When pembrolizumab was approved by US FDA for the treatment of all unresectable or metastatic cancers with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) irrespective of tissue origin 1, it triggered the importance of MSI testing outside traditional MSI-associated cancers such as colorectal cancer (CRC) and endometrial cancer (EC). With next-generation sequencing (NGS)-based molecular diagnostics tests readily available in the market, and more and more companies adopting to use NGS data for MSI assessment, the question remains… HOW DOES IT COMPARE TO OTHER MSI TESTING METHODS?
What Is MSI?
Microsatellites are short repeats in the DNA sequence consisting of 1-10 nucleotides that are repeated about 5-50 times. They occur at thousands of locations within an individual’s genome and due to their fragile nature, have about 10 times higher mutation rate compared to other areas of DNA.
The MMR system is in charge of correcting errors during DNA replication and when this system malfunctions, usually due to mutational inactivation or epigenetic silencing of the MMR genes such as MLH1, MSH2, MSH6 and PMS2, errors will accumulate in the microsatellite regions. This will expand or reduce the length of microsatellites, eventually causing MSI.
The Existing Methods For MSI Testing
MSI can be tested either directly or indirectly. For the latter, immunohistochemistry (IHC) is used to determine the expression of the abovementioned MMR genes. Loss of protein expression in any of the genes in the tumor cells is assumed to be associated with dMMR if the protein is expressed in normal cells. The direct testing, and also the gold standard for MSI assessment, is conducted using polymerase chain reaction (PCR) on five predefined microsatellites. A difference in the length of one out of the five regions tested is classified as MSI-Low (MSI-L) and if at least two regions are changed, it is called MSI-H. The IHC and PCR methods have a high concordance assessing MSI2
MSI Calculation Using NGS Data
Recently an alternative to the above has emerged where targeted NGS panels are used to directly assess the MSI status. This is done by examining the microsatellite regions present in the genes sequenced in the NGS panel. Ideally, the microsatellite length is obtained from the sequencing data of both the tumor and the matched normal genomes. When the difference between the length distributions in the two samples is significant, the locus is said to have an MSI event. However, methodologies have also emerged where comparison is done to a pool of existing normal samples and hence, sequencing of the matched normal sample is not required 34. Lastly, and similarly to tumor mutational burden (TMB) calculation, the panel size is of importance as enough microsatellite regions will need to be sequenced for an accurate assessment of the MSI status.
How Does NGS-Based MSI Detection Compare To Other Methods?
Various computational tools have been developed for MSI calculation from NGS data such as MANTIS, MOSAIC, mSINGS, MSIsensor and others 5 . Each of them has been compared to the PCR-based gold standard, identifying a high concordance between the two methods (>94% sensitivity and >97% specificity)6 7 8 9 10 . A clear difference to note is that while the PCR assay enables detection of microsatellite stable (MSS), MSI-L and MSI-H populations, most NGS-based tests report only the MSS and MSI-H classifications. Regardless, the current approval for checkpoint inhibitors is only for MSI-H and not MSI-L patients. As MSI patterns may vary from one cancer to another, an important consideration when assessing the accuracy of an NGS-based MSI detection method is to check whether validation was conducted in various tumor types including those outside CRC and EC. This will ensure of a more holistic computational tool that is not biased towards a specific tumor type.
For many patients, access to sufficient tumor material is limited and the biggest benefit of NGS-based MSI detection is that it eliminates the need for a separate test where sectioning of additional sample would be required. Together with full mutational profile of the sequenced genes, as well as TMB calculation, targeted panel NGS tests hence enable to infer an amplitude of therapy-relevant information from the precious specimens.
In addition, NGS-based profiling will facilitate efficient MSI assessment across all cancers. Nearly 70% of the major tumor types in the TCGA dataset include a subgroup of patients with MSI-H phenotype (Figure 1), making them eligible for checkpoint inhibition therapy. Therefore, MSI screening should become a standard for all metastatic cancers, so that the most optimal care can be provided to the patients.
Figure 1. MSI prevalence across 39 major tumor types from the TCGA dataset11
1. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-pembrolizumab-first-tissuesite-agnostic-indication (accessed 6 Mar 2020)
2. Haraldsdottir. JCO Precis Oncol. 2017
3. Bonneville et al. Methods Mol Biol. 2020
4. Trabucco et al. J Mol Diagn. 2019
5. Bonneville et al. Methods Mol Biol. 2020
6. Salipante et al. Clin Chem. 2014
7. Hause et al. Nat Med. 2016
8. Kautto et al. Oncotarget. 2017
9. Middha et al. JCO Precis Oncol. 2017
10. Trabucco et al. J Mol Diagn. 2019
11. Bonneville et al. JCO Precis Oncol. 2017