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).
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ABSTRACT |
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INTRODUCTION |
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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.
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SUBJECTS AND METHODS |
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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 1
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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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 magnification4x 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.
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RESULTS |
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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). 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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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, 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,
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,
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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Kaplan-Meier curves relating MVD in primary tumors and lymph node
metastases to disease-free survival and overall survival are shown in
Fig. 3, A-D. No significant association was
observed between MVD in the primary tumor and disease-free survival or
overall survival (Fig. 3,
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,
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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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.
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DISCUSSION |
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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).
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NOTES |
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The following institutions participated in the study (grants from the NCI [CA]):
Cancer and Leukemia Group B Statistical Office, Durham, NCS. George (CA33601); Columbia Presbyterian Medical Center, New York, NYR. R. Ellison (CA1201); Dana-Farber Cancer Institute, Boston, MAG. P. Canellos (CA32291); Dartmouth Medical School-Norris Cotton Cancer Center, Lebanon, NHL. H. Maurer (CA04326); Eastern Maine Medical Center Community Clinical Oncology Program (CCOP), BangorP. L. Brooks (CA35406); Long Island Jewish Medical Center, Lake Success, NYM. Citron (CA11028); Massachusetts General Hospital, BostonM. L. Grossbard (CA12449); McGill Department of Oncology, Montreal, PQ, CanadaB. Leyland-Jones (CA31809); Medical Center of Delaware CCOP, WilmingtonI. M. Berkowitz (CA45418); Mount Sinai School of Medicine, New York, NYJ. F. Holland (CA04457); North Shore University Hospital CCOP, Manhasset, NYV. Vinciguerra (CA35279); Rhode Island Hospital, ProvidenceL. A. Leone (CA08025); Roswell Park Cancer Institute, Buffalo, NYE. Levine (CA02599); Southern Nevada Cancer Research Foundation CCOP, Las VegasJ. Ellerton (CA35421); State University of New York (SUNY) Health Science Center at SyracuseS. L. Graziano (CA21060); SUNY Maimonides Medical Center, BrooklynS. Kopel (CA25119); University of California at San DiegoS. L. Seagren (CA11789); University of Maryland Cancer Center, BaltimoreD. Van Echo (CA31983); University of Massachusetts Medical Center, WorcesterM. Stewart (CA37135); University of Minnesota, MinneapolisB. A. Peterson (CA16450); University of Missouri/Ellis Fischel Cancer Center, ColumbiaM. C. Perry (CA12046); University of North Carolina at Chapel HillT. C. Shea (CA47559); Wake Forest University School of Medicine, Winston-Salem, NCD. D. Hurd (CA03927); Walter Reed Army Medical Center, Washington, DCJ. C. Byrd (CA26806); and Weill Medical College of Cornell University, New York, NYT. P. Szatrowski (CA07968).
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Manuscript received July 13, 1999; revised December 22, 1999; accepted January 4, 2000.
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