Dermatology: Practical and Conceptual Research | Dermatol Pract Concept 2017;7(3):4 21 DERMATOLOGY PRACTICAL & CONCEPTUAL www.derm101.com Successful treatment of plantar warts with intralesional bleomycin and electroporation: pilot prospective study Paola Pasquali1, Azael Freites-Martinez1, Salvador Gonzalez2,3, Enrico P. Spugnini4, Alfonso Baldi4,5 1 Dermatology Service, Pius Hospital De Valls, Tarragona, Spain 2 Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA 3 Medicine Department, Alcalá University, Madrid, Spain 4 Biopulse srl, Naples, Italy 5 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy Key words: warts, electroporation, bleomycin Citation: Pasquali P, Freites-Martinez A, Gonzales S, Spugnini EP, Baldi A. Successful treatment of plantar warts with intralesional bleomycin and electroporation: pilot prospective study. Dermatol Pract Concept 2017;7(3):4. DOI: https://doi.org/10.5826/dpc.0703a04 Received: February 22, 2017; Accepted: May 27, 2017; Published: July 31, 2017 Copyright: ©2017 Pasquali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: None. Competing interests: E.P. Spugnini and A. Baldi are stockholders in Biopulse s.r.l. All authors have contributed significantly to this publication. P.P., A.F.M., S.G. performed the clinical trial; E.P.S. and A.B. performed the statistical analysis; P.P., E.P.S. and A.B. wrote the manuscript. Corresponding author: Prof. Alfonso Baldi, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Via Vivaldi, 43 - 81100 Caserta, Italy. Email: alfonsobaldi@tiscali.it Background: Numerous studies have been performed to evaluate the efficacy of intralesional bleomy- cin for the treatment of warts with inconsistent result. Nevertheless, it is well known that the cytotox- icity of bleomycin can be enhanced in vivo by 300 to 700-fold by electroporation. Objective and Methods: In this article, we present an interventional, one-center, prospective case series, clinical trial of the effectiveness of intralesional bleomycin combined with electroporation for the treatment of plantar warts, in comparison to the use of intralesional bleomycin alone. Results: The study’s cohort included 12 men and 10 women, with a mean age of 53.8 years. A total of 22 warts were treated. In dividing the patients in two groups (complete remission against all the others) and analyzing the different outcomes in the two arms of patients, a statistical significant dif- ference was found (p=0.0015), proving a greater efficacy of the treatment with bleomycin combined with ECT as opposed to bleomycin alone. Electroporation was always well tolerated by the patients with no discomfort. Conclusions: This study serves as a basis for the application of novel protocols in the treatment of different benign and locally malignant skin lesion by means of electroporation. ABSTRACT 22 Research | Dermatol Pract Concept 2017;7(3):4 in comparison to the use of intralesional bleomycin alone (EUDRACT N° 2014-003339-21). Patients Twenty-two patients with shave biopsy proven warts were recruited to participate in this study. Participation was on a volunteer basis, according to the principles of the Declaration of Helsinki. After detailed clinical assessment of the patient, information of the patient on all available alternative treat- ment options for her/his disease, information on ECT (appli- cation and expected results in comparison to other modalities, expected side effects and their management), and the aim of the present study, written informed consent was obtained and a structured follow-up program was discussed. Patients were evaluated for eligibility to participate in this study according to the criteria listed in Table 1. There were no demographic restrictions for the inclusion. All the patients were recruited from the Dermatology Service, Pius Hospital de Valls, Tar- ragona, Spain. Patients were randomly assigned to one of the two arms of the study (bleomycin alone and bleomycin combined with ECT). Of note, we did not test and stratify for HPV subtype the patients. Therapeutic protocol Each patient signed a consent form. Clinical and dermo- scopic images of lesions were taken using a Canon Power- Shot G11 (Canon, Inc, Tokyo, Japan) camera followed by shave biopsy. Affected areas were anesthetized with sub- cutaneous lidocaine; block anesthesia was not performed. Overlying calluses were trimmed with a number 15 blade. Bleomycin in a concentration of 1 mg/cm3 was injected into verrucous foci at a depth of about 1.5 mm with a final vol- ume of 0.1 cc. Patients enrolled in the arm including the ECT treatment, were subjected to trains of 8 biphasic pulses with an interpulse of 10 microseconds and a 50+50 microseconds duration, generated by an electroporator (Onkodisruptor®, Biopulse S.r.l., Naples, Italy). The pulses were delivered at a voltage of 700 V/cm, with 1 Hz frequency (total treatment Introduction Cutaneous warts are very common among dermatological diseases and are of benign nature. However, they can be extremely difficult to treat and can significantly impact qual- ity of life [1,2]. Human papillomavirus, a double-stranded DNA virus, is the etiologic agent of warts. The most common sites of infection are the hands, feet, face, and the anogenital area [3]. Several different therapeutic approaches are possible, depending on lesion size, number and location, patient age and comorbidities, as well as adverse reactions associated with therapy [4]. Numerous studies have been performed to evaluate the efficacy of intralesional bleomycin for the treat- ment of warts with inconsistent result [5]. The uptake of this drug by the cells is slow and limited, because bleomycin can pass the cell membrane only through protein receptors due to its lipophobic nature [6]. In vitro studies, indeed, show evidence that less than 0.1% of bleo- mycin added to a culture medium becomes associated with the cell. Therefore, the high toxicity of bleomycin is weakened by its incapacity to freely diffuse through the cytoplasmic membrane [6]. Several studies have proved that the cyto- toxicity of bleomycin can be enhanced in vivo by 300- to 700-fold by electroporation [7]. Electrochemotherapy (ECT) is a loco-regional therapy that is based on the application of permeabilizing electric pulses on tumors or tumor beds after the administration (either systemic or intralesional) of a chemotherapy agent [6]. In veterinary medicine, ECT is cur- rently adopted as first-line therapy, generally in an adjuvant fashion, to improve the chemotherapeutic agent uptake by the neoplastic cells, thus resulting in better local control of the neoplastic disease [7]. In humans, its use is actually focused on palliation of cutaneous metastases of melanoma [8], but it is going to prove to be a valuable skin-directed therapy for a range of malignancies [9]. Among the numerous electro- poration protocols implemented, our research group recently proposed a novel protocol involving the adoption of bursts of rectangular and biphasic pulses with a selected period of repetition [10]. This schedule enabled decreased morbidity of the treated animals and human beings as well as improving the clinical outcome [10]. In this study, we investigate the potential benefits of com- bining intralesional bleomycin with ECT for the treatment of plantar warts of big dimensions, with respect to the treatment with bleomycin alone. Material and Methods Study Description This study is an interventional, one-center, prospective case series, clinical trial on the effectiveness of intralesional bleo- mycin combined with ECT for the treatment of plantar warts, TABLE 1. Selection criteria for the patients with warts Inclusion Criteria Exclusion criteria Histologically confirmed Previous Reynaud phenomenon Minimal size of the wart 0.5 cm Heart patients Plantar warts Another wart less than 3 cm Ability of patients to follow the instructions Previous bleomycin use Patients older than 18 Patients younger than 18 Research | Dermatol Pract Concept 2017;7(3):4 23 Statistical analysis Fisher exact test was used to assess any difference in terms of response rate at the two time points between the control arm and the experimental arm. SPSS software (version 17.00, SPSS, Chicago) was used for statistical analysis. A P-value of less than 0.05 was considered to indicate statistical signifi- cance. Results Patient demographics Characteristics of the patients enrolled in the protocol are depicted in Table 2. Briefly, the study’s cohort included 12 men and 10 women with ages ranging from 27 to 85 years, with a mean of 53.8 years. A total of 22 warts were treated, exclusively located on the feet. All patients were non-respond- ers of other treatment modalities and had had a treatment history of more than 6 months. Efficacy Table 2 summarizes the data obtained at 1 month and 3 months follow-up. Indeed, focusing on the 3-month follow- up, 78% (7/9) of patients treated with bleomycin coupled time: 1 ms per cm2 of treated area). The pulses have been delivered by using caliper electrodes. In Figure 1, panel A, an example of calipers application on a plantar wart is depicted. A pain scale (0, no pain; 10, maximum pain) was passed on each patient to evaluate pain during the procedure. Data collection Follow-up of the progress of the treatment process was sched- uled at day 30 and day 90 after the treatment. The documen- tation plan during follow up, including photo-documentation of the treated area, consisted of evaluation of local treatment outcome, de novo appearance of skin warts, development of any disorders from any other organic system, as well as long- time outcome of the scarring process at the site of ECT treat- ment (“cosmetic result”). Treatment outcome was evaluated according to endpoint results, at the first or second follow-up appointment, 1 or 3 months after the end of treatment. The endpoints were defined as follows: (1) “Cured” (= complete remission, CR): no wart is detectable (2) “Partial response” as 50% wart reduction (PR) (3) “Non-responding” (4) “Progressive disease”: larger final wart TABLE 2. Main clinical characteristics of the patients and outcome after one and three months Patient age Sex Treatment Outcome after 30 days Outcome after 90 days 70 Male BL PR PR 59 Female BL PR PR 59 Female BL PR PR 52 Female BL+E PR CR 51 Female BL PR PR 54 Male BL NR NR 45 Male BL CR PR 60 Female BL+E PR PR 62 Male BL PR PR 62 Male BL PR PR 39 Male BL+E NR NR 63 Female BL PR CR 45 Male BL+E CR CR 47 Female BL+E CR CR 61 Male BL PR PR 48 Male BL PR CR 85 Male BL+E CR CR 33 Female BL+E CR CR 68 Female BL PR PR 59 Male BL NR PR 27 Male BL+E PR CR 21 Male BL+E PR CR 24 Research | Dermatol Pract Concept 2017;7(3):4 pain is the most commonly described, while no systemic reactions have been linked with the use of bleomycin for warts [12]. Interestingly, a recent Cochrane meta-analysis has shown with ECT had complete remission and only 2 patients displayed partial remis- sion or no response. On the other hand, at 3-month follow-up 16% (2/13) of patients treated with bleomycin had complete remission, while 76% (10/13) of patients displayed partial remission and 8% (1/13) had no response. Divid- ing the two groups of patients (com- plete remission against all the others) and analyzing the different outcomes in the two arms of treatment, a statis- tical significant difference was found (p=0.0015), proving a greater efficacy of the treatment with bleomycin combined with ECT with respect to bleomycin alone. In Figure 1 (Panels from B to I) two cases with partial or complete resolution of the wart after one month of treatment are depicted. Side effects Not one of the patients had adverse sys- temic effects. The most notable local side effect was he pain during the injection of local anesthetic. None complained of pain during intralesional bleomy- cin injection or during ECT. Other side effects were relatively infrequent, occa- sionally including redness and tender- ness, and skin discoloration at the site of injection. One patient of arm A of the study reported pain on the third day after bleomycin injection. Concern- ing ECT treatment, it was always well tolerated by the patients without any discomfort. Discussion Bleomycin has been successfully used for the treatment of recalcitrant warts [11]. The most likely mechanism of action of this drug is the creation of unstable free radicals causing single-strand breaks and apoptosis [12]. Multiple bleomy- cin administration techniques have been studied with various success rates [11,13]. However, several intralesional bleomycin injections are generally nec- essary to obtain clearance of the warts [14]. Concerning side effects, injection that the treatment of choice for warts remains ambiguous, despite the proved efficacy of intralesional bleomycin [15]. Several studies have demonstrated that the efficacy of bleomycin can be greatly improved by coupling its injection with F G (Continued next page) Research | Dermatol Pract Concept 2017;7(3):4 25 References 1. Ciconte A, Campbell J, Tabrizi S, Garland S, Marks R. Warts are not merely blemishes on the skin: a study on the morbidity as- sociated with having viral cutaneous warts. Australas J Dermatol. 2003;44:169-173. 2. Gibbs S, Harvey I, Sterling J, Stark R. Local treatments for cutane- ous warts: systematic review. BMJ. 2002;325:461. 3. Cardoso JC, Calonje E. Cutaneous manifestations of human papillomaviruses: a review. Acta Dermatovenereol Alp Pannonica Adriat. 2011; 20:145-154. 4. Rivera A, Tyirng SK. Therapy of cutaneous human papillomavirus infections. Dermatol Ther. 2004;17:4411-4418. 5. Buchanan J, Nieland-Fisher N. Document responses from pa- tients regarding warts and current therapy. Arch Dermatol. 2004;140:487-488. 6. Spugnini EP, Azzarito T, Fais S, Fanciulli M, Baldi A. Electroche- motherapy as First Line Cancer Treatment: Experiences from Veterinary Medicine in Developing Novel Protocols. Curr Cancer Drug Targets. 2016;16:43-52. 7. Spugnini EP, Baldi A. Electrochemotherapy in veterinary on- cology: from rescue to first line therapy. Methods Mol Biol. 2014;1121:247-256. 8. Spugnini EP, Melillo A, Quagliuolo L, et al. Definition of novel electrochemotherapy parameters and validation of their in vitro and in vivo effectiveness. J Cell Physiol. 2014;229:1177-1181. 9. Campana LG, Testori A, Curatolo P, et al. Treatment efficacy with electrochemotherapy: A multi-institutional prospective observa- tional study on 376 patients with superficial tumors. Eur J Surg Oncol. 2016; 42:1914-1923. 10. Spugnini EP, Fais S, Azzarito T, Baldi A. Novel Instruments for the implementation of electrochemotherapy protocols: From bench side to veterinary clinic. J Cell Physiol. 2017;232:490-495. 11. Kruter L, Saggar V, Akhavan A, , et al. Intralesional bleomycin for warts: patient satisfaction and treatment outcomes. J Cutan Med Surg. 2015;19:470-476. electrical pulses enabling the cells to be more prone to incor- porate bleomycin [7]. Indeed, bleomycin is one of the most used drugs in ECT protocols for the treatment of tumors [8]. A major goal of this project was to demonstrate an increased efficacy in the treatment of plantar warts with intra- lesional bleomycin coupled with electroporation. Indeed, the results of our study show that electroporation coupled with bleomycin significantly improve the outcome of patients with respect to the patients treated with bleomycin alone. Never- theless, in our experimental protocol, only one intralesional injection of bleomycin was performed and, when coupled with electroporation, was sufficient to get complete remission in the majority of the patients with disappearance of the wart. This further supports the idea that electroporation is able to amplify the effect of bleomycin, since its efficacy in the treat- ment of warts is generally linked to multiple injections on the site of the wart [15]. The second goal of the project was to demonstrate that the application of electric voltage to the warts had no adverse side effects. Indeed, none of the patients suffered systemic side effects, and the only local adverse effects were the pain at the site of the injection and occasion- ally redness and tenderness of the skin. Electroporation was well tolerated by the patients without any discomfort. The main limitation of our study was the small number of patients enrolled in the protocol. Therefore, additional studies are necessary to confirm the efficacy of electroporation cou- pled with bleomycin in treating plantar warts. Nevertheless, the promising results obtained, together with the fact that the treatment was well tolerated by all the patients, represent the basis for the application of novel protocols for the treatment of different benign and locally malignant diseases of the skin by means of electroporation. Figure 1. Two cases of plantar wart with partial or complete resolution after one month from the treatment with bleomycin and electropora- tion. (A) The application of the electrodes on the plantar wart; (B) A plantar wart at presentation; (C) Partial resolution after one month form the treatment; (D) Dermoscopic appearance of the wart at presentation; (E) Dermosocopic appearance of the wart after partial remission; (F) A plantar wart at presentation; (G) Complete resolution after one month form the treatment; (H) Dermoscopic appearance of the wart at presentation; I) Dermosocopic appearance of the wart after complete remission. [Copyright: ©2017 Pasquali et al.] H I 26 Research | Dermatol Pract Concept 2017;7(3):4 in both external auditory canals using bleomycin injections. Clin Exp Otorhi- nolaryngol. 2015;8:295-297. 14. Dhar SB, Rashid MM, Islam A, Bhuiyan M. Intralesional bleomycin in the treat- ment of cutaneous warts: a randomized clinical trial comparing it with cryother- 12. Kollipara R, Ekhlassi E, Downing C, Guidry J, Lee M, Tyring SK. Advance- ments in pharmacotherapy for noncan- cerous manifestations of HPV. J Clin Med. 2015;4:832-846. 13. Lee JH, Burm JS, Yang WY, Kang SY, Byun SW. Treatment of verruca vulgaris apy. Indian J Dermatol Venereol Leprol. 2009;75:262-267. 15. Kwok CS, Gibbs S, Bennett C, Holland R, Abbott R. Topical treatments for cutane- ous warts. Cochrane Database Syst Rev. 2012;9:CD001781.