Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model

Abstract

While there is an increasing role of ultrasound for breast cancer screening in patients with dense breast, conventional anatomical ultrasound lacks sensitivity and specificity for early breast cancer detection. In this study, we assessed the potential of ultrasound molecular imaging using clinically translatable vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubbles (MB(VEGFR2)) to improve the diagnostic accuracy of ultrasound in earlier detection of breast cancer and ductal carcinoma in situ (DCIS) in a transgenic mouse model [FVB/N-Tg(MMTV-PyMT)634Mul]. In vivo binding specificity studies (n = 26 tumors) showed that ultrasound imaging signal was significantly higher (P < 0.001) using MB(VEGFR2) than nontargeted microbubbles and imaging signal significantly decreased (P < 0.001) by blocking antibodies. Ultrasound molecular imaging signal significantly increased (P < 0.001) when breast tissue (n = 315 glands) progressed from normal [1.65 ± 0.17 arbitrary units (a.u.)] to hyperplasia (4.21 ± 1.16), DCIS (15.95 ± 1.31), and invasive cancer (78.1 ± 6.31) and highly correlated with ex vivo VEGFR2 expression [R(2) = 0.84; 95% confidence interval (CI), 0.72-0.91; P < 0.001]. At an imaging signal threshold of 4.6 a.u., ultrasound molecular imaging differentiated benign from malignant entities with a sensitivity of 84% (95% CI, 78-88) and specificity of 89% (95% CI, 81-94). In a prospective screening trail (n = 63 glands), diagnostic performance of detecting DCIS and breast cancer was assessed and two independent readers correctly diagnosed malignant disease in more than 95% of cases and highly agreed between each other [intraclass correlation coefficient (ICC) = 0.98; 95% CI, 97-99]. These results suggest that VEGFR2-targeted ultrasound molecular imaging allows highly accurate detection of DCIS and breast cancer in transgenic mice and may be a promising approach for early breast cancer detection in women.

Figure 1

Summary of overall study design which included three parts. MBD = microbubble destruction.

Figure 2

Summary of ultrasound molecular imaging signals obtained from intra-animal comparison after intravenous administration of MB_VEGFR2, MB_Control and MB_VEGFR2 after blocking with anti-VEGFR2 antibodies (*P<0.001). Representative transverse B-mode images with overlaid VEGFR2-targeted ultrasound molecular imaging signals are shown from one mammary gland harboring invasive breast cancer. Green line represents region of interest (ROI). Scale bar = 1 mm.

Figure 3

Bar graph shows percentage distribution of four different histological findings (normal tissue, hyperplasia, DCIS, invasive breast cancer) in mammary glands of FVB/N-Tg(MMTV-PyMT)634Mul transgenic mouse model evaluated between age 4 and 10 weeks.

Figure 4

(A) Box plot summarizes VEGFR2-targeted ultrasound molecular imaging signals obtained in normal, hyperplasia, DCIS and invasive breast cancer of 315 mammary glands. Imaging signal increased with mammary gland tissue progressing through different histological stages and imaging signals significantly increased compared to preceding stage. *P = 0.021; **P < 0.001). Note, each box in the plot represent the 25^th and 75^th quartiles, the line inside each box identifies the median and the vertical lines outside indicate the largest and smallest values in the series of measurements excluding the outliers. The 5^th and 95^th percentile of the data are represented as outliers (dots). (B) Representative ultrasound and H&E images of mammary glands with four different histological stages. Transverse B-mode with overlaid VEGFR2-targeted ultrasound molecular imaging signal (second row) shows substantial increase of ultrasound molecular imaging signal in DCIS and invasive breast cancer compared to hyperplasia and normal mammary gland tissue (scale bar = 1 mm). Note that B-mode images alone (first row) do not allow characterization of four different histological types. H&E staining of the corresponding mammary glands is shown in third row (scale bar = 1 mm). Ten-fold magnified images from insets are shown in fourth row (scale bar = 0.1 mm).

Figure 5

Representative photomicrographs of mouse mammary glands with four different histological stages of breast cancer development. Mammary gland sections were stained for vascular endothelial cell marker CD31 (green, first column) and for VEGFR2 (red, second column). Merged images (yellow, third column) show expression of VEGFR2 on vascular endothelial cells. Note that number of microvessels and the VEGFR2 staining increased with breast cancer development (scale bar = 0.1 mm).

Figure 6

ROC curve for differentiating benign histological entities (normal and hyperplasia) from malignant entities (DCIS and invasive cancer) at molecular ultrasound imaging threshold level of 4.6 a.u. Colored scale bar on right represents VEGFR2-targeted ultrasound molecular imaging signal in arbitrary units (a.u).