Pan-cancer prevalence of microsatellite instability and Lynch syndrome in India 

Technical Summary: Pan-cancer Prevalence of Microsatellite Instability and Lynch Syndrome in India

Problem Statement
Lynch syndrome (LS), caused by germline pathogenic variants in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2, EPCAM), is a major hereditary cancer predisposition syndrome. However, it remains significantly underdiagnosed in India, particularly outside the context of colorectal cancer (CRC). Existing data on pan-cancer microsatellite instability (MSI), MMR deficiency (MMRd), and germline predisposition in the Indian population is limited. Current clinical guidelines in India lack universal implementation of MMR testing, and multicenter data spanning both colorectal and extracolonic index tumors with systematic germline confirmation is sparse. Furthermore, population-specific factors, such as founder variants, may alter the observed tumor spectrum and detection yields compared to European cohorts.

Methodology
The study employed a prospective, multicenter, hospital-based cohort design involving 519 patients with primary solid tumors recruited from 17 centers in India between 2019 and 2025. The cohort included diverse anatomical sites: colorectum, endometrium, ovaries, esophagus, stomach, liver, pancreas, appendix, prostate, cervix, and small intestine.

The study utilized a "tumour-first" screening workflow:

1. MSI Testing: All 519 tumors underwent MSI analysis using PCR-fragment length analysis (PCR-FLA) with five mononucleotide and two pentanucleotide markers. Tumors were classified as MSI-High (MSI-H) if ≥2 markers showed instability.
2. MMR Immunohistochemistry (IHC): MMR-IHC (for MLH1, PMS2, MSH2, MSH6) was performed on all non-CRC tumors, as PCR-FLA performance can be inferior in these tissues.
3. Somatic Differentiation: In MSI-H CRC cases, somatic BRAF V600E testing was performed via ARMS-PCR to distinguish sporadic cases (BRAF mutant) from potential LS cases (BRAF wild-type).
4. Germline Sequencing: Germline whole-exome sequencing (WES) was conducted on:
   • MSI-H, BRAF wild-type CRCs.
   • MMRd non-CRC tumors.
   • Variants were prioritized using Exomiser, filtered against population databases (gnomAD, 1000 Genomes), and classified according to ACMG-AMP guidelines.
5. Haplotype Analysis: For recurrent MLH1 variants, genome-wide genotyping and Identity-by-Descent (IBD) analysis were performed to estimate the Time to Most Recent Common Ancestor (TMRCA) and confirm founder status.
6. Cascade Testing: First-degree relatives of confirmed LS patients were offered germline testing.

Key Results

• Prevalence of MSI-H/MMRd: Among 519 tumors, 25.6% (133/519) were identified as MSI-H or MMRd. The highest proportions were observed in CRC, endometrial, and gastric tumors.
• Lynch Syndrome Yield: Of the 133 MSI-H/MMRd tumors, 49.6% (66/133) harbored pathogenic or likely pathogenic (P/LP) germline variants consistent with LS.
• Tumor Site Distribution: CRC showed the highest LS yield (20% of all CRCs), followed by endometrial (9.3%) and ovarian cancers. Notably, 11% of LS patients presented with a primary tumor other than CRC.
• Gene Distribution: MLH1 accounted for the majority of LS diagnoses (69.7%, 46/66), followed by MSH2 (18.2%).
• Founder Variants: Two recurrent MLH1 variants, c.306G>T and c.156delA, together accounted for 36.4% (24/66) of all LS cases.
  • MLH1 c.306G>T was previously identified as a founder variant in India.
  • MLH1 c.156delA was identified as a novel founder variant. Haplotype analysis of 24 carriers (including 7 first-degree relatives) indicated a shared haplotype originating in Gujarat, with an estimated TMRCA of approximately 1,750 years (70 generations).
• Assay Concordance: In extracolonic tumors where both PCR-FLA and MMR-IHC were available (n=208), concordance was 93.8% (Cohen's κ=0.71). Discordant cases (n=13) where IHC indicated MMRd but PCR showed MSS were confirmed to carry LS germline variants, suggesting higher sensitivity of IHC in non-CRC contexts.
• Demographics: The mean age at diagnosis was 52.9 ± 13.9 years. 16.9% of patients reported a family history of cancer.

Significance and Claims
The authors claim that this study establishes the clinical utility of a pan-cancer, tumor-first approach for LS detection in the Indian clinical setting. Key claims include:

1. Diagnostic Utility: The high yield of LS (49.6%) among MSI-H/MMRd tumors supports the implementation of reflex germline testing for all such cases in India, regardless of tumor site.
2. Population Specificity: The identification of MLH1 c.156delA as a novel founder variant, alongside the known c.306G>T, highlights the necessity for regionally informed diagnostic pathways. These two variants alone explain over one-third of LS cases in this cohort, suggesting that targeted testing could improve efficiency.
3. Screening Strategy: The study supports the use of combined tumor-based screening (MSI and/or MMR-IHC) in pan-cancer workflows. The high concordance between PCR-FLA and MMR-IHC in extracolonic tumors validates the use of these assays to trigger germline testing.
4. Clinical Impact: Early detection of LS through this approach enables tailored surveillance, chemoprevention, risk-reducing surgeries, and access to immunotherapies for MSI-H tumors.
5. Limitations: The authors modestly note that as a hospital-based cohort rather than a population-based study, the reported frequencies should not be interpreted as national prevalence estimates. They also acknowledge that recruitment was geographically skewed toward Gujarat (where founder variants are concentrated) and that the cohort was enriched for CRCs, which may influence the observed tumor spectrum.

In conclusion, the paper argues for the broader implementation of universal MMR testing and subsequent germline confirmation in India to address the underdiagnosis of Lynch syndrome and improve patient management.