Genetic amplification of the vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in human osteosarcoma

Abstract

Background: Osteosarcoma is the most common primary tumor of bone. It is a highly vascular and extremely destructive malignancy that mainly affects children and young adults. The authors conducted microarray-based comparative genomic hybridization (aCGH) and pathway analyses to gain a systemic view of pathway alterations in the genetically altered genes.

Methods: Recurrent amplified and deleted genes that were detected by aCGH were subjected to an analysis based on the Kyoto Encyclopedia of Genes and Genomes to identify the altered pathways. Among the enriched pathways, vascular endothelial growth factor (VEGF) pathway genes collectively were amplified, and alterations of this pathway were validated by fluorescence in situ hybridization (FISH) and immunohistochemistry analyses in 58 formalin-fixed, paraffin-embedded osteosarcoma archival tissues that had clinical follow-up information.

Results: The pathway enrichment analyses of the aCGH data revealed that VEGF pathway genes, including the VEGFA gene itself, were amplified significantly in osteosarcoma. Genetic amplification of the VEGFA gene, both focally and in larger fragment, was validated by FISH analysis. It is noteworthy that amplification of the VEGFA gene and elevated expression of the VEGFA protein were associated significantly with microvascular density and adverse tumor-free survival in patients with osteosarcoma.

Conclusions: The authors report for the first time that VEGF pathway genes, including the VEGFA gene, are amplified in osteosarcoma. Amplification of the VEGFA gene is not only an important mechanism for elevated VEGFA protein expression but also is a poor prognostic factor for tumor-free survival. Combined classification of VEGFA gene amplification and positive VEGFA protein expression may provide a more accurate stratification method of selecting anti-VEGF therapy for patients with osteosarcoma.

Figure 1. Chromosomal and gene aberrations of 20 human osteosarcomas

(A) The recurrence pattern of copy number alterations (CNAs) in two aCGH datasets (GSE19180 and GSE9654). The x-axis denotes chromosome numbers; the y-axis shows frequency of aberration of gains (positive) and losses (negative) for each measured aCGH probe arranged based on their genomic coordinates along the x-axis. Dashed lines indicate the thresholds for significant recurrent aberrations. Measured sequences with aberration frequency exceeding the thresholds are color-coded to emphasize the locations of significantly recurrent aberrations. Red denotes significantly recurrent amplification, and green denotes significantly recurrent deletion. Gray represents nonsignificant recurrence of aberrations. (B) Heatmap of genetic aberrations corresponding to CNAs shown in A. The columns denote the measured aCGH probes which are arranged based on their genomic coordinates in chromosome order; the rows show the case ID of the 20 OS samples. Red denotes the probe is amplified in corresponding sample, and green denotes deleted. Black represents no copy number aberrations.

Figure 2. Genes with copy number aberrations in the VEGF pathway

A. The copy number aberration status of genes involved in VEGF pathway in 20 OS samples. Red denotes amplification, and green denotes deletion. Black represents no copy number aberration. Significantly altered genes in the VEGF pathway are indicated on the right, and samples are indicated on the bottom. B. The visualization of the location of altered genes in the VEGF pathway. Pink indicates genes with significantly recurrent amplification, and green denotes genes with significantly recurrent deletion.

Figure 3. Validation of VEGFA gene amplification by FISH

A: VEGF gene copy number aberrations in osteosarcomas. Sample IDs of the 20 OS samples in aCGH datasets GSE9654 and GSE19180 are indicated on the bottom. OS1-19 represent the case IDs in GSE9654 and S6272-6285 represent the case IDs in GSE19180. Scatters denote the copy number change of VEGFA gene. Lines in black and grey color denote the regional copy number value estimated by the circular binary segmentation (CBS) algorithm. Black lines denote significant amplification or deletion, whereas grey lines denote non-significant amplification or deletion. Twelve samples show amplification of the VEGF gene. B: Location of the CEP 6 probe and VEGFA gene FISH probe. The green arrow shows the FISH CEP 6 probe located to the chromosome 6 centromere. The right red line shows the location of the VEGFA gene FISH probe. C, D, and E: Validation of the different patterns of VEGFA gene status in aCGH and FISH assay. The aCGH probes of chromosome 6 are ordered on the basis of their genomic position (x-axis). Scatters denote the copy number change (y-axis) of probes. Dashed lines indicate the thresholds for significant aberrations. Blue lines denote regional copy number value estimated by CBS. Blue lines above or below the break line indicate amplification or deletion in the corresponding region. The dashed red line and the green arrows (left) denote the location of centromere 6. The red dots and arrows (left) denote four probes corresponding to the VEGFA gene and its location on chromosome 6 in aCGH analysis. CEP 6 FISH probe showing a green signal indicates the chromosome 6 centromere in FISH detection and is indicated with green arrows in the right panels. The VEGFA gene FISH probe shows an orange signal representing the VEGFA gene copy number, which is indicated with red arrows in the right panels. C, Pattern 1: VEGFA gene amplifications with ploysomy 6. Sample S6275 harbors ploysomy 6 with amplifications of the VEGFA gene. D, Pattern 2: focal amplification of the VEGFA gene. The genetic amplifications of the VEGFA gene are detected in local fragments in sample S6284. FISH detection shows that sample S6284 harbors disomy 6 with amplifications of the VEGFA gene. E: No VEGFA gene amplification. Two green signals with two orange signals suggest there is no VEGFA gene amplification in S6272.

Figure 4. Correlations of VEGFA gene amplification, VEGFA protein, microvascular density (MVD), and disease-free survival in human osteosarcoma

A: Strong positive expression of VEGFA protein was detected by immunohistochemical staining, and MVD was measured by scoring CD34-positive cells. Magnification, ×40. Upper panel: VEGFA protein; lower panel: CD34. B: Numbers of MVD in the VEGFA protein–positive cases were significantly higher than in the negative ones. C: Kaplan-Meier survival analysis showed that positive VEGF expression was significantly associated with adverse tumor-free survival. D: Concordance of VEGFA gene amplifications with VEGFA protein expression. E: Kaplan-Meier survival analysis showed that VEGFA gene amplifications were significantly associated with adverse tumor-free survival. F: Kaplan-Meier survival analysis showed that high VEGFA was significantly associated with adverse tumor-free survival.