Four methods to analyze H3K27M mutation in diffuse midline gliomas

The histone H3 K27M mutation has been frequently reported in the majority of diffuse midline gliomas, which is considered as a prognostic and predictive biomarker. A number of different methods and platforms including pyrosequencing (PSQ), sanger sequencing, immunohistochemistry (IHC), Mass array and NGS (Next Generation Sequencing) have been used to detect H3K27M mutation in diffuse midline gliomas. However, controversy remains about the most appropriate method to use for analyzing H3K27M status. The H3K27 M mutation status of a total of 50 diffuse midline gliomas was examined using PSQ, sanger sequencing, IHC and Mass array in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity and correlation with the other assays were calculated. Among 50 diffuse midline glioma cases, the H3K27M mutation was positive in 64 %, 66 %, 62 % and 62 % of the cases by PSQ, IHC, sanger sequencing and mass array, respectively. The sensitivity and specificity of IHC were 100 % and 94.4 %, respectively. The sensitivity and specificity of sanger sequencing and mass array were both 96.9 % and 100 %, respectively. This study demonstrated that IHC is an effective and rapid detection method for routine use in pathology laboratories for the identification of H3K27M mutation. A combination of IHC and sanger sequencing assays can provide 100 % sensitivity and specificity for the prediction of H3K27M status.