Abstract 173: Profiling T cell clonality in FFPE samples using a targeted sequencing approach: Critical parameters and limitations
Authors: Montel Valerie N. , Greene Stephanie B. , Tsai Jeff H. 
Published: September 2020

Abstract
With the rapid development of immuno-therapy approaches for cancer treatment, tools that characterize tumor-infiltrating immune cells fuel new opportunities to better understand the natural defense mechanisms against tumor cells. The AmpliSeqTM for Illumina Immune Repertoire Plus, TCR beta SR Panel is a targeted sequencing assay that assesses T cell clonal diversity by amplifying the CDR3 region of the TCRβ gene in both DNA or RNA. This study describes the performance of this assay on peripheral blood mononuclear cells (PBMC) as high-quality input as well as formalin-fixed paraffin embedded (FFPE) samples, using the MiXCR Immune Repertoire Analyzer app for clonotype analysis. Multiple replicate libraries prepared from RNA (n=6) and DNA (n=12) extracted from the same PBMC donor showed a minimum of 96.4% alignment, with high reproducibility between replicates for clonotypes (&lt;7% CV). Interestingly the RNA input had a 25x higher clonal diversity compared to its DNA counterpart, highlighting a fundamental difference between the two inputs to be taken into consideration for optimal sequencing requirements. Indeed, when we tested various inputs of DNA or RNA at different sequencing depths, we found that the clonotype count reaches saturation at a much lower read depth for DNA samples than for RNA. Looking at the top 50 clones ranked by abundance, we also noticed that the DNA assay only showed 58% common clones between 4 replicates whereas the respective RNA assay showed a much higher replicate reproducibility (96% of common clones). To further investigate the sensitivity of this assay, we modelled the biological representation of immune cells and nucleic acid quality in FFPE tumor samples by titrating heat-treated PBMC RNA (10%, 1% and 0.5%) into a TCRβ-negative RNA background. The Shannon index, a classic TCRβ clonal richness metric, showed that RNA quality was a more critical parameter than the input amount. Evenness (clone frequency), however, was robust to both parameters. Looking at the top 50 clones in four FFPE samples, we noticed a clear split between two distinct trace patterns: a very skewed frequency distribution with a couple of highly represented clones (“activated pattern”) versus a more even distribution, like PBMC controls. Except for the lymph node biopsy, all three skin biopsies showed a direct correlation between the plot pattern and the pathologist inflammation assessment. Taken together, with this limited dataset, this study shows that AmpliSeqTM for Illumina Immune Repertoire Plus, TCR beta SR Panel is a flexible assay that delivers T cell clonal diversity information, even in challenging samples like FFPE. While the data significance still remains to be established, its availability to the immuno-oncology research field opens up promising avenues for future cancer treatment discoveries.
Citation Format: Valerie N. Montel, Stephanie B. Greene, Jeff H. Tsai. Profiling T cell clonality in FFPE samples using a targeted sequencing approach: Critical parameters and limitations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 173.