Quantification of angiogenesis by the Chalkley method and its prognostic significance in epithelial ovarian cancer☆
Introduction
Angiogenesis means the formation of new capillaries from the existing vascular network, and is essential for tumour growth and metastatic capacity. Neovascularisation of a tumour is required to provide essential nutrients beyond the limit of simple diffusion, and allow for tumour growth beyond 2 mm3,1 although tumours may be able to grow also without neovascularisation if they find a suitable preexisting vascular bed.2 The regulation of angiogenesis is complex, and controlled by the balance between inhibitors and stimulators of angiogenesis.3
Association of angiogenesis with patient outcome has been studied widely in different carcinomas, including ovarian cancer, but mainly with relatively small materials, which have left the prognostic significance of tumour angiogenesis still controversial in epithelial ovarian cancer also in studies including more than 90 patients.4, 5, 6, 7 The discrepancies in results may originate at least partly from differences in the study materials. In addition, a lack of standardised immunohistochemical techniques hampers the comparison of studies. For example, antibodies to CD34,4, 5, 6, 8, 9, 10 CD31,7, 11, 12 von Willebrand factor (Factor VIII)13, 14 and Ulex15 have been used to detect vascular structures in prognostic ovarian carcinoma studies. CD34 is a cell surface protein that is selectively expressed by human hematopoietic progenitor cells and vascular endothelial cells,16, 17 and it has been shown to mark tumour vessels also in the solid cancers of ovary.18 Since Weidner and colleagues estimated the microvessel density in the most vascularised area (“hot-spot”) in their pioneering work in 1991,19 the same technique with slight modifications has been used widely to assess the prognostic value of angiogenesis in various types of carcinomas. Afterwards, the Chalkley method based on Chalkley eyepiece graticule was introduced to provide a quicker and more objective procedure for measuring tumour vascularity.20 Currently, the Chalkley assay with CD34 immunostaining has been suggested as a standard method for angiogenesis quantification in solid tumour sections in an international consensus report,21 although the basis for the consensus has been questioned by others.22 No previous studies on angiogenesis in ovarian cancer were found in the literature, analysed with the original Chalkley method. Therefore, we decided to study the expression of CD34 and its prognostic significance using the Chalkley method in epithelial ovarian cancer.
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Patients
We analysed 175 specimens from patients treated and diagnosed for epithelial ovarian malignancy at Kuopio University Hospital and Jyväskylä Central Hospital, Finland, between 1976 and 1992, with a follow-up until January 2004. Patients who were given any treatment before operation were excluded, as were also patients who were not operated on. Patients who died because of any post-operative complications (deaths during one month after operation) were not included in survival analyses. In
CD34 expression
A total of 175 primary ovarian cancers and their 32 metastases were analysed for CD34 expression by the Chalkley method. The median Chalkley count was 7.67 for primary tumours and 8.17 for metastases (range 4.00–19.00 and 6.00–15.00, respectively). Ninety one of 175 (52%) primary samples were categorised into low (Fig. 1A), and 84 samples (48%) into high (Fig. 1B) expression group. There was no significant difference in the Chalkley count between primary tumours and metastases (z = −1.9; p =
Discussion
While the Chalkley method has been suggested as a preferred method in estimating the prognostic significance of angiogenesis in breast cancer,24 and recommended for angiogenesis quantification in solid tumour sections in an international consensus report,21 its applicability in ovarian cancer has not been studied. In the present pilot study, the method turned out to be reliable, and indicated that the high Chalkley count is an independent prognostic marker for poor survival in epithelial
Conflict of interest statement
The authors have no conflicts of interests with relation to the study methodology, results, or other sections of the study.
Acknowledgements
We thank Mrs. Aija Parkkinen for expert technical assistance in histology, Mr. Torsten Lindgren, Mrs. Raija Törrönen and Mr. Alpo Pelttari for photographic facilities, and Ph.Lic. Vesa Kiviniemi and Pirjo Halonen for statistical advice. Valuable comments on the manuscript from Dr. Raija Tammi and Prof. Markku Tammi are greatfully acknowledged.
This study was supported by Finnish Cancer Foundation, The North Savo Cancer Fund, EVO funds of Kuopio University Hospital, Kuopio University Fund, The
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Supported by Finnish Cancer Foundation (V-MK), The North Savo Cancer Fund (K.A.S., S.M.S.), EVO funds of Kuopio University Hospital (K.A.S., M.A.A., S.M.S., K.M.H., V-M.K.), Kuopio University Fund (K.A.S., S.M.S.), The Finnish Cultural Fund (K.A.S., S.M.S.) and the Fund of Paavo Koistinen (K.A.S., S.M.S.).