Evaluation of Droplet Digital PCR Assay for the Detection of Microsatellite Instability in Colorectal, Gastric, and Endometrial Cancers.

Background: Microsatellite instability (MSI) is an important biomarker for the diagnosis of Lynch syndrome and for guiding immunotherapy in various solid tumors. Droplet digital PCR (ddPCR) has emerged as a highly sensitive method for detecting MSI, particularly in circulating tumor DNA (ctDNA).

This study aimed to evaluate the analytical and clinical performance of a ddPCR assay using three MSI markers (BAT-26, ACVR2A, and DEFB105A/B) in colorectal, gastric, and endometrial cancers. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 190 patients (83 colorectal, 44 gastric, and 63 endometrial cancers) and 21 plasma samples from patients with metastatic solid tumors were analyzed. MSI status determined by ddPCR was compared with conventional PCR using a pentaplex panel and immunohistochemistry (IHC).

Analytical performance, including limit of blank (LoB) and limit of detection (LoD), was evaluated using cell line DNA, and clinical cut-offs were established using receiver operating characteristic analysis. Results: The ddPCR assay demonstrated high analytical sensitivity, with LoD values of 0.075% for BAT-26, 0.1% for ACVR2A, and 0.025% for DEFB105A/B. Using optimized clinical cut-offs, the concordance rate between ddPCR and conventional PCR assays was 98.4% in tissue samples. Marker performance varied by cancer type, with reduced sensitivity observed in endometrial cancer.

In plasma samples, MSI-H was detected in 1 of 21 cases (4.8%), and the overall concordance rate with tissue-based MSI status was 94.7%. Conclusions: The ddPCR assay demonstrated high concordance with conventional MSI testing methods and showed potential as a sensitive tool for MSI detection in both tissue and plasma samples.

However, optimization of marker panels and establishment of sample-type-specific clinical cut-offs are required, particularly for ctDNA-based analysis. Further large-scale studies are needed to validate the clinical utility of ddPCR for MSI detection and monitoring.