Kay, Neil E.

Abstract The treatment of Chronic lymphocytic leukemia (CLL) has been revolutionized in recent years, however CLL is still incurable, and the leukemic cells often develop drug resistance. Previous studies show that the interaction of CLL cells with the bone marrow (BM) microenvironment promotes spontaneous and drug induced survival. But the nature of this interaction is still in need of a full understanding. To pursue this we discerned RNA profiles using RNA-seq in paired CLL cells isolated from untreated blood and BM (n=6) and in untreated patients CLL cells (n=4) cultured alone or cocultured with BM stromal cells (BMSCs). CLL cells from blood/BM and CLL cells cultured alone or with BMSCs were examined by Western blot (WB), untargeted metabolomics (LCMS+GCMS) and preclinical drug sensitivity assays. We found upregulation of 232 genes in CLL cells from BM vs the paired blood and 917 genes in cocultured CLL cells compared to CLL cells cultured alone (p&lt0.05, fold change &gt1.5). Here we detected only 13 genes that overlap between BM and cocultured CLL cells. When we analyzed the expression of these 13 genes in blood CLL cells from a cohort of 162 untreated patients by RNA-seq we found a positive relationship between 4 (PNP, C16orf54, MOB3A, CDK2AP2) with CLL patient clinical outcome including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Out of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine salvage pathway, showed the most significant association for patient’s OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine salvage pathway metabolites adenosine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis using blood CLL cells from untreated patients showed a variable PNP protein expression (high, low/no PNP) and induction in PNP protein levels were found in blood CLL cells expressing low/no PNP only when in contact with BM/BMSCs. But PNP inhibitor forodesine did not show any significant killing in high vs low/no PNP group. When we treated CLL cells from untreated patients with Bcl2 inhibitors venetoclax, S55746 and LP-118 (Bcl-2/Bcl-xl inhibitor, Newave), CLL cells showed increasing sensitivity to the Bcl2 inhibitors associated with their PNP expression (high&gtlow&gtno PNP). But a covalent and non-covalent BTKi LP-168 (Newave) cultured with CLL cells showed no difference in drug sensitivity associated with CLL PNP levels. In contrast CLL cells when treated with both venetoclax and LP-168, showed more drug sensitivity in the high PNP expressing CLL cells vs low/no PNP. These results indicate that active purine metabolism in CLL cells can contribute to differential novel agent drug responses of CLL patients. Future studies to understand the exact mechanism of how PNP relates to this differential drug sensitivity should be instructive in regard to alternate maneuvers to treat CLL. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Yi Chen, Fenlai Tan, Stephen P. Anthony, Yu Chen, Bing Dai, Yue Shen, Neil E. Kay. Role of purine metabolism in CLL cell pathobiology and CLL disease progression [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 297.

Abstract Chronic Lymphocytic Leukemia (CLL) exhibits a substantial familial risk that extends beyond CLL to other B-cell lymphomas (including follicular lymphoma and diffuse large B-cell lymphoma). Monoclonal B-cell Lymphocytosis (MBL), a precursor to CLL, is also associated with a significant risk for hematologic malignancies overall and for lymphoid malignancies, specifically. Moreover, previous research has shown an association between the CLL polygenic risk score (CLL-PRS) and common lymphoma subtypes. This study aims to further evaluate CLL-PRS associations with hematological cancer overall and by lineage while also considering MBL status a strong risk factor for lymphoid malignancies. Utilizing the Mayo Clinic Biobank, we selected individuals aged 40 years or older, residing in proximal counties, who had no prior hematologic malignancies, and with available biospecimens. MBL screening was done using 8-color flow cytometry, and MBL with CLL (CD5+/CD20dim) or atypical CLL (CD5+/CD20+) immunophenotype were included. CLL-PRS was calculated from 41 known susceptibility variants, with weights being the log of the odds ratios from the largest CLL genome-wide association study. Incident hematologic malignancies were ascertained through ICD codes and confirmed via medical record review. Hazard ratios (HR) and 95% confidence intervals (CIs) for associations with hematologic malignancies were estimated through Cox regression. Of 7,332 participants, 1,150 (16%) screened positive for MBL; median age was 67 years (range 40-101), and 37% male. Median follow-up was 2.9 years (range 0-13), and 56 hematologic cancers were identified. After adjusting for age and sex, the CLL-PRS was associated with increased risk of developing hematologic (HR=1.49, 95%CI=1.12-1.98, P=0.006) which was all attributable to lymphoid cancers (HR=1.79, 95%CI=1.26-2.56, P=0.001). When adjusting for baseline MBL status in the models, the CLL-PRS associations remained significant but were attenuated for hematological (HR=1.33, 95%CI=0.99-1.79, P=0.058) and lymphoid (HR=1.56, 95%CI=1.07-2.26, P=0.020) malignancies, while MBL status was also significantly associated with risk of hematologic (HR=2.76, 95%CI=1.55-4.91, P=0.001) and lymphoid (HR=3.61, 95%CI=1.78-3.32, P<0.001) malignancies. In this prospective cohort study of individuals screened for MBL, CLL-PRS was associated with an elevated risk of hematologic malignancies overall and within the lymphoid lineage, underscoring a shared genetic etiology across many B-cell lymphomas. However, the associations between CLL-PRS and hematologic malignancies were attenuated when accounting for MBL status or when analyzing among MBL-positive individuals, highlighting the role of MBL status in these associations. Further research with expanded cohorts is needed to unravel these complex genetic and clinical interactions. Citation Format: Raphael Mwangi, Dennis P. Robinson, Sara J. Achenbach, Geffen Kleinstern, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie Griffin, Nicholas J. Boddicker, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, James R. Cerhan, Tait D. Shanafelt, Susan L. Slager. Genetic associations with hematologic malignancies: An evaluation of CLL polygenic risk score and MBL status in the Mayo Clinic Biobank Cohort [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 6152.

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [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 3841.

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [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 3849.

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [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 5842.

Abstract High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Abstract Monoclonal B-cell lymphocytosis (MBL) progresses to chronic lymphocytic leukemia (CLL) requiring therapy at 1% to 5% per year. Improved prediction of progression would greatly benefit individuals with MBL. Patients with CLL separate into 3 distinct epigenetic subtypes (epitypes) with high prognostic significance, and recently the intermediate epitype has been shown to be enriched for high-risk immunoglobulin lambda variable (IGLV) 3-21 rearrangements, impacting outcomes for these patients. Here, we employed this combined strategy to generate the epigenetic and light chain immunoglobulin (ELCLV3-21) signature to classify 219 individuals with MBL. The ELCLV3-21 high-risk signature distinguished MBL individuals with a high probability of progression (39.9% and 71.1% at 5 and 10 years, respectively). ELCLV3-21 improved the accuracy of predicting time to therapy for individuals with MBL compared with other established prognostic indicators, including the CLL international prognostic index (c-statistic, 0.767 vs 0.668, respectively). Comparing ELCLV3-21 risk groups in MBL vs a cohort of 226 patients with CLL revealed ELCLV3-21 high-risk individuals with MBL had significantly shorter time to therapy (P = .003) and reduced overall survival (P = .03) compared with ELCLV3-21 low-risk individuals with CLL. These results highlight the power of the ELCLV3-21 approach to identify individuals with a higher likelihood of adverse clinical outcome and may provide a more accurate approach to classify individuals with small B-cell clones.

Despite notable advancements in treating chronic lymphocytic leukemia (CLL) with targeted therapies, a significant clinical challenge arises as a subset of CLL patients progress and develop Richter's transformation (RT), an aggressive form of lymphoma. While immune cell dysfunction and T cell defects are well-known characteristics of CLL, the alterations in immune cell subsets during CLL progression to RT remain largely unexplored. While CLL patients typically fail to respond to anti-PD1 pembrolizumab, a positive response has been observed in 20-40% of RT patients. Previously, our study involving three RT patients (1 responder and 2 progressors) revealed enriched regulatory T cells (Tregs) in the responders compared to progressors after pembrolizumab treatment (Iyer et al, ASH2020). Hence, we propose that dynamic changes in T cell subsets at baseline may be associated with response to pembrolizumab treatment. To this end, we performed single-cell RNA sequencing on sorted T cells (CD3 +) from PBMCs derived from 20 patients (responders (RES), n=5; stable disease (SD), n=5; progressive disease/progressors (PD), n=10). A total of 408,627 cells were captured, and an average of 1,393 genes were detected per cell per patient. Using a graph-based clustering approach (Seurat V4.3), we identified 19 clusters based on marker genes and transcriptional signatures and identified enriched pathways using Enrichr analysis. Our results showed that RES had an increased Th17 subset at baseline compared to PD (15% vs. 5%, p=0.01) and reduced Th2 T cells compared to SD (p=0.004) and PD (p=0.05). The Th17/Th2 ratio was higher in RES at baseline (Th17/Th2 R: 1.24, PD: 0.21, SD: 0.25, p0.05) and post-pembrolizumab (Th17/Th2 RES: 1.31, PD: 0.34, SD: 0.30, p0.05). Furthermore, RES had an increased fraction of proliferating CD4 T cells at baseline (4% RES vs. 0.4% SD and 0.4% PD, p=0.02) and post-treatment (1.9% RES vs. 0.8% PD and 0.34% SD, p=0.02). Although we observed an increasing trend in the fraction of Tregs in the RES at baseline [(7.6% RES vs.6.1% PD and 6.5% SD, p=0.4; ns) and post-treatment (10.3% RES vs. 8.3% PD and 5.8% SD, p= 0.2; ns] compared to PD and SD, the difference was not significant. Similarly, among CD8+ T cells, we observed an increasing trend in CD8+ effector T cell fraction in the responders at baseline (29.8% RES vs. 21% PD and 15.6% SD, p=0.9, ns) and post-treatment (30.1% RES vs. 21.2% PD and 19.9% SD, p=0.44, ns). However, the difference was not statistically significant. These findings highlight the dynamic changes in T cell subsets associated with pembrolizumab response. In terms of transcriptional changes in Tregs following pembrolizumab treatment among RES, PD, and SD, we observed dynamic changes on a few Treg marker genes, including TIGIT and TOX. TIGIT and TOX expression drastically decreased after treatment in RES (p < 0.0001), while expression did not change in PD and SD. There were no significant changes in the expression of FOXP3 or CD27 in RES, PD, and SD before and after the treatment. In CD8 effector T cells, GZMK, GZMA (granzymes), and PRF1 (perforin) are marker genes. We observed higher expression of GZMK and GZMA in RES (p <0.0001) at the baseline compared to SD and PD. In the proliferating CD4 T cells, we observed increased expression of IDH2 (Isocitrate dehydrogenase 2-key enzyme of TCA cycle) in RES at the baseline, as compared to PD and SD, indicating increased TCA cycle activity in the proliferating T cells. Pathway analysis of T cell clusters using the Enrichr database showed TNFA-IFNG-MYC targets are enriched in CD8 + cells, IL2-STAT5 signaling is upregulated in Th17, TREGs, and E2F cell cycle associated pathways enriched in CD4 + proliferation cluster. In conclusion, our study unveils significant baseline differences in CD4 subsets between RES and PD/SD subjects and dynamic changes in T cell subsets and transcriptional signatures. These findings offer potential predictors of pembrolizumab response in RT, such as increased Th17/Th2 ratio, enriched proliferating CD4 + T cells, and elevated TIGIT, TOX gene expression in Tregs, GZMK, and GZMA in CD8 effector cells. This deeper understanding of the immunological landscape in RT patients sheds light on the interplay between T cell subsets and treatment response in RT, paving the way for personalized therapeutic strategies. Further validation and exploration can lead to improved outcomes and tailored interventions for RT.

INTRODUCTION Richter transformation (RT) is the histologic transformation of CLL/SLL into an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). We previously reported an incidence of 0.5% per year of RT in patients with newly diagnosed CLL and an incidence of 1% per year following initiation of therapy (Parikh, BJH, 2013). It is unknown whether these incidences have changed in the novel agent era. METHODS We identified patients with previously untreated CLL/SLL in the Mayo Clinic CLL Database who were seen within 12 months of diagnosis. Only patients with biopsy-proven DLBCL with histopathology confirmation at Mayo Clinic were included. Cumulative incidence methodology was used to display the time to development of RT with death as a competing risk (both from time of initial CLL/SLL diagnosis, and from start of CLL directed therapy). We defined the time period prior to February 2014 (FDA approval of ibrutinib for CLL) as the pre-novel agent era and the time period starting February 2014 as the novel agent era. Using Cox regression analysis, we compared the incidence of RT, both after initial CLL diagnosis and after initiation of CLL therapy, between patients diagnosed with CLL in the pre-novel agent era versus the novel agent era. RESULTS Between 1/2000 and 4/2023, 3347 patients were seen at Mayo Clinic. The median age at diagnosis was 65 years (range 21-97 years), and 66% patients were male. Baseline characteristics of patients at the time of CLL and RT diagnoses between the two treatment eras were similar, including distance in miles of primary residence from Mayo Clinic ( Table 1). The median follow-up for the entire cohort was 5.6 years (9.5 years for the pre-novel agent era cohort and 3.2 years for the novel agent era cohort). A total of 82 patients were diagnosed with RT (69 in the pre-novel agent era and 13 in the novel agent era). Overall, the 1-, 5- and 10-year cumulative incidence rates for RT from the time of CLL/SLL diagnosis were 0.6% (95% CI: 0.4-0.9%), 1.8% (95% CI: 1.3-2.3%), and 3.0% (95% CI: 2.4-3.8%), respectively. Among the patients who received CLL/SLL therapy (n=1382), the 1-, 5-, and 10-year incidence rates for RT from the time of treatment start were 0.3% (95% CI: 0.1-0.8%), 3.3% (95% CI: 2.4-4.5%), and 4.9% (95% CI: 3.7-6.5%), respectively. Among 1981 patients diagnosed with CLL/SLL in the pre-novel agent era, the 1- and 5-year incidence rates for RT from the time of diagnosis were 0.6% (95% CI: 0.4-1.1%) and 2.1% (95% CI: 1.5-2.8%), respectively. Among 1366 patients diagnosed with CLL/SLL in the novel agent era, the 1- and 5-year incidence rates following CLL/SLL diagnosis were 0.5% (95% CI: 0.2-1.1%) and 1.1% (95% CI: 0.6-2.2%), respectively. The RT incidence from time of CLL/SLL diagnosis was not significantly different between the pre-novel agent and novel agent era patients (hazard ratio [HR] 1.6, 95%CI 0.9-3.0, p=0.13). Patients who received CLL/SLL treatment in the pre-novel agent era (n=926) and novel agent era (n=456) had subsequent 1-year incidence rates of RT of 0.3% (95% CI: 0.1-1.0%) and 0.2% (95% CI: 0.0-1.8%), respectively. The 5-year incidence rates for these groups were 3.7% (95% CI: 2.6-5.2%) and 1.9% (95% CI: 0.7-5.0%), respectively. Although an increased risk of RT after CLL/SLL directed therapy was observed in the pre-novel agent era compared to the novel agent era (HR 2.4, 95%CI: 0.9-6.1, p=0.07; Figure 1), this did not cross the threshold of statistial significance. Among patients diagnosed in the pre-novel agent era who developed RT (n=69), CLL treatment prior to RT included: none, n=19 (28%); chemoimmunotherapy only, n=36 (52%); and novel agent, n=14 (20%; includes 11 treated with prior chemoimmunotherapy also). In contrast, among patients diagnosed in the novel agent era who developed RT (n=13), CLL treatment prior to RT included: none, n=8 (62%); and novel agent, n=5 (38%). CONCLUSIONS Patients who received CLL/SLL directed treatment appear to have a lower risk of RT in the novel agent era. This may be driven by the fact that none of the patients who developed RT in the novel agent era received chemoimmunotherapy. Whether novel agent use for CLL/SLL suppresses the development of RT or avoids the risk associated with cytotoxic chemotherapy-induced clonal evolution requires further study. Although limited by length of follow-up, our findings challenge the notion that RT incidence will increase in parallel with CLL patients living longer with novel agent treatments.

Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor condition to chronic lymphocytic leukemia (CLL) characterized by the presence of circulating monoclonal B-cell populations in the peripheral blood of otherwise healthy individuals. MBL increases risk of several adverse clinical outcomes, including development of hematological malignancies. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis which affect large segments of DNA and include gains, losses, and copy number neutral loss of heterozygosity events. mCAs can be detected from SNP-array genotyping. Prior work has suggested mCAs as a potent risk factor for hematological malignancies. However, the nature of the relationship between mCAs and MBL is unknown. We sought to determine 1) the association between mCAs and MBL and 2) their combined effect on risk of hematological malignancies, in a large, prospectively collected, and well-annotated cohort of individuals with known MBL, mCA, and clinical outcomes. Methods: We examined the prevalence of mCA in a cohort of individuals from the Mayo Clinic Biobank who were screened for MBL using a sensitive eight-color flow cytometry assay. We classified MBL by the size of the clone relative to total B-cells, with high count (HC) defined as those individuals who had a percent clonal B-cell count ≥85% out of total B-cell count and low-count (LC) otherwise. Autosomal mCAs were detected from whole blood DNA collected at the time of MBL screening using a SNP-array-based assay. The MoChA pipeline was used to identify mCA events. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). To examine incident hematological cancers, we identified and validated all hematological cancers through medical record abstraction using the International Statistical Classification of Diseases codes. Cox regression was used to calculate hazard ratios (HR) and 95% CIs, stratified by MBL status or autosomal mCA status. All models were adjusted for age and sex. Results: We evaluated the prevalence of autosomal mCA in 3,764 individuals (792 individuals with MBL of whom 64 had HC-MBL and 2,972 without MBL). The prevalence of autosomal mCA was 7.7% ( n=288) with a median mCA cell fraction of 0.09 (range 0.01-1). Among those with and without MBL overall, the prevalence of mCA was 15.5% ( n=123) and 5.6% ( n=165), respectively (Figure 1). Compared to those without an autosomal mCA, individuals with an autosomal mCA were 2.4-fold more likely to have MBL (95% CI 1.9-3.2; P =2.0x10 -11; Figure 1). CLL-associated mCA (i.e., those commonly altered in CLL including trisomy 12 and deletion of 6q, 11q, 13q, and 17p) were present in 42 individuals, of whom 38 (90.5%) had MBL overall and 30 (71.4%) individuals had HC-MBL. Compared to those without a CLL-associated mCA, individuals with a CLL-associated mCA were 539.39-fold more likely to have HC-MBL (95% CI 190.3-1975.9; P <2.0x10 -16) and 6.7-fold more likely to have LC-MBL (95% CI 2.0-26.5; P =0.0031; Figure 1). Next, we evaluated the relationship between MBL and mCA with risk of developing a hematologic malignancy in 3,009 individuals with follow-up data (median 10 years). Forty-one individuals developed an incident hematological malignancy (68.3% lymphoid). When we evaluated the effect of autosomal mCA stratified by MBL status we observed elevated but non-significant risk of hematological malignancies (Figure 2). When we evaluated the effect of MBL stratified by autosomal mCA status we observed elevated and significant increased risk of developing a hematological malignancy (Figure 2). Conclusions: We found that the presence of an autosomal mCA is highly associated with having MBL. In particular, >90% of CLL-associated mCA carriers had MBL with ~7- and ~540-fold increased association for LC- and HC-MBL, respectively. These results suggest that almost all individuals in large biobanks with CLL-associated mCAs have undiagnosed MBL. However, not all individuals with MBL have an mCA. When evaluating risk of hematological malignancies, we found autosomal mCAs had >2-fold elevated but non-significant effects when stratified by MBL status while MBL remained a significant risk factor regardless of autosomal mCA status. Collectively these results support that MBL remains an important risk factor and suggests that mCAs hold potential to identify a subgroup of individuals at greater risk of developing a hematological malignancy.