Triple-negative Breast Cancer and PTEN (Phosphatase and Tensin Homologue) Loss Are Predictors of BRCA1 Germline Mutations in Women With Early-onset and Familial Breast Cancer, but Not in Women With Isolated Late-onset Breast Cancer

Sze-Yee Phuah; Lai-Meng Looi; Norhashimah Hassan; Anthony Rhodes; Sarah Dean; Nur AM Taib; Cheng-Har Yip; Soo-Hwang Teo

Disclosures

Breast Cancer Res. 2012;14(6):R142 

In This Article

Materials and Methods

MyBrCa Breast Cancer Cohort

The recruitment of breast cancer patients into the Malaysian Breast Cancer Genetic Study (MyBrCa) started in January 2003 at the University Malaya Medical Centre in Kuala Lumpur. All were histopathology-proven breast carcinomas. Histomorphologic and biomarker parameters were retrieved from the histopathology reports. Blood, demographic, and family-history data were collected from breast cancer patients who consented to participate in this study. This study was approved by the ethics committee of University Malaya Medical Centre.

Analysis of BRCA1 and BRCA2

From January 2003 to February 2012, 1,454 breast cancer patients were recruited into the MyBrCa study. Germline DNA samples were screened for BRCA1 and BRCA2 mutations for all women with (a) early-onset breast cancer (≤35 years of age) (35 with and 96 without family history of breast and ovarian cancer); (b) family history of breast or ovarian cancer in first- and second-degree relatives (193 women); or (c) isolated triple-negative breast cancer diagnosed at between 36 and 50 years old in the absence of family history (47 women). In addition, of the 432 women who were diagnosed aged 36 to 50 years with non-TNBC, 60 women with the highest risk were analyzed (bilateral breast cancer, breast and ovarian cancer in the index patient, family history of breast and ovarian cancer in third-degree or isolated breast cancer (≤45 years of age)). Mutation detection for germline BRCA1 and BRCA2 mutations was conducted by using direct DNA sequencing and multiple ligation-dependent probe amplification (MLPA), as previously described.[12,13]

Pathologic Analysis

For this cohort, patients were classified as having TNBC when we found <10% ER, <10% PR, and 0 or ≤2+ HER2 staining with immunohistochemistry. HER2 was not routinely tested for in all patients prior to 2006, and therefore, in 259 patients, HER2 status was unavailable. Of the 110 women for whom germline BRCA1 and BRCA2 mutations were analyzed, adequate archived paraffinized invasive tumor tissue for evaluation was available from 32 index patients and two relatives. All cases were tested with immunohistochemistry for cytokeratin 5/6 (D5/16B4; Dako Ltd.), cytokeratin 14 (LL02; Novocastra Labs), and PTEN (6H2.1; Dako). Cytokeratin 5/6 or cytokeratin 14 was defined as positive if >10% of invasive tumor cells showed cytoplasmic staining,[14] and PTEN was defined as negative if PTEN staining was undetectable in tumor cells in contrast to adjacent normal stromal cells.[6–8] Description of other pathologic features was conducted as previously described.[14]

Statistical Methods

Unconditional multiple linear logistic regression was used to model the probability that the proband was a mutation carrier as a function of her personal and family history and age at diagnosis, as described in.[15]

Manchester Score Analysis

Manchester score was calculated as previously described.[16] In brief, this system assigns scores depending on the type of cancer and age at diagnosis and developed such that a score of 15 was equivalent to a 10% chance of identifying a BRCA1 or BRCA2 mutation. The modified method[17–19] includes upward adjustments for TNBC (+4), estrogen-negative (+1), and high-grade invasive cancers (+2), and downward adjustments for human epidermal growth factor receptor 2 (HER2)-positive (-4) or estrogen-receptor positive (-1), lobular (-2), and low-grade noninvasive cancers (-2). For individuals in whom tumor tissue was available for PTEN staining, upward (+1) and downward (-1) adjustments were made for the absence or presence of PTEN staining, respectively.

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