High-Resolution Ultrasound Characterization of Local Scattering in Cancer Tissue.

Ultrasound (US) has afforded an approach to tissue characterization for more than a decade. The challenge is to reveal hidden patterns in the US data that describe tissue function and pathology that cannot be seen in conventional US images. Our group has developed a high-resolution analysis technique for tissue characterization termed H-scan US, an imaging method used to interpret the relative size of acoustic scatterers. In the present study, the objective was to compare local H-scan US image intensity with registered histologic measurements made directly at the cellular level. Human breast cancer cells (MDA-MB 231, American Type Culture Collection, Manassas, VA, USA) were orthotopically implanted into female mice (N = 5). Tumors were allowed to grow for approximately 4 wk before the study started. In vivo imaging of tumor tissue was performed using a US system (Vantage 256, Verasonics Inc., Kirkland, WA, USA) equipped with a broadband capacitive micromachined ultrasonic linear array transducer (Kolo Medical, San Jose, CA, USA). A 15-MHz center frequency was used for plane wave imaging with five angles for spatial compounding. H-scan US image reconstruction involved use of parallel convolution filters to measure the relative strength of backscattered US signals. Color codes were applied to filter outputs to form the final H-scan US image display. For histologic processing, US imaging cross-sections were carefully marked on the tumor surface, and tumors were excised and sliced along the same plane. By use of optical microscopy, whole tumor tissue sections were scanned and digitized after nuclear staining. US images were interpolated to have the same number of pixels as the histology images and then spatially aligned. Each nucleus from the histologic sections was automatically segmented using custom MATLAB software (The MathWorks Inc., Natick, MA, USA). Nuclear size and spacing from the histology images were then compared with local H-scan US image features. Overall, local H-scan US image intensity exhibited a significant correlation with both cancer cell nuclear size (R2 > 0.27, p < 0.001) and the inverse relationship with nuclear spacing (R2 > 0.17, p < 0.001).

Generalized mathematical framework for contrast-enhanced ultrasound imaging with pulse inversion spectral deconvolution.

A generalized mathematical framework for performing contrast-enhanced ultrasound (CEUS) imaging is introduced. Termed pulse inversion spectral deconvolution (PISD), this CEUS approach is founded on Gaussian derivative functions (GDFs). PISD pulses are used to form two inverted pulse sequences, which are then used to filter backscattered ultrasound (US) data for isolation of the nonlinear (NL) microbubble (MB) signal component. An US scanner equipped with a linear array transducer was used for data acquisition. With a vascular flow phantom perfused with MBs, data was collected using PISD and NL-based CEUS imaging. The role of wide-beam transmit aperture size (32 or 64 elements) was also evaluated using an US pulse frequency of 6.25 MHz. Image enhancement was quantified by a contrast-to-noise ratio (CNR). Preliminary in vivo data was collected in the hindlimb and kidney of healthy rats. Overall, in vitro wide-beam CEUS imaging using an aperture size of 64 elements yielded improved CNR values. Specifically, PISD-based CEUS imaging produced CNR values of 37.3 dB. For comparison, CNR values obtained using B-mode US or NL approaches were 2.1 and 12.1 dB, respectively. In vivo results demonstrated that PISD-based CEUS imaging improved vascular visualization compared to the NL imaging strategy.

Three-Dimensional H-Scan Ultrasound Imaging of Early Breast Cancer Response to Neoadjuvant Therapy in a Murine Model.

OBJECTIVES: Three-dimensional (3D) H-scan is a new ultrasound (US) technique that images the relative size of acoustic scatterers. The goal of this research was to evaluate use of 3D H-scan US imaging for monitoring early breast cancer response to neoadjuvant therapy using a preclinical murine model of breast cancer. MATERIALS AND METHODS: Preclinical studies were conducted using luciferase-positive breast cancer-bearing mice (n = 40). Anesthetized animals underwent US imaging at baseline before administration with an apoptosis-inducing drug or a saline control. Image data were acquired using a US scanner equipped with a volumetric transducer following either a shorter- or longer-term protocol. The later included bioluminescent imaging to quantify tumor cell viability. At termination, tumors were excised for ex vivo analysis. RESULTS: In vivo results showed that 3D H-scan US imaging is considerably more sensitive to tumor changes after apoptosis-inducing drug therapy as compared with traditional B-scan US. Although there was no difference at baseline (P > 0.99), H-scan US results from treated tumors exhibited progressive decreases in image intensity (up to 62.2% by day 3) that had a significant linear correlation with cancer cell nuclear size (R2 > 0.51, P < 0.001). Results were validated by histological data and a secondary longitudinal study with survival as the primary end point. DISCUSSION: Experimental results demonstrate that noninvasive 3D H-scan US imaging can detect an early breast tumor response to apoptosis-inducing drug therapy. Local in vivo H-scan US image intensity correlated with cancer cell nuclear size, which is one of the first observable changes of a cancer cell undergoing apoptosis and confirmed using histological techniques. Early imaging results seem to provide prognostic insight on longer-term tumor response. Overall, 3D H-scan US imaging is a promising technique that visualizes the entire tumor and detects breast cancer response at an early stage of therapy.

3-D H-Scan Ultrasound Imaging and Use of a Convolutional Neural Network for Scatterer Size Estimation.

H-Scan ultrasound (US) is a new imaging technology that estimates the relative size of acoustic scattering objects and structures. The purpose of this study was to introduce a three-dimensional (3-D) H-scan US imaging approach for scatterer size estimation in volume space. Using a programmable research scanner (Vantage 256, Verasonics Inc, Kirkland, WA, USA) equipped with a custom volumetric imaging transducer (4 DL7, Vermon, Tours, France), raw radiofrequency (RF) data was collected for offline processing to generate H-scan US volumes. A deep convolutional neural network (CNN) was modified and used to achieve voxel mapping from the input H-scan US image to underlying scatterer size. Preliminary studies were conducted using homogeneous gelatin-based tissue-mimicking phantom materials embedded with acoustic scatterers of varying size (15 to 250 µm) and concentrations (0.1 to 1%). Two additional phantoms were embedded with 63 or 125 µm-sized microspheres and used to test CNN estimation accuracy. In vitro results indicate that 3-D H-scan US imaging can visualize the spatial distribution of acoustic scatterers of varying size at different concentrations (R2 > 0.85, p < 0.03). The result of scatterer size estimation reveals that a CNN can achieve an average mapping accuracy of 93.3%. Overall, our preliminary in vitro findings reveal that 3-D H-scan US imaging allows the visualization of tissue scatterer patterns and incorporation of a CNN can be used to help estimate size of the acoustic scattering objects.

Adaptive attenuation correction during H-scan ultrasound imaging using K-means clustering.

H-scan ultrasound (US) imaging (where the 'H' stands for Hermite) is a novel non-invasive, low cost and real-time technology. Like traditional US, H-scan US suffers from frequency-dependent attenuation that must be corrected to have acceptable image quality for tissue characterization. The goal of this research was to develop a novel attenuation correction method based on adaptive K-means clustering. To properly isolate these signals, a lateral moving window approach applied to adaptively adjust GH filters based on the changing of RF vector spectrums. Then the signal isolated via the same filter will be combined together via overlap-add technology to keep the information loss minimum. Experimental data was collected using a Verasonics 256 US scanner equipped with a L11-4v linear array transducer. In vivo data indicates that H-scan US imaging after adaptive attenuation correction can optimally re-scale the GH kernels and match to the changing spectrum undergoing attenuation (i.e. high frequency shift). This approach produces H-scan US images with more uniform spatial intensity and outperforms global attenuation correction strategies. Overall, this approach will improve the ability of H-scan US imaging to estimate acoustic scatterer size and will improve its clinical use for tissue characterization when imaging complex tissues.

Contrast-enhanced ultrasound imaging using pulse inversion spectral deconvolution.

A contrast-enhanced ultrasound (CEUS) imaging approach, termed pulse inversion spectral deconvolution (PISD), is introduced. The approach uses two Gaussian-weighted Hermite polynomials to form two inverted pulse sequences. The two inversed pulses are then used to filter ultrasound (US) backscattered data and discrimination of the linear and nonlinear signal components. A research US scanner equipped with a linear array transducer was used for data acquisition. The receive data from all channels are shaped using plane wave imaging beamforming with angular compounding (from one to nine angles). In vitro data was collected with a tissue mimicking flow phantom perfused with an US contrast agent using PISD and traditional nonlinear (NLI) US imaging as comparison. The role of imaging frequency (between 4.5 and 6.25 MHz) and mechanical index (from 0.1 to 0.3) were evaluated. Preliminary in vivo data was collected in the hindlimb of three healthy mice. Preliminary experimental findings indicate that the PISD contrast-to-tissue ratio was improved nearly ten times compared to the NLI US imaging approach. Also, the spatial resolution was improved due to the effect of deconvolution and spatial angular compounding. Overall, PISD is a promising postprocessing technique for real-time CEUS imaging.

Impact of hydrostatic pressure on phase-change contrast agent activation by pulsed ultrasound.

A phase-change contrast agent (PCCA) can be activated from a liquid (nanodroplet) state using pulsed ultrasound (US) energy to form a larger highly echogenic microbubble (MB). PCCA activation is dependent on the ambient pressure of the surrounding media, so any increase in hydrostatic pressure demands higher US energies to phase transition. In this paper, the authors explore this basic relationship as a potential direction for noninvasive pressure measurement and foundation of a unique technology the authors are developing termed tumor interstitial pressure estimation using ultrasound (TIPE-US). TIPE-US was developed using a programmable US research scanner. A custom scan sequence interleaved pulsed US transmissions for both PCCA activation and detection. An automated US pressure sweep was applied, and US images were acquired at each increment. Various hydrostatic pressures were applied to PCCA samples. Pressurized samples were imaged using the TIPE-US system. The activation threshold required to convert PCCA from the liquid to gaseous state was recorded for various US and PCCA conditions. Given the relationship between the hydrostatic pressure applied to the PCCA and US energy needed for activation, phase transition can be used as a surrogate of hydrostatic pressure. Consistent with theoretical predictions, the PCCA activation threshold was lowered with increasing sample temperature and by decreasing the frequency of US exposure, but it was not impacted by PCCA concentration.

Real-time H-scan ultrasound imaging using a Verasonics research scanner.

H-scan ultrasound (US) is a new imaging technique that relies on matching a model that describes US image formation to the mathematics of a class of Gaussian-weighted Hermite polynomials (GH). In short, H-scan US (where the 'H' denotes Hermite or hue) is a tissue classification technique that images the relative size of acoustic scatterers. Herein, we detail development of a real-time H-scan US imaging technology that was implemented on a programmable US research scanner (Vantage 256, Verasonics Inc, Kirkland, WA). This custom US imaging system has a dual display for real-time visualization of both the H-scan and B-scan US images. This MATLAB-based (Mathworks Inc, Natick, MA) system includes a graphical user interface (GUI) for controlling the entire US scan sequence including the raw radio frequency (RF) data acquisition parameters, image processing, variable control of a parallel set of convolution filters used to derive the H-scan US signal, and data (cine loop) save. The system-level structure used for software-based image reconstruction and display is detailed. Imaging studies were conducted using a series of homogeneous and heterogeneous tissue-mimicking phantom materials embedded with monodisperse spherical US scatterers of size 15-40 µm in diameter. Relative to H-scan US image measurements from a phantom with 15 µm-sized scatterers, data from phantoms with the 30 and 40 µm-sized scatterers exhibited mean intensity increases of 5.2% and 11.6%, respectively. Overall, real-time H-scan US imaging is a promising approach for visualizing the relative size and distribution of acoustic scattering objects.

Monitoring Early Breast Cancer Response to Neoadjuvant Therapy Using H-Scan Ultrasound Imaging: Preliminary Preclinical Results.

OBJECTIVE: H-scan imaging is a new ultrasound technique used to visualize the relative size of acoustic scatterers. The purpose of this study was to evaluate the use of H-scan ultrasound imaging for monitoring early tumor response to neoadjuvant treatment using a preclinical breast cancer animal model. METHODS: Real-time H-scan ultrasound imaging was implemented on a programmable ultrasound scanner (Vantage 256; Verasonics Inc., Kirkland, WA) equipped with an L11-4v transducer. Bioluminescence and H-scan ultrasound was used to image luciferase-positive breast cancer-bearing mice at baseline and at 24, 48, and 168 hours after administration of a single dose of neoadjuvant (paclitaxel) or sham treatment. Animals were euthanized at 48 or 168 hours, and tumors underwent histologic processing to identify cancer cell proliferation and apoptosis. RESULTS: Baseline H-scan ultrasound images of control and therapy group tumors were comparable, but the latter exhibited significant changes over the 7-day study (P < .05). At termination, there was a marked difference between the H-scan ultrasound images of control and treated tumors (P < .05). Specifically, H-scan ultrasound images of treated tumors were more blue in hue than images obtained from control tumors. There was a significant linear correlation between the predominance of the blue hue found in the H-scan ultrasound images and intratumoral apoptotic activity (R2 > 0.40, P < .04). CONCLUSION: Preliminary preclinical results suggest that H-scan ultrasound imaging is a new and promising tissue characterization modality. H-scan ultrasound imaging may provide prognostic value when monitoring early tumor response to neoadjuvant treatment.

Spatial Angular Compounding Technique for H-Scan Ultrasound Imaging.

H-Scan is a new ultrasound imaging technique that relies on matching a model of pulse-echo formation to the mathematics of a class of Gaussian-weighted Hermite polynomials. This technique may be beneficial in the measurement of relative scatterer sizes and in cancer therapy, particularly for early response to drug treatment. Because current H-scan techniques use focused ultrasound data acquisitions, spatial resolution degrades away from the focal region and inherently affects relative scatterer size estimation. Although the resolution of ultrasound plane wave imaging can be inferior to that of traditional focused ultrasound approaches, the former exhibits a homogeneous spatial resolution throughout the image plane. The purpose of this study was to implement H-scan using plane wave imaging and investigate the impact of spatial angular compounding on H-scan image quality. Parallel convolution filters using two different Gaussian-weighted Hermite polynomials that describe ultrasound scattering events are applied to the radiofrequency data. The H-scan processing is done on each radiofrequency image plane before averaging to get the angular compounded image. The relative strength from each convolution is color-coded to represent relative scatterer size. Given results from a series of phantom materials, H-scan imaging with spatial angular compounding more accurately reflects the true scatterer size caused by reductions in the system point spread function and improved signal-to-noise ratio. Preliminary in vivo H-scan imaging of tumor-bearing animals suggests this modality may be useful for monitoring early response to chemotherapeutic treatment. Overall, H-scan imaging using ultrasound plane waves and spatial angular compounding is a promising approach for visualizing the relative size and distribution of acoustic scattering sources.

H-scan sensitivity to scattering size.

In the H-scan analysis and display, visualization of different scattering sizes and types is enabled by a matched filter approach involving different orders of Gaussian weighted Hermite functions. An important question with respect to clinical applications involves the change in H-scan outputs with respect to small changes in scatterer sizes. The sensitivity of H-scan outputs is analyzed using the theory of backscatter from a compressible sphere. Experimental corroboration is established using mono dispersed spherical scatterers in phantoms. With a 6-MHz center frequency broadband transducer, it is possible to visualize changes in scattering size in the order of 10 to [Formula: see text] in phantoms and also changes in ex vivo bovine liver tissue due to edema caused by hypotonic perfusion.