Gene panel testing for hereditary breast cancer

It is estimated that less than 5% of breast cancer occurs as a result of single gene predispositions; some 2% is explained by mutations in the well known BRCA1 or BRCA2 genes.1 The Australian Institute of Health and Welfare estimated that 15 740 people would be diagnosed with breast cancer in 2015,2 suggesting about 780 of these cancers would be due to genetic predisposition. While this number seems small, the people affected have many more family members (most of whom are unaffected) who can have their potentially high risk of breast cancer managed once it is identified.

Recognition of a familial predisposition to breast cancer led to a race for the “responsible” genes in the late 20th century. The BRCA1 gene was the first gene found to have very rare mutations associated with high penetrance of breast and ovarian cancers.3,4 These rare mutations were often identified in young women or in members of families with multiple cases of breast or ovarian cancer. In 1994, the BRCA2 gene was identified,5,6 and mutations in this gene were found to be associated with increased risk of several cancer types; breast and ovarian cancers were seen in families who also had a higher incidence of melanoma, pancreatic cancer and prostate cancer. A characteristic feature of mutations in BRCA2 is the increased risk of breast cancer in men.6

Twenty years later, the “missing heritability” of breast cancer remains elusive. A network of genetic services and familial cancer centres across Australia investigate families when there is an evident inherited predisposition to breast cancer. However, most of these families do not have mutations in either the BRCA1 or BRCA2 gene. Rare mutations in other genes have subsequently been recognised as playing a role, especially in cases where breast cancer is diagnosed at an early age.7-10

A new approach — multiple gene testing

To date, tests have been conducted as single gene tests in an iterative manner, one gene at a time, at very high cost. Although some information could be used to direct this testing (eg, specific histopathological features of the breast cancer, such as mitotic index and trabecular growth pattern),11 the testing process is slow and laborious, and most test results remain uninformative. However, the revolution in our capacity to conduct genetic analyses in very recent years has had a profound impact on how genetic testing services can be applied. Now, a panel of multiple genes can be screened for mutations in a single test, at considerably reduced cost, via the application of massively parallel sequencing technology. This approach applies across many disciplines, most notably cardiology; gene panel testing is now the standard of care in the cardiac genetic clinic at the Royal Melbourne Hospital.12

Increases in genetic technology capacity have, in parallel, enabled work that has identified additional breast cancer predisposition genes, and many other candidate breast cancer predisposition genes have been reported (however, these require further evidence). Today, gene panel tests for breast cancer predisposition are offered by numerous providers and service laboratories.10,13 However, few of these tested genes are bona fide breast cancer predisposition genes. Indeed, most of the genetic variation identified by these gene panel tests cannot be interpreted in terms of breast or ovarian cancer risk.

The PALB2 gene is an example of an important new breast cancer predisposition gene, as it has rare mutations that are associated with high penetrance for breast cancer.14,15 The PALB2 protein plays an essential role in interacting with the BRCA1 and BRCA2 proteins and enabling DNA repair. The risk of breast cancer for women with heterozygous germline mutations in the PALB2 gene is as much as eight to nine times as high as in the general population, depending on the age of the woman.15 Mutations in PALB2 account for about 2.4%14,16 of the familial aggregation of breast cancer in the Australian population, but occur at extremely low rates in the general population, making single gene testing for PALB2 cost-ineffective at a population health level.

The low yield of single gene testing and the potential for inclusion of other genes with low frequency mutations (such as PALB2) make a gene panel test approach ideal in the investigation of breast cancer predisposition. Genes that are regularly included in breast cancer susceptibility gene panels that are currently available through academic or commercial testing laboratories include BRCA1, BRCA2, PALB2, PTEN, STK11, TP53, ATM, BARD1, BRIP1, CDH1, CHEK2, MRE11A, MUTYH, NBN, NF1, RAD50 and RAD51C.10,13

The utility of gene panel testing is illustrated by the hypothetical case outlined in the Box.

Implications for breast cancer risk management

There is an evidence-based strategy for managing women who harbour high penetrance mutations in genes such as BRCA1 and BRCA2, and emerging evidence for the risk management of women with mutations in several other genes, including PALB2. For BRCA1 and BRCA2 mutation carriers, it is now known that bilateral salpingo-oophorectomy around the age of 40 years or before the natural menopause will halve the risk of breast cancer, while substantially lowering the risk of ovarian cancer.19 Likewise, chemoprevention and risk-reducing mastectomy are effective in reducing the risk of breast cancer in women with pathogenic mutations in these genes.20 In the absence of a mutation, where there is a theoretical 50% risk that each woman in such families will not have inherited the genetic predisposition, this irreversible and invasive approach is hard to justify.

Genetic testing is thus the only way to provide personalised risk assessment, through predictive testing of at-risk unaffected family members, although this approach depends on identifying a pathogenic mutation in the index case. Effective risk management is thus predicated on accurate risk estimation; as is release from high-risk cancer surveillance for those found not to be at high risk of cancer. The initial inability to find a pathogenic mutation in our illustrative case (Box) meant that this risk management strategy was not possible, whereas gene panel testing and the resulting detection of the PALB2 gene mutation enabled personalised management of the extended family.

Considerations in gene panel testing

The choice of genes to include in panel testing needs careful consideration. The ACCE framework, named for the acronym derived from its descriptors, calls for genetic tests to be underpinned by analytical validity, clinical validity, clinical utility and ethical issues.21

Clinical validity and utility

Clinical validity and consequent clinical utility are currently the most complex areas — is the genetic variation detectable in these genes associated with clear and quantifiable risk of disease? Initially, genetic testing entered clinical practice without much process, nor an evidence base, but spurred by an enthusiasm to reduce the risk of potentially predictable cancers. In contrast, adoption by clinical services of the newly recognised breast cancer predisposition genes, such as PALB2, has been slow, due to an increased awareness of the complexity of interpretation of genetic test results.22 In Australia, protocols for genetic testing and subsequent management based on genetic knowledge have been built over time and are now consolidated in the eviQ guidelines,18 used by clinical genetic and familial cancer services nationally.

It was important initially to ensure that panel testing was as good as the standard of care in finding mutations in BRCA1 and BRCA2, before considering potential additional benefits. In a recent study of 966 patients undergoing breast cancer genetic testing,23 deleterious BRCA1 or BRCA2 mutations were identified at equivalent rates between single gene testing and a multiple gene panel test that included BRCA1 and BRCA2. An additional 3.9% of patients tested by the multiple gene panel had pathogenic mutations identified in other breast cancer predisposition genes, most commonly PALB2, CHEK2 and ATM. In a retrospective review of 90 consecutive patients undergoing genetic testing by a multiple gene panel test,24 pathogenic mutations were identified in nine individuals: five in BRCA1 or BRCA2, and four in other breast cancer predisposition genes. As single gene testing has only identified mutations in BRCA1 and BRCA2 genes, multiple gene panel testing finds more clinically significant mutations.

However, gene panel tests also identify many variants of uncertain significance. Classification of variants is important. In short, there are sequence variants that are clearly disease-associated and classed as pathogenic mutations, while at the other end of the spectrum, much variation in DNA is clearly not associated with disease predisposition and can be discounted. A large grey zone exists in between, the so-called variants of uncertain significance, where classification is not possible, and this information cannot be used clinically. For this reason, interpretation of results, whether derived from gene panels or single gene testing, remains highly specialised and requires a team of skilled scientists, informaticians and genetic practitioners, along with research-derived evidence.

Ethical issues

Technology that allows analysis of many genes at once has a raft of associated ethical issues when applied in a diagnostic setting.25 It is prudent to consider the ability of such tests to produce secondary findings (previously also referred to as incidental findings), wherein genetic changes may be found that were not anticipated.

The recent confirmation in the High Court of Australia that the BRCA1 gene is not patentable has been welcomed, and represents a moral victory for the women and families concerned.26 Indeed, in 2013, following the ruling by the Supreme Court in the United States to invalidate patent claims made by Myriad Genetics, the BRCA1 and BRCA2 genes were included in existing panels that had hitherto excluded them and thus had reduced the comprehensiveness of the analyses being conducted.10

Conclusions

The use of new genetic technology can provide an affordable, effective way of investigating the heritability of breast cancer. Early studies suggest that the gene panel testing approach will detect all mutations found by conventional testing and increase the diagnostic yield through analysis of more genes. This gives more women and their families the opportunity to have personalised risk assessment and risk management, in a preventive approach to predictable breast cancer.

On a cautionary note, the consequences — that is, the clinical validity and utility of many of the genes listed — are not yet clear. Large scale studies through international collaborations into the genes included in these gene panels are essential to yield the information that will allow the safe and effective use of newer technologies and new genetic information in clinical practice.