Subject characteristics
A total of 9 samples from 9 gastric cancer patients, who were 6 advanced stage and 3 early stage controls were analyzed in this study. The pathological classifications of all tumor is adenocarcinoma. The 6 advanced gastric cancer contained lymph node metastasis and vascular invasion and so on. Anticancer treatment included chemotherapy and surgery therapy. These therapies were often used in combination in most patients. Early stage patients (33.3%) had received Oxaliplatin combined with S-1, and advanced stage patients (66.7%) had received Docetaxel combined with S-1. All the patients received the high throughtput sequencing and two of nine patients were detected P53/PTEN and PI3K mutation respectively. All the patients received the biochemical detects as the baseline examination. Detailed patient characteristics and outcomes are summarized in Supporting Information Tables 1 and 2.
Differences in TCR repertoires between early and advanced stage patients
Diversity of the TCR repertoire can be measured using the V- J and the CDR3 clonotype. We then compared TCR diversity between two groups. We identified a total of 50 distinct V gene segments and 13 distinct J gene segments from every sample. Of these fragments, the most frequent V gene segments were TRBV7-2 (23.11% early stage, 14.91% advanced stage) and TRBV7-8 (18.76% early stage, 14.24% advanced stage) were more expression in patients with early gastric cancer, whereas other fragments were more expression in advanced stage gastric cancer controls. Five of the most frequent J gene segments were TRBJ1-5(24.75% ) 、TRBJ 2–2 (23.68%,p ༜0.05)、TRBJ1-6 (13.95%)、TRBJ2-5 (12.89%) and TRBJ2-6 (1.88%) in advanced gastric cancer,whereas other fragments were more expression in early stage gastric cancer (Fig. 1A). As shown in Fig. 1B, a heat map was generated according to the usage frequency of V and J gene segments. Furthermore, we also detected the composition of V-J gene combinations and found a total of 671 distinct V-J gene combinations. V-J paired fragments exhibit more diversity in early stage gastric cancer group than advanced stage group (Fig. 1C, p = 0.028). Further, The composition of V-J gene combinations revealed significant usage differences in two groups, including TRBV3-2_TRBJ1-1、TRBV7-3_TRBJ1-1、TRBV7-8_TRBJ2-3、TRBV5-8_TRBJ1-5 (Fig. 1D,P༜0.05). Compared with early and advanced stage, the expression of V-J gene combinations were different in two groups (sFig. 1A and 1B). CDR3 clonotypes, which include a conserved cysteine in the V region and a conserved phenylalanine in the J region, determine the diversity of the TCRβ repertoire.We first investigated CDR3 variety and expression in two groups and found that advanced gastric cancer patients showed significantly lower V-D-J and CDR3 variety compared to early controls (Figs. 1E and 1F). The different expression levels of V-D-J and CDR3 were analyzed by crater plots. As shown in Figs. 1G and sFig 1C, there were differences in the expression levels of some segment combination between advanced and early gastric cancer. Analysis of V-D-J fragments revealed that 6 VDJ fragments exhibited differential expression between two groups (sFig. 1D). Furthermore, Rank-abundance and normalized Shannon diversity entropy were used to compare CDR3 diversity. All results suggested a remarkably decreased CDR3 diversity in advanced groups (Fig. 1H and sFig. 1E,F). These data demonstrated that the TCR repertoire in peripheral blood samples from gastric cancer patients could distinguish between early and advanced gastric cancer in several aspects. The advanced stage changed the usage of several key low-frequency CDR3 and lessened TCRβ diversity. The patients with advanced gastric cancer exhibit limited TCR repertoire diversity and that this diversity is further limited in patients with advanced stage. Moreover, these changes may implied an altered immune status associated with gastric cancer stage.
The prognosis value of TCR repertoires in the advanced gastric cancers
Next we continued to explore the prognostic value of the TCR repertoire. To determine whether patients with poor prognosis demonstrated differences in diversity and gene expression level, we identified six advanced stage patients. Among B1 and B3 who have longer DFS (37.2 and 26.5+ months respectively )were as control group. To be similar ,we identified a total of 48 distinct V gene segments and 13 distinct J gene segments from every sample. Of these J fragments exhibited different usage frequency between two groups, TRBJ2-2 was 49.04% in control group whereas 11.00% in case group. TRBV7-2 and TRBV7-8 counted of 49.92% in control group whereas 19.96% in case group (Fig. 2A). As shown in Fig. 2B, a heat map was generated according to the usage frequency of V and J gene segments. Further, The composition of V-J gene combinations revealed significant usage differences in two groups, TRBV5-6_TRBJ1-2 and TRBV6-1_TRBJ1-5 showed more frequency in control group especially TRBV6-1_TRBJ1-5 combinations; however, TRBV19_TRBJ 1–4 showed lower frequency in control group (Fig. 2C and 2D). However, cluster analysis of the V-J and V-D-J fragments showed that 4 VDJs and 2 VJ fragments showed different expression levels between shorter DFS gastric cancer patients and longer controls (Fig. 2E and sFig.2A). The difference in expression levels of V-J / V-D-J and CDR3 fragments was then analyzed by crater plots and heatmap. As shown in Figs. 2F and 2G, there was a difference in the amount of gene expression in gastric cancer with a long DFS, especially that some fragments of the CDR3 region have abnormally high expression(sFig. 2C). In addition, V-J paired also showed different expression between groups (sFig. 2B).These data suggest that peripheral blood CDR3 and VDJ expression provide valuable prognostic information. Differential expression of TCR profiles in peripheral blood samples of patients with advanced gastric cancer may predict prognosis. This results indicated that not only the ratios of common T-cell diversity gradually decreased, but their frequencies also gradually changed, some high-frequency TCR clones turned into low-frequency ones, while some lesion specific clones abnormally amplified.
Mutation and TCR repertoires among the gastric cancer
All the patients received the ctDNA detect and NGS detect.Among of these patients there were 2 patients were detected P53、PI3K and PTEN mutation (table 2), therefor the two patients were as the control group compared to other patients. We identified a total of 48 distinct V gene segments and 13 distinct J gene segments from every sample. Of these fragments, The most frequent J gene segments were TRBJ 1–6 (41.82% mutation group, 0.78% wild group ) and TRBJ 2–7 (22.85% mutation group, 4.74% wild group) were more frequencies in patients with mutation group, whereas other fragments were more expression in wild group; however, the frequent of V gene segments was similar in mutation and wild groups(Fig. 3A). Figure 3B demonstrated a heat map which was generated according to the usage frequency of V and J gene segments. As shown in sFigure 3A and 3B, there were differences in the expression levels and preferential usage of some J genes fragments between mutation and wild patients. The analysis of V-J and V-D-J fragments revealed that VDJ and VJ fragments combination exhibited differential usage frequencies between mutation and wild gastric cancer patients(Fig. 3C and sFig3C). Then the analysis of the differences in V-J/V-D-J and CDR3 expression was performed by the volcano plot. As shown in Fig. 3D、E and sFig3D there were difference expression in mutation and wild gastric cancer, especially CDR3 have down expression in wild gastric cancer; however there was no significant diversity of V-D-J and CDR3 clonotype in two groups(data not shown). These data demonstrate that the somatic mutation in peripheral blood samples from patients is not relation with clonotype number and diversity ; however, the there is relevant with expression and usage frequencies.