【作者】 汤绚丽；

【导师】 姚根有； 杨泽然；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 普通型胃腺癌伴神经内分泌(neuroendocrine,NE)分化,是指胃腺癌中有分化的神经内分泌细胞,但其数目在癌组织成分中所占的比例较小不足50%,且以单个细胞或细胞巢的形式分散存在,是弥漫神经内分泌系统的一部分。我们以往研究发现前列腺癌伴散在NE细胞分化约占38.1%,大肠癌和乳腺癌分别为41.5%和21%,且高分化癌NE细胞的发现率明显高于低分化癌。而普通型胃腺癌中,NE细胞分化约占39.6%,低分化腺癌明显高于高分化腺癌。这些差异是否与肿瘤所在部位的胚胎起源、肿瘤病因学、组织发生学的不同及遗传学改变有关,仍处于探索研究阶段。 与神经内分泌肿瘤相比,非神经内分泌肿瘤中NE细胞研究较少,特别是用分子遗传学方法研究普通型胃腺癌中单个NE细胞的起源方面尚未见报道。关于普通腺癌伴NE分化的发生机制存在许多不同的假说。目前多数人认为NE细胞和上皮细胞均来源于内胚层多潜能干细胞。在肿瘤发生及演进过程中,内胚层的多能干细胞受激素、局部微环境及基因组不稳定性的影响,使某些被抑制的基因组密码发生随机脱抑制,在RNA翻译水平选择性的激活两种以上的调节基因产生双向或多向分化。然而,这些推测仅建立在形态学的观察及体外实验的基础上,缺乏可靠性和再现性;且不同肿瘤组织中散在的NE细胞不论在形态上还是对肿瘤组织的生物学行为的影响上都存在很大区别,究竟散在的神经内分泌细胞是否存在染色体及相关基因水平的改变,它们是否为肿瘤的一种成分,尚不清楚。因此,我们对伴神经内分泌分化的普通型胃腺癌进行分子水平的研究,以期明确NE细胞的克隆性起源,对这一类型的胃肠道肿瘤做出新的评价。 激光显微切割技术的出现,使得我们能够从胃腺癌中获取单个散在的NE细胞。全基因组扩增技术是一项以微量(ng级)DNA为模板,通过Phi 29 DNA聚合酶强大的结合能力及3’—5’外切酶活性对全基因组进行高保真性地连续扩增,从而得到大量(μg级)基因组DNA的新技术,它弥补了模板量不足的问题。联合激光显微切割及全基因组放大,获取腺癌中NE细胞,提取DNA并与腺癌细胞进行分子水平改变的比较,在全基因组范围内寻找复制差异片段,推测其克隆起源。更多还原

【Abstract】 Adenocarcinoma with neuroendocrine differentiation (NED) has been defined as single and scattered neuroendocrine differentiated cells emerging in non-neuroendocrine organ with the number less than a half. These cells are found diffusely spread or nidulant among epithelial cells. Our previous study revealed that 38.1% of prostate carcinoma, 41.5% of colon cancer, 21% of breast cancer and 39.6% of gastric cancer have neuroendocrine differentiation. Interestingly, although closely relationship has been shown between NE cell numbers and tissue differentiation in those tumors, a negative correlation is only seen in gastric cancer. It’s unknown yet whether the differed correlate among different tumors is due to tumor embryogenesis, etiology, histogenesis and genetic changes.Compared with neuroendocrine tumor, studies of neuroendocrine differentiation in gastric adenocarcinoma are rare, especially by molecular biological methods. There are several hypothesis of genesis mechanism of neuroendocrine differentiaion. The common theory is that NE cells of the gastrointestinal tract originate from the multi-potential endodermal stem cell. Impacted by hormone, microenvironment and genomic instability in tumorigenesis and progression, some subdued genomic codes are randomly derepressed and selectively activate more than two regulatory gene in RNA translational level as a result of which endodermal multipotent stem cell generate bidirectional or multidirectional differentiation. However, scattered single neuroendocrine cell from different tumors has apparently different morphologic representation and biological behavior influences. Besidies, whether these neuroendocrine cells have chromosomal and genetic alterations, whether adenocarcinoma components and neuroendocrine cells expand from two distinct, intimately located precursors or arise from a single progenitor cell remain unclear. In order to have a better understanding of neuroendocrine cellular origin and to give putative new evaluations for planning a rational preventive and therapeutic strategy, in this thesis, a prospective study of neuroedndocrine differentiation in gastric adenocarcinomas was performed by molecular approach.Laser capture microdissection (LCM) technique allows the isolation of morphologically identified cell populations down to the single-cell level for genetic analysis and thus can circumventor ameliorate the problem of tissue heterogeneity. Whole genome amplification (WGA) is a method which uses Phi 29 DNA polymerase possessing high combining power and fidelity to generate microgram quantities of DNA starting with as little as nanogram quantities of genomic DNA. In this study, LCM and WGA were used to compare genomic characteristics of NE cells in tumor with adenocarcinoma cells, from the data of which the clonality of a NE cell was speculated.During stem cell differentiation, unbalanced mitosis induces the same genetic changes (such as gains, losses of parts of chromosomes and gene mutation) in matched adenocarcinoma and neuroendocrine cells in adenocarcinoma. This theory is applied in clonality analysis by microsatellite change (including loss of heterozygosity and microsatellite instability) and 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 these methods gives more precise results.Materials and methodsIn this study, 125 cases of gastric adenocarcinoma and corresponding non-neoplastic gastric mucosa tissues were obtained from People’s hospital of Zhejiang province between 1995 and 2000. Firstly, tissues were freshly cut, immunohistochemically stained by chromogranin A monoclonal antibodies to obtain large quantity of NE cell samples. Secondly, LCM and WGA were used in large quantity of NE cell samples to obtain single NE cell, adenocarcinoma components and normal mucosa components. Thirdly, genome-wide microsatellite abnormalities and p53 mutation were detected by PCR-SSLP and PCR-sequencing. Finally, LOH, MSI and p53 mutation detected in NE cells of tumor and adenocarcinoma components were compared and genetic evidence for clonality was proposed.Results1. According to immuno-LCM, of 125 well matched cases, 28.8% of gastric adenocarcinoma and 29.8% of non-neoplastic mucosa showed moderate-large NE cells, of which 14 were observed strong positive chromogranin A staining in both adenocarcinoma and normal mucosa. For the 6 cases showed the largest quantities of NE cells, LCM was performed and about 200 NE cells, 100 adenocarcinoma nests and non-neoplastic mucosal glands from each sample were precisely cut out under 150 power lens.2. According to microsatellite abnormalities, MSI was demonstrated more than LOH. Adenocarcinoma cells had more LOH and MSI than NE cells in tumor. Most LOH concentrated in 4q, 5q, lip, 17p, which contained gastrointestinal cancer-related gene, whilemost MSI concentrated in 7p, 8p, I2q, I3q, 18q. According to clonality analysis, case 5 and case 6 had highest concordance and lowest discordance in NE cells and adenocarcinoma cells with MSI or LOH while case 2 had highest discordance and no concordance. The concordance rate in six samples were 6>5>4>3>1>2.3. According to p53 mutation, exon 7, intron 7 and exon 8 mutations exhibited in adenocarcinoma cells in three samples (50%), while exon 7 and intron 7 mutations exhibited in NE cells in two samples (33.3%). According to sequencing, Exon 7 mutation was analyzed in codon 244 while exon 8 in 273 and intron 7 in 23222 site. Concordance mutations of NE cells and adenocarcinoma cells were demonstrated in two samples (case 4 and case 5) of exon 7 and intron 7, while discordance mutation exhibited only in adenocarcinoma cells in one sample (case 1) of exon 8. The concordant mutated tumors had poor differentiation and were in stage II1A of TNM. Other samples had no mutation detected.4. Of the six adenocarcinoma with neuroendocrine cells examined, according to microsatellite abnormalities and p53 mutation results, four of them were dual differentiated from multi-potential stem cells, those NE cells were probably one of the tumor components and had malignant biological behavior characterized by secreting hormone such as secoronin, gastrin to promote tumorous growth and invasion. The other two cases might arise from different clones, those NE cells were likely interstitial substance or products of caceration, but this result should be confirmed by using more samples.ConclusionThe clonal origins of scattered neuroendocrine cells in gastric adenocarcinoma are probably genetic heterogeneous.更多还原