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Loss of heterozygosity assay to support VUS (variant of unknown significance) reclassification in BRCA1 and BRCA2 genes


Germline variants in BRCA1/2 tumor suppressor genes impairing protein function, predisposes carriers to the hereditary breast and ovarian cancer (HBOC) syndrome. Loss of heterozygosity (LoH) as a second hit for inactivating wildtype allele is a common somatic event in breast tumorigenesis. A significant number of patients undergoing genetic testing for HBOC are identified with VUSs and represent a challenge for clinical practice and genetic counseling, since limits the clinical utility of a genetic test and impairs the correct patient management.


The proposal of this study is (1) to investigate LoH of BRCA1/2 VUSs in carries-matched tumors, and (2) to classify BRCA1/2 VUSs in probably pathogenic or probably benign.


BRCA1/2 variants reported as VUSs was obtained from three institutions A.C.Camargo Cancer Center (ACC), MD Anderson Cancer Center (MDA) and Hospital de Amor (HA). Variants were periodically revisited to get updated classification following ClinVar and American College of Medical Genetics (ACMG) guidelines. Allelic imbalance (mutant/wild-type alleles) in tumor FFPE tissues from HBOC syndrome spectrum, but not restrict to (breast, prostate and ovary tumors, other female genital tract tumors, neuroendocrine pancreas, thyroid and gastric tumors) were evaluated by amplicon deep NGS-sequencing. The DNA isolated from tissues containing at least 40% tumor cells was used for a target PCR reaction, amplicon library construction and sequencing in S5 Ion platform (ThermoFisher). Results were analyzed using the Integrative Genomics Viewer (IGV) software. LoH event supporting the loss of wild type allele was considered when the variant allele fraction (VAF) observed in tumor was superior to 60% or, when the VAF observed in tumor was superior to 10% comparing to the observed in paired leucocyte or saliva sample. This study was approved by ACC human IRB (n.2464/17).


A list of 349 VUSs was initially considerate. In the course of the study, after ClinVar and/or ACMG revision, 27 variants were reclassified as pathogenic (P)/probably pathogenic (PP) and 42 variants as benign (B)/probably benign (B), and 280 remained as VUS. A total of 200 BRCA1/2-carrier matched tumors was recovered from ACC (195) and HA (5) archives, which correspond to 131 unique variants (21, 26 and 84 - P/PP, B/PB and VUS, respectively). To date, we were able to analyze LoH in 88 tissues corresponding to 15 P/PP variants (9 in BRCA1 and 6 in BRCA2), 15 B/PB (6 in BRCA1 and 9 in BRCA2) and 43 VUSs (12 in BRCA1 and 31 in BRCA2). In P/PP variant group, as expected, LoH was observed in 100% of BRCA1 carrier-matched tumors (13/13) and in 75% of BRCA2 carrier-matched tumors (6/8). In B/PB variant group, LoH was detected only in 12.5% (1/8) and 23% (3/13) of BRCA1 and BRCA2 carrier-matched tumors, respectively. For the VUSs detected in BRCA1 (4) and BRAC2 (9) genes, LoH event in the matched tumors was present in 30.7% (4/13) and in 27.2% (9/33), respectively, supporting their reclassification to PP.


Our previous results showed that LoH analyses is a promising approach for supporting reclassification of VUSs to probably pathogenic, especially for VUS in BRCA1 gene.


variants of unknown significance, BRCA1/2 genes, loss of heterozygosity.

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Pesquisa básica / translacional


KARINA MIRANDA SANTIAGO, Carolina Maria Berra, Nicole Malachias Nacarato, Rafael Canfield Brianese, Giovana Tardin Torrezan, Marina De Brot Andrade, Pedro José Silva dos Santos, Edenir Inez Palmero, Banu Arun, Dirce Maria Carraro