Reduced expression of E-cadherin correlates with poor prognosis and unfavorable clinicopathological features in gastric carcinoma: a meta-analysis

Backgrounds: Gastric carcinoma (GC) is one of the most fatal human malignancies globally, with a median survival time less than 1 year. E-cadherin exerts a crucial role in the development and progression of GC as an adhesive, invasive suppressor gene. Whether reduced E-cadherin has an impact on prognosis, clinicopathological features for GC has been well studied, but no conclusive results has been obtained. Methods: Eligible studies and relevant data were obtained from PubMed, Elsevier, Embase, Cochrane Library and Web of Science databases until June 30, 2023. A fixed- or random-effects model was used to calculate pooled odds ratios (OR) and 95% confidence intervals (CI). Correlation of E-cadherin expression with overall survival (OS), clinicopathological features and risk factors were evaluated. Results: 36 studies fulfilled the selected criteria. 9048 cases were included. This meta-analysis showed that patients with GC with reduced E-cadherin had unfavourable clinicopathological features and poor OS. The pooled ORs of one-, three- and five-year OS were 0.38 (n = 25 studies, 95%CI: 0.25–0.57, Z = 4.61, P < 0.00001), 0.33 (n = 25 studies, 95% CI: 0.23–0.47, Z = 6.22, P < 0.00001), 0.27 (n = 22 studies, 95% CI: 0.18–0.41, Z = 6.23, P < 0.00001), respectively. Moreover, reduced E-cadherin expression significantly correlated with differentiation grade (OR = 0.29, 95% CI: 0.22–0.39, Z = 8.58, P < 0.00001), depth of invasion (OR = 0.49, 95% CI: 0.36–0.66, Z = 4.58, P < 0.00001), lymphatic node metastasis (OR = 0.49, 95% CI: 0.38–0.64, Z = 5.38, P < 0.00001), distant metastasis (OR = 2.24, 95% CI: 1.62–3.09, Z = 4.88, P < 0.00001), peritoneal metastasis (OR = 2.17, 95% CI: 1.39–3.39, Z = 3.40, P = 0.0007), TNM stage (OR = 0.41, 95% CI: 0.28–0.61, Z = 4.44, P < 0.00001), lymphatic vessel invasion (OR = 1.77, 95% CI: 1.11–2.82, Z = 2.39, P = 0.02), vascular invasion (OR = 1.55, 95% CI: 1.22–1.96, Z = 3.58, P = 0.0003), Lauren type (OR = 0.35, 95% CI: 0.21–0.57, Z = 4.14, P < 0.0001), Borrmann classification (OR = 0.50, 95% CI: 0.25–0.99, Z = 1.97, P = 0.048) and tumor size (≥5 cm vs. <5 cm: OR = 1.73, 95% CI: 1.34–2.23, Z = 4.19, P < 0.0001; ≥6 cm vs. <6 cm: OR = 2.29, 95% CI: 1.51–3.49, Z = 3.87, P = 0.0001). No significant association was observed between reduced E-cadherin expression and liver metastasis, perineural invasion, alcohol consumption, smoking status, familial history, Helicobacter pylori (HP) infection. Conclusions: The reduced expression of E-cadherin is significantly correlated with poor OS and unfavourable clinicopathological features in GC. The expression level of E-cadherin not only serves as a predictor for disease progression and prognosis in GC but also emerges as a novel therapeutic target.


INTRODUCTION
Gastric carcinoma (GC) is one of the most fatal human malignancies globally [1].It was reported that 1 million new patients suffer from GC annually [1].It was estimated that 784000 deaths were caused by GC globally in 2018 [1].Endoscopic mucosal resection or endoscopic submucosal dissection is adopted for patients with early GC.Gastrectomy with D2 lymphadenectomy is suitable for locally advanced GC.A comprehensive plan including chemotherapy, immunotherapy, anti-angiogenic therapy, and trastuzumab for Her2-positive GC, improves overall survival (OS).Nonetheless median OS is within 12 months.It is believed that Helicobacter pylori (HP) infection, dinking, hereditary tendency, salted and smoked food intake, and gastroesophageal reflux disease are risk factors for GC [2].There is an urgent need to understand genes involved in the initiation, progression, and prognosis of gastric cancer, which exhibits a high level of heterogeneity both at the molecular and phenotypic levels.E-cadherin (E-cad) is a member of Ca 2+ -dependent membrane glycoprotein, encoded by CDH1 gene which is crucial for preserving epithelial cell-cell junctions and cell polarity, and suppresses tumor growth, metastasis and invasion in numerous cancers comprising GC.E-cadherin exerts its effects on the Wnt-signaling pathway by negatively regulating the quantity of unbound β-catenin, which is indispensable in the pathogenesis of GC [3,4].Low E-cadherin expression in GC is attributed to mutation in the CDH1 gene on chromosome 16q22.1 [5], E-cadherin promoter hypermethylation [6], and transcriptional repression resulting from Snail [7] and Sip-1 [8] binding to the CDH1-E box.
As far as the correlations between E-cad expression and clinical characteristics, as well as prognoses for patients with GC are concerned, vast amounts of work have been done but study results exhibit great diversity and inconsistency.Furthermore, the quantity of participants recruited for each research is not sufficiently large.So, this article was conducted to systematically and comprehensively evaluate its correlations.

Data retrieval
The articles published before June 30, 2023 in the PubMed, Elsevier, Embase, Cochrane Library, and Web of Science databases were systematically searched.The terms used in the search were as follows: "E-Cadherin", "prognosis", and "stomach neoplasms".The reference lists of publications were retrieved by manual.Only English-language studies were encompassed in the selection process.

Criteria for inclusion and exclusion
Inclusion criteria: (1) Pathological diagnosis is GC; (2) Data about E-cadherin expression, OS, and clinical characteristics were comprehensive; (3) E-cadherin expression was detected by immunohistochemical staining, western blotting, immunofluorescence; (4) When multiple studies were published by a single author, only the one with the highest quality was included; (5) Study written in English was enrolled.
Exclusion criteria: (1) Abstracts, reviews, editorials, case reports, as well as letters; (2) Study subjects are cell lines, and animals; (3) Overlapping publication; (4) Information about E-cadherin expression, OS, as well as clinical characteristics was unavailable.

Data retrieval and compilation and evaluation of literature quality
Each study was evaluated and relevant characteristics were extracted by three reviewers (GLL, JYS and RYJ) independently.The data were presented as follows: (1) authors and publication time; (2) clinical characteristics; (3) level of evidence, (4) the rate of E-cadherin expression, (5) OS data (Table 1).Literature quality was evaluated by Newcastle-Ottawa scale (NOS) [9].

Statistical analysis
The Review Manager software (version 5.3) and Stata software (version 18) were utilized to generate pooled odds ratios (ORs) along with 95% confidence intervals (CIs) [10].The associations between E-cad expression P = 0.0001).No significant association was observed between reduced E-cadherin expression and liver metastasis, perineural invasion, alcohol consumption, smoking status, familial history, Helicobacter pylori (HP) infection.Conclusions: The reduced expression of E-cadherin is significantly correlated with poor OS and unfavourable clinicopathological features in GC.The expression level of E-cadherin not only serves as a predictor for disease progression and prognosis in GC but also emerges as a novel therapeutic target.and overall survival (OS), clinicopathological features, and risk factors were evaluated.Stratification based on study origin was conducted through subgroup analysis and meta-regression [9,10].Funnel plots and Egger's test were employed to evaluate publication bias.As the I² value exceeds 50%, there is considered to be significant heterogeneity.When the P-value is less than 0.05, it is deemed that statistical significance exists.

Selection of trials
Figure 1 demonstrates that 36 studies met the inclusion criteria and were enrolled for analysis of the prognostic value of E-cad expression, as well as its association with clinical characteristics and risk factors for GC (of the 1985 publications, 1921 studies were excluded due to incomplete content, 16 were excluded because they lacked sufficient data to calculate OS, and 12 were excluded as their data overlapped with those of other studies).

Study characteristics
Table 1 shows the data on E-cad expression, OS, clinical features, and risk factors from 36 enrolled studies eligible for the meta-analysis.A total of 9048 patients with GC were included, among whom 2998 patients exhibited lower levels of E-cad expression.The expression of E-cadherin in each study was determined by immunohistochemical staining, western blotting, immunofluorescence, or other methods, all conducted without subjective interference.

Impact of E-cadherin expression on OS
As indicated in Figures 2-4 and Table 2, there are predominant correlations between reduced E-cadherin  the one-, three-, five-year OS was 77%, 82%, 85% respectively.The results of subgroup analyses revealed that reduced E-cadherin was predominantly associated with three-, five-year OS of patients with GC in China, Japan and Korea, as well as one-year OS of patients with GC in Japan, as illustrated in Table 3.It was concluded that reduced E-cad had a worse impact on prognosis in GC.

Correlation of E-cadherin expression with risk factors
The associations of E-cadherin expression with risk factors, including alcohol consumption, smoking status,     2 and Supplementary Figures 3-6, E-cadherin expression is not correlated with alcohol consumption, smoking status, familial history and HP infection.

Publication bias
Egger's test manifests that there is not any publication bias for studies included in analysis of OS, risk factors, and clinicopathological parameters except differentiation grade (p = 0.0001).As shown in Supplementary Figures 7-26, the funnel plots for publication bias were symmetric except for some degree of asymmetry of studies involved in the analysis of differentiation grade (Supplementary Figure 27).

DISCUSSION
A personalized treatment plan, including surgery, chemotherapy, anti-angiogenic therapy, and immunotherapy, trastuzumab for Her2-positive GC, can help patients with GC improve their OS.
However, the median survival is within 12 months.It is demonstrated that E-cad is crucial for tumor   development, invasion, metastasis in GC.There is no consensus about impact of E-cadherin expression on prognosis and clinical characteristics of patients with GC.In this meta-analysis 9048 cases from 36 eligible studies were analyzed to elucidate its correlation.
OR is a measure of effect size commonly used in metaanalysis, particularly when dealing with dichotomous outcomes, which is also a statistic that quantifies the strength of outcome between the correlation of an exposure with an outcome.A pooled OR, is a single and overall estimate of the effect, which is obtained in a meta-analysis to combine the results from multiple studies.The resulting pooled OR provides a more precise and reliable estimate of the effect than any single study alone.
Recent researches have disclosed that decreased Ecadherin expression in GC ranges from 15.9% [37] to 85.4% [3] by IHC tests.This study denoted that the lower levels of E-cad in GC occur at the rate of 33.1%.Zhou et al., revealed that a normal state of E-cadherin expression is essential for the favourable prognosis of patients with GC [46].As demonstrated in this article,

Figure 16. Forest plot of the odds ratio for the correlation of E-cadherin expression with peritoneal metastasis. AGING
reduced expression of E-cadherin was significantly correlated with one-, three-, and five-year overall survival (OS) of patients with gastric cancer, especially in China, Korea, and Japan.No publication bias was observed in the subgroup analysis conducted in each of these regions.It is consistent with the result of Zhou et al.
Regarding clinicopathological parameters, this study found that lower levels of E-cad expression are predominantly correlated with deeper invasion, poor differentiation, higher TNM staging, distant metastasis, lymphatic node metastasis, peritoneal metastasis, vascular invasion, lymphatic vessel invasion, greater tumor size, diffuse type of Lauren classification, and Borrmann III+IV.No obvious association exists between lower E-cadherin level and liver metastasis and perineural invasion.A normal state of E-cadherin expression is key to favourable clinicopathological characteristics of GC.
It is believed that Helicobacter pylori (HP) infection, dinking, hereditary tendency, salted and smoked food intake, and gastroesophageal reflux disease are risk factors for GC [2].Worldwide incidence of distal GC related to HP seems to be on the rise.HP silences E-cad gene by secreting CgA and counteracting protein kinase C [49][50][51].Reduced E-cadherin expression is not pronouncedly correlated with alcohol consumption, smoking status, familial history, or HP infection in this meta-analysis.Some limitations deserve further attention in this study.Firstly, different antibody sources and dilutions bring bias into this meta-analysis.Secondly, there was heterogeneity in this study, as displayed in given tables and forest plots.A random-effects model was utilized to account for heterogeneity among studies.Subgroup analyses failed to clarify the source of heterogeneity.Thirdly, publication bias was present for differentiation grade.Fourthly, the inclusion of studies published in English may also introduce bias.
A conclusion can be drawn from this metaanalysis that the reduced expression of E-cadherin is significantly correlated with poor OS and unfavourable clinicopathological features in GC.The expression level of E-cadherin not only serves as a predictor for disease progression and prognosis in GC but also emerges as a novel therapeutic target.

Figure 1 .
Figure 1.Flow chart of literature search strategies.

Figure 2 .
Figure 2. Forest plot of the odds ratio for the correlation of E-cadherin expression with one-year overall survival.

Figure 3 .
Figure 3. Forest plot of the odds ratio for the correlation of E-cadherin expression with three-year overall survival.

Figure 4 .
Figure 4. Forest plot of the odds ratio for the correlation of E-cadherin expression with five-year overall survival.

Figure 5 .
Figure 5. Forest plot of the odds ratio for the correlation of E-cadherin expression with depth of invasion.

Figure 6 .
Figure 6.Forest plot of the odds ratio for the correlation of E-cadherin expression with lymphatic node metastasis.

Figure 7 .
Figure 7. Forest plot of the odds ratio for the correlation of E-cadherin expression with distant metastasis.

Figure 8 .
Figure 8. Forest plot of the odds ratio for the correlation of E-cadherin expression with lymphatic vessel invasion.

Figure 9 .
Figure 9. Forest plot of the odds ratio for the correlation of E-cadherin expression with vascular invasion.

Figure 10 .
Figure 10.Forest plot of the odds ratio for the correlation of E-cadherin expression with tumor size (≥5 cm vs. <5 cm).

Figure 11 .
Figure 11.Forest plot of the odds ratio for the correlation of E-cadherin expression with tumor size (≥6 cm vs. <6 cm).

Figure 12 .
Figure 12.Forest plot of the odds ratio for the correlation of E-cadherin expression with TNM stage.

Figure 13 .
Figure 13.Forest plot of the odds ratio for the correlation of E-cadherin expression with Lauren type.

Figure 14 .
Figure 14.Forest plot of the odds ratio for the correlation of E-cadherin expression with differentiation grade.

Figure 15 .
Figure 15.Forest plot of the odds ratio for the correlation of E-cadherin expression with Borrmann classification.

Table 1 . Characteristics of studies included in the meta-analysis. First author and year Country or region Mean age Gender (M/F) Level of evidence Stage Clinicopatholo gical features Method Provided- OS data No. of patients Reduced/total E-cadherin (%)
Abbreviations: IHC: immunohistochemistry test; D: differentiation grade; T: depth of invasion; M: distant metastasis; OS: overall survival; NR: not reported.

Table 2 . Correlation between E-cadherin expression and OS, clinicopathological feature, and risk factors for GC.
Abbreviations: Hp: Helicobacter pylori, RE: reduced E-cadherin expression, PE: preserved E-cadherin expression; OR: odds ratio; CI: confidence interval; TNM stage: depth of tumor invasion, lymphatic node metastasis, distant metastasis stage classification.

Table 3 . Subgroup analysis for E-cadherin expression with OS and lymphatic node metastasis in GC.
Abbreviations: OR: odds ratio; CI: confidence interval.