Comparing subharmonic imaging and immunohistochemical markers of angiogenesis

Ultrasound imaging studies of tumor angiogenesis are often conducted with pathological markers as the reference standard. There is, however, no established standard for how to analyze specimen markers and compare them to imaging parameters. Hence, this study compared different methods for obtaining tumor neovascularity parameters based on immunohistochemical markers to contrast-enhanced subharmonic ultrasound imaging (SHI). Breast cancer cells (MDA-MB-231) were implanted into the mammary fat pad of 85 athymic, nude, female rats. An ultrasound contrast agent was injected and SHI was performed using a Sonix RP scanner (Analogic Ultrasound, Richmond, BC, Canada) transmitting and receiving at 8 and 4 MHz, respectively. Tumor specimens were sliced corresponding to the imaging planes and stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2). Tumor neovascularity was assessed in 4 different ways 1) over the entire tumor, 2) in small sub-ROIs, 3) in the tumor periphery and centrally, and 4) in regions of maximum marker expression (so called hotspots). Of the 85 rats implanted 54 (64 %) exhibited tumor growth and 38 were successfully imaged. SHI depicted the tortuous morphology of tumor neovessels and delineated small areas of necrosis. SHI measures of tumor vascularity did not correlate with the immunohistochemical markers when assessed over the entire tumor area or over small sub-ROIs (p > 0.18). However, when the specimens were sub-dived into a central and a peripheral region, COX-2 and VEGF correlated with SHI in the periphery (r = -0.42; p = 0.005 and r = -0.32; p = 0.049, respectively). In conclusion, when comparing quantitative contrast measures of tumor neovascularity to immunohistochemical markers of angiogenesis in xenograft models it appears that sub-ROIs corresponding to the biologically active region (i.e., the tumor periphery) should be used to account for tumor heterogeneity and development.