Hong Kong Med J 2016 Apr;22(2):171–7 | Epub 14 Mar 2016
DOI: 10.12809/hkmj154634
A new paradigm of genetic testing for hereditary breast/ovarian cancers
Ava Kwong, FRCS (Edin), PhD; JW Chen, PhD; Vivian Y Shin, PhD
Breast Surgery Division, The University of Hong Kong, Pokfulam, Hong Kong
Corresponding author: Dr Ava Kwong (akwong@asiabreastregistry.com)
 Full paper in PDF
Introduction: Genetic risk factors and family history play an important role in breast cancer development. This review aimed to summarise the current genetic testing approach to hereditary breast/ovarian cancer.
Methods: A systematic literature review was performed by searching the PubMed database. Publications available online until January 2015 that addressed issues related to hereditary breast/ovarian cancer genetic counselling/testing were selected. The search terms used were “familial breast/ovarian cancer”, “susceptibility genes”, “genetic counselling”, and “genetic testing”. The data extracted for this review were analysed by the authors, with a focus on genetic testing for hereditary breast/ovarian cancer.
Results: Although a greater proportion of inherited breast/ovarian cancers are due to the BRCA1 and BRCA2 mutations, a number of new genes have emerged as susceptibility candidates, including rare germline mutations in high penetrance genes, such as TP53 and PTEN, and more frequent mutations in moderate/low penetrance genes, such as PALB2, CHEK2 and ATM. Multi-gene testing, if used appropriately, is generally a more cost- and time-effective method than single-gene testing, and may increase the number of patients who can be offered personal surveillance, risk-reduction options, and testing of high-risk family members.
Conclusions: Recent advances in molecular genetics testing have identified a number of susceptibility genes related to hereditary breast and/or ovarian cancers other than BRCA1 and BRCA2. The introduction of multi-gene testing for hereditary cancer has revolutionised the clinical management of high-risk patients and their families. Individuals with hereditary breast/ovarian cancer will benefit from genetic counselling/testing.
Breast cancer is one of the most common cancers and the second most common leading cause of cancer-related death among women with 1.67 million new cases diagnosed in 2012 (25% of all cancers).1 About 39% of these new cases are found in Asia.1 In the US, women have a 12% lifetime risk of developing breast cancer including women of young age. In addition, approximately 1 in 250 women in their 30s will develop breast cancer in the next 10 years.2 Assessment of an individual’s risk for breast cancer is complex, and based on different aspects such as personal lifestyle, environmental exposure, reproductive influences, and drug use. Genetic risk factors and family history, however, also play important roles in breast cancer development. Only 5% to 10% of breast cancer cases are characterised as hereditary and follow the autosomal dominant pattern of transmission.3 On the other hand, 15% to 20% of breast cancer cases are familial, referring to women who have two or more first- or second-degree relatives with the disease.4 5 6 Hereditary cancers follow a Mendelian inheritance pattern and tend to have an earlier age of onset. Familial cancers do not follow a specific inheritance pattern. Defects in the BRCA1 and BRCA2 genes are the most well-known high-risk factors among inherited breast cancers. Results from genome-wide association studies have broadened our knowledge over the last few years about the specific genes that contribute to familial breast cancer. Other genes such as TP53 and PTEN have also been identified to be associated with an increased risk of breast cancer.7 High-risk women are likely to benefit from genetic testing as there are now emerging targeted therapies and interventions that have been shown to improve outcome in mutation carriers.
A search of the medical literature was performed to identify the relevant studies and reviews on genetic testing for hereditary breast/ovarian cancer. The PubMed database was searched for publications available online until January 2015 that address the related issues; “familial breast/ovarian cancer”, “susceptibility genes”, “genetic counselling”, and “genetic testing” were used as the search terms.
High-penetrance genes
Hereditary breast and ovarian cancer syndrome (HBOC) refers to a germline mutation in either the BRCA1 or BRCA2 gene, and individuals who carry a mutation have an increased risk of developing cancers. BRCA1 and BRCA2 are tumour-suppressor genes that code for proteins that help repair damaged DNA and therefore play vital roles in securing the stability of the cell’s genetic material. Defects in these two genes may result in protein with malfunction, thus DNA damage may not be repaired properly. As a result, cells are prone to develop genetic mutations leading to cancer development. Scientists discovered in the 1990s that BRCA1 and BRCA2 are breast cancer susceptibility genes.8 9 Women have a 57% to 60% and 49% to 55% lifetime risk of developing breast cancer if they carry a BRCA1 or BRCA2 mutation, respectively.10 11 Women with mutations in the BRCA1 cancer susceptibility gene associated with HBOC have a 39% to 46% risk of developing ovarian cancer by the age of 70 years while approximately 10% to 27% BRCA2-positive women are at risk.12 13 14 The result of genetic testing for the BRCA mutation is important to decisions made about management of breast cancer. For example, a woman diagnosed with breast cancer and who harbours the BRCA1 or BRCA2 mutation has a greater risk of developing a second breast cancer in the contralateral breast, and this risk is age-related. Women diagnosed with breast cancer at a younger age have a higher risk of developing contralateral malignancy compared with those diagnosed at an older age.15 BRCA1 mutation carriers tend to have more triple-negative breast cancer (TNBC), medullary histopathology, somatic TP53 mutations, higher histological grade, and present at a younger age compared with women with sporadic breast cancers. Basal markers such as cytokeratin (CK14, CK5/6, CK17), osteonectin, and EGFR are more commonly expressed in BRCA1-positive tumours than in control tumours unselected for mutation status.16 17 18 The National Comprehensive Cancer Network (NCCN) annually updates guidelines with respect to genetic counselling and testing (www.nccn.org) and the most updated guidelines recommend it for individuals who meet the HBOC testing criteria. Guidelines are based on young age of onset, family history of breast cancer, specific histological types of breast cancer (TNBC), ovarian (epithelial and peritoneal), and prostate cancer (Gleason score ≥7). For details refer to NCCN guidelines (Genetic/Familial High-Risk Assessment: Breast and Ovarian), version 1.2016.19
Knowing the mutation status of germline BRCA1 and BRCA2, patients may be offered alternative screening and/or therapeutic interventions (Table 119), including intensive breast surveillance (magnetic resonance imaging [MRI] of the breasts in addition to standard breast imaging such as mammography and ultrasonography), mastectomy instead of breast conservation surgery, prophylactic mastectomy and salpingo-opherectomy, or the prescription of chemopreventive drugs and more recently the choice of chemotherapy as primary treatment, for example, carboplatin. A recent study has shown that treatment with carboplatin produces no advantage over docetaxel in patients with TNBC, although those with BRCA1 or BRCA2 mutation benefited from either drug.20 A number of targeted therapies, such as poly(ADP-ribose) polymerase inhibitors, have been shown to be effective in BRCA mutation carriers.21 22 The evolution of sequencing technologies enables parallel testing of multiple genes, leading to simultaneous analysis of breast cancer predisposition genes with either high or intermediate penetration. Multi-gene panel testing, however, has raised new issues regarding patient eligibility for gene testing other than BRCA1 and BRCA2, and more importantly, interpretation of genetic results.

Table 1. NCCN management of the hereditary breast and/or ovarian cancer syndrome19
(Adapted from the NCCN guidelines, version 1.2016)
One of the high penetrance genes is TP53, which is a tumour-suppressor gene that encodes the transcription factor protein p53. It is a ubiquitous protein implicated in preservation of an intact genome. It regulates cell cycle, DNA repair, apoptosis, cellular senescence, and metabolism. It has been shown to be involved in various kinds of cancer progression such as osteosarcomas, colon cancer, and lung cancer.23 24 25 26 27 28 Li-Fraumeni syndrome (LFS) is a rare but highly penetrance familial cancer syndrome that is characterised by germline TP53 mutations inherited in an autosomal dominant manner, in which 60% to 80% of LFS families carry a mutant TP53.29 In addition to soft-tissue sarcomas and osteosarcomas, LFS families are likely to exhibit a pattern of early-onset and multiple primary cancers including breast, brain, and adrenocortical tumours29 30; LFS is thought to account for approximately 1% of all breast cancers.31 32 Approximately 1% of women diagnosed with breast cancer before the age of 40 years carry a TP53 mutation.32 Breast cancer is the most frequent malignancy among female TP53 mutation carriers and accounts for up to one third of all cancers in LFS families.33 Although LFS is only responsible for a tiny fraction of breast cancers, women with LFS have a breast cancer risk of 56% by the age of 45 years and greater than 90% by the age of 60 years, and LFS accounts for a 60-fold increased risk for early-onset breast cancer compared with the general population.34 35 Women with LFS-related breast cancer are reported to have very early disease onset (20s or 30s) and a relatively advanced disease staging.36 37 38 Studies have shown that 3% to 8% of women who are diagnosed with breast cancer younger than 30 years without a significant family history of cancer have TP53 mutation.31 39 40 The NCCN has included early-onset breast cancer as one of the criteria for offering TP53 genetic testing, regardless of the family history of cancer. TP53 mutations can be tested either through sequencing the entire encoding region that identifies approximately 95% of TP53 mutations or just selected regions. Analysis of hot-spot regions located in exons 4-9 can detect approximately 90% of all TP53 mutations.19 41 42 When the TP53 mutation is present in an individual, breast screening and preventive guidelines are similar to those for BRCA mutation carriers. In addition, a full-body MRI scan is an option as a screening tool. Individuals with the following should be included for genetic testing of TP5319: early-onset breast cancer (≤35 years), a combination of diagnosis of a sarcoma at the age of <45 years, AND a first-degree relative diagnosed at the age of <45 years with cancer, multiple cancers (brain tumours, sarcomas, and leukaemia).
PTEN is a phosphatase tensin homologue located on chromosome 10q23.3 that plays a tumour-suppressive role due to its PI3K (phosphatidylinositol-3-kinase) phosphatase activity. Abnormal PTEN cannot activate cell cycle arrest and apoptosis and leads to uncontrolled cell survival.43 Germline PTEN mutations have been identified in a variety of disorders such as Cowden syndrome (CS) or PTEN hamartoma tumour syndrome. Affected individuals have multiple hamartomas in a variety of tissues with an increased risk of malignant transformation.44 Breast cancer is the most common tumour associated with CS. Although CS is responsible for <1% of all breast cancers, women with this syndrome have a 25% to 50% risk of developing breast cancer in a lifetime and are prone to early onset.45 46 The frequency of multifocal and bilateral disease is increased in CS-associated breast cancers compared with sporadic cases.47 48 Women with CS also have an increased risk (67%) of benign breast disease characterised by mammary hamartomas that can be multiple and bilateral.49 Similar to TP53 mutation carriers, PTEN mutation carriers are advised to have breast surveillance and interventions as recommended for BRCA mutation carriers. The testing criteria for CS are those who present with breast cancer, endometrial cancer, follicular thyroid cancer, multiple gastrointestinal hamartomas, ganglioneuromas, or other diseases including macrocephaly, macular pigmentation of glans penis, and mucocutaneous lesions.19
Moderate- and low-penetrance genes
PALB2 (partner and localiser of BRCA2) is involved in homologous recombination and double-strand break repair along with BRCA2.50 51 Loss-of-function mutations are associated with a 2 to 4 times higher risk than non-mutation carriers for familial breast cancer.52 53 54 A study analysed the risk of breast cancer among 362 members of 154 families who had deleterious PALB2 mutations.55 The results revealed that the risk of having breast cancer for female PALB2 mutation carriers was 8 to 9 times higher among those younger than 40 years, 6 to 8 times higher among those 40 to 60 years, and 5 times higher among those >60 years when compared with the general population. The estimated cumulative risk of breast cancer among female mutation carriers increased from 14% to 35% from the age of 50 to 70 years. In addition, the risk of breast cancer for PALB2 mutation carriers was significantly increased by familial factor.56 Thus, it has been advised that PALB2 mutation testing should be performed routinely to identify mutations in HBOC families since it may be of clinical relevance. This is increasingly being tested.
Other hereditary breast cancer susceptibility genes
There are other low-penetrance genes that are associated with hereditary breast cancer such as STK11, CDH1, and MMR genes, and that are responsible for Peutz-Jeghers syndrome, hereditary diffuse gastric cancer syndrome, and Lynch syndrome, respectively.57 58 59 Some moderate-penetrance genes such as CHEK2, ATM, BRIP1, RAD51C, RAD51D, BARD1, MRE11, RAD50, NBS1, and FANCM have been recognised as breast cancer susceptibility genes.60
The recent development of multi-gene testing for hereditary cancer has had a great impact on the clinical management and genetic counselling of high-risk patients and their families. The decision to use multi-gene testing should be no different than the rationale for testing a single gene. Multi-gene testing is more cost-effective than sequentially testing multiple genes associated with a phenotype. For example, young women diagnosed with breast cancer can be tested for mutations in BRCA1, BRCA2, and TP53. Detailed testing criteria for genes can be found in NCCN guidelines version 1.2016.19 Next-generation sequencing enables simultaneous analysis of a specific panel of genes, but there are limited outcome data on clinical interventions, particularly in lower-penetrance-gene-mutation–related breast cancers. Results of a multi-gene panel may pose difficulty in interpretation and clinical decisions. At present, multi-gene testing is largely performed for research purposes. There are limited data regarding the degree of cancer risk associated with some of the genes on the recurrent multi-gene test. There is a lack of well-established guidelines for risk management for carriers of mutations in some of the genes, which may lead to extra surveillance and surgeries.
Nonetheless multi-gene testing is more cost-effective and time-effective than single-gene testing, and provides a higher mutation detection rate. It may reduce the number of high-risk families with negative results of finding a gene mutation due to the increased coverage. The lifetime breast cancer risk estimates associated with gene mutations are listed in Table 2.10 13 36 56 58 61 62 63 64 65 66 67 68 69 70 71 72 73 74

Table 2. Estimated lifetime risk of breast cancer associated with selected susceptibility genes10 13 36 56 58 61 62 63 64 65 66 67 68 69 70 71 72 73 74
In Hong Kong, breast cancer is the most common cancer in the female population. The Hong Kong Hereditary Breast Cancer Family Registry was established in 2007. It functions as a data registry of hereditary breast, ovarian and prostate cancer families and is also an established charitable organisation that subsidises the cost of genetic testing for underprivileged individuals. More than 1900 patients with breast and/or ovarian cancer who satisfied the selection criteria have received genetic testing in Hong Kong. Each individual underwent thorough genetic counselling to ensure the implications of genetic testing were understood. Around 600 probands were screened for BRCA1 and BRCA2 mutations by bi-directional Sanger sequencing of all coding exons and multiplex ligation-dependent probe amplification.75 The sensitivity of identifying mutations is comparable with the gold-standard method with good bioinformatics support. Next-generation sequencing meets rigorous quality standards and can provide clinical sequencing results that are equivalent to those obtained from Sanger DNA sequencing analysis.76 We started employing next-generation DNA sequencing to expedite analysis workflow and expand the gene panel in 2011 to include TP53 and PTEN for sequencing. Cases with a negative result after screening with our in-house developed gene panel are further sequenced using 454 GS Junior System (Roche Life Sciences) or MiSeq (Illumina). Sequencing data are analysed by an in-house fully developed automatic bioinformatics pipeline. The mutation screening result of a 4-gene panel BRCA1, BRCA2, TP53, and PTEN in our recruited patients revealed that 9% carried such mutations. Nonetheless a number of clinically high-risk patients have tested negative for the above genes. This indicates that there is further potential in expanding the coverage to different lower-penetrance genes such as PALB2, which has recently been reported to be important to cause hereditary breast cancer in our testing strategy.56
Clinical assessment of an individual’s risk of hereditary cancer is based on the evaluation of family history, age of onset, and type of cancer. Advances in molecular genetics testing have identified a number of genes associated with inherited susceptibility to breast and/or ovarian cancers such as BRCA1, BRCA2, PTEN, and TP53. The recent introduction of next-generation sequencing technology and multi-gene panel testing for hereditary cancer has rapidly altered the clinical approach to high-risk patients and their families. Although there are still limitations, individuals with hereditary or familial breast/ovarian cancer are likely to benefit from strategies including prevention, screening, and targeted treatment. Suitable patients and families should be offered genetic counselling and testing.
This study was supported by The Hong Kong Hereditary Breast Cancer Family Registry, Hong Kong Sanatorium and Hospital, Dr. Ellen Li Charitable Foundation, the Kerry Group Kuok Foundation Limited and Health and Medical Research Fund (1123176).
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