Purpose/Background: cHL patients who receive standard therapy have a high rate of event-free and overall survival. However, some patients (~10%) will be refractory to initial therapy and up to 1/3 will relapse. Thus, improved methods of prognostication and new treatment targets are needed. High-throughput sequencing can identify recurrent somatic mutations that drive lymphomagenesis and impact treatment response. However, Hodgkin-Reed-Sternberg (HRS) cells have a low (~1%) abundance in cHL biopsies, creating a challenge for comprehensive and accurate detection of somatic mutations in bulk lymphoma biopsies. Genomic studies of cHL have characterized HRS somatic mutations through the analysis of malignant cells obtained using purification techniques, cell-free DNA, or DNA amplified through whole-genome amplification. We hypothesized that ultradeep sequencing of bulk lymphoma biopsies provides a more accessible approach to HRS characterization while also creating robust and reproducible data.

Methods: We performed exome sequencing on 32 fresh frozen samples from 31 cHL patients obtained prior to treatment (27) or after relapse (4) with paired normal skin samples (31). The Illumina HiSeq platform (2 x 150bp reads) was used with multiple independent library constructions and a 1,000X median coverage goal. Sequence data were aligned to GRCh38. SNVs and INDELs were called using multiple algorithms. We employed several variant filtering strategies, including manual review, to remove common polymorphisms and false positives. Because we discovered mutations with VAFs close to the platform error rate (~1%), we used an orthogonal sequencing strategy (Haloplex) to validate all somatic variants.

Results: We observed 4,020 somatic variants. On average, we observed 32 protein-coding mutations/case, excluding one hypermutated case in which 3,084 variants were observed. We identified a potential loss-of-function insertion in MSH6 that could explain the hypermutated phenotype. We achieved a 99% validation rate across the cohort for somatic variants discovered in exomes. We confirmed known recurrently mutated cHL genes (e.g., SOCS1 [43%], STAT6 [20%], TNFAIP3 [40%]). We identified several significantly recurrent mutated genes not well characterized in cHL, including IGLL5 [26%] and IL4R [13%]. All IL4R mutations are potential loss-of-function mutations that could result in greater activation of STAT6 through ablation of ITIM negative modulation. We identified an enrichment of SOCS1 and IGLL5 mutations that is likely the result of aberrant somatic hypermutation. Pathway analysis also identified an enrichment of mutations in MAPK pathways.

Conclusion: These data suggest that cHL somatic mutations can be confidently identified via ultradeep exome sequencing without cell purification. We show that cHL genomes harbor somatic variation that inform new targets for treatment and prognostication.

Citation Format: Felicia Gomez, Matthew Mosior, Zachary Skidmore, Alina Schmidt, Fernanda Rodrigues-Martins, Kilannin Krysiak, Cody Ramirez, Eric Duncavage, Grace Triska, Lee Trani, Nancy Bartlett, Amanda Cashen, Neha Mehta-Shah, Friederike Kreisel, Malachi Griffith, Todd Fehniger, Obi Griffith. Ultradeep sequencing of classical Hodgkin lymphoma (cHL) identifies recurrent somatic mutations and demonstrates the production of reproducible data from rare malignant cells [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-06.