Copy number gains in EGFR and copy number losses in PTEN are common events in osteosarcoma tumors

Abstract

Background: Osteosarcoma cell lines and tumors have been shown to express epidermal growth factor receptor (EGFR) and harbor amplifications at the EGFR locus. In this study, the authors further analyzed the genomic features of EGFR in osteosarcoma tumors and investigated whether they correlate with phosphatase and tensin homolog (PTEN) expression and copy number status.

Methods: EGFR and PTEN expression was assessed by immunohistochemistry (n = 28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix (Santa Clara, Calif) 50K single nucleotide polymorphism (SNP) arrays (n = 31) and fluorescence in situ hybridization (FISH) (n = 27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n = 34). In addition, EGFRvIII expression was assessed using reverse transcriptase polymerase chain reaction (n = 24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5-23 years) and median follow-up of 4.4 years.

Results: EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23 of 28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16 of 27 cases; 59%) showed gains at the EGFR locus using FISH (amplification in 4 of 27 [15%] and balanced chromosome 7 polysomy in 12 of 27 [44%]), and 12 cases showed deletions at the PTEN locus (biallelic deletions in 4 of 27 [15%] and monoallelic deletion in 9 of 27 [33%]). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected.

Conclusions: EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus.

Conflict of interest statement

Disclaimers:

None of the authors have any significant potential conflicts of interest to disclose.

- This study was presented, in part, at the 2007 Annual Meeting of the United States and Canadian Academy of Pathology.

(c) 2008 American Cancer Society.

Figures

Figure 1

Panel illustrating EGFR and PTEN expression in osteosarcoma using immunohistochemistry. For EGFR, assigned scores for these illustrated cases were 300, 100, and 0. For PTEN, assigned scores for these illustrated cases were 300, 100, and 0.

Figure 2

Results of 50K SNP array analysis in 31 osteosarcoma tumors demonstrate frequent genomic gains at 7p11.2, which includes the EGFR locus, and frequent genomic losses at 10q23.21, which includes the PTEN locus, using log2 ratio of tumor signal/mean signal of all 61 references for each SNP in each tumor case. In addition, loss of heterozygosity appears more common at 10q23.21 than at 7p11.2 (yellow: retention; dark blue: loss of heterozygosity; light blue: non-informative). These findings are matched with corresponding data (top panel) obtained using fluorescence in situ hybridization and immunohistochemistry where available. [A: amplification; CN: copy number; FISH: fluorescence in situ hybridization; IHC: immunohistochemistry; M: mixed pattern of monoallelic loss and polysomy; n: suboptimal hybridization; N: negative; p: balanced polysomy; P: positive; L1: monoallelic loss; L2: biallelic loss; LOH: loss of heterozygosity]

Figure 3

(A) Fluorescence in situ hybridization demonstrating osteosarcoma tumor with EGFR gene amplification (green signal: EGFR test probe at 7p11.2; red signal: control probe at 7q31.2). (B) Another osteosarcoma tumor harboring biallelic loss of PTEN (red signal: PTEN test probe at 10q23.21; green signal: control probe at 10p11.21); note the presence of chromosome 10 polysomy.

Figure 4

Agarose gel electrophoresis of RT-PCR products using EGFR exon 1 and exon 8 primers. The wild type EGFR mRNA is expected to yield a 929bp amplicon whereas the EGFRvIII mutant mRNA, in which exons 2 to 7 are deleted, is expected to yield a 128bp amplicon. None of the cases analyzed in our group showed expression of EGFRvIII.