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

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

Immune cell populations in the tumour environment following calcium electropora-tion for cutaneous metastasis: a histopathological study.

BACKGROUND AND PURPOSE: Calcium electroporation (CaEP) involves injecting calcium into tumour tissues and using electrical pulses to create membrane pores that induce cell death. This study assesses resultant immune responses and histopathological changes in patients with cutaneous metastases. PATIENTS/MATERIALS AND METHODS: The aimed cohort comprised 24 patients with metastases exceeding 5 mm. Tumours were treated once with CaEP (day 0) or twice (day 28). Biopsies were performed on days 0 and 2, with additional samples on days 7, 28, 30, 35, 60, and 90 if multiple tumours were treated. The primary endpoint was the change in tumour-infiltrating lymphocytes (TILs) two days post-treatment, with secondary endpoints evaluating local and systemic immune responses via histopathological analysis of immune markers, necrosis, and inflammation. RESULTS: Seventeen patients, with metastases primarily from breast cancer (14 patients), but also lung cancer (1), melanoma (1), and urothelial cancer (1), completed the study. Of the 49 lesions treated, no significant changes in TIL count or PD-L1 expression were observed. However, there was substantial necrosis and a decrease in FOXP3-expression (p = 0.0025) noted, with a slight increase in CD4+ cells but no changes in CD3, CD8, or CD20 expressions. Notably, four patients showed reduced tumour invasiveness, including one case of an abscopal response. INTERPRETATION: This exploratory study indicates that CaEP can be an effective anti-tumour therapy potentially enhancing immunity. Significant necrosis and decreased regulatory lymphocytes were observed, although TIL count remained unchanged. Several patients exhibited clinical signs of immune response following treatment.

Calcium electroporation in cutaneous metastases - A non-randomised phase II multicentre clinical trial.

BACKGROUND: Cutaneous metastases can cause distressing symptoms and be challenging to treat. Local therapies are essential in management. Calcium electroporation uses calcium and electrical pulses to selectively kill cancer cells. This multicentre study aimed to define response in cutaneous metastases across different cancer types. METHODS: Patients with tumours ≤3 cm of any histology were included (stable or progressing on current therapy ≥2 months), at three centres. Tumours were treated with 220 mM calcium chloride injection and manual application of eight 0.1 ms pulses with 1 kV/cm and 1Hz with a handheld electrode, in local or general anaesthesia. Clinical response was evaluated after 1, 2, 3, 4, 5, 6, and 12 months. Primary endpoint was response at two months. The overall response rate (ORR) was partial- and complete responses of treated tumours. MR-imaging and qualitative interviews were performed in respective subsets. RESULTS: Nineteen patients with disseminated cancer (breast n = 4, lung n = 5, pancreatic n = 1, colorectal n = 2, gastric n = 1, and endometrial cancer n = 1) were enrolled, and 58 metastases were treated (50 once, 8 retreated). The ORR was 36% (95% CI 22-53) after two months. Best ORR was 51% (CR 42%; PR 9%). Previous irradiation improved outcomes (p = 0.0004). Adverse events were minimal. Median pain score was reduced after two months (p = 0.017). Treatment may relieve symptoms according to qualitative interviews. MRI showed restriction in treated tissue. CONCLUSION: The majority of tumours were treated only once with calcium electroporation, achieving an ORR of 36% after two months and best ORR of 51%. Efficacy, symptom-relief and safety support calcium electroporation as a palliative treatment option for cutaneous metastases.

Qualitative Investigation of Experience and Quality of Life in Patients Treated with Calcium Electroporation for Cutaneous Metastases.

(1) Background: Calcium electroporation is a novel cancer treatment. It includes injecting calcium-solution and applying electric pulses to tumour tissue. Data on quality of life for patients with cutaneous metastases treated with calcium electroporation is limited. We evaluated quality of life in patients with skin metastases treated with calcium electroporation using qualitative interviews. (2) Methods: This investigation featured a subgroup from a non-randomised phase II study (CaEP-R) at Zealand University Hospital, Denmark, studying response to calcium electroporation in cutaneous metastasis (ClinicalTrials no. NCT04225767). Participants were interviewed at baseline before calcium electroporation treatment and after two months. Data was analysed phenomenologically; (3) Results: Interviews were conducted February 2020-November 2021. Nine patients were included, of which seven participated in both interviews. All seven patients expected treated tumours to disappear, symptom relief and minimal side effects. Most patients requested peer accounts. All patients found the treatment uncomfortable but acceptable; all thought their fears of electric pulses exceeded their experience. All would repeat the treatment if effective. Successful treatment had a positive effect on pain, symptomatic wounds, sleep, vigour and social inclination; (4) Conclusions: Calcium electroporation enhanced health-related quality of life by reducing symptoms and increasing social inclination. Peer accounts provide patients with a shortcut to confidence in treatment on top of doctors' recommendations.

Calcium Electroporation for Management of Cutaneous Metastases in HER2-Positive Breast Cancer: A Case Report.

We report a case of successful treatment of cutaneous metastases in HER2-positive breast cancer with calcium electroporation (CaEP), in addition to trastuzumab, over a period of 5 years. CaEP is performed in local or general anesthesia, by injecting calcium chloride intratumorally and then electroporating cells in the area. Using a handheld needle electrode, a series of short, high-voltage electric pulses are delivered, which transiently permeabilizes cell membranes, causing toxic intracellular calcium levels. The treatment causes cancer cell death, while normal cells are less affected, making the treatment useful for local management of cutaneous lesions. This case presents a 66-year-old female, who had mastectomy surgery followed by adjuvant chemo- and radiotherapy for an ER-negative, HER2-positive breast cancer on her right side in 2003, and a mastectomy followed by endocrine therapy for an ER-positive, HER2 normal breast cancer on her left side in 2006. In 2015, the patient presented local cutaneous recurrence of the ER-negative, HER2-positive breast cancer. The patient was treated with trastuzumab alone, trastuzumab emtansine (TDM1), and a combination of trastuzumab and CaEP. TDM1 was found to have a slightly better effect on the cutaneous metastases than trastuzumab, but the side effects of TDM1 were not acceptable to the patient. The combination of continuous HER2-inhibition and intermittent CaEP, when needed, has been effective in keeping the cutaneous metastases under control for 5 years, and presumably more tolerable for the patient than chemotherapy. An interesting finding was local sparing of calcium electroporated skin from new recurrences, otherwise seen in the general area, which could be a sign of local immunity. This warrants further studies investigating local immunomodulation following CaEP. The patient reported appreciation of a treatment option without chemotherapy, and satisfaction with the outcome of the combination of HER2 inhibition and CaEP treatment. CaEP treatment is currently phase II treatment, and mechanisms and possible applications still need investigation. This novel anticancer treatment could potentially benefit many patients, due to its efficacy, low cost, and accessibility. This case provides observations, which may inspire future trials with CaEP for skin metastases of HER2-positive breast cancer.

Electrochemotherapy with intravenous bleomycin for patients with cutaneous malignancies, across tumour histology: a systematic review.

BACKGROUND: Electrochemotherapy (ECT) is an established treatment for primary and secondary cutaneous tumours. The method combines chemotherapy with electroporation, thus increasing the cytotoxic effect of the chemotherapeutic drug. Bleomycin is the drug of choice for ECT, as it is already well established as a treatment for several cancer types and has the largest increase in efficacy after electroporation, enhancing the cytotoxic effect several hundred fold. The response rates of ECT have over the past 30 years been high and consistent. Case based reports point out that the efficacy possibly can be maintained even when the dose of bleomycin is reduced. Consequently in 2018, studies began investigating reducing the bleomycin dose. AIM: The purpose of this review is to summarise all data published using intravenous bleomycin for cutaneous malignancies and is to our knowledge the first review to examine the use of a reduced bleomycin dose in ECT. METHODS: This study is a systematic review. Fifty-five clinical studies investigating ECT with intravenous bleomycin for patients with cutaneous malignancies were included. RESULTS: Studies published from 1993 to 2021 investigating the effect of ECT include 3729 patients and indicate a consistent and high response with a mean objective response rate (ORR) of 81.5%. Interestingly, studies using lower doses of bleomycin observe a similar ORR (85.5%), opening the possibility that a lower dose may not be inferior. CONCLUSION: This study gives an overview of published studies on ECT with intravenous bleomycin for patients with cutaneous malignancies, including the use of a reduced bleomycin dose, as preparation for a randomised study.

Study protocol designed to investigate tumour response to calcium electroporation in cancers affecting the skin: a non-randomised phase II clinical trial.

INTRODUCTION: Skin malignancy is a distressing problem for many patients, and clinical management is challenging. This article describes the protocol for the Calcium Electroporation Response Study (CaEP-R) designed to investigate tumour response to calcium electroporation and is a descriptive guide to calcium electroporation treatment of malignant tumours in the skin. Calcium electroporation is a local treatment that induces supraphysiological intracellular calcium levels by intratumoural calcium administration and application of electrical pulses. The pulses create transient membrane pores allowing diffusion of non-permeant calcium ions into target cells. High calcium levels can kill cancer cells, while normal cells can restore homeostasis. Prior trials with smaller cohorts have found calcium electroporation to be safe and efficient. This trial aims to include a larger multiregional cohort of patients with different cancer diagnoses and also to investigate treatment areas using MRI as well as assess impact on quality of life. METHODS AND ANALYSIS: This non-randomised phase II multicentre study will investigate response to calcium electroporation in 30 patients with cutaneous or subcutaneous malignancy. Enrolment of 10 patients is planned at three centres: Zealand University Hospital, University Hospital of Southern Denmark and University Hospital Schleswig-Holstein. Response after 2 months was chosen as the primary endpoint based on short-term response rates observed in a prior clinical study. Secondary endpoints include response to treatment using MRI and change in quality of life assessed by questionnaires and qualitative interviews. ETHICS AND DISSEMINATION: The trial is approved by the Danish Medicines Agency and The Danish Regional Committee on Health Research Ethics. All included patients will receive active treatment (calcium electroporation). Patients can continue systemic treatment during the study, and side effects are expected to be limited. Data will be published in a peer-reviewed journal and made available to the public. TRIAL REGISTRATION NUMBERS: NCT04225767 and EudraCT no: 2019-004314-34.

Calcium Electroporation for Keloids: A First-in-Man Phase I Study.

BACKGROUND: Keloid scarring is a pathologic proliferation of scar tissue that often causes pruritus, pain, and disfigurement. Keloids can be difficult to treat and have a high risk of recurrence. Recent studies have shown promising results in the treatment of cutaneous metastases with intralesional calcium combined with electroporation (calcium electroporation). As calcium electroporation has shown limited side effects it has advantages when treating benign keloid lesions, and on this indication we performed a phase I study. METHODS: Patients with keloids were treated with at least 1 session of calcium electroporation and followed up for 2 years. Calcium was administered intralesionally (220 mM) followed by the application of eight 100-µs pulses (400 V) using linear-array electrodes and Cliniporator (IGEA, Italy). Treatment efficacy was evaluated clinically (size, shape, erythema), by patient self-assessment (pruritus, pain, other) and assessed histologically. RESULTS: Six patients were included in this small proof of concept study. Treatment was well tolerated, with all patients requesting further treatment. Two out of 6 patients experienced a decrease in keloid thickness over 30%. A mean reduction of 11% was observed in volume size, and a mean flattening of 22% was observed (not statistically significant). Five out of 6 patients reported decreased pain and pruritus. No serious adverse effects or recurrences were observed over a mean follow-up period of 338 days. CONCLUSION: In this first phase I clinical study on calcium electroporation for keloids, treatment was found to be safe with minor side effects. Overall, patients experienced symptom relief, and in some patients keloid thickness was reduced.

A Comprehensive Review of Calcium Electroporation -A Novel Cancer Treatment Modality.

Calcium electroporation is a potential novel anti-cancer treatment where high calcium concentrations are introduced into cells by electroporation, a method where short, high voltage pulses induce transient permeabilisation of the plasma membrane allowing passage of molecules into the cytosol. Calcium is a tightly regulated, ubiquitous second messenger involved in many cellular processes including cell death. Electroporation increases calcium uptake leading to acute and severe ATP depletion associated with cancer cell death. This comprehensive review describes published data about calcium electroporation applied in vitro, in vivo, and clinically from the first publication in 2012. Calcium electroporation has been shown to be a safe and efficient anti-cancer treatment in clinical studies with cutaneous metastases and recurrent head and neck cancer. Normal cells have been shown to be less affected by calcium electroporation than cancer cells and this difference might be partly induced by differences in membrane repair, expression of calcium transporters, and cellular structural changes. Interestingly, both clinical data and preclinical studies have indicated a systemic immune response induced by calcium electroporation. New cancer treatments are needed, and calcium electroporation represents an inexpensive and efficient treatment with few side effects, that could potentially be used worldwide and for different tumor types.