Breast Cancer Genetics: Syndromes, Genes, Pathology, Counseling, Testing, and Treatment

The Value of Genetic Testing

For individuals not yet diagnosed with cancer, genetic testing can be a powerful risk stratification tool. Lifetime breast cancer risk is 57% to 81% for women with pathogenic BRCA1 variants and 45% to 85% for BRCA2 variants. Ovarian cancer risk by age 70 years is 39% to 46% for BRCA1 variants and 10% to 27% for BRCA2 variants. Pathogenic variants in other genes may point to increased risk for gastric, thyroid, endometrial, or other cancers ( Table 10.1 ). Quantitative risk information can guide decisions about enhanced surveillance to diagnose cancers earlier, or prophylactic surgery or chemoprevention to reduce the risk of ever developing cancer. Pathogenic variants in most of the genes commonly tested today will be associated with at least a 20% lifetime breast cancer risk. These women all meet the risk threshold for consideration of enhanced surveillance with breast MRI.

Pathogenic variants in breast cancer predisposition genes can influence decisions about surgery, radiation therapy, and systemic treatments in the newly diagnosed breast cancer patient. With respect to surgery, second primary breast cancer risk is often elevated in carriers. This information is critical for deciding between breast conservation and unilateral or bilateral mastectomy. Most of the known breast cancer predisposition genes serve a role in DNA maintenance and repair. This knowledge can influence radiation therapy decisions. Though therapeutic whole breast radiation is not contraindicated in any other than homozygous ATM mutation carriers, it should be used with caution in TP53 mutation carriers. Some professional societies advise against accelerated partial breast irradiation for BRCA-associated breast cancer, but others do not. Pathogenic BRCA1/BRCA2 variants are increasingly recognized as markers of reduced sensitivity to taxol-based chemotherapy and increased sensitivity to platins and PARP inhibitors. Germline genetic testing serves as a predictive biomarker by identifying BRCA-associated breast and ovarian cancers eligible for PARP inhibition.

Role of the Cancer Genetics Counselor

Receiving the results of a genetic test can be emotionally challenging and can set in to motion a chain of events with significant quality-of-life implications. Some patients simply prefer not to know. Decisions about whether to undergo testing or not, what test to perform, and then what to do with the results are complex. This complexity is amplified in the era of multigene panel testing. Some have advocated genetic testing by primary care providers, or by those who diagnose and treat breast cancer. The ABOUT study, a survey of 11,159 women who underwent BRCA1/BRCA2 testing in 2012, found that only 37% received genetic counseling. Those who received genetic counseling had greater knowledge, understanding, and satisfaction than those who did not. In 2012 the American College of Surgeons Commission on Cancer accreditation program mandated that cancer risk assessment, genetic counseling, and genetic testing services be provided to patients by a qualified genetics professional either on site or by referral. This standard was reiterated in 2020 ( https://www.facs.org/quality-programs/cancer/coc/standards/2020 ). The US Preventive Services Task Force completed a systematic review of the literature and concluded that genetic counseling reduces distress, improves risk perception, and reduces intention for testing.

The National Society of Genetic Counselors has outlined the essential functions of the genetic counselor, which include: (1) estimating cancer risk based on personal and family medical history prior to genetic testing, (2) offering genetic testing when certain criteria are met, (3) fully informing the individual about the test and the possible results, and (4) disclosing test results in conjunction with a reestimation of cancer risk and a discussion about options for reducing risk. These steps should not be neglected when genetic testing is provided outside of the purview of a certified genetic counselor.

Identifying Mutation Carriers

Variants in BRCA1 and BRCA2 account for about half of the individuals with identifiable hereditary breast cancer predisposition. The prevalence (allelic frequency) of pathogenic BRCA1/BRCA2 variants is estimated at 0.13% to 0.26% for the general population and 1.3% to 2.7% for the Ashkenazi Jewish population. It is estimated that there are 350,000 to >500,000 individuals in the United States who carry a pathogenic variant in a breast cancer predisposition gene.

Genetic testing guidelines are directed at recognizing individuals who are reasonably likely to carry a pathogenic variant. This is currently largely determined by personal and family history unless there is a known variant in the family. Third-party payors usually adopt these guidelines, so financial constraints can limit testing outside of these guidelines.

NCCN guidelines are becoming more liberal, but also more complex. Testing recommendations are largely based on personal or family history of early onset breast cancer, ovarian cancer, pancreatic cancer, or prostate cancer (especially intraductal/cribriform, high-risk, or metastatic prostate cancer). NCCN guidelines also include testing criteria for TP53 (Li-Fraumeni Syndrome) and PTEN (Cowden Syndrome). There are nearly a dozen genes firmly linked to inherited breast cancer predisposition. Generating and maintaining detailed testing criteria for each one may not be practical, and a stepwise approach to genetic testing may be cost-prohibitive. In general, hereditary predisposition can be suspected based on early age at breast cancer diagnosis, multiple breast cancers in the same lineage, and the presence of associated cancers such as ovarian, pancreas, or prostate for hereditary breast/ovarian cancer syndrome, and gastric, melanoma, sarcoma, endometrial, or others for the other syndromes ( Table 10.1 ).

The probability that an individual carries a pathogenic variant in BRCA1 or BRCA2 can be estimated using family history models such as BRCAPRO, Tyrer-Cusick, or CanRisk(BOADICEA). NCCN guidelines recommend genetic testing when the pathogenic variant probability is >5% and support consideration of genetic testing when the probability is 2.5% to 5%.

The US Preventive Services Task Force recommends that primary care providers screen women for personal and family cancer histories that may suggest an inherited predisposition to breast cancer. Numerous screening tools are available. The simplest tool is shown in Table 10.2 . Despite these recommendations, detailed cancer family history screening is not routinely practiced. One approach for increasing genetic counseling referrals has been to provide physicians with education and tools to facilitate screening, but this approach has not been particularly successful. Many organizations have articulated guidelines for genetics referrals, but providers are often unaware of these guidelines.

The use of personal and family history as a filter for excluding some people from genetic testing is currently being challenged as it becomes apparent that this approach does not discriminate very well between carriers and noncarriers. This is because self-reported history is not always accurate and because many people come from small families. A recent study found that half of 306 women with breast cancer diagnosed before age 50 years had fewer than two first- or second-degree female relatives living past the age of 45 years. Nevertheless, pathogenic BRCA1/BRCA2 variants were identified in 14% of these women. In another study eight genetic counselors and the computer program BRCAPRO assessed a series of pedigrees from a genetic counseling clinic. Discrimination between carriers and noncarriers was only 10% to 28% better than the flip of coin. In yet another study a family history tool was used to screen 96,055 mammography patients, of whom 4% met genetic testing criteria. Of these, 55% accepted genetic counseling. This approach identified only 26% of the number of pathogenic variants expected based on the composition of the population. These studies consistently confirm that our family history approaches are not robust enough to exclude anyone from genetic testing. Indeed, variant prevalence studies consistently demonstrate that adherence to testing guidelines will miss a large number of pathogenic/likely-pathogenic variants identifiable on multigene panels. The American Society of Breast Surgeons now recommends that genetic testing be made available to anyone with a personal history of breast cancer.

However, delaying genetic testing until after breast cancer has developed precludes taking measures to reduce the risk of breast cancer or to improve the chances that breast cancer is diagnosed early. Some are advocating population-based testing so that resources can be directed toward those at greatest risk. Opponents to this strategy cite cost and difficulties in the interpretation of results for people without a significant family history. Recent data mitigate these concerns. For the cost concern, it has previously been estimated that the cost of the testing would need to be <$250 in order to get the cost per quality life year gained down to an acceptable $53,000. Multigene panel testing is now available for ≤$250. In addition, mathematical models suggest that more liberalized multigene panel testing is cost-effective, especially if it is employed in younger women. One of the first studies to look at cancer risk in mutation carriers identified through population testing included 8195 healthy Ashkenazi Jewish men. Pathogenic BRCA1/BRCA2 variants were found in 2.2%, resulting in the identification of 211 carriers among 629 female relatives. Among carriers identified in this fashion, breast cancer risk to age 80 years was 60% for BRCA1 variants and 40% for BRCA2 variants. Ovarian cancer risk was 53% for BRCA1 variants and 62% for BRCA2 variants. Since then, additional studies have confirmed that cancer risk for carriers identified through population-based multigene panel testing are similar to those for conventionally identified mutation carriers. Some health maintenance organizations have begun offering genetic testing to unselected patients as part of personalized wellness programs. This identifies mutation carriers before they develop cancer, permitting adoption of measures to avoid cancer or to diagnose it early.

The pathology laboratory may also have a role in the identification of carriers. Multigene panel testing was performed in 1824 triple-negative breast cancer (TNBC) patients unselected for family history. Pathogenic BRCA1 or BRCA2 variants were identified in 11.2% and pathogenic variants in other genes in 3.7%.

In summary, in the United States alone there are hundreds of thousands of individuals with unrecognized mutations in breast cancer predisposition genes. Testing guidelines unnecessarily erect barriers that end up excluding certain people from genetic testing. Support is growing for unrestricted genetic testing in order to identify mutation carriers before they develop cancer.

Sanger Sequencing

For over a decade Sanger sequencing was the primary laboratory test performed on DNA extracted from white blood cells or saliva samples to determine if an individual carried a variant in a known breast cancer predisposition gene. Sanger sequencing involves separating the two strands of DNA and then defining the region to be sequenced by allowing short complementary sequences (primers) to bind to the DNA at either end of the region of interest. Double-strand DNA is then rebuilt by filling in the gap between the primers with new nucleotides (A, C, T, and G). Included in the mix of new nucleotides are low concentrations of specially prepared As, Cs, Ts, and Gs that are labeled with a colored probe that will stop the elongation of the new DNA strand at that point. Several cycles of DNA synthesis in a test tube will produce a mixture of DNA strands of various lengths, each capped with a color marker representing the last nucleotide added to that particular strand. After many cycles of DNA synthesis, the mixture is separated by size using electrophoresis. The order in which the colored nucleotides exit the electrophoresis gel will establish the original sequence of nucleotides between the two primer pairs. One limitation of this approach is that only predefined, relatively short (300–900 nucleotides) segments of DNA between the primer pairs will be assessed. A gene like BRCA2 has 10,254 coding nucleotides, so more than a dozen sequencing reactions need to be set up just to assess the coding region. People decide whether other sequencing reactions need to be designed to also assess portions of the promoter region or introns that may contain disease-causing sequence variants. The point being that sequencing tests in use around the start of the 21st century may have missed important variants. Another limitation of this approach is that point mutations are recognized by observing two colors coming off of the electrophoresis gel at the same point (one representing the correct nucleotide and one representing the variant nucleotide). If one copy of a particular section of DNA is simply missing (a deletion), only one color will come off of the gel for that section (the normal sequence), and an important genomic alteration will be missed. This is why additional tests to identify insertions, deletions, or rearrangements of entire sections of the DNA have been gradually added to genetic testing protocols over time.

Next-Generation Sequencing

Massive parallel sequencing, or next-generation sequencing (NGS), became commercially available in 2005. There are several iterations of the technology, but in general DNA is minced up to generate short fragments that are then widely distributed across glass surfaces. Each short fragment is duplicated several times to create bundles of DNA with the same sequence. These appear as spots on an imaging screen. Reagents, which include color-labeled nucleotides, are sequentially added and washed away. Each spot is photographed between each cycle. The color of a given spot after any cycle corresponds to the specific nucleotide most recently bound to the immobilized DNA strands at that location. In this way, a nucleotide sequence is generated for each spot ( Fig. 10.2 ). Each sequencing test will include tens of thousands to millions of spots, each generating 50 to 100 base sequences. Translating this enormous dataset into nucleotide sequences for specific regions of the genome is a daunting task that is done by computers. First, each individual sequence is aligned to a standard human genome to determine exactly what part of the genome is represented. Because of the random nature of the initial DNA digestion and spot creation, each nucleotide of interest will occur several times on overlapping sequences. Statistical filters are used to assess the quality of nucleotide calls based on the number of replicates generated by the test and the consistency of the calls between replicates. Comparison of the test genome with the standard genome will reveal tens of thousands of sequence variants for any given individual. This very long variant list can be shortened by ignoring known polymorphisms and by focusing only on specific regions of interest (e.g., the BRCA1 gene). Nevertheless, NGS produces a wealth of sequence variants that poses a problem for interpretation. Technically, NGS is very reliable and will identify the same mutations identified by Sanger sequencing. NGS can identify large insertions, deletions, and rearrangements missed by Sanger sequencing but may have difficulty recognizing small rearrangements.

Large Rearrangements

All of the DNA sequencing technologies are highly sensitive and specific for recognizing single nucleotide changes and short insertions or deletions. Recognizing rearrangement of larger regions is more problematic. These large rearrangements account for up to 17% of pathogenic BRCA1/BRCA2 gene mutations in individuals of Near East/Middle Eastern ancestry and up to 22% for individuals with Latin American/Caribbean ancestry. In 2012 NCCN guidelines specified that BRCA1/BRCA2 gene testing should routinely include special tests for large rearrangements. Testing prior to this may not have included this special testing. NGS data files generally include enough information to recognize rearrangements if specific bioinformatics algorithms are used to look for them. Some test providers will supplement NGS “allele dose” calculations with additional tests specifically directed at identifying large rearrangements such as microarray comparative genomic hybridization (CGH) and multiplex ligation-dependent probe amplification analysis (MPLA). Thus far sensitivity for detecting these rearrangements seems high. Confirmatory testing using one of the classical assays is currently recommended when NGS suggests a large rearrangement.

Classifying Variants

DNA sequencing of any kind will always identify small differences between individuals or small differences compared to some standard genome. Each variant must be adjudicated based on available evidence. Variants that occur in greater than 1% of the population are generally considered polymorphisms and easily classified as almost certainly benign. Variants that have previously been definitively linked to breast cancer in family studies are more easily classified as almost certainly pathogenic. In the middle, however, is every shade of gray from “variant likely benign” to “variant likely pathogenic.” For some variants there is simply not enough information to make an educated guess. These are reported as “variants of uncertain significance” (VUSs).

The American College of Medical Genetics has published guidelines for classifying and reporting DNA sequence variations. There are five possible classifications based on the availability of published data and the type of DNA change ( Table 10.3 ). Classification is straightforward for variants that have previously been reported and for which there are quite a lot of data available. For everything else, it is people who weigh the evidence and initially assign a classification.

There are thousands of distinct rare variants in BRCA1 and BRCA2 , and the list is rapidly growing for many other genes because of increased use of multigene panel testing. Each new variant is initially classified somewhere along the spectrum of benign to pathogenic based on features of the specific nucleotide change. Certain types of mutations are more likely to be pathogenic than others. These include single nucleotide changes that result in an early stop codon and a shorter than normal protein (nonsense or truncating variants), as well as insertion or deletion of some number of nucleotides that is not a multiple of three. This will result in a shift in the reading frame (frameshift) and often an early stop codon. Missense variants are changes in one nucleotide that result in a different amino acid being added when the mRNA is read. Most of these variants are not pathogenic, though some are. Several additional lines of evidence can move the classification toward or away from pathogenic. Variants that have previously been observed in individuals that have other, definitely pathogenic variants in the same autosomal dominant gene are more likely neutral. A variant that occurs in a region that is normally coded exactly the same across many species (i.e., conserved) may be more likely to be pathogenic, as is a variant that causes a significant change in the type of amino acid that is added to the growing protein chain (e.g., switching from a hydrophilic to a hydrophobic amino acid). Variants that occur near the intron-exon boundary can affect the way that mRNA is spliced together and may also be pathogenic. Fig. 10.3 illustrates the two most common types of variants and explains the nomenclature found in test reports.

Various informatics tools are available to help classify variants. ClinVar is a publicly available list of sequence variations and their current interpretation that is maintained by the NIH ( https://www.ncbi.nlm.nih.gov/clinvar/ ). The Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) is an international group devoted to the collection and interpretation of rare variants in BRCA1 and BRCA2 . They have developed a software tool to estimate the probability of pathogenicity for any variant using combined evolutionary sequence conservation, family-based segregation and cancer history, tumor pathology, and RNA splicing effects. Some clinical laboratories use personal and family history weighting to assist in the classification of new variants.

Variants of Uncertain Significance

Every new variant identified in a known breast cancer predisposition gene has uncertain significance until it has been investigated, as described earlier. Many times at the conclusion of this investigation there are insufficient data to confidently place the variant anywhere along the benign-to-pathogenic spectrum. These variants are temporarily classified as VUSs. The natural history of most VUSs is definitive reclassification over time as new information becomes available. Sometimes this new information is proactively generated by testing affected and unaffected relatives from the same family, or by assessing the functional significance of the DNA change in the laboratory. Sometimes new information is passively acquired as the same variant is observed in more informative families. The point is that VUS rates are high when a new test begins identifying new variants, but this rate decreases as experience increases. This was observed with BRCA1 and BRCA2 testing, where the VUS rate was 7% to 15% in 2002 but had decreased to 2.9% by 2012. VUS rates are somewhat higher for non-White populations, but have declined from 22% to 46% to 2.6% to 7.8% in recent years.

The introduction of multigene panel testing has generated a torrent of new variants. VUS rates are currently 20% to 40% for genes that are less well-studied than BRCA1 and BRCA2 . One study that evaluated a 42-gene panel in 175 patients reported an average of 2.1 VUS calls per patient. This degree of uncertainty is understandably unsettling. It will take time to accumulate sufficient clinical and functional data to definitively reclassify these variants. To this end, the Prospective Registry of Multiplex Testing (PROMPT) was established. This is an online registry that collects variant and clinical information from patients who have had multigene panel testing ( https://promptstudy.info/ ).

Patients whose genetic test result returns a VUS are said to have had a noninformative result. In this case the test was not helpful, and the patient is managed based on the personal and family history. It has been reported that 24% of high-volume surgeons but 50% of low-volume surgeons incorrectly manage VUS patients the same as patients with pathogenic mutations. Patients with a VUS should be managed as though they never had genetic testing. Variant classifications can change over time as more data become available. It is advisable to establish and maintain procedures for recontacting patients if their variant is reclassified.