A Working Group of the European Society for Medical Oncology has determined that a decision tree on the sequential use of different tests in immunotherapy decision-making cannot be a general one for all cancers but should be designed on the basis of the specific tumor type.
Aldo Scarpa, MD, PhD
Since the first tumor-agnostic immunotherapy was approved in 2017, questions about optimal procedures for biomarker testing have become more pressing. A Working Group of the European Society for Medical Oncology (ESMO) has determined that a decision tree on the sequential use of different tests in immunotherapy decision-making cannot be a general one for all cancers but should be designed on the basis of the specific tumor type.
Through a new published recommendation, the ESMO Working Group has set a standard for the definition of microsatellite instability (MSI)/mismatch repair deficient (dMMR), tumor mutational burden (TMB), and PD-L1 expression, as well as the relationships among these tests.1
MSI has been an FDA-indicated biomarker for immunotherapy since 2017, when the PD-1 inhibitor pembrolizumab (Keytruda) was approved for patients with solid tumors found to be dMMR or MSI high (MSI-H).2 Other approvals since then, such as for the combination of ipilimumab (Yervoy) and nivolumab (Opdivo) in patients with MSI-H/dMMR metastatic colorectal cancer,3 have established the relevance of cancer therapies directed at this biomarker specifically rather than tumor histology alone.
“MSI is a diagnostic test for screening for the broad spectrum of Lynch syndrome[—associated] cancers, but it is also a prognostic test for gastrointestinal cancers and a predicative marker for immunotherapy,” Aldo Scarpa, MD, PhD, director of the Applied Research on Cancer–Network Research Centre for Applied Research on Cancer and chair of the Department of Pathology and Diagnostics at the University and Hospital Trust of Verona, Italy, said during a presentation of the working group’s data at the ESMO Immuno-Oncology Congress 2019, held December 11 to 14, 2019, in Geneva, Switzerland.4
Tumors that are dMMR accumulate mutations in regions of DNA called microsatellites, which are prone to mismatch errors; the result is the condition known as MSI. A hallmark of Lynch syndrome, MSI can occur in a variety of cancers, including colorectal, endometrial, and gastric.
To promote the use of clear and consistent terminology in future research, the collaborative group established standardized definitions for MSI-related terms from a systematic review of 18 articles related to MSI. These definitions are outlined in Table 1.1,4
Testing for MSI
Table 1. Definitions of MSI and MSI-Related Terms1,4 (Click to Enlarge)
The MMR proteins form heterodimers consisting of MLH1 and PMS2 or MSH2 and MSH6. MLH1 and MSH2 are considered the “obligatory partners” in these protein pairs; alterations in them interfere with dimerization and result in proteolytic degradation of both proteins in the heterodimer. However, the same is not necessarily true of the “secondary partners,” PMS2 and MSH6, because other proteins may substitute for them in the heterodimer. Therefore, functional heterodimers may be present despite PMS2 or MSH6 mutations.
For the spectrum of cancers associated with Lynch syndrome, immunohistochemistry (IHC) of all 4 MMR proteins is the first line of testing. Subsequently, MSI—polymerase chain reaction (PCR) molecular testing of specific microsatellite loci is indicated for cases in which IHC results are indeterminate, such as the loss of only 1 heterodimer subunit.
“When you have an alteration of a secondary partner, you may have an immunohistochemistry [result that] is not informative because you do not have the loss of both partners,” Scarpa said. “This is because PMS2 may be substituted for PMS1 or MLH3, and MSH6 may be substituted for MSH3. Alterations of secondary proteins may be compensated by other partners, and so the machinery is working.”
Normal cells with increased turnover—such as mucosal cells, lymphocytes, and stromal cells—must serve as an internal positive control for IHC testing. Determinant IHC findings include positive results, in which tumor and control cells exhibit nuclear staining of comparable intensity, and negative results, in which control cells have nuclear staining but tumor cells do not. However, cases with negative staining of controls or weaker staining of tumor cells compared with controls require verification by MSI-PCR, which has the highest specificity and sensitivity.
For cancers not associated with Lynch syndrome, the consensus group touted the advantages of next-generation sequencing (NGS), which can couple analyses of MSI and TMB3 (Table 21). In a study analyzing sequence data from 5930 cancer samples, investigators found MSI-H tumors in 14 of the 18 cancer types studied, with incidences ranging from about 30% for endometrial cancer to <1% for thyroid carcinoma.5 In a similar study, MSI was detected in 27 of 39 cancer types analyzed, mostly at low proportions; only 4 types had an MSI-H frequency >5%.6
“We cannot imagine [developing] these tests to analyze all these cases unless we use NGS,” Scarpa said. “In [cancers of] other organs outside Lynch syndrome, we do not understand how to work with immunohistochemistry [to determine] the real partners of MMR. [With NGS], you can analyze several dozen genes to get information about mutations, loss, amplifications, and copy-number alterations. Moreover, you have the mutational spectrum, which [refers to] the types of combinations of mutations that may happen through different mechanisms.”
Relating MSI to TMB and PD-L1 Expression
From the 18 articles selected for systematic review, the collaborative group attempted to identify associations with immunotherapy response among 3 biomarkers: high TMB, PD-L1 expression on tumor cells, and MSI. In total, 4186 patients were positive for at least 1 of the biomarkers, with the simultaneous presence of all 3 in 2.9% of cases.3 Significantly higher (P <.05) rates of positivity for all 3 biomarkers were observed in patients with colorectal and esophagogastric adenocarcinomas, at 12.8% and 14.6%, respectively.3
Notably, the majority of patients with colorectal cancer who had MSI also had high TMB, and vice versa. Positivity for both biomarkers (but not PD-L1) accounted for 31.4% of patients, indicating a high concordance between the 2 indices. Patients with MSI alone or high TMB alone accounted for just 1.7% and 8.7%, respectively, of patients with colorectal tumors. Patients with PD-L1 expression alone made up 43.6% of the population.3
Table 2. Recommendations for MSI Testing Methods in Different Cancer Types1 (Click to Enlarge)
In endometrial cancer, high TMB was almost always accompanied by MSI; however, the inverse correlation was not observed. Among patients with this cancer, 25.8% expressed both biomarkers (but not PD-L1), 19.9% had MSI only, and 4.9% had high TMB only. In 12.8% of patients, the tumors expressed all 3 biomarkers.
MSI was absent in melanoma cases and present in only a small number of cases of non—small cell lung cancers.
In all cancer types studied, PD-L1 expression was not found to necessarily co-occur with other biomarkers, and it was the most frequently occurring biomarker in most tumor types.
The consensus group concluded that clinicians should perform first-line MSI testing using IHC for all 4 MMR-associated proteins for the cancer types frequently associated with Lynch syndrome, which include endometrial, intestinal, gastric, and ovarian cancers, as well as esophageal adenocarcinoma and glioblastoma. In cases of indeterminate IHC results, second-line testing should be performed using a molecularly based assay, with MSI-PCR as the preferred method. For rare cancer types and those not associated with Lynch syndrome, MSI testing should be performed using NGS. The group also noted that although correlations with TMB and PD-L1 may be useful for future prognostication, MSI-H tumors do not need to express either of these biomarkers to necessitate use of immunotherapy.
Ongoing research advancing the role of MSI as a biomarker for immunotherapy responses and clarifying its relationship with other biomarkers will continue to elucidate the role of MSI in the treatment of solid tumors.
Investigators observed similar concordance in patients with esophagogastric adenocarcinomas, with 13.1% of tumors exhibiting both high TMB and MSI (but not PD-L1). High TMB or MSI alone was observed in 3.7% and 5.1% of patients, respectively. In total, 14.6% of patients in this population expressed all 3 biomarkers simultaneously.
To better understand the rationale behind testing methodologies, the collaborative group examined the mechanisms underlying DNA mismatch repair (MMR) across different solid tumors.