WHAT IS MSI?

Microsatellite (MS), also called Short Tandem Repeats (STRs) or Simple Sequence Repeat (SSRs), consists of 1-6 nucleotide sequences that are repeated. Small satellite DNA, which is primarily found near the ends of chromosomes and has tandem repeats of 15 to 65 nucleotides, has a different distribution pattern. MS is extensively dispersed, generally found close to the coding area, though it can also be found in places like the intron or the non-coding region.

It is generally accepted that the mechanism of MS formation involves DNA slippage during replication or a mismatch between the basic group of slippage strand and complementary strand during DNA replication and repair, which results in one or more repeating units being either lost or inserted. DNA replication errors can be fixed by the tissue’s regular DNA repair machinery, known as mismatch repair (MMR). However, the likelihood of gene mutation is enhanced because tumor cells lack MMR genes or because the replication repair process is flawed. It is clear that MSI plays a significant role in the occurrence and growth of cancers.

Microsatellite stability, low microsatellite instability, and high microsatellite instability can all be classified according to the frequency of MSI (MSS). Clinical research currently has a tendency to group MSS-L and MSS as one kind. Colorectal cancer (CRC) with no evident family genetic history and Lynch syndrome with non-polyposis with family genetic history can be distinguished based on the several molecular mechanisms of MSI. Early research findings revealed that sporadic colorectal cancer, which is brought on by epigenetic inactivation of gene expression, accounts for the majority of MSI cases.

VARIATIONS IN MSI

About 80–85% percent of those with colorectal cancer are not MSI-H/MMR deficient but are instead categorized as MSS also known as Microsatellite Stable. The term “cold” tumor has been used to describe MSS tumors. They are among the most genetically altered tumor forms in terms of the total number of mutations. Even though cancers with fewer genetic abnormalities, like stomach and head and neck cancers, have demonstrated responses, non-MSI tumors typically do not. These tumors frequently develop in an environment where the immune system is weakened. MSI-L is identified when one MSI marker exhibits instability while the others were microsatellite stable (MSS) and tumors lacked any evidence of instability.

MSI-H also indicates that a tumor has a higher range of instability. This happens when mismatch repair genes, which regulate DNA, don’t function properly. Mismatch Repair Genes (MMR) correct errors in DNA as cells divide. Areas of DNA could begin to become unstable as a result of the errors when MMR genes stop working efficiently.

An MSI screening test can determine whether there is a high level of instability, known as MSI-High, by looking for alterations in the DNA sequence between normal tissue and malignant tissue. About 15% of CRC tumors have an MSI-High status. Even though many MSI-High tumors are random, it is frequently found in tumors linked to the genetic condition Lynch syndrome (not due to a hereditary syndrome). MSI-High tumors are thought to exist in patients who test positive for the MSI.

To distinguish between hereditary and nonhereditary MSI-High, an additional test known as the Immunohistochemistry test is frequently used. If Lynch syndrome is hereditary, there is a chance that their family members will also have it, which increases the risk that they will develop colorectal or other tumors. Patients’ immediate families can consider Lynch syndrome testing in that case.

Tumors with high MSI can attract the immune system’s attention. These tumors frequently contain significant numbers of immune system cells when viewed under a microscope. The immune cells are only prevented from carrying out their duties completely. Numerous MSI-H tumor patients have responded well to immunotherapy treatments (or immune-checkpoint therapies). Therefore, before choosing a treatment, it is necessary to have an understanding of the MSI status.

IMMUNOHISTOCHEMISTRY

The presence of MSI can be inferred indirectly from the detection of MMR gene deletion. The MMR protein, which comprises hMLH1, hPMS2, hMSH2, and hMSH6, is detected using the IHC technique. If any of these MMR protein expressions are lacking, the outcome indicates MMR deficiency (dMMR). Proficient Mismatch Repair results from the expression of all four MMR proteins (pMMR). dMMR and MSI-H are comparable in most aspects. Some people believe that PCR can be replaced with IHC because it is so straightforward and useful. However, sometimes it was unable to identify both dMMR and MSI-H simultaneously.

METHODS OF DETECTION

Several methods can be used to detect microsatellite instability (MSI) or deficient DNA mismatch repair (dMMR). Polymerase chain reaction (PCR) and Immunohistochemistry (IHC) are the two most popular techniques. The industry-recognized gold standard approach for MSI detection is MSI testing by PCR.

1. MSI by PCR

   Since mononucleotide repeat microsatellite sequences are particularly susceptible to transcription mistakes, they make excellent targets for PCR amplification-based monitoring. Fragments from the tumor and matched normal samples are amplified using fluorescently labeled primers to identify MSI. Using capillary electrophoresis, the amplified fragments are separated by size, and the fluorescent labeling enables further separation based on the fluorescent tag’s color. The fluorescent markers can be multiplexed to enable the detection of several pieces of similar size in a single reaction. Tumors with this microsatellite instability are referred to as MSI-high or MSI-H. Size changes are an indicator of microsatellite instability.

2. MMR by IHC

   IHC, a histological technique that uses labeled, protein-specific antibodies to detect proteins in tissue samples, can be used to determine whether or not MMR proteins are present. A different tissue sample is used to identify each protein. The term “dMMR” (deficient in mismatch repair) is used to describe cancers that have lost one or more of the key mismatch repair proteins since it implies a defect in mismatch repair.

   MMR protein expression is not always a reliable indicator of MMR activity. While 5–10% of proteins retain their antigenicity when they are not functional, there can be a reduction in function without a corresponding loss of the protein in the cell.

3. MSI Analysis by Next Generation Sequencing (NGS)

   Microsatellite sequences are identified via NGS MSI analysis, which compares them to either matched normal tissue or a consensus sequence. Microsatellite sequence length variations suggest instability. The MSI-H or MSS (microsatellite stable) status is then determined by NGS using algorithms to determine whether the quantity of instability observed is significant. It may not be possible to make a call in some circumstances due to computational difficulties associated with NGS analysis of microsatellite homopolymers. Numerous methods are employed in the field because there is currently no agreement on the loci, data analysis pipeline, or cutoffs that should be used for MSI determination by NGS.

MSI TEST COST IN INDIA

In India, MSI testing is available at many specialized laboratories like 4baseCare, Promega, DNA Labs, etc., with prices typically ranging from ₹5,000 to ₹20,000 depending on the location and the provider. The cost of the test may vary depending on the type of testing method used, the turnaround time for results, result interpretation, etc.

The packages provided by Precision Oncology firm 4baseCare are created with an emphasis on accessibility and affordability. In addition to MSI testing, NGS-based Comprehensive Genomic Profiling panels like TARGT Indiegene Solid, TARGT Indiegene Liquid, TARGT Absolute, and TARGT Absolute+ cover a variety of well-known and novel genomic biomarkers that will help identify the key genes responsible for the patient’s particular cancer type, opening the door to immunotherapy and targeted therapy.

How can I book my Microsatellite instability Test in India?

Visit the website www.4basecare.com for booking any DNA or RNA tests, and you can write to us at info@4basecare.com. Our qualified professionals will reach out to you in the shortest possible and help you get clarified with all your queries related to genetic testing beforehand.

REFERENCES :

1. Microsatellite instability: A review of what the oncologist should know. (2020, January 13). BioMed Central.
2. What is MSI vs MSS? (2017, June 15). Fight Colorectal Cancer.
3. Methods of MSI detection. (n.d.). Promega Corporation.