R. M. Whitehouse

Prognostic value of angiogenesis in operable non-small cell lung cancer.

Tumour angiogenesis is an important factor for tumour growth and metastasis. Although some recent reports suggest that microvessel counts in non‐small cell lung cancer are related to a poor disease outcome, the results were not conclusive and were not compared with other molecular prognostic markers. In the present study, the vascular grade was assessed in 107 (T1,2–N0,1) operable non‐small cell lung carcinomas, using the JC70 monoclonal antibody to CD31. Three vascular grades were defined with appraisal by eye and by Chalkley counting: high (Chalkley score 7–12), medium (5–6), and low (2–4). There was a significant correlation between eye appraisal and Chalkley counting (P<0·0001). Vascular grade was not related to histology, grade, proliferation index (Ki67), or EGFR or p53 expression. Tumours from younger patients had a higher grade of angiogenesis (P=0·05). Apart from the vascular grade, none of the other factors examined was statistically related to lymph node metastasis (P<0·0001). A univariate analysis of survival showed that vascular grade was the most significant prognostic factor (P=0·0004), followed by N‐stage (P=0·001). In a multivariate analysis, N‐stage and vascular grade were not found to be independent prognostic factors, since they were strongly related to each other. Excluding N‐stage, vascular grade was the only independent prognostic factor (P=0·007). Kaplan–Meier survival curves showed a statistically significant worse prognosis for patients with high vascular grade, but no difference was observed between low and medium vascular grade. These data suggest that angiogenesis in operable non‐small cell lung cancer is a major prognostic factor for survival and, among the parameters tested, is the only factor related to cancer cell migration to lymph nodes. The integration of vascular grading in clinical trials on adjuvant chemotherapy and/or radiotherapy could substantially contribute in defining groups of operable patients who might benefit from cytotoxic treatment.

The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase is up-regulated in breast cancer epithelium and endothelium.

Tumour angiogenesis is a complex multistep process regulated by a number of angiogenic factors. One such factor, platelet-derived endothelial cell growth factor has recently been shown to be thymidine phosphorylase (TP). TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Although known to be generally elevated in tumours, the expression of this enzyme in breast carcinomas is unknown. Therefore, we used ribonuclease protection assays and immunohistochemistry to examine the expression of TP in 240 primary breast carcinomas. Nuclear and/or cytoplasmic TP expression was observed in the neoplastic tumour epithelium in 53% of tumours. Immunoreactivity was also often present in the stromal, inflammatory and endothelial cell elements. Although endothelial cell staining was usually focal, immunoreactivity was observed in 61% of tumours and was prominent at the tumour periphery, an area where tumour angiogenesis is most active. Tumour cell TP expression was significantly inversely correlated with grade (P = 0.05) and size (P = 0.003) but no association was observed with other tumour variables. These findings suggest that TP is important for remodelling the existing vasculature early in tumour development, consistent with its chemotactic non-mitogenic properties, and that additional angiogenic factors are more important for other angiogenic processes like endothelial cell proliferation. Relapse-free survival was higher in node-positive patients with elevated TP (P = 0.05) but not in other patient groups. This might be due to the potentiation of chemotherapeutic agents like methotrexate by TP. Therefore, this enzyme might be a prediction marker for response to chemotherapy.

Platelet-derived endothelial cell growth factor expression correlates with tumour angiogenesis and prognosis in non-small-cell lung cancer

Angiogenesis is a recently described prognostic factor in non-small-cell lung cancer. Platelet-derived endothelial cell growth factor (PD-ECGF), shown to be the enzyme thymidine phosphorylase (TP), induces angiogenesis in vitro and in vivo. High intracellular levels of the enzyme are associated with increased chemosensitivity to pyrimidine antimetabolites. PD-ECGF/TP expression was evaluated immunohistochemically in surgically resected specimens from 107 patients with operable non-small-cell lung cancer using the P-GF,44C monoclonal antibody. High expression of PD-ECGF/TP was found in 25% of cases and was associated with high vascular grade (P = 0.01). Fourteen of 32 (44%) high vascular grade tumours showed a positive reactivity for PD-ECGF/TP vs 13/75 (17%) of low/medium vascular grade. Positive expression was observed more frequently in T2-staged cases than in T1 (P = 0.04). While overall survival was not affected (P = 0.09), subset analysis revealed that node-negative patients with positive PD-ECGF/TP expression had a worse prognosis (P = 0.04). The results suggest that PD-ECGF/TP may be an important molecule involved in angiogenesis in non-small-cell lung cancer. Up-regulation of the enzyme defines a more aggressive tumour phenotype in patients with node-negative disease. Assessment of vascular grade and PD-ECGF/TP expression should be taken into account in the design of randomized trials assessing the role of adjuvant chemotherapy in non-small-cell lung cancer.

Distinct angiogenic patterns are associated with high-grade in situ ductal carcinomas of the breast.

Angiogenesis, the formation of new blood vessels from the existing vascular network, has been demonstrated to be an important prognostic factor in invasive breast carcinoma. The switch to an angiogenic phenotype represents a growth‐limiting step during carcinogenesis and might, in pre‐invasive lesions, indicate the risk for developing an invasive phenotype. The discrepancy between modern therapy options for invasive breast carcinomas and the relatively aggressive treatment of in situ lesions underlines the need for such prognostic factors for ductal in situ breast carcinomas (DCIS). Patterns of vascularity were examined in 75 formalin‐fixed, paraffin‐embedded DCIS by immunohistochemical staining of vessels using antibodies against Factor VIII‐related antigen. Histological classification was performed according to four different systems, based on architectural or cytonuclear features, or a combination of both. Two distinct vascular patterns were observed: a diffuse increase of stromal vascularity between duct lesions (pattern I), which was present alone in 8/75 (11 per cent), and a dense rim of microvessels adjacent to the basement membrane of individual ducts (pattern II), present alone in 12/75 (16 per cent). In total, 57 per cent (43/75) showed pattern I and 62 per cent (47/75) showed pattern II. There was a significant correlation between these patterns (P=0·0001; χ2=15·1), such that both were present in 35 (47 per cent). These different vascular patterns imply two angiogenic pathways: one pathway mediated by angiogenic factors released directly by tumour cells resulting in the rim of vessels and another generated indirectly via recruitment of accessory cells such as macrophage and endothelial cells, which themselves release other angiogenic factors, causing the increase of stromal vascularity. A significant increase in both stromal vascularity (pattern I) and the presence of a rim (pattern II) was observed in high‐grade DCIS lesions (P=0·005 and P=0·037). Indeed, all the patient relapses occurred in these high‐grade lesions, but due to the small number of patient events, no significant correlation of vascular pattern to survival was observed (P>0·05). This study suggests that distinct patterns of vascularity in DCIS might be useful for identifying patients who are at risk of relapse.

Upregulation of basic fibroblast growth factor in breast carcinoma and its relationship to vascular density, oestrogen receptor, epidermal growth factor receptor and survival

BACKGROUND Angiogenesis, the process whereby endothelial cells divide and migrate to form new blood capillaries, has been assessed in tumours by measuring microvessel density. High microvessel density is a significant adverse prognostic factor in breast cancer. The angiogenic factor, basic fibroblast growth factor (bFGF), has been associated with tumourigenesis and metastasis in several human cancers. There are few quantitative studies of bFGF expression in normal tissues compared to cancer. PATIENTS AND METHODS We have measured bFGF levels in 149 human primary breast carcinomas and assessed the findings in relation to microvessel density, oestrogen receptor (ER) and epidermal growth factor receptor (EGFR). Basic FGF levels were measured by ELISA. Western blotting and immunohistochemistry were carried out to confirm the presence of bFGF. RESULTS Levels of bFGF were more than 10-fold higher in tumour cytosols compared to reduction mammoplasty tissue and 3-fold compared to non neoplastic cytosols from the same breast as the tumour (P < 0.0001). Immunohistochemistry showed bFGF protein was localised exclusively in the stroma whereas no bFGF staining was observed in the epithelial cells. High bFGF levels were significantly related to high ER (P = 0.01). Similarly, high bFGF levels were significantly related to low grade (P = 0.046) and to small tumour size (P = 0.04). No significant relationship was observed between bFGF and microvessel count, EGFR or age. In univariate analysis and in a Cox proportional hazard model bFGF did not reach significance for overall or relapse free survival. CONCLUSIONS Our results show that although bFGF is elevated in breast carcinomas compared to normal breast tissue it is not related to microvessel density and it is not an independent predictor of survival in breast cancer patients. Basic FGF may be one of multiple factors that synergise with other growth factors such as VEGF to enhance angiogenesis.

Up-regulation of thymidine phosphorylase expression is associated with a discrete pattern of angiogenesis in ductal carcinomas in situ of the breast.

Angiogenesis is essential for tumour growth and metastasis. Although vascular density as a measure of angiogenesis is an important prognostic factor in invasive breast carcinoma, the mechanism of a switch to an angiogenic phenotype in ductal in situ breast carcinomas (DCIS) has yet to be identified. Nevertheless, two distinct vascular patterns have been reported in DCIS: a diffuse increase of stromal vascularity and a dense rim of microvessels close to the basement membrane of involved ducts. This suggests that tumour angiogenesis in invasive breast cancer arises from two different angiogenic pathways. Platelet‐derived endothelial cell growth factor, now known to be thymidine phosphorylase (TP), is a candidate for initiating one of these pathways, since it is important in remodelling the existing vasculature through its chemotactic non‐mitogenic properties and is expressed early in breast cancer development. The expression of TP was therefore examined in 75 formalin‐fixed, paraffin‐embedded specimens of DCIS by immunohistochemistry, using the monoclonal antibody PGF44c to detect TP. The results were correlated with blood vessel staining by polyclonal antibodies to von Willebrand factor (Factor VIII‐related antigen, FVIIIrAg) and other clinicopathological variables. TP expression was nuclear and/or cytoplasmic and was observed in all subtypes of DCIS. High TP expression was demonstrated in 36 per cent (27/75) of tumours. This was not limited to the neoplastic cells, but was also present in stroma, endothelium, and tumour‐associated macrophages. There was no correlation between high TP and DCIS subtype (P>0·05). There was a significant correlation between TP expression and the presence of a dense vascular rim (P=0·042; χ2=4·1), but not with an increase in stromal vascularity (P=0·800; χ2=0·1). There was no significant correlation between tumour TP expression and relapse‐free survival (P=0·662; χ2=0·2). These findings suggest that remodelling of the pre‐existing vascular network induced by TP is important in generating a dense rim of microvessels around DCIS.

Potential role of bcl‐2 as a suppressor of tumour angiogenesis in non‐small‐cell lung cancer

It has been reported that genes regulating apoptosis may play a role in tumoral angiogenesis. This study examined the relationship between tumour vascularization, a measure of tumour angiogenesis, and bcl‐2 and p53 expression in operable non‐small‐cell lung cancer (NSCLC). The relationship between bcl‐2, p53 and tumour vascularization and epidermal‐growth‐factor‐receptor(EGFR) and c‐erbB‐2 expression was also studied. Tissue sections from resected tumour specimens of 107 NSCLC patients were evaluated immunohistochemically for vascular grade and bcl‐2, p53, EGFR and c‐erbB‐2 expression. bcl‐2 expression was found in 20/107 (19%) cases and was associated with squamous‐cell histology (p = 0.03). A strong inverse relationship was found between bcl‐2 expression and vascular grade (p = 0.005). All c‐erbB‐2‐positive cases were negative for bcl‐2 expression (p = 0.01). Overall no association was found between c‐erbB‐2 expression and vascular grade. However, in bcl‐2‐negative cases positive c‐erbB‐2 expression correlated with low angiogenesis (p = 0.05). No relationship was found between p53 and EGFR expression and bcl‐2, c‐erbB‐2 or vascular grade. The improved prognosis reported in bcl‐2‐positive NSCLC may be related to low tumour vascularization. The results suggest that the anti‐apoptotic gene bcl‐2 plays a role in regulating tumour angiogenesis. Since normal lung epithelium expresses bcl‐2, a sequence of tumour progression involving loss of bcl‐2, then activation of c‐erbB‐2 or increase in tumour vascularization is proposed. Int. J. Cancer 74:565–570, 1997.© 1997 Wiley‐Liss, Inc.

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

SummaryAngiogenesis 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.

Neural network prediction of relapse in breast cancer patients

When a woman diagnosed as having breast cancer has a tumour removed, it is important to try and predict whether she is likely to relapse within, say, the next three years. In this paper, the performance of a neural network classifier trained on a number of prognostic indicators is shown to be better than that of the clinical experts working with the same information. To obtain meaningful statistics with the relatively small dataset available, the network is trained using a modified form of the leave-one-out method. A procedure is also introduced for investigating how much independentinformation each input parameter contributes. This shows that, in this type of retrospective study, the type of therapy given to the woman does not significantly affect the networks prediction of whether or not she will relapse within three years. Finally, since this problem, in common with many other medical problems, is plagued by a shortage of data, the final section of the paper reports on an investigation of whether or not multi-centre databases might be feasible.