Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to the rare and aggressive mast cell leukemia (MCL). Somatic mutations in the KIT receptor kinase (most frequently, D816V) can be detected in >90% of patients and are thought to play an important pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL case who came to our attention in 2012. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified mast cells (MCs) isolated from bone marrow at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA-seq on an HiSeq1000 (Illumina, San Diego CA). High resolution karyotyping was also performed with Cytoscan HD arrays (Affymetrix, Santa Clara CA). Among the mutated genes detected in MCs but not in saliva by WES, SETD2 stood out among others because two loss-of-function mutations (a nonsense and a frameshift mutation) inactivating both alleles of the gene were identified. Western Blotting (WB) confirmed the expression of the truncated SETD2 isoform resulting from the nonsense mutation. The SETD2 gene encodes a histone methyltransferase responsible for trimethylation of Lysine 36 of histone H3 (H3K36Me3), a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. Loss of the highly conserved WW and SRI domains was predicted to impair SETD2 binding with RNA polymeraseII and hNRNP L, as confirmed by co-immunoprecipitation. Accordingly, RNA-seq showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes as well as non-canonical splice isoforms. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the role of SETD2-dependent H3K36Me3 in DNA repair and genome stability, Cytoscan HD arrays and WES showed that several losses at many chromosomal loci, together with more than 70 additional point mutations, undetectable at diagnosis, were acquired at the time of progression. Absence of SETD2 protein expression and/or reduced H3K36Me3 were detected in 3/3 additional MCLs and in 6/8 aggressive SMs so far screened. Our data point to epigenetic regulation and/or DNA repair as candidate pathways deserving further investigation in SM - in an attempt to elucidate the mechanisms underlying enhanced clinical aggressiveness and to identify more effective treatment modalities. Molecular characterization of 10 additional MCLs and aggressive SMs is ongoing. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12(L. Bolondi)

Citation Format: Simona Soverini, Caterina De Benedittis, Michela Rondoni, Manuela Mancini, Cristina Papayannidis, Luca Zazzeroni, Viviana Guadagnuolo, Elisa Zago, Francesca Griggio, Alberto Ferrarini, Marianna Garonzi, Massimo Delledonne, Giorgina Specchia, Roberta Zanotti, Omar Perbellini, Livio Pagano, Michele Cavo, Peter Valent, Giovanni Martinelli. Integrated molecular characterization of mast cell leukemia reveals recurrent inactivation of the SETD2 tumor suppressor gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2015-3957