Zampiga, Valentina

Abstract The success of precision cancer prevention and treatment hinges on accurate discrimination between benign and pathogenic germline alleles in cancer susceptibility genes. Variants of uncertain clinical significance (VUS) present a challenge for cancer risk assessment and functional data will be essential to resolve many VUS. To address this, we developed a cloud-based environment to collate, curate, integrate and analyze all published functional data related to BRCA1 and BRCA2 missense VUS. Then, functional evidence criteria for pathogenicity were determined for each assay using the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) classification guidelines and ClinGen criteria. Our approach involved retrieving published articles reporting one or more functional assays of BRCA1/2 missense variants reporting variant impact on various biochemical and cell biological functions. Functional results were recorded using original authors' thresholds and categories, then converted to ordinal variables to harmonize: [0 = no functional impact], [1 = intermediate impact], and [2 = functional impact]. For BRCA1, we updated our previous published integration (Genet Med. 2021;23:306-315) with 14 additional publications, collating results from 53 instances of functional assays reporting functional data on 3,219 unique missense variants. For BRCA2, 28 publications were identified reporting data from 141 instances of functional assays on 4,759 unique missense variants. Utilizing a panel of 542 and 396 known pathogenic and benign reference variants for BRCA1 and BRCA2, respectively, we determined the sensitivity, specificity, and ACMG/AMP odds of pathogenicity for each instance of the assay. The remaining variants were assigned a relevant functional assay code weight for each assay instance. Our study successfully derived ACMG/AMP evidence strength from functional data for 3,040 BRCA1 (94% of 3,219) and 2,704 BRCA2 (57% of 4,759) missense variants. This comprehensive work underscores the utility of functional data in informing classification of BRCA1 and BRCA2 missense VUS. Citation Format: Paulo Lyra, Lucas Dalcolmo, Michael Parsons, Thales Nepomuceno, Samuel Brito, Nam Phuong N. Nguyen, Geise de Oliveira, Joao Paulo da Siva, Laura Caleca, Tanisha Taneja, Chunling Hu, Marcy Richardson, Maria Rossing, Aditi Hazra, Alexandra Martins, Sandrine Caputo, Gael A. Millot, Joanne NgeowYuen Yie, Marcelo A. Carvalho, Melissa Cline, Paolo Radice, Rachael Carlsen, Romy Mesman, Valentina Zampiga, Rehan Villani, Vijay Josef, Shyam Sharan, Kyriaki Michailidou, Amanda B. Spurdle, Fergus Couch, Maaike P.G. Vreeswijk, Alvaro N. Monteiro. Integration of functional data to classify BRCA1/2 missense variants: An ENIGMA project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7325.

Abstract Purpose: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). Experimental Design: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1–CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case–control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. Results: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)–like (N = 226). We then examined their association with breast cancer risk in the case–control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35–3.41), 1.57 (95% CI, 1.41–1.75), and 1.19 (95% CI, 1.08–1.31), respectively. The meta-analysis of population-specific datasets showed similar results. Conclusions: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.