Electrochemotherapy for colorectal cancer using endoscopic electroporation: a phase 1 clinical study.

Background and study aims Electrochemotherapy is an anticancer treatment that uses electric pulses to facilitate uptake of chemotherapeutic drugs in tumor cells and has proven to have a high local cytotoxic effect with minimal adverse events. Electrochemotherapy has mostly been used in treatment of cutaneous metastases but development of a new endoscopic electrode device has made treatment of colorectal tumors possible. This first-in-man multicenter phase I study investigated safety and efficacy of electrochemotherapy using endoscopic electroporation in patients with colorectal tumors. Patients and methods Seven patients with colorectal tumors who were deemed ineligible for or had declined standard treatment were included. They were treated with bleomycin either intratumorally or intravenously and the electric pulses were delivered through the endoscopic electrode device. Safety and efficacy were assessed clinically and by scans immediately after treatment and adverse events were reported. Response was evaluated up to 6 months after treatment by scans (magnetic resonance imaging or computed tomography) and endoscopic examinations. Results Seven patients aged 62 to 88 years with multiple comorbidities were included and had one or two treatments each. Post-treatment scans showed tumor responses in the treated areas and no damage to surrounding tissues. Only a few grade one adverse events were reported. Three patients had preoperative rectal bleeding, of which two reported cessation of bleeding and one reported decreased bleeding. Conclusion This first-in-man study shows that electrochemotherapy for colorectal tumors using the endoscopic electrode device can induce local tumor response and is safe also for fragile elderly patients with comorbidities.

Electrochemotherapy with cisplatin increases survival and induces immunogenic responses in murine models of lung cancer and colorectal cancer.

Electrochemotherapy is an emerging treatment modality for cancer patients which can effectively reduce tumour burden and induce immunogenic cell death. Electrochemotherapy is most commonly used with bleomycin as the drug of choice, here we examine the efficacy of electrochemotherapy with cisplatin. Electrochemotherapy with cisplatin was found to effectively reduce tumour growth in a range of murine models and induce significant intratumoural recruitment of myeloid and humoral immune cells. Following the observations of immune system mobilisation, we have shown an ability of electrochemotherapy to reduce metastatic potential as determined by tumour burden in the lung, and to exert an abscopal effect by reducing growth at distal untreated secondary tumours.

Electrochemotherapy - Emerging applications technical advances, new indications, combined approaches, and multi-institutional collaboration.

The treatment of tumors with electrochemotherapy (ECT) has surged over the past decade. Thanks to the transient cell membrane permeabilization induced by the short electric pulses used by ECT, cancer cells are exposed to otherwise poorly permeant chemotherapy agents, with consequent increased cytotoxicity. The codification of the procedure in 2006 led to a broad diffusion of the procedure, mainly in Europe, and since then, the progressive clinical experience, together with the emerging technologies, have extended the range of its application. Herein, we review the key advances in the ECT field since the European Standard Operating Procedures on ECT (ESOPE) 2006 guidelines and discuss the emerging clinical data on the new ECT indications. First, technical developments have improved ECT equipment, with custom electrode probes and dedicated tools supporting individual treatment planning in anatomically challenging tumors. Second, the feasibility and short-term efficacy of ECT has been established in deep-seated tumors, including bone metastases, liver malignancies, and pancreatic and prostate cancers (long-needle variable electrode geometry ECT), and gastrointestinal tumors (endoscopic ECT). Moreover, pioneering studies indicate lung and brain tumors as suitable future targets. A further advance relates to new combination strategies with immunotherapy, gene electro transfer (GET), calcium EP, and radiotherapy. Finally and fourth, cross-institutional collaborative groups have been established to refine procedural guidelines, promote clinical research, and explore new indications.

Combination of electroporation delivered metabolic modulators with low-dose chemotherapy in osteosarcoma.

BACKGROUND: Osteosarcoma accounts for roughly 60% of all malignant bone tumors in children and young adults. The five-year survival rate for localized tumors after surgery and chemotherapy is approximately 70% whilst it drastically reduces to 15-30% in metastatic cases. Metabolic modulation is known to increase sensitivity of cancers to chemotherapy. A novel treatment strategy in Osteosarcoma is needed to battle this devastating malady. RESULTS: Electroporation-delivered metabolic modulators were more effective in halting the cell cycle of Osteosarcoma cells and this negatively affects their ability to recover and proliferate, as shown in colony formation assays. Electroporation-delivered metabolic modulators increase the sensitivity of Osteosarcoma cells to chemotherapy and this combination reduces their survivability. CONCLUSION: This novel treatment approach highlights the efficacy of electroporation in the delivery of metabolic modulators in Osteosarcoma cells, and increased sensitivity to chemotherapy allowing for a lower dose to be therapeutic. METHODS: Metabolic modulations of two Osteosarcoma cell lines were performed with clinically available modulators delivered using electroporation, and its combination with low-dose Cisplatin. The effects of Dicholoroacetic acid, 2-Deoxy-D-glucose and Metformin on cell cycle and recovery of Osteosarcoma cells were assessed. Their sensitivity to chemotherapy was also assessed when treated in combination with electroporation-delivered metabolic modulators.

Expressional changes in stemness markers post electrochemotherapy in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. The capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Cancer stem cells exhibit great tumorigenicity and are closely correlated with drug resistance and tumor recurrence. The aim of our study was to illustrate electrochemotherapy as an effective treatment for pancreatic cancer along with the expression change in stemness genes (Nanog, Sox2 and Oct3/4) in pancreatic cancer cells post electrochemotherapy with bleomycin, cisplatin and oxaliplatin. Our results showed the enhanced expression of Nanog and decreased expression level of Oct3/4 after electrochemotherpy. We thus propose that these stemness markerS may have important roles in the initiation and/or recurrence of pancreatic cancer, and consequently may serve as important molecular diagnostics and/or therapeutic targets for the development of novel treatment strategies in pancreatic cancer patients. In conclusion, targeting these stemness factors could potentially improve electrochemotherapy as a treatment and preventing recurrence.

Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases.

Electrochemotherapy is now in routine clinical use to treat cutaneous metastases of any histology, and is listed in national and international guidelines for cutaneous metastases and primary skin cancer. Electrochemotherapy is used by dermatologists, surgeons, and oncologists, and for different degrees and manifestations of metastases to skin and primary skin tumours not amenable to surgery. This treatment utilises electric pulses to permeabilize cell membranes in tumours, thus allowing a dramatic increase of the cytotoxicity of anti-cancer agents. Response rates, often after only one treatment, are very high across all tumour types. The most frequent indications are cutaneous metastases from malignant melanoma and breast cancer. In 2006, standard operating procedures (SOPs) were written for this novel technology, greatly facilitating introduction and dissemination of the therapy. Since then considerable experience has been obtained treating a wider range of tumour histologies and increasing size of tumours which was not originally thought possible. A pan-European expert panel drawn from a range of disciplines from dermatology, general surgery, head and neck surgery, plastic surgery, and oncology met to form a consensus opinion to update the SOPs based on the experience obtained. This paper contains these updated recommendations for indications for electrochemotherapy, pre-treatment information and evaluation, treatment choices, as well as follow-up.

ICOS activation in combination with electrochemotherapy generates effective anti-cancer immunological responses in murine models of primary, secondary and metastatic disease.

Electrochemotherapy is an evolving therapy which has recently been shown to induce an immunogenic form of cell death. It is hypothesized that the immunogenic cell death induced by electrochemotherapy may compliment the responses seen with anti-cancer immunotherapies. We therefore examined the effect of electrochemotherapy in combination with ICOS activation, which promotes the activity of previously activated T cells. In comparison to either monotherapy which resulted in no curative outcomes in any model, in a CT26 primary tumour 50% of mice were cured, with 100% of cured mice surviving tumour rechallenge. In a dual flank CT26 model mimicking secondary disease 20% of mice were cured, and 30% of mice were cured using an aggressively metastatic Lewis Lung Carcinoma model. We have shown the novel combination of electrochemotherapy with ICOS activation can inhibit local and distal tumour growth, including total tumour clearance with long lasting immunological memory.

Clinical evaluation of macrophages in cancer: role in treatment, modulation and challenges.

The focus of immunotherapeutics has been placed firmly on anti-tumour T cell responses. Significant progress has been made in the treatment of both local and systemic malignancies, but low response rates and rising toxicities are limiting this approach. Advancements in the understanding of tumour immunology are opening up a new range of therapeutic targets, including immunosuppressive factors in the tumour microenvironment. Macrophages are a heterogeneous group of cells that have roles in innate and adaptive immunity and tissue repair, but become co-opted by tumours to support tumour growth, survival, metastasis and immunosuppression. Macrophages also support tumour resistance to conventional therapy. In preclinical models, interference with macrophage migration, macrophage depletion and macrophage re-education have all been shown to reduce tumour growth and support anti-tumour immune responses. Here we discuss the role of macrophages in prognosis and sensitivity to therapy, while examining the significant progress which has been made in modulating the behaviour of these cells in cancer patients.

Calcium electroporation induces tumor eradication, long-lasting immunity and cytokine responses in the CT26 colon cancer mouse model.

Electroporation is used in cancer treatment because of its ability to increase local cytotoxicity of e.g. bleomycin (electrochemotherapy) and calcium (calcium electroporation). Calcium electroporation is a novel anticancer treatment that selectively kills cancer cells by necrosis, a cell death pathway that stimulates the immune system due to high release of antigens and "danger signals." In this exploratory study, we aimed to investigate whether calcium electroporation could initiate an anticancer immune response similar to electrochemotherapy. To this end, we treated immunocompetent balb/c mice with CT26 colon tumors with calcium electroporation, electrochemotherapy, or ultrasound-based delivery of calcium or bleomycin. High treatment efficiency was observed with 100% complete remission in all four groups (12/12 with complete remission in each treatment group). In addition, none of the surviving mice from these groups formed new tumors when re-challenged with CT26 cancer cells 100-d post treatment, whereas mice challenged with different cancer cells (4T1 breast cancer) all developed tumors. Treatment of immunodeficient mice with calcium electroporation and electrochemotherapy showed no long-lasting tumor response. Calcium electroporation and electrochemotherapy was associated with a release of High Mobility Group Box 1 protein (HMGB1) in vitro (p = 0.029) and a significant increase of the overall systemic level of pro-inflammatory cytokines in serum from the treated mice (p < 0.003). These findings indicate that calcium electroporation as well as electrochemotherapy could have a role as immune stimulators in future treatments.

Effective treatment of intractable cutaneous metastases of breast cancer with electrochemotherapy: Ten-year audit of single centre experience.

PURPOSE: Electrochemotherapy (ECT) is the application of electric pulses to tumour tissue to render the cell membranes permeable to usually impermeant hydrophilic anti-cancer drugs, thereby enhancing cytotoxic effects. We sought to ascertain whether ECT can be an effective palliative treatment for cutaneous metastases of breast cancer. METHODS: This work reports data from the European Standard Operating Procedures for Electrochemotherapy trial (EudraCT Number: 2004-002183-18). In combination with systemic and/or intratumoural bleomycin, optimised electric pulses were delivered to locally recurrent or metastatic cutaneous breast cancer lesions. Follow-up continued until December 2014. RESULTS: Between February 2004 and December 2014, twenty-four patients were treated. All patients had received prior multimodal therapy. In total, the patient cohort had, or developed, 242 lesions. Two hundred and 36 lesions were treated, with 34 lost to follow-up. An objective response was seen in 161 of 202 lesions (79.7%), with a complete response observed in 130 (64.3%). Thirty-nine lesions (19.3%) did not respond, while 2 (1%) progressed following ECT. 17 (73.9%) patients received two or fewer treatments. A minimum of a partial response was seen in at least 50% of treated lesions in 18 of the 24 (75%) patients. Smaller lesions were more likely to have an objective response (Chi-square test for trend, p < 0.001). CONCLUSIONS: Electrochemotherapy is an effective treatment for cutaneous breast cancer lesions that have proven refractory to standard therapies. As smaller lesions were found to be more responsive, we suggest that ECT should be considered as an early treatment modality, within multimodal treatment strategies.

Antitumour responses induced by a cell-based Reovirus vaccine in murine lung and melanoma models.

BACKGROUND: The ever increasing knowledge in the areas of cell biology, the immune system and the mechanisms of cancer are allowing a new phase of immunotherapy to develop. The aim of cancer vaccination is to activate the host immune system and some success has been observed particularly in the use of the BCG vaccine for bladder cancer as an immunostimulant. Reovirus, an orphan virus, has proven itself as an oncolytic virus in vitro and in vivo. Over 80 % of tumour cell lines have been found to be susceptible to Reovirus infection and it is currently in phase III clinical trials. It has been shown to induce immune responses to tumours with very low toxicities. METHODS: In this study, Reovirus was examined in two main approaches in vivo, in mice, using the melanoma B16F10 and Lewis Lung Carcinoma (LLC) models. Initially, mice were treated intratumourally (IT) with Reovirus and the immune responses determined by cytokine analysis. Mice were also vaccinated using a cell-based Reovirus vaccine and subsequently exposed to a tumourigenic dose of cells (B16F10 or LLC). Using the same cell-based Reovirus vaccine, established tumours were treated and subsequent immune responses and virus retrieval investigated. RESULTS: Upregulation of several cytokines was observed following treatment and replication-competent virus was also retrieved from treated tumours. Varying levels of cytokine upregulation were observed and no replication-competent virus was retrieved in vaccine-treated mice. Prolongation of survival and delayed tumour growth were observed in all models and an immune response to Reovirus, either using Reovirus alone or a cell-based vaccine was also observed in all mice. CONCLUSION: This study provides evidence of immune response to tumours using a cell-based Reovirus vaccine in both tumour models investigated, B16F10 and LLC, cytokine induction was observed with prolongation of survival in almost all cases which may suggest a new method for using Reovirus in the clinic.

Glycolysis inhibition as a cancer treatment and its role in an anti-tumour immune response.

Increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for ATP generation. Altered energy metabolism is a biochemical fingerprint of cancer cells that represents one of the "hallmarks of cancer". The immune system can prevent tumour growth by eliminating cancer cells but this editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour growth. In this review we look at the glycolysis pathway as a target for cancer treatments. We also examine the interplay between the glycolysis modulation and the immune response as an anti-cancer therapy.

Preclinical evaluation of an endoscopic electroporation system.

BACKGROUND AND STUDY AIMS: Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. METHODS: Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. RESULTS: Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. CONCLUSIONS: Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.

Local tumour ablative therapies: opportunities for maximising immune engagement and activation.

The relationship between cancer and the immune system is a complex one. The immune system can prevent tumour growth by eliminating cancer cells but this editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour growth. In light of this, the focus of cancer treatment should be to maximise cancer elimination and the prevention of escape mechanisms. In this review we will examine current and emerging ablative treatment modalities that induce Immunogenic Cell Death (ICD), a special type of cell death that allows for immune cell involvement and the generation of an anti-tumour specific immune response. When paired with immune modulating agents, capable of potentiating the immune response and reversing the immune-suppressive environment created by tumours, we may be looking at the future of anti-cancer therapy.

Releasing pressure in tumors: what do we know so far and where do we go from here? A review.

Tumor interstitial pressure is a fundamental feature of cancer biology. Elevation in tumor pressure affects the efficacy of cancer treatment. It causes heterogenous intratumoral distribution of drugs and macromolecules. It also causes the development of hypoxia within tumor bulk, leading to reduced efficacy of therapeutic drugs and radiotherapy. Tumor pressure has been associated with increased metastatic potential and poor prognosis in some tumors. The formation of increased pressure in solid tumors is multifactorial. Factors known to affect tumor pressure include hyperpermeable tortuous tumor vasculatures, the lack of functional intratumoral lymphatic vessels, abnormal tumor microenvironment, and the solid stress exerted by proliferating tumor cells. Reducing this pressure is known to enhance the uptake and homogenous distribution of many therapies. Pharmacologic and biologic agents have been shown to reduce tumor pressure. These include antiangiogenic therapy, vasodilatory agents, antilymphogenic therapy, and proteolytic enzymes. Physical manipulation has been shown to cause reduction in tumor pressure. These include irradiation, hyperbaric oxygen therapy, hyper- or hypothermic therapy, and photodynamic therapy. This review explores the methods to reduce tumor pressure that may open up new avenues in cancer treatment.

Development and characterization of an enhanced nonviral expression vector for electroporation cancer treatment.

Nonviral plasmid DNA gene therapy represents a promising approach for the treatment of many diseases including cancer. Intracellular delivery of DNA can be achieved with the application of electroporation, which facilitates the initial transport of exogenous DNA across the cell membrane into the cytoplasm. However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches. To overcome these expression difficulties, we developed a proof-of-concept vector enhanced expression vector (EEV), which incorporates elements from viral systems including nuclear localization sequences and a viral replicase from the Semliki Forest virus. The replicase allows for cytoplasmic mRNA expression and bypasses the need for nuclear localization to generate high levels of gene expression. We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types. Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model. Using a novel endoscopic electroporation system, EndoVe, we demonstrate and compare, for the first time, both standard cytomegalovirus (CMV) promoter-driven plasmid and EEV gene expression in intraluminal porcine tissues. Our EEV plasmid displays reliable and superior expression capability, and due to its inherent induced oncolytic activity in transfected cells, it may enhance the efficacy and safety of several cancer immunogene therapy approaches.

Review of current thermal ablation treatment for lung cancer and the potential of electrochemotherapy as a means for treatment of lung tumours.

Lung cancer remains the most common cancer diagnosed worldwide and has one of the lowest survival rates of all cancers. Surgery remains the only curative treatment option but because most patients are either diagnosed at advanced stages or are unfit for surgery, less than a third of all lung cancer patients will undergo a surgical resection. Thermal ablation has emerged as an alternative option in patients who are unfit to undergo surgery. Thermal ablative therapies used in clinical practice to date include Radiofrequency Ablation (RFA), Microwave Ablation (MWA) and Cryoablation This article will focus on the advantages and limitations of thermal ablative therapy and investigates the potential of a relatively new treatment modality, Electrochemotherapy (ECT), as a novel treatment for lung cancer.

Can non-viral technologies knockdown the barriers to siRNA delivery and achieve the next generation of cancer therapeutics?

Cancer is one of the most wide-spread diseases of modern times, with an estimated increase in the number of patients diagnosed worldwide, from 11.3 million in 2007 to 15.5 million in 2030 (www.who.int). In many cases, due to the delay in diagnosis and high increase of relapse, survival rates are low. Current therapies, including surgery, radiation and chemotherapy, have made significant progress, but they have many limitations and are far from ideal. Although immunotherapy has recently offered great promise as a new approach in cancer treatment, it is still very much in its infancy and more information on this approach is required before it can be widely applied. For these reasons effective, safe and patient-acceptable cancer therapy is still largely an unmet clinical need. Recent knowledge of the genetic basis of the disease opens up the potential for cancer gene therapeutics based on siRNA. However, the future of such gene-based therapeutics is dependent on achieving successful delivery. Extensive research is ongoing regarding the design and assessment of non-viral delivery technologies for siRNA to treat a wide range of cancers. Preliminary results on the first human Phase I trial for solid tumours, using a targeted non-viral vector, illustrate the enormous therapeutic benefits once the issue of delivery is resolved. In this review the genes regulating cancer will be discussed and potential therapeutic targets will be identified. The physiological and biochemical changes caused by tumours, and the potential to exploit this knowledge to produce bio-responsive 'smart' delivery systems, will be evaluated. This review will also provide a critical and comprehensive overview of the different non-viral formulation strategies under investigation for siRNA delivery, with particular emphasis on those designed to exploit the physiological environment of the disease site. In addition, a section of the review will be dedicated to pre-clinical animal models used to evaluate the stability, safety and efficacy of the delivery systems.

Sonoporation mediated immunogene therapy of solid tumors.

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm(2) at 20% duty cycle for 5 min (60 J/cm(2)). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches.

Electrochemotherapy as an adjunct or alternative to other treatments for unresectable or in-transit melanoma.

Treatment of recurrent or in-transit unresectable melanoma continues to be a major therapeutic challenge. Electrochemotherapy (ECT) is a therapeutic option for those patients whose lesions are not suitable for surgical resection and who have exhausted all other treatment modalities. ECT combines electroporation of tumor cells with the administration either of intravenous or intratumoral antineoplastic drugs, such as bleomycin or cisplatin. The cell membranes are thus rendered permeant to these impermeant hydrophilic drugs with a several hundred-fold increase in intracellular delivery and cytotoxicity. ECT is an effective treatment in the palliative management of unresectable recurrent disease with overall objective response rates of approximately 80-90%. ECT technology continues to evolve allowing application to deeper lesions. By combining ECT with tumor-specific immunostimulating approaches, such as perilesional IL-2, CpG oligonucleotides or prior immunogene therapy, there is a promise of both local and systemic control of this disease.