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The rising incidence of small, asymptomatic renal tumours discovered usmg abdominal imaging during the investigation of unrelated symptoms has fuelled the desire for new therapies which avoid surgical excision. Extracorporeal High Intensity Focused Ultrasound (HIFU) was proposed as one of these modalities but so far clinical research has been ,~." inconclusive. The present work was designed to improve these clii teal outcomes through the conduct of further clinical trials, laboratory based research and the translation of new technology into existing HIFU devices. A Phase II clinical trial of patients (n=13) with newly diagnosed <4cm renal tumours (clinical stage T1a) was designed, peer reviewed and received ethical approval (Ox REC 09/H0606104). Ten of 13 patients underwent renal HIFU using a clinical HIFU device (Model JCIJC200, HAIFU, China). One patient could not be treated due to poor tumour visualisation after anaesthesia and two patients could not be treated as they became unwell before or during anaesthesia. Histological evidence of HIFU ablation in either tumour or normal renal parenchyma was seen in all ten patients. Evidence of sub-total tumour ablation was seen in 8/10 of patients. Grade 1 «50%), 2 (50-90%) & 3 (90-99%) ablation was achieved in 4/10, 3/1 0 & 3/1 0 patients respectively but complete (100%) tumour ablation was not possible. HIFU treatment caused minimal morbidity - no Grade III- V (Clavien-Dindo) complications related to HIFU treatment occurred. Grade I skin pain and induration was seen in 9/1 0 patients; Grade II skin pain occurred in a single patient. Patient demographics, imaging and tumour characteristics were used to design parameters to improve patient selection for renal HIFU. The tumour location, thickness of peri-nephric fat and renal nephrometry score were useful predictors of successful screening for treatment. Page /ii Dr R. W Ritchie Nutiield Department of Surgical Sciences - TT 2012 Abstract Diligent use of these factors could limit unnecessary treatments and Improve ablation outcomes. , It is well known that ultrasound imaging of small renal masses can be challenging. Ultrasound imaging often deteriorates further during HIFU as the abdominal wall and fat tissues swell and cause increased attenuation. This loss of imaging quality was clearly demonstrated in this clinical trial and resulted in the early termination of treatment, before ,#,J' ... ~ .•.. endpoints were reached, in a number of cases. The current clinical method for monitoring the success of HIFU ablation using hyperecho analysis of B-mode ultrasound images is also questionable. Laboratory based studies using ex-vivo bovine liver subjected to HIFU confirmed that hyperecho monitoring had low sensitivity, predictive values and overall accuracy. A novel method of HIFU monitoring - passive mapping of the emissions received from acoustic cavitation activity and other sources of non-linearity during HIFU treatment - is believed to represent a significant opportunity to improve feedback. This technique uses the passively received signature of cavity activity which, when time-reversed, gives high- resolution images of the precise location of the activity. Laboratory-based ex-vivo work, using a commercially available ultrasound system (z.one, Zonare, USA), demonstrates its superiority over hyperecho monitoring. Indeed, thresholds could be applied to successfully predict HIFU ablation with high sensitivity and specificity. This technique was successfully translated into the clinical setting through the design of a Passive Acoustic Mapping (P AM) device. Custom-built receiving elements were applied without limiting the function of the existing HIFU devices. Both pre-clinical and ethically- Page [iii Dr R. W Ritchie Nuffield Department of Surgical Sciences - TT 2012 Abstract approved clinical studies demonstrated its safe integration without significant impact on the device energy output or treatment accuracy. Using similar passive beamfonning algorithms, acoustic cavitation activity was successfully mapped and corresponded with the location of thermal ablation in both ex-vivo tissue phantoms and during clinical HIFU therapy. ,~-' It is believed that the development of new patient selection paral~~tel's will elimil?ate target those patients who are most suitable for renal HIFU - small tumours, minimal peri-nephric fat & low nephrometry score .. The use of P AM will lead to a significant improvement in the efficacy of treatment. It can be successfully applied to existing devices and predicts the location and extent ofHIFU ablation with greater accuracy that existing techniques.

Regensburg, Univ., Diss., 2008

The deposition of ultrasonic energy in tissue can cause tissue damage due to local heating. For pressures above a critical threshold, cavitation will occur in tissue and bubbles will be created. These oscillating bubbles can induce a much larger thermal energy deposition in the local region. Traditionally, clinicians and researchers have not exploited this bubble-enhanced heating since cavitation behavior is erratic and very difficult to control. The present work is an attempt to control and utilize this bubble-enhanced heating. First, by applying appropriate bubble dynamic models, limits on the asymptotic bubble size distribution are obtained for different driving pressures at 1 MHz. The size distributions are bounded by two thresholds: the bubble shape instability threshold and the rectified diffusion threshold. The growth rate of bubbles in this region is also given, and the resulting time evolution of the heating in a given insonation scenario is modeled. In addition, some experimental results have been obtained to investigate the bubble-enhanced heating in an agar and graphite based tissue- mimicking material. Heating as a function of dissolved gas concentrations in the tissue phantom is investigated. Bubble-based contrast agents are introduced to investigate the effect on the bubble-enhanced heating, and to control the initial bubble size distribution. The mechanisms of cavitation-related bubble heating are investigated, and a heating model is established using our understanding of the bubble dynamics. By fitting appropriate bubble densities in the ultrasound field, the peak temperature changes are simulated. The results for required bubble density are given. Finally, a simple bubbly liquid model is presented to estimate the shielding effects which may be important even for low void fraction during high intensity focused ultrasound (HIFU) treatment.

A complete understanding of high-intensity focused ultrasound-induced temperature changes in tissue requires insight into all potential mechanisms for heat deposition. Applications of therapeutic ultrasound often utilize acoustic pressures capable of producing cavitation activity. Recognizing the ability of bubbles to transfer acoustic energy into heat generation, a study of the role bubbles play in tissue hyperthermia becomes necessary. These bubbles are typically less than 50μm. This dissertation examines the contribution of bubbles and their motion to an enhanced heating effect observed in a tissue-mimicking phantom. A series of experiments established a relationship between bubble activity and an enhanced temperature rise in the phantom by simultaneously measuring both the temperature change and acoustic emissions from bubbles. It was found that a strong correlation exists between the onset of the enhanced heating effect and observable cavitation activity. In addition, the likelihood of observing the enhanced heating effect was largely unaffected by the insonation duration for all but the shortest of insonation times, 0.1 seconds. Numerical simulations were used investigate the relative importance of two candidate mechanisms for heat deposition from bubbles as a means to quantify the number of bubbles required to produce the enhanced temperature rise. The energy deposition from viscous dissipation and the absorption of radiated sound from bubbles were considered as a function of the bubble size and the viscosity of the surrounding medium. Although both mechanisms were capable of producing the level of energy required for the enhanced heating effect, it was found that inertial cavitation, associated with high acoustic radiation and low viscous dissipation, coincided with the the nature of the cavitation best detected by the experimental system. The number of bubbles required to account for the enhanced heating effect was determined through the numerical study to be on the order of 150 or less.

Given the great promise of High Intensity Focused Ultrasound (HIFU) as a therapeutic modality, the aim of the present study is to develop and optimise a technique that uses externally applied focused ultrasound energy and remote, ultrasound-based treatment monitoring to destroy subcutaneous fat safely, effectively and non-invasively. Based on initial cavitation and temperature measurements performed ex vivo in excised porcine fat at four different frequencies (0.5, 1.1, 1.6 & 3.4MHz) over a range of pressure amplitudes and exposure durations, it was concluded that 0.5MHz is the optimal frequency for this application since it is capable of instigating inertial cavitation at relatively modest pressures while enhancing focal heat deposition. Histological assessment of tissue treated above the cavitation threshold at 0.5MHz both ex vivo and in vivo demonstrated damage to adipocytes and connective tissue. Furthermore, a good correlation was identified between the energy of broadband emissions detected by the passive cavitation detector (PCD) and the focal temperature rise at 0.5MHz during ex vivo experimentation, which could be exploited as a tool for non-invasive monitoring of successful treatment delivery. In addition, localisation of cavitation activity by means of passive cavitation detection was achieved and shown to provide a strong indicator of the location of induced histological damage. Based on the specific requirements identified during initial experimentation, an application-specific HIFU transducer, cavitation detector and real-time treatment monitoring software was developed and tested ex vivo. This treatment system was found capable of producing extensive damage to adipocytes and collagen confined to the subcutaneous fat layer at the desired treatment depth, which coincided with the location of cavitation activity as displayed by the real-time treatment monitoring software.

Malignant or benign tumors may be ablated with high‐intensity focused ultrasound (HIFU). This technique, known as focused ultrasound surgery (FUS), has been actively investigated for decades, but slow to be implemented and difficult to control due to lack of real‐time feedback during ablation. Two methods of imaging and monitoring HIFU lesions during formation were implemented simultaneously, in order to investigate the efficacy of each and to increase confidence in the detection of the lesion. The first, Acousto‐Optic Imaging (AOI) detects the increasing optical absorption and scattering in the lesion. The intensity of a diffuse optical field in illuminated tissue is mapped at the spatial resolution of an ultrasound focal spot, using the acousto‐optic effect. The second, Harmonic Motion Imaging (HMI), detects the changing stiffness in the lesion. The HIFU beam is modulated to force oscillatory motion in the tissue, and the amplitude of this motion, measured by ultrasound pulse‐echo techniques, is influenced by the stiffness. Experiments were performed on store‐bought chicken breast and freshly slaughtered bovine liver. The AOI results correlated with the onset and relative size of forming lesions much better than prior knowledge of the HIFU power and duration. For HMI, a significant artifact was discovered due to acoustic nonlinearity. The artifact was mitigated by adjusting the phase of the HIFU and imaging pulses. A more detailed model of the HMI process than previously published was made using finite element analysis. The model showed that the amplitude of harmonic motion was primarily affected by increases in acoustic attenuation and stiffness as the lesion formed and the interaction of these effects was complex and often counteracted each other. Further biological variability in tissue properties meant that changes in motion were masked by sample‐to‐sample variation. The HMI experiments predicted lesion formation in only about a quarter of the lesions made. In simultaneous AOI/HMI experiments it appeared that AOI was a more robust method for lesion detection. / Bernard M. Gordon Center for Subsurface and Imaging Systems (CenSSIS) via the NSF ERC award number EEC‐9986821.

acase@tulane.edu / Introduction: High-Intensity Focused Ultrasound (HIFU) is the only noninvasive method available today for thermal ablation of tumors. HIFU-induced rapid heating and mechanical disruption of tissue, not only has a direct destructive effect on tumors, but also provides a noninvasive way for targeted release of chemotherapeutic drugs from drug delivery vehicles such as temperature sensitive liposomes (SfTSLs). The objective of this work was to evaluate the synergistic treatment of Sorafenib-loaded TSLs (SfTSLs) and HIFU via in vitro analysis of cell viability and proliferation using an aggressive human liver cancer cell line and corresponding in vivo analysis of tumor growth and survival using a human xenograft mouse model. Materials and Methods: Liposomes were developed using 70% Dipalmitoylphosphatidylcholine, 20% L-a-Phosphatidylcholinehydrogenated Soy, and 10% Cholesterol using thin film hydration method to encapsulate Sorafenib at 10μM. Pellets of Hep3B human liver cancer cells (100 μl, 2.7 million cells/ml) were placed in a 0.2 ml thin-wall PCR tube to mimic dense tumor aggregation. Cell pellets were then inoculated with HIFU alone, SfTSLs, or exposed to a combination of HIFU and SfTSLs. The focused ultrasound signal was generated by a 1.1 MHz transducer with acoustic power ranging from 4.1 W to 12.0 W. Cell viability and proliferation experiments were conducted to measure cancer cell damage at 24, 48, 72, and 96 h post treatment via Annexin V/PI and WST-8 staining. In our in vivo study, 1.0×106 Hep3B cells in Matrigel were injected into left and right flanks of athymic nude mice. Tumors were allowed to grow to 8-10 mm size and then separated into the following treatment groups: HIFU alone, SfTSLs (50 μl) alone, SfTSLs + HIFU, and sham. Tumor sizes were measured by caliper every day and a diagnostic ultrasound system was used pre-treatment, 5 days, 14 days, and prior to sacrificing. Tumors were grouped and processed at 5 days, 14 days, or placed in a survival study to evaluate whether treatment facilitated longer lifespans. Tumor tissues were collected for H&E staining and evaluated by a blinded pathologist post euthanasia. Results and Discussion: Our in vitro data indicate that Hep3B cells exposed to both SfTSLs and HIFU have a significantly lower viability and proliferation rate than untreated cells or the cells treated with only SfTSLs or HIFU. According to our in vivo study, tumor growth in the SfTSLs + HIFU group was reduced as compared to Sham, SfTSLs only, or HIFU only groups. Conclusions: The results of our in vitro and in vivo experiments clearly indicate that chemotherapeutic drug-loaded SfTSLs and HIFU can be an effective therapy for locally aggressive liver cancer. This combination treatment leads to more cellular damage, reduction in tumor growth, and better survival. / 1 / Gray Halliburton

Background: Histotripsy is a non-thermal, non-invasive high-intensity focused ultrasound (HIFU) ablative technique that causes mechanical fragmentation of tissue, resulting in liquefied acellular debris with histologically clear demarcated boundaries between treated and non-treated tissues. The acellular debris may include tumor antigens with preserved immunogenicity and the potential to generate systemic immune response against tumor cells. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to STS in humans. Long-term tumor control requires complete removal with extensive surgical resection, which in many cases is not feasible. As a result, there is need for alternative therapies. Objectives: The primary objective of this study was to demonstrate safety and feasibility of histotripsy in a small animal model of spontaneous STS. The secondary objective was to characterize the impact of histotripsy on the immunologic response. Materials and methods: Pet dogs diagnosed with spontaneous STS were recruited. CT scan of the chest, abdomen, and the tumor was performed for staging and treatment planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, owner reports, and CBC/serum biochemistry. Partial tumor ablation was performed using a 500 kHz prototype histotripsy system. A spherical treatment zone of up to 3 cm diameter in each tumor was treated with histotripsy according to the patient-specific treatment plan using 1-2 cycle pulses applied at a pulse repetition frequency (PRF) of 500 Hz. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-6 days post-treatment. Tumor microenvironment (TME) gene expression was evaluated with the Nanostring Canine IO panel, and the systemic immune response was evaluated using multiplex serum cytokine levels. Results: Ten dogs were recruited and treated. Tumor histologies included 3 grade III STS, 4 grade II STS, 2 grade I STS, and 1 malignant mesenchymoma. Six dogs were alive, three dogs were euthanized due to disease progression, and one dog was lost to follow up. Histotripsy-related complications were generally self-limiting, with only one patient having increased cutaneous injury score from 1 to 2 (scale 1-5) post-treatment, likely due to prefocal cavitation at the skin. No significant adverse events impacting patient outcome were noted in any of the patients. Visible histotripsy cavitation bubble clouds were seen on real-time ultrasound imaging in nine of ten treatments. Post-treatment histopathology indicated sharply defined regions of ablation that were clearly identifiable grossly and histologically in all samples. Treatment zones were characterized by loss of cell viability, hyalinization, and acute hemorrhage. Post-treatment contrast-enhanced CT images revealed clear, demarcated regions of histotripsy ablated tissue in seven of ten patients. Differential gene expression analysis identified 79 genes with at least 2-fold change following treatment. Genes associated with inflammation, immune cell migration, and immune cell interactions were the highest upregulated. Amongst the gene set analyses, the myeloid compartment gene sets obtained the highest significance score. There were no statistically significant differences between pre- and post-treatment cytokine concentrations for any of the analytes. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs diagnosed with STS. Histotripsy induced pro-inflammatory changes within the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Histotripsy has a potential to be a precise, non-invasive treatment for canine STS. / Master of Science / Histotripsy is a non-thermal high-intensity focused ultrasound (HIFU) ablative technique that uses controlled acoustic cavitation to cause mechanical fragmentation of tissue. To date, there are no reports investigating histotripsy for the treatment of soft tissue sarcoma (STS). This study aimed to investigate the in vivo feasibility of ablating STS with histotripsy and to characterize the impact of partial histotripsy ablation on the acute immunologic response in canine patients with spontaneous STS. CT of the chest, abdomen, and the tumor was performed for staging and treatment-planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, through owner reports, and CBC/serum biochemistry. A custom 500 kHz histotripsy system was used to treat ten dogs with naturally occurring STS. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-days post-treatment. Safety was determined by monitoring vital signs during treatment and post-treatment physical examinations, routine lab work, and owners' reports. Ablation was characterized using radiologic and histopathologic analyses. Systemic immunological impact was evaluated by measuring changes in cytokine concentrations, and tumor microenvironment changes were evaluated by characterizing changes in infiltration with tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) using multiplex immunohistochemistry and differential gene expression. Results showed histotripsy ablation can achieve safe and effective tumor ablation in all ten dogs. Immunological results showed histotripsy induced pro-inflammatory changes in the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Overall, this study demonstrates histotripsy's potential as a precise, non-invasive treatment for STS.

Sabe-se que o aumento na temperatura do tecido tumoral pode aumentar a eficiência de técnicas convencionais de combate ao câncer (radioterapia e quimioterapia). Além disso, a variação de temperatura em tumores pode ser uma forma de tratamento alternativo à cirurgia, feito por meio do fornecimento de calor direcionado às células cancerosas e preservando o tecido sadio. Para maior eficácia e segurança no emprego de técnicas que utilizam fornecimento de calor ao tecido biológico, é necessário o monitoramento da temperatura tecidual para garantir que a morte celular por ablação térmica seja limitada ao tecido alvo, minimizando os danos aos tecidos adjacentes. A imagem fotoacústica é uma técnica baseada no efeito fotoacústico, o qual consiste na absorção de radiação eletromagnética pelo tecido e, devido à expansão termoelástica, na geração de ondas acústicas. A amplitude da onda de pressão gerada pelo efeito fotoacústico possui dependência com a temperatura do meio pelo parâmetro de Grueneisen, que depende das propriedades mecânicas do material. Portanto, mudanças na amplitude do sinal fotoacústico carregam informações a respeito da variação na temperatura do material. Neste trabalho, a dependência da amplitude do sinal fotoacústico com a temperatura foi estudada em um material simulador de tecido biológico (phantom) em condições similares a de tratamentos por hipertermia Nesse caso, imagens fotoacústicas foram adquiridas para cada grau de temperatura em uma faixa de 36 até 41 ºC durante o procedimento de aquecimento por banho térmico. Mudanças na amplitude e fase do sinal fotoacústico foram avaliadas através da aplicação de algoritmos de speckle tracking. Para estimar a variação na amplitude do sinal também foram utilizados e avaliados diferentes métodos de comparação. Os resultados são apresentados por imagens fotoacústicas termais produzidas pela aplicação de um fator de calibração aos mapas de variação relativa da amplitude do sinal em função da temperatura do meio. Finalmente, avaliamos um experimento de hipertemia por ultrassom focalizado de alta intensidade (High Intensity Therapeutic Ultrasound - HITU) em uma amostra de músculo suíno. Nesse caso foram geradas imagens termais fotoacústicas e imagens termais produzidas pela mudança de fase do sinal pulso-eco de ultrassom. Os resultados sugerem uma maneira não invasiva de calcular a distribuição da variação de temperatura do meio que pode ser aplicada para monitoramento durante tratamentos que utilizam o fornecimento de calor ao tecido biológico. / Several studies have shown that elevating the temperature of tumoral tissue improves standard cancer treatments success rate (radiotherapy and chemotherapy). This procedure can also be a therapy to cancer by delivering heat and killing cancer cells while healthy tissues are preserved. For improved efficiency and security in heat applications, it is important to monitor tissue temperature during treatments. Photoacoustic (PA) pressure wave amplitude has a temperature dependence given by the sample mechanical properties (Gruenesein parameter). These changes in photoacoustic signal amplitude carry information about temperature variation in tissue. Therefore, PA has been proposed as an imaging technique to monitor temperature during hyperthermia. In this study, PA images were acquired for temperatures ranging from 36ºC to 41ºC using a tissue-mimicking phantom immersed in a temperature controlled thermal bath. Relative amplitude variation was calculated using speckle tracking algorithms using four different methods to estimate these variations in PA signal amplitude. The results are presented as PA-based thermal images, generated using a calibration factor to the percentage variations in the amplitude maps. Finally, PA-based and ultrasound-based thermal images were acquired during heating by high intensity focused ultrasound (High Intensity Therapeutic Ultrasound - HITU) in a porcine muscle. The results suggest a non-invasive way to monitor temperature during hyperthermia procedures.