Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma

Abstract

Background: Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets.

Methods: We used a genome-wide screening method - array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study.

Results: Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (PRDM16), 6p21.1 (CDC5L, HSPCB, NFKBIE), 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 (PMP22 MYCD, SOX1,ELAC27). We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases).

Conclusions: This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones.

Figure 1

Frequency of DNA copy number changes identified by array CGH in 48 osteosarcomas. The gains and losses are depicted as green and red color bars respectively. Clones are arranged from chromosome 1 to 22 and within each chromosome on the basis of UCSF mapping position.

Figure 2

A representative chromosome profiles showing homozygous deletions (A, E) and high-level amplifications (B- E). The clones showing homozygous deletions from 3p14.1 (RP11-89A12) and 17q12 (RP11-79O9) in tumor 06 (A, E) and high-level amplifications from 6p12-p21 in tumor 248(B), 8q12 in tumors 06 and 341 and 8q23-q24 in tumor 06, (C), 12q13-q15 in tumor 48, (D) and 17p11.2 in tumor 06(E), are shown as log ratios (Y-axis). The clones on each chromosome are arranged (pter to qter) on the basis of UCSC mapping positions.

Figure 3

Ideogram showing recurrent homozygous deletions (left) and high-level amplifications (right) identified by array CGH in 48 cases. The gene(s) contained within the BAC clone are shown in parentheses of the respective clones. Gene(s) present in the BAC clones were identified using UCSC browser by downloading gene table (refFlat) from human gene assembly, July 2003. The ideograms for chromosomes 6, 8, 12 and 17 are shown separately.

Figure 4

FISH validation of some of the high-level amplifications (6p12.1, 8q24.3 and 17p11.2) identified by array CGH. Interphase cells hybridized with centromere 6 (red)/RP11-81F7 (green) in case 274 (A), RP11-89K10 (red) in case 527 (B) and RP11-189D22 (red) in case 364 (C). The ploidy of these cases was determined based on the modal chromosome number of the respective cases, e.g. diploid (case 426) triploid (cases 274 and 364), and tetraploid (case 527).