Clinical trial NCT02352259
Conditions: Liver Metastases
Keywords: Liver metastases of colorectal cancer Electrochemotherapy Bleomycin Electrochemotherapy
Sponsor: Institute of Oncology Ljubljana
Start date: 2013-09-01; Completion date: 2018-10-01
Enrollment: 23 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The study is prospective, phase II study, The primary objective of the study is evaluation of the feasibility and safety of intraoperative electrochemotherapy of colorectal liver metastases. The secondary objective is to determine the efficacy of electrochemotherapy treatment, based on histological and radiological evaluation of treated metastases. The endpoints are: toxicity according to the Common Terminology Criteria for Adverse Events (CTCAE) ver. 4.0 and response rate measured by percentage of vital tumor cells and mRECIST criteria.

Detailed description:
The study is an extension of previously concluded and published study on electrochemotherapy of colorectal liver metastases (Edhemovic et al. JSO 2014). In this extension 15 patients will be included. Four groups of patients will be included. The patients from groups 1 and 2 are patients with up to 3 unresectable liver metastases, demanding too excessive resection, or untreatable by standard thermal ablative methods due to the close proximity of major blood vessels. Electrochemotherapy will be offered to these patients as the only treatment option. In case of multiple, both resectable and non-resectable liver metastases, the latter will be treated with electrochemotherapy, whereas other metastases will be resected or treated by RFA. These groups of patients are:

1. Patients with one to three (1-3) metachronous liver metastases of colorectal cancer, not larger than 5 cm, that are positioned in unresectable liver area, near blood vessels, but in otherwise operable patients.
2. Recurrent liver metastases of colorectal cancer, not more than 3 and not larger than 5 cm in diameter, each. For those patients any other treatment could represent unacceptable risk due to possibility of insufficient liver residual after resection or less effective radiofrequency ablation due to the proximity of blood vessels.

   Groups 3 and 4 include patients with intent to cure within standard of care using two-stage surgical approach. This two-stage surgical approach will allow adding electrochemotherapy during the first operation and tissue collection for histological analysis during the second operation. Adding electrochemotherapy to these patients will not affect their treatment within standard of care recommended in the current guidelines. These groups of patients are:
3. Patients with synchronous metastases, but their general condition and extent of the disease will not allow simultaneous removal of the primary tumor and metastases. During the first operation, the primary tumor will be removed (colorectal resection) and some of the liver metastases will be treated by electrochemotherapy. About 6 weeks later, during the second operation for liver metastases, both treated and non-treated metastases will be removed with liver resection.
4. Patients with bilateral, multiple, metachronous metastases in whom standard treatment includ two-stage liver resection, due to the extent of the disease and/or their general condition. During the first operation, right portal vein will be ligated and metastases on the left side will be excised or ablated with radiofrequency ablation. At the same time, up to three metastases on the right side were treated with electrochemotherapy. During the second operation, both treated and non-treated metastases on the right side will be removed with right hemihepatectomy.

Electrochemotherapy will be offered to the patients also when they refuse standard treatment.

Depending on the position of metastases, appropriate electrodes will be selected; hexagonal needle electrodes with fixed geometry for metastases not larger than 3 cm in diameter, where their lower edge is located up to 3 cm below the liver capsule. ECT will be performed based on standard operating procedures for treatment of cutaneous and subcutaneous tumors / metastases with ECT.

Individual electrodes, positioned according to the prepared treatment plan will be used for metastases up to 5 cm in diameter, or located near vena cava or large hepatic or portal veins.

Electrochemotherapy will be performed within 8-28 min after intravenous in bolus administration of bleomycin (15 mg/m2).

Triggering of electric pulses will be synchronized with ECG signals, through the ECG triggering device AccuSync to avoid delivery of pulses in vulnerable period of the heart.

All patients will be treated after the procedure has been thoroughly described to them, and have signed informed consent.

Arms: arm 1: None
Interventions: intervention 1: Electrochemotherapy intervention 2: Cliniporator Vitae® intervention 3: Bleomycin PHC 15 e. (United States Pharmacopeia - USP)
Intervention descriptions: intervention 1: Treatment procedure: anesthesia, positioning of electrodes, intravenous in bolus administration of bleomycin, within 8-30 min after administration of bleomycin application of electric pulses, removal of electrodes. intervention 2: Positioning of electrodes, within 8-30 min after administration of bleomycin application of electric pulses (8 pulses, duration 100 microseconds, frequency 4 Hz with amplitude adequate to cover the whole treated lesion with electric field necessary for reversible plasma membrane permeabilization), removal of electrodes. intervention 3: Intravenous in bolus administration of bleomycin (15 mg/m2), 8 minutes before application of electric pulses

Protocol documents:
Prot_SAP_001.pdf: 
 
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. 
 
 

ICF_000.pdf: 
 
Consent for participating in a clinical study 
1/6 
 
Protocol 2.0, 30 March 2013 
 
 Consent for participating in a clinical study 
 
 
Treating liver metastases using electrochemotherapy 
 
What do I need to know about the clinical study? 
You are invited to participate in a clinical study (hereinafter: ‘the study’) in which the research team 
will evaluate the efficacy and potential toxicity of treating colorectal liver metastases with 
electrochemotherapy. The study is led by Assist. Dr Ibrahim Edhemović, MSc, and coordinated by Prof. 
Dr Gregor Serša, PhD. Other health professionals employed at the Institute of Oncology Ljubljana and 
the University Medical Centre Ljubljana, as well as researchers from the Faculty of Electrical 
Engineering, University of Ljubljana, also be involved in the study. 
The study is the continuation of a previous clinical study with the same name, approved by the 
National Medical Ethics Committee of the Republic of Slovenia on 22 October 2008, and involving a 
larger number of patients. 
It is important that you carefully read and understand this document before signing the consent. The 
document sets out the purpose, procedures, benefits, risks, possible adverse effects, and precautions 
related to the study. If you are not completely honest with your doctor about your medical condition, 
participating in the study may be detrimental to you. If this document contains any terms or 
expressions that you do not understand, please ask your doctor or healthcare professional 
participating in the study to explain them to you before signing. 
Participation in the study is voluntary and you can refuse to participate. Furthermore, you are free to 
withdraw from study whenever and without giving a reason – this will not affect your further care or 
relationship with your doctor at that institution.  
Details of the study are described below. It is important that you understand the information provided, 
as this is the only way you can make an informed decision whether to participate or not. If you decide 
to participate, you will receive a copy of the signed consent.  
 
What is the purpose of the study?  
The purpose of the study is to determine the efficacy of bleomycin-based electrochemotherapy and 
potential toxicity during surgery on colorectal liver metastases. 
 
Why was I asked to participate in the study? 
We suggest treatment of colorectal liver metastases using electrochemotherapy because you have 
been identified as having one of the following conditions: 
a) Colorectal metastases that are not suitable for surgical removal or treatment with standard 
ablation techniques due to their anatomical position. 


 
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b) Progression of the disease in the form of recurring colorectal liver metastases for which 
another type of treatment would be either risky or expected to be less effective. 
c) Colorectal metastases arising simultaneously with the primary tumour. Because your general 
health does not allow simultaneous surgical removal of both the primary tumour and liver 
metastases, two surgeries are planned for you according to the standard treatment protocol: 
removal of the primary tumour during the first operation and removal of liver metastases 
during the second operation. During the first operation, i.e. when the primary tumour is 
removed, electrochemotherapy would be used to treat liver metastases because we expect a 
reduction in their size or their complete disappearance, which would facilitate the second 
operation or even make it unnecessary. 
d) Colorectal metastases for which two operations are planned due to the extent of the disease. 
During the first operation, the hepatic vein would be ligated to allow the left lobe of the liver 
to grow to such an extent that by the second operation, the removal of the right liver lobe 
would be safe (which is not the case now). Some of the metastases on the right liver lobe 
would be treated with electrochemotherapy during the first operation, while others would 
not. During the second operation, the right liver lobe would be removed along with all the 
metastases.  
In this case, electrochemotherapy treatment would not affect the course of the disease; your 
treatment would be in line with the standard protocol, completely uninterrupted and 
unchanged. By analyzing the electrochemotherapy-treated metastases, we would gain new 
information and knowledge that would help future patients with the same disease. 
 
How long will the cooperation last?  
Your participation in the study, including the screening period, will last approximately 4 months. 
 
How will the treatment take place? 
You will receive treatment while under general anaesthesia. The treatment involves an intravenous 
injection of the chemotherapeutic agent bleomycin and, after approximately 8 minutes, application 
of electrical pulses via sterile stainless-steel electrodes inserted into the metastases.  
 
Appointments and procedures during the study: 
During the study, you will have at least 7 appointments over a period of 4 months. 
The treatment consists of the following: 
 1st appointment: screening (up to 2 weeks before treatment) 
If you agree to participate in the study, your doctor will examine you to see if you are a viable 
candidate. You and your doctor will also discuss any previous illnesses and treatments. 
The examination will consist of the following. 
 General medical examination: measuring the electrical activity of the heart (ECG). 
 Venous blood collection for haematological and biochemical examinations and coagulation tests. 


 
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 Radiological examinations: you will be referred to computed tomography with perfusion (CT 
perfusion), dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) or dynamic 
contrast-enhanced ultrasound (DCE-US). 
 You will complete a quality-of-life questionnaire. 
 2nd appointment (1 day before treatment) 
 General medical examination: measuring the electrical activity of the heart (ECG). 
 Venous blood collection for haematological and biochemical examinations and coagulation tests. 
 
 3rd appointment (1 week after treatment) 
 General medical examination. 
 Venous blood collection for haematological and biochemical examinations and coagulation tests. 
 You will complete a quality-of-life questionnaire. 
 
 4th appointment (1 month after treatment) 
 General medical examination. 
 Venous blood collection for biochemical analysis. 
 Radiological examinations: you will be referred to CT perfusion, DCE-MRI or DCE-US. 
 You will complete a quality-of-life questionnaire. 
 
 5th appointment (2 months after treatment) 
 General medical examination. 
 Venous blood collection for biochemical analysis. 
 Radiological examinations: you will be referred to DCE-US. 
 
 6th appointment (3 months after treatment) 
 General medical examination. 
 Radiological examinations: you will be referred to DCE-MRI. 
 
 7th appointment (4 months after treatment) 
 General medical examination. 
 Venous blood collection for biochemical analysis. 
 Radiological examinations: you will be referred to CT perfusion and DCE-US. 
 
What are the potential benefits of the study for me? 


 
Consent for participating in a clinical study 
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Protocol 2.0, 30 March 2013 
 
Electrochemotherapy is used as a standard method to treat cutaneous tumours. Our experience 
gained from having treated 16 patients also shows the effectiveness of electrochemotherapy in 
treating liver metastases. 
We cannot guarantee that the treatment will be effective in your case, but we hope that we will 
achieve at least a reduction in the size of the metastases. 
 
What are the potential risks or adverse effects associated with the study?  
As with any medicine or treatment, adverse effects may occur during treatment, so we cannot 
guarantee that you will not experience any adverse effects during the study.  
It is important that you inform the doctor monitoring you during the study of any symptoms or a new 
medicine prescribed by another doctor.  
 
What are my responsibilities if I decide to participate in the study? 
You will need to sign a consent form for participating in the study.  
You will need to visit your medical institution at least 7 times and undergo the treatments and 
procedures described in this document. 
You will not be allowed to participate in another study while participating in this one. 
Before taking any medicine that you have not taken before, you will need to consult the doctor 
monitoring you during the study. 
You will need to report any changes in your general well-being to the doctor monitoring you during 
the study.  
 
What are my options if I choose not to participate in the study? 
If you do not wish to participate in this study, you will continue to be treated by your doctor. Your 
health care and treatment will not be compromised. You are free to withdraw from study at any time. 
Doing so will not affect your further medical care. 
 
What if new information becomes available during the study? 
Sometimes, new information about the treatment in question comes up during the study. In this case, 
your doctor will inform and discuss with you whether you wish to continue participating in the study. 
If you decide to withdraw from the study, your doctor will offer you other treatment options. If you 
decide to continue participating in the study, you will be asked to confirm your continued participation 
in the study by signing an updated consent form. 
 
How will my privacy be protected? 
In this study, your personal information and information about your medical condition will be 
documented anonymously (without your name, using only the number you will be assigned upon 
entering the study).  


 
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Protocol 2.0, 30 March 2013 
 
In accordance with Slovenian legislation, all data collected during the study will be kept strictly 
confidential. The results of the study, including photographs of tumour lesions, will be available for 
scientific evaluation purposes and will be published anonymously in health publications. 
 
Voluntary participation and termination of participation: 
Participation in the study is voluntary. You can choose to withdraw from the study at any time. Doing 
so will not affect your current or future medical care. To withdraw from the study, please contact 
Assist. Ibrahim Edhemović, MD, MSc, on +386 1 5879 953.  
Please note that if you withdraw from the study, your doctor will still be able to use the information 
gathered while you were still part of the study. 
Your participation in the study may be terminated without your consent if you fail to follow the 
instructions and protocol. Further, we may exclude you from the study without your consent if the 
treatment being studied is detrimental to you and has adverse effects. 
 
Will I need to pay to participate in the study? 
The treatment is free of charge. 
 
Who may I contact if I have a question about harm or a reaction associated with the study?  
You have the right to ask questions about the study and to have them answered. 
If you have any questions about participating in the study or think that you have been harmed in the 
study or that you have a reaction to the treatment being tested, please contact: 
Assist. Ibrahim Edhemović, MSc, MD by phone: +386 1 5879 953 or by email: iedhemovic@onko-i.si 
 
Who may I contact to report a breach of data protection policy:  
If you believe that there has been a breach of your personal data, please contact the principal 
investigator: 
Assist. Ibrahim Edhemović, MSc, MD by phone: +386 1 5879 953 or by email: iedhemovic@onko-i.si 
 
Who may I contact if I have a question about my patient rights?  
The study has been reviewed and approved by the Committee for Expert Evaluation of Clinical 
Research Protocols at the Institute of Oncology Ljubljana. If you have questions about your patient 
rights, you may contact (including anonymously):  
Assist. Prof. Cvetka Grašič Kuhar, MD, PhD on +386 1 5879 283 or at  
cgrasic@onko-i.si 
 
 


 
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Patient consent for participating in the study 
 
I, the undersigned, hereby agree to participate in the study “Treating liver metastases using 
electrochemotherapy”. I understand that my refusing to participate in the study will not 
affect my health care. 
 
I have read the description of the study. The study and the starting points have been 
explained to me in language I understand. I am aware that participation is voluntary. I am 
sufficiently familiar with the purpose, methods, risks and benefits of the study. 
 
I understand that the doctor in charge of the study will inform me of any new findings that 
have arisen during the study that may affect my consent. 
 
I allow my study-related medical information to be used and passed on to health authorities 
and institutions. I consent to the use and disclosure of my personal health information as 
described in this document.  
 
I understand that I may refuse to participate in the study. I further understand that I am free 
to withdraw from the study at any time and without giving a reason, and that this will not 
affect my further care or relationship with my doctor. 
 
I declare that I have read this document, have had the opportunity to ask as many questions 
as I wanted, and have received satisfactory answers thereto. I understand that I have not 
waived any legal rights I have as a participant in the study. 
 
I am aware that I will receive a copy of the signed voluntary consent. 
 
I allow samples of metastases to be stored for histological analyses, and photographic records 
to be published for scientific purposes. 
 Yes  No 
I have read the whole document. I have asked all the questions I had. I agree to participate in this 
study. 
Name of the patient/subject in block letters 
Signature of the patient/subject 
Date (DD/MM/YYYY) 
Name of the person obtaining the consent in block letters 
Signature of the person obtaining the consent 
Date (DD/MM/YYYY) 
Name of the person responsible for the study in block letters 
Signature of the person responsible for the study 
Date (DD/MM/YYYY)