Differential expression of neuronal genes defines subtypes of disseminated neuroblastoma with favorable and unfavorable outcome.

PURPOSE: Identification of molecular characteristics of spontaneously regressing stage IVS and progressing stage IV neuroblastoma to improve discrimination of patients with metastatic disease following favorable and unfavorable clinical courses. EXPERIMENTAL DESIGN: Serial analysis of gene expression profiles were generated from five stage IVS and three stage IV neuroblastoma. Differential expression of candidate genes was evaluated by real-time quantitative reverse transcription-PCR in 76 pretreatment tumor samples (stage IVS n=27 and stage IV n=49). Gene expression-based outcome prediction was determined by Prediction Analysis for Microarrays using 38 tumors as a training set and 38 tumors as a test set. RESULTS: Comparison of serial analysis of gene expression profiles from stage IV and IVS neuroblastoma revealed approximately 500 differentially expressed transcripts. Genes related to neuronal differentiation were observed more frequently in stage IVS tumors as determined by associating transcripts to Gene Ontology annotations. Forty-one candidate genes were evaluated by quantitative reverse transcription-PCR and 18 were confirmed to be differentially expressed (P

Characterization of a complex genomic alteration on chromosome 2p that leads to four alternatively spliced fusion transcripts in the neuroblastoma cell lines IMR-5, IMR-5/75 and IMR-32.

Genetic aberrations in neuroblastoma (NB) have been extensively characterized over the last years. Alterations of the short arm of chromosome 2 (2p) have been of particular interest, since amplification of the MYCN oncogene on 2p24 is associated with an adverse outcome in NB patients. Here, we report on the characterization of a novel genomic rearrangement involving genetic material from 2p13 and 2p24 in NB cell lines that was discovered based on a serial analysis of gene expression (SAGE) profile of the MYCN-amplified NB cell line IMR-5. By analysis of a highly expressed SAGE tag not matching a Unigene cluster we identified four alternatively spliced corresponding transcripts, each of which consisted of the first 14 exons of the anthrax toxin receptor 1 gene (2p13.1) and varying combinations of exons of an unidentified gene located 1.3 Mb telomeric of MYCN (2p24.3) that was termed novel neuroblastoma gene 1. By Southern Blotting, Fluorescent In Situ Hybridization and Long Distance Inverse-PCR we disclosed that these transcripts result from a genomic alteration including material from distinct regions of chromosome 2p and four genomic breakpoints that are joined by short sequences of unknown origin. Furthermore, we show that this rearrangement lies within the homogeneous staining regions (HSR) in IMR-32 cells and is prevalent in both IMR-32 cells and their sub-clones IMR-5 and IMR-5/75, but not in a panel of 70 primary NB tumors. Our work is the first study discovering a fusion transcript based on a SAGE profile and for the first time precisely describes the DNA sequence of amplified breakpoint regions in NB.

Quantitative real-time PCR for quick simultaneous determination of therapy-stratifying markers MYCN amplification, deletion 1p and 11q.

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.

Reliable transcript quantification by real-time reverse transcriptase-polymerase chain reaction in primary neuroblastoma using normalization to averaged expression levels of the control genes HPRT1 and SDHA.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) represents a sensitive and efficient technique to determine expression levels of target genes in multiple samples and is increasingly used in clinical oncology to evaluate the patient's outcome or to detect minimal residual disease. Normalization of raw data are required to obtain comparable results between different specimens and is usually achieved by correlating transcript abundances of target genes with those of a single control gene with putatively stable expression levels. In this study, expression stability of six supposed control genes was evaluated in 64 samples of primary neuroblastoma and HPRT1 and SDHA mRNA levels were shown to exhibit the least expression variability among the samples. Because application of more than one control gene may enhance reliability of real-time RT-PCR results, various normalization factors consisting of the geometrical mean of multiple control gene expression values were calculated and evaluated by mRNA quantification of 14 target genes. Comparison with transcript levels determined by oligonucleotide-array expression analysis revealed that target gene mRNA quantification became most consistent after normalization to averaged expression levels of HPRT1 and SDHA. This normalization factor was in addition demonstrated to be not associated with stage of disease or MYCN amplification status of the tumor. Thus, these data indicate that the geometrical mean of HPRT1 and SDHA transcript levels represents a suitable internal control for biological and clinical studies investigating differential gene expression in primary neuroblastoma by real-time RT-PCR.