Analysis of the Genomic Heterogeneity in Hodgkin Lymphoma Using Next Generation Sequencing
Authors: Mata Elena , Díaz-López Antonio , Martín-Moreno Ana M , Montalbán Carlos , Mestre Maria J , Santonja Carlos , Burgos Fernando , Menárguez Javier , Piris Miguel A , García Juan F 
Published: October 2019

Abstract
Classical Hodgkin´s lymphoma (cHL) is a clonal proliferation of the characteristic Hodgkin and Reed Sternberg cells (HRS), diluted in a reactive inflammatory background, and characterized by a defective B cell immunophenotype. Few studies have been aimed to the identification of the driver mutations in this disease, and only occasional gene mutations in members of the NF-kappaB and JAK/STAT pathways, and, more recently B2M, have been previously described.
We analyzed 57 cHL tumor samples (FFPE) using massive parallel sequencing (Ion Torrent™ semiconductor technology). To increase coverage a previous tumor cell enrichment process by punch tissue cores from selected tumor-rich areas was implemented. In addition, we sequenced 7 cHL cell lines using the same technical approach. Bioinformatics analysis, including alignment with germline sequences and SNPs filtering, were done using the Torrent Suite Software and Variant Caller. Pathogenic prediction of single nucleotide variants (SNVs) and mutation interpretation were performed using the Alamut and Provean softwares. All experiments were done in duplicate to minimize false positive rates.
Overall, the results show very high genomic heterogeneity with a range of 10-400 SNVs per sample, most of them (~60%) missense type. We found a relatively large number of genes recurrently mutated at low frequency and only a few genes mutated in up to 15-20% of the patients, reflecting a high level of genomic instability in the neoplasm. Specific mutations in genes previously described in cHL (NFKBIA, TNFRSF14) and in diffuse large B-cell lymphomas (CARD11, STAT6, CREBBP, CMYB) were consistently found, as well as new SNVs in genes not previously described (BTK, NFKB2). Mutations affecting selected genes (B2M, CARD11, NFKBIA, CSF2RB, and STAT3) in cell lines were additionally validated by Sanger sequencing.
Of note, we found high prevalence of mutations affecting BTK and the BCR pathway, suggesting some dependencies of active BCR signaling albeit the absence of BCR expression by HRS cells. Consistent with this interpretation, incubationof a panel of cHL cellular models with either Ibrutininb or AVL-292, selective-BTK inhibitors, in vitro constrains cell proliferation and causes cell death. Interestingly, the concentration to induce cell apoptosis in cHL was comparable to other cell models of diffuse large B-cell and mantle cell lymphoma sustaining the requirement of an active BCR signaling for cHL cell viability.
In conclusion, cHL is characterized by high genomic instability, including numerous mutations in genes related with B-cell function and specific signaling pathways. Pathogenic predictions of the different SNVs identify potential driver mutations that can be associated with the pathogenesis of the disease and might represent new therapeutic targets.

Disclosures
No relevant conflicts of interest to declare.