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EJSO 40 (2014) 61e66 www.ejso.com

Electrochemotherapy as new standard of care treatment for cutaneous Kaposis

sarcoma
G. Di Monta a,*, C. Carac
o a, L. Benedetto a, S. La Padula a, U. Marone a, M.L. Tornesello b,
F.M. Buonaguro b, E. Simeone c, P.A. Ascierto c, N. Mozzillo a
a
Department of Surgery Melanoma, Soft Tissues, Head and Neck, Skin Cancers, Istituto Nazionale dei Tumori Fondazione Pascale Napoli 80131 Naples,
Italy
b
Molecular Biology and Viral Oncology, Istituto Nazionale dei Tumori Fondazione Pascale Napoli 80131 Naples, Italy
c
Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Istituto Nazionale dei Tumori Fondazione Pascale Napoli 80131 Naples, Italy

Accepted 1 September 2013

Available online 12 September 2013

Abstract
Background: Electrochemotherapy (ECT) is a novel modality for the treatment of skin nodules and cutaneous or subcutaneous tumors that
allows delivery of low and non-permeant drug into cells. The aim of this prospective single-center study was to evaluate ECT efficacy in the
local treatment of Classic Kaposis sarcoma (CKS) skin localization stage IeII sec. Brambilla et al.
Methods: Nineteen consecutive patients affected by classic KS were included in this study. All patients underwent blood sampling and con-
current incisional biopsy for histological diagnosis and Kaposis sarcoma related herpes virus 8 (HHV-8) molecular analysis. ECT treatment
of KS cutaneous lesions were performed according to the European Standard Operating Procedures of Electrochemotherapy (ESOPE).
The primary endpoint of the study was the evaluation of ECT efficacy in the treatment of KS skin nodules and the assessment of HHV-
8 viral load in the peripheral blood following the ECT therapy.
Results: Complete response (CR) was observed in 14 (73.6%) patients after first ECT session, while 3 (15.7%) and 2 (10.5%) out of 19
patients received a second and a third ECT treatment, respectively. Clinical response dragged out the whole follow-up period that ranged
between 6 and 31 months with a median of 16 months.
Conclusions: Clinical management of CKS skin localizations still represents a challenging task for surgeons and oncologists. Therefore,
according to this and other authors recent experiences, ECT is claimed to become the new standard of care as first line treatment strategy
for stage IeII CKS patients.
2013 Elsevier Ltd. All rights reserved.

Keywords: Kaposis sarcoma; Electrochemotherapy; HHV-8

Introduction (HHV-8), were consistently identified in AIDS-associated

Kaposis sarcoma (KS) was first described in 1872 by the
Hungarian dermatologist Moritz Kaposi.1 It has been
defined as a mesenchymal tumor involving blood and
lymphatic vessels.2 Kaposis sarcoma is characterized by
four different clinical and epidemiological settings3: 1)
epidemic or AIDS-associated KS, 2) endemic or African
KS, 3) iatrogenic post-transplantation KS, and 4) Mediterra-
nean or classic KS (CKS). In the late 1994, DNA sequences
of a new KS-associated virus, the human herpesvirus type 8
* Corresponding author. Via Massimo Stanzione, 18, 80129 Naples,
Italy. Tel.: 39 0815903460; fax: 39 0815903810.
E-mail address: gidimonta@libero.it (G. Di Monta).
KS.4 This virus has been demonstrated to be the etiological
agent of all clinic-epidemiological forms of KS.5e7 HHV-8,
however, is necessary but not sufficient to cause KS and
other factors such as immunosuppression have been shown
to play a major role in tumor development.8
Classic KS mostly affects elderly Eastern European Jew-
ish or Mediterranean men (male/female ratio: 10e15/1).
Lesions occur mostly on the skin and subcutaneous tissue
of the lower limbs and, more rarely, in internal organs.9
Classic KS is usually chronic, persisting over many years,
but is not life threatening. The risk factors for CKS include
advanced age, diabetes, and the use of corticosteroid medi-
cation.10,11 Age and corticosteroid medication are also
associated with CKS progression.12 If immunosenescence

0748-7983/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejso.2013.09.002
62 G. Di Monta et al. / EJSO 40 (2014) 61e66

cannot be modified, then an immunodeficiency (exogenous present in the current literature on this issue.22 The aim
or endogenous) that could potentially be corrected should of our study is to evaluate if ECT can be considered as a
always be sought. Clinically, KS is characterized by multi- first line therapy and a new standard of care in patients
ple firm, purple-blue or reddish-brown plaques and nodules, with stage IeII CKS.
often associated with venous stasis, lymphedema, and/or
hyperkeratosis.13 Classic KS is usually indolent and slowly Material and methods
progressive (mean survival, 10e15 years).14 However, the
clinical course of CKS may be characterized by lymph Patients and samples
node and visceral involvement and by local complications
that may seriously impair quality of life. These more This was a prospective, single-center study including
aggressive forms require systemic therapy with antiprolifer- nineteen consecutive patients with classic KS lesions of
ative drugs. Clinico-histologic findings are identical for all the inferior limbs (16 males and 3 females) that were
variants. Skin lesions often cause pain and disfigurement referred to the National Cancer Institute of Naples from
and may lead to functional disability. A recent CKS staging January 2010 to June 2012. Brambilla CKS staging system
system based on objective criteria has been proposed by based on objective criteria, was accounted to select pa-
Brambilla et al.15 Because classic KS frequently occurs in tients. Twelve patients had stage I characteristics while
elderly patients, a repeatable and safe therapeutic procedure the remaining 7 cases were classified as stage II (Table 1).
that acts quickly on multiple lesions represents a relevant Each patient was asked to give a written informed con-
opportunity. Classic KS often represent a therapeutic chal- sent to participate to the study and was invited to fill an
lenge, due to the number, localization and size of the le- epidemiologic questionnaire regarding lifestyle, risk factors
sions. For localized forms, the available options are and anamnestic data. Furthermore, demographic features
radiotherapy, surgical excision, laser, cryosurgery intrale- including origin, age at onset, gender of the patient, as
sional injections and topical treatments with cytotoxic well as clinical features such as localization of lesions,
drugs. Unfortunately, none of them has been proven to be treatment modalities, results and tumor recurrence at the
superior to the others.16 Although many options are avail- time of observation were also recorded. All patients under-
able, standard therapeutic guideline hasnt been stated for went concurrent incisional biopsy and blood sampling for
this condition. histological examination and HHV-8 DNA viral load deter-
Electrochemotherapy (ECT) is a recent therapeutic mination at the time of enrollment. A second blood sample
method used in primary and metastatic skin tumors. It is a was obtained at a six-month follow-up visit after the end of
safe procedure that can be considered for KS patients, espe- ECT treatment to monitor the HHV-8 viral load. Each cuta-
cially when multiple lesions are present. The rationale neous biopsy was divided in two sections, one processed
behind this technique is that electroporation, obtained by for pathological examination and the other was stored in
the application of electric fields, temporarily increases the RNAlater stabilizing solution (Ambion, Austin, TX) at
permeability of cell membrane by creating transient pores, 80  C. Ten ml of fresh blood was processed within one
thus allowing the direct diffusion of different molecules hour for PBMCs isolation by Ficoll density gradient
within cells.17 It combines the administration of highly cyto-
Table 1
toxic drugs (like bleomycin or cisplatin) followed by the ap-
Patients characteristics.
plications of high intensity electric pulses in tumor lesions
Patient no. Sex, age (years) Localization Response Stage
on the skin or subcutaneous tissue. At the appropriate pulse
parameters, pore formation on the cell membrane allows 01 F, 85 Right foot CR I
02 F, 74 Lower limb CR I
low permeant drugs like bleomycin or cisplatin to enter
03 F, 63 Lower limbs bilateral CR II
the cell and thus locally increase thereby their toxicity: up 04 M, 44 Lower limbs bilateral CR II
to 10,000 times for bleomycin and 80 times for cisplatin.18 05 M, 69 Foot CR I
In addition to drug-induced cell killing, electroporation is 06 M, 61 Foot CR I
responsible for changes in the tumor region. A vascular 07 M, 74 Lower limbs bilateral CR II
08 M, 72 Foot CR I
lock, consisting in a reflex constriction of vessels after elec-
09 M, 75 Foot CR I
tric pulse delivery, produces a temporary reduction in perfu- 10 M, 83 Foot CR I
sion of tumor tissue and an interstitial edema. This effect 11 M, 59 Foot CR I
appears to last longer in tumor tissue compared with normal 12 M, 84 Lower limbs CR II
tissue. For this reason, the cytotoxic drugs must be adminis- 13 M, 65 Left foot CR I
14 M, 58 Genitalia CR II
tered prior to electroporation. Furthermore, other vascular
15 M, 73 Lower limbs CR II
effects exerted by ECT include endothelial cell destruction 16 M, 68 Lower limbs CR I
and neovascular reorganization due to a local reduction in 17 M, 66 Foot CR I
angiogenic factors production.19e21 18 M, 70 Lower limbs CR II
The use of ECT in a KS patient was first reported by 19 M, 73 Lower limb CR I
Heller et al., in 1998 and to date only few reports are CR: complete response.
 G. Di Monta et al. / EJSO 40 (2014) 61e66
separation and was stored in RNAlater stabilizing solution
at 80  C. All cases included in the study presented histo-
pathologically confirmed KS lesions and were negative for
HIV-1/2 antibodies by macro enzyme immunoassay. All pa-
tients underwent tumor staging by lymph node and abdom-
inal ultrasound as well as chest X-ray.
Electrochemotherapy treatment regimen
ECT treatment of KS cutaneous lesions was performed ac-
cording to the European Standard Operating procedures of
Electrochemotherapy (ESOPE).23 The primary endpoint of
the study was to evaluate the efficacy of ECT in the treatment
of KS skin nodules. Treatment outcome was evaluated accord-
ing to the Response Evaluation Criteria in Solid Tumors (RE-
CIST-Guidelines).24 The term complete response means
clearance of KS lesions on later visits when compared with
the first lesions on admission. Partial response equals atleast
30 percent decrease in the sum of the longest diameter of target
lesions whereas stable disease involves less than 30 percent
decrease. The terms complete and partial response as well as
stable disease implicate the absence of new lesions or of pro-
gressive lesions. The electric pulse generator used in this study
was the CE certified medical device Cliniporator (IGEA
S.p.A., Carpi, Modena, Italy). The delivery devices were nee-
dle electrodes which were inserted at subcutaneous level
directly into deep tumor tissues and surrounding areas, so
that the entire tumor tissue could lie within the electric field,
or plate electrodes which were applied onto the skin surface
of superficial cutaneous lesions. Electric pulses were adminis-
tered in a time interval of 8e28 min after intravenous injection
of bleomycin at the dose of 15,000 IU/m2, in bolus, in a time
interval of about 40e45 s. Bleomycin exerts its cytotoxic ac-
tivity by inducing single- and double-stranded DNA breaks,
which leads to cell-cycle arrest, apoptosis, and mitotic cell
death. The main drug-related contraindications for ECT treat-
ment were: kidney failure (creatinine level <150 mmol/L);
known allergy to the drug; interstitial lung fibrosis (bleomy-
cin); cumulative bleomycin dose more than 400,000 UI/m2.
The procedure was performed either under loco-regional or
under general anesthesia. Local anesthesia was used for iso-
lated lesions; general or epidural anesthesia for nodules which
were too numerous or bulky or too painful to be treated under
local anesthesia. The treatment was repeated in patients who
presented multiple lesion, difficult to treat in a single session
or if a complete response was not achieved at a first ECTappli-
cation. Our intent to treat was curative (disappearance of the
treated nodules) for all patients.
Nucleic acids extraction and HHV-8 amplification by
real time PCR
Genomic DNA was extracted from tissue biopsies and
PBMCs according to published procedures.25 In particular,
all samples were digested by proteinase K treatment
(150 mg ml1 at 56  C for 2 h) in 100e500 ml of lysis buffer
63
(10 mM TriseHCl, pH 7.6, 5 mM EDTA, 150 mM NaCl,
1% SDS), followed by phenolechloroformeisoamyl
alcohol (25:24:1) extraction and ethanol precipitation in
0.3 M sodium acetate (pH 4.6). All DNA samples were sub-
jected to the amplification of HHV-8 ORF26 DNA se-
quences by real time polymerase chain reaction (PCR)
using primer sequences previously described.26 The PCR
reactions were performed in a total volume of 25 mL of
iQ SYBR Green Supermix, containing 50 mM KCl,
20 mM TriseHCL, pH 8.4, 0.2 mM of each dNTP and
25 units/mL iTaq DNA polymerase, 3 mM MgCl2, 10 nM
SYBR Green I (Bio-Rad Laboratories, Inc), 3 mmol/L of
each primer, and 100 nge500 ng of genomic DNA. All ex-
periments were performed on the CFX96 Real Time Sys-
tem (Bio-rad Laboratories, Inc). Dilution series (10 to 106
copies) of genomic DNA extracted from HHV-8-infected
BCBL-1 cell line were used to construct the standard curve.
Eight weeks after ECT a new blood sample was taken
for every patients to evaluate after treatment viral
parameters.
Results
This study included a total of 19 patients (3 females and
16 males) affected by classic KS, with median age at the
diagnosis of 70 years (range 44e85).
According to RECIST guidelines a response to the first
ECT treatment, scored at 4 weeks, was obtained in all
patients (Figs. 1 and 2). Complete response (CR) was
observed in 14 (73.6%) out of 19 patients after first ECT
session, while 3 (15.7%) and 2 (10.5%) patients received
respectively a second and a third ECT treatment. The
mean interval between two treatment was 145 days. Clin-
ical response dragged out the whole follow-up period that
ranged between 3 and 28 months with a median of 13
months. The clinical response to ECT was evaluated 4
weeks after treatment and monitored every 3 months. All
responsive patients showed a progressive improvement of
pretreatment symptoms as local pain or functional limita-
tion. All patients showed no residual disease after the last
ECT session.
Overall the treatment was well tolerated with a very low
complication rate. Pain and erythema to the treated and sur-
rounding area were among the most commonly reported
side effects. They were considered tolerable by most pa-
tients. The erythema usually disappeared in a few days
and a crust over the treated areas was generally present
up to 2 weeks post-treatment.
Treated lesions healed without scar, due to the fact that
the collagenic extracellular matrix and proteins are not de-
naturated by ECT, unlike other physical ablation technolo-
gies, thus allowing a faster healing of the wound.
HHV-8 DNA viral load was evaluated by Real-Time
PCR targeting the HHV-8 ORF26 in DNA samples
extracted from KS biopsies and PBMCs obtained from
nine patients. All nine tumor biopsies were positive for
64 G. Di Monta et al. / EJSO 40 (2014) 61e66

Figure 1. Kaposis sarcoma of the leg; a) pre-operative view b) four weeks after ECT treatment c) six months after ECT.

HHV-8 sequences with viral loads ranging from 2 to 50
copies per 104 cell. Eight out of nine PBMCs samples
were HHV-8 positive with viral loads ranging from 0.5 to
25 copies per 104 cells. Two patients out of three, moni-
tored over a six-month period, were still positive for
HHV-8 DNA sequences following ECT therapy.
Discussion
Classic KS skin localizations still represents a chal-
lenging task for surgeons. The appearance of widespread
cutaneous tumoral nodules is a distressing situation for
many KS patients. The presence of these lesions may un-
favorably impact with patients quality of life, in partic-
ular if ulceration or bleeding occurs. Extremely variable
clinical display of this pathology determined a lack of
consensus on standardized line of treatment. Brambilla
et al. had recently developed a CKS staging classification
based on objective criteria.15In our cases ECT with bleo-
mycin administration proved successful in the local con-
trol of KS skin nodules where other approaches, such as
surgery or radiotherapy, would have been hazardous due

Figure 2. Kaposis sarcoma of inferior limb; a) pre-operative view b) eight weeks after ECT treatment.
 G. Di Monta et al. / EJSO 40 (2014) 61e66
to the high risk of ulceration, bleeding, infection and de-
layed healing. The therapeutic response occurred in the
absence of any increased morbidity for the patients, in
tissue-sparing and low-risk conditions due to the minimal
doses of bleomycin, and absence of surgical wounds.
The HHV-8 viral loads of nine KS patients ranged be-
tween 2 and 50 copies per 104 cells before ECT treatment,
and in three patients evaluated six months following the
therapy the viral load did not change significantly. This is
not surprising as the complete clearance of HHV-8 from
circulating cells is strongly associated with immune recon-
stitution which can be partially achieved by the local treat-
ment in a short period of time.
Our study demonstrates for the first time that ECT may
be considered to be used as a first line therapy for patients
with CKS.
ECT has significantly higher effectiveness than chemo-
therapy alone (bleomycin or cisplatin) applied at the same
cumulative doses as in ECT.27 The main advantages of
ECT include: 1) high success rate in local tumor control
after a single session; 2) no damage to healthy peripheral
tissue (using low doses of chemotherapeutic agents ECT
is very specific for dividing tumor cells, sparing the sur-
rounding normal tissue); 3) no protein denaturation, so
that tumor antigens are not destroyed and may elicit an
immune response; 4) excellent safety profile (in clinical
use, no serious adverse events were reported in associa-
tion with ECT); 5) advantageous cost/benefit ratio: the
technology and the drugs used, in particular bleomycin,
do not require large investments; 6) improvement of pa-
tients quality of life. ECT is well accepted by the pa-
tients, (the most common side effects consist in
erythema, electrodes tattoo, erosion or ulceration with
scars healing, slight edema and pain, and are well
accepted by the patients). In conclusion ECT has a very
low morbidity rate. The procedure is safe, simple, eco-
nomic and high effective requiring a short learning phase.
It has been demonstrated to improve the quality of life in
patients with CKS, independently of life expectancy. Thus
we suggest to consider this option for skin limited CKS,
including stage I and stage II disease (slow evolution).
In our series the procedure alone was capable to provide
complete skin lesions healing. According to this and other
authors recent experiences, ECT is questioned to become
the new standard of care as first line treatment strategy
for CKS patients.
Financial support
None.
Conflict of interest statement
All authors disclose no financial and personal relation-
ships with other people or organizations that could inappro-
priately influence their work.
65
Acknowledgments
We thank Mario Malinconico, for technical assistance in
sample selection, retrieval and processing.
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