Microvessel Density Quantification in Gastric Cancer: Comparing Methods for Standard Measures

The quantification of angiogenic and linfangiogenic factors has been explored in an attempt to predict the prognosis of various malignancies. In gastric cancer (GC), a promising parameter is the microvessel density (MVD). Objective: The aim of our study is to evaluate the different methods used for its quantification. Methods: 52 cases of GC were labeled by immunohistochemistry for CD34, CD105 and D2-40. The quantification of the microvasculature was performed for each marker by counting microvessels (mv) in three “hot spots”, using three different microscopic magnifications (100x, 200x and 400x). MVD was then calculated by dividing the number of vessels by the microscopic field area (measured in mm2) and compared between the three different evaluations. Results: the MVD obtained for CD34 was 203 mv/mm2 (100x), 311 mv/mm2 (200x) and 490 mv/mm2 (400x). The MVD score for CD105 was 127 mv/mm2 (100x), 213 mv/mm2 (200x) and 347 mv/mm2 (400x). The MVD obtained for D2-40 was 35 mv/mm2 (100x), 69 mv/mm2 (200x) and 170 mv/mm2 (400x). We found that MVD obtained in 100x magnification was lower than 200x, which was lower than in 400x. Those differences were statistically significant and occurred in a proportional way for all three markers. MVD obtained for CD34 was higher than for CD105. The MVD for lymphatics obtained by D2-40 was lower than the MVD for CD34 and CD105. Conclusion: Our results show that the lack of standardized methods for assessing angiogenesis and lymphangiogenesis in GC can produce variations in the MDV value, impairing the reproducibility of the results and the comparison between different studies and populations. It is necessary to standardize the MVD determination methods to compare results and confirm its prognostic value in GC and in other types of tumors. Citation: Gresta LT, Júnior IAR, Cabral MMDÁ (2014) Microvessel Density Quantification in Gastric Cancer: Comparing Methods for Standard Measures. J Cancer Sci Ther 6: 401-405. doi:10.4172/1948-5956.1000299 Volume 6(10) 401-405 (2014) 402 J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Gastrointestinal Cancer Material and Methods This is a cross-sectional study of a series of cases of primary GC with IHC study. We selected 52 cases of GC retrospectively from the database of the Gastrointestinal Pathology research laboratory from Hospital das Clinicas of Federal University of Minas Gerais. All patients underwent a gastrectomy and lymphadenectomy for GC in the same institution. The IHC study was performed in tumor sections using streptavidinbiotin peroxidase (Dako LSAB® + System) with CD34, CD105 and D240 markers. All slides were pre-treated in a buffer at 98°C in a steamer for 20 minutes for antigen retrieval. Blockage of endogenous peroxidase and avidin protein for 15 minutes each were carried out. The D2-40, CD34 and CD105 primary antibodies were incubated for 30 minutes at room temperature. The slides were revealed with diaminobenzidine. The background staining was performed with hematoxylin. Positive and negative controls were included in all reactions. The quantification of the microvasculature was performed according to the method described by Weidner et al. [1]. The three hot spots were initially detected at lower magnification (40x) and selected in each marker. Those areas were captured and digitized for morphometric image analysis. The microvessel count was performed in the same hot spot area using the following microscopic magnifications: 100x, 200x and 400x. Isolated endothelial cells or groups of cells highlighted by the endothelial markers, with or without lumen, were considered as individual vessels and counted manually using the software Image J (Figure 1). The mean values of microvessel count from the three hot spots in each magnification and each marker was calculated. The MVD was then determined dividing the number of vessels (mean microvessel count) by the microscopic field area of each magnification (in mm2) for each marker. The method used is illustrated in Figure 2.