The genomic landscape of Vk*MYC myeloma highlights shared pathways of transformation between mice and humans.

Multiple myeloma (MM) is a heterogeneous disease characterized by frequent MYC translocations. Sporadic MYC activation in the germinal center of genetically engineered Vk*MYC mice is sufficient to induce plasma cell tumors in which a variety of secondary mutations are spontaneously acquired and selected over time. Analysis of 119 Vk*MYC myeloma reveals recurrent copy number alterations, structural variations, chromothripsis, driver mutations, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational activity, and a progressive decrease in immunoglobulin transcription that inversely correlates with proliferation. Moreover, we identify frequent insertional mutagenesis by endogenous retro-elements as a murine specific mechanism to activate NF-kB and IL6 signaling pathways shared with human MM. Despite the increased genomic complexity associated with progression, advanced tumors remain dependent on MYC. In summary, here we credential the Vk*MYC mouse as a unique resource to explore MM genomic evolution and describe a fully annotated collection of diverse and immortalized murine MM tumors.

Tumor mutational load is prognostic for progression to therapy among high-count monoclonal B-cell lymphocytosis.

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.

Prediction of outcomes for high-count monoclonal B lymphocytosis using an epigenetic and immunogenetic signature.

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.

Superior detection rate of plasma cell FISH using FACS-FISH.

OBJECTIVES: Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. METHODS: The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. RESULTS: Fluorescence-activated cell sorting-FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. CONCLUSIONS: Fluorescence-activated cell sorting-FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.

MicroRNA and long non-coding RNA analysis in IgM monoclonal gammopathies reveals epigenetic influence on cellular functions and oncogenesis.

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The Vk*MYC Mouse Model recapitulates human multiple myeloma evolution and genomic diversity.

Despite advancements in profiling multiple myeloma (MM) and its precursor conditions, there is limited information on mechanisms underlying disease progression. Clincal efforts designed to deconvolute such mechanisms are challenged by the long lead time between monoclonal gammopathy and its transformation to MM. MM mouse models represent an opportunity to overcome this temporal limitation. Here, we profile the genomic landscape of 118 genetically engineered Vk*MYC MM and reveal that it recapitulates the genomic heterogenenity and life history of human MM. We observed recurrent copy number alterations, structural variations, chromothripsis, driver mutations, APOBEC mutational activity, and a progressive decrease in immunoglobulin transcription that inversely correlates with proliferation. Moreover, we identified frequent insertional mutagenesis by endogenous retro-elements as a murine specific mechanism to activate NF-kB and IL6 signaling pathways shared with human MM. Despite the increased genomic complexity associated with progression, advanced tumors remain dependent on MYC expression, that drives the progression of monoclonal gammopathy to MM.

Differential prognosis of single and multiple TP53 abnormalities in high-count MBL and untreated CLL.

TP53 aberrations, including mutations and deletion of 17p13, are important adverse prognostic markers in chronic lymphocytic leukemia (CLL) but are less studied in high count monoclonal B-cell lymphocytosis (HCMBL), an asymptomatic pre-malignant stage of CLL. Here we estimated the prevalence and impact of TP53 aberrations in 1,230 newly diagnosed treatment-naïve individuals (849 CLL, 381 HCMBL). We defined TP53 state as: wild-type (no TP53 mutations and normal 17p), single-hit (del(17p) or one TP53 mutation), or multi-hit (TP53 mutation and del(17p), TP53 mutation and loss of heterozygosity, or multiple TP53 mutations). Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time to first treatment and overall survival by TP53 state. We found 64 (7.5%) CLL patients and 17 (4.5%) HCMBL individuals had TP53 mutations with variant allele fraction >10%. Del(17p) was present in 58 (6.8%) of CLL and 11 (2.9%) of HCMBL cases. Most individuals had wild-type (N=1,128, 91.7%) TP53 state, followed by multi-hit (N=55, 4.5%) and then single-hit (N=47, 3.8%) TP53 state. The risk of shorter time to therapy and death increased with the number of TP53 abnormalities. Compared to wild-type patients, multi-hit patients had 3-fold and single-hit patients had 1.5-fold increased risk of requiring therapy. Multi-hit patients also had 2.9-fold increased risk of death compared to wild-type. These results remained stable after accounting for other known poor prognostic factors. Both TP53 mutations and del(17p) may provide important prognostic information for HCMBL and CLL that would be missed if only one were measured.

BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Clonal Hematopoiesis of Indeterminate Potential Is Associated With Coronary Microvascular Dysfunction In Early Nonobstructive Coronary Artery Disease.

BACKGROUND: Clonal hematopoiesis (CH) of indeterminate potential (CHIP) is a risk factor for cardiovascular disease. The relationship between CHIP and coronary microvascular dysfunction (CMD) is unknown. The current study examines the association between CHIP and CH with CMD and the potential relationships in risk for adverse cardiovascular outcomes. METHODS: In this retrospective observational study, targeted next-generation sequencing was performed for 177 participants with no coronary artery disease who presented with chest pain and underwent routine coronary functional angiogram. Patients with somatic mutations in leukemia-associated driver genes in hematopoietic stem and progenitor cells were examined; CHIP was considered at a variant allele fraction ≥2%; CH was considered at a variant allele fraction ≥1%. CMD was defined as coronary flow reserve to intracoronary adenosine of ≤2. Major adverse cardiovascular events considered were myocardial infarction, coronary revascularization, or stroke. RESULTS: A total of 177 participants were examined. Mean follow-up was 12±7 years. A total of 17 patients had CHIP and 28 had CH. Cases with CMD (n=19) were compared with controls with no CMD (n=158). Cases were 56±9 years, were 68% women, and had more CHIP (27%; P=0.028) and CH (42%; P=0.001) than controls. CMD was associated with independent risk for major adverse cardiovascular events (hazard ratio, 3.89 [95% CI, 1.21-12.56]; P=0.023), and 32% of this risk was mediated by CH. The risk mediated by CH was ≈0.5× as large as the direct effect of CMD on major adverse cardiovascular events. CONCLUSIONS: In humans, we observe patients with CMD are more likely to have CHIP, and nearly one-third of major adverse cardiovascular events in CMD are mediated by CH.

Molecular Clusters and Tumor-Immune Drivers of IgM Monoclonal Gammopathies.

PURPOSE: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. EXPERIMENTAL DESIGN: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. RESULTS: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. CONCLUSIONS: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.

Clinical outcomes in patients with chronic lymphocytic leukemia with disease progression on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) with disease progression on ibrutinib have worse outcomes compared to patients stopping ibrutinib due to toxicity. A better understanding of expected outcomes in these patients is necessary to establish a benchmark for evaluating novel agents currently available and in development. We evaluated outcomes of 144 patients with CLL treated at Mayo Clinic with 2018 iwCLL disease progression on ibrutinib. The median overall survival (OS) for the entire cohort was 25.5 months; it was 29.8 months and 8.3 months among patients with CLL progression (n = 104) and Richter transformation (n = 38), respectively. Longer OS was observed among patients with CLL progression who had received ibrutinib in the frontline compared to relapsed/refractory setting (not reached versus 28.5 months; p = 0.04), but was similar amongst patients treated with 1, 2, or ≥3 prior lines (18.5, 30.9, and 26.0 months, respectively, p = 0.24). Among patients with CLL disease progression on ibrutinib, OS was significantly longer when next-line treatment was chimeric antigen receptor T-cell therapy (median not reached) or venetoclax-based treatment (median 29.8 months) compared to other approved treatments, such as chemoimmunotherapy, phosphoinositide 3'-kinase inhibitors, and anti-CD20 monoclonal antibodies (9.1 months; p = 0.03). These findings suggest an unmet need for this growing patient population.

Transcriptional profiles define drug refractory disease in myeloma.

Identifying biomarkers associated with disease progression and drug resistance are important for personalized care. We investigated the expression of 121 curated genes, related to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) responsiveness. We analyzed 28 human multiple myeloma (MM) cell lines with known drug sensitivities and 130 primary MM patient samples collected at different disease stages, including newly diagnosed (ND), on therapy (OT), and relapsed and refractory (RR, collected within 12 months before the patients' death) timepoints. Our findings led to the identification of a subset of genes linked to clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs. Finally, we built a seven-gene model (MM-IMiD and PI sensitivity-7 genes [IP-7]) using digital gene expression profiling data that significantly separates ND patients from IMiD- and PI-refractory RR patients. Using this model, we retrospectively analyzed RNA sequcencing (RNAseq) data from the Mulltiple Myeloma Research Foundation (MMRF) CoMMpass (n = 578) and Mayo Clinic MM patient registry (n = 487) to divide patients into probabilities of responder and nonresponder, which subsequently correlated with overall survival, disease stage, and number of prior treatments. Our findings suggest that this model may be useful in predicting acquired resistance to treatments containing IMiDs and/or PIs.

Progression and survival of MBL: a screening study of 10 139 individuals.

Monoclonal B-cell lymphocytosis (MBL) is a common hematological premalignant condition that is understudied in screening cohorts. MBL can be classified into low-count (LC) and high-count (HC) types based on the size of the B-cell clone. Using the Mayo Clinic Biobank, we screened for MBL and evaluated its association with future hematologic malignancy and overall survival (OS). We had a two-stage study design including discovery and validation cohorts. We screened for MBL using an eight-color flow-cytometry assay. Medical records were abstracted for hematological cancers and death. We used Cox regression to evaluate associations and estimate hazard ratios and 95% confidence intervals (CIs), adjusting for age and sex. We identified 1712 (17%) individuals with MBL (95% LC-MBL), and the median follow-up time for OS was 34.4 months with 621 individuals who died. We did not observe an association with OS among individuals with LC-MBL (P = .78) but did among HC-MBL (hazard ratio, 1.8; 95% CI, 1.1-3.1; P = .03). Among the discovery cohort with a median of 10.0 years follow-up, 31 individuals developed hematological cancers with two-thirds being lymphoid malignancies. MBL was associated with 3.6-fold risk of hematological cancer compared to controls (95% CI, 1.7-7.7; P < .001) and 7.7-fold increased risk for lymphoid malignancies (95% CI:3.1-19.2; P < .001). LC-MBL was associated with 4.3-fold risk of lymphoid malignancies (95% CI, 1.4-12.7; P = .009); HC-MBL had a 74-fold increased risk (95% CI, 22-246; P < .001). In this large screening cohort, we observed similar survival among individuals with and without LC-MBL, yet individuals with LC-MBL have a fourfold increased risk of lymphoid malignancies. Accumulating evidence indicates that there are clinical consequences to LC-MBL, a condition that affects 8 to 10 million adults in the United States.

Combined ibrutinib and venetoclax for treatment of patients with ibrutinib-resistant or double-refractory chronic lymphocytic leukaemia.

Patients with chronic lymphocytic leukaemia (CLL) disease progression on ibrutinib or after sequential ibrutinib and venetoclax-based treatments (double-refractory) have poor outcomes. In this retrospective study, we analysed outcomes with combined ibrutinib and venetoclax treatment in these groups of patients. The median treatment-free and overall survival for 22 patients with prior progression on ibrutinib (venetoclax-naïve) were 23.7 and 47.1 months respectively. In 11 patients with double-refractory CLL, the median treatment-free and overall survival were 11.2 and 27.0 months respectively. The combination of ibrutinib and venetoclax may help bridge the current gap in options for patients with disease refractory to the most commonly used novel agents.

B cell receptor signaling drives APOBEC3 expression via direct enhancer regulation in chronic lymphocytic leukemia B cells.

Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes by regulating the activity of their enhancers. BTKi treatment reduces enrichment of enhancer marks (H3K4me1 and H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. We also find that enhancer-regulated APOBEC3 expression contributes to replication stress in malignant B cells. In total we demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in an NFATc1-dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.

Long-term outcomes for ibrutinib-rituximab and chemoimmunotherapy in CLL: updated results of the E1912 trial.

Herein, we present the long-term follow-up of the randomized E1912 trial comparing the long-term efficacy of ibrutinib-rituximab (IR) therapy to fludarabine, cyclophosphamide, and rituximab (FCR) and describe the tolerability of continuous ibrutinib. The E1912 trial enrolled 529 treatment-naïve patients aged ≤70 years with chronic lymphocytic leukemia (CLL). Patients were randomly assigned (2:1 ratio) to receive IR or 6 cycles of FCR. With a median follow-up of 5.8 years, median progression-free survival (PFS) is superior for IR (hazard ratio [HR], 0.37; P < .001). IR improved PFS relative to FCR in patients with both immunoglobulin heavy chain variable region (IGHV) gene mutated CLL (HR: 0.27; P < .001) and IGHV unmutated CLL (HR: 0.27; P < .001). Among the 354 patients randomized to IR, 214 (60.5%) currently remain on ibrutinib. Among the 138 IR-treated patients who discontinued treatment, 37 (10.5% of patients who started IR) discontinued therapy due to disease progression or death, 77 (21.9% of patients who started IR) discontinued therapy for adverse events (AEs)/complications, and 24 (6.8% of patients who started IR) withdrew for other reasons. Progression was uncommon among patients able to remain on ibrutinib. The median time from ibrutinib discontinuation to disease progression or death among those who discontinued treatment for a reason other than progression was 25 months. Sustained improvement in overall survival (OS) was observed for patients in the IR arm (HR, 0.47; P = .018). In conclusion, IR therapy offers superior PFS relative to FCR in patients with IGHV mutated or unmutated CLL, as well as superior OS. Continuous ibrutinib therapy is tolerated beyond 5 years in the majority of CLL patients. This trial was registered at www.clinicaltrials.gov as #NCT02048813.

Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers.

BACKGROUND AND AIMS: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. APPROACH AND RESULTS: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. CONCLUSIONS: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.