| Esophageal carcinoma (EC) is one of the six most common malignant diseases worldwide. The dramatic geographic distribution is the striking characteristic for EC, the ratios for EC incidence between the high- and low-risk areas could be as great as 500:1. Moreover, EC has a very poor prognosis, the five-year survival rate for the late or advanced stage is only 10%. Linzhou (formerly Linxian) and Huixian of Henan province, Northern China, have been well recognized as the highest incidence area for EC in the world, with a mortality of 161/100,000 for male and 103/100,000 for female. Gastric cardia adenocarcinoma (GCA) is also one of the most common malignant diseases in northern China, especially in Linxian and Anyang, the geographic distribution for GCA is very consistent with EC. Over the past twenty years, the tendency of incidence of distal gastric tumor was obviously decreasing, on the other hand, the tendency of incidence of adenocarcinoma at esophageal and gastric junction was obviously increasing. EC and GCA remains a leading cause of cancer-related deaths in these areas. Previous studies have been suggested that multistep genetic alterations in nucleotide level and chromosome level may underlie carcinogenesis in EC and GCA. Some possible EC or GCA -related gene may be located in the affected chromosomal arms. But very little information is available on the genetic alterations occurring in EC and GCA in high-incidence area for esophageal carcinoma in LinxianHenan.Compatative genomic hybridization (CGH) is a relatively new molecular cytogenetic technique. It combines fluorescent hybridization in situ with digital image analysis and allows the identification of the entire genome for regional variations of DNA sequence copy number (gain, loss, and amplification of DNA sequences) in a single experiment. This technique can detect recurrent copy number changes and may highlight chromosomal regions containing genes that contribute to cancer development and /or progression. In this study, using CGH technique, we wanted to document the spectrum of tumor genome regions of DNA sequences gains and losses in EC and GCA and to reveal and locate novel sites of genetic alteration in EC and GCA -related genes and demonstrate the carcinogenesis mechanism in EC and GCA.II. 2.1 Material and methods II.2.1.1 EC and GCA samplesAll the 37 cases with EC (18 male and 19 female, with an average age of 56 ± 9 year) and 30 cases with GCA (23 male and 7 female, with an average age of 56 ±9) were obtained at the time of surgical resection at the Department of Surgery, Yiaochun Hospital, between September in 2001 and March in 2002. All the patients came from the same area of Linzhou, Henan. Tumor tissue specimens were frozen immediately and kept in a freezer at -80 ℃ until use. All the patients did not receive any preoperative radiotherapy and/or chemotherapy. Of the EC, all samples were confirmed as esophageal squamous cell carcinoma (SCC) by pathological examination, including 8 cases with well-differentiated type, 21 with moderate-differentiated, and 8 with poorly-differentiated. Of the GCA, all samples were confirmed as GCA by pathological examination, including 6 case with well-differentiated type, 12 with moderate-differentiated and poorly-differentiated, respectively.II. 2.1.2 DNA extractionThe frozen sample was embedded in OTC and cryosected into slides under -20 ℃ and fixed with 95% alcohol. Tumor cells were selected under dissected microscopy onthe basis of estimated tumor cells to more than 80%. Grenomic DNA was extracted from the dissected tumor cells by proteinase K/sodium dodecyl sulfate digestion followed by phenol/chloroform/alcohol extraction. Normal reference DNA was prepared from human placenta.II.2.1.3 Slide preparation with metaphase chromosome spreads5 ml peripheral blood were extracted from healthy donors. The blood sample (1ml) was cultured for 72h (5% CO2 incubator) in RPMI1640 (20% fetal bovine serum) 10ml, PHA 120 u 1 (48 u g) . Blood cells were harvested b...
|
PDF Full Text Request