Detailed genomic mapping and expression analyses of 12p amplifications in pancreatic carcinomas reveal a 3.5‐Mb target region for amplification

Abstract

Previous cytogenetic and comparative genomic hybridization (CGH) analyses have shown that the gain of chromosome arm 12p is frequent in pancreatic carcinomas. We investigated 15 pancreatic carcinoma cell lines using CGH, fluorescence in situ hybridization (FISH), and semiquantitative polymerase chain reaction (PCR) to characterize 12p amplifications in detail. The CGH analysis revealed gains of 12p in four of the cell lines and local amplification within 12p11-12 in six cell lines. By FISH analysis, using precisely mapped YAC clones, the commonly amplified region was found to be approximately 5 Mb. The amplified segment extended from YAC 753f12, covering the KRAS2 locus, to YAC 891f1, close to the centromere. A semiquantitative PCR methodology was used to estimate genomic copy numbers of 14 precisely mapped expressed sequence tags (ESTs) and sequence-tagged sites, located within this interval. The level of amplification ranged from two- to 12-fold. The produced gene copy profiles revealed a 3.5-Mb segment with various local amplifications. This region includes KRAS2 and ranges from D12S1617 to sts-N38796. Two of the cell lines (primary and metastatic tumor from the same patient) showed amplification peaks within the distal region of this segment, two had peaks within the proximal region, one showed subpeaks in both regions, and one displayed amplification of the entire region. Chromosome segment-specific cDNA array analysis of 29 expressed sequences within the whole interval between D12S1617 and sts-N38796 indicated overexpression of four ESTs, two corresponding to DEC2 and PPFIBP1, and two to ESTs with unknown function. Expression analysis of these and of KRAS2 showed specific overexpression in the six cell lines with local 12p amplifications. These findings indicate two target regions within the 3.5-Mb segment in 12p11-12, one proximal including PPFIBP1, and one distal including KRAS2.

Microarray analyses reveal strong influence of DNA copy number alterations on the transcriptional patterns in pancreatic cancer: implications for the interpretation of genomic amplifications

“…We previously identified BHLBH3 (DEC2), KRAS2 and PPFIBP1 as possible target genes for 12p amplifications in pancreatic carcinomas (Heidenblad et al, 2002). In the present study, as many as 20 additional putative target genes/ESTs were identified in this region (Figure 2), four of which (EKI1, BCAT, CMAS, and SURB7) have also been shown to be overexpressed in testicular germ cell tumors with similar amplifications (Rodriguez et al, 2003).…”

“…For RT-PCR validations, including MET and MYC, two multiplex PCR reactions were performed for each gene, one including ACTB, and the other GAPD as internal standard. PCR conditions conformed to standard procedures, and quantification was performed by phosphorimaging as described previously (Heidenblad et al, 2002). The expression levels were calculated as the mean of the ratios between the assayed genes and internal standards, normalized against the reference.…”

12p-Amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH

“…It is of interest to note that a similar situation seems to exist in pancreatic carcinomas (Heidenblad et al, 2002). Investigation of, specifically, the 12p11.2-p12.1 region is of relevance for all (T)GCTs.…”

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

“…One prior report identified EWS-FLI1 increasing TERT activity independently of DNA binding; however, no isoform analysis was performed (56). These four genes were recognized as putative contributors to oncogenesis based on the published literature (57)(58)(59)(60)(61)(62)(63)(64)(65). None of these genes were alternatively spliced by WT EWS reduction.…”

“…PPFIBP1 is a scaffolding protein where the reduction of EWS-FLI1 leads to the inclusion of exon 19, which is translated to a region between the first and second sterile alpha motif (SAM) protein interaction domain, which is critical for CASK binding (73). PPFIBP1 has been implicated in both brain and pancreatic cancer; however, the relationship between oncogenesis and specific isoforms is not well characterized (60,64). One of the significant challenges for physicians treating patients with ES is therapeutic stratification at the time of diagnosis, beyond metastatic disease (74,75).…”