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Clinical Classification of BRCA1 and BRCA2 DNA Sequence Variants: The Value of Cytokeratin Profiles and Evolutionary Analysis—A Report From the kConFab Investigators

Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Suzanne Parry, Leonard M. Da Silva, Ross Brinkworth, John L. Hopper, Melissa A. Brown, Davit Babikyan, Georgia Chenevix-Trench, Sean V. Tavtigian, David E. Goldgar

From the Queensland Institute of Medical Research; School of Medicine, and School of Molecular and Microbial Sciences, University of Queensland, Brisbane; Centre for Genetic Epidemiology, University of Melbourne and Peter MacCallum Cancer Centre, Melbourne, Australia; International Agency for Research on Cancer, Lyon, France; and the Department of Dermatology, University of Utah, Salt Lake City, UT

Corresponding author: Amanda B. Spurdle, PhD, Queensland Institute of Medical Research, c/o Royal Brisbane Hospital Post Office, Herston, Queensland 4029, Australia; e-mail: Amanda.Spurdle{at}qimr.edu.au

Purpose: Rare missense substitutions and in-frame deletions of BRCA1 and BRCA2 genes present a challenge for genetic counseling of individuals carrying such unclassified variants. We assessed the value of tumor immunohistochemical markers in conjunction with genetic and evolutionary approaches for investigating the clinical significance of unclassified variants.

Patients and Methods: We studied 10 BRCA1 and 12 BRCA2 variants identified in Australian families with breast cancer. Analyses assumed a prior probability based on revised cross-species sequence alignment methods assessing amino acid evolutionary conservation and position, combined with likelihoods from data on co-occurrence with pathogenic mutations in the same gene, segregation analysis, and immunohistochemistry. We specifically explored the value of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 as tumor markers of BRCA1 mutation status.

Results: Posterior probabilities classified 72% of variants. BRCA1 variants IVS18+1 G>T (del exon 18) and 5632 T >A (V1838E) were classified as pathogenic, with >99% posterior probability of being deleterious, and tumor histopathology was particularly important for their classification. BRCA2 variant classification was improved over previous studies, largely by incorporating the prior probability of pathogenicity based on amino acid cross-species sequence alignments.

Conclusion: Variant classification was considerably improved by analysis of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumor expression, and use of updated methods estimating the clinical relevance of amino acid evolutionary conservation and position. These methodologies may assist genetic counseling of individuals with unclassified sequence variants.

The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania, and South Australia, and the Cancer Foundation of Western Australia. The kConFab clinical follow-up study was funded by NHMRC Grants No. 145684 and 288704; and by a grant from the Susan G. Komen Breast Cancer Foundation, and the NHMRC. J.L.H. and G.C-T. are NHMRC senior principal research fellows; A.B.S. is funded by an NHMRC career development award; L.Da.S. is supported by a fellowship from the Ludwig Institute for Cancer Research; D.B. is a recipient of a post-doctoral fellowship from the International Agency for Research on Cancer; and S.V.T. and D.E.G. were supported in part by the INHERIT BRCAs programme from the Canadian Institute for Health Research, and a subaward agreement from the Mayo Clinic, Rochester, MN.

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