Casey N. Ta

Hollow silica and silica-boron nano/microparticles for contrast-enhanced ultrasound to detect small tumors

Diagnosing tumors at an early stage when they are easily curable and may not require systemic chemotherapy remains a challenge to clinicians. In order to improve early cancer detection, gas filled hollow boron-doped silica particles have been developed, which can be used for ultrasound-guided breast conservation therapy. The particles are synthesized using a polystyrene template and subsequently calcinated to create hollow, rigid nanoporous microspheres. The microshells are filled with perfluoropentane vapor. Studies were performed in phantoms to optimize particle concentration, injection dose, and the ultrasound settings such as pulse frequency and mechanical index. In vitro studies have shown that these particles can be continuously imaged by US up to 48 min and their signal lifetime persisted for 5 days. These particles could potentially be given by intravenous injection and, in conjunction with contrast-enhanced ultrasound, be utilized as a screening tool to detect smaller breast cancers before they are detectible by traditional mammography.

Automating tumor classification with pixel-by-pixel contrast-enhanced ultrasound perfusion kinetics

Contrast-enhanced ultrasound (CEUS) enables highly specific time-resolved imaging of vasculature by intravenous injection of ∼2 μm gas filled microbubbles. To develop a quantitative automated diagnosis of breast tumors with CEUS, breast tumors were induced in rats by administration of N-ethyl-N-nitrosourea. A bolus injection of microbubbles was administered and CEUS videos of each tumor were acquired for at least 3 min. The time-intensity curve of each pixel within a region of interest (ROI) was analyzed to measure kinetic parameters associated with the wash-in, peak enhancement, and wash-out phases of microbubble bolus injections since it was expected that the aberrant vascularity of malignant tumors will result in faster and more diverse perfusion kinetics versus those of benign lesions. Parameters were classified using linear discriminant analysis to differentiate between benign and malignant tumors and improve diagnostic accuracy. Preliminary results with a small dataset (10 tumors, 19 videos) show 100% accuracy with fivefold cross-validation testing using as few as two choice variables for training and validation. Several of the parameters which provided the best differentiation between malignant and benign tumors employed comparative analysis of all the pixels in the ROI including enhancement coverage, fractional enhancement coverage times, and the standard deviation of the envelope curve difference normalized to the mean of the peak frame. Analysis of combinations of five variables demonstrated that pixel-by-pixel analysis produced the most robust information for tumor diagnostics and achieved 5 times greater separation of benign and malignant cases than ROI-based analysis.

Integrated processing of contrast pulse sequencing ultrasound imaging for enhanced active contrast of hollow gas filled silica nanoshells and microshells

In recent years, there have been increasing developments in the field of contrast-enhanced ultrasound both in the creation of new contrast agents and in imaging modalities. These contrast agents have been employed to study tumor vasculature in order to improve cancer detection and diagnosis. An in vivo study is presented of ultrasound imaging of gas filled hollow silica microshells and nanoshells which have been delivered intraperitoneally to an IGROV-1 tumor bearing mouse. In contrast to microbubbles, this formulation of microshells provided strong ultrasound imaging signals by shell disruption and release of gas. Imaging of the microshells in an animal model was facilitated by novel image processing. Although the particle signal could be identified by eye under live imaging, high background obfuscated the particle signal in still images and near the borders of the tumor with live images. Image processing techniques were developed that employed the transient nature of the particle signal to selectively filter out the background signal. By applying image registration, high-pass, median, threshold, and motion filtering, a short video clip of the particle signal was compressed into a single image, thereby resolving the silica shells within the tumor.

Quantitative automated image analysis system with automated debris filtering for the detection of breast carcinoma cells.

Objective: To develop an intraoperative method for margin status evaluation during breast conservation therapy (BCT) using an automated analysis of imprint cytology specimens. Study Design: Imprint cytology samples were prospectively taken from 47 patients undergoing either BCT or breast reduction surgery. Touch preparations from BCT patients were taken on cut sections through the tumor to generate positive margin controls. For breast reduction patients, slide imprints were taken at cuts through the center of excised tissue. Analysis results from the presented technique were compared against standard pathologic diagnosis. Slides were stained with cytokeratin and Hoechst, imaged with an automated fluorescent microscope, and analyzed with a fast algorithm to automate discrimination between epithelial cells and noncellular debris. Results: The accuracy of the automated analysis was 95% for identifying invasive cancers compared against final pathologic diagnosis. The overall sensitivity was 87% while specificity was 100% (no false positives). This is comparable to the best reported results from manual examination of intraoperative imprint cytology slides while reducing the need for direct input from a cytopathologist. Conclusion: This work demonstrates a proof of concept for developing a highly accurate and automated system for the intraoperative evaluation of margin status to guide surgical decisions and lower positive margin rates.

Extended Lifetime In Vivo Pulse Stimulated Ultrasound Imaging

An on-demand long-lived ultrasound contrast agent that can be activated with single pulse stimulated imaging (SPSI) has been developed using hard shell liquid perfluoropentane filled silica 500-nm nanoparticles for tumor ultrasound imaging. SPSI was tested on LnCAP prostate tumor models in mice; tumor localization was observed after intravenous (IV) injection of the contrast agent. Consistent with enhanced permeability and retention, the silica nanoparticles displayed an extended imaging lifetime of 3.3±1 days (mean±standard deviation). With added tumor specific folate functionalization, the useful lifetime was extended to 12 ± 2 days; in contrast to ligand-based tumor targeting, the effect of the ligands in this application is enhanced nanoparticle retention by the tumor. This paper demonstrates for the first time that IV injected functionalized silica contrast agents can be imaged with an in vivo lifetime ~500 times longer than current microbubble-based contrast agents. Such functionalized long-lived contrast agents may lead to new applications in tumor monitoring and therapy.

Ultrasound Responsive Macrophase-Segregated Microcomposite Films for in Vivo Biosensing

Ultrasound imaging is a safe, low-cost, and in situ method for detecting in vivo medical devices. A poly(methyl-2-cyanoacrylate) film containing 2 μm boron-doped, calcined, porous silica microshells was developed as an ultrasound imaging marker for multiple medical devices. A macrophase separation drove the gas-filled porous silica microshells to the top surface of the polymer film by controlled curing of the cyanoacrylate glue and the amount of microshell loading. A thin film of polymer blocked the wall pores of the microshells to seal air in their hollow core, which served as an ultrasound contrast agent. The ultrasound activity disappeared when curing conditions were modified to prevent the macrophase segregation. Phase segregated films were attached to multiple surgical tools and needles and gave strong color Doppler signals in vitro and in vivo with the use of a clinical ultrasound imaging instrument. Postprocessing of the simultaneous color Doppler and B-mode images can be used for autonomous identification of implanted surgical items by correlating the two images. The thin films were also hydrophobic, thereby extending the lifetime of ultrasound signals to hours of imaging in tissues by preventing liquid penetration. This technology can be used as a coating to guide the placement of implantable medical devices or used to image and help remove retained surgical items.

2-Tier In-Plane Motion Correction and Out-of-Plane Motion Filtering for Contrast-Enhanced Ultrasound

ObjectivesContrast-enhanced ultrasound (CEUS) cines of focal liver lesions (FLLs) can be quantitatively analyzed to measure tumor perfusion on a pixel-by-pixel basis for diagnostic indication. However, CEUS cines acquired freehand and during free breathing cause nonuniform in-plane and out-of-plane motion from frame to frame. These motions create fluctuations in the time-intensity curves (TICs), reducing the accuracy of quantitative measurements. Out-of-plane motion cannot be corrected by image registration in 2-dimensional CEUS and degrades the quality of in-plane motion correction (IPMC). A 2-tier IPMC strategy and adaptive out-of-plane motion filter (OPMF) are proposed to provide a stable correction of nonuniform motion to reduce the impact of motion on quantitative analyses. Materials and MethodsA total of 22 cines of FLLs were imaged with dual B-mode and contrast specific imaging to acquire a 3-minute TIC. B-mode images were analyzed for motion, and the motion correction was applied to both B-mode and contrast images. For IPMC, the main reference frame was automatically selected for each cine, and subreference frames were selected in each respiratory cycle and sequentially registered toward the main reference frame. All other frames were sequentially registered toward the local subreference frame. Four OPMFs were developed and tested: subsample normalized correlation (NC), subsample sum of absolute differences, mean frame NC, and histogram. The frames that were most dissimilar to the OPMF reference frame using 1 of the 4 above criteria in each respiratory cycle were adaptively removed by thresholding against the low-pass filter of the similarity curve. Out-of-plane motion filter was quantitatively evaluated by an out-of-plane motion metric (OPMM) that measured normalized variance in the high-pass filtered TIC within the tumor region-of-interest with low OPMM being the goal. Results for IPMC and OPMF were qualitatively evaluated by 2 blinded observers who ranked the motion in the cines before and after various combinations of motion correction steps. ResultsQuantitative measurements showed that 2-tier IPMC and OPMF improved imaging stability. With IPMC, the NC B-mode metric increased from 0.504 ± 0.149 to 0.585 ± 0.145 over all cines (P < 0.001). Two-tier IPMC also produced better fits on the contrast-specific TIC than industry standard IPMC techniques did (P < 0.02). In-plane motion correction and OPMF were shown to improve goodness of fit for pixel-by-pixel analysis (P < 0.001). Out-of-plane motion filter reduced variance in the contrast-specific signal as shown by a median decrease of 49.8% in the OPMM. Two-tier IPMC and OPMF were also shown to qualitatively reduce motion. Observers consistently ranked cines with IPMC higher than the same cine before IPMC (P < 0.001) as well as ranked cines with OPMF higher than when they were uncorrected. ConclusionThe 2-tier sequential IPMC and adaptive OPMF significantly reduced motion in 3-minute CEUS cines of FLLs, thereby overcoming the challenges of drift and irregular breathing motion in long cines. The 2-tier IPMC strategy provided stable motion correction tolerant of out-of-plane motion throughout the cine by sequentially registering subreference frames that bypassed the motion cycles, thereby overcoming the lack of a nearly stationary reference point in long cines. Out-of-plane motion filter reduced apparent motion by adaptively removing frames imaged off-plane from the automatically selected OPMF reference frame, thereby tolerating nonuniform breathing motion. Selection of the best OPMF by minimizing OPMM effectively reduced motion under a wide variety of motion patterns applicable to clinical CEUS. These semiautomated processes only required user input for region-of-interest selection and can improve the accuracy of quantitative perfusion measurements.

Focal Liver Lesions: Computer-aided Diagnosis by Using Contrast-enhanced US Cine Recordings

Purpose To assess the performance of computer-aided diagnosis (CAD) systems and to determine the dominant ultrasonographic (US) features when classifying benign versus malignant focal liver lesions (FLLs) by using contrast material-enhanced US cine clips. Materials and Methods One hundred six US data sets in all subjects enrolled by three centers from a multicenter trial that included 54 malignant, 51 benign, and one indeterminate FLL were retrospectively analyzed. The 105 benign or malignant lesions were confirmed at histologic examination, contrast-enhanced computed tomography (CT), dynamic contrast-enhanced magnetic resonance (MR) imaging, and/or 6 or more months of clinical follow-up. Data sets included 3-minute cine clips that were automatically corrected for in-plane motion and automatically filtered out frames acquired off plane. B-mode and contrast-specific features were automatically extracted on a pixel-by-pixel basis and analyzed by using an artificial neural network (ANN) and a support vector machine (SVM). Areas under the receiver operating characteristic curve (AUCs) for CAD were compared with those for one experienced and one inexperienced blinded reader. A third observer graded cine quality to assess its effects on CAD performance. Results CAD, the inexperienced observer, and the experienced observer were able to analyze 95, 100, and 102 cine clips, respectively. The AUCs for the SVM, ANN, and experienced and inexperienced observers were 0.883 (95% confidence interval [CI]: 0.793, 0.940), 0.829 (95% CI: 0.724, 0.901), 0.843 (95% CI: 0.756, 0.903), and 0.702 (95% CI: 0.586, 0.782), respectively; only the difference between SVM and the inexperienced observer was statistically significant. Accuracy improved from 71.3% (67 of 94; 95% CI: 60.6%, 79.8%) to 87.7% (57 of 65; 95% CI: 78.5%, 93.8%) and from 80.9% (76 of 94; 95% CI: 72.3%, 88.3%) to 90.3% (65 of 72; 95% CI: 80.6%, 95.8%) when CAD was in agreement with the inexperienced reader and when it was in agreement with the experienced reader, respectively. B-mode heterogeneity and contrast material washout were the most discriminating features selected by CAD for all iterations. CAD selected time-based time-intensity curve (TIC) features 99.0% (207 of 209) of the time to classify FLLs, versus 1.0% (two of 209) of the time for intensity-based features. None of the 15 video-quality criteria had a statistically significant effect on CAD accuracy-all P values were greater than the Holm-Sidak α-level correction for multiple comparisons. Conclusion CAD systems classified benign and malignant FLLs with an accuracy similar to that of an expert reader. CAD improved the accuracy of both readers. Time-based features of TIC were more discriminating than intensity-based features.

Automated Intra-Operative Analysis of Breast Cancer.

Background: Multiple operations are a major problem for breast conservation therapy. Accurate interpretation of intra-operative tumor margins can limit multiple re-excision procedures in order to obtain negative margins while maintaining a good cosmetic result from BCT. Intra-operative touch preparations have been used in the past but can be difficult to interpret without an experienced cytopathologist.Objective: To examine the reliability of fluorescently labeled intra-operative touch preps in conjunction with automated analysis in correctly identifying cancer cells compared to final pathologic diagnosis.Methods: Touch preps were propctively performed on 38 consecutive cases of patients undergoing breast surgery. The surgical cases included 15 invasive tumors, 9 ductal carcinoma in situ, 5 prophylactic mastectomies, and 9 normal tissues from breast reduction operations. Tumors were grossly located by a surgical pathologist and a cross sectional cut was made at the suspected tumor center. Touch preps with poly-l-lysine coated slides were then taken on the tissue surfaces exposed from the cross sectional cut. Additionally, the location where the touch preps were performed was isolated in a cassette and separated for later comparison. In cases of benign disease, touch preps were performed at the center of the excised tissue. The slides were fixed in 95% ethanol and stained with a Cytokeratin, an epithelial cell marker, and Hoechst, a nuclear stain. The prepared slides were then imaged with an automated fluorescent microscope, and suspected cancer cells were detected through standard image processing and statistical techniques.Results: The overall accuracy of our automated fluorescence analysis was 95% for identifying invasive or pre-invasive cancer compared final pathologic diagnosis. The overall specificity was 100% (there were no false positives). The sensitivity for ductal carcinoma in situ was 75% and invasive cancer was 90%. Overall sensitivity was 87% which is comparable to the best reported results from manual examination of touch preparations.Conclusion: Automated analysis of fluorescently stained touch prep slides is highly accurate in identifying cancer with no false positives. The immunofluorescence stains and automated microscopy may help the pathologist to quickly identify cancer cells in fresh breast tissue during BCT operations, and, thus, limit breast re-excisions in the future. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6016.