Application of a simplified comparative genomic hybridization technique to screen for gene amplification in pediatric...

Conventional cytogenetic analysis of solid tumors is technically very demanding and requires a large number of viable cells. The technique of comparative genomic hybridization (CGH) circumvents these difficulties and has been shown to be particularly useful for identifying new gene amplifications. We have simplified the CGH technique for the detection of amplifications by utilizing a single labeling approach in which labeled tumor DNA is mixed with unlabeled normal human DNA and hybridized to normal metaphases on a slide. To examine the consistency and sensitivity of the method, initial experiments were performed using a retinoblastoma (RB) cell line and five pediatric solid tumors known to contain an amplification. The technique was easy to use and sensitive enough to detect low-level amplifications. The RB cell line showed reproducible signals at 2p24, indicative of amplified sequences, on both homologues in 95% of the metaphases (> 30) examined. Amplifications of the MYCN gene (2p24) were detected in three alveolar rhabdomyosarcomas and one medulloblastoma. CGH was then applied to six tumors in a prospective fashion, before data about specific gene amplification were available. In two, amplification of the MDM2 gene (12q13-14) was identified using CGH and later confirmed by Southern blot analysis. Four tumors negative for MDM2 and MYCN amplifications by CGH analysis were also negative by Southern blot analysis. Gene amplification as low as fourfold was detected in one tumor and the overall pattern of gene amplification detected by CGH in these tumors was not complex, involving just one amplification site for each case. Therefore, this simplified CGH technique is suitable for routine screening of pediatric solid tumors for amplifications when genetic studies are important but sample sizes are small and dividing cells are infrequent or unavailable.