Using next-generation sequencing (NGS) platform to diagnose pathogenic germline BRCA1/2 mutations from archival tumor specimens

Published:August 31, 2019DOI:


      • Archival tumor testing using an NGS platform can detect about 70% of germline BRCA1/2 pathogenic variants.
      • No false positive findings were identified for germline BRCA1/2 pathogenic variants.
      • Tumor testing can potentially diagnose BRCA1/2 mutation to facilitate predictive testing in cancer-free family members.
      • False positive results were noted in 4 genes with the exception of ATM, being highest in TP53 followed by PTEN.
      • Tumor testing will not be a reliable test to diagnose Cowden syndrome or Li-Fraumeni syndrome with current technology.



      Clinical genetic testing to diagnose germline mutations often requires blood sample or saliva smear from a cancer-affected individual. This rules out testing in families when cancer-affected individuals are deceased. We explored the use of a next-generation sequencing (NGS) platform to diagnose germline pathogenic mutations from tumors.


      Archival tumors (ovarian = 26, breast = 25, others = 9) were retrieved from 60 cancer patients who have undergone multi-gene panel blood testing. Genomic DNA was extracted and sequenced for BRCA1/2 using a NGS platform. 41/60 specimens were sequenced for 5 other genes (APC, ATM, PALB2, PTEN, TP53). Tumor testing and results interpretation were performed blinded to the blood test result.


      All 38 patients with no BRCA1/2 mutations on blood testing were correctly tested negative on tumor. Tumor testing correctly diagnosed BRCA1/2 pathogenic mutations in 15/22 (68%) patients while in 7/22 (32%) patients, the mutation was either detected but incorrectly classified as VUS (n = 3) or not detected at all (n = 4). Overall concordance rate for tumor and blood testing for BRCA1/2 mutations was 88%, with 0% false positive and 32% false negative rate for pathogenic mutations. Tumor testing correctly diagnosed 1/2 pathogenic germline ATM mutation, 1/1 pathogenic germline PALB2 mutation and 2/2 pathogenic germline TP53 mutations. False positive germline mutations were diagnosed in 4 genes at a rate of 2.4%–10.3% (APC = 2.4%, PALB2 = 2.4%, PTEN = 4.9%, TP53 = 10.3%).


      Tumor testing for BRCA1/2 germline mutations using an NGS platform is fairly reliable with no false positive findings, and correctly diagnosed more than two-thirds of pathogenic germline BRCA1/2 mutations. However, it is not reliable to diagnose pathogenic germline mutations in genes frequently mutated in sporadic cancers, such as PTEN and TP53.


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