Somatic Variants Research Articles

Abstract 6299: Strainy: Multi-allelic phasing and assembly of bacterial strains and tumor clones using long reads

Abstract INTRODUCTION: Metagenomic sequencing of human microbiome and other complex microbial communities reveals extensive heterogeneity on sub-species levels. Detecting these heterogeneities (and strains) in the human microbiome is particularly important because different strains of the same bacterial species often display distinct phenotypes, such as pathogenicity in humans and antimicrobial resistance. For the context of cancer, studies show that the host microbiome may slow or contribute to the development and progression of cancer as well as the patient response to certain cancer therapies. However, reconstruction of heterogeneous bacterial species (represented by multiple strains) from a single metagenomic sample is challenging. Here we present an algorithm for phasing and assembly of closely related strains from long reads, which could also be applied to phased somatic variants in cancer. METHODS: The goal of the Strainy phasing procedure is to cluster long reads based on their strain (or haplotype) of origin. Given a set of all reads aligned to a unitig, Strainy builds a connection graph, where nodes correspond to aligned reads, and edges connect reads that share the same SNP genotypes. Then, Strainy iteratively uses a community detection algorithm to partition the connection graph into densely connected components that are assembled into strain unitigs. Finally, Strainy uses the overlap graph approach to extend strain unitigs and integrate them back to the original de novo assembly graph. RESULTS: We benchmark Strainy against Hifiasm-meta, Strainberry, metaFlye and metaMDBG using several simulated, mock and real datasets with ONT and PacBio HiFi reads.On simulated and mock metagenomic datasets, Strainy phased and reconstructed a substantially higher portion of unique strain sequence compared to the other tools, while having fewer errors. We then applied Strainy to untangle strains in metagenomic sequencing of activated sludge from an anaerobic digester bacterial community previously sequenced with Nanopre R9, R10 and PacBio HiFi. The number of recovered strains per bacterial species varied from 1 to 4. On average, 8.6 heterozygous structural variants per bacterial species were recovered. Non-synonymous to synonymous substitutions rates (dN/dS) revealed a few hotspots with evidence of selection that were specific to different species. Strainy represents a first practical algorithmic framework for multi-allelic phasing with long reads. Tumor genomes often contain multiple clones defined by characteristic somatic variants. Phasing of somatic variants into clonal haplotypes is therefore a promising approach to better characterize clonal heterogeneity in cancer. We tested Strainy for multi-allelic phasing on a mix of multiple human genomes and successfully phased distinct haplotypes. Motivated by the promising results, we are working on extending our approach to cancer clone phasing. Citation Format: Ataberk Donmez, Ekaterina Kazantseva, Maria Frolova, Mihai Pop, Mikhail Kolmogorov. Strainy: Multi-allelic phasing and assembly of bacterial strains and tumor clones using long reads [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 6299.

Abstract 5925: Circulating cell-free methylated tumor DNA measurements correlate with plasma VAF-based tumor fraction estimates

Abstract Introduction: Cell-free DNA (cfDNA) consists of circulating tumor DNA (ctDNA) and DNA shed by non-tumor cells such as immune and endothelial cells. Tumor fraction (TF) is the proportion of ctDNA present in cfDNA. TF is often estimated by summary statistics on somatic variants such as the mean or maximum variant allele fraction (VAF). The Northstar Response (NSR) assay quantifies ctDNA by accurately and precisely counting methylated cfDNA molecules across over 500 loci uniquely methylated in tumor tissue but not in any non-cancerous cells. We hypothesize that the NSR quantification of plasma methylated DNA, via the Tumor Methylation Score (TMS), correlates with tumor fraction as estimated by VAF. Methods: A pan-cancer cohort of 271 longitudinal blood samples across 74 patients was gathered and run on the Northstar Select (NSS), an 84-gene liquid biopsy panel for somatic variant detection, and NSR assays. Samples were required to have positive detection in both assays to ensure precise TF measurements (max(VAF)>0.01%, TMS>25 methylated molecules). Tumor fractions were estimated by removing germline variants and evaluating the max(VAF) and sum(VAF), with the latter measurement paralleling the summation of methylated molecules used by the TMS. Due to the high variability of tumor mutational burden (TMB) and tumor methylation levels between tumors, correlations between VAF and TMS were calculated on a per-patient basis for patients with at least 3 longitudinal samples. Results: TMS ranged from 25 to 253k molecules with a mean of 16k; sum(VAF) ranged from 0.06 to 782 with a mean of 67; max(VAF) ranged from 0.06% to 100% with a mean of 34%. TMS correlated most strongly with sum(VAF) with mean(R2)=0.77, median(R2)=0.96, whereas for max(VAF), mean(R2)=0.67, median(R2)=0.91. A linear regression model between max(VAF) and TMS demonstrated a mean increase of 1.3k methylated molecules per unit percent increase in max(VAF). Conclusions: Previous studies have demonstrated that VAF decreases are prognostic to targeted therapy response including immune checkpoint blockade and tyrosine kinase inhibitors. However, VAF-based tumor burden measurements are limited by the paucity of somatic variants. In contrast, aberrant DNA methylation is a well-known hallmark of cancer that is an abundant source of ctDNA. The strong, linear correlation between TMS and VAF shown here suggests that TMS is an effective measure of ctDNA burden and can similarly be used to monitor response to therapy. Furthermore, the large amount of methylated DNA in patient samples (mean=16k molecules) suggests that epigenetic approaches can be highly sensitive even at very low tumor fraction (<1%). Citation Format: Lee Davis McDaniel, David Tsao. Circulating cell-free methylated tumor DNA measurements correlate with plasma VAF-based tumor fraction estimates [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 5925.

Abstract 4905: Mosaic chromosomal alterations overlapping hotspot and coldspot sites of somatic structural variation are associated with increased odds of hematological malignancy

Abstract BACKGROUND: Clonal hematopoiesis (CH) occurs when hematopoietic cells acquire somatic mutations and proliferate to generate clones in blood. CH can be driven by mosaic chromosomal alterations (mCAs), which are large somatic structural variants, and is associated with increased risk of blood malignancy. We conducted a genome-wide search for sites significantly more or less impacted by mCA events, termed hotspots and coldspots. We tested whether the odds of blood malignancy in participants harboring mCAs overlapping hotspots or coldspots differed from the overall association between mCAs and blood malignancy. METHODS: This study utilized two cohorts of the Canadian Partnership for Tomorrow’s Health, including the Ontario Health Study (OHS; n=7070) and CARTaGENE (n=28,639). Participants were genotyped with the UK Biobank Axiom or Infinium Global Screening Array and completed a baseline cancer questionnaire. All mCA calling was performed using MoChA software. We identified hotspot and coldspot sites of autosomal mCA accumulation using binomial tests scaled by chromosome and array type. We determined whether the number of overlapping mCAs at each query site was greater or less than would be expected under the null expectation. Then, we calculated the prevalence of cancer in participants with or without mCAs overlapping hotspots/coldspots and conducted Fisher’s exact tests to generate odds ratios. RESULTS: In OHS, participants with an mCA had a significantly greater odds of having a hematological malignancy at baseline (OR=7.29, 95% CI=3.72-13.38, p=7.84e-08). They were also more likely to have a cancer diagnosis of any type (OR=1.63, 95% CI=1.14-2.30, p=5.59e-03). Participants with an mCA overlapping a hotspot site had a further increased odds of harboring hematological malignancy (OR=9.79, 95% CI=3.94-21.29, p=5.05-e06). For participants with an mCA overlapping a coldspot site, the OR reached 20.52 (95% CI=3.68-76.85, p=7.36e-04). In CARTaGENE, participants with an mCA were also at increased odds of hematological malignancy (OR=11.31, 95% CI=6.78-18.17, p=9.77e-16), and any cancer diagnosis (OR=1.93, 95% CI=1.53-2.41, p=5.95e-08). Those carrying an mCA overlapping a hotspot site were 14.6 times more likely to have a hematological malignancy (95% CI=7.94-25.29, p=3.30e-13), and those with an mCA overlapping a coldspot site were 14.2 times more likely (95% CI=5.0-32.9, p=7.63e-06). CONCLUSIONS: These results suggest that there may be genomic locations at which somatic structural variation has a larger impact on the development of blood malignancy, relative to other regions. Further work is needed to characterize the functional consequences of somatic mutation at these key regions and explore whether these associations of hotspots, coldspots, and cancer incidence are conserved across a range of tissues. Citation Format: Jasmine Ryu Won Kang, Vanessa Bruat, Kimberly Skead, Mawusse Agbessi, June Kim, Elias Gbeha, Marie-Julie Fave, Philip Awadalla. Mosaic chromosomal alterations overlapping hotspot and coldspot sites of somatic structural variation are associated with increased odds of hematological malignancy [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 4905.

Abstract 5050: Comparative analysis of somatic copy number variations and allelic imbalance in whole-genome bisulfite sequencing: challenges and insights

Goal: To assess the feasibility, reliability, and limitations of using Whole Genome Bisulfite Sequencing (WGBS) for detecting somatic copy number variation (SCNV) and tumor-specific allelic imbalance (AI). Background: In cancer genomics, the use of WGBS for SCNV and AI detection enables simultaneous analysis of genetic variations and methylation landscapes, potentially providing insights into the interplay between somatic alterations and DNA methylation in tumorigenesis. However, WGBS data is inherently noisier compared to whole-genome sequencing (WGS) or microarray data due to artifacts introduced during bisulfite conversion. These challenges, including difficulties in read alignment, genotype calling, and phasing, the lack of strand complementarity, and the need to distinguish true C>T SNPs from bisulfite-induced changes, collectively complicate SCNV and AI detection. Method: We analyzed WGBS data from The Cancer Genome Atlas (TCGA), including 39 tumor samples and 8 adjacent normal tissues across 9 tumor types. SCNV detection was performed using GATK-CNV, while AI callings were conducted with BisSNP, BISCUIT, Revelio (with Unified Genotyper), and BSsnp. Confusion matrices were used to compare GATK-CNV (WGBS) and hapLOH (microarray) at the base, gene, and arm levels. F1 score, specificity, and sensitivity were calculated to evaluate their performance across different genomic scales. Additionally, AI identified from WGBS data using BisSNP, BISCUIT, Revelio (with Unified Genotyper), and BSsnp were compared to those detected from array and WGS data using hapLOH/seq. To determine detection thresholds and refine results, sequencing data were downsampled, with filters applied to exclude low-quality data and retain high-confidence SNPs and genotypes. Result: GATK-CNV exhibited variable performance across samples, with most samples showing high true positives and low false positives, indicating good sensitivity for detecting SCNVs and capturing a genome burden similar to hapLOHseq data. However, for samples with a high number of events, normalization disrupts event callings, resulting in inconsistencies in SCNV identification compared to array data. For AI detection, Revelio (with Unified Genotyper) showed the best overall performance among the tools evaluated. Downsampling analysis showed that reducing sequencing coverage impaired event detection, becoming less effective at lower coverage levels. BSsnp, BISCUIT, and BisSNP produced widespread and inconsistent variant calls, and initial filtering for high-quality SNPs showed limited improvement in precision. This study highlights the potential and challenges of WGBS as a tool for analyzing SCNV/AI alongside DNA methylation with phased data, offering insights into tumorigenesis and advancements in cancer diagnostics and treatment. Citation Format: Yiru Gu, Jian Gu, Paul Scheet, Justin Wong. Comparative analysis of somatic copy number variations and allelic imbalance in whole-genome bisulfite sequencing: challenges and insights [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 5050.

Abstract 6639: Optimized selection of tumor-informed MRD panels enhances sensitivity of ctDNA detection

Abstract Circulating tumor DNA (ctDNA) is a highly sensitive and specific biomarker for molecular residual disease (MRD) and can be by identified by the presence of tumor-specific somatic variants. We have developed a second-generation hybrid-capture-based tumor-informed MRD assay to detect the presence and quantity of ctDNA in plasma-derived cell-free DNA (cfDNA). Our assay surveys the whole tumor genome to identify the superset of high-confidence somatic variant targets that can be used to distinguish ctDNA from non-tumor cfDNA. Each panel consists of up to 1000 targets that are selected based on target-specific and tumor-specific features that impact the representation of tumor variants or the effective molecular depth of a panel, such as somatic call confidence, zygosity, copy number, background error rate and expected molecular depth following capture and sequencing. cfDNA captured using a patient-matched panel undergoes targeted sequencing and that sequence data is fit to a statistical model that evaluates the presence and fraction of ctDNA in the sample. Here, we evaluated the performance of our target-ranking algorithm using contrived mixtures of matched tumor and normal cell lines, with tumor DNA concentrations ranging from 5 to 10, 000 parts per million (ppm) and samples processed across multiple days, reagent lots, operators and instruments. Identical sets of contrived samples were analyzed using subsets of panel targets, including top-ranking and bottom-ranking target sets. We found the Limit of Detection with 95% sensitivity (LoD95) for top-ranking targets was 40% of the LoD95 for low-ranking targets, demonstrating the effectiveness of our ranking algorithm in stratifying high and low sensitivity targets. In conclusion, the constituents of tumor-informed MRD panels have a measurable impact on the sensitivity of ctDNA detection and, with optimization, can enhance the resolution of residual disease detection. Citation Format: Ashley Acevedo, Ravi Patel, Kyle Trettin, Nafei Xu, Genevieve Gould, Dale Muzzey. Optimized selection of tumor-informed MRD panels enhances sensitivity of ctDNA detection [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 6639.

Abstract 1977: Implementation of a comprehensive clinical cfDNA analysis platform for aqueous humor liquid biopsy in retinoblastoma

Introduction: The development of liquid biopsy testing based on sequencing analysis of cell-free DNA (cfDNA) has the potential to transform cancer care in pediatric tumors, which are characterized by a wide array of genomic alterations including copy number alterations (CNAs), sequence variants, epigenetic alterations, and structural rearrangements in tumor-associated genes. In patients with retinoblastoma where direct tumor biopsy is not possible due to the risk of tumor spread, aqueous humor (AH) is a rich source of cfDNA. Comprehensive cfDNA testing, combining a low pass whole genome sequencing (LP-WGS) assay to detect CNAs and a custom targeted sequencing panel (TSP) to detect mutations, can provide a highly effective platform to inform diagnosis, risk stratification, response to therapy, and surveillance in patients with retinoblastoma. LP-WGS Results: We validated a liquid biopsy LP-WGS assay, LBSeq4Kids, to detect CNAs in cfDNA from plasma, cerebrospinal fluid, or AH. Clinical testing has been performed for the past two years for diagnostic evaluation and monitoring response to therapy using AH from retinoblastoma patients. Thirty-six children with unilateral or bilateral retinoblastoma (44 eyes) underwent LBSeq4Kids LP-WGS analysis. CNAs were detected in 30/32 (94%) samples at diagnosis or recurrence. Serial sampling of 22/44 (50%) eyes was performed using a range of 1-10 samples per patient. Seventeen of those 22 (77%) eyes demonstrated clearance of CNAs with treatment. Five eyes showed persistence of abnormal profiles, necessitating enucleation of 2 eyes. Treatment and evaluation are ongoing for the remaining three eyes. Targeted Sequencing Panel Results: We are validating a custom cfDNA TSP to detect single nucleotide variants and small indels in the coding sequence of 136 genes and fusions involving EWSR1, FOXO1, and BRAF for patients with ocular disease, solid tumors and brain tumors. To date, 17 children (21 eyes) diagnosed with retinoblastoma have had TSP testing using AH. All eleven (100%) patients with germline variants demonstrated the same RB1 alterations in the AH using the panel. Six of these patients (55%) had a second somatic alteration in RB1 detected with the panel. Two of 11 (18%) with known germline RB1 mutations also demonstrated mutations in BCOR, ARID1A, or MSH6. Somatic variants in RB1, ARID1A and BCOR were detected in the remaining six patients with sporadic retinoblastoma. Targeted sequencing of the AH surveillance samples from these patients is in progress. Conclusion: Here we demonstrate clinical utility for aqueous humor liquid biopsy molecular testing as a complementary means of diagnosis and monitoring treatment response in patients with retinoblastoma. Continued surveillance of these patients is ongoing to monitor disease status and rule out recurrence, as well as identify novel prognostic biomarkers. Citation Format: Laura A. Kagami, Eirini Christodoulou, Venkata Yellapantula, Dong Xu, Cindy Fong, Dejerianne Ostrow, Liya Xu, Jesse Berry, Jaclyn A. Biegel. Implementation of a comprehensive clinical cfDNA analysis platform for aqueous humor liquid biopsy in retinoblastoma [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 1977.

Abstract 5802: Somatic variant predictors of cutaneous immune-related adverse events in cancer patients treated with immunotherapy

Cutaneous immune-related adverse events (cirAEs) affect nearly one-third of patients treated with immune checkpoint inhibitors (ICIs). Identifying patients at high risk for cirAE can help guide monitoring and therapeutic management. Whether tumor somatic mutations are associated with the risk of cirAE development remains unknown. We analyzed tumor somatic single nucleotide variants (SNVs) from a discovery cohort of 733 patients with stage III/IV melanoma receiving ICIs at Dana Farber Cancer Institute and Massachusetts General Hospital. CirAE development was determined by chart review. SNVs were detected by OncoPanel v1-3. Nonsynonymous SNVs were used to calculate tumor mutational burden (TMB) and determine gene mutation status. Logistic regression models were used to test the association between gene mutation status and the binary outcome cirAE, while Cox regression models were used to examine the association between TMB or gene mutation status with time to cirAE development. Models were adjusted for demographic covariates (age at ICI, sex, self-reported race/ethnicity) and clinical covariates (melanoma subtype and ICI type). To validate significant associations identified in the discovery cohort, we used an independent cohort of 792 non-melanoma cancer patients treated with ICIs. Consistent with prior studies, TMB-high (>10 mutations/Mb) was significantly associated with improved overall survival (hazard ratio (HR) [95% CI] = 0.72 [0.53, 0.98]) in melanoma. However, TMB-high was not associated with cirAE development (HR = 0.92 [0.69, 1.22]). Binary logistic regression revealed that somatic mutations in PRKCI (OR = 2.76 [1.27, 6.33]), STAG2 (OR = 2.25 [1.09, 4.80]), and CXCR4 (OR = 2.56 [1.02, 6.80]) were associated with increased odds of cirAE. Furthermore, time-to-event Cox regression identified that somatic mutations in PRKCI, STAG2, and CXCR4 as well as five additional genes (MEN1, AKT3, FANCG, DAXX, H3F3B) were significantly associated with increased risk of cirAE development (nominal p-values<.05). Mutations in CXCR4 replicated in the validation cohort as significantly associated with time to cirAE (HR = 4.02 [1.22, 13.21]) adjusting for cancer type, ICI type, and demographics. Of the CXCR4 mutations identified in patients with cirAE, 5/15 (33%) localized to the C-terminus, were predicted deleterious by SIFT and PolyPhen2, and altered key residues known to be involved in CXCR4 degradation, suggesting a mechanistic link between CXCR4 gain-of-function mutations and increased risk of cirAE. We identified somatic mutations in several genes as significant predictors of cirAE development in melanoma. The association between mutations in CXCR4 and cirAE was replicated in a pan-cancer validation cohort, providing mechanistic insight into cirAE development across cancers. Citation Format: Ninghui Hao, Jenny Lai, Crystal Chang, Ahmad Rajeh, Chuck Lin, Cameron Moseley, Arjun Mahajan, Matthew Tran, Coleen Valery, Christopher J. Thang, Guihong Wan, Alexander Gusev, Yevgeniy R. Semenov. Somatic variant predictors of cutaneous immune-related adverse events in cancer patients treated with immunotherapy [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 5802.

Abstract 5895: Development and analytical validation of a highly sensitive tumor fraction estimation method using blood samples without matched normal controls

Abstract Background: Tumor fraction (TF) in cell-free DNA (cfDNA) is a quantitative indicator of circulating tumor DNA (ctDNA) and has become an important biomarker for disease monitoring and tracking therapeutic responses. We developed a longitudinal TF estimation method that does not require matched normal control samples and analytically validated its ability to accurately estimate TF when longitudinal blood samples are available. Methods: Both reference materials and clinical blood samples were used in the analytical validation. We first determined the limit of detection (LoD) of TF for our PredicineCARE cfDNA assay at targeted sequencing depth of 20, 000x using serially diluted reference materials with 12 gradient values from 0 to 4% TF. We further used a sequentially diluted clinical sample (with known TF) with healthy donor cfDNA to a gradient of expected TF values. We evaluated the variant calling and TF calculation algorithm by treating each dilution separately or treating the immediately nearby samples as longitudinal samples. We further evaluated our longitudinal-aware analysis method on 20 clinical subjects tested with PredicineCARE cfDNA assay with ≥2 time points to evaluate the improvement in TF estimation compared to individual estimation. Results: With the titrated reference materials and manufacturer-labelled somatic variants, we achieved an LoD of 0.01% TF for 30ng cfDNA input and a sequencing depth at 20, 000x using PredicineCARE cfDNA assay. When approaching the LoD, the number of reads for the somatic variants becomes more stochastic, which resulted in big variances in the calculated TF values. When independently estimating the TF without referring to matched normal or longitudinal samples, the LoD was 0.5% TF. When the longitudinal sample information was included, the LoD went down to 0.01% TF, contingent on the number of trackable variants. Our simulated results showed that when there are 4 or more tracking variants between longitudinal samples, the LoD can go down to 0.01% TF most of the time. We further applied this longitudinal sample aware dynamic TF estimation method to 20 clinical subjects tested with PredicineCARE cfDNA assay and with ≥2 time points, most showed improved TF estimation with ≥50% of the subjects having questionable somatic/germline calls if called separately. Conclusions: We developed a dynamic TF estimation methodology that leverages longitudinal samples to improve somatic/germline variant calling and exclude CHIP mutations. Our results demonstrated that with longitudinal samples, the accuracy in TF estimation can approach the ideal situation where all somatic/germline status of the variants are known. Notably, longer interval between time points and a higher number of tracking somatic mutations (≥2) in longitudinal samples generally enhance the effectiveness of this dynamic estimation methodology. Citation Format: Junmei Wang, Wanshu He, Lisha Zhu, Yong Huang, Xiaohong Wang, Shidong Jia, Pan Du. Development and analytical validation of a highly sensitive tumor fraction estimation method using blood samples without matched normal controls [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 5895.

Abstract 691: Decentralized pan-cancer tumor profiling: a comprehensive performance evaluation of the MSK-IMPACT® Assay with SOPHiA DDMTM

Abstract Background: Molecular profiling of solid tumors through FFPE (Formalin-Fixed Paraffin-Embedded) tissue samples is crucial for personalized cancer treatment. MSK-IMPACT® (Memorial Sloan Kettering - Integrated Mutation Profiling of Actionable Cancer Targets) is a well-established, high-throughput genomic assay designed to identify known cancer-driving alterations in solid tumors from FFPE tissue samples. SOPHiA GENETICS is developing a decentralized version of this assay, integrating key features of MSK-IMPACT® including comprehensive genomic profiling, the utilization of hybrid capture-based next-generation sequencing, and the identification of actionable somatic mutations. This report presents the results from a performance evaluation study conducted during the development of this product. Methods: A cohort of 167 FFPE solid tumor samples and matched normal tissue were collected from cancer patients and processed at MSKCC (Memorial Sloan Kettering Cancer Center) according to the validated standard operating procedures for MSK-IMPACT®, which involves DNA extraction, library preparation, hybrid capture targeting 505 actionable cancer genes, and sequencing on an Illumina platform. In parallel, SOPHiA GENETICS processed the same samples using proprietary enrichment techniques, and sequencing was performed on Illumina platforms. Somatic variants were identified using a robust bioinformatics pipeline, and the results were compared with those obtained by MSKCC to assess the accuracy of the decentralized “MSK-IMPACT® powered with SOPHiA DDM™” solution. Results: A total of 1736 somatic mutations were detected, with variant allele fractions (VAF) as low as 1.1% and a median of 5 variants with coding impact per sample. The positive percent agreement (PPA) for all detected variants with the original MSK-IMPACT® assay is 99.3% for SNV/INDEL with a PPA >98% down to 2.5-6% VAF. Furthermore, PPA for CNVs is 94.6%, and overall percent agreement for MSI and TMB of 99.4% and 98.8%, respectively. Conclusions: The findings affirm that the decentralized “MSK-IMPACT® powered with SOPHiA DDM™” solution delivers a high level of concordance with the established results by MSK-IMPACT®. Future studies are planned to further verify the analytical and clinical performance and to evaluate the utility in routine clinical practice. This solution equips laboratories with a premier, comprehensive testing capability that identifies SNV/INDEL, CNV, DNA fusion, and the critical immuno-oncology biomarkers TMB and MSI. Citation Format: Julien Pontis, Fuad Mohammad, Anjali Anjali, Yvan Wenger, Florian Klemm, Anita F. Bowman, Brian Houck-Loomis, Jacklyn Casanova-Murphy, A. Rose Brannon, Micheal F. Berger, Zhenyu Xu. Decentralized pan-cancer tumor profiling: a comprehensive performance evaluation of the MSK-IMPACT Assay with SOPHiA DDM [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 691.

Abstract 3305: Notch mutations in subjects with recurrent or metastatic adenoid cystic carcinoma treated with rivoceranib from study RM-202

Abstract Background: Activating NOTCH mutations are present in approximately 20% of metastatic adenoid cystic carcinomas (ACC) leading to an aggressive tumor phenotype and poor clinical prognosis, yet less common pathogenic NOTCH mutations are poorly characterized. RM-202 was an international, single-arm, open-label phase 2 trial of oral VEGFR-2 tyrosine kinase inhibitor, rivoceranib, in patients with histologically confirmed recurrent or metastatic (R/M) ACC (Hanna et al. 2023 Clin Cancer Res 29(22): 4555). Herein, we report the mutational analysis NOTCH genes 1-4 (NOTCH1-4) in a subset of ACC samples from RM-202. Methods: Patients (n=72) enrolled in RM-202 received oral rivoceranib 700 mg once daily for a 28-day cycle. The co-primary endpoints were objective response rate (ORR) assessed by both investigator and a blinded independent review committee (BIRC). RNA seq was performed on ACC tumor samples from 27 patients. Complete coding sequences were analyzed with NOTCH1-4 variants identified and cross-referenced with Tempus and public databases to categorize mutations according to pathogenicity. Results: The investigator and BIRC assessed objective response rates were 15.3% (95%CI, 7.9-25.7) and 9.7% (95% CI, 4.0-19.0), respectively. The median duration of response was 14.9 months (95% CI, 4.9-17.3) and median progression free survival was 9.0 months (95% CI, 7.3-11.5). Grade 3 treatment related adverse events occurred in 70% of patients, with hypertension (42.5%) and stomatitis (7.5%) being the most common. A total of 477 variants were identified in NOTCH1-4 from 27 RM-202 ACC tumor samples, of which 463 (97%) mutants had been previously reported by Tempus DNA sequencing of germline and somatic variants across multiple tumor types. Three pathogenic NOTCH1 mutations (two nonsense mutations and 1 in-frame deletion) were identified across 2 tumor samples. Two of these mutations had not been previously reported in ACC. Forty-five additional non-synonymous variants were identified in NOTCH1-4. Of those, NOTCH4 L16 mutations occurred in 17/27 (63%) of tumors, with 12/27 (44%) tumors harboring L16 deletions, 4/27 (15%) of tumors with L16 duplications, and 1/27 (4%) that had both an L16 deletion and an L16 duplication. The frequency of L16 mutant sequences in tumors ranged from 8%-67% suggesting subclonal variation and potential pathogenicity. Conclusions: Previously unreported pathogenic NOTCH1-4 mutations were identified in ACC tumor samples from the RM-202 phase II trial. High rates of NOTCH4 L16 mutations suggest a novel mechanism of Notch pathway activation in ACC and represents a potential therapeutic target in this rare disease. Citation Format: Ari Rosenberg, James Bugni, Lissa Nazal, Chris Galloway, Kristin Ryan. Notch mutations in subjects with recurrent or metastatic adenoid cystic carcinoma treated with rivoceranib from study RM-202 [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 3305.

Abstract 7171: Comparison of clinical next generation sequencing and whole genome sequencing for acute myeloid leukemia characterization and classification

Abstract Acute myeloid leukemia (AML) is a hematopoietic neoplasm characterized by uncontrolled proliferation of myeloid blast cells. AML is the most common acute leukemia, with over 20, 000 new U.S. cases diagnosed annually. The 5-year survival rate varies greatly by age, from 63% in younger patients to 11% in older patients. AML is a genetically heterogeneous disease, with over 25 genes implicated in classification. Clinical next-generation sequencing (NGS) is crucial for AML management, aiding in relapse risk assessment, therapy guidance, and understanding of the genetic landscape. In AML, this typically involves sequencing a panel of 50-500 genes. In practice, only diagnostic samples are tested, without germline controls. In research settings, whole genome sequencing (WGS) is performed on paired diagnosis and remission samples, with remission serving as a germline control. This study assessed the accuracy and utility of AML clinical NGS reports by comparing with matched WGS data. We explored the benefits of germline controls and how discrepancies impact risk classification. 65 AML patients treated in the Northwell Health System and enrolled in an IRB-approved biospecimen study were analyzed. Clinical NGS testing was performed using FoundationOne Heme (n=51) or GenPath OnkoSight (n=14). We analyzed mutation data from clinical NGS reports (median 12 variants per report; range 1-26), identifying DNMT3A, FLT3, TP53, and NPM1 as the most frequently mutated genes, consistent with prior studies. Matched WGS data from 15 patients classified clinically reported variants as somatic (27%; n=57), germline (65%; n=135), or not observed (8%; n=17), using variant allele frequencies (VAFs). There was a strong but imperfect correlation between somatic/germline labels and known/unknown status, provided in the clinical NGS reports: 89% of germline variants were of unknown significance (n=120), while 83% of somatic variants were of known significance (n=43). Clinical NGS reports lacked data such as population variant frequency, pathogenicity scores, and VAFs. Germline and unknown significance variants had higher population frequencies and lower pathogenicity scores than somatic and known significance variants (p<0.0001). European LeukemiaNet (ELN) risk groups assigned using clinical NGS data (Adverse: 51%, n=33; Favorable: 29%, n=19) were affected by the inclusion of germline or unobserved variants; excluding those reclassified two patients from Adverse to Intermediate risk. This study finds general agreement between AML clinical NGS and WGS in the overlapping genes. The clinical NGS reports studied here lack annotation of germline and subclonal variant status. We highlight the limitations of clinical NGS without germline controls and its implications for AML classification, clinical decision making, and broader scientific understanding. Citation Format: Annette A. Prah, Derek Van Delden, Joan Alexander, Asya Stepansky, Erin Boyle, Nicholas Chiorazzi, Jonathan Kolitz, Stephanie Boisclair, Steven L. Allen, David Chitty, Andrea B. Moffitt. Comparison of clinical next generation sequencing and whole genome sequencing for acute myeloid leukemia characterization and classification [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 7171.

Abstract 6624: Comprehensive, multi-omic detection of somatic variants from the GIAB HG008 matched tumor-normal pair using highly accurate long- and short-read whole-genome sequencing

Abstract Disentangling the molecular drivers of cancer progression requires a precise understanding of the somatic alterations that take place at the DNA level during tumor development. These include not only small changes like SNVs and indels, but also structural variants, changes in repetitive elements, differential methylation, as well as the haplotype context in which these changes occur. Existing short-read sequencing methods using sequencing by synthesis (SBS) chemistry lack the read length to characterize large structural variants or to span longer repetitive regions as well as to phase variants into haplotypes, while simultaneously lacking the sequencing accuracy to distinguish very low frequency somatic variants from background sequencing error or to sequence through shorter repetitive elements like microsatellites. Long-read PacBio HiFi data addresses the first set of challenges by accurately sequencing long fragments up to 25 kb, while highly accurate short-read sequencing by binding (SBB) chemistry from PacBio addresses the latter by improving raw sequencing accuracy by orders of magnitude, including in homopolymer regions which make up most microsatellites in the human genome. Here we apply these highly accurate long- and short-read sequencing technologies from PacBio to perform whole-genome sequencing of the newly described HG008 matched tumor-normal pair from the Genome in a Bottle (GIAB) consortium. This reference sample includes an adherent, epithelial-like pancreatic adenocarcinoma (PDAC) cell line as the tumor material, with the matched normal obtained from adjacent duodenal and pancreatic tissue. We perform whole-genome sequencing with both PacBio HiFi (35X normal, 80X tumor) and SBB (50X normal, 100X tumor), followed by alignment and tumor-normal variant calling for all variant types. These accurate long- and short-read sequencing technologies offer a more robust and comprehensive picture of somatic variation in this reference sample and contribute to developing this novel benchmark. Citation Format: Alexandra Sockell, Khi Pin Chua, Christopher Kingsley, Dan Nasko, Matthew Boitano, Young Kim, Melanie Wescott, Ian McLaughlin, Primo Baybayan, Jennifer McDaniel, Andrew Shaver, Justin M. Zook, Aaron Wenger. Comprehensive, multi-omic detection of somatic variants from the GIAB HG008 matched tumor-normal pair using highly accurate long- and short-read whole-genome sequencing [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 6624.

Abstract 7471: Mutational profiles of pancreatic cancer patients from Eastern Asia using somatic exome sequencing

Abstract Introduction: Pancreatic adenocarcinoma (PC) is one of the most lethal diseases. The incidence of PC is rising in the Asia-Pacific population, with some of the highest rates in Japan. As such, the utilization of molecular testing has increased, especially for advanced disease, to identify molecular drivers and therapeutic targets. Methods: 146 patients from Japan (ages 45-81) who were diagnosed with PC underwent exome-based, tumor molecular profiling (TMP) (Labcorp Genetics Inc). 96/146 (65.8%) patients had unresectable tumors. Results were correlated with clinical information. Variants were interpreted according to the AMP/ASCO/CAP guidelines. Signaling pathway analysis was conducted using the Reactome database, with significance determined by binomial test with corrected p-values (FDR; Benjamini-Hochberg procedure). Fisher’s exact test was utilized for statistical assessment of clinical parameters. Results: TMP detected 404 Tier 1/2 somatic variants in 135 (92.5%) patients. Genes known to be frequently mutated in PC were identified in the cohort: KRAS (122/146; 83.6%), TP53 (108/146; 74.0%), SMAD4 (35/146; 24.0%) and CDKN2A (25/146; 17.1%). A potential enrichment of hotspot U2AF1 (5/146; 3.4%) codon 34 mutations was identified, compared to published frequencies in PC in cancer databases (1%) that trended toward statistical significance (p=0.07). Pathway analysis identified a statistically significant enrichment of mutations in several kinase signaling pathways including PI3K/AKT (p-value: 2.1e-11; FDR: 9.8e-09), ERBB2 (p-value: 5.3e-07; FDR: 1.2e-04), and RAS/MAPK (p-value: 4.2e-06; FDR: 5.5e-04). Mutations associated with homologous recombination or mismatch repair pathways were statistically associated with early age of onset (<50 years of age; p= 0.036). Hotspot KRAS mutations were associated with unresectability (p=0.002), late tumor stage (stages 3-4, p=0.026), and metastasis (p=0.010). Conclusion: Alterations enriched in targetable pathways were identified in the patient cohort in addition to statistically significant findings associated with more aggressive PC. TMP testing can aid in the identification of alterations that are pertinent to the identification of relevant therapies and clinical trials as well as identify PC patients with potentially more aggressive disease. Citation Format: Jacquelyn Reuther, Taro Shibuki, Megan Hawley, Hideaki Bando, Keir Violaceae, Makoto Ueno, Kenji Ikezawa, Takuji Okusaka, Teiichi Sugiura, Keiko Kamei, Yoshito Komatsu, Michiaki Unno, Hiroki Yukami, Takuji Iwashita, Emily Westheimer, Kumiko Umemoto, Robert Daber, Takayuki Yoshino, Jianhua Zhao, Masafumi Ikeda. Mutational profiles of pancreatic cancer patients from Eastern Asia using somatic exome sequencing [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 7471.

Abstract 1005: Germline-somatic interactions in non-small cell lung cancer (NSCLC) patients from Latin America

Abstract Motivation: Tumors of non-small cell lung cancer (NSCLC) patients are heterogeneous entities. Genomic landscapes are influenced by genetic constitution, environmental exposures and the complex interaction between the two. In particular, it has been reported that germline variability specific to Latin American (LA) populations play an important role modulating the presence of acquired somatic alterations in EGFR and KRAS, two of the most clinically relevant actionable genes for lung cancer (LC). The influence of these specific genetic differences appears to be independent of smoking status, a key exogenous agent also known to influence LC genomic heterogeneity. However, LA patients are strikingly diverse and further studies are needed to better understand the influence of germline-somatic interaction on tumor variability across populations with different patterns of environmental exposures. Methodology: The population derived from the study protocol Characterization and Validation of Molecular Diagnostic Technologies for LC Patients from Chile, Brazil, and Peru (clinicaltrials.gov NCT03220230). The recruitment period was July 2015 to October 2018 and encompassed 37 centers. From NSCLC samples, genomics profiles were assessed using the Oncomine Focus Assay (OFA), with QC-approved assays obtained for 1864 subjects. Relevant covariates were interrogated at enrollment. In the absence of matched normal tissue samples, an in-house algorithm was developed to distinguish germline from somatic variation. Descriptive statistics, Principal Component Analyses (PCA) and logistic regression were used to evaluate germline-somatic interactions. Results and Conclusions: The in-house algorithm detected 738 unique germline variants and 6636 unique somatic alterations. The majority of the germline variants (80.8%, n=597) are reported in other populations with a Minor Allele Frequency (MAF) ≥ 1%; only a minority (19.1%, n=141) report a MAF either <1% or unknown. These rare germline variants are present in 98 study participants (5.3%) and are more common in the group of Chilean smoker patients with adenocarcinoma and advanced disease, and without a personal and family history of cancer (FHC). Statistical significance was found for cancer stage (stage IV: 80.8% [n=63] vs. 70.8% [n=823], p-value<0.05) and FHC (positive: 20.5% [n=16] vs. 31.9% [n=371], p-value<0.05). Regarding the relationship between germline and somatic alterations, we found a positive association for EGFR, PIK3CA, ALK, FGFR4 and ERBB2 and a negative association for the remaining genes (n=46). These findings provide novel insights into the influence of inherited genetic variability on the clinical presentation and somatic landscape of an underrepresented population. Identification of the underlying germline alleles can improve our understanding of the affected biological pathways for the subset of LCs driven by these alterations. Citation Format: Javiera Garrido, Evelin González, Alejandro Blanco, Gonzalo Sepúlveda-Hermosilla, Matías Freire, Solange Rivas, Katherine Marcelain, Gareth I. Owen, Carolina Ibañez, Alejandro Corvalán, Marcelo Garrido, Rodrigo Assar, Rodrigo Lizana, Javier Cáceres-Molina, Diego Ampuero, Liliana Ramos, Paola Pérez, Osvaldo Aren, Cristina Fernández, María Loreto Spencer, Jacqueline Flores, Giuliano Bernal, Mónica Ahumada Olea, Germán Rasse, Carolina Sánchez, Maria Galli de Amorim, Emmanuel Dias-Neto, Helano C. Freitas, NIRVANA team, Ricardo Armisén. Germline-somatic interactions in non-small cell lung cancer (NSCLC) patients from Latin America [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 1005.

Abstract 442: Dual KAT6/7 inhibition disrupts epigenetic programs that promote tumor evolution and adaptive drug resistance

Abstract The formation of cancer and responsiveness to therapy is governed by a constantly evolving balance between somatic variation and dynamic epigenetic regulation. This evolution has been recognized as a significant obstacle confronting the development of durable cancer therapies. Thus, targeting epigenetic reprogramming in human cancers is a key focus for the design of new drugs to potentially deliver robust and durable anti-tumor activity. Lysine acetyltransferases (KATs) are a promising class of epigenetic targets that operate within interchangeable multi-subunit complexes to regulate cell cycle, pluripotency and lineage determination. Within this class, KAT6A and KAT7 have been recognized as mechanistically intertwined modulators of stem cell identity and lineage commitment through partially overlapping control of chromatin organization specified by histone H3 acetylation of lysine 23 (H3K23) and lysine 14 (H3K14). We noted multiple lines of orthogonal genetic evidence for a “paralog” relationship between these enzymes to support cancer cell autonomous survival in lineage-enriched subsets of the CCLE associated with predictive molecular features. These observations prompted evaluation of concomitant inhibition of KAT6A and KAT7 as a therapeutic approach in biomarker-defined patient populations. Here, we report an equipotent KAT6/KAT7 dual inhibitor with high selectivity versus the structurally similar KAT family members KAT5 and KAT8. We observed synergistic impact on cell viability and pharmacodynamic (PD) modulation of H3K23/H3K14 acetylation in biomarker positive breast, lung, and esophageal cancers with dual KAT6/7 inhibition versus selective inhibition of KAT6 or KAT7. In vitro transcriptomic evaluation of dual KAT6/7 drug effects in ER+ metastatic breast cancer models recapitulated convergent paralog behavior observed using genetic suppression with RNAi. Quantitative transcriptomic and epigenomic profiling of cancer cell responses to dual KAT6/7 inhibition confirmed downregulation of gene expression programs supporting lineage specification, stemness, and WNT signaling. In vivo efficacy studies in 8p11-amplified breast and lung xenografts confirmed enhanced tumor growth inhibition and PD modulation responses for dual KAT6/7 versus selective KAT6 inhibition. Notably, dual KAT6/7 inhibition also promoted improved durable and stable anti-tumor responses in endocrine therapy resistant and ESR1-mutant breast cancer patient derived xenografts that could not be achieved with selective KAT6 inhibition alone. Taken together, these observations support evaluation of dual KAT6/7 inhibition in biomarker-selected patient populations. Citation Format: Manav Gupta, Pranav Gupta, Scott R. Tyler, Nour Ghaddar, Steven L. Spivak, Zabrisky Roland, Katelyn N. Lukas, Ivan G. Shabalin, Jonathon S. Ryan, Carl Schultz, Daniel M. Walden, Santosh Keshipeddy, Muzaffar Alam, Daniel Severance, Anjali Bisaria, Kelly S. Tego, Parker Y. Jameson, Mason J. Appel, Brittany Cruzan, Jay Prakash Jain, Michael E. Dalziel, Sunjay Sethi, Rebeca Choy, Richard Zang, Xin Linghu, Yuchen Bai, Peter Teriete, Diana M. Muñoz, Christian R. Frey, Claire L. Neilan, Josh Taygerly, Jacob Burch-Konda, Paul A. Barsanti, Michael A. White, Brian T. Jones. Dual KAT6/7 inhibition disrupts epigenetic programs that promote tumor evolution and adaptive drug resistance [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 442.

Abstract 1066: OncoKBTM, MSK’s precision oncology knowledge base: 2024 updates

OncoKB, Memorial Sloan Kettering Cancer Center’s (MSK) precision oncology knowledge base, contains evidence-based information about the oncogenic effect and therapeutic implications of somatic alterations in cancer. OncoKB currently includes annotation for >8000 alterations in 900 cancer-associated genes and is the only somatic cancer variant database partially recognized by the US-FDA. OncoKB supports variant interpretation in the cBioPortal for Cancer Genomics, is used to annotate >17,000 MSK patient sequencing reports annually and is publicly available through its website (www.oncokb.org). Access to OncoKB, including its web-based API, is freely available to users in an academic setting, while users from commercial and hospital settings require a fee-based license. OncoKB’s Therapeutic Levels of Evidence system classifies variants based on tumor type-specific sensitivity or resistance to targeted therapies. To date, OncoKB includes 53 Level 1 genes as well as MSI-H and TMB-H (included in the FDA drug label), 7 Level 2 genes (included in professional guidelines), 14 Level 3A genes (predictive of response in well-powered clinical studies), 10 Level 4 genes (predictive of response based on compelling biological evidence), and 12 R1/R2 resistance genes. In 2024, OncoKB updated its content to include notable precision oncology drug development changes. OncoKB promoted BRAF fusions to Level 1 following inclusion as patient eligibility criteria in the FDA drug label for tovorafenib (low-grade glioma). Additionally, OncoKB included KRAS G12C in colorectal cancer and IDH1 mutations in myelodysplastic syndromes as Level 1 following FDA approval of adagrasib + cetuximab and ivosidenib, respectively. NCCN guidelines for colorectal cancer and small bowel cancer listed immune-checkpoint inhibitors for tumors harboring POLE or POLD1 oncogenic exonuclease domain missense mutations, making them Level 2 in these indications. Lastly, novel biomarkers including FBXW7 and PPP2R1A alterations (endometrial and ovarian cancer), SMARCA4 mutations (non-small cell lung cancer and esophageal adenocarcinoma) and MTAP deletions (all solid tumors) were included in OncoKB based on compelling preclinical and emerging clinical evidence in association with lunresertib + camonsertib, PRT3789, and AMG193 and MRTX1719, respectively. In sum, 17 novel clinically actionable biomarkers (Levels 1-4) and 26 follow-on precision oncology therapies for existing leveled biomarkers were added to OncoKB in 2024. Additionally in 2024, OncoKB prepared to incorporate germline variants into the knowledge base, a feature expected to be publicly available in 2025. OncoKB also implemented major software updates to support data integration into the EPIC platform. Future OncoKB efforts are focused on whole genome/exome curation, inclusion of biomarkers for non-NGS-based precision oncology therapies, and the development of a clinical trial matching system. Citation Format: Moriah H. Nissan, Sarah P. Suehnholz, Hongxin Zhang, Ritika Kundra, Calvin Lu, Amanda Dhaneshwar, Nicole Fernandez, Kelly Cavender, Benjamin Preiser, John Konecny, Kseniya Petrova, Mark Ewalt, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Anoop Balakrishnan-Rema, Aijazuddin Syed, A. Rose Brannon, Ahmet Dogan, Diana Mandelker, Zsofia Stadler, Alexander Drilon, David B. Solit, Ross Levine, Nikolaus Schultz, Debyani Chakravarty. OncoKBTM, MSK’s precision oncology knowledge base: 2024 updates [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 1066.

Abstract 3901: Charting copy number gain histories from mutational divergence among multi-samples

Abstract In cancer, a somatic copy number gain (SCNG) on a tumor chromosomal region is often assumed to result from a series of minimum number of copy number events, but this parsimonious-trajectory assumption may be violated in reality. For example, an SCNG could develop through a complex trajectory involving a combination of gain and loss events, rather than exclusively through gains. While prior efforts have been made to infer the SCNG trajectories from patient data, the presence of complex trajectories remains to be validated. Furthermore, these methods are limited by either only utilizing SCNG profiles, or inferring trajectories from only a single sample. Here, we hypothesize that SCNG trajectories can be reflected by somatic single nucleotide variants (SSNVs) shared between samples from the same patient. By analyzing multi-regional whole genome sequencing data of tumor specimens, we found evidence where the multiplicities of shared SSNVs between samples pinpoint complex SCNG trajectories. Here, the SSNV multiplicity refers to the number of alleles harboring the corresponding SSNV in the dominating clone of a tumor sample. We also define the joint multiplicity distribution as the proportion of shared SSNVs that occur at different allelic states (multiplicities) across distinct tumor samples. We developed a probabilistic computational pipeline to automate the identification of non-parsimonious multi-sample SCNG trajectories utilizing shared SSNV multiplicities. Extending an existing method GRITIC from single sample to multi-sample setting, this pipeline enumerates all trajectories leading to SCNGs states in multiple samples, and characterizes the probability of observing the joint multiplicity distribution in the real patient data given each possible trajectory. We applied the method on prostate cancer and breast cancer datasets, and identified conclusive cases of complex trajectories on genomic regions affected by SCNGs. On regions showing different copy number states in multiple samples, we identified cases experiencing both gains and losses. On regions showing the same copy number states in multiple samples, we also identified a few of them experiencing independent acquisition of SCNGs in different samples. Our findings call for a stochastic modeling of SCNGs dynamics during tumor evolution. Citation Format: Yunong Xia, Ruping Sun. Charting copy number gain histories from mutational divergence among multi-samples [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 3901.

Abstract 5002: Multi-omics insights into the role of clonal hematopoiesis in multiple myeloma

Abstract Multiple myeloma (MM) is a cancer involving the abnormal proliferation of malignant plasma cells in the bone marrow (BM). Clonal hematopoiesis (CH), a condition involving the clonal expansion of blood cells due to somatic mutations, is commonly observed with aging. However, its precise role in the pathogenesis of MM remains unclear. This study hypothesizes that CH influences MM cells and the microenvironment through paracrine mechanisms, thereby contributing to disease progression and treatment resistance. Using UK Biobank data, somatic CH variants were called, excluding those with a variant allele frequency (VAF) < 0.02, and analyzed them against ICD-10 codes. Hazard ratios (HR) were computed through Cox proportional regression, adjusting for covariates. Single-cell multi-omics (scDNA) sequencing was performed on BM aspirates from two MM patients, followed by cell type annotation and CH mutation subtyping using the MissionBio Mosaic pipeline. Exosomes were extracted from BM samples of 30 MM patients, and exosome small RNA sequencing was conducted. Differential expression of miRNAs was identified, and a computational tool was used to predict and integrate the target genes and pathways regulated by the differentially expressed miRNAs. Single-cell RNA (scRNA) sequencing was performed on BM sample from 12 MM patients, with data analyzed using the Seurat pipeline. Proteomics analysis using UK Biobank data was performed on a cohort of 190 MM patients identified based on ICD-10 codes. Differentially expressed protein and pathway analyses were conducted using OlinkAnalyze R package. Survival analysis conducted using the UK Biobank cohort revealed that the presence of CH was significantly correlated with MM diagnosis (HR: 1.64, CI: 1.35-2, P < 0.001), and CH in MM impacted EFS (HR: 1.61, CI: 1.25-2.1, P < 0.001). The results of the scDNA analysis revealed that approximately 88% of CH-mutated cells were distributed within myeloid cells, suggesting potential indirect effects on MM through paracrine interactions. Exosomal miRNA analysis of 30 MM patients revealed a significant reduction in miRNAs targeting the MAPK and JAK-STAT pathways (KEGG, P < 0.005), indicating dysregulation of oncogenic and inflammatory signaling in individuals with CH. scRNA analysis identified the activation of inflammatory pathways in CD14+ monocytes of individuals with TET2 and DNMT3A mutations, including the MAPK pathway (GOBP, adjP < 0.05) and cytokine interactions (KEGG, adjP < 0.05). Proteomics analysis using Olink data from 190 MM patients in the UK Biobank further validated the activation of inflammatory and oncogenic pathways, including MAPK pathway (GOBP, adjP < 0.05), STAT pathway, and cytokine-cytokine receptor interactions (KEGG, adjP < 0.05), in CH-positive MM cases. These findings highlight the potential for CH mutations, particularly in TET2 and DNMT3A, to exacerbate MM progression through inflammatory paracrine mechanisms. Citation Format: Jeongmin Park, Ja Min Byun, Dong-Yeop Shin, Junshik Hong, Sung-Soo Yoon, Siddhartha Jaiswal, Youngil Koh. Multi-omics insights into the role of clonal hematopoiesis in 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 5002.

Abstract 3892: Multi-sample whole genome sequencing unveils complex mutational dynamics and clonal evolutionary patterns in young onset breast cancer from Nigeria

Abstract Background: Breast cancer (BC) is a heterogenous disease, and the rising global epidemic of premenopausal BC remains poorly understood. Mutational landscape and evolutionary dynamics of BC from diverse geography and populations are largely unknown, preventing the global acceleration of progress towards precision prevention. Methods: To examine etiology and heterogeneity of BC in indigenous Africans, 43 multi-region tumor samples and blood samples from 18 Nigerian women with BC (mean age 53 +/-12.7) were analyzed. Whole Genome Sequencing (WGS) was performed to identify somatic single nucleotide variants, insertions and deletions (ID), copy number alterations (CN), and structural variants (SV). Five mutational signature types (single base substitutions (SBS), double base substitutions (DBS), ID, CN, and SV), were analyzed and compared to COSMIC human cancer signatures. Multi-DPclust was used to classify mutations to clonal and subclonal cluster and create mutational phylogenetic trees. Results: Driver gene analysis highlighted key driver genes, such as TP53, GATA3, and PIK3CA, corroborating prior findings. The most common signature was clock-like (CL) SBS5, followed by APOBEC-related SBS2 and SBS13. Other notable signatures included hypoxia-linked SBS18 and HRD-related DBS13. Signature profiles showed moderate heterogeneity across samples, with distinct patterns observed across mutational clusters. Intra-cluster correlation coefficients for signatures within samples from same patient range from 0.37 to 0.99 (median 0.84). TP53 and GATA3 mutations are common in clonal (early) clusters, with GATA3 often appearing in consecutive subclones. The CL SBS5 signature was prevalent early, while SBS18, linked to hypoxia, became prominent late, highlighting evolving mutational processes across disease progression. Distinct BC subtypes also displayed unique mutational profiles. HR+/HER2+ tumors exhibited higher levels of signature SBS91, while HR-/HER2- tumors exhibited higher levels of the HRD signature SBS3 compared to the rest of the tumors. HR-/HER2- tumors demonstrated lower levels of the mismatch repair-related signature ID1 but higher levels of the HRD signature ID6 and TOP2A signature ID8. Significant differences were observed in SV signatures, with higher SV9 signature activities in HR+/HER2+ tumors and elevated BRCA-related SV3 signature activities in HR-/HER2- tumors. Heterogeneity was also evident in the DBS and CN mutational signatures, further highlighting tumor complexity. Conclusion: This analysis sheds light on the diverse mutational dynamics within and across molecular subtypes, providing insights into the mutational evolution of BC in a non-screen detected young onset population. Ongoing work integrating WGS and transcriptome data will be presented at the conference. Citation Format: Avraam Tapinos, Toshio Yoshimatsu, Ilona Siljander, Mustapha A. Ajan, Ayodele Sanni, Atara Ntekim, Abayomi Odetunde, Elisabeth Sveen, Jeffrey Mueller, Galina Khramtsova, Sulin Wu, Dorothy Nyamai, Mihai Giurcanu, Dezheng Huo, Yonglan Zheng, David C. Wedge, Olufunmilayo I. Olopade. Multi-sample whole genome sequencing unveils complex mutational dynamics and clonal evolutionary patterns in young onset breast cancer from Nigeria [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 3892.

Abstract 2780: Topoisomerase IIb binding underlies frequently mutated elements in cancer genomes

Abstract Type-II topoisomerases resolve topological stress in DNA through controlled double-strand breaks. While TOP2A is a chemotherapy target in proliferating cells, the ubiquitously expressed TOP2B is a potential off-target. Here we explore roles of TOP2B in mutagenesis by generating DNA-binding maps of TOP2B, CTCF, and RAD21 in human cancer samples and analyzing these maps for driver mutations and mutational processes in 6500 whole cancer genomes. TOP2B-CTCF-RAD21 and TOP2B-RAD21 sites are enriched in somatic mutations and structural variants (SVs), especially at evolutionary conserved sites displaying high transcription and long-range chromatin interactions. TOP2B binding underlies SVs and hotspot mutations in cancer-driving genes such as TP53, MYC, FOXA1, and VHL, and many cis-regulatory elements. We show that the TOP2B-bound mutational hotspot at RMRP drives tumor initiation and growth in vivo. These data highlight TOP2B as a protector of the genome from topological challenges whose aberrant activity promotes driver and passenger mutations in cancer genomes. Citation Format: Jüri Reimand, 1 Christian A. Lee, 1 Robin H. Oh, 2 Zoe P. Klein, 1 Nina Adler, 1 Sana Akhtar Alvi, 3 Ellen Langille, 2 Elisa Pasini, 4 Kevin C. Cheng, 1 Diala Abd-Rabbo, 1 Huayun Hou, 3 Ricky Tsai, 2 Mamatha Bhat, 1 Daniel Schramek, 2 Michael D. Wilson, 3 Liis Uusküla-Reimand3. Topoisomerase IIb binding underlies frequently mutated elements in cancer genomes [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 2780.