Detection of Single-Copy Chromosome 17q Gain in Human Neuroblastomas Using Real-Time Quantitative Polymerase Chain Reaction

摘要

Regional genomic alterations resulting from single-copy allelic loss or gain have been well characterized in many human cancers and are often of prognostic relevance. Unbalanced gain of 17q material is common in malignant human neuroblastomas and typically results from unbalanced translocations. Unbalanced 17q gain may be an independent predictor of disease outcome, but technical difficulties with quantifying such gain using fluorescent in situ hybridization gives this method limited clinical applicability. We now describe a duplex genomic DNA-based quantitative polymerase chain reaction assay to determine the presence or absence of unbalanced gain of chromosome 17q in primary neuroblastoma specimens. The technique was first refined and validated in a panel of nine human neuroblastoma-derived cell lines by direct comparison with dual-color fluorescent in situ hybridization. Prospective blinded comparison of quantitative polymerase chain reaction and fluorescence in situ hybridization in 40 human neuroblastoma primary tumor samples showed a sensitivity of 96% and 100% specificity for detecting unbalanced 17q gain when a relative 17q copy number ratio of 1.3 was used to define unbalanced gain. Tumors with ratios >1.3 were highly associated with malignant tumor phenotypic features such as metastatic disease (P MYCN amplification (P = .008). These data suggest that quantitative polymerase chain reaction determination of 17q status is feasible and highly specific in primary tumor samples. Sensitivity may be limited because of the inherent complexity of both the chromosomal rearrangements and heterogeneity of some tumor samples. Taken together, quantitative polymerase chain reaction can be used as a high-throughput screening tool for 17q aberrations, but a subset of samples may also require fluorescence in situ hybridization analysis in an attempt to conclusively determine 17q allelic status.