Abstract 145: Comparison of whole transcriptome sequencing immune repertoire sequencing using RNA for tumor milieu analysis
Authors: Song Seulki , Park Hyejoo , Kim Daeyoon , Kim Sheehyun , Yun Hongseok , Lee Sungyoung , Koh Youngil , Yoon Sung-Soo 
Published: August 2019

Abstract
Introduction Recent rapid methodological advancements of techniques and reduction of sequencing cost have opened the way for high-throughput immune repertoire sequencing to broaden understanding of the adaptive immune system in cancer. T cell receptor (TCR) repertoire and clonotype analysis are essential for characterizing the tumor-associated T-cells. Current immune repertoire assays identify specific alignments of TCR, B cell receptor (BCR), and CDR3 using both whole transcriptome sequencing (WTS) and immune repertoire sequencing. We tried to compare the characteristics of WTS and repertoire sequencing for assessing immune status in cancer.
Materials and Methods A total of 32 RNA specimen from multiple myeloma was obtained from the Seoul National University Hospital. The mononuclear cell (MNC) layer of bone marrow specimens from multiple myeloma (MM) patients was isolated and stained with CD138 microbeads. By analyzing the degree of fluorescence of CD138-PE, RNA from CD138 negative fraction was extracted from the cells, which represents immune milieu of MM bone marrow. The immune repertoire sequencing from ArcherDX and the WTS was performed with the RNA. We investigated the clone diversity, top 10 clonotype proportion with respect to total clone per reads, and V-D-J region recombination usage analysis from each sample using MiXCR, tcR, and VDJtools.
Results WTS and immune repertoire sequencing of CD138 negative RNAs from the 32 MM patients were compared. First, the mean number of clones was significantly higher in repertoire sequenced data compared to WTS data; mean clone number of WTS was 215.48 and was 139,981 for repertoire sequencing. Furthermore, when the proportion of the clone in each samples was listed by its clonal proportion size, the order of clone count were significantly different according to the sequencing methods. For example, the immune repertoire sequencing result of MM_46 sample showed that it contains the 10th largest clones, but WTS sequencing suggested MM_46 sample have the lowest 5th clone. Also, there was also a significant difference in the results of two sequencing data for the top 10 clonotype proportion related to immune adaptation. Finally, in the results of V-D-J region usage analysis, the ratio was completely different and the usage patterns of TCR genes are different according to the sequencing platform.
Conclusion The paradigm of most recent immune system analysis tools has been the use of high throughput repertoire sequencing methods that can yield unprecedented quantitative insights into lymphocyte diversity. Although there are a number of repertoire analyses performed in WTS, caution is required in future data analysis because it shows inaccurate aspects in comparison with immune repertoire sequencing. Immunoassays are preferably performed using repertoire sequencing data.
Citation Format: Seulki Song, Hyejoo Park, Daeyoon Kim, Sheehyun Kim, Hongseok Yun, Sungyoung Lee, Youngil Koh, Sung-Soo Yoon. Comparison of whole transcriptome sequencing immune repertoire sequencing using RNA for tumor milieu analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 145.