Treatment of Liver Metastases With Electrochemotherapy (ECTJ) Phase II
Phase: PHASE2
Conditions: Liver Metastases
Sponsor: Institute of Oncology Ljubljana

 
Clinical Study Extension Protocol 
 
 
Protocol 2.0, 25 March 2013 
 
 
Zdravljenje jetrnih zasevkov z elektrokemoterapijo 
 
Treatment of liver metastases with electrochemotherapy  
ClinicalTrials.gov identification number: NCT01264952 (Phase I), NCT02352259 (Phase II) 
EudraCT identification number: 2008-008290-54 
Acronym: ECTJ 
 
Principal investigator:  
Assist. Dr Ibrahim Edhemović, MSc 
Coordinator: 
Prof. Dr Gregor Serša 
Institution: 
Institute of Oncology Ljubljana 
Participating institutions: 
University of Ljubljana, Faculty of Electrical 
Engineering, Laboratory of Biocybernetics (Prof. 
Dr Damijan Miklavčič) 
University Medical Centre Ljubljana (head 
physician Vojka Gorjup, MSc) 
Phone number: 
5879 953 (Assist. Ibrahim Edhemović, MSc)  
5879 434 (Prof. Gregor Serša, PhD) 
E-mail: 
iedhemovic@onko-i.si  
gsersa@onko-i.si 
 
 
 
 

 
Clinical Study Extension Protocol 
 
 
 
Protocol 2.0, 25 March 2013 
Index 
 
1 PURPOSE OF EXTENDING THE STUDY 
3 
2 STARTING POINTS 
4 
Principles of electroporation and electrochemotherapy (ECT) 
4 
Clinical applications of ECT 
4 
Treatment of liver tumours 
5 
Planning ECT treatment 
5 
Electroporation pulse synchronization using electrocardiogram (ECG) 
6 
3 PATIENT SELECTION 
9 
3.1 Inclusion criteria 
9 
3.2 Exclusion criteria 
10 
3.3 Examinations and tests before treatment 
10 
4 TREATMENT PLAN 
11 
5 TREATMENT: ELECTROCHEMOTHERAPY (ECT) 
12 
5.1 Chemotherapeutic agents 
12 
5.2 Electroporation 
13 
5.2.1 Electric pulse generator 
13 
5.2.2 Electrodes 
13 
5.2.3 Electroporation pulses 
14 
5.3 Synchronization of electroporation pulses with ECG and Holter ECG recording 
14 
6 FOLLOW-UP AFTER TREATMENT 
15 
6.1. Effects of treating colorectal liver metastases using ECT 
15 
6.2 Effects of electroporation pulses, BLM or ECT as a whole on heart function 
16 
6.3 ECT adverse effects will be recorded in line with the criteria of the National Cancer Institute 
(NCI) 
16 
7 DATA PROCESSING 
17 
8 STATISTICAL ANALYSISI 
17 

 
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1 PURPOSE OF EXTENDING THE STUDY 
 
The purpose of extending the study was to evaluate the efficacy of bleomycin-based 
electrochemotherapy in treating colorectal liver metastases in a phase II clinical study. 
 
The purpose will be achieved by: 
 
applying treatment in a controlled manner based on pre-clinical experience and the clinical 
experience of the ESOPE project; 
 
evaluating procedures and results achieved; 
 
preparing standardized procedures; 
 
disseminating these new approaches to treatment using written and audio-visual materials as 
well as by attending and organizing expert meetings.  
 
The study is designed as an institutional study (Institute of Oncology Ljubljana) conducted in 
cooperation with the Faculty of Electrical Engineering (University of Ljubljana), University Medical 
Centre Ljubljana and industrial partner, IGEA srl., Carpi, Italy, which will provide technical support 
for the study. An additional 15 patients who meet the inclusion criteria will be included in the 
extended part of the study. 
 
The study will be carried out within the P3-0003 “Razvoj in ovrednotenje novih terapij za zdravljenje 
malignih tumorjev” (Development and evaluation of new therapies for treating malignant tumours) 
research programme headed by Prof. Gregor Serša, PhD. 
 
Principal investigator: 
 
Ibrahim Edhemović, MSc (surgeon) 
Coordinator:  
Prof. Gregor Serša, PhD (researcher) 
Colleagues: 
 
Institute of Oncology Ljubljana 
 
 
Prof. Eldar Gadžijev, PhD (surgeon) 
Prof. Marko Snoj, PhD (surgeon) 
Assist. Prof. Janja Ocvirk, PhD (internist) 
Gorana Gasljević, PhD (pathologist) 
Assist. Prof. Maja Mušič, PhD (radiologist) 
Prof. Maja Čemažar, PhD (researcher) 
Tjaša Pečnik Vavpotič (research nurse) 
 
Faculty of Electrical Engineering, University of Ljubljana 
Prof. Damijan Miklavčič, PhD (researcher) 
Prof. Tomaž Jarm, PhD (researcher) 
Barbara Mali, PhD (researcher) 
Denis Pavliha (researcher) 
Bor Kos (researcher) 
Marija Marčan (researcher) 
 
University Medical Centre Ljubljana 

 
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Head physician Vojka Gorjup, MSc (cardiologist) 
2 STARTING POINTS 
Principles of electroporation and electrochemotherapy (ECT) 
By exposing a cell to an external electric field, transmembrane voltage is established on the cell 
membrane. When the threshold value is exceeded, this voltage causes electroporation of the cell 
membrane, i.e. the formation of hydrophilic pores, which allow the increased passage of molecules 
into the cell.1  
The principle of electrochemotherapy (ECT) is based on exposing the tumour to electroporation pulses 
delivered by specific electrodes. This allows the passage of previously applied hydrophilic cytostatics, 
e.g. bleomycin and cisplatin, into cells.2 Because the intake of cytostatics is increased only in areas 
exposed to electroporation pulses, ECT is a local treatment method. In addition to the direct action of 
cytostatics on tumour cells, ECT has an indirect vascular-disrupting effect, because cytostatics act on 
the endothelial cells in tumour vasculature.3 Doses of cytostatics used in ECT are extremely low, 
resulting in minimal or no systemic toxicity.4 Furthermore, the application of electroporation pulses 
reduces blood flow in the tumour, which keeps the cytostatics in the tumour for a longer period of 
time, thus making the treatment more effective.3 
Clinical applications of ECT 
In the early 1990s, our team took an active part in pioneering clinical studies on treating cutaneous 
and subcutaneous tumours with ECT.5 According to the findings of these first clinical trials, 80% of 
objective responses of treated tumour nodules are achieved using ECT.6 Our team also helped draw 
up and publish standard surgical procedures for treating cutaneous and subcutaneous tumours using 
ECT. Today, ECT is a well-established method for treating malignant melanoma metastases, especially 
as part of palliative care, in 15 European countries. In 2011, 2,000 patients were treated with ECT and, 
according to several databases, ECT is now used in 100 oncology centres.  
In the first recorded clinical case of treating a deep-seated tumour with ECT, melanoma metastases in 
thigh muscles were treated.7 Experience in treating other types of soft tissue tumours with ECT is 
good, but limited to a few clinical cases and studies involving a small number of patients.8-11 In addition 
to treating deep-seated soft tissue tumours, ECT is also used to treat deep-seated tumours in internal 
organs. Brain and liver metastases have already been shown to be effective targets in ECT.12–14 

 
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Treatment of liver tumours 
Colorectal cancer is the most common cancer and the second most common cause of cancer death in 
Europe, taking into account both sexes15. At the time of diagnosis, more than 23% of patients have 
liver metastases,16 while after the first treatment, metastases occur in a further 20% of patients.17 In 
Slovenia, the incidence of colorectal cancer metastases that are eligible for surgical treatment is from 
12 to 15 per 100,000 population, and is higher than the incidence of other liver metastases. In these 
cases, radical liver resection is the only promising treatment method, having an almost 50% 5-year 
survival, but due to the scale of the disease, it is only feasible in a minority of cases.18 The distant form 
of the disease is usually treated with systemic chemotherapy or biological drugs, which are only 
partially successful while producing high systemic toxicity. Where individual small metastases are 
present, patients are offered a local form of treatment: a thermoablation method called 
radiofrequency ablation (RFA). Metastases that grow into the inferior vena cava or into the area of 
large hepatic and portal veins, and on which RFA has small or no effect due to cooling, are particularly 
problematic. Therefore, new, more effective approaches to liver metastases treatment are being 
sought for such patients. 
A phase I/II clinical study is currently underway at Institute of Oncology Ljubljana to assess the safety 
and efficacy of treatment for colorectal liver metastases using ECT. We wish to include an additional 
fifteen (15) patients within phase II using this protocol. To date, 17 patients have been included in the 
clinical study, and preliminary results in 16 of these patients indicate that treating colorectal liver 
metastases and one HCC patient using ECT is a safe method for treating tumours even in hard-to-reach 
locations. Preliminary MRI results in all patients testify to radiological changes similar to those 
observed after RFA treatment. The response to ECT treatment was complete in 50% of patients and 
partial in the remaining 50% of patients. Preliminary histological analysis results for all ECT-treated 
metastases after surgical removal indicate progressive degenerative changes within metastases and a 
statistically significant reduction in viable tumour tissue in ECT-treated metastases compared to non-
ECT-treated metastases (p <0.001). We were the first to publish the technological part of the 
procedure in the clinical case.14 
Planning ECT treatment 
ECT treatment of deep-seated tumours in internal organs requires planning based on diagnostic 
radiology imaging. Electrode type is selected based on the precise location and measured size of the 

 
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tumour nodules; for metastases located up to 3 cm (lower edge of the metastasis) below the liver 
surface, which are more easily accessible to ECT, short needle electrodes with a fixed hexagonal 
geometric arrangement are appropriate, while for hard-to-reach and deep-seated metastases, long 
needle electrodes with an arbitrary geometric arrangement have recently been made available. In line 
with technological advances and the development of new electrode types, experts at the Italian 
company IGEA designed a new electric pulse generator called the Cliniporator VITAE for treating larger 
and deep-seated tumours, such as liver metastases. The generator meets the essential requirements 
for consumer safety, and health or environmental protection as defined by EU guidelines or 
regulations, as it has the CE mark. 
The distribution of the electric field within the tissue is an important predictor for the success of ECT 
tumour treatment, as the change in the transmembrane potential of the cell membrane, which is a 
prerequisite for successful electroporation, is directly proportional to the electric field strength.19 
Numerical modelling is currently the only effective method for predicting electric field distribution in 
tissue, as its structure is, by definition, inhomogeneous, nonlinear, and in some cases anisotropic. 
When using long needle electrodes with an arbitrary geometric arrangement, it is necessary to 
determine the depth of electrode insertion into the tumour nodule, and the voltage, length and 
frequency of the supplied electroporation pulses based on numerical modelling in order to achieve 
optimal coverage of tumour nodules with an electric field.7 
Electroporation pulse synchronization using electrocardiogram (ECG) 
In ECT treatment, the electric current reaches the surroundings of the treated area due to the high 
electrical conductivity of the tissue. This increases the likelihood of possible interaction between 
electroporation pulses and heart function when treating deep-seated tumours in internal organs due 
to the anatomical proximity of the heart. In our clinical study, we used a synchronization procedure 
built into the electroporation pulse generator (Cliniporator VITAE) to avoid cardiac dysfunction caused 
by ECT. So far, this procedure has proven to be effective in preventing cardiac pacing during the 
ventricular sensitivity period.14 Nevertheless, further research is urgently needed to verify the 
effectiveness of the existing synchronization procedure and to determine any short- or long-term 
effects of electroporation pulses on heart function.  
Preliminary results suggest that ECT does not seriously affect cardiac function, but there are 
statistically significant changes in some parameters of heart rate variability during and after therapy 
relative to pre-therapy parameter values.  

 
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The analysis of ECG signals recorded during ECT applied to liver tumours shows short-term effects on 
cardiac function, even though the delivery of electroporation pulses was synchronized with the ECG. 
The effects were expressed as a temporary shortening of the corrected QT interval and an increase in 
short-term heart rate variability. The analysis of ECG signals recorded before and after ECT was applied 
to liver tumours indicates that there are longer-term effects of ECT on cardiac function, which are 
expressed as increased heart rate or shortened RR interval and as reduced long-term heart rate 
variability parameters (low-frequency component). We find that these changes are most likely at least 
partly attributable to the effects of analgesics and other medications received by patients in intensive 
care, and to postoperative pain, and not to the effects of electrochemotherapy itself, although these 
results have yet to be confirmed. These results could be confirmed by capturing ECG signals before, 
during, and after abdominal surgery, which would include procedures similar to those performed 
during ECT of liver tumours. The only difference should be that ECT is not performed during these 
surgical procedures. The ECG signals captured in this way would serve as a control and would allow 
these assumptions to be confirmed. 
 
1. 
Kotnik T, Pucihar G, Miklavcic D. Induced transmembrane voltage and its correlation with electroporation-
mediated molecular transport. J Membr Biol 2010; 236(1): 3–13. 
2. 
Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z. Electrochemotherapy with cisplatin: potentiation 
of local cisplatin antitumour effectiveness by application of electric pulses in cancer patients. Eur J Cancer 1998; 34(8): 
1213–8. 
3. 
Sersa G, Jarm T, Kotnik T, Coer A, Podkrajsek M, Sentjurc M et al. Vascular disrupting action of electroporation 
and electrochemotherapy with bleomycin in murine sarcoma. Br J Cancer 2008; 98(2): 388–98. 
4. 
Marty M, Sersa G, Garbay J, Gehl J, Collins C, Snoj M et al. Electrochemotherapy – An easy, highly effective and 
safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of 
Electrochemotherapy) study. Ejc Supplements 2006; 4(11): 3–13. 
5. 
Rudolf Z, Štabuc R, Čemažar M, Miklavčič D, Vodovnik L, Serša G. Electrochemotherapy with bleomycin. The first 
clinical experience in malignant melanoma patients. Radiol Oncol 1995; 29: 229–35. 
6. 
Sersa G, Miklavcic D. Electrochemotherapy of tumours. J Vis Exp 2008; (22). 
7. 
Miklavcic D, Snoj M, Zupanic A, Kos B, Cemazar M, Kropivnik M et al. Towards treatment planning and treatment 
of deep-seated solid tumors by electrochemotherapy. Biomed Eng Online 2010; 9: 10. 
8. 
Shimizu T, Nikaido T, Gomyo H, Yoshimura Y, Horiuchi A, Isobe K et al. Electrochemotherapy for digital 
chondrosarcoma. J Orthop Sci 2003; 8(2): 248–51. 
9. 
de Bree R, Tijink BM, van Groeningen CJ, Leemans CR. Electroporation therapy in soft tissue sarcoma: a 
potentially effective novel treatment. Sarcoma 2006; 2006: 85234. 
10. 
Curatolo P, Quaglino P, Marenco F, Mancini M, Nardò T, Mortera C et al. Electrochemotherapy in the treatment 
of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial. Ann Surg Oncol 2012; 19(1): 192–8. 
11. 
Campana LG, Mocellin S, Basso M, Puccetti O, De Salvo GL, Chiarion-Sileni V et al. Bleomycin-based 
electrochemotherapy: clinical outcome from a single institution's experience with 52 patients. Ann Surg Oncol 2009; 16(1): 
191–9. 
12. 
Soden DM, Larkin JO, Collins CG, Tangney M, Aarons S, Piggott J et al. Successful application of targeted 
electrochemotherapy using novel flexible electrodes and low dose bleomycin to solid tumours. Cancer Lett 2006; 232(2): 
300–10. 

 
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13. 
Agerholm-Larsen B, Iversen HK, Ibsen P, Moller JM, Mahmood F, Jensen KS et al. Preclinical validation of 
electrochemotherapy as an effective treatment for brain tumors. Cancer Res 2011; 71(11): 3753–62. 
14. 
Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A et al. Electrochemotherapy: a new 
technological approach in treatment of metastases in the liver. Technol Cancer Res Treat 2011; 10(5): 475–85. 
15. 
Ferlay J, Parkin DM, Steliarova-Foucher E. Estimates of cancer incidence and mortality in Europe in 2008. Eur J 
Cancer 2010; 46(4): 765–81. 
16. 
Gatta G, Capocaccia R, Sant M, Bell CM, Coebergh JW, Damhuis RA et al. Understanding variations in survival for 
colorectal cancer in Europe: a EUROCARE high resolution study. Gut 2000; 47(4): 533–8. 
17. 
Lyass S, Zamir G, Matot I, Goitein D, Eid A, Jurim O. Combined colon and hepatic resection for synchronous 
colorectal liver metastases. J Surg Oncol 2001; 78(1): 17–21. 
18. 
Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR et al. Recurrence and outcomes following hepatic 
resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg 2004; 
239(6): 818–25; discussion 825–7. 
19. 
Miklavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G. The importance of electric field distribution for 
effective in vivo electroporation of tissues. Biophys J 1998; 74(5): 2152–8. 
 
 
 
 

 
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3 PATIENT SELECTION 
The suitability of patients for inclusion in the study will be assessed using inclusion and exclusion 
criteria. 
3.1 Inclusion criteria 
1. Patients with one to three (1–3) metachronous colorectal liver metastases, up to 5 cm in size, 
that are difficult to resect due to their position (likelihood of insufficient healthy liver after 
resection) or inaccessible for treatment with standard ablation techniques because they are 
located on large hepatic vascular structures (portal veins, hepatic veins or their inflow into the 
inferior vena cava, immediate vicinity of vena cava), but are otherwise operable.  
2. Patients with up to three (3) recurring colorectal liver metastases, each with a maximum 
diameter not exceeding five (5) cm, and for which other treatment is either unacceptably risky 
due to possible insufficient residual liver volume after resection or expected to be less 
effective due to the proximity of veins in RFA. 
Patients in groups 1 and 2 are patients in whom standard treatment options (systemic and/or 
surgical) have been exhausted. ECT will be offered to these patients as the only therapeutic 
option. If a patient has a large number of metastases that are unresectable and at the same 
time unsuitable for RFA, the metastases will be treated with ECT, while any other metastases 
will be resected or treated with RFA. 
3. Patients with synchronous colorectal liver metastases, in whom—due to their poor general 
health or the extent of the disease—first the removal of the primary tumour is planned, and 
after 2–3 months, surgery on the metastases, with systemic treatment taking place in the 
meantime. ECT would be used to treat metastases up to 5 cm in maximum diameter during 
the first surgical procedure, i.e. when the primary tumour is removed. 
4. Patients with bilateral metachronous colorectal liver metastases who require two-stage 
surgery due to the extent of the disease, i.e. right portal vein ligation and excision or RFA of 
metastases in the left hepatic lobe at the first stage and right hepatic lobe resection at the 
second stage. During the right portal vein ligation surgery, the metastases should be accessible 
in order to apply electroporation pulses without major manipulation and mobilization of the 
liver, so as not to create additional problems for the second planned procedure, i.e. the 
removal of metastases by resecting the right hepatic lobe. 
Patients in groups 3 and 4 are potentially curable using standard treatment. The fact that these 
patients will be operated on twice as part of standard treatment will be used to apply ECT 
during the first procedure and to resect treated and untreated metastases during the second. 
The added ECT used on these patients will not affect the standard treatment recommended in 
the current guidelines. 
5. The patient is offered ECT treatment even if the patient refuses standard treatment options. 
6. Histologically and/or cytologically confirmed colorectal cancer. 
7. Age above 18. 
8. Life expectancy over 3 months. 
9. Performance status (PS) ≥ 70 according to Karnofsky PS or ≥ 2 according to WHO 
recommendations. 
10. Depending on the type of active substance, 2–5 weeks have passed since the last treatment, 
if any. 

 
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11. The patient must understand the treatment process and any side effects. 
12. The patient must be able to give consent to participate in a clinical trial (‘informed consent’). 
13. Prior to inclusion in the study, the patient should be treated by a multidisciplinary 
gastrointestinal tumour team. 
 
3.2 Exclusion criteria 
1. Any previously confirmed form of cancer other than surgically treated non-invasive uterine 
cancer or basal cell carcinoma treated with surgery or radiotherapy. 
2. Proven visceral and/or bone metastases or diffuse metastases. 
3. Life-threatening infection and/or heart failure and/or liver failure and/or other life-
threatening systemic conditions. 
4. Ascites. 
5. Significantly reduced lung function. 
6. Age under 18. 
7. Coagulation system disorders. 
8. Received cumulative dose of bleomycin ≥ 250 mg/m2. 
9. Allergic reactions to previous treatment with bleomycin. 
10. Chronic decrease in renal function (creatinine > 150 µmol/L). 
11. Epilepsy 
12. Heart rhythm disorders. 
13. Implanted pacemaker or defibrillator. 
14. Pregnancy. 
15. Patients who are unable to understand the purpose of the study. 
 
3.3 Examinations and tests before treatment 
Two weeks before inclusion in the study, the following will be performed: 
 
clinical examination, anamnesis 
 
radiological imaging techniques to discover liver metastases: 
- 
dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 
- 
dynamic contrast-enhanced ultrasound (DCE-US) 
- 
computed tomography (CT) perfusion 
 
Testing for systemic metastases 
o CT of the lungs 
o other examinations depending on the clinical picture (bone scintigraphy, PET/CT) 
 
One week before inclusion in the study, the following will be performed: 

 
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 
laboratory tests: 
- 
haematological tests with a complete blood count 
- 
coagulation tests 
- 
biochemical tests: electrolytes, creatinine, transaminases 
- 
immunohistochemical examinations: tumour markers 
 
ECG 
In patients for whom two-stage surgery is planned, the following will be performed:  
 
histological analysis of liver metastases and comparison of pathohistological changes between 
treated and untreated metastases. 
 
4 TREATMENT PLAN 
When there are liver metastases (up to 3 cm in maximum diameter) with the lower edge located up 
to 3 cm below the liver capsule, ECT using short needle electrodes with a hexagonal geometric 
arrangement will be performed in line with standard surgical procedures for treating cutaneous and 
subcutaneous tumours/metastases with ECT. 
When there are larger liver metastases (up to 5 cm) located near the vena cava or along large hepatic 
or portal veins, ECT using long needle electrodes with an arbitrary geometric arrangement will be 
performed. Using numerical modelling, we will prepare a treatment plan to determine the optimal 
position of the electrodes and the required electric current voltage for optimal electroporation of the 
target tissue. The preparation of a treatment plan using numerical modelling will take place in two 
basic steps (see below) based on radiological images captured up to 2 weeks before the procedure. 
1. Decomposing medical images and constructing a model: 
- 
importing medical radiology images into an appropriate program, 
- 
determining important areas,  
- 
pre-treating and decomposing images, 
- 
constructing a 3D model.  
2. Planning treatment: 
- 
The 3D model will be supplemented with electrodes supplying electroporation pulses, and 
with corresponding tissue properties and boundary conditions based on the calculation 
using the finite element method and the calculation processed by automatic algorithms 
for determining the criterion function, 

 
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- 
the possibility of statistical and probability functions to determine the probability of 
electroporation will be examined, the effect probability being a function of the electric 
field strength applied. 
 
 
Figure 1: Schematic representation of electrode placement in the middle and the periphery of a metastasis in healthy 
liver tissue. 
 
5 TREATMENT: ELECTROCHEMOTHERAPY (ECT) 
Patients with multiple bilateral metastases scheduled for two-stage surgery depending on their 
general condition and extent of the disease will have their right portal vein ligated and up to 3 
metastases treated on the right hepatic lobe with up to 3 cm in maximum diameter using ECT during 
the first surgical procedure. On the left, metastases will be treated using established methods, such 
as surgery and RFA. During the second procedure, the entire right half of the liver will be removed. 
In patients with liver metastases in areas where, due to the proximity of the main hepatic veins, 
surgery is not possible and the success of other ablation techniques cannot be predicted, ECT will be 
performed. 
 
5.1 Chemotherapeutic agents  
- 
The cytostatic used will be bleomycin (BLM) manufactured by Heinrich Mack Nachf. GmbH & Co. 
KG, Illertissen, Germany. 
- 
BLM will be dissolved in saline solution and administered intravenously with a bolus injection at a 
dose of 15 mg/m2. 
- 
8–28 min after BLM application, electroporation pulses will be delivered to the metastases.  
 

 
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5.2 Electroporation 
5.2.1 Electric pulse generator 
- 
We will use the Cliniporator VITAE electric pulse generator manufactured by IGEA, Carpi, Italy, 
designed to treat larger and deep-seated tumours, such as liver metastases. 
- 
It has the CE mark and is authorized for use in a clinical setting. 
5.2.2 Electrodes 
Two types of electrodes will be used: 
a) short needle electrodes with a fixed hexagonal geometric arrangement: 
- 
for metastases with their lower edge located up to 3 cm below the liver capsule;  
- 
ECT will be performed in line with standard surgical procedures for cutaneous and 
subcutaneous tumours/metastases; 
 
b) long needle electrodes with an arbitrary geometric arrangement:  
- 
for deeper-seated tumours and tumours located ≥ 3 cm below the liver capsule;  
- 
to capture the safety margin, the required number of electrodes (presumably 
from 4 to 6) will be inserted into the metastasis and its surroundings using 
ultrasound guidance in line with the treatment plan prepared based on numerical 
modelling before the procedure. 
 
 
Figure 2 Short needle electrodes with a fixed hexagonal geometric arrangement (a), long needle electrodes with an 
arbitrary geometric arrangement (b) and Cliniporator VITAE (c). 

 
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5.2.3 Electroporation pulses 
- 
Between each pair of electrodes, 8 rectangular electroporation pulses (length: 100 µs) will 
be supplied to the metastases in two trains, each containing 4 electroporation pulses of 
reversed polarity and a repeating frequency of 1000 Hz. 
- 
The delivery of individual pulse trains will be synchronized with the occurrence of 
successive heartbeats. 
- 
When using long needle electrodes with an arbitrary geometric arrangement, the voltage 
of electroporation pulses for each electrode pair will be determined on the basis of 
numerical modelling, and when using short needle electrodes with a fixed hexagonal 
geometric arrangement, a voltage of 730 V will be applied. 
- 
If excessive electric current (> 50 A) makes it impossible to supply a sufficiently high 
voltage, a larger number of lower-voltage electroporation pulses will be supplied. 
 
5.3 Synchronization of electroporation pulses with ECG and Holter ECG recording 
- 
To ensure synchronization, the Cliniporator VITAE will be connected to an AccuSync 42 external 
unit, which produces trigger pulses based on the measured ECG signal to deliver electroporation 
pulses on the R wave of each heartbeat. 
- 
For interoperative patient monitoring, patients will be connected to AccuSync via three ECG 
electrodes independently of ECG capture. 
- 
On the selected ECG lead (standard leads I, II, III; the most suitable lead is chosen depending the 
intensity of the R wave), AccuSync detects the R wave of individual heartbeats in the early phase 
of increasing R wave slope, and produces a trigger pulse.  
- 
The trigger pulse is brought to the Cliniporator VITAE, which uses its built-in synchronization 
algorithm for a further verification of trigger pulse suitability, and, meeting a series of 
conditions, produces electroporation pulses with a 50 ms delay behind the trigger pulse to avoid 
ventricular sensitivity (T wave range). 
- 
ECG signal and trigger pulses will be captured and stored for later analysis of the synchronization 
algorithm function and of any immediate effects of treatment on heart function. 
 
6 FOLLOW-UP AFTER TREATMENT 

 
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Follow-up will be carried out at three levels. First, defining the early and late effects of treating 
colorectal liver metastases using ECT; second, evaluating short- and/or long-term effects of 
electroporation pulses, BLM or ECT as a whole on heart function; and third, recording adverse effects 
of ECT. 
 
6.1. Effects of treating colorectal liver metastases using ECT 
- 
Early treatment effects will be assessed using DCE-US or CT based on changes in tumour 
blood flow after ECT compared to pre-ECT blood flow. 
- 
Late effects will be assessed based on modified RECIST criteria, taking into account the 
change in the size and density of treated tumour nodules before and after ECT. These will 
be assessed based on DCE-MRI images; the volume of the metastases will be calculated 
using the formula 𝑉= 
𝑎 × 𝑏2 × 6
𝜋
, where a is the shorter and b the longer metastasis 
diameter. 
Based on the assessment of the late ECT effects, the response to treatment will be evaluated in 
line with the WHO classification: 
 
Complete response (CR): complete disappearance of the tumour nodule 
 
Partial response (PR): reduction of tumour nodule size by more than 50% 
 
No change (NC): decrease in tumour nodule size by less than 50% or increase in tumour nodule 
size by less than 25% 
 
Progressive disease (PD): growth of the tumour nodule by more than 25% 
- 
The direct effect will be assessed by histological analysis of those metastases that will be 
surgically removed. 
- 
EORTC Quality of Life questionnaire according to QLQ C30 standards 
 
 
 
EXAMINATION/TEST 
Screening 
period 
until D −14 
D −1  
 
D 7 
 
D 30 
 
D 60 
 
D 90 
 
D 120  
 
clinical examination, 
anamnesis 
X 
X 
X 
X 
X 
X 
X 
ECG 
X 
X 
 
 
 
 
 

 
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DCE-MRI 
X 
 
 
X 
 
X 
 
DCE-US 
X 
 
 
X 
X 
 
X 
CT perfusion 
X 
 
 
X 
 
 
X 
Blood count 
X 
X 
X 
 
 
 
 
Coagulation tests 
X 
X 
X 
 
 
 
 
Biochemical blood analysis 
X 
X 
X 
X 
X 
 
X 
EORTC QLQ C30 
questionnaire 
X 
 
X 
X 
 
 
 
*D = day 
 
6.2 Effects of electroporation pulses, BLM or ECT as a whole on heart function 
- 
To study the short-term effect, we will use the ECG signal captured during surgery, which 
will cover the period before, during and immediately after ECT, including BLM injection 
and electroporation pulses. 
- 
To study possible late and/or long-term effects, ECG signal using a standard Holter system 
will be recorded in 24-hour periods immediately before and after surgery. 5–10 patients 
will be included. ECG recordings will also be performed on 10–15 patients who will 
undergo abdominal surgery without ECT at the Institute of Oncology Ljubljana.  
 
6.3 ECT adverse effects will be recorded in line with the criteria of the National Cancer Institute 
(NCI) 
An adverse effect is an undesirable and unexpected sign, symptom or disease, regardless of the cause, 
which develops or worsens during the study and which includes pathological clinical observations and 
deviations in the values of laboratory-measured parameters. The latter are considered a side effect 
only if they require additional treatment or result in premature withdrawal of the patient from the 
study.  
We will record any side effects that occur during the study and up to 30 days after the end of 
treatment. If a serious adverse reaction occurs more than 30 days after the end of treatment, the 
principal investigator will decide whether the complication is study-related or not. 
We will record the start and end time of any adverse effect, and the cause-and-effect relationship 
between the adverse effect and ECT, cancer or other diseases, as well as assessing the severity of the 
adverse effect and describing the actions that will need to be taken. 

 
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The causal relationship between the adverse effect and the ECT will be assessed as follows: 
 
Not related to the study. 
 
Probably not related to the study. 
 
Possibly related to the study. 
 
Probably related to the study. 
 
Certainly related to the study. 
The severity of side effects will be assessed using the following grades: 
 
Grade 1 – mild  
 
Grade 2 – moderate  
 
Grade 3 – a difficult complication requiring medical care 
 
Grade 4 – a life-threatening condition requiring immediate medical assistance 
 
Grade 5 – death 
If a serious adverse effect is recorded during the study, the principal investigator will notify the 
responsible person at the National Medical Ethics Committee of the Republic of Slovenia in writing 
within 24 hours. A serious adverse effect is a complication that results in the following: 
 
death, 
 
a life-threatening condition requiring immediate medical assistance, 
 
hospitalization or extended hospitalization, 
 
prolonged inability of the patient to perform any task that they were able to perform before 
the onset of the complication, 
 
any event or condition which endangers the life or health of the patient and which, according 
to a medical assessment, could lead to any of the serious complications referred to above. 
 
7 DATA PROCESSING 
The results of the proposed clinical trial will be analyzed descriptively by describing the antitumour 
effect, any adverse effects and the patients’ quality of life after treatment of colorectal liver 
metastases using ECT. 
8 STATISTICAL ANALSIS 
All data will be entered into a Microsoft Access 2010 database, which will be used for all calculations 
except for statistical analysis, which will be performed with GraphPad Software (La Jolla, CA, USA). The 
log-rank (Mantel-Cox) test will be performed on the Kaplan-Meier estimates. A chi‐squared test will 

 
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be used for the statistical comparison of response according to tumor location. A two‐tailed P value 
less than 0.05 will be considered to be statistically significant.