Abstract Variants of unknown significance (VUS) occur in approximately 10% of BRCA1 and BRCA2 genetic testing. Definition of VUS as pathogenic or of benign potential is a crucial step in the reporting and counseling process. Thus limiting the clinical utility of a genetic test and impairs the correct patient management. BRCA1 and 2 are tumor suppressor genes and loss of heterozygosity (LoH) as a second hit for inactivating wildtype allele is a relatively common event in tumor. Previously, in a multi-institutional effort, AC Camargo Cancer Center (Brazil), Barretos Cancer Hospital (Brazil) and UTMD Anderson Cancer Center (USA) compiled a total of 332 BRCA1/2 VUS supposedly from unrelated carriers (46 of BRCA1 and 286 of BRCA2). From them, 256 were distinct VUS (37 in BRCA1 and 219 in BRCA2). Breast cancer sub-classification were collected from medical records and in silico analyzes were performed using four software (SIFT, Polyphen2, CADD and REVEL). Based on this analysis, VUS were categorized into five classes: very high, high, medium, low and very low risk of pathogenicity, according to agreement level among the four in silico software. However, all these analysis are not sufficient for supporting the reclassification of VUS. Therefore we proposed to analyze LoH in matched tumor tissue to assess the pathogenic potential of BRCA1 and 2 VUS. Normal (leucocytes or normal breast tumor tissue) samples, when available, were used for correcting PCR amplification bias between the two alleles. DNA was extracted and subjected to PCR reaction. Amplicons libraries were performed according to manufacturing protocol (Ion Plus Fragment Library Kit) and sequenced on the Ion Proton™ System (ThermoFisher Scientific). Specific variants were called in the TVC program (ThermoFisher Scientific) with standard criteria. A total of 12 VUS were investigated (5 in BRCA1 and 7 in BRCA2). LoH was detected in 7 (60%) tumor samples (3 in BRCA1 and 4 in BRCA2). Of these, 4 VUS were classified as very high or high risk of pathogenicity [2 in BRCA1 (BR1p.Y1703C; BR1p.S1655P) and 2 in BRCA2 (BR2p.S2670L; BR2p.E3002K)]. Variant of allele frequency (VAF) of 94, 65, 70 and 75%, respectively, were detected in tumor tissues. Additionally, recent functional assays described these 4 VUS as non-functional or deleterious variants showing complete agreement with the current LoH results. According to ACMG guidelines considering all available data, LoH, deleterious function and concordant pathogenicity probability. These 4 VUS might be reclassified as pathogenic variants. On the other hand, the same combined analysis did not allow reclassification of 3 other VUS. Although VAF were 74, 73 and 70% respectively, they were previously classified as very or low risk of pathogenicity and there are no functional assays describing them until now. Our results showed that LoH analyses can substantially contribute to VUS reclassification, improving the management and counseling of BRCA1/2 VUS-carriers. Citation Format: Dirce M Carraro, Edenir I Palmero, Carolina M Berra, Giovana T Torrezan, Rafael C Brianese, Gabriela C Fernandes, Angelica M Gutierrez Barrera, Henrique C Galvo, Maria NC Formiga, Banu K Arun. Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-09-04.