Clonality Analysis Of Neuroendocrine Cells In Gastric Adenocarinoma And Clinicopathologic Significance

Neuroendocrine(NE) differentiation is a common phenomenon in adenocarcinomas. The definition of NE is the appearance of single or nidulant NE cells in the cancer tissue, accounting for＜50%of the total cell number.According to our previous data,NE differentiation occurs in 41.5%of colon cancer,39.6%of gastric cancer,38.1%of prostate carcinoma,21%of breast cancer and 17.9%of pancreatic carcinoma.Differ from the rest of investigated cancers,where NE was associated with poor differentiation, NE in gastric adenocarcinoma was more frequently observed in well differentiated ones than in poorly differentiated ones.However,it is not known whether NE is derived from embryogenesis,histogenesis,or genetic changes associated with tumor etiology.Nor is known the functional contribution of NE towards gastric cancer development and prognosis.We thus employed laser capture microdissection technique to capture NE cells and utilized molecular and genetic approaches to study the originality of NE cells and their association with gastric cancer biology.Studies of NE in gastric adenocarcinoma are relatively few.Clonality about the neuroendocrine differentiation cells in gastric adenocarcinoma and related genes are unclear.The most common theory is that neuroendocrine cells are derived from the multi-potential stem cells.NE differentiation was initiated by hormonal change, microenvironmental change and genomic instability.Some subdued genomic codes are randomly depressed and selectively activated by more than two regulatary genes during RNA translation,as a result the multi-potential stem cell generate biladifferentiationl or multidifferentiation.Despite of the apparently different morphological representation of NE cells in the tumor mass,it is largely unknown whether these neuroendocrine cells have chromosomal or genetic alterations.Moreover,it is not clear whether NE cells present as tumor or a stroma component.Neuroendocrine cells from gastric adenocarcinoma are harvested by Laser capture microdissection(LCM),which ensured the cell purity.Whole genome amplification(WGA)(QIAGEN) method was then employed to compare genomic characteristics of neuroendocrine cells with adenocarcinoma cells,for the identification of the clonality of neuroendocrine cells.Wild type p53 acts is an anti-oncogene in normal tissues,important in DNA reparing and cell cycle regulating.Tumourigenesis is closely associated with p53 mutation or loses of functions.MLH1 gene is an important member of the mismatch repair genes family.It has the function of DNA mismatch repairing,enhancing DNA replication fidelity,maintaining genomic stability,and reducing the spontaneous mutation.E-cadherin(epithelial cadherin) is a kind of calcium-dependent transmembrane glycoprotein,essential for the establishment of cell polarity and the maintenance of epithelial cells in normal form.Tumor suppressor gene PTEN is involved in DNA damage repairing,cell cycle regulation,apoptosis,cell-cell interaction and inhibiting angiogenesis.Mutation or deletion of PTEN will lead to the hampered regulation of apoptosis,the promotion of cells into cell cycle,and unlimited cell proliferation.Genetic changes(such as gain,loss of parts of chromosome or gene mutation) in allelic gene are induced by unbalanced mitosis during stem cell differentiation.Those genetic changes could be used for the analysis of the clonality of cells.They could be detected by microsatellite change(including loss of heterozygosity (LOH) and microsatellite instability(MSI)),gene mutation,and comparative genomic hybridization.LOH and MSI have strong sensitivity but poor specificity,while gene mutation has strong specificity but poor sensitivity.The suitable combination of the two methods can give more precise results.In order to have a better understanding of their origination and give a clear evaluation to clinical doctors,we performed a prospective study on neuroedndocrine differentiation in gastric adenocarcinomas by molecular methods.In this study we used microsatellite instability,LOH,p53 mutation to evaluate the clonality of neuroendocrine differentiation in gastric adenocarcinoma. Immunohistochemistry was performed on tissue microarray to detect the expression of CGA,p53,MLH1,E-cadherin and PTEN in gastric cancer.The expression of these indicators,clinicopathological parameters and patient follow-up data was combined to comprehensively analyze the association between gastric cancer diagnosis and prognosis and NE differentiation.Materials and methodsIn this study,120 cases of gastric adenocarcinoma and corresponding nonneoplastic gastric mucosa tissues were obtained from the People's hospital of Zhejiang province between 2001 and 2003.First,frozen section-IHC samples were selected with large quantity of neuroendocrine cells.Second,LCM were used to get aim cells from gastric adenocarcinoma and WGA was applied to get large quantity of DNA for further study,Third,genome-wide microsatellite abnormalities(MSI and LOH ) and p53 mutation were detected by PCR-SSCP-silver staining and PCR-sequencing in order to identify the clonality of NE cells.220 cases of gastric adenocarcinoma and 31 nonneoplastic gastric mucosa tissues were obtained from the first affiliated hospital of Zhejiang University and the Pople's hospital of Shaoxing City.Immunohistochemistry was performed on tissue microarray to detect the expression of CGA,p53,MLH1, E-cadherin and PTEN in gastric cancer.The expression of these indicators, clinicopathological parameters and patient follow-up data was combined to comprehensively analyze the association between gastric cancer diagnosis and prognosis and NE differentiation.Results1,Thirty samples from a tatol of 120 containing large quantity of neuroendocrine cells were chosen for Laser capture microdissection.LCM was performed and about 500 neuroendocrine cells were precisely cut from each sample.2,Microsatellite analysis:The total incidence rate of MSI is 27.4%,while LOH is 17.9%,less than MSI.The incidence rate in Adenocarcinomas and NE cell is similar. There is no significant relationship between the incidence rate of MSI or LOH and clinicopathologic characteristics.According to the coincidence of microsatellite changes,case 2,3,5,6,11,12,18,24,27,30 had highest concordance in two kinds of cells,the other samples also had similar microsatellite changes except case7 and 10.3,p53 mutaion:most mutaions were detected in exon 7 and exon 8.Concordant mutations exhibited in sample 4,14,21 and 27,there were different mutations in two kinds of cells in case 7.In case 17,mutation took place only in adenocarcinima cells. p53 mutation took place 6 in adenocarcinomas(20.0%),5 in NE cells(16.7%).Clinical pathological data display that mutation cases exhibit in poorly-differentiated andⅢtoⅣof TNM stage,p53 mutation was closely related with degree of differentiation,TNM stage,vessel invasion and lymph node metastasis.4,According to Microsatellite changes and p53 mutation analysis,we conclude that NE cells and adenocarcinoma cells in 27 of these 30 cases originate from the same stem cell,NE cell is a component of tumor,The rest 3 cases showed different originality, which warranted further research.5,The expression rate of CGA in gastric carcinoma is 47.7%,and is correlated with poor differentiation degree.In p53 positive cases,46.5%with NE differentiation, and is positively related with invasion depth,negatively related with survival,cases with CGA positive have poor prognosis.6,45.0%of these cases are p53 positive expression in gastric carcinoma,which is much higher than normal tissue.There is a significant positive relationship between p53 expression and TNM stage,invasion depth.In gastric cancer with NE differentiation, there is a positive relationship between p53 expression and invasion depth and distant metastasis.Survival analysis shows that cases with p53 positive expression have poor prognosis.7,The expression rate of E-cadherin is 15.5%,which is lower than normal tissue, and it's expression is negatively correlated with TNM stage and vessel invasion. E-cadherin positive cases have well prognosis.8,In gastric carcinoma the expression rate of MLH1 is 15.9%,correlating with TNM stage.Survival analysis shows that patients with MLH1 positive expression have well prognosis.9,The expression rate of PTEN is 27.3%,bur there is no significant relationship between PTEN expression and clinicopathologic characteristics or survival.Conclusion1,The origination of neuroendocrine differentiation cells and adenocarcinoma cells may be concordant.2,NE differentiation and expression of p53 contribute to the infiltration and metastasis of gastric adenocarcinoma and may be predictor of poor prognosis.3,Expression of MLH1 and E-cadherin could inhibit gastric progression,may provide theoretical guidance for the estimation of prognosis.