Treatment of vulvar cancer recurrence with electrochemotherapy: a case-control study.

BACKGROUND: Electrochemotherapy (ECT) is a combined treatment method based on electroporation and simultaneous chemotherapy. In cases where radiotherapy has previously been used, surgery is often the only treatment option for vulvar cancer recurrence with potential resection of clitoris, vagina, urethra or anal sphincter. The unique advantage of ECT is its selectivity for cancer cells while sparing the surrounding healthy tissue. The aim of the study was to compare the ECT treatment of vulvar cancer recurrence for non-palliative purposes with surgical treatment. MATERIALS AND METHODS: Eleven patients with single vulvar cancer recurrence were treated with ECT and followed up for 12 months. As a control group, 15 patients with single vulvar cancer recurrence were treated with wide local excision. The following data were collected, analyzed and compared: Age, body mass index, comorbidities, histological type, location and size of vulvar cancer recurrence, treatment history, details of procedures and hospital stay. RESULTS: The probability curves for local tumor control did not differ between the ECT group and the surgical group (p = 0.694). The mean hospital stay and the mean duration of procedure were statistically significantly shorter in the ECT group (p < 0.001). There were no statistically significant differences between the ECT and surgical groups in terms of mean body mass index, associated diseases, previous treatments, presence of lichen sclerosus, p16 status, gradus, anatomical site of the tumor, and type of anesthesia. CONCLUSION: In this case-control study, treatment of vulvar cancer recurrence with ECT for non-palliative purposes was comparable to surgical treatment in terms of effectiveness. The results need to be confirmed in larger randomized trials.

An Explorative Study on Calcium Electroporation for Low-risk Basal Cell Carcinoma.

In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.

The equivalence of different types of electric pulses for electrochemotherapy with cisplatin - an in vitro study.

BACKGROUND: Electrochemotherapy (ECT) is a treatment involving the administration of chemotherapeutics drugs followed by the application of 8 square monopolar pulses of 100 µs duration at a repetition frequency of 1 Hz or 5000 Hz. However, there is increasing interest in using alternative types of pulses for ECT. The use of high-frequency short bipolar pulses has been shown to mitigate pain and muscle contractions. Conversely, the use of millisecond pulses is interesting when combining ECT with gene electrotransfer for the uptake of DNA-encoding proteins that stimulate the immune response with the aim of converting ECT from a local to systemic treatment. Therefore, the aim of this study was to investigate how alternative types of pulses affect the efficiency of the ECT. MATERIALS AND METHODS: We performed in vitro experiments, exposing Chinese hamster ovary (CHO) cells to conventional ECT pulses, high-frequency bipolar pulses, and millisecond pulses in the presence of different concentrations of cisplatin. We determined cisplatin uptake by inductively coupled plasma mass spectrometry and cisplatin cytotoxicity by the clonogenic assay. RESULTS: We observed that the three tested types of pulses potentiate the uptake and cytotoxicity of cisplatin in an equivalent manner, provided that the electric field is properly adjusted for each pulse type. Furthermore, we quantified that the number of cisplatin molecules, resulting in the eradication of most cells, was 2-7 × 107 per cell. CONCLUSIONS: High-frequency bipolar pulses and millisecond pulses can potentially be used in ECT to reduce pain and muscle contraction and increase the effect of the immune response in combination with gene electrotransfer, respectively.

Integrated Time Nanosecond Pulse Irreversible Electroporation (INSPIRE): Assessment of Dose, Temperature, and Voltage on Experimental and Clinical Treatment Outcomes.

OBJECTIVE: This study sought to investigate a novel strategy using temperature-controlled delivery of nanosecond pulsed electric fields as an alternative to the 50-100 microsecond pulses used for irreversible electroporation. METHODS: INSPIRE treatments were carried out at two temperatures in 3D tumor models using doses between 0.001 s and 0.1 s. The resulting treatment zones were quantified using viability staining and lethal electric field intensities were determined numerically. Computational modeling was then used to determine parameters necessary for INSPIRE treatments to achieve equivalent treatment zones to clinical electroporation treatments and evaluate the potential for these treatments to induce deleterious thermal damage. RESULTS: Lethal thresholds between 1109 and 709 V/cm were found for nominal 0.01 s treatments with pulses between 350 ns and 2000 ns at physiological temperatures. Further increases in dose resulted in significant decreases in lethal thresholds. Given these experimental results, treatment zones comparable to clinical electroporation are possible by increasing the dose and voltage used with nanosecond duration pulses. Temperature-controlled simulations indicate minimal thermal cell death while achieving equivalent treatment volumes to clinical electroporation. CONCLUSION: Nanosecond electrical pulses can achieve comparable outcomes to traditional electroporation provided sufficient electrical doses or voltages are applied. The use of temperature-controlled delivery may minimize thermal damage during treatment. SIGNIFICANCE: Intense muscle stimulation and the need for cardiac gating have limited irreversible electroporation. Nanosecond pulses can alleviate these challenges, but traditionally have produced significantly smaller treatment zones. This study suggests that larger ablation volumes may be possible with the INSPIRE approach and that future in vivo studies are warranted.

Intraoperative electrochemotherapy of the posterior resection surface after pancreaticoduodenectomy: Preliminary results of a hybrid approach treatment of pancreatic cancer.

BACKGROUND: Despite extensive research in recent decades, pancreatic cancer continues to be among the most lethal forms of cancer, with no substantial increase in survival rates. Local recurrences account for approximately 30 per cent of all disease recurrences. With the intent to improve survival, we designed a novel, hybrid treatment strategy consisting of surgical resection and additional intraoperative electrochemotherapy of the posterior resection surface. We present the study protocols and preliminary findings of a prospective pilot study investigating this treatment approach. METHODS: Consenting patients with resectable pancreatic head ductal adenocarcinoma who met the inclusion criteria were enrolled in the study. After surgical resection, electrochemotherapy with bleomycin was performed using plate electrodes to cover the area between anatomical landmarks. RESULTS: Electrochemotherapy of the posterior resection surface was feasible in all 7 patients. We observed pancreatic fistula grade B in only one patient; all other noted complications were Clavien-Dindo grade 2 or less. The hospital mortality was 0%. CONCLUSIONS: Our preliminary results suggest that a hybrid approach combining surgery with intraoperative electrochemotherapy is safe and feasible.

Negative effects of cancellation during nanosecond range High-Frequency calcium based electrochemotherapy in vitro.

Drug delivery using nanosecond pulsed electric fields is a new branch of electroporation-based treatments, which potentially can substitute European standard operating procedures for electrochemotherapy. In this work, for the first time, we characterize the effects of ultra-fast repetition frequency (1-2.5 MHz) nanosecond pulses (5-9 kV/cm, 200 and 400 ns) in the context of nano-electrochemotherapy with calcium. Additionally, we investigate the feasibility of bipolar symmetric (↑200 ns + ↓200 ns) and asymmetric (↑200 ns + ↓400 ns) nanosecond protocols for calcium delivery. The effects of bipolar cancellation and the influence of interphase delay (200 ns) are overviewed. Human lung cancer cell lines A549 and H69AR were used as a model. It was shown that unipolar pulses delivered at high frequency are effective for electrochemotherapy with a significant improvement in efficiency when the delay between separate pulses is reduced. Bipolar symmetric pulses trigger the cancellation phenomenon limiting applications for drug delivery and can be compensated by the asymmetry of the pulse (↑200 ns + ↓400 ns or ↑400 ns + ↓200 ns). The results of this study can be successfully used to derive a new generation of nsPEF protocols for successful electrochemotherapy treatments.

Evaluation of the safety and feasibility of electrochemotherapy with intravenous bleomycin as local treatment of bladder cancer in dogs.

Local treatment of canine urothelial carcinoma (UC) of the bladder is a challenge. More than 90% of the cases invade the muscular layer, more than 50% develop on bladder sites with a difficult surgical approach and often requiring radical surgical procedures. This study aims to evaluate the safety and feasibility of electrochemotherapy (ECT) with intravenous bleomycin (BLM) as a local therapy for bladder UC. This prospective study included 21 dogs with spontaneous bladder UC. Regional/distant metastases and neoplastic infiltration of the serosa was considered the main exclusion criteria. We had no deaths during ECT or in the immediate postoperative period, and no suture dehiscence. Most dogs (19/21) developed mild adverse effects, whereas two dogs developed ureteral stenosis. Complete response (CR) was achieved in 62% of the cases (13/21), while partial response (PR) was achieved in 24% (5/21). The median survival and disease-free survival times were 284 and 270 days, respectively. Overall survival was significantly better in the dogs who achieved a CR. In conclusion, ECT was well-tolerated in dogs with UC, demonstrating its safety and feasibility. These data pave the way for new studies aimed at evaluating the effectiveness of ECT in canine bladder UC as a translational model for human disease.

Effects of pulse repetition rate in static electrochemotherapy models.

Electroporation alters cell membrane structure and tissue electrical properties by short and intense pulsed electric fields (PEF). Static mathematical models are often used to explain the change in electrical properties of tissues caused by electroporation. Electric pulse repetition rate may play an important role, as tissue dielectric dispersion, electroporation dynamics, and Joule heating may affect the electrical properties. In this work, we investigate the effects on the magnitude of the electric current when the repetition rate of the standard electrochemotherapy protocol is increased. Liver, oral mucosa, and muscle tissues were studied. Ex vivo animal experiments show that the magnitude of the electric current increases when the repetition rate is changed from 1 Hz to 5 kHz (10.8% for liver, 5.8% for oral mucosa, and 4.7% for muscle). Although a correction factor could reduce the error to less than 1%, dynamic models seem to be necessary to analyze different protocol signatures. Authors should be aware that static models and experimental results can only be compared if they use exactly the same PEF signature. The repetition rate is a key information to consider in the pretreatment computer study because the current at 1 Hz PEF differs from a 5 kHz PEF.

Influence of electric field, blood velocity, and pharmacokinetics on electrochemotherapy efficiency.

The convective delivery of chemotherapeutic drugs in cancerous tissues is directly proportional to the blood perfusion rate, which in turns can be transiently reduced by the application of high-voltage and short-duration electric pulses due to vessel vasoconstriction. However, electric pulses can also increase vessel wall and cell membrane permeabilities, boosting the extravasation and cell internalization of drug. These opposite effects, as well as possible adverse impacts on the viability of tissues and endothelial cells, suggest the importance of conducting in silico studies about the influence of physical parameters involved in electric-mediated drug transport. In the present work, the global method of approximate particular solutions for axisymmetric domains, together with two solution schemes (Gauss-Seidel iterative and linearization+successive over-relaxation), is applied for the simulation of drug transport in electroporated cancer tissues, using a continuum tumor cord approach and considering both the electropermeabilization and vasoconstriction phenomena. The developed global method of approximate particular solutions algorithm is validated with numerical and experimental results previously published, obtaining a satisfactory accuracy and convergence. Then, a parametric study about the influence of electric field magnitude and inlet blood velocity on the internalization efficacy, drug distribution uniformity, and cell-kill capacity of the treatment, as expressed by the number of internalized moles into viable cells, homogeneity of exposure to bound intracellular drug, and cell survival fraction, respectively, is analyzed for three pharmacokinetic profiles, namely one-short tri-exponential, mono-exponential, and uniform. According to numerical results, the trade-off between vasoconstriction and electropermeabilization effects and, consequently, the influence of electric field magnitude and inlet blood velocity on the assessment parameters considered here (efficacy, uniformity, and cell-kill capacity) is different for each pharmacokinetic profile deemed.

Electrochemotherapy vs radiotherapy in the treatment of primary cutaneous malignancies or cutaneous metastases from primary solid organ malignancies: A systematic review and narrative synthesis.

BACKGROUND: Electrochemotherapy has gained international traction and commendation in national guidelines as an effective tool in the management of cutaneous malignancies not amenable to surgical resection. Despite this, no level 5 evidence exists comparing it to radiotherapy in the treatment of cutaneous malignancies. This systematic review aimed to examine the literature directly and indirectly comparing electrochemotherapy and radiotherapy in the treatment of primary cutaneous malignancies or cutaneous metastases from primary solid organ malignancies. MATERIALS & METHODS: The protocol for this review was registered on the PROSPERO International Prospective Register of Systematic Reviews with the protocol ID CRD42021285415. Searches of MEDLINE, Embase, CINAHL, CENTRAL and ClinicalTrials.gov databases were undertaken from database inception to 28 December 2021. Studies in humans comparing treatment with electrochemotherapy to radiotherapy and reporting tumour response with a minimum four week follow-up were eligible. Risk of bias was assessed using the ROBINS-I tool. Results are provided as a narrative synthesis. RESULTS: Two case series with a total of 92 patients were identified as relevant to this study. Both case series examined patients with cutaneous squamous cell carcinoma. One case series examined elderly patients with predominantly head/neck lesions. The other examined younger patients with predominantly limb lesions who had cutaneous squamous cell carcinoma directly attributable to a rare skin condition. CONCLUSION: There is little literature presenting comparative data for electrochemotherapy and radiotherapy in the treatment of primary cutaneous malignancies or cutaneous metastases. Included studies were marred by serious risk of bias particularly due to confounding. The inherent bias and heterogeneity of the included studies precluded synthesis of a consolidated comparison of clinical outcomes between the two therapies. Further research is required in this domain in the form of clinical trials and observational studies to inform guidelines for electrochemotherapy treatment.

Research frontiers of electroporation-based applications in cancer treatment: a bibliometric analysis.

OBJECTIVES: Electroporation, the breakdown of the biomembrane induced by external electric fields, has increasingly become a research hotspot for its promising related methods in various kinds of cancers. CONTENT: In this article, we utilized CiteSpace 6.1.R2 to perform a bibliometric analysis on the research foundation and frontier of electroporation-based applications in cancer therapy. A total of 3,966 bibliographic records were retrieved from the Web of Science Core Collection for the bibliometric analysis. Sersa G. and Mir L. M. are the most indispensable researchers in this field, and the University of Ljubljana of Slovenia is a prominent institution. By analyzing references and keywords, we found that, with a lower recurrence rate, fewer severe adverse events, and a higher success rate, irreversible electroporation, gene electrotransfer, and electrochemotherapy are the three main research directions that may influence the future treatment protocol of cancers. SUMMARY: This article visualized relevant data to synthesize scientific research on electroporation-based cancer therapy, providing helpful suggestions for further investigations on electroporation. OUTLOOK: Although electroporation-based technologies have been proven as promising tools for cancer treatment, its radical mechanism is still opaque and their commercialization and universalization need further efforts from peers.

Electrochemotherapy of Melanoma Cutaneous Metastases in Organ Transplant Recipients: A Systematic Review of Preclinical and Clinical Studies.

Cutaneous melanoma is a highly aggressive form of skin cancer. The development of immune checkpoint inhibitors (ICIs) has revolutionized the management of advanced melanoma, led to durable responses, and improved overall survival. However, the success of ICIs in melanoma treatment is influenced by the tumor microenvironment (TME) which plays a critical role in regulating the immune response to the tumor. Understanding the mechanisms underlying this interaction is crucial to optimizing the efficiency of ICIs. Electrochemotherapy (ECT) has been shown to enhance the efficacy of ICIs in melanoma treatment by inducing tumor cell death and facilitating the release of tumor antigens which can subsequently be recognized and targeted by the immune system. Moreover, ECT has been reported to modulate the TME, leading to increased infiltration of immune cells and a more favorable immunological profile. In this review, we summarize the available knowledge of changes in TME after ECT of melanoma cutaneous metastasis and highlight the differences in tumor-infiltrating immune cells between immunocompetent and immunosuppressed organisms. In addition, we showed that ECT can be an effective and safe procedure for organ transplant recipients. Furthermore, repeated ECT may enhance immune activation and probably induce a bystander effect by trained immunity.

Treatment of spontaneous canine mast cell tumors by electrochemotherapy combined with IL-12 gene electrotransfer: Comparison of intratumoral and peritumoral application of IL-12.

The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria.

Engineering high post-electroporation viabilities and transfection efficiencies for elongated cells on suspended nanofiber networks.

The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.

PIRET-A Platform for Treatment Planning in Electroporation-Based Therapies.

Tissue electroporation is the basis of several therapies. Electroporation is performed by briefly exposing tissues to high electric fields. It is generally accepted that electroporation is effective where an electric field magnitude threshold is overreached. However, it is difficult to preoperatively estimate the field distribution because it is highly dependent on anatomy and treatment parameters. OBJECTIVE: We developed PIRET, a platform to predict the treatment volume in electroporation-based therapies. METHODS: The platform seamlessly integrates tools to build patient-specific models where the electric field is simulated to predict the treatment volume. Patient anatomy is segmented from medical images and 3D reconstruction aids in placing the electrodes and setting up treatment parameters. RESULTS: Four canine patients that had been treated with high-frequency irreversible electroporation were retrospectively planned with PIRET and with a workflow commonly used in previous studies, which uses different general-purpose segmentation (3D Slicer) and modeling software (3Matic and COMSOL Multiphysics). PIRET outperformed the other workflow by 65 minutes (× 1.7 faster), thanks to the improved user experience during treatment setup and model building. Both approaches computed similarly accurate electric field distributions, with average Dice scores higher than 0.93. CONCLUSION: A platform which integrates all the required tools for electroporation treatment planning is presented. Treatment plan can be performed rapidly with minimal user interaction in a stand-alone platform. SIGNIFICANCE: This platform is, to the best of our knowledge, the most complete software for treatment planning of irreversible electroporation. It can potentially be used for other electroporation applications.

Electrochemotherapy as an Effective Alternative in the Treatment of Local Advanced Oral Squamous Cell Carcinoma: A Retrospective Analysis of Treated Cases.

Advanced oral squamous cell carcinomas represent a major challenge for maxillofacial surgeons, oncologists and radiation therapists. They also account for a large share of healthcare costs. They respond little and/or poorly to conventional therapies (surgery, radiotherapy and chemotherapy). Electrochemotherapy is a new method used as a palliative treatment in patients with advanced cancer of the neck/head region who are not eligible for standard therapies. It combines the use of cytotoxic drugs with the physical principle of electroporation; it effectively controls the tumour locally and preserves organ function. To date, ECT has been little used for oral mucosal tumours, as this is difficult to access for electrodes. We report six cases of advanced oral squamous cell carcinoma treated with electrochemotherapy. This study aims to assess the debulking effect of cancer via ECT in patients with advanced oral squamous cell carcinoma. It also aims to assess the safety and tolerability of this treatment.

Treatment of vulvar cancer recurrences with electrochemotherapy - a detailed analysis of possible causes for unsuccessful treatment.

BACKGROUND: Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological cancers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. PATIENT AND METHODS: A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to standard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. RESULTS: Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histological analysis showed minimal presence of blood vessels in the tumor. Thus, low perfusion may reduce drug delivery and lead to a lower response rate because of the minor antitumor effectiveness of vascular disruption. In this case, no immune response in the tumor was elicited by electrochemotherapy. CONCLUSIONS: In this case, of nonresponsive vulvar recurrence treated by electrochemotherapy, we analyzed possible factors that could predict treatment failure. Based on histological analysis, low vascularization of the tumor was observed, which hampered drug delivery and distribution and resulted in no vascular disrupting action of electro-chemotherapy. All these factors could contribute to ineffective treatment with electrochemotherapy.

Electrochemotherapy with intravenous bleomycin for heavily pre-treated vulvar cancer patients.

OBJECTIVE: The management of vulvar cancer recurrences is complicated by patients' advanced age and comorbidities. Bleomycin-based electrochemotherapy is a potential treatment option in this setting. However, no data on long-term outcomes are available. Therefore, a multicenter observational study was designed to evaluate the 5-year results in these patients. METHODS: Data about patients and tumor characteristics, electrochemotherapy cycles, clinical response, and follow-up were recorded. Treatment procedures were performed according to the European Standard Operating Procedures of Electrochemotherapy (ESOPE) guidelines. Response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. RESULTS: Fifty-one patients (mean age 82.31±7.28 years) with squamous cell vulvar cancer underwent electrochemotherapy (median number of sessions 1; range 1-4). 20 patients had complete response and 32% of these were disease-free after 2 years (median progression-free survival 16.8 months). In 13 patients with partial response the median progression-free survival was 15.36 months, while patients with stable or progressive disease showed tumor relapse after 6.95 and 3.26 months, respectively (p<0.001). Median overall survival was 18.77, 13.07, 6.73, and 11.13 months in patients with complete response, partial response, stable disease, and progressive disease, respectively (p=0.001). CONCLUSION: Long-term follow-up of vulvar cancer patients showed reasonable tumor control after electrochemotherapy and improved progression-free survival and overall survival in responder subjects compared with non-responders. Further studies aimed at improving local response after electrochemotherapy are warranted. Thus, this approach represents a potential alternative for these patients.

Electrochemotherapy of melanoma: What we know and what is unexplored?

The cell membrane can be permeabilized when subjected to calibrated short electric pulses. This membrane alteration can be reversible, leaving cell viability unaffected. This set of events is called electroporation (EP). It is now used in clinical applications to introduce hydrophilic drugs into the cytoplasm. One of the EP applications is electrochemotherapy (ECT), in which EP is used for the selective delivery of drugs administered to treat cancer. The combination of EP with chemotherapy allows local cancer treatment, lowering the drug dose and reducing the side effects of systemic chemotherapy. Nowadays, bleomycin-based ECT (BLM-ECT) is a safe treatment for cutaneous tumors and skin metastases with established standard operating procedures. Additionally, there is emerging evidence that BLM-ECT may be particularly effective in combination with immunotherapies, acting synergistically and producing enhanced systemic anti-tumor effects. Still, to make it the first-choice therapy in patients with metastatic melanoma, further studies are needed to establish the relative effectiveness of ECT. Analyzing the EP phenomenon and the objective complexity of the associated effects at the cell level, we came across a problem that has not yet been investigated in increasing the therapeutic effectiveness of ECT. The profile and kinetics of extracellular vesicles (EVs) released from cells subjected to EP have not been analyzed. The exact nature of these EVs is unknown.

Study on B16 Cell Cytotoxicity by High Frequency Reversible Electroporation With Bleomycin That Induces Hallmarks of Immunogenic Death.

Hundreds of high frequency bipolar pulse bursts with ∼1 µs have been suggested to alleviate muscle contractions and pain during the irreversible electroporation (IRE) tumor treatment. This study is performed to verify whether eight bursts of high frequency reversible electroporation pulses (HFREs) with bleomycin could be used for electrochemotherapy (ECT) tumor treatment. Firstly, in vitro experiments on B16 cells are performed to determine the cytotoxicity of the HFREs with bleomycin. The result indicates that the protocol of HFREs with bleomycin has a significant killing effect compared with only bleomycin, in which the used HFRE pulses are set to induce high membrane permeabilization while maintaining high cell viability. The immunogenic cell death (ICD) that generates danger associated molecular patterns (DAMPs) could trigger an adaptive immune response against tumors. We demonstrated that HFREs with bleomycin could trigger the hallmarks of ICD with obvious up-regulation of DAMPs, including ATP, HMGB1, and CRT. The ICD process may begin at 3 h but perform at 6 h after HFREs with bleomycin stimulation. The in vivo experiment on mice tumor treatment also showed that the protocol of HFREs with bleomycin could inhibit tumor growth with more cytotoxic CD8+ T cells infiltration. The results obtained from in vitro and in vitro experiments preliminary confirmed that the HFREs with bleomycin could be used for ECT tumor treatment associated with the hallmarks of ICD and preliminary trigger the adaptive immune response.