Clonality Analysis Of Neuroendocrine Cells And Related Genes In Gastric Adenocarcinoma

Neuroendocrine differentiation are common events in adenocarcinomas. Usually they appear as single or nidulant in epithelial cells, and less than a half in number. Our past study showed that colon carcinoma with neuroendocrine differentiation take part in 41.5%, prostate carcinoma 38.1%, mammary adenocarcinoma 21%, gastric adenocarcioma 39.6% and pancreatic carcinoma 17.9%. The relationship between neuroendocrine differentiation and their primary cancers is intimate, except for gastric adenocarcioma, there are more neuroendocrine cells in well differentiated adenocarciomas than in poorly differentiated ones. In all the cancers above, it's unknown yet whether the correlation among different tumors is due to tumor embryogenesis, etiology, histogenesis or genetic changes.Compared with neuroendocrine tumors, statistics of non- neuroendocrine tumors are relatively few, especially for gastric adenocarcinoma. Clonality about the neuroendocrine differentiation cells in gastric adenocarcinoma and neuroendocrine differentiation related genes are unclear. The most common theory is that neuroendocrine cells are originate from the multi-potential stem cell. During tumorigenesis and progression, impacted by hormone, microenvironment 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. However, scattered neuroendocrine cells from different tumors have apparently different morphological representation and influence the biological behavior of primary tumors. Whether these neuroendocrine cells have chromosomal or genetic alterations; if they present as a stroma of adenocarcinoma are still unclear. In order to have a better understanding of their origination and give a clear evaluation to clinical doctors, We performed a prospective study on neuroedndocrinedifferentiation in gastric adenocarcinomas by molecular methods. In this study we used microsatellite instability, LOH, mutation to eveluate the clonality of neuroendocrine differentiation in gastric adenocarcinoma and to find out some related genes.We harvested neuroendocrine cells from gastric adenocarcinoma by Laser capture microdissection (LCM). By using this technique morphological identified cells down to the single-cell level can be isolated for genetic analysis without polluted by neighbour cells, thus solving the problem of tissue heterogeneity. Whole genome amplification (WGA) is a new method developed by the QIAGEN company, it contains a special DNATaq named Phi 29 DNA polymerase, which can extend genomic DNA at room temperature, it's not only possessing high combining power but also generating microgram quantities of DNA from nanogram DNA fidelity. In this study, LCM and WGA were performed to compare genomic characteristics of neuroendocrine cells with adenocarcinoma cells, in order to speculate the clonality of neuroendocrine cells in gastric adenocarcinoma. And to show some genes related to neuroendocrine differentiation in gastric adenocarcinoma.During stem cell differentiation, unbalanced mitosis induce the same genetic changes (such as gain, loss of parts of chromosome or gene mutation) in allelic gene. This theory is applied to analysis the clonality of different cells, by using microsatellite change (including loss of heterozygosity and microsatellite instability) and gene mutation, comparative genomic hybridization. Loss of heterozygosity (LOH) and microsatellite instability (MSI) are commonly used and have strong sensitivity but poor specificity for extensive markers, while gene mutation has strong specificity but poor sensitivity. The suitable combination of the two methods can give more precise results.Recently, studies showed that normal cells need two mechanism to regulate their proliferation, the positive signal which promots cell proliferation and the negative signal acts as the opposite. Oncogene give positive signals and anti-oncogene may give negative ones. Wild type p53, PTEN, APC act as anti-oncogene. In normal tissues, they take part in DNA reparing, cell cycle regulating, programe cell death processing and blood vessel formating. When mutated they lost the above functions. As a result cells proliferation unbounded. In this study we want to evaluated the function of the three genes by using the methods of mutation and LOH.Materials and methodsIn this study, 125 cases of gastric adenocarcinoma were obtained from the People's hospital of Zhejiang province between 2001 and 2003. First, to screen samples have large quantity of neuroendocrine cells by frozen section-IHC,. Second, LCM were used to get aim cells from gastric adenocarcinoma and WGA was applied to get large quantity of DNA for our study, Third, genome-wide microsatellite abnormalities and p53 mutation were detected by PCR-SSLP and PCR-sequencing. Finally, to have a further study about the related genes,such as p53, PTEN and APC.Results1. According to immunohistochemistry, 30 cases were chosed from 125 well samples which contain the largeest quantity of neuroendocrine cells for Laser capture microdissection. LCM was performed and about 500 neuroendocrine cells were precisely cut from each sample.2. According to microsatellite instability, there are more MSI than LOH. Adenocarcinomas had more LOH and MSI than NE cell. Most LOH concentrated in 4q, 5q, 11p, 17p, which contains gastrointestinal tumor-related genes, while most MSI concentrated in 7p, 8p, 12q, 13q, 18q. According to clonality analysis, case 6, 10, 14, 15,16 had highest concordance LOH, while case 5, 14 ,29 had highest concordance and lowest discordance with MSI or LOH ,bute case 2 and case 7 had highest discordance and no concordance.3. According to p53 mutaion, exon 7, intron 7 and exon 8 mutation were detected, 13 in adenocarcinomas (43.3%), 12 in NE cells(40%). While concordance mutations of exon 8 exhibited in 3 samples, intron 7 exhibited in 2 samples and exon 7 exhibited in 4 samples in NE and adenocarcinoma cells. According to sequencing, Exon 7 mutation located at codon 244 while exon 8 at 273 and intron 7 at 23222 site. Mutation of p53 in NE cells, Of which 11 cases exhibit in in poorly-differentiated and III to IV of TNM stage.4. Mutation of PTEN, of the 30 cases mutation of PTEN in neuroendocrine differentiation cells are 8(26.7%). Of which 0 in well-differentiated cases, and 7in III-IV of TNM.5. LOH and MSI of gene PTEN and APC, The highest MSI of PTEN is site D10S2394, about 40%, the highest site of LOH in NE cells is D10S541, about 16.7%. The highest MSI of APC is site D5S1965 about 43.3%, the lowest site of LOH in NE cells is D5S346 about 0.Conclusion1 , The origination of neuroendocrine differentiation cells and adenocarcinoma cells may fromthe same stem cell. 2, Mutation or LOH of P53, PTEN , APC may contribute to the neuroendocrine differentiation ingastric adenocarcinoma.