The Effect of Reduced Bleomycin in Electrochemotherapy Treatment

Introduction

Electroporation Electroporation is a method in which the cell is exposed to an external electric field resulting in increased permeability of the cell membrane \[1\]. Electroporation is used to introduce drugs into cells, without affecting intracellular organelles or cell viability. By inducing this response, drugs that are normally non-permeant, e.g. hydrophilic chemotherapy such as bleomycin, will enter the cell by diffusion thus enhancing the cytotoxic effect \[2, 3\]. The combination of electroporation and chemotherapy is known as electrochemotherapy (ECT). The application of the electric pulses causes vasoconstriction, inducing drug entrapment due to reduced blood flow - this is termed the vascular lock \[4, 5\]. In addition, electroporation also has a vascular-disrupting effect, when combined with chemotherapy \[4\]. ECT damages tumour vasculature leading to an additional cascade of tumour cell death due to lack of oxygen, nutrients, and waste product accumulation \[5\]. These vascular changes are more prolonged in tumours than in normal tissue \[6\].

Electrochemotherapy ECT is the combination of chemotherapy and electroporation, in which electric pulses are administered to a tumour after intravenous or intratumoural injection of chemotherapy. ECT has been shown to be effective in the treatment of cutaneous malignancies and is established as standard treatment for cutaneous primary and secondary skin tumours and ulcerating malignant wounds \[7, 8\]. ECT is often used as a one time treatment, but can be repeated if necessary.

Bleomycin is the drug of choice for ECT; it has low toxicity to normal cells and the largest increase in efficacy after electroporation enhancing the cytotoxic effect with a factor 300 to 5.000 \[9\]. ECT is performed in general or local anaesthesia depending on tumour location and size. Patient preference and institutional practice is also taken into account \[8\].

Bleomycin is an antineoplastic drug derived from Streptomyces verticillus and is used in the treatment of a variety of malignancies, such as lymphoma and testicular cancer \[11\]. Bleomycin generates single- and double-strand DNA breaks, i.e., one molecule of bleomycin can cause 10-15 DNA strand breaks \[12\]. Due to its hydrophilic nature, the entry of bleomycin into cells is restricted and occurs via an endocytotic process \[13\]. Bleomycin causes cell death in two ways, depending on the doses used: 1) if a lower amount of bleomycin is used (thousands of bleomycin molecules) the cell arrests in the G2-M phase, enlarges and becomes polynucleated and dies slowly by necrosis; 2) if a larger amount is used (millions of bleomycin molecules), pseudo-apoptosis kills the cell within a few minutes \[12, 14\].

Recently, it has been shown that the disaccharide moiety of bleomycin facilitates uptake by cancer cells. It is suggested that the upregulation of receptors associated with enhanced glycolysis in tumour cells could be the reason for bleomycin targeting tumour cells selectively \[51\].

Bleomycin is degraded by an enzyme - bleomycin hydrolase -, found in various normal tissues \[11\]. A lower concentration of this enzyme is found in the skin and lungs, which can lead to increased harms in these organs \[15, 16\]. The Danish Medicines Agency (DMA) states in its product summary that half of patients receiving bleomycin experience harms related to the skin (erythema, hyperpigmentation, striae or soreness) and one tenth experience pulmonary reactions with a minority (1%) dying from pulmonary fibrosis \[15\]. Some studies have observed that the risk of developing pulmonary toxicity from bleomycin is increased with age, drug dose, and concomitant oxygen therapy and thoracic radiation therapy \[16\].

Since harms are associated with accumulative bleomycin dose, it is desirable to investigate if the dose can be reduced to minimize risk of harms and maintain the same efficacy. This may also enable some ineligible fragile patients to become eligible for ECT treatment, e.g., lung cancer patients with a higher risk of developing pulmonary toxicity.

Cutaneous malignancies A significant proportion of cancer patients develop cutaneous metastases (\~10%) that often become exuding, bleeding or odorous \[17, 18\]. Thus, the metastases may influence patients' quality of life \[19, 49\]. Cutaneous metastases negatively affect patients, i.e., self-esteem, body image, and sexuality with feelings tied to frustration and loss of power \[21, 22\]. Most cancer patients receive chemotherapy or immunotherapy, but some cancers do not respond to these standard treatments \[23\]. ECT may be used for such cancers enhancing palliation and quality of life \[3, 24\]. The most frequently seen cutaneous metastases originate from breast cancer and occur in the chest region, but may arise anywhere on the body and originate from most cancer types \[20\]. The cutaneous malignancies may vary in size from a few millimetres to very large and may be focal or extensive.

Clinical experience with electrochemotherapy In 1993, Belehradek and colleagues published the first clinical trial of ECT using bleomycin in 8 patients indicating that the treatment was well-tolerated by patients and with a clinical complete rumour response of 57% \[25\]. In 2003 Gothelf and colleagues reviewed the 11 clinical trials completed between 1993 and 2001, involving a total of 96 patients with 411 tumours/metastases treated with different approaches \[26\]. The overall response (OR) varied between 15% and 100%. For the 11 ECT studies investigating bleomycin and ECT treatment on cutaneous malignancies the OR has been between 22% and 100%. Three studies stand out, with an OR of 22%, 28% and 45%, respectively \[27-29\]. The study by Domenge from 1996, was one of the first studies conducted on ECT and included only seven patients \[27\]. In the studies by Matthiessen and Kreuter larger tumours (\>3 cm) were included. It is well-known that the size of the tumour affects the response of the ECT treatment \[29\]. When excluding these studies the OR varies from 58% to 100%. The largest ECT bleomycin study by Clover and colleagues from 2020 included 987 patients with 2,483 tumours \[30\] and showed that different tumours have different ORs; for example, basal cell carcinoma (BCC) and Kaposi's sarcoma (KS) show the highest OR with 96% and 98%, respectively; while breast cancer metastases, malignant melanoma (MM) and squamous cell carcinoma (SCC) have lower responses of 77%, 82% and 80%, respectively. This study also observed an effect of tumour size on the overall tumour response. The highest response rates were observed in small tumours (\<3 cm). In small skin metastases (\<2 cm), no significant OR difference was observed between intravenous vs. intratumoural administration of bleomycin, however, for larger skin metastases (\>2cm), the OR was significantly (p\<0.05) higher with intravenous administration (57% vs. 48%) \[30\]. Thus, our trial will be stratified according to tumour/metastasis size.

Clinical experience with reduced bleomycin In 1996, one of the first clinical ECT bleomycin studies with six patients used a bleomycin dose of 10.000 IU/m2 \[31\] (see Table 1), since medical and surgical oncologists believed that a dose of 22,700 IU/m2 - used in the first clinical trials - was too high. This is the only study using this dose and from 1998 the standard dose of 15.000 IU/m2 was used in most studies.

In 2016, a case report of a patient receiving ECT with bleomycin with a 50% reduced dose (7.500 IU/m2) due to renal dysfunction was published. In this case a complete tumour response was observed \[32\].

From 2013 to 2016, Rotunno identified via the International Network for Sharing Practices on Electrochemotherapy (InspECT) database 57 patients treated with ECT and reduced bleomycin (7.500, 10.000 or 13.500 IU/m2) \[33\] due to e.g., reduced renal function and high age. Reduced bleomycin showed similar tumour response to a standard bleomycin dose (15.000 IU/m2), with an OR of 64% to 82 %. Thus, the authors of the study suggested that reduced bleomycin doses could be as effective as the standard treatment - especially in patients with impaired renal function or patients eligible to multiple ECT cycles. It was concluded that further research in order to find a personalized bleomycin dose is needed \[33\].

In 2018 and 2021, two studies concluded that a reduced dose of bleomycin is a feasible treatment option for elderly patients, with equal efficacy to standard dose treatment (OR 87% to 100%) and should be considered as a treatment in patients with comorbidities \[34, 35\]. In addition, in a study by Groselj et al. from 2018 it was suggested that the cosmetic outcome was improved with the use of a reduced dose of bleomycin compared to the standard dose due to shorter healing and less development of fibrous scarring tissue \[36\].

Rationale for 50% bleomycin reduction in this study:

Based on former literature \[32-36\] we find it realistic to reduce the bleomycin dose with 50% and still gain a sufficient overall tumour response after ECT treatment. For time efficiency purposes, in this study we have decided to investigate a 50% reduced dose of bleomycin only. Randomisation to several bleomycin dose levels would subsequently result in the need of a significant increase in the number of included patients and/or referring hospital departments, which would be time consuming and difficult to complete within a manageable time frame.

Methods Trial design The study design as a non-inferiority study and is a parallel two-armed double-blinded randomized clinical trial, where the patients are stratified due to the size of their biggest cutaneous tumour (≤ 3 cm or \> 3 cm) and afterwards randomized (1:1) to receive a standard dose bleomycin or a 50% reduced dose bleomycin.

Blinding is performed by the pharmacy mixing the allotted dose regimen after randomization, both patients and treating staff will be blinded to the bleomycin dose. Unblinding will be performed according to procedure if need be. Unblinding of all results will take place after last patients evaluation of primary endpoint.

The study will be conducted at two centers in Denmark: At the department of Clinical Oncology and Palliative Care, Zealand University Hospital, Roskilde and at the Department of Oncology, Copenhagen University Hospital, Herlev Gentofte Hospital. The three year study period is expected to start in October 2024.

Study population Patients will be referred from oncologic departments and must be advised about treatment alternatives. It must be evaluated whether the patient will benefit from the treatment, given the localisation and size of the tumour/tumours, the patient's general condition and expected survival. The purpose, possible risks and benefits should be discussed in collaboration with the patient.

Indications for participating in this trial includes:

* Biopsy verified cutaneous malignancies of any histology, which are symptomatic due to bleeding, ulceration, oozing, odour, or pain * Primary skin cancers, including recurrent tumours, where other treatment modalities have failed, are not possible or not appropriate * Patients who are receiving systematic anti-neoplastic therapy, but where cutaneous metastases are progressing or not responding despite satisfactory response to systemic therapy in internal organs * Patient preference for electrochemotherapy, after other treatment possibilities have been thoroughly explained to the patient For eligibility criteria, please refer to the section about eligibility criteria.

Pre-treatment examination and pain management:

The pre-treatment examination will be performed up to one month prior to the procedure and will include diagnosis of primary tumour, TNM (tumour, node, and metastasis) classification, and previous anti-neoplastic treatments. Under previous treatments, it must be noted which nodu

• Treatment response for normal dose group, reduced dose group, and for all treated tumors at optimnal time points using the mRECIST criteria. [Time frame: The follow up is mandatory for the patients after 2 weeks, after 3 months and 12 months. Follow up is optional at 1 month, 2 moths, 4 moths and 6 months. At every visit the tumours size will be measured.]
• Aesthetic outcome for normal dose group, reduced dose group and for all tumours 3 months after treatment and 1 year after treatment, using the Vancouver Scar Scale. [Time frame: Assessed 3 months after treatment and 1 year after treatment.]
• Aesthetic outcome for normal dose group, reduced dose group and for all tumors assessed at 3 month and 12 month, using Patient and Observer Scar Assessment Scale (POSAS). [Time frame: 3 months ater treatment and 1 year after treatment.]
• Complete and partial remissions for all patients treated (patient level) [Time frame: Before treatment (baseline) compared to 3 months after treatment.]
• Biopsy at 12 months (optional) for histological examination of malignancy presence (HE stain, and staining for specific tumour markers can be included). [Time frame: 12 months]
• All-cause mortality and cancer related mortality within 12 months. [Time frame: Study period of 12 months.]
• Quality of life before treatment and at approximately 3 months measured by EORTC-QLQ-C30 [Time frame: Questionaires will be performed before electrochemotherapy, and at 3 and 12 months after the treatment. .]
• Quality of life before treatment and 3 months post-treatment measured in 16 patients with semi-structured qualitative interviews. [Time frame: Qualitative interviews will be performed before electrochemotherapy and 3 months post-treatment in the same patients.]
• Relation between tumour histology and tumor response rates at 3 months and 12 months. [Time frame: Response rate will be calculated by comparing measurement at baseline and at respectively 3 months, and at 12 months.]
• Response rates in tumours according to size [Time frame: Baseline measurement will be compared to measurements at respectively 3 months and 12 months.]
• Side effects according to CTCAE according to treatment dose - data from normal dose group, reduced dose group, and for the whole population will be registered and compared. [Time frame: Will be meassured at every visit up to one year]
• The presence of pain (if any) associated with the cutaneous malignancies will be assessed using Numeric Rating Scale (NRS). [Time frame: Will be meassured at every visit up to 12 months.]