A compact VEGF signature associated with distant metastases and poor outcomes

Abstract

Background: Tumor metastases pose the greatest threat to a patient's survival, and thus, understanding the biology of disseminated cancer cells is critical for developing effective therapies.

Methods: Microarrays and immunohistochemistry were used to analyze primary breast tumors, regional (lymph node) metastases, and distant metastases in order to identify biological features associated with distant metastases.

Results: When compared with each other, primary tumors and regional metastases showed statistically indistinguishable gene expression patterns. Supervised analyses comparing patients with distant metastases versus primary tumors or regional metastases showed that the distant metastases were distinct and distinguished by the lack of expression of fibroblast/mesenchymal genes, and by the high expression of a 13-gene profile (that is, the 'vascular endothelial growth factor (VEGF) profile') that included VEGF, ANGPTL4, ADM and the monocarboxylic acid transporter SLC16A3. At least 8 out of 13 of these genes contained HIF1alpha binding sites, many are known to be HIF1alpha-regulated, and expression of the VEGF profile correlated with HIF1alpha IHC positivity. The VEGF profile also showed prognostic significance on tests of sets of patients with breast and lung cancer and glioblastomas, and was an independent predictor of outcomes in primary breast cancers when tested in models that contained other prognostic gene expression profiles and clinical variables.

Conclusion: These data identify a compact in vivo hypoxia signature that tends to be present in distant metastasis samples, and which portends a poor outcome in multiple tumor types.This signature suggests that the response to hypoxia includes the ability to promote new blood and lymphatic vessel formation, and that the dual targeting of multiple cell types and pathways will be needed to prevent metastatic spread.

Figure 1

Kaplan-Meier survival plots. Kaplan-Meier survival plots according to MetScore status (A and B) and according to intrinsic subtype (C and D) across the 146 patient UNC training data set.

Figure 2

One-way average linkage hierarchical cluster analysis. One-way average linkage hierarchical cluster analysis of the gene set associated with MetScore status. One hundred and ninety-five microarrays, representing 146 tumors and 10 normal breast samples were analyzed using the 1195 gene MetScore gene set. Overview of the complete cluster diagram (the full cluster diagram can be found as Additional file 2). The tumors were ordered according to their MetScore, and then according to their increasing correlation to the Metscore3 centroid within each group. Clinical regional node status, distant metastasis status, ER, PR, and intrinsic subtype are shown. A) FOS-JUN gene expression cluster, B) fibroblast/mesenchymal cell cluster, C) CXCL12 gene expression cluster, D), immune-cell/HLA cluster, E) VEGF profile.

Figure 3

In situ hybridization. In Situ hybridization to localize gene transcripts using a representative tumor for A) Adrenomedulin (ADM), B) Angiopoetin-like 4 (ANGPLT4), and C) Vascular Endothelial Growth Factor A (VEGF). Magnification 200×.

Figure 4

Univariate Kaplan-Meier survival plots. Univariate Kaplan-Meier survival plots of survival for patients stratified using the VEGF profile on the A) UNC training data set, B) NKI test data set, C) Bhattacharjee et al lung carcinoma data set [29], and D) Nutt et al glioblastoma data set [30]. Note: two genes ANGPTL4 and C14ORF58 were not found on Affymetrix platforms for C and D.

Figure 5

VEGF profile, glycolysis and HIF1α gene expression analyses. A) Gene expression for the VEGF profile (plus average values), for the six glycolysis genes and glycolysis centroid, HIF1α and fibroblast centroids are shown across the 146 patient UNC training data set with the tumors ordered according to their VEGF profile average values. B) Similar analysis as presented in A except the data set is the NKI patient test set.

Figure 6

H&E images of a primary breast tumor taken from a MetScore 3 patient. Showing a prominent admixed stromal component comprised of fibroblasts and myofibroblasts in the primary tumor. The fibroblast/myofibroblast component is markedly diminished in the distant metastatic sites (B and C) as compared with the primary tumor (A). Magnification 200×.