Abstract 4750: Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma

Abstract

Abstract Introduction: Most genomic studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines due to the difficulty of isolating sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. One approach to evaluate primary cases has been to microdissect HRS cells from fresh-frozen tissue biopsies, which has been used for gene expression profiling and array comparative genomic hybridization to assess copy number alterations. However, laser capture microdissection is technically challenging, does not provide a pure tumor cell population, and yields very small amounts of degraded nucleic acids that may not be adequate for whole-transcriptome level RNA sequencing. Methods: We used a flow cytometric cell isolation method, which has enabled rapid isolation of thousands of viable HRS cells from primary cHL tumors (Fromm, et al., Am J Clin Pathol 126, 2006). Here we combined flow cytometry using CD64, CD95, CD30, CD5, CD20, CD15, CD40, and CD45, with the Arcturus PicoPure RNA Isolation kit to isolate high quality RNA from viable HRS cells and intratumor B cells from nine primary cases of CHL respectively. We applied the Clontech SMARTer Ultra Low Input RNA Kit followed by Kapa Library Preparation kit to generate what is to our knowledge the first full transcriptome RNA sequence data from primary cases of cHL. Four cell lines were sequenced using the same methodology. We performed differential expression and gene fusion analyses, and conducted a search for activated pathways. Results: We report the expression signature of purified primary HRS cells. PCA analysis showed that these segregate from HRS cell lines and primary B cells. We confirmed at the RNA level overexpression of relevant amplified genes for which inhibitors are available (AURKA and EZH2), and underexpression of transcripts encoded by genes previously found to be deleted (for example ATM). Pathway analysis revealed extensive downregulation of antigen presentation, consistent with our previous exome sequence analysis showing mutations in beta-2 microglobulin as the most common genetic alteration. Results of the mutational and transcriptional landscape of the HRS cells will be presented. Conclusions: Transcriptome-level analysis of flow-sorted HRS cells allows for expanded study of CHL pathogenesis, new target identification based on altered signaling pathways and potentially individualized approaches to CHL therapy. Citation Format: Jonathan Reichel, Joshua Brody, Olivier Elemento, Raul Rabadan, Ethel Cesarman. Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2015-4750