Somatic Variants Research Articles

Abstract 5351: The landscape of somatic mutations and copy-number alterations in the Indian Cohort of Prostate Cancer Patients

Abstract In India, prostate cancer (PCa) has the third highest incidence rate among males and is often diagnosed at the advanced stage. PCa is known to be molecularly heterogeneous, and an in-depth characterization of its subtypes by geographical ethnicities may aid in the understanding of its etiology and disparities. Hence, we perform molecular characterization of PCa patients from India. Integrative genomics studies categorized PCa into molecular subtypes based on genetic aberrations (e.g., somatic mutations, INDEL, etc.). Although genetic factors are known to impact PCa pathobiology, genomic profiling of Indian PCa patients and its clinical utility remain largely unexplored. Thus, in a prospective study, we performed whole exome sequencing (WES) of treatment-naive primary PCa (N=90) and benign prostatic hyperplasia specimens (N=14) along with matched blood. Our findings reveal distinct genetic alterations unique to the Indian cohort, wherein an elevated somatic tumor mutational burden and an increased percentage of clinically actionable alterations were noted. About 80% of the somatic variants (called using Mutect2) were found to be missense mutations with a high recurrence of C>T transitions, a signature of age-related human malignancies. Several novel pathogenic alterations affecting critical domains of the cancer-associated genes (e.g., APC, BRCA2, PI3KCA, etc.) were found, which have not been reported in the dbSNP or COSMIC databases. Systematic comparison of our findings with primary PCa data available at The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) revealed distinct genomic alterations in Indian PCa patients: specifically, 35% of the patients showed presumed actionable lesions in the RTK-Ras pathway genes, while 10% had mutations in the PI3K pathway genes, and 21% of all the specimens harbored putative inactivating mutations of genes involved in the homologous recombination repair pathway. We also found major differences in the mutational frequency of some driver genes, such as ATRX, PIK3C2G, TAL1, etc., compared to the TCGA-PRAD cohort. Furthermore, GISTIC2 analysis revealed significant arm-level amplification at 8q (MYC, GLI4), 7q (GNB2, PDIA4), and 16p (TELO2, TSC2) regions and deletion at 8p (NKX3.1, MSR1), 9p (CDKN2A, CDKN2B), and 13q (RB1, BRCA2) regions. We also identified recurrent focal amplifications at 9q, 14q, and 11q, and deletions at 16q, 10q, and 5q loci. Further, integrating WES data with transcriptome data would be helpful in categorizing PCa patients based on gene fusion status and prioritizing functionally impactful and actionable variants. Collectively, our findings reveal molecular heterogeneity among PCa patients from India, leading to the identification of clinically relevant candidates that need to be further validated and would help in strategizing therapies and clinical management of this disease. Citation Format: Tanay Biswas, Pankaj Vats, Vipul Bhatia, Atin Singhai, Alok Srivastava, Abhishek Chauhan, Manzoor Ahmad, Piyush Tripathi, Sanjeev Mehrotra, Anjali Tewari, Nuzhat Husain, Apul Goel, Nallasivam Palanisamy, Bushra Ateeq. The landscape of somatic mutations and copy-number alterations in the Indian Cohort of Prostate Cancer Patients [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 5351.

Abstract 1089: Developing subtype-specific pediatric cancer molecular targets by aggregating diverse genomic data resources to the pediatric cancer (PeCan) knowledge base portal

Abstract Knowledge about molecular targets for pediatric cancer has accelerated exponentially in recent years thanks to the increased application of multi-omics profiling in both research and clinical settings. The efficacy of genomic-based clinical interventions may depend on whether observed genomic abnormalities are fundamental to the pathogenesis of a specific cancer subtype. At present such information is limited in pediatric cancer due to the rapid evolution of subtype discovery and classification as well as disease heterogeneity owing to the presence of many rare cancer types. To develop a comprehensive resource of molecular targets in pediatric cancer subtypes, we have integrated somatic variants in pediatric cancer samples (n=∼5, 500) currently hosted in PeCan Portal (http://pecan.stjude.coud) with those (n=∼1, 400) profiled by NCI’s Childhood Cancer Data Initiative (CCDI) molecular characterization program. To leverage the recently developed WHO CNS5 classification of CNS tumors, we reclassified 2, 167 relevant samples based on molecular drivers and histology as a major update to our pediatric cancer ontology definitions, which have recently become a shared standard in the CCDI community. To determine driver genes in disease pathways relevant to specific cancer types, we analyzed the pathogenicity of SNVs and indels, CNVs, SVs and gene fusions and integrated the findings with driver genes reported in 176 publications. A total of 469 distinct driver genes were mapped to 40 pathways across the 22 major pediatric cancer types, which include 3 types of hematological malignancies (HM), 10 types of CNS tumor and 9 types of solid tumor (ST). Pathways with driver genes disrupted across the broad spectrum of cancer types include cell cycle, chromatin remodeling, PI-3K, RAS signaling and telomere maintenance. By contrast the vast majority (>90%) of the transcription factors (TF) are lineage-specific— among the 128 TF genes identified as driver genes, only 11 affect multiple lineages. These include MYC/MYCN which affect HM, ST and CNS tumors; ERG, EWSR1, FLI1 and WT1 which affect HM and ST; and KLF4, MED12, MN1, MYB and TCF4 which affect CNS and HM. These results highlight the importance of developing an infrastructure for exploring mutation prevalence in driver genes and pathways at the cancer subtype level, which will be supported in the upcoming release of the PeCan portal. This, coupled with new features for dynamic exploration of mutational signatures, global expression maps and gene-level transcription across the cancer subtype classification tree, will greatly expand our knowledge of molecular targets and vulnerabilities in pediatric cancer, paving the way for novel therapeutic advancements Citation Format: David B. Finkelstein, Delaram Rahbarinia, Ramzi Alsallaq, Stephanie Sandor, Michael Macias, Jian Wang, Sue Qiu, Brian Curran, Michael Edmonson, Andrew Thrasher, Jobin Sunny, James Madson, Scott Foy, Brent Orr, Mark R. Wilkinson, Clay McLeod, Michael Rusch, Xiaotu Ma, Jinghui Zhang. Developing subtype-specific pediatric cancer molecular targets by aggregating diverse genomic data resources to the pediatric cancer (PeCan) knowledge base portal [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 1089.

Abstract 4090: Analytical validation of PanTracer LBx performance, a comprehensive pan-solid tumor liquid biopsy assay

Abstract Introduction: Pan-solid tumor comprehensive genomic profiling (CGP) assays have gradually replaced single gene and smaller, tumor-specific panels to identify actionable somatic mutations in patients with advanced cancer. While tissue-based CGP remains important for clinical decisions, invasive biopsies can be challenging in terms of material quantity/quality and/or risk to patients. Plasma-based CGP assays provide the potential for rescuing cases where tissue fails, providing faster results to enable treatment decisions, monitoring treatment through repeated sampling, or capturing tumor heterogeneity. As such, CGP via liquid biopsy (LBx) represents an asset to patients and the options for such testing spans several to hundreds of genes. Here we describe initial results of the analytical validation (AV) process of a large deep sequencing panel aiming to establish the initial performance characteristics of the PanTracer LBx CGP assay. We also provide initial preliminary comparison data to an established highly sensitive targeted panel. Methods: PanTracer LBx, a next-generation sequencing (NGS) assay based on TSO500 chemistry, is designed to detect key classes of somatic alterations across solid tumors, such as SNVs and small InDels (517 genes), CNVs (59 genes), fusions (23 genes) and key immune signatures. AV was performed in a CAP/CLIA certified laboratory across the above mutation classes to determine key assay performance characteristics: limit of detection (LoD), limit of blank (LoB), precision and analytical sensitivity/specificity using clinical samples with orthogonal data. Results: LoD90, was assessed by measuring the detection of somatic variants present in SeraSeq complete ctDNA reference material at different %VAF levels (0.1%-2%) and DNA concentrations. Using a Probit regression model, LoD90 for SNVs and InDels was determined as 0.17% and 0.31%, respectively, and 1.23x for CNVs. LoB for SNVs/InDels and CNVs, assessed by calculating the cumulative per gene specificity across 21 healthy donors subjected to sequencing, was 0%. Analytical sensitivity and specificity were determined by sequencing late-stage cancer samples and comparing results to those obtained with an amplicon-based assay (InVisionFirstTM-Lung). Positive and negative percent agreement with the orthogonal method was 96.9% and 99.3%, respectively. Precision (inter-assay, -lot and -operator) was >95%. Conclusions: Performance evaluation of PanTracer LBx assay met all pre-specified AV acceptance criteria. Fusion LoD90 analysis is ongoing and will be presented during the meeting with additional results including input metrics and preliminary clinical validation. Based on a large panel targeting different classes of somatic variants across multiple genes, it offers results consistent with a smaller, established targeted panel with the addition of more biomarkers that may be informative or actionable. Citation Format: Christodoulos Pipinikas, Faqiang Wu, Daniel Whang, Rebecca Hong, Samuel Koo, Qinqin Zha, Jieying Chu, Rachel Schell, Jiannan Guo, Giovanni Marsico, Tim Forshew, Amber Chevalier, Andrew Lukowiak, Heng Xie, Gregory Jones. Analytical validation of PanTracer LBx performance, a comprehensive pan-solid tumor liquid biopsy assay [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 4090.

Abstract 179: Spatial and multiomics analysis reveals immune interactions as key drivers of immunotherapy outcomes in melanoma patients with in-transit metastases

Abstract Background: In-transit metastasis (ITM) of melanoma occurs between the primary tumor and draining lymph nodes. ITM can be managed by by adjuvant immunotherapy after resection, or immunotherapy alone when unresectable. Nevertheless, nearly half of ITM patients are resistant, which constitutes a clinical challenge. Multiomic spatial profiling offers insights into features influencing immunotherapy response, but the tumor microenvironment (TME) and biological determinants of ITM remain poorly defined. This study aims to characterize molecular and spatial TME attributes in ITM patients to improve understanding of immunotherapy response and resistance. Methods: Fifty-four samples from ITM patients treated with immunotherapy were collected at pre-treatment (PRE) and progression (PROG). Patients with a complete or partial response in the advanced setting, or recurrence-free survival over 12 months in the adjuvant setting, were classified as "responsive," and others as "resistant". Single-cell sequencing and 40-marker imaging were conducted on 18 matched tumor dissociates and FFPE samples at PRE (n=3 resistant, n=5 responsive) and PROG (n=10). Whole genome sequencing on 12 fresh frozen samples (n=4 resistant, n=8 responsive at PRE) were used to identify somatic variants. Results: While regional immune cell proportions were broadly similar across response groups, PRE responders showed increased intra- and peri-tumoral T cells, and surprisingly, reduced CD141+ dendritic cells (cDC1s) compared to resistant patients (P=0.024). K-means clustering identified 7 spatially resolved cellular neighborhoods (CNs) across all ITMs with heterogeneous cellular constitution; nevertheless, lymphocyte-enriched CNs were more dispersed in PRE responders and interacted with other immune and tumor CNs, while remaining peripheral in PRE resistant tumors. Despite having a lower DC density, pairwise interaction analysis highlighted significant interactions between cDC1s and CD14+ macrophages in PRE responders, and between macrophages and IDO1+ CD4+ T cells or Granzyme B+ CD8+ T cells (P<0.05, log2 fold change>0.2), suggesting enhanced macrophage-driven local T cell stimulation in the absence of DCs. Single-cell transcriptomics analysis comfirmed the reduced proportion of cDC1s in PRE responders compared to resistant ITM (P=0.006). Genomic profiling revealed EREG mutations exclusively in resistant ITMs, which has been linked to immunoregulationand tumor invasion through the EGFR pathway. Ongoing analysis explores interactions among DCs, macrophages, T cells, B cells, melanocytes, and endothelial cells to elucidate TME dynamics associated with immunotherapy outcomes. Conclusion: These findings deepen our understanding of tumor-immune interactions in ITM patients and inform personalized immunotherapy approaches tailored to their unique TME. Citation Format: Xinyu Bai, Camelia Quek, Ghamdan Al-Eryani, Sonny Ramkomuth, Louise Baldwin, Jiabao Tian, Nurudeen A. Adegoke, Ismael A. Vergara, Felicity Newell, Kate Harvey, John Reeves, Andrew J. Spillane, Nicola Waddell, Serigne N. Lo, Alexander M. Menzies, Ines P. da Silva, Umaimainthan Palendira, Georgina V. Long, Richard A. Scolyer, Alexander Swarbrick, James S. Wilmott. Spatial and multiomics analysis reveals immune interactions as key drivers of immunotherapy outcomes in melanoma patients with in-transit metastases [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 179.

Abstract 6288: Precise characterization of complex repeats in cancer genomes

The characterization of somatic variation, especially in complex genomic regions, is crucial for understanding the molecular drivers of cancer progression. Accurate PacBio long-read sequencing (HiFi) enables detection of all variant classes, from simple SNVs and INDELs up to complex structural variation, tandem repeats, and changes in epigenetic signatures. Complex and repetitive regions, while fully sequenced by HiFi reads, remain bioinformatically challenging, requiring tailored solutions. Here, we describe new tools to genotype understudied repetitive regions in cancer genomes, a task that has historically posed significant challenges to short-read sequencing.Our software tool, TRGT, resolves the sequence and methylation patterns of complex repetitive regions at a single-nucleotide resolution. A recent algorithmic improvement (vclust module) enables us to type expanded hyper-polymorphic regions, termed variation clusters. Variation clusters usually result in many incorrect variant calls in computational pipelines. By analyzing a variation cluster as a single region, rather than many small variants, we can accurately measure genetic variation. To demonstrate the importance of variation clusters in cancer, we comprehensively resolved repeat regions in five cell lines and 50 clinical tumor samples (with matched healthy tissue). We identified 5,500 new variation clusters across the tumors and an average of 46,220 (out of 4 million, 1%) loci that are contracted or expanded in a tumor compared to its matched normal tissue. We find 3% of these loci can be located within genes found in the Compendium of Cancer Genes, suggesting potentially oncogenic significance. In addition, using TRGT, we were able to detect microsatellite instability (MSI) in a tumor with a compound heterozygous coding mutation in MSH2. Lastly, we further analyzed haplotypes in selected variation clusters to detect and resolve sequences in somatic mosaicism. By incorporating this new class of variants—complex repeats—into our analysis, we significantly enhanced the capability of HiFi reads to detect a comprehensive spectrum of genomic variations. The inclusion of complex repeat genotyping with PacBio HiFi reads will enable a more detailed understanding of cancer genetics and open new avenues for research and clinical applications. Citation Format: Khi Pin Chua, Egor Dolzhenko, Zev N. Kronenberg, Seiya Imoto, Seiichi Mori, Michael A. Eberle. Precise characterization of complex repeats 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 6288.

Abstract 1956: Comparison of somatic variations between circulating tumor DNA and paired tissue in 1,111 pan-cancer patients

Abstract Circulating tumor DNA (ctDNA) offers a noninvasive method to assess tumor-specific mutations. This retrospective study investigated the concordance of genomic variations between ctDNA and tissue and its influence factors. Paired blood and tissue samples were collected from 1, 111 patients with ten tumor types and underwent sequencing of 425 genes. We evaluated the relationship between the concentration of cell-free DNA (cfDNA), sensitivity of ctDNA, mutation landscape, variant allele frequency, and various clinical parameters. The median concentration of cfDNA and overall sensitivity of ctDNA was 16.8 ng/ml and 61.5%, which correlated positively with clinical stage and tumor diameter and negatively with treatment response. The overall concordance rate of genomic alterations between ctDNA and tissue was 24.6%. The top five altered genes and pathway-based disruption signatures identified by ctDNA profiling were similar to tissue. For mutations with a normalized allele frequency less than 0.2, the proportion of ctDNA-specific mutations was higher than concordant mutations (61.5% and 9.7%). Advanced clinical stage, large tumor diameter, poor treatment response, the presence of liver metastasis, and impaired liver function lead to a high concordance rate. Patients with ctDNA-positive status, subclonal mutations, and more ctDNA-specific mutations had shorter overall survival than those with ctDNA-negative status, no subclonal mutations, and fewer ctDNA-specific mutations, respectively. In conclusion, a higher concordance rate of somatic variations between ctDNA and tissue indicated an advanced clinical stage, larger tumor size, worse treatment response, and presence of liver metastasis. Subclonal or more ctDNA-specific mutations detected were associated with poor prognosis. Citation Format: Meng Zhang, Yi Feng, Xue Du, Changda Qu, Meizhu Meng, Meiying Ye, Min Liang, Ziran Yang, Wenjuan Gong, Xingyu Ma, Jialiang Guo, Wenmei Li, Chenxuan Wang, Qiuxiang Ou, Sha Wang, Haimeng Tang, Shuqin Jia. Comparison of somatic variations between circulating tumor DNA and paired tissue in , pan-cancer patients [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 1956.

Identifying genetic errors of immunity due to mosaicism.

Inborn errors of immunity are monogenic disorders of the immune system that lead to immune deficiency and/or dysregulation in patients. Identification of precise genetic causes of disease aids diagnosis and advances our understanding of the human immune system; however, a significant portion of patients lack a molecular diagnosis. Somatic mosaicism, genetic changes in a subset of cells, is emerging as an important mechanism of immune disease in both young and older patients. Here, we review the current landscape of somatic genetic errors of immunity and methods for the detection and validation of somatic variants.

Integration of clinical data with results from next-generation sequencing technology used to detect somatic and germline variants in patients with high-grade serous ovarian cancer

Ovarian cancer represents a significant global health issue, posing a significant challenge for the field of medicine. One strategy to improve the prognosis for women around the world is to implement modern technologies and apply the concept of personalized medicine. This study employed an in-depth case study of patients diagnosed with high-grade serous ovarian cancer. Advanced genetic testing methods and a comprehensive review of the patient’s medical history were utilized to identify new potential markers for early detection of the disease and eligibility for treatment. In this study, next-generation sequencing (NGS) technology was utilized for analysis. Tissue panel tests were performed to detect somatic mutations, while whole exome sequencing (WES) was conducted on blood samples to detect germline mutations. The results obtained were then analyzed in the context of the patient’s medical history to identify patients with a familial predisposition to cancer and to look for an association with comorbidities. The utilization of genetic testing and the analysis of patients’ medical histories facilitated the identification of somatic and germline variants in genes associated with carcinogenesis. This approach led to the identification of ovarian cancer-specific and novel variants. Furthermore, germline variants associated with comorbidities were identified. The utilization of contemporary molecular biology methodologies can markedly enhance the diagnostic accuracy of patients and allow the development of new diagnostic tests.

TET2 gene mutation status associated with poor prognosis of transition zone prostate cancer: a retrospective cohort study based on whole exome sequencing and machine learning models.

Prostate cancer (PCa) in the transition zone (TZ) is uncommon and often poses challenges for early diagnosis, but its genomic determinants and therapeutic vulnerabilities remain poorly characterized. Tumor mutational landscape was characterized in nine patients with TZ PCa, identifying somatic variants through whole-exome sequencing (WES). Novel candidate variants relevant to driver gene were selected using rare-variant burden analysis. Kaplan-Meier curves with log-rank testing and Cox regression models were applied to evaluate the prognostic significance of selected mutant driver gene and clinicopathological characteristics in a cohort of 132 patients with TZ PCa. Significant prognostic determinants were integrated into a validated nomogram for individualized prediction of 3-, 4-, and 5-year biochemical recurrence-free survival (BRFS) and overall survival (OS) probabilities. Eight machine learning algorithms were employed to develop BRFS and OS prediction models in a cohort. A total of 5,036 somatic single nucleotide variants (SNVs) and 587 somatic insertion and deletion (INDELs) were discovered. Among eight driver gene mutations which were verified through Sanger sequencing, TET2 gene, with high mutation frequency and potential targeted drug relevance (bromodomain inhibitors and DOT1L inhibitors) was selected for further validation. Retrospective cohort study demonstrated that TET2 mutant status was significantly associated with Gleason score (p = 0.004) and distant metastasis (p = 0.002). Furthermore, TET2 mutant status was significantly correlated with inferior BRFS and OS and served as an independent predictor. Comparative evaluation of eight algorithms revealed the GBM model achieved superior discriminative ability for BRFS (AUC for 3-year: 0.752, 4-year: 0.786, 5-year: 0.796). The predictive model based on the GBM machine learning algorithm achieved the best predictive performance for OS (AUC for 3-year: 0.838, 4-year: 0.915, 5-year: 0.868). The constructed predictive nomogram provided evidence that TET2 mutant status integration conferred statistically significant improvements in model accuracy and clinical predictive value. Our study elucidated the distinct genetic basis of prostate cancer in the transition zone and identified TET2 mutation as an independent prognostic determinant in TZ PCa. However, the limited sample size of this study necessitates cautious interpretation of these findings, and further validation in larger cohorts is warranted to confirm their generalizability.

A Rare Case of Lhermitte Duclos Disease Associated with Somatic PTEN and Germline SUFU Variants.

Lhermitte-Duclos disease (LDD) is a rare dysplastic gangliocytoma of the cerebellum, typically manifesting as a hamartomatous lesion in the posterior fossa. Currently, LDD has been only linked to PTEN pathogenic variants, with the PI3K/AKT/mTOR pathway acting as the primary signaling cascade responsible for its pathogenesis. We present a case of LDD in which a novel germline heterozygous splice site variant (c.183-2A > G) in the SUFU gene and a somatic heterozygous missense variant (c.389 G > A) in the PTEN gene, identified from tumor tissue were detected by targeted next-generation sequencing (NGS). SUFU, a tumor suppressor gene, primarily inhibits the hedgehog (Hh) signaling pathway and furthermore influences the AKT/mTOR pathway. Pathogenic variants in SUFU have been linked to medulloblastoma, and their potential role in LDD remains under investigation. Given that both conditions involve granule cell progenitors and are influenced by impaired Hh signaling, they may share a similar developmental path. This is the first research indicating that SUFU may play a role in the etiology of LDD, despite SUFU variants being associated with several central nervous system malignancies. The SUFU variant was shown to disrupt splicing via Sanger sequencing and gel electrophoresis of RNA extracted from blood. Analysis of DNA from tumor tissue using the TWIST Exome 2.0 Panel revealed de novo pathogenic SUFU (c.183-2A > G) and PTEN (c.389G > A) variants. This paper establishes an initial link between LDD and germline SUFU along with somatic PTEN variants identified from tumor tissue, providing novel insights into the molecular pathogenesis of this rare condition.

Analysis of Concurrent Intracholecystic Papillary Neoplasms and Biliary Intraepithelial Neoplasia Reveals Distinct Histologic and Molecular Profiles.

Intracholecystic papillary neoplasms (ICPNs) and biliary intraepithelial neoplasia (BilIN) are presumed precursors to gallbladder adenocarcinomas, but their relationship is incompletely understood. To perform morphologic and molecular characterization of concurrent ICPNs, nonpolypoid mucosa, and adenocarcinomas to determine whether these lesions are related at the DNA level. Background mucosa and 36 ICPNs were graded by a pathologist blinded to original diagnoses. Separate areas of ICPNs, BilIN (n = 5), nondysplastic adjacent mucosa (n = 8), and invasive adenocarcinoma (n = 3) were amplified and sequenced on a next-generation sequencer. Data were manually curated to identify pathogenic somatic variants. High-grade ICPNs were associated with low-grade (n = 1) or high-grade (n = 3) BilIN or no dysplasia (n = 5). Fifteen were associated with invasive adenocarcinoma. Low-grade ICPNs were associated with low-grade BilIN (n = 3) or no dysplasia (n = 9). Pathogenic variants included CTNNB1 (catenin beta 1) exon 3 (7); TP53 (tumor protein p53) (6); APC (APC regulator of WNT signaling pathway) (2); RB1 (RB transcriptional corepressor 1) (1); KRAS (KRAS proto-oncogene, GTPase) (1); and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) (1). Pathogenic variants in ICPNs were not detected in BilIN or nondysplastic mucosa. Mutations in invasive cancers included TP53, PIK3CA, and RB1, concordant with the ICPN, but not with BilIN, in all 3 cases. BilIN in the background of ICPNs was of the same or lower grade than ICPNs. Synchronous ICPNs and BilIN lacked concordant somatic mutations. Mutations in adenocarcinomas aligned with the ICPNs. This suggests that ICPN and BilIN are independent at the DNA level and that the presence of ICPNs may not imply risk for subsequent flat dysplasia elsewhere in the biliary tree.

Cohesins: Crossroad Between Cornelia de Lange Spectrum and Cancer Predisposition

ABSTRACTThe cohesin complex plays crucial roles in DNA repair, chromatid separation, and gene transcription regulation. Pathogenic variants in cohesins or dysfunctional transcriptional regulators lead to cohesinopathies, a broader group of disorders including Cornelia de Lange Spectrum (CdLSp), for which the prevalence of cancer cases remains unclear. Here, we aimed to assess the prevalence of oncological events in CdLSp and elucidate the role of cohesin variants in cancer predisposition. We developed a custom next‐generation sequencing (NGS) panel targeting predisposition and pathogenic genes, which we applied on N = 120 samples of pediatric patients with acute lymphoblastic leukemia (ALL), identifying 11 out of 229 total—10 germline and 1 somatic—variants in cohesin genes. Data of N = 205 brain tumors were extracted by bioinformatic analysis of data from open‐source databases carrying 19 somatic variants. In a cohort of 54 CdLSp patients, the largest cohort from a single center, with a median age of 13 years, the hypothesis of an increased prevalence of cancer in CdLSp was not confirmed. Our findings highlight a significant involvement of germline NIPBL variants in CdLSp, whereas RAD21 and STAG1/2 are predominantly found as somatic variants in neoplasms. However, a distinct genetic or molecular pattern distinguishing variants leading to CdLSp from tumors was not identified. Hence, we advocate for further investigation into the relationship between cohesin variants and cancer predisposition in a larger cohort of patients, with a longer observation time and including different types of malignancies, with more focus on epigenetic approaches.

Effective Utilization of a Customized Targeted Hybrid Capture RNA Sequencing in the Routine Molecular Categorization of Adolescent and Adult B-Lineage Acute Lymphoblastic Leukemia: A Real-World Experience.

Recent World Health Organization (WHO) and International Consensus Classifications have introduced numerous molecular entities in B-lineage acute lymphoblastic leukemia (B-ALL), necessitating comprehensive genomic characterization by detecting gene fusions, expression, mutations, and exon deletions. While whole-genome plus transcriptome sequencing is the ideal strategy, it remains cost-prohibitive for routine use. This study reports a cost-effective and reasonably efficient alternate approach integrating a customized targeted hybrid capture RNA sequencing (RNAseq) into the routine workup. A total of 95 consecutive adolescent/adult B-ALL cases negative for common chimeric gene fusions (CGF) (BCR::ABL1, KMT2A::AFF1, TCF3::PBX1, and ETV6::RUNX1) were analyzed using a customized 69-gene targeted RNAseq panel. In total, three fusion detection pipelines, the Trinity Cancer Transcriptome Analysis Toolkit (CTAT) Mutations pipeline, and the Toblerone alignment tool were employed, and the results were compared with fluorescence in situ hybridization (FISH)/multiplex ligation-dependent probe amplification (MLPA) testing. RNAseq identified fusions in 43% of cases (including BCR::ABL1-like: 15.8% and IGH::DUX4: 10.5%), demonstrating superior detection of cryptic intrachromosomal rearrangements. Somatic variants were detected in 30% of cases (including rat sarcoma (RAS) pathway and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) variants in 18% and 5.3% respectively), and IKZF1 deletions were detected in 25% (77% concordance with MLPA). The integration of targeted RNAseq and comprehensive bioinformatic analysis with flow-cytometry-based ploidy analysis and FISH-based IGH rearrangements helped categorize 79% of common CGF-negative B-ALL. The BCR::ABL1/BCR::ABL1-like group showed a higher frequency of pathogenic IKZF1 deletions (50% versus 21.7%; p = 0.011), measurable residual disease (92% versus 51%; p = 0.009), and poorer overall survival (8.6 versus 22.8 months; p = 0.07). Effective utilization of RNAseq data by comprehensive bioinformatic analysis to test fusions, mutations, and deletions, supported by only minimal supplementary FISH testing, provides a practical, cost-effective solution for the molecular characterization of B-ALL in real-world scenarios until a single alternative and cost-effective test is available.

Genomics guiding personalized first-line immunotherapy response in lung and bladder tumors

BackgroundImmune checkpoint inhibitors (ICI) have revolutionized cancer treatment, particularly in advanced non-small cell lung cancer (NSCLC) and muscle-invasive bladder cancer (MIBC). However, identifying reliable predictive biomarkers for ICI response remains a significant challenge. In this study, we analyzed real-world cohorts of advanced NSCLC and MIBC patients treated with ICI as first-line therapy.MethodsTumor samples underwent Whole Genome Sequencing (WGS) to identify specific somatic variants and assess tumor mutational burden (TMB). Additionally, mutational signature extraction and pathway enrichment analyses were performed to uncover the underlying mechanisms of ICI response. We also characterized HLA-I haplotypes and investigated LINE-1 retrotransposition.ResultsDistinct mutation patterns were identified in patients who responded to treatment, suggesting potential biomarkers for predicting ICI effectiveness. In NSCLC, tumor mutational burden (TMB) did not differ significantly between responders and non-responders, while in MIBC, higher TMB was linked to better responses. Specific mutational signatures and HLA haplotypes were associated with ICI response in both cancers. Pathway analysis showed that NSCLC responders had active inflammatory and immune pathways, while pathways enriched in non-responders related to FGFR3 and neural crest differentiation, associated to resistance mechanisms. In MIBC, responders had alterations in DNA repair, leading to more neoantigens and a stronger ICI response. Importantly, for the first time, we found that LINE-1 activation was positively linked to ICI response, especially in MIBC.ConclusionThese findings reveal promising biomarkers and mechanistic insights, offering a new perspective on predicting ICI response and opening up exciting possibilities for more personalized immunotherapy strategies in NSCLC and MIBC.

Severus detects somatic structural variation and complex rearrangements in cancer genomes using long-read sequencing

Severus detects somatic structural variation and complex rearrangements in cancer genomes using long-read sequencing

ATM-dependent DNA damage response constrains cell growth and drives clonal hematopoiesis in telomere biology disorders.

Telomere biology disorders (TBDs) are genetic diseases caused by defective telomere maintenance. TBD patients often develop bone marrow failure and have an increased risk of myeloid neoplasms. To better understand the factors underlying hematopoietic outcomes in TBD, we comprehensively evaluated acquired genetic alterations in hematopoietic cells from 166 pediatric and adult TBD patients. Of these patients, 47.6% (28.8% of children, 56.1% of adults) had clonal hematopoiesis. Recurrent somatic alterations involved telomere maintenance genes (7.6%), spliceosome genes (10.4%, mainly U2AF1 p.S34), and chromosomal alterations (20.2%), including 1q gain (5.9%). Somatic variants affecting the DNA damage response (DDR) were identified in 21.5% of patients, including 20 presumed loss-of-function variants in ataxia-telangiectasia mutated (ATM). Using multimodal approaches, including single-cell sequencing, assays of ATM activation, telomere dysfunction-induced foci analysis, and cell-growth assays, we demonstrate telomere dysfunction-induced activation of the ATM-dependent DDR pathway with increased senescence and apoptosis in TBD patient cells. Pharmacologic ATM inhibition, modeling the effects of somatic ATM variants, selectively improved TBD cell fitness by allowing cells to bypass DDR-mediated senescence without detectably inducing chromosomal instability. Our results indicate that ATM-dependent DDR induced by telomere dysfunction is a key contributor to TBD pathogenesis and suggest dampening hyperactive ATM-dependent DDR as a potential therapeutic intervention.

Clinical and Neuropsychological Phenotyping of Individuals With Somatic Variants in Neurodevelopmental Disorders.

Somatic variants in brain-related genes can cause neurodevelopmental disorders, but detailed characterizations of their clinical phenotypes, neurobehavioral profiles, and comparisons with individuals with germline variants are limited. Using data from the Simons Searchlight natural history cohort, which uses standardized parent-report data collection methods, we identified individuals with neurodevelopmental disorders caused by pathogenic somatic variants and examined their phenotypic data. We further used results from standardized measurements of adaptive functioning, social behavior, and emotional and behavioral problems to compare individuals with somatic variants with those with germline variants. We identified 15 probands with pathogenic or likely pathogenic somatic variants in the Simons Searchlight cohort. For 8 individuals with detailed phenotype information, symptoms included developmental delay or language delay (n = 8), hypotonia (n = 5), autism spectrum disorder (n = 4), and epilepsy (n = 3). Individuals with mosaic variants showed a range of severity in their scores on standardized measurements of adaptive functioning, social behavior, and emotional and behavioral problems. In particular, some individuals with mosaic variants showed impairments that were similar in severity or more severe compared with individuals with germline variants in the same gene. This study improves our understanding of the clinical phenotypes and neuropsychological profiles of individuals with mosaic pathogenic variants in neurodevelopmental disorders.

Clinical Impact of Somatic Genomic Variants of Oncogenes and Tumor Suppressor Genes in Previously Treated Advanced Non-Small Cell Lung Cancer.

Next-generation sequencing in non-small cell lung cancer (NSCLC) identifies somatic genomic variants (SGVs) in cancer susceptibility genes (CSGs). We hypothesized that SGVs would be associated with poorer overall survival (OS) but greater benefit with immune checkpoint inhibitors over chemotherapy. We investigated the prevalence and predictive value of SGVs, using data from OAK and POPLAR trials comparing atezolizumab with docetaxel. We curated a list of SGVs (excluding TP53, EGFR, ALK, and ROS1) on the basis of CSGs associated with tumorigenesis. We classified participants as SGV mutant or wild-type using baseline plasma analyzed by the FoundationOne Liquid CDx assay. Cox regression analyses and interaction tests between SGV status and treatment were performed. Of 762 participants, 29% harbored an SGV. The SGV mutant group had worse OS (hazard ratio [HR], 1.28, 95% CI, 1.06 to 1.54), and within each treatment arm (docetaxel: HR, 1.31; atezolizumab: HR, 1.27). In the atezolizumab arm, the SGV mutant group compared with wild-type had worse OS in the PD-L1 high (HR, 1.31 [95% CI, 0.59 to 2.91]) and low (HR, 1.38 [95% CI, 0.98 to 1.93]) subgroups. SGV with missense, splice, and nonsense mutations had significantly worse OS than wild-type in the docetaxel arm (log-rank P = .01) but not in the atezolizumab arm (log-rank P = .33). SGV status did not predict greater OS benefit with atezolizumab over docetaxel (interaction P = .67). In advanced NSCLC after chemotherapy progression, plasma-detected SGVs are common, and associated with inferior OS. Plasma SGV status should be considered as a stratification factor in future trials.

The interplay between germline and somatic variants in alpha-1 anti-trypsin deficiency liver disease.

The interplay between germline and somatic variants in alpha-1 anti-trypsin deficiency liver disease.

Somatic NAP1L1 p.D349E promotes cardiac hypertrophy through cGAS-STING-IFN signaling

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, often caused by sarcomere gene mutations, though many sporadic cases remain genetically unexplained. Here we show that the somatic variant NAP1L1 p.D349E was involved in cardiac hypertrophy in sporadic HCM patients. Through next generation sequencing, we found that somatic variant NAP1L1 p.D349E was recurrent in the cardiomyocytes of gene-elusive sporadic HCM patients. Subsequent in vivo and in vitro functional analysis confirmed that NAP1L1 p.D349E contributes to HCM by triggering an innate immunity response. This mutation destabilizes nucleosome formation, causing DNA to leak into the cytoplasm. This leakage activates a key immune pathway, cGAS-STING, which leads to the release of inflammatory molecules and promotes heart muscle thickening. Our findings reveal a new mechanism driving HCM and suggest that somatic variants could be important in understanding and management of HCM.