Quantitative real-time PCR for quick simultaneous determination of therapy-stratifying markers MYCN amplification,...

Amplification of the oncogene MYCN as well as deletions in 1p and 11q are important prognostic and in part therapy-stratifying factors in human neuroblastoma. Due to the increasing clinical relevance of these molecular markers, accurate and fast assessment of the status of MYCN, 1p, and 11q is essential. As 2 techniques are recommended to avoid artefacts and to circumvent technical limitations, we developed a real-time q-PCR assay using genomic DNA from frozen and paraffin-embedded tissue as template as an alternative to LOH analyses and Southern blot (SB) and in addition to fluorescence in situ hybridization (FISH). Determination of deletion or amplification was achieved by comparing the copy number of a target gene (TG from the region of interest) to an unaffected reference gene (RG) within the same chromosome. PCR raw data were normalized to a serial dilution standard curve and a ratio TG/RG was created. The ratio to define a deletion was set as 0.5 (= expected ratio 1 TG copy/2 RG copies), the amplification threshold was set as >10.0. Data were compared to results obtained by FISH and were consistent in 10 of 13 (77%) tumors with deletion 1p, 18 of 20 (90%) with deletion 11q, 12 of 12 (100%) with MYCN amplification, and 146 of 151 (97%) samples without any aberration. Three tumors with aberrations in 1p and 2 tumors with aberrations in 11q were detectable by FISH but not by PCR. Three cases indicated a deletion 11q, 1 tumor a deletion 1p by PCR only. Specificity was 98% for 1p and MYCN each and 92% for 11q. Sensitivity was 77% for 1p, 90% for 11q, and 100% for MYCN. The discrepant results were mostly caused by heterogeneous cell populations of the investigated tissue; the use of real-time q-PCR for the detection of chromosomal aberrances in NB enables a fast and reliable assessment of the 3 most relevant chromosomal aberrations simultaneously. As the assay does not require reference tissue, can be performed with small amounts of DNA, and allows the investigation of paraffin-embedded material for the MYCN-status, it can be regarded alternative to LOH or SB analyses and in addition to FISH.