Comparing Quantitative Immunohistochemical Markers of Angiogenesis to Contrast-Enhanced Subharmonic Imaging.

OBJECTIVES: 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.

Comparison of photoacoustically derived hemoglobin and oxygenation measurements with contrast-enhanced ultrasound estimated vascularity and immunohistochemical staining in a breast cancer model.

In this preliminary study, we compared two noninvasive techniques for imaging intratumoral physiological conditions to immunohistochemical staining in a murine breast cancer model. MDA-MB-231 tumors were implanted in the mammary pad of 11 nude rats. Ultrasound and photoacoustic (PA) scanning were performed using a Vevo 2100 scanner (Visualsonics, Toronto, Canada). Contrast-enhanced ultrasound (CEUS) was used to create maximum intensity projections as a measure of tumor vascularity. PAs were used to determine total hemoglobin signal (HbT), oxygenation levels in detected blood (SO2 Avg), and oxygenation levels over the entire tumor area (SO2 Tot). Tumors were then stained for vascular endothelial growth factor (VEGF), cyclooxygenase-2 (Cox-2), and the platelet endothelial cell adhesion molecule CD31. Correlations between findings were analyzed using Pearson's coefficient. Significant correlation was observed between CEUS-derived vascularity measurements and both PA indicators of blood volume (r = 0.49 for HbT, r = 0.50 for SO2 Tot). Cox-2 showed significant negative correlation with SO2 Avg (r = -0.49, p = 0.020) and SO2 Tot (r = -0.43, p = 0.047), while CD31 showed significant negative correlation with CEUS-derived vascularity (r = -0.47, p = 0.036). However, no significant correlation was observed between VEGF expression and any imaging modality (p > 0.08). Photoacoustically derived HbT and SO2 Tot may be a good indicator of tumor fractional vascularity. While CEUS correlates with CD31 expression, photoacoustically derived SO2 Avg appears to be a better predictor of Cox-2 expression.

Contrast-enhanced sonography for detection of secondary lymph nodes in a melanoma tumor animal model.

OBJECTIVES: To investigate the use of contrast-enhanced ultrasound imaging (US) for detection of secondary lymph nodes (LNs) in a naturally occurring melanoma swine model compared to surgery and pathologic assessment. METHODS: Twenty-seven Sinclair swine were studied. The perfluorobutane microbubble contrast agent Sonazoid (GE Healthcare, Oslo, Norway) was administered (1.0 mL total dose) around the melanoma, and contrast-enhanced US was used to localize contrast-enhanced sentinel lymph nodes (SLNs). Then Sonazoid (dose, 0.25-1.0 mL) was injected into the SLNs to detect contrast-enhanced efferent lymphatic channels and secondary LNs. After peritumoral injection of blue dye, a surgeon (blinded to the contrast-enhanced US results) performed a radical LN dissection. Contrast-enhanced US was used to guide removal of any enhanced secondary LNs left after radical LN dissection. Clustered conditional logistic regression analyzed the benefit of contrast-enhanced US-directed secondary LN dissection over radical LN dissection using pathologic findings as the reference standard. RESULTS: A total of 268 secondary LNs were resected, with 59 (22%) containing metastases. Contrast-enhanced US detected 92 secondary LNs; 248 were identified by radical LN dissection; and 68 were identified by both methods. Metastases were detected in 20% (51 of 248) and 40% (37 of 92) of the secondary LNs identified by radical LN dissection and contrast-enhanced US, respectively. Thus, secondary LNs detected by contrast-enhanced US were nearly 5 times more likely to contain metastases than secondary LNs removed by radical LN dissection (odds ratio, 4.8; P < .0001). Twenty-two of the 180 secondary LNs (12%) identified only by radical LN dissection contained metastases, whereas contrast-enhanced US identified 20 secondary LNs after the surgeon completed the radical LN dissection, of which 8 (40%) contained metastases. CONCLUSIONS: Secondary LNs can be detected by using contrast-enhanced US after injection of Sonazoid into SLNs. Secondary LNs detected with contrast-enhanced US are significantly more likely to contain metastases than those removed by radical LN dissection.

Static and dynamic cumulative maximum intensity display mode for subharmonic breast imaging: a comparative study with mammographic and conventional ultrasound techniques.

OBJECTIVE: The purpose of this study was to test the efficacy of static and dynamic cumulative maximum intensity (CMI) subharmonic imaging (SHI) in breast ultrasound studies. METHODS: Contrast-enhanced SHI was performed in 14 women using a modified LOGIQ 9 scanner (GE Healthcare, Milwaukee, WI) transmitting/receiving at 4.4/2.2 MHz. Following mammography, baseline scans of gray scale ultrasound and power Doppler imaging (PDI) were performed. Contrast-enhanced PDI and gray scale SHI were performed after contrast agent administration. Static CMI-SHI is a composite image summarizing blood flow over multiple frames using the maximum intensity projection technique. The dynamic CMI-SHI mode depicts the gradual inflow pattern of the contrast agent in blood vessels. Both CMI-SHI modes were set up using a new automated sum-absolute-difference-based block-matching algorithm to reduce noise and blurring and compensate for motion artifacts. Evaluation of the imaging modes for detecting breast cancer was done by an experienced radiologist, blinded to histopathologic findings. Sensitivity, specificity, and receiver operating characteristic (ROC) analyses were computed and compared for all ultrasound imaging modes and mammography. Results Of the 16 lesions, 4 were malignant. The area under the ROC curve (A(z)) for the diagnosis of breast cancer was 0.64 for gray scale and PDI, 0.67 for contrast-enhanced PDI, 0.76 for mammography, 0.78 for SHI, and 0.75 for static CMI-SHI. For the dynamic CMI-SHI mode, the A(z) increased to 0.90, and this was significantly better than mammography (P = .03). CONCLUSIONS: The new dynamic CMI-SHI mode produced the highest A(z) for the diagnosis of breast cancer compared to conventional techniques and thus appears to improve diagnosis of breast cancer relative to conventional techniques, albeit based on a limited patient population.

Preclinical acute toxicology study of surfactant-stabilized ultrasound contrast agents in adult rats.

Gas-filled microbubbles are used as contrast agents in diagnostic ultrasound imaging. A preclinical, acute toxicity study of 2 surfactant-stabilized ultrasound contrast agents (ST68 and ST44) was conducted. Subjects were 104 Sprague-Dawley rats (experimental doses, 0.1, 0.2, 0.8, and 1.0 mL/kg; control, 1.0 mL/kg saline) that were studied for 14 days after contrast; clinical signs, weight, blood, and urine were evaluated. Histopathology was performed following euthanasia. Of the 40 animals receiving ST44, 4 died prematurely and a dose dependency was demonstrated (P = .011), whereas in the ST68 groups only 1 death occurred (no dose dependency; P = .48). Only the weight of rats injected with ST44 varied significantly (P = .0003). This dependency was also found for 3 of 5 urine parameters and 4 of 36 blood parameters (P < .05). For ST68, only 1 urine parameter showed significance (P < .0001). Giant cell infiltration in the lungs was significantly higher than controls in the ST44 0.1 mL/kg and the ST68 0.8-1.0 mL/kg groups (P < .01). It is concluded that the prudent choice for future nonrodent, toxicology studies and potentially for human clinical trials is ST68 (given the deaths in the ST44 groups).

Subharmonic-Aided Pressure Estimation for Monitoring Interstitial Fluid Pressure in Tumors: Calibration and Treatment with Paclitaxel in Breast Cancer Xenografts.

Interstitial fluid pressure (IFP) in rats with breast cancer xenografts was non-invasively estimated using subharmonic-aided pressure estimation (SHAPE) versus an invasive pressure monitor. Moreover, monitoring of IFP changes after chemotherapy was assessed. Eighty-nine rats (calibration n = 25, treatment n = 64) were injected with 5 × 106 breast cancer cells (MDA-MB-231). Radiofrequency signals were acquired (39 rats successfully imaged) with a Sonix RP scanner (BK Ultrasound, Richmond, BC, Canada) using a linear array (L9-4, transmit/receive: 8/4 MHz) after administration of Definity (Lantheus Medical Imaging, North Billerica, MA, USA; 180 µL/kg) and compared with readings from an invasive pressure monitor (Stryker, Berkshire, UK). An inverse linear relationship was established between tumor IFP and SHAPE (y = -1.06x + 28.27, r = -0.69, p = 0.01) in the calibration group. Use of this relationship in the treatment group resulted in r = 0.74 (p < 0.05) between measured (pressure monitor) and SHAPE-estimated IFP (average error: 6.24 mmHg). No significant before/after differences were observed with respect to paclitaxel treatment (5 mg/kg, Mayne Pharma, Paramus, NJ, USA) with either method (p ≥ 0.15).

Contrast-Enhanced Subharmonic and Harmonic Ultrasound of Renal Masses Undergoing Percutaneous Cryoablation.

RATIONALE AND OBJECTIVES: The objective of this study was to evaluate and compare contrast-enhanced subharmonic and harmonic ultrasound as tools for characterizing solid renal masses and monitoring their response to cryoablation therapy. MATERIALS AND METHODS: Sixteen patients undergoing percutaneous ablation of a renal mass provided informed consent to undergo ultrasound examinations the morning before and approximately 4 months after cryoablation. Ultrasound contrast parameters during pretreatment imaging were compared to biopsy results obtained during ablation (n = 13). Posttreatment changes were evaluated by a radiologist and compared to contrast-enhanced magnetic resonance imaging (MRI)/computed tomography (CT) follow-up. RESULTS: All masses initially showed heterogeneous enhancement with both subharmonic and harmonic ultrasound. Early contrast washout in the mass relative to the cortex was observed in 6 of 9 malignant and 0 of 4 benign lesions in subharmonic mode and 8 of 9 malignant and 1 of 4 benign lesions in harmonic imaging. In cases where the lesion was adequately visualized at follow-up (n = 12), subharmonic and harmonic ultrasound showed accuracies of 83% and 75%, respectively, in predicting treatment outcome. Although harmonic imaging showed less overall error, no significant differences (P > .29) in ablation cavity volumes were observed between MRI/CT and either contrast-imaging mode. CONCLUSIONS: Subharmonic and harmonic contrast-enhanced ultrasound may be a safe and accurate imaging alternative for characterizing renal masses and evaluating their response to cryoablation therapy. Although subharmonic imaging was more accurate in detecting effective cryoablation, harmonic imaging was superior in quantifying ablation cavity volumes.

High and low frequency subharmonic imaging of angiogenesis in a murine breast cancer model.

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 5×10(6) 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 LF 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 HF 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.

Correlation of ultrasound contrast agent derived blood flow parameters with immunohistochemical angiogenesis markers in murine xenograft tumor models.

PURPOSE: In this study we used temporal analysis of ultrasound contrast agent (UCA) estimate blood flow dynamics and demonstrate their improved correlation to angiogenesis markers relative to previously reported, non-temporal fractional vascularity estimates. MATERIALS AND METHODS: Breast tumor (NMU) or glioma (C6) cells were implanted in either the abdomen or thigh of 144 rats. After 6, 8 or 10 days, rats received a bolus UCA injection of Optison (GE Healthcare, Princeton, NJ; 0.4 ml/kg) during power Doppler imaging (PDI), harmonic imaging (HI), and microflow imaging (MFI) using an Aplio ultrasound scanner with 7.5 MHz linear array (Toshiba America Medical Systems, Tustin, CA). Time-intensity curves of contrast wash-in were constructed on a pixel-by-pixel basis and averaged to calculate maximum intensity, time to peak, perfusion, and time integrated intensity (TII). Tumors were then stained for four immunohistochemical markers (bFGF, CD31, COX-2, and VEGF). Correlations between temporal parameters and the angiogenesis markers were investigated for each imaging mode. Effects of tumor model and implant location on these correlations were also investigated. RESULTS: Significant correlation over the entire dataset was only observed between TII and VEGF for all three imaging modes (R=-0.35, -0.54, -0.32 for PDI, HI and MFI, respectively; p<0.0001). Tumor type and location affected these correlations, with the strongest correlation of TII to VEGF found to be with implanted C6 cells (R=-0.43, -0.54, -0.52 for PDI, HI and MFI, respectively; p<0.0002). CONCLUSIONS: While UCA-derived temporal blood flow parameters were found to correlate strongly with VEGF expression, these correlations were also found to be influenced by both tumor type and implant location.

Contrast enhanced maximum intensity projection ultrasound imaging for assessing angiogenesis in murine glioma and breast tumor models: A comparative study.

The purpose of this study was to prospectively compare noninvasive, quantitative measures of vascularity obtained from four contrast enhanced ultrasound (US) techniques to four invasive immunohistochemical markers of tumor angiogenesis in a large group of murine xenografts. Glioma (C6) or breast cancer (NMU) cells were implanted in 144 rats. The contrast agent Optison (GE Healthcare, Princeton, NJ) was injected in a tail vein (dose: 0.4ml/kg). Power Doppler imaging (PDI), pulse-subtraction harmonic imaging (PSHI), flash-echo imaging (FEI), and Microflow imaging (MFI; a technique creating maximum intensity projection images over time) was performed with an Aplio scanner (Toshiba America Medical Systems, Tustin, CA) and a 7.5MHz linear array. Fractional tumor neovascularity was calculated from digital clips of contrast US, while the relative area stained was calculated from specimens. Results were compared using a factorial, repeated measures ANOVA, linear regression and z-tests. The tortuous morphology of tumor neovessels was visualized better with MFI than with the other US modes. Cell line, implantation method and contrast US imaging technique were significant parameters in the ANOVA model (p<0.05). The strongest correlation determined by linear regression in the C6 model was between PSHI and percent area stained with CD31 (r=0.37, p<0.0001). In the NMU model the strongest correlation was between FEI and COX-2 (r=0.46, p<0.0001). There were no statistically significant differences between correlations obtained with the various US methods (p>0.05). In conclusion, the largest study of contrast US of murine xenografts to date has been conducted and quantitative contrast enhanced US measures of tumor neovascularity in glioma and breast cancer xenograft models appear to provide a noninvasive marker for angiogenesis; although the best method for monitoring angiogenesis was not conclusively established.

Comparing differential tissue harmonic imaging with tissue harmonic and fundamental gray scale imaging of the liver.

OBJECTIVE: The purpose of this study was to compare fundamental gray scale sonography, tissue harmonic imaging (THI), and differential tissue harmonic imaging (DTHI) for depicting normal and abnormal livers. METHODS: The in vitro lateral resolution of DTHI, THI, and sonography was assessed in a phantom. Sagittal and transverse images of right and left hepatic lobes of 5 volunteers and 20 patients and images of 27 liver lesions were also acquired. Three independent blinded readers scored all randomized images for noise, detail resolution, image quality, and margin (for lesions) on a 7-point scale. Next, images from the same location obtained with all 3 modes were compared blindly side by side and rated by all readers. Contrast-to-noise ratios were calculated for the lesions, and the depth of penetration (centimeters) was determined for all images. RESULTS: In vitro, the lateral resolution of DTHI was significantly better than fundamental sonography (P = .009) and showed a trend toward significance versus THI (P = .06). In the far field, DTHI performed better than both modes (P < .04). In vivo, 450 images were scored, and for all parameters, DTHI and THI did better than sonography (P < .002). Differential tissue harmonic imaging scored significantly higher than THI with regard to detail resolution and image quality (P < .001). The average increase in penetration with THI and DTHI was around 0.6 cm relative to sonography (P < .0001). The contrast-to-noise ratio for DTHI showed a trend toward significance versus THI (P = .06). Side-by-side comparisons rated DTHI better than THI and sonography in 54% of the cases (P < .007). CONCLUSIONS: Tissue harmonic imaging and DTHI do better than fundamental sonography for hepatic imaging, with DTHI being rated the best of the 3 techniques.