Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

This protocol was approved by the Ethical Committee of the Ministry of Agriculture, Forestry and Food and follows its guidelines. The protocol was validated on more than 80 dogs and 20 cats.

NOTE: ECT treatment consists of the administration of cisplatin or bleomycin and exposure of tumor nodules to electric pulses.

1. Patient Selection

NOTE: Patient selection allows the practitioner to determine whether the patient is suitable or not to benefit from this treatment.

1. Evaluate the history of the patient (general data about age, breed, sex and presence of any concurrent diseases, tumor type, evolution of the tumor with time, previous treatments and its results, recurrence and speed of recurrence, owner's expectations about the successfulness of the therapy).
2. Perform a physical examination of the patient
   1. Evaluate the tumor in detail in order to make a possible clinical differential diagnosis and perform a fine needle aspirate for cytology evaluation and possible tissue biopsy for histology evaluation in order to determine the treatment area: if a potentially invasive tumor is presented treat the wide margins (make the decision about the size of the treated margins according to recommendations for any other treatment for the same type of tumor, e.g., mast cell tumor 2r ≤ 1 cm, 1 cm margin should be treated; r – radius).
   2. Evaluate the number and size of the nodules in order to prepare a treatment approach.
3. General examination
   1. Implement a full general examination in order to make staging of the disease and check:
      1. If there is no other concurrent disease which could precludes the treatment, if the patient has a history of any chronic infection (according to epidemiology in a certain area) or immune-mediated disease which could alter hematological or coagulation changes (perform the specific tests), If the animal is able to withstand a series of deep sedations and/or general anesthesia, If the patient has sensitivity to cisplatin (standard dosage is 60-70 mg/m² body surface area / 21 d) or bleomycin or has already received a cumulative dose of bleomycin exceeding 400.000 IU/m².
   2. Use diagnostic imaging techniques (radiography of the thorax and ultrasound examination of the abdomen) in order to check any possible metastases according to the tumor type.

2. Laboratory Analysis

1. Blood work
   1. Perform complete hematology analyses with a biochemistry panel of blood urea nitrogen (BUN), creatinine, alanine-aminotransferase (ALT), alkaline phosphatase (AF) and calcium to exclude advanced chronic renal and hepatic diseases and possible most frequent paraneoplastic syndromes in order to determine the clinical stage which may have an effect on the prognosis.
   2. Do an additional specific blood analysis:
      1. If the patient is given any medicine which could potentially alter changes in coagulation ability, check the coagulation profile.
      2. If using invasive treatment (needle electrodes), check the platelet number; the number of platelets should be >100 or in case it is <100, the coagulation profile (PT, APTT, fibrinogen, D-dimer) should be normal.
      3. In specific cases according to neoplastic disease and the clinical status of the patient check the total proteins, albumin, phosphors, sodium (Na), potassium (K), chloride (Cl) and lipase concentration.
2. Tumor(s) histological analysis
   1. Histology analysis is not necessary if the cytology diagnosis was performed previously. In the case of certain tumor types where the histological grade is a crucial prognostic factor (e.g., mast cell tumors) perform hematoxylin and eosin staining¹⁶.
      NOTE: The biopsy (incisional or "punch") can be made before ECT during the same visit. In any case, it is good clinical practice to diagnose the tumor type.
      NOTE: After the case is fully evaluated a thorough discussion and clarification with the animal's owner is needed. Explain to the owner what ECT is and what could be expected from ECT in terms of clinical results. Discuss with the owner the treatment course: the number of ECT sessions under general anesthesia or deep sedation, total duration of treatment, cost of treatment, possibilities of recurrence. Obtain the owner's written consent in the case of patient inclusion within a clinical trial of ECT, if the patient is actually eligible.

3. Choice of Treatment Modality

NOTE: For ECT in dogs use cisplatin and bleomycin. For ECT in cats use bleomycin only, as cisplatin is contraindicated in cats.

1. Treat each tumor separately. Determine the location and size of each nodule and the number of nodules to outline the treatment protocol.
2. Calculate the volume of tumor nodules using the formula V = ab²π/6 ("a" is the larger diameter of the tumor nodule and "b" the diameter of the tumor nodule perpendicular to "a") or V = abcπ/6 ("a", "b" and "c" are three perpendicular diameters of the tumor nodule) .

4. Choice of Operating Modality

1. Selection of electrodes
   NOTE: It is important to choose the optimal electrode type as electrode configuration affects the electrical field distribution in the tissue. Two types of electrode are used: plate and needle electrodes. Plate electrodes have parallel stainless steel plates: the inner distance between the electrodes depends on the size of the tumor and the capacity of the generator of electric pulses. Usually the distance between the electrodes is 6-8 mm. Needle electrodes can be arranged in rows (parallel array) or in hexagonal geometry. The hexagonal electrode has a central needle and 6 needles in a circular array. The pulses are applied between the electrodes in the circular array and between them and the central one. The pulses are applied in both directions between each pair of electrodes. Choose the type of electrode according to the depth of the tumor nodule and provide appropriate electroporation in the whole desired treated area. Choose the electrodes according to the particular target tissue. In general, both types of electrodes can be used for the treatment.
2. Selection and preparation of cisplatin or bleomycin
   NOTE: ECT consists of intratumoral administration of cisplatin or bleomycin or intravenous administration of bleomycin and exposure of tumor nodules to electric pulses.
   1. Select an appropriate cytostatic and the route of its administration.
   2. Dissolve cisplatin (cis-diamminedichloroplatinum II), if obtained as a powder, in distilled water at a concentration of 1 mg/ml (otherwise pharmaceutically prepared solutions could be used). Give cisplatin intratumorally.
   3. Dissolve bleomycin in a physiological saline at a concentration of 3 mg/ml and give intratumorally or intravenously.
3. Anesthesia
   NOTE: Depending on operating modality (Table 1) deep sedation or general anesthesia can be used.
   1. Evaluate the patient according to The American Society of Anesthesiologists' (ASA) physical status classification and according to tumor type because of possible paraneoplastic syndromes.
4. Deep sedation
   1. Use medetomidine (0.08 mg/kg) or double combination medetomidine (0.025 mg/kg) with butorphanol tartrate (0.1 mg/kg) in dogs or follow standard procedures for deep sedation in the specific hospital.
5. General anesthesia
   1. Premedicate with acepromazine (0.02 mg/kg) and methadone (2 mg/kg).
   2. Thirty min later induce general anesthesia with thiopental (5 mg/kg) or propofol (1 mg/kg).
   3. Place endotracheal tube and maintain anesthesia with isoflurane in oxygen delivered via a T-piece anesthetic circuit.
   4. During anesthesia use fluid support treatment with Hartmann's solution at a rate of 10 ml/kg/hr and analgesia with a single dose of a non-steroid analgetic drug (e.g., carprofen 2-4 mg/kg).
   5. Use eye ointment to prevent dryness while under anesthesia. After treatment, supervise the patient until it regains sufficient consciousness to sternal recumbency. Keep the patient in the clinic for about 2 to 4 hr.
      NOTE: Standard procedures for general anesthesia in the specific hospital can also be followed.
6. Operating environment
   NOTE: Personnel operating are trained ECT operators (DVM), anesthetists and assistants.
   1. Use any ambulance room when ECT under deep sedation is performed or any surgical room where general anesthesia is allowed.
   2. For sedation and/or general anesthesia use all required equipment according to the procedures for anesthesia.
   3. For the ECT procedure see all required materials in the Materials List.
7. Operator and surrounding personnel protection
   NOTE: Droplet exposure of the skin to antineoplastic drugs rarely causes acute problems. Cisplatin and bleomycin are not absorbed through intact skin. Cisplatin may cause only irritation. Cisplatin and bleomycin leakage could occur when injecting firm nodules or tumors. In case of needle stick injury, wash with abundance of water and take care of the wound according to the standard medical practice.
   1. Protect the operator and surrounding assistants with masks and gloves. In case of skin exposure to cisplatin or bleomycin solution, rinse immediately and abundantly with water.
   2. Set up biosafety measures according to a quality insurance program complying with local regulations about anticancer drug usage, if any.
8. Generators of electric pulses
   NOTE: Applications of electric pulses can be carried out with the generator of electric pulses. Commonly, the apparatus is a square-wave pulse generator with integrated software and a touch-screen interface to select pre-designed electrical pulses. It is equipped with a hand-held electrode holder with a pulse-activating button attached to the generator by a cord. The delivery of electric pulses can be controlled by a foot switch. The generator of electric pulses should meet European Union standards: 2006/95/CE from 12/12/2006 concerning electronic material used within specific voltage limits, 2004/108/CE from 12/15/2004 concerning electromagnetic compatibility and should be compliant with the FDA's regulations.
9. Procedure of application of electric pulses
   NOTE: The application of electric pulses follows the intratumoral administration of cisplatin or bleomycin, or intravenous administration of bleomycin. When administering intratumorally cytostatic should be applied slowly to prevent leakage. For intravenously administration cytostatics should be applied in a bolus lasting approximately 30 sec. A water-based gel is used to achieve good contact between the skin and the electrodes, either plate or needle, and to achieve less heterogeneous electric field distribution. The conductivity of gels should be between 0.5 to 1 S/m ^17,18. The electric conductivity of the gel used was 0.5 S/m. The plate electrodes should be placed in such a way that the tumor is encompassed between them. Furthermore, to achieve better distribution of the electric field the application of electric pulses should be performed twice in a perpendicular direction. If the tumor is larger than the distance between the electrodes, the application of electrodes should start from the margins of the tumor (including the safety margin) and progress toward the center of the tumor. In the case of needle electrodes, care should be taken that all the needles are inserted into the tissue to achieve the appropriate distribution of the electric field. The application of a gel is not needed. Otherwise, the procedure is the same as in the case of plate electrodes.
   1. Depilate the skin in the area of application of electric pulses
   2. Administer cisplatin or bleomycin. If cytostatic is administered intravenously wait for 8 min and the time interval for electric pulses delivery is 20 min. If cytostatic is administered intratumorally start with the application of electric pulses immediately (within 1 min after injection).
   3. Before application of electric pulses apply a water-based gel onto the depilated skin.
   4. For plate electrodes use these pulse parameters: pulse duration 100 µsec, amplitude to electrode distance ratio 1,300 V/cm, frequency 1 Hz or 5 kHz. Deliver each run of 8 unipolar electric pulses in two trains of four pulses, with 1 sec interval, in two perpendicular directions.
   5. For needle electrodes with rows use pulse parameters: pulse duration 100 µsec, amplitude to electrode distance ratio 1,000 V/cm, frequency 1 or 5 kHz. Each run of unipolar electric pulses consists of eight pulses.
   6. For needle electrodes, arrange in a hexagonal configuration and use pulse parameters: pulse duration 100 μsec; the hexagonal electrode has a fixed voltage of 730 V (if the distance between the electrodes is 7 mm; 1,040 V/cm), with a frequency of 5 kHz and the number of pulses depends on the number of needles. In case of 7 needles 24 pulses are applied
10. Disposal of contaminated material.
    1. Dispose of all contaminated materials (syringes, needles, vials, gauzes, gloves) in a container intended for incineration.

5. Follow-up

NOTE: At each visit, tumor nodules are measured with a Vernier caliper and photographed. These visits also serve in order to evaluate the possible local and systemic side effects. Response to the treatment is scored after 4 weeks based on the criteria of "Response evaluation criteria in solid tumors" (RECIST)¹⁹ as a complete response (CR) — disappearance of the nodule, partial response (PR) — at least a 30% decrease in the sum of the diameters of the target lesion, taking as a reference the baseline sum diameters, progressive disease (PD) — at least a 20% increase in the sum of the diameters of the target lesions, taking as a reference the smallest sum being studied, stable disease (SD) — neither a sufficient shrinkage to qualify for PR, nor a sufficient increase to qualify for PD, taken as a reference the smallest sum diameters. For all response definitions a minimum 4-week duration is required for qualifying each type of response. Observation time is calculated as the interval between the date of the first treatment and the date of the last examination of the patient. Possible side effects are determined according to the Veterinary Cooperative Oncology Group toxicity scale (VCOG – CTCAE)²⁰ at each follow-up examination.

1. Schedule an appointment 1 week after the therapy. Schedule an appointment 2 weeks after the therapy. Schedule an appointment 4 weeks after the therapy. After 4 weeks schedule an appointment once a month.