Genomic Hybridization Analysis Of Genetic Aberrations In Biliary Tract Carcinomas

Background:Patients with biliary tract carcinoma which including gallbladder carcinoma and bile duct carcinoma characteristically have no signs or symptoms in the early stages of their cancers. Accordingly, patients are frequently not diagnosed until late in the disease process, leading to a poor prognosis. For better cancer therapy, in-depth study and knowledge of the biological characteristics of the disease is required. Genetic abnormalities are reported in biliary tract carcinoma including several tumor genes such as ras, c-erbB-2, bcl-2, c-myc, bax, HER-2/neu, c-met and Fas , as well as inactivation of several tumor suppress genes including p53, pl6, nm23, p27, DPC4 and Rb. However, the molecular genetic approaches used in these studies were restricted to the analysis of single or fewer genes and could not assess other genetic changes elsewhere in the tumor genome. A global overview of genomic change can be obtained using Comparative genomic hybridization (CGH). 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 of DNA sequences) in a single experiment. This technique can detect recurrent copy number changers and may highlight chromosomal regions containing genes that contribute to cancer development and/or progression.The technology of microarray (gene chip) is a new technique of gene analysis, going with practicing human genome project. Due to its low cost, high sensitivity and high flux, gene chip is one of the important tools for the study of genome, which is obviously better than the previous research model of single gene. The network mechanisms of gene expression regulation are thoroughly studied in the level of whole genome using gene chip.The aim of the experiment is to detect genetic aberrations on character of DNA copy in biliary tract carcinomas with comparative genomic hybridization , document the spectrum of tumor genome regions of DNA sequences gains and losses in GBC and BDC , reveal and locate novel sites of genetic alteration in GBC and BDC-related genes and demonstrate the carcinogenesis mechanism in GBC and BDC. The differential expression genes in gallbladder carcinoma is to find out by profiling cDNA microarray combining with the result of profile CGH simultaneously and to provide data for the candidate gene marker and therapy target and to elucidate cytogenetic changes characteristic of GBC.Methods:All the 28 cases with GBC and 18 cases with BDC were obtained at the time of surgical resection at the department of surgery, second affiliated hospital of Zhejiang University and Sir Run Shaw Shaw hospital of Zhejiang University, between October 2003 to August 2005. All patients were diagnosed by pathological examination and were not accepted any preoperative radiotherapy and/or chemotherapy. Tumor DNA was isolated by Proteinase K digestion and pHenol/chloroform/isoamylalcohol extraction and normal reference DNA were prepared from healthy man perblood lympHocyte, then tumor DNA and reference DNA were labeled by nick translation with SpectrumGreen-dUTP and SpectrumRed-dUTP respectively, and simultaneously hybridized to normal metapHase chromosomes in 1:1. After hybridization, under fluorescent microscope fixing with CCD camera, the tumor DNA is exhibited as green, the normal control DNA is exhibited as red, the fluorescence ratio between tumor and normal DNA along each reference chromosome is measured and analyzed.Five metapHases were analyzed to generate fluorescence ratio profiles in each case and image analyses were carried out using Quips CGH analysis software (Vysis). 2.Differential expression genes of GBC: The total RNA of 4 cases were extracted and detected from the fresh specimens of GBC and normal control respectively. Hybridization with BiostarH-40s type profiling cDNA microarray was performed after the RNA was labeled with fluorescent probe. The image scanning and data analysis were performed subsequently. The differential expression genes of GBC was validated with the result of profile of CGH at the same specimen and to find out the most possible gene related to the mechanism of GBC.Results:1. CGH results: Using CGH technique, the results showed that all the 28 GBCspecimens had chromosomal aberrations in different degrees. Total 157 DNA copy number gains and 114 DNA copy number losses were found in 28 GBC specimens, with an average of DNA copy number gains and DNA copy number losses per patient as 5.6 and 4.1, respectively, hi GBC, the ratio more than 20% of chromosomal profile of DNA copy number gains was characterized as follows: 7p(54%,15/28),7q(50%, 14/28),8q(46%,l 3/28),l 7q(36%, 10/28),5p(36%, 10/28), 11 q(32%,9/28), 1 q(29%,8/28), 9q(21%,6/28),16q(21%,6/28), which the minimal overlapping regions on chromosome were located on 7pl2-pl5, 7q31-q35, 8q21.3-q24.1, 17q21-q24, 5p 12-p 15.1,11 q 12-q22, 1 q23-q24,9q 11 -q 13,16q 11 -q 12. The chromosomal profile of DNA copy number losses was characterized as follows (from high to low): 17p(43%, 12/28),9p(36%, 10/28),5q(29%,8/28),6q(25%,7/28),3p(l 8%,5/28), 15p( 18%, 5/28),13p(18%,5/28), which the minimal overlapping region on chromosome were located on 17pl2-pl3,9p21-p23, 5q23-q32, 6ql6-q22, 3pl3-p21,15pll.2-pter, 13pl2-pl3oIn BDC, total 89 DNA copy number gains and 67 DNA copy number losses were found in 18 BDC samples, with an average of DNA copy number gains and DNA copy number losses per patient as 4.9 and 3.7, respectively. For BDC, chromosomal profile of DNA copy number gains was characterized as follows: themost frequently detected gain was on chromosome arms 8q (50%, 9/18) , and they were followed by (from high to low) 20q (44%, 8/18), 5p (39%, 7/18), 7p (33%,6/18), lq(28%,5/18),19q(28%,5/18),llq(28%,5/18),13q(28%,5/18),2q(22%,4/18), 15q(17%, 3/18), 7q(17%,3/18),which the minimal overlapping regions on chromosome were located on 8q21-q24.2, 20qll.2-ql3.2, 5pl3-pl5.2, 17q21-q24 , Iql2-q24, 19ql3.3-ql3.4, Ilql4-q24, 13ql3-q33, 2q21-q22, 15q26, 7q33-q36 . The chromosomal profile of DNA copy number losses in BDC was characterized as follows: the most frequently detected loss was on 3p (44%, 8/18), the following detected DNA copy number loss (from high to low) was on 18q(39%,7/18), 17p(39%, 7/18), 8p(33%,6/18), 9p(33%,6/18), 6q(22%,4/18), 5q(17%,3/18), lp(17%,3/18), 4q(17%,3/18), which the minimal overlapping regions on chromosome were located on 3pll-pl4, 18q22-qter, 17pl2-13, 8p22-p23, 9p21-p23, 6q23-qter, 5q34-qter, Ip35-pter,4q26-p28<,Statistically, for DNA copy number gains on chromosome lq, 5p, 8q 17q and losses on chromosome 5q,9p,17p ,there were no evident differences between GBC and BDC(p>0.05). In contrast, gains of 7p and 7q were more predominant in GBC(p<0.05),meanwhile the gain of 20q and losses of 3p,18q were more predominant in BDC(p<0.05).2. Microarray results: 61 differential expression genes were definited in all the 4specimen by single microarray, which was involved in several aspect of tumor development. There were 17 genes down-regulated and 44 genes up-regulated. 21 differential expression genes were identified through screening the results of microarray and the results profile CGH on 4 cases of GBC simultaneously. There were 2 oncogene and 1 tumor suppressor genes;2 genes about apoptosis;1 gene about Synthesis, repair and reconstruction of DNA;4 genes about signal transformation;4 genes about metabolism, 3 gene about protein translation and others 4 gene.Conclusion:1 It has been recognized that DNA copy number gains is related with oncogene amplification, on the contrast, DNA copy number loss is related with inactivation oftumor suppressor gene. The present study had shown that the most frequently detected DNA copy number gain in GBC was on chromosome arms 7p, 7q and 8q and DNA copy number loss in GBC was on chromosome arms 17p and 9p;which suggest that 7p, 7q and 8q regions may contain GBC-related oncogenes and 17p and 9p regions may contain promising GBC-related tumor suppressor genes. Furthermore, the gains and losses of chromosomal regions identified in this study provide important theoretic information for identifying and cloning novel GBC-related oncogenes and tumor suppressor genes.2. The present study demonstrated that the most frequently detected copy number gain in BDC was on chromosome arms 8q, the second frequently detected gain was on chromosome 20q and 5p;the most frequently detected copy number loss in BDC was on chromosome arms 3p, the second detected losses were on chromosome 17p and 18q, which the important findings suggest that chromosome 8q and 5p may contain BDC -related oncogenes, 3p , chromosome 17p and 18q may contain BDC -related tumor suppressor genes. Furthermore, the gains and losses of chromosomal regions identified in this study provide important theoretic information for identifying and cloning novel BDC -related oncogenes and tumor suppressor genes. 3.Our results demonstrated a number of common genetic imbalances in both tumors such as gain of the lq, 5p, 8q and 17q, and loss of the 5q,9p and 17p region, which could be considered relevant events in biliary tract cancer formation and progression. Characteristic abnormal changes for GBC were gain of 7p,7q, meanwhile for BDC were gain of 20q and loss of 3p and 18q, which had been shown differential molecular and genetics background between two tumors.4.Twenty-one genes the most possibility gene related to GBC were be analyzed by cDNA microarray and CGH, which may be provided the data for next exploring the mechanism of GBC.