Liquid Biopsy Biomarkers for Endoscopic Surveillance in Lynch Syndrome

Rationale: Lynch syndrome is caused by germline pathogenic variants in one of the DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, or EPCAM through MSH2 inactivation). Tumors arising in LS carriers characteristically exhibit microsatellite instability, which develops early during colorectal carcinogenesis, together with loss of expression of one or more MMR proteins on immunohistochemistry. MMR deficiency predisposes to frameshift mutations at coding mononucleotide repeats, and these mutations have recently been detected in the circulation of patients with MSI-high/MMR-deficient tumors.

In Italy, more than 50,000 new colorectal cancer cases are diagnosed each year, approximately 3-5% of which are associated with LS. Although LS is relatively uncommon, it is associated with a markedly increased lifetime risk of CRC, including an approximately 14-fold higher risk of developing CRC before the age of 50 compared with the general population, as well as an increased risk of metachronous tumors. Colonoscopic surveillance starting at 20-25 years of age and repeated every 1-2 years is currently the only established strategy to reduce CRC morbidity and mortality. However, colonoscopy is invasive, costly, and may become burdensome after years of lifelong surveillance, potentially affecting adherence. Moreover, interval cancers may develop between scheduled examinations.

Minimally invasive biomarkers capable of detecting early neoplastic changes could improve risk stratification, personalize surveillance intervals, and increase patient compliance. Circulating tumor-derived nucleic acids, including circulating tumor DNA and microRNAs, represent promising biomarkers for CRC detection and monitoring. MSI and MMR-related frameshift mutations arise early during tumorigenesis and may be detected by liquid biopsy, while circulating miRNAs are highly stable and become dysregulated during early CRC development. Stool represents a non-invasive source of tumor-derived DNA, miRNAs, and microbial signals directly related to colorectal epithelial biology. Analysis of fecal DNA may improve the detection of early MMR-deficient lesions by identifying characteristic frameshift mutations, while fecal miRNA and gut microbiome profiling may provide complementary signatures associated with colorectal carcinogenesis.

Preliminary institutional data from study INT 118/21 support the feasibility of liquid biopsy-based MSI analysis and miRNA profiling in LS patients undergoing endoscopic surveillance. These findings provide the rationale for a larger prospective observational study integrating plasma-, stool-, and urine-based molecular biomarkers with standard surveillance colonoscopy.

Study design and procedures: this is a prospective, longitudinal, observational study enrolling adult patients (≥18 years) with genetically confirmed Lynch Syndrome followed at the Unit of Hereditary Digestive Tract Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Participants are followed for 24 months and undergo surveillance colonoscopy at baseline (T0), after 12 months (T1), and after 24 months (T2), according to international guideline recommendations. At each scheduled colonoscopy visit, blood, urine, and stool samples are collected before bowel preparation and/or the endoscopic procedure. In addition, venous blood samples are collected every 3 months throughout the study, for a total of 12 blood collections per participant. A clinical evaluation is performed at each scheduled endoscopic visit. Dietary habits are assessed at T0, T1, and T2 using the validated EPIC dietary questionnaire.

Colonoscopy findings and, when available, histopathological results are recorded in a dedicated study database. Formalin-fixed paraffin-embedded tissue specimens obtained during routine clinical care are collected for possible additional translational analyses, including immunohistochemical assessment of MMR protein expression and/or MSI testing, when appropriate. Additional colonoscopies may be performed if clinically indicated.

MSI analysis is performed by digital PCR using the BAT25, BAT26, NR21, NR24, and Mono27 loci on ctDNA extracted from plasma samples. Plasma and stool miRNA profiling is performed by next-generation sequencing. Frameshift mutations are investigated in DNA extracted from stool samples using targeted sequencing of recurrent MMR deficiency-associated coding mononucleotide repeat mutations previously identified in MSI-high tumors. Stool microbiome analysis is performed by shotgun sequencing. Cell-free DNA extracted from urine samples is stored at -20°C for future exploratory analyses.

Objective: To evaluate the feasibility and diagnostic performance of plasma-, stool-, and urine-based molecular biomarkers for the early detection of colorectal precancerous and cancerous lesions in individuals with Lynch syndrome undergoing routine endoscopic surveillance, including MSI analysis in plasma ctDNA, plasma and stool miRNA profiling, fecal ctDNA frameshift mutation analysis, stool microbiome profiling, and exploratory urine cell-free DNA banking.