John H. Rossmeisl

Successful Treatment of a Large Soft Tissue Sarcoma With Irreversible Electroporation

Introduction Irreversible electroporation (IRE) is a promising technique for the focal treatment of pathologic tissues that involves placing minimally invasive electrodes within the targeted region. A series of short, intense electric pulses are then applied to destabilize the cell membrane, presumably by creating nanopores, inducing cell death in a nonthermal manner. The unique therapeutic mechanism of IRE does not rely on tissue temperature changes, as with hyperthermic or cryoablative procedures. Therefore, IRE preserves the extracellular matrix, major tissue vasculature, and other sensitive structures. Treated regions resolve rapidly, with submillimeter resolution between treated and unaffected cells, and are predictable with numerical modeling. Treatments promote an immune response, are unaltered by blood flow, can be administered quickly (approximately 5 minutes), and can be visualized in real time. IRE has been studied extensively in healthy tissue, and tumors have been treated with IRE in mice. IRE has been attempted in humans for prostate, lung, kidney, and liver cancers. Human treatments revealed negligible postablation pain and the ability to apply the pulses in proximity to vital structures. Overall, assessment of the therapeutic efficacy of IRE remains in its infancy. We hypothesize that IRE treatments can be designed and implemented to successfully treat soft tissue malignancies, including large and complex tumors, a crucial step for translation of the technology into routine clinical use. Here we report our treatment of a focal histiocytic sarcoma of the coxofemoral joint in a canine patient. Follow-up examinations demonstrated prolonged relief of cancerassociated pain, preservation of pelvic limb function, and complete tumor regression 6 months after initial treatment.

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

BackgroundTherapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.MethodsA combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.ResultsNo visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.ConclusionsH-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.

A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure

BackgroundIrreversible electroporation (IRE) is a new minimally invasive technique to kill undesirable tissue in a non-thermal manner. In order to maximize the benefits from an IRE procedure, the pulse parameters and electrode configuration must be optimized to achieve complete coverage of the targeted tissue while preventing thermal damage due to excessive Joule heating.MethodsWe developed numerical simulations of typical protocols based on a previously published computed tomographic (CT) guided in vivo procedure. These models were adapted to assess the effects of temperature, electroporation, pulse duration, and repetition rate on the volumes of tissue undergoing IRE alone or in superposition with thermal damage.ResultsNine different combinations of voltage and pulse frequency were investigated, five of which resulted in IRE alone while four produced IRE in superposition with thermal damage.ConclusionsThe parametric study evaluated the influence of pulse frequency and applied voltage on treatment volumes, and refined a proposed method to delineate IRE from thermal damage. We confirm that determining an IRE treatment protocol requires incorporating all the physical effects of electroporation, and that these effects may have significant implications in treatment planning and outcome assessment. The goal of the manuscript is to provide the reader with the numerical methods to assess multiple-pulse electroporation treatment protocols in order to isolate IRE from thermal damage and capitalize on the benefits of a non-thermal mode of tissue ablation.

MAGNETIC RESONANCE IMAGING FEATURES OF INTRACRANIAL ASTROCYTOMAS AND OLIGODENDROGLIOMAS IN DOGS

Astrocytomas and oligodendrogliomas represent one third of histologically confirmed canine brain tumors. Our purpose was to describe the magnetic resonance (MR) imaging features of histologically confirmed canine intracranial astrocytomas and oligodendrogliomas and to examine for MR features that differentiate these tumor types. Thirty animals with confirmed astrocytoma (14) or oligodendroglioma (16) were studied. All oligodendrogliomas and 12 astrocytomas were located in the cerebrum or thalamus, with the remainder of astrocytomas in the cerebellum or caudal brainstem. Most (27/30) tumors were associated with both gray and white matter. The signal characteristics of both tumor types were hypointense on T1-weighted images (12 each) and hyperintense on T2-weighted images (11/14 astrocytomas, 12/16 oligodendrogliomas). For astrocytomas and oligodendrogliomas, respectively, common findings were contrast enhancement (10/13, 11/15), ring-like contrast enhancement (6/10, 9/11), cystic regions within the mass (7/14, 12/16), and hemorrhage (4/14, 6/16). Oligodendrogliomas were significantly more likely to contact the brain surface (meninges) than astrocytomas (14/16, 7/14, respectively, P=0.046). Contact with the lateral ventricle was the most common finding, occurring in 13/14 astrocytomas and 14/16 oligodendrogliomas. No MR features were identified that reliably distinguished between these two tumor types. Contrast enhancement was more common in high-grade tumors (III or IV) than low-grade tumors (II, P=0.008).

Survival time following hospital discharge in dogs with palliatively treated primary brain tumors

OBJECTIVE To analyze survival time and identify prognostic factors associated with outcome following discharge in dogs with primary brain tumors treated palliatively. DESIGN Prospective case series. ANIMALS 51 dogs with 5 histopathologic types of brain tumors. PROCEDURES Owners with dogs examined from 2004 to 2008 were invited to participate if dogs had CT or MRI evidence of a brain mass that was histopathologically confirmed as a neoplasm upon death, dogs survived for ≥ 48 hours after hospital discharge, and treatments following discharge were limited to administration of prednisone or phenobarbital. Prognostic factors, including signalment, clinical signs (including duration), tumor type, tumor location, degree of peritumoral edema, lesion burden, and prescribed treatment, were evaluated. Survival time was estimated and animal- and tumor-specific variables evaluated as potential prognostic factors. RESULTS The median survival time in all dogs was 69 days (95% confidence interval [CI], 18 to 201 days). Multivariate analyses identified neuroanatomic location as the only significant prognostic variable, with the survival time of dogs with infratentorial tumors (n = 18) being significantly shorter (median, 28 days; 95% CI, 19 to 68 days) than survival time of dogs with supratentorial (33) tumors (median, 178 days; 95% CI, 119 to 270 days). Seizures were the most common clinical sign associated with supratentorial tumors (24/33 [73%]) and central vestibular dysfunction with infratentorial tumors (12/18). CONCLUSIONS AND CLINICAL RELEVANCE Dogs with palliatively treated primary brain tumors, particularly those with tumors in the cerebellum, pons, or medulla, had a poor prognosis. However, dogs with supratentorial tumors had survival times > 3 months.

Vestibular Disease in Dogs and Cats

The vestibular system is the major sensory (special proprioceptive) system that, along with the general proprioceptive and visual systems, maintains balance. Clinical signs of vestibular disease include asymmetric ataxia, head tilt, and pathologic nystagmus. Neuroanatomic localization of observed vestibular signs to either the peripheral or central components of the vestibular system is paramount to the management of the patient with vestibular dysfunction, as the etiology, diagnostic approaches, and prognoses are dependent on the neuroanatomic diagnosis. This article reviews functional vestibular neuroanatomy as well as the diagnosis and treatment of common causes of small animal vestibular disease.

7.0-T magnetic resonance imaging characterization of acute blood-brain-barrier disruption achieved with intracranial irreversible electroporation.

The blood-brain-barrier (BBB) presents a significant obstacle to the delivery of systemically administered chemotherapeutics for the treatment of brain cancer. Irreversible electroporation (IRE) is an emerging technology that uses pulsed electric fields for the non-thermal ablation of tumors. We hypothesized that there is a minimal electric field at which BBB disruption occurs surrounding an IRE-induced zone of ablation and that this transient response can be measured using gadolinium (Gd) uptake as a surrogate marker for BBB disruption. The study was performed in a Good Laboratory Practices (GLP) compliant facility and had Institutional Animal Care and Use Committee (IACUC) approval. IRE ablations were performed in vivo in normal rat brain (n = 21) with 1-mm electrodes (0.45 mm diameter) separated by an edge-to-edge distance of 4 mm. We used an ECM830 pulse generator to deliver ninety 50-μs pulse treatments (0, 200, 400, 600, 800, and 1000 V/cm) at 1 Hz. The effects of applied electric fields and timing of Gd administration (−5, +5, +15, and +30 min) was assessed by systematically characterizing IRE-induced regions of cell death and BBB disruption with 7.0-T magnetic resonance imaging (MRI) and histopathologic evaluations. Statistical analysis on the effect of applied electric field and Gd timing was conducted via Fit of Least Squares with α = 0.05 and linear regression analysis. The focal nature of IRE treatment was confirmed with 3D MRI reconstructions with linear correlations between volume of ablation and electric field. Our results also demonstrated that IRE is an ablation technique that kills brain tissue in a focal manner depicted by MRI (n = 16) and transiently disrupts the BBB adjacent to the ablated area in a voltage-dependent manner as seen with Evans Blue (n = 5) and Gd administration.

Improved Local and Systemic Anti-Tumor Efficacy for Irreversible Electroporation in Immunocompetent versus Immunodeficient Mice

Irreversible electroporation (IRE) is a non-thermal focal ablation technique that uses a series of brief but intense electric pulses delivered into a targeted region of tissue, killing the cells by irrecoverably disrupting cellular membrane integrity. This study investigates if there is an improved local anti-tumor response in immunocompetent (IC) BALB/c versus immunodeficient (ID) nude mice, including the potential for a systemic protective effect against rechallenge. Subcutaneous murine renal carcinoma tumors were treated with an IRE pulsing protocol that used 60% of the predicted voltage required to invoke complete regressions in the ID mice. Tumors were followed for 34 days following treatment for 11 treated mice from each strain, and 7 controls from each strain. Mouse survival based on tumor burden and the progression-free disease period was substantially longer in the treated IC mice relative to the treated ID mice and sham controls for both strains. Treated IC mice were rechallenged with the same cell line 18 days after treatment, where growth of the second tumors was shown to be significantly reduced or prevented entirely. There was robust CD3+ cell infiltration in some treated BALB/C mice, with immunocytes focused at the transition between viable and dead tumor. There was no difference in the low immunocyte presence for untreated tumors, nude mice, and matrigel-only injections in both strains. These findings suggest IRE therapy may have greater therapeutic efficacy in immunocompetent patients than what has been suggested by immunodeficient models, and that IRE may invoke a systemic response beyond the targeted ablation region.

New Agents for Targeting of IL-13RA2 Expressed in Primary Human and Canine Brain Tumors

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts.

Clinical signs, risk factors, and outcomes associated with bromide toxicosis (bromism) in dogs with idiopathic epilepsy

OBJECTIVE To evaluate clinical signs, risk factors, and outcomes associated with bromide toxicosis (bromism) in dogs with idiopathic epilepsy treated with potassium or sodium bromide. DESIGN Retrospective case-control study. ANIMALS 83 clinically ill epileptic dogs with (cases; n = 31) and without (controls; 52) bromism. PROCEDURES Medical records were reviewed for information regarding signalment, epilepsy history, treatment, diet, clinicopathologic test results, concurrent diseases, clinical signs, and outcome. Case and control dogs were matched by the veterinary hospitals from which they were referred and by month of admission. A presumptive diagnosis of bromism was made in case dogs when treatment for primary clinical signs was limited to induction of diuresis or reduction in the dose of bromide administered, and this diagnosis was supported by serum bromide concentrations. Potential risk factors for bromism were identified via univariate and subsequent multivariate logistic regression analyses. RESULTS Common clinical signs of bromism included alterations in consciousness, ataxia, and upper and lower motor neuron tetraparesis and paraparesis. The multivariate analysis identified bromide dose at admission to the hospital as the only factor significantly associated with bromism. In all dogs with bromism, treatment via dose reduction or facilitated renal excretion of bromide resulted in rapid clinical improvement, although breakthrough seizures happened during treatment in 8 of 31 (26%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE Bromism is a clinically heterogeneous, dose-dependent neurotoxicosis that is largely reversible with treatment. Regular serial monitoring of serum bromide concentrations is recommended to optimize anticonvulsant treatment in dogs with idiopathic epilepsy.