CAP Authoritative Voice: What to deal with when MSI and MMR test results are inconsistent

In July 2022, the American Association of Pathologists (CAP) released Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guidelines in archives of Pathology and Laboratory Medicine The From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer guidelines are designed to help oncologists and pathologists accurately determine whether a patient is eligible for immune checkpoint inhibitors[1

There is finally an official explanation

In 1993, Jacobs et al.

With the increasing application of clinical PD-1/PD-L1 immune checkpoint inhibitors, in order to allow more patients to benefit from immunotherapy, how to accurately identify patients with MSI-H or dMMR tumors when MSI and MMR are inconsistent is still clinically difficult

Literature reports that the consistency between MSI / MMR can reach more than 90%, due to the principle and experimental operation, there is an objective inconsistency between the PCR method and IHC detection results, the following analysis of the detection principle and common inconsistencies between MSI and MMR, so as to improve the clinical understanding of MSI and MMR detection, make accurate judgments, and make it better serve clinical diagnosis and treatment

First, MSI and MMR

Microsatellites refer to short tandem repeats

MMR system and MSI relationship[2]

Second, the introduction of MSI/MMR detection methods

Immunohistochemical detection of MMR: the expression of four common MMR proteins (MLH1, MSH2, PMS2, MSH6) is mainly detected, and the MMR status

Detection of MSI by fluorescent PCR-capillary electrophoresis: It is currently recognized that MSI detection is the "gold standard" method for MSI detection by comparing the number of microsatellite instability sites in tumor samples, and then determining the number of microsatellite instability sites in tumor samples

NGS method to detect MSI: The MSI algorithm usually determines the unstable state of the site by characterizing the difference between the number of reads corresponding to the length of different repeat sequences of the selected marker sites in MSI-H and MSS, and determines the MSI state of the sample by whether the proportion of unstable sites exceeds the established threshold
.
At present, when detecting MSI in NGS, due to the complexity of the technology, it is not easy to standardize, there is no unified standard for detection sites and cut-off values, and due to economic burden and waste of resources, NGS is not recommended for MSI detection
alone.

Third, MSI and MMR test results are common inconsistencies and possible causes

Studies have shown that MMR-IHC and MSI-PCR have a consistency
of 90%-97.
5%.
However, due to the different detection principles of the two methods, MMR stain heterogeneity, the functional compensation mechanism of the MMR system, mmR gene missense mutations, and chemotherapy may lead to inconsistencies
between MSI and MMR results.

Mismatch Repair System Normal (pMMR)/MSI-H

(1) In the heterogeneous expression region of MMR protein, tumors with positive expression of MMR protein may also have MMR gene mutations and methylation, which are manifested as MSI-H[4].

(2) When the gene encoding the MMR protein is missenseed, the MMR protein function is abnormal but may not affect its antigen structure, in this case, the IHC can detect the expression of the MMR protein, but its mismatch repair function is abnormal, and the MSI detection result is MSI-H [5].

(3) Since MMR involves but is not limited to a series of genes such as MLH1, MSH2, MSH6, PMS2, MSH3, PMS1, etc.
, mutations in mmR genes that have not been detected by IHC or MMR genes that have not yet been identified may lead to MMR defects, manifested as MSI-H [6].

(4) Mutations in the exonuclease region of the POLE gene have been reported to explain the pMMR/MSI-H phenomenon of <1% [5,7].

dMMR/Microsatellite Stabilization (MSS)

(1) MMR system function compensation mechanism has MMR expression missing but MSS

Since MMR immunohistochemistry only detects 4 proteins commonly, but due to functional compensation in the MMR system, protein expression loss may occur, but the MSS status is unchanged
.
Common compensation mechanisms are PMS2 and PMS1 complementary, MSH6 and MSH3 complementary
.
The most common inconsistency is the coexistence
of MSH6 expression loss with MSS status.

Part of the reason stems from MSH3's functional compensation
of the MSH6 protein.
When the MSH6 protein is damaged, MSH2/MSH3 continues to work and DNA mismatch repair is corrected
.

At the same time, because MSH6 mainly recognizes the mismatch of single bases, the use of MSI markers at double nucleotide sites may miss the detection of MSH6 defects, so it is recommended to use a label containing only single nucleotide sites to maximize the detection rate of MSH6 carriers [8].

(2) Neoadjuvant chemoradiotherapy may affect MMR protein expression

Rectal cancer after neoadjuvant chemoradiotherapy may lose MSH6 stain almost completely or have only nucleolar staining; About 30% of rectal cancers after neoadjuvant chemoradiotherapy have decreased PMS2 staining, manifested by MSS
.
If staining is ambiguous at this point, IHC staining with biopsy specimens before chemoradiotherapy usually solves this problem [9].

(3) There have been articles reporting methylation of mlH1 promoters or pathogenic mutations in mlH1 genes that can explain the absence of most MLH1 proteins and the phenomenon of MSS [5,7].

In addition to the above, tumor heterogeneity, immunohistochemical staining and quality control may cause MMR and MSI to be inconsistent
.
The latest CAP guidelines state that when there is an MSI inconsistency with an MMR result, the inconsistent results should be reviewed to ensure that the inconsistent result is not due to an error
in interpretation.
Therefore, in order to screen more truly positive patients and benefit more patients, it is recommended that THE COMBINED TEST
OF MMR and MSI be carried out before the screening of LS, the treatment guidance of tumor patients and the treatment of immune checkpoint inhibitors.

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