Tumor angiogenesis is the formation or growth of new vessels from the existing vascular network towards and within the tumor. It is driven by production of tumor and/ or host derived angiogenic factors. Angiogenesis play an important role in the growth, prognosis and metastasis of solid tumors, so new prognostic factors are being researched in Non-Hodgkin's lymphoma tumor in order to determine the prognosis of the disease. Angiogenesis can be quantitated by staining histological sections with antibodies that specifically identify endothelial cells. One of the most antibodies used is CD34 antibody, which is a sensitive marker for vascular endothelium. The number of microvessels can be estimated by many methods like hot spot method and the use of computerized automatic vessel quantification using computerized
image analysis system for estimation of many vessel parameters like microvessel density and area fraction.
Aim of the study The aim of this study is: 1. Assessment of angiogenesis in Non-Hodgkin's lymphoma (NHLs) using CD34 by measuring the microvessel density (MVD) and by the use of computerized image analysis system. 2. To investigate the correlation between microvessels and various clinicopathological parameters(age,sex,site&grade) in NHLs. Materials and Methods A total of 42 cases of Non-Hodgkin's lymphoma patients were randomly included in this study;all formalin fixed paraffin embedded tissues were retrived from archived files of Department of Pathology of Al-Kadhemia hospital ,the teaching laboratories of Medical City hospital &private pathology laboratories. This study was conducted from October 2005 -April 2006. For each case 3 sections of 5 μm thickness were taken, the first being stained with Hematoxyline and Eosin stain and the others were stained immunohistochemically with CD34 antibodies. Clinical information regarding age, site, and the grade of the disease was obtained from the available histological reports.
Counting the number of microvessels was done by two means; hot spot counting method and computerized image analysis system. Scanning of the tumor sections at (X400magnification), then identification of areas of highest neovascularization (hotspots) was done. Three hot spot areas were selected and the mean number of microvessels density was calculated. The microvessel density and area fraction was estimated using the computerized image analysis. Results In this study, a high significant statistical correlation was found in the microvessel density (in both hot spot method and computerized automated counting method) regarding tumor grade, the results showed a statistically significant difference between low and high grade NHLs, when classified according to the National Cancer Institute Working Formulation (NCIWF) .Mean MVD in lowgrade tumors were(68.37پ}5.6), intermediate-grade tumors were(116.61پ}13.7), and in high-grade tumor were(119.89پ}13.1).So the (p =0.008).No differences were found in vessel density between Working Formulation intermediate and high grade lymphomas. So the (p =0.866) .In regard to the area fraction,also there is a significant difference between low and high grade NHLs. No differences were found in vessel density between intermediate and high grade lymphomas We did not find a significant relationship between MVD and age, sex, and site(nodal & extranodal) involvement. The mean MVD for age ≤60 years were (103.07پ}9.19),while it was (121.04پ}14.44) in NHL patients with age >60 years .So the (p-value=0.26).
The mean MVD for sex male was (111.34پ}9.39), while it was(110.64پ}16.14) in females.So the (p-value = 0.97). The mean MVD for site with nodal involvement was (118.54پ}11.79), while it was (97.72پ}7.63) in NHL patients with extranodal involvement.So the (p = 0.23) In regard to area fraction no significant correlations were found between AF(area fraction)% and and age, sex, and site(nodal & extranodal) involvement. Conclusion 1. Assessment of tumor vascularity (MVDs) by immunohistochemistry using CD34 antibodies is useful in quantifying angiogenesis in Non-Hodgkin's lymphoma. 2. The use of computerized image analysis system for the quantitative assessment of angiogenesis is quite reliable and reproducible method. 3. Higher MVDs are correlated with the tumor grade according to the Working Formulation. 4. Statistically no significant correlation were found between MVDs and age,sex,and site (nodal&extranodal) of the tumor.