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

Abstract

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 mm 2 ) and compared between the three different evaluations. . 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. Results 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. Material and MethodsThis 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 D2-40 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 v...

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“…Every single endothelial cell stained for CD31 found solitary in the tumor stroma, isolated from adjacent vessels with visible lumens, was counted separately and considered to represent a countable microvessel. The MVD was expressed as the number of blood vessels per 1 mm 2 , and based on the average value all cases were divided into two groups, the one with low or high MVD [18].…”

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“…The stained slides were scanned at low magnification, and three sections with the highest concentration of microvessel hot spots were selected for further assessment. The average vessel count of the selected areas at ×400 magnification was then calculated as CD31-positive density of each specimen, with the aid of Image J software, using cell counter plugin (National Institute of Health [NIH], Java image processing program, USA) [29].…”