Quantitative Ultrasound Monitoring of Breast Tumour Response to Neoadjuvant Chemotherapy: Comparison of Results Among Clinical Scanners.

Quantitative ultrasound (QUS) techniques have been demonstrated to detect cell death in vitro and in vivo. Recently, multi-feature classification models have been incorporated into QUS texture-feature analysis methods to increase further the sensitivity and specificity of detecting treatment response in locally advanced breast cancer patients. To effectively incorporate these analytic methods into clinical applications, QUS and texture-feature estimations should be independent of data acquisition systems. The study here investigated the consistencies of QUS and texture-feature estimation techniques relative to several factors. These included the ultrasound system properties, the effects of tissue heterogeneity and the effects of these factors on the monitoring of response to neoadjuvant chemotherapy. Specifically, tumour-response-detection performance based on QUS and texture parameters using two clinical ultrasound systems was compared. Observed variations in data between the systems were small and the results exhibited good agreement in tumour response predictions obtained from both ultrasound systems. The results obtained in this study suggest that tissue heterogeneity was a dominant feature in the parameters measured with the two different ultrasound systems; whereas differences in ultrasound system beam properties only exhibited a minor impact on texture features. The McNemar statistical test performed on tumour response prediction results from the two systems did not reveal significant differences. Overall, the results in this study demonstrate the potential to achieve reliable and consistent QUS and texture-based analyses across different ultrasound imaging platforms.

Ultrasound microbubble potentiated enhancement of hyperthermia-effect in tumours.

It is now well established that for tumour growth and survival, tumour vasculature is an important element. Studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment causes extensive endothelial cell death leading to tumour vascular disruption. The subsequent rapid vascular collapse translates to overall increases in tumour response to various therapies. In this study, we explored USMB involvement in the enhancement of hyperthermia (HT) treatment effects. Human prostate tumour (PC3) xenografts were grown in mice and were treated with USMB, HT, or with a combination of the two treatments. Treatment parameters consisted of ultrasound pressures of 0 to 740 kPa, the use of perfluorocarbon-filled microbubbles administered intravenously, and an HT temperature of 43°C delivered for various times (0-50 minutes). Single and multiple repeated treatments were evaluated. Tumour response was monitored 24 hours after treatments and tumour growth was monitored for up to over 30 days for a single treatment and 4 weeks for multiple treatments. Tumours exposed to USMB combined with HT exhibited enhanced cell death (p<0.05) and decreased vasculature (p<0.05) compared to untreated tumours or those treated with either USMB alone or HT alone within 24 hours. Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and cluster of differentiation 31 (CD31) staining were used to assess cell death and vascular content, respectively. Further, tumours receiving a single combined USMB and HT treatment exhibited decreased tumour volumes (p<0.05) compared to those receiving either treatment alone when monitored over the duration of 30 days. Additionally, tumour response monitored weekly up to 4 weeks demonstrated a reduced vascular index and tumour volume, increased fibrosis and lesser number of proliferating cells with combined treatment of USMB and HT. Thus in this study, we characterize a novel therapeutic approach that combines USMB with HT to enhance treatment responses in a prostate cancer xenograft model in vivo.

A priori prediction of breast tumour response to chemotherapy using quantitative ultrasound imaging and artificial neural networks.

We demonstrate the clinical utility of combining quantitative ultrasound (QUS) imaging of the breast with an artificial neural network (ANN) classifier to predict the response of breast cancer patients to neoadjuvant chemotherapy (NAC) administration prior to the start of treatment. Using a 6 MHz ultrasound system, radiofrequency (RF) ultrasound data were acquired from 100 patients with biopsy-confirmed locally advanced breast cancer prior to the start of NAC. Quantitative ultrasound mean parameter intensity and texture features were computed from the tumour core and margin, and were compared to the clinical/pathological response and 5-year recurrence-free survival (RFS) of patients. A multi-parametric QUS model in conjunction with an ANN classifier predicted patient response with 96 ± 6% accuracy, and a 0.96 ± 0.08 area under the receiver operating characteristic curve (AUC), compared to 65 ± 10 % accuracy and 0.67 ± 0.14 AUC achieved using a K-Nearest Neighbour (KNN) algorithm. A separate ANN model predicted patient RFS with 85 ± 7% accuracy, and a 0.89 ± 0.11 AUC, whereas the KNN methodology achieved a 58 ± 6 % accuracy and a 0.64 ± 0.09 AUC. The application of ANN for classifying patient response based on tumour QUS features performs well in terms of predicting response to chemotherapy. The findings here provide a framework for developing personalized a priori chemotherapy selection for patients that are candidates for NAC, potentially resulting in improved patient treatment outcomes and prognosis.

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy.

High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to characterize vascular and tumour response effects simultaneously, as well as a therapeutic modality to complement radiation therapy.

Breast-Lesion Characterization using Textural Features of Quantitative Ultrasound Parametric Maps.

This study evaluated, for the first time, the efficacy of quantitative ultrasound (QUS) spectral parametric maps in conjunction with texture-analysis techniques to differentiate non-invasively benign versus malignant breast lesions. Ultrasound B-mode images and radiofrequency data were acquired from 78 patients with suspicious breast lesions. QUS spectral-analysis techniques were performed on radiofrequency data to generate parametric maps of mid-band fit, spectral slope, spectral intercept, spacing among scatterers, average scatterer diameter, and average acoustic concentration. Texture-analysis techniques were applied to determine imaging biomarkers consisting of mean, contrast, correlation, energy and homogeneity features of parametric maps. These biomarkers were utilized to classify benign versus malignant lesions with leave-one-patient-out cross-validation. Results were compared to histopathology findings from biopsy specimens and radiology reports on MR images to evaluate the accuracy of technique. Among the biomarkers investigated, one mean-value parameter and 14 textural features demonstrated statistically significant differences (p < 0.05) between the two lesion types. A hybrid biomarker developed using a stepwise feature selection method could classify the legions with a sensitivity of 96%, a specificity of 84%, and an AUC of 0.97. Findings from this study pave the way towards adapting novel QUS-based frameworks for breast cancer screening and rapid diagnosis in clinic.

Effect of chromatin structure on quantitative ultrasound parameters.

High-frequency ultrasound (~20 MHz) techniques were investigated using in vitro and ex vivo models to determine whether alterations in chromatin structure are responsible for ultrasound backscatter changes in biological samples. Acute myeloid leukemia (AML) cells and their isolated nuclei were exposed to various chromatin altering treatments. These included 10 different ionic environments, DNA cleaving and unfolding agents, as well as DNA condensing agents. Raw radiofrequency (RF) data was used to generate quantitative ultrasound parameters from spectral and form factor analyses. Chromatin structure was evaluated using electron microscopy. Results indicated that trends in quantitative ultrasound parameters mirrored trends in biophysical chromatin structure parameters. In general, higher ordered states of chromatin compaction resulted in increases to ultrasound paramaters of midband fit, spectral intercept, and estimated scatterer concentration, while samples with decondensed forms of chromatin followed an opposite trend. Experiments with isolated nuclei demonstrated that chromatin changes alone were sufficient to account for these observations. Experiments with ex vivo samples indicated similar effects of chromatin structure changes. The results obtained in this research provide a mechanistic explanation for ultrasound investigations studying scattering from cells and tissues undergoing biological processes affecting chromatin.

Assessment of tumor response to radiation and vascular targeting therapy in mice using quantitative ultrasound spectroscopy.

PURPOSE: It is now recognized that the tumor vasculature is in part responsible for regulating tumor responses to radiation therapy. However, the extent to which radiation-based vascular damage contributes to tumor cell death remains unknown. In this work, quantitative ultrasound spectroscopy (QUS) methods were used to investigate the acute responses of tumors to radiation-based vascular treatments. METHODS: Tumor xenografts (MDA-MB-231) were treated with single radiation doses of 2 or 8 Gy alone, or in combination with pharmacological agents that modulate vascular radiosensitivity. The midband fit, the slope, and the 0-MHz intercept QUS parameters were obtained from a linear-regression fit to the averaged power spectrum of frequency-dependent ultrasound backscatter and were used to quantify acute tumor responses following treatment administration. Power spectrums were extracted from raw volumetric radio-frequency ultrasound data obtained before and 24 h following treatment administration. These parameters have previously been correlated to tumor cell death. Staining using in situ end labeling, carbonic anhydrase 9 and cluster of differentiation 31 of tumor sections were used to assess cell death, oxygenation, and vasculature distributions, respectively. RESULTS: Results indicate a significant midband fit QUS parameter increases of 3.2 ± 0.3 dBr and 5.4 ± 0.5 dBr for tumors treated with 2 and 8 Gy radiation combined with the antiangiogenic agent Sunitinib, respectively. In contrast, tumors treated with radiation alone demonstrated a significant midband fit increase of 4.4 ± 0.3 dBr at 8 Gy only. Preadministration of basic fibroblast growth factor, an endothelial radioprotector, acted to minimize tumor response following single large doses of radiation. Immunohistochemical analysis was in general agreement with QUS findings; an R(2) of 0.9 was observed when quantified cell death was correlated with changes in midband fit. CONCLUSIONS: Results from QUS analysis presented in this study confirm that acute tumor response is linked to a vascular effect following high doses of radiation therapy. Overall, this is in agreement with previous reports suggesting that acute tumor radiation response is regulated by a vascular-driven response. Data also suggest that Sunitinib may enhance tumor radiosensitivity through a vascular remodeling process, and that QUS may be sensitive to changes in tissue properties associated with vascular remodeling. Finally, the work also demonstrates the ability of QUS methods to monitor response to radiation-based vascular strategies.

Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model.

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (γH2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy.

Ultrasound-mediated microbubble enhancement of radiation therapy studied using three-dimensional high-frequency power Doppler ultrasound.

Tumor responses to high-dose (>8 Gy) radiation therapy are tightly connected to endothelial cell death. In the study described here, we investigated whether ultrasound-activated microbubbles can locally enhance tumor response to radiation treatments of 2 and 8 Gy by mechanically perturbing the endothelial lining of tumors. We evaluated vascular changes resulting from combined microbubble and radiation treatments using high-frequency 3-D power Doppler ultrasound in a breast cancer xenograft model. We compared treatment effects and monitored vasculature damage 3 hours, 24 hours and 7 days after treatment delivery. Mice treated with 2 Gy radiation and ultrasound-activated microbubbles exhibited a decrease in vascular index to 48 ± 10% at 24 hours, whereas vascular indices of mice treated with 2 Gy radiation alone or microbubbles alone were relatively unchanged at 95 ± 14% and 78 ± 14%, respectively. These results suggest that ultrasound-activated microbubbles enhance the effects of 2 Gy radiation through a synergistic mechanism, resulting in alterations of tumor blood flow. This novel therapy may potentiate lower radiation doses to preferentially target endothelial cells, thus reducing effects on neighboring normal tissue and increasing the efficacy of cancer treatments.

Perspectives in Implementing Radiogenomics to Radiotherapy.

Genomics is the study of all nucleotide sequences, including structural genes, regulatory sequences and noncoding DNA segments, in the chromosomes of an organism. Radiogenomics focuses on the underlying genetic causes of cellular responses to radiation and its applications to radiation oncology practice. The purpose of this review is to introduce the technology behind radiogenomics and discuss how it may impact radiotherapy practice for practitioners. A literature search was conducted on PubMed using the key words genomics, genes, pharmacogenomics, radiogenomics, dose painting and dose sculpting. A review of the literature shows much promise for future radiation oncology health care practices. Current findings from radiogenomics research require much work in translational research via rigorous testing, evaluation and validation to corroborate data and research methodologies. This review consolidates the theoretical basis of radiogenomics research and discusses the considerations of genomics technology in the context of how it will impact radiotherapy practice. The promise of radiogenomics applications holds a future role in identifying tissue-specific radiation resistance and tolerance by identifying genes responsible for radiobiological response.

Practical Considerations in Cone Beam and Ultrasound IGRT Systems in Prostate Localization: A Review of the Literature.

BACKGROUND: The use of image-guided radiotherapy (IGRT) is gaining in popularity for radiotherapy clinicians as a means of adaptive tumour targeting. With the increased use of intensity-modulated radiation therapy (IMRT), IGRT has been looked at as an important partnering tool in resolving the problem of prostate displacement prior to radiotherapy administration. Online correction tools such as kilovoltage cone beam computed tomography (kV-CBCT) and ultrasound (US) image-guided radiotherapy have been the focus of many studies that examine the feasibility, validity, and usefulness of these technologies. PURPOSE: The purpose of this article is to provide a comprehensive review of kV-CBCT and US-IGRT modalities and the practical considerations for radiation therapists and radiotherapy departments when using kilovoltage-CBCT and US-IGRT. CONCLUSION: The findings of this review indicate that both modalities are adequate for achieving image guidance in radiotherapy delivery; however, both modalities would require that the user have specific knowledge when applying these IGRT techniques and that the implementation of these technologies warrant further study to see the organizational impact to the radiotherapy department.