AACR 2014 --Hua Fang

Abstract Number: 3568
Presentation Title: Quantitative t cell receptor (tcr) repertoire analysis by next-generation sequencing (ngs) in non-small cell lung cancer patients treated with therapeutic cancer peptide vaccines
Presentation Time: Tuesday, Apr 08, 2014, 8:00 AM -12:00 PM
Location: Hall A-E, Poster Section 23
Poster Board Number: 9
Author Block: Hua Fang1, Rui Yamaguchi2, Xiao Liu1, Yataro Daigo3, Satoru Miyano2, Yusuke Nakamura11University of Chicago, Chicago, IL; 2The University of Tokyo, Tokyo, Japan; 3Shiga University of Medical Science Hospital, Otsu, Japan
Abstract Body:

Cancer immunotherapy is expected to become one of the major treatment options of cancer, and there is lots of enthusiasm for the development of therapeutic cancer vaccines. To better understand the molecular mechanism of action of therapeutic cancer peptide vaccines and also to better monitor patients’ immunological response as a predictive biomarker, a fast, sensitive, comprehensive, and quantitative method to characterize T lymphocytes is highly desired. However, TCR repertoire diversity and clonotype follow-up after cancer vaccination represent a major challenge due to the enormous complexity of the TCR that characterizes individual T cell clones. Here, we demonstrated a new high throughput sequencing method to investigate the detailed genetic profiling of the TCR repertoire of millions of peripheral T lymphocytes from non-small cell lung cancer (NSCLC) patients who had been treated with cancer vaccine cocktail of peptides derived from CDCA1, URLC10, and KIF20A. The new technology of cDNA sequences of TCR α and β chains using next generation DNA sequencer (Ion Torrent Personal Genome Machine (PGM) with 400bp sequencing chemistry) enabled us to assess the comprehensive TCR α and β repertoire information in an unbiased way. We also developed a novel algorithm ‘VDJ decomposition with soft-clipping’ that allowed us to identify the rearranged V-(D)-J regions in both TCR α and β chains, to analyze non-template nucleotides added at the junction sites, and also to determine the exact length and sequence of the complement determining region 3 (CDR3). In total, 16 samples from 5 NSCLC patients with different time points (before and after treatment) were analyzed for both TCR α and β repertoire. We obtained average 239,689 (range: 103,851 - 329,817) VJ identified reads for TCR α sequences and 499,210 (range: 193,753 - 982,040) VDJ identified reads for TCR β sequences. Significant increases of specific TCRβ CDR3 sequences were observed in patients showing stable condition after vaccine treatment. In patient #1 with long stable disease (SD) after treatment, two enriched clones of CASSSLQNIQYF and CASSPSVGLAGVAQKETQYF were present after vaccine treatment, suggesting those clones might represent peptide-specific cytotoxic T lymphocytes. Similarly, enriched CDR3 clones of CASTVRQKDGYTF and CSVGAYRGETQYF were observed in patient #3 and #5 at SD condition. In conclusion, this newly-developed NGS technology with the novel algorism should contribute to the better understanding of the immune responses in patients treated with immunotherapy. Our approach can also be applied for the investigation of a wide range of immune-related disorders including rejection after organ transplantation.