APPLICATION OF A SIMPLIFIED COMPARATIVE GENOMIC HYBRIDIZATION TECHNIQUE TO SCREEN FOR GENE AMPLIFICATION IN PEDIATRIC SOLID TUMORS

Conventional cytogenetic analysis of solid tumors is technically very demanding and requires a large number of viable cells. The technique o f comparative genomic hybridization (CGH) circumvents these difficulti es and has been shown to be particularly useful for identifying new ge ne amplifications. We have simplified the CGH technique for the detect ion of amplifications by utilizing a single labeling approach in which labeled tumor DNA is mixed with unlabeled normal human DNA and hybrid ized to normal metaphases on a slide. To examine the consistency and s ensitivity of the method, initial experiments were performed using a r etinoblastoma (RB) cell line and five pediatric solid tumors known to contain an amplification. The technique was easy to we and sensitive e nough to detect low-level amplifications. The RB cell line showed repr oducible signals at 2p24, indicative of amplified amplified sequences, on both homologues in 95 % of the metaphases (>30) examined. Amplific ations of the MYCN gene (2p24) were detected in three alveolar rhabdom yosarcomas and one medulloblastoma. CGH was then applied to six tumors in a prospective fashion before data about specific gene amplificatio n were available. In two, amplification of the MDM2 gene (12q13-14) wa s 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 lo w as fourfold was detected in one tumor and the overall pattern of gen e amplification detected by CGH in these tumors was not complex, invol ving just one amplification site for each case. Therefore, this simpli fied CGH technique is suitable for routine screening of pediatric soli d tumors for amplifications when genetic studies are important but sam ple sizes are small and dividing cells are infrequent or unavailable.