Biomarker Basics: A Look at MSI's Growing Role

Antonia Sepulveda, MD, PhD

The FDA’s recent approval of 2 immunotherapies for the treatment of certain patients with tumors that exhibit microsatellite instability (MSI) has propelled the emerging biomarker into clinical practice, prompting calls for broader genetic testing. The body of research into the optimal use of MSI testing is growing, and clinicians will no doubt encounter evolving data about its clinical utility.

In May, the FDA granted accelerated approval for pembrolizumab (Keytruda) for the treatment of adult and pediatric patients with unresectable or metastatic MSI-H or deficient mismatch repair (dMMR) refractory solid tumors for whom there are no alternative treatment options, and for patients with MSI-H or dMMR colorectal cancer (CRC) who are resistant to fluoropyrimidine, oxaliplatin, and irinotecan. In August, nivolumab (Opdivo) gained an accelerated approval for adult and pediatric patients with MSI-H or dMMR metastatic CRC (mCRC) that has progressed after standard chemotherapy.

Microsatellites, or tandem repeats of short DNA sequences, are abundant throughout the genome.¹ Tumors can develop along the MSI pathway, which is driven by a defective MMR mechanism, explained Zsofia K. Stadler, MD, a medical oncologist at Memorial Sloan Kettering Cancer Center (MSK) in New York City. “Their DNA is mismatched but it is not fixed,” she said in an interview with OncologyLive^®. “Therefore, throughout the entire tumor genome, you get the introduction of small point mutations within the tumors; these are called microsatellites. These essentially become unstable in these tumors, which is MSI.”

Although MSI testing has become an established part of the workup for patients with CRC, researchers are increasingly finding that MSI plays a role in other tumor types. “MSI may occur in a broad spectrum of tumors,” said Antonia R. Sepulveda, MD, PhD, a professor of pathology and cell biology and vice chair for translational research at Columbia University in New York City, in an interview. She noted that, in patients with Lynch syndrome, MSI may be a factor in endometrial, ovarian, skin, brain, and upper gastrointestinal tumors.

“Then there are sporadic types of MSI tumors. In every tumor type, they’re going to be of much lower frequency,” she said. “For example, we’ll find MSI tumors in prostate cancers and bladder cancers, but very infrequently in other cancers, like sarcomas. Nonetheless, especially for patients who might have had other risk factors, like previous therapies and/or enhanced exposure to carcinogens, we’re still not clear about what [the MSI rate] might be. Occasionally in lower frequencies, we might find MSI across the board in most tumors.”

In sporadic cases, MSI is caused by inactivation of MMR genes (eg, MLH1, MSH2, MSH3, MSH6, and PMS2) through somatic mutations, with increased cancer risk in individuals with inherited germline mutations (ie, Lynch syndrome).² Hypermethylation of the MLH1 promoter, epigenetic inactivation of MSH2, or downregulation of MMR genes by microRNAs can also cause MSI.^3-5 When MSI occurs within coding regions, the reading frame can be altered, resulting in functionally impaired proteins.⁶

Typical tumors may have approximately 100 mutations in the exome; however, MSI-positive CRCs, for example, characteristically have thousands of mutations in the coding regions of tumor cells.⁷ MSI-positive tumors also have higher numbers of tumor infiltrating lymphocytes (TILs), such as cytotoxic T cells, which is thought to be a result of increased neoantigen production induced by the high mutational load. These neoantigens are recognized as foreign, driving an inflammatory response.⁸

Measurement and Classification of MSI

As a compensatory response to this inflammation, MSI-positive tumors upregulate molecules such as the immune checkpoint protein PD-L1, which allows cells to evade the immune system. A phase I study in treatment-refractory MSI-positive tumors reported responses to a PD-1 blockade with pembrolizumab, demonstrating that reversing this inhibitory pathway leads to durable immune-mediated tumor control by reactivating TILs.⁹MSI can be classified as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). Using MSI testing, tumors are identified as MSI-H when 30% or more of the repeats are unstable, as MSI-L if fewer than 30% of the repeats are unstable, and as MSS if no repeats are unstable.¹⁰

MSI and/or immunohistochemistry (IHC) testing facilitates targeting the gene sequencing of MMR genes and screening out individuals unlikely to have Lynch syndrome.11 MSI testing detects an abnormal number of microsatellite repeats, indicating that cancer likely arose from cells with defective MMR genes. IHC testing detects the presence or absence of protein products of the MMR genes, of which a missing protein suggests a mutation in the gene that codes for that protein.

Polymerase chain reaction—based MSI testing amplifies DNA at several microsatellite sites from a tumor tissue sample and must be compared with normal DNA from each patient.¹² MSI testing can profile the Bethesda markers (often 2 mononucleotide and 3 dinucleotide microsatellite loci) or 5 mononucleotide markers as recommended by the National Cancer Institute/International Collaborative Group on Hereditary Non-polyposis Colorectal Cancer (NCI/ICG/HNPCC), but this method is time consuming and expensive.^13,14

A recent study evaluated the use of a single mononucleotide marker, CAT25, and reported high specificity and sensitivity for identifying tumors with MMR deficiency; however, this method would need to be evaluated in various ethnic populations to ensure consistent results.14 MSI testing has 80% to 91% sensitivity among tumors with MLH1 or MSH2 mutations and 55% to 77% sensitivity among MSH6 or PMS2 mutations.¹¹ Based on the composition of the MSI panel, specificity may reach 90%.

IHC is a complementary testing strategy, wherein most MSI-H tumors demonstrate a loss of protein expression for at least 1 MMR gene. Loss of protein expression within tumors is helpful for identifying the corresponding genes to target for mutation analysis.¹⁵ IHC testing has a sensitivity of 83%, regardless of the MMR gene involved, with a specificity of 89%.¹¹ It is convenient, inexpensive, and may detect loss of MSH6 protein that can be missed via MSI testing.¹⁶ IHC staining patterns may vary, however, leading to uncertainty in the interpretation of results.

MSI and IHC are often used synergistically to detect cases that may be missed by either test alone; however, they can be used separately based on individual clinical barriers such as cost and specimen availability.¹⁷ A recent study demonstrated the equivalence of MSI testing and MMR IHC in a cohort of patients with endometrial carcinomas, supporting previously reported concordance rates of over 92%.^16,18,19

A recent NRG Oncology and Gynecologic Oncology Group Study evaluated the combination of MSI testing, MLH1 methylation analysis, and IHC in patients with endometrial cancer, identifying patients with Lynch syndrome who would not have been considered for mutation testing if only IHC and MSI were used.²⁰

MSI Detection in Various Cancer Types

MSI testing is usually ordered at the time of CRC diagnosis to identify patients with Lynch syndrome, to determine prognosis, or to direct adjuvant chemotherapy.²¹ When asked whether providers should initiate MSI testing at diagnosis, Luis A. Diaz, MD, head of the Division of Solid Tumor Oncology at MSK, said, “That remains to be seen, but I think, right now, anyone with metastatic disease who has progression on standard-of-care therapy should have their MSI-H status checked because it can have a profound impact.”MSI occurs in approximately 15% of sporadic CRCs in the United States.¹³ It has also been reported in glioblastomas, lymphomas, and tumors in the stomach, ovaries, endometrium, and urinary tract.²² MSI-prone tumor types include colon adenocarcinoma, esophageal carcinoma, rectal adenocarcinoma, stomach adenocarcinoma, and uterine corpus endometrial carcinoma; these types contained the majority of MSI events in a recent molecular analysis study.¹

Another recent study evaluated MSI presence and the number of unstable loci in 18 tumor types.²³ MSI-positive tumors were identified in 14 of these cancer types, with the highest proportion of MSI-H cases observed in endometrial, gastric, rectal, and colon cancers (Figure).

Prognostic Importance of MSI

According to Diaz, the incidence of MSI-H expression in other tumor types is “hard to pinpoint but if we look across all metastatic tumors, we think it’s about 4%. In the United States, that’s around 30,000 patients. If we look worldwide, it’s probably closer to 500,000 patients. If we move into the earlier stages, the numbers go up.A systematic review of 31 studies and 12,782 patients with MSI tumors showed favorable prognoses in terms of overall survival (OS) and disease-free survival, which was dependent on stage.²⁴

MSI-H was associated with worse outcomes and tumors with more aggressive pathological features in stage III versus stage I/II CRC.²⁵ These findings supported previous research reporting reduced progression-free survival (PFS) and OS in patients with advanced dMMR CRC.²⁶ The presence of MSI in CRC has also been demonstrated to be predictive of nonresponse to 5-fluorouracil— based adjuvant chemotherapy for early-stage disease.²⁷ Furthermore, emerging data suggest that MMR status may be predictive in patients with advanced disease being considered for anti—PD-1/ PD-L1 immune checkpoint inhibitor therapy.^9,28,29

MSI as Immune Checkpoint Biomarker

The prognostic value of MSI levels in other malignancies has not been established clearly. In a study of gastric cancer, 11% of patients undergoing surgery exhibited MSI-H tumors, and these patients demonstrated significantly longer OS compared with MSS patients.³⁰ However, another study revealed no clear effects of MSI status on OS in patients with gastric cancer.³¹ A study of patients with invasive upper urinary tract transitional cell carcinomas reported an MSI-H rate of 20%, with a better prognosis reported for these patients with MSI-H tumors.³² MSI-H was found to be a marker of poor prognosis in patients with early-stage endometrial endometrioid adenocarcinomas.³³Recent phase II studies evaluating the PD-1 inhibitor pembrolizumab in patients with dMMR mCRC, MMR-proficient mCRC, and other dMMR-cancers reported an ORR and PFS of 40% and 78%, respectively, for dMMR CRC, and 0% and 11% for MMR-proficient mCRC.^9,34-36 Both median PFS and OS were significantly longer in dMMR CRC compared with MMR-proficient mCRC. Furthermore, significantly more somatic mutations were found in dMMR versus MMR-proficient tumors, and higher somatic mutation loads were significantly associated with prolonged OS. Together, these results support dMMR status as a predictor of pembrolizumab efficacy.

These results, combined with those from KEYNOTE-016, evaluated a total of 149 patients with MSI-H/dMMR cancer, demonstrating an ORR with pembrolizumab of 36% in patients with CRC and 46% in patients with other tumor types. Among the responders, 78% had responses that lasted for 6 months.⁹ In all, 90 patients had CRC and 59 patients had 1 of 14 other tumor types.

“What was remarkable was we discovered that not only did colon cancers respond excellently, but also endometrial cancers, gastric cancers, small intestine cancers, pancreatic cancers, brain tumors, prostate cancers, and more, as we kept adding patients who are mismatch deficient across any tumor type and we started seeing responses,” explained Diaz, the lead investigator, in an interview. “The thinking has started to change. Perhaps this is a genetic alteration that is a more powerful indicator than where the tumor comes from.”

On May 23, the FDA granted an accelerated approval of pembrolizumab for adult and pediatric patients with unresectable or metastatic MSI-H or dMMR refractory solid tumors for whom there are no alternative treatment options, as well as patients with MSI-H or dMMR CRC who are resistant to fluoropyrimidine, oxaliplatin, and irinotecan.³⁷

“This is an important first for the cancer community,” said Richard Pazdur, MD, acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research and director of the FDA’s Oncology Center of Excellence.³⁷ “Until now, the FDA has approved cancer treatments based on where in the body the cancer started—for example, lung or breast cancers. We have now approved a drug based on a tumor’s biomarker without regard to the tumor’s original location.”

“What’s impressive about the FDA approval is that it is not only across all tumor types, but it is in adults and children,” noted Diaz. “It’s the first time that we’re no longer looking at tumors by the site of origin, but rather by the genetic lesion. This is incredibly exciting and is a historic approval by the FDA. In my opinion, it will knock down the walls of how we think about cancer in the future.”

Similarly, nivolumab, also a PD-1 inhibitor, is showing efficacy in MSI-H/dMMR tumors. The phase II Checkmate-142 study evaluated nivolumab at 3 mg/kg in patients with MSI-H or dMMR mCRC. Overall, 32% patients (24 of 74) responded to nivolumab, including 2.7% (n = 2) with a complete response.³⁸ Among patients who had progressed after receiving prior treatment with fluoropyrimidine-, oxaliplatin-, or irinotecan-based chemotherapy (n = 53), 28% responded to nivolumab.

On August 1, the FDA granted an accelerated approval for nivolumab as a treatment for patients with MSI-H or dMMR mCRC after progression on standard chemotherapy.

Further exploration of PD-1/PD-L1 pathway inhibitors for patients with MSI-H tumors is ongoing (Table). Trials evaluating the PD-L1 inhibitor atezolizumab (Tecentriq) combined with bevacizumab (Avastin) and cobimetinib (Cotellic) provide initial promise for combination strategies, which may extend the indication of immune checkpoint inhibitors to patients with MMR-proficient mCRC.^39,40

A recent study evaluated PD-L1 expression in samples from 78 MSI-H gastric cancers, reporting PD-L1 expression in 61.5% of samples, including 9% with expression on tumors and 60.3% with expression on immune cells.⁴¹ A significantly lower risk of lymph node metastasis and lower tumor stage was found in samples with immune cell PD-L1 expression compared with samples without PD-L1 expression. Immune cell PD-L1 expression was also a significant prognostic factor for increased OS compared with PD-L1—negative samples.

A rare, clinically important cohort of clear cell ovarian cancers (CCOCs) with MSI was recently described to exhibit significantly higher numbers of CD8-positive and PD-1—positive TILs compared with MSS CCOCs.⁴² These findings indicate that these CCOCs are highly immunogenic and may be very responsive to immune checkpoint inhibitors, leading to alternative treatment options for these patients and warranting routine testing of CCOCs for MSI.

^amFOLFOX6 is composed of oxaliplatin IV over 2 hours on day 1 of courses 1-10, leucovorin calcium IV over 2 hours on day 1, and fluorouracil IV over 46-48 hours on days 1 and 2. ^bNot yet open for participant recruitment. CRC indicates colorectal cancer; dMMR, deficient mismatch repair; mCRC, metastatic colorectal cancer; FOLFOX, oxaliplatin, leucovorin and fluorouracil; MSI-H, microsatellite instability-high; MSS, microsatellite stable; RT, radiation therapy

The Future of MSI as a Biomarker

The approval of pembrolizumab as the first anticancer therapy based on biomarker status rather than primary tumor site has heightened calls for extending next-generation sequencing (NGS) to a broader patient population.

Gordon B. Mills, MD, PhD, said in an interview that the pembrolizumab decision, along with other tumor-agnostic developments in the field, demonstrates the value of conducting multipanel NGS testing, which is hampered now by cost and reimbursement barriers.

“We hope that we’re going to be able to get some traction for more broad testing in a multiplex manner that will benefit patients,” said Mills, who is chair of the Department of Systems Biology and head of the Kleberg Center for Molecular Markers at The University of Texas MD Anderson Cancer Center in Houston.

“If you’ve a got a curable disease by surgery, you probably don’t need this,” he said, referring to NGS testing. “But all patients where there’s a potential to help their outcome by saying that we could find MSI or we could find a trick fusion, and those would be then susceptible to drugs that we have available— you need to cover this.”

Diaz agreed that more patients should be tested for MSI status. “I think with [the approval of pembrolizumab], we’re seeing a change in how we think about cancer. It’s going to take some time to reach everyone and to be accepted,” said Diaz. “The challenge is to get everyone tested and I think that is the first step.”

Other researchers have noted the potential for MSI status as a biomarker. “Notably, some of [the] extra-colonic cancers, such as endometrial carcinoma, gastric carcinoma, and ovarian carcinoma, also exhibit [an] MSI-H profile, and it is possible that MSI status may eventually become an important prognostic and predictive marker in tumors beyond the colon,” noted Liisa Chang, MA, MB, BChir, of Medway NHS Foundation Trust, Gillingham, Kent, United Kingdom, and colleagues.¹² “At the present moment, multiple clinical trials are ongoing, and in the near future, our knowledge about MSI in targeted immunotherapy will increase as a consequence of the completion of these ongoing studies.”

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