Radiol Oncol 2023; 57(1): 121-126. doi: 10.2478/raon-2023-0010 121 research article Treatment of vulvar cancer recurrences with electrochemotherapy – a detailed analysis of possible causes for unsuccessful treatment Gregor Vivod1,2, Tanja Jesenko2,3, Gorana Gasljevic4, Nina Kovacevic1,2,5, Masa Bosnjak3,6, Gregor Sersa3,7; Sebastjan Merlo1,2,8, Maja Cemazar3,9 1 Department of Gynecological Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Medical Faculty Ljubljana, University of Ljubljana, Ljubljana, Slovenia 3 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Faculty of Health Care Angela Boškin, Jesenice, Slovenia 6 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 7 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 8 Medical Faculty, University of Maribor, Maribor, Slovenia 9 Faculty of Health Sciences, University of Primorska, Izola, Slovenia Radiol Oncol 2023; 57(1): 121-126. Received 21 December 2022 Accepted 11 January 2023 Correspondence to: Assist. Prof. Sebastjan Merlo, M.D., Department of Gynecological Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: smerlo@onko-i.si and Prof. Maja Čemažar, Ph.D., Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: mcemazar@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. 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 can- cers 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 stand- ard 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 histo- logical 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 pos- sible 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. Key words: electrochemotherapy; bleomycin; vulvar cancer; recurrence Introduction Vulvar cancer is the fourth most common gyneco- logical cancer, with an incidence of 2.6 per 100,000 women per year.1 The treatment of vulvar cancer usually involves a combination of surgery and radiotherapy. Systemic treatment is rarely used. Most often, surgery includes radical vulvectomy Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer122 and bilateral lymph groin node dissection or sen- tinel lymph node biopsy.2 Radiotherapy can be used as adjuvant therapy after initial surgery or as part of primary therapy in locally advanced disease. Most recurrences of vulvar cancer occur locally near the surgical margins or in the con- tralateral lymph groin region. The therapeutic mo- dalities used depend on the location, the extent of recurrence and previously used radiotherapy or concomitant chemoradiotherapy.3 The emerging treatment modality for vulvar cancer recurrence is electrochemotherapy. Literature review of electrochemotherapy Electrochemotherapy is a local ablative therapy that uses the application of reversible electric pulses to the tumor to permeabilize the cell mem- brane, hence enabling the entry of cytotoxic drugs into the cells.4 It is most commonly used for the treatment of superficial tumors such as melanoma, sarcoma, squamous cell carcinoma, basal cell car- cinoma, skin metastases from breast cancer and others.5,6 It can also be utilized for the treatment of deep-seated tumors such as primary hepatocellu- lar carcinoma, colorectal cancer, unresectable colo- rectal liver metastases or pancreatic carcinoma.7-12 It is conducted following standard operating pro- cedures, and the method is now used in nearly 180 cancer centers around the world.6 Only a small number of papers describing the use of electrochemotherapy for the palliative treat- ment of gynecological cancers and mostly vulvar squamous cell carcinoma have been presented.13,14 Safety and local efficacy after electrochemothera- py with bleomycin in locoregional cutaneous re- currences of vulvar carcinomas previously treated with chemotherapy, radiotherapy and surgery or unsuitable for standard treatments have been demonstrated.15-19 The effectiveness of the clini- cal cases and studies is presented in Table 1. The success rate of such tumors is 80%, which is lower than the response rate of other skin tumors treated with electrochemotherapy.13,16,18,20-22 The biological predictors of unsuccessful treat- ment have already been reviewed.5 The clinical predictive factors of the tumor response were identified to be the size of the lesions and previ- ous treatment as well as the tumor type. However, the biology behind this process has still not been explored. Indicated were the intrinsic tumor sen- sitivity and tumor stroma, where the vasculariza- tion of the tumors might be the most important factor, in addition to the involvement of the im- mune response. The importance of the vasculature and vascular perfusion of tumors has already been shown to have an important role in the responsive- ness of tumors to electrochemotherapy due to its role in drug delivery and tumor response due to the vascular disrupting action of electrochemo- therapy.23 However, its importance in the response in clinical cases has not yet been discussed. With the aim of better understanding the pathophysiol- ogy of possible causes for unsuccessful treatment with electrochemotherapy in vulvar cancer recur- rences, we present a case report of a 75-year-old woman with vulvar cancer recurrence in whom treatment with electrochemotherapy was ineffec- tive and analyzed the possible causes of failure of such a treatment. A case of unresponsive tumor A 75-year-old woman was diagnosed with recur- rence of vulvar cancer in the clitoral region. At the age of 70, simple vulvectomy of the left la- bium major and sentinel node biopsy (SNB) were TABLE 1. Review of studies evaluating electrochemotherapy in vulvar cancer First author, year published Included no. of patients Average age Histology Response of vulvar cancer OR NR Perrone, 201320 8 84y 8 SCC 6/8 (75%) 2/8 (25%) Perrone, 201521 25 85y 25 SCC 20/25 (80%) 5/20 (20%) Pellegrino, 201618 10 68y 9 SCC1 Paget’s 6/10 (60%) 4/10 (40%) Perrone, 201913 55 79y 57 SCC 3 Paget’s 1 melanoma 46/55 (84%) 9/55 (16%) Corrado, 202016 15 83y 14 SCC 1 CS 12/15 (80%) 3/15 (20%) CS = carcinosarcoma; No = number; NR = no response; OR = objective response; SCC = squamous cell carcinoma; y = year(s) Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer 123 performed because of vulvar squamous cell car- cinoma. The sentinel inguinal nodes were nega- tive, and the tumor was removed with free surgi- cal margins. Five years after primary treatment, recurrence of vulvar squamous cell carcinoma in the clitoral region was diagnosed on regular fol- low up (Figure 1A). Inguinal and distal metasta- ses were excluded after clinical assessment and imaging diagnostics. The patient was presented at the Interinstitutional Tumor Board, which de- cided that electrochemotherapy is a safe and vi- able treatment approach before eventual surgical treatment. The tumor board comprised medical oncologists, radiotherapists, and gynecological on- cologists. The electrochemotherapy protocol was approved by the Institutional Medical Board and Slovenian National Ethical Committee (Number 0120-262/2021/3). Electrochemotherapy with bleo- mycin was performed according to the standard operating procedure.6 The patient underwent re- gional anesthesia. Bleomycin was administered at a dose of 15000 IU/m2 (Bleomycin medac, Medac GmbH, Germany). Eight minutes after intravenous administration of the drug, electric pulses were applied to the tumor in a way that covered all tu- mor nodule, including the safety margin of ~ 1 cm. Hexagonal geometry needle electrodes were used, and electric pulses were generated by Cliniporator (IGEA S.P.A., Italy). Altogether, 7 applications of electric pulses were delivered, and their delivery was verified on the screen of the generator (cur- rent > 1.5 A). The patient was discharged from the hospital the day after electrochemotherapy with no pain and no symptoms of any disturbance. At regular follow-ups one and two months after elec- trochemotherapy, we observed no clinical changes in the tumor (Figure 1B). Histologic analysis of repeated biopsy showed the presence of vulvar squamous cell carcinoma. Wide excision of the tumor was performed with free surgical margins. Eighteen months after vulvar cancer recurrence treatment, there were no visible signs of recur- rence or progression of the disease. Histological examination of the first excisional biopsy performed in 2016 showed well-differ- entiated squamous cell carcinoma arising in the background of differentiated vulvar intraepithe- lial neoplasm (VIN) (Figure 2A). The absence of high-risk HPV was proven by the negative immu- nohistochemical reaction to p16. Staining for p16 was negative in the invasive component as well as in precancerous lesions. Additionally, two sentinel lymph nodes were excised and found to be nega- tive. The patient again underwent an excisional biopsy in 2018 showing differentiated VIN and in 2021 after electrochemotherapy. Histological ex- amination of the post electrochemotherapy biopsy (Figure 2B) showed residual, well to moderately differentiated squamous cell carcinoma, measur- ing approximately 1.3 cm in the largest diameter, invading 0.4 cm in depth. There was no lympho- vascular or perineural invasion present. In the sur- rounding parenchyma, there were some throm- bosed and recanalized blood vessels. Additionally, an immunohistochemical panel consisting of anti CD3, CD20, CD68 PGM1, CD163, and ERG antibod- ies (markers for T and B lymphocytes, macrophag- es and blood vessels) was stained on the primary biopsy from 2016 and the post electrochemothera- py biopsy from 2021. Negligible number of cells on the primary as well as post-electrochemotherapy biopsy was stained positive for CD20, therefore we did not include these sections into analysis. For other markers, five different fields of immu- nohistochemically stained sections were captured with a DP72 CCD camera connected to a BX- 51microscope (Olympus,Hamburg,Germany) and analyzed with AxioVision program (Carl Zeiss, Jena, Germany) to determine the number of posi- tive regions per section. These were then averaged and t-test were performed to determine the sta- FIGURE 1. (A) Local recurrence of vulvar cancer. (B) No response to treatment two months after electrochemotherapy. FIGURE 2. Hematoxylin and eosin stained sections of primary (A) and post- electrochemotherapy (B) biopsy. Scale bar represents 50 μm. A A B B Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer124 A B C D FIGURE 3. Immunohistologically stained sections for lymphocytes CD3 (A), macrophages CD68 PMG1 (B) and CD163 (C), and blood vessels ERG (D), from primary biopsy (Primary Bx) and post-electrochemotherapy biopsy (post-ECT Bx) samples. Scale bar represents 50 μm. The number of positively stained regions ± standard error of the mean (SEM) is presented. Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer 125 tistical significance using GraphPad Prism 9 (La Jolla, CA, USA). Detailed analysis showed signifi- cant differences only in the amount of small blood vessels being more numerous in the first pretreat- ment biopsy in comparison to the posttreatment biopsy (Figure 3). On the other hand, no difference was found in the number of CD3-positive lympho- cytes or CD68-positive (pan macrophages marker) or CD163-positive M2 macrophages between the primary biopsy and post electrochemotherapy bi- opsy. Discussion Response to electrochemotherapy is evaluated in accordance with the modified Response Evaluation Criteria in Solid Tumors (RECIST).24 Complete re- sponse for squamous cell carcinoma is observed in 63% of cases, and objective response is observed in 80%.22 For vulvar cancer, stable disease and pro- gression of disease are observed in 16 to 40% of cases.14 There are many clinical factors contributing to predicting the response to treatment with electro- chemotherapy. The possibility for unsuccessful treatment increases with tumor size, and a marked drop in response rate occurs after chemotherapy or in previously irradiated tissue compared with nonirradiated tissue.22 There are, although, differences in the response rate of different tumor histologies; i.e., melanoma was the most resistant, and basal cell carcinoma was the most sensitive to electrochemotherapy.22 The underlying biological factors have not yet been fully explored. As indicated in the review5, stromal factors may play a significant role. Vasculature has already been shown in preclinical studies to play a crucial role in the perfusion of tumors and conse- quently in drug delivery to tumors.23 To overcome this obstacle in less perfused tumors, intratumoral drug delivery could be an approach to overcome insufficient drug delivery. The vascular component and its destruction by electrochemotherapy can be a significant factor in the tumor response. The vascular disrupting effect of electrochemotherapy is based on the apoptosis of endothelial cells in small vessels, where the abrogation of the blood flow induces hypoxia in tumors and consequently indirect tumor cell death. The bigger vessels, as demonstrated in a study on electrochemotherapy of normal liver in pigs, are not affected by elec- trochemotherapy. The histological analysis was performed 2 and 7 days after electrochemotherapy and no thrombosis or other clinically significant damage to large blood vessels and bile ducts in the liver was observed.25 Another biological factor may contribute to non-responsiveness, tumor cells in low oxygenated parts of tumors are more aggres- sive and are more resistant to therapy, leading also to higher recurrence rate of the treated tumors.26,27 Therefore, in less responsive tumors, lower vascu- larity and slow perfusion could be predictors of a lower response rate. In two clinical studies on elec- trochemotherapy of liver tumors, one on colorectal liver metastases and the other on hepatocellular carcinoma, the effect of vascularity was evident.7,8 A clear difference in the response rate of these two tumor types was observed, although the treat- ment was performed in the same way, even by the same team of experts. Based on the known tumor histology features, colorectal metastases are less perfused tumors than hepatocellular carcinoma. Based on this, it can be deduced that the vascular component is important factor contributing to the response of the tumors. Namely, the vasculature is important for drug delivery and distribution and the vascular disruptive component of electro- chemotherapy. Clinical cases of nonresponsive tumors to elec- trochemotherapy are lacking. This is the first de- tailed analyzed case of recurrent vulvar cancer that has not responded to electrochemotherapy. The analysis of the immune component of the tu- mor stroma showed no significant changes after electrochemotherapy, with lymphocytes present in the margin of the tumor, while macrophages were also distributed in the tumors; however, we can presume that their phenotype was M2, as there were no antitumor effects. In responsive tumors, at least some increase in immune cell infiltration would be expected since electrochemotherapy in- duces immunogenic cell death.28,29 On the other hand, the vascularization of the treated lesion was minimal, which may indicate a poor response to electrochemotherapy due to the minimal delivery of bleomycin after intravenous administration to the tumor site, lack of distribution around the tu- mor and absence of vascular disrupting action of electrochemotherapy. This case clearly indicates that we have to search for biological determinants of failure of electrochemotherapy. As already suggested, the explanations for the heterogeneity in tumor re- sponse may reside in the altered vasculature that occurs with tumor growth and the difference in cell susceptibility or aggressiveness in the hypoxic environment.5 Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer126 In conclusion, we propose to analyze the tu- mor vasculature with pathohistological biopsy or ultrasound prior to electrochemotherapy. The investigation of tumor vasculature may allow us to predict the treatment response of vulvar cancer with electrochemotherapy, which will help to de- termine the best individual treatment option and may ultimately improve patient outcomes. Acknowledgement The authors acknowledge the financial support from the state budget by the Slovenian Research Agency, program no. P3-0003. References 1. United States Cancer Statistics (USCS). U.S. Cancer Statistics Data Visualizations Tool. 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