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For a tumor to grow beyond a limited volume of 1-2 mm(3), the tumor cells must not only proliferate, but they must be able to induce the growth of new capillary blood vessels from the host. As early as 1971, it was proposed that tumor growth was dependent on angiogenesis; and, that tumor cells and blood vessels composed a highly integrated ecosystem, that endothelial cells could be switched from a resting state to one of rapid growth by a diffusible signal from tumor cells, and that anti-angiogenesis may become an effective anti-cancer therapy. Indeed, now there is considerable indirect and direct evidence to show that tumor growth is angiogenesis dependent, that tumor cells can produce diffusible angiogenic regulatory molecules, and that angiogenesis inhibitors can slow or prevent tumor growth, and that angiogenesis is a relevant target for anti-cancer therapy. Measuring intratumoral microvessel density (iMVD) in vascular "hot spots" has been shown to correlate with aggressive tumor behavior. This chapter reviews the techniques available for measuring iMVD.

Purpose: The objective of this study was to evaluate tumor angiogenesis as measured by microvessel density (MVD) as an independent prognostic factor in patients with nasopharyngeal carcinoma (NPC) treated with radiotherapy alone., Methods and Materials: Eligible patients included those with NPC treated with radiotherapy. Paraffin blocks of the primary tumor had a hematoxylin and eosin-stained section prepared at the block face. One representative section for tumor was stained for factor VIII-related antigen using a standard immunoperoxidase staining technique. MVD was determined by light microscopy in areas of invasive tumor containing the highest numbers of capillaries and microvessels per area. Individual microvessel counts were made on a 200x field within the area of most intense tumor neovascularization. Results were expressed as the highest number of microvessels identified within any single 200x field. Using a breakpoint of MVD <60 vs. > or =60, the distributions between the two MVD groups were compared by the method of Gray. Overall survival rates were estimated by the Kaplan-Meier method and compared by the log-rank test. A multivariate Cox proportional hazard model was employed to examine the relationship between MVD and disease outcomes while adjusting for other concomitant variables., Results: One hundred sixty-six were eligible, of whom 123 had values determined for MVD. The MVD values ranged from 9 to 243 with a median of 70. In the multivariate analysis of overall survival, distant metastases, and local-regional failure, MVD did not significantly improve the model containing T stage, N stage, age, radiation dose, and World Health Organization class., Conclusions: We found no significant differences in overall survival, time to distant metastasis, or time to local-regional failure using a breakpoint of MVD <60 vs. MVD > or =60. The study was perhaps limited by the small size of the NPC samples.

The purpose of this investigation was to evaluate the relationship between tumor angiogenesis and nuclear p53 accumulation in invasive bladder cancer. We studied 161 patients with invasive transitional cell carcinoma of the bladder who had previously undergone radical cystectomy. Analysis was performed to determine the presence of p53 nuclear accumulation and extent of tumor-associated angiogenesis. p53 status identified a group of patients at high risk for tumor progression (p53-altered tumors), and microvessel density determinations added additional prognostic information by identifying a subset of aggressive tumors within the wild-type p53 subgroup. At 5 years, patients with tumors exhibiting no evidence of p53 alterations and low microvessel counts demonstrated 3% recurrence and 88% survival, compared to 43% recurrence and 59% overall survival for patients with intermediate vessel counts and 61% recurrence and 43% overall survival for patients with the highest vessel counts (P < 0.001 and P = 0.003, respectively). Angiogenesis also provides additional prognostic information to patients with tumors that demonstrate p53 alterations. An association between angiogenesis and p53 status did exist (P = 0. 05); however, 27% of the tumors that showed no evidence of p53 alterations exhibited high microvessel counts, and 26% of tumors with evidence of p53 alterations had low microvessel counts. Tumor-associated angiogenesis adds additional useful prognostic information to that which is obtained from p53 status in patients with invasive transitional cell carcinoma of the bladder. Although an association between p53 status and the degree of angiogenesis was identified, other factors appear to play a role in the regulation of tumor-induced neovasularization.

Background: This study was conducted to determine the prognostic significance of tumor angiogenesis, c-erbB2, epidermal growth factor receptor, Ki-67, and p53 for patients with nasopharyngeal carcinoma., Methods: In this retrospective study, the authors retrieved clinical data and made immunohistochemical preparations on archival tissue specimens from 30 patients with nasopharyngeal carcinoma treated with radiation therapy at the University of California at San Francisco between 1956 and 1990. The pretreatment clinical and immunohistochemical data were correlated with cervical lymph node metastasis at diagnosis, and endpoints including development of locoregional failure, distant metastasis, disease free survival, and overall survival., Results: Intense tumor angiogenesis, defined as microvessel counts for 60 or more microvessels per 200 x field correlated with the development of distant metastasis (P = 0.03), shorter overall survival (P = 0.02), and disease free survival (P = 0.05). Strong c-erbB2 expression correlated with a shorter overall survival (P = 0.02) and disease free survival (P = 0.02). Stage IV disease (P = 0.04) and the presence of cervical lymph node metastasis (P = 0.05) correlated with a shorter overall survival. A duration of symptoms of fewer than 6 months correlated with a shorter disease free survival (P = 0.05). No association was observed between any of the biologic prognostic factors and lymph node involvement or locoregional failure., Conclusions: The results of this study show that the tumor angiogenesis and c-erbB2 expression are significant prognostic indicators for patients with nasopharyngeal carcinoma. The presence of cervical lymph node metastasis, Stage IV disease, and a duration of symptoms fewer than 6 months also indicate a poor prognosis. The authors did not find any prognostic significance for epidermal growth factor receptor, Ki-67, or p53. These results suggest that tumor microvessel counts and c-erbB2 may be useful in identifying patients who may benefit from systemic chemotherapy or other novel treatment strategies.

Purpose: To determine the relationship between capillary density, a recognized surrogate of tumor angiogenesis, and magnetic resonance (MR) imaging-derived estimates of plasma volume (PV) and microvascular permeability in two mammary carcinoma models., Materials and Methods: Dynamic spin-echo imaging was performed by using albumin-(gadolinium-diethylenetriaminepentaacetic acid)34, a prototype blood-pool contrast medium, in 14 rats with a subcutaneously implanted slow- or fast-growing subtype of R3230 mammary carcinoma. Data were fitted to an established two-compartment kinetic model to estimate PV and permeability., Results: MR imaging-derived tumor PVs and permeabilities increased exponentially with increasing capillary density. MR imaging-derived microvascular characteristics correlated strongly with histologic capillary density, with an r2 of .85., Conclusion: Contrast medium-enhanced MR imaging may prove useful in estimating angiogenic activity in carcinomas. MR imaging may be superior to histologic assay because it is noninvasive, can be used to "sample" the entire tumor, and reflects both anatomic and physiologic characteristics.

Background: Tumor angiogenesis is necessary for tumor growth and metastasis, and increasing intratumoral microvessel density (MVD) in prostate carcinoma has been shown in several studies to be associated with increasing tumor stage. But, the relationships of endothelial cell proliferation to intratumoral MVD, tumor cell proliferation, and Gleason's score remain unknown in prostate carcinoma., Experimental Design: Using a double-immunolabeling technique (paraffin-reactive MIB1 Ab to determine Ki67 labeling index (Ki67LI) and anti-CD34 to quantify microvessels), we immunostained 20 prostatic carcinomas and adjacent benign prostate hyperplasia (BH) and four cases of trauma-induced granulation tissue. We correlated intratumoral endothelial cell proliferation with intratumoral MVD, tumor cell proliferation, and Gleason's score, and we compared these measurements with endothelial cell proliferation, MVD, and epithelial cell proliferation in adjacent BH., Results: The intratumoral endothelial cell proliferation index (mean 0.15%) and intratumoral MVD measured 30-fold (p = 0.00007) and 1.9-fold (p = 0.000003) greater than that of adjacent BH, respectively. However, the intratumoral endothelial cell proliferation index did not correlate with intratumoral MVD, tumor cell proliferation, or Gleason's score. Nor did intratumoral MVD correlate with tumor cell proliferation. In comparison, the mean MVD and endothelial cell proliferation rates in granulation tissue were 199 and 6.50%, respectively, the latter 43-fold greater than the mean intratumoral endothelial cell proliferation index (p = 0.00023)., Conclusions: Endothelial cells are actively proliferating in prostatic carcinoma, and their proliferation appears independent of intratumoral MVD and tumor cell proliferation. Yet, the relatively low intratumoral hyperplasia, suggests that endothelial cell migration and capillary remodeling play an important role in neovascularization of prostatic carcinoma, especially when compared with granulation tissue, which showed a 43-fold greater endothelial cell proliferation index. Moreover, the lack of correlation of intratumoral endothelial cell proliferation and intratumoral MVD with tumor cell proliferation suggests that tumor angiogenesis and tumor-cell proliferation are regulated by different mechanisms.

Neovascularization of tumor tissue (tumor angiogenesis) is considered essential for tumor growth, proliferation and eventually metastasis. Microvessel density or count, a measure of tumor angiogenesis, correlates with clinical outcome in skin, breast, lung and prostate carcinomas. To determine whether an association of tumor angiogenesis and nodal metastasis exists in invasive bladder cancer, microvessel counts in 41 primary invasive stages (T2 to 4,NX,M0) bladder cancers were assessed. Microvessels were identified by immunostaining of endothelial cells for factor VIII-related antigen. Microvessels were scored in selected areas showing active neovascularization, either counting a 200 x field (0.74 mm.2) or by using a 10 x 10 square ocular grid (0.16 mm.2). The microvessel count correlated with the presence of occult lymph node metastases (p < 0.0001) by both techniques. The mean microvessel count in 27 patients without lymph node metastases was 56.2 microvessels per 200 x field (standard deviation [SD] 29.5, range 7 to 130) or 28.6 microvessels per grid (SD 14.4, range 4 to 65). The 14 patients with histologically proved lymph node metastases showed mean 138.1 microvessels per 200 x fields (SD 37.9, range 82 to 202) or 74.7 microvessels per grid (SD 14.4, range 43 to 115). Good correlation was noted between area and grid counting (r = 0.97). Tumor T stage, grade and the presence of vascular or lymphatic invasion did not correlate with the presence of lymph node metastases (p = 0.41, 0.59 and 0.26, respectively). Microvessel count may provide important information regarding the risk of occult metastasis in patients with invasive bladder carcinomas.

Abundant evidence has shown that tumor growth and metastasis are dependent upon tumor angiogenesis (TA). TA refers to the growth of new vessels toward and within the tumor. Until TA occurs, tumors grow no larger than 2-4 mm in diameter. Also, TA is necessary at the beginning and at the end of the metastatic cascade of events. Thus, it seems reasonable that increasing intratumoral microvascular density (iMVD) might correlate with greater tumor aggressiveness, such as a higher frequency of metastases and/or decreased survival. Indeed, in 1991 my colleagues and I reported a statistically significant association between greater incidence of metastases in patients with breast carcinoma and increasing iMVD. Microvessel density was measured with a light microscope in a single area of invasive tumor (200x field or 0.74 mm2) representative of the highest microvessel density (neovascular "hot spot"). This was done after endothelial cells, lining the microvessels, had been highlighted with anti-factor VIII-related antigen/von Willebrand's factor (F8RA/vWF). Subsequent studies by other investigators, using either anti-F8RA/vWF or other relatively vessel-specific reagents such as anti-CD31, have shown that the association of greater tumor aggressiveness with increasing iMVD exists not only in breast carcinoma, but also in other solid tumors. This article reviews the methods of highlighting intratumoral vessels and describes the techniques for counting these vessels for assessing iMVD.

In the present study we update previous results on the prognostic value of intratumoral microvessel density (IMD), determined immunocytochemically using the monoclonal antibody CD-31 and a standard streptavidin-immunoperoxidase technique, published in the J Clin Oncol 12:454-466, 1994. This study was undertaken in those 211 node-negative breast cancer (NNBC) cases of that series of which we had pathological material available to determine all the prognostic indicators. The median period of follow-up has been extended to 78 and 80 months for relapse-free survival (RFS) and overall survival (OS), respectively, and new biological indicators (i.e. Ki-67 labeling and 67 kDa laminin receptor expression) were included in the analysis. The main results obtained are: i) a confirmation that IMD is not associated with the other biological markers studied, i.e. expression of p53 protein, c-erbB-2 protein, 67 kDa laminin receptor, and cell kinetics; IMD was weakly associated only with histological grade (p = 0.053); ii) IMD remains a highly significant prognostic factor for RFS and OS (p < 0.0001 and p = 0.018, respectively) in univariate analysis; iii) in multivariate analysis on RFS, IMD (likelihood ratio test (LRT) = 30.16; p < 0.0001), 67 kDa laminin receptor (LRT = 9.80; p = 0.0017), the IMD/67 kDa laminin receptor interaction (LRT = 8.62; p = 0.0033), tumor size (LRT = 8.56; p = 0.0034), and p53 protein (LRT = 4.96; p = 0.025) are significant and independent prognostic indicators. For OS, only tumor size (LRT = 8.34; p = 0.0038), menopausal status (LRT = 5.16; p = 0.023), p53 protein (LRT = 4.37; p = 0.036), and IMD (LRT = 4.05; p = 0.044) retain a significant and independent prognostic value. The results of this study confirm the prognostic importance on RFS of the variables previously tested, but not of peritumoral lymphatic vessel invasion. A novel finding is that 67 kDa laminin receptor and the IMD/67 kDa laminin receptor interaction are also significant and independent variables. For OS, the results confirm that both IMD and tumor size are significant and independent variables. With prolonged follow-up the novel finding that emerges is the prognostic importance of menopausal status and p53 protein. This new information could be useful for a more accurate selection of high-risk NNBC patients who require careful follow-up and may benefit from adjuvant therapy.

A more accurate method of detecting nodal disease in squamous cell carcinoma of the tongue is needed so that treatment of the neck with its associated morbidity can safely be reserved for patients who actually have metastatic disease. Tumor angiogenesis and the expression of the p53 antigen--which have each been shown to be predictive of metastasis in breast and colon cancer, respectively--are examined for their ability to predict neck metastasis in tongue cancer. Fifty-seven patients with T1 and T2 squamous cell carcinoma of the oral tongue, whose neck disease was examined by dissection or by 2-year follow-up, were studied. Twenty-eight patients (49%) were node positive and 29 patients (51%) were node negative. The primary tumors were immunohistochemically stained for the p53 antigen and for factor VIII, which allowed the blood vessels within the tumor to be quantitated. The mean vessel counts per x200 high-power field were 59.8 and 61.5 for node-positive and node-negative patients, respectively (p = 0.8). Node-positive patients showed overexpression of p53 43% of the time, vs. 61% for node-negative patients (p = 0.17). Multivariate analysis confirmed that no difference in tumor angiogenesis or the expression of the p53 antigen was found between tumors that had metastasized and those that had not. Therefore neither tumor angiogenesis nor the p53 tumor marker is clinically useful in determining lymph node metastasis in these patients.

Tumor angiogenesis is essential for tumor growth and metastasis, and intratumoral microvessel density correlates with prognosis in breast carcinoma. Yet, how intratumoral microvessel density correlates with tumor cell and intratumoral endothelial cell proliferation remains incompletely understood. To this end, we stained 57 formalin-fixed, paraffin-embedded breast carcinomas with antibody MIB1 to determine tumor cell Ki67 labeling index and with anti-CD34 to observe microvessels. We correlated the tumor cell Ki67 labeling index and mitotic figure index with intratumoral microvessel density. Using a double labeling technique combining antibody MIB1 and anti-CD34, we measured intratumoral endothelial cell proliferation in 20 of these cases and correlated these findings with tumor cell Ki67 labeling index, mitotic figure index, and intratumoral microvessel density. The intratumoral Ki67-labeling index was 45-fold greater (P < 0.000001) than that of microvessels in adjacent benign breast. Yet, endothelial cell Ki67 labeling index did not correlate with intratumoral microvessel density, tumor cell Ki67 labeling index, or mitotic figure index nor did intratumoral microvessel density correlate with tumor cell Ki67 labeling index or mitotic figure index. These findings suggest that, although endothelial cells are actively proliferating within the tumor, intratumoral microvessel density and intratumoral endothelial cell proliferation are independent of each other and of tumor cell proliferation. Thus, intratumoral microvessel density, endothelial cell proliferation, and tumor cell proliferation may be regulated by separate mechanisms.

Purpose: To determine the absolute and relative value of microvessel density (MVD), p53 and c-erbB-2 protein expression, peritumoral lymphatic vessel invasion (PLVI), and conventional prognosticators in predicting relapse-free (RFS) and overall survival (OS) rates in patients with node-negative breast carcinoma (NNBC)., Patients and Methods: We monitored 254 consecutive patients with NNBC for a median of 62 months. Intratumoral MVD was measured after microvessels were immunostained using anti-CD31 antibody. p53 and c-erbB-2 protein and hormone receptors were also determined immunocytochemically. Results were analyzed by both univariate and multivariate statistical analysis., Results: Univariate analysis showed that MVD was significantly predictive of both RFS (odds ratio [OR], 8.30; P = .0001) and OS (OR, 4.50; P = .012) when tested as a continuous or dichotomous variable. Likewise, tumor size (OR, 3.16; P = .0012), PLVI (OR, 4.36; P = .0009), estrogen receptor (ER) status (OR, 2.35; P = .016), progesterone receptor (PR) status (OR, 2.00; P = .017), and expression of p53 protein (OR, 2.82; P = .004) were significantly associated with RFS. Tumor size (OR, 3.80; P = .0038) and expression of p53 protein (OR, 2.58; P = .024) were significantly associated with OS by univariate analysis. Multivariate analysis showed that MVD (P = .0004), p53 protein expression (P = .0063), tumor size (P = .0144), and PLVI (P = .0033) were all significant and independent prognostic factors for RFS. However, only tumor size (P = .004) and MVD (P = .047) were independent predictors for OS. c-erbB2 expression was not associated with outcome by either univariate or multivariate analysis., Conclusion: MVD, p53 expression, PLVI, and tumor size are independent prognostic indicators of recurrence, which are useful in selection of high-risk NNBC patients who may be eligible to receive adjuvant therapies.

Cervical dysplasia, or cervical intraepithelial neoplasia (CIN), is a premalignant precursor to cervical cancer. This study was designed to determine whether dysplastic lesions are angiogenic. Tissue sections from 23 surgical specimens were immunohistochemically stained for factor VIII antigen, a marker for endothelial cells. The results demonstrate that a region of neovascularization develops along the basement membrane subtending dysplastic epithelium when compared to adjacent normal epithelium. Comparison of microvessel counts underlying low grade lesions (condyloma and CIN I) with microvessel counts of CIN III lesions shows a statistically significant increase in the more advanced lesions. In a subset of the high grade lesions, large vascular structures are also noted in the upper layers of the epithelium, suggesting that a second stage of neovascularization consists of extension of stromal vascular papillae into the dysplastic lesions toward the surface of the epithelium. There is no statistical correlation between the amount of inflammation and the angiogenic ratio for each lesion, implying that angiogenesis is not secondary to the inflammatory response evoked by the lesion. The human papillomavirus type present in four CIN III lesions was determined by in situ hybridization; the amount of angiogenesis appears to be independent of the human papillomavirus type.

Recent studies have shown that women with invasive breast carcinoma having high microvessel density (MVD) (a measure of tumor angiogenesis) or epidermal growth factor receptor (EGFR) expression have increased risk for metastasis and/or decreased survival. To determine if MVD and EGFR expression provide additive prognostic information, we analyzed these two prognostic indicators in the primary invasive breast carcinomas from 165 consecutive patients, who were followed for a median of 51 months. Univariate analysis showed a highly significant (p < 0.001) association of MVD with overall and relapse-free survival in all patients, including both node-negative and node-positive groups (see JNCI 84:1875-1887, 1992). For EGFR, although univariate analysis suggested that women with tumors showing EGFR expression relapsed earlier and, perhaps, died earlier, the differences were not statistically significant. Multivariate analysis, in contrast, revealed that determination of EGFR did provide significant additional information to that already provided by MVD for predicting relapse-free survival in all women (p = 0.001) and in the subset of node-positive women (p = 0.007). Among node-negative women, however, the contribution of EGFR expression in predicting relapse-free survival was not significant (p = 0.12). Likewise, EGFR measurement did not provide significant additional information beyond that of MVD alone for predicting overall survival. Thus, EGFR provides additional prognostic information to determination of MVD, but only for relapse-free survival in node-positive women with invasive breast carcinoma.

The first requirement of a new prognostic indicator is that it should possess a clear biological significance. Indeed, much evidence shows that tumor growth and metastasis depend on neovascularization. Tumor angiogenesis (TA) refers to the growth of new vessels toward and within the tumor; unless tumor neovascularization occurs, cell proliferation reaches a steady state, and the tumor grows no larger than about 2 mm greatest diameter. Moreover, for tumor cells to metastasize, they must gain access to the vasculature from the primary tumor, survive the circulation, localize in the target organ, and induce angiogenesis in that target organ. TA is necessary both at the beginning and at the end of the metastatic cascade of events. Recently, my colleagues and I showed that a statistically significant correlation exists between incidence of metastases and microvessel density (MVD) of primary invasive breast carcinomas. Now, subsequent studies have shown that the association of prognosis with MVD exists not only in breast carcinoma but also in non-small-cell lung carcinoma, prostate carcinoma, and head-and-neck carcinoma. This article reviews the concepts and mechanisms of TA, the evidence supporting its role in growth and metastasis of solid tumors, and how measuring MVD within invasive tumors correlates with factor VIII-related antigen, blood vessel.

Tumor growth and metastasis require angiogenesis; and microvessel density, a measure of tumor angiogenesis, correlates with metastasis in breast and lung carcinoma. To determine how microvessel density correlated with metastasis in prostate carcinoma, we counted microvessels within the initial invasive carcinomas of 74 patients (29 with metastasis, 45 without). Microvessels were highlighted by immunostaining endothelial cells for factor VIII-related antigen. Without knowledge of the patient's cancer stage, microvessels were counted in a 200 field (0.739 mm2) in the most active areas of neovascularization. The mean microvessel count in tumors from patients with metastases was 76.8 microvessels per 200 field (median, 66; standard deviation, 44.6). The counts within carcinomas from patients without metastasis were significantly lower, 39.2 (median, 36; standard deviation, 18.6) (P < 0.0001). Microvessel counts increased with increasing Gleason's score (P < 0.0001), but this increase was present predominantly in the poorly differentiated tumors. Although Gleason's score also correlated with metastasis (P = 0.01), multivariate analysis showed that Gleason's score added no additional information to that provided by microvessel count alone. Assay of microvessel density within invasive tumors may prove valuable in selecting patients for aggressive adjuvant therapies in early prostate carcinoma.

Background: Axillary lymph node status has been the most important prognostic factor in operable breast carcinoma, but it does not fully account for the varied disease outcome. More accurate prognostic indicators would help in selection of patients at high risk for disease recurrence and death who are candidates for systemic adjuvant therapy. Our recent findings indicated that microvessel density (count or grade) in invasive breast carcinoma (a measure of tumor angiogenesis) is associated with metastasis and thus may be a prognostic indicator., Purpose: This study was designed to further define the relationship of microvessel density with overall and relapse-free survival and with other reported prognostic indicators in breast carcinoma., Methods: In a prospective, blinded study of 165 consecutive patients, the microvessels within primary invasive breast carcinoma were highlighted by immunocytochemical staining to detect factor VIII-related antigen. Using light microscopy, we counted microvessels per 200x field in the most active areas of neovascularization and graded microvessel density. These findings were correlated, by univariate and multivariate analyses, with overall and relapse-free survival, axillary node status, and other prognostic indicators (median follow-up, 51 months)., Results: There was a highly significant (P < or = .001) association of microvessel density with overall survival and relapse-free survival in all patients, including node-negative and node-positive subsets. All patients with breast carcinomas having more than 100 microvessels per 200x field experienced tumor recurrence within 33 months of diagnosis, compared with less than 5% of the patients with breast carcinoma having 33 or fewer microvessels per 200x field. Moreover, microvessel density was the only statistically significant predictor of overall survival among node-negative women (P < .001). Only microvessel density (P < .001) and histologic grade (P = .04) showed statistically significant correlations with relapse-free survival in the node-negative subset., Conclusions: Microvessel density in the area of the most intense neovascularization in invasive breast carcinoma is an independent and highly significant prognostic indicator for overall and relapse-free survival in patients with early-stage breast carcinoma (I or II by International Union Against Cancer criteria)., Implications: Such an indicator would be useful in selection of those node-negative patients with breast carcinoma who are at high risk for having occult metastasis at presentation. These patients could then be given systemic adjuvant therapy.

Background: Experimental evidence suggests that the growth of a tumor beyond a certain size requires angiogenesis, which may also permit metastasis. To investigate how tumor angiogenesis correlates with metastases in breast carcinoma, we counted microvessels (capillaries and venules) and graded the density of microvessels within the initial invasive carcinomas of 49 patients (30 with metastases and 19 without)., Methods: Using light microscopy, we highlighted the vessels by staining their endothelial cells immunocytochemically for factor VIII. The microvessels were carefully counted (per 200x field), and their density was graded (1 to 4+), in the most active areas of neovascularization, without knowledge of the outcome in the patient, the presence or absence of metastases, or any other pertinent variable., Results: Both microvessel counts and density grades correlated with metastatic disease. The mean (+/- SD) count and grade in the patients with metastases were 101 +/- 49.3 and 2.95 +/- 1.00 vessels, respectively. The corresponding values in the patients without metastases were significantly lower--45 +/- 21.1 and 1.38 +/- 0.82 (P = 0.003 and P less than or equal to 0.001, respectively). For each 10-microvessel increase in the count per 200x field, there was a 1.59-fold increase in the risk of metastasis (95 percent confidence interval, 1.19 to 2.12; P = 0.003). The microvessel count and density grade also correlated with distant metastases. For each 10-microvessel increase in the vessel count per 200x field, there was a 1.17-fold increase in the risk of distant metastasis (95 percent confidence interval, 1.02 to 1.34; P = 0.029)., Conclusions: The number of microvessels per 200x field in the areas of most intensive neovascularization in an invasive breast carcinoma may be an independent predictor of metastatic disease either in axillary lymph nodes or at distant sites (or both). Assessment of tumor angiogenesis may therefore prove valuable in selecting patients with early breast carcinoma for aggressive therapy.