One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

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Copyright: © 2024 Bendixen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Déclaration de conflit d'intérêts

I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.K.B., E.E.H., S.K.E., M.B., U.B.C., and R.K.P. received salaries from Innovation Fund Denmark. The funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors’ salaries and research materials. K.K.B., S.FP., E.E.H., S.K.E., M.B., U.B.C., and R.K.P. were employed at PentaBase A/S during this study. PentaBase A/S is the manufacturer and seller of MicroSight® MSI 1-step HRM Analysis, which is the assay of interest in this study. K.K.B., E.E.H., S.K.E., U.B.C., and R.K.P. are all founders of a patent application covering the novel technology which MicroSight® MSI 1-step HRM Analysis is based on [International Publication number: WO2020229510A1]. The remaining authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.