An estimated 5-10% of cancers have a heritable component, and there are a growing number of hereditary cancer syndromes.1-5 Identifying pathogenic variants in genes associated with hereditary cancer syndromes can uncover genomic mechanisms that have predictive, diagnostic, and prognostic utility to patients and are used to better their management.6-8 Pathogenic variants in germline genes have been associated with an increased lifetime risk of hereditary breast and ovarian cancer (HBOC), colorectal cancer (CRC), as well as other cancers, such as endometrial, pancreatic, prostate, and melanoma. Traditionally, testing of genes associated with hereditary cancers was performed based on specific gene-disease relationships and an individual’s personal or family history, often in a single-gene reflex fashion. However, the growing number of genes known to be associated with hereditary cancer syndromes and the overlap between clinical presentations has challenged this paradigm.
The application of NGS technology has facilitated multi-gene panel testing for definitive genes associated with many hereditary cancer syndromes. NGS has been shown to be more efficient than single-gene sequential testing, and is becoming a routine component of the diagnostic process.1,9-11 For example, BRCA1 and BRCA2 (BRCA1/2) have historically been the most frequently tested genes in HBOC. Yet, it is now estimated that more than half of the individuals with hereditary breast cancer carry pathogenic variants in genes other than BRCA1/2.10,12-14 Breast cancer is also a component of several other hereditary cancer syndromes, such as Li-Fraumeni syndrome, Cowden syndrome, hereditary diffuse gastric cancer and Peutz-Jeghers syndrome.15-19 Studies estimate that approximately 30% of all CRC cases are an inherited form of disease20-22 and nearly 5% are associated with highly penetrant hereditary clinical presentations. Lynch syndrome (LS), previously known as hereditary non-polyposis colorectal cancer (HNPCC), is the most common hereditary CRC syndrome accounting for 2-3% of all CRC. It is caused by germline pathogenic variants in 5 mismatch repair genes, MHL1, MSH2, MSH6, EPCAM and PMS2. Traditionally, a testing cascade of microsatellite instability (MSI) analysis and/or immunohistochemistry was performed followed by testing of individual single genes. However, NGS allows for a majority of the genes to be tested simultaneously, reducing the time to diagnosis and reducing costs.11,23 The National Comprehensive Cancer Network (NCCN) guidelines have also expanded to incorporate testing of multiple genes into medical management recommendations.5,24-26 The established Centers for Medicare and Medicaid Services (CMS) NCD 90.2 confirms testing using NGS to be both reasonable and necessary in Medicare beneficiaries.
Clinical Indications and Risk Factors
Although inherited cancer syndromes each have their own clinical criteria for testing, there are some findings that are associated more frequently with hereditary cancers when compared to those that are acquired including: diagnosis at an earlier age than what is typically seen for that cancer type, 2 or more affected close blood relatives (first-, second- and third-degree relatives) on the same side of the family with the same type of cancer, multiple affected generations within 1 family. Additional findings include multiple cancer types occurring in the same individual, cancers that develop bilaterally, and presence of congenital conditions known to be associated with a particular cancer syndrome.3,27
Additional factors are specific to certain cancer types. For example, factors that significantly predict for a germline BRCA1/2 pathogenic variant in breast cancer include Ashkenazi Jewish heritage, triple-negative breast cancer, tumor histologic grade 3, and diagnosis prior to the age of 50.12 Multi-gene testing should also be considered in individuals with CRC with adenomatous polyposis, colonic polyposis with an unknown histology, or when more than 1 syndrome may explain the presentation. For example, 10 or more adenomas are more likely to be associated with pathogenic mutations in genes that cause classical familial adenomatous polyposis (FAP) or attenuated FAP or MUTYH-associated polyposis (MAP). Whereas 2 or more hamartomatous polyps are indicative of Peutz-Jeghers, Juvenile polyposis, and Cowden/PTEN hamartoma syndromes.5 The risk of melanoma is influenced by sensitivity to Ultraviolet (UV) radiation, sunburns during childhood, and sunlight exposure.28,29 The NCCN has defined criteria for which testing of well-established germline genes is appropriate for a variety of inherited cancers.5,24-26
NGS is currently the most common methodology utilized for hereditary cancer gene testing. NGS is not a specific test, but a sequencing methodology utilized to capture genomic information. Unlike Sanger sequencing (the prior standard technology) that typically provides sequence information for a single DNA strand/molecule, NGS allows for massively parallel sequencing of millions of DNA molecules concurrently.30,31 This allows for capturing many relevant genomic targets simultaneously, usually by utilizing technologies, such as by polymerase chain reaction (PCR) amplification or hybrid capture. As such, NGS tests for use in germline cancer are often comprised of gene panels whose content is either relevant to a specific cancer type or condition, or a larger panel of genes that can be used for multiple cancer types.
NGS tests can vary significantly for many reasons. While NGS defines a broad methodology for massively parallel sequencing, different technologies that have different strengths, weaknesses, and technical limitations or liabilities are available.32 The most common sequencing platforms in clinical use today sequence by synthesis similar to Sanger sequencing; these platforms utilize different chemistries, signal amplification, and detection methods. Gene panels can include only the portions of genes that contain the most critical clinically relevant information, or be comprehensive, containing entire exonic gene regions (coding regions), introns (non-coding regions), and even sequence ribonucleic acid (RNA) for detecting abnormal transcripts. Downstream from the pre-analytic processes mentioned above, the bioinformatics used to process and assess the resultant sequencing reads and identify variants/mutations can yield different results based on the software used and what types of variants the test is attempting to detect. These software tools must take the resultant sequencing file (generally starting with the FASTQ format), align all possible sequences with a reference genome (BAM/SAM), and identify variants from the reference (typically a VCF file). Once such variants are identified, they must be assessed for validity and subsequently, for their clinical relevance.
The types of genomic information reported can vary, as tests can uncover a myriad of genomic alterations, such as single nucleotide variants (SNVs), Insertions/Deletions (INDELs), Copy Number Alterations ([CNAs]; these can be simply amplifications at a single locus or chromosomal gains and losses), Structural Alterations ([SAs]; inversions, insertions, translocations) and abnormal RNA splice site variants. All these variant classes have demonstrated clinical utility in germline testing. Additionally, NGS testing is highly complex and requires expertise from handling the specimen, running the complex equipment, understanding the required bioinformatics, interpreting the findings and creating an actionable medical report. Guidelines for validating clinical NGS panel tests and bioinformatics pipelines have been published.33-35 Variant interpretation is also a crucial component of NGS testing in hereditary cancers. The American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) have published standards and guidelines for both sequencing and copy number variant interpretation that have been widely adopted by clinical laboratories around the world.36-38
Multi-gene hereditary cancer panels offered by diagnostic clinical laboratories vary on the number of genes that are included. The Clinical Genome Resource (ClinGen) has developed a clinical validity framework that evaluates evidence in the literature and determines the strength of association of genes with disease using a point-based system. Genes that have been repeatedly demonstrated in both the research and clinical diagnostic settings to be associated with a particular disorder with no contradicting evidence are considered definitive.39 Lee, et al., and Seifert, et al., describe the assessment of genes frequency tested in hereditary breast and ovarian, and colorectal cancer and polyposis multi-gene panels, respectively, using the ClinGen Gene Curation framework.40,41 These assessments considered multiple lines of evidence including case reports, familial and case-control association studies, and segregation data and describe genes that are considered definitive for these cancer types.