EVALUATION OF ANGIOGENESIS IN IDIOPATHIC MYELOFIBROSIS WITH CD31 AND CD34 BY LIGHT MICROSCOPE AND COMPUTER-AIDED IMAGE ANALYSIS SYSTEM

Background: Myelofibrosis with myeloid metaplasia (MMM) is a clonal stem cell disease with varying degrees of fibrosis in the marrow. Vasculogenesis is the formation of new blood vessels from pre-existing vessels during adult life; it plays a critical role in neoplastic development, progression and metastasization and has been shown to be an adverse prognostic factor in many solid tumors. It is becoming increasingly evident that angiogenesis plays a key role in the pathophysiology of hematologic malignancies by estimating bone marrow microvessel density and by measuring circulating angiogenic factors. MMM is probably the disease with the more pronounced angiogenesis among Chronic Myeloproliferative Disorders. Objective: 1. Evaluation of angiogenesis in Idiopathic Myelofibrosis (IMF) via immunohistochemical staining for CD31 and CD34 and quantifying them by Computerized Image Analysis System and Light Microscope. 2. To investigate whether angiogenesis can be considered as a marker for disease activity. Methods: This cross sectional study was conducted on 31 formalin fixed paraffin embedded blocks of Idiopathic Myelofibrosis cases along with 10 age matched control cases having no abnormal bone marrow pathology, from January 2006 to June 2006. The sections from bone marrow biopsies were processed routinely and stained with Hematoxylin and Eosin (H&E) and with immunohistochemical stain for CD31 and CD34 markers. The bone marrow Microvessel Density of IMF and control case was assessed by the means of Computer Aided Image Analysis System and by visual count using light microscope. Results: This study revealed that there was a significant increase in microvessel density in IMF cases, using both the computerized method and the visual count by light microscope, with both CD31 & CD34 compared to control group (P < 0.05). Also this study showed that the increase in angiogenesis was positively correlated with the Dupreiz score prognostic system and the age of the patients. On the other hand; no significant correlation was found between angiogenesis and the following parameters: sex of the patient, Hb value, WBCs count, peripheral blood platelets count. Conclusion: This study has showed that angiogenesis was an integral component of the bone marrow stromal reaction in MMM and it was closely related to many prognostic parameters; thus bone marrow angiogenesis can be used as a tool to assess the disease activity. Key word: Idiopathic myelofibrosis, angiogenesis, Computeriz image analysis. IRAQI J MED SCI, 2010; VOL.8 (3):25-33 Introduction Idiopathic Myelofibrosis (IMF) or as also called Myelofibrosis with Myeloid Dept. Pathology and Forensic Medicine, College of Medicine, Al-Nahrain University, 2 Al-Kadhmyia Teaching Hospital . Address Correspondence to: Dr. Subuh Salim AL Mudallal , Email: subhmudallal@yahoo.com Received: 5 October 2009, Accepted: 21 April 2010. Metaplasia (MMM) is one of the Philadelphia-negative chronic myeloid disorders and is stem cell disorder characterized by clonal megakaryocytic hyperplasia, a leukoerythroblastic peripheral blood smear, extramedullary hematopoiesis, and secondary bone marrow fibrosis. Angiogenesis, or the formation of new blood vessels, is an important Angiogenesis in idiopathic myelofibrosis. Subuh S. AL Mudallal et al. Iraqi Journal of Medical Sciences 26 process in health and disease and may be integral to solid tumor growth and metastasis. The concentration of these vessels has been shown to be increased in various hematologic disorders, and it may provide useful prognostic informations (2. Indeed with the use of immunohistochemistry, various antibody markers for endothelial cells have been used to identify intratumoral vessels, the most commonly used markers are CD31 and CD34 (7, . Quantification and analysis of the degree of intratumoral angiogenesis is mostly done by estimating the microvessel density (MVD) which is maximal number of blood vessels per unit area of section . The recent introduction of morphometric analysis using fully automatic Computerized Image Analysis (CIA) has offered objectivity, increase precision compared with direct visual appraisal and makes statistical analysis easier, as it is possible now to measure MVD and the intercapillary distance (ICD) . Subjects, Materials, and Methods This cross sectional study was conducted on 31 formalin fixed paraffin embedded blocks of Idiopathic Myelofibrosis cases along with 10 age matched control cases having normal bone marrow. The cases were collected from the archives of the histopathology laboratory in Al Kadhimiya teaching hospital; Al Yarmook Center for Blood Disorders; the Teaching Laboratory of Medical City Hospital, and from private laboratories from January 2006 to June 2006. The selection criteria of Idiopathic myelofibrosis cases was based on the biopsies reports which was examined by a specialized haematologist, and other diagnostic criteria of IMF; which include bone marrow fibrosis, atypical megakaryocytic hyperplasia, and peripheral blood leukoerythroblastosis, in addition to clinical & physical findings. Whereas the bone marrow biopsies of control cases showed no bone marrow abnormalities and no excess in fibrous tissue. From each specimen, 3 sections of 5 μm thicknesses were taken, the first was stained with Hematoxyline and Eosin stain and the other two were stained immunohistochemically with CD31 and CD34 antibodies. Both patients and controls sections were examined for bone marrow microvessel density at x100, x200, and x400 magnification, and five hot spot areas (areas of highest neovascularization) in each CD31 and CD34 stained sections were selected and the mean number of microvessels (MVD) was measured,and the same field was examined by light microscope (LM) and by computerized image analysis system (CIA)and was compared with that of normal reactive bone marrow specimens. For performing visual count by LM, each of the study slide was first scanned at 100× magnification, and 5 hot spot areas were defined and the number of blood vessels were determined at 400× magnification, and that number was divided by the tissue area of that field (tissue area using Olympus microscope at 400× is 0.1885 mm) yielding microvessel visual count/area (MVC/Area) parameter which is comparable to the MVD estimated by CIA system and this was used for statistical analysis . During the counting process, large vessels, tortuous vessels, and vessels in the periosteum or bone and open Angiogenesis in idiopathic myelofibrosis. Subuh S. AL Mudallal et al. Iraqi Journal of Medical Sciences 27 sinusoids were excluded. Areas of staining with no discrete breaks were counted as single vessels, and the presence of a Lumen was not required. The Computerized image analysis (CIA) system used in this study is an automatic morphometry machine that analyse semi-automatic features using computerized pixel counting. Microvessel surface area was determined and expressed as the percent of the area showing the stained blood vessel in relation to the total tissue area which is the Microvessel Density (MVD) . Lille Scoring System (Dupriez Score) (12) was done for all the patients; accordingly, three distinct prognostic groups were identified depending on three adverse prognostic factors as shown in the following table: Table 1: The Lille Scoring System (Dupriez score) for Predicting Survival in MMM . Number of adverse prognostic factors* Risk Group Median Survival(months)