To further study amplification of the 12q chromosomal region in RMS, we used data from The Cancer Genome Atlas to compare our findings in RMS with amplification of this region in other cancer categories. Amplification of this region has been previously described in glioblastoma multiforme (GBM), dedifferentiated (DD) liposarcoma (LPS), and lung adenocarcinoma (LUAD). Cases with both copy number and RNA sequencing data were chosen for subsequent analyses. We focused on focal amplification events in the 12q13-q15 chromosomal region in RMS, GBM, LUAD and LPS categories. The frequency of 12q13-q14 amplification was: FP RMS (32%), FN RMS (3%), GBM (17%), LUAD (5%), DDLPS (90%), and myxoid LPS (MLPS) (4%). For the 12q15 amplification event, the frequencies of amplification were: FP RMS (4%), FN RMS (23%), GBM (9%), LUAD (5%), DDLPS (94%), and MLPS (4%). Based on these findings, FP-RMS and FN-RMS cases were used to assess 12q13-q14 and 12q15 amplification events, respectively, in the RMS category. In the LPS category, since there were very few DDLPS cases without 12q13- q15 amplification, the MLPS subtype, which is generally not amplified in this region, was used to provide amplicon-negative cases for comparison to amplicon-positive DDLPS cases. We developed a statistical approach to define the common 12q13-q14 and 12q15 amplification events in these cancers. To statistically define a high confidence region of amplification, we divided the chromosomal regions of interest into multiple segments, determined the copy number of each segment and assessed whether there were statistically relevant differences in copy number between amplicon-positive and amplicon-negative samples within each segment. Our analysis of 12q13-q14 amplification in FP RMS, GBM, LUAD, and DDLPS revealed high confidence regions of amplification ranging from 0.5 Mb in GBM to 1.6 Mb in LUAD. In our analysis of these amplified regions, we observed an overlap of 0.2 Mb across the four tumor categories, which contained 15 genes, including CDK4. In addition, there was a 0.1 Mb region of amplification common to GBM and RMS; and a 0.2 Mb region of amplification common to GBM, LPS, and LUAD. Finally, our results show that amplicons in DDLPS and LUAD extended further distally, while the amplicon in FP RMS extended further proximally. In particular, there was a 0.5 Mb amplified region specific to FP RMS and a 0.9 Mb amplified region specific to LUAD. We analyzed RNAseq data to assess if genes in the amplified region are overexpressed in these cases. For the 12q13-q14 amplicons, 18 genes in GBM, 15 genes in LUAD, 15 genes in LPS, and 14 genes in RMS were significantly overexpressed in amplified samples. Of note, CDK4 and six nearby genes within the 0.2 Mb overlapping amplified region were overexpressed in amplicon-positive samples in all four cancer categories. Two genes were preferentially overexpressed in in amplified cases of GBM, LUAD, and LPS and contained within the region specifically amplified in these three tumor categories. Furthermore, four genes were differentially expressed only in amplified FP RMS and map to the corresponding amplified region. In a comparable analysis of 12q15 amplification, the consistently amplified reason was largest in LUAD (3.8 Mb), and smallest in GBM (0.2 Mb). A 0.2 Mb region of overlap across all four tumor categories overlaps four genes, including MDM2. In addition to this region, there was a 0.8 Mb region of amplification common to LPS, LUAD and RMS. Finally, the high confidence region of 12q15 amplification in LUAD extended further proximally, and as a result, there was a 1.8 Mb region of LUAD-specific amplification. In our correlation of copy number with expression, two genes in GBM, 12 genes in LUAD, 8 genes in LPS, and 10 genes in FN RMS were significantly overexpressed in amplified compared to non-amplified samples. MDM2 and CPM were the only genes overexpressed in amplicon-positive samples in all four tumor types and contained within the 0.2 Mb amplified region common to all four tumor types. In association with the region of amplification common to FN RMS, LUAD, and DDLPS, there were at least three additional genes from this region that are overexpressed in amplified cases of these three tumor types. Finally, two genes were only overexpressed in amplified LUAD cases, which is consistent with the localization of these genes to the region of LUAD-specific amplification.
