Citations

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 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.

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.

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value <0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value<0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p<0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p<0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

INTRODUCTION We aim to identify factors that impact outcomes of venetoclax for patients with CLL treated in routine practice at a tertiary center. We report on the use of venetoclax in the most frequently encountered disease scenarios: first-line, relapsed/Bruton tyrosine kinase inhibitor (BTKi)-naïve, and relapsed/BTKi-exposed. METHODS We identified patients who received venetoclax therapy for CLL (between 4/2012-4/2023) from the Mayo Clinic CLL Database. Undetectable measurable residual disease (uMRD) was defined as <1 CLL cell per 10,000 leukocytes using 8-color flow cytometry on peripheral blood (PB) or bone marrow (BM). Overall survival (OS) was defined as the time from venetoclax start until date of death or last known to be alive. Treatment-free survival (TFS) after venetoclax was defined as the time from venetoclax start until the earliest of date of next treatment, or death. Kaplan-Meier was used to display OS and TFS. Multivariable Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS A total of 155 patients received venetoclax: firstline therapy (in combination with obinutuzumab, n=55) and relapsed CLL (n=100; 17 had relapsed/BTKi-naïve CLL, and 83 had previously received BTKi [55 with progression after BTKi], relapsed/BTKi-exposed). The median follow-up for the cohorts of first-line therapy, relapsed/BTKi-naïve, and relapsed/BTKi-exposed was 12.9 months, 37.0 months, and 27.6 months, respectively. Baseline characteristics at the time of venetoclax initiation for all patients are shown in Table 1. The median TFS for the overall cohort was 39.0 months. The median OS was 54.6 months. Among patients treated with venetoclax as first-line therapy (n=55), the 2-year TFS ( Figure 1) and 2-year OS rates were both 91%. MRD testing was performed in 28 patients and was uMRD in 23 (82%) patients (only PB assessed, n=7; only BM assessed, n=2; PB and BM assessed, n=14). Detectable MRD was identified in 3 patients, and 2 patients had discordant results (PB uMRD and BM detectable disease). Among patients treated with venetoclax in the relapsed/BTKi-naïve setting (n=17), the 2-year TFS rate was 73% ( Figure 1) and the 2-year OS rate was 100%. MRD testing was performed in 7 patients and was uMRD in all 7 (100%) (only PB assessed, n=3; only BM assessed, n=2; PB and BM assessed, n=2). The median time to first uMRD result was 14.1 months. Among relapsed/BTKi-exposed venetoclax-treated patients (n=83), the median TFS was 26.9 months ( Figure 1), and the median OS was 39.4 months.In this subgroup, the median TFS for patients with (n=55) and without (n=28) prior disease progression on prior BTKi were 22.3 and 42.3 months, respectively. Median TFS with venetoclax monotherapy (n=30) was 24.0 months, venetoclax in combination with rituximab (n=37) was 26.9 months, and venetoclax in combination with obinutuzumab (n=16) was 39.0 months. BTKi-exposed patients that were chemotherapy-naïve (n=27) and chemotherapy-exposed (n=56) had median TFS of 29.1 and 24.0 months, respectively. MRD testing was performed in 28 patients and was uMRD in 16 (57%) patients (only PB assessed, n=9; only BM assessed, n=2; PB and BM assessed, n=5). Detectable MRD was identified in 11 patients, and 1 had discordant results (PB uMRD and BM detectable disease). The median time to first uMRD result was 11.5 months. TP53 disruption, unmutated IGHV genes, older age, complex karyotype (CK; defined as more than 3 chromosomal aberrations on CpG stimulated karyotype), and disease progression on prior BTKi were associated with shorter TFS in the overall cohort on univariate analysis. TP53 disruption, older age, CK, and disease progression on prior BTKi were associated with shorter OS in the overall cohort on univariate analysis. Multivariable analysis was performed by including only those patients where all variables significant in univariable analysis were available (OS model n=65, TFS model n=53). In these models, only CK was significantly associated with shorter TFS (HR 8.5; 95%CI 2.5-29.1; P<0.001) and shorter OS (HR 4.1; 95%CI 1.2-14; P=0.03). CONCLUSIONS Patients with BTKi-exposed CLL, particularly those with prior disease progression on BTKi, had worse outcomes. Our study identified CK as one of the most important baseline predictors of adverse TFS and OS in the overall cohort of patients, supporting karyotype assessment for prognostication prior to venetoclax treatment.

Introduction Multiple myeloma (MM) patients often become unresponsive to immunomodulatory drugs (IMiDs) over time. Research has indicated that the cellular ability to counteract oxidative stress plays a crucial role in determining the sensitivity to IMiDs in MM. This study aims to comprehend the metabolic pathways responsible for MM's antioxidative capability and develop new treatments to augment the effectiveness of IMiDs. Methods We experimented using MM cells to investigate how different nutrient conditions affect cell proliferation and drug response. We utilized stable isotope-labeled amino acids and mass spectrometry to analyze the metabolites in these cells. We evaluated cell viability through the MTT assay, the CellTiter-Glo® Luminescent Assay, and direct cell counting. Furthermore, we conducted Western blots to measure the levels of various proteins expressed in MM cells grown under different nutrient conditions. To create CRBN knockout cells, we employed the CRBN CRISPR/Cas9 KO plasmid, and for the overexpression of CRBN in MM cells, we used a lentivirus system. Results Our research findings reveal that the production of cellular cysteine enhances the cell's ability to counteract oxidative damage, leading to resistance to IMiDs even with the presence of the wild-type CRBN protein. However, MM cells not producing enough cysteine rely on consuming extracellular cystine, which increases sensitivity to lenalidomide and reduces antioxidative capacity. Lenalidomide-sensitive cells acquire more cystine from the extracellular environment than resistant cells. The analysis of cysteine uptake in various MM cell lines using C13-labeled cysteine revealed that the lenalidomide-sensitive cell line, OCIMY5-CRBN, consumed more cysteine from the media, and the presence of L-glutamine further increased this uptake. Lenalidomide treatment increased the catabolism of cysteine into taurine in sensitive cell lines, confirming the link between lenalidomide's molecular action and cysteine catabolism. Our study also identified that lenalidomide treatment led to an accumulation of CDO protein, which adds molecular oxygen to cysteine, transforming it into cysteine sulfinic acid. Our study suggests that CRBN may play a role in maintaining intracellular CDO protein levels, and the accumulation of CDO protein induced by lenalidomide is the molecular factor responsible for cystine catabolism and sensitivity to IMiDs in multiple myeloma. We found that by inhibiting intracellular cysteine biosynthesis, we could improve the effectiveness of IMiD against MM. One drug that can achieve this is 5-aza-2-deoxycytidine (DAC), which acts as a DNA methylation inhibitor. Additionally, combining DAC with IMiDs improves their effectiveness, even in cell lines that are resistant to treatment. Furthermore, we attempted to inhibit cysteine biosynthesis by promoting the synthesis of methionine from homocysteine with the supplementation of 5-methyltetrahydrofolate. We discovered that treatment with 5-methyltetrahydrofolate further enhanced the sensitivity of IMiDs in resistant MM cell lines. Our results confirm that interfering with intracellular cysteine biosynthesis is a promising therapeutic approach for MM and can improve the effectiveness of IMiDs. Conclusions The biosynthesis of L-Cysteine is vital for the cellular ability to resist oxidation. Cancer cells that create high levels of cysteine within the cell from methionine generate a more significant amount of reduced cysteine, which can act as antioxidants and make MM cells resistant to IMiDs. On the other hand, cells that cannot produce enough cysteine increase their consumption of extracellular cystine and are more susceptible to IMiDs. Cells that consume an oxidized form of cystine from outside the cell require a reduction process inside the cell, which further reduces their ability to resist oxidation. Additionally, the accumulation of CDO protein induced by lenalidomide can enhance the breakdown of intracellular cysteine and cystine uptake in vulnerable cells. Overall, MM cells that rely on extracellular cysteine are sensitive to IMiDs. Therefore, interfering with intracellular cysteine biosynthesis is a better option for myeloma treatment and enhancing the sensitivity of IMiDs.

Presence of deletion17p (del17p) in frontline (1L) and relapsed/refractory (R/R) CLL has been associated with shorter time to treatment initiation, early relapse after therapy and inferior overall survival (OS) (Mato et.al., Haemotologica 2022). However, there exists notable clinical heterogeneity in terms of outcome within this group (Tam et.al., Blood 2009, Stephens et.al; AJH 2015). Previous reports in 1L and R/R CLL have identified percentage of del17p nuclei by fluorescence in situ hybridization (FISH), having TP53 mutations on the second allele, copy number alterations and complex karyotype (CK) as additional risk factors within the del17p population (Yu et al., CCR 2017; Baliakas et al., Blood 2019). Here we profile a cohort of wait and watch (W&W, n=14), 1L treated (n=36) and R/R CLL (n=3) patients by whole genome sequencing (WGS) and RNA sequencing (RNAseq) to gain insights into genomic factors and gene expression pathways contributing to the high-risk nature of del17p across the spectrum of CLL patients. Using the Mayo Clinic CLL Database, we identified 53 patients with del17p (identified by FISH testing in routine clinical practice at diagnosis) who had stored peripheral blood mononuclear cells (PBMCs) available. RNA and DNA from matched peripheral blood (tumor) and DNA from CD19- (germline) samples at diagnosis (n=50) and relapse (n=3) was extracted using Qiagen RNeasy (RNA) and Purene (DNA) kits and subjected to RNAseq and WGS respectively. Additional FISH assays (del11q, trisomy 12, del13q) and CK by chromosomal banding analysis (CBA) were performed. Baseline clinical characteristics, time to first treatment, types of treatment, and survival were abstracted from the CLL Database. The Mayo Clinic IRB approved this study. Copy number analysis identified deletions in 3p, 4p, 8p, 9p, 9q and gains in 2p, 8q, 15q and 17q chromosomal lesions occurring in patients. We explored the association of clinical and genetic factors, and genomic instability signatures with survival to identify high-risk features in del17p patients. Both total number of deletions by WGS and number of chromosomal aberrations by CBA as a continuous variable were significantly associated with unfavorable OS in the entire cohort (n=53) in a univariate analysis (Hazard ratio, HR= 2.29, p= 0.00054 and HR= 2.33, p=0.0054 respectively). However, there was no significant association of number of gained genes by WGS or aneuploidy scores with survival (HR=0.74, p=0.37). In patients with CK data (n=41), deletion burden, but not gains, were highly correlated to chromosomal aberrations by CBA. Using cutpointr package for identifying deletion threshold corresponding to CK≥ 3, we defined patients with deletion burden ≥ 514 as enhanced-high risk (EHR; median OS = 4.83 years) compared to the group with high-risk (HR; median OS=9.94 years) (p=0.025). There was no significant difference in number of deleted genes between WW and treated patients. The EHR group is enriched in patients with high clonal cell fraction of del17p by FISH, unmutated IGHV and CK≥ 3. The EHR group remains significant in a multi-variable analysis with IGHV-mutation status, ZAP70, and age (HR: 5.16, 95% CI: 1.04 - 25.6, p=0.04). Mutational analysis identified 35 known CLL driver mutations with TP53 (86%), ATM (15%), NOTCH1(13%), MGA (13%), CHD2 and MED12(11%) as the top mutated genes. Total number of driver genes mutated was not significantly different between EHR and HR subgroups, but the EHR subset was significantly enriched with mutations in NOTCH1 (21%), and MGA (21%) as compared to no mutations in the HR group in these genes. Deleted genes (≥ 514) was prognostic in bi-allelic TP53 aberration patients as well (p=0.021) Differential expression gene-set enrichment analysis indicated a significant enrichment of DNA repair, cellular response to DNA damage and downregulation of inflammatory responses in the EHR group. CK is a known prognostic marker which includes both chromosomal losses and gains. In this small cohort of del17p we were able to show the prognostic value of deletions only, but not gains, as captured by WGS. Using genome wide sequencing, we identify increasing genomic deletions as a feature of enhanced-high risk del17p. While deletion burden cut-offs identified here are specific to our research method and require further validation in additional independent cohorts, EHR subgroup remained significant after adjusting for other known prognostic variables .

INTRODUCTION Chronic lymphocytic leukemia (CLL) and SLL represent a single entity in current classifications per the WHO-HAEM5 and ICC. Both are characterized by malignant B cells with an identical immunophenotype, but they are distinguished by the predominant site of disease burden, such as peripheral blood for CLL and nodal compartments for SLL. Most clinical trials and retrospective studies involving CLL/SLL do not separate out SLL patients. In this study, we report reasons for treatment initiation and outcomes in a cohort of SLL patients seen at our institution. METHODS Using the Mayo Clinic CLL Database, we retrospectively identified patients with SLL who received first line therapy between 12/1995 and 2/2023. Patients were classified as having SLL if they met at least one of the following criteria for diagnosis: 1) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute clonal B cell count of < 5 x 109/L; or 2) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute lymphocyte count of < 11 x 109/L (if absolute clonal B cell count was not available). Although the presence of anemia (hemoglobin < 11 g/dL) and thrombocytopenia (platelets < 100 x 109/L) are considered as exclusion criteria for a diagnosis of SLL, patients were included if these cytopenia were due to autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) with < 50% bone marrow involvement by SLL (bone marrow biopsy confirmed). Patients treated for SLL with extranodal involvement were also included. Indications for SLL treatment were abstracted by chart review and included one of the following: 1) symptomatic or progressive lymphadenopathy and/or splenomegaly, 2) autoimmune cytopenias, 3) extranodal/direct organ infiltration; and 4) other (e.g., cryoglobulinemia, paraneoplastic, non-infiltrative renal disease such as minimal change disease). Overall survival (OS) was defined as the time from treatment start until date of death or last known to be alive. Treatment-free survival (TFS) was defined as the time from treatment start until the earliest of date of next treatment, or death. Kaplan-Meier method was used to display OS and TFS. Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS We identified 182 patients with SLL who received first line treatment. The median age at treatment start was 66 years, and 132 (73%) were male. Unmutated IGHV was identified in 60/82 (73%) patients and peripheral blood fluorescent in situ hybridization (FISH) assessed pre-treatment start showed del13q in 10%, trisomy 12 in 43%, del11q in 7%, del17p in 8%, and no abnormalities in 33%. TP53 disruption (del17p and/or TP53 mutation) was present in 7/89 (8%) patients prior to first therapy. The median follow-up after diagnosis was 6.9 years. The indications for treatment were grouped as follows: symptomatic or progressive lymphadenopathy/splenomegaly (n=130), extranodal/direct organ infiltration (n=30), AIHA/ITP (n=10), and other (n=12). For the entire cohort, the median TFS was 2.9 years and the median OS was 12.3 years. The median TFS by the treatment indication groups were 3.3, 5.1, 1.6, and 5.3 years, respectively (Figure 1). The median OS by treatment indication groups were 11.1, 10.5, 8.8, and 19.9 years, respectively. Among the 160 patients with symptomatic or progressive lymphadenopathy/splenomegaly or extranodal disease, the treatments administered include: mAb alone (n=35), chemotherapy alone (n=33), chemoimmunotherapy (n=57), targeted agent (n=33; Bruton tyrosine kinase inhibitor [BTKi] based n=28; venetoclax-based n=3, BTKi + venetoclax n=2), and other (n=2). The median TFS by treatment groups were 2.4, 2.0, 3.8, and 6.0 years (Figure 2), respectively (excluding the small “other” subgroup of 2 patients). The median OS for these groups were 9.5, 9.7, 15.0, and not evaluable, respectively. Only older age at treatment start predicted for shorter TFS (HR 1.02; 95%CI 1.002-1.038; p=0.03) and shorter OS (HR 1.05; 95%CI 1.03-1.07; p<0.01). CONCLUSIONS In this study of patients with SLL who received therapy, the most frequent indications for treatment included symptomatic lymphadenopathy and/or splenomegaly, followed by extranodal disease. Trisomy 12 was the most frequently detected abnormality on peripheral blood FISH testing. Treatment with novel agents achieved durable TFS and OS.