Association of tumor angiogenesis with bone marrow micrometastases in breast cancer patients.

BACKGROUND AND PURPOSE: The microscopic detection of tumor cells (micrometastases) in bone marrow and the extent of blood vessel formation (angiogenesis) in primary tumor specimens are recognized as independent prognostic markers in patients with breast cancer. Since micrometastases occur as a consequence of interaction between the neoplastic cells and the tumor neovasculature, we have examined the relationship between these markers to determine whether the degree of angiogenesis is related to the presence of micrometastases. METHODS: Micrometastases were identified in bone marrow aspirates collected from multiple sites in 214 breast cancer patients prior to surgery (mastectomy or lumpectomy). Tumor cells were detected through an examination of epithelial membrane antigen expression and an analysis of cell morphology. Tumor vascularity was graded semiquantitatively or quantitatively (Chalkley point count) after immunohistochemical staining of the CD31 antigen expressed by the endothelial cells. The reproducibility and accuracy of the vascular grading were validated by use of kappa statistics. Associations between micrometastases and clinicopathologic characteristics, including angiogenesis, were examined using chi-squared and logistic regression techniques. All tests of statistical significance were two-sided. RESULTS: Of the 214 patients, 42 (20%) were positive for bone marrow micrometastases and 75 (35%) had tumors of high vascular grade. There was 86% agreement between vascular grades assessed twice for 35 tumors (kappa statistic = 0.66); for 22 evaluated tumors, there was absolute concordance between vascular grade and Chalkley point count. There were significant positive associations between tumor angiogenesis and micrometastasis (P = .01), tumor grade (P = .003), and estrogen receptor expression (P = .007); however, no significant associations were observed with tumor size (P = .9), lymph node status (P = .33), vascular invasion (peritumoral blood or lymph vessels) (P = .9), menopausal status (P = .17), or age (P = .12). Adjusting for confounding factors, multivariate analysis showed that only tumor angiogenesis (odds ratio = 2.7; P = .016) and vascular invasion (odds ratio = 2.7; P = .012) were significant determinants for the presence of micrometastases. CONCLUSIONS: This study suggests that an assessment of tumor angiogenesis and vascular invasion gives a reliable indication of the likelihood of the presence of bone marrow micrometastases in patients with breast cancer and that both processes contribute to metastases.

Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.

Angiogenesis is a key process in tumor growth and metastasis and is a major independent prognostic factor in breast cancer. A range of cytokines stimulate the tumor neovasculature, and tumor-associated macrophages have been shown recently to produce several important angiogenic factors. We have quantified macrophage infiltration using Chalkley count morphometry in a series of invasive breast carcinomas to investigate the relationship between tumor-associated macrophage infiltration and tumor angiogenesis, and prognosis. There was a significant positive correlation between high vascular grade and increased macrophage index (P = 0.03), and a strong relationship was observed between increased macrophage counts and reduced relapse-free survival (P = 0.006) and reduced overall survival (P = 0.004) as an independent prognostic variable. These data indicate a role for macrophages in angiogenesis and prognosis in breast cancer and that this cell type may represent an important target for immunoinhibitory therapy in breast cancer.

Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma.

PURPOSE: To assess the frequency of expression and the prognostic significance of a hypoxia-regulated marker, carbonic anhydrase IX (CA IX), in a cohort of patients with invasive breast cancer. PATIENTS AND METHODS: CA IX expression was evaluated by immunohistochemistry with a murine monoclonal antibody, M75, in a series of 103 women treated surgically for invasive breast cancer. The majority of patients were treated with adjuvant hormonal or chemotherapy. The frequency of CA IX expression, its association with recognized prognostic factors, and the relationship with outcome was evaluated by univariate and multivariate statistical analyses. RESULTS: CA IX expression was present in 49 (48%) of 103 cases. The level of CA IX expression was found to be significantly associated with tumor necrosis (P <.001), higher grade (P =.02), and negative estrogen receptor status (P <.001). Furthermore, CA IX expression was associated with a higher relapse rate (P =.004) and a worse overall survival (P =.001). By multivariate analysis, CA IX was also shown to be an independent predictive factor for overall survival (hazard ratio, 2.61; 95% confidence interval, 1.01 to 6.75, P =.05). CONCLUSION: CA IX expression was associated with worse relapse-free survival and overall survival in an unselected cohort of patients with invasive breast carcinoma. The potential role of CA IX as a marker of hypoxia within breast carcinomas was also indicated by a significant association with necrosis. Further work assessing its prognostic significance in breast cancer is warranted, particularly interactions with radiotherapy and chemotherapy resistance.

Cytokine networks in solid human tumors: regulation of angiogenesis.

The isolation, purification, and molecular cloning of an increasing number of cytokines and their receptors have allowed major advances in our understanding of the relevance of these proteins to the pathobiology and treatment of such human diseases as neoplasia. Cytokines produced by the multiple cell types present within the microenvironment of solid tumors from a complex, dynamic network, in which they have overlapping properties, induce other cytokines and alter the expression of soluble and cell surface-bound cytokine receptors. A broad number of such intratumoral cytokines have multiple effects on tumor progression. These include direct and indirect effects both on tumor cell growth and metastatic behaviors and on such cells in the stromal compartment as fibroblasts, infiltrating immune cells, and endothelial cells in the microvasculature. Here, we review the sites of production and multifaceted role of several key cytokines in the stimulation of a new blood supply within growing neoplasms. The clinical implications and new therapeutic targets suggested by this rapidly emerging picture of the cellular and molecular mechanisms subserving tumor angiogenesis are also discussed.

Bcl-2 and p53 expression in node-negative breast carcinoma: a study with long-term follow-up.

Bcl-2 and p53 gene products (Bcl-2, p53) are important regulators of apoptosis and cell proliferation, and their immunohistochemical expression may help to identify high-risk breast cancer patients. The authors evaluated p53 and Bcl-2 immunoreactivity in 178 node-negative breast cancers (NNBC) with long-term follow-up (median, 60 months). Bcl-2 was seen in 111 (62%) cases, and was significantly associated with small tumor size, nonductal morphology, low tumor grade, estrogen-receptor (ER) positivity, and p53 negativity. p53 overexpression (ie, > 15% reactive nuclei) was observed in 31 (17%) cases, and was associated with lower age, large tumor size, ductal morphology, high tumor grade, negative ER status, and lack of Bcl-2 immunoreactivity. In univariate analysis, the variables associated with short relapse-free survival (RFS) were large tumor size (P = .002), high histological grade (P = .01), high mitotic count (P = .03), and high Nottingham prognostic index (NPI) (P = .0002). In multivariate analysis (final model), only the NPI was of independent prognostic value concerning RFS.

Expression of aminopeptidase-n (CD 13) in normal tissues and malignant neoplasms of epithelial and lymphoid origin.

AIMS: To provide a detailed knowledge of the distribution of the CD13 molecule, also known as the protease aminopeptidase-N, on both normal tissues and malignant neoplasms of epithelial and lymphoid origin. METHODS: CD13 antigen was examined by immunocytochemistry, using a recently produced antibody (VS5E) alongside a commercially available anti-CD13 monoclonal antibody. The VS5E recognising CD13 was produced by immunising a doxorubicin resistant breast cancer cell line (MCF-7-ADr). A striking feature of this antibody was that it stained the doxorubicin resistant cells but not the parental cell line. Both antibodies were tested on a broad range of normal tissues and three common types of epithelial malignancy (colon n = 28, lung n = 30, breast n = 35), and 12 cases of Hodgkin's and 52 of non-Hodgkin's lymphomas. RESULTS: CD13 was expressed on many tissue and cell types outside the haematopoietic system. In particular it was present on breast epithelium and in 20% (seven of 35) of breast carcinomas, but absent in normal and neoplastic colonic and bronchial tissues and lymphomas. CONCLUSIONS: This study provides not only detailed information about the expression of the CD13 antigen, but also raises the important possibility that CD13 expression may correlate with drug resistance in breast carcinomas.

The prognostic role of angiogenesis in breast cancer.

Angiogenesis is of key importance in the process of tumour progression in a number of tumour types including breast cancer. Breast cancer angiogenesis has been the most extensively studied and now serves as a paradigm for understanding the biology of angiogenesis and its effects on tumour outcome and patient prognosis. The first study to examine intra-tumoural microvessel density (IMD) immunohistochemically, and relate it to tumour outcome, was carried out by Weidner and colleagues in 1991 using an antibody against factor-8 related antigen as an endothelial marker in a series of breast cancers. They found a near linear relationship between increased microvessel counts and metastasis. This work defined the standard methodology still used today for the morphometric assessment of IMD, such that by evaluating only three so called "hotspot" areas it was possible to determine the metastatic potential of a tumour. The biological rationale behind this was that these highly angiogenic areas were those most likely to be the easiest point of entry for tumour cells into the systemic circulation. Since this initial work there have been many studies which confirm these findings and have related angiogenesis to poor prognosis using a variety of antibodies including those to CD31 and CD34. Angiogenesis is also potentially a unique target for anti-tumour therapy, and much research is being carried out in this area, including blockade of angiogenic signalling pathways and the therapeutic use of antiangiogenic factors.

Prognostic significance of microvessel density and other variables in Japanese and British patients with primary invasive breast cancer.

The purpose of this study is to investigate the associations of microvessel density (MVD) and other pathological variables with survival, and whether they accounted for survival differences between Japanese and British patients. One hundred seventy-three Japanese and 184 British patients were included in the study. British patients were significantly older (56.3+/-11.4 years vs 52.5+/-12.9 years; P<0.01) and had smaller tumours (2.2+/-1.3 vs 2.7+/-1.8 cm; P<0.01), which were more frequently oestrogen receptor positive (78.8 vs 57.2%, P<0.01), had more grade III tumours (29.9 vs 21.4%, P=0.04) and more infiltrating lobular carcinomas (13.6 vs 4.0%, P<0.01) and a higher MVD compared with Japanese patients (57.9+/-19.8 vs 53.2+/-18.6; P=0.01). However, no difference in the prevalence of lymph-node metastasis was found between them (39.1 vs 37.5%, P=0.75). Younger British patients (age <50 years) had the highest MVD compared with Japanese and older British patients (P<0.01). Japanese patients were proportionately more likely to receive chemotherapy than endocrine therapy (P<0.01). British patients had a significantly worse relapse-free survival and overall survival compared with Japanese patients, after statistical adjustment for variables (hazard ratio=2.1, 2.4, P<0.01, P<0.01, respectively), especially, in T2 stage, low MVD and older subgroup (HR: 3.6, 5.0; 3.1, 3.3; 3.2, 3.9, respectively), but only in ER negative cases (P=0.04, P=0.01, respectively). The present study shows that MVD contributes to the Japanese-British disparity in breast cancer. However, the MVD variability did not explain the survival differences between Japanese and British patients.

The expression and cellular localization of phosphorylated VEGFR2 in lymphoma and non-neoplastic lymphadenopathy: an immunohistochemical study.

AIMS: To study the expression of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2), a membrane-bound tyrosine kinase receptor to vascular endothelial growth factor, in lymphoma and non-neoplastic lymphadenopathy. METHODS AND RESULTS: Archival cases (89 cases of lymphoma and 17 cases of non-neoplastic lymphadenopathy) were studied immunohistochemically with three monoclonal antibodies to the different autophosphorylation sites in the cytoplasmic tail of the receptor. There was increased expression of this receptor in lymphoma and particularly in all cases of peripheral T-cell lymphoma. In this category, there was nuclear re-location of this receptor. CONCLUSIONS: This very interesting finding raises the possibility that VEGFR2 may be involved in the transcriptional regulation of this disease. Small molecule inhibitors to this receptor may therefore be a useful adjunct in the therapy of this disease.

A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity.

This study was undertaken to determine the highly sensitive method for detecting tumour lymphatic vessels in all the fields of each slide (LV), lymphatic microvessel density (LMVD) and lymphatic vessel invasion (LVI) and to compare them with other prognostic parameters using immunohistochemical staining with polyclonal (PCAB) and monoclonal antibodies (MCAB) to the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and the pan-endothelial marker factor VIII in a series of 67 human breast cancers. In all LYVE-1-stained sections, LV (some of which contained red blood cells) were frequently found localised in extralobular stroma, dermis, connective tissue stroma and adjacent to artery and vein, but were rare within the intralobular stroma or the tumour body (3/67 cases) or areas of widespread invasion. In contrast small blood vessels were observed in intra- and extralobular stroma in the factor VIII-stained sections. Quantitation of vessel numbers revealed that LYVE-1/PCAB detected a significantly larger number of LV than either H&E or LYVE-1/MCAB (P<0.0001). LYVE-1/PCAB detected LVI in 25/67 cases (37.3%) and their presence was significantly associated with both lymph node metastasis (chi(2)=4.698, P=0.0248) and unfavourable overall survival (OS) (P=0.0453), while not relapse- free survival (RFS) (P=0.2948). LMVD had no influence for RFS and OS (P=0.4879, P=0.1463, respectively). Our study demonstrates that immunohistochemistry with LYVE-1/PCAB is a highly sensitive method for detecting tumour LV/LVI in breast cancer and LVI is a useful prognostic indicator for lymphatic tumour dissemination.

Correlation of numerical chromosome 11 and 17 imbalance with metastasis of primary breast cancer to lymph nodes.

Abnormalities of chromosomes 11 and 17 have been widely reported in invasive carcinoma of the breast. Interphase cytogenetics using pericentromeric repeat probes allows the evaluation of numerical chromosomal aberrations in tumour cell populations. We have developed a method for interphase cytogenetics on fine needle aspirates taken from breast tumours and have applied it to the analysis of chromosomes 11 and 17 in 49 cases of invasive adenocarcinoma. Frequency distributions of signal number were generated for each case and no correlation was found between modal signal number and tumour size at presentation, nodal status or tumour differentiation. In 14 cases, two copies of each of chromosomes 11 and 17 were present, and in 14, the number of chromosomes 11 and 17 were equal but abnormal. In 14 cases, the chromosome 11 number was greater than chromosome 17 and in 7 cases, the chromosome 17 number was greater than chromosome 11. Chromosome inequality correlated with the presence of lymph node metastases or disseminated disease at presentation and the absence of in situ carcinoma. There was no relationship with the presence of vascular invasion. These data suggest that numerical chromosome 11 and 17 imbalance may indicate the ability of breast cancers to metastasize rather than invade vessels. The pattern of numerical chromosome abnormality described may define a subgroup of tumours with a greater tendency for metastasis.

Variations in the occurrence of silver-staining nucleolar organizer regions (AgNORs) in non-proliferating and proliferating tissues.

Previous studies on the subject of silver-staining nucleolar organizer regions (AgNORs) as indicators of precise proliferative status of tissues have sometimes resulted in ambiguity. The studies, however, have most frequently addressed themselves to the prognosis of neoplasias, with the aim of using AgNORs principally to distinguish between benign and malignant tumours. This investigation was to determine a base-line relationship of AgNOR clusters to proliferation and thus concentrated on normally proliferative tissues and conditionally renewing tissues after appropriate stimulation. Two murine transplantable tumours were also examined as examples of frank malignancy. As an example of the former, variations in AgNOR clusters were noted in the small intestine of man, mouse, and rat. The conditionally renewing systems of liver, prostate, and salivary glands were stimulated into proliferation by two-thirds partial hepatectomy, castration followed by treatment with testosterone, and isoproterenol treatment, respectively, in rat models; the murine sarcoma SaF and carcinoma CaNT provided relatively simple malignant tumours for AgNOR investigation. Proliferation was monitored by noting labelling indices after injection with bromodeoxyuridine (BrdUrd) in vivo followed by immunocytochemical visualization of S-phase cells. In all tissues, an increase in the size of AgNOR clusters rather than their number correlated positively with elevated labelling, particularly with the emergence of silver-staining regions of 2-3 microns visible diameter. Thus, increased AgNOR cluster size (diameter) as representative of AgNOR cluster/nucleolus volume was found to be dependent on proliferative activity in a range of normal and neoplastic tissues.

bcl-2 in normal human breast and carcinoma, association with oestrogen receptor-positive, epidermal growth factor receptor-negative tumours and in situ cancer.

The role of bcl-2 expression in solid tumours is as yet undefined. It was, therefore, the purpose of this study to investigate expression of bcl-2 protein in 111 human breast carcinomas using immunohistochemistry and the monoclonal antibody bcl-2 124. Expression was then compared with the established indicators of prognosis and biological behaviour in malignant breast disease. No relationship could be observed between bcl-2 and node status, tumour size, differentiation, type or age at excision. However, a strong positive relationship was seen between bcl-2 and oestrogen receptor (ER), with 70 of 88 (80%) bcl-2-positive tumours being ER positive also, compared with seven of 23 (30%) bcl-2-negative tumours being ER positive (P < 0.0001). The converse was found when bcl-2 was compared with epidermal growth factor receptor (EGFR). A strong negative relationship was observed, with 26 of 88 (30%) bcl-2-positive tumours being EGFR positive, compared with 16 of 23 (70%) bcl-2-negative tumours being EGFR positive (P = 0.001), raising the possibility that bcl-2 is an ER-regulated gene. An inverse relationship was also found between bcl-2 and the oncogenes c-erbB-2 and p53. Thus, loss of bcl-2 expression in breast cancer is associated with a range of molecular markers of poor prognosis and may define part of an ER-negative, EGFR-positive phenotype.

Necrosis correlates with high vascular density and focal macrophage infiltration in invasive carcinoma of the breast.

Necrosis is a common feature of invasive carcinoma of the breast and is caused by chronic ischaemia leading to infarction. Although necrosis was previously assumed to be due to a generally poor blood supply in the tumour, in this study we show that it is present in tumours with focal areas of high vascular density situated away from the actual sites of necrosis. This may account, in part, for the previous observation that necrosis is linked to poor prognosis in this disease. Highly angiogenic tumours often display blood vessel shunting from one tumour area to another, which further exacerbates ischaemia and the formation of tumour necrosis. We have recently demonstrated that high focal microphage infiltration into breast tumours is significantly associated with increased tumour angiogenesis and poor prognosis and that the macrophages accumulate in poorly vascularized, hypoxic areas within breast tumours. In order to investigate the interactions of macrophages with chronic ischaemia (as reflected by the presence of necrosis) and angiogenesis in breast tumours, we quantified the levels of these three biological parameters in a series of 109 consecutive invasive breast carcinomas. We found that the degree of tumour necrosis was correlated with both microphage infiltration (Mann-Whitney U, P-value = 0.0009; chi-square, P-value = 0.01) and angiogenesis (Mann-Whitney U P-value = 0.0008, chi square P-value = 0.03). It was also observed that necrosis was a feature of tumours possessing an aggressive phenotype, i.e. high tumour grade (chi-square, P-value < 0.001), larger size (Mann-Whitney U, P-value = 0.003) and low oestrogen receptor status (Mann-Whitney U, P-value = 0.008; chi-square, P-value < 0.008). We suggest, therefore, that aggressive tumours rapidly outgrow their vascular supply in certain areas, leading to areas of prolonged hypoxia within the tumour and, subsequently, to necrosis. This, in turn, may attract macrophages into the tumour, which then contribute to the angiogenic process, giving rise to an association between high levels of angiogenesis and extensive necrosis.

Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer.

Angiogenesis is esential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and is an important component of the angiogenic stimulus in a range of human neoplasias. In addition to its mitogenic activities, VEGF has also been found to stimulate migration in macrophages via the flt-1 VEGF receptor. It has previously been shown that increased focal tumour macrophage infiltration is associated with increased angiogenesis and worsened relapse-free and overall survival in breast cancer. Macrophages are able to stimulate angiogenesis by their production of a range of factors including VEGF, tumour necrosis factor-alpha (TNF-alpha), and thymidine phosphorylase (TP). Thus, in breast cancer, VEGF could have a dual role in the regulation of angiogenesis, by direct mitogenic stimulation of endothelial cells, and also indirectly by attracting macrophages into avascular tumours. The purpose of this study was to localize VEGF protein in a series of 96 consecutive primary breast carcinomas and to determine its relationship to focal macrophage infiltration (macrophage index). These two variables were also compared with the pathological features of the tumours, as well as oestrogen receptor (ER), epidermal growth factor receptor (EGFR), microvessel density, macrophage index, and survival. An inverse relationship (p=0.0006) was noted between VEGF and EGFR, with high VEGF expression correlating with low EGFR levels. In the EGFR-negative group of cases (n=56), positive associations were observed between VEGF expression and macrophage index (p=0.005), ER (p=0.05), p53 (p=0. 006), tumour grade (p=0.02), and tumour necrosis (p=0.03). Macrophage counts were higher in EGFR-positive tumours (p=0.0006) and no associations were found between VEGF expression and increased microvessel density. These results show that in breast cancers there are two types of macrophage infiltrates, one associated with the presence of EGFR and low VEGF expression in tumours and the other with high VEGF expression in EGFR-negative tumours. VEGF expression may be an important factor in the recruitment of tumour-associated macrophages into breast carcinomas and may thus have an additional, indirect, pathway of angiogenic stimulation in this type of tumour.

Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis.

In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel 'hot spots' has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the 'hot spots' assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity. A Chalkley count was also performed on a further 211 invasive breast carcinomas. Significant correlations were observed between manual microvessel density and luminal perimeter (r = 0.6, P = 0.0004), luminal area (r = 0.56, P = 0.002), and microvessel number (r = 0.57, P = 0.0009) by computerized analysis. There were also significant correlations between the microscopic hot spots of 0.155 mm2 and 0.848 mm2 for microvessel number (r = 0.81, P < 0.00005), luminal perimeter (r = 0.78, P < 0.00005), and luminal area (r = 0.65, P = 0.0001). In addition, a significant correlation was observed between microvessel density and both subjective vascular grade (P = 0.002) and Chalkley count (P = 0.0001). A significant reduction in overall survival was observed between patients stratified by Chalkley count in both a univariate (P = 0.02) and a multivariate (P = 0.05) analysis in the 211 invasive breast carcinomas. These findings show that Chalkley counting is a rapid method of quantifying tumour angiogenesis and gives independent prognostic information which might be useful in diagnostic practice.

Immunohistochemical assay for epidermal growth factor receptor on paraffin-embedded sections: validation against ligand-binding assay and clinical relevance in breast cancer.

Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embedded sections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay.

The prognostic value of quantitative angiogenesis in breast cancer and role of adhesion molecule expression in tumor endothelium.

Angiogenesis is the formation of new capillaries from the existing vascular network and is essential for tumor growth and metastases. Increased microvessel density in breast cancer is associated with lymph node metastasis and reduced survival. We have assessed tumor vascularity in 211 breast carcinomas using a more rapid technique based on a Chalkley point eyepiece graticule. We confirmed using this method a significant reduction in overall survival between patients stratified by Chalkley count in both a univariate (p = 0.02) and multivariate (p = 0.05) analysis. Since studies have suggested that cell adhesion molecules (CAMs) might be important in the angiogenic process, and interaction of neoplastic cells with this neovasculature is a significant step in tumor metastasis, we have also examined the expression of CAMs in a subset of these tumors (n = 64). Using immunohistochemistry we observed widespread and intense staining on the endothelium of tumor-associated vessels for PECAM (100%), ICAM 1 (69%), and E- and P-selectins (52% and 59% of cases respectively). Endothelial expression of the selectins was more prominent at the tumor periphery. Immunoreactivity of ICAM-1 (34%), PECAM (1.6%), and E- and P-selectins (7% and 37% of cases respectively) was also observed on the neoplastic element of the tumors.