, Sara J. Achenbach, Sameer A. Parikh, Geffen Kleinstern, Aaron D. Norman, Kari G. Rabe, Connie E. Lesnick, Timothy G. Call, Janet E. Olson, James R. Cerhan, Neil E. Kay, Celine M. Vachon, Esteban Braggio, Curtis A. Hanson, Tait D. Shanafelt, Christian L. Baum, Susan L. Slager. Incidence of squamous cell carcinoma (SCC) in a large screening cohort of monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5256.

Abstract Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.