Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.
Whole genome sequencing (WGS) has emerged as a tool to characterize the entire genomic landscape of cancer and capture potential new targets and predictive biomarkers. Using state-of-the-art whole-genome and transcriptome sequencing (WGTS) analysis we interrogated a pan cancer cohort to nominate new treatment options. WGS was performed on 414 tumor/germline pairs from our pan cancer cohort of 355 patients, of which 103 had results of other commercial NGS assays. RNAseq data was available in 96 samples. We employed the Isabl GxT analytic platform and manually curated single base substitution (COSMIC v3) mutational signatures and structural variants (SVs). WGTS results were correlated with available clinico-pathologic data. High throughput drug screening that included PARP inhibitors and functional validation were performed in 61 matched patient-derived tumor organoids (PDTO). We found 5,129 SVs affecting at least one cancer gene in 88% of cases. High-confidence homologous recombination deficiency (HRD), determined by curation of mutational signatures and mutations/SVs affecting oncogenes and tumor suppressor genes, was detected in 8.3% of solid tumors (e.g., prostate, pancreas, ovarian, uterine, breast, gallbladder, and stomach carcinomas). HRD status included tumors with BRCA1/2 wildtype and SVs impacting BRCA1/2, RAD51, ATM and other HRD genes. CDK12-loss associated genomic instability was detected in 7 cases including 3 cases of prostate adenocarcinoma. 93 gene fusions involving cancer genes were detected including known and novel fusions of potential significance (e.g., SLC45A3::ALK in metastatic prostate adenocarcinoma, TMEM106A::BRCA1 in serious ovarian carcinoma, ESR1::EP300 in metastatic breast cancer). Some tumor cohorts demonstrated enrichment of drivers e.g., FGFR1 amplification in male breast cancer, rearrangements at the TERT locus in adrenocortical carcinoma, enrichment for repeat-mediated deletion attributed to mismatch repair (MMR) in gynecological carcinosarcoma, and SVs affecting FHIT in gastric adenocarcinoma. Drug screening of PDTO and functional testing with RAD51 immunofluorescence assay validated high-confidence HRD annotation. One example is a BRCA1/2 wildtype pancreatic adenocarcinoma with increased tandem duplications and SV (inversion) impacting BRCA2, and high-confidence HRD by our novel WGS classifier. Drug screening of the corresponding PDTO showed an enhanced response to Olaparib, in contrast to cases without HRD that did not. We have used state-of-the-art analysis of WGTS to improve the detection of targets and significant biomarkers in a pan cancer cohort, including HRD, CDK12-type genomic instability, pathogenic germline mutations and potential targetable novel fusions. A robust and clinically relevant ex vivo preclinical model allowed us to validate a WGS-based classifier that detects HRD even in cancers with wildtype BRCA1/2. Citation Format: Majd Al Assaad, Hui-Hsuan Kuo, Jones Nauseef, Max F. Levine, Gunes Gundem, Juan S. Medina-Martínez, Jyothi Manohar, Michael Sigouros, Ahmed Elsaeed, David Wilkes, Elli Papaemmanuil, Eloise Chapman, Scott T. Tagawa, Allyson Ocean, Cora N. Sternberg, Eleni Andreopoulou, Manish Shah, Andrea Sboner, Kevin Holcomb, David Nanus, Laura Martin, Olivier Elemento, Juan Miguel Mosquera. Whole genome and transcriptome sequencing of a pan cancer cohort unearths novel therapeutic avenues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3437.
CUP is a relatively common diagnosis that accounts for 3-9% of all cancers. The prognosis is poor with median survival of approximately 9 months. The identification of the primary tumor and therapy targets could improve the survival of these patients. We employed the BostonGene Tumor PortraitTM platform to interrogate 19 CUP cases. Trained on >19,000 samples and validated on 28,000 samples from independent datasets, the machine-learning based algorithm integrates whole-exome and RNA sequencing (WES and RNAseq) analysis to characterize cancer drivers, the tumor microenvironment, potential targets, tumor composition, molecular signatures, and site of origin (94% sensitivity and 99% specificity). The predicted tissue of origin was considered acceptable when it was compatible with tumor histopathology and immunoprofile, and included in the differential diagnosis of radiologic studies. The discovered biomarkers and possible treatments were discussed during our multidisciplinary precision oncology meeting. 16 out of 19 CUP cases (84%) had an acceptable predicted tissue of origin. Two cases lacked clinical evidence to support the predicted primary tumor, and 1 case failed RNAseq. Lung accounted for most of the sites of origin (31%) followed by gastrointestinal (15%) and breast cancers (8%). Other diagnoses included melanoma, uterine, bladder, and renal carcinomas, among others. Except for one case, a clinically significant biomarker or target was found. Those included relevant mutational signatures (e.g., homologous recombination deficiency, DNA mismatch repair), genetic characteristics (high tumor mutational burden (TMB), microsatellite instability), activating alterations (FGFR1, MYC, ERBB2 amplifications; NCOA4::RET fusion), loss of function in tumor suppressor genes (TP53, FANCA, ATM), and gene overexpression (ER). Further, microenvironment analysis characterized the tumor immune infiltrate and the level of RNA expression of PD-L1, PD-L2, and CTLA4. These oncology biomarkers and potential targets are of significant value independent of tissue of origin. In addition, mutations in NF1, KRAS, TP53, MSH2, BRCA1, and PTEN were found and validated by commercially available targeted NGS panels. Based on CUP tumor profiling by this platform, positive treatment response has been observed in 3 out of 4 CUP patients thus far, e.g., one patient with metastatic disease that showed high TMB and immune infiltrated microenvironment treated with Ipilimumab and Nivolumab had a sustained response. Therapy in oncology is often determined by the tissue origin, making CUP a therapeutic challenge. In this study, we demonstrate the application of an integrative WES and RNAseq platform to not only predict the site of origin, but also to identify relevant biomarkers and therapeutic targets in CUP. Citation Format: Majd Al Assaad, Michael Sigouros, Jyothi Manohar, Daniela Guevara, Zoia Antysheva, Daria Kiriy, Alexandra Boyko, Naira Samarina, Nara Shin, Viktor Svekolkin, Svetlana Podsvirova, Noel English, Alaina Villarreal, Brianna McKenna, Cagdas Tazearslan, Diana Shamsutdinova, Vladimir Kushnarev, Troy Kane, David Wilkes, Manish Shah, Barbara Ma, Scott T. Tagawa, David Nanus, Jones Nauseef, Olivier Elemento, Juan Miguel Mosquera, Cora N. Sternberg. Cancer of unknown Primary (CUP): Beyond the identification of the site of origin by an integrative genomic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2143.
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