In all, 1,790 distinct mutations, polymorphisms, and variants in the BRCA1 gene and 2,000 in BRCA2 have been reported to the Breast Cancer Information Core (BIC) database, respectively (July 2014).24 Approximately 53–55% of these are private mutations, are only detected in single families. Mutations are distributed across the entire coding sequences. The most common types of pathogenic mutations are small deletions or insertions or nonsense mutations resulting in protein truncation leading to non-functional protein. Mutations affecting splice-sites as well as large genomic rearrangements are also observed in both genes.8,25 Missense mutations, silent mutations, and polymorphisms are also frequently identified; however, the clinical interpretation of their pathogenic potential is often difficult. Also, variants such as small in-frame insertions and deletions and possible splice-site alterations are problematic for precise cancer-risk estimation. Almost 1,800 distinct sequence variants found in BRCA1 and BRCA2 are classified as having unknown clinical significance (unclassified variants, UVs). To assess the clinical significance of individually rare sequence variants is challenging, as existing methods require a high number of occurrences of the specific variant. In 2009, the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium consortium was established with the purpose of evaluating the clinical significance of rare sequence variants by pooling genetic and associated clinical and histopathological information from a world-wide network of laboratories to gather sufficient data and resources to facilitate the classification of UVs.26
A germline mutation in BRCA1 or BRCA2 only represents the first hit in the classical Knudson’s two-hit hypothesis, whereas the second inactivating somatic mutation often involves deletion of the wild-type allele, termed loss of heterozygosity (LOH). LOH has been reported to be present in the majority (>80%) of tumors arising from mutation carriers.27,28 In contrast, small somatic mutations involving a single or few bases are very rare.29 Another somatic inactivation mechanism, epigenetic silencing by promoter methylation, has been reported of BRCA1 in 9–13% of sporadic breast tumors, an up to 42% in non-BRCA1/2 hereditary breast tumors leading to reduced BRCA1 expression.30–34 In contrast, BRCA1promoter methylations are rare in tumors from BRCA1 and BRCA2 mutation carriers,35 and BRCA2promoter methylation in general is seldom observed in both sporadic and hereditary breast cancers.36
Familial non-BRCA1/2 Breast Cancer
Several rare gene variants have been described to confer an increased risk of breast cancer, involving high-penetrance genes such as TP53, CDH1, PTEN, STK11, RAD51C, and RAD51D and the low/moderate-penetrance genes such as ATM, CHEK2, BRIP1, and PALB2, among others (reviewed by Vargas et al).37 In general, most of these genes are involved in the maintenance of genomic integrity and DNA repair mechanisms, and many are associated with multiple cancer syndromes such as Li–Fraumeni syndrome (TP53), Cowden syndrome (PTEN), and Peutz–Jeghers syndrome (STK11/LKB1).38–40 Furthermore, a number of common low-penetrance breast cancer alleles have recently been identified by genome-wide association studies (GWAS), including 10q26, 16q12, 2q35, 8q24, 5p12, 11p15, 5q11, and 2q33.41–43
Low- and moderate-penetrant genes/loci can only explain a minor fraction of the remaining non-BRCA1/2families that show high incidence of breast cancer. Despite intensive research, genetic linkage analysis, GWAS, and most recently, next-generation sequencing (NGS) exome studies have failed to identify other common high-penetrance breast cancer susceptibility genes, such as BRCA1 and BRCA2, and more than 70% of the genetic predisposition to breast cancer remains unexplained. No single high-penetrance gene is likely to account for a larger fraction of the remaining familial aggregation.44–49 Instead, the remaining predisposition is expected to be a mixture of rare high-risk variants and polygenic mechanisms involving more common and/or rare low-penetrance alleles or rare moderate-penetrance genes, acting in concert to confer a high breast cancer-risk.38 However, very recently germline mutations in RAD51C have been linked to high cancer-risk in a small number of hereditary breast and ovarian cancer (HBOC) families, supporting the hypothesis that some proportion of the remaining predisposition may be caused by rare high-risk alleles.50
Eventually, exogenous factors such as oral contraceptive, hormone replacement therapy, alcohol consumption, overweight, and physical inactivity are all known breast cancer-risk factors.51 An unknown fraction of these families with apparently strong family history could be attributable to such environmental risk factors or could, as breast cancer is a common disease, be random aggregation of sporadic breast cancer cases.
Pathological Characteristics of Hereditary Breast Cancer
The majority of invasive breast cancers arising in BRCA1 and BRCA2 carriers are invasive ductal carcinomas (IDC) (>80%). A higher frequency of BRCA1 tumors are classified as medullary carcinomas compared to sporadic tumors (9% versus 2%).10,52 Medullary carcinomas are poorly differentiated, high-grade carcinomas with diffuse lymphocytic infiltrate but with a remarkably favorable prognosis, probably because of low incidence of lymph node metastasis.53 Notably, 11% of medullary carcinomas carryBRCA1 germline mutations.54 By contrast, excess of invasive lobular and tubular carcinomas has been reported for BRCA2 relative to BRCA1 tumors.10,55 BRCA1 tumors are more frequently high-grade compared to sporadic tumors. They have a higher number of mitosis, and show a high frequency of necrotic areas and a higher proportion of continuous pushing margins and lymphocytic infiltration. All these features point toward a more aggressive tumor type.56,57 Most BRCA2 tumors are grade 2/3 with high mitotic rates. Continuous pushing margins are also characteristic of BRCA2 tumors.
Breast tumors express a number of immunohistochemical (IHC) markers providing both prognostic and predictive information. The estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (EGFR) 2 (HER2) are among the most important IHC markers. Among sporadic tumors, 70% are ER-positive and 50% are PR-positive, and HER2-overexpression is observed in approximately 15% of the cases. ER-positive tumors respond better to endocrine anti-estrogen treatment, whereas tumors overexpressing HER2 respond well to targeted therapy such as trastuzumab (Herceptin). Approximately 20% of all breast cancer cases are negative for ER, PR, and HER2, known as “triple-negative” (TN) cancers. The prognosis of TN tumors is very poor, not only because these tumors seem to be more aggressive than other breast cancers but also because endocrine and anti-HER2 therapies are ineffective, leaving chemotherapy as the only treatment option available.58,59
A recent study examining pathology data from 4,325 BRCA1 and 2,568 BRCA2 mutation carriers reported that 78% of tumors arising in BRCA1 carriers were ER-negative, while only 23% of tumors arising inBRCA2 mutation carriers were ER-negative. Furthermore, HER2-overexpression was only observed in approximate 10% of the tumors from mutation carriers. Consequently, 69% of the BRCA1 tumors were TN, which was true for only 16% of the BRCA2 tumors.10 The relation between BRCA1 mutations and low expression of the hormone receptors is significantly different from sporadic tumors even when adjusting for the younger age of the BRCA1 patients. The majority of BRCA1 tumors exhibit a basal/myoepithelial phenotype by expressing several basal markers including the cytokeratins CK5/CK6, CK14, caveolin, vimentin, laminin, p-cadherin, oesteonectin, and the EGFR.56,60 It has also been reported that BRCA1tumors stained more often p53-positive compared to sporadic, and this probably reflects the higher frequencies and distinct patterns of somatic TP53 mutations that are found among BRCA1 tumors.61,62 AsBRCA1 or BRCA2 inactivation leads to cell cycle arrest because of activation of p53, mutations in the TP53gene have been suggested as a mechanism to escape cell cycle arrest.63 Several attempts have been conducted where IHC profiles have been applied in combination with morphological characteristics to identify patients with a high risk of carrying a BRCA1 or BRCA2 mutation for clinical classification of unclassified sequence variants, but with mixed success.60,64–66
In contrast to BRCA1 tumors, BRCA2 tumors seem to be more similar to sporadic tumors with relation to the expression of IHC markers. Most BRCA2 breast tumors show a luminal phenotype by overexpressing ER and PR and the cytokeratins CK8 and CK18.64