Citations

Abstract Background Mosaic chromosomal alterations (mCAs) are large-scale somatic gains, losses, or copy-neutral loss of heterozygosity (CNLOH) detectable by genotyping of peripheral blood DNA. They represent a form of clonal hematopoiesis and are observed in 0.5-5% of cancer-free individuals. The presence of any autosomal mCA has been associated with >3-fold risk of hematological malignancies. In particular, mCAs occurring in regions related to lymphoid malignancies have an 11-fold increased risk of lymphoid malignancies and a 68.6-fold risk for chronic lymphocytic leukemia (CLL). Monoclonal B-cell lymphocytosis (MBL), the precursor state to CLL, is defined by <5×10⁹/L clonal B cells in otherwise healthy individuals and is classified as low-count (<0.5x109/L) or high-count (0.5-5x10⁹/L) based on clonal B-cell count. MBL is also associated with increased risk of lymphoid malignancy (beyond CLL), with 4.3-fold and 74-fold risk in individuals with low- and high-count MBL, respectively. We previously reported a significant enrichment of autosomal mCAs in MBL, demonstrating a 1.7-fold association in those with low-count MBL and a 50.5-fold association in those with high-count MBL. Whether mCAs independently predict lymphoid malignancy risk after accounting for MBL status is unknown. Here, we evaluated the impact of mCAs on risk of incident lymphoid malignancies in individuals with MBL and in individuals screened negative for MBL. Methods CLL-type MBL (CD5 positive) was identified using flow cytometry assay on peripheral blood. Participants were age ≥40, had no prior hematologic malignancy, and were enrolled through the Mayo Clinic Biobank or the Division of Hematology at Mayo Clinic, Rochester. Participants were followed for incident lymphoid malignancy, including CLL, with diagnoses confirmed by medical record review. mCAs with cell fraction >1% were detected from array-based genotyping of whole blood DNA. Cox proportional hazards models, adjusted for age and sex, estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of developing an incident lymphoid malignancy in individuals with autosomal mCA versus those without an autosomal mCA. Results In 4,331 participants with available MBL and mCA status, the median age was 67 years, and 45% were male. Flow cytometry identified 3,231 individuals who screened negative for MBL, 754 individuals with low-count MBL, and 346 individuals with high-count MBL. Autosomal mCAs were present in 6.5% of individuals without MBL, 7.6% with low-count MBL, and 71.1% with high-count MBL. Over a median follow-up of 6.9 years, 237 individuals developed an incident lymphoid malignancy. The most common types were CLL, multiple myeloma, and diffuse large B-cell lymphoma. Ignoring MBL status, the presence of an autosomal mCA was associated with increased risk of lymphoid malignancy (HR = 2.65; 95% CI: 1.45–4.86; P = 0.002), replicating previous results. However, after excluding individuals with MBL, this association attenuated; those with mCA alone did not have a significantly elevated risk (HR = 1.60; 95% CI: 0.57-4.52; P = 0.38) compared to individuals without MBL or mCA. We next examined the effect of mCAs among individuals with MBL. In those with low-count MBL, the most frequent mCA events were 13q loss (2.1%), 12 gain (1.9%), and 4q CNLOH (1.2%), and individuals with an mCA had a borderline significant risk of developing a lymphoid malignancy (HR = 2.78; 95% CI: 1.01–7.67; P = 0.049) compared to those without an mCA. Similarly, in those with high-count MBL, the most frequent mCA events were 13q loss/CNLOH (31.2%), 12 gain (13.0%), and 11q loss (4.9%), and the presence of an mCA conferred an increased risk compared to those without mCA (HR = 1.51; 95% CI: 1.09-2.08; P = 0.012). Conclusions Autosomal mCAs were more frequent in individuals with high-count MBL compared to those with low-count or no MBL. mCAs showed limited predictive value for lymphoid malignancy among individuals without MBL. In contrast, in individuals with low-count MBL, autosomal mCAs emerged as a potential biomarker for identifying those at risk of developing a lymphoid malignancy. In high-count MBL, mCAs were also a biomarker, but were driven by known chromosomal abnormalities identified through clinical testing. These findings suggest that the utility of mCAs as a predictive marker for lymphoid malignancy is contingent on the presence of MBL and may inform risk stratification and surveillance strategies in this population.

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage as morphology and cytogenetics cannot reliably distinguish them. In this study, using genomic data from 374 patients with MGUS or SMM (277 training, 97 validation), to our knowledge, we demonstrate for the first time the ability to identify malignant transformation in MGUS and SMM. We introduce the concept of genomic MM and genomic MGUS to differentiate the subsets of MGUS and SMM that are biologically malignant with genomic features indistinguishable from MM from the subset that is premalignant and unlikely to progress to malignancy. Importantly, we find that most SMM has biological features of malignant transformation indistinguishable from MM. As expected, this subset that we consider having genomic MM is associated with a high risk of progression to MM although some patients remained progression-free beyond 5 years. Conversely, 60% of MGUS and 10% of SMM have no evidence of malignant transformation (genomic MGUS), with no progression during follow-up. Integration of genomic features with the 2/20/20 International Myeloma Working Group model significantly improved the prediction of progression among genomic MM. These findings support the use of genomic criteria to refine the classification and the risk stratification in myeloma precursor conditions.

SummaryIn individuals with high‐count monoclonal B‐cell lymphocytosis (MBL), we investigated if lymphadenopathy or splenomegaly found by imaging was associated with shorter time to first chronic lymphocytic leukaemia (CLL) therapy (TTFT) and overall survival (OS). Individuals with MBL seen at Mayo Clinic (2002–2019) were retrospectively divided into three cohorts based on imaging studies within 1 year of diagnosis: no imaging studies (Cohort A); imaging with no evidence of lymphadenopathy/splenomegaly (Cohort B); imaging with evidence of lymphadenopathy/splenomegaly (Cohort C). We compared baseline characteristics, TTFT and OS across the MBL cohorts to a cohort of individuals with small lymphocytic lymphoma (SLL). A total of 1078 patients were included: 640 with MBL and 438 with SLL. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated immunoglobulin heavy chain variable region (IGHV) (43% vs. 25% p = 0.016), high‐risk fluorescence in situ hybridization (FISH) (del17p and del11q in 15% vs. 4%, p = 0.038) and higher expression of CD38 (31% vs. 16%; p = 0.011). After adjusting for sex and CLL‐International Prognostic Index (IPI), lymphadenopathy/splenomegaly was associated with a shorter TTFT (hazard ratio [HR] = 2.04, 95% confidence interval [CI]: 1.02–4.04, p = 0.042) but not OS (HR = 1.09, 95% CI: 0.62–1.92, p = 0.775). Lymphadenopathy/splenomegaly on imaging in individuals with high‐count MBL is associated with a more unfavourable risk profile and shorter time to first CLL‐directed therapy.

Abstract Monoclonal antibodies targeting CD38 are a therapeutic mainstay in multiple myeloma (MM). Although they have contributed to improved outcomes, most patients still experience disease relapse, and little is known about tumor-intrinsic mechanisms of resistance to these drugs. Antigen escape has been implicated as a mechanism of tumor-cell evasion in immunotherapy. Yet, it is unknown whether MM cells can develop permanent resistance to anti-CD38 antibodies by acquiring genomic events leading to biallelic disruption of the CD38 gene locus. Here, we analyzed whole-genome and whole-exome sequencing data from patients 701 newly diagnosed MM, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse after anti-CD38 antibodies. We report a loss of CD38 in 10 of 50 patients (20%) after CD38 therapy, 3 of whom exhibited a loss of both copies. Two of these cases showed convergent evolution in which distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that 2 variants, L153H and C275Y, decreased binding affinity and antibody-dependent cellular cytotoxicity of the commercial antibodies daratumumab and isatuximab. However, a third mutation, R140G, conferred selective resistance to daratumumab, while retaining sensitivity to isatuximab. Clinically, patients with MM are often rechallenged with CD38 antibodies after disease progression and these data suggest that next-generation sequencing may play a role in subsequent treatment selection for a subset of patients.

Abstract Extramedullary disease (EMD) is associated with an inferior prognosis and lower response rates to conventional multiple myeloma (MM)–directed therapies compared to MM without EMD. A deeper understanding of the molecular landscape and underlying drivers of EMD is essential to identify potential targets for novel therapeutic strategies. To address this, we performed whole-exome sequencing on EMD tumor tissue from 18 unique patients and bone marrow aspirates (BMAs) from 20 patients at the time of EMD development. Notably, paired EMD and BMA samples were collected from 6 patients at the point of EMD diagnosis, allowing for direct comparison of molecular profiles. Our analysis revealed a near-universal presence of mutations within the MAPK pathway in EMD samples (94%), compared to BMAs (60%; odds ratio, 10.7; P = .02; q < 0.1). Additionally, mutations in established driver genes (NRAS, KRAS, and BRAF) were common and frequently clonal, suggesting their central role in EMD pathogenesis. We also identified alterations in genes associated with cell adhesion and migration (ROBO1, ROBO2, and FAT1) and the SWI/SNF complex and epigenetic regulators (ARID1A, KMT2C, KMT2D, and EP300), although these were predominantly subclonal. Furthermore, we frequently detected biallelic alterations in the tumor suppressor genes MAX (22%), a binding partner for MYC, and CDKN2C (17%). Genomic complexity was significantly higher in EMD samples than BMAs, as evidenced by increased tumor mutational burden and the enrichment of 1q gain/amplifications. These findings underscore the distinct molecular profile of EMD compared to BMA and highlight the genomically complex and heterogeneous nature of extramedullary disease in MM.

Abstract Most disease-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWASs) reside in noncoding regions. There is increasing evidence that they play regulatory roles through epigenetic mechanisms by altering local chromatin state, 3D chromatin structure, and looping interactions. We and others have identified 42 CLL risk loci from GWASs. At 12 risk loci located outside of gene promoters, we previously demonstrated that functional SNPs are highly enriched in enhancers and super-enhancers that mostly target BCL2 family genes and transcriptional regulators. However, the study was limited by using only public epigenomic data generated in lymphoblastoid cell lines (LCL) and normal immune related cell types. To gain a better understanding of how the functional SNPs alter local chromatin state and modulate target gene expression in CLL, we generated RNA-seq data (n=588) and transposase-accessible chromatin with sequencing (ATAC-seq) data (n=69) in CLL tumor B cells. We performed molecular quantitative trait loci (QTL) analysis with index and correlated SNPs (R2 0.5) at 41 risk loci (excluding the HLA locus). The analyses identified cis expression QTLs (eQTLs), within 1Mb of genes’ transcription start site, at 25 risk loci and chromatin accessibility QTLs (caQTLs, within peak) at 11 risk loci, with both eQTLs and caQTLs at 8 risk loci. We identified a total of 54 eGenes, for which the expression is affected by nearby SNPs within 1Mb, including 40 novel eGenes at 16 CLL risk loci not previously reported. Notably, based on publicly available chromosome interaction data from CLL, LCL, and normal B cells, for over 70% of the novel eGenes, their promoters showed looping interactions with the regions carrying the eQTLs. Further, 13 SNPs were identified as both caQTL and eQTL, with the associated novel eGenes including CD81 at 11p15.5, IPCEF1 at 6q25.2, as well as CXXC1 and MBD1 at 18q21.1. The result suggests that these SNPs play a role in altering chromatin accessibility contributed to gene expression variation. At 11p15.5 risk locus, SNP rs2651823 (A/G) represents a novel caQTL and eQTL. The alternative G allele is associated with increased chromatin accessibility and CD81 expression. Hi-C data from GM12878 and CD20+ B cells supported interaction between the open chromatin region carrying rs2651823 and CD81 promoter. CD81 facilitates the trafficking of CD19 to the surface of activated B cells, and CD81-null mice showed impaired immune response. In a mouse model, CD81 contributes to tumor progression and metastasis in Lewis lung carcinoma. In summary, our molecular QTL analysis revealed that functional SNPs, located in CLL risk loci, modulate chromatin accessibility and expression of downstream target genes that could impact CLL risk. Citation Format: Huihuang Yan, Zhiquan Wang, Mingma Hoel, Cristine Allmer, Chantal McCabe, Daniel O’Brien, Dennis Robinson, James Cerhan, Sameer Parikh, Esteban Braggio, Susan Slager. Molecular QTL analyses identify functional variants at inherited risk loci in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2763.

Abstract Despite the advent of novel targeted therapies with high overall response rates, chronic lymphocytic leukemia (CLL) is still largely incurable due to the development of resistance. The bone marrow microenvironment (BMME) plays an important role in the survival and drug resistance in CLL. BM stromal cells (BMSCs) are considered a key component of BMME. Our long-term goal is to gain further insights of BMSCs-CLL crosstalk influencing both survival and drug resistance in CLL. To understand the global biologic changes that occur in leukemic CLL cells when in contact with BMSCs, we assessed RNA-seq in paired CLL cells isolated from untreated blood and BM and in untreated patients’ CLL cells cultured alone or with BMSCs. CLL cells from blood/BM and cultured alone/with BMSCs were also examined by Immunoprecipitation (IP), Western blot (WB), untargeted metabolomics (LCMS+GCMS), and preclinical drug sensitivity assays. We detected only 13 upregulated genes that overlap between CLL cells from BM and cocultured with BMSCs (p&lt0.05, fold change &gt1.5). When we analyzed the expression of these 13 genes in CLL cells from a cohort of 162 untreated patients by RNA-seq, we found associations between 4 genes (PNP, C16orf54, MOB3A, CDK2AP2) and clinical outcomes including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine pathway, had the most significant association with OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine pathway metabolites adenosine, adenine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis showed variable baseline PNP protein expression with 2 cohorts: low /no (L) and high (H) PNP using CLL blood cells. An induction in PNP protein levels were noticed in CLL cells expressing only LPNP when cocultured. Given the association of HPNP with adverse clinical outcomes, we tested for L vs. HPNP cohorts drug sensitivity to known BTK inhibitors(is) or Bcl2is. Treatment of CLL cells from untreated patients with Bcl2is venetoclax, S55746, and LP-118 (Bcl2-Bclxli, Newave) showed increased sensitivity to the Bcl2/Bclxlis in HPNP but not in LPNP cells. In addition, BTKis showed no difference in drug sensitivity associated with CLL PNP levels. To explain this discrepancy, WB and IP analyses were conducted in L vs. HPNP leukemic cells for apoptotic profiles, where we found increased phosphorylated-Bcl2(S70) and an increased association of Bcl2 with Bax in LPNP-expressing CLL cells compared to HPNP. These findings support increased resistance to apoptosis afforded by inhibiting Bcl2 for LPNP vs. HPNP-expressing CLL cells. Our future goals are to continue to understand the prognostic significance of the 4 genes with adverse clinical outcomes and to investigate how best to exploit the peculiar, enhanced sensitivity to Bcl2/Bclxlis found in the HPNP CLL cells. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, Brianna M. Finke, Heather C. Darby, 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. CLL drug resistance- A multifaceted mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4423.

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is a precursor to multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor to either myeloid or lymphoid malignancy, depending on whether the mutation is in a gene associated with lymphoid (L-CHIP) or myeloid (M-CHIP) outcome. Little is known about the association between CHIP and MGUS and their combined effect on risk of MM. We examined this question within Mayo Clinic Biobank cohort of participants aged 50 years or older with no prior history of hematological malignancies. Using whole blood, individuals were screened for MGUS using a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry assay. M-CHIP and L-CHIP were defined based on mutations in 56 genes associated with myeloid malignancies or mutations in 235 genes associated with lymphoid malignancies, respectively, using whole-exome-sequencing. Incident MM was identified using International Classification of Diseases (ICD) codes and confirmed via medical record review. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals for the association of CHIP and MGUS. Cox proportional hazards regression was used to estimate hazard ratios (HR), with time defined as date between blood sample and the first incident MM, date of death, or 12/31/2023. All models were adjusted for age and sex. Of the 16, 479 individuals (median age 65 years, 45% male) screened, 12.3% were positive for MGUS, 9.8% were positive for M-CHIP, and 1.5% were positive for L-CHIP. There was no evidence of an association between M-CHIP and MGUS (OR=0.97, 95% CI:0.83-1.12) nor L-CHIP and MGUS (OR=1.12, 95% CI:0.78-1.57), including with any MGUS isotype (IgA, IgG, or IgM; P-values>0.22). Next, we investigated the association of these precursors on risk of MM. Median follow-up was 11.7 years (range 0.1-15.1), and 46 individuals developed incident MM. Both MGUS (HR=29.7, 95% CI: 14.1-62.4) and L-CHIP (HR=4.1, 95% CI: 1.3-13.2) were significantly associated with incident MM, but not M-CHIP (HR-1.0, 95% CI: 0.4-2.5). Investigating the combined effect of MGUS and L-CHIP, we found those with MGUS alone had a 27.5-fold (95% CI: 13.0-58.2) increased risk of incident MM compared to those with no precursors. No individuals with L-CHIP only developed MM, but those with both L-CHIP and MGUS had a 129.4-fold (95% CI: 34.4-486.4) increased risk of incident MM. In the largest cohort with screened MGUS and CHIP, we found no evidence of an association between either definition of CHIP with MGUS. However, individuals with both L-CHIP and MGUS were at the highest risk of developing MM. Citation Format: Nicholas J. Boddicker, Cristine Allmer, Danelle H. Moonen, Aaron D. Norman, Angelica Macauda, Alyssa Clay-Gilmour, Janet E. Olson, Mrinal S. Patnaik, Vincent Rajkumar, Esteban Braggio, David L. Murray, Shaji Kumar, Susan L. Slager, Celine M. Vachon. The association between MGUS and CHIP and their impact on multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4917.

Genome-wide association studies (GWAS) of multiple myeloma (MM) in individuals of genetic European ancestry (EA) have identified 35 susceptibility loci. Co-heritability analyses have revealed a strong genetic correlation between MM and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS). Previous research validated the association between a polygenic risk score (PRS) based on 23 MM risk loci and the risk of MGUS. Went and colleagues recently reported 12 new MM risk loci. We constructed a MM-PRS using all 35 MM risk variants and assessed its association with MGUS. Additionally, among individuals with MGUS, we assessed associations of MM-PRS with established prognostic factors [M-protein (<0.2 g/dL; 0.2-1.5 g/dL; >1.5 g/dL); isotype (IgG, IgA, IgM); free light chain (FLC) ratio (Normal (0.26-1.65), Abnormal (<0.26 or >1.65))]. Our study population included 1,723 individuals with MGUS seen at Mayo Clinic, Rochester, MN and 14,175 controls from the Mayo Clinic Biobank who lived in the 27-county region around Rochester, MN, and screened negative for MGUS using MALDI-TOF MS. The majority of MGUS patients and controls were of EA. We used logistic or multinomial regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs). All models were adjusted for age, sex, and the first three principal components. The MM-PRS was significantly associated with MGUS when assessed continuously (OR, 1.28 per standard deviation (SD); 95% CI, 1.22-1.35). When comparing to the middle quintile of the MM-PRS distribution, individuals in the highest quintile had 1.41-fold increased risk of MGUS (95% CI, 1.21-1.64), and individuals in the lowest quintile had 0.68 decreased risk of MGUS (95% CI, 0.57-0.81). Of the 35 SNPs evaluated, 27 demonstrated consistent directions of effect for risk of MGUS with published risk estimates for MM. The effect of MM-PRS differed by isotype (P = 2.2 x 105), with higher MM-PRS associated with IgA MGUS (OR, 1.40 per SD; 95% CI, 1.08-1.81) and a lower MM-PRS associated with IgM (OR, 0.66 per SD; 95% CI, 0.52-0.84) compared to IgG MGUS. The MM-PRS was higher in MGUS patients with M-protein > 1.5 g/dL compared to M-protein of size 0.2-1.5 g/dL, although not statistically significant (OR, 1.41 per SD; 95% CI, 0.95-2.08); there were no differences in the MM-PRS between MGUS patients with M-protein size <0.2 g/dL compared to 0.2-1.5 g/dL (OR, 1.01 per SD;95% CI, 0.81-1.26). There were also no differences in MM-PRS by FLC ratio, (OR, 0.95 per SD; 95% CI, 0.852-1.10 for abnormal vs. normal). Our findings of an association of the MM-PRS with risk of MGUS provides further evidence for shared heritability between MGUS and MM. Future studies are needed to examine whether the expanded MM-PRS adds to existing models for MGUS progression. Citation Format: Angelica Macauda, Alyssa Clay-Gilmour, Cristine Allmer, Danelle Moonen, Aaron D. Normann, Nicholas J. Boddicker, Joselle M. Cook, Linda Baughn, Elizabeth E. Brown, Vincent S. Rajkumar, Esteban Braggio, David L. Murray, Susan L. Slager, Shaji Kumar, Celine M. Vachon. Implications of multiple myeloma polygenic risk scores (PRS) for MGUS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2274.