We measured neovascularization, epidermal growth factor receptor, and c-erbB-2 expression in a consecutive series of 233 surgically resected axillary lymph node-negative breast cancer patients with a long-term follow-up to define the usefulness of these parameters as independent prognostic indicators of overall survival (OAS). Microvessel count (MVC), as a measure of neovascularization, was determined using a monoclonal antibody against human factor VIII-related antigen. The median MVC of 20 (range, 4–76) was used as a cutoff value for discriminating between low and high vascularized tumors. Epidermal growth factor receptor and c-erbB-2 expression were evaluated by immunohistochemistry. Tumors were considered positive if >10% of the cells showed specific membrane staining. OAS curves were estimated by the Kaplan-Meier method. The indepen-dent prognostic effect of each variable was determined with the Cox proportional hazards model. High MVC (P = 0.04), high nuclear grade (P = 0.005), and high S-phase (P = 0.02) significantly affected OAS at univariate analysis. In a Cox multivariate analysis, the characteristics with an independent prognostic effect on OAS were: MVC (relative hazard, 2.12; 95% confidence interval, 1.18–3.81; P = 0.01) and nuclear grade (relative hazard, 2.83; 95% confidence interval, 1.12–7.17; P = 0.01). These results demonstrate that quantification of neovascularization adds useful independent prognostic information on survival in node-negative breast cancer patients with long-term follow-up.

Although recent data indicate a general benefit in giving adjuvant systemic therapy to all operable breast cancer patients regardless of axillary lymph node status (1, 2), only 25–30% of node-negative breast cancer patients are expected to relapse following surgery. Therefore, a great effort has been made in recent years to identify the proportion of patients that needs adjuvant therapy, thus sparing toxicity to those node-negative patients who could be considered cured by surgery alone. In this respect, the prognostic effect of several biological parameters, including oncogenes, growth factors and growth factor receptors, tumor suppressor genes, and cancer cell proliferation markers (3, 4, 5, 6, 7, 8, 9, 10), has been investigated in node-negative breast cancer patients to identify those at high risk of relapse following surgery. However, conflicting results on the independent prognostic role of these variables have been reported (11).

In recent years, the critical role of tumor-induced neovascularization in neoplastic development, progression, and metastasis has been elucidated (12, 13, 14). The formation of novel blood vessels is an important step in cancer development and progression because it is essential for providing an adequate oxygen and nutrient supply to the growing tumor mass and for initiating metastatic spreading (15). Tumor-induced angiogenesis is a complex, multistep process, requiring the production and local release of various endothelial cell growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor, that are synthesized by both tumor and normal host cells (16). A reliable histological estimate of such a process is the immunohistochemical detection and quantitation of novel blood vessels in tumor specimens that can be performed in both fresh and paraffin-embedded tissues (17). In fact, the MVC3 in the most intense areas of neovascularization has been suggested as a prognostic marker in patients with different human cancers, including malignant melanoma (18) and breast (19), head and neck (20), prostatic, ovarian (21), gastric, and lung carcinomas (22). A correlation between a high MVC and more advanced disease or a worse outcome has generally been observed in breast cancer (23, 24, 25, 26, 27, 28, 29). However, most of the data available on the prognostic role of MVC have been obtained in retrospective studies often with small series of patients. Furthermore, conflicting results have been reported in node-negative breast cancer patients.

EGFR and c-erbB-2 are directly involved in regulating breast cancer development and progression; therefore, their expression has been evaluated as potential indicator of prognosis (4, 5, 6, 7, 8, 9, 10). Although several pilot or preliminary reports have suggested an independent prognostic effect of EGFR and c-erbB-2 on survival of node-negative breast cancer patients, no consensus has been reached on the routine use of their measurement in the clinical management of these women (11).

In the present study, we quantified tumor-induced neovascularization, EGFR, and c-erbB-2 in a consecutive series of 233 surgically resected axillary lymph node-negative breast cancer patients with a long-term follow-up to investigate the usefulness of these variables as prognostic indicators of OAS.

Patients and Tumor Characteristics.

From 1978 to 1985, 233 pre- and postmenopausal women with node-negative early breast cancer were referred to our Institution. Primary treatment consisted of either radical or modified mastectomy or quadrantectomy followed by radiation therapy on the residual breast. Complete axillary lymph node dissection was performed in all patients. Overall survival was defined as the time elapsed from the date of surgery and the date of death or the last follow-up. As of June 30, 1998, the median follow-up period for living patients was 11.4 years (range, 1–19 years). A series of pathological features such as tumor size, histological type, and nuclear grade was obtained from routine histological examination. Tumor size was defined according to the postsurgical TNM classification and categorized as T ≤ 2 cm and > 2 cm. Nuclear grade was scored as described previously (30, 31). The following biological characteristics were evaluated: ER content, expression of EGFR and c-erbB-2, tumor proliferation index as measured by flow cytometric evaluation of SPF, and tumor DNA ploidy. ER content was assayed using the dextran-coated charcoal technique (32) and was determined in 132 patients for which an adequate amount of fresh tumor tissue was available. Tumors were considered ER positive if the specific hormone binding was ≥ 10 fmol/mg of cytosol protein. Flow cytometric evaluation of tumor DNA content and proliferative activity was performed on nuclear suspensions obtained from 50-μm paraffin-embedded sections by a FACScan flow-cytometer (Becton Dickinson, San Jose, CA). DNA content and SPF were determined as reported previously (33). A SPF of 6% was considered as the cutoff value to distinguish between tumors with high or low proliferation, as determined previously in a larger series of node-positive and node-negative breast cancer patients (33).

Immunohistochemical Evaluation of EGFR and c-erbB-2.

EGFR and c-erbB-2 expression was determined by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (5 μm) as described previously (31). The following antibodies were used: MAb 1, an anti-c-erbB-2 mouse MAb against the extracellular domain of the p185c-erbB-2 protein (MAb-1), and C216, an anti-EGFR mouse MAb that was generated using human EGFR as an immunogen. Both antibodies were purchased from Triton, Alameda, CA. Only cell membrane staining was considered, and tumors were scored as positive when >10% of the cancer cells were stained.

Immunohistochemical Evaluation of Neoangiogenesis.

New blood vessels were detected by the immunohistochemical staining technique first described by Weidner et al.(19). Briefly, 5-μm thick sections, representative of invasive carcinoma, were cut from formalin-fixed and paraffin-embedded tissues, pretreated with trypsin, incubated with a MAb raised against human factor VIII-related antigen (Dako, Copenhagen, Denmark) and stained with a standard immunoperoxidase method (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA). Each slide was first scanned at low power (×10–100 magnification), and the area with the higher number of new vessels was identified (hot spot). This region was then scanned at ×250 microscope magnification (0.37 mm2). Five fields were analyzed, and for each of them the number of stained blood vessels was counted. For individual tumors, the MVC was scored by averaging the five fields counts. The median MVC of 20 was used as a cutoff value for discriminating between low and high vascularized tumors.

Statistical Methods.

The association between the different pathological and biological characteristics was studied by the use of contingency tables. Statistical significance was evaluated by the Pearson χ2 test. Univariate analysis for the role of each prognostic variable on OAS was estimated according to the Kaplan-Meier product-limit method (34). The statistical significance of the differences in survival distribution among prognostic groups was evaluated by the log-rank test (35). Multivariate analysis was carried out by the Cox proportional hazards regression model (36). Covariate selection was performed by a stepwise procedure using a maximum likelihood ratio test for backward elimination. Relative hazards with 95% confidence intervals were estimated. All P values represent two-sided tests of statistical significance. All statistical analyses were performed with the BMDP statistical package (BMDP Statistical Software, Los Angeles, CA).

Clinicopathological and Biological Characteristics.

Study participants included 233 surgically resected axillary lymph node-negative breast cancer patients referred to our Institution from January 1, 1978, to December 31, 1985. After surgery, 135 the 233 patients (57.5%) entered the Gruppo Universitario Napoletano-1 study (37) and were randomly assigned to receive 30 mg/day tamoxifen for 2 years (54 patients) or no additional therapy (81 patients); the remaining patients did not receive any additional therapy. As of June 30, 1998, the median follow-up for living patients was 11.4 years (range, 1–19 years). At the time of analysis, 179 patients (76.8%) were still alive, whereas 54 patients (23.2%) were dead. Table 1 shows the distribution of patients according to clinicopathological parameters. Heterogeneous MVC distribution, ranging between 4 and 76 microvessels/tumor sample with a median of 20, was observed. A specific immunostaining indicating EGFR or c-erbB-2 expression was found in 94 of 228 (41.2%) and in 61 of 233 cases (26.2%), respectively. Flow cytometry was performed on nuclear suspensions obtained from paraffin-embedded tumor samples. Interpretable histograms for evaluation of DNA ploidy (coefficient of variation of the G0-G1 peak ≤ 7) were obtained in 194 of 233 (83.3%) specimens, whereas SPF measurement was possible in 163 of 233 (70%) cases.

Correlations between Clinicopathological and Biological Characteristics.

High MVC was significantly associated only with c-erbB-2 expression. No significant correlation between MVC, EGFR, c-erbB-2, and the other considered variables was observed.

Univariate Analysis of Association of Clinicopathological and Biological Characteristics with Overall Survival.

As shown in Table 2, high MVC, defined by using the median cutoff value of 20 microvessels/tumor sample (P = 0.04), high nuclear grade (P = 0.005), and high SPF (P = 0.02) were significantly correlated with a worse OAS. Age, tumor size, EGFR or c-erbB-2 expression, ploidy, ER status, and treatment did not affect OAS. Fig. 1 shows the survival curves according to MVC.

Multivariate Analysis of Association of Clinicopathological and Biological Characteristics with Overall Survival.

A multivariate analysis was performed by a Cox proportional hazards model with the following covariates: tumor size, nuclear grade, MVC, age, c-erbB-2 expression, EGFR expression, and adjuvant therapy (Cox 1: starting model; n = 223; deaths = 53). Using a backward selection, we eliminated covariates without significant influence on OAS to produce a more parsimonious model (Cox 1: final model). After this process, only MVC, tumor size, and nuclear grade were retained into the model. These variables and their estimated parameters are listed in Table 3.

A second Cox model (Cox 2) was fitted in a subgroup of patients for whom SPF was available (n = 161; deaths = 38). After inclusion of SPF among the covariates of the model, MVC maintained its independent prognostic effect on survival (Table 4).

The identification of prognostic variables that significantly affect the outcome of early breast cancer patients has been a very intense area of clinical investigation during the last 10 years. In this respect, a great effort has been made to define subsets of axillary lymph node-negative breast cancer patients with different risks of relapse.

The increasing body of experimental and clinical evidence on the key role of neovascularization in the development and progression of human solid tumors has prompted the evaluation of specific markers of novel blood vessel formation to allow the immunohistochemical quantitation of neovascularization and the study of its potential prognostic relevance. The majority of these studies have shown that MVC correlates significantly with various pathological and biological features generally associated with a more aggressive disease, such as tumor size, nodal status, and histological and/or nuclear grade (23, 26, 29). More importantly, in some of these reports demonstrated that MVC is an independent prognosticator for disease-free survival and OAS (24, 26, 27, 28, 29). However, the reproducibility of these results has been questioned (38). The major concerns have been the use of different antibodies to detect endothelial cells, such as anti-factor VIII-related antigen, and anti-CD31 or -CD34 MAbs; the variability of MVC and of scoring systems between different pathologists and, in some cases, among regions within each tumor; and the inadequacy of patient numbers and follow-up length in most studies.

In the present study, the prognostic role of neovascularization, assessed by immunodetection of factor VIII-related antigen, has been evaluated in a large series (233 cases) of consecutive node-negative breast cancer patients with a long-term follow-up (median for living patients, 11.4 years) for whom several pathological and biological parameters were determined. Among the biological variables measured were the expression of two growth factor receptors, such as EGFR and c-erbB2, and the flow cytometric evaluation of tumor cell proliferation because several studies have suggested a prognostic role of these parameters in node-positive and, in some cases, in node-negative breast cancer patients (3, 4, 5, 6, 7, 8, 9, 10). In our study, nuclear grade, SPF, and MVC were the only characteristics that significantly affected patient survival in univariate analysis. More importantly, in multivariate analysis, MVC was an independent prognostic indicator of OAS.

A significant correlation between enhanced neoangiogenesis and worse prognosis has been demonstrated in node-negative breast cancer patients by at least three different studies that used different antibodies and counting techniques to detect neovessel formation and that had various follow-up lengths. Gasparini et al.(26) evaluated neoangiogenesis using an anti-CD31 MAb in 254 node-negative patients with a median follow-up of 62 months. Fox et al.(24) measured microvessels using the anti-CD31 MAb in 109 women with a follow-up of 25 months. Finally, Heimann et al.(29) performed MVC using an anti-CD34 MAb in 167 patients with a median follow-up of 15.4 years. In contrast to these studies, others have failed to find any correlation between MVC and node-negative breast cancer patient survival. Among these are the studies by Van Hoef et al.(39), who used an anti-factor VIII-related antigen antibody in a series of 93 patients with a 12-year median follow-up period, and by Axelsson et al.(40), who determined MVC with an anti-CD31 MAb in 110 women with a median follow-up of 11.5 years.

Our study failed to demonstrate a prognostic role for the expression of EGFR and c-erbB-2 in this series of patients. Conflicting results have been reported on the association between the expression of these growth factor receptors and patient outcome. The majority of the published studies have suggested that c-erbB-2 expression correlates with a worse prognosis in node-positive, but not node-negative, breast cancer patients. Because node-positive patients usually receive adjuvant treatment, these results may be an expression of an interaction between c-erbB-2 and treatment rather than of a plain prognostic effect (7, 31).

The majority of studies that have evaluated the prognostic effect of EGFR have suggested a correlation with a worse disease-free survival or OAS. However, the number of node-negative patients in many series was too small and the length of follow-up too short to reach conclusive evidence of the prognostic role of EGFR (10).

In conclusion, the results of our study strongly support the clinical relevance of measuring tumor-induced neovascularization in node-negative as well as node-positive breast cancer patients. To our knowledge, this is the largest reported study on the prognostic role of MVC in a consecutive series of node-negative breast cancer patients with a sufficiently long follow-up period. The present results provide further support to the hypothesis that breast cancer patients could benefit of tumor vasculature-directed therapy. The ongoing clinical development of specific antiangiogenic drugs that are able to selectively block tumor-induced endothelial cell proliferation (41) could allow, in a near future, the design of novel adjuvant regimens with antiangiogenic drugs used in combination with conventional cytotoxic or hormonal treatment. In fact, antiangiogenic drugs and chemotherapeutic agents have different cellular targets (neovessels and cancer cells, respectively), different mechanisms of action, and are not cross-resistant (42). In this respect, preclinical studies have demonstrated a potentiation of the antitumor effect by combining antiangiogenic agents with chemotherapy (43).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by grants from Associazione Italiana Ricerca sul Cancro, Consiglio Nazionale delle Ricerche-Applicazioni Cliniche della Ricerca Oncologica, and Ministero dell’Università e della Ricerca Scientifica e Tecnologica.

                
3

The abbreviations used are: MVC, microvessel count; EGFR, epidermal growth factor receptor; OAS, overall survival; ER, estrogen receptor; SPF, S-phase fraction; MAb, monoclonal antibody.

Fig. 1.

Kaplan-Meier estimates for OAS according to MVC.

Fig. 1.

Kaplan-Meier estimates for OAS according to MVC.

Close modal
Table 1

Patient characteristics

VariableNo. of patients%
Age (years)   
 54 (median)   
 30–90 (range)   
Menopausal status   
 Pre 84 36.1 
 Post 149 63.9 
Tumor size   
 ≤2 cm 112 48.1 
 >2 cm 117 50.2 
 Unknown 1.7 
Histologic type   
 DICa 167 71.7 
 LIC 13 5.6 
 Others 53 22.7 
Nuclear grade   
 Low 46 19.7 
 High 187 80.3 
ER   
 Negative 46 19.7 
 Positive 86 36.9 
 Unknown 101 43.4 
c-erbB-2   
 Negative 172 73.8 
 Positive 61 26.2 
EGFR   
 Negative 134 57.5 
 Positive 94 40.3 
 Unknown 2.2 
Ploidy   
 Euploidy 65 27.9 
 Aneuploidy 129 55.4 
 Unknown 39 16.7 
S-phase fraction   
 Low 45 19.3 
 High 118 50.6 
 Unknown 70 30.1 
Adjuvant therapy   
 No 179 76.8 
 Tamoxifen 54 23.2 
Microvessel count   
 20 (median)   
 4–76 (range)   
VariableNo. of patients%
Age (years)   
 54 (median)   
 30–90 (range)   
Menopausal status   
 Pre 84 36.1 
 Post 149 63.9 
Tumor size   
 ≤2 cm 112 48.1 
 >2 cm 117 50.2 
 Unknown 1.7 
Histologic type   
 DICa 167 71.7 
 LIC 13 5.6 
 Others 53 22.7 
Nuclear grade   
 Low 46 19.7 
 High 187 80.3 
ER   
 Negative 46 19.7 
 Positive 86 36.9 
 Unknown 101 43.4 
c-erbB-2   
 Negative 172 73.8 
 Positive 61 26.2 
EGFR   
 Negative 134 57.5 
 Positive 94 40.3 
 Unknown 2.2 
Ploidy   
 Euploidy 65 27.9 
 Aneuploidy 129 55.4 
 Unknown 39 16.7 
S-phase fraction   
 Low 45 19.3 
 High 118 50.6 
 Unknown 70 30.1 
Adjuvant therapy   
 No 179 76.8 
 Tamoxifen 54 23.2 
Microvessel count   
 20 (median)   
 4–76 (range)   
a

DIC, ductal infiltrating carcinoma; LIC, lobular infiltrating carcinoma.

Table 2

Univariate analysis for overall survival

VariableO/EaP
MVC  0.04 
 ≤20 vessels 0.77  
 >20 vessels 1.25  
Tumor size  0.06 
 T ≤2 cm 0.82  
 T >2 cm 1.31  
ER  0.15 
 Negative 0.64  
 Positive 1.18  
Nuclear grade  0.005 
 Low 0.40  
 High 1.18  
c-erbB-2  0.54 
 Negative 0.97  
 Positive 1.09  
EGFR  0.75 
 Negative 1.00  
 Positive 1.01  
Ploidy  0.42 
 Euploidy 0.84  
 Aneuploidy 1.08  
S-phase fraction  0.02 
 Low 0.45  
 High 1.23  
Age  0.79 
 ≤50 years 0.90  
 >50 years 1.06  
Adjuvant therapy  0.75 
 No 1.02  
 Tamoxifen 0.95  
VariableO/EaP
MVC  0.04 
 ≤20 vessels 0.77  
 >20 vessels 1.25  
Tumor size  0.06 
 T ≤2 cm 0.82  
 T >2 cm 1.31  
ER  0.15 
 Negative 0.64  
 Positive 1.18  
Nuclear grade  0.005 
 Low 0.40  
 High 1.18  
c-erbB-2  0.54 
 Negative 0.97  
 Positive 1.09  
EGFR  0.75 
 Negative 1.00  
 Positive 1.01  
Ploidy  0.42 
 Euploidy 0.84  
 Aneuploidy 1.08  
S-phase fraction  0.02 
 Low 0.45  
 High 1.23  
Age  0.79 
 ≤50 years 0.90  
 >50 years 1.06  
Adjuvant therapy  0.75 
 No 1.02  
 Tamoxifen 0.95  
a

O/E, observed/expected ratio of death.

Table 3

Multivariate analysis for overall survival (Cox 1: final model)

Variableβ (SE)RH (95% CI)aP
MVC 0.7519 (0.2995) 2.12 (1.18–3.81) 0.01 
Tumor size 0.5531 (0.2910) 1.74 (0.98–3.07) 0.06 
Nuclear grade 1.0399 (0.4745) 2.83 (1.12–7.17) 0.01 
Variableβ (SE)RH (95% CI)aP
MVC 0.7519 (0.2995) 2.12 (1.18–3.81) 0.01 
Tumor size 0.5531 (0.2910) 1.74 (0.98–3.07) 0.06 
Nuclear grade 1.0399 (0.4745) 2.83 (1.12–7.17) 0.01 
a

RH, relative hazard; CI, confidence interval.

Table 4

Multivariate analysis for overall survival (Cox 2: final model)

Variableβ (SE)RH (95% CI)aP
MVC 0.8914 (0.3568) 2.44 (1.21–4.91) 0.03 
Tumor size 0.5901 (0.2953) 1.80 (1.01–3.22) 0.04 
Nuclear grade 0.8914 (0.6266) 2.44 (0.71–8.33) 0.11 
S-phase 0.8524 (0.5097) 2.34 (0.86–6.37) 0.07 
Variableβ (SE)RH (95% CI)aP
MVC 0.8914 (0.3568) 2.44 (1.21–4.91) 0.03 
Tumor size 0.5901 (0.2953) 1.80 (1.01–3.22) 0.04 
Nuclear grade 0.8914 (0.6266) 2.44 (0.71–8.33) 0.11 
S-phase 0.8524 (0.5097) 2.34 (0.86–6.37) 0.07 
a

RH, relative hazard; CI, confidence interval.

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