Association of PD-1 + Treg/PD-1 + CD8 ratio and tertiary lymphoid structures with prognosis and response in advanced gastric cancer patients receiving preoperative treatment

Abstract

Background: Recent studies have highlighted the distinct ratio of PD-1 + Treg/PD-1 + CD8 for prognosis prediction. However, it remains unclear about the association of this ratio and tertiary lymphoid structures (TLS) with prognosis and response to neoadjuvant or conversion therapy in advanced gastric cancer.

Methods: Firstly, fresh postoperative samples from 68 gastric cancer patients in Renji Hospital were collected. Meanwhile, immune cell infiltration as well as clinical prognosis analysis were conducted. Subsequently, we further systematically evaluated flow cytometry analysis of tumor samples and TLS expression in 38 gastric cancer patients with different response situations after neoadjuvant therapy. Also, a Renji conversion therapy cohort including 10 patients with complete matching samples before and after treatment was established to receive RNA sequencing analysis and multiplex immunohistochemistry (mIHC) tests. The corresponding TLS score and immune cell infiltration were further compared based on therapeutic response variations.

Results: In general, the ratio of PD-1 + Treg/PD-1 + CD8>1 could be regarded as an independent predictor of prognosis in advanced gastric cancer patients. Moreover, PD-1 + Treg/PD-1 + CD8 < 1 and high expression of TLS could indicate better neoadjuvant therapy response and extended survival time in advanved gastric cancer patients. Besides, PD-1 + Treg/PD-1 + CD8 _low &TLS _high group could predict better progression free survival time (PFS) in complete response (CR) subgroup. In response group after conversion therapy, the number of PD-1 + CD8 + T cells significantly increased, mainly occurring outside the TLSs. Meanwhile, the TLSs were also considerably activated as we could observed.

Conclusions: This study underlined that combining PD-1 + Treg/PD-1 + CD8 ratio and TLS were significantly associated with prognosis and preoperative treatment response in advanced gastric cancer. Inspiringly, these indicators have the potential to elucidate the immune balance of advanced gastric cancer patients and can accurately guide subsequent therapeutic strategies.

Keywords: Conversion therapy; Gastric cancer (GC); Neoadjuvant therapy; PD-1 + Treg/PD-1 + CD8 ratio; Tertiary lymphoid structure (TLS).

Fig. 1

Study flowchart for the analysis

Fig. 2

The correlation between T cell ratios and prognosis of gastric cancer patients at different stages. (A): Representative images of the proportion of PD-1 + Treg、PD-1 + CD8、PD-1 + CD4 in Stage I and Stage III gastric cancer; (B): The proportion of PD-1 + Treg/PD-1+CD8、PD-1 + Treg/PD-1+CD4、PD-1 + CD4/PD-1+CD8 between Stage I and Stage II-IV gastric cancer; (C): The Correlation of PD-1 + Treg/PD-1 + CD8、PD-1 + Treg/PD-1 + CD4、PD-1 + CD4/PD-1 + CD8 with GC patients’ overall survival. Kaplan–Meier survival curves for OS based on PD-1 + Treg/PD-1 + CD8、PD-1 + Treg/PD-1 + CD4、PD-1 + CD4/PD-1 + CD8 in total GC patients in Stage I and Stage II-IV GC patients (cutoff values are ratios>1 and median, respectively.); (D): Forest plot displaying the multivariate regression analysis of risk factors affecting 55 advanced GC. (* and ** represented P-values less than 0.05 and 0.01, respectively. ns: no statistical significance)

Fig. 3

The correlation of PD-1 + Treg/PD-1 + CD8 ratio&TLS and neoadjuvant response as well as survival in advanved gastric cancer patients. (A): Representative images of the proportion of PD-1 + Treg、PD-1 + CD8 in responders and non-responders; (B): The proportion of PD-1 + Treg、PD-1 + CD8、PD-1 + Treg/PD-1+CD8 in responders and non-responders; (C):The tertiary lymphoid structures expression in gastric cancer by immunohistochemistry staining (×5). The representative images of responders and non-responders. (The right panel:×20). (D): The Correlation of PD-1 + Treg/PD-1 + CD8、TLS、PD-1 + Treg/PD-1 + CD8 _low and TLS _high with GC patients’ overall survival; (E): The Correlation of PD-1 + Treg/PD-1 + CD8、TLS、PD-1 + Treg/PD-1 + CD8 _low and TLS _high with GC patients’ progression-free survival; (F): The Correlation of PD-1 + Treg/PD-1 + CD8、TLS、PD-1 + Treg/PD-1 + CD8 _low and TLS _high with 14 CR patients’ progression-free survival. (*, **, and *** represent P-values less than 0.05, 0.01, and 0.001, respectively.ns: no statistical significance)

Fig. 4

Representative CT images and fluorescence images between responders and non-responders. (A): Representative CT images and fluorescence images of responders before and after conversion treatment; (B): Representative CT images and fluorescence images of non-responders before and after conversion treatment (The red arrows indicate TLS); (C): The density of CD3+、CD8+、PD-1+、FOXP3 + and CD20 + cells between pre- and post-conversion therapy in responders; (D): The density of PD-1 + CD3+, PD-1 + CD8+, PD-1 + FOXP3 + and PD-1 + CD20 + cells between pre- and post-conversion therapy in responders; (E):The TLS counts between pre- and post-conversion therapy in responders. (*represented P-values less than 0.05, respectively. ns: no statistical significance)

Fig. 5

Distribution of immune cell subsets inside and outside TLS. (A): Characteristics of immune cell subsets inside TLS by multicolor immunofluorescence staining; (B): The density of CD3、CD8、Treg and CD20 between TLS and nTLS before and after treatment. (C): The density of PD-1 + CD3、PD-1 + CD8、PD-1 + Treg and PD-1 + CD20 between TLS and nTLS before and after treatment. (D): Cell subsets in TLS/nTLS density ratio before and after treatment. (*, **and*** represented P-values less than 0.05, 0.01and 0.001, respectively. ns: no statistical significance)

Fig. 6

Related immune analysis between prior- and post-conversion therapy. (A): The Box diagram of immunoactivity analysis; (B): Abundency boxplot of immune cell subsets between groups; (C): Heat map of infiltration analysis of immune cell subsets between groups; (D): Boxplot of T cells’ ability to kill tumor cells between groups; (E): Box map of tertiary lymphoid structure correlation features. (* and ** represented P-values less than 0.05 and 0.01, respectively. ns: no statistical significance)

Fig. 7

The model diagram of immune balance in advanced gastric cancer tumor microenvironment