| Aims Esophageal carcinoma is one of the most common malignant neoplasms in China. Development and progression of esophageal carcinoma is driven by the malfunction of specific genes (i.e., overexpression of oncogenes or inactivation of tumor suppressor genes). 3q gains were observed in most cases of esophageal carcinoma by comparative genomic hybridization (CGH). However, there was some limitation by CGH that was unable to detect gene amplification less than 1 Mb, and FISH can locate the gene gains accurately. In this study, 35 cases of esophageal carcinoma, 18 mucosa adjacent to the carcinoma and esophageal carcinoma cell line EC9706 were performed by fluorescence in situ hybridization (FISH). Six genes in 3q were chosen as probes, including two candidate oncogenes: MUC4 and Lamp3; two translation- and replication-associated genes: eIF4Gl and RFC4; one candidate tumor suppress gene: hDlg; one p53 family member: p63. We then explored the relationship between gene copy number changes and clinicopathological features, and tried to find new genes that had close relationship with histogenesis and progression of esophageal carcinoma.Methods Eighteen cases of fresh esophageal carcinoma tissues and mucosa adjacent to carcinoma were fixed in Carnoy's liquid, then embedded in paraffin. The other 17 cases were surgically removed samples from esophageal carcinoma, fixed in 10% formalin. All cases were cut in 7 serial sections, one for HE staining, six for FISH. Metaphase chromosome of esophageal carcinoma cell line EC9706 and normal peripheral blood lymphocytes were prapared. Plasmid DNA were extracted from Bacillus coli. Bacterial artificial chromosome (BAC) was labeled by nick translation with Biotin-16-dUTP, DNA probes were mixed and precipitated with unlabeled Cot-1 DNA. The hybridization mixture was added on slides and hybridized for 24 hours at 37 in a moist chamber. After hybridization, the DNA probes were detected by avidin-FITC, biotinylated goat-anti-avidin, and a second layer ofavidin-FITC. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Two gray pictures were taken by CCD through fluorescence microscope, then dealed by Metamorph system: red one represented nuclei and green one represented the detected gene signals. One hundred to 200 nuclei were counted, more than 5% of nuclei that contained more than 2 green signals were judged as gene amplification.Results All the 6 genes had two signals in normal peripheral blood lymphocytes. Amplification of MUC4 was detected with 4 signals in one nucleus in esophageal carcinoma cell line EC9706. All the 6 genes were observed with amplification in 35/35 (100%) esophageal carcinoma cases with different rate: MUC4 was observed with the highest amplification rate, one nucleus mainly contained 4 signals in 34/35 cases (97.1%), one poorly-differentiated case (2.9%) mainly contained more than 4 signals in one nucleus. Lamp3, eIF4Gl, RFC4, p63 and hDlg genes were detected with low copy numbers amplification in 35/35 cases, one nucleus mainly contained 3 signals. Among 18 mucosa adjacent to carcimoma, amplification of MUC4 was observed in 5/9 (55.6%) mucosa with atypical hyperplasia, but none of the other 9 cases without atypical hyperplasia. There was significant difference between MUC4 gene amplification in esophageal carcinoma and mucosa adjacent to carcinoma (P<0.01). The other 5 genes were not observed any amplification in mucosa adjacent to carcinoma. Further analysis found that the amplification of MUC4, Lamp3, eDF4Gl, RFC4, p63 and hDlg genes had no correlation with TNM staging, differentiation, lymph node metastases, age and gender of patients (P>0.05) . In comparison with samples fixed by formalin, samples fixed by Carney's liquid had less fluorescent background and enhanced signals.Conclusions Amplification of MUC4, p63, Lamp3, eIF4Gl, RFC4 and hDlg genes all are observed in esophageal carcimoma, in which these genes in 3q, MUC4 are detected with the highest amplification rate. MUC4 may have close relationship with histogenesis of esophag...
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