CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Laboratory tests that indicate the possibility of Lynch syndrome are MSH2, PMS2, and MSH6 IHC loss; MLH1 IHC loss that is not associated with MLH1 gene methylation; and MSI-high with concurrent absence of MLH1 gene methylation. “It is recommended that the pathologist communicate directly this finding with the patient’s oncologist, in addition to the written communication in the pathology report,” he said.
The clinical significance of true discordance is unclear, Dr. Broaddus said. For example, it is unknown if an endometrial cancer patient with MMR IHC loss and microsatellite stability and another endometrial cancer patient with MMR IHC loss and MSI-high would have comparable responses to immune checkpoint blockade therapy. “Certainly, some of the data present already in published studies could be mined retrospectively to get this information.”
In two studies the expert panel found that central review detected a substantial number of interpretive errors made initially with MMR IHC (Kim JH, et al. Cancer Res Treat. 2020;52[4]:1135–1144; Overman MJ, et al. Lancet Oncol. 2017;18[9]:1182–1191).
The second draft good practice statement says when indeterminate results are identified, an orthogonal assay should be performed or the same assay repeated using a different tumor block. It also advises developing a robust peer-review process for such cases.
“For mismatch repair immunohistochemistry,” Dr. Broaddus said, “I have often found that biopsies perform much better when the matching surgical specimen has unclear IHC results. This may be because the surgical specimen has been anoxic too long, wasn’t fixed optimally, or had some problem during the processing cycle.” He cited an example from his practice in which a colon cancer was identified initially as having loss of MLH1. The patient was young, so germline testing was performed, and no MLH1 mutation was identified. When asked to review it, he noted “the tumor was negative for MLH1, but also the adjacent stromal cells did not have convincingly nuclear positive expression of MLH1.” When MLH1 IHC was performed again using a different tumor block, the tumor was strongly positive for MLH1, “and this patient did not need germline testing for this gene.”
The third draft good practice statement addresses subclonal, or heterogeneous, IHC loss of MMR protein and says it’s uncertain whether these patients respond to immune checkpoint blockade.
“If a more definitive result is needed to place a patient in a clinical trial involving immune checkpoint blockade, it is recommended that a microdissected area of tumor that shows complete loss of mismatch repair protein be analyzed for microsatellite instability by the PCR-based approach, or an NGS-based approach if it’s a GI cancer.”
Dr. Broaddus calls it “distressingly common” for interpretive errors in MMR by IHC to be identified in a study’s central review. In one large study, up to one in five cases identified as MMR deficient by IHC at initial review were found to be MMR intact after central review. “And importantly, this is presumably after the patients were started on immune checkpoint blockade therapy.” Immune checkpoint blockade likely will be a pillar of cancer therapy for years to come, “so this is not going to be a problem that goes away.” A challenge for pathologists, then, is to provide more formal training in interpretation of MMR by IHC. “Ideally, this training should begin in residency and fellowship.”Another challenge: Immunohistochemistry, PCR-based MSI analysis, NGS-based MSI analysis, and tumor mutation burden as assessed by NGS are not interchangeable assays, he said. In colon cancers the results of each often but do not always overlap, and for cancers outside the GI tract, “this overlap is definitely less frequent.”
Numerous studies have examined the concordance or discordance between MMR by IHC and PCR-based MSI analysis, he noted. In one study of 591 colorectal and endometrial cancers (primarily colorectal) nearly 12 percent of cases identified as MSI-high had intact IHC expression of MMR proteins (Bartley AN, et al. Cancer Prev Res. 2012;5[2]:320–327). “Some of these cases, but certainly not all, ended up being patients with MLH1 germline mutations that resulted in expression of a nonfunctional MLH1 truncated protein.”
In a study of 938 endometrial cancer patients, 13 percent had MSI-high cancers with no evidence of loss of an MMR protein by IHC (Goodfellow PJ, et al. J Clin Oncol. 2015;33[36]:4301–4308). “Recall that because mutations in MSH6 are more common in Lynch-associated endometrial cancer, MSI-low is a more common finding than in colorectal cancer. In colorectal cancer, for the most part, we believe patients with MSI-low tumors can be treated the same as microsatellite stable patients—no evidence of Lynch syndrome and not eligible for immune checkpoint blockade therapy.” But in that study, for 19 of the MSI-low cases that retained MMR protein expression, “we do not have germline sequencing data because of insufficient funding. So who knows how many of the 19 actually had germline mutations in a Lynch gene?”
In a prospective study of 192 endometrial cancer patients, IHC and PCR-based MSI discordances were identified in about three percent of patients, he said. Five patients had microsatellite stable tumors and loss of MMR protein by IHC, and one had an MSI-high result with intact MMR protein by IHC. In three of the MSS cases, the source of discordance was likely heterogeneous expression of MLH1 and PMS2 by IHC (Bruegl AS, et al. Cancer Prev Res. 2017;10[2]:108–115).
Testing discordances have been seen in other cancer types also, but the evidence is weaker, he said. “The patient numbers are admittedly much smaller. This represents a problem but also an opportunity for pathologists to provide the published evidence moving forward.”
He cited a case of a young patient with Lynch syndrome who had a germline MSH2 mutation and colon cancer, endometrial cancer, and thyroid anaplastic carcinoma. All three tumor types showed loss of MSH2 by IHC. The colon cancer was MSI-high; the thyroid carcinoma was MSI-low. “There was insufficient endometrial tissue to perform MSI analysis. This again highlights that there can be discordance between immunohistochemistry and MSI results.”
The clinical significance of low-level microsatellite instability outside the GI tract is uncertain, Dr. Broaddus said. MSI-low CRCs typically are treated as sporadic microsatellite stable cancers, and “there is some evidence to support this approach.” But for endometrial cancer, MSI-low likely has a different meaning. “It is well established that endometrial cancers associated with MSH6 have a higher incidence of MSI-low.”
In one example, a 51-year-old patient with endometrial cancer had intact MMR by IHC and an MSI-low cancer. “Her family history of cancer was modest,” with no young relatives with Lynch-like tumors. Her oncologist suspected Lynch syndrome because the tumor was centered in the lower uterine segment, and the oncologist was aware of evidence demonstrating that a high percentage of lower-uterine-segment–based endometrial cancers are associated with Lynch syndrome. The patient “was indeed shown to have a deleterious germline MSH6 mutation,” an MMR defect that would have been missed had IHC been the only screening approach. “The presence of MSI-low by itself is unlikely to classify most cancer types as MMR deficient for the purposes of clinical trials of immune checkpoint inhibitors,” Dr. Broaddus said.
Another challenge (he prefers to call them opportunities) moving forward, then, is that the optimal assessment for identifying cancer patients most likely to respond to immune checkpoint blockade therapy may involve multiple modalities. “We have so far only considered these tests in isolation.” The best biomarker-of-treatment outcome, he said, might be a combination: high TMB as determined by NGS and MSI-high, or MSI-high and high numbers of tumor-infiltrating lymphocytes, “and there are numerous other combinations you could consider.”
In addition, these analyses have been considered previously as having only binary outputs. A tumor assessed by IHC, for instance, is either MMR deficient or intact. TMB is considered high or low, as is MSI, and PD-L1 is considered positive or negative. “Should we contemplate instead that these outputs be considered as continuous variables?” This is an emerging concept, he said, but published evidence indirectly supports it.
Other opportunities for pathologists: The relationship between NGS-based MSI-high and mismatch repair IHC in cancer types outside the GI tract must be better defined, Dr. Broaddus said. And determining the utility of a staged approach: assess PD-L1 IHC first, then MMR IHC, then MSI by NGS. “We would need to gather the evidence for individual cancer types.”
If one testing modality does not identify an MMR defect, he said, is there utility in performing additional tests? If NGS doesn’t detect MSI-high in a colorectal cancer, for example, is it worthwhile to perform another test, such as MMR IHC, to more definitively determine whether the tumor is microsatellite stable?
“Currently, we have no evidence to support or not support this type of approach.”
Charna Albert is CAP TODAY associate contributing editor.