Laboratory Investigation Angiogenic patterns and their quantitation in high grade astrocytic tumors Suash Sharma 1 , Mehar C. Sharma 1 , Deepak Kumar Gupta 2 and Chitra Sarkar 1 1 Department of Pathology, All India Institute of Medical Sciences, 110029, New Delhi, India; 2 Department of Neu- rosurgery, All India Institute of Medical Sciences, 110029, New Delhi, India Key words: angiogenic patterns, astrocytoma grade III, astrocytoma grade IV, glioblastoma, intratumoral microvascular density, MIB1, topoisomerase II alpha Summary Background: The objectives of this study on high grade astrocytic tumors were (i) to establish differences, if any, between grades III & IV tumors among angiogenic parameters, both qualitative and quantitative, and (ii) to correlate angiogenic parameters with proliferation indices, namely T2a and MIB1 labeling indices. Design: Twenty nine consecutive cases of WHO grades III (11) and IV (18) astrocytic tumors diagnosed in the year-2004 were studied, using H&E and CD34, MIB1 and T2a immunostaining by streptavidin biotin technique. Angiogenic patterns were studied and parameters quantitated using Image Pro Plus software (four hotspots) on CD34 immunostained sections to determine intratumoral microvessel density (iMVD), microvascular area (MVA), aspect, mean diameter (MD) and fractal dimension (FD). Results: Main angiogenic patterns of capillary (18) and glomeruloid (9) types were best developed in glioblas- tomas. Statistically significant differences (P<0.05) were seen between grades III and IV in iMVD, aspect, MD and FD, but not in angiogenic patterns or MVA (P = 0.27). Statistically significant differences (P<0.05) were seen between glioblastomas with glomeruloid vs. capillary types in iMVD and FD, but not in MVA, aspect and mean vessel diameter. T2a values correlated with MIB1 labeling indices (R = 0.965, P<0.001). Intratumoral endothelial MIB1 LI was significantly higher in grade IV as compared to grade III, but did not correlate with angiogenic parameters. No correlation of angiogenic patterns and proliferation indices was noted (R = )0.221, P = 0.26). Limited follow up data showed all recurrent grade IV tumors to be of glomeruloid type. Conclusion: Increased angiogenesis in grade IV, as compared to grade III, astrocytic tumors is characterized by an increased number/density of vessels: an increase in vessels characterized by disproportionate lengthening and likely associated with the infiltrative properties of the tumors; and an increase in pliable, irregularly shaped or structured vessels. In addition, there is a greater frequency of glomeruloid structures indicating inadequate direc- tional migration of the newly formed vessels. The lack of correlation of these angiogenesis parameters with the MIB1 and T2a proliferation indices reflects the complexity of angiogenesis parameters in high grade gliomas. Further studies are needed to determine the usefulness of the angiogenic parameters in the improved diagnosis (grading) and prognosis of astrocytic tumors. Introduction It has been well established that the degree of vascularity correlates with increasing malignancy in astrocytic brain tumors, so that glioblastomas are the most vascularized among astrocytic tumors [1–3]. It is notable that the WHO classification takes into account microvascular proliferation (multilayered endothelial proliferation), along with other criteria such as mitoses and necrosis for assigning higher grades to astrocytic tumors [4,5]. However, other vascular parameters such as microvessel density and area do not form the basis of histologic grading under WHO schema. Recently, microvessel density [6,7], microvessel size and shape related param- eters, such as branching counts [2], and angiogenic patterns [8,9] have been found to correlate with patient survival. It has been shown that low content of unevenly distributed glomeruloid and high content of evenly dis- tributed capillary-like microvessels were associated with a statistically significant longer survival in glioblastomas [9]. It was also noted that microvessel density and apoptotic index were higher in those glioblastomas that showed prominent classic vascular pattern as compared to those with bizarre vascular patterns, but MIB1 labeling index was not significantly different in the two groups [9]. In addition to MIB1, DNA topoisomerase II alpha (t2a), a marker of proliferation and chemosensi- tivity, restricted to S and G2/M phases, correlates with prognosis in gliomas [10,11]. The present study was undertaken to study grades III and IV astrocytic tumors in terms of angiogenic parameters and T2a and MIB1 labeling indices with the following objectives: Firstly, to establish differences, if any, between grades III & IV astrocytic tumors with respect to angiogenic parameters, both qualitative and quantitative. Second, to correlate angiogenic parameters with proliferation indices such as T2a and MIB1 label- ing indices in high grade astrocytic tumors. Journal of Neuro-Oncology (2006) 79: 19–30 Ó Springer 2006 DOI 10.1007/s11060-006-9120-6