Assessment of vascular endothelial growth factor in formalin fixed, paraffin embedded colon cancer specimens by means of a well-based reverse phase protein array

Abstract

Background: Vascular endothelial growth factor (VEGF) is a critical pro-angiogenic factor, found in a number of cancers, and a target of therapy. It is typically assessed by immunohistochemistry (IHC) in clinical research. However, IHC is not a quantitative assay and is rarely reproducible. We compared VEGF levels in colon cancer by IHC and a quantitative immunoassay on proteins isolated from formalin fixed, paraffin embedded tissues.

Results: VEGF expression was studied by means of a well-based reverse phase protein array (RPPA) and immunohistochemistry in 69 colon cancer cases, and compared with various clinicopathologic factors. Protein lysates derived from formalin fixed, paraffin embedded tissue contained measurable immunoreactive VEGF molecules. The VEGF expression level of well differentiated colon cancer was significantly higher than those with moderately and poorly differentiated carcinomas by immunohistochemistry (P = 0.04) and well-based RPPA (P = 0.04). VEGF quantification by well-based RPPA also demonstrated an association with nodal metastasis status (P = 0.05). In addition, the normalized VEGF value by well-based RPPA correlated (r = 0.283, P = 0.018). Furthermore, subgroup analysis by histologic type revealed that adenocarcinoma cases showed significant correlation (r = 0.315, P = 0.031) between well-based RPPA and IHC.

Conclusions: The well-based RPPA method is a high throughput and sensitive approach, is an excellent tool for quantification of marker proteins. Notably, this method may be helpful for more objective evaluation of protein expression in cancer patients.

Keywords: Colon cancer; Formalin-fixed paraffin-embedded; Immunohistochemistry; Reverse-phase protein array; Vascular endothelial growth factor.

Figure 1

VEGF expression profiling by well-based reverse phase protein array and western blotting. We extracted total proteins from 2 different colon FFPE tissue specimens. (A) Western blotting shows different VEGF expression pattern. For reliable signal quantitation on same blot, we reprobed the membrane with anti-GAPDH antibodies. Quantative anlysis was performed using the ImageQuant program. (B) VEGF and GAPDH expressional signals were measured using well-based reverse phase protein array. Relative expressional signal of VEGF was displayed as a ratio of VEGF to GAPDH. Light bar and black bar represent results of western blotting and well-based reverse phase protein array (RPPA), respectively. The bar graph shows the average ± SD of two independent experiments.

Figure 2

Immunohistochemical analysis of VEGF expression in colon tissues. Representative images (final magnification, × 40) of VEGF expression are shown in normal colonic epithelia (A), adenocarcinoma (B), mucinous adenocarcinoma (C). VEGF expression in tumor cells of adenocarcinoma and mucinous carcinoma were strong, while it was weak in normal colonic epithelia.

Figure 3

Relative expression rate of VEGF and its association with histopathologic differentiation. Box plots of relative expression rates are calculated by immunohistochemistry (A) or well-based RPPA (B). Relative expressional signal of VEGF was displayed as a ratio of VEGF to GAPDH. Patients with well differentiated carcinomas had significantly higher expression than those with poorly and moderately differentiated carcinomas. *P < 0.05, ANOVA post-hoc Duncan test.

Figure 4

Reliability of well-based RPPA and its correlation with immunohistochemistry. Scatter diagrams are presented by unnormalized VEGF value vs. immunohistochemical VEGF score (A) and normalized VEGF value vs. IHC VEGF score (B). There was a statistically meaningful correlation between expressional signals in the normalized VEGF value by well-based RPPA and immunohistochemical VEGF score (r = 0.283, P = 0.018) whereas the unormalized VEGF signal was expressed negative correlation (r = -0.350, P = 0.003). By histologic subgroup analysis, adenocarcinoma type (C) was only showed good correlation (r = 0.315, P = 0.031) with IHC assessment compare to that of mucinous adenocarcinoma (r = -0.086, P = 0.703, D).