Validation Of Modeplex Technology For The Determination Of Pole Hotspot Mutations In Endometrial Carcinoma Samples

According to the molecular classification based on TCGA data, endometrial cancer can be separated into 4 groups harboring prognostic and therapeutic implications: 1) Ultramutated (with mutations in POLE gene), 2) Hypermutated, 3) Copy Number Low and 4) Copy Number High. To bring this classification into clinical practice, some authors have proposed a simplified scheme using three immunohistochemical markers (p53, MSH6, and PMS2) and a molecular test for detecting mutations on POLE. The lack of a cost-effective Gold Standard has prompted the development of new techniques such as MODAPLEX (BIOTYPE). In this study, we intend to explore the diagnostic performance of this technology in order to study POLE “Hotspot” mutations in endometrial carcinoma samples. The number of recruited cases has been 258 ECs. From each case a tumor paraffin block has been selected. Sections of 10 μm thickness, have been obtained, subsequently performing DNA isolation and quantification. Cases with a DNA concentration under 10 ng/μl have been excluded, the rest have been tested by MODAPLEX. Every positive result has been reconfirmed by Sanger sequencing. The pilot phase is composed of 105 EC, diagnosed between 2016 and 2020 and with available molecular classification. The second phase includes a retrospective cohort of 30 EC diagnosed between 2000 and 2015, with the aim of checking diagnostic performance of the test when applied to samples with a lesser DNA quality. Finally, we have recruited a prospective cohort, made up of EC diagnosed from the end of 2020 to the present and in which the molecular classification has not been yet performed. A total of 258 samples have been finally submitted to the test. Regarding the first two phases, 30 positive and 103 negative cases have been detected with just a false negative. In this subset, the test have revealed sensitivity and specificity values of 97% and 100%, and positive/negative predictive values of 100% and 99%. Out of 123 prospective cases, the test have detected 16 positive samples. All of them subsequently confirmed by Sanger sequencing. MODAPLEX is a promising technology that allows the determination of the main “Hotspot” mutations in POLE gene in a fast, practical and efficient way.