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

Abstract

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.

Keywords: MRI; calcium; cancer; electroporation; metastases; qualitative interviews; skin; tumour response.

Conflict of interest statement

Competing interests: JG and SKF are coinventors of a patent regarding calcium electroporation. Therapeutic applications of calcium electroporation to effectively induce tumour necrosis. Granted. PCT/DK2012/050496

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.

Figures

Figure 1

Schematic overview of known effects of calcium electroporation on normal cells and cancer cells. Electroporation pulse treatment creates transient pores in both cancer cells (bottom row) and normal cells (top row). In a high calcium concentration environment, the intracellular calcium level is greatly increased immediately after treatment. After a few minutes, the plasma membrane reseals and both normal and malignant cells are overloaded with Ca2+. Ca2+ extrusion from the cytoplasm is carried out by Na+-Ca2+ exchangers, plasma membrane Ca2+-ATPase pumps and sarco-endoplasmic reticulum Ca2+-ATPase pumps that transport Ca2+ into the extracellular space and into the endoplasmic reticulum lumen, respectively while consuming ATP in both normal and cancer cells. Suppression of free cytosolic calcium is further facilitated by buffering in mitochondrial compartments and binding to Ca2+-binding proteins. Calcium pumps and channels may be upregulated or downregulated in malignant cells, impairing calcium homeostasis. Furthermore, cancer cells can have weakened membrane repair mechanisms. Ca2+ overload induces mitochondrial dysfunction and critical ATP depletion in cancer cells. These properties may act as mechanisms of calcium electroporation-induced cell death. As illustrated, electroporation may also cause cellular swelling. Following calcium electroporation, normal cells have the ability to restore homeostasis, while cancer cells are prone to necrotise. Cancer cell necrosis leads to release of a danger signal as well as cell remnants that may induce a local and/or systemic immune response.

Figure 2

Pretreatment and follow-up photography. Guide to overview photo of numbered target lesions (A) and photo of target lesion centrally in the image with longest diameter horizontally. Tumour marked proximally (violet) and adhesive ruler for scale 1 cm below the tumour at the bottom of the image (B). Tumours are numbered according to clinician preference, preferably with the most symptomatic tumour marked as tumour 1.

Figure 3

Treatment area. Local anaesthetic applied peritumourally in a rhomboid manner in an appropriate distance from the target area. For a total tumour treatment, the aim is to treat all visible and/or palpable tumours with a 3 mm margin of clinically normal skin included in the target area. Blue-grey: anaesthetised area; rose: tumour; violet: target margin.

Figure 4

Equipment for calcium electroporation. (A) Anaesthesia is necessary, either local anaesthesia or other (depending on tumour location and size). (B) Calcium is always administered by local injection and using, for example, 1 mL syringes ensures easy and steady administration. (C) Electric pulse delivery is performed by needle electrodes that can penetrate so that the bottom of the tumour can be covered. The linear array electrode is preferred due to superior results for smaller tumours. (D) A square wave pulse generator (electroporator) enables precise delivery of pulses of the planned treatment sequence, in this case eight pulses of 0.1 ms with a voltage of 400 V (corresponding to 1 kV/cm applied voltage to electrode distance ratio, as used for the linear array electrode).

Figure 5

Calcium electroporation procedure. (A) The target area is defined as the area that is clinically visualised as tumour +a margin. (B) When performing the local anaesthesia it is important to provide coverage of the margin as well as a zone around the margin so that the electrodes may be inserted without discomfort. Adding further local anaesthetic below the tumour can also be helpful, in particular when treating larger lesions. (C) The calculated intratoumoral (i.t.) dose of calcium is injected into the tumour in a parallel fashion throughout the tumour. The margin area is then supplemented with calcium until the calcium is evenly distributed throughout the entire target area. (D) The electrode is inserted so that needles reach just beyond the deepest part of the tumour, and a pulse sequence is applied. The electrode can then be subsequently inserted in a systematic way to cover the entire tumour volume, as indicated in figure part E. As can be seen, the treatment area then covers the tumour with treatment margin.

Figure 6

Cancer location in skin layers. Tumours may present as subcutaneous or cutaneous and involve some or all skin layers, for example, subcutaneous fat (A), dermis (B) and/or epidermis (C). If the epidermis is involved, the tumours may present as ulcerating or fungating wounds. Intact skin versus necrosis after CaEP. In most cases, subcutaneous tumours (A) will not ulcerate after calcium electroporation, and skin will often appear intact after treatment. If treated tumours are situated as in figure part B, there may or may not be ulceration after treatment, depending on many factors such as patient healing potential and degree of invasion of the upper skin layers. Ulcerating or fungating tumours (C) may develop a necrotic, often crusted wound following treatment.

Figure 7

Distribution of electric field. (A) The linear array needle electrodes have needles of 0.7 mm that can be extended up to 3 cm. (B) A diagram of field distribution adapted from Gehl et al (BBA 1999) shows that the distance between arrays is 4 mm, and the distance between needles in the array is approximately 2 mm. At the needle insertion points, high fields will be present that may lead to irreversible electroporation. In the zone for reversible electroporation cell death will be due to the effect of internalised calcium.