T Cell Receptor β-chain Repertoire Analysis Of Esophageal Squamous Cell Carcinoma

Background:Oesophageal cancer is one of the most frequency cancer worldwide, an estimated 455,800 new oesophageal cancer cases and 400,200 deaths occurred in 2012 worldwide. The highest rates are found in Eastern Asia and in Eastern and Southern Africa, especially in China. The 2 main types of oesophageal cancer are squamous cell carcinoma and adenocarcinoma,oesophageal squamous cell carcinoma (ESCC) is the generally type in China. ESCC has a generally poor prognosis due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of TILs response which depends on the interaction between antigens and T cell receptor (TCR).Objective:Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated ESCC samples can provide insights into the clonality and heterogeneity of intratumoral T cells in ESCC, a tumor type that can display extensive genetic intratumor heterogeneity(ITH).Materials and Methods:Our study included tumour, adjacent normal tissue and peripheral blood specimens collected from seven patients diagnosed with primary ESCC at the time of tumour debulking surgery. Every tumer tissue included at least four sites.In parallel, turner infiltration by CD3 T cells was evaluated by immunohistochemistry. For each sample, DNA was extracted directly and TCR β CDR3 region was amplified by Multiplex PCR and sequenced by Illumina Hiseq2000 platform. To evaluate the contribution of sampling noise and sequencing error introduced by PCR and sequencing method relative to genuine spatial TCR β repertoire variability, we performed replicate PCR reactions in all four tumour samples from Patient 1.Results:The total number of unique TCRb reads detected in each sample was similar, distributed among a median of 99,899 (interquartile range,75,525-155,647) unique TCRb CDR3 sequences for each tissue sample from seven patients. The TCR β repertoires of the tumour tissues were not significantly more oligoclonal than their matched other tissue types (mean clonality 0.74 for tumour samples vs. 0.75 for other tissue types; P=0.275 by unpaired t-test). The average fraction of the top 100 TCRb sequences was 35.8％in tumour,31.4％in normal tissue and 31.5％in blood; and they were not significantly different (one-way ANOVA, P=0.402). Only 13-33％(mean 26%, std. dev.0.06) of the 100 T cell clones detected with the highest frequency in each tumour sample were ubiquitous, whereas 67-87％(mean 74%, std. dev.0.06) of the T cell clones were heterogeneous.Conclusion:(1) Intratumoural TIL populations in ESCC could be highly polyclonal. (2) Expanded T cell clones within tumours may mainly be reacting to tumour antigens. (3) TIL populations found in ESCC were spatially heterogeneous. (4) Ubiquitous T cells clones could be activated by tumour specific antigen.