Demystifying DNA Variant Reporting

As the understanding of the pathogenesis of hematologic malignancies advances, molecular profiling by next-generation sequencing (NGS) is playing larger role in the diagnosis, prognosis, and treatment of cancers. But in some cases, the utility of the information gleaned from the technique is limited by clinicians’ and molecular pathologists’ ability to consistently analyze and interpret the results.

“One of the barriers to adopting this technology in practice is the amount of data that’s returned,” said Rafael Bejar MD, PhD, Associate Professor of Medicine at the University of California San Diego Health. “It’s not like getting a platelet count where you get a value and, if you are appropriately trained, you know how to interpret that. These genetic sequencing tests generate a lot of data – some useful, some not. Teasing out the useful information can be difficult.”

Another challenge is time: both the time required for the clinician and pathologists to keep up with gene-specific research findings, and the time to return test results once a sample is obtained from the patient. In some cases, NGS results can take more than 2 weeks to be returned to the ordering physician.

However, NGS is now commonplace and a routine aspect of patient diagnosis and management across hematology. ASH Clinical News spoke with Dr. Bejar and other experts about using NGS in the management of patients with hematologic malignancies and the challenges that need to be overcome in order to realize the full benefit of this technique.

The Promise of NGS

John Pfeifer, MD, PhD, Professor of Pathology and Immunology at the Washington University School of Medicine in St. Louis, says it was “a double win” for pathologists when NGS was popularized for clinical use about a decade ago. “You could sequence a lot of genes at the same time, which allowed you to get a definitive answer to your clinical question before you ran out of tissue,” he said. “Secondly, it enabled you to do that with a level of sensitivity and specificity that exceeded the technologies that were already there.”

The utility of NGS starts with confirming a patient’s diagnosis, Dr. Bejar said. Then, it can help physicians classify the disease. “If a patient has certain genetic markers, they fit into a different category than if they have other genetic markers,” he said.

Those categories can help physicians set expectations for the course of the disease. Some mutations are considered adverse, others favorable. The risk level of a patient’s mutations can help determine how aggressive their initial treatment strategy should be.

Patients whose sequences suggest they have higher-risk disease will get more intensive therapy, like hematopoietic cell transplantation, as soon as possible. Patients with lower-risk disease, on the other hand, may be treated more conservatively. “We may initially offer more supportive care or even observation for this group of patients,” Dr. Bejar explained.

In some cases, the mutations can predict which therapies the disease is most likely to respond to. For example, about one-quarter of patients with acute myeloid leukemia (AML) have an FLT3 mutation which leads to a more aggressive form of leukemia in which the body to produces more abnormal white cells.1 However, specific drugs such as FLT3 inhibitors can improve prognosis in these patients – but only if the treating clinician knows the patient has the mutation. In patients with chronic myeloid leukemia, an ABL kinase mutation makes the disease resistant to tyrosine kinase inhibitors (TKIs), and may allow the clinician to choose a specific drug from among the 5 TKIs currently approved for the disease.2

However, the use of NGS doesn’t end when treatment starts, Dr. Bejar said. “There is an emerging role for performing mutation testing later on after the diagnosis,” he noted. “For AML in particular, there’s a lot of interest in using mutation detection as a way of detecting residual disease in patients who have responded to therapy.”

“[Next-generation] sequencing tests generate a lot of data – some useful, some not. Teasing out the useful information can be difficult.”

—Rafael Bejar, MD, PhD

Currently, doctors consider patients to be in complete remission if a pathologist is unable to find any malignant cells in a small sample of the bone marrow. However, Dr. Bejar explained that, by using NGS, pathologists can sequence the blood of the bone marrow to look for mutations associated with residual disease. This would detect evidence of “measurable residual disease” that might be missed in the bone marrow samples.

Ensuring Reliability and Accuracy

Before NGS becomes a routine part of patient care, the results it generates need to be trustworthy and accurate. There are hundreds of laboratories and companies that conduct NGS, but, as Julie Eggington, MS, PhD, CEO of the Center of Genomic Interpretation, told ASH Clinical News, not all are created equal.

While Dr. Eggington said that any lab performing NGS must be Clinical Laboratory Improvement Amendments (CLIA)–accredited, “a CLIA accreditation is basically pointless for NGS tests.” The U.S. government legislated CLIA accreditation in 1988, long before NGS was even imagined.

CLIA accreditation establishes quality standards for laboratory testing performed on specimens from humans, such as blood, body fluid, and tissue. However, she noted, in the context of labs performing NGS, the certification mainly demonstrates that a lab isn’t using expired chemicals for tests, and not that interpretation of results is correct.

In April 2019, the Clinical Laboratory Improvement Advisory Committee, a group managed by the Centers for Disease Control and Prevention, convened a workgroup on NGS to consider recommendations for assuring the quality of NGS-based testing in clinical laboratory settings.3 The group outlined challenges in performing NGS test validations and quality control and assurance, but has not issued a set of NGS-specific standards for accreditation.

The College of American Pathologists (CAP) and the Association for Molecular Pathology (AMP) published a set of five worksheets that “translate regulatory specifications into concrete instructions that guide the user through the entire life cycle of an NGS test.”4 The guidance focuses on variant detection in the setting of inherited disease and help labs document NGS protocols, but Dr. Eggington said even these aren’t able to keep pace with the progress being made in NGS, especially in the context of malignancy.

“While choosing a CAP-accredited lab is better than choosing a lab that only has CLIA accreditation, it still doesn’t mean that the test is worthwhile,” she explained. “I can generate protocols and put them in binders and pass inspection to ensure that I can reproduce the results, but it doesn’t mean that my results or interpretations are accurate.”

Laboratories might try to overcorrect for lack of quality by putting as much information in the NGS test report as they can. “Some doctors really like that because just about every patient case they send in will come back with a recommendation,” said Dr. Eggington. “The problem is that the majority of the recommendations will be useless, containing false positives and dead leads. It is a waste of time for the patients and a waste of money for the health care system.”

Even if two separate laboratories both seem to be high-quality, they may report conflicting results. Dr. Pfeifer provided this example: Several years ago, his team sequenced a patient whose care team had requested a second opinion. Even though he considered the first lab that conducted the NGS to be a high-quality lab, his lab still found a mutation that the first one didn’t.

“The previous lab was running a very narrow hotspot-based test,” he explained. “Although we had a hotspot-based assay, our target area was a little bigger. So, the variant we found was in a part of the DNA that we tested and they didn’t.”

The two tests were virtually identical from a technical and an analytic point of view, Dr. Pfeifer continued, “it’s just that, because the tests were designed differently, they produced two different sets of information.”

Quantifying Quality

Laboratories may test for anywhere from one or two gene mutations in certain parts of the genome to hundreds of genes, depending on the clinical question that needs to be answered. “We require our clinical colleagues to tell us what they want us to do,” said Dr. Pfeifer. “At last count, there are about 8 billion base pairs in the human genome. That’s an awful lot. Somebody has to tell the sequencing laboratory what to look for.”

But, for clinicians, that answer isn’t always clear. Dr. Bejar said that pathologists can look for somatic mutations or mutations that happen over and over again. “Recurrence is a good marker of a disease-related mutation,” he said. Some mutations are well-known and understood, but “then there is a hazy area where you find a mutation in a gene that isn’t very common and you are uncertain about its relevance to this particular disease. We call those ‘variants of undetermined significance.’”

“The limiting factor is the amount of human effort, research, and time it takes to understand how genes are modified in the disease and learn about how they interact. It’s a huge undertaking.”

—John Pfeifer, MD, PhD

New markers are constantly being discovered and validated, so a working group made up of members of AMP, CAP, the American College of Medical Genetics and Genomics, and the American Society of Clinical Oncology devised a tier-based system to denote the quality of evidence behind each finding.5

Tier 1, “variants of strong clinical significance,” are the most reliable. These are mutations with FDA-approved therapeutics and published guidelines based on well-powered studies.

Tier 2, “variants of potential clinical significance,” are the next step down. These are mutations which have a body of evidence suggesting they are pathologic, but where that body of evidence may include primarily preclinical work or small clinical studies.

Things start to get fuzzier around tier 3, “variants of unknown clinical significance.” Mutations in this category are not frequently observed in the general community or specific subsets and the data for their involvement in the disease process is not yet convincing.

Lastly, tier 4, “benign or likely benign variants,” are those that tend to be found frequently throughout the general community or subpopulation and for which there is no evidence of an association with cancer. Not all investigators favor this tiered system.

Outstanding Questions

As researchers continue to make discoveries regarding the multitude of possible disease-related genes, others are working on building massive databases to help pathologists compare their testing results to up-to-date research. “Those databases are huge, they’re hard to create, and they need to be updated daily,” said Dr. Pfeifer.

There also is no single coordinated database effort. Investigators have established databases for inherited diseases, databases for cancer, specialty disease databases, public and private databases … the list goes on. “In fact, you get the best data if you query half a dozen databases, rather than just one,” Dr. Pfeifer added. “Then again, the problem is the databases will have a different annotation for the clinical relevance of the same variant.” An American Society of Hematology (ASH)–sponsored Somatic Mutations Working Group is helping evaluate which genes and mutations are most important for hematological malignancies.

Reporting the clinical significance is further complicated when pathologists must interpret combinations of genes – not just individual variants.

Lastly, as is often the case with new technologies, expense is an issue. Though the cost of NGS-based testing has come down in recent years, some say the price can still be prohibitive.

Dr. Bejar emphasized that, while he is sensitive to the expense, researchers get a lot of “bang for their buck” with NGS tests, based on the amount of information physicians receive. Dr. Pfeifer added that payers will need to start demanding quality and accuracy to drive down cost.

When it comes to making the most of NGS, though, Dr. Pfeifer said that “the limiting factor is the amount of human effort, the research, the time it takes to ask questions about the disease process, understand how genes are modified in the disease, and learn about how they interact. It’s a huge undertaking.” —By Emma Yasinski


  1. Antar AI, Otrock ZK, Jabbour E, et al. FLT3 inhibitors in acute myeloid leukemia: Ten frequently asked questions. Leukemia. 2020;34:682-696.
  2. Patel AB, O’Hare T, Deininger MW. Mechanisms of resistance to ABL kinase inhibition in CML and the development of next generation ABL kinase inhibitors. Hematol Oncol Clin North Am. 2017;31(4):589-612.
  3. Centers for Disease Control and Prevention. Clinical Laboratory Improvement Advisory Committee (CLIAC) Next Generation Sequencing (NGS) Workgroup Summary Report. Accessed November 11, 2020, from
  4. College of American Pathologists. Next Generation Sequencing (NGS) Worksheets. Accessed November 11, 2020, from
  5. Li M, Datto M, Duncavage E, et al. Clinical implementation of the standards and guidelines for the interpretation and reporting of sequence variants in cancer: a Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017;19(1):4-23.