To extend our studies of the 12q13-q14 amplification in fusion-positive rhabdomyosarcoma (RMS), we used data from The Cancer Genome Atlas (TCGA) to compare our findings in RMS with amplification of this chromosomal region in other cancer categories. In particular, we identified multiple cases with 12q13-q14 amplification in glioblastoma multiforme (GBM), dedifferentiated liposarcoma (dLPS), and lung adenocarcinoma (LAC). Whereas our previous studies identified this amplicon in 25% of cases of PAX3-FOXO1-positive RMS, analysis of the TCGA data found 12q13-q14 amplification in 7% of LAC, 20% of GBM, and 86% of dLPS. We then used a statistical approach to delineate the consistently amplified region in each cancer category. In this analysis, a large genomic region surrounding the amplified region was split into numerous intervals, and the copy number in each interval was then assessed in amplicon-positive and amplicon-negative samples. Significant copy number differences in each interval were then evaluated by Mann-Whitney U tests with Bonferroni corrections. This analysis identified a common 0.24 Mb region of amplification (containing 13 genes, including CDK4) across the four cancer categories. In addition to the commonly amplified regions, the region of amplification extended in the centromeric direction in fusion-positive RMS and extended in the telomeric direction in GBM, dLPS, and LAC. To evaluate the expression consequences of these amplification events, RNA-Seq data was analyzed for differential expression between amplicon-positive and amplicon-negative samples in each of the cancer categories, and the similar statistical methods as described above were used to determine significant expression differences. This analysis revealed that seven genes within the common overlap region were overexpressed in association with this amplification event in all four categories. In addition, there were four overexpressed genes specifically associated with the extended region of amplification in fusion-positive RMS and three overexpressed genes specifically associated with the extended region of amplification in GBM, LAD, and dLPS. These findings indicate that cytogenetically similar amplification events actually consist of a common core amplified region and additional extended regions of amplification that are specific to certain cancer categories. Our previous studies of the 2p24 amplification event in fusion-positive RMS indicated that MYCN was amplified and over-expressed in association with this amplicon. The functional significance of high MYCN expression in fusion-positive RMS was demonstrated in studies in which we show that the combination of exogenous MYCN and PAX3-FOXO1 stimulated oncogenic transformation and rapid tumorigenesis of human myoblasts, though neither high MYCN nor high PAX3-FOXO1 expression was sufficient to cause these oncogenic effects. To explore the effect of different levels of MYCN expression and to modulate MYCN expression during experiments, we designed an inducible MYCN expression construct by cloning the MYCN cDNA and a hygromycin resistance gene into a lentiviral doxycycline-inducible expression construct. The resulting construct showed doxycycline induction of high levels of MYCN mRNA and protein in transduced myoblasts. However, in the absence of doxycycline induction, these cells showed significantly higher MYCN expression than cells transduced with the empty expression construct. This low level of leakiness was sufficient to permit oncogenic transformation in the presence of PAX3-FOXO1. To derive transduced cells with lower basal levels of MYCN expression, we performed several independent transduction experiments and analyzed the transduced replicates to find the population with the lowest basal MYCN expression and then obtained multiple subclones from this population. Though very low basal MYCN expression was found in specific subclones, this basal level increased as the cells were passed,indicating that there is a strong selection for high basal MYCN expression that complicated these planned studies.
