| Summary: | 碩士 === 國立彰化師範大學 === 生物學系 === 90 === Abstract
Early diagnosis of micrometastasis of cancer patients is always the major topic in cancer clinic. It is reasonable to assume that if we can detect cancer cells in peripheral blood of cancer patients, implying that they have high probability to have cancer metastasis. The phenomena of loss of heterozygosity (LOH) and microsatellite instability (MSI) in cancer cells are derived from the accumulation of genetic alterations. Both have been widely accepted as biomarkers of cancer cells. For the purpose of identifying the biomarkers for detecting micrometastasis of cancer cells, we designed the present study to determine the presence of tumor DNA in the plasma of patients with hepatocellular carcinomas (HCC), gastric cancer (GC), and breast cancer (BC), characterized by LOH in 18 microsatellite markers. DNA extracted from tumor tissues, neighboring normal tissues, blood cells and plasma of cancer patients was amplified with fluorescent primers specific for the microsatellite markers. PCR amplicons were separated on the ABI-3100 16 capillary array electrophoresis instrument and analyzed with GeneScan analysis software v.3.7. According to the ratios of heterozygosity, LOH in tumor and plasma DNA, microsatellite markers were classified as four grades. Markers with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≧30 % were classified as the first grade; those with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≦30 % were classified as the second grade; those with heterozygosity≧80 % and LOH in tumor DNA≦50 % were classified as the third grade; while those with heterozygosity≦80 % were classified as the fourth grade, which were considered as unsuitable markers. Three markers (LPL, D16S413 and TP53) were classified as the first grade markers in BC, which together detected 85.3 % (29/34) of tumor DNA with LOH, and 41.2 % (14/34) of plasma DNA with LOH. In HCC, TP53 were the only first grade marker, which detected 44.9 % (22/49) of tumor DNA with LOH, and 24.5 % (12/49) of plasma DNA. None was qualified as the first and second grade markers in GC. No association was found between the presence of LOH in plasma and clinicopathological parameters, such as tumor size, tumor stage, tumor grade, the expression of estrogen receptor, progesterone receptor, c-erbB-2 oncoprotein and p53. However, our method was significantly better than dissected lymph node histochemical method in detecting micrometastasis. As a conclusion, we have provided a specific and noninvasive method, which can be applied as a diagnostic technique for early diagnosis and prognostic follow-up tests for cancer.
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