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Cancer Genetics Overview (PDQ®)

Clinical Sequencing


Broad-scale genome sequencing approaches, including multigene panel testing, whole-exome sequencing (WES), and whole-genome sequencing (WGS), are rapidly being developed and incorporated into a spectrum of clinical oncologic settings, including cancer therapeutics and cancer risk assessment. Several institutions and companies offer tumor sequencing, and some are developing “precision medicine” programs that sequence tumor genomes to identify driver genetic alterations that are targetable for therapeutic benefit to patients.[1-3] Many of these tumor-based approaches use germline DNA sequences as a reference to discriminate between DNA changes only within the tumor and those that are potentially inherited. In the genetic counseling and cancer risk assessment –setting, the use of gene panel testing to evaluate inherited cancer risk is becoming more common and may become routine in the near future, with institutions and companies offering gene panel testing to detect alterations in a host of cancer risk–associated genes.

These advances in gene sequencing technologies also identify variants in genes related to the primary indication for ordering genetic sequence testing, along with findings not related to the disorder being tested. The latter genetic findings, termed incidental or secondary findings, are currently a source of significant clinical, ethical, legal, and counseling debate. This section was created to provide information about genomic sequencing technologies in the context of clinical sequencing and highlights additional areas of clinical uncertainty for which further research and approaches are needed.


DNA sequencing technologies have undergone rapid evolution, particularly since 2005 when massively parallel sequencing, or next-generation sequencing (NGS), was introduced.[4]

Automated Sanger sequencing is considered the first generation of sequencing technology.[5] Sanger cancer gene sequencing uses polymerase chain reaction (PCR) amplification of genetic regions of interest followed by sequencing of PCR products using fluorescently labeled terminators, capillary electrophoresis separation of products, and laser signal detection of nucleotide sequence.[6,7] While this is an accurate sequencing technology, the main limitations of Sanger sequencing include low throughput, a limited ability to sequence more than a few genes at a time, and the inability to detect structural rearrangements.[6]

NGS refers to high throughput DNA sequencing technologies that are capable of processing multiple DNA sequences in parallel.[7] Although platforms differ in template generation and sequence interrogation, the overall approach to NGS technologies involves shearing and immobilizing DNA template molecules onto a solid surface, which allows separation of molecules for simultaneous sequencing reactions (millions to billions) to be performed in a parallel fashion.[6,8] Thus, the major advantages of NGS technologies include the ability to sequence thousands of genes at one time, a lower cost, and the ability to detect multiple types of genomic alterations such as insertions, deletions, copy number alterations, and rearrangements.[6] Limitations include the possibility that specific gene regions may be missed, turnaround time can be lengthy (although it is decreasing), and informatics support to handle massive amounts of genetic data has lagged behind the sequencing capability. A well-recognized bottleneck to utilizing NGS data is the need for advanced computational infrastructure to preserve, process, and analyze the vast amount of genetic data. The magnitude of the variants obtained from NGS is exponential; bioinformatics approaches need to evaluate genetic variants for predicted functional consequence in disease biology. There is also a need for user-friendly bioinformatics pipelines to analyze and integrate genetic data to influence the scientific and medical community.[7,9]

The following terms need to be defined to understand the clinical application of NGS testing and implications of results reported.

  • Germline alteration: A gene change in a reproductive cell (egg or sperm) that becomes incorporated into the DNA of every cell in the body of the offspring. A germline mutation is passed on from parent to offspring. Also called hereditary mutation. (NCI Dictionary of Cancer Terms)
  • Somatic alteration: An alteration in DNA that occurs after conception. Somatic mutations can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. (NCI Dictionary of Cancer Terms)
  • Incidental findings: Genetic test results that are not related to the indication for ordering the sequencing but that may be of medical value or utility to the ordering physician, the patient, or his or her family. (Adapted from [10])
  • Actionable genetic alteration: The presence or absence of a genetic mutation in a tumor or the germline that can be used to inform clinical management. (Adapted from [1]).

Emerging Clinical Application

NGS has multiple potential clinical applications. In oncology, the two dominant applications are: 1) the assessment of somatic alterations in tumors to inform prognosis and/or targeted therapeutics; and 2) the assessment of the germline to identify cancer risk alleles.

Somatic testing

There are multiple approaches to tumor testing for somatic alterations. With targeted gene panel testing, a number of different genes can be assessed simultaneously. These targeted panels can differ substantially in the genes that are included, and they can be tailored to individual tumor types. Targeted gene panels limit the data to be analyzed and include only known genes, which makes the interpretation more straightforward than in whole exome or whole genome techniques. In addition, greater depth of coverage is possible with targeted panels, compared with WES or WGS. Depth of coverage refers to the number of times a nucleotide has been sequenced; a greater depth of coverage has fewer sequencing errors. Deep coverage also aids in differentiating sequencing errors from single nucleotide polymorphisms.

WES and WGS are far more extensive techniques and aim to uncover mutations in known genes and in genes not suspected a priori. The discovery of a mutation that is unexpected for a particular tumor type can lead to the use of a directed therapeutic, which could improve patient outcomes. WES generates sequence data of the coding regions of the genome (representing approximately 1% of the human genome), rather than the entire genome (WGS). Consequently, WES is less expensive than WGS.

Noncoding variants can be identified using WGS but cannot be identified using WES. The use of WGS is limited by cost and the vast bioinformatics needed for interpretation. Although the costs of sequencing have dropped precipitously, the analysis remains formidable.[11]

Although the goal of WES and WGS is to improve patient care by detecting actionable genetic mutations (mutations that can be targeted therapeutically), a number of issues warrant consideration. This testing may detect deleterious mutations, variants of uncertain significance, or no detectable abnormalities. In addition, deleterious mutations can be found in genes that are thought to be clearly related to tumorigenesis but can also be detected in genes with unclear relevance (particularly with WES and WGS approaches). Variants of uncertain significance have unclear implications as they may, or may not, disrupt the function of the protein. The definition of actionable can vary, but often this term is used when an aberration, if found, would lead to recommendations against certain treatments (such as mutations in ras) for which a clinical trial is available, or for which there is a known targeted drug. Although there are case reports of success with this approach, it is unlikely to be straightforward. Studies are ongoing.

Some commercial and single-institution assays test only the tumor. Clearly deleterious mutations found in important genes in the tumor can be somatic but could also be from the germline. In situations in which somatic analysis is paired with a germline analysis, it is known whether the alteration is inherited; when somatic analysis only is performed, it is not known. Reported allelic frequencies of the mutation in the somatic analysis can provide clues, but they are not always reported by laboratories and do not give a definitive answer. As many medical professionals do not have a deep understanding of genetics, guidance as to when to proceed to germline testing would be valuable.

Germline testing

The goal of germline testing is to identify mutations associated with an inherited risk of cancer and to guide cancer risk–management decisions. Also, germline testing can aid in some management decisions at the time of diagnosis (e.g., decisions about colectomy in Lynch syndrome–related colon cancer and contralateral mastectomy in BRCA1/2 mutation carriers). In addition, there are emerging data that germline status may help determine systemic therapy (e.g., the use of cisplatin or PARP inhibitors in BRCA1/2-related cancer).

To date, most germline genetic testing has been performed in a targeted manner, looking for the gene(s) associated with a clinical picture (e.g., BRCA1 and BRCA2 in hereditary breast and ovarian cancer; or the mismatch repair [MMR] genes in Lynch syndrome). However, multiple targeted gene panels now available commercially or within an institution contain different sets of genes. Some are targeted to all cancers, others to specific cancers (e.g., breast, colon, or prostate cancers). The genes on the panels include high penetrance genes related to the specific tumor (such as BRCA1/2 on a breast cancer panel); high penetrance genes related to a different type of cancer but with a more moderate risk for the tumor of reference (such as CDH1 or MSH6 on a breast cancer panel); and moderate penetrance genes for which clinical utility is uncertain (such as NBN on a breast cancer panel). Because multiple genes are included on these panels, it is anticipated that many, and perhaps most, individuals undergoing testing on using these panels will be found to have at least one variant of unknown significance. As it is not possible to do standard pretest counseling models for a panel of 20 genes, new counseling models are needed. Ethical issues of whether patients can opt out of specific results (such as TP53 or CDH1 in breast cancer) and how this would be done in clinical practice are unresolved.

WES for inherited cancer susceptibility is also commercially available. Incidental findings are likely and management of such findings is evolving.

Governance, Interpretation, and Institutional Oversight of Next-Generation Sequencing (NGS)

Several layers of complexity exist in managing NGS in the clinical setting. At the purely technical level, improvements in the sequencing technique have allowed for sequencing across the entire genome, not merely the exome. As the costs decrease, exomic and genomic sequencing of tumor and of normal tissue can be expected to become more routine.

With routine use of WGS, major challenges in interpretation emerge. Foremost is the matter of determining which sequence variations in known cancer predisposition genes are pathologic, which are harmless, and which variations require further evaluation as to their significance. This is not a new challenge. Various groups are developing processes for the interpretation and curation of a growing database of variants and their significance. For example, the International Society for Gastrointestinal Hereditary Tumors has developed such a process for the MMR genes in concert with the Human Variome Project and International Mismatch Repair Consortium.

This process, which has been put in place for the known MMR genes, may serve as a framework for the emerging challenge of interpreting the significance of sequence variations in genes of uncertain or unknown function in regulation of neoplastic progression or other diseases. Larger cancer predisposition panels have been developed by commercial laboratories, with their own process for interpretation. To the extent that increasingly larger panels include genes of unknown significance, governance of the interpretation process requires that academic institutions offering their own panels or using external proprietary panels develop a deliberative process for managing the quality assurance for test performance (including Clinical Laboratory Improvement Amendments [CLIA], where appropriate) and interpretation.

The American College of Medical Genetics and Genomics (ACMG) has issued the following guidelines for achieving accountability in interpreting and reporting incidental variants: (Adapted from [10])

  • Constitutional mutations should be reported by the laboratory to the ordering clinician, regardless of the indication for which the clinical sequencing was ordered and regardless of patient age. This includes the normal sample of a tumor-normal sequenced dyad.[10]
  • Only variants previously reported and a recognized cause of the disorder, or variants previously unreported but of the type expected to cause the disorder, should be reported.[10]
  • It is the responsibility of the ordering clinician to provide comprehensive pretest and posttest counseling and alert patients to the possibility that clinical sequencing may generate incidental findings requiring further evaluation.[10]
  • Clinicians should be familiar with basic attributes and limitations of clinical sequencing.[10]
  • Given the complexity of genomic information, the clinical geneticist should be consulted at the appropriate time, which may include ordering, interpreting, and communicating about genomic testing.[10]
  • ACMG, together with content experts and other professional organizations, should continue to refine and update such guidelines annually.[10]

These ACMG guidelines have come under debate when the complexities and implications of reporting incidental and secondary germline findings for clinical laboratories, oncologists, and patients are considered.[12] Specifically, concerns remain that the routine reporting of germline mutations in the context of tumor sequencing would require laboratories to conduct results review with germline and tumor genome expertise, which would be expected to increase costs, laboratory efforts, and turnaround time for results reporting. The nature of discussions between oncologists and patients would be altered to include the multiple facets involved with germline testing and potential results. Pre- and post-test discussions would also potentially require involvement of genetic counselors and geneticists, who are a limited resource in oncology practices. Recent expert comment stated that more data are needed about the benefits of return of incidental germline findings to cancer patients undergoing tumor sequencing, citing a need for recommendations by experts in the oncology and genetics communities.[12]

It is still very early in the development processes for oversight at the institutional level. As an example, at one high-volume cancer center, the following process has been used:

  1. When considering evaluation of tumor tissue for research purposes, patients are asked to consent to undergo tests of very large panels of somatic tissue that are paired with corresponding panels for constitutional tissue. The key research undertakings involve evaluating for differences in prognosis and treatment response according to mutation patterns in tumors.
  2. The consent process outlines the possibility that mutations suggesting underlying tumor susceptibility may be identified. Patients are asked whether they want to receive this information, should an actionable mutation be identified. Patients also receive expedited genetic counseling process (via counselors associated with research protocol). If a patient has chosen not to receive such actionable mutation information, findings will nevertheless be reported to the patient’s institutional primary care physician (PCP). Unresolved to date are the circumstances in which a patient may be approached again by his or her PCP regarding pertinent findings, notwithstanding a previous decline to receive such information.
  3. When potentially significant results are found, the specific test may be repeated in a CLIA lab.
  4. A committee was formed to review variants to determine whether they are considered pathogenic or otherwise actionable.
  5. A second committee was formed to provide oversight for the conduct of such protocols, in the interest of deliberately evolving the processes above in a manner consistent with the anticipated routine performance of such panels in cancer patients, balanced against the need for patient autonomy and appropriately detailed informed consent.

Informed Consent Issues Arising From the Application of NGS

Informed consent for the sequencing of highly penetrant disease genes has been conducted since the mid 1990s in the contexts of known or suspected inherited diseases within selected families. However, the best methods and approaches for educating and counseling individuals about the potential benefits, limitations, and harms of genetic testing to facilitate informed decisions have not been fully elucidated or adequately tested. New informed consent challenges arise as NGS technologies are applied in clinical and research settings. Challenges to facilitating informed consent include the following:

  • Providing a person-centered appreciation for the breadth and diversity of medical risks potentially identified through genomic sequencing. Establishing informed consent may be particularly challenging in medically underserved populations with less familiarity with the concept of disease risk and in individuals with no previous knowledge, experience, or context of the disease(s) for which they are identified to be at increased risk.
  • Sequencing for diseases without clear management algorithms or identified best practices.
  • Interpretation of variants of possible but unclear pathogenicity.
  • Communicating genetic information that may be of relevance for other family members.
  • Additional challenges are anticipated as health care providers not trained in genetic/genomic medicine order and receive results on behalf of their patients with the expectation to return and manage medically actionable results.


Advances in genetic sequencing technologies have dramatically reduced the cost of sequencing an individual's full genome or exome. WGS and WES are increasingly being employed in the clinical setting in testing for both somatic and germline mutations. In addition, multiple-gene panel tests are now available commercially or within an institution. Considerable debate surrounds the clinical, ethical, legal, and counseling aspects associated with NGS and gene panels. Future research is warranted to address these issues.


  1. Dancey JE, Bedard PL, Onetto N, et al.: The genetic basis for cancer treatment decisions. Cell 148 (3): 409-20, 2012. [PUBMED Abstract]
  2. Meric-Bernstam F, Farhangfar C, Mendelsohn J, et al.: Building a personalized medicine infrastructure at a major cancer center. J Clin Oncol 31 (15): 1849-57, 2013. [PUBMED Abstract]
  3. Sleijfer S, Bogaerts J, Siu LL: Designing transformative clinical trials in the cancer genome era. J Clin Oncol 31 (15): 1834-41, 2013. [PUBMED Abstract]
  4. National Human Genome Research Institute: DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP). 2014. Available online. Last accessed November 24, 2014.
  5. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74 (12): 5463-7, 1977. [PUBMED Abstract]
  6. MacConaill LE: Existing and emerging technologies for tumor genomic profiling. J Clin Oncol 31 (15): 1815-24, 2013. [PUBMED Abstract]
  7. Rizzo JM, Buck MJ: Key principles and clinical applications of "next-generation" DNA sequencing. Cancer Prev Res (Phila) 5 (7): 887-900, 2012. [PUBMED Abstract]
  8. Linnarsson S: Recent advances in DNA sequencing methods - general principles of sample preparation. Exp Cell Res 316 (8): 1339-43, 2010. [PUBMED Abstract]
  9. Fernald GH, Capriotti E, Daneshjou R, et al.: Bioinformatics challenges for personalized medicine. Bioinformatics 27 (13): 1741-8, 2011. [PUBMED Abstract]
  10. Green RC, Berg JS, Grody WW, et al.: ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. Genet Med 15 (7): 565-74, 2013. [PUBMED Abstract]
  11. Mardis ER: The $1,000 genome, the $100,000 analysis? Genome Med 2 (11): 84, 2010. [PUBMED Abstract]
  12. Parsons DW, Roy A, Plon SE, et al.: Clinical tumor sequencing: an incidental casualty of the American College of Medical Genetics and Genomics recommendations for reporting of incidental findings. J Clin Oncol 32 (21): 2203-5, 2014. [PUBMED Abstract]
  • Updated: March 31, 2015