Impact of breast cancer surgery on angiogenesis circulating biomarkers: a prospective longitudinal study

Abstract

Background: Debate about the potential effects that surgery might have on cancer cells dormancy and angiogenesis prompted us to investigate the impact of breast surgery on circulating angiogenesis modulating gene transcripts and proteins.

Methods: Blood samples from 10 female patients diagnosed with breast cancer and 6 with fibroadenoma were collected before surgery and post-operatively on days 3 and 7 (breast cancer patients only). A set of 84 angiogenesis-associated transcripts were assessed using quantitative PCR arrays, and circulating protein levels (vascular endothelial growth factor A (VEGFA), IL8 and fibroblast growth factor 2 (FGF2) were measured using ELISA in the same samples. The results were investigated against clinicopathological data and patient outcome.

Results: Plasma levels of VEGFA and IL8 after surgery were significantly elevated in the breast cancer group compared to the control group (P = 0.038 and P = 0.021, respectively). In the cohort of breast cancer patients, VEGFA increased on day 3 (P = 0.038) and declined on day 7 (P= 0.017), while IL8 did not change on day 3 but showed a significant decline on day 7 (P = 0.02). FGF2 levels did not change significantly over time. Regarding gene transcripts, we detected upregulation of a significant number of angiogenesis-specific genes in patients with breast cancer versus controls: sphingosine kinase 1(SPHK1), epidermal growth factor (EGF), vascular endothelial growth factor C (VEGFC), neuropilin 1 (NRP1), fibroblast growth factor (FGF1), laminin alpha 5 (LAMA5), collagen type IV alpha 3 (COL4A3), IL8, ephrin B2 (EFNB2), ephrin A3 (EFNA3), tyrosine endothelial kinase (TEK), integrin beta 3 (ITGB3), AKT1, thrombospondin 1 (THBS1), chemokine (C-C motif) ligand 11 (CCL11) and TIMP metallopeptidase inhibitor 3 (TIMP3). Surgery induced an altered expression in several keygenes in breast cancer patients. We identified an upregulation of COL4A3 and downregulation of chemokine (C-X-C motif) ligand 9 (CXCL9), EGF, FGF1, Kinase insert domain receptor (KDR), Placental growth factor (PGF), TIMP3 and VEGFC.

Conclusion: Breast cancer patients have a different expression profile of circulating angiogenesis biomarkers compared to patients with fibroadenoma. Moreover, mastectomy promotes a transient increase of VEGFA and a shift in the expression patterns of a broad panel of angiogenesis-related circulating gene transcripts.

Figure 1

Aligned plot of repeated measures of vascular endothelial growth factor A (VEGFA) with matched values stacked in subcolumns. The outlier refers to a patient with locally advanced disease (T₂N₂) who eventually relapsed.

Figure 2

Circulating vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and IL8 levels measured at different time points before and after surgery. (A) Both groups of patients. (B) Breast cancer patients only. Values are shown as means± SEM. D3,sampling on postoperative day 3; D7,sampling on postoperative day 7. Only significant P values are shown and refer to paired t tests.

Figure 3

Scatter plot analysis and heat map of all patient samples preoperatively. The heat map demonstrates comparative expression of all 84 genes. Significantly upregulated genes (for example, SPHK1) are depicted in a more intense red color (F09), while significantly downregulated genes are shown with a more intense green color (for example, CXCL10 in B02). The graph below plots the log₁₀ of normalized gene expression levels between the control group (x-axis) and the breast cancer group (y-axis). Genes with a more than twofold upregulation in the breast cancer group are depicted in the upper left corner with red dots, while genes which are more than twofold downregulated are represented in the lower right section with green dots (these genes are analytically listed in the adjacent table).

Figure 4

Scatter plot analysis and heat map of all patient samples on postoperative day 3. Significantly upregulated genes (for example, SPHK1) are depicted in a more intense red color (F09), while significantly downregulated genes are shown with a more intense green color (for example, CXCL10 in B02). The graph below plots the log₁₀ of normalized gene expression levels between the control group (x-axis) and the breast cancer group (y-axis). Genes with a more than twofold upregulation in the breast cancer group are depicted in the upper left corner with red dots, while genes which are more than twofold downregulated are represented in the lower right section with green dots (these genes are analytically listed in the adjacent table).