High-sensitivity microsatellite instability assessment for the detection of mismatch repair defects in normal tissue of biallelic germline mismatch repair mutation carriers

Abstract

Introduction: Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary cancer syndromes associated with mismatch repair (MMR) deficiency. Tumours show microsatellite instability (MSI), also reported at low levels in non-neoplastic tissues. Our aim was to evaluate the performance of high-sensitivity MSI (hs-MSI) assessment for the identification of LS and CMMRD in non-neoplastic tissues.

Materials and methods: Blood DNA samples from 131 individuals were grouped into three cohorts: baseline (22 controls), training (11 CMMRD, 48 LS and 15 controls) and validation (18 CMMRD and 18 controls). Custom next generation sequencing panel and bioinformatics pipeline were used to detect insertions and deletions in microsatellite markers. An hs-MSI score was calculated representing the percentage of unstable markers.

Results: The hs-MSI score was significantly higher in CMMRD blood samples when compared with controls in the training cohort (p<0.001). This finding was confirmed in the validation set, reaching 100% specificity and sensitivity. Higher hs-MSI scores were detected in biallelic MSH2 carriers (n=5) compared with MSH6 carriers (n=15). The hs-MSI analysis did not detect a difference between LS and control blood samples (p=0.564).

Conclusions: The hs-MSI approach is a valuable tool for CMMRD diagnosis, especially in suspected patients harbouring MMR variants of unknown significance or non-detected biallelic germline mutations.

Keywords: constitutional mismatch repair deficiency; highly sensitive methodologies; lynch syndrome; microsatellite instability; next generation sequencing.

Figure 1

hs-MSI analysis in the training and validation cohorts. Monomorphic microsatellite markers frequently mutated in MSI-H tumours (n=186) analysed using the mutational load analysis method. (A) MSI score in blood DNA samples from LS (median=0.85, IQR=0.55–1.65, range=0.00–3.33), CMMRD (median=23.58, IQR=21.33–25.49, range=14.84–59.22) and healthy individuals (median=1.1, IQR=0.54–1.65, range=0.00–3.89) from the training set. Significant differences were observed between patients with CMMRD and negative controls (***p=1.24e-05), while no differences were found between patients with LS and negative controls (ns, non-significant, p=0.564). Dashed line indicates the threshold for hs-MSI detection in blood samples. (B) MSI score in blinded samples from the validation cohort. Patients with CMMRD (median=26.28, IQR=19.14–38.37, range=10.56–76.50) and negative controls (median=0.57, IQR=0–1.11, range=0–1.79) were discriminated with no overlapping (hatched area) (***p=2.784e-07). Dashed line indicates the threshold for hs-MSI detection. CMMRD, constitutional mismatch repair deficiency; hs-MSI, high-sensitivity microsatellite instability; LS, Lynch syndrome.

Figure 2

Characterisation of the hs-MSI observed in CMMRD samples. Monomorphic microsatellite markers (selected as frequently mutated in MSI-H tumours) have been analysed (n=186). (A) MSI score in CMMRD blood samples plotted against patient age at blood sampling. No correlation was observed (dashed line, r=−0.04, p=0.823). (B) MSI score in CMMRD blood samples plotted against age of cancer onset. No correlation was observed (dashed line, r=−0.15, p=0.491). (C) MSI score in CMMRD samples plotted against the germline mutated MMR gene. Samples from the same family are indicated by the same symbol. White dots inside symbols indicate samples from the same individual. The buccal mucosa sample is indicated by an arrow. Statistically significant differences between affected genes are indicated (**p<0.005). CMMRD, constitutional mismatch repair deficiency; hs-MSI, high-sensitivity microsatellite instability; MMR, mismatch repair; MSI-H, tumours with high instability.