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In cancer sequencing, a new lingua franca

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Karen Titus

February 2017—NGS has taken its NBS, or next big step: a newly published joint consensus guideline on how to interpret and report sequence variants in cancer (Li MM, et al. J Mol Diagn. 2017;19[1]:4–23). With these 20 pages of best practices for making next-generation sequencing a regular part of cancer diagnostics, the field is moving, essentially, from frontier town to gated community.

Dr. Neal Lindeman (left) with Eliezer Van Allen, MD, of the Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital. The consensus reached by the CAP, AMP, and ASCO is aimed at “any laboratory that’s involved in performing multiplex genetic analysis for cancer samples and cancer patients,” Dr. Lindeman says.

Dr. Neal Lindeman (left) with Eliezer Van Allen, MD, of the Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital. The consensus reached by the CAP, AMP, and ASCO is aimed at “any laboratory that’s involved in performing multiplex genetic analysis for cancer samples and cancer patients,” Dr. Lindeman says.

Broadly speaking, it’s part of the evolutionary process in diagnostic methods. “We’ve seen this in the molecular field—with BCR-ABL, for instance, a long time ago,” says Neal Lindeman, MD, a guideline author and associate professor of pathology, Harvard Medical School, and director of molecular diagnostics, Brigham and Women’s Hospital, Boston. For many years, he recalls, different laboratories used different housekeeping genes before a standardized approach emerged. This sort of creative license was long common in coagulation testing as well, he says. “And you see it still with some immunoassays in clinical chemistry.”

The time is ripe to develop concrete guidance for NGS, says lead author Marilyn Li, MD, who chaired the working group. (The guideline was put together by representatives from the Association for Molecular Pathology, the American Society of Clinical Oncology, and the CAP.) As NGS has become more widespread, and as each laboratory makes in-house adjustments, the landscape has come to resemble the city of Babel, post-Tower, with different labs using different words to describe what they’re doing. “That makes communication difficult not only among pathologists and oncologists, but even within the same group,” she says. “You need a common language.”

Just as critical, adds Dr. Lindeman, is that the language needs to be understood. “Standard nomenclature is very hard to read if you’re not an expert in the field,” says Dr. Lindeman, whose particular guideline assignment, so to speak, was overseeing the section on reporting. “My overriding principle, which is a little hard to argue with—although sometimes people do—is that the most important thing about a report is that it be easy to read and understand.”

“Sometimes in pathology, we communicate to each other,” he continues. “And we need to keep in mind that these reports are going out to our colleagues in medical and surgical oncology, and often cancer patients.” The audience isn’t looking for Shakespeare, in other words. Arthur Miller will work just fine.

Dr. Lindeman even offers a tongue-twister of his own to get the point across: The reports need to be “interpretable in the vernacular.”

“You can’t just put all of them (variants) in a report and let the physicians figure out the significance,” says Marilyn Li, MD.

The only guideline available prior to this was the American College of Medical Genetics and Genomics/AMP guideline for germline variant interpretation. As the authors began their work, they conducted two surveys, one to see how labs are currently interpreting results (67 responses) and one to see how they are reporting variants (44 responses).

Those results, as well as a review of the literature, various databases, and the existing germline document, made it clear: There was a real need for a somatic variant guideline. “Not only because we’re looking at different diseases, but also there are different focuses for each,” says Dr. Li, who is a professor of pathology and laboratory medicine, and a professor of pediatrics, Perelman School of Medicine, University of Pennsylvania.

The numbers can explode rapidly. Germline testing usually looks at one mutation, which might be heterozygous or homozygous. Cancer, of course, requires looking at multiple genes with multiple mutations. Add in multiple levels, and different types of variants—point mutations, copy number changes, structural changes that can lead to fusion genes, etc.—as well as two genomes (tumor and normal) and multiple tumor clones, and it should be evident why the working group thought a new guideline was in order.

If there was any lingering doubt, Dr. Li says it was banished from her mind when the guideline was presented at the Association for Molecular Pathology meeting last November, shortly before its publication. “It was on a Saturday. And early in the morning, at 7:00,” she recalls. The room, with a capacity of 600-plus people, was packed to overflowing.

The survey results gave the participants pause, she says, even if there were no real surprises, and launched animated discussions. “To me—and this was a no-brainer—if you find a relevant germline variant, you report that. But others say, ‘We’re doing cancer testing. Why should we worry about germline?’ So those were some of the issues we encountered, even while creating the guideline,” says Dr. Li, who is also director of cancer genomic diagnostics and vice chief, Division of Genomic Diagnostics, Children’s Hospital of Philadelphia.

Another notable finding, she says, was that some laboratories use a one percent cutoff for population databases, while other labs use much higher or lower percentages, with each generating a different set of variants. Laboratories also use different testing cutoffs, ranging from five to 10 percent, or sometimes even higher.

The reporting tiers resembled another variety show. Some laboratories opt for the simplified, two-tiered, “mutation or benign” approach, says Dr. Li. Other labs use three, four, or five. “It’s all over the place.” Some laboratories even provide subtiers.

Taking this all in, Dr. Lindeman says, “It seemed like every center had its own language, almost, in terms of what different variants mean in different contexts.”

Though all these differences point to the need for more standardization, Dr. Li is careful to note that the guideline does leave room for laboratory directors to use their own discretion and professional judgment. In somatic testing and for most tumors, for example, her laboratory’s variant allele frequency (mutation fraction) cutoff is five percent. But in cases where testing is done to follow residual disease, laboratories may want to set a lower cutoff rate, such as one percent or lower. For those that do that, she adds, “Then you have to validate your test accordingly. You have to sequence much deeper and may need additional strategies to eliminate false-positives and make sure you can rely on it to detect variants at that level.”

Once it knew the lay of the land, the group got to work.

The highlight of the guideline, for Dr. Li, is the tier-based categorization and reporting system. “We feel this will allow us to weigh all available evidence,” she says.

The guideline stratifies the variants into four levels based on clinical significance:

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