Jung Yeon Kim, 139-707 Department of Pathology, Inje University Sanggye Paik Hospital, 761-1 Sanggye 7-dong, Nowon-gu, Seoul, Korea. e-mail: email@example.com
Kim JY, Bae BN, Kwon JE, Kim HJ, Park K. Prognostic significance of epidermal growth factor receptor and vascular endothelial growth factor receptor in colorectal adenocarcinoma, APMIS 2011; 119: 449–59.
The purpose of this study was to evaluate the association between the expression of growth factors and the clinicopathological variables of colorectal adenocarcinoma. Immunohistochemistry and fluorescence in situ hybridization (FISH) were used to evaluate the amplification and expression of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), VEGF-D, VEGF receptor (VEGFR)-2, VEGFR-3, transforming growth factor (TGF)-β1, and insulin-like growth factor-1 receptor (IGF-1R) in a tissue microarray of 292 colorectal adenocarcinomas. The expression of EGFR, VEGF, VEGF-D, VEGFR-2 and VEGFR-3 was detected in 5.1%, 10.0%, 6.8%, 5.2%, and 57.2%. EGFR expression was associated with angioinvasion (p < 0.05) and lymph node metastasis (p < 0.005). VEGFR-3 expression was higher in the rectum than in the colon (p < 0.05). VEGF expression correlated with VEGF-D (p < 0.05) and VEGFR-3 (p < 0.005) expression, while VEGF-D expression showed no significant association with VEGFR-2 or VEGFR-3. EGFR amplification was present in 10.6% and was not associated with EGFR protein expression. VEGFR-2 and VEGFR-3 expression levels were related to poor patient survival. Stage, perineural invasion, and lymph node metastasis were independent prognostic factors based on a Cox analysis. VEGFR-2 and VEGFR-3 expression are markers of a poor prognosis in patients with surgically resected colorectal adenocarcinoma, whereas EGFR has a minor influence.
Growth factors and their receptors are critical regulators of cancer progression and neovascularization (1, 2). In particular, epidermal growth factor receptor (EGFR) regulates cell proliferation and plays a key role in cellular stress responses to chemotherapy and radiotherapy (3). EGFR has been investigated as a target for anti-neoplastic therapy. Mutations that activate EGFR are related to an increased response rate and survival in patients treated with EGFR tyrosine kinase inhibitors (4–8). In addition, the transactivation of EGFR is thought to regulate early transforming growth factor (TGF)-β signaling and may represent a novel pathway to control TGF-β-mediated gene expression (9). In animal models, EGFR inhibition enhances TGF-β-mediated apoptosis by increasing the oxidative stress response in cells (10). In addition, both the anti-proliferative and pro-apoptotic effects of EGFR inhibition are augmented through the inhibition of the insulin-like growth factor 1 receptor (IGF-1R) cascade in colorectal cancer cells (11). IGF-1R plays a major role in promoting the oncogenic process.
Lymphatic vessel formation is important for metastasis, with the lymph node being the first site of metastasis for most tumors. Among the factors involved in lymphangiogenesis and lymph node metastasis vascular endothelial growth factor (VEGF) is one of the most powerful angiogenic factors (12). The VEGF family consists of five isoforms, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PLGF) (13). VEGF-A, also known as the vascular permeability factor or VEGF, acts as not only a mitogenic and permeability factor but also a survival factor through the activation of intracellular signaling cascades, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase-Akt pathways (14). VEGF-C and its closely related homolog VEGF-D exert their effects on endothelial cells via the vascular endothelial growth factor receptor (VEGFR). There are three known membrane receptors, VEGFR-1/FLt-1, VEGFR-2/Flk-1, and VEGFR-3/Flt-4, with VEGFR-2 being the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF (15). VEGFR-3 is expressed in lymphatics and some fenestrated vascular endothelium supporting lymphangiogenesis (16, 17). In addition, VEGFR-3 is expressed in some tumor cells, where its overexpression is related to an increased tumor stage owing to an autocrine/paracrine loop that promotes tumor cell survival and proliferation (18). Accordingly, the VEGF/VEGFR signaling pathway has been targeted in the treatment of solid tumors, and VEGFR tyrosine kinase inhibitors have been shown to prevent both angiogenesis and tumor progression in animal models (19). Furthermore, the inclusion of bevacizumab, a VEGF-specific antibody, in standard chemotherapy treatment regimens has improved survival rates in lung cancer patients (20).
The expression of EGFR, VEGF, and their receptors correlates with angiogenesis and progressive tumor growth in specific types of human carcinomas (21–25). However, research in this area with regard to colorectal adenocarcinoma, a leading cause of cancer-related deaths, has been limited. The purpose of this study was to characterize the expression of growth factor and growth factor receptors and to evaluate the associations with clinical and pathological variables of colorectal adenocarcinoma.
Patients and tumor samples
We studied 292 patients with primary human colorectal carcinoma, who were treated between 1998 and 2003 at the Sanggye Paik Hospital, Seoul, Korea. The use of human tissue was approved by our institutional review board. The study included 154 (52.7%) men and 138 (47.3%) women, with a mean age of 61.0 ± 12.3 years (range: 25–86). The tumors were located in the rectum, including the rectosigmoid colon in 109 patients (37.3%) and in the colon in 183 patients (62.7%). All tumors were adenocarcinomas and one case showed partial neuroendocrine feature. The majority of the tumors were moderately differentiated (245 patients), and 12 cases of mucinous carcinoma were observed. The pathological T stage was primarily pT3 (243 patients). No patients had undergone preoperative chemotherapy or radiotherapy. Twenty-five patients had distant metastases at the time of or following surgery. The median follow-up period was 48.0 months (range: 10–84) for all patients and 55.0 months for survivors. Tumor specimens were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin.
Tissue microarray (TMA) block construction and immunohistochemistry
We constructed TMA blocks, as described previously (26). All slides were reviewed, the most representative tumor blocks were selected, and the invasive front including tumor was chosen for microarray block. We acquired two cylindrical tissue cores from different sites with a diameter of 2 mm from donor blocks and transferred to holes created in a recipient block. Sections (4-μm thickness) of the microarray blocks were cut, and immunohistochemically stained: using anti-EGFR (Dakocytomation, Carpinteria, CA, USA), anti-VEGF (Dakocytomation), anti-VEGF-D (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGFR-2 (Sigma-Aldrich, St. Louis, MO, USA), anti-VEGFR-3 (Novocastra, Newcastle, UK), anti-TGF-β1 (Springbio, Fremont, CA, USA), and anti-IGF-1R (Chemicon, Billerica, MA, USA) antibodies. Endogenous peroxidase activity was quenched by incubation in 0.03% H2O2 in alcohol for 30 min. Then deparaffinized tissue sections were placed in 10 mM citrate buffer (pH 6.0) and heated for antigen retrieval. After a 1-h incubation with a primary antibody at room temperature, the sections were incubated with a biotinylated horse anti-mouse IgG secondary antibody for 30 min. Immunoreactive proteins were detected with avidin-conjugated horseradish peroxidase (Dakocytomation) and the color was developed using 3-3′-diaminobenzidine (DAB; ScyTek, Logan, UT, USA). Mayer’s hematoxylin was used as a counterstain. Two sets of microarray blocks were stained. Positive and negative control staining were used in each assay run. Two pathologists (JYK and JEK) evaluated TMA staining separately and semi-quantitatively. Consensus was achieved in discrepant cases. If any discrepancy was noted in the TMA staining, standard-sectioned carcinomas were stained and re-evaluated.
For EGFR staining, a four-point scoring system, similar to the HER-2 scoring system in gastric cancer, was used (Fig. 1) (27); 0 (negative, no reactivity or membranous reactivity in <10% of tumor cells); 1 (negative, faint/barely perceptible membranous reactivity in >10% of tumor cells or cells are reactive only in part of their membrane); 2 (equivocal, weak to moderate complete or basolateral membranous reactivity in >10% of tumor cells); and 3 (positive, moderate to strong complete or basolateral membranous reactivity in >10% of tumor cells). For the other proteins, the area of moderate or strong cytoplasmic staining was determined as a percentage and scored as follows: 0 (staining in less than 10% of tumor cells); 1 (staining in 10–50% of tumor cells); and 2 (staining in more than 50% of tumor cells). Cases with a score of 1 or 2 were classified as positive.
Fluorescence in situ hybridization (FISH) for gene amplification analysis
The EGFR amplification was measured in TMA block sections using a standard two-color FISH technique. The EGFR copy numbers were estimated using centromere (CEP)-7 in the predominant tumor cell population. The gene level was calculated from the ratio of orange (EGFR) to green (CEP-7) signals in morphologically intact and non-overlapping nuclei. A minimum of a twofold increase in the EGFR/CEP-7 signal ratio was deemed as a definitive gene amplification (28).
Associations between the expression levels of growth factors, and growth factor receptors and the clinicopathological factors were analyzed using the chi-squared test, Fisher’s exact test, and Pearson’s correlation coefficient. Overall survival was calculated using the Kaplan–Meier method, and differences between survival rates were compared using the log-rank test. The Cox proportional hazards model was used to identify prognostic factors influencing survival. The p-values <0.05 were taken to indicate statistical significance. All statistical analyses were performed using sas software (SAS Institute, Inc., Cary, NC, USA).
The EGFR, VEGF, VEGF-D, VEGFR-2, and VEGFR-3 protein expression was detected in 5.1%, 10.0%, 6.8%, 5.2%, and 57.2% of the adenocarcinoma tissue samples, respectively (Table 1). Missing numbers were due to loss of TMA core or disappearance of tumor cells in serial sectioning. TGF-β1 was expressed in one case, and IGF-1R was not expressed in any of the cases of colorectal adenocarcinoma. EGFR expression was associated with both angioinvasion (p < 0.05), and lymph node metastasis (p < 0.005) (Table 2). VEGFR-3 positivity was higher in the rectum than in the colon (p < 0.05). Neither EGFR nor VEGFR-3 was associated with any other clinicopathological factors, including gender, tumor size, tumor stage, tumor location, lymphatic invasion, perineural invasion, microscopic type, and degree of differentiation. VEGF expression was associated with VEGFR-3 (p < 0.005, Table 3) and VEGF-D expression (p < 0.005). VEGF-D expression showed no association with either VEGFR-2 or VEGFR-3. FISH for EGFR was performed in 239 cases, and 25 cases were positive (10.6%). We observed no correlation between EGFR amplification and EGFR protein expression (Table 4).
Table 1. The protein expression of growth factors and growth factor receptors in colorectal adenocarcinoma
The expression of VEGFR-2 and VEGFR-3 was associated with poor patient survival (Fig. 2). Similarly, patients expressing EGFR and VEGF showed a tendency for shorter survival times compared with non-expressing patients. EGFR amplification did not correlate with patient survival. The co-expression of VEGF plus VEGFR-2, VEGF-D plus VEGFR-3, and VEGFR-2 plus VEGFR-3 showed an association with poor patient survival (Fig. 3). Tumor stage, perineural invasion, lymph node metastasis, and VEGFR-2 and VEGFR-3 expression were independent prognostic factors based on a Cox regression analysis. Lymphatic invasion and angioinvasion were additional prognostic factors in the log-rank test (Table 5).
Table 5. Prognostic factors in colorectal adenocarcinoma
In the present study, we observed a relatively lower incidence of EGFR over-expression (5.1%) and higher incidence of EGFR amplification (10.6%) compared with the results of previous studies (29, 30). The strict criteria for EGFR assessment used in our study were based on an evaluation of HER-2, recently optimized in gastric cancer, and cytoplasmic staining was not considered as EGFR positive. This strict assessment may account for the discrepancies between our data and previous studies. We observed no positive correlation between EGFR over-expression and EGFR amplification, which is consistent with previous reports (28, 31). This suggests that EGFR expression is regulated by transcriptional and post-translational events or other regulatory mechanism, rather than by gene amplification. These findings demonstrate the need to determine the EGFR protein expression level, as opposed to the EGFR gene copy number, when considering the clinical use of anti-EGFR therapeutics (28).
The clinical implications of EGFR gene expression remain controversial. EGFR overexpression has been associated with poor clinical outcome (32, 33), and a high EGFR copy number has been described as a prognostic factor for poor survival and a predictive factor for the effectiveness of erlotinib in lung cancer survival (34). Previous studies have shown greater survival benefits of erlotinib therapy in patients with EGFR-expressing tumors and tumors having a high EGFR gene copy number (35). Others have reported that patients with EGFR amplification have a significantly longer time to progression and longer survival than patients without EGFR amplification (36). In addition, EGFR amplification has been proposed as a stronger predictive marker for survival than mutational status (37). High EGFR mRNA expression levels were associated with an improved response and longer survival in patients with colorectal cancer who received irinotecan therapy (38). The patients in this study were not treated with irinotecan or erlotinib and we found no correlation between EGFR expression, or amplification, and survival. Further studies are required to confirm these findings in colorectal adenocarcinoma.
In the present study, we demonstrate a correlation between VEGF and VEGF-D expression and between VEGF and VEGFR-3 expression, although no direct correlation between VEGF-D and VEGFR-3 expression was observed. VEGF-A typically binds to VEGFR-1 and/or VEGFR-2, whereas VEGF-B and PLGF interact only with VEGFR-1. VEGF-C and VEGF-D can bind VEGFR-2 and VEGFR-3, and VEGF-A interacts with VEGF-C and VEGF-D, thereby communicating with VEGFR-2 and VEGFR-3 (39). This reciprocal interaction may explain the differences in VEGF and VEGFR-3 expression observed in this study.
The high expression rates of VEGF-A and VEGFR-3 in tumor cells have been shown to correlate with nodal metastasis, and the VEGF-A–VEGFR-3 axis may be of interest in targeted therapy to prevent lymph node metastasis (17). A significant correlation has been reported between VEGFR-3 expression in endothelial cells and nodal status in non-small cell lung cancer, but no correlation between VEGFR-3 expression in tumor cells and nodal status has been reported (40). In addition, blocking VEGFR-3 signaling with monoclonal antibodies has been shown to prevent lymphatic metastasis (41). A correlation between VEGF-D expression in tumor cells and lymph node metastasis has been described; however, lower levels of VEGF-D expression in tumor tissues have also been reported (42). The present study did not show any relationship between lymph node status or lymphovascular invasion and VEGF or VEGFR expression. This study was performed in carcinoma cells rather than in lymphatic vessels, and thus further studies are required to clarify any association.
Previous studies have demonstrated significantly higher VEGFR protein levels in acute myelogenous leukemia compared with myelodysplastic syndrome, suggesting that increased expression is associated with advanced stage (43). Furthermore, metastatic melanoma cells display high VEGFR-3 expression levels that gradually increase with the tumor stage (44). Previous studies have shown that serum VEGFR-3 levels are increased in patients with a high tumor burden (13, 44), and after the tumor results in metastases, VEGFR-3 plays a less central role in tumor growth (13).
In the present study, the expression of VEGFR-2 and/or VEGFR-3 was associated with poor patient survival, consistent with previous reports (45–47). Cediranib, which blocks signaling from VEGFR-2 and VEGFR-3, can inhibit lymphatic hyperplasia and lymphatic metastasis, and inhibits primary tumor growth through an antiangiogenic mechanism (41). The inhibition of VEGFR-3 more potently suppressed regional and distant metastases, compared with the inactivation of VEGFR-2. A correlation of VEGF-D and/or VEGFR-3 with an unfavorable outcome has been observed in colorectal, ovarian, gastric, and breast cancer, while other studies have shown no such relationships (48). The simultaneous inhibition of several tyrosine kinase receptors is thought to optimize the overall therapeutic benefit associated with molecularly targeted anticancer agents (49). In the present study, the combined expression of VEGFR-2/VEGF and VEGFR-3/VEGF-D was related to poor patient survival, whereas VEGF or VEGF-D expression alone showed no relationship.
A critical concern in this study was whether minute tissue sample of TMA blocks was representative of heterogeneous donor tumors. A single sample per tumor can provide meaningful data if the sample is large enough (50, 51). As the number of core samples increases from one to two, three, five and nine, the concordance rate increases from 92% to 96%, 98%, 99.5% and 99.97% (52). So we selected two cores from different parts of the tumor, especially the invasive front. We compared TMA staining results with those of 10 cases of standard-sectioned colorectal carcinoma using the same immunohistochemical staining method. The results were almost in concordance with TMA results with only minute regional variations.
In conclusion, the expression profiles of both VEGF and VEGFR may be of prognostic value in colorectal cancer and may help in identifying efficient responses to antiangiogenic therapies. We demonstrate that VEGFR-2 and VEGFR-3 expression determined immunohistochemically are markers for a poor prognosis in patients with surgically resected colorectal adenocarcinoma, whereas EGFR expression may have only a minor influence.
This work was supported by a 2009 Inje University research grant. The authors declare that they have no conflict of interest.