Changes In Tumor Infiltrating Lymphocytes Of Peripheral Blood And Tissue During Chemotherapy In Patients With Gastric Cancer

To study the state of systemic immunity and local immunity before and during chemotherapy in patients with gastric adenocarcinoma. From 2017 to 2018 at the Tashkent city branch of the Republican specialized scientific and practical medical center of oncology and radiology 20 primary metastatic patients with gastric adenocarcinoma received chemotherapy. The sampling of biological material (peripheral blood, tumor tissue) was carried out twice (before treatment and during the first control examination, after 3 courses). The percentage of the degree of infiltration of tumor tissue by lymphocytes (CD45+CD14-TILs) was estimated by flow cytometry; T cells (CD3+CD19-TILs); B cells (CD3-CD19+TILs); NK cells (CD3-CD16+CD56+TILs); CD16 and CD8 effector cells and their cytotoxic potential (CTP) (CD16+Perforin+TILs; CD16CTPTILs), (CD8+Perforin+TILs; CD8CTPTILs); regulatory T cells - NKT cells (CD3+CD16+CD56+TILs), CD4 (CD4+CD25+CD127-TILs) and CD8 (CD8+CD11b-CD28-TILs) regulatory cells and these parameters of systemic results. The factor of a favorable prognosis for PFS in patients with metastatic gastric cancer in the peripheral blood was an increase in the number of CD8 + T-regulatory cells (5.1% - 12.1%, p = 0.019), and in tumor tissue - an increase in the perforin potential of effector CD16 cells (0.5% - 4.9%, p = 0.030) and their cytotoxic potential (13.2% - 55.7%, p = 0.011). When assessing the changes in the indices of local immunity during chemotherapy, it was noted a negative effect of an increase of T cells (22.0% - -9.7%, p = 0.012), NKT - cells (207.9% - -13.8%, p = 0.002) and CD4 + T-regulatory cells (190.7% - -25.2%, p = 0.002). In contrast, an increase in the level of effector CD16 cells during chemotherapy increases the likelihood of surviving PFS - 9 months (-69.5% - 9.1%, p = 0.013). Indicators of local and systemic immunity serve as additional prognostic factors for gastric cancer.


INTRODUCTION
Gastric cancer (GC) ranks 5th place among oncology diseases (1,313,000 cases) and is the 3rd cause of death from cancer (819,000 deaths) in the world [1]. Currently, there is no doubt that a malignant tumor is a dynamic system, considered in combination with all morphological components that form its microenvironment: stromal cells, cells of the immune system, blood and lymphatic vessels and extracellular matrix [1,2]. Tumorinfiltrating lymphocytes (TILs) are the subject of active research [3][4][5].They play a key role in the concept of "antitumor immunity". In addition, the cells of systemic immunity are responsible for both suppressing tumor growth [6] and, on the contrary, correlate with a poor prognosis [7,8]. Thus, over the past 20-30 years, great success has been achieved in the treatment of gastric cancer. An important step in the development of new therapeutic agents was the understanding of the role of prognostic and predictive factors, including the significance of the subpopulation composition of immunocompetent cells and tumor biology.
The aim of this work was the comprehensively study the structure of tumor-infiltrating lymphocytes (TILs), systemic immunity before and during treatment in metastatic patients with gastric adenocarcinoma.

MATERIALS AND METHODS
The prospective study included patients with metastatic gastric cancer (GC) who were treated at the Tashkent city branch of the Republican specialized scientific and practical medical center of oncology and radiology in the period from 2017 to 2018. Patients underwent 9 two-week cycles of chemotherapy (CTx) of the 1st line or 6 threeweek courses with subsequent observation until the disease progressed. Analysis of the immune status by morphological material and peripheral blood was carried out twice (before the start of treatment and after 3-4 courses of chemotherapy). The main criteria for the inclusion of patients in the study: age over 18

FLOW CYTOMETRIC ANALYSIS
The structure of subpopulations of immunocompetent cells was assessed by binding to monoclonal antibodies of various specificities by multivariable quantitative analysis on a FACSCalibur flow cytometer (BD Biosciences). For each sample, at least 500-5000 cells were analyzed in a CD45+gate. DotPlot analysis of cytograms was used with the commercial BD CellQuest PRO software (BD Biosciences). Further processing of the FSC files of primary cytometric data was performed using the WinMDI software package, version 2.8.

STATISTICAL PROCESSING OF RESULTS
Statistical processing of the material and calculations of indicators were carried out using the statistical software package Statistica for Windows v. 10  Differences were considered significant at p <0.05 (acceptable level of α-error 5%). The degree of relationship between the parameters was assessed using the Spearman correlation analysis. Determination of the boundaries with the optimal ratio of sensitivity and specificity was performed by constructing the ROC curve.

Patient characteristics
The study included 20 patients with mGC -9 (45%) men and 11 (55%) women. The age of the patients ranged from 44 to 64 years (mean age 58). Depending on the division of patients into age groups -up to 45 years old; 46-60 years old; over 60 years old, patients mainly belonged to the 3rd group -9 (45%), respectively. In patients with mGC, the tumor was poorly differentiated -12 (60%) and represented by the intestinal type Lauren 12 (60%). HER2neu status was positive in 6 (30%) patients. In 20 patients, no disturbances in the repair system were noted (MSS). The average follow-up time for patients was 16.4±6.2 months. (from 0.7 to 23.6 months, median 18.5 months). In the surgical group, 2 patients (4%) had a high level of MSI.
Study of cellular immunity in peripheral blood.
The results are shown in Table 1. Thus, the percentage of effector CD16+cells and their cytotoxic potential before chemotherapy was higher than during treatment (p= 0.031; p=0.009). However, after 3-4 courses of chemotherapy, there is a statistically significant increase in the percentage of T-regulatory cells with the phenotypes CD4+CD25+ CD127-(p=0.007) and CD8+CD11b-CD28 (p=0.031).
To assess the possibility of predicting early progression in gastric cancer patients according to blood parameters already before the start of treatment, we compared the results of the study of systemic cellular immunity in subgroups where the progression of the disease was recorded up to 6 months and later (Table 2). It follows from the table that a high level of CD8 T-regulatory cells with the CD8+CD11b-CD28-phenotype before treatment is a predictor of a favorable prognosis (p = 0.019).
The nature of changes in indicators during chemotherapy can also be informative for predicting early progression, so we compared the magnitude of this change depending on the timing of progression.
However, in patients examined in dynamics, early progression was not noted (up to 6 months). Therefore, to analyze the dynamics of indicators, we have chosen the border of the progression time frame in 9 months. Among the "linear" structure of lymphocytes, the median of T and B cells increases in patients with progression before and after 9 months, 8  The results are shown in Table 3.  To assess the ability to predict early progression in breast cancer patients already before starting treatment, we similarly compared the results of a study of local immunity in subgroups where disease progression was recorded up to 6 months and later (Table 4). Thus, a low level of perforin potential of effector CD16 cells (p=0.030) and their cytotoxic potential (p=0.011) before chemotherapy is an unfavorable factor and may indicate a higher risk and probability of progression up to 6 months.
When assessing the nature of changes in indicators during chemotherapy to predict early progression, we also chose the 9-month progression time limit. It was noted that patients with progression up to 9 months are characterized by a decrease in the degree of infiltration of tumor tissue with lymphocytes, NK cells, perforin potential of effector CD8 cells and cytotoxic potential of CD16 (-7.5% -18.1%, p=0.031) and CD8-cells, as well as CD8+T regulatory cells, and, on the contrary, an increase in T cells (22.0% --9.7%, p=0.012), CD8+CTP, perforin potential of effector CD16 cells, NKT cells (207, 9% --13.8%, p=0.002) and CD4+ T-regulatory cells (190.7% --25.2%, p=0.002).
However, in patients with progression after 9 months, there is an increase in the degree of infiltration of tumor tissue with lymphocytes, NK cells, perforin potential of effector CD8 cells and cytotoxic potential of CD16 and CD8 cells and CD8+ T regulatory cells, and the level of T cells, CTLs of CD8+ cells , the perforin potential of effector CD16 cells, NKT cells and CD4+ T regulatory cells decreases.
Thus, patients with gastric cancer with progression up to 9 months are characterized by a statistically significant increase in T cells (p=0.012), NKT cells (p=0.002), and CD4+ T regulatory cells (p=0.002). On the contrary, an increase in the perforin potential of effector CD16 cells (p=0.030) and their cytotoxic potential (p=0.011) is a favorable prognostic factor and increases the likelihood of achieving long-term PFS.
These changes in the indices of local and systemic immunity in dynamics indicate an increase in cytotoxic CD8+ lymphocytes, NKT cells and CD4+ T regulatory cells in peripheral blood and tumor tissue during treatment.
However, a high level of CD8+ T regulatory cells in peripheral blood and a low level of perforin potential of effector CD16 cells and their cytotoxic potential in tumor tissue prior to treatment indicate a low and high risk and probability of progression up to 6 months, respectively. At the same time, an increase in the level of effector CD16 cells in peripheral blood and tumor tissue before and during treatment is a favorable prognostic factor and increases the likelihood of progression after 9 months.
On the contrary, an increase in T cells, NKT cells, and CD4+ T regulatory cells in tumor tissue is characteristic of patients with gastric cancer with a probability of progression up to 9 months.

DISCUSSION
Cancer development is a complex process that depends on the interaction of individual cells in the tumor, the microenvironment, and the immune system, which can both stimulate and suppress tumor growth and invasion [9].
On the contrary, the content of regulatory minor populations of peripheral blood lymphocytes with phenotypes The increase of T-lymphocytes is due to the activation of T-cell immunity, but in our study there was only a slight tendency to their growth after 3-4 courses of chemotherapy, from 24.7% to 31.4%. However, when assessing the magnitude of changes in systemic immunity indicators before and during chemotherapy from the "linear" structure of peripheral blood, only T-cells (CD3+CD19-) have a tendency to their increase. In addition, in patients with a high level of CD3+CD8+CTLs and a low level of CD4+CD25+CD127-Tregs, an increase in OS was noted (p=0.012% and p=0.048%, respectively).
According to a number of studies in patients with various types of neoplasms, a correlation was found between CD3+CD19-T cells and CD3+ CD8+CTLs and a favorable prognosis [143;155]. However, according to He Q. et al., The content of CD3+CD19-T cells and CD3+CD4+CTLs after chemotherapy did not change and did not correlate with the clinical course of the disease, which is consistent with the results of our study [10].
Accordingly, the correlation between CD4+CD25+CD127-Tregs and clinical outcome points to their key role in the prognosis of gastric cancer patients and may serve as a biomarker for identifying patients with the best response to neoadjuvant chemotherapy.In comparing the results of the study of systemic cellular immunity in subgroups where the progression of the disease was recorded in terms of up to 6 months and later, it was revealed that a high level of CD8 T-regulatory cells before treatment is a predictor of a favorable prognosis, 12.1% versus 5.1% , (p=0.019).
Our results are consistent with a recent study of the subpopulation composition of peripheral blood lymphocytes in patients with head and neck squamous cell carcinoma (HNSCC). When analyzing 29 patients with early (I-II stages, n = 16) and late (III-IV stages, n = 13) stages of HNSCC, it was revealed that patients with early stages of HNSCC (group A) had a statistically significant increase in the percentage of CD8+CD11b-CD28 lymphocytes, 40.0% -28.6% [11]. According to the results of the analysis Zabotina T.N. and others in the analysis of 29 patients with early and late stages of HNSCC in patients with I-II stages there was an increase in the content of CD16 cells compared with III, IV stages, 22.1% versus 18.0% [11].
Accordingly, an increase in the level of effector CD16 cells with the CD16+Perforin+ phenotype during chemotherapy increases progressionfree survival, which is also consistent with our results.
Taking into account our results and the data of foreign researchers, it can be assumed that in the peripheral blood of patients with gastric cancer, the implementation of effector functions during chemotherapy occurs at the expense of CD16 cells, thereby increasing the likelihood of surviving PFS -9 months.
A number of studies on the distribution of lymphocyte subpopulations in tumor tissue have shown that TILs are associated with the