Next-generation sequencing (NGS) is a breakthrough technology that renders decoding genetic information easier, faster, and less expensive than conventional methods such as Sanger sequencing. The process enables simultaneous and massively parallel analysis of genetic material from multiple patients across multiple genes in a single run. Whereas in the past few decades, it took several weeks, intensive labor, and high cost to sequence a single gene in one patient, NGS can achieve the same results with much less time, effort, and cost by sequencing many genes from many patients at one time.
Vast Resources Are Available
The introduction of NGS to clinical laboratory testing has been largely supported by the wealth of genetic information deposited in publicly available population databases (see SIDEBAR 1). These databases catalogue the increasing number of single nucleotide variants or small copy number variants that are common in the general population (usually at a frequency of greater than 1%), and are considered to be benign. The knowledge of benign variants has allowed further research into pathogenic variants, usually occurring at a frequency less than 1% in the general population, and are more likely to cause disease.
The catalog of pathogenic variants also is stored in readily available patient databases (see SIDEBAR 2). The realization of the extensive variation in our genomes and the implication of their changes in disease have been instrumental in our ability to interpret massive numbers of sequencing variants. With the aid of nationally funded mutation databases such as ClinVar, it is easier to determine whether variants that differ from the reference standard have any effect on health. Such resources have been developed to provide variation-phenotype relationships, including supporting evidence of submitted interpretations.
Germline and Somatic Testing
Clinical information derived from NGS data can be attributed to two categories: germline or somatic. Germline variants are constitutional in nature and enable diagnosis, prognosis, and often treatment of the afflicted patient (eg, inherited metabolic disorders or anti-epileptic drug therapy). Information derived from germline NGS can range from a panel of a handful of genes to the whole genome, depending on the clinical history and utility of the test, the diagnostic odyssey, and the economic situation. Additionally, these variants will undoubtedly have an impact on recurrence risk and family planning considerations. Therefore, it is imperative to ensure germline NGS testing is accompanied by pre- and post-test genetic counseling for pedigree analysis, test interpretation, and patient consent.
It is important to acknowledge that every clinical laboratory testing program aiming at utilizing NGS for somatic tumor analysis needs to establish a program to address incidental germline findings (ie, a germline predisposition to cancer). That said, this article is dedicated primarily to a discussion of somatic NGS testing.
Somatic Variants and Oncology
Somatic variants detected by NGS are derived from the study of a patient’s tumor tissue. The information therein can give insight into the mutational changes that characterize the tested tumor. Cancer is universally a genetic disease and it often mimics a constellation of orphan diseases with unique molecular signatures, such as the BRAF V600 mutations in melanoma or ERBB2 amplification in breast cancer. Furthermore, cancer genomes are genetically complex and display an innate ability to continuously mutate and evade treatment. It is therefore generally acknowledged that the most successful fight against cancer remains the adoption and broad utilization of screening programs for early detection before tumors have a chance to accumulate excessive genetic changes.
Mutations in cancer can be either drivers or passengers, with the identification of the former being most essential when choosing therapies. Mutations in cancer can decrease the translation of a tumor suppressor gene product, increase the production of an oncogene, or simply create a new gene function that allows the cell to grow and divide uncontrollably. Expanding knowledge of these molecular disease mechanisms has been made clinically valuable with the advance of targeted therapy and companion diagnostics, whereby the presence of a certain DNA variant would render the tumor susceptible (sensitive) to a specific drug. Recently, NGS has become generally accepted as the most efficient test methodology for cancer genomes, especially with the realization that molecular alterations not only initiate, but also drive tumor growth, metastasis, and eventually, resistance to therapy.
Clinical Application of NGS
Implementing NGS tumor testing in a clinical genomic laboratory is advantageous not only because of the significant advancements made in recent years to this sequencing technology, but also the range of treatment options it enables, particularly when standard chemotherapy proves ineffective. NGS applications in the clinical setting range from sequencing entire tumor genomes to targeted diagnostic gene panels, which can be applied to both solid tumors and hematologic malignancies using formalin-fixed paraffin-embedded (FFPE) tissue or bone marrow specimens, respectively. The number of genes included in panels can differ among testing laboratories and panel-content decisions usually relate to the establishment of clinical utility, an understanding of the ensuing economic burden on the patient and the payor, and ultimately, the read capacity of the NGS platform validated for tumor testing.
Choosing among the commercially available NGS platforms to validate to a laboratory’s specific operations can be a challenge in terms of technological sophistication and cost. Although there are several platforms to choose from, the decision should be directly related to the clinical laboratory’s workflow and structure, its clinical demand, and the availability of a bioinformatics support system to enable the analysis and interpretation of the massive sequencing data generated from each run.
At Legacy Health, an Oregon-based, seven-hospital system (including a children’s hospital) serving the Portland metropolitan region and its surroundings, we have implemented tumor NGS testing which utilizes a panel of 12 actionable genes, targeting the five most common and actionable adult cancers in the US: breast, ovarian, colon, lung, and melanoma. This technology has given us the ability to batch samples from multiple indications, helping to optimize the cost of the sequencing run. This panel was designed to detect single nucleotide variants and small insertions and deletions, covering approximately 770 well-vetted hotspots in 12 actionable genes. Our choice of NGS platform was attributable to several key components: The entire system would be provided by a single vendor, allowing continuity and standardization throughout the operation, especially in relation to tracking reagent lots and software updates, our familiarity with the vendor’s DNA manipulation expertise, and the availability of an integrated bioinformatics pipeline for the interpretation of the data.
As our understanding of cancer genetics evolves and new variants are discovered, it is important to be able to access a database that contains comprehensive and up-to-date interpretations of variant significance. For example, in 2016, The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) together published an evidence-based guideline which establishes recommendations for EGFR and ALK testing, helping to guide targeted therapies. At Legacy Health, we want to ensure the use of a database that encompasses the latest guideline information, affording patients best and proven care practice. It is important that the database provides access to a continuously updated library of information for appropriate variant interpretation.
Plan Ahead for Implementation
In any clinical laboratory, the establishment of NGS testing should be preceded by a planning algorithm whereby the laboratory determines what specimen type(s) will be tested and what diagnostic/prognostic information will be evaluated and reported. The laboratory can either choose a small set of core genes for which extensive information is available regarding targeted therapy and clinical trials, or opt for a larger number of genes or target sequences where evidence regarding pathogenicity is still growing. Both approaches offer benefits, but following extensive discussions with our system pathologists and oncologists, our decision to use a small panel of actionable genes deemed necessary and sufficient has allowed for more direct action in clinical management, and therefore a wider acceptance of the utility of our NGS testing.
Validation of any NGS workflow can be extensive and it is imperative that the laboratory work directly with oncologists and surgeons, and participate in tumor boards to gauge clinical need, help in results interpretation, and establish appropriate referral patterns. NGS workflow is generally divided into several steps comprising sample preparation, library preparation, sequencing, and sequence analysis. Each step needs to be validated, and the validation components will include analysis of commercially available reference specimens, inter-laboratory comparison of sequencing results, precision and reproducibility, and limit of detection.
All laboratory programs seeking to implement NGS clinical testing should give strong consideration to the following five factors:
- Integrate the experience and expertise of molecular geneticists and pathologists into the interdisciplinary tumor board
- Establish a testing methodology that is compatible with validated specimen types
- Incorporate QC metrics into NGS workflow to help mitigate the cost of sequencing
- Develop a robust bioinformatics pipeline for variant interpretation
- Ensure compliance with laboratory accreditation agencies (eg, CAP, CLIA)
Incorporating the above steps should substantially enable NGS and a shift of emphasis in medicine from reaction to prevention. The promise of this technology includes improved diagnoses, preemption of disease progression, customized disease-prevention strategies, greater drug therapy efficacy, and avoidance of prescription drugs with predictable side effects. Last, an NGS approach will help eliminate trial-and-error inefficiencies that inflate health care costs.
Yassmine Akkari, PhD, FACMG, is the scientific director for cytogenetics, the technical director for molecular pathology, and manager of genetics laboratory operations at Legacy Laboratory Services in Portland, Oregon.
- The National Center for Biotechnology Information (NCBI): dbSNP Short Genetic Variations Database
- The Exome Aggregation Consortium: ExAC Browser
- The International Genome Sample Resource:
1000 Genomes Project
- The NHLBI GO Exome Sequencing Project:
Exome Varian Server
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