Abstract Background: Malignant pleural mesothelioma (MPM) is a very aggressive neoplasm linked to asbestos exposure. In spite of research efforts to develop more effective therapeutic approaches, its prognosis remains poor, because of the absence of curative treatment. The identification of molecular profiles by omic analyses is a powerful approach to better define MPM subclasses and new targeted therapies. A recent transcriptomic analysis allowed to define a robust MPM classification and to identify two subgroups, C1 and C2, linked to histology and survival. The main finding was to separate epitheloid MPM, the most frequent histologic subtype, within these two subgroups. Epitheloid MPM with a worse survival prognosis were classified in the C2 subgroup. To better define the heterogeneity of MPM and establish a more precise classification, we determined genetic alterations in C1 and C2 MPM. Material and Methods: Using a gene candidate approach, we performed Next-Generation Sequencing (Miseq, Illumina) in 84 MPM including 49 MPM primary cultures and 35 frozen tumors to identify genetic alterations in specific genes with a 200X depth. We selected 22 genes including key altered genes in mesothelial carcinogenesis (NF2, CDKN2A, CDKN2B BAP1, TP53 and LATS2) previously sequenced by Sanger method, genes mutated at low frequency in MPM (KRAS, HRAS, EGFR⋯) and genes recently suggested as altered in MPM (CUL1, KMT2D, SETD2⋯). Results: Previous mutations identified by Sanger sequencing in our collection have been confirmed. The results showed an enrichment in C>T transitions, which was already observed in an exome sequencing study in MPM. We identified 93 variants inducing protein structure modification. Among them, 70% were deleterious to the function of the protein (deletion/insertion, splice-site mutation, substitutions nonsense and damaging missense predicted by SIFT or Polyphen). For most of the genes, the mutation frequency was consistent with literature data. The highest alteration frequency was found in CDKN2A, CDKN2B, NF2 and BAP1 genes (over 25%), lower frequencies were found in TP53, LATS2, KMT2D and SETD2 genes (5% to 20%). The alteration frequency of four genes was different between C1 and C2 tumor subgroups. BAP1 mutations were more frequent in C1 MPM, as we showed previously. We also identified new relevant mutations notably in the methyltransferases SETD2 and KMT2D that are also associated to C1 MPM. Mutations in TP53 were significantly associated with C2 MPM. Conclusion: We defined the genetic alterations signatures of C1 and C2 tumor subgroups. Mutations in enzymes involved in chromatin organization seem to be characteristic of the C1 subgroup. Interestingly, TP53 mutations, which are known to provide tumor aggressiveness, are found in the C2 subgroup gathering patients with the worse prognosis. Ongoing analysis of 83 additional MPM would allow to confirm statistically all the associations we observed. Citation Format: Lisa Quetel, Robin Tranchant, Clément Meiller, Sandrine Imbeaud, Annie Renier, Françoise Le Pimpec-Barthes, Jessica Zucman-Rossi, Marie-Claude Jaurand, Didier Jean. Genetic alterations in molecular tumor subgroups of malignant pleural mesothelioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 112.