Clinical Value and Quality Control of MSI (Fluorescent PCR Melting Curve Method) Kits in Tumor Diagnosis and Treatment

Clinical significance: From Lynch syndrome screening to immunotherapy efficacy prediction

Microsatellite instability is a direct reflection of defective DNA mismatch repair function. The MSI (fluorescence PCR melting curve method) kit is widely used for genetic screening of Lynch syndrome, molecular typing of colorectal cancer and endometrial cancer, and prediction of the efficacy of PD-1/PD-L1 immune checkpoint inhibitors. Studies have shown that patients with solid tumors exhibiting high microsatellite instability (MSI-H) can benefit from immunotherapy regardless of the primary tumor site; therefore, accurate interpretation of the kit is directly related to treatment decisions. This method is particularly suitable for FFPE samples, and results are generally reported in three categories: MSI-H (≥2 unstable markers), MSI-L (1 unstable marker), and MSS (no unstable markers). Clinical reports should specify the loci examined and the interpretation algorithm to facilitate cross-center data comparison.

Internal quality control and standardized operation

To ensure the reliability of MSI (fluorescence PCR melting curve method) kit test results, laboratories should establish a complete quality control system: each batch of experiments should include a positive control (known MSI-H cell line or sample), a negative control (normal tissue or MSS sample), and a no-template control. The background fluorescence level of the melting curve peak shape and the coefficient of variation of the Tm value must be controlled within the range specified by the manufacturer. The tumor cell content in samples is generally required to be ≥20% (some kits can accept as low as 5%); below this threshold, false negatives may occur. For borderline results, it is recommended to re-section the tissue block or use supplementary methods (such as immunohistochemical detection of MMR proteins) for verification.

Common challenges and recommended solutions

Challenges in clinical practice include: decreased amplification efficiency due to DNA degradation in FFPE samples, difficulty in obtaining paired normal and tumor tissue samples, and polymorphic variation of some single nucleotide markers in specific populations. Countermeasures include: selecting extraction kits optimized for freeze-thaw cycles; introducing a manual review step in the interpretation software; when paired normal samples are unavailable, referring to family analysis or using population-corrected baseline thresholds. The marker combination configured in the kit should cover at least 5 mononucleotide repeat loci (the Bethesda or Pentaplex panel is recommended) to reduce classification bias caused by locus selection. It is recommended that laboratories regularly track updates to kit manufacturers' instructions and external quality assessment feedback, and continuously optimize the experimental workflow.