Erik N.K. Cressman

Molecular mechanisms of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) typically has poor prognosis, because it is often diagnosed at an advanced stage. Heterogeneous phenotypic and genetic traits of affected individuals and a wide range of risk factors have classified it a complex disease. HCC is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. Therefore, development of novel, effective therapies is of prime importance. Extensive research over the past decade has identified a number of molecular biomarkers as well as cellular networks and signaling pathways affected in liver cancer. Recent studies using a combination of “omics” technologies, microRNA studies, combinatorial chemistry, and bioinformatics are providing new insights into the gene expression and protein profiles during various stages of the disease. In this review, we discuss the contribution of these newer approaches toward an understanding of molecular mechanisms of HCC and for the development of novel cancer therapeutics. (HEPATOLOGY 2008;48:2047‐2063.)

Hepatic differentiation of porcine induced pluripotent stem cells in vitro.

Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders.

Animal models of cancer in interventional radiology

Animal models will play an increasingly important role in oncology research, especially for solid tumours such as hepatocellular carcinoma that are resistant to chemotherapy. Many models have been used, but there is a need for increased awareness of the limitations of these models and also a need for guidance for future model development.

Thermochemical ablation in an ex-vivo porcine liver model using acetic acid and sodium hydroxide: proof of concept.

PURPOSE To establish proof of concept in tissue, using the exothermic neutralization reaction of acetic acid and sodium hydroxide in ex vivo porcine liver and to conduct an initial probe into the relationships of volume and concentration of reagents to temperatures and the areas affected. MATERIALS AND METHODS A total of 0.5 mL or 2 mL of either 5 mole/L or 10 mole/L acid and base solutions was injected simultaneously into the periphery of ex vivo porcine liver using a prototype injection device. Tissue temperature was recorded at the injection site for 5 minutes using a type T thermocouple temperature probe inserted parallel to and near the tip of the injection device. The injections were repeated for infrared thermography, and ablated tissues were sectioned quickly and imaged. A gross photograph was captured in each case to provide correlation. RESULTS Maximum temperatures (17°C baseline) ranged from 42.1° ± α3.34°C to 61.7° ± α10°C (P<.05) when injecting 0.5 mL of 5 mole/L reactants and 2 mL of 10 mole/L reactants, respectively. The maximum temperature measured by infrared imaging ranged from 31°-47°C. Using an infrared viewing scale from 19°-40°C, the cross-sectional area of tissue heating above baseline measured from 1.07 cm(2)± 0.45 to 4.95 cm(2)± 0.28 (P <05). CONCLUSIONS The reaction of acetic acid and sodium hydroxide releases significant heat energy at the site of injection, and histologic changes are consistent with coagulation necrosis. Increased reagent concentration and volume were associated with larger temperature changes and larger areas of hyperthermia at gross pathology and infrared imaging.

A Hydrophobic Gel Phantom for Study of Thermochemical Ablation: Initial Results Using a Weak Acid and Weak Base

PURPOSE To develop a model for study of exothermic chemical reactions potentially useful for tissue ablation. MATERIALS AND METHODS Seven gelatins ranging from 0.5% to 30% wt/vol with and without 15% or 30% caps and several commercial gels were evaluated. Baseline temperature measurements were taken. Acetic acid and ammonium hydroxide were sequentially injected over periods of 10-15 seconds in 1-mL aliquots, forming a discrete aqueous reaction chamber. Congo red pH indicator was included to assess the reaction. A thermocouple allowed data collection at completion of injection and every 15 seconds for 5 minutes. Injections were performed in triplicate, and average temperatures for each time point were reported. RESULTS Gelatins fractured or refluxed even at the lowest concentrations tested. Most commercial gels proved too viscous and likewise led to reflux along the needle tract. A mineral oil-based gel was selected because of its ability to form a chamber without reflux or fracture and its clear colorless character, hydrophobic nature, chemical stability, viscosity, specific gravity, and cost. Temperatures during the first 60 seconds of the neutralization reaction showed an immediate increase that correlated well with concentration. CONCLUSIONS The oil gel phantom is a safe, useful, readily available, inexpensive model to study mixing behaviors and maximum heating potentials for reactions that may prove useful in thermochemical tissue ablation for oncologic interventions. Measurable temperature changes occurred even at the lowest concentrations, and higher concentrations produced a greater release of heat energy.

Stem Cell Origins and Animal Models of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor that almost always occurs within a preexisting background of chronic liver disease and cirrhosis. Currently, medical therapy is not effective in treating most HCC, and the only hope of cure is either resection or liver transplantation. A small minority of patients is eligible for these therapies, which entail major morbidity at the very least. In spite of immense scientific advances during the past 3 decades, patient survival has improved very little. In order to reduce morbidity and mortality from HCC, improvements in early diagnosis and development of novel local and systemic therapies for advanced disease are essential, in addition to efforts geared towards primary prevention. Studies with experimental animal models that closely mimic human disease are very valuable in understanding physiological, cellular and molecular mechanisms underlying the disease. Furthermore, appropriate animal models have the potential to increase our understanding of the effects of image-guided minimally invasive therapies and thereby help to improve such therapies. In this review, we examine the evidence for stem cell origins of such tumors, critically evaluate existing models and reflect on how to develop new models for minimally invasive, image-guided treatment of HCC.

Concentration and volume effects in thermochemical ablation in vivo: Results in a porcine model

Purpose: To explore the effects of volume and concentration in thermochemical ablation using an in vivo porcine model. Methods: Twelve swine 60–75 kg were used in this institutionally approved study. A needle design prototype coaxial device for reagent injections and a thermocouple were inserted into surgically exposed liver. Simultaneously, an acid and base (acetic acid and NaOH) were injected at 4 mL/min based on a 3 × 3 matrix with concentration (5, 10, and 15 mol/L) and volume on the axes (total volumes of 1, 2, and 4 mL). Three animals (centre grid position) strengthened the statistical analysis. Each animal received four identical injections (total 48). Temperatures and heart rate were recorded. Livers were formalin-fixed after sacrifice. After sectioning, coagulation zones were analysed by two observers. Area and slice thickness were used to calculate the volume, surface area, and sphericity for each treatment. Results: Coagulation volumes ranged from 2.95 ± 0.29 to 14.72 ± 1.42 mL with a maximum of 18.3 mL. Highest peak temperature was 105°C with temperatures ranging 43.5 ± 2.6°C to 91.0 ± 6.5°C. There was no association between conditions and sphericity or heart rate. Conclusions: The method can be used successfully to ablate tissue in vivo. By neutralising acid in situ and releasing heat and a salt, this technique improves considerably upon the use of acetic acid used alone. Peak temperatures exceeded accepted coagulation thresholds even if the only mechanism operating was hyperthermia. Reagent concentrations and volumes increased the amount of the coagulum but not in a linear fashion.

In Vitro Thermal Profile Suitability Assessment of Acids and Bases for Thermochemical Ablation: Underlying Principles

PURPOSE To measure and compare temperature changes in a recently developed gel phantom for thermochemical ablation as a function of reagent strength and concentration with several acids and bases. MATERIALS AND METHODS Aliquots (0.5-1 mL) of hydrochloric acid or acetic acid and sodium hydroxide or aqueous ammonia were injected for 5 seconds into a hydrophobic gel phantom. Stepwise increments in concentration were used to survey the temperature changes caused by these reactions. Injections were performed in triplicate, measured with a thermocouple probe, and plotted as functions of concentration and time. RESULTS Maximum temperatures were reached almost immediately in all cases, reaching 75 degrees C-110 degrees C at the higher concentrations. The highest temperatures were seen with hydrochloric acid and either base. More concentrated solutions of sodium hydroxide tended to mix incompletely, such that experiments at 9 M and higher were difficult to perform consistently. CONCLUSIONS Higher concentrations for any reagent resulted in higher temperatures. Stronger acid and base combinations resulted in higher temperatures versus weak acid and base combinations at the same concentration. Maximum temperatures obtained are in a range known to cause tissue coagulation, and all combinations tested therefore appeared suitable for further investigation in thermochemical ablation. Because of the loss of the reaction chamber shape at higher concentrations of stronger agents, the phantom does not allow complete characterization under these circumstances. Adequate mixing of reagents to maximize heating potential and avoid systemic exposure to unreacted acid and base must be addressed if the method is to be safely employed in tissues. In addition, understanding factors that control lesion shape in a more realistic tissue model will be critical.

A new heat source for thermochemical ablation based on redox chemistry: initial studies using permanganate.

Purpose: To evaluate exothermic permanganate redox chemistry for utility in tumour ablation. Materials and methods: Sodium permanganate (1–3 mL, 1 or 2 M) and glycerol (1 mL, 1 M) were injected in triplicate into a beaker using three injection orders: (1) simultaneous, (2) glycerol injection then permanganate injection, (3) permanganate injection then glycerol injection. Selected experiments were repeated with glucose, sucrose, dextrin, maltodextrin, different polysaccharides, and polyvinyl alcohol as substrates. Simultaneous injections of permanganate (0.5 and 1 mL, 2 M) and glucose (1 mL, 1 M) into explanted porcine muscle were also performed. Temperatures were recorded with a thermocouple probe or an infrared camera. Results: With optimal conditions of 2 M permanganate and 1 M glycerol at 1 mL each, an average maximum temperature of 97.4°C was obtained for all three injection orders. When glucose and sucrose were used as the substrates, similar temperatures were achieved but at different rates. Dextrin and maltodextrin (180 g/L) led to maximum temperatures of 42.5° and 51.1°C respectively when simultaneously injected with permanganate (1 mL, 2 M). Polysaccharides and polyvinyl alcohols under the same conditions resulted in a minimal temperature increase. Intramuscular injections of glucose (0.5 mL, 1 M) and permanganate (0.5 mL, 2 M) reached an average temperature of 76.5°C at the lesion site. Conclusions: The permanganate redox reaction is a powerful system with potential to reach tumouricidal temperatures. The reagent volumes, concentrations, injection order, and substrate choice allow a measure of control over the reaction.

In vivo comparison of simultaneous versus sequential injection technique for thermochemical ablation in a porcine model

Purpose: To investigate simultaneous and sequential injection thermochemical ablation in a porcine model, and compare them to sham and acid-only ablation. Materials and methods: This IACUC-approved study involved 11 pigs in an acute setting. Ultrasound was used to guide placement of a thermocouple probe and coaxial device designed for thermochemical ablation. Solutions of 10 M acetic acid and NaOH were used in the study. Four injections per pig were performed in identical order at a total rate of 4 mL/min: saline sham, simultaneous, sequential, and acid only. Volume and sphericity of zones of coagulation were measured. Fixed specimens were examined by H&E stain. Results: Average coagulation volumes were 11.2 mL (simultaneous), 19.0 mL (sequential) and 4.4 mL (acid). The highest temperature, 81.3°C, was obtained with simultaneous injection. Average temperatures were 61.1°C (simultaneous), 47.7°C (sequential) and 39.5°C (acid only). Sphericity coefficients (0.83–0.89) had no statistically significant difference among conditions. Conclusions: Thermochemical ablation produced substantial volumes of coagulated tissues relative to the amounts of reagents injected, considerably greater than acid alone in either technique employed. The largest volumes were obtained with sequential injection, yet this came at a price in one case of cardiac arrest. Simultaneous injection yielded the highest recorded temperatures and may be tolerated as well as or better than acid injection alone. Although this pilot study did not show a clear advantage for either sequential or simultaneous methods, the results indicate that thermochemical ablation is attractive for further investigation with regard to both safety and efficacy.