Evaluation of IgG, IgM, CD4+ and CD8+ T cells during neoadjuvant chemotherapy with Tezio and Apatinib in gastric cancer patients.

This experiment was carried out to study the effect the neoadjuvant chemotherapy with Tezio and Apatinib on the treatment of advanced gastric cancer and its impact on the postoperative immune function of patients' function. The patients with advanced gastric cancer who were treated in gastrointestinal surgery in our hospital from January 2017 to December 2018 were divided into two groups. The new chemotherapy and the traditional chemotherapy groups. The new chemotherapy group received neoadjuvant chemotherapy of Tezio combined with Apatinib before surgery, and the control group received the traditional first-line chemotherapy regimen of oxaliplatin combined with capecitabine. During the chemotherapy, the adverse complications of the two groups of patients were recorded, and the RECIST1.1 evaluation system was used to evaluate the chemotherapy effect of the patients after chemotherapy. At the same time, the R0 resection rate of the two groups of patients with gastric cancer radical surgery was recorded and compared. The fasting venous blood of the patients was collected before the chemotherapy, after the end of chemotherapy, and one week after the operation. The content of immunoglobulin G and M (IgG and IgM) was measured. The flow cytometry was used to detect CD4 + T cells and CD8+ T cells. A total of 64 patients were enrolled in this study, including 26 patients in the new chemotherapy group. Chi-square test showed that the incidence of bone marrow suppression (Leukopenia, thrombocytopenia and anemia) and liver dysfunction in the new chemotherapy group were lower than that in the traditional chemotherapy group (all P <0.05), but the surgical R0 resection rate in the new chemotherapy group was higher than that in the traditional chemotherapy group (P <0.05). Wilcoxon rank-sum test and chi-square test found that the chemotherapy effect of the new chemotherapy group was better than that of the traditional chemotherapy group, and the results of repeated measures analysis of variance showed that the IgG, IgM and CD4+/CD8+ T cell ratios of the patients in the new chemotherapy group were higher than that of traditional chemotherapy group (all P <0.05). Apatinib combined with Tezio's preoperative neoadjuvant chemotherapy can improve the chemotherapy effect of advanced gastric cancer, increase the rate of surgical R0 resection, and reduce the patient's immunosuppressive status during treatment.


Introduction
In the world, gastric cancer causes 720000 deaths every year in the world, and the trend is increasing, and the new cases of gastric cancer in China can account for 42.5% of the new cases in the world every year (1)(2)(3). The early symptoms of gastric cancer are mild and lack of specificity, so it is difficult for early diagnosis and early treatment. Many patients have developed into the advanced stage or even metastasis at the time of treatment, which leads to the failure of radical gastrectomy (4)(5)(6). Before the operation, neoadjuvant chemotherapy is often used to reduce the tumor stage and then select an opportunity for surgery, so as to improve the surgical R0 resection rate and improve the prognosis of patients (7,8). Although a variety of non-specific chemotherapy drugs including paclitaxel, oxaliplatin and S-1 have the effect of killing tumor cells, they have serious side effects such as inhibiting the immune system and hematopoiesis of the body, affecting the prognosis of patients (9,10). Therefore, it is urgent to develop and try to use targeted chemotherapy drugs including Apatinib in the clinic. Vascular endothelial growth factor, VEGF) and its receptors play an important role in the angiogenesis of tumor development. Apatinib can competitively bind to vascular endothelial growth factor receptor-2 (VEG-FR-2), block the downstream signal pathway and inhibit tumor angiogenesis, and play a role in inhibiting tumor growth (11,12). But up to now, the role of Apatinib in preoperative chemotherapy of advanced gastric cancer is still lack of sufficient medical evidence. Therefore, Tezio and Apatinib were used in this study before the operation to observe the effect of chemotherapy in the advanced stage and to study the influence of Tezio and Apatinib on the patients' immune function after the operation.

General information
From January 2017 to December 2018, 64 patients with advanced gastric cancer were selected from the gastrointestinal surgery of our hospital, with an average age of (45.98 ± 7.94) years. Inclusion criteria: ① gastric cancer was first diagnosed and treated in our hospital by means of CT and gastroscopic biopsy; ② the stage of gastric cancer was (T3-4N0-3M0) and no distant metastasis occurred; ③ the patient was in the good physical condition and could tolerate preoperative chemotherapy (ECoG score was 0-2). Exclusion criteria: ① patients with HIV and other serious diseases affecting the immune system; ② patients with drug allergy used in this study; ③ patients with severe cardiovascular or endocrine diseases; ④ Nrs2002 scoring system shows patients with nutritional risk requiring intervention (NRS2002 ≥ 3 points). With the permission of the hospital's ethics committee, all patients and their families signed informed consent.

Implementation of preoperative chemotherapy
The patients were divided into two groups: neochemotherapy group (n = 26) and traditional chemotherapy group (n = 38). Neochemotherapy group (n = 26) received neoadjuvant chemotherapy of Tezio combined with Apatinib before the operation, the control group received oxaliplatin + capecitabine conventional first-line chemotherapy. Common adverse complications were monitored during chemotherapy. Chemotherapy was suspended for 2-3 days or reduced the chemotherapy dose when the patient cannot tolerate chemotherapy.
At the end of chemotherapy, according to the actual situation of the patients, we choose to carry out radical gastrectomy, which is performed by senior doctors in our hospital.

Collection of adverse complications and operation effect
During chemotherapy, the adverse complications of the two groups were recorded, including myelosuppression, diarrhea, hand-foot syndrome, malignant vomiting and other adverse reactions. CT was performed before chemotherapy and after 4 cycles of preoperative chemotherapy. According to the RECIST1.1 evaluation standard of solid tumor effect (13), the chemotherapy effect of the patients was evaluated: ① Complete response (CR): the lesion completely disappeared; ② Partial response (PR): the lesion diameter was more than 30% less than the preoperative baseline level; ③ Progressive Disease: the diameter of lesions increased by more than 20% or new lesions appeared; ④ Stable disease (SD): between the disease progress and partial remission. Postoperative specimens were sent to the department of pathology to evaluate the surgical effect: R0: no tumor cells were left under the microscope; R1: microscopically, there are residual tumor cells.

Comparison of serum antibody level and proportion of immune cells in patients
Five -ten ml (2 tubes) of fasting venous blood were collected from patients before chemotherapy, after chemotherapy and one week after operation respectively. One tube of the blood sample was centrifuged at 4 ℃ and 3000 rpm to separate serum. Then the content of immunoglobulin G and m (IgG and IgM) were measured by immunoturbidimetry. 200 L whole blood was collected from another tube of the blood sample. According to the instruction manual, FITC labeled anti-human CD4 antibody (Anti-CD4-FITC; ab59474; Abcam) and PE-labeled anti-human CD4 antibody (anti-CD8-PE; ab39853, Abcam) were added successively. The blood sample was beaten and mixed and incubated at room temperature away from light for 30 min. 200 µL of hemolysin to lyse the red blood cells were added. After centrifugation at 1000 RPM for 5min at 4℃, the supernatant was removed and washed with 1 mL sterile PBS for 3 times. At last, 500 µLPBS was added to the cell precipitation, and flow cytometer (EPICS-XL, Beckman counter) was used for detection, and Flow Jo software was used to analyze the results.

Statistical analysis
The counting data are expressed by rate or percentage (%), and the measurement data of normal distribution are expressed by mean ± standard deviation. T-test and chi-square tests were used to analyze the differences between the two groups in general clinical data. Wilcoxon rank-sum test was used to compare the difference in chemotherapy effect between the two groups, and the chi-square test was used to analyze the difference in surgical R0 resection rate and chemotherapy adverse reactions between the two groups. The serum antibody level and the ratio of CD4 + T / CD8 + T in the two groups were also measured by repeated ANOVA. When p < 0.05, the difference was statistically significant.

Comparison of general data
The general clinical data of the two groups of patients are shown in Table 1. The results of the t-test and chi-square analysis showed that there were no statistical differences between the two groups in gender, age, postoperative pathological type and gastric cancer staging (all P > 0.05).

Comparison of chemotherapy and surgical effects
AS shown in Figure 1A, B, Wilcoxon rank-sum test and chi-square test respectively showed that the chemotherapy effect of the new chemotherapy group was better than that of the traditional chemotherapy group, and the surgical R0 resection rate of the group was higher than that of the traditional chemotherapy group (all P < 0.05). Representative abdominal CT results of 2 patients in the new chemotherapy group before and after chemotherapy were shown in figure 2. After chemotherapy, gastric tumors in the new chemotherapy group significantly decreased in volume and tumor diameter.

Comparison of immune function of patients after operation
Serum antibody (IgM, IgG) and CD4 + / CD8 + were recorded in the traditional chemotherapy group and neochemotherapy group, respectively, as shown in Table 3 and Table 4. The serum antibody (IgM, IgG) and CD4 + / CD8 + in the conventional chemotherapy group and the new chemotherapy group showed a downward trend during the observation period, and the difference was statistically significant in different periods (all P < 0.05). The serum antibody (IgM, IgG) and CD4 + / CD8 + in the conventional chemotherapy group were significantly lower than those in the new chemotherapy group (P < 0.05). The analysis of interaction shows that there is an interaction between different chemotherapy schemes and treatment time, and the difference is statistically significant (all P interaction < 0.05), details are showed in Table 3 and Table 4.

Discussion
Lack of physical examination awareness and lack of specific symptoms are the main reasons for the development of advanced gastric cancer in some patients (15,16). Direct surgical treatment for advanced gastric cancer is not effective, some patients are difficult to achieve surgical R0 resection, so preoperative chemotherapy is often needed to reduce the stage of patients' tumor after elective surgery (17). Apatinib has a strong inhibitory effect on tumor angiogenesis and has been proved to have a therapeutic effect on a variety of tumors, including cervical cancer, gastric cancer and so on (18,19). In this study, Apatinib combined with Tezio was selected for preoperative chemotherapy, and it was found that compared with the conventional preoperative chemotherapy, Apatinib combined with Tezio could improve the chemotherapy effect of patients, and improve the chemotherapy effect and postoperative immunosuppression.
Tumor can stimulate neovascularization in the process of growth and provide nutrients for metabolism. In this process, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) on the endothelial cell membrane combine with and transfer cell proliferation signal, promote the generation of capillaries and participate in tumor growth (20,21). Apatinib is a small molecular inhibitor targeting VEGFR-2. It can competitively bind to VEGFR-2, block the signal pathway related to the downstream cell proliferation after binding with VEGF, and play a role in inhibiting the growth of tumor cells (18). Previous studies have found that Apatinib can prolong the median survival time and reduce the risk of death in patients with gastric cancer who failed in the secondline chemotherapy, showing a good application prospect (22). This study also suggests that, compared with the conventional preoperative chemotherapy regimen of oxaliplatin combined with capecitabine, CT results after chemotherapy show that Apatinib combined with Tezio will increase the proportion of CP and PR after chemotherapy, and improve the surgical R0 resection rate of patients. Wilcoxon's rank-sum test showed that the incidence of myelosuppression (leukocytes, platelets and anemia) and liver dysfunction in the chemotherapy regimen of Apatinib combined with Tegio was low, but there was no advantage in diarrhea, hand-foot syndrome and malignant vomiting. It is speculated that the reason may be that VEGF is highly expressed only in the process of tumor growth. The chemotherapy plan of Apatinib combined with Tezio will target to inhibit neovascularization and kill tumor cells at the same time. The action of Apatinib targeting tumor cells will reduce the use of pan-specific chemotherapy drugs to some extent.   Therefore, it will have some advantages in reducing adverse complications during chemotherapy. However, small molecule targeted drugs also have diarrhea and hand-foot syndrome, so they cannot reduce the occurrence of some chemotherapy complications (23). In clinical practice, in order to ensure the smooth progress of chemotherapy, it is necessary to properly screen the patients with preoperative chemotherapy, to ensure that the general situation of patients can be tolerated and conduct preoperative chemotherapy, and to deal with the complications in time during the treatment process.
Oxaliplatin and capecitabine are both highly cytotoxic chemotherapy drugs. The former can inhibit tumor DNA replication by binding with G covalent bond in DNA, while the latter can play an anti-tumor role by generating 5-FU through metabolism in vivo (24,25). However, these drugs can not only inhibit tumor growth, but also inhibit the immune system of the body, leading to the decline of humoral immunity and cellular immunity of patients, and affect the killing effect of the autoimmune system on tumor cells, which is not conducive to the prognosis of patients (26). IgM and IgA are produced by B-lymphocytes and participate in many important immune processes such as anti-infection and anti-virus immunity (27). The proportion of CD4 + / CD8 + T cells is also an important indicator of the immune state of the body. The decrease in the proportion indicates that the immune function of the body is in a state of inhibition. Severe immunosuppression will make it difficult for the body to remove the residual tumor cells after surgery and increase the risk of recurrence. The results of this study show that different chemotherapy regimens can lead to the decrease of IgM, IgA and CD4 + / CD8 + T cell ratio in the course of treatment, which suggests that the cytotoxic effect of chemotherapy drugs can inhibit the normal immune system of the body during the treatment. However, the decrease degree of each index of Apatinib combined with Tezio group was lower than that of oxaliplatin and capecitabine group, which further indicated that the targeting mechanism of molecularly targeted drugs could improve the inhibition effect of traditional cytotoxic drugs on the immune function of the body, which was conducive to the resistance of patients for pathogen infection and the reduction of postoperative tumor recurrence. In general, different aspects of gastric cancer treatment should be considered.
There are also deficiencies in this study. Due to the small number of people included in this study, this study did not discuss the differences in the therapeutic effect of different drug doses. In addition, due to the limitation of study time, the patients were not followed up after the surgery in this study. It is impossible to know whether Apatinib combined with Tezio can improve the longterm prognosis of patients.
In summary, the preoperative neoadjuvant chemotherapy regimen of Apatinib combined with Tezio can improve the chemotherapy effect of advanced gastric cancer, improve the R0 resection rate and reduce the immunosuppressive status of patients during treatment.