Immunotherapy Knowledge: How to detect MSI/MMR?

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Immunotherapy Knowledge: How to detect MSI/MMR?

Immunotherapy Knowledge: How to detect MSI/MMR? In 2017, the U.S. FDA announced: Accelerated approval of PD-1 antibody-pembrolizumab (also known as “K drug”) for the determination of high microsatellite instability or mismatch repair gene defects (MSI-H/dMMR) For the treatment of solid tumors, K drug has also become the first anti-tumor immune drug that “looks at the marker regardless of the location”.

So, what exactly is MSI/MMR? Which patients should be tested? This article attempts to answer this question.

01. What is MSI/MMR?

Microsatellites are DNA sequences with simple repeating units.

MSI (microsatellite instability): “Microsatellite instability”

Refers to any change in the length of a microsatellite caused by the insertion or deletion of a repeat unit in a tumor compared with normal tissues, and the appearance of new microsatellite alleles.

MMR (mismatch repair): “Mismatch repair”

System members include proteins such as MLH1, MSH2, MSH6 and PMS2. In the process of DNA replication, small DNA mismatch errors may occasionally occur, which can be recognized by these proteins and then cut, and new strands are synthesized for repair.

The entire genome has more than 100,000 short tandem repeat regions called “microsatellites”, which are prone to slippage errors during the copying process, so they rely heavily on MMR system repair.

When the above 4 proteins are abnormal, MMR defects (deficient MMR, dMMR) are caused, and microsatellite replication errors cannot be detected and corrected, resulting in diffuse MSI. Although most of the microsatellites are located in the non-coding region, misplaced mutations can cause frameshift mutations, causing abnormalities in tumor-related genes, and inducing cancer.

High MSI (MSI-H) plays a decisive role in approximately 15% of CRC. In addition, MSI-H can also cause endometrial cancer, ovarian cancer, gastric cancer and other tumors.

dMMR is common in two situations:

Germline mutations in the MMR gene, this condition is called Lynch Syndrome (Lynch Syndrome), which often occurs in a family of hereditary clusters of malignant tumors [1];
Sporadic cases caused by inactivation of MMR gene epigenetic modification are more common, usually accompanied by CpG island methylation phenotype (CIMP), and 50% of cases also have BRAFV600E activating mutations. Conversely, having CIMP and BRAFV600E mutations usually excludes Lynch syndrome [2,3].

From this we can see that dMMR is clinically equivalent to MSI-H, but in some cases, dMMR and MSI-H cannot be detected at the same time.

For example, dMMR caused by MSH6 mutation causes a low rate of MSI, which may not meet the criteria for diagnosis of MSI-H; and MSI-H-positive tumors occasionally come from MMR pathway proteins that have not been discovered so far. Therefore, although the two detections have a high coincidence rate and are often confused in clinical practice, they cannot be absolutely equal [4].

02. MSI/MMR detection method

At present, two main methods are used to detect MSI/MMR in clinical practice: immunohistochemistry (IHC) to detect abnormal MMR proteins, or polymerase chain reaction (PCR) to detect MSI. Next generation sequencing (NGS) is a new detection method that has emerged in recent years.

1) IHC

IHC staining analysis of MMR protein expression is the most commonly used method. It mainly detects 4 known MMR proteins (MLH1, MSH2, MSH6 and PMS2), and the positive expression is located in the nucleus:
If ≥ 1 of the 4 proteins is not expressed, the tumor may be MSI-H [5];
All 4 proteins are positive for pMMR (complete mismatch repair function).

IHC is cheap and easy to do, but it is possible to miss some abnormalities caused by other MMR proteins, and due to the heterogeneity of the tumor, the scores of the entire tumor may be inconsistent.

2) PCR

PCR usually detects BAT25, BAT26, D2S123, D5S346 and D17S250 alleles.
MSI over 30% (more than 2 out of 5 sites) is MSI-H;
Less than 30% (1 site) is considered as microsatellite low instability (MSI-L);
If there is no instability, it is microsatellite stability (MSS) [6].

PCR is the gold standard for testing, which can more objectively evaluate functional dMMR activity, but it requires high laboratory conditions and is relatively expensive.

In general, the sensitivity and specificity of IHC and PCR methods for detecting MSI/MMR are very good, and the two methods have a high degree of consistency (>95%) [7].

3) NGS

In recent years, target area sequencing (NGS panel), whole exome sequencing (WES), and whole genome sequencing (WGS) of the second-generation sequencing platform have begun to be applied to MSI detection.

NGS is suitable for patients who need to detect tumor driver genes and/or treatment-related gene mutations at the same time.

The NCCN guidelines recommend that MSI/MMR testing can only be performed in clinical laboratories accredited by the Clinical Laboratory Improvement Act of the United States (CLIA).

03. The meaning of MSI/MMR

MSI/MMR is of great significance for the diagnosis, prognostic judgment and treatment options of various tumors.

1) Lynch syndrome screening

As mentioned earlier, Lynch syndrome is due to MMR germline mutations that cause individuals to be prone to malignant tumors such as CRC and endometrial cancer. For patients with the above-mentioned family history of tumors or with early-onset tumors, it is necessary to screen themselves and their family members for Lynch syndrome, which can significantly reduce the morbidity and mortality of tumors.

2) Prognostic judgment

MSI can reflect the prognosis. The prognosis of MSI for CRC depends on the stage. MSI-H is an indicator of good prognosis for stage II CRC[8]. Although only 4% of metastatic CRC (mCRC) are MSI-H, the prognosis of these tumor patients does not improve. Especially patients with BRAFV600E mutation [9].

3) Guide treatment

MSI can guide follow-up treatment. MSI-H stage II CRC has a good prognosis and is not sensitive to fluorouracils[10]. Therefore, NCCN guidelines and CSCO colorectal cancer diagnosis and treatment guidelines recommend that all stage II CRCs be tested for MSI/MMR, and MSI-H is present. Of patients are not recommended to add adjuvant chemotherapy to the treatment.

In addition to chemotherapy, MSI can also reflect the response of mCRC patients to targeted drugs. The molecular analysis of the CALGB/SWAOG 80405 study found that patients with MSI-H can benefit from bevacizumab rather than cetuximab, and that receiving bevacizumab has a lower risk of death than cetuximab by 87 %[11]. Of course, this needs more research to confirm.

MMR/MSI can also predict the efficacy of immunotherapy. MSI-H/dMMR tumors have many mutations and are widely immunogenic, so they respond well to PD-1/PD-L1 inhibitors.

In the KEYNOTE-177 study, patients with advanced colorectal cancer with MSI-H/dMMR were randomized to receive pembrolizumab or chemotherapy. The median PFS of the two groups were 16.5 months and 8.2 months, respectively, which was significantly different [15 ]. Based on this result, on June 29 this year, the FDA approved pembrolizumab as the first-line treatment for patients with MSI-H/dMMR advanced colorectal cancer.

In the KEYNOTE-016 study, the ORR of CRC patients with MSI-H/dMMR treated with pembrolizumab above second-line treatment reached 36%, and the ORR of other tumor types reached 46%. Therefore, in 2017, the FDA approved K drug for CRC patients with MSI-H/dMMR who progressed after standard chemotherapy and in patients with metastatic MSI-H/dMMR solid tumors after treatment failure [12].

Another immunological drug, nivolumab, also achieved a response rate of 32% in patients with multi-drug resistant MSI-H/dMMR mCRC. Drug O was also approved for accelerated approval for MSI-H/dMMR CRC in the same year. Patients were treated with fluorouracil, oxaliplatin and irinotecan after progression [13].

Conclusion:

Existing evidence shows that in addition to screening for tumor genetic risks and guiding the treatment of CRC and other tumors, MSI/MMR testing can benefit all tumor patients who may have dMMR status, so it has increasingly become a pan-tumor biomarker and deserves attention .

(source:internet, reference only)

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