Nadeem Riaz

AlphaMissense for Identifying Pathogenic Missense Mutations in DNA Damage Repair Genes in Cancer

PURPOSE AlphaMissense is a new artificial intelligence–based approach to predicting the pathogenicity of missense variants. However, whether its predictions can be directly applied to clinical decision making remains unclear. This study aimed to evaluate the accuracy of AlphaMissense predictions for DNA damage repair (DDR) genes using genomic and clinical characteristics. METHODS Sequencing data from 56,965 patients with cancer who underwent Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing between April 2015 and March 2023 and data from The Cancer Genome Atlas (TCGA)/Pan-Cancer Analysis of Whole Genomes (PCAWG) were analyzed. AlphaMissense pathogenicity was evaluated in six commonly mutated DDR genes. Missense mutations were classified into three categories on the basis of AlphaMissense and OncoKB: known pathogenic, newly identified pathogenic by AlphaMissense, or benign. RESULTS In the MSK-IMPACT cohort, 1,182 (17.5%) of 6,743 unique DDR gene missense mutations were newly identified as pathogenic. In breast, ovarian, pancreatic, and prostate cancers, homologous recombination (HR)–deficiency signatures were more common in tumors with new pathogenic missense mutations in BRCA1/2 , PALB2 , and RAD51C than in benign missense mutations (66.7% v 35.2%, P = .021). In the TCGA/PCAWG data set, HR-deficiency signatures were also more frequent in tumors with new pathogenic mutations than in wild-type (46.2% v 19.2%, P = .036). For tumors with POLE missense mutations, there were no significant differences in tumor mutation burden and POLE-associated signatures between new pathogenic and benign mutations. Patients with new pathogenic ATM missense mutations had fewer TP53 mutations (30.5% v 54.6%, P < .001) and showed improved irradiated tumor control (hazard ratio, 0.58 [95% CI, 0.35 to 0.95]; P = .03) compared with those with benign missense mutations. CONCLUSION Our findings suggest that AlphaMissense can help identify previously unknown pathogenic DDR gene mutations, but its accuracy is gene-dependent. AlphaMissense prediction still requires additional confirmation with clinical and functional validation.

Evolution and Co-occurrence of PI3K Pathway Gene Mutations in Endometrial Carcinoma Molecular Subtypes at the Single-Cell Level

Abstract Purpose: The PI3K pathway is altered in >85% of endometrioid endometrial carcinomas (EEC), with multiple mutations commonly co-occurring. Yet, the therapeutic effects of single-agent PI3K pathway inhibitors have been limited. We used single-cell sequencing to determine whether co-occurring PTEN, PIK3CA, and/or PIK3R1 somatic mutations in EECs stratified by molecular subtype originated through convergent or linear evolution. Experimental Design: Banked frozen EECs with co-occurring PI3K pathway mutations of no specific molecular profile (NSMP; n = 5), mismatch repair–deficient (MMRd; n = 3), and POLE (n = 3) subtypes were selected for single-nucleus DNA sequencing targeting hotspot variants of 64 cancer-related genes and the PTEN, PIK3R1, and PIK3CA coding sequences. EEC cell lines and nonmalignant samples were used to define error rates and filter false-positive calls. Results: Single-nucleus analyses (n = 50,009 cells) revealed that in NSMP EECs, the co-occurring PIK3CA, PIK3R1, and/or PTEN mutations affected nearly all cells through linear evolution. MMRd EECs displayed higher levels of genetic heterogeneity, harboring PI3K pathway gene mutations in subsets of cells ranging from 3.9% to 96%. POLE EECs had the highest level of clonal diversity and harbored multiple, minor subclonal structures in all cases, through convergent evolution. We found a clear distinction between nearly clonal PI3K pathway gene alterations (>95%) and multiple, minor mutually exclusive subclones only affecting 1.4% to 27% of the tumor cells sequenced. Conclusions: Our exploratory, hypothesis-generating analysis suggests that PI3K pathway alterations evolve distinctly in MMRd/POLE compared with NSMP EECs, which may have therapeutic consequences. Further studies on the signaling output and PI3K pathway inhibitor response in EECs with subclonal PI3K pathway alterations are warranted.

Germline RAD51B variants confer susceptibility to breast and ovarian cancers deficient in homologous recombination.

Pathogenic germline mutations in the RAD51 paralog genes RAD51C and RAD51D, are known to confer susceptibility to ovarian and triple-negative breast cancer. Here, we investigated whether germline loss-of-function variants affecting another RAD51 paralog gene, RAD51B, are also associated with breast and ovarian cancer. Among 3422 consecutively accrued breast and ovarian cancer patients consented to tumor/germline sequencing, the observed carrier frequency of loss-of-function germline RAD51B variants was significantly higher than control cases from the gnomAD population database (0.26% vs 0.09%), with an odds ratio of 2.69 (95% CI: 1.4-5.3). Furthermore, we demonstrate that tumors harboring biallelic RAD51B alteration are deficient in homologous recombination DNA repair deficiency (HRD), as evidenced by analysis of sequencing data and in vitro functional assays. Our findings suggest that RAD51B should be considered as an addition to clinical germline testing panels for breast and ovarian cancer susceptibility.