喉鳞癌9p13-23区域微卫星杂合性缺失精细作图及其临床意义

Detailed Mapping and Clinical Significance of Loss of Heterozygosity on 9p 13-23 in Laryngeal Squamous Cell Carcinoma by Microsatellite Analysis

背景与目的:微卫星(microsatellite)是广泛分布于生物基因组中的DNA重复序列,与许多重要基因紧密连锁。在肿瘤中微卫星象抑癌基因一样常常发生杂合性缺失(lossofheterozygosity,LOH)。在某些特定肿瘤中有些微卫星位点存在高频发的LOH“热点”,而与该肿瘤发生、发展相关的抑癌基因很可能就隐藏在这些“热点”附近。微卫星LOH的研究是寻找新的抑癌基因的重要方法。本研究旨在探讨喉鳞癌9p13-23区域微卫星LOH的热点及其与喉鳞癌患者临床病理特征的关系。方法:采用显微切割法从喉鳞癌组织石蜡切片中挑取肿瘤组织,苯酚氯仿抽提肿瘤组织和外周血淋巴细胞基因组DNA,应用9p13-23区域13个多态性微卫星标记对42例喉鳞癌组织及其相应的外周血淋巴细胞DNA进行PCR扩增和变性凝胶电泳,并对频发微卫星LOH的发生与喉鳞癌患者临床病理特征的关系进行比较分析。结果:(1)42例喉鳞癌组织9p13-23区域微卫星LOH的发生率是97.6%(41/42),LOH发生率最高者是位于9p22-23的D9S162(89.5%),其次是位于9p21的D9S171(80.0%),与p16基因紧密连锁的D9S1748的LOH发生率仅50.0%;(2)等位基因缺失作图发现42例喉鳞癌组织在9p13-23上存在两个明显的LOH较小区域,分别位于9p21的D9S161～D9S171之间和9p22-23的IFNA～D9S162之间;(3)年龄<60岁。

BACKGROUND &OBJECTIVE:Microsatellites are the repeated DNA sequences scattered widely within the biological genomes and closely linked with many important genes. In carcinogenesis, microsatellites often display loss of heterozygosity(LOH) as tumor suppressor genes. Some microsatellite loci often exist in the hot spots of LOH at high frequency in some specific maligances. The tumor suppressor genes, which are associated with the development and progression of the tumor, possibly harbor in the vicinity of these hot spots. Therefore, the study of LOH by microsatellite analysis is an important way to detect the putative tumor suppressor genes. This study was designed to refine the hot spots of LOH on 9p13 23 in laryngeal squamous cell carcinoma and compare the correlation between the incidence of microsatellite LOH and the clinicopathological parameters. METHODS:Tumor tissues were obtained from paraffin embedded sections with microdissection. Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol chloroform.Polymerase chain reaction(PCR) amplification and denaturing gel electrophoresis were performed on a set of 42 laryngeal squamous cell carcinoma and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13 23. The correlation was analyzed between microsatellite LOH at the high frequency on 9p13 23 and clinicopathological characteristics in the patients with squamous cell carcinoma of larynx. RESULTS:(1)Of the 42 laryngeal cancers, 41(97.6%) showed LOH in at least one of the microsatellite markers tested on 9p13 23. The most frequently deleted marker was D9S162 in 17 of the 19 (89.5%) informative samples. The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80.0%). LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50.0%). (2)Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161 D9S171 on 9p21 and IFNA D9S162 on 9p22 23. (3) Multiple LOH (≥4) on 9p21 23 was found more frequently in the patients under 60 years, with supraglottic squamous cell carcinoma or cervical lymph node metastasis than those over 60 years, with glottic squamous cell carcinoma or without cervical lymph node metastasis (P< 0.01,P< 0.01,P< 0.05, respectively). On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21 23 in 42 squamous cell carcinoma of larynx. CONCLUSION:These findings imply the presence of at least two putative tumor suppressor genes on 9p13 23 in laryngeal squamous cell carcinoma. Multiple genetic alterations are probably implicated in supraglottic squamous cell carcinoma with cervical lymph node metastasis in younger patients.