Royal, R. E., Topalian, S. L., Kammula, U. S., Restifo, N. P., Zheng, Z., Nahvi, A., de Vries, C. R., Rogers-Freezer, L. J., Mavroukakis, S. A., and Rosenberg, S. A. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes.

N., Sazegar, H., Chodon, T., Nelson, S. F., McArthur, G., Sosman, J. A., Ribas, A., and Lo, R. S. (2010). Melanomas acquire resistance to B-RAF(V600E) inhibition by

N., Queirolo, P., Lundgren, L., Mikhailov, S., Roman, L., Verschraegen, C., Humphrey, R., Ibrahim, R., de Pril, V., Hoos, A., and Wolchok, J. D. (2010). Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study. *Ann Oncol*, 21(8), 1712-1717 O'Day, S. J., Pavlick, A. C., Albertini, M. R., Hamid, O., Schalch, H., Lang, Z., Ling, J., Mata,

M., Reddy, M., and Foster, B. (2011). Clinical and pharmacologic evaluation of two

dose levels of intetumumab (CNTO 95) in patients with melanoma or angiosarcoma. *Invest New Drugs*, 2011 Feb 18.[Epub ahead of print]

Promising Experimental Therapies for Metastatic Melanoma 205

Ugurel, S., Hildenbrand, R., Zimpfer, A., La Rosee, P., Paschka, P., Sucker, A., Keikavoussi,

overcome by cotargeting MEK and IGF-1R/PI3K. *Cancer Cell*, 18(6), 683-695 Vivanco, I., and Sawyers, C. L. (2002). The phosphatidylinositol 3-Kinase AKT pathway in

Weber, J., Thompson, J. A., Hamid, O., Minor, D., Amin, A., Ron, I., Ridolfi, R., Assi, H.,

Wilhelm, S. M., Carter, C., Tang, L., Wilkie, D., McNabola, A., Rong, H., Chen, C., Zhang, X.,

Wolchok JD, H. A., O'Day S, Weber JS, Hamid O, Lebbé C, Maio M, Binder M, Bohnsack O,

Wolchok, J. D., Neyns, B., Linette, G., Negrier, S., Lutzky, J., Thomas, L., Waterfield, W.,

Yang, J. C., Hughes, M., Kammula, U., Royal, R., Sherry, R. M., Topalian, S. L., Suri, K. B.,

multicentre, phase 2, dose-ranging study. *Lancet Oncol*, 11(2), 155-164 Wyman, K., Atkins, M. B., Prieto, V., Eton, O., McDermott, D. F., Hubbard, F., Byrnes, C.,

breast cancer: a proof-of-concept trial. *Lancet*, 376(9737), 235-244

human cancer. *Nat Rev Cancer*, 2(7), 489-501

5598

*Res*, 64(19), 7099-7109

*Res*, 15(23), 7412-7420

efficacy. *Cancer*, 106(9), 2005-2011

Earl, H., Wickens, M., and Carmichael, J. (2010). Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced

P., Becker, J. C., Rittgen, W., Hochhaus, A., and Schadendorf, D. (2005). Lack of clinical efficacy of imatinib in metastatic melanoma. *Br J Cancer*, 92(8), 1398-1405 Villanueva, J., Vultur, A., Lee, J. T., Somasundaram, R., Fukunaga-Kalabis, M., Cipolla, A.

K., Wubbenhorst, B., Xu, X., Gimotty, P. A., Kee, D., Santiago-Walker, A. E., Letrero, R., D'Andrea, K., Pushparajan, A., Hayden, J. E., Brown, K. D., Laquerre, S., McArthur, G. A., Sosman, J. A., Nathanson, K. L., and Herlyn, M. (2010). Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be

Maraveyas, A., Berman, D., Siegel, J., and O'Day, S. J. (2009). A randomized, double-blind, placebo-controlled, phase II study comparing the tolerability and efficacy of ipilimumab administered with or without prophylactic budesonide in patients with unresectable stage III or IV melanoma. *Clin Cancer Res*, 15(17), 5591-

Vincent, P., McHugh, M., Cao, Y., Shujath, J., Gawlak, S., Eveleigh, D., Rowley, B., Liu, L., Adnane, L., Lynch, M., Auclair, D., Taylor, I., Gedrich, R., Voznesensky, A., Riedl, B., Post, L. E., Bollag, G., and Trail, P. A. (2004). BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. *Cancer*

Nichol G, Humphrey R, Hodi FS. (2009). Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria. *Clin Cancer*

Schadendorf, D., Smylie, M., Guthrie, T., Jr., Grob, J. J., Chesney, J., Chin, K., Chen, K., Hoos, A., O'Day, S. J., and Lebbe, C. (2010). Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind,

Sanders, K., and Sosman, J. A. (2006). Multicenter Phase II trial of high-dose imatinib mesylate in metastatic melanoma: significant toxicity with no clinical

Levy, C., Allen, T., Mavroukakis, S., Lowy, I., White, D. E., and Rosenberg, S. A. (2007). Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis. *J Immunother*, 30(8), 825-830 Yang, S., Dudley, M. E., Rosenberg, S. A., and Morgan, R. A. (2010). A simplified method for

the clinical-scale generation of central memory-like CD8+ T cells after transduction

Sampson, J. H., Carter, J. H., Jr., Friedman, A. H., and Seigler, H. F. (1998). Demographics,

Schartz, N. E., Farges, C., Madelaine, I., Bruzzoni, H., Calvo, F., Hoos, A., and Lebbe, C.

Serrone, L., Zeuli, M., Sega, F. M., and Cognetti, F. (2000). Dacarbazine-based chemotherapy

Shao, Y., and Aplin, A. E. (2010). Akt3-mediated resistance to apoptosis in B-RAF-targeted

Shields, J. M., Thomas, N. E., Cregger, M., Berger, A. J., Leslie, M., Torrice, C., Hao, H.,

Sinnberg, T., Lasithiotakis, K., Niessner, H., Schittek, B., Flaherty, K. T., Kulms, D., Maczey,

Stahl, J. M., Sharma, A., Cheung, M., Zimmerman, M., Cheng, J. Q., Bosenberg, M. W.,

Staibano, S., Pepe, S., Lo Muzio, L., Somma, P., Mascolo, M., Argenziano, G., Scalvenzi, M.,

Strome, S. E., Dong, H., Tamura, H., Voss, S. G., Flies, D. B., Tamada, K., Salomao, D.,

Sznol, M., Powderly, J.D. , Smith, D.C , Brahmer, J.R. , Drake, C.G. , McDermott, D.F. ,

Trikha, M., Zhou, Z., Nemeth, J. A., Chen, Q., Sharp, C., Emmell, E., Giles-Komar, J., and

Tsao, H., Atkins, M. B. & Sober, A. J. (2004). Management of cutaneous melanoma. *N Engl J*

Tutt, A., Robson, M., Garber, J. E., Domchek, S. M., Audeh, M. W., Weitzel, J. N.,

melanoma. *J Neurosurg*, 88(1), 11-20

19(1), 21-34

7010

with ipilimumab. *Melanoma Res*, 20(3), 247-50

melanoma cells. *Cancer Res*, 70(16), 6670-6681

type of melanoma. *Cancer Res*, 67(4), 1502-1512

cell carcinoma. *Cancer Res*, 63(19), 6501-6505

*Dermatol*, 129(6), 1500-1515

*Pathol*, 36(7), 724-731

*abstr 2506)*

110(3), 326-335

*Med*, 351(10), 998-1012

prognosis, and therapy in 702 patients with brain metastases from malignant

(2010). Complete regression of a previously untreated melanoma brain metastasis

for metastatic melanoma: thirty-year experience overview. *J Exp Clin Cancer Res*,

Penland, S., Arbiser, J., Scott, G., Zhou, T., Bar-Eli, M., Bear, J. E., Der, C. J., Kaufmann, W. K., Rimm, D. L., and Sharpless, N. E. (2007). Lack of extracellular signal-regulated kinase mitogen-activated protein kinase signaling shows a new

E., Campos, M., Gogel, J., Garbe, C., and Meier, F. (2009). Inhibition of PI3K-AKTmTOR signaling sensitizes melanoma cells to cisplatin and temozolomide. *J Invest*

Kester, M., Sandirasegarane, L., and Robertson, G. P. (2004). Deregulated Akt3 activity promotes development of malignant melanoma. *Cancer Res*, 64(19), 7002-

Salvatore, G., Fabbrocini, G., Molea, G., Bianco, A. R., Carlomagno, C., and De Rosa, G. (2005). Poly(adenosine diphosphate-ribose) polymerase 1 expression in malignant melanomas from photoexposed areas of the head and neck region. *Hum*

Cheville, J., Hirano, F., Lin, W., Kasperbauer, J. L., Ballman, K. V., and Chen, L. (2003). B7-H1 blockade augments adoptive T-cell immunotherapy for squamous

Lawrence, P.D. , Wolchok, J.D. , Topalian, S.L. , Lowy, I. (2010). Safety and antitumor activity of biweekly MDX-1106 (Anti-PD-1, BMS-936558/ONO-4538) in patients with advanced refractory malignancies. *J Clin Oncol 28:15s, 2010 (suppl;*

Nakada, M. T. (2004). CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo. *Int J Cancer*,

Friedlander, M., Arun, B., Loman, N., Schmutzler, R. K., Wardley, A., Mitchell, G.,

Earl, H., Wickens, M., and Carmichael, J. (2010). Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. *Lancet*, 376(9737), 235-244

**Part 4**

**Electrochemotherapy**

with lentiviral vectors encoding antitumor antigen T-cell receptors. *J Immunother*, 33(6), 648-658

Yeh, T. C., Marsh, V., Bernat, B. A., Ballard, J., Colwell, H., Evans, R. J., Parry, J., Smith, D., Brandhuber, B. J., Gross, S., Marlow, A., Hurley, B., Lyssikatos, J., Lee, P. A., Winkler, J. D., Koch, K., and Wallace, E. (2007). Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. *Clin Cancer Res*, 13(5), 1576-1583

**Part 4**

**Electrochemotherapy**

206 Treatment of Metastatic Melanoma

Yeh, T. C., Marsh, V., Bernat, B. A., Ballard, J., Colwell, H., Evans, R. J., Parry, J., Smith, D.,

kinase kinase 1/2 inhibitor. *Clin Cancer Res*, 13(5), 1576-1583

33(6), 648-658

with lentiviral vectors encoding antitumor antigen T-cell receptors. *J Immunother*,

Brandhuber, B. J., Gross, S., Marlow, A., Hurley, B., Lyssikatos, J., Lee, P. A., Winkler, J. D., Koch, K., and Wallace, E. (2007). Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein

**10**

*1USA 2Ireland*

**The Role of Electrochemotherapy in the**

*1Division of Surgical Oncology, Department of Surgery, University of Miami,*

*2Cork Cancer Research Centre, Leslie C. Quick Laboratory, BioSciences Institute, University College Cork and Department of Surgery, Mercy University Hospital, Cork,*

About 68,130 new melanomas will be diagnosed in the United States during 2010 (38,870 men: 29,260 women) and of those 8,700 people will die of the disease (5,670 men; 3,030 women). The death rate has been dropping since the 1990s for those younger than 50, but has remained stable or is rising for older individuals. However, the incidence of melanoma has been increasing for at least 30 years, and this trend has become more pronounced in

Malignant Melanoma is the seventh most common type of cancer, but it is the first cause of death from cutaneous skin cancers2. It has been estimated that the lifetime risk of developing malignant melanoma is 2% (1 in 50) for Caucasians, 0.1% (1 in 1,000) for those of

When melanoma is detected in advanced stages, it carries a dismal prognosis, with a mean survival of about 8 months and a 5-year survival as low as 5% 4-6. The disease spreads both by the lymphogenous and the haematogenous routes and can metastasize to virtually any organ in the body. When secondary tumours emerge, these usually follow a sequential pattern to regional lymph node basins, followed by distant sites including skin, subcutaneous tissue, lung, liver, brain, bone and other viscera5-6. Local recurrence, intransit metastases and satellitosis (cutaneous metastases within 2 cm of original lesion) represent the same dissemination process4 in the dermal lymphatics. When the patients present with cutaneous metastases, they are considered to have stage IIIB disease7. Cutaneous metastases occur in 2-20% of patients, depending on tumor thickness 6, 8-9 and can occur either during the early or late phase of the disease 10. In many instances they can represent the first site of recurrence after surgical excision of the primary tumor 11 . The majority (70-80%) of recurrences are diagnosed within the first 3 years of initial diagnosis, and the median time to the presence of in-transit disease could range between 3 to 16 months4. The recurrences present as local or in-transit disease in 20-28%, regional disease in 26-60% and as distant metastases in 15-50% of patients. Even though local recurrence is

**1. Introduction**

young white females and in older white men1.

black descent, and 0.5% (1 in 200) for Hispanics3 .

**Treatment of Malignant Melanoma**

Mecker G. Möller1, Slawomir Salwa2,

*Miller School of Medicine, Miami, Florida*

Declan M. Soden2 and Gerald C. O'Sullivan2

### **The Role of Electrochemotherapy in the Treatment of Malignant Melanoma**

Mecker G. Möller1, Slawomir Salwa2, Declan M. Soden2 and Gerald C. O'Sullivan2 *1Division of Surgical Oncology, Department of Surgery, University of Miami, Miller School of Medicine, Miami, Florida 2Cork Cancer Research Centre, Leslie C. Quick Laboratory, BioSciences Institute, University College Cork and Department of Surgery, Mercy University Hospital, Cork, 1USA 2Ireland*

#### **1. Introduction**

About 68,130 new melanomas will be diagnosed in the United States during 2010 (38,870 men: 29,260 women) and of those 8,700 people will die of the disease (5,670 men; 3,030 women). The death rate has been dropping since the 1990s for those younger than 50, but has remained stable or is rising for older individuals. However, the incidence of melanoma has been increasing for at least 30 years, and this trend has become more pronounced in young white females and in older white men1.

Malignant Melanoma is the seventh most common type of cancer, but it is the first cause of death from cutaneous skin cancers2. It has been estimated that the lifetime risk of developing malignant melanoma is 2% (1 in 50) for Caucasians, 0.1% (1 in 1,000) for those of black descent, and 0.5% (1 in 200) for Hispanics3 .

When melanoma is detected in advanced stages, it carries a dismal prognosis, with a mean survival of about 8 months and a 5-year survival as low as 5% 4-6. The disease spreads both by the lymphogenous and the haematogenous routes and can metastasize to virtually any organ in the body. When secondary tumours emerge, these usually follow a sequential pattern to regional lymph node basins, followed by distant sites including skin, subcutaneous tissue, lung, liver, brain, bone and other viscera5-6. Local recurrence, intransit metastases and satellitosis (cutaneous metastases within 2 cm of original lesion) represent the same dissemination process4 in the dermal lymphatics. When the patients present with cutaneous metastases, they are considered to have stage IIIB disease7. Cutaneous metastases occur in 2-20% of patients, depending on tumor thickness 6, 8-9 and can occur either during the early or late phase of the disease 10. In many instances they can represent the first site of recurrence after surgical excision of the primary tumor 11 . The majority (70-80%) of recurrences are diagnosed within the first 3 years of initial diagnosis, and the median time to the presence of in-transit disease could range between 3 to 16 months4. The recurrences present as local or in-transit disease in 20-28%, regional disease in 26-60% and as distant metastases in 15-50% of patients. Even though local recurrence is

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 211

tails are embedded within the plasma membrane. This porosity is transient and reversible if

Electroporation (EP) of tumour nodules allow the permeation, into the cancer cells, of poorly permeable antineoplastic drugs, such as bleomycin or cisplatin which are given either

The temporary permeability of the cell membrane caused by the electric pulses facilitates a potent localized effect and magnifies the drugs cytotoxicity by orders of magnitude 18, 39, 46-50.

After several studies of different cytotoxic drugs, two have been identified as the best candidates for ECT: Bleomycin and Cisplatin 17, 45. Of importance in these studies EP or the drugs on their own do not influence the growth of tumours, yet their combination have potent tumoricidal effects18, 51-53 ( See Figure 1). An advantage of ECT is that it requires much lower doses of the cytotoxic drug for optimal effects than those usually used for systemic treatments. In addition there is little in the way of collateral damage or complications, since the the cell killing is confined to the tissues affected by the electric field. Bleomycin intercalates into the cellular chromatin causing single and double stranded breaks in DNA resulting in a mitotic cell death by pseudoapoptosis. ( Figure 1 B,C & D). On the other hand, Cisplatin creates an apoptosis effect on the cell 18, 46. This cytolytic activity is potentiated more than 1,000 fold for Bleomycin and 100 fold for Cisplatin by the addition of EP 12, 19, 48-49.

The electric pulses produce a transient state of hypoperfusion by local reflex vasoconstriction at the arteriolar level (lasting 1-2 minutes) and a phase of interstitial edema (that resolves with membrane resealing). However the effect may last longer (12 hours to 5 days) in rapidly dividing tumor cells and is more prominent in tumours with a less mature endothelial lining and higher interstitial pressure. This phenomenon mediated by the sympathetic nervous system is termed the "vascular lock" and it has implications for timing of drug administration18-19. After application of the electric current, there is a retention of drugs already in the tumour but there is also an impairment of entry of drugs from the circulation. Thus when the cytotoxic drugs are administered systemically sufficient time should be allowed to achieve optimal intratumoral drug concentration prior to application of EP. ECT produces other vascular influences, which are believed to be secondary to a reduction in local angiogenic factor production, such as endothelial cell destruction and neovascular reorganization 46, 54-55. The combined vascular influences have been successfully exploited in the treatment of bleeding melanomas 56-57– which may be a difficult to manage

problem, sometimes refractory and fatal, in patients with unresectable disease.

The electric pulses may be applied to the tumors either by plate electrodes on the skin surface or by needle electrodes inserted into the lesion (figure2). The electric field distribution is determined by the geometry of the electrodes. Regardless of the type of electrode the electric field is highest around and between the electrodes 17-18. The current pulse generators in use are the Cliniporator (IGEA, Carpi, Italy ) (FIG 2) and the MedpulserTM (Inovio Biomedical Corporation, CA, USA). It appears that plate electrodes are

**4. Technique of electrochemotherapy**

short duration high amplitude square wave electrical pulses are optimized.18

systemically or intratumorally (IT)18, 39, 43-46

**Types of Drugs**

**The Vascular Lock**

**1. The Equipment**

not yet considered stage IV disease, the prognosis is poor with 5 year survival rates of less than 50% 9,12. The risk of developing metastases may be predicted at excision of the primary lesion and determinants include, increasing thickness of the tumour, the anatomical location, histological subtype and gender 13. In general tumours of the trunk, non cutaneous and subungual regions behave more aggressively than those of the extremity 14-16 .

#### **2. Treatment limitations for recurrent or in-transit melanoma**

Treating extensive cutaneous/subcutaneous nodules or in-transit disease is a clinical challenge because of common unresectability and relative insensitivity to conventional systemic therapies6. These recurrences provide a significant psychological burden for patients whose quality of life is negatively affected by the symptoms caused by the tumor, such as pain, bleeding, ulceration, and malodorous discharge 4, 17-18. Many patients suffering from in-transit disease also manifest with systemic disease, within 6-13 months of the onset of the local lesions. Their life expectancy is foreshortened and is determined by factors such as the burden of the loco-regional disease, the interval from initial treatment and their immune status 4.

The aim of the treatment should be the elimination of both local and systemic disease with the benefit of improved life expectancy and quality of life, while minimizing toxicities or deformities. It is important to note that metastatic cells have disseminated prior to surgical intervention, outlining the critical need for the development of adjuvant therapeutic strategies19. Thus the treatment of unresectable and in-transit disease is both systemic and loco/regional. The options include surgery, which is the most effective method for limited recurrent or in-transit disease, systemic chemotherapy12,21, radiation therapy22, cryosurgery23; carbon dioxide laser ablation,12,24-26, intralesional therapies12,21 with the Bacille Calmette-Guerin vaccine27, TNFerade a non replicant adenovirus complex, expressing the Tumor Necrosis Factor alpha (TNF-α) gene (hTNF-α cDNA)12,28, and Interleukin-229, Cell Vaccine and Immunotherapy 30-33, Interferon alpha34-36, Regional therapies such as hyperthermic isolated limb perfusion (HILP) and Isolated limb infusion (ILP)37, Novel Molecular Therapies with c-Kit inhibitors (Imatinib), C-RAF-inhibitors (Sorafenib) 38, and blocking B-RAF regulation of mitogen-activated protein kinase pathway (MAPK) 38. In more recent years electroporation with anticancer drugs which is termed electrochemotherapy (ECT) has been successfully applied for local tumour control 17, 19, 39. In general malignant melanoma is refractory to systemic treatments and survival after treatment of regional cutaneous metastases has been reported to be between 20-28% at 10 years6,9,40. However all current treatment modalities have individual limitations and variable response rates 4, 12, 19, 41-42.

#### **3. Electrochemotherapy (ECT)**

#### **Definition and physiological effect:**

The cell membrane represents a physical barrier to the intracellular transfer of hydrophilic drugs, macromolecules, nucleotides and peptides. The movement of polar and hydrophilic molecules across the membrane is restricted by the phospholipid bilayer18 Studies suggest that exposure to sufficiently strong and long electrical fields, could rearrange the lipid bilayer, by reorienting the hydrophillic heads in pore-like fashion, while the hydrophobic tails are embedded within the plasma membrane. This porosity is transient and reversible if short duration high amplitude square wave electrical pulses are optimized.18

Electroporation (EP) of tumour nodules allow the permeation, into the cancer cells, of poorly permeable antineoplastic drugs, such as bleomycin or cisplatin which are given either systemically or intratumorally (IT)18, 39, 43-46

The temporary permeability of the cell membrane caused by the electric pulses facilitates a potent localized effect and magnifies the drugs cytotoxicity by orders of magnitude 18, 39, 46-50.

#### **Types of Drugs**

210 Treatment of Metastatic Melanoma

not yet considered stage IV disease, the prognosis is poor with 5 year survival rates of less than 50% 9,12. The risk of developing metastases may be predicted at excision of the primary lesion and determinants include, increasing thickness of the tumour, the anatomical location, histological subtype and gender 13. In general tumours of the trunk, non cutaneous and subungual regions behave more aggressively than those of the

Treating extensive cutaneous/subcutaneous nodules or in-transit disease is a clinical challenge because of common unresectability and relative insensitivity to conventional systemic therapies6. These recurrences provide a significant psychological burden for patients whose quality of life is negatively affected by the symptoms caused by the tumor, such as pain, bleeding, ulceration, and malodorous discharge 4, 17-18. Many patients suffering from in-transit disease also manifest with systemic disease, within 6-13 months of the onset of the local lesions. Their life expectancy is foreshortened and is determined by factors such as the burden of the loco-regional disease, the interval from initial treatment and their

The aim of the treatment should be the elimination of both local and systemic disease with the benefit of improved life expectancy and quality of life, while minimizing toxicities or deformities. It is important to note that metastatic cells have disseminated prior to surgical intervention, outlining the critical need for the development of adjuvant therapeutic strategies19. Thus the treatment of unresectable and in-transit disease is both systemic and loco/regional. The options include surgery, which is the most effective method for limited recurrent or in-transit disease, systemic chemotherapy12,21, radiation therapy22, cryosurgery23; carbon dioxide laser ablation,12,24-26, intralesional therapies12,21 with the Bacille Calmette-Guerin vaccine27, TNFerade a non replicant adenovirus complex, expressing the Tumor Necrosis Factor alpha (TNF-α) gene (hTNF-α cDNA)12,28, and Interleukin-229, Cell Vaccine and Immunotherapy 30-33, Interferon alpha34-36, Regional therapies such as hyperthermic isolated limb perfusion (HILP) and Isolated limb infusion (ILP)37, Novel Molecular Therapies with c-Kit inhibitors (Imatinib), C-RAF-inhibitors (Sorafenib) 38, and blocking B-RAF regulation of mitogen-activated protein kinase pathway (MAPK) 38. In more recent years electroporation with anticancer drugs which is termed electrochemotherapy (ECT) has been successfully applied for local tumour control 17, 19, 39. In general malignant melanoma is refractory to systemic treatments and survival after treatment of regional cutaneous metastases has been reported to be between 20-28% at 10 years6,9,40. However all current treatment modalities have individual limitations and variable response rates 4, 12, 19,

The cell membrane represents a physical barrier to the intracellular transfer of hydrophilic drugs, macromolecules, nucleotides and peptides. The movement of polar and hydrophilic molecules across the membrane is restricted by the phospholipid bilayer18 Studies suggest that exposure to sufficiently strong and long electrical fields, could rearrange the lipid bilayer, by reorienting the hydrophillic heads in pore-like fashion, while the hydrophobic

**2. Treatment limitations for recurrent or in-transit melanoma**

extremity 14-16 .

immune status 4.

41-42.

**3. Electrochemotherapy (ECT) Definition and physiological effect:**  After several studies of different cytotoxic drugs, two have been identified as the best candidates for ECT: Bleomycin and Cisplatin 17, 45. Of importance in these studies EP or the drugs on their own do not influence the growth of tumours, yet their combination have potent tumoricidal effects18, 51-53 ( See Figure 1). An advantage of ECT is that it requires much lower doses of the cytotoxic drug for optimal effects than those usually used for systemic treatments. In addition there is little in the way of collateral damage or complications, since the the cell killing is confined to the tissues affected by the electric field. Bleomycin intercalates into the cellular chromatin causing single and double stranded breaks in DNA resulting in a mitotic cell death by pseudoapoptosis. ( Figure 1 B,C & D). On the other hand, Cisplatin creates an apoptosis effect on the cell 18, 46. This cytolytic activity is potentiated more than 1,000 fold for Bleomycin and 100 fold for Cisplatin by the addition of EP 12, 19, 48-49.

#### **The Vascular Lock**

The electric pulses produce a transient state of hypoperfusion by local reflex vasoconstriction at the arteriolar level (lasting 1-2 minutes) and a phase of interstitial edema (that resolves with membrane resealing). However the effect may last longer (12 hours to 5 days) in rapidly dividing tumor cells and is more prominent in tumours with a less mature endothelial lining and higher interstitial pressure. This phenomenon mediated by the sympathetic nervous system is termed the "vascular lock" and it has implications for timing of drug administration18-19. After application of the electric current, there is a retention of drugs already in the tumour but there is also an impairment of entry of drugs from the circulation. Thus when the cytotoxic drugs are administered systemically sufficient time should be allowed to achieve optimal intratumoral drug concentration prior to application of EP. ECT produces other vascular influences, which are believed to be secondary to a reduction in local angiogenic factor production, such as endothelial cell destruction and neovascular reorganization 46, 54-55. The combined vascular influences have been successfully exploited in the treatment of bleeding melanomas 56-57– which may be a difficult to manage problem, sometimes refractory and fatal, in patients with unresectable disease.

#### **4. Technique of electrochemotherapy**

#### **1. The Equipment**

The electric pulses may be applied to the tumors either by plate electrodes on the skin surface or by needle electrodes inserted into the lesion (figure2). The electric field distribution is determined by the geometry of the electrodes. Regardless of the type of electrode the electric field is highest around and between the electrodes 17-18. The current pulse generators in use are the Cliniporator (IGEA, Carpi, Italy ) (FIG 2) and the MedpulserTM (Inovio Biomedical Corporation, CA, USA). It appears that plate electrodes are

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 213

more suitable for use in superficial skin lesions, while needle electrodes are used for deeper seated lesions, such as exophytic and thick lesions (maximum depth 3 cm)52. A disadvantage of plate electrodes over the needle type is the potential skin damage that may be generated by the higher impedance/resistance of the skin, especially when treating larger affected areas19, 58. Care must be taken to avoid inserting the needle electrodes into the healthy tissue surrounding the tumors, which may also result in local subcutaneous burns 39. There are three types of electrodes in common usage *(FIG 2 ).* Type I electrodes consist of two parallel stainless-steel plate electrodes, used for superficial lesions and do not penetrate the skin. Type II electrodes are used for smaller lesions and consist of two rows of eight needles with 4 mm distance between them, while Type III electrodes are recommended for larger lesions (>1cm), with the needles in a hexagonal configuration. The needles are inserted encircling the tumor and down to the subcutaneous tissue, slightly deeper than tumor depth 17, 19, 52, 59 . It is recommended to elevate or tent the skin at the time of delivering the electric current , if the electrodes are to be inserted in a superficial or shallow subcutaneous areas such as near

the knees, tibial tuborosities, the scalp, or close to any other osseous structures52

Fig. 2. (A) The ECT pulse generator. B) Note an entrainment of 8 square wave pulses.

C) Electrodes type1 (plates) and (D) type 3 (Needle array).

Fig. 1. A. Growth curves of experimental cancers in mice, which demonstrate that electropermeabilisation or intratumoural bleomycin alone, have no influence on tumour growth but when used together completely ablate the tumour. B) Positive Tunel stain 48 hrs post ECT indicating tumor cell death by apoptosis. C &D Tumour before showing normal cellularity and D 48 hrs post ECT showing regions of denucleation

A C

B D

Fig. 1. A. Growth curves of experimental cancers in mice, which demonstrate that electropermeabilisation or intratumoural bleomycin alone, have no influence on tumour growth but when used together completely ablate the tumour. B) Positive Tunel stain 48 hrs post ECT indicating tumor cell death by apoptosis. C &D Tumour before showing normal

cellularity and D 48 hrs post ECT showing regions of denucleation

more suitable for use in superficial skin lesions, while needle electrodes are used for deeper seated lesions, such as exophytic and thick lesions (maximum depth 3 cm)52. A disadvantage of plate electrodes over the needle type is the potential skin damage that may be generated by the higher impedance/resistance of the skin, especially when treating larger affected areas19, 58. Care must be taken to avoid inserting the needle electrodes into the healthy tissue surrounding the tumors, which may also result in local subcutaneous burns 39. There are three types of electrodes in common usage *(FIG 2 ).* Type I electrodes consist of two parallel stainless-steel plate electrodes, used for superficial lesions and do not penetrate the skin. Type II electrodes are used for smaller lesions and consist of two rows of eight needles with 4 mm distance between them, while Type III electrodes are recommended for larger lesions (>1cm), with the needles in a hexagonal configuration. The needles are inserted encircling the tumor and down to the subcutaneous tissue, slightly deeper than tumor depth 17, 19, 52, 59 . It is recommended to elevate or tent the skin at the time of delivering the electric current , if the electrodes are to be inserted in a superficial or shallow subcutaneous areas such as near the knees, tibial tuborosities, the scalp, or close to any other osseous structures52

Fig. 2. (A) The ECT pulse generator. B) Note an entrainment of 8 square wave pulses. C) Electrodes type1 (plates) and (D) type 3 (Needle array).

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 215

administered. The cumulative dose should not exceed 400,000 IU/m2 due to the cumulative risk of lung fibrosis46,52. If the cumulative dose surpasses 60,000 IU /m2, objective respiratory function tests should be made at intervals and the drug discontinued if the respiratory diffusion capacity is abnormal60. The IT dose of Cisplatin is given as follow: for nodules less than 0.5 cm3 a dose of 2mg/cm3, for nodules between 0.5-1 cm3, a dose of

> **Bleomycin dose (1000 IU/mL concentration)**

The rationale for reducing the intralesional dose per cm3 when treating areas with larger tumor burdens is to reduce the risk of systemic toxicities of the absorbed drug without

Prophylactic antibiotics against skin flora should be given intravenously prior to the start of

The required time for the procedure is short, with a median treatment duration of 25 minutes32. Repeated treatments are usually well tolerated by the patients 11, 65-66 and these

Prior to each treatment session patients should have an electrocardiogram (ECG), blood work for evaluation of renal function, coagulation and electrolyte values. All lesions should be photographed and the tumor burden documented. Cellular debris may be released from the tumour during electroporation and these may interfere with the clearance of cytotoxic drugs by the kidney. Thus, when using IV Bleomycin, the serum creatinine should be maintained at less than 150mol/L to ensure proper renal clearance. Physiological monitoring includes visual display of O2 saturation, pulse rate, blood pressure, continuous ECG tracing and respiratory parameters. Acetominophen or an anti-histaminic medication may be given to prevent the mild febrile reaction that may occur in the early post procedural

3. Keep in mind correct timing of drug administration and application of the electric

5. Communicate with awake patients and assisting staff, prior to delivery of electric

can usually be performed at 1-6 weekly intervals, without evident resistance 45.

1. Test the device and the electrodes prior to beginning the procedure

6. Check electroporator traces and waves to confirm proper current delivery

2. Prep and drape following sterile technique principles

1 mL/cm3 tumor 0.5 mL/ cm3 tumor 0.25 mL/ cm3 tumor

**Cisplastin dose (2mg/mL concentration)**

1mg/cm3 and for nodules larger than 1 cm3 a dose of 0.5mg/cm3 is given. (Table 1)

**Tumor Volume (V=ab2π/6)**

<0.5 cm3

>0.5 < 1 cm3 >1 cm3

compromising local efficacy19.

**5. Patient monitoring**

**Technical pearls**

current

current

Table 1. Tumor volume and IT drug dose concentration

period when Bleomycin is administered 46, 59.

4. Protect osseous surfaces by tenting the skin

7. Do not overlap treating fields with normal tissue 8. Dress wounds according to presenting symptoms

the procedure, especially when lesions are ulcerated or necrotic.

#### **2. Anesthesia**

The procedure can be performed in the outpatient or ambulatory setting, under local anesthesia in association with conscious sedation. General anesthesia may be preferred for larger tumors, or tumors located in prior irradiated or fibrotic tissues where infiltration of local anesthetic may be painful and less likely to diffuse and thus achieve adequate pain control and for those located too close to vascular or bony structures. The dose of local anesthetic should be recorded and should not exceed the maximum allowed per body weight for Lidocaine without epinephrin (3 mg/kg ) 52, 59-60. In addition, a mixture of 02/air with Fi02 of 40 % is administered to the patients during conscious sedation.

#### **3. The Electric Current**

Permeabilization occurs in the cell when the internal transmembrane potential has surpassed the critical value between 200-300mV. The extent of the electroporative effect depends on the number and duration of the electric pulses18,55. Two pulse parameters have been evaluated - exponentially decaying pulses and square wave pulses. Of the two, square wave pulses are preferred as they permit independent control of the length and amplitude. The pulse parameters selected for treatment depend on the type of electrode 18, 52.

#### **Pulse parameters**

Ideally for Type I (plate) electrodes, pulse parameters of 8 square waves with amplitude of 1300 V/cm, duration of 100 µs, and frequency of 1Hz are used. The current should be delivered in two perpendicular directions.

For type II (needle) electrodes the voltage amplitude may be reduced to 1000V/cm. The pulses are administered simultaneously between the needle pairs.

For Type III, the needles are positioned hexagonally and 96 pulses are given together (12 pairs x 8 pulses) at a frequency of 5 kHz 61.

Due to the already described vascular lock phenomenon, it is recommended that the electric current is applied between 8 to 28 minutes after IV administration of the drug62, or immediately (within 2-10 minutes) after intratumoral administration 19, 59, 63-64.

#### **4. Drug Dosage Recommendations**

Initial studies compared the routes of administration of Bleomycin suggested advantages for the intralesional over intravenous administration (77% vs. 45 % complete responses) 45 but later the prospective multi-institutional ESOPE (European Standard Operating Procedures of Electrochemotherapy) study in 2006, concluded that IV or IT Bleomycin were comparable when given to tumors of volumes less than 0.5 cm3 52. For Cisplatin, the studies have shown that IT is more effective than the IV route, with CR rates of 82% vs. 48% respectively19, 45. In the ESOPE study local tumor control was achieved in up to 88% of tumors treated with IV Bleomycin, 73% with IT Bleomycin and 75% with IT Cisplatin52.

If a lack of uniformity of drug distribution within the tumor is anticipated either by the presence of large and extensive disease, harder fibrotic tumor nodules, lymphedema or limb fibrosis, the IV route would be more suitable 52. The Intratumoral route is more feasible for those less perfused nodules located in previously pretreated areas19.

When Bleomycin is given IV the dose is 15,000 IU/m2 of body surface area in a bolus lasting 30-45 seconds. But when the IT route is chosen, both drugs are given in a dose calculation based on tumor burden. The IT dose of Bleomycin based on tumor size is calculated as follows: for tumor nodules less than 0.5 cm3 a dose of 1000 IU/cm3, tumor nodules between 0.5 and 1 cm3 a dose of 500 IU/cm3 and for tumors larger than 1 cm3 a dose of 250IU/cm3 is administered. The cumulative dose should not exceed 400,000 IU/m2 due to the cumulative risk of lung fibrosis46,52. If the cumulative dose surpasses 60,000 IU /m2, objective respiratory function tests should be made at intervals and the drug discontinued if the respiratory diffusion capacity is abnormal60. The IT dose of Cisplatin is given as follow: for nodules less than 0.5 cm3 a dose of 2mg/cm3, for nodules between 0.5-1 cm3, a dose of 1mg/cm3 and for nodules larger than 1 cm3 a dose of 0.5mg/cm3 is given. (Table 1)

Table 1. Tumor volume and IT drug dose concentration

The rationale for reducing the intralesional dose per cm3 when treating areas with larger tumor burdens is to reduce the risk of systemic toxicities of the absorbed drug without compromising local efficacy19.

 Prophylactic antibiotics against skin flora should be given intravenously prior to the start of the procedure, especially when lesions are ulcerated or necrotic.

The required time for the procedure is short, with a median treatment duration of 25 minutes32. Repeated treatments are usually well tolerated by the patients 11, 65-66 and these can usually be performed at 1-6 weekly intervals, without evident resistance 45.

#### **5. Patient monitoring**

214 Treatment of Metastatic Melanoma

The procedure can be performed in the outpatient or ambulatory setting, under local anesthesia in association with conscious sedation. General anesthesia may be preferred for larger tumors, or tumors located in prior irradiated or fibrotic tissues where infiltration of local anesthetic may be painful and less likely to diffuse and thus achieve adequate pain control and for those located too close to vascular or bony structures. The dose of local anesthetic should be recorded and should not exceed the maximum allowed per body weight for Lidocaine without epinephrin (3 mg/kg ) 52, 59-60. In addition, a mixture of 02/air

Permeabilization occurs in the cell when the internal transmembrane potential has surpassed the critical value between 200-300mV. The extent of the electroporative effect depends on the number and duration of the electric pulses18,55. Two pulse parameters have been evaluated - exponentially decaying pulses and square wave pulses. Of the two, square wave pulses are preferred as they permit independent control of the length and amplitude.

Ideally for Type I (plate) electrodes, pulse parameters of 8 square waves with amplitude of 1300 V/cm, duration of 100 µs, and frequency of 1Hz are used. The current should be

For type II (needle) electrodes the voltage amplitude may be reduced to 1000V/cm. The

For Type III, the needles are positioned hexagonally and 96 pulses are given together (12

Due to the already described vascular lock phenomenon, it is recommended that the electric current is applied between 8 to 28 minutes after IV administration of the drug62, or

Initial studies compared the routes of administration of Bleomycin suggested advantages for the intralesional over intravenous administration (77% vs. 45 % complete responses) 45 but later the prospective multi-institutional ESOPE (European Standard Operating Procedures of Electrochemotherapy) study in 2006, concluded that IV or IT Bleomycin were comparable when given to tumors of volumes less than 0.5 cm3 52. For Cisplatin, the studies have shown that IT is more effective than the IV route, with CR rates of 82% vs. 48% respectively19, 45. In the ESOPE study local tumor control was achieved in up to 88% of tumors treated with IV

If a lack of uniformity of drug distribution within the tumor is anticipated either by the presence of large and extensive disease, harder fibrotic tumor nodules, lymphedema or limb fibrosis, the IV route would be more suitable 52. The Intratumoral route is more feasible for

When Bleomycin is given IV the dose is 15,000 IU/m2 of body surface area in a bolus lasting 30-45 seconds. But when the IT route is chosen, both drugs are given in a dose calculation based on tumor burden. The IT dose of Bleomycin based on tumor size is calculated as follows: for tumor nodules less than 0.5 cm3 a dose of 1000 IU/cm3, tumor nodules between 0.5 and 1 cm3 a dose of 500 IU/cm3 and for tumors larger than 1 cm3 a dose of 250IU/cm3 is

with Fi02 of 40 % is administered to the patients during conscious sedation.

The pulse parameters selected for treatment depend on the type of electrode 18, 52.

immediately (within 2-10 minutes) after intratumoral administration 19, 59, 63-64.

pulses are administered simultaneously between the needle pairs.

Bleomycin, 73% with IT Bleomycin and 75% with IT Cisplatin52.

those less perfused nodules located in previously pretreated areas19.

**2. Anesthesia**

**3. The Electric Current**

**Pulse parameters**

delivered in two perpendicular directions.

pairs x 8 pulses) at a frequency of 5 kHz 61.

**4. Drug Dosage Recommendations**

Prior to each treatment session patients should have an electrocardiogram (ECG), blood work for evaluation of renal function, coagulation and electrolyte values. All lesions should be photographed and the tumor burden documented. Cellular debris may be released from the tumour during electroporation and these may interfere with the clearance of cytotoxic drugs by the kidney. Thus, when using IV Bleomycin, the serum creatinine should be maintained at less than 150mol/L to ensure proper renal clearance. Physiological monitoring includes visual display of O2 saturation, pulse rate, blood pressure, continuous ECG tracing and respiratory parameters. Acetominophen or an anti-histaminic medication may be given to prevent the mild febrile reaction that may occur in the early post procedural period when Bleomycin is administered 46, 59.

#### **Technical pearls**

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 217

These results were independent of the drug used or the route of administration chosen. Additionally subsequent studies 11, 49, 66, 89-92 of ECT evaluating its effect in the treatment of melanoma and other skin cancers continue to demonstrate the efficacy of the treatment, with

Repeated treatments are feasible as demonstrated by Campana et al.90 and Quaglino et al.11 producing additional clinical responses in patients who had initial non or partial responses or who presented with new recurrent lesions. In the Campana90 study 34 patients out of 52 were diagnosed with unresectable melanoma, but the response rate for the entire cohort of patients treated with either IT or IV Bleomycin improved significantly from a CR of 50%, up

There are approximately 60 institutions in Europe and in the United States that continue to investigate and offer ECT as a palliative treatment for a variety of unresectable tumors, including melanoma; and in an occasional report it has been used as an alternative curative

In patients with unresectable recurrent or in-transit melanoma disease who are not candidates for standard surgical or medical treatment, ECT is now an important therapeutic option18,43,49,75,84,94. These cases include those with unresectable disease due to the extensive number of nodules or lesions located in compromising anatomic areas, such as those around joints, nerves, distal leg and in previously operated fields. Encouraging results with long

Fig. 3. A) An Exophytic type recurrent malignant melanoma after isolated limb perfusion.

response rates comparable to the earlier studies, ranging from 46-100%.

to 83 % after the third ECT treatments.

B) Six months following treatment by ECT .

**6. ECT in the treatment of advanced melanoma**

term remissions have been documented 17, 45, 78, 93, 95-96. ( Figure 3)

therapy 93.

#### **5. ECT development and clinical applications in melanoma**

Neumann in 198267 published the first paper regarding EP as a method to transfer genes into mammalian and bacterial cells. There were other *in vitro* 65, 68 and *in vivo* studies in early and late 1980's using EP in combination with drugs69, but the first clinical trials demonstrating its effectiveness over Bleomycin alone for the treatment of a diversity of cutaneous tumors of the head and neck region, were published in early 90's by Mir et al47 and Belehradek et al.70 from the Institute Gustave Roussy, in Villejuif France. These first 8 patients were treated for squamous cell carcinoma tumors and a complete response (CR) was observed in 57% of the lesions. These early publications encouraged other investigators to expand the principles of this technique to other tumor types including basal cell carcinomas, Kaposi sarcoma, and melanoma metastases 6, 62, 71-74. By the mid 90's, Rudolf et al.71 and Heller et al. 73 published the results of an initial small group (5) of melanoma patients that underwent treatment with ECT and IV Bleomycin. They reported overall response rates (OR) in 92% and 50% of 24 and 10 metastatic nodules respectively19. That same year, Glass et al. 75 from the University of South Florida in Tampa, USA, reported on the first study using ECT with IT Bleomycin in melanoma metastases obtaining OR rates of 92% (78% CR and 14 % PR) in 20 metastatic lesion – results similar to those obtained by the IV route in the previous studies. Later in 1998, the same group of investigators published the results of a bigger cohort of patients with a variety of cutaneous and subcutaneous malignant nodules. Twelve of 34 patients had 84 metastatic melanoma nodules with documented OR rates up to 99% (89% complete response (CR) and 10 % partial response (PR))76. That same year, the combined data produced by five institutions in USA, France and Slovenia was published by Mir et al.39 In this study, twenty patients with metastatic melanoma lesions showed responses in 131 (92%) of 142 lesions, with CR of 53% and PR of 39%. A major finding of this study was that the results were comparable among the institutions even though their treatment protocols and the route used for administering the Bleomycin were not standardized. Additional small studies 56, 77-79 using ECT with IT Bleomycin for melanoma lesions continued to show good responses, with OR rates of 71 to 100% (CR 23%-100% and PR 0%-62%). Rols et al.80 in 2000 continued to demonstrate OR's of 93% using ECT and IV Bleomycin in the treatment of 54 metastatic melanoma nodules.

Sersa et al.81-82 introduced Cisplatin as a therapeutic option in 1998. In their studies they reported CR rates of 100% for 2 patients with 13 lesions treated with IT Cisplatin, but low CR rates of 11 % in 9 patients with 27 lesions treated using the IV route. Additional studies were published using this drug by the IT route only 61, 82-83 with OR rates ranging from 81% to 100% (CR 0%-70 % and PR 6%-100%).

The main issue with most of the studies of the 1990's and early 2000's was the utilization of a variety of treatment protocols with different pulse parameters and pulse generators in combination with different electrode types and drug administration routes 6, 39, 44, 47, 52, 56, 58, 63, 70, 72-73, 75-78, 81-88. But in 2006 the results of a pivotal prospective non-randomized multicenter ESOPE study 52 were published, thus providing recommendations for a standardized protocol for the procedure. The study included 102 patients, 61 evaluated for response and 41 for toxicity respectively. The protocol allowed for administration of Bleomycin either IV or IT, or with Cisplatin IT, and included several histological types of lesions. Ninety-eight lesions from 20 melanoma patients were evaluated - The OR rates were 81% with a CR of 66%. The results confirmed the effectiveness of ECT in the treatment of lesions of different histology, demonstrating an 85% objective response rate and a CR rate of 74% for all lesions.

Neumann in 198267 published the first paper regarding EP as a method to transfer genes into mammalian and bacterial cells. There were other *in vitro* 65, 68 and *in vivo* studies in early and late 1980's using EP in combination with drugs69, but the first clinical trials demonstrating its effectiveness over Bleomycin alone for the treatment of a diversity of cutaneous tumors of the head and neck region, were published in early 90's by Mir et al47 and Belehradek et al.70 from the Institute Gustave Roussy, in Villejuif France. These first 8 patients were treated for squamous cell carcinoma tumors and a complete response (CR) was observed in 57% of the lesions. These early publications encouraged other investigators to expand the principles of this technique to other tumor types including basal cell carcinomas, Kaposi sarcoma, and melanoma metastases 6, 62, 71-74. By the mid 90's, Rudolf et al.71 and Heller et al. 73 published the results of an initial small group (5) of melanoma patients that underwent treatment with ECT and IV Bleomycin. They reported overall response rates (OR) in 92% and 50% of 24 and 10 metastatic nodules respectively19. That same year, Glass et al. 75 from the University of South Florida in Tampa, USA, reported on the first study using ECT with IT Bleomycin in melanoma metastases obtaining OR rates of 92% (78% CR and 14 % PR) in 20 metastatic lesion – results similar to those obtained by the IV route in the previous studies. Later in 1998, the same group of investigators published the results of a bigger cohort of patients with a variety of cutaneous and subcutaneous malignant nodules. Twelve of 34 patients had 84 metastatic melanoma nodules with documented OR rates up to 99% (89% complete response (CR) and 10 % partial response (PR))76. That same year, the combined data produced by five institutions in USA, France and Slovenia was published by Mir et al.39 In this study, twenty patients with metastatic melanoma lesions showed responses in 131 (92%) of 142 lesions, with CR of 53% and PR of 39%. A major finding of this study was that the results were comparable among the institutions even though their treatment protocols and the route used for administering the Bleomycin were not standardized. Additional small studies 56, 77-79 using ECT with IT Bleomycin for melanoma lesions continued to show good responses, with OR rates of 71 to 100% (CR 23%-100% and PR 0%-62%). Rols et al.80 in 2000 continued to demonstrate OR's of 93% using ECT and IV Bleomycin in the treatment of

Sersa et al.81-82 introduced Cisplatin as a therapeutic option in 1998. In their studies they reported CR rates of 100% for 2 patients with 13 lesions treated with IT Cisplatin, but low CR rates of 11 % in 9 patients with 27 lesions treated using the IV route. Additional studies were published using this drug by the IT route only 61, 82-83 with OR rates ranging from 81%

The main issue with most of the studies of the 1990's and early 2000's was the utilization of a variety of treatment protocols with different pulse parameters and pulse generators in combination with different electrode types and drug administration routes 6, 39, 44, 47, 52, 56, 58, 63, 70, 72-73, 75-78, 81-88. But in 2006 the results of a pivotal prospective non-randomized multicenter ESOPE study 52 were published, thus providing recommendations for a standardized protocol for the procedure. The study included 102 patients, 61 evaluated for response and 41 for toxicity respectively. The protocol allowed for administration of Bleomycin either IV or IT, or with Cisplatin IT, and included several histological types of lesions. Ninety-eight lesions from 20 melanoma patients were evaluated - The OR rates were 81% with a CR of 66%. The results confirmed the effectiveness of ECT in the treatment of lesions of different histology, demonstrating an 85% objective response rate and a CR rate of 74% for all lesions.

**5. ECT development and clinical applications in melanoma**

54 metastatic melanoma nodules.

to 100% (CR 0%-70 % and PR 6%-100%).

These results were independent of the drug used or the route of administration chosen. Additionally subsequent studies 11, 49, 66, 89-92 of ECT evaluating its effect in the treatment of melanoma and other skin cancers continue to demonstrate the efficacy of the treatment, with response rates comparable to the earlier studies, ranging from 46-100%.

Repeated treatments are feasible as demonstrated by Campana et al.90 and Quaglino et al.11 producing additional clinical responses in patients who had initial non or partial responses or who presented with new recurrent lesions. In the Campana90 study 34 patients out of 52 were diagnosed with unresectable melanoma, but the response rate for the entire cohort of patients treated with either IT or IV Bleomycin improved significantly from a CR of 50%, up to 83 % after the third ECT treatments.

There are approximately 60 institutions in Europe and in the United States that continue to investigate and offer ECT as a palliative treatment for a variety of unresectable tumors, including melanoma; and in an occasional report it has been used as an alternative curative therapy 93.

#### **6. ECT in the treatment of advanced melanoma**

In patients with unresectable recurrent or in-transit melanoma disease who are not candidates for standard surgical or medical treatment, ECT is now an important therapeutic option18,43,49,75,84,94. These cases include those with unresectable disease due to the extensive number of nodules or lesions located in compromising anatomic areas, such as those around joints, nerves, distal leg and in previously operated fields. Encouraging results with long term remissions have been documented 17, 45, 78, 93, 95-96. ( Figure 3)

Fig. 3. A) An Exophytic type recurrent malignant melanoma after isolated limb perfusion. B) Six months following treatment by ECT .

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 219

Its effectiveness has been proven when providing palliative treatment for hemorrhagic and painful tumor nodules 56-57, 79, 97-98. This is a benefit believed to be secondary to the "vascular lock" phenomenon The vasoconstriction at the arteriolar level produces an imediate and dramatic reduction of perfusion of the malignant lesions, thus controlling the bleeding 18, 45, 98. ECT can also be useful as a neoadjuvant treatment for cytoreduction and organ sparing treatment. Its benefits has been reported in patients with perineal melanoma treated with

ECT can also be suitable for and more tolerable by those patients with a prohibitive surgical risk due to significant comorbidities52 because the length of the procedure is relatively short 52 and patients are able to tolerate multiple sessions. In some anatomical locations ECT could

ECT in combination with cytokine therapy or gene coding immunotherapies has advanced to clinical trials in advanced melanoma. In a phase II study, patients treated with injections of low dose perilesional IL-2 and ECT with Bleomycin, the cytotoxic T lymphocytes response against the known melanoma antigens initially decreased after treatment to reappear when IL-2 was stopped. The tumor-specific peripheral T cells could be detected later in the lesions. The authors theorized that cell death produced by ECT may have attracted and primed denditric cells with the tumor antigens, which later migrated to the draining lymph node basin, and elicited a T cell response against those antigens expressed

The first Human phase I trial of in vivo DNA electroporation of recurrent malignant melanoma was published in the USA in 2009101. EP with dose escalation of the interleukin-12 plasmid (in vivo DNA EP) was used in the treatment of 24 patients with stage III B/C or IV disease. It resulted in significant necrosis of melanoma cells and regression of the majority of treated lesions. In addition, clinical regression of untreated lesions suggested the induction of systemic anti tumor immune responses. The treatment was found to be safe with no significant reported toxicities . While additional studies in larger cohorts of patients are still necessary, to obtain reproducibility of these results, these data show a new method of

There are no studies comparing ECT with other surgical modalities, however when taking into consideration the learning curve of other complex regional treatment modalities, the techniques of ECT are considered to be "user friendly" and easy to teach and learn. This technique can also be highly advantageous and useful in countries or hospitals where other

inducing potent tumour specific immune responses individual to the patient.

3. Lower chemotherapy doses needed, minimizing the toxicity profile of the drug

4. There is no protein denaturation, which may elicit an undesireable immune response

6. Good cost/benefit ratio profile: ambulatory setting, lower cost for drugs, and minimal

Cisplatin 85 and for a sphincter saving procedure in anal melanoma 83.

provide good and sometimes better cosmetic results than surgery 93.

by the melanoma100.

modalities or resources are limited.45

1. Excellent local tumor control rates (80-90%)

5. High safety profile without severe side effects

7. Treatments are well tolerated by patients 8. Improvement in the perceived quality of life

2. Minimal risk of damage to healthy surrounding tissue

**7. Advantages of ECT**

against self antigens

equipment needed.

IV: Intravenous, IT: Intratumoral, CRR: Complete Response Rate, ORR: Overall Response Rate, PR: Partial Response, CR: Complete Response

RETC: Repeated Electrochemotherapy, LTF: Lost to follow up, ns: No specified, MM: Malignant melanoma

\*: Patients with different histological types

Table 2. Summary of most relevant studies using ECT for unresectable or In-Transit Melanoma

Rudolf 1995135 2 24 Bleomycin IV 92 92 Heller 1996137 3 10 Bleomycin IV 30 50 Glass 1996140 5 23 Bleomycin IT 78 96 Heller 1998141 12 84 Bleomycin IT 75 99 Kubota 1998144 1 8 Bleomycin IT 100 100 Mir 199827 20 142 Bleomycin IV 53 92 Sersa 1998146 2 13 Cisplastin IT 100 100 Sersa 2000147 9 27 Cisplastin IV 11 48 Sersa 2000153 10 82 Cisplastin IT 80 87 Rols 2000145 4 55 Bleomycin IV 9 93 Gehl 2000128 1 9 Bleomycin IT 100 100

<sup>2001143</sup> 2 13 Bleomycin IT 23 85 Sersa 2003149 14 211 Cisplastin IT 70 81 Byrne 2005144 21 52 Bleomycin IT 63 71 Snoj 2005148 1 1 Cisplastin IT 0 100

Snoj 2006165 1 16 Cisplastin IT 100 100

Tauceri 2007157 3 ns Bleomycin IT ns ns

<sup>257</sup>Bleomycin IT/IV

IV: Intravenous, IT: Intratumoral, CRR: Complete Response Rate, ORR: Overall Response Rate, PR:

RETC: Repeated Electrochemotherapy, LTF: Lost to follow up, ns: No specified, MM: Malignant

Table 2. Summary of most relevant studies using ECT for unresectable or In-Transit

*(2 pts LTF)* 56 Bleomycin IT or IV

Marty 2006124 20 98 Bleomycin IT/IV\*

30

160

373 608

12 *(7 per protocol)*

14 *(7 pts RETC)*

34 MM *\*(52 cohort 27 RETC)*

Partial Response, CR: Complete Response

\*: Patients with different histological types

**Patients Nodules Drug/route CRR % ORR**

23 Bleomycin IT <sup>36</sup>

*(RETC x4)* 224 Bleomycin IV 100 100

73 Bleomycin IV <sup>50</sup>

larger 3 cm 66 81

for nodules > 3 cm 0 <sup>50</sup>

74

58

ns 50 66 **(PR+CR)%**

46 82

93 93

ns 74 90

**Author No.**

Rodriguez

Gaudy 2006159

Qualigno 200812

Campana 2009158

melanoma

Melanoma

Larkin 2007121 <sup>4</sup>

Snoj 2007164 <sup>1</sup>

Its effectiveness has been proven when providing palliative treatment for hemorrhagic and painful tumor nodules 56-57, 79, 97-98. This is a benefit believed to be secondary to the "vascular lock" phenomenon The vasoconstriction at the arteriolar level produces an imediate and dramatic reduction of perfusion of the malignant lesions, thus controlling the bleeding 18, 45, 98. ECT can also be useful as a neoadjuvant treatment for cytoreduction and organ sparing treatment. Its benefits has been reported in patients with perineal melanoma treated with Cisplatin 85 and for a sphincter saving procedure in anal melanoma 83.

ECT can also be suitable for and more tolerable by those patients with a prohibitive surgical risk due to significant comorbidities52 because the length of the procedure is relatively short 52 and patients are able to tolerate multiple sessions. In some anatomical locations ECT could provide good and sometimes better cosmetic results than surgery 93.

ECT in combination with cytokine therapy or gene coding immunotherapies has advanced to clinical trials in advanced melanoma. In a phase II study, patients treated with injections of low dose perilesional IL-2 and ECT with Bleomycin, the cytotoxic T lymphocytes response against the known melanoma antigens initially decreased after treatment to reappear when IL-2 was stopped. The tumor-specific peripheral T cells could be detected later in the lesions. The authors theorized that cell death produced by ECT may have attracted and primed denditric cells with the tumor antigens, which later migrated to the draining lymph node basin, and elicited a T cell response against those antigens expressed by the melanoma100.

The first Human phase I trial of in vivo DNA electroporation of recurrent malignant melanoma was published in the USA in 2009101. EP with dose escalation of the interleukin-12 plasmid (in vivo DNA EP) was used in the treatment of 24 patients with stage III B/C or IV disease. It resulted in significant necrosis of melanoma cells and regression of the majority of treated lesions. In addition, clinical regression of untreated lesions suggested the induction of systemic anti tumor immune responses. The treatment was found to be safe with no significant reported toxicities . While additional studies in larger cohorts of patients are still necessary, to obtain reproducibility of these results, these data show a new method of inducing potent tumour specific immune responses individual to the patient.

There are no studies comparing ECT with other surgical modalities, however when taking into consideration the learning curve of other complex regional treatment modalities, the techniques of ECT are considered to be "user friendly" and easy to teach and learn. This technique can also be highly advantageous and useful in countries or hospitals where other modalities or resources are limited.45

#### **7. Advantages of ECT**

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 221

erythematous reaction usually recedes within a few days11. Local tumor necrosis has been reported in 42% of cases66. Delayed wound healing that may take several weeks or months to resolve, and epidermal erosions, and hematomas have been reported as rare events66,73,75. The injection site reactions appear to be low (Type I and II) in the Wiebendirk toxicity

Transient muscle spasms myoclonus, secondary to muscle stimulation by the electrical pulses, have been reported in 25% with lower intensity contractions in up to 78% of patients52,66. Some authors advocate the administration of diazepam to alleviate these particular symptoms73,75. Interestingly the majority of patients are willing to continue

Bioengineering developments and evolution of the technique continue to expand the applications of ECT as an alternative treatment for tumors that are inaccessible to current electrical probes. A redeveloped electroporator generator provides more flexibility to deliver the electric current at different phases of the cardiac cycle and the facility to connect several electrode probes around the tumor to deliver the electric pulses in synchrony. The other development is related to the type of electrodes. Longer array electrodes are now available to treat larger and deeper seated tumors, which in the past was a limitation of the procedure. These longer electrodes are insulated proximally to prevent short circuiting of current and to protect the normal tissues that are transgressed en-route to the tumor. These new devices have recently been applied clinically19, 102-103. Kos et al. 103 have proposed an algorithmic computer optimized analysis to treat deeper tumors, to minimize errors and to

Another novelty is the creation of finger applicators which allows the application of electrodes into lesions located in difficult to reach anatomic locations, such as the inside of the oral cavity. These finger electrodes have been already tested in the treatment of melanoma of

New endoluminal electrodes to reach internal lesions within the gastrointestinal tract have been used in animal models, as well as tumors transplanted into rabbit liver and murine models 87, 104-106. These studies are encouraging and have demonstrated both *in vitro* and *in vivo* (human solid tumor masses in nude mouse models), that the use of flexible electrodes is

There is still the need to create harder needle probes for use in those difficult to treat subcutaneous and cutaneous tumors. These needles must be capable of penetrating into hard fibrotic tissue, while minimizing the bleeding risks and maintaining an adequate

Higher amplitude electric pulses or "nanopulses" are currently being evaluated. The use of shorter pulse durations in the nanosecond range are believed to create smaller pores that allow ions but not large molecules to penetrate the membrane. These higher electric fields increase the possibility of producing non resealable pores, thus producing the effect of irreversible electroporation, and consequently allowing the cells to lose their cytoplasm with concomitant cell death. This principle is being used for tumor ablation, palliation or both 43,

the oral mucosa, and head and neck regions, with complete tumor regression 90.

treatment if indicated, since the side effects are tolerable19.

**11. What the future holds**

maximize treatment benefits.

safe, feasible and reproducible.

**Nanopulses**

electrical distance between the probes.

**Equipment evolution**

scale90.

#### **8. Patient selection and limitations of the procedure**

The contraindications of the ECT could be divided into drug and procedure related.

A. Drug related contraindications60:

B. Procedure related contraindications43:

#### **9. Limitations**

With the current available electrodes, ECT has limitations when treating deep seated tumors43. Tumors larger than 3 cm2 appear to have lower response rates (CR 73 %) to ECT 11,49,90 as compared to nodules smaller than 1cm2 (CR 98%). These findings were not affected by either the cutaneous or subcutaneous location of the nodules 11.

When tumor nodules are located in irradiated or fibrotic tissues the needle electrode penetration may be problematic with a suboptimal delivery of the electrical current or drugs 90. Nonetheless, if optimal needle penetration is achieved, ECT is equally effective in irradiated as in non-irradiated tissue52. If extensive disease (more than 15 lesions) is present repeated sessions may be necessary. In aggressive disease – while undergoing ECT new cutaneous nodules may emerge but palliative retreatment is worthwhile eventhough the systemic disease progresses rapidly60.

This treatment modality has not been studied in randomized trials with other treatment techniques, such as ablative and perfusion or infusion procedures, or radiation therapy. More studies with longer follow up are still needed to evaluate disease free survival and to compare ECT to surgical excision, not only in the palliative setting but as a curative alternative in those patients unsuitable or unfit for a surgical procedure19.

#### **10. Toxicity and side effects**

ECT has a low toxicity profile with limited side effects as compared to other regional therapies such as HILP or ILP 41. However one of the limitations of fully assessing the toxicities of the treatment is the inconsistency of the large majority of the published studies documenting complications.

The systemic dose of Bleomycin is one twentieth of that used in the majority of chemotherapeutic regimens and thus the systemic side effects appear to be limited to nausea 18, 90. There has been two reported cases of post procedure lypothymia90.

The most common local side effects reported by the majority of patients are pain (75%) and erythema limited to the tumor and surrounding treated tissue 11, 45, 52, 66. Most of the patients considered those symptoms tolerable as documented by the ESOPE study 52. The erythematous reaction usually recedes within a few days11. Local tumor necrosis has been reported in 42% of cases66. Delayed wound healing that may take several weeks or months to resolve, and epidermal erosions, and hematomas have been reported as rare events66,73,75. The injection site reactions appear to be low (Type I and II) in the Wiebendirk toxicity scale90.

Transient muscle spasms myoclonus, secondary to muscle stimulation by the electrical pulses, have been reported in 25% with lower intensity contractions in up to 78% of patients52,66. Some authors advocate the administration of diazepam to alleviate these particular symptoms73,75. Interestingly the majority of patients are willing to continue treatment if indicated, since the side effects are tolerable19.

### **11. What the future holds**

#### **Equipment evolution**

220 Treatment of Metastatic Melanoma

1. For safety reasons ECT should not be used in patients with implanted electric devices

2. In patients who may carry a higher risk of bleeding such as those on anticoagulants or

With the current available electrodes, ECT has limitations when treating deep seated tumors43. Tumors larger than 3 cm2 appear to have lower response rates (CR 73 %) to ECT 11,49,90 as compared to nodules smaller than 1cm2 (CR 98%). These findings were not

When tumor nodules are located in irradiated or fibrotic tissues the needle electrode penetration may be problematic with a suboptimal delivery of the electrical current or drugs 90. Nonetheless, if optimal needle penetration is achieved, ECT is equally effective in irradiated as in non-irradiated tissue52. If extensive disease (more than 15 lesions) is present repeated sessions may be necessary. In aggressive disease – while undergoing ECT new cutaneous nodules may emerge but palliative retreatment is worthwhile eventhough the

This treatment modality has not been studied in randomized trials with other treatment techniques, such as ablative and perfusion or infusion procedures, or radiation therapy. More studies with longer follow up are still needed to evaluate disease free survival and to compare ECT to surgical excision, not only in the palliative setting but as a curative

ECT has a low toxicity profile with limited side effects as compared to other regional therapies such as HILP or ILP 41. However one of the limitations of fully assessing the toxicities of the treatment is the inconsistency of the large majority of the published studies

The systemic dose of Bleomycin is one twentieth of that used in the majority of chemotherapeutic regimens and thus the systemic side effects appear to be limited to

The most common local side effects reported by the majority of patients are pain (75%) and erythema limited to the tumor and surrounding treated tissue 11, 45, 52, 66. Most of the patients considered those symptoms tolerable as documented by the ESOPE study 52. The

nausea 18, 90. There has been two reported cases of post procedure lypothymia90.

The contraindications of the ECT could be divided into drug and procedure related.

**8. Patient selection and limitations of the procedure**

2. Interstitial lung fibrosis, if Bleomycin is going to be used

with increased INR and platelets count < 70,000.

4. When the cumulative dose of Bleomycin has reached >400,000 UI/m2

affected by either the cutaneous or subcutaneous location of the nodules 11.

alternative in those patients unsuitable or unfit for a surgical procedure19.

A. Drug related contraindications60:

1. Known allergy to the drug to be administered

3. Kidney failure or limited renal function

B. Procedure related contraindications43:

systemic disease progresses rapidly60.

**10. Toxicity and side effects**

documenting complications.

such as pacemakers

**9. Limitations**

Bioengineering developments and evolution of the technique continue to expand the applications of ECT as an alternative treatment for tumors that are inaccessible to current electrical probes. A redeveloped electroporator generator provides more flexibility to deliver the electric current at different phases of the cardiac cycle and the facility to connect several electrode probes around the tumor to deliver the electric pulses in synchrony. The other development is related to the type of electrodes. Longer array electrodes are now available to treat larger and deeper seated tumors, which in the past was a limitation of the procedure. These longer electrodes are insulated proximally to prevent short circuiting of current and to protect the normal tissues that are transgressed en-route to the tumor. These new devices have recently been applied clinically19, 102-103. Kos et al. 103 have proposed an algorithmic computer optimized analysis to treat deeper tumors, to minimize errors and to maximize treatment benefits.

Another novelty is the creation of finger applicators which allows the application of electrodes into lesions located in difficult to reach anatomic locations, such as the inside of the oral cavity. These finger electrodes have been already tested in the treatment of melanoma of the oral mucosa, and head and neck regions, with complete tumor regression 90.

New endoluminal electrodes to reach internal lesions within the gastrointestinal tract have been used in animal models, as well as tumors transplanted into rabbit liver and murine models 87, 104-106. These studies are encouraging and have demonstrated both *in vitro* and *in vivo* (human solid tumor masses in nude mouse models), that the use of flexible electrodes is safe, feasible and reproducible.

There is still the need to create harder needle probes for use in those difficult to treat subcutaneous and cutaneous tumors. These needles must be capable of penetrating into hard fibrotic tissue, while minimizing the bleeding risks and maintaining an adequate electrical distance between the probes.

#### **Nanopulses**

Higher amplitude electric pulses or "nanopulses" are currently being evaluated. The use of shorter pulse durations in the nanosecond range are believed to create smaller pores that allow ions but not large molecules to penetrate the membrane. These higher electric fields increase the possibility of producing non resealable pores, thus producing the effect of irreversible electroporation, and consequently allowing the cells to lose their cytoplasm with concomitant cell death. This principle is being used for tumor ablation, palliation or both 43,

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 223

higher risk malignant melanomas by means of immunogene or cytokine therapies, hence establishing tumour specific responses which could prevent recurrence and eradicate

ECT with Bleomycin or Cisplatin is an effective treatment in the palliative management of unresectable recurrent cutaneous or subcutaneous melanoma metastases or in-transit disease, with OR rates of approximately 80-90%. ECT should now be considered as part of the armamentarium for treatment of loco regional advanced melanoma. The technology of ECT continues to evolve allowing for the treatment of metastatic lesions in other organs or anatomic regions. The principles of EP are already being applied in the clinical setting for the delivery of targeted therapies such as gene transfer and immunotherapy. These therapies along with ECT have the potential not only for local, but for distant treatment of tumors such as those of malignant melanoma, by stimulating a self-driven immune response

However studies evaluating long term follow up results of ECT in melanoma are still needed prior to considering it as an option for curative intent. ECT has been proven to be an

(http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessi

http://www.cancer.org/Research/CancerFactsFigures/CancerFactsFigures/cance

http://www.cancer.org/docroot/CRI/content/CRI\_2\_4\_1X\_What\_are\_the\_key\_st

[3] American Cancer Society: Detailed Guide: Skin Cancer - Melanoma. What Are the Key

[4] Hoekstra HJ. The European approach to in-transit melanoma lesions. *Int J Hyperthermia.*

[5] Yeatman TJ, Nicolson GL. Molecular basis of tumor progression: mechanisms of organspecific tumor metastasis. *Semin Surg Oncol.* May-Jun 1993;9(3):256-263. [6] Glass LF, Fenske NA, Jaroszeski M, et al. Bleomycin-mediated electrochemotherapy of

[7] Balch CM, Buzaid AC, Soong SJ, et al. Final version of the American Joint Committee on

[8] Karakousis CP, Choe KJ, Holyoke ED. Biologic behavior and treatment of intransit

[9] Ross MI, Balch CM. The current management of cutaneous melanoma. *Adv Surg.*

[10] Meier F, Will S, Ellwanger U, et al. Metastatic pathways and time courses in the orderly progression of cutaneous melanoma. *Br J Dermatol.* Jul 2002;147(1):62-70.

metastasis of melanoma. *Surg Gynecol Obstet.* Jan 1980;150(1):29-32.

Cancer staging system for cutaneous melanoma. *J Clin Oncol.* Aug 15

basal cell carcinoma. *J Am Acad Dermatol.* Jan 1996;34(1):82-86.

excellent palliative option in treatment of recurrent unresectable or in-transit disease.

disseminated micometastases.

to achieve systemic control of the disease

onal/. Accessed 18 March 2011.

Statistics About Melanoma? . 2009;

May 2008;24(3):227-237.

2001;19(16):3635-3648.

1991;24:139-200.

atistics\_for\_melanoma\_50.asp?sitearea=.

[2] American Cancer Society: Cancer facts and figures 2010. 2010;

r-facts-and-figures-2010. Accessed 21 March, 2011.

**12. Summary**

**13. References**  [1] Institute NC.

107-109. However in the treatment of melanoma, the advantages of these higher electric fields are not immediately evident.

ECT combination with Gene transfer, immunology and nanomolecules

Several studies suggest that the immune system is also involved in the mechanisms of response to ECT treatment and that this could be exploited for systemic disease control.

In a murine model 114, ECT followed by CpG oligonucleotide injection locally, produced an enhancement of the complete regression responses of tumors from 43 to 100 % , while also triggering systemic antitumor phenomenon with specific immune memory. Activation of dendritic cells released from the tumors are believed to be involved in this response with a recruitment of CD11c and CD11b receptors and an increase of TLR9 expression .

EP in combination with gene transfer is termed "Gene Electrotransfer ". It uses gene coding plasmids in order to transfer intracellularly a combination of genes, to either knock down the expression of a particular gene, or to stimulate temporary patterns of gene expression). The technique allows for avoidance of the biohazard issues intrinsic to viral vectors 101, 115-116. Scientists at the Cork Cancer Research Centar in Ireland have investigated in an *in vivo* murine model, the application of EP and local gene therapy for malignant tumors. A plasmid coding for two immunogenes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the B7-1 co-stimulatory immune molecule were delivered by EP. This resulted in the complete regression of the majority (60%) of non immunogenic tumors, while eliciting a tumour specific systemic response that hindered the metastatic growth in the liver in a 100 % of the mice. This was a durable potent response with tumor specificity. When the remaining non responders non responders tumors were excised, an improved survival was observed when compared to the control groups, thus suggesting that the use of neoadjuvant electroporation and gene therapy given at an appropriate time interval prior to tumour excision or ablation could prevent the surfacing of metastatic disease 117-119. Regulatory T cell depletion at the time of electrogenetherapy stimulated improved complete response rates from 60 to near 100% suggesting a potential for improving efficacy of electroporation based immunogene therapy.

ECT with injection of TNF-α intra or peritumorally and suboptimal doses of Bleomycin in mice might have a positive immunomodulatory effect, and possibly adds a systemic component to the localized ECT treatment120 . It has been noted that EP with INF-α used to treat Mycosis Fungoides (subcutaneous lymphoma) lesions produced a 100 % CR 121 The cytotoxic action of INF-α was attributed to its increased tumoral concentration and the prolonged time of action produced by the EP. ECT either with INF-α or TNF-α might permit the usage of less toxic doses, while enhancing clinical local and systemic immunological response rates and minimizing systemic side effects19.

ECT, radiotherapy and activation of bioreductive drugs

The "vascular lock" principle of EP has the potential to be used to "activate" bioreductive drugs such as Tirapazamine against neoplastic cells 43, due to its vascular disrupting effect of tumor blood supply. In addition ECT has been shown to have a synergistic effect with radiotherapy in preclinical investigations, opening the possibility to use it as a radiosensitising tool for the palliation of subcutaneous lesion43, 122.

The continued development of improved diagnostic methods will allow for earlier diagnosis of metastases, and possibly open the opportunities for *in situ* neoadjuvant treatment of higher risk malignant melanomas by means of immunogene or cytokine therapies, hence establishing tumour specific responses which could prevent recurrence and eradicate disseminated micometastases.

### **12. Summary**

222 Treatment of Metastatic Melanoma

107-109. However in the treatment of melanoma, the advantages of these higher electric fields

Several studies suggest that the immune system is also involved in the mechanisms of response to ECT treatment and that this could be exploited for systemic disease control. In a murine model 114, ECT followed by CpG oligonucleotide injection locally, produced an enhancement of the complete regression responses of tumors from 43 to 100 % , while also triggering systemic antitumor phenomenon with specific immune memory. Activation of dendritic cells released from the tumors are believed to be involved in this response with a

EP in combination with gene transfer is termed "Gene Electrotransfer ". It uses gene coding plasmids in order to transfer intracellularly a combination of genes, to either knock down the expression of a particular gene, or to stimulate temporary patterns of gene expression). The technique allows for avoidance of the biohazard issues intrinsic to viral vectors 101, 115-116. Scientists at the Cork Cancer Research Centar in Ireland have investigated in an *in vivo* murine model, the application of EP and local gene therapy for malignant tumors. A plasmid coding for two immunogenes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the B7-1 co-stimulatory immune molecule were delivered by EP. This resulted in the complete regression of the majority (60%) of non immunogenic tumors, while eliciting a tumour specific systemic response that hindered the metastatic growth in the liver in a 100 % of the mice. This was a durable potent response with tumor specificity. When the remaining non responders non responders tumors were excised, an improved survival was observed when compared to the control groups, thus suggesting that the use of neoadjuvant electroporation and gene therapy given at an appropriate time interval prior to tumour excision or ablation could prevent the surfacing of metastatic disease 117-119. Regulatory T cell depletion at the time of electrogenetherapy stimulated improved complete response rates from 60 to near 100% suggesting a potential for improving efficacy of electroporation based

ECT with injection of TNF-α intra or peritumorally and suboptimal doses of Bleomycin in mice might have a positive immunomodulatory effect, and possibly adds a systemic component to the localized ECT treatment120 . It has been noted that EP with INF-α used to treat Mycosis Fungoides (subcutaneous lymphoma) lesions produced a 100 % CR 121 The cytotoxic action of INF-α was attributed to its increased tumoral concentration and the prolonged time of action produced by the EP. ECT either with INF-α or TNF-α might permit the usage of less toxic doses, while enhancing clinical local and systemic immunological

The "vascular lock" principle of EP has the potential to be used to "activate" bioreductive drugs such as Tirapazamine against neoplastic cells 43, due to its vascular disrupting effect of tumor blood supply. In addition ECT has been shown to have a synergistic effect with radiotherapy in preclinical investigations, opening the possibility to use it as a

The continued development of improved diagnostic methods will allow for earlier diagnosis of metastases, and possibly open the opportunities for *in situ* neoadjuvant treatment of

ECT combination with Gene transfer, immunology and nanomolecules

recruitment of CD11c and CD11b receptors and an increase of TLR9 expression .

are not immediately evident.

immunogene therapy.

response rates and minimizing systemic side effects19. ECT, radiotherapy and activation of bioreductive drugs

radiosensitising tool for the palliation of subcutaneous lesion43, 122.

ECT with Bleomycin or Cisplatin is an effective treatment in the palliative management of unresectable recurrent cutaneous or subcutaneous melanoma metastases or in-transit disease, with OR rates of approximately 80-90%. ECT should now be considered as part of the armamentarium for treatment of loco regional advanced melanoma. The technology of ECT continues to evolve allowing for the treatment of metastatic lesions in other organs or anatomic regions. The principles of EP are already being applied in the clinical setting for the delivery of targeted therapies such as gene transfer and immunotherapy. These therapies along with ECT have the potential not only for local, but for distant treatment of tumors such as those of malignant melanoma, by stimulating a self-driven immune response to achieve systemic control of the disease

However studies evaluating long term follow up results of ECT in melanoma are still needed prior to considering it as an option for curative intent. ECT has been proven to be an excellent palliative option in treatment of recurrent unresectable or in-transit disease.

#### **13. References**

[1] Institute NC.

(http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessi onal/. Accessed 18 March 2011.

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 225

[29] Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 therapy

[30] Terando AM, Faries MB, Morton DL. Vaccine therapy for melanoma: current status and

[31] Yun CO, Nolan KF, Beecham EJ, Reisfeld RA, Junghans RP. Targeting of T lymphocytes

[32] Lotem M, Machlenkin A, Hamburger T, et al. Autologous melanoma vaccine induces

[34] Eggermont AM, Testori A, Marsden J, et al. Utility of adjuvant systemic therapy in

[35] Sondak VK, Flaherty LE. Adjuvant therapy of melanoma: is pegylated interferon alfa-2b

[36] Schadendorf D, Algarra SM, Bastholt L, et al. Immunotherapy of distant metastatic

[37] Creech O, Jr., Krementz ET, Ryan RF, Winblad JN. Chemotherapy of cancer: regional perfusion utilizing an extracorporeal circuit. *Ann Surg.* Oct 1958;148(4):616-632. [38] Smalley KS, Nathanson KL, Flaherty KT. Genetic subgrouping of melanoma reveals new opportunities for targeted therapy. *Cancer Res.* Apr 15 2009;69(8):3241-3244. [39] Mir LM, Glass LF, Sersa G, et al. Effective treatment of cutaneous and subcutaneous

[40] Ross MI. Surgery and other local-regional modalities for all stages of melanoma. *Curr*

[41] Moller MG, Lewis JM, Dessureault S, Zager JS. Toxicities associated with hyperthermic

[42] Eggermont AM, Gore M. Randomized adjuvant therapy trials in melanoma: surgical

[43] Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M. Electrochemotherapy in

[44] Glass LF, Jaroszeski M, Gilbert R, Reintgen DS, Heller R. Intralesional bleomycin-

[45] Sersa G. The state-of-the-art of electrochemotherapy before the ESOPE study;

[46] Mir LM. Bases and rationale of electrochemotherapy. *European Journal of Cancer.*

[47] Mir LM, Belehradek M, Domenge C, et al. [Electrochemotherapy, a new antitumor

[48] Orlowski S, Belehradek J, Jr., Paoletti C, Mir LM. Transient electropermeabilization of

cells in culture. Increase of the cytotoxicity of anticancer drugs. *Biochem Pharmacol.*

malignant tumours by electrochemotherapy. *Br J Cancer.* Jun 1998;77(12):2336-2342.

isolated limb perfusion and isolated limb infusion in the treatment of melanoma

mediated electrochemotherapy in 20 patients with basal cell carcinoma. *J Am Acad*

[33] Gilboa E. DC-based cancer vaccines. *J Clin Invest.* May 2007;117(5):1195-1203.

what we've been waiting for? *Lancet.* Jul 12 2008;372(9633):89-90.

1985 and 1993. *J Clin Oncol.* Jul 1999;17(7):2105-2116.

melanoma. *Ann Oncol.* Aug 2009;20 Suppl 6:vi30-34.

disease. *Ann Oncol.* Aug 2009;20 Suppl 6:vi41-50.

and sarcoma. *Int J Hyperthermia.* May 2008;24(3):275-289.

treatment of tumours. *Eur J Surg Oncol.* Feb 2008;34(2):232-240.

advantages and clinical uses. *Eur J Cancer. Supplement.* 2006(4):8.

treatment: first clinical trial]. *C R Acad Sci III.* 1991;313(13):613-618.

and systemic. *Semin Oncol.* Dec 2007;34(6):509-515.

*Neoplasia.* Sep-Oct 2000;2(5):449-459.

*Opin Oncol.* Mar 1994;6(2):197-203.

*Dermatol.* Oct 1997;37(4):596-599.

*Supplement.* 2006;4:38-44.

Dec 15 1988;37(24):4727-4733.

2009;15(15):4968-4977.

future directions. *Vaccine.* Sep 27 2007;25 Suppl 2:B4-16.

for patients with metastatic melanoma: analysis of 270 patients treated between

to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors.

antitumor and self-reactive immune responses that affect patient survival and depend on MHC class II expression on vaccine cells. *Clin Cancer Res.* Aug 1

[11] Quaglino P, Mortera C, Osella-Abate S, et al. Electrochemotherapy with intravenous

[12] Gimbel MI, Delman KA, Zager JS. Therapy for unresectable recurrent and in-transit

[13] Balch CM, Soong SJ, Gershenwald JE, et al. Prognostic factors analysis of 17,600

[15] Garbe C, Buttner P, Bertz J, et al. Primary cutaneous melanoma. Prognostic classification of anatomic location. *Cancer.* May 15 1995;75(10):2492-2498. [16] Garbe C, Buttner P, Bertz J, et al. Primary cutaneous melanoma. Identification of

[17] Byrne CM, Thompson JF. Role of electrochemotherapy in the treatment of metastatic

[18] Sadadcharam M, Soden DM, O'Sullivan G C. Electrochemotherapy: an emerging cancer

[19] Moller MG, Salwa S, Soden DM, O'Sullivan GC. Electrochemotherapy as an adjunct or

[20] Testori A, Rutkowski P, Marsden J, et al. Surgery and radiotherapy in the treatment of

[21] Atkins MB, Hsu J, Lee S, et al. Phase III trial comparing concurrent biochemotherapy

[22] Barranco SC, Romsdahl MM, Humphrey RM. The radiation response of human malignant melanoma cells grown in vitro. *Cancer Res.* Jun 1971;31(6):830-833. [23] Scala M, Gipponi M, Queirolo P, et al. Cryosurgery for advanced malignant melanoma

[24] Hill S, Thomas JM. Use of the carbon dioxide laser to manage cutaneous metastases

[25] Kandamany N, Mahaffey P. Carbon dioxide laser ablation as first-line management of

[26] Hayes AJ, Clark MA, Harries M, Thomas JM. Management of in-transit metastases from

[27] Morton DL, Eilber FR, Holmes EC, et al. BCG immunotherapy of malignant melanoma: summary of a seven-year experience. *Ann Surg.* Oct 1974;180(4):635-643. [28] Rasmussen H, Rasmussen C, Lempicki M, et al. TNFerade Biologic: preclinical

in-transit cutaneous malignant melanoma metastases. *Lasers Med Sci.* May

toxicology of a novel adenovector with a radiation-inducible promoter, carrying the human tumor necrosis factor alpha gene. *Cancer Gene Ther.* Nov 2002;9(11):951-957.

of the facial skin. A case report. *In Vivo.* Jan-Feb 2006;20(1):153-156.

cutaneous malignant melanoma. *Br J Surg.* Jun 2004;91(6):673-682.

from malignant melanoma. *Br J Surg.* Apr 1996;83(4):509-512.

cutaneous melanoma. *Ann Oncol.* Aug 2009;20 Suppl 6:vi22-29.

treatment. *Int J Hyperthermia.* May 2008;24(3):263-273.

*Anticancer Ther.* Nov 2009;9(11):1611-1630.

*J Clin Oncol.* Dec 10 2008;26(35):5748-5754.

melanoma staging system. *J Clin Oncol.* Aug 15 2001;19(16):3622-3634. [14] Weinstock MA. Epidemiology and prognosis of anorectal melanoma. *Gastroenterology.*

extremity melanoma. *Cancer Control.* Jul 2008;15(3):225-232.

Aug 2008;15(8):2215-2222.

Jan 1993;104(1):174-178.

May 15 1995;75(10):2484-2491.

*Ther.* May 2006;6(5):671-678.

2009;24(3):411-414.

bleomycin in the local treatment of skin melanoma metastases. *Ann Surg Oncol.*

melanoma patients: validation of the American Joint Committee on Cancer

prognostic groups and estimation of individual prognosis for 5093 patients. *Cancer.*

melanoma and other metastatic and primary skin tumors. *Expert Rev Anticancer*

alternative to other treatments for unresectable or in-transit melanoma. *Expert Rev*

with cisplatin, vinblastine, dacarbazine, interleukin-2, and interferon alfa-2b with cisplatin, vinblastine, and dacarbazine alone in patients with metastatic malignant melanoma (E3695): a trial coordinated by the Eastern Cooperative Oncology Group.

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 227

[66] Gaudy C, Richard MA, Folchetti G, Bonerandi JJ, Grob JJ. Randomized controlled study

[67] Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. *EMBO J.* 1982;1(7):841-845. [68] Melvik JE, Pettersen EO, Gordon PB, Seglen PO. Increase in cis-

[69] Okino M, Mohri H. Effects of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. *Jpn J Cancer Res.* Dec 1987;78(12):1319-1321. [70] Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek J, Jr., Mir LM.

[71] Rudolf Z, Stabuc B, Cemazar M, Miklavcic F, Vodovnik L, Sersa G.

[72] Rols MP, Bachaud JM, Giraud P, Chevreau C, Roche H, Teissie J. Electrochemotherapy

[73] Heller R, Jaroszeski MJ, Glass LF, et al. Phase I/II trial for the treatment of cutaneous

[74] Sersa G, Cufer T, Cemazar M, Rebersek M, Zvonimir R. Electrochemotherapy with

[75] Glass LF, Pepine ML, Fenske NA, Jaroszeski M, Reintgen DS, Heller R. Bleomycin-

[76] Heller R, Jaroszeski MJ, Reintgen DS, et al. Treatment of cutaneous and subcutaneous

[77] Rodriguez-Cuevas S, Barroso-Bravo S, Almanza-Estrada J, Cristobal-Martinez L,

[78] Byrne CM, Thompson JF, Johnston H, et al. Treatment of metastatic melanoma using

[79] Kubota Y, Mir LM, Nakada T, Sasagawa I, Suzuki H, Aoyama N. Successful treatment of metastatic skin lesions with electrochemotherapy. *J Urol.* Oct 1998;160(4):1426. [80] Giraud P, Bachaud JM, Teissie J, Rols MP. Effects of electrochemotherapy on cutaneous

[81] Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z. Electrochemotherapy

*Cutan Med Surg.* May-Jun 2006;10(3):115-121.

*Cancer.* Dec 15 1993;72(12):3694-3700.

474.

971.

melanoma patients. *Radiol Oncol.* 1995(29):6.

review. *Tumori.* Mar-Apr 2000;86(2):163-165.

1996;132(11):1353-1357.

1998;83(1):148-157.

2001;32(4):273-276.

2005;15(1):45-51.

1996;36(5):1285.

cells. *Eur J Cancer Clin Oncol.* Dec 1986;22(12):1523-1530.

of electrochemotherapy in the local treatment of skin metastases of melanoma. *J*

dichlorodiammineplatinum (II) cytotoxicity upon reversible electropermeabilization of the plasma membrane in cultured human NHIK 3025

Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial.

Electrochemotherapy with bleomycin: the first clinical experiencie in malignant

of cutaneous metastases in malignant melanoma. *Melanoma Res.* Oct 2000;10(5):468-

and subcutaneous tumors using electrochemotherapy. *Cancer.* Mar 1 1996;77(5):964-

bleomycin in the treatment of hypernephroma metastasis: case report and literature

mediated electrochemotherapy of metastatic melanoma. *Arch Dermatol.* Nov

tumors with electrochemotherapy using intralesional bleomycin. *Cancer.* Jul 1

Gonzalez-Rodriguez E. Electrochemotherapy in primary and metastatic skin tumors: phase II trial using intralesional bleomycin. *Arch Med Res.* Jul-Aug

electroporation therapy with bleomycin (electrochemotherapy). *Melanoma Res.* Feb

metastases of human malignant melanoma. *Int J Radiat Oncol Biol Phys.* Dec 1

with cisplatin: potentiation of local cisplatin antitumour effectiveness by

[49] Larkin JO, Collins CG, Aarons S, et al. Electrochemotherapy: aspects of preclinical development and early clinical experience. *Ann Surg.* Mar 2007;245(3):469-479. [50] Weaver JC. Electroporation theory. Concepts and mechanisms. *Methods Mol Biol.*

[51] Belehradek J, Jr., Orlowski S, Poddevin B, Paoletti C, Mir LM. Electrochemotherapy of spontaneous mammary tumours in mice. *Eur J Cancer.* 1991;27(1):73-76. [52] Marty M SG, Garbay JM, Gehl J, .Collins CG, Snoj M, Billard V, Geertsen PF, Larkin JO,

[53] Sersa G, Cemazar M, Miklavcic D. Antitumor effectiveness of electrochemotherapy with

[54] Gehl J. Electroporation for drug and gene delivery in the clinic: doctors go electric.

[55] Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene

[56] Gehl J, Geertsen PF. Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy. *Melanoma Res.* Dec 2000;10(6):585-589. [57] Snoj M, Cemazar M, Srnovrsnik T, Kosir SP, Sersa G. Limb sparing treatment of

[58] Hofmann GA, Dev SB, Nanda GS, Rabussay D. Electroporation therapy of solid tumors.

[59] Mir L, Gehl J, Sersa G, Collins CG, Garbay J, Billard V, Geertsen PF, Rudolf Z, OSullivan

[60] Testori A, Tosti G, Martinoli C, et al. Electrochemotherapy for cutaneous and

[61] Sersa G CM, Rudolf Z. . Electrochemotherapy: advantages and drawbacks in treatment

[62] Domenge C, Orlowski S, Luboinski B, et al. Antitumor electrochemotherapy: new

[63] Heller R, Jaroszeski M, Perrott R, Messina J, Gilbert R. Effective treatment of B16

[64] Cemazar M, Milacic R, Miklavcic D, Dolzan V, Sersa G. Intratumoral cisplatin

dependence and platinum content. *Anticancer Drugs.* Jul 1998;9(6):525-530. [65] Mir LM, Banoun H, Paoletti C. Introduction of definite amounts of nonpermeant

advances in the clinical protocol. *Cancer.* Mar 1 1996;77(5):956-963.

therapy and research. *Acta Physiol Scand.* Apr 2003;177(4):437-447.

*European Journal of Cancer. Supplement.* 2006;4(11):3-13.

*Crit Rev Ther Drug Carrier Syst.* 1999;16(6):523-569.

of cancer patients. *Cancer Therapy.* 2003;1:133-142.

*Methods Mol Biol.* 2008;423:351-359.

2009;95(3):398-402.

2006;4:14-25.

2010;23(6):651-661.

*Res.* Feb 1997;7(1):10-18.

*Exp Cell Res.* Mar 1988;175(1):15-25.

Miklavcic D, Paulovic I, Pavlin-Kosir SM, Cemazar M, Morsli N, Soden DM, Rudolf Z, Robert C , O'Sullivan GC, Mir LM. Electrochemotherapy -An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases : Results of ESOPE (european standard operating procedures of electrochemotherapy) study.

cis-diamminedichloroplatinum(II) in mice. *Cancer Res.* Aug 1 1995;55(15):3450-3455.

bleeding melanoma recurrence by electrochemotherapy. *Tumori.* May-Jun

GC, Marty M. Standard operating procedures of hte electrochemotherapy: Instructions for the use of bleomycin and cisplastin administered either systemically or locally and electric pulses delivered by CliniporatorTM by means of ivansive or non-invasive electrodes. *European Journal of Cancer. Supplement.*

subcutaneous tumor lesions: a novel therapeutic approach. *Dermatol Ther.* Nov

melanoma by direct delivery of bleomycin using electrochemotherapy. *Melanoma*

administration in electrochemotherapy: antitumor effectiveness, sequence

molecules into living cells after electropermeabilization: direct access to the cytosol.

1995;55:3-28.

The Role of Electrochemotherapy in the Treatment of Malignant Melanoma 229

[97] Giardino R, Fini M, Bonazzi V, Cadossi R, Nicolini A, Carpi A. Electrochemotherapy a

[98] Kaehler KC, Egberts F, Hauschild A. Electrochemotherapy in symptomatic melanoma

[99] Heller LC, Heller R. Electroporation gene therapy preclinical and clinical trials for

[100] Andersen MH, Gehl J, Reker S, et al. Dynamic changes of specific T cell responses to

[101] Daud AI, DeConti RC, Andrews S, et al. Phase I trial of interleukin-12 plasmid

[102] Miklavcic D, Snoj M, Zupanic A, et al. Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy. *Biomed Eng Online.* 2010;9:10. [103] Kos B, Zupanic A, Kotnik T, Snoj M, Sersa G, Miklavcic D. Robustness of treatment

[104] Orlowski S, An D, Belehradek J, Jr., Mir LM. Antimetastatic effects of

[105] Ramirez LH, Orlowski S, An D, et al. Electrochemotherapy on liver tumours in rabbits.

[106] Soden D, Larkin J, Collins C, et al. The development of novel flexible electrode arrays

[107] Edd JF, Horowitz L, Davalos RV, Mir LM, Rubinsky B. In vivo results of a new focal

[108] Nuccitelli R, Pliquett U, Chen X, et al. Nanosecond pulsed electric fields cause

[109] Frey W, White JA, Price RO, et al. Plasma membrane voltage changes during

[110] Mir LM, Orlowski S, Belehradek J, Jr., Paoletti C. Electrochemotherapy potentiation of

[111] Mir LM, Orlowski S, Poddevin B, Belehradek J, Jr. Electrochemotherapy tumor

[112] Mir LM, Roth C, Orlowski S, et al. [Potentiation of the antitumoral effect of

[113] Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, Heller R. In vivo electroporation of

murine Lewis lung tumor. *Anticancer Drugs.* Jul 1998;9(6):551-556.

subcutaneous tissues. *Biomed Pharmacother.* Sep 2006;60(8):458-462.

melanoma. *Curr Gene Ther.* Aug 2010;10(4):312-317.

*Surg.* Jul 2010;36(7):1200-1202.

2008;26(36):5896-5903.

2010;236(1):147-153.

2006;53(7):1409-1415.

*Br J Cancer.* Jun 1998;77(12):2104-2111.

*Cytokine Netw.* May-Jun 1992;3(3):331-334.

interleukin 2]. *C R Acad Sci III.* 1992;314(12):539-544.

459.

3550.

360.

72.

novel approach to the treatment of metastatic nodules on the skin and

skin metastases: intraindividual comparison with conventional surgery. *Dermatol*

melanoma correlate with IL-2 administration. *Semin Cancer Biol.* Dec 2003;13(6):449-

electroporation in patients with metastatic melanoma. *J Clin Oncol.* Dec 20

planning for electrochemotherapy of deep-seated tumors. *J Membr Biol.* Jul

electrochemotherapy and of histoincompatible interleukin-2-secreting cells in the

for the electrochemotherapy of solid tumour tissue. (Potential for endoscopic treatment of inaccessible cancers). *Conf Proc IEEE Eng Med Biol Soc.* 2004;5:3547-

tissue ablation technique: irreversible electroporation. *IEEE Trans Biomed Eng.* Jul

melanomas to self-destruct. *Biochem Biophys Res Commun.* May 5 2006;343(2):351-

nanosecond pulsed electric field exposure. *Biophys J.* May 15 2006;90(10):3608-3615.

antitumour effect of bleomycin by local electric pulses. *Eur J Cancer.* 1991;27(1):68-

treatment is improved by interleukin-2 stimulation of the host's defenses. *Eur*

electrochemotherapy by immunotherapy with allogeneic cells producing

plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas

application of electric pulses in cancer patients. *Eur J Cancer.* Jul 1998;34(8):1213- 1218.

[82] Sersa G, Stabuc B, Cemazar M, Miklavcic D, Rudolf Z. Electrochemotherapy with

[83] Snoj M, Rudolf Z, Cemazar M, Jancar B, Sersa G. Successful sphincter-saving treatment

[84] Heller R. Treatment of cutaneous nodules using electrochemotherapy. *J Fla Med Assoc.*

[85] Kubota Y, Tomita Y, Tsukigi M, Kurachi H, Motoyama T, Mir LM. A case of perineal

[86] Sersa G, Stabuc B, Cemazar M, Miklavcic D, Rudolf Z. Electrochemotherapy with

[87] Soden DM, Larkin JO, Collins CG, et al. Successful application of targeted

[88] Rabussay DP, Nanda GS, Goldfarb PM. Enhancing the effectiveness of drug-based

[89] Tauceri F, Mura G, Roseano M, Framarini M, Ridolfi L, Verdecchia GM. Surgery and

[90] Campana LG, Mocellin S, Basso M, et al. Bleomycin-based electrochemotherapy: clinical

[91] Landstrom FJ, Nilsson CO, Crafoord S, Reizenstein JA, Adamsson GB, Lofgren LA.

[92] Landstrom FJ, Nilsson CO, Reizenstein JA, Nordqvist K, Adamsson GB, Lofgren AL.

[93] Sersa G, Cemazar M, Snoj M. Electrochemotherapy of tumours. *Curr Oncol.* Mar

[94] Heller R, Gilbert R, Jaroszeski MJ. Clinical applications of electrochemotherapy. *Adv*

[95] Snoj M, Cemazar M, Slekovec Kolar B, Sersa G. Effective treatment of multiple

[96] Snoj MR, Z. Paulin-Kosir, S. Cemanzar, M, Snoj, R. Sersa, G. . Long lasting complete

adjuvant brachytherapy. *Anticancer Drugs.* Mar 2005;16(3):345-348.

skin metastases. *Melanoma Res.* Aug 2000;10(4):381-385.

solid tumours. *Cancer Lett.* Feb 8 2006;232(2):300-310.

patients. *Langenbecks Arch Surg.* Mar 4 2008.

*Drug Deliv Rev.* Jan 4 1999;35(1):119-129.

1218.

2000;6(3):863-867.

Feb 1995;82(2):147-150.

Apr 2005;15(2):133-134.

*Treat.* Feb 2002;1(1):71-82.

2009;16(1):191-199.

2009;16(2):34-35.

2007;48(3):391-395.

2006;4(11):2.

2010.

Aug 2010;36(8):1245-1250.

application of electric pulses in cancer patients. *Eur J Cancer.* Jul 1998;34(8):1213-

cisplatin: clinical experience in malignant melanoma patients. *Clin Cancer Res.* Mar

of anorectal malignant melanoma with electrochemotherapy, local excision and

malignant melanoma successfully treated with electrochemotherapy. *Melanoma Res.*

cisplatin: the systemic antitumour effectiveness of cisplatin can be potentiated locally by the application of electric pulses in the treatment of malignant melanoma

electrochemotherapy using novel flexible electrodes and low dose bleomycin to

cancer therapy by electroporation (electropermeabilization). *Technol Cancer Res*

adjuvant therapies in the treatment of stage IV melanoma: our experience in 84

outcome from a single institution's experience with 52 patients. *Ann Surg Oncol.* Jan

Electroporation therapy of skin cancer in the head and neck area. *Dermatol Surg.*

Electroporation therapy for T1 and T2 oral tongue cancer. *Acta Otolaryngol.* Dec 29

unresectable skin melanoma metastases by electrochemotherapy. *Croat Med J.* Jun

response in melanoma treated by electrochemotherapy. *Eur J Cancer Suppl.*

**11**

*USA*

*Norfolk, Virginia,*

**Pulse Power Ablation of Melanoma with**

Stephen J. Beebe, Wentia E. Ford, Wei Ren and Xinhua Chen *Old Dominion University / Frank Reidy Research Center for Bioelectrics*

Melanocytes are cells that originate in the neural crest. In the dermis, their well characterized role is to produce melanin and through interactions with keratinocytes transfer this pigment to determine skin color and protect the largest organ in the body, the skin, from ultraviolet light. As an effective free radical scavenger, melanin protects against reactive oxygen species that would otherwise damage DNA [Rózanowska et al., 1999]. Melanocytes may also have other roles such as immune, neuroendocrine, and signaling functions through interactions with cells other than keratinocytes, such as lymphocytes, mast cells and endothelial cells [see Tsatmali et al., 2002 for review]. However, keratinocytes regulate melanocyte number, differentiation and melanin production in response to UV radiation. It may be that the resilience of melanocytes to protect the skin, their extraordinary regenerative capacity and their origin as neural crest migratory cells makes them one of the most deadly forms of metastatic skin cancers when they undergo tumorogenesis. It is known that there is a great deal of common cellular and genetic events among embryonic development, tissue regeneration and cancer. Further, typical self-renewal and migration capacity of cancer are shared with embryonic and regenerative cells [White and Zon, 2008]. The recapitulation of embryonic genetic programs is facilitated by overexposure to extreme sunlight (UVA and UVB) or tanning bed light (mostly UVA). The American Cancer Society estimated that in the US in 2009, 68,720 new cases of melanoma (188 new cases /day) will be diagnosed and 8,650 people will die from the disease (24 melanoma deaths /day). The continued increase in melanoma is a significant cause of morbidity and mortality in the Western world. Thus, metastatic melanoma remains a persistent therapeutic challenge. There are limited successes in preventing this often fatal disease and there are even fewer successes

Presently available treatment strategies have had limited impact on progression and overall survival of patients with melanoma. In addition to surgery, currently approved treatments for metastatic melanoma include chemotherapy, radiation and/or immunotherapy. Chemotherapy is mostly limited to dacarbazine, cisplatin, carmustine and vinblastin. The addition of systemic immunotherapy with IL-2 and/or INFά to these chemotherapeutic

**1. Introduction**

in developing a cure.

**2. Standard melanoma therapies**

**Nanosecond Pulsed Electric Fields**

combined with electrochemotherapy induces long-term antitumour immunity. *Melanoma Res.* Dec 2000;10(6):577-583.

Websites:

http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessi onal/page4

[204] National Comprenhensive Cancer Network. Melanoma 2009 [cited 2009 August 4, 2009.]; Available from:

http://www.nccn.org/professionals/physician\_gls/PDF/melanoma.pdf

### **Pulse Power Ablation of Melanoma with Nanosecond Pulsed Electric Fields**

Stephen J. Beebe, Wentia E. Ford, Wei Ren and Xinhua Chen *Old Dominion University / Frank Reidy Research Center for Bioelectrics Norfolk, Virginia, USA*

#### **1. Introduction**

230 Treatment of Metastatic Melanoma

[114] Roux S, Bernat C, Al-Sakere B, et al. Tumor destruction using electrochemotherapy

[115] Heller LC, Heller R. In vivo electroporation for gene therapy. *Hum Gene Ther.* Sep

[116] Cemazar M, Golzio M, Sersa G, Rols MP, Teissie J. Electrically-assisted nucleic acids

[117] Cashman JP, Larkin JO, Casey G, et al. Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers. *Cancer Lett.* Jan 5 2008. [118] Collins CG, Tangney M, Larkin JO, et al. Local gene therapy of solid tumors with GM-

[119] Tangney M, Casey G, Larkin JO, et al. Non-viral in vivo immune gene therapy of

[120] Sersa G, Cemazar M, Menart V, Gaberc-Porekar V, Miklavcic D. Anti-tumor

[121] Peycheva E, Daskalov I, Tsoneva I. Electrochemotherapy of Mycosis fungoides by

[122] Kranjc S, Cemazar M, Grosel A, Sentjurc M, Sersa G. Radiosensitising effect of

[201] American Cancer Society: Cancer Facts and Figures 2009. Atlanta, Ga: American

[202] American Cancer Society: Detailed Guide: Skin Cancer - Melanoma. What Are the Key

[203] National Cancer Institute. Melanoma Treatment (PDQ)R. Staging Information. 2009

[204] National Comprenhensive Cancer Network. Melanoma 2009 [cited 2009 August 4,

http://www.nccn.org/professionals/physician\_gls/PDF/melanoma.pdf

*Melanoma Res.* Dec 2000;10(6):577-583.

*Immunother.* Nov 2006;55(11):1443-1450.

atistics\_for\_melanoma\_50.asp?sitearea=

[cited August 4, 2009.]; Available from:

SA-1 tumors in mice. *Cancer Lett.* Jun 3 1997;116(1):85-92.

interferon-alpha. *Bioelectrochemistry.* May 2007;70(2):283-286.

2006;17(9):890-897.

2006;13(12):1061-1071.

*BMC Cancer.* 2005;5:115.

Presentation.asp

onal/page4

2009.]; Available from:

3825.

Websites:

*Cancer Immunol Immunother.* Sep 2008;57(9):1291-1300.

combined with electrochemotherapy induces long-term antitumour immunity.

followed by CpG oligodeoxynucleotide injection induces distant tumor responses.

delivery to tissues in vivo: where do we stand? *Curr Pharm Des.* 2006;12(29):3817-

CSF and B7-1 eradicates both treated and distal tumors. *Cancer Gene Ther.* Dec

cancer: combined strategies for treatment of systemic disease. *Cancer Immunol*

effectiveness of electrochemotherapy with bleomycin is increased by TNF-alpha on

electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice.

Cancer Society, 2009. See a dermatologist. Skin cancer is very treatable when caught early. Also available online. 2009 [cited 2009 May 25, 2009]; Available from: http://www.cancer.org/docroot/PRO/content/PRO\_1\_1\_Cancer\_Statistics\_2009\_

Statistics About Melanoma? 2009 May 14, 2009 [cited 2009 May 25]; Available from: http://www.cancer.org/docroot/CRI/content/CRI\_2\_4\_1X\_What\_are\_the\_key\_st

http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessi

Melanocytes are cells that originate in the neural crest. In the dermis, their well characterized role is to produce melanin and through interactions with keratinocytes transfer this pigment to determine skin color and protect the largest organ in the body, the skin, from ultraviolet light. As an effective free radical scavenger, melanin protects against reactive oxygen species that would otherwise damage DNA [Rózanowska et al., 1999]. Melanocytes may also have other roles such as immune, neuroendocrine, and signaling functions through interactions with cells other than keratinocytes, such as lymphocytes, mast cells and endothelial cells [see Tsatmali et al., 2002 for review]. However, keratinocytes regulate melanocyte number, differentiation and melanin production in response to UV radiation. It may be that the resilience of melanocytes to protect the skin, their extraordinary regenerative capacity and their origin as neural crest migratory cells makes them one of the most deadly forms of metastatic skin cancers when they undergo tumorogenesis. It is known that there is a great deal of common cellular and genetic events among embryonic development, tissue regeneration and cancer. Further, typical self-renewal and migration capacity of cancer are shared with embryonic and regenerative cells [White and Zon, 2008]. The recapitulation of embryonic genetic programs is facilitated by overexposure to extreme sunlight (UVA and UVB) or tanning bed light (mostly UVA). The American Cancer Society estimated that in the US in 2009, 68,720 new cases of melanoma (188 new cases /day) will be diagnosed and 8,650 people will die from the disease (24 melanoma deaths /day). The continued increase in melanoma is a significant cause of morbidity and mortality in the Western world. Thus, metastatic melanoma remains a persistent therapeutic challenge. There are limited successes in preventing this often fatal disease and there are even fewer successes in developing a cure.

#### **2. Standard melanoma therapies**

Presently available treatment strategies have had limited impact on progression and overall survival of patients with melanoma. In addition to surgery, currently approved treatments for metastatic melanoma include chemotherapy, radiation and/or immunotherapy. Chemotherapy is mostly limited to dacarbazine, cisplatin, carmustine and vinblastin. The addition of systemic immunotherapy with IL-2 and/or INFά to these chemotherapeutic

Pulse Power Ablation of Melanoma with Nanosecond Pulsed Electric Fields 233

that attempt to "escape" the modification, which may also lead to resistance and recurrences. These issues may complicate uses of targeted medicine in melanoma

Although other causes of cancer are known, mutations that often lead to malignant growth and metastasis frequently occur in protein kinases [Blume-Jensen and Hunter, 2001; Chenga and Force, 2010]. Protein tyrosine kinases transduce extracellular signals into intracellular functions that regulate a wide array of cellular activity. Tumorigenesis is often driven by constitutively active protein kinases that modulate cell cycle, angiogenesis or apoptosis. Two general classes of protein kinase inhibitors include monoclonal antibodies or small molecular weight inhibitors that are directed against specific protein tyrosine kinase receptors or their ligands. A common site for protein kinase inhibitor drug design is the ATP binding site because transfer of phosphate to substrates cannot occur without the gammaphosphate from ATP. However, the binding sites are so highly conserved among kinases that kinase genes can be identified without phosphotransferase activity by the presence of specific sequences that define ATP binding. Two major concerns in designing these drugs are issues of specificity and potency. Specificity is one issue because of the ATP binding site conservation among all protein kinases. Given the broad range of kinase functions, it becomes important to inhibit kinases involved in tumorigenesis without affecting other kinases. Potency is the other concern because ATP is at relative high concentrations in the cell (milliMolar) and an orally active small molecular weight drug must have a high affinity to be an effective inhibitor. Kinase inhibitors are divided into three major classes based on binding to their ATP site [Chenga and Force, 2010]. Type I inhibitors have high affinity for the ATP binding pocket. Therefore type I kinase inhibitors generally have broad kinase specificity. Type II kinase inhibitor (e.g., imatinib) are more selective because they not only have specificity for the ATP binding site but also sites adjacent the ATP binding site. In addition, unlike type I inhibitors, type II inhibitors can bind to kinases in their inactive conformation. Type III kinase inhibitors (e.g., families of mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitors) are more selective but more

difficult to design because they bind to sites distant from the ATP binding pocket.

melanomas, should be analyzed for their *KIT* status [Satzger et al., 2010].

A number of monoclonal antibodies and targeted small molecular weight protein tyrosine kinase inhibitors are in clinical trials for the treatment of melanoma. Regarding the hallmarks of cancer, these agents are directed to prevent limitless replicative potential and/or self-sufficiency in growth signals, evasion of immune surveillance, sustained angiogenesis, and evasion of apoptosis. Because protein tyrosine kinases are involved in many of these hallmarks, many of these agents are multi-protein tyrosine kinase inhibitors that are directed towards more than one of these hallmarks and kinases so have overlapping targets. For example, Sunitinib, Sorafenib and Imatinib target VEGFRs and/or PDGFRs and can act as anti-angiogenesis agents. In addition, these drugs can also act on either c-Kit and/or Raf-1/B-Raf and are directed towards limitless replicative potential and/or selfsufficiency in growth signals. Current data indicate that melanomas with activating c-KIT mutations and possibly also with KIT gene amplifications respond to therapy with tyrosine kinase inhibitors blocking c-KIT. It was suggested that subgroups of patients with metastatic melanoma prone to *KIT* mutations, such as primary mucosal and acrolentiginous

A significant number of melanomas are mutated at V600E in BRAF (~60%), an oncogene that is a signal transduction enzyme near the origin or proximal end of a cascade of phosphorylation events that promotes proliferation through the Raf/Mek/Erk pathway and

treatments.

agents resulted in major toxicities. INFά is approved for use but is not included as a standard of care due to minimal impact on overall survival and significant toxicities [Amaravadi et al., 2007; Gogas et al., 2007; Tarhini et al., 2006]. Unfortunately, metastatic melanoma is one of the most resistant cancers to a wide range of treatment modalities including single-agents and combination chemotherapy, immunotherapy, chemoimmunotherapy and a host of immune stimulators [Riker et al., 2007].

#### **3. Targeted melanoma therapies**

One of the major problems that cancer therapeutics face today is coping with a diversity of cancer diseases and melanoma is no exception. However, Hanahan and Weinberg [2000, 2011] reasoned that since all mammalian cells express the same molecular mechanisms for proliferation, differentiation and death, most if not all cancers should share a limited number of common molecular, biochemical and cellular traits that govern their behavior. This is insightful since cancers exhibit hundreds of genotypes and subtypes of tumors can be found in specific organs. In addition, different mutations can be found within the same tumor. In order to promote the development of cancer research into a more logical science, to provide more focused characterization of cancer and to manage this array of diseases, Hanahan and Weinberg [2000,2011] initially defined six major hallmarks of cancer that exhibit physiological anomalies that control cell homeostasis and proliferation. These include self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. They later included reprogramming of energy metabolism and evasion of immune detection as cancer hallmarks [2011]. Kroemer and Pouyssegur [2007] included evasion of immune surveillance as a seventh hallmark. Luo et al. [7] expanded these classic hallmarks to include stress phenotypes of tumorigenesis and defined a large class of non-oncogenes that are essential for cancer cell survival. Treatment strategies to develop targeted, mutation-specific small molecule drugs or monoclonal antibodies to treat melanomas is an outcome of an evolving understanding of the molecular mechanisms of melanoma in the perspective of these defined cancer hallmarks.

A number of new agents for advanced melanoma are being tested in clinical trials. Considering the hallmarks of cancer, the most recent new drugs for treating melanoma have found some successes in the inhibition of self sufficiency of growth signals, immune surveillance evasion, sustained angiogenesis and evasion of apoptosis. These agents are directed towards proteins that are involved in cell signaling pathways that are responsible for cell division and proliferation, immune responses, blood vessel formation and programmed cell death. Several considerations are appropriate when taking targeted drugs into account. First, targeted therapies are generally effective for individuals identified with a specific mutation, so patients can be screened before treatment to determine if they have the specific mutation for which the drug is designed to affect. This exemplifies the impact of personalized medicine on oncology and if successful will become a common procedure. This can be achieved by relatively simple and inexpensive procedures. The second concern is issues of heterogeneity within a patient's melanoma. While a specific mutation may be determined in a biopsy sample, the targeted mutation may not be present in all of the patient's melanoma cells. This provides a potential means for resistance and recurrences. A third issue is the continued "pressure" exerted or relieved by a targeted agent on cell signaling in the affected cancer cells. Such events modify signaling dynamics with responses

agents resulted in major toxicities. INFά is approved for use but is not included as a standard of care due to minimal impact on overall survival and significant toxicities [Amaravadi et al., 2007; Gogas et al., 2007; Tarhini et al., 2006]. Unfortunately, metastatic melanoma is one of the most resistant cancers to a wide range of treatment modalities including single-agents and combination chemotherapy, immunotherapy,