Electrochemotherapy Associated with Calcium Electroporation in Metastatic Feline Cutaneous Malignant Melanoma

Background: Calcium electroporation (CaEP) is a novel therapeutic treatment that has been studied for cancer due to its selective killing cancer cells by necrosis and danger signals. Besides that, electrochemotherapy (ECT) is an effective local treatment that involves the administration of chemotherapeutic drugs followed by delivery of electrical pulses to the tumor. The combination with ECT and CaEP has been reported in literature suggesting that additional response of immune system could have been enhanced by electroporation with calcium. This case, report on the successful treatment with CaEP combined with ECT for treatment of a regional metastasis in a feline model of malignant melanoma. Case: A 9-year-old, mixed breed cat was referred to the veterinary clinic with a 2-month history of cutaneous peripalpebral plaque lesion (0.19 cm3) and a submandibular lymph node enlargement (0.5 cm3). Incisional biopsy of the cutaneous lesion and fine-needle aspiration of submandibular lymph node confirmed a cutaneous melanoma with submandibular lymph node metastasis. Tumor staging was set in T1N1M0 according to WHO staging criteria. ECT for the primary lesion and lymph node metastasis was proposed. For the ECT, bleomycin (15,000 UI/m2) application was performed intravenous followed by electroporation (8 pulses of 100 μs at 1000 V/cm, and 1 Hz) using a needle array electrode consisted of two parallel rows with six needles in each row. At 28-day post-ECT complete remission of the primary tumor and metastatic foci was achieved. However, 120 days after ECT, recurrence was observed in submandibular and retropharyngeal lymph nodes. A second ECT approach was performed adding to bleomycin the intra lymph nodal application of calcium gluconate. The dose of calcium gluconate was diluted in an isotonic 0.9% NaCl solution resulting in a low concentration at 9 mM, injected in both metastatic lymph nodes (submandibular total volume: 1.4 ml; retropharyngeal total volume: 0.5 mL) and pulses were delivered immediately after drug administration. No systemic adverse effects were observed. Local adverse effects were considered mild as transitory edema and ulceration post procedure. One-week post-ECT+CaEP, complete remission of local metastasis occurred. However, the patient achieved five months disease free interval, and died during a surgical approach, achieving nine months of survival time. Discussion: Feline cutaneous malignant melanoma is an aggressive disease with a short survival time for the patients with mean of 4.5 months. CaEP is a novel anticancer treatment that has been study in the past years due to its selective killing cancer cells by necrosis and danger signals. The CaEP induces supraphysiological calcium influx into neoplastic cells leading to acute ATP depletion and necrosis of tumor cells. This use could be an interesting therapeutic choice for both human and veterinary medicine. In this patient, it was demonstrated a good clinical response with its use, showing temporarily tumor remission from the case presented with disease free interval of five months when compared to other report of two months. This description showed that ECT associated with CaEP improved outcome of regional melanoma lymph node metastasis in a cat. However, further investigations are needed to understand the use of CaEP in patients with metastasis as well as evaluate the use of both modalities to determine its synergistic effect.


INTRODUCTION
Calcium electroporation (CaEP) is a novel therapeutic perspective for patients with cutaneous metastasis of malignant melanoma [3] and remission of metastatic melanoma foci [4]. Its approach leads to supraphysiological calcium influx into neoplastic cells leading to acute ATP depletion killing cancer cells by necrosis and cellular danger signals (expression of ATP, calrecticulin and HMGB1) [7]. Besides this, electrochemotherapy is an effective local treatment that involves the administration of chemotherapeutic drugs followed by delivery of electrical pulses (electroporation) to the tumor.
This technique is mainly used on skin tumors, cutaneous metastasis of several tumors and head and neck cancers [9,13]. New evidences suggested on small skin metastasis that CaEP has a similar objective response rate compared to ECT with limited side effects [3]. The combination with ECT and CaEP has been reported in a dog with oral melanoma with resolution of regional metastasis 30 days after treatment, suggesting that additional response of immune system could have been enhanced by electroporation with calcium [8]. Taking into theses previous considerations, this report a description of ECT combined with CaEP for treatment of a regional metastasis in a cat with malignant melanoma.

CASE
The animal included were client-owned and written owner consent was obtained. A 9-years-old, mixed breed cat was referred to the veterinary clinic with a 2-month history of cutaneous peripalpebral plaque lesion (0.19 cm³) and a submandibular lymph node enlargement (0.5 cm³). Tumor volume was calculated as ab²π/6 ('a' is the larger diameter of the tumor nodule, and 'b' is the diameter of the tumor nodule perpendicular to 'a') [14]. Incisional biopsy of the cutaneous lesion and fine-needle aspiration (FNA) of submandibular lymph node were performed and confirmed a cutaneous melanoma with submandibular lymph node metastasis. Abdominal ultrasound, three-view thoracic radiograph, complete blood count and chemistry panel were performed and revealed no abnormalities. Tumor staging was set in T1N1M0 according to WHO staging criteria. Since the owner declined the surgical excision associated with systemic chemotherapy, ECT for the primary lesion ( Figure 1A) and lymph node metastasis ( Figure 1C) was proposed. Electric pulses were administered in time intervals of 8-28 min after 5 min of intravenously injecting bleomycin sulfate 1 [Cinaleo ® ) at 15,000 IU/m² in bolus 1.05 mL] under general anesthesia. The cat was premedicated with pethidine 2 [Pethidine ® -0.3 mg/kg] injected intramuscularly; anesthetic induction was performed with propofol 2 [Provive ® -3 mg/kg] and ketamine 3 [Ketamine ® -1 mg/kg] in the same syringe applied intravenously (IV) followed by endotracheal intubation; anesthesia was maintained with isoflurane 4 [Isoforine ® ]. The electroporation parameters consisted of eight unipolar square electric pulses with frequencies of 1 Hz and 1000 V/ cm, lasting 100 μs each and delivered by six needle electrodes arranged in rows (parallel array) with distances of 0.3 mm between them using pulse generator LC BK-100 (São Paulo, SP, Brazil). All the extension of the primary tumor as well as the regional metastasis was covered with the needles. Antitumor response was performed by clinical examination according to RECIST guideline defined as measurable tumor lesions in at least one dimension with a minimum size of 10 mm caliper measurement [1] At 28-day post-ECT complete remission of the primary tumor and metastatic foci was achieved (Figure 1B and D). CR of lymph node was considered based on its reduction < 10 mm (at baseline examination 0.5 cm³) and also negative FNA. The subject was followedup monthly by clinical examination (measurement of lymph node was classified as non-measurable due to < 10 mm in its axis) and 120 days after ECT, submandibular and retropharyngeal lymph nodes enlargement was noted 0.4 cm³ and 2.77 cm³, respectively. FNA of both lymph nodes confirmed tumor recurrence metastasis ( Figure 1E). Then, a second ECT approach was performed as mentioned above, however, based on the previous human literature [3,4,7] it was proposed adding to bleomycin the calcium gluconate 5 [Calcium gluconate ® 10%]. The dose of calcium gluconate was diluted in an isotonic 0.9% NaCl solution resulting in a low concentration at 9 mM, injected in both metastatic lymph nodes (submandibular total volume: 1.4 mL; retropharyngeal total volume: 0.5 mL) and pulses were delivered immediately after drug administration.
One-week post-second ECT and CaEP, PR was observed in retropharyngeal lymph node (1.31 cm³) and CR in submandibular lymph node (non-measurable). No systemic adverse effects were observed. Local adverse effects were considered mild as transitory edema and ulceration post-ECT and CaEP that resolved in 21 days after use of cefovexin 6 [Convenia ® -0.1 mg/ kg SC single dose] plus non-steroidal anti-inflammatory Piroxicam 7 [Anflene ® 0.3 mg/kg once daily for 7 days]. The patient achieved CR at 28-day post-second ECT+CaEP observed by non-measurable lymph nodes and negative FNA with scar and fibrous tissue locally ( Figure 1F). Clinical outcome can be observed in Table  1. Five months disease free interval was achieved (after second ECT and CaEP), however, at this moment, recurrence was observed in the retropharyngeal lymph node confirmed by FNA (9 months after first session). The owner declined a new ECT session, and submitted the patient to surgical excision, but during surgery a cardio-respiratory arrest occurred, and patient died presenting overall survival of 9 months. The owner declined a post-mortem examination.

DISCUSSION
Feline cutaneous malignant melanoma is an aggressive disease with a short survival time for the patients (mean 4.5 months) [15]. On the other hand, canine cutaneous melanoma seems to be less aggressive and metastatic [11] than feline cutaneous melanoma [12,15] prognosis for feline cutaneous melanoma seems to be poor, as is seen in a metastatic human melanomas and cats could be an interesting spontaneous model for human cutaneous metastatic melanomas. According to the previous literature, the mean survival time after surgical removal of cutaneous metastatic melanoma in cats is 4.5 months [15], resembling human cutaneous melanoma.
Due the poor prognosis in this case, the owner rejected surgical treatment and accepted a the ECT as a more conservative therapeutic approach. Recently, it was observed in canine oral malignant melanoma that ECT generates an increase of cytotoxic T-cells alongside a decrease of regulatory T-cells post-treatment, indicating that activation of immune system occurs delaying metastatic lesions [10]. CaEP is a novel anticancer treatment that has been study in the past years due to its selective killing cancer cells by necrosis and danger signals (expression of ATP, calrecticulin and HMGB1) [7]. The CaEP induces supraphysiological calcium influx into neoplastic cells leading to acute ATP depletion and necrosis of tumor cells [7]. However, since this is singular case report, we were not able to correlate immune system alteration with the CaEP.
The use of CaEP could be an interesting therapeutic choice for both human and veterinary medicine. Previous in vitro studies showed the effect of CaEP and bleomycin electroporation on three different cancer cell spheroids (breast, cancer and colon cancer) and normal dermal fibroblast spheroids, showing spheroid size reduction in all three cancers cells but not in normal cell spheroids [5]. These interesting results indicated that normal cells seem less sensitive to CaEP [5]. The first randomized clinical trial in humans with cutaneous metastases treated with CaEP or ECT using bleomycin, showed similar results between both techniques [8].  However, seems that tumor cells are more sensitive to bleomycin than normal ones [2]. Thus, need to be conducted to better understand the complex mechanism behind the CaEP [3][4][5][6][7] A previous study compared the use of bleomycin with calcium compounds as calcium chloride and calcium glubionate [5]. These authors demonstrated decreased cell viability in three cancer cell lines independent of calcium compound [5], indicating the use of different calcium compounds associated with CaEP. Based on this fact, it was use calcium gluconate due the readiness way to be acquired. It was demonstrated a good clinical response with its use, showing temporarily tumor remission from the case presented (disease free-interval of five months) when compared to other report (two months) [8]. A previous case report recorded the use of ECT and CaEP in canine melanoma, observing similar results [8], as well as observed in another study using combination of ECT and calcium electroporation, suggesting that this approach could be used as a strategy to enhance immune system response and improve survival in patients with disseminated malignant melanoma [4].
This description showed that ECT associated with CaEP improved outcome of regional melanoma lymph node metastasis in a cat. Further investigations are needed to understand the use of CaEP in patients with metastasis as well as evaluate the use of both modalities to determine its synergistic effect.