Abstract Endemic Burkitt lymphoma (eBL) constitutes the commonest cancer in children in Developing Countries, while the sporadic (sBL) and immunodeficiency associated BL (ID-BL) forms are mainly encountered in Western Countries. The molecular hallmark of three BL variants is the translocation of MYC proto-oncogene to the immunoglobulin-heavy [t(8;14)(q24;q32)] or one of the light chain genes [t(2;8)(p12; q24) and t(8;22)(q24; q11)], leading to constitutive MYC activation. However, additional genetic events contributes to BL pathogenesis, most of which have been studies in sBL only. Here, we performed RNA Sequencing aiming to identify genetic changes possibly cooperating with MYC in the pathogenesis of eBL. We studied by RNA Sequencing (Illumina HiScanSQ) 21 eBL cases, collected at different African Institutions as discovery set. Total RNA was extracted with Trizol and libraries were prepared according to TruSeq RNA sample preparation v2 protocol. Sequence variants were obtained using the SAVI (Statistical Algorithm for Variant Identification) algorithm independently for each sample. Candidate somatic mutations were obtained by eliminating common germline variants and recurrence. To validate single-nucleotide variants (SNVs) we performed Sanger sequencing on these 21 cases. Further, we studied these SNVs by Sequenom Technology and Sanger sequencing on additional 24 eBL cases as validation set. 41 sBL and 8 ID-BL were also considered as controls. We found 66 genes affected by 219 total SNVs with different frequency in 21 samples (range 2-22 for sample). We then focused on genes mutated in at least three samples and predicted to induce somatic protein-changing. We identified 25 genes affected by 172 total SNVs, that were recurrently mutated (min. 3/21 samples; max 11/21 samples). These included genes previously known to be involved in sBL, such as TP53, MYC, ID3, PCBP1 and TCF3 as well as genes involved in other lymphomas such as DDX3X and RHOA. The remaining 18 genes (AGAP6, APBB1IP, ARID1A, ASPSCR1, AVEN, CAD, CCNF, GPATCH4, HERC2, KPNA2, MTBP, MTERFD1, NEK9, NUP133, PARP1, POLQ, SCFD2, TIGD1) were previously not related to BL, and resulted to be involved in several important molecular pathways as cell cycle progression, apoptosis, matrix remodelling, angiogenesis. Sanger sequencing confirmed such SNVs with 100% accuracy in 21 cases of discovery set. Subsequently, we studied them in 24 independent cases of validation set by both Sanger sequencing and by mass spectrometry (Sequenom). We found that most eBL cases carried additional SNVs rather than MYC translocations. Noteworthy, we failed to find these mutations in sBL, confirming the concept that different pathogenetic events may contribute to the pathogenesis of BL subtypes. In conclusion , we discovered new SNVs that might have a significant role in the pathogenesis of eBL. Functional experiments are required to definitely assess their impact. Citation Format: Maria Antonella Laginestra, Francesco Abate, Maryam Etebari, Giulia De Falco, Fabio Fuligni, Maura Rossi, Sakellarios Zairis, Maria Rosaria Sapienza, Anna Gazzola, Claudia Mannu, Federica Melle, Claudio Agostinelli, Mohsen Navari, Cristiana Bellan, Sara Gazaneo, Lucia Mundo, Martin Ogwang, Valeria Calbi, Lorenzo Leoncini, Stefano A. Pileri, Raul Rabadan, Pier Paolo Piccaluga. Identification of single-nucleotide variants by high-throughput RNA sequencing in endemic Burkitt Lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2014-5173