Abstract 2448: Deep exome sequencing reveals recurrent somatic mutations in Hodgkin's lymphoma
Authors: Gomez Felicia , Krysiak Kilannin , Ramirez Cody , Bartlett Nancy , Cashen Amanda , Kreisel Friederike , Duncavage Eric , Griffith Malachi , Fehniger Todd , Griffith Obi 
Published: July 2017

High-throughput sequencing provides insights into cancer pathogenesis. The application of genomics technologies to cancers characterized by rare cell populations is a challenge. DNA extracted from bulk tumor samples is a mixture of malignant and non-malignant cells. This is a problem in cancers defined by rare cell populations because it complicates the task of identifying true somatic mutations. Hodgkin lymphoma (HL) exemplifies this challenge. The pathologic hallmark of HL is Hodgkin-Reed-Sternberg (HRS) cells. HRS cells have a low abundance within an affected lymph node (1-30% of tumor microenvironment). Because HRS cells are rare, a limited number of studies have described characteristic genomic events. These studies have examined HL cell lines, and, recently, a small number of flow-sorted HRS-cells (Reichel et. al. 2015). We hypothesized that recurrent somatic mutations can be identified in HRS cells using bulk HL biopsies and ultra deep exome sequencing.

The Illumina HiSeq X,which can generate&gt; 1.6 Tb of sequence data per run, is a applicable tool for the discovery of somatic mutations in HL. Frozen tumor (lymph node)/normal (skin) pairs from 31 patients with HL were examined. Three KAPA libraries were constructed per sample. The libraries were pooled, size selected, and captured using an IDT xGen capture reagent. The pools were sequenced across eight lanes of a HiSeq X. A total of 7.04 x 1012 bases were sequenced across all samples with an average of 1.10 x 1011 bases per sample. The mean depth of coverage achieved across all samples was 783.4x (425.9x - 1091.9x). Given the low abundance of HRS cells we anticipated most somatic sites would have a VAF of 1% -5%. Given the level of coverage we anticipate that most somatic variants will have &gt;5 reads of support.

Instrument data were processed using the McDonnell Genome Institute somatic variant calling pipeline that includes 5 SNV callers and 3 indel callers. We used these steps to filter variants: min. 50x coverage, max. 5000x coverage, min. 1% tumor VAF, max. 5%, normal VAF, and min. 5 variant reads in the tumor. Here we report preliminary data on mutations in five pilot samples. The remaining 26 will be presented at the meeting. We confirmed 7 previously identified recurrent mutations in HL (Reichel et al. 2015). Within our pilot data 13 recurrently mutated genes were found. The most interesting of our recurrent genes is TNFAIP3, which was the second most frequently mutated gene in Reichel et al. 2015. These data suggest that ultra deep sequencing of tumor samples containing rare HRS cells can identify recurrent somatic mutations. Thus, ultra deep exome sequencing may be a useful discovery tool for rare tumor populations, and will improve our understanding of HL.

Citation Format: Felicia Gomez, Kilannin Krysiak, Cody Ramirez, Nancy Bartlett, Amanda Cashen, Friederike Kreisel, Eric Duncavage, Malachi Griffith, Todd Fehniger, Obi Griffith. Deep exome sequencing reveals recurrent somatic mutations in Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2017-2448