752 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 46, NO. 6, JUNE 1999

Electroporation Therapy: A New Approachfor the Treatment of Head and Neck CancerG. A. Hofmann,* Senior Member, IEEE, S. B. Dev, S. Dimmer, Member IEEE, and G. S. Nanda

Abstract Electroporation can deliver exogenous moleculeslike drugs and genes into cells by pulsed electric fields througha temporary increase in cell membrane permeability. This ef-fect is being used for the treatment of cancer by intratumoralinjection of low dosage of an otherwise marginally effectivechemotherapeutic drug, bleomycin. Application of a pulsed elec-tric field results in substantially higher uptake of the drugand enhanced killing of the cancer cells than is possible byconventional methods. The MedPulser, a new treatment systemfor local electroporation therapy (EPT) of head and neck tumorswas developed and is described in this paper. EPT with bleomycinhas been found to be very effective in killing cancer cells in vitro,in mouse tumor xenografts in vivo, and in tumors in humans. Tenhead and neck cancer patients with recurring or unresponsivetumors were enrolled in a Phase I/II clinical trial. Treatmentof the entire tumor mass in each of eight patients resulted infive complete responses confirmed by biopsy and MRI, and threepartial responses ( 50% shrinkage). Two additional patientswho received partial treatment of their tumor mass had localresponse where treated, but no overall lesion remission. Durationof the complete responses ranges from 210 months to date. Allpatients tolerated the treatment well with no significant local orsystemic adverse effects.

Index Terms Bleomycin, electrochemotherapy, electropora-tion therapy, head and neck cancer.

I. CANCERS OF THE HEAD AND NECK REGION

SQUAMOUS cell carcinoma of the head and neck is thesixth most frequently occurring cancer, with over 400 000cases projected annually worldwide [1]. In the United Statesthere are approximately 53 000 cases, resulting in 23 000deaths annually [2]. The larynx is the most common tumorsite, followed by the oral cavity, pharynx and salivary glands.Although head and neck cancers (HNCs) constitute a smallportion of all malignancies, pronounced functional deficits andcosmetic deformities associated with these diseases, or as aresult of surgery, heighten their relative morbidity.Interventional procedures currently performed for the treat-

ment of HNC include surgery, radiation therapy, and/or sys-temic chemotherapy. Surgery and radiation therapies are themajor curative modalities. Chemotherapy may be potentiallyeffective as an adjuvant to these modes and enhance curerates. However, the advanced stage and poor nutrition ofmost subjects referred for chemotherapy result in limited

Manuscript received January 12, 1998; revised November 19, 1998. Asteriskindicates corresponding author.*G. A. Hofmann is with Genetronics, Inc., 11199 Sorrento Valley Road,

San Diego, CA 92121 USA (e-mail: gah@cts.com).S. B. Dev, S. Dimmer, and G. S. Nanda are with Genetronics, Inc., San

Diego, CA 92121 USA.Publisher Item Identifier S 0018-9294(99)04329-3.

effectiveness. Use of chemotherapy when combined with shortelectrical pulses could become an effective new treatment forthe loco-regional management of HNC.

II. ELECTROPORATION AND ELECTROPORATION THERAPYElectroporation is a physical process of inducing transient

pores in cell membranes by the application of short electricpulses. Many biotechnological advances involving applicationof electrical pulses for the transformation and fusion of cellshave been reported since Sale and Hamilton first showed in1967 that electric fields could cause the death and lysis of cells[3]. Further studies revealed that the membrane permeabilitybarrier might be weakened temporarily and reversibly, bysubjecting cells to well controlled, high-intensity electric fieldpulses [4], [5]. This electrical pulse-induced permeabilizationof cellular membranes, generally referred to as electroporation,is ascribed to the formation of transient permeable structuresor micropores in the cell membrane [4][6]. One observableeffect of cell membrane electroporation is a significant increasein the exchange of molecules between the cell interior and ex-terior. The extent of cell membrane permeabilization achievedwith electroporation depends upon a variety of electrical,physical, chemical and biological parameters [4][11]. Amongthese factors are the strength and duration of the electricfield pulses, the size of the target cell, and the size andchemical properties of the entity crossing the membrane barrier[4][11]. The transient, reversible nature of this electricalpermeabilization of membranes provides a novel method ofgaining greater access to the cytosol.Electroporation has been employed since the early 1980s

as a research tool in molecular biology, wherein cell mem-branes are permeabilized to facilitate the cellular uptake ofvarious drugs, genetic material, proteins, other micro andmacromolecules and liposomes [10], [11]. This technology isnow being used for biomedical applications such as electropo-ration therapy, transdermal drug delivery, cellular drug carriersystems and electro gene therapy [12][15]. Electroporationtherapy involves combining chemotherapeutic drugs with elec-trical pulses for treatment of malignancies. The first report ofan in vivo application of electric field pulses in combinationwith chemotherapeutic drugs was published by Okino et al.,in 1987, which they termed electrical impulse chemotherapy[16]. Subsequent in vitro and in vivo studies of electroporationtherapy for treatment of various cancers in animal modelsshowed very promising results which paved the way for humanclinical studies [17][25].

00189294/99$10.00 1999 IEEE

HOFMANN et al.: NEW APPROACH FOR THE TREATMENT OF HNC 753

(a)

(b)

(c)Fig. 1. Schematic representation of the electroporation therapy system and its application. (a) The MedPulser consisting of the pulse generator and disposableneedle array electrode applicator. (b) Steps of treating a tumor by electroporation therapy. First, bleomycin is injected into the tumor. After 10 min, the needlearray is inserted into the tumor and the MedPulser is activated. The MedPulser generates electrical fields between opposing pairs of needles; betweeneach pair of needles two pulses of opposite polarity are applied. In a six-needle array, one treatment cycle consists of a total of six pulses between threepairs of needles in two polarities. The field switching is done automatically by the MedPulser and one treatment cycle is completed within approximately1.25 s. (c) Schematic representation of the effect of electroporation on the uptake of bleomycin into a cell. Without electroporation, the cell membrane islargely impermeable to the drug. Electroporation induces pores through which the drug enters the cell. After electroporation the cell membrane re-seals,keeping the drug inside the cell. Bleomycin causes DNA damage leading to cell death.

754 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 46, NO. 6, JUNE 1999

Fig. 2. Different needle array electrode applicators for HNC treatment with electroporation therapy.

There are many potential advantages of electroporationtherapy over conventional treatments. It can be performedat relatively low, nontoxic drug concentrations as a sin-gle treatment with minimal side effects; it may be suitablefor nonresectable tumors and can save functional tissues: Itmay be very suitable for cancers near critical organs wheresurgery and/or radiation therapy can lead to a permanentloss of function and it may be effective where conventionalchemotherapy, surgery, and radiation therapy have failed [23],[24]. Furthermore, this treatment modality has the potential ofovercoming the multidrug resistance problem and it has alsobeen reported to reduce the number of metastatic nodules whenthe primary tumor was treated [20].

III. THE ELECTROPORATION THERAPY SYSTEMThe EPT system is composed of a remote (foot pedal)

activated electric pulse generator, the MedPulser, whichdelivers square wave electrical pulses of fixed voltage andduration, and a sterile, disposable needle array electrode ap-plicator. Fig. 1(a)(c) shows the operating principles of theEPT system. After administration of the drug, the needlearray is inserted into the tumor and the MedPulser isactivated. The MedPulser automatically and sequentiallyactivates opposing needle electrode pairs so that the electricfield is rotated from pulse to pulse. In addition, pulses betweenneedle pairs are delivered in both polarities, i.e., the polarityof two opposing needles is changed between two subsequentpulses. The operation of the system is very simple; the mostappropriate electrode applicator for the specific tumor sizeand anatomical location to be treated needs to be selected

and connected to the instrument. A unique sensing elementis embedded in the electrical connector of the applicator toautomatically set the appropriate electrical parameters and thepulsing scheme. A variety of electrode applicator sizes andconfigurations are available to the clinician, varying in needlenumber, needle length, tip diameter, and tip angle (Fig. 2).The instrument incorporates features to assure that therapy

has been properly administered. These features include internalinstrument checks of the therapy electrical parameters beforea ready indication is given to the user. The remote acti-vation foot pedal controls the actual application of a therapysequence. The clinician can control the entire therapy sequencewith the foot pedal without compromising hand freedom.Audio and visual indications are given to the user to confirmsuccessful completion of therapy.The voltage and pulse duration set in the MedPulser for

each needle array applicator have been determined by takinginto consideration theoretical field calculations, experimenta-tion carried out in cell cultures, subcutaneous tumor modelsin animals, as well as human clinical studies.

IV. EFFICACY OF VARIOUS NEEDLE ARRAY CONFIGURATIONSFor successful electroporation therapy a minimum electric

field strength is required to be generated across the tumor in thepresence of a chemotherapeutic drug. It was shown [26] thatthe field strength necessary to achieve effective electroporationof tumor cells is in excess of 600 V/cm.Theoretical calculations of the electric field strength gener-

ated by needle arrays have been carried out using a softwarepackage (E3, Field Precision, Albuquerque, New Mexico). The

HOFMANN et al.: NEW APPROACH FOR THE TREATMENT OF HNC 755

Fig. 3. Simulated isofield contour plots generated by a six-needle array (1-cm diameter) when fields are generated between opposing pairs of needlesas follows: AB and ED; BC and FE; and CD and FA. The positions of the needle electrodes are indicated by open circles. Applied voltage: 1130 V.The dotted area is exposed to field strengths of at least 600 V/cm.

program calculates the electrostatic fields in three dimensionsby solving Poisson equations. The data generated from theseprograms are used to generate isofield contour plots in thefollowing way: The field was first calculated between one pairof opposing needles. The contour lines of constant electric fieldstrength were then plotted, and the plots of additional needlepairs superimposed after rotating the arrangement 60 forhexagonal arrays, and 90 for square arrays. Continuous linescorresponding to the 600 V/cm field strength and 100 V/cmfield strength were then outlined on this combined plot tocreate the contour diagrams shown in Figs. 3 and 4. It isapparent that the strength of the electric field decreases rapidlywith distance from the needle array, and reaches low valuesof 10100 V/cm at a distance roughly equal to the diameterof a needle array itself. It can be seen that the whole crosssection of the needle array is subjected to a minimum field of600 V/cm at the voltages selected for the MedPulser for theappropriate needle array.Experimental evidence indicates that, with the possible

exception of tissue granulosis/fibrosis of muscle, EPT innormal tissue such as skin, muscle, nerve and blood vessels

at or above 600 V/cm resulted in transient necrosis, inflam-mation and granulosis/fibrosis which were reversible within56 days with no functional sequelae (Heller, et al., personalcommunication).In successful clinical studies conducted at the Moffitt Cancer

Center, in Tampa, FL, the electric field strengths in the centerof the needle arrays, used at fixed voltages, were calculatedto range from 6301356 V/cm. As a result, the MedPulserSystem has been designed to assure that it generates centerfield strengths within the needle arrays of the various electrodeapplicators of no less than 780 V/cm, providing a safety marginabove the minimum effective field strength of 600 V/cm.

V. EPT SUITABLE DRUGBleomycin, the preferred EPT drug, is an antitumor agent

commonly used in combination with other antitumor drugs.It is a glycopeptide antibiotic of 1450 molecular weight,which is very water soluble. In the absence of electropo-ration, bleomycin penetrates the cell membrane with poorefficiency. Once inside the cell, the basic mechanism ofaction of bleomycin is scission of both single and double-

756 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 46, NO. 6, JUNE 1999

Fig. 4. Simulation of isofield contour plots generated by four modular four-needle array units (0.87-cm distance between needles). The electric pulse is appliedbetween opposite pairs of needles AB and CD, then switched to AC and BD for the first square. The sequence is then repeated for other square modules. Theapplied voltage is 1500 V. Positions of the needle electrodes are indicated by open circles. The field strength within the dotted area is at least 600 V/cm.

stranded DNA and cleavage of RNA [27]. It has also beenassociated with apoptosis and p53 induction [28]. Bleomycinis the only drug amongst the many drugs tested, which hasproduced excellent results when used in vitro, in vivo inanimal models, and in human pilot studies in conjunctionwith electroporation for treatment of various cancers [17][25].What makes bleomycin a unique EPT drug is not completelyunderstood. Some of the properties of bleomycin which mayrelate to its remarkable effectiveness are as follows: 1) It is alarge molecule compared to other typical antineoplastic agents(MW 1450). 2) It is a highly charged molecule. 3) It isvery hydrophilic, log ( partition coefficient inoctanol/water). 4) It is a strong chelating agent. 5) It generateshighly reactive free radicals. 6) It is known to stimulate releaseof cytokines (like tumor necrosis factors) from macrophages.7) It is known to cause cell membrane damage, and 8) Cellshave been shown to have receptors for bleomycin.In order to rule out the possibility that the structure of

bleomycin is changed by electroporation, the characteristicsof bleomycin solutions pulsed in vitro were studied usinghigh-pressure liquid chromatography (HPLC). Analysis of

bleomycin solutions by HPLC after electroporation, underconditions similar to those used for the electroporation therapy,did not show any change in the elution profiles for bleomycin.The results indicate that electric pulses of the strength used inEPT do not change the characteristics of the major bleomycincomponents.

VI. EPT STUDIESIn order to demonstrate the enhancing effect of electropo-

ration on the effectiveness of bleomycin, we studied the cyto-toxic effect of bleomycin on a cell line of human squamous cellcarcinoma of tongue (SCC-25) both alone and in combinationwith electric field pulses in vitro, using the Medpulser and asix-needle array applicator. The electrical parameters used forEPT of SCC-25 cells were those that show 80% survival ofcells. These parameters are purposefully selected to show thecombined effect of the drug and the pulse rather than that of thepulse alone. Electric fields of high intensity may be beneficialin killing the cells by lysis but may not be very practical in aclinical situation. The response of SCC-25 cells to treatmentwith various concentrations of bleomycin alone (D E ), or

HOFMANN et al.: NEW APPROACH FOR THE TREATMENT OF HNC 757

Fig. 5. Survival of SCC-25 cells after EPT. D = Drug, E = Electric field ( denote presence or absence, respectively). The SCC-25 cells were seeded ina 96-well microplate with various concentrations of bleomycin. The electrical pulses (559 V, 6 100 s at 4 Hz) were generated by the MedPulser anddelivered to the cells using a six-needle array applicator (0.5-cm diameter) inserted in each well. The cell survival was measured by an XTT cell proliferationassay 20 h after EPT. The assay is based on metabolic conversion of XTT, a tetrazolium dye, to formazan, which is measured spectrophotometrically at450 nm. The cell survival values are relative values calculated from the OD values of the sample, [ODsample

]; a control with 100% cell survival, [OD100

];and a control with 0% cell survival, [OD0

], using the formula: % cell survival = ([ODsample

] [OD0

])/([OD100

] [OD0

]) 100. Each data pointrepresents the mean standard error derived from four experiments, each with n = 6.

in combination with electrical pulses (D E ) is shown inFig. 5. It can be seen that at all concentrations investigated,the cytotoxicity of bleomycin is substantially greater whencombined with electrical pulses. Bleomycin is poorly perme-able to the cell membrane. Therefore, at lower concentrationsand relatively shorter time of treatment with SCC-25 cells, itseffect on their viability is not very pronounced. IC (con-centration inhibiting 50% of cell population) of bleomycin,when combined with electrical pulses is reduced by a factorof nearly 400 compared to bleomycin alone. The implication ofthis observation is that the concentration of bleomycin can bereduced by nearly a factor of 400 to achieve the same amountof tumor cell killing as without an electric field, provided thesein vitro results are valid also in vivo.In vivo, the cytotoxicity of bleomycin on human larynx

(HEp-2) epidermoid carcinoma, xenografted subcutaneouslyin nude mice, was significantly enhanced by combining itwith electrical pulses. The treatment consisted of intratu-moral injection of bleomycin followed by 6 100 s squarewave electrical pulses of 1130 V applied to a 1-cm-diametersix-needle array. Results showed complete tumor regression in83% of the treated mice on day 67 after treatment. Histopathol-ogy of samples from the treated sites of animals with 100%tumor regression showed complete absence of tumor cells [29].

VII. CLINICAL TREATMENT OF HEADAND NECK CANCER WITH EPT

The first clinical trial of EPT was conducted by Mir etal., on patients who had already undergone conventional

cancer treatment for head and neck squamous cell carcinomas[23]. The treatment protocol involved intravenous injection ofbleomycin (10 mg/m ) followed by pulsing the tumor withparallel plate electrodes. Of the 40 nodules treated, 23 showedcomplete regression, six partial regression and 11 no changes.EPT did not show any adverse effect. Since then, studies atthe Moffitt Cancer Center in Tampa, FL, sponsored by Gen-etronics, Inc., using intratumoral injection of bleomycin with avasoconstrictor, combined with the application of needle arraysfor pulsing, have resulted in improved outcomes [13], [24].In a Phase I/II clinical study sponsored by Genetronics,

Inc., and performed at Rush-Presbyterian-St. Lukes MedicalCenter in Chicago, ten patients with HNC (eight squamouscell carcinomas and two adenocarcinomas) were treated withelectroporation therapy with intratumoral administration ofbleomycin [30]. Baseline tumor characteristics and experimen-tal details for tumor treatment are summarized in Table I. Theinclusion criteria were as follows:1) HNC that is not amenable to conventional surgery,

radiation and/or chemotherapy treatment;2) Recurring HNC unresponsive to prior radiation and/or

chemotherapy;3) Stage IV HNC with positive surgical margins that would

be immeasurably mutilating and/or cause excessive mor-bidity (e.g., total glossectomy, orbital exoenteration,total laryngectomy, loss of facial structures such as nose,lips or jaws, and/or tumors causing stroke or blindnessif further resection was undertaken).

758 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 46, NO. 6, JUNE 1999

TABLE IEPT OF HUMAN HNC

Study Design StudyPopulationTumor

Histology(n)1

BleomycinDose & Route

ofAdministration

ElectroporationSettings and Device

UsedEfficacy Results(Tumor Response)

Study Design:Open-label,nonrandomized,single center

Age:Mean: 70.7Range: 37-95

Gender:Six male,Four female

Diagnosis:Head and neckcancerunamenable orunresponsive toconventionaltherapy

SCC - 8AD - 2

Dose: 1 U/cm3 oftumor volume

Route:Intratumor

Settings:Six pulsesField Strength:725-1100 V/cm99 s/pulse

Device:T-820 pulse generatorMedPulser

Six-needle arrayelectrodes

SCCCR: 4/8PR: 2/8NR: 2/8

ADCR: 1/2PR: 1/2NR: 0/2

TOTALCR: 5/10PR: 3/10NR: 2/10

number of tumors treated with bleomycin-EPTSCC squamous cell carcinomaAD adenocarcinomaCR complete responsePR partial responseNR no response

Bleomycin (4 U/ml) was administered intratumorally(1 U/cm tumor volume) followed by electroporation usingthe MedPulser system. In eight of the patients the entiretumor mass was electroporated; in two of the patients onlypart of the tumor was treated by electroporation.All patients tolerated the EPT treatment well with no

significant local or systemic adverse effects. Typical EPTtreatment reactions included necrosis, sloughing, eschar andinduration of the tumor suggestive of tumor response andhealing with minimal, transient reactions to the perilesionaltissue within the electroporation treatment field.The two patients who received partial treatment of their

tumor mass had local response at the EPT-treated site but nooverall lesion remission. Treatment of the entire tumor massin eight patients resulted in five biopsy and MRI confirmedcomplete responses and three partial ( 50% tumor shrinkage)responses. None of the five subjects who demonstrated acomplete response to treatment has shown evidence of tumorrecurrence. Duration of the complete response ranged from210 months to date (mean 6 mo).

VIII. SUMMARY AND OUTLOOKElectroporation therapy on a limited number of patients

has shown the potential of becoming an effective treatmentof carcinoma of head and neck with minimal side effects.Presently, an expanded multicenter FDA-approved Phase IIclinical trial for EPT of HNC is underway.One can reasonably expect that EPT might be useful in the

treatment of other cancer indications.

ACKNOWLEDGMENTThe Phase I/II clinical study, sponsored by Genetronics Inc.,

was performed at the Rush-Presbyterian-St. Lukes MedicalCenter, Chicago, IL, with W. R. Panje, MD, as the investigator.

The authors would like to thank Dr. D. Rabussay for hiscomments and critical reading of this paper.

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G. A. Hofmann (M79SM80) received thedoctorate degree in physics from the Max-Planck-Institute for Plasma Physics, Garching, Germany.He is Founder, Chairman, and Chief Scientific

Officer of Genetronics, Inc., San Diego, CA.and holds 49 patents and has several otherspending, including many related to electroporation,electrofusion, and drug/gene delivery systems andapplicators. His research interests are in the effectsof pulsed electromagnetic fields on biologicalsystems.

Dr. Hofmann was named Inventor of the Year by the San Diego IntellectualProperty Law Association for his patent on electroincorporation.

S. B. Dev received the doctorate degree in polymerphysics from Chelsea College, London, U.K.He is Principal Scientist, Genetronics, Inc., San

Diego, CA. Prior to joining Genetronics, Inc. in1988, he was a Visiting Scientist at MassachusettsInstitute of Technology, Cambridge, where he didresearch on conformation of proteins, nucleic acids,and polysaccharides by various spectroscopic tech-niques. Since 1992, he has been involved with theGenetronics electroporation-mediated drug deliveryand gene therapy program in the areas of cancer,

cardiology, and gene therapy.

S. Dimmer (S84M85) received the B.S. degreein electrical engineering from San Diego State Uni-versity, San Diego, CA, in 1985.He is Director of Engineering, Genetronics, Inc.,

San Diego, CA. Prior to joining Genetronics, Inc.,he developed medical instrumentation for severaldifferent biomedical companies. He is currently en-gaged in developing commercial medical devices forGenetronics electroporation mediated drug deliveryprograms.

G. S. Nanda received the B.Sc. degree in microbi-ology and the M.Sc. degree in biophysics from theUniversity of Bombay, Bombay, India, in 1987 and1989, respectively. He was awarded a fellowship bythe University Grants Commission of India to carryout his doctoral studies at Bhabha Atomic ResearchCentre, Bombay, India, for which he received thePh.D. degree in biophysics from the University ofBombay in 1994.He is Staff Scientist with Genetronics, Inc., San

Diego, CA, and is currently working on electropo-ration and gene therapy projects in the Drug Delivery Division. His researchinterests are in bioengineering of cells for biotechnological and medicalapplications.