Russell Leek

Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer.

Animal models suggest a role for new vessel formation (angiogenesis) in tumours with metastatic potential, and there is some evidence that this is true for human tumours. What is needed is a sensitive and specific label for endothelial cells, and one candidate would be a monoclonal antibody to platelet/endothelial cell adhesion molecule (PECAM). We have counted microvessels in 103 primary breast cancers using the JC70 antibody to PECAM (or CD31). We compared our findings with various pathological indicators (lymph node status and tumour grade, size, and type and markers (oestrogen receptor, and c-erbB-2 expression and detection of mutant p53). Tumours showed significantly higher vascularisation than normal breast tissue and the number of blood vessels/mm2 was significantly associated with node metastasis. Only 2 out of 50 tumours with 99 vessel/mm2 or less were node positive whereas 31 out of 39 tumours with counts above 140/mm2 were positive (p < 0.0001). Tumour size and grade also correlated with node metastasis and vascularisation also increased with the size of the primary and with poor differentiation. However, within each subgroup of size or differentiation tumours without node involvement had much lower vascular counts, and multivariate analysis showed that vascular count alone explains the association of size and grade with node metastasis. Other markers, conventional or novel, did not correlate with vascularisation. Even with the short follow-up in this series, vascular counts correlated with early death. These results suggest that angiogenesis is closely linked to metastasis, that it is acquired at a critical density of vessels, and that this process occurs as tumours enlarge or become more poorly differentiated. Counting of newly formed microvessels stained with endothelium-specific antibodies may prove to be a useful tool in the early detection of metastatic potential and in the selection of patients for whom anti-angiogenesis drugs might be beneficial.

Quantification of Regulatory T Cells Enables the Identification of High-Risk Breast Cancer Patients and Those at Risk of Late Relapse

PURPOSE To assess the clinical significance of tumor-infiltrating FOXP3-positive regulatory T cells (TR) in breast cancer patients with long-term follow-up. PATIENTS AND METHODS FOXP3-positive TR were detected by immunohistochemistry with our new, extensively characterized FOXP3 monoclonal antibody, 236A/E7. Numbers of FOXP3-positive lymphocytes in tissue microarray cores from pure ductal carcinoma in situ (DCIS; n = 62), invasive breast cancer (n = 237) or from comparable areas of normal terminal duct lobular breast tissue (n = 10) were determined. A median cutoff of > or = 15 defined patients with high numbers of TR. RESULTS TR numbers were significantly higher in in situ and invasive breast carcinomas than in normal breast; invasive tumors have significantly higher numbers than DCIS (P = .001). High numbers of FOXP3-positive TR identified patients with DCIS at increased risk of relapse (P = .04) and patients with invasive tumors with both shorter relapse-free (P = .004) and overall survival (P = .007). High TR numbers were present in high-grade tumors (P < or = .001), in patients with lymph node involvement (P = .01), and in estrogen receptor (ER) -negative tumors (P = .001). Importantly, high numbers of TR within ER-positive tumors identified high-risk patients (P = .005). Unlike conventional clinicopathologic factors, high numbers of FOXP3-positive TR can identify patients at risk of relapse after 5 years. CONCLUSION These findings indicate that quantification of FOXP3-positive TR in breast tumors is valuable for assessing disease prognosis and progression, and that TR are an important therapeutic target for breast cancer. FOXP3-positive TR represent a novel marker for identifying late-relapse patients who may benefit from aromatase therapy after standard tamoxifen treatment.

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

SummaryNecrosis 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.

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.

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.

Tumor-Associated Macrophages in Breast Cancer

Neoplastic cells form only one part of a complex network of cell types that make up a breast tumor. The normal cell types that make up the nonneoplastic components of tumors include fibroblasts, endothelium, and inflammatory cells, such as tumor associated macrophages (TAMs). TAMs have the potential to carry out both anti- and protumor activities. In their antitumor role TAMs can present tumor antigens to cytotoxic T-cells and are capable of being directly cytotoxic to neoplastic cells. Conversely, TAMs are also able to promote tumor growth directly by secreting breast tumor mitogens, such as epidermal growth factor, and indirectly by stimulating tumor angiogenesis and metastasis. Recent studies have indicated that in breast cancers the protumor role of TAMs is dominant, and that TAMs may be executing a “wound healing” type of process in response to stimuli found in the tumor microenvironment, such as hypoxia. As such, TAMs may provide opportunities for future therapeutic interventions.

Delta-like 4 Notch Ligand Regulates Tumor Angiogenesis, Improves Tumor Vascular Function, and Promotes Tumor Growth In vivo

The vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. However, clinical trials targeting the VEGF pathway are often ineffective, suggesting that other factors/pathways are also important in tumor angiogenesis. We have previously shown that the Notch ligand Delta-like 4 (DLL4) is up-regulated in tumor vasculature. Here, we show that DLL4, when expressed in tumor cells, functions as a negative regulator of tumor angiogenesis by reducing the number of blood vessels in all five types of xenografts, but acts as a positive driver for tumor growth in two of them (human glioblastoma and prostate cancer). The growth of in vivo models was not related to the effects on growth in vitro. DLL4 expressed in the tumor cells activated Notch signaling in host stromal/endothelial cells, increased blood vessel size, and improved vascular function within tumors. The promotion of tumor growth was, to some extent, due to a reduction of tumor hypoxia and apoptosis. DLL4-expressing tumor cells responded to anti-VEGF therapy with bevacizumab. A soluble form of DLL4 (D4ECD-Fc) blocked tumor growth in both bevacizumab-sensitive and bevacizumab-resistant tumors by disrupting vascular function despite increased tumor vessel density. In addition, we show that DLL4 is up-regulated in tumor cells and tumor endothelial cells of human glioblastoma. Our findings provide a rational basis for the development of novel antiangiogenic strategies via blockade of DLL4/Notch signaling and suggest that combined approaches for interrupting both DLL4 and VEGF pathways may improve antiangiogenic therapy.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

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‐α (TNF‐α), 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. Copyright

Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages.

Studies over the past 20 years have established that the development of new capillaries from an existing vascular network (a process called angiogenesis) is an essential component of tumor growth. Malignant tumors do not grow beyond 2–3 mm3 in size unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products. Tumor angiogenesis also provides an essential exit route for metastasizing tumor cells from the tumor to the bloodstream. Indeed, extensive neovascularization is a poor prognostic factor in several forms of human cancer. Angiogenesis is a complex, multistep process driven by many local signals within the tumor. This involves the degradation of the extracellular matrix around a local venule after the release of collagenases and proteases, the proliferation and migration of capillary endothelial cells, and their differentiation into functioning capillaries. Cytokines produced by various cell types present within the microenvironment of solid tumors form a complex, dynamic network in which they have multiple effects on tumor progression. Herein we review our work on the presence, and possible regulatory influence on tumor angiogenesis, of a number of these cytokines within invasive breast carcinomas. We have combined immunocyto‐chemistry with a single cell cytokine release assay called the reverse hemolytic plaque assay to investigate the cellular sources of the key angiogenic cytokines, vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor‐α. Tumor‐associated macrophages in the stromal compartment of these tumors and/or malignant epithelial cells were seen to be a major producer cell for these cytokines, whereas tumor necrosis factor‐α receptors were expressed by leukocytes, malignant cells, and endothelial cells in tumor blood vessels. J. Leukoc. Biol 57: 747–751; 1995.