A novel cyclic adenosine monophosphate analog induces hypercalcemia via production of 1,25-dihydroxyvitamin D in patients with solid tumors.

The treatment of cancer patients with conventional chemotherapy is sometimes associated with severe systemic toxicity and only a minimal survival benefit. Because of this, new less toxic and more efficacious treatments have been sought. 8-Chloro-cAMP (8-Cl-cAMP) is one of a new generation of anticancer drugs that act at the level of signal transduction. In preclinical models, 8-Cl-cAMP modulates protein kinase A (PKA) leading to growth inhibition and increased differentiation of cancer cells. 8-Cl-cAMP was given to 16 patients with advanced cancer as an infusion via an indwelling subclavian venous catheter. We showed that 8-Cl-cAMP had a parathyroid hormone-like effect leading to increased synthesis of renal 1,25-dihydroxyvitamin D [up to 14 times the baseline value, median 3.6 times; P = 0.00001 (Student's paired t test)]. This produced the dose-limiting toxicity of reversible hypercalcemia that could not be controlled by the administration of either pamidronate or dexamethasone. The treatment was otherwise well tolerated, and other cAMP-dependent pathways (cortisol and TSH) were not affected, emphasizing the marked differences between organs in their sensitivity to this cAMP analog. Our results have shown that 8-Cl-cAMP is biologically active, and it is feasible that if the hypercalcemia can be controlled, then this drug may have a role as a single agent, or as a short infusion between cycles of chemotherapy.

Predicting distant failure in nasopharyngeal cancer.

In a prospective study of 78 patients with nasopharyngeal cancer, we examined the prognostic significance of T stage, histology, parapharyngeal involvement, and lymph node dimensions, size, and level concerning distant metastasis. AU patients were treated with radical radiotherapy alone and completed 3 to 7 years of follow-up. In univariate analysis of time to metastasis, there was a significant difference stratifying for T stage (T1 and 2 versus T3 and 4), node dimensions (less than 6 versus more than or equal to 6 cm), neck level (above versus below the thyroid notch), and parapharyngeal involvement, but not for bilaterality of lymphadenopathy. Histology was an important prognostic factor related to distant metastasis since none of the 24 World Health Organization class I cases showed distant metastasis versus 14 (26%) of 54 patients with World Health Organization class II/III carcinoma. A multivariate duration model of time to metastasis within the later histologic group suggested that lymph node dimensions, node level, and T stage were the most important factors related to distant metastases, with the hazard ratios being 3.98, 3.23, and 1.76, respectively. Multivariate analysis within the T3- to T4-stage group showed that node dimension was the only significant variable, with an associated hazard ratio of 4.09. Cases with upper-neck lymphadenopathy and node dimensions of less than 6 cm had a distant metastasis rate of <5%. We conclude that adjuvant chemotherapy for nasopharyngeal cancer is justified in T3- and T4-staged cases with nonkeratinizing or undifferentiated histology and with lymph nodes larger than 6 cm and/or located below the thyroid notch.

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 1 and 62 per cent (47/75) showed pattern II. There was a significant correlation between these patterns (P = 0.0001; chi 2 = 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.

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; chi 2 = 4.1), but not with an increase in stromal vascularity (P = 0.800; chi 2 = 0.1). There was no significant correlation between tumour TP expression and relapse-free survival (P = 0.662; chi 2 = 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.

Relationship of elevated tumour thymidine phosphorylase in node-positive breast carcinomas to the effects of adjuvant CMF.

BACKGROUND: Thymidine phosphorylase (TP) catalyses the reversible phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. High expression of TP in cell lines potentiates the effects of the cytotoxic drugs 5-fluorouracil and methotrexate, both of which are used in the cyclophosphamide, 5-fluorouracil and methotrexate (CMF) treatment regimen of breast cancer. PATIENTS AND METHODS: We therefore examined the expression of this enzyme in 328 invasive breast carcinomas using immunohistochemistry and assessed whether the expression of this enzyme by the tumour predicts patients response to CMF in node-positive patients. RESULTS: Whereas no significant difference in either relapse-free survival (RFS) (P = 0.2) or overall survival (OS) (P = 0.07) was observed between TP-negative and -positive tumours in non-treated patients, there was a significant increase in both RFS (P = 0.02) and OS (P = 0.02) in patients treated with CMF in TP-positive compared with TP-negative tumours. A multivariate analysis of the 134 node-positive patients demonstrated that in ductal carcinomas, TP was an independent variable for OS. CONCLUSIONS: This pilot study suggests that patients with TP-positive tumours have a significant survival benefit when treated with CMF and supports the hypothesis that TP enhances tumour sensitivity to the anti-metabolites 5-fluorouracil and methotrexate.

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.

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.

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.

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.

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.