Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors.
BACKGROUND
Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas.


METHODS
Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5-192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest.


RESULTS
Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed.


CONCLUSIONS
We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.
Savola
Suvi
S
Department of Pathology, Haartman Institute and HUSLAB, University of Helsinki, Helsinki, Finland. suvi.savola@helsinki.fi
Klami
Arto
A
Tripathi
Abhishek
A
Niini
Tarja
T
Serra
Massimo
M
Picci
Piero
P
Kaski
Samuel
S
Zambelli
Diana
D
Scotlandi
Katia
K
Knuutila
Sakari
S
D000293:Adolescent; D001859:Bone Neoplasms / Q000235:genetics*; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D002648:Child; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002890:Chromosomes, Human, Pair 20 / Q000235:genetics; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics; D018572:Disease-Free Survival; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D053208:Kaplan-Meier Estimate; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000556:secondary; D015996:Survival Rate; D055815:Young Adult
C492515:ANKRD11 protein, human; D002148:Calmodulin-Binding Proteins; C500986:EWSR1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C051610:hepatoma-derived growth factor