Genetic Defects Affecting Lymphocyte Cytotoxicity in Epstein-Barr Virus-Associated T/NK-Cell Lymphoproliferative Diseases

Background: EBV-associated T/NK-cell lymphoproliferative diseases (EBV⁺T/NK-LPDs) are a variety of diseases that characterized by clonal expansion of the EBV-infected T or NK cells. In a broad sense, they have a vast spectrum from reactive to neoplastic processes, and from indolent to aggressive or fulminant forms. Such diseases have overlapping clinical manifestations, and are associated with poor outcomes, which usually lead to diagnostic confusion and treatment dilemma. The hallmark of those diseases was refractory elevated EBV DNA titers in target tissues, suggesting genetic defects of the anti-EBV immune response might involve in the pathogenesis.

Methods: We retrospectively enrolled 127 adult patients with EBV⁺T/NK-LPDs, including EBV-associated hemophagocytic lymphohistiocytosis (EBV⁺HLH, n=43), chronic active EBV infection of T/NK-cell type (CAEBV-T/NK, n=27), extranodal NK/T-cell lymphoma of nasal type (ENKTL, n=19), and aggressive NK-cell leukemia (ANKL, n=38). Genomic DNA was isolated from peripheral blood mononuclear cells. Targeted high-throughput sequencing based on Ion Torrent Personal Genome Machine (PGM) together with Sanger sequencing were performed in all the samples, which covering the coding sequences in 13 genes associated with lymphocyte cytotoxicity. Those 13 genes included GZMB , PRF1 , UNC13D, STX11 , STXBP2 , RAB27A , LYST, AP3B1 , SH2D1A , XIAP , MAGT1 , ITK , and CD27 . Probably pathogenic germline variants were filtered by in silico methods and validated by pedigree studies.

Results: A total of 282 probably germline variants were initially identified in this study. According to our filtering strategy, 35 variants were eventually evaluated to be probably pathogenic. Those 35 variants were detected 62 times totally, affecting 59 genes of 47 patients. Mutation patterns were demonstrated in Figure 1. Mutation frequency were 21/43 (49%) in EBV⁺HLH, 13/27 (49%) in CAEBV-T/NK, 5/19 (26%) in ENKTL, and 8/38 (21%) in ANKL. Of 62 detected mutations, 57 (93%) were overwhelmingly missense mutations, 4 were truncated mutations, and 1 was deep intronic mutation. 3/4 truncated mutations were detected in EBV⁺HLH. Mutations were most frequently detected in UNC13D , PRF1 , LYST , and ITK . No mutation was found in STX11 , SH2D1A or MAGT1 . All mutations were heterozygous or hemizygous. Monoallelic heterozygous mutations were quite frequent than expected, accounting for 93% (55/59) mutated genes. Only two mutated genes presented biallelic compound heterozygous, which were both detected in EBV⁺HLH. In addition, di/trigenic synergistic mutations were significantly common in EBV⁺HLH, accounting for 8/21 (38%) mutated EBV⁺HLH cases. However, di/trigenic mutations were rarely in other clinical categories.

Conclusions: Genetic defects affecting lymphocyte cytotoxicity were prevalent in adult EBV⁺T/NK-LPDs, especially in EBV⁺HLH and CAEBV-T/NK. A majority of mutations detected in adult EBV⁺T/NK-LPDs were "milder" type of mutations represented monogenic, monoallelic, and missense, rather than "severer" type of mutations represented di/trigenic, biallelic, and truncated. Accumulation of partial genetic defects affecting lymphocyte cytotoxicity increases the likelihood of developing adult EBV⁺T/NK-LPDs, and the severity of genetic defects was associated with the clinical phenotype of onset. Those provided us a novel insight into the pathogenesis of EBV⁺T/NK-LPDs, and indicate that such spectrum of diseases might be partly a biologic continuum rather than completely discrete entities.

Figure 1

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Figure 1

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