Navigating Variants of Uncertain Significance in Cancer


Cancer is a complex disease with a variety of genetic origins.1 More and more patients with cancer-or those who are at higher risk of developing cancer-can receive NGS-based tests to determine if they have actionable genetic mutations. For example, this testing provides some patients, like the roughly 5% of people with cancer who have a point mutation in the KRAS gene, with results that enable better clinical management.2 However, nearly 20% of genetic tests identify one or more variants of uncertain significance (VUS), which are results that cannot be interpreted as benign or pathological based on current evidence.3,4 When this happens, patients and clinicians may be left wondering: What now?

In honor of National Cancer Research Month (NCRM), we will explore how VUS are identified, how clinicians can effectively respond to these test results, and technologies and methods that could help future assessment of VUS.

Identification Process

In oncology settings, VUS are germline or somatic changes in a gene’s DNA sequence, and clinicians need more evidence to determine whether or not these mutations are related to disease growth and progression.

In the American College of Medical Genetics and Genomics (ACMG) classification system, VUS are Class 3 mutations and can be subdivided into low-, medium-, or high-probability.5  ACMG considers factors such as population frequency, computational predictive algorithms, functional assays, conservation analysis, and disease-specific databases.

VUS lack evidence of clinical association at the time of reporting, and genetic testing can be multi-faceted. Therefore, ACMG recommends that the referring healthcare provider and the clinical laboratory work collaboratively in the testing process. Additional monitoring of the patient may be warranted, although further clinical action should be based solely on existing evidence.6

Powerful NGS technology enables multiple genes to be tested at once for applications like hereditary cancer screening or tumor genomic profiling. The large amount of genetic code analyzed by these panels, while a boon to discovery, yields more frequent VUS than traditional Sanger or single gene panels.

The prevalence of VUS will vary depending on the NGS panel used, cancer type, variant calling criteria, and available research, among other factors. However, a 2023 study of 1,057 patients who received testing for hereditary breast and ovarian cancer and Lynch syndrome found that 11.9% had at least one VUS result.

The Complexity of Reclassification

To understand the implications of a patient’s test result, healthcare providers should understand that VUS could fall into one of several categories:

  • A variant in a gene related to the cancer in question but with insufficient evidence of pathogenicity
  • A variant in an unrelated gene but with potential to be associated with the patient’s phenotype
  • A variant that lacks evidence of pathogenicity found incidentally in a disease-causing gene unrelated to the patient’s condition

In addition to the sheer amount of genetic data generated by NGS, building quality evidence for a gene’s pathogenicity takes time. So research often lags behind variant identification. If enough evidence becomes available, VUS may eventually be reclassified. And in the case of reclassification, most VUS are downgraded.

A 2018 study of 1.67 million hereditary cancer tests found that 7.7% of identified VUS were reclassified.7 Of those, 91.2% of reclassified VUS were eventually downgraded as “benign” or “likely benign”, while 8.7% were upgraded as “pathogenic” or “likely pathogenic”. This latter group, while smaller, justifies additional research into VUS.

Confirming the clinical significance (or insignificance) of a VUS requires combining multiple lines of evidence from available strategies, including:

  • Population-based frequency data
  • Family member evaluation
  • Functional studies
  • Computational predictive algorithms
  • Clinical correlation
  • Longitudinal data and cohort studies

Given the resources required to power this evaluation, VUS reclassification takes time.8 Plus, VUS suspected of pathogenicity may need additional confirmation as upgrading them could inform potentially irreversible care decisions.

The frequency of VUS in NGS testing poses a clinical challenge. Although not actionable at the time of testing, these results may enable changes to clinical management if reclassified with new strong evidence.

Communication is Crucial to VUS Response

Clinical institutions and laboratories that perform and report tests should aim for transparent communication of which criteria are used to identify VUS, and expectations for if and how potential reclassification is reported. Healthcare institutions may need to leverage proactive approaches to stay on top of VUS research and guidelines to ensure timely and appropriate care decisions.

Receiving a VUS test result can understandably leave a patient feeling uncertain and anxious during an already stressful experience. In a 2019 study, patients reported that receiving clear and consistent information from their healthcare providers about their test result helped manage uncertainty, while seeking information and reading their genetic testing report did not.9

Pre- and post-test genetic counseling guidelines are evolving to help healthcare providers educate their patients about VUS in the setting of large multigene NGS panel testing.10  Overall, next steps after testing depends on a multifactorial assessment of the patient’s personal and family history and disease course.

Future Directions for Research

The challenges with VUS reporting and interpretation will persist as long as research cannot keep up with rapid variant identification driven by NGS. Fortunately, new technologies and methods can help detangle the messy web of VUS in cancer and other genetic conditions.

CRISPR-Cas9 genome editing technology has emerged as a powerful tool for assessing the functional impact of VUS. 11 Initial studies have focused on developing CRISPR protocols for assessing variants in the BRCA1 and BRCA2 breast-cancer predisposing genes. In that regard, seqWell’s transposase-based library prep solutions can help with quality control and support the development of safer, more effective gene editing.12,13

VUS will continue to emerge in NGS. The power lies in how researchers and clinicians can respond to these uncertainties. Better computational tools, high-throughput functional assays, and more studies including diverse genetic populations can collectively bridge the gap between variant identification and classification so patients with cancer can feel as supported as possible throughout their care journey.

This blog is the second and final published in honor of NCRM. Click here to read the first blog titled “NGS in Cancer Research: Unlocking New Insights and Fueling Progress”.