Next-gen sequencing finds further clinical utility in oncology

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Dr. Pritchard

“This was a really surprising finding,” he added. “We thought until recently that germline mutations in these genes were rare in prostate cancer. While that’s true in prostate cancer overall, in the small subset of men with metastatic disease a much higher percentage have a somatic mutation in mismatch repair genes. Already in the NCCN [National Comprehensive Cancer Network] guidelines there is a recommendation to consider germline genetic testing in any man with metastatic prostate cancer.”

Because of the clinical value of detecting tumors with defective DNA repair genes, clinical testing in Dr. Pritchard’s laboratory includes up-front tumor and germline paired sequencing with a 60-gene DNA repair-focused NGS panel that includes full exon and intron coverage, copy number and structural variant analysis (introns are critical at the DNA level to detect rearrangements), microsatellite instability by NGS, and loss of heterozygosity analysis.

For the next application of NGS, Dr. Pritchard described tumor sequencing in a Lynch syndrome workup. He talked first about sequencing after a negative germline test and then about sequencing as first-line screening.

If a colorectal tumor is positive either for microsatellite instability or by IHC and negative for BRAF V600E/MLH1 methylation, germline testing is done. If germline testing is negative, “we can do a tumor-based confirmation test” to exclude Lynch, Dr. Pritchard said.

It is this context that provides a new clinical utility for NGS, which can sometimes explain a positive screening test with a negative germline result. About two to three percent of colorectal cancer with a positive initial screen but a negative germline result have double somatic mutations in one of the four mismatch repair genes. This is about the same frequency as true Lynch syndrome in screen-positive CRC, a fact that is increasingly clinically important with universal tumor-based Lynch screening.

These screen-positive, germline-negative cases are sometimes called “Lynch like,” a term Dr. Pritchard considers confusing. “I prefer to call them double somatic,” he tells CAP TODAY. “That’s really a new concept driven by universal Lynch screening.” Double somatic mismatch repair genetic alterations explain up to 75 percent of so-called Lynch-like cases.

“At the University of Washington we do a lot of clinical testing in this setting, almost always after a negative germline test,” Dr. Pritchard says. If germline testing at an outside laboratory comes up negative, the specimen is sent to Dr. Pritchard’s laboratory for further workup, where paired tumor and germline sequencing is done with a panel they developed called ColoSeq Tumor. The test includes full sequencing of mismatch repair genes, including introns, to maximize the sensitivity for germline mutations and to improve detection of structural variation that is common in cancers.

To demonstrate the spectrum of results with samples that are positive on initial screening but negative on germline examination, Dr. Pritchard showed results from an NGS panel analysis of over 300 consecutive patients with Lynch-like syndrome referred to their laboratory for further workup. (Such patients make up only three percent of all screened CRC patients.) Double somatic mutations are found in about 75 percent. Missed Lynch syndrome is discovered in seven percent of cases. False IHC is uncovered in three percent of patients. In five percent the MLH1 mutation is found, while 10 percent are unexplained.
“The good news,” he tells CAP TODAY, “is that this testing works to reassure up to three-fourths of these patients that they don’t have Lynch syndrome but double somatic mutations that explain their positive screening results.”

Can tumor NGS be used to replace IHC and MSI-PCR as first-line screening in a Lynch workup? Dr. Pritchard asks. “If we could, it would simplify testing.” Traditional screening employs a complex algorithm, while NGS detects MSI, mismatch repair mutation status, as well as BRAF, KRAS, and NRAS mutations all in one test. If the test is positive, that provides the etiology of the tumor. If negative, there’s no need for further testing.

To explore this possibility, Dr. Pritchard and collaborators in genetics and pathology at Ohio State University are analyzing CRC cases by a tumor-only NGS panel interpreted by blinded expert review.

Use of NGS as first-line screening is “more conceptual” at this time, Dr. Pritchard says, adding that the data are currently preliminary. Tumor NGS may be able to replace traditional Lynch screening in the future, he said in an interview. “We are increasingly doing NGS on colorectal cancers to guide therapy and to qualify patients for targeted therapy. This is a brand-new area and we are excited about it.” He thinks it is going to be the future. “But first we need more data.”

Cost may already be favorable for NGS, he says, given the total cost of the many individual assays. Turnaround time needs to be addressed.

The third new area in which somatic NGS can provide clinical utility is in helping with variant classification in patients with germline variants of unknown significance (VUS).

“We are seeing increasing tumor testing to clarify Lynch-like cases as standard of care,” Dr. Pritchard said. The same tests are sometimes used in patients with germline VUS in mismatch repair genes that might explain IHC results. He is “extra cautious,” he says, in using tumor sequencing results to reclassify VUS findings: “We don’t yet use tumor sequencing data from a patient as a sole basis to reclassify a germline VUS from that same patient because there are not yet guidelines.”

Consider a patient with a germline VUS in MSH2, a Lynch syndrome gene, whose tumor has loss of MSH2 protein by IHC and microsatellite instability, which supports that the MSH2 variant may be causative. Evidence may be seen of second allele inactivation at the MSH2 locus, either loss of heterozygosity or a second hit. “All of these clues add a lot of information that this germline variant may be pathogenic,” Dr. Pritchard says. “Nonetheless, when we have this experience in practice, we have been very cautious how we handle it in clinical reporting.” Reports do sometimes say tumor findings make it more probable that this germline alteration is pathogenic while emphasizing that the VUS is not formally reclassified.

Tumor testing might help with a germline VUS that is considered highly probably pathogenic, or, conversely, one that is close to being benign. Still, Dr. Pritchard cautions, “There are many pitfalls here. Proceed carefully.” There could be a missed germline or somatic mutation or double somatic mutations in cis configuration.

Dr. Pritchard reported their experience in 40 patients who had a germline mismatch repair VUS. After tumor sequencing, four were reclassified to likely pathogenic and one to likely benign. Tumor sequencing provided only part of the basis for reclassification.

“Germline variant classification guidelines should incorporate somatic data,” he concluded. “Guideline committees are currently working on this.”

He shared a case to illustrate how somatic findings can inform germline variant classification: a woman in her 40s with CRC whose father was diagnosed with CRC in his 50s. She had a VUS germline variant—a very rare deep intronic variant in the MSH2 gene. That same MSH2 deep intronic variant was observed twice as a somatic mutation in colorectal cancer in other patients who had loss of MSH2 and MSH6 that could not be explained by any other mutation. Her final diagnosis was Lynch syndrome caused by an inherited pathogenic deep intronic mutation in MSH2 that results in the introduction of a cryptic exon and a premature frameshift.
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William Check is a writer in Ft. Lauderdale, Fla.