Original contributionAutomated immunofluorescence analysis defines microvessel area as a prognostic parameter in clear cell renal cell cancer☆
Introduction
Clear cell renal cell cancer (ccRCC) is the most common subtype of renal cell cancer (RCC). The VHL gene is inactivated in ccRCC of patients with the von Hippel-Lindau (VHL) syndrome and in most sporadic ccRCCs. Inactivation of the VHL tumor suppressor protein (pVHL) through mutation or inhibition of expression leads to constitutive hypoxia-inducible transcription factor (HIF) activation, an event that is believed to contribute to the regulation of vascular endothelial growth factor (VEGF). VEGF is an angiogenetic factor that may be involved in tumor growth and metastasis. It is postulated that ccRCC is therefore characterized by rich neovascularization and an often prominent vascular network around tumor cells. They often metastasize via the vascular route, suggesting tumor angiogenesis as an important parameter for ccRCC progression.
Microvessel density (MVD) defined as the number of small vessels for a given tumor area is used as a measure of angiogenesis in solid tumors [1]. MVD is frequently determined by using a method introduced by Weidner [1], which defines rules for reproducible microvessel counting. For several carcinomas including breast [2], prostate [3], bladder [4], colon [5], stomach [6], ovary [7], and melanoma [8], angiogenesis is independently associated with metastasis and lower disease-specific survival.
In contrast, in ccRCC the relationship between vascularity as measured by MVD and prognosis are conflicting. An association between high MVD and poor prognosis was reported by Joo et al [9] and Fukata et al [10]. Other groups have observed an association between high MVD and better prognosis [11], [12], [13], [14], [15], whereas some groups failed to confirm the association between MVD and prognosis [16], [17]. These discrepancies may be attributable to interobserver variability in precisely determining MVD in ccRCC [13], [18]. The method used by Weidner is semiquantitative, depending on the experience of the pathologist and on poorly controlled variables, including quality of endothelial cell immunohistochemistry. Furthermore, the method may not account for the complexity of microvessel architecture in ccRCC with its extremely high degree of vascular branching and irregularity.
Recently, an automated quantitative analysis (AQUA) system has been introduced by Camp and colleagues [19], [20]. The fluorescence-based AQUA system automatically measures and localizes disease-specific variations in protein expression within tissues. It is objective and reproducible, with features lacking in manual biomarker evaluation. AQUA can be applied to standard tissue samples or tissue microarrays (TMAs), allowing automated high-throughput quantification of biomarkers [21].
Consequently, in this study we evaluate the use of the AQUA system to measure vascularization in ccRCC. In contrast to MVD, which is determined by manual counting according to Weidner [1], we established the area of microvessels (microvessel area [MVA]) as a new parameter for the measurement of vascularization by AQUA. We developed a computer-aided approach to determine angiogenesis in primary ccRCCs by using an antibody against the endothelial marker CD34 and defined a ccRCC-specific epithelial/stromal mask with epithelial membrane antigen (EMA), CD10, and vimentin. The results were compared with prognostic parameters of ccRCC (tumor grade, stage, size, metastasis, presence of necrosis) and cancer-specific survival. The AQUA approach allowed for fast, objective, straightforward, and highly reproducible measurement of tumor vascularity in a high-throughput setting. An association between a large area of microvessels and better patient prognosis was confirmed with TMAs.
Section snippets
Patients and TMAs
RCC patients were identified from the files of the Institute for Surgical Pathology, Department of Pathology of the University Hospital Zurich, Switzerland, and from the Institute for Pathology, Kantonsspital St. Gallen, Switzerland. All samples were collected from patients, who gave their consent, with prior institutional review board approval at each respective institution. All RCCs were histologically reevaluated by a pathologist (HM) and selected for the study on the basis of hematoxylin
MVA and MVD
Immunofluorescent staining for the endothelial marker CD34 revealed heterogeneously distributed microvessels in ccRCC with an extremely high degree of vascular branching and irregularities. Representative CD34-stained sections of ccRCCs with small, medium, and large MVA values are shown in Fig. 1.
The MVA as determined by AQUA ranged from 0% to 30.3% (mean ± SD, 10.1% ± 6.3%). A large MVA (>10%) was associated with histologic grade (P < .001), tumor stage (P = .008), presence of metastasis (P =
Discussion
Tumor growth is dependent on angiogenesis. There is evidence to suggest an association between tumor vascularity and patient survival in a variety of malignant neoplasms. An increased MVD has been associated with early progression in a number of tumors [2], [3], [4], [5], [6], [7], [8]. Such an association is likely due to increased nutrient transfer in hypervascular tumors, promoting the establishment of more rapidly proliferating tumor cells. Indeed, it has been shown for various malignancies
Acknowledgments
We thank Xiao-Wei Sun for expert and diligent technical support.
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This work was supported by the NIH NCI P50 CA090381Prostate SPORE at the Dana-Farber/Harvard Cancer Center (MAR), the Swiss National Science Foundation 323880-103145/1 (HM), and the Swiss Foundation for Medical-Biological Grants SSMBS, SNF no. 1168 (KM).