Radiol Oncol 2023; 57(2): 141-149. doi: 10.2478/raon-2023-0029 141 review Bleomycin electrosclerotherapy (BEST) for the treatment of vascular malformations. An International Network for Sharing Practices on Electrochemotherapy (InspECT) study group report Tobian Muir 1 , Giulia Bertino 2 , Ales Groselj 3,4 , Lakshmi Ratnam 5 , Erika Kis 6 , Joy Odili 7 , Ian McCafferty 8 , Walter A Wohlgemuth 9 , Maja Cemazar 10,11 , Aljosa Krt 11 , Masa Bosnjak 10 , Alessandro Zanasi 13 , Michela Battista 13 , Francesca de Terlizzi 13 , Luca G Campana 14 , Gregor Sersa 10,15 1 Department of Reconstructive Plastic Surgery, James Cook University Hospital, Middlesbrough, United Kingdom 2 Department of Otolaryngology Head Neck Surgery, University of Pavia, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo Foundation, Pavia, Italy, 3 Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 5 Department of Interventional Radiology, St George’s University Hospitals NHS Foundation Trust, London, United Kingdom 6 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary 7 Department of Plastic Surgery, St. Georges University Hospitals NHS Trust, London, United Kingdom 8 Birmingham Women’s and Children’s Hospital NHS Foundation Trust, Birmingham, United Kingdom 9 Universitätsklinik und Poliklinik für Radiologie, Universitätsmedizin Halle, Halle, Germany 10 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 11 Faculty of Health Sciences, University of Primorska, Slovenia 12 Department of Otorhinolaryngology, Izola General Hospital, Izola, Slovenia 13 IGEA S.p.A., Clinical Biophysics Lab. Carpi, Modena, Italy 14 Department of Surgery, Manchester University NHS Foundation Trust, Manchester, United Kingdom 15 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(2): 141-149. Received 26 May 2023 Accepted 3 June 2023 Correspondence to: Mr. Tobian Muir, Department of Reconstructive Plastic Surgery, James Cook University Hospital, Middlesbrough, United Kingdom. E-mail: tobian.muir@nhs.net and Prof. Gregor Sersa, Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Biomedical applications of electroporation are expanding out of the field of oncology into vaccina- tion, treatment of arrhythmias and now in the treatment of vascular malformations. Bleomycin is a widely used scle- rosing agent in the treatment of various vascular malformations. The application of electric pulses in addition to bleo- mycin enhances the effectiveness of the drug, as demonstrated by electrochemotherapy, which utilizes bleomycin in the treatment of tumors. The same principle is used in bleomycin electrosclerotherapy (BEST). The approach seems to be effective in the treatment of low-flow (venous and lymphatic) and, potentially, even high-flow (arteriovenous) malformations. Although there are only a few published reports to date, the surgical community is interested, and an increasing number of centers are applying BEST in the treatment of vascular malformations. Within the International Network for Sharing Practices on Electrochemotherapy (InspECT) consortium, a dedicated working group has been constituted to develop standard operating procedures for BEST and foster clinical trials. Conclusions. By treatment standardization and successful completion of clinical trials demonstrating the effective- ness and safety of the approach, higher quality data and better clinical outcomes may be achieved. Key words: vascular malformations; bleomycin electrosclerotherapy; bleomycin; electrochemotherapy Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 142 Classification of vascular malformations Vascular malformations are a rare condition caused by abnormally developed blood vessels. They can occur anywhere in the body and range from simple and benign to complex conditions, with an incidence of around 1.5% in the general population. The latest and most used categorization is the International Society for the Study of Vascular Anomalies (ISSVA) classification (Table 1). 1 This classification divides vascular anomalies into two main categories: tumors (true proliferative neo- plasms) and malformations (morphogenetic de- fects). These two categories are further subcatego- rized: tumors are divided into benign, locally ag- gressive/borderline and malignant, whereas mal- formations are subdivided into simple, combined or associated with other anomalies. Clinically, vas- cular anomalies can also be divided into low-flow and high-flow malformations. 2 Treatment of vascular malformations Current approaches vary depending on the type and anatomical location of the vascular malforma- tion. Treatment options include observation, scle- rotherapy, laser therapy, embolization, and sur- gery. 3,4 Observation is recommended for asymp- tomatic superficial or low-flow malformations that pose no immediate risk to the patient and are stable in size. Sclerotherapy involves the injection of sclerosing agents, such as bleomycin, or other agents (pingyangmycin, absolute ethanol, ethanol- amine oleate, polidocanol, doxycycline, cyanoacr- ylate, sodium morrhuate and sodium tetradecyl sulfate (STS)). 5,6 Laser therapy is used to treat su- perficial vascular malformations and involves the use of a laser to heat the affected area and reduce vessel size. Embolization is a minimally invasive procedure in which small particles, metal coils, or solidifying liquid agents are injected into the mal- formation to block the flow of blood and reduce its size. This treatment is typically used for high- flow malformations. Surgery may be necessary for high-flow malformations that are difficult to treat with the other methods mentioned. Depending on the size and anatomical location, the surgeon may only remove part of the lesion .7 The use of bleomycin combined with electroporation (electrochemotherapy) in oncology There are several biomedical applications of electroporation. Reversible and irreversible elec- troporation are distinguished by the amplitude, timing and number of electric pulse applications. Irreversible electroporation is based on irreversi- ble disruption of the cell membrane causing desta- bilization of cell physiology to the extent that cells die either by apoptosis, necrosis or even immuno- genic cell death. 8 In contrast, reversible electropo- ration does not cause cell death but temporarily disrupts the cell membrane in such a way that it becomes permeable for molecules that have ham- pered transport through the membrane. 9–12 This TABLE 1. International Society for the Study of Vascular Anomalies (ISSVA) classification for vascular anomalies 2018 VASCULAR TUMORS VASCULAR MALFORMATIONS Benign Locally aggressive Malignant Simple Combined Infantile hemangioma Kaposiform hemangioendothelioma Epithelioid hemangioendothelioma Angiosarcoma Capillary malformation (CM) CVM, CLM Congenital hemangioma Retiform hemangioendothelioma Lymphatic malformation (LM) LVM. CLVM Tufted hemangioma PILA. Dabska tumor Venous malformation (VM) CAVM Spindle-cell hemangioma Composite hemangioendothelioma Arteriovenous malformation (AVM) CLAVM Epithelioid hemangioma Kaposi sarcoma Arteriovenous fistula Pyogenic granuloma AVM = arteriovenous malformation; CAVM = capillary arteriovenous malformation; CLAVM = capillary lymphatic arteriovenous malformation; CLM = capillary lymphatic malformations; CLVM = capillary lymphatic venous malformation; CM = capillary malformation; CVM = capillary venous malformations; LM = lymphatic malformation; LVM = lymphatic venous malformation; PILA – papillary intralymphatic angioendothelioma; VM - venous malformation Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 143 phenomenon can be exploited for enhanced drug, DNA or RNA delivery into cells. If we deliver nu- cleic acids, it is called gene electrotransfer, which can be used for cancer immune-gene therapy; for example, if introduced, DNA or mRNA encodes immunomodulatory molecules. It can also be used for vaccination purposes. 13,14 The biomedical ap- plications of gene electrotransfer can be used in the treatment of cancer, as well as other diseases, including vaccination for infectious diseases such as SARS CoV-2 virus. 15 In the case of electrochemo- therapy, electroporation is used to enhance the de- livery of cytotoxic molecules for cancer treatment. The principle is to inject cytotoxic drugs such as bleomycin or cisplatin into the cancer patient and apply electric pulses at the site of the tumor, where enhanced drug uptake is desired. 16 Therefore, elec- trochemotherapy is a local treatment since drug cytotoxicity is enhanced only at the site of electric pulse applications. This approach is being used widely in the treatment of either cutaneous tumors or deep-seated tumors in internal organs, such as liver and pancreas. 16 Several electrodes were de- signed that are best suited for the delivery of elec- tric pulses to specific anatomical locations. Due to its simple principle, i.e., the use of highly cyto- toxic drugs and the application of electric pulses for its enhanced cytotoxicity, electrochemotherapy is effective on tumors of different histological ori- gins. Its objective response rate ranges between 70-80%. 17 Electrochemotherapy is listed in many national and international guidelines as a local ablative therapy and is applied in more than 200 centers across Europe. 16 Electrochemotherapy mechanisms of action There are three underlying mechanisms of elec- trochemotherapy. The first is enhanced drug de- livery to tumor cells, which die due to the cytotox- icity of the drugs, either by apoptosis or necrosis. This is predominantly related to the drug used and its mode of action. Bleomycin, for example, induces mitotic cell death, which induces slow resolution of the tumor mass. Experimental data on tumors in mice demonstrate that the drug concentration in tumor cells after electroporation is increased 10 times or more, depending on the tumor type, in the case of bleomycin electrochemotherapy. 18 The second mechanism is the induction of the immune response due to immunogenic tumor cell death induced by the drug. It is well known that certain ablative therapies induce immunogenic cell death that can attract and boost the immune response of the organism. This mechanism has been described in radiation therapy, thermal ab- lative techniques, and electrochemotherapy. 10,12,19 Several groups have investigated the role of im- munogenic cell death in the effectiveness of elec- trochemotherapy. Now, the experimental data indicate that the response of the tumors varies depending on the immunogenicity of the tumors; more immunogenic tumors respond better to elec- trochemotherapy than less immunogenic tumors, which was linked to more pronounced immuno- genic cell death after electrochemotherapy. 10,12,20 Additionally, clinical data on the treatment of melanoma demonstrate that local treatment of cutaneous metastases can boost or interact with treatment using immune checkpoint inhibitors, such as pembrolizumab. 21 Patients treated with both electrochemotherapy and immune check- point inhibitors had lower disease progression rates and longer survival than those who received pembrolizumab only. The third mechanism is the vascular disrupting effect of electrochemotherapy. In early preclinical research, it was established that the application of electric pulses only temporarily abrogates blood flow within tumors. This phenomenon was termed vascular lock and lasts less than an hour. 22,23 Furthermore, the effect is enhanced when the drug is present during application of the electric pulses. Investigations have shown that it results in vascular disruption that occurs within hours in tumors. Endothelial cells start to die, blood flow is obstructed, and secondary tumor cell death is induced within days due to induced tumor hy- poxia. 24–27 The phenomenon is predominantly con- fined to the tumor vasculature, sparing the normal vasculature around the tumors. The reason for this is because of the high proliferation rate of endothe- lial cells in tumors compared to the vasculature in normal tissues, where the endothelial proliferation rate is very slow. The vascular disrupting effect of electrochemotherapy is not fully understood. To date, we do not know in what proportion this vas- cular disrupting effect contributes to the overall effectiveness of electrochemotherapy in specific tumor types. We know that it is dependent on the distribution and extent of tumor vascularization, and better vascularized tumors respond better to electrochemotherapy. 28,29 Preclinical data also indi- cate that tumor perfusion influences the effective- ness of the treatment, with well perfused tumors showing an improved response. 30 Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 144 Combination of bleomycin with reversible electroporation for treatment of vascular malformations Electrochemotherapy is safely applied to palliate bleeding cutaneous metastases and treat vascular tumors e.g., Kaposi sarcoma, superficial angio- sarcoma 31-34 and highly vascularized liver metas- tases. 35,36 Bleeding after the insertion of needle electrodes quickly stops due to vascular lock. 37 Additionally, both superficial and liver tumors themselves also do not bleed after electrochemo- therapy due to the vascular disrupting effect. These observations indicate that electrochemo- therapy indeed exerts vascular effects, the above- mentioned vascular lock and the vascular disrupt- ing effect. Furthermore, several reports indicate that electrochemotherapy can be safely applied to control or treat bleeding tumors. 37 Of note, bleed- ing stops almost immediately after the application of electric pulses; therefore, treatment of bleeding tumors is also one of the indications for electro- chemotherapy (Figure 1). These data support the potential advantage of bleomycin electrosclerotherapy in the treatment of vascular malformations. Bleomycin is already one of the most frequently used sclerotherapy agents in the treatment of these lesions. 5,6 Therefore, the combination with electric pulses may only add to the effectiveness of bleomycin since it would in- crease the uptake of the drug into the endothelial lining of the affected blood vessels. In many cases, blood vessels are abnormal and endothelial cell proliferation is higher than that in normal blood vessels. 38 Therefore, the vasculature in vascular malformations is impaired as it is in tumors, and electrochemotherapy is supposed to be effective in both. Histological evidence from liver biopsies in- dicates that venules are more sensitive to elec- trochemotherapy than arterioles in normal liver parenchyma. 39 This would indicate that venous malformations would have been more susceptible to BEST. We recently performed a study on pigs inves- tigating vascular changes in large blood vessels such as the portal vein, inferior vena cava, and lineal vein. In this study, the vessels were directly exposed to electroporation and electrochemo- therapy by the application of electric pulses us- ing plate electrodes that embraced the vessels. Electrochemotherapy may temporarily disrupt the endothelial lining and disrupt the vasa vasorum of the vessels (unpublished data). This effect may, however, be a desired one in the treatment of all vascular malformations, specifically because we have not observed thrombi formation in the treat- ed vessels in the pig model. All this evidence supports the use of bleomycin electroporation in the treatment of vascular mal- formations, also called bleomycin electrosclero- therapy (BEST). In principle, BEST could be a safe and effective approach for the treatment of vascu- lar malformations; however, more clinical data are needed to confirm this approach. To date, there are only a handful of clinical reports on the treatment of vascular malformations with BEST. Finally, and importantly, the technique needs to be standard- ized through the development of dedicated stand- ard operating procedures. FIGURE 1. Treatment of vascularized melanoma metastasis by electrochemotherapy. (A) Highly vascularized tumor before treatment. (B) Bleeding due to electrode insertion after application of electric pulses to the tumor. (C) Bleeding stopped immediately after electric pulse application. A B C Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 145 Overview of current clinical BEST reports Publications on BEST are still limited in number. Table 2 summarizes the clinical studies and case reports published thus far. 40–45 In summary, dif- ferent types of vascular malformations have been treated, with favorable clinical outcomes. Most of the studies used intralesional bleomycin, either di- luted or mixed with lidocaine. Bleomycin dosage varied between studies, but in most reports, it was lower than in traditional sclerotherapy. In addi- tion, the number of treatments required was much lower when BEST was used than when bleomycin was used alone. Drug dosage, the number of treat- ments needed, and route of drug administration are all aspects that need to be explored to develop recommendations for the future use of BEST. Another relevant aspect of BEST is the applica- tion of electric pulses. Due to the blood accumu- lated in the malformation, the electrical conduc- tivity of the treated tissue is high. Therefore, it is assumed that the coverage of the target lesion with electric pulses does not need to be so strict as in electrochemotherapy. In this regard, some clini- cians who perform BEST on patients report that fewer applications of electric pulses to the lesions are needed. This aspect that requires clarification in future studies. Studies on BEST should also report the num- ber and amplitudes of pulse applications and the electrical parameters (current). The predominantly used generator is from one producer, and vary- ing electrodes specific to this generator are used. Usually, in each electric pulse application 8 pulses of 1000−1300 V/cm in frequency 5 kHz are applied. Since different electrodes are used for different clinical situations, the reports should describe which electrodes were used. Furthermore, when standard operating procedures for BEST will be prepared, recommendations for the use of specific types of electrodes should also be prepared. BEST standardization To date, there are no standardized guidelines for BEST. As a result, each center applies the treatment according to local protocols and clinical experi- ence. Therefore, standardization of some proce- dural aspects is needed. The spread of a new technology depends on its safety. Safety aspects have already been cor- roborated, since there are already some reports of significant morbidity with bleomycin only, but not with BEST treatment. 46 We must be aware that cy- totoxic drugs are used for the treatment of benign disease, that can be spilled and are sometimes also used in very young patients. This safety aspect is also related to the experience in image-guided ble- omycin and electric pulse application (Figure 2). Currently, BEST must be practiced in the frame- work of clinical studies, where patient referral and suitability for BEST treatment need to be defined. The patients need to be informed about other treatment options and their suitability for the treatment evaluated by a multidisciplinary team (MDT). It is expected that in the early stage of develop- ment, BEST will be considered in recurrences after previous treatments, whereas subsequently more precise indications for specific types of vascular malformations will be individuated. FIGURE 2. Patient treated by BEST. Axial T2-weighted, fat-saturated MRI with hyperintense (arrow) gluteal venous malformation before treatment (A). Axial T2-weighted, fat-saturated MRI of the same region 4 months after treatment. The main part of the venous malformation is occluded (B). Photography before (C) and after the treatment (D). A B C D Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 146 Other relevant procedural aspects that need to be clarified include drug injection, questions of dosage, the need for general anesthesia, electrode placement and delivery of electric pulses. Exclusion criteria need to be defined as well, such as pregnancy, lactation and allergy or hyper- sensitivity to bleomycin, or abnormal respiratory parameters. The application of electric pulses, especially with needle electrodes, is painful. Therefore, local or general anesthesia is needed. In this regard, the experience accumulated with electrochemo- therapy can be informative. Generally, the choice of the type of anesthesia is at the discretion of each center. However, there is a recent report that continuous intravenous sedation is also an option that requires less anesthetic and is much shorter in time. 47 Another important issue is the route of bleo- mycin injection. In electrochemotherapy, the pre- ferred route is intravenous injection; however, in BEST treatment, this is a limited option since in- travenous sclerotherapy with bleomycin is not the standard of care. Instead, direct injection is pre- ferred, possibly under image guidance and poten- tially with a lower dose to avoid systemic toxicity. In electrochemotherapy, the bleomycin dose in intravenous or intratumoral administration is defined. 48 In elderly patients older than 65 years or with renal impairment, the intravenously ad- ministered dose can be decreased by 1/3. 48 In BEST treatment, the dose can be substantially decreased. The lowest effective dose of bleomycin for BEST treatment still needs to be established. The con- centration of bleomycin in the solution needs to be standardized. Another peculiar aspect is that the volume of the drug solution is dependent on the type and volume of the malformation. Furthermore, it de- pends on whether bleomycin is diluted either with foam, lidocaine or contrast agent and whether there is drainage from the malformation that needs to be stopped, either by compression or in- travascular techniques. By all means, the concen- tration and volume of the drug injected needs to be recorded and reported. The dose and route of administration may also differ in the case of fast or slow flow malformations. The treatment can be repeated up to a cumulative dose for bleomycin of 400 000 IU. 48 The interval between the intralesional bleo- mycin injection and the application of electric TABLE 2. Clinical studies and case reports using bleomycin electrosclerotherapy 40–45 Reference Type of malformation N of pts Bleomycin dose and concentration Electrodes used N of pulse applications Response Comment McMorrow et al., Br J Oral and Maxillofacial Surg 2017 44 Venous malformation 1 Reduced dose: 1/3 of the standard dose Not reported Not reported Considerable improvement after 6 unsuccessful sessions with bleomycin Case report with poor respiratory function Horbach et al., Dermatologic Surgery 2020 45 Hypertrophic capillary malformations 5 pts. (out of 20 planned) 0.25 mg or units/cm 3 Plate & needle Not reported 7-8 weeks DEROI (changes in colorimetry) Flux in ROI (in Perfusion Units) Randomized controlled pilot trial Dalmady et al., Pediatrics 2020 43 Lymphatic malformation 1 0.5 mg/kg (5.4 mg) Needle 1 st session: 68 applications 2 nd session: 74 applications 63% growth-corrected volume decrease. No recurrence at 18 months Follow up Case report Wohlgemuth et al., Journal of Vascular Surgery 2021 40 Venous malformations 17 pts. (20 lesions) Calculated based on the size of the lesion. C = 0.25 mg/mL Intralesional injection (25% concentration of standard bleomycin sclerotherapy) Needle & finger Not reported 3-month post-therapy Changes in volume MRI: Volume reduction,%: > 90% 9 lesions > 70% in < 90% 6 lesions > 50% in < 70% 2 lesions < 35% 2 lesions No response 1 lesion Retrospective observational case study Kostusiak et al., Dermatologic Surgery 2022 42 Various vascular malformations 30 pts. VM: 17 AVM: 3 CVM LM: 2 Mixed: 2 Calculated based on the size of the lesion. Bleomycin mixed with 1 mL plain 1% lidocaine Dose not reported Needle & finger Not reported 17 Complete Response 7: significant improvement 1: moderate improvement 1: minor response 1: no response 3: active follow up Prospective observational case study Electrosclerotherapy offered to non-responding patients to standard bleomycin Krt et al., Front Oncol 2022 41 Arteriovenous malformation 1 750 IU BLM intralesional Plate 15 CR 18 months after BEST Case report AVM = arteriovenous malformation; BLM = bleomycin; CVM = capillary venous malformations; VM - venous malformation; LM = lymphatic malformation; Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 147 pulses needs to be short, while the drug is pre- sent in the treated tissue. The one-minute interval would be enough. A similar situation is also ob- served in electrochemotherapy, where the time in- terval is different when bleomycin is injected either intratumorally or intravenously. In the case of in- travenous injection, the time interval is 8 minutes, and after intratumoral injection, the time interval is just 1-3 minutes. 48 The choice of the electrodes used is dependent on many factors. The type, lo- cation and size of the malformation are the most important factors. Needle electrodes with shorter or longer needles in fixed geometry are available. Some centers have concerns about so-called hex- agonal electrodes since they deliver many electric pulses between the needles with a high risk of skin hyperpigmentation at the puncture sites. Some malformations require so-called variable geom- etry electrodes. These are single long needle elec- trodes that can be placed separately and can cover deeper mostly subfascial malformations. Vascular malformations do not need to be cov- ered entirely with electric pulses, in contrast to electrochemotherapy. In the case of BEST, damage to the endothelium and vessels needs to be done throughout the malformation but not necessarily with dense and complete coverage of the whole lesion, since the goal is to improve the symptoms more than eradicating the lesion. In this way, the risk of tissue swelling and mucosal ulceration could be reduced. BEST treatment can be performed as a day case procedure unless pain, bleeding or swelling is an- ticipated. Generally, follow-up is recommended at approximately three-month intervals. These are recommendations and considerations for BEST treatment according to the experiences gained by the authors of this manuscript. The members of this working group will continue to share experiences and discuss results to identify the procedural aspects associated with the best re- sults. When a more formal consensus is reached, we will propose our best practice in the form of standard operating procedures (SOPs). Role of InspECT in BEST applications InspECT is an international network of 42 clinical centers using electrochemotherapy for the treat- ment of cancer. This is the largest group of experts on electroporation-based treatments. Some of them are acquainted with BEST and report a posi- tive experience as other external centers. Together, these centers form a network that can promote BEST worldwide. A dedicated working group for vascular malformations has been formed within InspECT. This group will promote clinical studies with BEST and seek collaboration with other cent- ers. This paper aims to raise interest and aware- ness in the treatment of vascular malformations with BEST and provide an overview of the current status of development of this approach. Future directions and conclusions The number of clinicians using BEST to treat vascu- lar malformations is growing. Due to the first and positive experiences in various vascular malfor- mations, BEST application is being practiced in an increasing number of centers throughout Europe and the UK. This article summarizes the rationale and underlying mechanisms of BEST, along with the initial clinical experiences. Additionally, it highlights the main controversial procedural as- pects and the need for dedicated SOPs. Acknowledgments The authors acknowledge the financial support from the state budget by the Slovenian Research Agency, program no. P3-0003. References 1. Classification International Society for the Study of Vascular Anomalies. [Internet]. [cited 2023 May 16]. Available at: https://www.issva.org/clas- sification 2. Wu IC, Orbach DB. Neurointerventional management of high-flow vascular malformations of the head and neck. Neuroimaging Clin N Am 2009; 19: 219-40. doi: 10.1016/j.nic.2009.01.005. 3. Sadick M, Wohlgemuth WA, Huelse R, Lange B, Henzler T, Schoenberg SO, et al. Interdisciplinary management of head and neck vascular anomalies: clinical presentation, diagnostic findings and minimalinvasive therapies. Eur J Radiol Open 2017; 4: 63-8. doi: 10.1016/j.ejro.2017.05.001 4. Hage AN, Beecham Chick JF, Srinivasa RN, Bundy JJ, Chauhan NR, Acord M, et al. Treatment of venous malformations: the data, where we are, and how it is done. Tech Vasc Interv Radiol 2018; 21: 45-54. doi: 10.1053/j. tvir.2018.03.001 5. Horbach SER, Lokhorst MM, Saeed P , de Goüyon Matignon de Pontouraude CMF, Rothová A, Van Der Horst CMAM. Sclerotherapy for low-flow vascular malformations of the head and neck: a systematic review of sclerosing agents. J Plast Reconstr Aesthet Surg 2016; 69: 295-304. doi: 10.1016/j. bjps.2015.10.045 6. Maria L De, Sanctis P De, Balakrishnan K, Tollefson M, Brinjikji W. Sclerotherapy for venous malformations of head and neck: systematic review and meta-analysis. Neurointervention 2020; 15: 4. doi: 10.5469/ neuroint.2019.00213 Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 148 7. Johnson AB, Richter GT. Surgical considerations in vascular malformations. Tech Vasc Interv Radiol 2019; 22: 100635. doi: 10.1016/j.tvir.2019.100635 8. Wang YJ, Fletcher R, Yu J, Zhang L. Immunogenic effects of chemotherapy- induced tumor cell death. Genes Dis 2018; 5: 194. doi: 10.1016/j.gen- dis.2018.05.003 9. Sersa G, Ursic K, Cemazar M, Heller R, Bosnjak M, Campana LG. Biological factors of the tumour response to electrochemotherapy: review of the evi- dence and a research roadmap. Eur J Surg Oncol 2021; 47: 1836-1846. doi: 10.1016/j.ejso.2021.03.229 10. Ursic K, Kos S, Kamensek U, Cemazar M, Miceska S, Markelc B, et al. Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune sta- tus. J Control Release 2021; 332: 623-35. doi: 10.1016/j.jconrel.2021.03.009 11. Yarmush ML, Golberg A, Sersa G, Kotnik T, Miklavcic D. Electroporation- based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng 2014; 16: 295-320. doi: 10.1146/annurev-bio- eng-071813-104622 12. Kesar U, Markelc B, Jesenko T, Valentinuzzi KU, Cemazar M, Strojan P , et al. Effects of electrochemotherapy on immunologically important modifica- tions in tumor cells. Vaccines 2023; 11: 925. doi: 10.3390/vaccines11050925 13. Sersa G, Teissie J, Cemazar M, Signori E, Kamensek U, Marshall G, et al. Electrochemotherapy of tumors as in situ vaccination boosted by immuno- gene electrotransfer. Cancer Immunol Immunother 2015; 64: 1315-27. doi: 10.1007/s00262-015-1724-2 14. Groselj A, Bosnjak M, Jesenko T, Cemazar M, Markelc B, Strojan P, et al. Treatment of skin tumors with intratumoral interleukin 12 gene electro- transfer in the head and neck region: a first-in-human clinical trial protocol. Radiol Oncol 2022; 56: 398-408. doi: 10.2478/raon-2022-0021 15. Conforti A, Marra E, Palombo F , Roscilli G, Ravà M, Fumagalli V, et al. COVID- eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models. Mol Ther 2022; 30: 311- 26. doi: 10.1016/j.ymthe.2021.09.011 16. Campana LG, Edhemovic I, Soden D, Perrone AM, Scarpa M, Campanacci L, et al. Electrochemotherapy − emerging applications technical advances, new indications, combined approaches, and multi-institutional collabora- tion. Eur J Surg Oncol 2019; 45: 92-102. doi: 10.1016/j.ejso.2018.11.023 17. Clover AJP, de Terlizzi F, Bertino G, Curatolo P, Odili J, Campana LG, et al. Electrochemotherapy in the treatment of cutaneous malignancy: outcomes and subgroup analysis from the cumulative results from the pan-European International Network for Sharing Practice in Electrochemotherapy data- base for 2482 lesions in 987 patients (2008–2019). Eur J Cancer 2020; 138: 30-40. doi: 10.1016/j.ejca.2020.06.020 18. Mir LM, Orlowski S, Belehradek J, Paoletti C. Electrochemotherapy potentia- tion of antitumour effect of bleomycin by local electric pulses. Eur J Cancer 1991; 27: 68-72. doi: 10.1016/0277-5379(91)90064-K 19. Wang M, Duan Y, Yang M, Guo Y, Li F, Wang J, et al. The analysis of im- munogenic cell death induced by ablation at different temperatures in hepatocellular carcinoma cells. Front Cell Dev Biol 2023; 11: 1146195. doi: 10.3389/fcell.2023.1146195 20. Polajzer T, Jarm T, Miklavcic D. Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro. Radiol Oncol 2020; 54: 317-328. doi: 10.2478/raon-2020-0047 21. Campana LG, Peric B, Mascherini M, Spina R, Kunte C, Kis E, et al. Combination of pembrolizumab with electrochemotherapy in cutaneous metastases from melanoma: a comparative retrospective study from the inspect and slovenian cancer registry. Cancers 2021; 13: 4289. doi: 10.3390/ cancers13174289 22. Sersa G, Cemazar M, Parkins CS, Chaplin DJ. Tumour blood flow changes induced by application of electric pulses. Eur J Cancer 1999; 35: 672-7. doi: 10.1016/S0959-8049(98)00426-2 23. Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in vivo electropora- tion: characterization and consequences for drug and gene delivery. Biochim Biophys Acta Gen Subj 2002; 1569: 51-8. doi: 10.1016/S0304- 4165(01)00233-1 24. Jarm T, Cemazar M, Miklavcic D, Sersa G. Antivascular effects of electro- chemotherapy: implications in treatment of bleeding metastases. Expert Rev Anticancer Ther 2010; 10: 729-46. doi: 10.1586/era.10.43. 25. Markelc B, Sersa G, Cemazar M. Differential mechanisms associated with vascular disrupting action of electrochemotherapy: intravital microscopy on the level of single normal and tumor blood vessels. PLoS One 2013; 8: e59557 doi: 10.1371/journal.pone.0059557 26. Markelc B, Bellard E, Sersa G, Jesenko T, Pelofy S, Teissié J, et al. Increased permeability of blood vessels after reversible electroporation is facilitated by alterations in endothelial cell-to-cell junctions. J Control Release 2018; 276: 30-41. doi: 10.1016/j.jconrel.2018.02.032 27. Sersa G, Jarm T, Kotnik T, Coer A, Podkrajsek M, Sentjurc M, et al. Vascular disrupting action of electroporation and electrochemotherapy with bleo- mycin in murine sarcoma. Br J Cancer 2008; 98: 388-98. doi: 10.1038/ sj.bjc.6604168 28. Djokic M, Cemazar M, Popovic P, Kos B, Dezman R, Bosnjak M, et al. Electrochemotherapy as treatment option for hepatocellular carcinoma, a prospective pilot study. Eur J Surg Oncol 2018; 44: 651-657. doi: 10.1016/j. ejso.2018.01.090 29. Edhemovic I, Brecelj E, Cemazar M, Boc N, Trotovsek B, Djokic M, et al. Intraoperative electrochemotherapy of colorectal liver metastases: a pro- spective phase II study. Eur J Surg Oncl 2020; 46: 1628-1633. doi: 10.1016/j. ejso.2020.04.037 30. Groselj A, Kranjc S, Bosnjak M, Krzan M, Kosjek T, Prevc A, et al. Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy. Basic Clin Pharmacol Toxicol 2018; 123: 247-256. doi: 10.1111/bcpt.13012 31. Curatolo P, Careri R, Simioni A, Campana LG. Cryotherapy, imiquimod, and electrochemotherapy are effective options for kaposi sarcoma: a call for standardization to allow for comparisons and informed decisions. J Cutan Med Surg 2020; 24: 218-9. doi: 10.1177/1203475419893302 32. Lalanda R, Bartolo J, Carvalhal S, Abecasis N, Farricha V, Sofia R, et al. Cutaneous and subcutaneous Kaposi’s sarcoma lesions treated with elec- trochemotherapy. Int J Dermatol 2023; 62: 115-119. doi: 10.1111/ijd.16261 33. Guida M, Campana LG, Curatolo P, Strippoli S, Bonadies A, Grilz G, et al. Local treatment with electrochemotherapy of superficial angiosarcomas: efficacy and safety results from a multi-institutional retrospective study. J Surg Oncol 2016; 114: 246-53. doi: 10.1002/jso.24287 34. Campana LG, Kis E, Bottyán K, Orlando A, de Terlizzi F, Mitsala G, et al. Electrochemotherapy for advanced cutaneous angiosarcoma: a European register-based cohort study from the International Network for Sharing Practices of Electrochemotherapy (InspECT). Int J Surg 2019; 72: 34-42. doi: 10.1016/J.IJSU.2019.10.013 35. Djokic M, Cemazar M, Bosnjak M, Dezman R, Badovinac D, Miklavcic D, et al. A Prospective phase II study evaluating intraoperative electrochemo- therapy of hepatocellular carcinoma. Cancers 2020; 12: 3778. doi: 10.3390/ cancers12123778 36. Spiliotis AE, Holländer S, Rudzitis-Auth J, Wagenpfeil G, Eisele R, Nika S, et al. Evaluation of electrochemotherapy with bleomycin in the treatment of colorectal hepatic metastases in a rat model. Cancers 2023; 15: 1598. doi: 10.3390/cancers15051598 37. Gehl J, Geertsen PF. Palliation of haemorrhaging and ulcerated cutaneous tumours using electrochemotherapy. Eur J Cancer Supp 2006; 4: 35-7. doi: 10.1016/j.ejcsup.2006.07.007 38. Lampejo AO, Ghavimi SAA, Hägerling R, Agarwal S, Murfee WL. Lymphatic/ blood vessel plasticity: motivation for a future research area based on present and past observations. Am J Physiol Heart Circ Physiol 2023; 324: H109-21. doi: 10.1152/ajpheart.00612.2022 39. Gasljevic G, Edhemovic I, Cemazar M, Brecelj E, Gadzijev EM, Music MM, et al. Histopathological findings in colorectal liver metastases after elec- trochemotherapy. PLoS One 2017; 12: e0180709. doi: 10.1371/journal. pone.0180709 40. Wohlgemuth WA, Müller-Wille R, Meyer L, Wildgruber M, Guntau M, Heydt S von der, et al. Bleomycin electrosclerotherapy in therapy-resistant venous malformations of the body. J Vasc Surg Venous Lymphat Disord 2021; 9: 731-9. doi: 10.1016/J.JVSV.2020.09.009 41. Krt A, Cemazar M, Lovric D, Sersa G, Jamsek C, Groselj A. Combining su- perselective catheterization and electrochemotherapy: a new technological approach to the treatment of high-flow head and neck vascular malforma- tions. Front Oncol 2022; 12: 1025270. doi: 10.3389/FONC.2022.1025270 Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 149 42. Kostusiak M, Murugan S, Muir T. Bleomycin electrosclerotherapy treatment in the management of vascular malformations. Dermatol Surg 2022; 48: 67-71. doi: 10.1097/DSS.0000000000003220 43. Dalmády S, Csoma Z, Besenyi Z, Bottyán K, Oláh J, Kemény L, et al. New treatment option for capillary lymphangioma: bleomycin-based electro- chemotherapy of an infant. Pediatrics 2020; 146: e20200566. doi: 10.1542/ PEDS.2020-0566 44. McMorrow L, Shaikh M, Kessell G, Muir T. Bleomycin electrosclerotherapy: new treatment to manage vascular malformations. Br J Oral Maxillofac Surg 2017; 55: 977-9. doi: 10.1016/J.BJOMS.2017.10.002 45. Horbach SER, Wolkerstorfer A, Jolink F, Bloemen PR, van der Horst CMAM. Electrosclerotherapy as a novel treatment option for hypertrophic capillary malformations: a randomized controlled pilot trial. Dermatol Surg 2020; 46: 491-8. doi: 10.1097/DSS.0000000000002191 46. Ge V, Banakh I, Tiruvoipati R, Haji K. Bleomycin-induced pulmonary toxicity and treatment with infliximab: a case report. Clin Case Rep 2018; 6: 2011-4. doi: 10.1002/CCR3.1790 47. Benedik J, Ogorevc B, Brezar SK, Cemazar M, Sersa G, Groselj A. Comparison of general anesthesia and continuous intravenous sedation for elec- trochemotherapy of head and neck skin lesions. Front Oncol 2022; 12: 1011721. doi: 10.3389/FONC.2022.1011721 48. Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cu- taneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602