MMR, MSI testing guideline nears finish line

Charna Albert

December 2021—No single assay can capture all cancer patients with DNA mismatch repair deficiency, and in determining a patient’s eligibility for immune checkpoint inhibitor therapy, assays for MMR deficiency, microsatellite instability, and tumor mutation burden should not be considered interchangeable, say the authors of a forthcoming CAP guideline on MMR and MSI testing.

The guideline expert panel considered three tests: immunohistochemistry for the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2; PCR-based MSI analysis; and next-generation-sequencing–based MSI analysis. When guideline proceedings began in early 2018, the FDA had not yet approved high TMB as a biomarker for immune checkpoint inhibitor therapy. Thus, the expert panel also considered whether high TMB can be used as a surrogate for MMR deficiency or high levels of MSI.

Expert and advisory panel members have approved a working final draft of the guideline, which at the end of November was being reviewed by an independent review panel established by the CAP. “We’re getting closer to the finish line,” Russell Broaddus, MD, PhD, professor in the Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, said in a CAP21 session on the guideline’s development. Once approved by the review panel, the guideline will be submitted to the Archives of Pathology & Laboratory Medicine.

“The main question we set out to answer with this guideline,” Dr. Broaddus said, “is ‘what clinical test best identifies defects in DNA mismatch repair?’” The clinical trial that accompanied the FDA’s 2017 approval of the checkpoint inhibitors across all cancer types did not distinguish sufficiently between MMR deficiency and high levels of MSI, he said. “And contrary to what many in the field may believe, MMR deficiency is not always synonymous with MSI-high. They may overlap in certain tumor types, primarily colorectal cancer.” And while it’s standard of care to screen colorectal and endometrial cancer patients for Lynch syndrome with MMR by IHC or PCR-based MSI analysis, MMR deficiency occurs in significant numbers of cancers outside the colon and endometrium.

In gathering evidence for the guideline, “we were relatively confident there would be sufficient literature for colorectal and endometrial cancer,” Dr. Broaddus said. But because they were less certain of the available evidence for other cancer types, they examined the diagnostic characteristics of the tests when predicting germline Lynch mutations, “recognizing this may be an imperfect surrogate for immune checkpoint inhibitors.”

The guideline expert panel developed the following draft guideline statements on MMR by IHC, MSI by PCR, and MSI by NGS:

1. In colorectal cancer patients being considered for immune checkpoint blockade therapy, pathologists should use MMR by IHC and/or MSI by PCR for detection of DNA MMR defects. Although MMR by IHC or MSI by PCR are preferred, a validated MSI by NGS assay also may be used (validated against MMR IHC or MSI by PCR and showing equivalency).

“This is a strong recommendation,” Dr. Broaddus said. “For colorectal cancer there was by far the most published evidence to evaluate compared to any other cancer type. Mismatch repair immunohistochemistry, PCR-based MSI analysis, and MSI by next-generation sequencing are nearly comparable methods in the detection of mismatch repair or MSI defects.”

2. In gastroesophageal and small bowel cancer patients being considered for immune checkpoint blockade therapy, pathologists should use MMR by IHC and/or MSI by PCR over MSI by NGS. This recommendation does not include esophageal squamous cell carcinoma.

“Again, this is a strong recommendation,” he said. Once sufficient evidence has accumulated, it’s likely that MSI by NGS will be shown to be comparable to MMR by IHC and PCR-based MSI for these two cancer types.

3. In endometrial cancer patients being considered for immune checkpoint blockade therapy, pathologists should use MMR by IHC over MSI by PCR or NGS to detect DNA MMR defects (strong recommendation).

4.  In patients with cancer types other than CRC, GEA, small bowel, and endometrial, pathologists should test for DNA MMR, though the optimal approach for detecting defects has not been established. “The expert panel found that only colorectal cancer, gastroesophageal adenocarcinoma, small bowel cancer, and endometrial cancer had sufficient evidence to formulate a specific recommendation. For all other cancer types, we recommend an assay be performed to detect MSI or mismatch repair defects, but there is no specific guidance as to which assay is best,” he said.

PCR- and NGS-based methods for determining MSI perform well for colorectal cancer and other GI cancers, possibly because many of these assays first were optimized for detection of MSI in colorectal cancer, Dr. Broaddus said. But PCR-based MSI approaches have significantly lower performance metrics in cancer types outside the GI tract. Evidence shows that when PCR-based or NGS-based methods are optimized for an individual cancer type, performance metrics improve. “This implies there may not be a universal PCR-based approach, which seems daunting for most clinical labs testing a wide variety of cancer types.” Thus, the expert panel concluded that MMR by IHC may be the preferred testing approach for cancer types outside the GI tract.

Detecting MSI in endometrial cancers may be more difficult than in colorectal cancers, Dr. Broaddus said. In an early study of eight families with known MLH1 or MSH2 mutations, microsatellite stable was seen in only 11 percent of colon cancers but in 23 percent of endometrial cancers (Kuismanen SA, et al. Am J Pathol. 2002;160[6]:1953–1958). Two other studies highlight another problem with MSI analysis in endometrial cancer. The chromatogram of a representative MSI-high colorectal cancer demonstrating loss of MMR proteins by IHC compared with the normal control shows that “for each microsatellite there are a lot more peaks in the tumor compared to the normal,” which makes detection of MSI in colon cancers typically straightforward. But in a representative endometrial cancer also demonstrating loss of MMR proteins by IHC, each microsatellite in the tumor has far fewer additional peaks in the tumor than in the normal tissue (Wang Y, et al. J Mol Diagn. 2017;19[1]:57–64; Wu X, et al. Mod Pathol. 2019;32[5]:650–658). “This minimal microsatellite shift is quite easy for an operator to miss,” he said. “By most standard PCR-based MSI assays, we may be missing subtle shifts in the microsatellites, thus classifying some tumors as MSI-low and microsatellite stable when they should be MSI-high.”

A study comparing MSI methods in prostate cancer “continues the theme from endometrial cancer,” Dr. Broaddus said, “that the traditional [PCR-based] five-marker panel for MSI detection that many clinical laboratories use may not be optimal for cancer types outside the GI tract.” In the study, Hempelmann, et al., identified 29 MMR-deficient and 62 MMR-intact prostate cancers by targeted sequencing of DNA MMR genes using Large-Panel NGS, MSIplus (an 18-marker NGS panel), and the traditional PCR-based five-marker panel. MSI-PCR was associated with the most false-negatives, while both NGS approaches were associated with better detection of true MSI-high cases (Hempelmann JA, et al. J Immunother Cancer. 2018;6[1]:29).

But a 2019 study, he said, “highlights a possible pitfall of the NGS-based approach for assessing microsatellite instability” (Trabucco SE, et al. J Mol Diagn. 2019;21[6]:1053–1066). Most NGS-based approaches employ a scoring system that includes an indeterminate range in which the MSI call is more uncertain. As tumor purity decreases, the MSI score decreases, and it becomes more difficult to distinguish MSI-intermediate from MSI-high. “Many of the cancer types we test by NGS-based approaches fall into the 20 to 40 percent tumor purity range,” he said of his own experience. In that range, mutations can be identified accurately by NGS, “but it becomes increasingly difficult to classify tumors as MSI-high or microsatellite stable, and thus we may get more tumors in the microsatellite-indeterminate range.”

The expert panel encountered methodological problems in the literature on NGS-based approaches to MSI detection, Dr. Broaddus said. In one study of 100 cancers, 73 of which were colorectal, the authors reported 98 percent concordance between MSI-NGS and MSI-PCR. But 15 of the 100 tumors had inconclusive results by MSI-NGS. “Thus, the true concordance is nowhere close to 98 percent.”

In another example, a study reported in JAMA Oncology summarizes “the seemingly impressive data for a different approach for detecting MSI-high” using NGS, Dr. Broaddus said, but the expert panel found, by “digging deeply into the methodology,” important information: In 313 of the 1,346 patients for whom the NGS assay was performed, MSI could not be accurately assessed, and for another 28 patients, MSI results were indeterminate. For 25 percent of the patients, then, there were no MSI results. “This was a common finding,” he said of several studies that reported results for MSI by NGS, and “a real limitation to using this approach broadly.”

Draft guideline statement No. 5 indicates that for all cancer patients being considered for immune checkpoint blockade therapy, tumor mutation burden should not be used as a surrogate for the detection of DNA MMR defects. “This is a strong recommendation,” Dr. Broaddus said.

A meta-analysis of the correlation between high TMB and objective response rate with immune checkpoint blockade therapy in 27 tumor types demonstrated broadly that the higher the TMB, the more likely the patient will respond to the therapy (Yarchoan M, et al. N Engl J Med. 2017;377[25]:2500–2501). “I have encountered numerous pathologists and oncologists who believe high tumor mutation burden is synonymous with microsatellite instability-high, and this may be because of the known findings that high levels of microsatellite instability are commonly associated with larger numbers of tumor mutations.”

But many studies show that high TMB is not always associated with MMR defects or MSI-high, he said, noting it appears to have some cancer type specificity. In CRC there is strong concordance between high TMB and MSI-high, but melanoma and non-small cell carcinoma—two cancer types with the highest TMB traditionally—rarely have high levels of MSI (Vanderwalde A, et al. Cancer Med. 2018;7[3]:746–756; Chalmers ZR, et al. Genome Med. 2017;9[1]:34). “So there is some disconnect in tumor mutation burden and MSI-high.”

The final draft guideline statement is as follows: For cancer patients being considered for immune checkpoint blockade therapy, if an MMR deficiency is identified, pathologists should recommend follow-up evaluation for Lynch syndrome. “This is a strong recommendation,” Dr. Broaddus said. “For colorectal and endometrial cancer patients, this communication is likely already occurring in the context of standard-of-care mismatch repair immunohistochemistry or MSI screening. However, many different cancer types have been reported in Lynch patients, and oncologists caring for these patients may not be thinking about the possibility of a hereditary cancer syndrome.”