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, and 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 Dermatol- ogy, University of Utah, Salt Lake City, UT. Submitted June 26, 2007; accepted December 5, 2007. 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 Medi- cal 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 con- flicts of interest and author contribu- tions are found at the end of this article. Corresponding author: Amanda B. Spurdle, PhD, Queensland Institute of Medical Research, c/o Royal Brisbane Hospital Post Office, Herston, Queens- land 4029, Australia; e-mail: Amanda.Spurdle@qimr.edu.au. © 2008 by American Society of Clinical Oncology 0732-183X/08/2610-1657/$20.00 DOI: 10.1200/JCO.2007.13.2779 A B S T R A C T 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 immunohisto- chemistry. 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+1G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 counsel- ing of individuals with unclassified sequence variants. J Clin Oncol 26:1657-1663. © 2008 by American Society of Clinical Oncology INTRODUCTION Screening of the breast cancer susceptibility genes BRCA1 and BRCA2 identifies numerous nucleotide sequence changes of varying clinical significance. The effect of rare changes predicted to cause mis- sense substitutions or in-frame exon deletions is of- ten not clear, and presents a challenge in the clinical setting. The scale of the problem is considerable, with approximately 30% of BRCA1 and 60% of BRCA2 entries in the Breast Cancer Information Core database for BRCA1 and BRCA2 variation 1 described as unclassified variants (UVs). An integrated approach to classification of UVs in BRCA1 and BRCA2 into high-risk mutations and neutral variants was developed to define a reliable protocol for prediction of the clinical significance of UVs. 2 This multifactorial likelihood model used data on co-occurrence of the UV with pathogenic mutations in the same gene, segregation in families, and amino acid physicochemical properties and evolutionary conservation. The model was used to estimate the odds of causality, a ratio of the likeli- hood of the observed data under the hypothesis of causality to that under the hypothesis of neutrality. Because the model cannot distinguish between vari- ants that are truly benign and those that might be associated with modest risk, neutral variants are sometimes alternatively termed to be of low/little clinical significance (neutral/LCS). We recently re- vised the model to take into account relevant fea- tures of BRCA1- and BRCA2-associated tumors, JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T VOLUME 26  NUMBER 10  APRIL 1 2008 © 2008 by American Society of Clinical Oncology 1657 Downloaded from ascopubs.org by 52.90.158.48 on June 20, 2022 from 052.090.158.048 Copyright © 2022 American Society of Clinical Oncology. All rights reserved.