|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
To clarify the mechanism of tumor progression, we carried out a lineage analysis between the subclones within individual tumors based on the genomic changes that irreversibly accumulate in tumor cells. Though CGH is suitable for such analyses because it enable us to analyze breakpoints, there still remain several methodological problems. Based on the solution to the problems, we established a new protocol in the first year and applied it to difuse-type gastric carcinoma in the second year.
DOP-PCR is currently used for the amplification of small amounts of DNA from microdissected tissue. The PCR product can be used for CGH. In the first year we tried to assess the sensitivity of CGH with or without DOP-PCR in detection of loss/gain of varying copy numbers in near-tetraploid Kato-III cells, to which varying proportions of normal cells were added. The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA. It was found that the amplification process scarcely affected the CGH results, whereas the labeling process gave less false positive in nick translation labeling than DOP-PCR labeling.
We applied improved protocol of DOP-PCR-CGH to multiple samples microdissected from 19 diffuse-type GCs including 8 early cancers. We discriminated earlier stemline changes (8q+, 8p+, 1q+, 17p-, etc.) with the breakpoints common to all the samples from later sideline changes (2q+, 11q+, 17q-, 21q-, etc.) in individual tumors. Between early and advanced cancers, the changes were largely common except 7p+, 15q+, 3p-, and 18q-, which were largely sideline changes and more frequently detected in advanced cancers (p < 0.05). On the contrary, 4q+ was preferentially seen in early cancers. Our results support the notion that early and advanced diffuse-type GCs are of a genetically single lineage ; the majority of advanced cancers derive from early cancers.