Power Doppler ultrasound and contrast-enhanced ultrasound demonstrate non-invasive tumour vascular response to anti-vascular therapy in canine cancer patients

Abstract

Combretastatin A4-phosphate (CA4P) is an anti-vascular agent which selectively shuts down blood supply in tumours, resulting in extensive tumour necrosis. The aim of this study was to assess in vivo, non-invasive ultrasound techniques for the early evaluation of tumour perfusion following CA4P treatment of spontaneous tumours. Eight dogs that bore spontaneous tumours were enrolled and were subsequently treated with a single dose of intravenous CA4P. Perfusion of tumours was evaluated by power Doppler ultrasound (PDUS) pre-treatment (0 h), during the injection (10 min, 20 min, 30 min) and after CA4P infusion (24 and 72 h). Vascularity index (VI) of the tumour tissue was quantitatively analysed and accuracy was verified by correlation analysis with the results of immunohistochemical evaluation of microvessel density (MVD). Central and peripheral perfusion was evaluated by contrast-enhanced ultrasound (CEUS) pre-treatment and at 72 h post-treatment. Post-treatment, PDUS demonstrated a significant decrease in VI within 10 min of CA4P infusion. CEUS parameters demonstrated a significant decrease in blood velocity and volume in the central aspect of the tumour. Histology revealed a 4.4-fold reduction (p < 0.001, 95% CI [2.2,9.4]) in MVD and a 4.1-fold increase (p = 0.003, 95% CI [1.4,11.8]) in necrotic tumour tissue. A strong correlation between PDUS results and immunohistochemical results was found (Pearson R² = 0.957, p < 0.001). Furthermore, the findings of PDUS were supported by the objective results of the CEUS analyses. These data suggest a role for ultrasound in real-time, non-invasive monitoring of tumour vascular response as an early indicator of CA4P treatment efficacy.

Figure 1

Tumour vascularization as measured by power Doppler ultrasound of a mammary gland adenocarcinoma (dog 1). (A) before intravenous CA4P treatment, (B) 24 h after treatment, (C) 72 h after treatment at matched locations. A decrease in central tumour vascularization is clearly noticeable (Scale bar = 3 cm).

Figure 2

Tumour evaluation after CA4P treatment using contrast-enhanced ultrasound (CEUS) of a mammary gland adenocarcinoma at a matched location (dog 1). The left side of the image corresponds to the CEUS image of the tumour, the right side to the B-mode ultrasound. (A) before intravenous CA4P treatment, (B) 24 h after treatment. There is a large diffuse uptake of microbubble contrast throughout the entire tumour before treatment. In contrast, 24 h after treatment, uptake of microbubble contrast has greatly diminished in the central tumour, indicating a decrease in central tumour vascularization (Scale bar = 3 cm).

Figure 3

Regions of interest were manually drawn on the contrast-enhanced ultrasound (CEUS) image of Fig. 2: Yellow encompasses the entire tumour, green outlines the peripheral one-third radius, and magenta contains the centre two-thirds radius of the tumour. (C) Before intravenous CA4P treatment, (D) 24 h after treatment (Scale bar = 3 cm).

Figure 4

Tumour evaluation after CA4P treatment of a mastocytoma using contrast-enhanced ultrasound (CEUS) of 3 regions of interest (ROI) (dog 5). Time-intensity curves of ROI_whole (yellow), ROI_periphery (green), and ROI_centre (magenta). (A) before treatment, (B) 72 h after treatment. An obvious decrease in tumour vascularization is noted by CEUS in all three ROI’s, but is most prominent in tumour centre.

Figure 5

Histopathological section of the central part of a chondrosarcoma (dog 3), 72 h after intravenous CA4P administration. The vessel has been obstructed by a thrombus (asterisk) and necrosis of the endothelial cell lining (arrowheads) is visible (Haemotoxylin and Eosin. Scale bar = 50 µm).

Figure 6

Microvessel density counting in a mammary gland adenocarcinoma (dog 7), (A) Two weeks before CA4P treatment, (B) 72 h after CA4P treatment. A decrease in microvessel density is clearly noticeable (Anti-von Willebrand Factor. Scale bar = 100 µm).

Figure 7

Scatter plot revealing the correlation between the power Doppler ultrasound (PDUS) and the microvessel density (R² = 0.957; p < 0.001). The X- and Y-axes are expressed as the ratios between pre- and post CA4P measurements.

Figure 8

A representative slide of microvessel density counting in a mammary gland adenocarcinoma (dog 7) before treatment with CA4P, before (A) and after (B) application of an H-DAB filter (Anti-von Willebrand Factor. Scale bar = 100 µm).