Driver Mutations Affecting Natural Killer/T Cell Lymphoma
Authors: DONG Gehong , Gong Qiang , Wang Jinhui , Wu Xiwei , Li Yuping , Fend Leticia Quintanilla-Martinez , Wang Jingwen , Liu Hong-gang , McKeithan Timothy W. , Chan Wing Chung 
Published: October 2019

Abstract
Natural killer/T cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma that is rare overall but has a higher prevalence in Chinese and Hispanic populations. Recent sequencing efforts have improved the understanding of the disease and revealed a number of genes that may drive the pathogenesis of NKTCL. These efforts were largely limited by the number of cases studied. Based on previously reported whole exome sequencing data in NKTCL and other lymphoid malignancies and on the frequency and potential biological significance of the mutant, we designed a 334-gene panel and sequenced 105 NKTCLs. We characterized single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations (CNAs) that may have pathogenic roles.
Tumor DNA was isolated from 74 Chinese and 31 Hispanic NKTCL patients. For 7 of these, matched normal DNA was extracted from peripheral blood. The 334 genes in the custom panel were captured using the Agilent SureSelect platform, and the exons of these genes were sequenced on an Illumina HiSeq 2500 instrument. For cases without matched normal samples, we discriminated between somatic and germline variants with an in-house machine learning algorithm which is based on information of public variant databases, variant annotations, and sequencing statistics, and followed by manual inspection. CNAs were determined based on the untargeted sequences using an R package, CopywriteR.
We identified a total of 479 SNVs and 113 indels in 155 genes in 102 out of 105 patients. Mutations in STAT3 and JAK3 were identified in 30% and 7% of the cases, respectively. The hotspot mutations Y640F, S614R, and G618R in the SH2 domain of STAT3 and A573V of the JH2 domain of JAK3 were observed in most of these cases. 46% of the cases harbored one or more mutations in epigenetic regulators ARID1A, EP300, MLL2, MLL3, and TET2. Transcription co-repressors BCOR and NCOR2 were mutated in 25 of the cases. 13% of the cases contained mutations in HLA-A or CIITA, most of which were inactivating, suggesting possible defects in immune surveillance. DDX3X and TP53 mutations were detected in 19% and 10% of the cases, respectively. TP53 mutations were mutually exclusive with mutations in DDX3X and, except for one case, with STAT3 also. MGA mutations, which were present in 12% of the cases, were usually concurrent with STAT3 mutations. The mutational profiles of Chinese and Hispanic cases were similar, and only CD58 mutations were significantly more common in Hispanic patients (p=0.03). Comparison between NKTCL with localized and systemic presentation demonstrated that CCR7 was mutated more frequently in systemic cases (p=0.02). Prognostic analysis identified EP300 mutations as a potential marker for poorer survival (p=0.01). Frequent copy loss on chromosome 6q was observed and PRDM1, which is present on 6q, was mutated in 10% of the cases.
In summary, we identified the profile of mutated genes in a large series of NKTCL. Most of the genes have been previously reported to be mutated in NKTCL, but the frequencies for some of the mutants were quite different. Interestingly, the mutational profiles of NKTCL in Chinese and Hispanic patients were very similar despite the geographic differences. Mutations leading to abnormal activation of the JAK-STAT3 pathway and abnormal chromatin modification were highly prevalent. BCOR, DDX3X and TP53 mutations were also frequent. Variants in CCR7 and EP300 were potential markers for systemic disease and poor prognosis, respectively.
Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Overall survival curves of cases with and without mutations in EP300. Figure. Overall survival curves of cases with and without mutations in EP300.

Disclosures
No relevant conflicts of interest to declare.