Originally published as JCO Early Release 10.1200/JCO.2008.20.5211 on July 20 2009

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Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

From the Department of Pathology, University of Colorado Health Sciences Center; and Department of Preventive Medicine, Creighton University Medical School, Tissue Genetics, Aurora, CO.

Corresponding author: Jeffrey T. Holt, MD, University of Colorado, Dept of Pathology, 12801 E 17th Ave, MS 8104, PO Box 6511, Aurora, CO 80045; e-mail: jeff.holt{at}uchsc.edu.

Purpose Mutations in the BRCA2 gene are dominantly inherited but cause cancers when the wild-type allele has loss of heterozygosity (LOH) within the cancer. Because most disease-associated BRCA2 mutations are protein-truncating mutations, a test for truncated BRCA2 proteins should identify most BRCA2 hereditary cancers.

Methods We have developed a tissue truncation test to identify truncated BRCA2 proteins in breast cancer tissue biopsies in vivo that does not use amplification or genetic manipulations. N-terminal and C-terminal antibodies are used to visualize protein truncation by demonstrating that the beginning of the protein is present but the end (ie, terminus) is absent.

Results A quantitative C-terminal immunostaining score or a C-terminal to N-terminal truncation ratio correctly classified 20 of 21 breast cancers arising in BRCA2 mutation carriers and 57 of 58 cancers arising outside the context of a multiple-case breast cancer family. This represents a sensitivity of 95% and a specificity of 98%. Because of the presence of C-terminal BRCA2 protein and atypical clinical features of the misclassified cancer in a BRCA2 mutation carrier, we performed polymerase chain reaction and sequence analyses on this cancer. The results showed continued presence of the BRCA2 wild-type allele in the cancer, which indicated that intact BRCA2 protein was present in this cancer.

Conclusion This immunohistochemistry-based test (which takes only 4 hours) appears to identify BRCA2 hereditary cancer with high accuracy. The test also appears to diagnose the biochemical loss of BRCA2 protein in cancers (ie, BRCA2-mutant genotype), which will usually but not always agree with the presence of a germline BRCA2 mutation found by susceptibility testing by DNA sequencing of blood samples.

Supported by Grants No. R41CA128233 (J.T.H.) and CA086389 (H.T.L.).

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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