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Expression of VEGF-C and its association with clinicopathological features among patients with gastric cancer undergoing gastric surgery at Tongji medical hospital, China.

Objectives: Gastric cancer shows epidemiological variability according to geographic locations, these differences have been demonstrated both regionally and within countries. There is no baseline protocol regarding using VEGF-C as tumor marker in local setting therefore it was necessary to conduct this study, the current study conducted to analyze the correlation exists between VEGF-C and clinicopathological parameters in patients with gastric cancer undergoing a surgical procedure at Tongji medical hospital. Results: 161samples analyzed were from Chinese patients who had undergone either palliative or curative surgical procedure between October 2015 and September 2017 at Tongji medical hospital. I mmunohistochemistry was method was used to analyses presence of VEGF-C in tissues. Among 161 gastric cancer patients, 101 (62.7%) gastric samples showed strong VEGF-C expression and 60 (37.3%) gastric samples showed weak VEGF-C expression. Using multivariate logistic regression analysis, tumor grade, invasion of lymph nodes and TNM staging were statistically significantly found to associate with strong expression of VEGF-C. VEGF-C strong expression was significantly seen more in adenocarcinoma of gastric cancer, and Therefore, VEGF-C may be used as a biological marker for assessing the biological characteristics of gastric.


Introduction
Gastric cancer (GC) exhibits epidemiological variability by geographic location, and these differences have been demonstrated both regionally and within countries [1]. Gastric cancer is positioned as the third most frequent cause of death in patients with cancer and is ranked fifth in the most common types of cancer globally [2]. Despite the dramatic decrease in gastric cancer's incidence in other countries, China has seen an increase in both younger and older groups, though there is a decreased incidence in women [3]. A study showed that the age of onset of developing gastric cancer among the Chinese population is earlier than that of Westerners. Also, the mortality and incidence of gastric cancer in China varies from province to province, it is more common in the north than the south [4,5].
The disease is curable when it presents itself at an early stage, at which point surgical manipulations are still possible, and radiotherapy or adjuvant chemotherapy are part of curative management [6].
However, patients with late-stage gastric cancer are left with a poor prognosis despite the sophisticated and modern technologies used in the management of gastric cancer [7].
In gastric cancer patients, VEGF subfamilies' status has demonstrated correlation with various clinicopathological parameters. Another study showed that strong expression of VEGF-C was significantly associated with lymphatic involvement, TNM staging and vascular involvement in gastric cancer (p<0.01) furthermore, the same study showed that VEGF-C was not seen to be associated with gender, age at time of surgery, tumor size, or tumor location [12]. Similar studies demonstrated that VEGF-C strong expression on gastric tissue was positively associated with lymphatic system invasion, invasion of lymph tissues and lymph nodes [13][14][15]. In addition, expression of vascular endothelial growth factor-C seen in an earlier stage of gastric cancer was significantly correlated with lymphatic invasion, thus it can be clinically helpful to predict the outcomes of minimal or more extensive surgical manipulations and nodal clearance in GC patients [16].
This study was conducted to find out expression of VEGF-C and its association with clinicopathological features among patients with gastric cancer undergoing gastric surgery at Tongji Medical Hospital, (TMH). Findings from this study can be used to establish management protocols for VEGF-C, as a molecular tumor marker in patients with gastric cancer at Tongji hospital.

Study design
This was an analytical cross-sectional study among patients undergoing surgery at TMH during the period of October 2015 to September 2017. This study was conducted in the oncology and pathology department at Tongji medical hospital in Wuhan, Hubei, China, a department with the capacity of 4,000-6,000 beds.

Sampling:
Convenient sampling was method deployed in this study . Tissue samples of patient who attended at Tongji Medical Hospital in oncology and pathology department were collected after filling precoded questionnaire. A precoded questionnaire was used to obtain information of the participants. The questionnaire was divided into demographic characteristics, clinical characteristics, histopathological features and staining analysis. All tissue sample of patients above 19 years of age of both sexes who 4 underwent curative or palliative surgery were included into the study. Patients whose biopsy sample for histopathological analysis could not be obtained and didn't consent for the study were excluded from the study. Their sample were taken for further analysis of VEGF-C by immunohistochemistry. To ensure internal validity of the study precoded questionnaire was tested. XY and BM were trained how to fill the questionnaire.

Specimen Collection
Tissue samples analyzed in this study were collected from Chinese patients (n=161) who had undergone either a palliative surgical procedure or a therapeutic surgical procedure between October VEGF -C scoring according to immunohistochemistry Two pathologists who were unaware of the clinical outcome of the patients did a pathological analysis of immunohistochemistry. The analysis of the staining was exclusively restricted to tumor cell reactions. VEGF-C stained results were classified, according to the intensity of color and percentage of epithelial cells that showed specific immunoreactivity into 0, 1, 2, and 3 designations. If the summation of intensity and percentage was in the 0-2 ranges, it was considered a weak expression of VEGF-C, while a range of 3-6 was regarded as a strong expression of VEGF-C [18,19,20].

Statistical data analysis
Data were entered into Excel, imported for analysis into Stat software version 13.v, and analyzed according to the objectives. Continuous variables were analyzed using mean and standard deviation.
For categorical variables frequency and proportion was used. Chi-square test was used for comparison between the categorical variables groups. The odds ratio was used to measure strength of the association between predictor and outcome variables. A p-value of less than 0.05 was reported as significant. Predictor variables, which were found to be significant on univariate analysis, were subjected to multivariate logistic regression analysis to test the significance of the association between these variables and the outcome. Adjusted odds ratio presented at 95% Confidence Interval and P-value less than 0.05 were considered statistically significant.

Populations studied and demographic characteristics
During the study, 1260 patients underwent gastric surgeries at TMH. Out of these, 268 were found to be positive for VEGF. Of these, 107 were excluded from the study because of failure to meet inclusion criteria. Thus, 161 patients were studied. Among the 161 gastric cancer patients, 101 (62.7%) gastric samples were regarded as strong VEGF-C expression group and 60 (37.3%) were regarded as weak VEGF-C expression group. (Figure 1). Patients' ages ranged from 28 years to 81 years. The mean was 58 years, and the standard deviation was 10.44 years. The distribution between females and males was 29 females (18%) and 132 males (82%).  Table 1 below shows clinical characteristics associated with expression of VEGF-C according to univariate analysis. There was no statistically significant association of VEGF-C expression in relation to age and sex. There was also no significant association between VEGF-C, 6 smoking and alcohol intake (Table 1). There was a statistically significant association of VEGF -C expression and Bormann classification.
Tumors with strong VEGF-C expression were 4.86 more likely to be ulcerative type of gastric cancer than those presented with weak VEGF-C expression (OR:4.86,CI;1.10-7.11, P=0.013). There was a statistically significant association between strong VEGF-C expression and tumor grade (P=0.005).
Strong VEGF-C expression were 4.88 more likely to be found in poorly differentiated gastric tumors than tumors with weak VEGF-C expression (OR=4.44,95% CI; 2.55-9.89). VEGF-C expression was also significantly associated with lymphatic involvement (P= 0.001). Malignancies with strong VEGF-C expression were 3.34 more likely to involve lymphatic system than those with weak VEGF-C However, no statistically significant association was found between tumor location, tumor size, tumor stage, vascular involvement, histological classification and VEGF-C expression ( Table 2).  (Table S1). This study demonstrates that vascular endothelial growth factor C does correlate with some clinicopathological parameters; and it also has a negative relationship with some of the clinicopathological settings. In the present study, VEGF-C expression was found to be significantly associated with lymphatic nodal invasion, and this result is supported by research done by Cristina et al. [21]. Yong Dai et al. [22] in his study also reported correlation of VEGF-C and lymph node metastasis in gastric cancer and recommended the use of VEGF-C in evaluating prognosis in gastric cancer patients. Lin Wang et al also reported a significant correlation between VEGF-C strongexpression, TNM staging, tumor grading, and lymphatic node invasion [23]. This is also supported by another study [24], VEGF-C has been implicated to be a lymphangiogenic factor, but exact mechanisms are not precise [25]. Previous clinical studies have established that strong expression of VEGF-C is well established in primary tumors and correlates with increased migration of tumor cells to regional lymph nodes in different human carcinomas (26)(27)(28)(29)(30)(31)(32)(33). Yonemura et al. [34] and Kabashima et al. [35] showed that VEGF-C expression was correlated with lymphatic nodal invasion.
Also, this study results showed there was no association found between the size of gastric tumor, gender, age, and VEGF-C strong-expression. This finding is in agreement with Lin Wang et al. [23].Yong Dai et al . [22] also reported no significant correlation of VEGF-C and age, sex. Unlike the present study which reported statistically significant association of VEGF-C expression and tumor grade also Yong Dai et al. [22] found no significant difference of VEGF -C and tumor grade. This study found no association between VEGF-C expression and tumor location. This finding is consistent with Bin Wang et al. [36], in his study he demonstrated no significant association of proximal, middle and distal gastric cancer and VEGF-C expression.
In our study, tumor grading, lymph node invasion and TNM stage in multivariate logistic regression analysis were found to be statistically significantly associated with strong VEGF-C expression.

Conclusion
8 VEGF-C expression was seen significantly more in adenocarcinoma histological type of gastric cancer.
In multivariate analysis, VEGF-C was found to be statistically significantly associated with tumor grade, lymph node invasion and TNM stage. In conclusion, VEGF-C may be of use as a tumor marker for assessing the biological characteristics in gastric cancer patients presenting with adenocarcinoma types of gastric malignancies.
Limitations of the study: 1.
The study population included only those of Asian 2.
Lauren classification was not used in this study.  Figure 1 Flow chart diagram 17 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table S1.docx