Japanese Journal of Clinical Oncology Advance Access published online on June 17, 2009
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyp066
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© The Author (2009). Published by Oxford University Press. All rights reserved
Vascular Endothelial Growth Factor Receptor Expression as a Prognostic Marker for Survival in Colorectal Cancer
1 Gastrointestinal Oncology Division, National Cancer Center Hospital
2 Clinical Laboratory Division, National Cancer Center Hospital, Tokyo, Japan
For reprints and all correspondence: Yasuhide Yamada, Gastrointestinal Oncology Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: yayamada{at}ncc.go.jp
Received October 3, 2008; accepted May 20, 2009
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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Objective: Vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, -R2 and -R3 play important roles in tumor angiogenesis and are associated with poor prognosis in several solid tumors. However, their functional significance remains unclarified. Here, we investigated the associations between the expression of these receptors and the clinical outcomes of colorectal cancer (CRC) patients.
Methods: An immunohistochemical approach was used to detect VEGF-R1, -R2 and -R3 expression in 91 CRC patients who underwent surgery and received chemotherapy at the National Cancer Center Hospital. Statistical analysis was performed to determine the prognostic significance of these biomarkers.
Results: Immunoreactivity for VEGF-R2 and -R3 was localized in microvessels and that for VEGF-R1 in cancer cells and stromal microvessels. VEGF-R1 staining in cancer cells (>10% staining) was found in 84 patients (92%) and in stromal vessels in 75 patients (82%). VEGF-R2 staining in tumor vessels (>10% staining) was found in 84 patients (92%), whereas VEGF-R3 staining was found in 85 patients (93%). Strong positive staining (>60% staining) of VEGF-R1 in tumor cells, and VEGF-R1, -R2 and -R3 in vessels was identified in 58 (64%), 33 (36%), 52 (57%) and 60 (66%) patients, respectively. Univariate analysis revealed that VEGF-R1 strong positive staining correlated with shorter post-operative survival in patients with Stage II/III disease (P = 0.01), but neither VEGF-R2 nor R3 expression correlated with survival.
Conclusions: VEGF-R1, -R2 and -R3 were highly expressed in CRC cells and stromal vessels. VEGF-R1 strong positive staining correlated with shorter survival after CRC surgery.
Key Words: VEGF • VEGF-R1 • VEGF-R2 • VEGF-R3 • colorectal cancer • prognostic factor
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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Angiogenesis plays an important role in cancer invasion and metastasis. Vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) represent important regulators of angiogenesis, and their increased expression has been documented in various cancer cell lines (1) and tissues (2,3). In the treatment of colorectal cancer (CRC) (4) and lung cancer (5), the efficacy of combining cytotoxic agents and bevacizumab, a monoclonal anti-VEGF antibody, has been reported. Some drugs that block the tyrosine kinase of VEGF-R are also being developed. However, the mechanisms controlling the expression of VEGF and VEGF-R and angiogenesis have not yet been fully elucidated. Moreover, the roles of VEGF and VEGF-R as prognostic markers and their usefulness in predicting the efficacy of anti-angiogenic agents have not been clarified to date.
In CRC, the expression of VEGF ligands and subtypes (i.e. VEGF-A, -B, -C, -D and -E) correlates with cancer stage (6,7) and prognosis (8,9), and the expression of soluble VEGF-R1 is a prognostic predictor (10). CRC cell lines have also been reported to express VEGF-R (11,12); however, the distribution, frequency and prognostic values of VEGF-R expression in CRC have not yet been clarified. This study investigated the relationships between VEGF-R expression and prognosis of primary CRC patients.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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Patients
Subjects were randomly selected from patients histologically diagnosed with CRC. Inclusion criteria were as follows: no prior chemotherapy or adjuvant/neoadjuvant chemotherapy; primary colorectal adenocarcinoma specimens were obtained by surgical resection before the start of chemotherapy at the National Cancer Center Hospital (NCCH); received 5-fluorouracil (5-FU)-based first-line chemotherapy for the treatment of recurrent or residual tumors at NCCH from January 1995 to December 2003; and therapeutic effects and prognoses were confirmed. Tissue samples were collected retrospectively from patients who met these criteria. Written informed consent was obtained before treatment and evaluation of tumor samples.
Immunohistochemical Staining
Serial 4 µm-thick sections were prepared from formalin-fixed paraffin-embedded tissue. One block that included the site of deepest invasion was selected from each specimen after examining the slides of the surgical specimens stained with hematoxylin and eosin. Tissue sections were dewaxed in xylene and rehydrated through graded alcohol. Antigen retrieval was performed by incubating tissue sections in target retrieval solution (Dako Japan, Tokyo, Japan) for 40 min in water bath at 95°C and cooling for at least 20 min.
After quenching endogenous peroxidase with peroxidase blocking reagent (Dako Japan) for 5 min and washing with Tris-buffered saline containing Tween 20, tissue sections were incubated with the primary antibody (Table 1).
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Immunoreaction was detected using the following secondary antibody systems: CSA-II (Dako Japan) for VEGF-R1, -R2 and -R3; Envison+ kit (Dako Japan) for CD34 and D2-40 according to the instructions of the manufacturer. Sections were counterstained using Mayer's hematoxylin. As the negative controls, the primary antibody solution was substituted with a buffer containing goat IgG1 (VEGF-R1, -R2 and -R3) or mouse IgG1 (CD34 and D2-40).
Immunostaining Evaluation
The entire specimen was initially examined at low magnification (x40), and positive cells and vessels were counted in areas showing strong staining at higher magnification (x100). Immunostaining was assessed in three fields of view, and the average ratio was calculated. The percentage of vessels was defined as the ratio of positive vessels to the total number of CD34- and D2-40-positive vessels. Ratios >10% were considered significant (positive), and strong positive staining was defined as 
60%. The cut-off value of strong positive staining (60%) was defined based on the median value. Microvessel densities (MVDs) of CD34- or D2-40-positive vessels were determined similarly to previous studies (13,14). However, MVD was quantified at lower magnification (x100) to compare VEGF receptors. Two investigators independently evaluated the immunostaining results without knowledge of clinical data.
Statistical Analysis
Statistical analysis was performed using SPSS version 11 software (SPSS Japan Inc., Tokyo, Japan). Spearman's rank correlation was used to assess the relationships between VEGF-R1, -R2, -R3, CD34 and D2-40. The 
2 test was used to evaluate the relationships between expression of biomarkers and therapeutic effect. The Mann–Whitney test was used to examine the association of biomarkers with clinicopathological factors [i.e. age, sex, histological type (well-differentiated vs. others) and metastasis (lymph node metastasis and distant metastasis)]. Each factor and overall survival were determined by the Kaplan–Meier method and analyzed using the log-rank test. Multivariate analysis was performed using a Cox proportional hazard model.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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Clinicopathological Characteristics
The clinicopathological characteristics of the patients are shown in Table 2. All patients underwent surgery to remove the primary lesion. At the time of primary resection, the tumor stage based on the TNM classification was II or III in 32 patients and distal metastasis (Stage IV) was confirmed in 59 patients. Well-differentiated carcinoma was found in 21 patients, moderately differentiated carcinoma was found in 63 patients and poorly differentiated or mucinous adenocarcinoma was identified in 7 patients histopathologically. All patients received chemotherapy, and first-line chemotherapy comprised 5-FU and leucovorin in 69 patients and other agents in the remaining 22 patients (all 5-FU-based chemotherapy). The median follow-up time was 28.5 months (range: 5.5–88.1 months).
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MVD of CD34-/D2-40-positive Vessels
The average MVD of CD34-positive vessels was 103 (44–247) and that of D2-40-positive vessels was 16 (1–43) at x100 magnification (Fig. 1A and B). Neither CD34 MVD nor D2-40 MVD was correlated with clinicopathological factors. In addition, CD34 or D2-40 MVD showed no correlation with survival from surgery or chemotherapy.
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Expression of VEGF-R1, -R2 and -R3
VEGF-R1 was stained on the tumor cell surface and stromal vessels (Fig. 1C and D). Specifically, VEGF-R1 was stained in tumor cells and vessels in 84 (92%) and 75 (82%) patients, respectively, and was strongly positively stained in tumor cells and vessels in 58 (64%) and 33 (36%) patients, respectively (Table 3).
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VEGF-R2 and -R3 showed immunoreactivity mainly in tumor stromal vessels (Fig. 1F and H). VEGF-R2 was positively stained in 84 patients (92%) and strongly positively stained in 52 patients (57%). VEGF-R3 was positively stained in 85 patients (93%) and strongly positively stained in 60 patients (66%). Some CD34+ or D2-40+ vessels were immunoreactive with VEGF-R2 or -R3. VEGF-R3 was expressed not only in D2-40+ vessels, but also in CD34+ vessels. For tumor cells, VEGF-R2 and -R3 were stained in 5 (5%) and 22 (24%) patients, respectively, and were strongly stained in only 2 (2%) and 9 (10%) patients, respectively. VEGF-R1, -R2 and -R3 were not uniformly stained in some cases; however, no characteristic patterns were detected.
Correlation Between VEGF-R1, -R2 and -R3 and Clinicopathological Factors
Marked correlation was not found between VEGF-R1 staining and VEGF-R2 or -R3. A slight correlation was identified between VEGF-R2 and -R3 staining in vessels (Spearman's rank correlation coefficient: 
= 0.487, P < 0.001). Significant correlation was not found between MVD (CD34 or D2-40) and staining of VEGF receptors. The 2 test showed no correlation between clinicopathological factors [i.e. age, sex, histological type (well differentiated vs. others) and metastasis (lymph node metastasis and distant metastasis)] and strong staining of VEGF receptors.
Relationship of VEGF-R Expression with Fluoropyrimidines
First-line chemotherapy based on 5-FU was administered to all patients, and the therapeutic effects were favorable (complete response or partial response) in 41 patients and unfavorable (non-responders, stable disease or progressive disease) in 50 patients. The 2 test showed no relationship between strong staining of VEGF receptors (R1, R2 and R3) and fluoropyrimidine efficacy (responder vs. non-responders) (P = 0.67, 0.67 and 0.19, respectively).
The relationship of survival with VEGF-R1, -R2 and -R3 staining was also assessed after chemotherapy. The median survival time after chemotherapy was 18.3 months for patients showing VEGF-R1strong positive staining and 22.6 months for other patients; 16.8 months for patients showing VEGF-R2 strong positive staining and 22.7 months for other patients; 18.5 months for patients showing VEGF-R3 strong positive staining and 21.1 months for other patients. Although the patients showing strong positive staining for VEGF receptors had poorer prognosis, no significant differences existed.
Relationship of VEGF-R1 Expression with Survival after Surgery in Stage II/III CRC
Among the 32 Stage II/III CRC patients, VEGF-R expression and survival after surgery were investigated. The median survival time of patients showing VEGF-R1 strong positive staining was 34.5 months and that of other patients was 47.4 months (P = 0.014, Fig. 2). Multivariate analysis showed VEGF-R1 strong positive staining as an independent poor prognostic factor for survival [hazard ratio, 2.85 (95% confidence interval; 1.16–6.99)] (Table 4). The median survival time of patients showing VEGF-R2 strong positive staining was 35.4 and that of other patients was 39.7 months, with no significant difference. The median survival time of patients showing VEGF-R3 strong positive staining was 35.4 and that of other patients was 39.7 months, with no significant difference.
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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This study showed that VEGF-R1 is mainly expressed in primary CRC cells, whereas VEGF-R2 and -R3 are mainly expressed in stromal vessels. Previous studies have shown that VEGF-R1 is expressed in CRC cell lines, such as HT-29 and KM12L4 (11,12) and an immunohistochemical study has also shown that VEGF-R1 was stained in tumor cells (11). These findings suggest that VEGF-R1 is mainly expressed in cancer cells and plays an important role in cancer proliferation. Takahashi et al. (15) reported no correlation between the expression of flt-1 (VEGF-R1) and clinicopathological factors after examining 52 patients with CRC and 10 patients with colon adenoma. Similar to the previous study, our study showed that VEGF-R1 strong staining did not correlate with clinicopathological findings. On the other hand, among Stage II/III patients, VEGF-R1 strong positive staining was an independent marker for poor prognosis. Yamaguchi et al. (10) reported that soluble VEGF-R1 expression was correlated with favorable prognosis. Some studies have reported that the expressions of VEGF, a VEGF-R1 ligand and the VEGF-A subtype correlated with advanced stage (6,7) and poor prognosis (8,9). Others have similarly shown that VEGF-R1 is important for proliferation of vascular endothelial cells (16) and migration of tumor cells (12,17). VEGF-R1 expression theoretically leads to tumor vessel proliferation and cell migration and causes cancer invasion and metastasis, thus we believe that VEGF-R1 strong positive staining is correlated with poor prognosis following surgery. The finding that VEGF-R1 was not correlated with MVD suggests that VEGF-R1 expressed on tumor cells might be more important for migration than proliferation of vascular endothelial cells. Here, the number of Stage II/III patients was small, and all patients received chemotherapy for recurrences. We therefore plan to examine a larger number of patients with Stage II/III CRC in the future.
VEGF-R2 and -R3 were mainly stained in intratumoral stromal vessels. Some CD34+ vessels were immunoreactive with VEGF-R2, suggesting that VEGF-R2 is mainly expressed in vascular endothelial cells. Meanwhile, VEGF-R3 has been thought to be expressed in lymphatic endothelia and involved in lymphangiogenesis. Here, VEGF-R3 was expressed not only in D2-40+ vessels, but also in CD34+ vessels. White et al. (18) also reported that VEGF-R3 was expressed in some CD31+ vessels, suggesting that VEGF-R3 is expressed in some lymphatic and vascular endothelial cells in the tumor stroma.
VEGF-R2 and -R3 showed no significant correlation with clinicopathological factors and prognosis. It was previously shown that VEGF-R2 expression was higher in metastatic tumors than in non-metastatic tumors in CRC, head and neck cancer (19) and breast cancer (20). Meanwhile, Yonemura et al. (21) have shown that VEGF-R3 expression demonstrated no correlation with lymph node metastasis and malignancy in gastric cancer. Importantly, a large number of patients are required to extensively clarify the interactions among VEGF-R subtypes and their clinical effects on angiogenesis and lymphangiogenesis.
Bevacizumab is a VEGF-neutralizing antibody and chemotherapy with bevacizumab and cytotoxic agents has been shown to prolong survival of CRC patients (4,22). Bevacizumab is regarded as an agent that suppresses cancer proliferation by directly blocking angiogenesis via the inhibition of VEGF, VEGF-R1 and -R2 as well as NP1/NP2 signal transduction. In breast cancer, HER-2 receptor expression was found in 
20–30% of affected patients, and studies have shown that trastuzumab, a monoclonal antibody against the HER-2 receptor, is significantly effective against HER-2-overexpressing breast cancer (23,24), with HER-2 receptor expression considered as one of the therapeutic criteria. On the other hand, fluoropyrimidine induces cell death by impairing nucleic acid synthesis and it also exerts slight effects on angiogenesis. Here, the expression of VEGF receptors showed no correlation with the therapeutic effects of fluoropyrimidine, although VEGF-R1 strong staining was correlated with shorter survival from surgery. We are going to evaluate the correlation between VEGF-R expression and the therapeutic effects of molecular-targeting agents, such as bevacizumab, containing regimens.
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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None declared.
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Acknowledgements |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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We are grateful to Dr Takuya Honda, Ms Hideko Morita, Ms Mari Araake, Ms Hiromi Orita and Ms Eri Onishi for technical assistance and help in collecting and organizing clinical samples.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementAcknowledgementsReferences |
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1 Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res (1986) 46:5629–32.
2 Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Senger DR, et al. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res (1993) 53:4727–35.
3 Brown LF, Berse B, Jackman RW, Tognazzi K, Guidi AJ, Dvorak HF, et al. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol (1995) 26:86–91.[CrossRef][Web of Science][Medline]
4 Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irinotecan, fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med (2004) 350:2335–42.
5 Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxel-carboplatin alone or with bevacizumab for non small cell lung cancer. N Engl J Med (2006) 355:2542–50.
6 Kumar H, Heer K, Lee PWR, Duthie GS, MacDonald AW, Greenman J, et al. Preoperative serum vascular endothelial growth factor can predict stage in colorectal cancer. Clin Cancer Res (1998) 4:1279–85.[Abstract]
7 Tokunaga T, Oshika Y, Abe Y, Ozeki Y, Sadahiro S, Kijima H, et al. Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br J Cancer (1998) 77:998–1002.[Web of Science][Medline]
8 Ishigami SI, Arii S, Furutani M, Niwano M, Harada T, Mizumoto M, et al. Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer. Br J Cancer (1998) 78:1379–84.[Web of Science][Medline]
9 Ellis LM, Takahashi Y, Liu W, Shaheen RM. Vascular endothelial growth factor in human colon cancer: Biology and therapeutic implications. Oncologist (2000) 5:11–5.
10 Yamaguchi T, Bando H, Mori T, Takahashi K, Matsumoto H, Yasutome M, et al. Overexpression of soluble vascular endothelial growth factor receptor 1 in colorectal cancer: association with progression and prognosis. Cancer Sci (2007) 98:405–10.[CrossRef][Medline]
11 Fan F, Wey JS, McCarty MF, Belcheva A, Liu W, Bauer TW, et al. Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene (2005) 24:2647–53.[CrossRef][Web of Science][Medline]
12 Lesslie DP III, Summy JM, Parikh NU, Fan F, Sawyer TK, Metcalf CA III, et al. Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases. Br J Cancer (2006) 94:1710–7.[Web of Science][Medline]
13 Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med (1991) 324:1–8.[Abstract]
14 Choi WW, Lewis MM, Lawson D, Yin-Goen Q, Birdsong GG, Cotsonis GA, et al. Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression. Mod Pathol (2005) 18:143–52.[CrossRef][Web of Science][Medline]
15 Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res (1995) 55:3964–8.
16 Sibuya M, Welsh LC. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res (2006) 312:549–60.[CrossRef][Web of Science][Medline]
17 Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood (1996) 87:3336–43.
18 White JD, Hewett PW, Kosuge D, McCulloch T, Enholm BC, Carmichael J, et al. Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma. Cancer Res (2002) 62:1669–75.
19 Moriyama M, Kumagai S, Kawashiri S, Kojima K, Kakihara K, Yamamoto E. Immunohistochemical study of tumour angiogenesis in oral squamous cell carcinoma. Oral Oncol (1997) 33:369–74.[Web of Science][Medline]
20 Valtola R, Salven P, Heikkaila P, Taipale J, Joensuu H, Rehn M, et al. VEGF-R3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am J Pathol (1999) 154:1381–90.
21 Yonemura Y, Endo Y, Fujita H, Fushida S, Ninomiya I, Bandou E, et al. Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin Cancer Res (1999) 5:1823–9.
22 Kabbinavar FF, Schulz J, McCleod M, Patel T, Hamm JT, Hecht JR, et al. Addition of bevacizumab to bolus fluorouracil and leucovorin in first-line metastatic colorectal cancer: results of a randomized phase II trial. J Clin Oncol (2005) 23:3697–705.
23 Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol (2002) 20:719–26.
24 Seidman AD, Fornier MN, Esteva FJ, Tan L, Kaptain S, Bach A, et al. Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification. J Clin Oncol (2001) 19:2587–95.
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