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This project compared quantifiable measures of tumor vascularity obtained from contrast-enhanced high frequency (HF) and low frequency (LF) subharmonic ultrasound imaging (SHI) to 3 immunohistochemical markers of angiogenesis in a murine breast cancer model (since angiogenesis is an important marker of malignancy and the target of many novel cancer treatments). Nineteen athymic, nude, female rats were implanted with 5×106 breast cancer cells (MDA-MB-231) in the mammary fat pad. The contrast agent Definity (Lantheus Medical Imaging, N Billerica, MA) was injected in a tail vein (dose: 180µl/kg) and low frequency pulse-inversion SHI was performed with a modified Sonix RP scanner (Analogic Ultrasound, Richmond, BC, Canada) using a L9-4 linear array (transmitting/receiving at 8/4MHz in SHI mode) followed by high frequency imaging with a Vevo 2100 scanner (Visualsonics, Toronto, ON, Canada) using a MS250 linear array transmitting and receiving at 24MHz. The radiofrequency data was filtered using a 4th order IIR Butterworth bandpass filter (11–13MHz) to isolate the subharmonic signal. After the experiments, specimens were stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). Fractional tumor vascularity was calculated as contrast enhanced pixels over all tumor pixels for SHI, while the relative area stained over total tumor area was calculated from specimens. Results were compared using linear regression analysis. Out of 19 rats, 16 showed tumor growth (84%) and 11 of them were successfully imaged. HF SHI demonstrated better resolution, but weaker signals than LF SHI (0.06±0.017 vs. 0.39±0.059; p<0.001). The strongest overall correlation in this breast cancer model was between HF SHI and VEGF (r=−0.38; p=0.03). In conclusion, quantifiable measures of tumor neovascularity derived from contrast-enhanced HF SHI appear to be a better method than LF SHI for monitoring angiogenesis in a murine xenograft model of breast cancer (corresponding in particular to the expression of VEGF); albeit based on a limited sample size.

Ability to visualize breast lesion vascularity and quantify the vascular heterogeneity using contrast-enhanced threedimensional (3D) harmonic (HI) and subharmonic (SHI) ultrasound imaging was investigated in a clinical population. Patients (n = 134) identified with breast lesions on mammography were scanned using power Doppler imaging (PDI), contrastenhanced 3D HI and 3D SHI on a modified Logiq 9 scanner (GE Healthcare). Ultrasound contrast agent (UCA) flow was identified in 4D View TM (GE Medical Systems) and used to generate a map of time-intensity curves for each lesion volume. Time-points corresponding to baseline, peak intensity and washout of UCA were identified to develop vascular heterogeneity plots. Vascularity was observed with PDI in 82 lesions (61 benign and 21 malignant). 3D HI showed flow in 8 lesions (5 benign and 3 malignant), while 3D SHI visualized flow in 68 (49 benign and 19 malignant). Analysis of vascular heterogeneity in the 3D SHI volumes found benign lesions having a significant difference in vascularity betweencentral and peripheral sections (1.71 ± 0.96 vs. 1.13 ± 0.79 dB, p < 0.001, respectively) whereas malignant lesions showed no difference (1.66 ± 1.39 vs. 1.24 ± 1.14 dB, p = 0.24), indicative of more vascular coverage. These preliminary results suggest quantitative evaluation of vascular heterogeneity in breast lesions using contrast-enhanced 3D SHI is feasible and able to detect variations in vascularity between central and peripheral sections for benign and malignant lesions. Finally, 3D parametric volumes were produced for perfusion (PER) and area under the curve (AUC).

UCA flow in 11 (8 benign and 3 malignant) and 92 (71 benign and 21 malignant) lesions, respectively. Analysis of vascular heterogeneity was only performed in 3D SHI volumes with benign lesions having a significant difference in vascularity between central and peripheral sections (1.7160.96 vs. 1.1360.79; p,0.001) whereas malignant lesions showed no difference (1.6661.39 vs. 1.2461.1; p50.24), indicative of more vascular coverage. Parametric volumes were able to isolate the UCA flow signal from the surrounding tissue and generate a representation of lesion vascularity. Specifically, PER showed improved sensitivity to vascularity compared to AUC, which was susceptible to motion artifacts Conclusions: Our preliminary results suggest that contrast-enhanced 3D SHI is able to provide visualization of vascularity and vascular heterogeneity via TIC and parametric volumes in breast lesions.

ngiogenesis is the growth of new blood vessels and is a fundamental physiologic process for reproduction, development, and wound repair. 1,2 Under normal circumstances, both the proangiogenic and antiangiogenic factors of normal cells strike a perfect balance for controlled angiogenesis. Abnormal rapid proliferation of blood vessels (so-called pathologic angiogenesis)Aditi Gupta, MS, Mark A. Forsberg, BS, Kelly Dulin, BS, Samantha Jaffe, BS, Jaydev K. Dave, PhD, Valgerdur G. Halldorsdottir, PhD, Andrew Marshall, MS, Anya I. Forsberg, John R. Eisenbrey, PhD, Priscilla Machado, MD, Traci B. Fox, EdD, Ji-Bin Liu, MD, Flemming Forsberg, PhD Received May 11, 2015, Electrical and Electronics Engineers;:1156-1159 Gupta A, Dulin K, Jaffe S, et al. Prog RSNA 2014; abstract PHS13616; and Gupta A, Jaffe S, Forsberg MA, et al. J Ultrasound Med 2014; 33(suppl) ORIGINAL RESEARCHObjectives-Different methods for obtaining tumor neovascularity parameters based on immunohistochemical markers were compared to contrast-enhanced subharmonic imaging (SHI).Methods-Eighty-five athymic nude female rats were implanted with 5 × 10 6 breast cancer cells (MDA-MB-231) in the mammary fat pad. The contrast agent Definity (Lantheus Medical Imaging, North Billerica, MA) was injected, and SHI was performed using a modified Sonix RP scanner (Analogic Ultrasound, Richmond, British Columbia, Canada) with a L9-4 linear array (transmitting/receiving frequencies, 8/4 MHz). Afterward, specimens were stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase 2 (COX-2). Tumor neovascularity was assessed in 4 different ways using a histomorphometry system (×100 magnification: (1) over the entire tumor; (2) in small sub-regions of interest (ROIs); (3) in the tumor periphery and centrally; and (4) in 3 regions of maximum marker expression (so-called hot spots).Results from specimens and from SHI were compared by linear regression.Results-Fifty-four rats (64%) showed tumor growth, and 38 were successfully imaged. Subharmonic imaging depicted the tortuous morphologic characteristics of tumor neovessels and delineated small areas of necrosis. The immunohistochemical markers did not correlate with SHI measures over the entire tumor area or over small sub-ROIs (P > .18). However, when the specimens were subdivided into central and peripheral regions, COX-2 and VEGF correlated with SHI in the periphery (r = -0.42; P = .005; and r = -0.32; P = .049, respectively).Conclusions-When comparing quantitative contrast measures of tumor neovascularity to immunohistochemical markers of angiogenesis in xenograft models, ROIs corresponding to the biologically active region should be used to account for tumor heterogeneity.