The trial will be conducted in accordance with the official version of the Declaration of Helsinki and in agreement with The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use directions for Good Clinical Practice and the respective rules and regulations in Denmark and Germany.
As the treatment is safe and has not led to disease progression or increased tumour growth compared with controls in any preclinical or clinical studies, we expect CaEP treatment to be safe in treatment of cutaneous or subcutaneous malignant tumours of any histology.2 3 11 13 21 22 25 26 31 32 Adverse events and serious adverse events (SAEs) will be evaluated and graded according to CTCAE V.4.0. In view of the severity of metastatic cancer disease, there are certain conditions defined as SAEs but not reported as such in this study, for example, voluntary hospitalisation and surgery as treatment of the underlying cancer.
The trial is approved by the European Medicines Agency and Danish Medicines Agency and pending approval from relevant authorities in Germany. The trial is approved by the Danish Regional Committee on Health Research Ethics (Den Videnskabsetiske Komite for Region Sjælland), 13 December 2019 (case no: SJ-810), Data Protection Agency (no. REG-115–2019) and this trial was registered on EudraCT and ClinicalTrials.gov. Participation in the study requires signed informed consent. The study started on 11 Febuary 2020, and the first patient was included in the study on 18 February 2020. Data will be published in a peer-reviewed journal. Deidentified participant data are available from the corresponding author on reasonable request. Reuse of data requires approval from the pertinent ethics committee. The results of the trial will be made available to the public by open access publication followed up with summaries posted on institution websites and other publically accessible sources.
Patient and public involvement
Patients and the public were not involved in the writing of the protocol in this study. A patient and public research panel will be engaged in the discussion of the outcomes of both response to therapy and the QoL analysis.
After consent and inclusion in the study, data concerning medical history including cancer diagnosis, TNM classification, past and ongoing treatments, demographic data, comorbidity, radiological data, pathology, clinical photos, ECGs, blood tests (including haemoglobin, blood cell count, serum-calcium and C reactive protein) and list of prescription medicine will be recorded. Relevant data will be stored on the case report form and on a clinical trial management system (EasyTrial ApS, Aalborg, Denmark).
Definition of treatment target and photography
The visible and/or palpable target area is assessed clinically and marked (figure 2).
The treated areas will be photographed prior to treatment on day 0 and at follow-up 1, 2, 3, 4, 6 and 12 months after treatment. At each visit, an overview photo of the treatment sites will be taken, as well as photos of each target tumour (figure 2).
Patient QoL is planned for evaluation before treatment, after 2 months and after 1 year, through using the EORTC-certified QLQ-C15-PAL Core Questionnaire. A subset of patients treated at Zealand University Hospital will participate in a qualitative interview at month 0 (before treatment), 2 and 12, in order to assess treatment impact on the QoL of patients with cancer treated with CaEP. The qualitative interview seeks to explore patients’ physical and emotional well-being before and after treatment as well as expectations and experience in relation to treatment. The interviews will be conducted as semistructured face-to-face interviews as well as phone calls, both based on interview guides.33 The recorded interviews will be transcribed and analysed to describe physical and emotional well-being experienced by the patients.
Paracetamol 1 g and/or lidocaine cream may be prescribed 1 hour preoperatively as a prophylactic analgesic for mild pain. CaEP will be performed using local anaesthesia with injection of lidocaine-epinephrine 1% peritumourally, although other anaesthesia may be used. A sufficient amount of local anaesthetics will be applied, which is comparative with other small local surgical procedures (figure 3).
The maximum recommended dose of local anaesthesia will not be exceeded (30 mL for lidocaine-epinephrine 1%). The procedure may be performed in general anaesthesia and muscular relaxant administered, according to standard procedure of the institute. The equipment used in the CaEP procedure is described in figure 4.
Calcium dose and tumour volume
Participants will be given a maximum of 20 mL calcium solution of 220 mmol/L administered intratumourally, equalling a total calcium dose of 4.4 mmol. The calcium dose in this trial will be analogous to the first phase II trials testing CaEP on small cutaneous tumours.2 3 Ten millilitres of calcium chloride 500 mM is suspended in 12.7 mL isotonic NaCl solution to a calcium dose of 220 mmol/L. Mixing is performed bedside and countersigned by an observer. The dose to tumour volume ratio is an adapted calculation from the European Standard Operating Procedure of Electrochemotherapy (ESOPE) guidelines23 29: (1) tumour with a diameter <0.5 cm: 1 mL/cm3 tumour tissue; and (2) with a diameter from 0.5 cm to 3 cm: 0.5 mL/cm3 of tumour tissue.
Tumour volume will be calculated according to ESOPE by the following formula: V=ab2 π / 6 (a=the longest tumour diameter in cm; b=the longest diameter perpendicular to a).
It is important that the entire tumour volume and surrounding tissue is treated, thus we have chosen to define a treatment margin of 3 mm around the tumour when injecting calcium in the electroporation treatment area (figure 3). Calcium will be administered intratumourally by manual injection with linear application on needle retirement spaced around 5 mm to ensure an equal distribution of calcium in the tumour (figure 5). As the volume of peripheral tissue is infinitely variable (figure 6A–C), adequate volume of injected calcium chloride should be individually assessed by the treating clinician.
For practical purposes, a calcium solution (220 mmol/L) dose corresponding to half of tumour volume (mL) is distributed in the margin area. Due to differences in tumour situation in the cutaneous and subcutaneous layers, standardised dose of the tumour margin area can be a challenge (figure 6a–c). The intratumourally injected volume and total injected dose for each tumour are noted.
Assuming a standard whole body extracellular volume of 15 L (dependent on patient size) and a total calcium concentration of 2.2–2.55 mmol/L of which approximately half (1.18–1.32 mmol/L) is unbound and metabolically active, a normal extracellular store of calcium is ~33–35 mmol of which 18–20 mmol is unbound.34 Administering a maximum dose of 4.4 mmol calcium under the circumstances described previously would lead to an increase in total calcium concentration from 2.55 mmol/L to 2.66 mmol/L. Although total calcium concentrations would rise, the concentration of free unbound calcium is tightly regulated by a buffer system primarily controlled by albumin, ensuring homeostasis.34 Free extracellular calcium concentrations can rise, under normal circumstances, to 1.4 mmol/L with no apparent symptoms. Symptoms of hypercalcaemia may occur when free calcium exceeds 1.6 mmol/L and includes increased thirst, frequent urination and abdominal pain (CTCAE 4.0). As previously mentioned, we expect the injected calcium to act locally, with an insignificant systemic rise in unbound calcium as observed in a previous study 6 hours post-CaEP for recurrent head and neck cancer, injecting up to 13 mL 225 mmol/mL CaCl2 intratoumorally (i.t.)27 The method is deemed safe with local injection of up to 360 mg CaCl2 (or 40 mL CaCl2 220 mmol/L solution). Moreover, electroporation causes a local hypoperfusion through vasoconstriction; thus, the injected calcium will linger in the electroporated area, trapped intracellularly as membranes reseal (figure 1).22
Applied electric pulses
We use linear array needle electrodes, which are superior in treatment of smaller tumours in the skin, to plate and hexagonal needle electrodes.35 Pulses will be administered immediately after calcium injection, repositioning the electrode in an adjacent fashion to ensure an even distribution of treatment throughout the tumour (figure 5). The needle application leaves small barely visible punctuations of the skin further allowing the treating clinician to keep track of treatment. The distributed electric field of a linear array needle electrode is illustrated in figure 7.
We use the CE-certified Cliniporator (IGEA, Carpi, Italy) square wave pulse generator for electroporation, which delivers a series of eight consecutive pulses of 0.1 ms each with an amplitude of 1 kV/cm and a frequency of 1 Hz. For the linear array electrodes used in this study, 400V are applied. It has been observed that the linear array electrode led to a higher response rate in small cutaneous tumours,35 which supports the use of this electrode. The maximum current delivered during treatment as measured by the pulse generator will be noted for each tumour.
Magnetic resonance imaging
On a subset of patients, MRI will be used to assess response before (from 30 min up to 24 hours) and immediately after treatment (within 30 min up to 4 hours), as well as after 2 months using DW-MRI as a method to monitor electroporated tissue, using the ADC). ADC is a measure of the magnitude of diffusion of water molecules within tissue, clinically calculated using DW-MRI and expressed in units of mm2/s.36–38
The side effects of CaEP for cutaneous and subcutaneous metastases less than 3 cm in diameter have been described in previous studies.2 3 When the electrical pulse is given, there is a short-term contraction of the underlying muscles. Patients treated under local anaesthesia can experience muscle contraction as mildly unpleasant. Patients under general anaesthesia will not register this contraction. The electric pulses last less than a second, and muscle contractions last for only the period in which the electric pulses are given. Experiences from treating small tumours with ECT and CaEP show that the treated area can initially become erythematous and swollen, but symptoms usually fade within hours to days. The area can subsequently become necrotic but generally heals within 6–10 weeks. In a few cases, infection may occur in the treated area. In case of infection, the patient may require antibiotics according to local guidelines.2 3
The treated area will usually require a bandage the first few days postoperatively because of slight oozing. Any postoperative pain will be treated according to local guidelines. A doctor will control the treated area at follow-up.
Patients are encouraged to take prescribed medications before, during and after treatment to make results more translational to a real-world setting.
Any anticoagulant medication is continued, as the minor blood spill in treatment of tumours up to 3 cm in diameter is expected to be manageable.
Evaluation of treatment response
Treatment response is assessed by clinical measurement as described previously in the endpoint sections. If treated areas present crusted wounds, the crust and any residual tumour are measured, and clinical responsiveness are noted. To evaluate long-term response, punch biopsies (preferably 4 mm) will be taken from the centre and edge of the treated areas 1 year after treatment. Biopsies of the tumour area will be stored as formalin-fixated, paraffin-embedded tissue blocks. Analyses will assess tumour and surrounding tissue response to treatment histological analyses (eg, percent tumour cells in sample and fibrosis). Samples will not be taken before 12-month follow-up.
Sign of systemic immunological response will be investigated from routine scans (MRI, PET-CT, etc) before and after treatment in the inclusion period by monitoring tumour size and TNM stage. Response rates and response duration according to tumour histology will be compared as well as the rate of response for each individual patient.
Statistical power calculation and definition of cohort
This is a phase II non-controlled study. The primary endpoint is to evaluate tumour response (overall response, which includes PRs and CRs) 2 months after CaEP treatment evaluated by clinical examination and documented by photography. The appropriate sample size is calculated to determine the minimum number of subjects that need to be enrolled in this study in order to have sufficient statistical power in detecting CaEP treatment effect.
Based on the response rate from the initial but small study with CaEP treatment, we predicted a mean CR rate of 66% and an estimated SD of 36%.2 In order to define our cohort, a response rate of no treatment is estimated to be 0%. To detect a 66% difference with a power of 90%, eight patients should be included. As we aim to support earlier studies in a real-world setting, 10 patients from each involved cancer centre will be included. Because our primary end point is follow-up after 2 months, we cautiously predict that 2 patients out of 10 will not be evaluable for the primary endpoint. We thereby expect 24 evaluable patients at primary end point with 95% CI. Off-study patients will not lead to further inclusion.
At least one tumour will be treated in each patient (up to seven tumours per patient may be included), and the response rate across all treated tumours will be calculated.