Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors

Abstract

Background: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6.

Methods: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4 bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay.

Results: Using the criteria of ≥ 1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI) = 91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI = 91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI = 80.6-97.5%) and a specificity of 97.9% (95% CI = 92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs).

Conclusions: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

Keywords: Colorectal cancer; DNA mismatch repair; Endometrial cancer; Hypermutated tumors; Microsatellite instability.

Fig. 1

Frequency of allele-size distribution (in base pairs) for the four individual markers in the test set. Allele-size distribution from 212 pMMR tumors and their corresponding normal mucosa, 45 dMLH1, 45 dMSH2, and 15 dMSH6 tumors. For each marker, gray shading indicates the adjusted QMVR

Fig. 2

The performance of the Tetraplex system in the test set. A colored circle denotes a positive for allelic variation (or MSI), and an empty circle indicates a negative for variation (or microsatellite stable) in this specific allele

Fig. 3

Frequency of allele-size distribution (in base pairs) for the four individual markers in the validation set. Allele-size distribution from 97 pMMR ECs: 23 dMLH1, eight dMSH2, eight dMSH6, and two PMS2-deficient ECs. For each marker, gray shading indicates the adjusted QMVR

Fig. 4

The performance of the Tetraplex system on the validation set. A colored circle denotes a positive for allelic variation (or MSI), and an empty circle indicates a negative for variation (or microsatellite stable) in this specific allele