REPORTS

Association of Angiogenesis in Lymph Node Metastases With Outcome of Breast Cancer

Anthony J. Guidi, Donald A. Berry, Gloria Broadwater, Marjorie Perloff, Larry Norton, Maurice P. Barcos, Daniel F. Hayes

Affiliations of authors: A. J. Guidi, North Shore Medical Center, Salem, MA; D. A. Berry, The University of Texas M. D. Anderson Cancer Center, Houston; G. Broadwater, Cancer and Leukemia Group B Statistical Center, Durham, NC; M. Perloff, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD; L. Norton, Memorial Sloan-Kettering Cancer Center, New York, NY; M. P. Barcos, Roswell Park Cancer Institute, Buffalo, NY; D. F. Hayes, Breast Cancer Program, Lombardi Cancer Center, Georgetown University, Washington, DC.

Correspondence to: Daniel F. Hayes, M.D., Breast Cancer Program, Lombardi Cancer Center, Georgetown University, 3970 Reservoir Rd., N.W., RB504E, Washington, DC 20007 (e-mail: hayesdf{at}gunet.georgetown.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Microvessel density (MVD) is a measure of the extent of new blood vessel growth or angiogenesis, which is required for tumor progression. Increased MVD in primary breast cancers appears to adversely affect disease-free survival and overall survival in patients with breast cancer. However, the clinical implications of angiogenesis in breast cancer metastases have not been well studied. The purpose of this study was to compare intratumoral MVD in primary breast cancer tissues with MVD in axillary lymph node metastases and to evaluate the relationships among primary- and metastatic-tumor MVD, disease-free survival, and overall survival in patients with lymph node-positive, stage II breast cancer who were treated with adjuvant chemotherapy in Cancer and Leukemia Group B Protocol 8082. METHODS: Immunostaining for factor VIII-related antigen was performed on tissue sections from 47 primary tumors and 91 axillary lymph nodes containing metastases from 110 patients with lymph node-positive breast cancer. Sections were examined for the presence or absence of focal areas of relatively intense neovascularization (vascular hot spots), and a quantitative assessment of intratumoral MVD was performed. RESULTS: The presence of vascular hot spots in axillary lymph node metastases, but not primary breast cancers, was associated with statistically significantly decreased disease-free survival (P = .006) and overall survival (P = .004) by univariate analysis. Similarly, increased MVD in metastases, but not in primary tumors, was statistically significantly associated with diminished overall survival in these patients (P = .02). In multivariate analysis, the number of positive axillary lymph nodes and the presence of vascular hot spots in axillary lymph node metastases predicted decreased disease-free survival (P = .0001 and .02, respectively) and overall survival (P = .0001 and .007, respectively). All P values were two-sided. CONCLUSION: This pilot study suggests that assessing neovascularization in axillary lymph node metastases may provide clinically useful information regarding survival in patients with primary breast cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Tumor progression is critically dependent on angiogenesis (1,2). Methods that quantify the degree of tumor angiogenesis have been shown to provide important prognostic information for patients with a variety of solid tumors, including breast cancers. A number of investigators (3-8) have shown that patients with invasive breast cancers with increased microvessel density (MVD) have a higher likelihood of harboring metastatic disease and have decreased disease-free survival and overall survival compared with patients with less vascular tumors.

To date, studies relating MVD to outcome in patients with breast cancer have focused on neovascularization in primary tumors. Although it is known that metastatic tumors are also capable of inducing a vascular stroma, the clinical significance of angiogenesis associated with metastatic tumor deposits has not been determined. The purpose of this study was to compare intratumoral MVD in primary breast cancer tissue and in axillary lymph node metastases and to evaluate the relationships among MVD in primary tumors and lymph node metastases, disease-free survival, and overall survival in patients with breast cancer that has metastasized to axillary lymph nodes.


    SUBJECTS AND METHODS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Patients

The study population represents a subset of patients enrolled in the Cancer and Leukemia Group B (CALGB) Protocol 8082. The overall results of Protocol 8082 have been published elsewhere (9). In clinical study 8082, a total of 945 women with stage II, lymph node-positive breast cancer were initially treated with a combination of cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, and prednisone (CMFVP). Patients were then randomly assigned to one of two CMFVP regimens that differed by schedule and cumulative dose and then again randomly assigned to either continued CMFVP or to a four-drug regimen consisting of a combination of vinblastine, doxorubicin, thiotepa, and fluoxymesterone (VATH).

Two-hundred thirty-four sections cut from formalin-fixed, paraffin-embedded tissue blocks from a subset (n = 180) of the original study case subjects were available for immunohistochemical staining with anti-factor VIII antiserum. Patient enrollment in Protocol 8082 was accompanied by signed informed consent. Archived specimens were analyzed under the auspices of a minimal-risk protocol approved by the Institutional Review Board at the Dana-Farber Cancer Institute (Boston, MA). Subjects for this study were selected solely on the criterion of tissue availability. These sections had been cut and stored at room temperature for 5-10 years before staining and analysis. Seventy-three sections were from primary tumors and 161 sections were from ipsilateral axillary lymph nodes containing metastases. In 54 cases, sections from both the primary tumor and the axillary lymph node metastasis from the same patient were available for immunostaining.Table 1Go provides a comparison of patients from whom sections were available and staining was adequate (see below) for this study of angiogenesis, with the remaining patients entered in the original study population from whom sections were not available.


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Table 1. Characteristics of 110 patients in this study for whom unstained sections were available and allowed adequate immunohistochemical staining and comparison (see P values) with other patients enrolled in CALGB Protocol 8082 for whom unstained sections were not available *

 
Immunohistochemistry

Staining for factor VIII-related antigen was performed by use of a rabbit polyclonal antibody (Dako Corp., Carpenteria, CA) at a 1 : 400 dilution on the Ventana 320 automated stainer (Ventana Medical Systems, Tucson AZ). The peroxidase-antiperoxidase technique was used following predigestion with proteinase 2 (Ventana Medical Systems), and diaminobenzidine was used as the chromogen. Sections were counterstained lightly with hematoxylin. The immunostained sections were screened for residual tumor, and the quality of factor VIII staining was evaluated by use of microvessels in surrounding benign breast parenchyma and adipose tissue as internal controls.

Assessment of Neovascularization

The factor VIII-stained sections were evaluated by one pathologist (A. J. Guidi), who was blinded to patient treatment assignment and outcome. Each section was evaluated for acceptable immunostaining by use of blood vessels in benign breast tissue as an internal positive control. Specimens with unacceptable immunostaining (i.e., in which no immunostaining in normal vessels could be identified) were excluded. Both nonquantitative and quantitative assessments of neovascularization were performed. Most, but not all, tumors exhibited one or more focal regions of relatively intense neovascularization (i.e., vascular "hot spots") obvious on low-power screening (40x magnification—4x objective lens; 10x ocular lens). In some cases, the vascular hot spots were characterized by a complex arborizing network of vessels that completely encircled tumor cells; in other cases, the hot spots were characterized by numerous individual vessels. Vascular hot spots evident on low-power scanning were scored as present or absent.

The quantitative vessel counts were performed according to the method described by Weidner et al. (3,6). MVD counts for both primary and metastatic tumors were performed in the areas of the most intense neovascularization. To improve the accuracy of the vessel counts, a four-quadrant crosshair eyepiece was used. Areas of MVD were counted in a minimum of five 200x fields (20x objective lens and 10x ocular lens; 0.74 mm2 per field). If multiple vascular hot spots were present, counts were performed in each hot spot. If vascular hot spots were not obvious at low power, a careful high-magnification scanning was necessary to identify the areas of highest MVD. In these cases, it was often necessary to count in more than five fields to ensure that the most vascular field was included in the analysis. Microvessels were defined as any discrete factor VIII-positive endothelial cell or endothelial cell aggregate, with or without definable lumina. The highest MVD per field count was used in the analysis.

Statistical Analysis

Overall survival and disease-free survival were estimated with the use of the Kaplan-Meier product-limit method. Cox proportional hazards univariate analysis was used to model overall survival and disease-free survival, and the log-rank test was applied to compare two overall or disease-free distributions for the Kaplan-Meier curve plots. Disease-free survival was calculated from the time of study entry to disease progression or death, whichever occurred first. Overall survival was calculated from the time of study entry to death. In both cases, patients who were event free at the date of last follow-up were censored at that time. Multivariate Cox regression models were performed to relate various prognostic variables with disease-free survival and overall survival. MVD data were plotted and observed to conform to a normal distribution. MVD, hot spots, estrogen receptor status, and menopausal status were analyzed as dichotomous variables, except in the Pearson correlation. Age, tumor size, and the number of positive lymph nodes were analyzed as continuous variables. Correlations between MVD measurements (as a continuous variable) were analyzed by calculating a Pearson correlation coefficient. Fisher's exact test or the chi-squared tests were used to examine the relationships among categorical variables. Student's t test was used to test for differences in age, and the nonparametric Wilcoxon test was used to test for differences in the number of positive lymph nodes and tumor size. P values less than .05 were considered to be statistically significant; all P values were two-sided.


    RESULTS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Of the 234 slides immunostained for factor VIII, residual invasive carcinoma could not be identified in 32 specimens. Of the remaining 202, a total of 64 sections (32%) were excluded from analysis because of unacceptably low-factor VIII immunoreactivity (determined by analysis of vessel staining in surrounding normal tissue). This phenomenon may be partly explained by the prolonged period of slide storage, which ranged from 5 to 10 years at room temperature (10). In all, MVD counts were performed on 138 sections from 110 patients. Forty-seven sections were from primary tumors, and 91 sections were from axillary lymph nodes. In 28 patients, satisfactorily stained sections of both the primary and metastatic tumors from the same patients were available for microvessel quantitation. The median length of follow-up for the 110 evaluable patients in this study was 16 years.

Angiogenesis in Primary Breast Cancer Tissue Versus Lymph Node Metastases

No statistically significant difference was identified in the likelihood of finding vascular hot spots in primary breast cancer tissues (34 [72%] of 47) versus axillary lymph node metastases (57 [63%] of 91) (P = .16; Fisher's exact test). Similarly, no significant difference in median MVD counts was observed between primary and metastatic tumors: The mean (± standard deviation [SD]) MVD count for primary tumors was 138 ± 7.4 vessels/field (range, 54-255 vessels/field), and the mean (±SD) MVD count for lymph node metastases was 135 ± 6.9 vessels/field (range, 37-310 vessels/field) (P = .77; Student's t test).

Among the 28 patients for whom MVD for both primary tumors and lymph node metastases could be determined, a weakly positive correlation between the primary and lymph node MVD measures was observed that was not statistically significant (Pearson correlation coefficient = .34; P = .08) (Fig. 1)Go. In 13 (46%) of the 28 patients, there was a discrepancy of more than 25% between the primary tumor and the lymph node tumor MVD scores. The proportion of patients in whom the axillary lymph node tumor was more vascular than the primary tumor (15 [54%] of 28) was essentially the same as the proportion of cases in which the primary tumor was more vascular than that in the lymph node (12 [43%] of 28) (two-sided P = .41 for comparison of proportions). In one matched set, MVD was identical in the primary and the axillary metastatic tissues. In summary, since there was only a weak correlation (Pearson correlation coefficient = .34; P = .08) between hot spots of the primary tissue and hot spots in the axillary lymph node metastases, we included these 28 patients in both subsets (primary and lymph node) for the analyses of MVD in both primary and lymph node tumors.



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Fig. 1. Comparison of microvessel density (MVD) (vessels/field) in primary breast cancer tissue and MVD in axillary lymph node metastases. In 28 patients, MVD was assessed by immunohistochemical staining for factor VIII in both primary cancer and axillary lymph node metastases (Pearson correlation coefficient = .34; two-sided P = .08).

 
No significant association was observed between indicators of angiogenesis in lymph node metastases (either MVD or the presence of vascular hot spots) and the number of positive lymph nodes (Spearman's correlation coefficient = .0019; P = .99).

Relationship Between Vascular Hot Spots and Survival

Kaplan-Meier curves relating the presence or absence of vascular hot spots in primary breast cancer tissue and axillary lymph node metastases to disease-free survival and overall survival are shown in Fig. 2,Go A-D. No statistically significant association was observed between the presence of vascular hot spots in primary tumor tissue and disease-free survival or overall survival (Fig. 2,Go A and B) (n = 47; P = .38 and .55, respectively). In contrast, the presence of vascular hot spots in lymph node metastases was statistically significantly associated with both decreased disease-free survival and overall survival in these patients (Fig. 2,Go C and D) (n = 91; P = .006 and .004, respectively). This association between vascular hot spots and decreased survival was similar in patients with one to three tumor-positive lymph nodes and in patients with four or more tumor-positive lymph nodes (data not shown).



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Fig. 2. Patient outcome related to the presence or the absence of an angiogenic hot spot in primary breast cancer tissue compared with patient outcome related to the presence ("Yes") or the absence ("No") of an angiogenic hot spot in axillary lymph node metastases. Panels A and B: Primary breast cancer tissue—disease-free survival (A) and overall survival (B) related to the presence or the absence of a hot spot in the primary tumor (n = 47). Panels C and D: Axillary lymph node metastasis—disease-free survival (C) and overall survival (D) related to the presence or the absence of a hot spot in lymph node metastasis (n = 91). Dotted line—no hot spot; solid line—hot spot identified. The number of patients at risk and the 95% confidence intervals (C.I.s) at each time point are listed under each graph. MVD = microvessel density; NA = C.I.s are too broad to calculate and are, therefore, deemed not applicable.

 
Relationship Between MVD and Survival

Kaplan-Meier curves relating MVD in primary tumors and lymph node metastases to disease-free survival and overall survival are shown in Fig. 3,Go A-D. No significant association was observed between MVD in the primary tumor and disease-free survival or overall survival (Fig. 3,Go A and B) (n = 47; P = .75 and .79, respectively). In contrast, patients with high MVD in the lymph node metastases had a significantly decreased overall survival compared with patients with less vascular tumors (Fig. 3,Go C and D) (n = 91; P = .022). A similar trend was observed with high MVD in lymph node metastases and decreased disease-free survival, but the results were not statistically significant (P = .089). This association between MVD in the lymph node tumors and decreased survival was similar in patients with one to three positive lymph nodes and in patients with four or more positive lymph nodes (data not shown).



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Fig. 3. Patient outcome related to microvessel density (MVD) in primary breast cancer tissue compared with outcome related to MVD in axillary lymph node metastases. Panels A and B: Primary breast cancer tissue—disease-free survival (A) and overall survival (B) related to MVD in primary tumor (n = 47). Panels C and D: Axillary lymph-node metastasis—disease-free survival (C) and overall survival (D) related to MVD in lymph node metastases (n = 91). Dotted line—low MVD; solid line—high MVD. The number of patients at risk and the 95% confidence intervals (C.I.s) at each time point are listed under each graph. NA = C.I.s are too broad to calculate and are, therefore, deemed not applicable.

 
Metastasis-Associated Angiogenesis and Survival: Multivariate Analysis

In a Cox proportional hazards multivariate analysis, the number of positive axillary lymph nodes and the presence of vascular hot spots in axillary lymph node metastases were predictors of decreased disease-free survival (P =.0001 and .02, respectively) and overall survival (P = .0001 and .007, respectively). The association between increased MVD in lymph node metastases and decreased overall survival was no longer statistically significant (P = .06). The strongest predictor of survival in all Cox multivariate models was the number of positive lymph nodes (P<.0001 for all comparisons). The proportional hazards assumption was met.


    DISCUSSION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
The ability of tumors to induce a vascular stroma is a critical requirement for tumor progression at all stages of breast cancer development (1,2). In many cases, angiogenesis begins before stromal invasion by tumor cells (11,12). Once invasion has occurred, angiogenesis is necessary for tumor growth and metastatic potential (1). Moreover, after tumor cells spread via lymphatic or vascular channels to distant sites, metastatic tumor deposits must then induce their own vascular stroma to grow and, potentially, to give rise to secondary metastases (13). To date, most studies evaluating angiogenesis in breast cancer have focused on the clinical implications of angiogenesis in primary invasive breast cancers. Most, but not all, studies have demonstrated that increased MVD surrounding primary tumors is associated with decreased disease-free survival and overall survival in patients with lymph node-negative and lymph node-positive breast cancer (3-8,14-16).

The results of this study confirm that metastases can exhibit neovascularization (17). These data are preliminary and are limited by patient selection and technical difficulties. However, they also suggest that assessing angiogenesis in lymph node metastases might provide useful information regarding disease-free survival and overall survival in patients with breast cancer.

Results from three previously published studies (18-20) have suggested that neovascularization in primary tumors is greater than in their associated distant metastases. However, none of these studies examined the relationship between angiogenesis in primary and lymph node metastases and/or whether one or the other is more strongly related to prognosis. Nonetheless, the results of this study and of the previously published studies suggest that metastatic clones do not necessarily carry the phenotype of the respective primary cancer. In this study, for the 28 patients from whom materials were available to evaluate angiogenesis in both the primary tumor and the corresponding axillary lymph node metastases, the relationship between MVD scores for each site was weak. In addition, where discrepancies between MVD in the primary and lymph node tumor were apparent, no trend was observed regarding higher counts in either the primary or the metastatic tumor. Although discrepancies between MVD in the primary and lymph node tumors may, in part, be explained by sampling error, it is possible that, in some cases, there is a true difference in the regional balance of angiogenesis-stimulatory and angiogenesis-inhibitory cytokines. Such an imbalance might result in different levels of neovascularization in primary and metastatic tumors. Brown et al. (21), using messenger RNA in situ hybridization, reported that the tumor cells of all examples of in situ, invasive, and metastatic ductal breast carcinomas studied produced high levels of the angiogenic cytokine vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF). The degree of VPF/VEGF expression by tumor cells has been shown by a number of investigators (22-25) to be associated with MVD in both in situ and invasive breast cancers. The mechanisms responsible for the discrepant neovascularization observed between some primary and metastatic tumors observed in this study, however, remain to be determined.

In this study, patients with metastatic tumors characterized by areas of intense neovascularization (i.e., vascular hot spots) and relatively high MVD scores for lymph node metastases were associated with decreased survival compared with patients with less vascular metastatic tumors. However, neither vascular hot spots nor MVD scores in the primary tumor were statistically significantly associated with patient survival in this study population. The relatively small number of primary tumors that were available for this study and the selected nature of this patient population preclude a definitive conclusion that MVD in metastases may be more prognostic than that of the primary tumor or that lymph node micrometastatic neovascularization has clinical utility (26). Nonetheless, these results raise the hypothesis that angiogenic activity in metastatic tumors rather than primary tumors might more reliably predict prognosis in patients with primary breast cancer.

The results of the multivariate analyses illustrate that the number of axillary lymph nodes involved with metastatic breast cancer remains the single most important prognostic factor in early disease, among those we measured. However, these results suggest that the neovascular phenotype in metastatic deposits may also be prognostic, consistent with current theories regarding the importance of angiogenesis in the biology of metastasis. Additional studies of larger numbers of patients are required to evaluate these findings further.

Neovascularization is presumably prognostic because it is associated with the invasive and metastatic processes of the tumor (27). Other investigators (28) have proposed that MVD might also have predictive value with regard to benefit from specific systemic therapy. Evaluation of the predictive strength of a tumor marker is best performed within the context of a randomized trial in which the outcome of patients who received one type of therapy is compared with that of patients who received another relative to tumor marker-defined subgroups (29,30). However, because all patients in this study received chemotherapy and because our population is a small subset of the entire population randomly assigned to arms of CALGB Protocol 8082, we are unable to comment on whether MVD levels were predictive of outcome in those patients who received VATH therapy versus those who did not.

In conclusion, the results of this study suggest that efforts to identify tumor-related prognostic factors in patients with metastatic breast cancer should not be limited to examining biologic characteristics of the primary tumor alone. Rather, they suggest that clinically relevant information may also be determined by evaluating biologic features of metastatic lesions, such as tumor angiogenesis. Efforts to study the relationship between the mechanisms responsible for angiogenesis in primary and metastatic tumors will, hopefully, lead to additional insights, not only in improved prediction of prognosis but also ultimately in the development of therapeutic interventions that can be utilized during all stages of breast cancer development (8,28).


    NOTES
 
Supported by Public Health Service grants CA31946 (to R. L. Schilsky), CA64507 (to D. F. Hayes), CA77651 (L. Norton), CA02599 (M. P. Barcos), and CA770597 (D. F. Hayes) from the National Cancer Institute (NCI), National Institutes of Health, Department of Health and Human Services; and by the Fashion Footwear Association of New York, Shoes-on-Sale®.

The following institutions participated in the study (grants from the NCI [CA]):

Cancer and Leukemia Group B Statistical Office, Durham, NC—S. George (CA33601); Columbia Presbyterian Medical Center, New York, NY—R. R. Ellison (CA1201); Dana-Farber Cancer Institute, Boston, MA—G. P. Canellos (CA32291); Dartmouth Medical School-Norris Cotton Cancer Center, Lebanon, NH—L. H. Maurer (CA04326); Eastern Maine Medical Center Community Clinical Oncology Program (CCOP), Bangor—P. L. Brooks (CA35406); Long Island Jewish Medical Center, Lake Success, NY—M. Citron (CA11028); Massachusetts General Hospital, Boston—M. L. Grossbard (CA12449); McGill Department of Oncology, Montreal, PQ, Canada—B. Leyland-Jones (CA31809); Medical Center of Delaware CCOP, Wilmington—I. M. Berkowitz (CA45418); Mount Sinai School of Medicine, New York, NY—J. F. Holland (CA04457); North Shore University Hospital CCOP, Manhasset, NY—V. Vinciguerra (CA35279); Rhode Island Hospital, Providence—L. A. Leone (CA08025); Roswell Park Cancer Institute, Buffalo, NY—E. Levine (CA02599); Southern Nevada Cancer Research Foundation CCOP, Las Vegas—J. Ellerton (CA35421); State University of New York (SUNY) Health Science Center at Syracuse—S. L. Graziano (CA21060); SUNY Maimonides Medical Center, Brooklyn—S. Kopel (CA25119); University of California at San Diego—S. L. Seagren (CA11789); University of Maryland Cancer Center, Baltimore—D. Van Echo (CA31983); University of Massachusetts Medical Center, Worcester—M. Stewart (CA37135); University of Minnesota, Minneapolis—B. A. Peterson (CA16450); University of Missouri/Ellis Fischel Cancer Center, Columbia—M. C. Perry (CA12046); University of North Carolina at Chapel Hill—T. C. Shea (CA47559); Wake Forest University School of Medicine, Winston-Salem, NC—D. D. Hurd (CA03927); Walter Reed Army Medical Center, Washington, DC—J. C. Byrd (CA26806); and Weill Medical College of Cornell University, New York, NY—T. P. Szatrowski (CA07968).


    REFERENCES
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 

1 Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990; 82:4-6.[Medline]

2 Blood CH, Zetter BR. Tumor interactions with the vasculature: angiogenesis and tumor metastasis. Biochim Biophys Acta 1990; 1032:89-118.[Medline]

3 Weidner N, Semple P, Welch W, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med 1991; 324:1-8.[Abstract]

4 Bosari S, Lee AK, DeLellis RA, Wiley BD, Heatley GJ, Silverman ML. Microvessel quantitation and prognosis in invasive breast carcinoma. Hum Pathol 1992; 23:755-61.[Medline]

5 Horak E, Leek R, Klenk N, LeJeune S, Smith K, Stuart N, et al. Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer. Lancet 1992; 340:1120-4.[Medline]

6 Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred E, Meli S, et al. Tumor angiogenesis: a new significant and independent prognostic indicator in early stage breast carcinoma. J Natl Cancer Inst 1992; 84:1875-87.[Abstract]

7 Gasparini G, Weidner N, Bevilacqua P, Maluta S, Dalla Palma P, Caffo O, et al. Tumor microvessel density, p53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinoma. J Clin Oncol 1994; 12:454-66.[Abstract]

8 Hayes DF. Angiogenesis and breast cancer In: Shapiro CL, Henderson IC, editors. Hematology/Oncology clinics of North America. Philadelphia (PA): Saunders; 1994. p. 51-71.

9 Perloff M, Norton L, Korzun A, Wood W, Carey R, Gottlieb A, et al. Postsurgical adjuvant chemotherapy of stage II breast carcinoma with or without crossover to a non-cross-resistant regimen: a Cancer and Leukemia Group B study. J Clin Oncol 1996; 14:1589-98.[Abstract]

10 Jacobs T, Prioleau J, Stillman I, Schnitt S. Loss of tumor marker immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst 1996; 88:1054-9.[Abstract/Free Full Text]

11 Guidi A, Schnitt S. Angiogenesis in preinvasive lesions of the breast. Breast 1996; 2:364-9.

12 Guidi AJ, Fischer L, Harris JR, Schnitt SJ. Microvessel density and distribution in ductal carcinoma in situ of the breast. J Natl Cancer Inst 1994; 86:614-9.[Abstract]

13 Folkman J. Angiogenesis and breast cancer. J Clin Oncol 1994; 21:441-3.

14 Axelsson K, Ljung BM, Moore D, Thor A, Chew K, Edgerton S, et al. Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma. J Natl Cancer Inst 1995; 87:997-1008.[Abstract]

15 Costello P, McCann A, Carney D, Dervan P. Prognostic significance of microvessel density in lymph node negative breast carcinoma. Hum Pathol 1995; 26:1181-4.[Medline]

16 Goulding H, Abdul Rashid N, Robertson J, Bell J, Elston C, Blamey R, et al. Assessment of angiogenesis in breast carcinoma: an important factor in prognosis? Hum Pathol 1995; 26:1196-200.[Medline]

17 Brown LF, Guidi AJ, Schnitt SJ, Van De Water L, Iruela-Arispe ML, Yeo TK, et al. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin Cancer Res 1999; 5:1041-56.[Abstract/Free Full Text]

18 Miliaras D, Kamas A, Kalekou H. Angiogenesis in invasive breast carcinoma: is it associated with parameters of prognostic significance? Histopathology 1995; 26:165-9.[Medline]

19 Hillen HF, Hak LE, Joosten-Achjanie SR, Arends JW. Microvessel density in unknown primary tumors. Int J Cancer 1997; 74:81-5.[Medline]

20 Mooteri S, Rubin D, Leurgans S, Jakate S, Drab E, Saclarides T. Tumor angiogenesis in primary and metastatic colorectal cancers. Dis Colon Rectum 1996; 39:1073-80.[Medline]

21 Brown LF, Berse B, Jackman R, Tognazzi K, Guidi A, Dvorak H, et al. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol 1995; 26:86-91.[Medline]

22 Guidi AJ, Schitt S, Fischer L, Tognazzi K, Harris J, Dvorak H, et al. Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in patients with ductal carcinoma in situ of the breast. Cancer 1997; 80:1945-53.[Medline]

23 Toi M, Hoshina S, Takayanagi T, Tominaga T. Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res 1994; 85:1045-9.[Medline]

24 Toi M, Kondo S, Suzuki H, Yamamoto Y, Inada K, Imazawa T, et al. Quantitative analysis of vascular endothelial growth factor in primary breast cancer. Cancer 1996; 77:1101-6.[Medline]

25 Anan K, Morisaki T, Katano M, Ikubo A, Kitsuki H, Uchiyama A, et al. Vascular endothelial growth factor and platelet derived growth factor are potential angiogenic and metastatic factors in human breast cancer. Surgery 1996; 119:333-9.[Medline]

26 Hayes DF, Bast R, Desch CE, Fritsche H, Kemeny NE, Jessup J, et al. A tumor marker utility grading system (TMUGS): a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996; 88:1456-66.[Abstract/Free Full Text]

27 Folkman J. What is the role of angiogenesis in metastasis from cutaneous melanoma? Eur J Cancer Clin Oncol 1987; 23:361-3.[Medline]

28 Gasparini G, Harris A. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol 1995; 13:765-82.[Abstract]

29 Gasparini G, Pozza F, Harris AL. Evaluating the potential usefulness of new prognostic and predictive indicators in node-negative breast cancer patients. J Natl Cancer Inst 1993; 85:1206-19.[Abstract]

30 Hayes DF, Trock B, Harris A. Assessing the clinical impact of prognostic factors: when is "statistically significant" clinically useful? Breast Cancer Res Treat 1998; 52:305-19.[Medline]

Manuscript received July 13, 1999; revised December 22, 1999; accepted January 4, 2000.


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