Studies Of Loss Of Heterozygosity Of Chromosome 3p In Non-small Cell Lung Cancer By Microdissection

Objective: To establishing the stable technology of microdissection-PCR from formalin-fixed and paraffin-embedding tissue(PET) sections for the retrospective molecular biology research,To, investigate the frequency of loss of heterozygosity (LOH) at several 3p regions in 53 non-small cell lung cancer (NSCLC) tissues,in order to study the role of chromosome 3p allele loss in the pathogenesis of lung cancer, and provide the important experimental basis of the molecular pathological diagonosis of lung cancer.Methods: Concluding two parts.part one: To find the fittest DNA extraction method of microdessection-PCR technology for following experiments, we compared four different DNA extration-PCR methods(direct PCR, phenol-chloroform DNA extraction-PCR, DNA extraction kit-PCR, and proteinase K digestion-PCR); To evaluate the longest archived time of PET which can get satified amplification,we microdissected the archival PET secton from 1992 to 1997 respectively; To observe the longest length fragment which can get satisfied amplification ,we respectively amplified five different length fragments(110bp, 268bp, 536bp, 989bp, and 1327bp).part two:53 archival, paraffin-embedded, surgically resected lung cancer specimens and 54 preneoplastic/preinvasive microdissected respiratory epithelial samples from 10 squamous cell carcinomas were studied. Precisely identified malignant and non-malignant lymphonodes were microdissected from stained slides and analyzed for allele loss using polymerase chain reaction-based assays for dinucleotide repeat polymorphisms at ten chromosome 3p loci (3p25,3p22-p24.3,3p21-p23,3p21.3,3p21.3,3p14.2and3p12).Results: 1,Method of proteinase K digestion was of choice in DNA extraction.2,Archived time of PET should be less than 13 years and 11 years if amplifying about 110bp and 268bp fragment length,separately. 3, Amplified fragment length should be less than 536bp to get satisfied amplification. 4,Allele losses at one or more 3p regions in the NSCLC were very frequent and often multiple.Allelic losses involving one or more 3p regions were detected in nearly all (98.1%)NSCLC tissues. The 3p21.3 region had the highest frequency of LOH(76.5%), followed by 3p 12(66.7%),3p14(66%), 3p22-p24.3(65.4%), 3p21-p23(58.3%),3p25(57.5%). 5,Chromosome 3p Allele loss frequently occurs in histologically normal and abnormal epitheium accompanying lung cancers,with the frequency of 62.5% and 63.2% separately. 6,Allele loss at one or more 3p regions was very frequent in the two major types of non-small cell lung cancer (100% of 25 squamous cell cancers,and 95.2% of 21 adenocarcinomas).The frequency of chromosome 3p allele loss in squamous cell cancers is significantly higher than adenocarcinomas in every tested 3p region,with 3p25(P=0.006), 3p22-p24.3(P=0.004), 3p21-p23(P=0.001), 3p21.3(P=0.000), 3p14.2(P=0.001), and 3p12(P=0.000). we found no correlation between chromosome 3p allelic loss and amount of clinicalpathological stage or sex. 7,The frequency of chromosome 3p allele loss in lung cancer with family history is much more.Conclusions: 1,We successfully established the stable microdissection-PCR technology from PET sections.This advancing scientific approach was suitable for detecting MSI and LOH of precise dissected tissues. 2,Chromosome 3p allele loss is almost universal in lung cancers and usually occurs at mutiple 3p chromosomal sites, and frequently occurs in histologically normal and abnormal epithelium accompanying lung cancer.The frequency of allele loss in Chromosome3p21.3 region is highest in lung cancer, may play a important role in the pathogenesis of lung cancer. 3,The frequency of chromosome 3p allele loss in squamous cell cancers is significantly higher than others.Increasing severity of histological change(from mildly abnormal to dysplasia to CIS) was, in general, characterized by increasing frequency of allelic loss in the chromosome 3p regions. There was no correlation between chromosome 3p allelic loss and amount of sex. 4,The patients with lung cancer family history usually have a predisposition to chromosome 3p allele losses.