Study Of The Association Of Tumor Deposits With Tumour-inltrating Lymphocytes And Prognosis In Gastric Cancer Patients

Background: To investigate the relationship between tumour deposits(TDs) with the clinicopathological characteristics,prognosis of gastric cancer and tumour-inltrating lymphocytes( TILs(cid:0). Methods: The pathological ndings of 369 patients with gastric cancer were retrospectively analysed to observe the expression of TDs, and the levels of stromal TILs . The relationships between TDs status, clinicopathological characteristics, and TILs inltration level were compared using the chi-square test, and rank data were tested using the rank sum test. Kaplan-Meier was used for survival analysis, and the log-rank test was used to determine the differences in survival curves between groups. The prognostic value of TDs was assessed using multivariate Cox proportional hazards regression analysis. Results: TDs were signicantly associated with sex, Lymphovascular invasion, Perineural invasion, pathological TNM stage, and clinical stage (all P<0.05). TILs levels were lower in TDs(+) group and higher in TDs(-) group. TDs(+) group had poor Disease-free survival, cancer-specic survival , and overall survival as compared with TDs(-) groups. Conclusions: TDs is negatively correlated with TILs , and TDs+ was an Independent predictors of the prognosis of gastric cancer. frequently observed and is an indicator of the aggressive characteristics of GC. The presence of TD is a strong and independent prognostic factor and should be incorporated into staging strategies in GC. Regarding the study on the number, size, and prognosis of TDs.Benoit et al. found that the number of TDs ≥ 4 had a lower DFS in rectal cancer [7]. In the present study, we investigated the relationships among the number of TDs, maximum diameter of TDs, and prognosis of gastric cancer. The results showed that the number of TDs was closely related to DFS, CSS, and OS of gastric cancer, and there was a signicant difference in survival between the two groups. But in our study ,the maximum diameter of TDs was not related to prognosis,this is similar to Raul’s study,suggesting that pathologists need to pay more attention to the number of TDs when observing the sections. The critical value of TDs should be veried by larger sample studies.


Introduction
Gastric cancer is an important cancer worldwide, with the fourth highest mortality rate [1]. At present, the prognosis of gastric cancer patients and the formulation of treatment plans mainly rely on clinicopathological parameters and molecular indicators [2,3], but due to the heterogeneity of gastric cancer, none of these indicators can fully accurately re ect the prognosis of gastric cancer patients. Thus, it is especially important to study and enrich new prognostic indicators. Tumour deposits (TDs) were rst described as colorectal cancer mesenteric satellites in 1935 [4], and according to the American Joint Cancer Committee (AJCC) TNM staging system (8th ed.) for colorectal cancer, TDs were clearly de ned as discrete tumour nodules within the lymphatic drainage area of primary cancer and without identi able lymph node tissue or identi able vascular or neural structures [5]. In colorectal cancer, TDs are included in staging treatment because they have been shown to be an independent prognostic factor [6,7]. However, TDs have not been included in the pathological staging of gastric cancer due to limited research evidence. Only a few studies in the literature have found TDs to be strongly associated with a poor prognosis in gastric cancer [2,8,9]. In addition, tumor immune response has gradually become a hot issue in recent years as the study of tumor microenvironment has been intensi ed. However, the relationship between tumor deposition and tumor microenvironment has been rarely reported,tumorin ltrating lymphocytes, as an important component of tumor microenvironment, are an important mechanism for the body to cope with tumor cells and induce tumor immune response [10,11]. Therefore, this study aimed to investigate the relationship between TDs and TILs and prognosis, and provide new ideas for the diagnosis and treatment of gastric cancer.

Interpretation of TDs
Two independent pathologists separately reviewed pathological sections of gastric cancer using a double-blind method, and disagreements were con rmed by a third expert.Positive TD(TDs+) is de ned as discrete tumor nodules within the lymph drainage area of the primary carcinoma without identi able lymph node tissue or identi able vascular or neural structure. [5].

Interpretation of TILs
The two independent pathologists assessed the percentage of stromal TILs in the central tumor and invasive margin of gastric cancer foci using a double-blind method and the assessment methods recommended by the 2014 International Working Group on Tumour-In ltrating Lymphocytes [12]. The methods for interpretation were as follows: (1) determine the extent of TIL assessment (TILs within the tumour border, including border locations, were assessed, and extratumoural and intraepithelial TILs, peritumoral tertiary lymphatic structures, and extensive necrosis or brosis were not assessed); (2) target the area of stromal TILs in the tumour (intraepithelial TILs were not assessed); (3) scan the entire eld of view at low magni cation; (4) determine the type of in ltrating cells, and count only single nucleated cells (lymphocytes and plasma cells); and (5) derive a percentage based on the ratio of the area occupied by stromal TILs to the total area of the interstitium, from which two groups were classi ed: a group with a low-to-medium TILs, with a percentage of < 40%, and a group with a high TILs, with a percentage of 40-90%. Averages were taken after the evaluation of multiview observations and were not focused on the hotspot view with the most in ltration.

Follow-up information
Follow-up started on the rst day after surgery, and the median follow-up time was 27 (1-54) mon, with reviews per month for 3 y post-surgery, every 6 mon for 3-5 y post-surgery, and every 12 mon after 5 years post-surgery. Follow-up information was obtained by telephonic follow-up. Disease-free survival (DFS) was de ned as the time from the start of follow-up until disease recurrence, metastasis, or progression; cancer-speci c survival (CSS) was de ned as the time from the start of follow-up to death due to gastric cancer; and overall survival (OS) was de ned as the time from the start of follow-up to the patient's death due to any other cause.

Statistical processing
All data were statistically analysed using SPSS 21.0 software (IBM Corporation, Armonk, NY, USA). The relationships between TDs status,with clinicopathological characteristics, and TILs in ltration level were compared using the chi-square test, and rank data were tested using the rank sum test. Kaplan-Meier was used for survival analysis, and the log-rank test was used to determine the differences in survival curves between groups. The prognostic value of TDs was assessed using multivariate Cox proportional hazards regression analysis. P < 0.05 indicated a statistically signi cant difference.

Relationships between tumour deposits and the clinicopathological characteristics
Of the 369 gastric cancer cases, 81 had TDs+ (22.0%) (Fig. 1). TDs were signi cantly associated with sex, Lymphovascular invasion(LVI), Perineural invasion(PNI), pathological TNM stage, and clinical stage (all P < 0.05), whereas there was no statistical difference between age, Histologic grade, Lauren's classi cation, and mismatch repair gene(MMR) (all P > 0.05) ( Table 1). For further analysis, the optimal cut-off values for the number and maximum diameter of TDs were selected based on the receiver operating characteristic curve (ROC), and the samples were divided into the number of TDs (< 4 and ≥ 4) and the maximum diameter of TDs (< 7 mm and ≥ 7 mm). The number of TDs was signi cantly associated with pathological N stage (P < 0.05).The maximum diameter of TDs was signi cantly correlated with Lauren's classi cation (P < 0.05) . Table 1 Association between tumor deposits clinicopathologic characteristics of gastric cancer.

2.Relationships between tumour deposits and the prognosis of gastric cancer
The TDs + group had lower DFS, CSS, and OS compared to the TDs-group (Fig. 2), and TDs + was an independent prognostic factor for DFS, CSS, and OS (Table 2). TDs ≥ 4 had lower DFS, CSS, and OS (P < 0.05) (Fig. 3) .The maximum diameter of TDs was not statistically signi cant with prognosis (P > 0.05) (Fig. 4) .

Relationships between tumour deposits and TILs
Among the 369 gastric cancer cases, 279 showed a low-to-medium TILs (75.6%), and 90 were high TILs (24.4%) (Fig. 5). In the TDs + group, there were 74 cases in the low-to-medium TILs and 7 cases in the high TILs. In the TDs-group, there were 205 cases in the low-to-medium TILs and 83 cases in the high TILs, The differences between the two groups were statistically signi cant (P < 0.05) ( Table 3).

Discussion
This study analyzed the relationship between TDs and clinicopathologic characteristics and prognosis of gastric cancer. The results found that 22.0% of the 369 gastric cancer samples was 22.0%, TDs was signi cantly associated with gender, Lymphovascular invasion, Perineural invasion, pathological TNM and clinical stages, and signi cant survival differences between TDs + and TDs-. TDs was an independent prognostic factor of DFS, CSS, OS of gastric cancer.Based on this basis, this topic further studies the relationship between TDs and TILs in the tumor microenvironment, and the results found that the TDs is negatively related to the TILs, suggesting that there may be a complex relationship between TDs and tumor microenvironment, TDs and TILs may interact and affect the prognosis of patients with gastric cancer.
In 1935, Gabriel et al. rst identi ed and reported TDs in colorectal cancer specimens, which they thought were the results of cancer cell dissemination along blood vessels [4]. The 8th edition of the AJCC/Union for International Cancer Control de nes TDs as discrete tumor nodules within the lymph drainage area of the primary carcinoma without identi able lymph node tissue or identi able vascular or neural structure [5]. In the pN staging of colorectal cancer, the absence of regional lymph node metastasis along with the presence of TDs within the subplasma and mesenteric tissues is classi ed as N1c. If both regional lymph node metastasis and TDs are present, the presence of TDs has no effect on staging, and the incidence of TDs in colorectal cancer ranges from 5-45% and is associated with a poor prognosis in colorectal cancer [13][14][15]. Previous studies have found that TDs are present not only in colorectal cancer but also in other solid malignancies, such as gastric, bile duct, and pancreatic cancers [2,16]. Currently, although a few studies have shown that the presence of TDs is an independent prognostic factor for a poor prognosis in gastric cancer [9,10], the mechanism of TDs formation is unclear.For colorectal cancer, the importance of TDs has been recognized and has been included in category N in the 7th edition of the TNM staging system for colorectal cancer. However, in the 8th edition of the TNM staging system, TDs are considered as a metastatic lymph node in gastric cancer, which is contrary to the ndings of the current study. A recent retrospective study that included 7,445 gastric cancer cases showed that the incidence of TDs ranging from 10.6-36.7% (mean: 20.9%) [3]. Liang studied 1,034 gastric cancer patients, of whom 240 (23.21%) had TDs + and found that TDs were an independent prognostic factor for gastric cancer patients [2], which is similar to our ndings. Therefore, the present study demonstrates that TDs is frequently observed and is an indicator of the aggressive characteristics of GC. The presence of TD is a strong and independent prognostic factor and should be incorporated into staging strategies in GC.
Regarding the study on the number, size, and prognosis of TDs.Benoit et al. found that the number of TDs ≥ 4 had a lower DFS in rectal cancer [7]. In the present study, we investigated the relationships among the number of TDs, maximum diameter of TDs, and prognosis of gastric cancer. The results showed that the number of TDs was closely related to DFS, CSS, and OS of gastric cancer, and there was a signi cant difference in survival between the two groups. But in our study ,the maximum diameter of TDs was not related to prognosis,this is similar to Raul's study,suggesting that pathologists need to pay more attention to the number of TDs when observing the sections. The critical value of TDs should be veri ed by larger sample studies.
TILs are T lymphocytes, B lymphocytes, and NK cells that accumulate in the area of the tumour lesion and are at the forefront of the immune response and regulatory role in the tumour immune mechanism [17].Studies have shown that the antitumor immune effect of TILs is mainly cellular, on the one hand, dendritic cells present the major histocompatibility complex molecules of captured tumor neoantigens to T cells, leading to the activation of effector T cells and killing of tumor cells, which in turn secrete suppressive cytokines and have antitumor effects [18]; however, in the majority of cancer patients, the immune system cannot However, in the majority of cancer patients, the immune system fails to function effectively: it may be due to the failure of the immune system to recognize the tumor antigen and treat the tumor antigen as its own, i.e., immune tolerance; the inability of the effector T cells to in ltrate into the tumor lesion, or the suppressor (or immunosuppressive cells) in the tumor microenvironment inhibiting the function of effector cells [19]. In addition, the immune system, while removing tumor cells, also "reshapes" the characteristics of tumor cells to make them more malignant and more resistant to immune attack, i.e., "immune editing" [20]. Therefore, the immune system has a "double-edged sword" role in the process of tumor cell development, because TIL is a major player in tumor immunity, and there are many different subgroups of TILs, and the role of different subgroups in tumor development varies greatly, so the impact on tumor is also different.In the present study [21], TDs have been shown to be an independent prognostic factor for gastric cancer patients, and TDs + was associated with poor prognosis in gastric cancer, which is consistent with previous studies [9,10]. And the present study also found that TDs were negatively correlated with TILs, and TILs levels were lower in TDs(+) group and higher in TDs(-) group.From which we can conclude that in the tumor microenvironment of gastric cancer, TDs and TILs interact with each other to regulate the development of gastric cancer, thus affecting gastric cancer prognosis of patients. However, the mechanism of the interaction between TDs and TILs has not yet been elucidated, and more studies are needed to explore it in the future.
However,there is one limitation that require further discussion. the ndings of this retrospective study from a single Chinese institution may not be generalizable to other settings.Therefore, these ndings should be considered only for hypothesis generation and require additional validation with more extensive studies.
Conclusions our study found that TDs were signi cantly negatively correlated with TILs, TDs was an Independent predictors of the prognosis of gastric cancer, provides potential biological indicators for the diagnosis and treatment of gastric cancer, and enriches the basis of anti-tumor immunotherapy.