Genotypic analysis of esophageal squamous cell carcinoma by molecular cytogenetics and real-time quantitative polymerase chain reaction.
We performed an integrated cytogenetic study using a combination of comparative genomic hybridization (CGH), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) to analyze chromosomal aberrations associated with 8 human esophageal squamous cell carcinoma (EC-SCC) cell lines, and used real-time quantitative PCR (Q-PCR) to study the copy number changes of two candidate genes of chromosome 3q, PIK3CA and TP63, in 20 primary tumors of EC-SCC. The pooled CGH results revealed frequent gain abnormalities on chromosome arms 1p, 1q, 3q, 5p, 6p, 7p, 7q, 8q, 9q, 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18p, 19q, 20q, 22q, and Xq, while frequent losses were found on 3p, 4, 5q, 6q, 7q, 9p, and 18q. SKY detected 195 translocations, 13 deletions and 2 duplications. Among the 374 breakpoints, most clustered at the centromeric regions, such as 8q10, 13q10, 7q10, 9q10, 14q10, 15q10, 16q10, 21q10, and 22q10, but also at other regions, including 3q (3q21, 3q22, 3q25), 7p (7p22, 7p14, 7p12), 7q (7q21, 7q31, 7q32), 8q (8q21.1, 8q23), 11q (11q21, 11q24), 13q (13q14) and 18q (18q21). There was a good correlation between the number of aberrations identified by CGH and SKY (r=0.667; p=0.035). Combined CGH and SKY analyses indicated that chromosomes 3, 7, 9, 11, 14, 16, 18, 19, 20, and 22 harbored higher frequency of chromosomal aberrations than expected. FISH using BAC clones containing oncogene PIK3CA and TP63 found that both genes were amplified in 6 and 5 cell lines, respectively. Q-PCR analysis of primary tumors revealed amplification of PIK3CA and TP63 in 100% and 80% of the cases. Average copy number of PIK3CA per haploid genome was greater than that of TP63 (6.27 vs 2.73), and the difference showed statistical significance (p<0.001). Combination of CGH, SKY and FISH could reveal detailed chromosomal changes associated with esophageal cancer cells, and Q-PCR could assess the change of the candidate genes in clinical samples in a high throughput way.
Yen
Chueh-Chuan
CC
Division of Medical Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan. linch@ym.edu.tw
Chen
Yann-Jang
YJ
Lu
Kai-Hsi
KH
Hsia
Jiun-Yi
JY
Chen
Jung-Ta
JT
Hu
Cheng-Po
CP
Chen
Po-Min
PM
Liu
Jin-Hwang
JH
Chiou
Tzeon-Jye
TJ
Wang
Wei-Shu
WS
Yang
Muh-Hwa
MH
Chao
Ta-Chung
TC
Lin
Chi-Hung
CH
D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D002872:Chromosome Deletion; D022202:Chromosomes, Artificial, Bacterial; D004938:Esophageal Neoplasms / Q000235:genetics*; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008677:Metaphase; D020133:Reverse Transcriptase Polymerase Chain Reaction