Establishment Of Repetitive Multicolour Fluorescence In Situ Hybridization And Study In Esophageal Squamous Cell Carcinomas

Objective: Numerical and structural chromosome aberrations are the defining characteristic of human cancer. In a genome-wide search for molecular alterations underlying tumorigenesis in esophageal squamous cell carcinoma(ESCC), the patterns of aberrations at chromosomal level might be an essential guide, as has been shown for many other human neoplasms. Due to technical problems, the chromosome morphology of solid tumors in general, and epithelial tumors in particular, is often poor, and as a consequence, the putative genetic events that might be important in ESCC remain undefined. To understand the chromosomal aberrations of ESCC, two investigations were carried out in this project: establishment of repetitive multicolour fluorescence in situ hybridization (RM-FISH) and primary culture technique, and identification of chromosomal imbalances involved in ESCC cell lines.Methods: The 24 microdissected chromosomes (22 pairs of autosomes plus the two sex chromosomes) were subjected to DOP-PCR, and the PCR products were tagged with different combination of fluorescein isothiocyanate (FITC), Cy3 and Cy5. Four pools of 6-colour painting probes were designed, and in situ hybridization was performed for four times on the same specimen. FISH images were captured with an epifluorescence microscope equipped with a CCD camera and 4-position filter wheel. By RM-FISH combining with inverted DAPI banding, we analyzed the chromosomal alteration of 3 ESCC cell lines. The karyotype description was according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). Tumor specimens were obtained at the time of surgery from 15 patients with ESCC to processed metaphase preparation, and analyzed metaphase preparation of a primary ESCC by RM-FISH.Results: 1) We developed a modified repetitive multicolour-FISH protocol. 2) RM-FISH technique combining with inverted 4,6-diamidino-2-phenylindole (DAPI) banding identified the karyotype in three ESCC cell lines and found a number of chromosome aberrations including deleting, inserting, translocation and mark chromosomes. Chromosomes 1, 5, 11 have higher probabilities of undergoing structural and numerical aberrations; we observed loss of 3p, gain of 3q and loss of Y in all cell lines; i(3q) in two cell lines (KYSE 410-4, KYSE 410-1); of 42 translocations, chromosomes 5 and 9 have the highest...