A tumour-first molecular diagnostic strategy combining MMR/MSI testing and germline NGS improves risk stratification for lynch syndrome in colorectal and endometrial cancer

Although most colorectal (CRC) and endometrial cancers (EC) are sporadic, a clinically relevant proportion results from hereditary cancer predisposition syndromes, particularly Lynch syndrome (LS). Next-generation sequencing (NGS) enables comprehensive germline testing; however, optimal diagnostic pathways for clinical practice remain under discussion. A germline NGS panel targeting 12 hereditary cancer predisposition genes ( MLH1 , MSH2 , EPCAM , MSH3 , MSH6 , PMS2 , APC , MUTYH , BMPR1A , SMAD4 , STK11 , PTEN ) was applied to 430 individuals (340 cancer patients, 5 individuals with polyposis without cancer, and 85 unaffected family members) from the Polish (Lower Silesia) population. MLPA analysis was performed in 48 selected cases. Tumour-based data from 183 CRC/EC cases included MMR/MSI status, KRAS / NRAS / BRAF mutations, MLH1 promoter methylation, and POLE analysis in EC. NGS identified germline pathogenic/likely pathogenic variants (PV/LPV) in 32/430 individuals (7.44%), predominantly in MMR genes (19/32; 59.38%). Among tumour-tested cases, all germline PV/LPV carriers were detected within the MMRd/MSI-H group (16/132; 12.12%). The highest enrichment was observed in tumours with isolated MSH6 loss (5/6; 83.33%). No PV/LPV were identified in MMRp/MSS tumours or MLH1 -methylated MMRd/MSI-H tumours. Our findings support a stepwise tumour-first strategy integrating MMR assessment, MLH1 methylation/ BRAF analysis, and targeted germline NGS referral to improve hereditary CRC/EC diagnostics.