Investigation of Histotripsy for Non-Invasive Treatment of Liver Tumor

Objective

The main objective of this research is to develop histotripsy for the non-invasive treatment of hepatocellular carcinoma (HCC). HCC tumors progress with local expansion, intrahepatic spread, and distant metastases. We are investigating if histotripsy can safely and completely ablate subcutaneous and orthotopic liver tumors. Further, we are studying the impact of this non-invasive treatment on tumor recurrence, immune response and metastatic potential.

Background

HCC has been the fastest growing cancer in the United States over the past decade. Current treatment modalities include surgical resection, radiation, chemotherapy, and liver ablation. Radiation and chemotherapy are associated with many side effects including nausea, weakness, fatigue, toxicity and may require multiple sessions. Surgical resection is recommended only for 10-30% liver cancer patients, most are not eligible due to complications including tumor morphology, location, associated disease, and physiological condition. Current liver ablation methods including radiofrequency ablation (RFA) and high intensity focused ultrasound (HIFU) are primarily thermal-based and possess inherent limitations such as inconsistent ablation, inability to treat larger or multi-nodular tumors, limited or no real-time imaging feedback and heat sink effect (cooling effect from the blood flow near major blood vessels) through the densely vascular liver . Despite the availability of multiple treatment options, the incidence of HCC in the US has more than tripled since 1980, and the five-year survival rate at just 17%. Thus, there is an unmet clinical need that warrants reliable, effective and life-prolonging treatments for HCC patients. To overcome these limitations, we are developing histotripsy for non-invasive liver cancer ablation.

Rodent histotripsy experimental setup

A 1 MHz custom-built 8 element focused transducer designed for rodent experiments is used to generate histotripsy pulses, driven by a custom high-voltage pulser developed in-house. A field-programmable gate array (FPGA) development board (DE0-Nano Terasic Technology, HsinChu, Taiwan) specifically programmed for histotripsy therapy pulsing is connected to the pulser, allowing the therapy transducer to output 1-2 cycle histotripsy pulses. A 20 MHz probe (L40-8/12, Ultrasonix, Vancouver, Canada) is coaxially positioned with the therapy transducer for real time targeting feedback. The transducer and imaging probe are submerged in a tank of degassed water while mounted to a motorized 3-axis positioning system, which allows for scanning and treating user-defined volumes.

Effectiveness of non-invasive subcutaneous tumor ablation in vivo

Histotripsy was performed to treat subcutaneous tumors in a human HCC xenograft murine model. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were delivered using a custom built 1 MHz therapy transducer attached to a motorized positioner, which scanned the  transducer focus to traverse the targeted tumor volume, guided by real-time US imaging. Tumor ablation effectiveness was assessed by obtaining T1, T2 and T2* weighted MR images. Post euthanasia, tissue samples were harvested for histology. Histology confirmed the fractionation of targeted region with a sharp boundary separating it from untreated tissue. In the survival study, we observed improved survival outcomes and effective tumor volume reduction post treatment on MRI as the homogenate and edema were resorbed within 2-3 weeks. However, as the tumor was subcutaneous, it was not possible to set adequate treatment margin and since the mice were immune-compromised, any residual viable tumor cells eventually developed into tumor regrowth at 3-9 weeks after histotripsy. Our study successfully demonstrated the potential of histotripsy for non-invasive HCC ablation in a subcutaneous murine model.

Non-invasive orthotopic tumor ablation in an immune-competent model

In this ongoing investigation, histotripsy is used for treating orthotopic liver tumors in an immune-competent N1S1 rat liver tumor model. Tumors have been targeted either partially or completely using 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa), guided by real-time US imaging. Tumor ablation effectiveness is assessed by T2 weighted MR images. In our preliminary study, rats have been survived for 2 months after a single histotripsy treatment session with no signs of recurrence, even in cases where only a partial tumor volume was treated.

Evaluation of histotripsy in a metastatic tumor model

We will be using the McA-RH7777 metastatic rodent liver tumor model to study the impact of histotripsy on metastatic potential. Since histotripsy destroys tumor cells to form acellular homogenate, it is important to investigate if there are viable tumor cells entering into circulation post-histotripsy that could induce a significant metastatic response. Based on preliminary data so far, we hypothesize that the post-histotripsy treatment debris released into circulation does not promote metastasis.

In vivo Porcine liver ablation

We have obtained substantial in vivo data in porcine normal liver^1-4 to show the following: (1) consistent cellular disruption in the target through intact ribs and other tissue without damage along the energy delivery path; (2) sharp margins <1 mm without significant inflammation; (3) capability to ablate tumors of any shape, size, and nodule number; (4) complete ablation of a 60 mL liver volume in 60 minutes (at a speed of 1mL/min) with decreasing ablation times expected after parameter optimization. Additionally, we have obtained the following data in a murine subcutaneous HCC model and rodent liver model⁵: 1) effective tumor volume reduction and significantly lengthened survival time; 2) Histotripsy-produced acellular homogenate completely absorbed in one month; 3) significant histotripsy-stimulated immune response at the local tumor site and distant organs.

In vivo histotripsy liver ablation in the porcine liver. A) Focal cavitation generated by histotripsy shows as the bright spot on ultrasound image during treatment. B) Histology of the histotripsy ablation zone, with the magnified view shows no intact cells within the ablation zone (C) and a sharp margin (D). E) Intact vessels >300 um remain within the ablation zone, as vessels and bile ducts are more resistant to histotripsy-induced damage. E) MR image of a 60 mL liver volume (equivalent to ~5 cm diameter) was treated within an hour by mechanically moving the histotripsy focus to cover the target volume.

Evaluation of immune response to histotripsy

We are currently investigating the impact of histotripsy on immune response in immune-competent bilateral subcutaneous mice tumor models (B16GP33 melanoma, Hepa1-6 liver). We hypothesize that histotripsy treatment induces a measurable local and systemic adaptive immune response to tumor antigen. To characterize the immune response, we are measuring the immune cell populations, CD4+ and CD8+ T cell responsiveness to tumor antigen both locally and systemically, and especially determining any abscopal effects. In the future we may extend the immunological investigation to orthotopic rodent tumor models and also study the impact of histotripsy on tumor re-challenge.

References

1. Vlaisavljevich E, Kim Y, Allen S, Owens G, Pelletier S, Cain C, Ives K, Xu Z. Image-guided non-invasive ultrasound liver ablation using histotripsy: feasibility study in an in vivo porcine model. Ultrasound Med Biol. 2013;39(8):1398-409. PMCID: 3709011.
2. Vlaisavljevich E, Owens G, Lundt J, Teofilovic D, Ives K, Duryea A, Bertolina J, Welling TH, Xu Z. Non-Invasive Liver Ablation Using Histotripsy: Preclinical Safety Study in an In Vivo Porcine Model. Ultrasound Med Biol. 2017;43(6):1237-51.
3. Kim Y, Vlaisavljevich E, Owens GE, Allen SP, Cain CA, Xu Z. In vivo transcostal histotripsy therapy without aberration correction. Phys Med Biol. 2014;59(11):2553-68.
4. Smolock AR, Cristescu MM, Vlaisavljevich E, Gendron-Fitzpatrick A, Green C, Cannata J, Ziemlewicz TJ, Lee FT, Jr. Robotically Assisted Sonic Therapy as a Noninvasive Nonthermal Ablation Modality: Proof of Concept in a Porcine Liver Model. Radiology. 2018;287(2):485-93.
5. Vlaisavljevich E, Greve J, Cheng X, Ives K, Shi J, Jin L, Arvidson A, Hall T, Welling TH, Owens G, Roberts W, Xu Z. Non-Invasive Ultrasound Liver Ablation Using Histotripsy: Chronic Study in an In Vivo Rodent Model. Ultrasound Med Biol. 2016;42(8):1890-902. PMCID: PMC4912895
6. Kim Y, Hall TL, Xu Z, Cain CA. Transcranial Histotripsy Therapy: A Feasibility Study. IEEE Trans Ultrason Ferroelectr Freq Control. 2014;61(4):582-93.