|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1996 : ¥1,100,000 (Direct Cost : ¥1,100,000)
First, the quality and quantity of the DNA extracted from formalin fixxed and microdissected tissue fragments were examined. Two to three ng DNA could be extracted from 200 to 400 cells which were scrached from 3 mum-thick formalin fixed materials. Theoretically, one nucleus is thought to contain 12 to 15 pg DNA,this estimation are consistent with the former results. Templates extracted from 10 to 20 cells could be successfully amplified by polymerase chain reaction.
Using extracted DNAs by this tissue microdissection method, the following analyzes were done :
(1) Small sized pulmonary adenocarcinomas, including 40 of early stage adenocarcinomas and 30 of early advanced adenocarcinomas were examined for loss of heterozygosity (LOH) on 2p, 3p, 9p, 17p, 17q using microsatellite markers. The frequencies of LOH were 19.8% in in early stage adenocar cinomas and 26.8% in early advanced adenocarcinomas. These results indicated that multiple allelic loss are one of the characteristic abnormalities of very early stage of pulmonary adenocarcinogenesis.
(2) DNA fingerprints qenerated by a single arbitrary primer were compared between normal and tumor tissues of the same individuals which were fixed with methanol and microdissected. Loss of sequence of chromosome 7 was detected in 41.7% of adenocarcinomas and that of chromosome 22 in 84.6% of small cell carcinomas. Gains of sequences in chromosome 1,8,13 were detected in over 40% of adenocarcinomas and chromosome 2 in 63.3% of squamous cell carcinomas. LOH of chromosome 22 in small cell carcinomas were confirmed by microsatellite PCR analysis and suggested that LOH on chromosome 22q13.3 is very frequent event in small cell carcinoma.