The Clinicopathological And Molecular Features Of Early Gastric Adenocarcinoma With A Crawling Growth Pattern

Part Ⅰ:Clinicopathological and immunophenotypic study of early gastric cancer with a crawling growth patternBackgroundGastric cancer (GC) is a malignant epithelial tumor caused by the combined effects of many factors.The incidence and mortality of gastric cancer in China rank third.According to the depth of invasion,gastric cancer is divided into early gastric cancer(EGC) and advanced gastric cancer.Compared to advanced gastric cancer,early gastric cancer has a higher five-year survival rate,more treatment options and a better prognosis.Thus,accurate diagnosis and early treatment of gastric cancer are of particular importance in the early stages of the disease.A typical Crawling-type gastric adenocarcinoma (CRA) is an early gastric cancer with low-atypia and mild morphologic features that are difficult to be found in clinical diagnosis,but aggressive with poor prognosis.CRA is usually located in the middle of the lamina propria of the gastric mucosa and grows laterally.This laterally disseminated growth pattern is manifested by the high fusion of gastric glands.Currently,the diagnosis of CRA depends mainly on its morphological characteristics,and there are no specific diagnostic and differential diagnosis indicators for CRA.CRA has an extremely low-grade cytological atypia with a low nuclear/cytoplasmic ratio,minimal nuclear atypia,indistinct nucleolus,and delicate chromatin.Architecturally,it appears as glands recapitulate the shape of the letters "X,Y,H,W" on low-power view under the microscope.Therefore,it’s also known graphically as "letter cancer." In addition,almost all tumor glands of EGAR include goblet cells,which can mimic reactive intestinal metaplasia (IM),making the diagnosis and differential diagnosis difficult.Long-term observational study by our group found that most CRAs grow mixed with high-atypia tubular adenocarcinoma,poorly differentiated adenocarcinoma,signet-ring cell carcinoma (SRCC),papillary adenocarcinoma and other tumor components,whereas CRAs without other tumor components are very rare.We have named early gastric adenocarcinoma with a ≥ 5% share of typical CRA as early gastric adenocarcinoma with a crawling growth pattern (EGAR),with or without the presence of other tumor components.EGAR is more invasive and has a poor prognosis.The main manifestations are:Meanwhile,EGAR often contains aggressive tumor components such as poorly differentiated adenocarcinoma,signet-ring cell carcinoma and papillary carcinoma,and the surface of CRA tumor components in EGAR is often covered with normal gastric concave epithelium.Endoscopically,EGAR usually presents as an ill-defined lesion with an indistinct border,and the lateral resection margin involvement rate in ESD for EGAR is high.Purpose1.By observing and analyzing the clinicopathologic and immunophenotypic features of EGAR,we can understand the general characteristics of EGAR,improve the diagnostic rate of EGAR and search for molecular indicators that may be useful in EGAR diagnosis and prognostic assessment;2.By observing and studying the morphological characteristics of EGAR,we can master the progression direction of low-atypia early gastric cancer,improve the accuracy of pathological diagnosis of EGAR in the early stage of low-atypia gastric adenocarcinoma,and search for molecular markers that can block the development of CRA to EGAR and advanced gastric cancer.Methods and resultsIn this study,133 EGAR and 133 early non-EGAR with high-atypia tubular adenocarcinoma were included.All samples were collected from patients who underwent radical gastrectomy or ESD in the Second Hospital of Shandong University between 2016 and 2022.The pathological sections were reviewed and the following studies were carried out:1.Through statistical analysis of the clinicopathologic data of the cases,the clinicopathologic data of the patients’ age,gender,lesion location,macroscopic type,tumor diameter,depth of invasion and histological type were analyzed.We found that EGAR was more common in middle-aged and elderly men.Compared with early nonEGAR with high-atypia tubular adenocarcinoma,EGAR is more likely to have poorly differentiated adenocarcinoma components (poorly differentiated adenocarcinoma and/or signet-ring cell carcinoma) and papillary adenocarcinoma.The lower 1/3 of the stomach is the most common site for signet-ring cell carcinoma and papillary adenocarcinoma of EGAR.Female EGAR patients are more likely to have poorly differentiated adenocarcinoma components.The possibility of submucosal infiltration increased after the presence of poorly differentiated adenocarcinoma components in EGAR.The proportion of CRA in EGAR decreased when there were poorly differentiated adenocarcinoma components or submucosal infiltration.Both CRA and papillary adenocarcinoma are likely to occur in EGAR.The CRA usually appears in the middle of the lamina propria,while papillary adenocarcinoma usually appears on the surface,and the two are closely related and need to be further studied independently;2.Chi-square test analysis showed that the positive rate of horizontal margin of EGAR after ESD treatment was higher than that of early non-EGAR with high-atypia tubular adenocarcinoma;3.Through analysis of Restricted cubic spline (RCS) curve model,we found that there is a correlation between tumor diameter of EGAR and presence of poorly differentiated adenocarcinoma components:When tumor diameter of EGAR exceeds 2.2 cm,the risk of presence of poorly differentiated adenocarcinoma components of EGAR increases;4.Immunohistochemistry staining of EGAR showed that the expression of MUC2 in EGAR was significantly lower than that in paracancer IM epithelium,and the difference was statistically significant.The MUC5AC cytoplasmic expression was significantly positive in EGAR than in early non-EGAR with high-atypia tubular adenocarcinoma.However,there was no difference in the expression of MUC6.The expression of mucin MUC2,MUC5AC and MUC6 can be observed simultaneously in EGAR.P53 was expressed wild-type more often in EGAR.Conclusions and significance1.EGAR has unique histopathologic characteristics.Endoscopists and pathologists should be alert to the presence of poorly differentiated adenocarcinoma components when EGAR is found in endoscopic biopsy and tumor diameter is estimated to be greater than 2.2 cm.2.The poorly differentiated adenocarcinoma components in EGAR may be derived from the CRA neoplastic epithelium,and CRA may transform into poorly differentiated adenocarcinoma components and infiltrate submucosa.CRA may be the precursor of highly invasive gastric cancer and one of the sources of poorly differentiated and highly invasive gastric cancer.However,the relationship between CRA and papillary adenocarcinoma needs further investigation.3.The immunophenotype suggests that EGAR has the characteristics of gastric and intestinal mixed (GI) gastric cancer.When morphological diagnosis is difficult,immunohistochemistry staining of mucin MUC2 and MUC5AC can assist in more accurate diagnosis of EGAR.The above studies help to clarify the clinicopathological and molecular characteristics of EGAR,and provide new ideas for improving the diagnosis rate of this disease,effective treatment and improving the prognosis of patients.Part Ⅱ:The regulatory effect and molecular mechanism of MUC2 on gastric cancer cells based on the study of early gastric adenocarcinoma with a crawling growth patternBackgroundThe incidence and death rate of gastric cancer is increasing yearly worldwide.With the implementation of the national precision medicine plan,more and more attention has been paid to the revelation of the key regulatory nodes in the early stage of disease occurrence.Therefore,it is necessary to explore the law of the occurrence and development of gastric cancer,the key molecular targets in its occurrence process,and early practical molecular diagnostic markers,which will provide a theoretical basis for preventing and controlling gastric cancer.Mucin is the main component of mucous on the surface of human gastrointestinal tract,which can protect and lubricate the gastrointestinal mucosa.MUC2 belongs to secreted gel-forming mucin,which is usually expressed in the intestinal mucosal epithelium and increased in the stomach only when intestinal metaplasia or gastric cancer occurs in the gastric mucosa.Our previous study found that when EGAR was present,MUC2 expression increased,and its expression intensity was different from that of IM.Therefore,we speculated that MUC2 might be related to the occurrence and development of gastric cancer.Studies have shown that the expression of MUC2 is upregulated in mucous carcinoma of lung and cervix.The literature indicates an abnormal expression of MUC2 in about 25% of gastric cancer,and it is suggested that this gene may be involved in the pathogenesis of gastric cancer.Other studies have reported that MUC2 expression is associated with poorer prognosis in gastric signetring cell carcinoma patients.However,the mechanism by which MUC2 plays a role in gastric cancer remains unclear.Therefore,further exploring the function of MUC2 in gastric cancer is of great significance.The transforming growth factor beta (TGF-β) signaling pathway is related to various biological functions such as cell growth,cell differentiation,apoptosis and cell homeostasis.TGF-β receptor (TGF-βR) plays an important role in the TGF-β/Smad signaling pathway,and its low expression is closely associated with the occurrence of gastric cancer.It has been reported that TGF-β induces the up-regulation of bacterial MUC2 transcription through TGF-βRII,leading to inflammation.Knockout of the SMAD4 gene in AGS led to decreased expression of endogenous MUC2.However,the correlation between MUC2 and TGF-β/Smad signaling pathway in tumor tissue remains unclear.In vitro experiments were conducted to verify their expression and correlation in gastric cancer cells,and further explore their relationship with the progression of gastric cancer and biological behavior,as well as their regulatory mechanism.Purpose1.To explore the effect of mucin MUC2 on gastric cancer cells;2.To clarify the molecular mechanism of MUC2 regulating the occurrence and development of gastric cancer,and to provide reference for the diagnosis and treatment of gastric cancer.Methods and resultsGastric cancer cell was transfected with small interference by siRNA transient transfection of MKN45 to knock down MUC2,Western blot and RT-qPCR to verify the knockdown efficiency.The effects of MUC2 on the proliferative ability of MKN45 cells were detected by plate cloning and EdU assay.The effects of MUC2 on the migration and invasion ability of MKN45 cells were detected by Tranwell assay.The effect of MUC2 on TGF-β/Smad signaling pathway was detected by Western blot.1.Results of RT-qPCR and Western blot showed that siRNA could effectively reduce the expression of MUC2 in gastric cancer cells MKN45;2.Plate cloning experiments showed that the clonogenesis ability of MKN45 decreased after MUC2 was knocked down.EdU results showed that the proliferative ability of MKN45 decreased after MUC2 knockdown.Transwell experiment confirmed that the migration and invasion ability of MKN45 decreased after MUC2 knockdown;3.Protein expression levels of Smad2/3,Smad4,TGF-βⅠ and TGF-βⅡ decreased after MUC2 was knocked down in MKN45 cells.Conclusions and significance1.MUC2 can promote the proliferation,migration and invasion of gastric cancer cells,suggesting that MUC2 may be involved in the occurrence and development of gastric cancer.2.The regulation of TGF-β/Smad signaling pathway in gastric cancer cells depends on MUC2.