Elek Trochelektrochemotherpiae Mother Pia

Curative Electrochemotherapy in the Head and Neck Area
To Noah, Gabriella, Elias and Mikaela
rebro Studies in Medicine 117
FREDRIK LANDSTRM
Fredrik Landstrm, 2015

Title: Curative Electrochemotherapy in the Head and Neck Area.
Publisher: rebro University 2015
www.oru.se/publikationer-avhandlingar
Print: rebro University, Repro 02/2015
ISSN 1652-4063
ISBN 978-91-7529-061-4
Abstract
Fredrik Landstrm (2015): Curative Electrochemotherapy in the Head and
Neck Area. rebro Studies in Medicine 117.
Electrochemotherapy (ECT) is a local cancer treatment modality in
which the local intracellular accumulation of chemotherapeutic agents is
enhanced by a local electric field. The effect of ECT is caused by a direct
cytotoxic effect on the cancer cells themselves as well as effects of the
tumor vasculature. The most common agent used is bleomycin. Today,
clinical ECT is mostly used in palliative treatment of skin metastases.
In this thesis the long-term follow up after ECT with intratumoral bleomycin in 26 patients with T1 and T2 head and neck cancer and nonmelanoma skin cancer was investigated. The primary outcome was local
control and safety of treatment. Secondary outcome was survival and functional assessment. A possible selective effect in vitro of ECT on survival in
different human cell-types, normal and malignant, was also investigated.
The local control rate in the 19 head and neck cancer patients treated
with curative intent was 100% in the 60 month follow-up period. Six
patients were cured by ECT as a mono-modality treatment and six by
ECT and adjuvant radiotherapy. Seven patients died, three from intercurrent disease and four from region recurrence making the tumorspecific survival 75%. The safety and functional outcome was very good
in the fifteen patients treated with oral tongue cancer but poor in the
patients with tumors in the floor of mouth, bucca and tongue base.
Four of the six patients with non-melanoma skin cancer had a complete response 24 months after treatment with ECT alone. The treatment
was also organ and function sparing in three patients. One patient had a
persistent tumor and one patient had a recurrence 30 months after
treatment.
There was also evidence for cell-type selectivity of ECT with bleomycin on cell survival in vitro. The survival was significantly higher in fibroblasts compared to endothelial and squamous cell carcinoma cells.
ECT as a curative treatment merits further investigation.
Keywords: electrochemotherapy, curative, squamous cell carcinoma, basal
cell carcinoma, bleomycin, local control, functional outcome, quality of
life, cell-type, selectivity.
Fredrik Landstrm, School of Medicine,
rebro University, SE-701 82 rebro, Sweden,
fredrik.landstrom@regionorebrolan.se
Table of Contents
ABBREVIATIONS .................................................................9
ORIGINAL PAPERS ............................................................11
INTRODUCTION ...............................................................13
The cell membrane ............................................................... 13
The membrane potential....................................................... 16
Electroporation .................................................................... 17
ECT in vitro ......................................................................... 21
ECT in vivo .......................................................................... 24
Effects of ECT on muscle .................................................. 26
Effects of ECT on the skin ................................................ 27
Effects of ECT on the vascular system ............................... 27
Effects of ECT on the heart ............................................... 30
Effects of ECT on the nervous system ............................... 30
Effects of ECT on the immune system ............................... 30
THE TREATED TUMORS ..................................................32
Non-melanoma skin cancer in the head and neck area ......... 32
Head and neck squamous cell carcinoma.............................. 34
Tumor vascularization and cancer stem cells ........................ 37
CLINICAL ECT ...................................................................39
ECT in clinical practice ........................................................ 39
Clinical efficacy of ECT .................................................... 39
The ESOPE guidelines ....................................................... 39
ECT in non-melanoma skin cancer ................................... 40
ECT in head and neck cancer ............................................ 41
From treatment to healing................................................. 42
The EU-CCBE-2003 and EU-HNBE-2003 trials ................... 44
The MedPulser system .................................................... 45
THE PRESENT INVESTIGATION ......................................47
Aims .................................................................................... 47
The overall aims of this thesis ........................................... 47
Specific aims for each paper .............................................. 47
Materials .............................................................................. 48
Study group ...................................................................... 48
Cells ................................................................................. 50
Methods ............................................................................... 51
ECT protocol (Papers I, II, III and IV)............................... 51
Additional treatment (Papers I, II, III and IV) .................. 51
Assessment of local control (Papers I, II, III, IV)................ 52
Assessment of treatment safety (Papers I, II and III) .......... 52
Assessment of functional outcome (Paper I) ...................... 52
Assessment of functional outcome (Papers II and III) ........ 52
Assessment of Quality of life (Paper IV) ............................ 53
ECT protocol (Paper V) .................................................... 53
Assessment of cell survival (Paper V) ................................ 53
Statistical methods ............................................................ 54
Results ................................................................................. 55
Paper I .............................................................................. 55
Paper II ............................................................................ 56
Paper III ........................................................................... 58
Paper IV ........................................................................... 60
Paper V ............................................................................ 63
Discussion ............................................................................ 68
Methodological limitations and strengths ......................... 68
Papers I, II, III and IV...................................................... 68
Paper V ............................................................................ 69
Efficacy of ECT ................................................................ 69
Safety of ECT ................................................................... 71
Functional and quality of life outcome after ECT.............. 72
Selectivity of ECT ............................................................. 73
Costs ................................................................................ 74
Treatment recommendations............................................. 74
Future perspectives ........................................................... 76
Conclusions ......................................................................... 78
ACKNOWLEDGEMENTS .................................................. 79
REFERENCES ..................................................................... 82
APPENDIX 1
APPENDIX 2
Abbreviations
BCC
BLM
CAL-27
CER
CR
CRR
CRT
CSC
CSCC
CT
CUP
CV
Da
Vm
DWD
DWOD
ECOG
ECT
EGFR
EORTC
E
EP
EPT
ESOPE
FB
FOM
G
Gy
HNC
HNSCC
HPV
HUVEC
I
IGRT
IMRT
FREDRIK LANDSTRM
Basal Cell Carcinoma

Bleomycin
Squamous Cell
Cytotoxicity Enhancement Ratio
Complete Response
Complete Response Rate
Controlled Randomized Trial
Cancer Stem Cells
Cutaneous Squamous Cell Carcinoma
Computed Tomography
Cancer of Unknown Primary
Crystal Violet staining method
Dalton
Induced membrane potential
Dead With Disease
Dead Without evidence of Disease
Eastern Cooperative Oncology Group
Electrochemotherapy
Epidermal Growth Factor Receptor
European Organisation for Research and
Treatment of Cancer
Electric Field Strength
Electroporation
Electroporation Therapy
European Standard Operation Procedures
for Electrochemotherapy
Fibroblast
Floor of Mouth
Electric Conductance
Gray
Head and Neck Cancer
Head and Neck Squamous Cell Carcinoma
Human Papilloma Virus
Human Umbilical Vein Endothelial Cells
Electric Current
Image Guided RadioTherapy
Intensity Modulated RadioTherapy
IRE
IT
IU
IV
NED
NMSC
ORN
PBL
PDT
PSSHN
QLQ-H&N35
RR
RT
SC
SCC
SCC-4
SEM
TNM
U
Vrev
Virrev
Vm
VMAT
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FREDRIK LANDSTRM
Irreversible Electroporation
Intratumoral
International Units
Intravenous
No Evidence of Disease
Non Melanoma Skin Cancer
Osteoradionecrosis
Phospholipid Bilayer
Photodynamic Therapy
Performance Status Scale for Head and
Neck cancer
Quality of Life Questionnaire-Head and
Neck 35
Regional Recurrence
Radiotherapy
Stratum Corneum
Squamous Cell Carcinoma
Squamous Cell Carcinoma Cell Line
Standard Error of Mean
Tumor, Node, Metastasis classification of
malignant tumors
Units
Threshold for Reversible Electroporation
Threshold for Irreversible Electroporation
Resting Membrane Potential
VoluMetric Arc Therapy
Original papers
This thesis is based on the following papers, which are referred to in this
text by their roman numerals.
I. Electroporation therapy of skin cancer in the head and neck area.
Landstrm FJ, Nilsson CO, Crafoord S, Reizenstein JA, Adamsson GB,
Lfgren LA.
Dermatol Surg. 2010 Aug; 36(8): 1245-50.
II. Electroporation therapy for T1 and T2 oral tongue cancer.

Landstrm FJ, Nilsson CO, Reizenstein JA, Nordqvist K, Adamsson GB,
Lfgren AL.
Acta Otolaryngol. 2011 Jun; 131(6): 660-4.
III. Electrochemotherapy possible benefits and limitations to its use in

the head and neck region. Landstrm FJ, Nilsson CO, Reizenstein JA,
Adamsson GB, Lfgren AL, Mller C. Acta Otolaryngol. 2015; 135: 9095
IV. Long-term follow-up in patients treated with curative electrochemotherapy. Landstrm FJ, Reizenstein JA, Adamsson GB, Lfgren AL, von
Beckerath M, Mller C.
Submitted to Acta Otolaryngologica.
V. Electrochemotherapy evidence for cell-type selectivity in vitro.

Landstrm FJ, Ivarsson M, Koskela A, Magnuson A, von Beckerath M,
Mller C.
Submitted to Bioelectrochemistry.
FREDRIK LANDSTRM
11
Introduction
Electrochemotherapy (ECT) (1), also known as electroporation therapy
(EPT), is a local cancer treatment modality where the intracellular accumulation of a chemotherapeutic agent is enhanced in the presence of an
electrical field by the phenomenon known as electroporation (EP) (2).
My interest in ECT began in 2005 as a sub investigator in two ECT trials.
In this thesis the treatment outcome after ECT in twenty patients with
head and neck cancer and in six patients with non-melanoma skin cancer
will be reported as well as evidence of cell-type selectivity of ECT in vitro.
The cell membrane

A basic knowledge of the structure and function of the plasma cell membrane is essential to understand the theory behind ECT. The cell membrane surrounding all cells is only 5 nm thick but maintains the cell integrity and hence its survival (3).
The biochemical and biophysical properties of the membrane depends
on its molecular composition of which the three basic components are
phospholipids, cholesterol and proteins (Figure 1).
Figure 1. The cell membrane with its main components, phospholipid molecules,
proteins and cholesterol (4).
FREDRIK LANDSTRM
13
Examples of common phospholipids in human cell membranes are shown

in Figure 2; they all share the same basic structure; an aquaphilic head
and a lipophilic tail made up of fatty acids (5).
These ampiphilic chemical properties make the phospholipid molecules
spontaneously assemble into a bilayer when placed in water-based solutions, reseal the bilayer when it is torn and also facilitates the transport of
some molecules while restricting others. This permeability is determined
both by the size and the electrical properties of the molecules.
Nonpolar molecules like oxygen (32 Da) and carbon dioxide (42 Da)
and small polar molecules like water (18 Da) can diffuse freely across the
phospholipid bilayer (PBL) while the permeability of larger especially polar molecules is restricted (2). The PBL is highly impermeable to even
small ions (for instance Na+ and K+)(3).
The cell membrane is not a static structure; instead the PBL is in a fluid
state where the individual phospholipids can diffuse and rotate freely. This
fluidity is biologically important and determined especially by the chemical compositions (saturation, chemical bonds) of the fatty acids in the
phospholipid molecules (3, 5). Cholesterol helps stabilize the membranes
by restricting the movement of the fatty acids thus decreasing the deformability of the membrane (5).
Figure 2. Chemical structure of the common phospholipids phosphatidylserine,

phosphatidylethanolamine and phosphatidylcholine with lipophilic fatty acids
(tails) and hydrophilic groups (heads)(6).
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FREDRIK LANDSTRM
Membrane proteins can somewhat oversimplified be divided into three

categories; transport proteins, receptor proteins and glycoproteins (Fig. 1).
The receptor proteins are important in cell signalling and the glycoproteins
in recognition and adhesion but are, as far as presently known, of limited
interest in the understanding of ECT. The transport proteins, on the other
hand, are very important in the electrochemical processes and the
transport of cytotoxic agents fundamental to understanding ECT.
The transport proteins can be classified as being either channels or carriers. Channels are multipass transmembrane proteins that allow hydrophilic and charged ions and molecules to diffuse through the PBL driven
by their individual concentration gradients (Figure 3)(3). For example,
even though water can diffuse through the PBL most of its diffusion takes
place in special transport protein channels called aquaporins (3, 7).
Carriers, on the other hand, are membrane proteins that actively bind
to the molecule and transport it across the PBL, carriers can mediate this
transport without energy or it can actively transport molecules against the
chemical gradient, these transport proteins are often called pumps and are
energy dependent (Figure 3). The Na+/K+-pump, for instance, transports
three Na+ ions out of the cell for every two K+ ions transported in thus
maintaining the osmotic balance and cell volume (3).
Figure 3. The different mechanisms for ion and polar molecule transport across
the cell membrane, diffusion, facilitated diffusion via carrier proteins and energy
dependent transport (for example the Na/K pump) Modified from Alberts (3).
The cell membrane is asymmetric; the outside and the inside do not have
the same composition mirroring the different environments on the inside
FREDRIK LANDSTRM
15
and the outside of the cell. For example, the negatively charged phospholipid phosphatidylserine is restricted to the inner, cytoplasmic side of the
membrane in normal human cells but is seen in the outer membrane of
apoptotic cells serving as a signal to macrophages for phagocytosis(8). The
inside of the cell membrane is also in direct contact with the cytoskeleton
important in, for instance cell movement and cell division (3).
The membrane potential

Due to the semipermeable properties of the PBL and the actions of the
membrane transport proteins (channels and carriers) described earlier the
intracellular and the extracellular side of the membrane have different
chemical compositions. This also leads to a difference in electrical charge
over the membrane; the so-called membrane potential Vm.
The name is somewhat misleading since it is not a potential but the potential difference (or voltage) between the inside and the outside of the cell
membrane, however since the name is used so extensively in the literature
we use it in this text as well. The Vm is generated by both the active and
passive transport systems of the cell membrane.
K+ is the most important ion in this process although both Na+ and Clions participate. K+ is pumped into the cells in exchange for Na+ leading to
a K+ concentration gradient; the K+ ions can diffuse through specific protein channels (leak channels).
However, since there is an intracellular accumulation of large negatively
charged molecules unable to move across the PLM (the fixed anions) there
is an electrostatic force that balances the concentration gradient of K+, the
membrane potential is the potential difference across the membrane when
this electrochemical gradient is zero.
In normal cells the Vm is between -40 and -90 mV (3). The Vm and its
very important role in the physiology of the nervous, neuromuscular and
cardiovascular systems are well known. There is also a growing
knowledge about and interest in the biological significance of the Vm in
other areas, for example in cell division and differentiation (9, 10). Proliferating cells, both tumor and non-tumor, seems to have a lower Vm than
non-proliferating cells (Figure 4) (9).
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FREDRIK LANDSTRM
Figure 4. The membrane potential Vm in tumor and non-tumor cells. Note the
differences in Vm between dividing and non-dividing cell (9).
Electroporation
The cell membrane has two basic electrical properties; it is both an insulator by restricting the flow of charged molecules and a capacitor since it
due to its thinness allows electrostatic forces to act between opposite
charges on both sides on the membrane (11). In general, the induced
membrane potential Vm when an external electrical field E is applied can
be described by the equation: Vm = f E r cos , where f is a factor related
to the form of the cell, r is the cell radius and the angle relative the external electrical field E (Figure 5) (12). It causes hyperpolarization in the
pole facing the positive electrode and depolarization in the pole facing the
negative electrode.
FREDRIK LANDSTRM
17
Figure 5. The induced membrane potential Vm by an external electrical field E in

a cell with radius r and angle causes hyperpolarization in the anode pole and
depolarization in the cathode pole of the cell. Vm varies between a maximal value
for = 0 and 0 for = 90. The degree of permeabilization is bigger in the depolarized pole allowing for transport of larger molecules like DNA (13, 14).
The induced potential Vm is superimposed on the resting membrane potential Vm. When a certain threshold Vrev for Vm + Vm is reached the permeability and the conductance (permeability of charged molecules) of the
cell membrane suddenly increases (14). This phenomenon is called dielectric breakdown or more commonly electroporation and has been studied
for 40 years (15). The threshold Vrev is in the range of 200mV-1V (16-18).
Vrev is first reached in the membrane facing the anode since here both Vm
and Vm have the same vectorial direction. At the cathode side these directions are opposite requiring a higher Vm to reach Vrev (18).
The equation above also shows that a stronger electrical field E is needed to electroporate cells with a smaller radius. Most mammalian cells have
a radius between 5 and 15 m, this could be compared to an axon with a
typical diameter of 0.5 m requiring an electric field at least ten times
stronger to achieve EP.
The exact mechanism of EP is not known but is thought to involve rearrangements of the membrane phospholipid molecules leading, within na-
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FREDRIK LANDSTRM
noseconds, to formation of perforating hydrophilic pores filled with

water molecules (2, 11) (Figure 6).
Figure 6. Electroporation hypothesis. Changes in the PLM induced by an electrical

field leading to pore formation (19).
This leads to a drastic increase in the transmembrane transport of ions and

large polar molecules. The area of permeabilization has been shown to be
larger in the cell pole facing the anode but that the degree of permeabilization is greater in the pole facing the cathode leading to increased transport
of larger molecules, for instance DNA, in this area (Figure 5) (14, 18).
When the external field is removed and if the sum Vm + Vm is lower
than the threshold Virrev the cell membrane, within seconds to minutes
depending on the temperature, can recover by membrane resealing with
intact cell integrity resulting in so-called reversible EP. The cell survival in
reversible electroporation depends on the energy level of the cell, if the
energy level is too low the cell will not be able to regulate its ion homeostasis (especially Ca2+) leading to cell death by either apoptosis or necrosis
(20). If Virrev is reached no resealing is achieved leading to cell death, so
called irreversible EP (21). Different electrical parameters can be used to
achieve different biological effects leading to different applications of EP
such as gene transfer, Irreversible Electroporation (IRE) and ECT (22)
(Figure 7). For instance, pulsed electrical fields with specific lengths and
number of pulses instead of static fields can be used to combine efficient
pore formation (permeabilization) with a high degree of cell survival
(23). This is important in both gene transfer and ECT where cell death due
to electrocution is unwanted (Figures 7 and 8).
FREDRIK LANDSTRM
19
Figure 7. Summary of different EP applications in relation to electrical field

strength and duration (22).
On the other hand, in IRE high field strengths is combined with repetitive
pulses to achieve cell death (Figure 7). IRE is a new cancer treatment modality currently investigated for treatment of liver and pancreas cancer, it
will not be further discussed in this thesis (24).
Figure 8. The area of reversible EP with electrical parameters used for ECT (100
s, 1000 V/cm) and gene delivery (longer pulses, lower field strength). Below the
breakdown voltage there is no increased permeability. With higher fields or longer
pulses EP becomes irreversible leading to cell death (25).
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FREDRIK LANDSTRM
Also, recently the investigation of very short (nanosecond) pulsed electrical

fields (nsPEF) has shown that with sufficient field strengths apoptosis with
externalization of phosphatidylserine can be achieved (Figure 7) (8). Cell
death due to heating is probably not an important factor in EP with normal parameters, in mathematical models field strengths of 4kV/cm with
pulse duration of 100 s is required to increase the temperature from 37 to
42 C in vivo (22). This field strength is almost 4 times higher than the
usual strength used in ECT (1-1.2 kV/cm)
ECT in vitro
One of the applications of EP is to facilitate the transport of low-permeant
or non-permeant molecules across the cell membrane of which ECT, the
internalization of chemotherapeutic agents, is a special case. In ECT the
goal is to permeabilize all cancer cells in order to internalize the chemotherapeutic agents used without killing the cells in the EP process because
when Virrev is reached all exposed cells (cancer and normal) die without
selectivity.
Depending on the chemotherapeutic agent used reversible EP can allow
for a selective effect where relatively more cancer cells than normal cells
die. When considering chemotherapeutic agents suitable for ECT from
what we know about the permeability of the cell membrane we could
make the assumption that the potentiation of EP for large polar molecules
with intracellular targets would be greater than for smaller, non-polar
molecules. This is also the case; bleomycin (BLM) and cisplatin are the
two chemotherapeutic agents that are enhanced by EP the most (Figure
10) (23, 26). BLM was isolated from the bacteria Streptomyces verticillis
in 1966 and is a water-soluble glycopeptide with a molecular weight of
approximately 1500 Da (27) (Figure 9). It is currently used in, for instance, the treatment of squamous cell carcinoma (SCC), testicular cancer
and lymphomas. It does not pass the blood-brain barrier and is eliminated
by the kidneys. Its half-life is approximately 3 hours with intramuscular or
intravenous injection. Although the exact mechanism of BLM cytotoxicity
is not known DNA is the primary target. BLM causes cleavage in both
DNA strands leading to both single and double strand breaks. This process requires oxygen and metals, usually Fe2+ (27). There are probably
other mechanisms of BLM injury, for instance induction of reactive oxygen species (ROS) and lipid peroxidation (28). Cells in the G2 and M
phases are more sensitive to BLM cytotoxicity.
FREDRIK LANDSTRM
21
Figure 9. Bleomycin and cisplatin molecules. Note the difference in size between
the non-permeant bleomycin and the low-permeant cisplatin molecules.
BLM causes two distinct types of cell death depending on the number of
intracellular BLM molecules. With a few hundred to a few thousand internalized molecules the cells die of a slow process called mitotic cell death
(mitotic catastrophe) (Figure 10). With several thousand or more intracellular BLM molecules the cells die of a much faster (pseudo)apoptotic process (27, 29). If the cell is not killed by BLM its cell cycle can stay in arrest
in the G2 or M-phase (Figure 10).
Figure 10. The two types of BLM mediated cell death, the slow mitotic catastrophe and the fast (pseudo)apoptotic death. Cells that survive can stay in cell cycle
arrest (G2 or M-phase) (30)
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FREDRIK LANDSTRM
Due to the size and polarity of BLM molecules its diffusion across the
plasma membrane is so effectively restricted it can be classified as a nonpermeant molecule; instead it is internalized by endocytosis with the help
of a transport protein (27, 31). Even with this mechanism less than 0.1 %
of BLM in medium is transported into the cells (32). EP has been shown to
greatly enhance the effect of BLM in vitro by increasing its permeability in
several studies (23, 26, 33-35).
The Cytoxicity Enhancement Ratio for EP is defined by the following
formula: (The drug dose needed to kill 50% of cells without EP) / (The
dose needed to kill 50% of cells with EP). The CER for BLM in vitro varies between 300 and 5000 and seems to be cell-type dependent as shown
in Table 1 (23, 26, 33, 34).
Table 1. Cytotoxicity enhancement ratios in vitro of ECT with bleomycin in three
different cell lines. The difference in CER for the DC3-F could probably be explained by the different field strengths used (23, 26, 33, 34).
Cell line
Cell type
SCC-25
Human tongue SCC
DC3-F
Chinese hamster fibroblast
HMEC-1
Human endothelial cell
CER
400
300-700
5000
SCC, Squamous cell carcinoma
BLM can be inactivated by the intracellular enzyme bleomycin hydrolase;

the level of this enzyme is especially low in skin and lung tissue (28).
One of the assumptions of a selective effect of ECT with BLM relies on
the fact that highly differentiated cells, for instance muscle and nerve cells,
do not divide and the mitotic cell death seen with lower doses of BLM in
dividing cells does not lead to death in these non-dividing cells. However,
these cells can still be killed by the pseudoapoptotic process with higher
BLM doses described above leading to a loss of selectivity (36).
On the other hand, endothelial cells show a high degree of sensitivity to
BLM and this is thought to be one of the mechanisms in the often-lethal
pulmonary fibrosis seen with intravenous administration of BLM (37).
This effect is dose-dependent (> 400,000 IU) and seems to be age-related.
Cisplatin, the drug besides BLM most commonly used in ECT is a platinum-based chemotherapeutic drug that is polar but much smaller than
FREDRIK LANDSTRM
23
BLM and hence a low-permeant rather than non-permeant drug (Figure

9). It is, for example, used in combination with RT in treatment of head
and neck cancer. Not surprisingly, the potentiation of cisplatin by EP is
lower than for BLM with CER 2.3 in vitro (23). On the other hand, the
cytotoxic effects of the non-polar lipophilic drugs daunorubicin, doxorubicin, etoposide and paclitaxel are not enhanced by EP with CER = 1(23).
BLM was the only drug used in the ECT treatment of the patients in this
thesis as well as in the cell studies.
As seen before different electrical parameters can cause different effects
in the exposed cells. When using eight 100 s square wave pulses of an
electrical field with 1200 V/cm cell survival and cell permeabilization rates
of more than 90 % in vitro can be achieved (23, 38). These are the parameters most commonly used in clinical ECT.
ECT in vivo
The cellular environment in tissues is very different from the environment
in solutions. Both the distribution of the chemotherapeutic agents and the
electrical field that are fairly uniform in vitro are much more complex in
vivo. With ECT in vivo the drug can be administered either intratumorally
(IT) or intravenously (IV). With IV administration the drug has to be distributed in adequate concentrations throughout the tumor and the margins
before EP, which will be discussed later. The blood vessels also transport
the drug from the tumor area thereby limiting the time window for treatment.
The tumor and the surrounding normal tissues have different electrical
conductivity with well-vascularized tumors having a higher conductivity
than less vascularized normal tissues. A high conductance (G) increases the
current (I) flowing between the electrodes and reduces the electrical field
strength (E) in accordance with Ohms law (E = I/G) (39).
In order to electroporate all the cells within a volume all cells must be
exposed to a field high enough to reach the threshold level Vrev for EP
without reaching the threshold Virrev for irreversible EP. The electrode
design for in vivo EP has to take these requirements into consideration to
achieve effective treatment results. Plate electrodes adequate for in vitro
EP are not as usable in the treatment of solid tumors. This has led to development of needle electrodes capable of penetrating tumors and normal
tissue to achieve wide margins on all sides of the tumor. As discussed before, cells that are not electroporated have a very low intracellular BLM
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FREDRIK LANDSTRM
content therefore there is a risk of leaving viable cancer cells if the tumor
area is not covered by a sufficiently high electrical field (39, 40).
In Figure 11 a simulation of the electrical field distribution in two
planes with a two-electrode model is shown (39). Note that the field immediately outside the needles is lower than Vrev threshold thereby avoiding
collateral damage of normal tissue. Furthermore, the effective electrical
field at the depth of the electrodes is actually at a lower depth than the
electrode tips themselves (Figure 11). Therefore it is important to insert
the needles deep enough to ensure adequate EP of the deep border of the
tumor.
Figure 11. Simulation of electrical field distribution (E) in two planes with needle
electrodes with distance 3 mm and 300 V applied. The tumor area is the marked
by a circle. The field is highest around the needles. (Erev = reversible electroporation threshold, Eirrev = irreversible electroporation threshold) (39).
If the electrodes are placed in two rows the cells the cells are electroporated in only one direction whereas in a hexagon array the cells can be
electroporated sequentially from several directions if the field is applied in
opposite electrodes pairwise. The electric field distribution with the hexagon needle electrode array is shown in Figure 12 (41), this was the geometry used in the treatment of all the patients in this thesis. The electrode
FREDRIK LANDSTRM
25
polarity was sequentially changed counter-clockwise in a pairwise 2x2

array (Figure 12). This array has been shown to have a significantly better
effect than the 3x3 array on tumors in vivo (40).
If the electrode distance increases, the field distribution will become
more inhomogeneous leading to inefficient EP (39). With large tumors it is
therefore necessary to apply the electrode array in an overlapping sequence to ensure adequate EP in both the tumor tissue and the margin.
Figure 12. Simulation of the electrical field distribution with a hexagonal needle
electrode array with diameter 8.66 mm (d) and 2x2 polarity. The black ring marks
a 5 mm treatment area. The model is linear making it scalable to higher values of
U. This was the electrode geometry and polarity used in this thesis (41).
Effects of ECT on the muscle

The effect of ECT with BLM on normal tissues have been investigated for
muscle in vivo in rat hind limbs showing necrosis with higher doses of
BLM but with normal limb function (42). EP alone causes muscle contractions related to the field strength and the pulse repetition frequency (43).
Traditionally a pulse repetition of 1 Hz (1 pulse per second) was used.
Since the field strength cannot be lowered without compromising the efficiency of EP the only parameter that can be changed is the frequency. The
frequency of tetanic muscle contractions is 100 Hz and an increase of the
pulse frequency above this value results in just one contraction. It has been
showed in vivo that an efficient EP can be achieved with frequencies up to
5 kHz (43). This is used in modern clinical EP systems.
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FREDRIK LANDSTRM
Effects of ECT on the skin

The skin consists of three layers, the epidermis, the dermis and the subcutis. The outermost layer of epidermis, stratum corneum (SC), consists of a
layer of 15-20 dead cells provides the important barrier function of the
skin. The SC also has a much greater resistance to electrical currents than
the other parts of the skin (44). This, in accordance with Ohms law, generates much higher electrical field strengths in the SC than in the underlying layers. This could lead to insufficient EP of tumors penetrating several
skin layers. Needle electrodes have been shown to distribute the electrical
field more evenly than plate electrodes throughout the skin in an animal
model (45).
Effects of ECT on the vascular system

Both EP alone and ECT with BLM have well documented effects on the
blood flow in both normal and tumor tissue in vivo (46-48). EP alone
causes a 2-phased reduction in blood flow. Phase I is an immediate vasoconstriction of the afferent arterioles (Figure 13). Phase II is a reduction in
blood flow measurable within 1-2 hours after EP, then a slow increase in
the blood flow with a normalization within 24-48 hours (Figure 13)(46).
This is thought to be a combined effect of the vasoconstriction and swelling of the endothelial cells reducing the diameter of the blood vessels.
This reversible effect called the vascular lock mechanism leads to capturing of ECT drugs already present in the tumor but also restricts the
inflow of drugs given systemically. The critical time window for EP after
IV BLM administration seems to be between 8 and 28 minutes (49). The
optimal time frame of EP following IT drug injection is not known. For
the patients treated in this thesis the time from BLM injection to the start
of EP was 10 minutes. EP was completed within an additional 10-15
minutes in all patients. In ECT with BLM there is a dose-dependent decrease in blood flow resulting in a cessation of the blood flow within 1248 hours with high BLM doses (Figure 13) (47). This irreversible effect
called the vascular disrupting mechanism of ECT is probably related to
endothelial cell death. As seen before, BLM is highly cytotoxic to endothelial cells (37). ECT can enhance this cytotoxicity 5000-fold (33).
Also, because blood has high conductivity the vessel wall where endothelial cells are located is exposed to very high electrical field strengths
(50). The antivascular effect is probably of great clinical significance in the
overall effect of ECT. It is, for instance, used in treatment of haemorrhaging metastases (51). Since the cytotoxic effect of BLM is dependent on
FREDRIK LANDSTRM
27
oxygen, the antivascular mechanisms can also, theoretically, have negative

effects on the treatment. A summary of the antivascular mechanisms is
shown in Figure 14.
Figure 13. Blood flow in subcutaneous Sa-1 tumours in A/J mice measured with
laser Doppler flow meter in two different time frames following exposure to BLM,
EP alone and ECT with BLM(47, 48, 50).
28
FREDRIK LANDSTRM
Figure 14. Summary of the antivascular effects of EP and ECT. EP alone causes a
reversible decrease in the blood flow the so-called vascular lock effect while
ECT with BLM causes an irreversible decrease and cessation of the blood flow, the
vascular disrupting effect of ECT(50).
FREDRIK LANDSTRM
29
Effects of ECT on the heart

Although EP is thought to be an important mechanism in defibrillation of
arrhythmias (52) the effect of ECT on heart function has not been thoroughly investigated. In 14 patients treated with ECT ECGs recorded during treatment were analysed. Electroporation drugs (BLM and cisplatin)
were injected IT to avoid systemic effects. In six of the patents the treated
tumors were located in the thorax. Transient increases in the R-R interval
were seen but no manifest pathological changes (53).
In our own experience a case of repeated 10-20 second asystoles in a
patient (not in this thesis) treated with ECT in the proximity of the carotid
bifurcation was seen. The outcome in that case was good without clinical
evidence of myocardial (normal postoperative ECG) or cerebral damage.
Effects of ECT on the nervous system

The nervous system is designed to transmit electrical signals and ECT with
its strong electrical fields seems to be able to cause both structural and
functional damage to the nervous system. The evidence that electroconvulsive therapy in treatment of depression can cause cognitive side effects
seems to support this (54). However no such side effects have been reported with ECT.
In an IRE pig model (90 pulses, 70 s, 1.5 kV/cm) the sciatic nerve was
treated. No histological nerve damage could be detected 2 months after
this treatment (55).
In this thesis a case of an epileptic seizure six months after treatment of
a BCC in the skin of left temporal area is reported. EP has been proposed
as one mechanism in the delayed neurological damage seen in lightning
and electrical injury (56).
Effects of ECT on the immune system

In ECT the tumor tissue is not resected but instead left to deteriorate leading to an accumulation of cancer antigens that at least in theory could
stimulate an immune response. There is evidence in vivo suggesting a role
of the immune system in the efficacy of ECT.
A group of immunocompetent mice had a significantly better outcome
than an immunodeficient group in ECT treatment of sarcoma. Furthermore, administration of interleukin-2, an important regulator of leukocyte
function, increased the tumor response (57). Also, immunocompetent mice
inoculated with a colon cancer cell line treated with ECT showed significantly higher response rates than immunodeficient mice. The immuno30
FREDRIK LANDSTRM
competent mice also rejected re-inoculation of that specific cancer cell line
implying a development of an immune response (58). A case report of the
long-term complete response in a patient with advanced malignant melanoma seven years after ECT with BLM and systemic IL-2 administration
was published in 2006 (59).
FREDRIK LANDSTRM
31
The treated tumors

Non-melanoma skin cancer in the head and neck area
Non-melanoma skin cancer (NMSC) is a very heterogeneous cancer
group, from low aggressive basal cell carcinomas (BCC) to highly aggressive tumors with high metastatic potential (i.e. Merkel cell carcinoma).
Basal cell carcinoma BCC is the most common NMSC and the most
common cancer in Sweden with approximately 40 000 new cases per year
(60). The head and neck region is the site most commonly affected with
70-80% of all cases (61). Intermittent strong UV exposure is the major
risk factor in developing BCC and other risk factors include radiotherapy
(RT) and immunosuppression (transplant patients, HIV)(62).
BCC usually develops after the fourth decade but there are also hereditary conditions like Gorlins and Bazex-Dupr-Christols syndromes in
which the affected patients continuously develop new BCC tumors from
an early age. The primary defect in both sporadic and syndrome-related
BCC seems to be an up regulation in the membrane based hedgehog signalling pathway that is part of the control system of proliferation and
apoptosis(62).
There are four main clinical subtypes of BCC. Nodular (Glas type IA)
an superficial BCC (Glas IB) are low aggressive tumors while infiltrative
(Glas II) and morpheaform BCC (Glas III) are aggressive tumors usually in
the head and neck area with ill-defined tumor borders and infiltrative
growth in cartilage, muscle, nerve and bone (62). Metatypical BCC is an
infiltrative BCC showing squamous differentiation that have a greater
potential to metastasize, something thats usually very rare in BCC (62).
Surgery is the primary treatment of BCC with, for example, Mohs micrographic technique (63). Other treatment options include photodynamic
therapy and radiotherapy, especially in unresectable tumors. There is also
a hedgehog pathway suppressor, vismodegib (Erivedge), available for
advanced cases. The recurrence rate after surgical resection is between 1
and 10 % and is especially high in the mask areas of the face reflecting
the difficulty of getting adequate margins, especially in close proximity to
the eyes and the nose (Figure 15)(64).
32
FREDRIK LANDSTRM
Figure 15. There is a high risk of recurrence for NMSC in the mask areas of the
face.
Cutaneous squamous cell carcinoma (CSCC) is the third most common

cancer in Sweden with 5718 new cases in 2012 (65). The head and neck
area is most commonly affected with approximately two thirds of all cases
(62). CSCC usually appears in older age groups.
A high cumulative UV exposure is the main risk factor in the development of CSCC (62). Like in BCC immunosuppression is a risk factor that
not only increases the risk of developing CSCC but also seems to lead to
more aggressive tumors. Other risk factors include previous RT, chronic
inflammation and arsenic exposure. Patients with the hereditary conditions xeroderma pigmentosum and oculocutaneos albinism also have elevated risks of developing CSCC (62).
CSCC often develops in areas with actinic keratosis, skin lesions with
chronic UV damage, in a process called field cancerization. The carcinogenesis is thought to involve a UV induced mutation resulting in inactivation of the TP53 gene coding for the tumor suppressor protein p53 and an
increased expression of the epidermal growth factor receptor (EGFR)
promoting increased cell proliferation (66).
CSCC tumors with high-risk of recurrence and metastasis are; tumors
with earlier recurrence, diameter > 2 cm, thickness > 2mm, location in the
mask areas of the face (Figure 1), poorly differentiated histopathology
and perineural invasion(67, 68). The overall risk of metastasis, especially
to the regional lymph nodes, is approximately 4-5 %. However in the
FREDRIK LANDSTRM
33
high-risk tumors described above the local or/and regional recurrence rate
can be as high as 80 % in the first two years (68).
The overall prognosis of CSCC is good but decreases dramatically with
metastatic disease (34.4 % overall 5-year survival)(67). Because of the
high incidence of CSCC the total mortality is as high as for malignant
melanomas, oropharyngeal cancer and renal cancer (62). Surgery is the
first line treatment for primary CSCC resulting in cure rates of 95% (68).
Mohs surgery could offer an even better outcome, especially with high-risk
tumors (69). RT can be performed as an adjuvant treatment in high-risk
tumors or in patients with positive margins after surgery, for salvage or
palliation in recurrent disease or as a primary treatment for unresectable
tumors. However, in locally advanced CSCC the cure rate with RT alone
is only 58% (70).
There are promising results in treating lip SCC with brachytherapy (71).
Photodynamic therapy (PDT) is another non-surgical treatment option for
superficial lesions. Local treatment of actinic keratosis with 5-fluorouracil
and imiquimod (Aldara) as well as PDT has been shown to prevent field
cancerization (68, 72). In summary, although the overall prognosis for
NMSC in the head and neck area is good the risk of recurrence and treatment morbidity is strongly related to the tumor location (mask areas).
Since the incidence of NMSC is so high the total mortality and morbidity
rivals that of more aggressive cancer groups.
Head and neck squamous cell carcinoma

Head and neck cancer is a diverse group of tumours often having little
more in common than the region in which they develop. For instance,
salivary gland malignancies, malignant melanomas in the nasal cavity and
sarcomas in the head and neck all have different prognosis and treatment
strategies.
However, SCC is by far the most common histopathology in head and
neck cancer. Head and neck squamous cell carcinoma (HNSCC) is currently the sixth most common cancer type worldwide with approximately
650.000 new cases and 350 000 deaths annually (73). The sites most frequently affected are the larynx, the oropharynx and the oral cavity. The
2012 incidence of head and neck cancer in Sweden for the most common
locales is shown in Table 2 (65).
34
FREDRIK LANDSTRM
Table 2. Total incidence of head and neck cancer in the most common locales in
Sweden in 2012. (Cancer incidence in Sweden 2012, Socialstyrelsen 2014) (65)
Localization
Male
Female
Sum
Lip
95
68
163
Tongue
150
111
261
Floor of mouth
Mouth, unspecified location
35
24
59
93
99
192
Oropharynx
224
84
308
Larynx
138
37
175
Alcohol and tobacco consumption, especially smoking, have historically

been the main risk factors in developing HNSCC. Today, however, there
is a worldwide increase in human papilloma virus (HPV)-induced HNSCC
in the oropharynx, as well as a decrease in smoking habits especially in
industrialized countries (74, 75). The role of HPV in HNSCC of other
locations than the oropharynx remains unclear. As with CSCC the carcinogenesis often involves mutations in the TP53 gene leading to a decrease
in p53 activity and overexpression of the EGFR-receptor in these tumours
leading to loss of control of cell proliferation. The carcinogenesis in HPVinduced cancer is different from alcohol and tobacco-induced cancer; it
involves inactivation of the tumor suppressor genes TP53 and the retinoblastoma suppressor gene by viral oncoproteins rather than mutations
of the genes themselves (76). The concept of field cancerization described
earlier with CSCC is equally applicable in the development of HNSCC, if
not more so. It was named by Danely P. Slaughter to describe multiple
primary carcinomas arising in the oral cavity from areas with histologically altered mucosa (77).
There is a growing knowledge about the genetic alterations and mutations in these precancerous lesions and their role in the development of
multiple primary malignancies (78). There is also a growing knowledge
about cancer stem cells and their role in local and regional recurrence (79).
The most important prognostic factor in HNSCC is regional recurrence;
the spread of SCC is usually lymphogenic with neck node involvement.
Risk factors for regional metastasis in primary tongue cancer is, for inFREDRIK LANDSTRM
35
stance, a higher T- stage, tumor infiltration depth > 5 mm, poorly differentiated histology and perineural invasion (80, 81).
Although the last decades have seen an increased knowledge about the
molecular biology of HNSCC, surgery and RT still remains the primary
treatment modalities. The surgical techniques have evolved with introduction of, for example, transoral laser surgery and robotic surgery increasing
the tumor access with minimally invasive approaches. The postoperative
morbidity has decreased with the introduction of free flap reconstruction
(76).
New RT techniques like intensity modulated RT (IMRT), volumetric
modulated arc therapy (VMAT), image guided RT IGRT and the development of brachytherapy have found applications in the treatment of
HNSCC and combinations of RT and platinum-based chemotherapy have
been beneficial in the treatment of advanced tumors and for organ preservation. There is also the emerging field of targeted therapy with antibodies
like cetuximab targeting the EGFR receptor.
The progress in imaging techniques has been substantial with the introduction of, for instance, FDG-PET, diffusion-weighed MRI and officebased ultrasound. There is also an ongoing vaccination program for HPV
expected to decrease the incidence of HPV-induced HNSCC. All this
should have led to a significant increase in the long-term survival and increased quality of life of the patients with HNSCC.
Although there have been a trend of significantly increased survival in
patients with tonsil and base of tongue cancer in the last 50 years in Sweden the survival in patients with tongue cancer has improved only modestly (82). Furthermore, patients with oral cavity cancer treated with a combination of surgery and RT show a significant decrease in quality of life
measurements even five years after treatment (83). There is also the increased risk of developing second primary tumors in previously treated
areas, especially in the oral cavity (84).
The TNM tumor classification system is helpful in predicting the prognosis and useful in treatment decisions. However, in HNSCC there are
other important factors such as smoking and HPV status that also determines the outcome and this may lead to more differentiated treatment
strategies. Patients with recurrent HNSCC after multimodality treatment
remain a group with a very bleak prognosis; there is a need for more
treatment strategies for both salvage and palliation.
36
FREDRIK LANDSTRM
Tumor vascularization and cancer stem cells

A steady supply of nutrients and oxygen are required for cell survival and
function. In normal tissues angiogenesis, the development of new blood
vessels from existing ones, are controlled by a balance of inhibiting and
inducing molecules. Angiogenesis is very important in both primary tumors and metastases; the delicate balance between induction and inhibition seen in normal tissues is lost. The tumor angiogenesis also seems to
depend on other cell types, fibroblasts, keratinocytes and monocytes that
secrete vascular inducing molecules (85).
This leads to a circulation in solid tumors that is very different from
normal tissues in both structure and function. The tumor blood vessels are
often irregularly shaped and chaotically arranged leading to poor perfusion and oxygenation. The perimeter of the tumor often has a better perfusion than the interior. This is a challenge in treatment with RT where
adequate oxygenation is required and in systemic chemotherapy where
inadequate circulation leads to sub lethal doses. It could also be a cause of
failure in ECT since oxygen is needed for BLM cytotoxicity.
The perimeter of the tumor is also where the cancer stem cells (CSC) are
located (Figure 16)(79).
Figure 16. Cancer stem cells are often found in the perivascular niche in the
periphery of the tumor where they interact with adjacent normal cells, especially
endothelial cells (86).
FREDRIK LANDSTRM
37
These slow-growing cells are less sensitive to RT and chemotherapy than

normal cancer cells and are thought to be important in local, regional
and distant recurrences sometimes even after considerable time. Hypoxia,
in addition to reducing the effects of RT and chemotherapy, also appears
to promote these cells (87). CSC seems to be dependent on the adjacent
stromal cells, especially endothelial cells, for its function and survival.
Selective ablation of endothelial cells has been shown to reduce the number of CSCs in vivo (88). Since ECT with BLM seems to have a very
strong anti-endothelial effect a hypothesis of a combined effect of ECT on
CSC both directly and indirectly via endothelial cell death could be
formulated.
38
FREDRIK LANDSTRM
Clinical ECT
ECT in clinical practice
Today, ECT is probably mostly used for palliative treatment of cutaneous
metastases. There are, currently, two systems approved for use in Europe,
the Cliniporator (IGEA, Carpi, Italy) and the Sennex (BionMed, Saarbrcken, Germany) systems. Both offer a variety of electrode applicators
for skin treatment but at the present time no flexible applicators are available.
Clinical efficacy of ECT

The efficacy of ECT in the clinical setting has been shown in several small
controlled trials (some randomized) where tumors (often in the same patient) were treated with either ECT or drug/EP alone (89-92). Several case
studies have shown the efficacy of ECT for cutaneous metastases of different histopathology (HNSCC, malignant melanoma, breast cancer, adenocarcinoma, Kaposis sarcoma) (49, 93, 94). Due to its antivascular effect it
has also been successfully used in palliation of haemorrhaging metastases
of malignant melanoma (51, 95).
In 2013, Mali et al published a meta-analysis of 44 case series, including papers I and II in this thesis, showing that the efficacy was significantly
higher with IT than IV drug administration (96-98). There was, however,
no statistically significant difference between IT BLM and cisplatin administration on the response rate. Kaposis sarcoma and BCC showed the
highest response rate, followed by adenocarcinomas, melanomas and SCC
(96).
Another meta-analysis of 9 case series, including paper I in this thesis,
was published by Mali et al in 2013 (99). There was a significantly lower
response rate in skin tumors (both primary and metastases) larger than 3 cm.
The ESOPE guidelines

In 2006 based on the multicentre ESOPE (European Standard Operation
Procedures for Electrochemotherapy) study the ESOPE guidelines were
published, providing an algorithm for treatment of multiple cutaneous and
subcutaneous metastatic nodules (100). In the ESOPE study standard operating procedures for treatment was used and the outcome showed a 73.7
% complete response rate (CRR) in 171 cutaneous and subcutaneous me-
FREDRIK LANDSTRM
39
tastases of different histopathology in 41 patients after a 5 month median

follow-up of period (101).
The ESOPE guidelines provide treatment stratification based on the
clinical presentation to electrode selection, drug selection and dose (IV
cisplatin or IV/IT BLM) and anaesthesia (local, regional IV or general).
Compared to the BLM dose used in this thesis the IT dose used in the
ESOPE protocol is significantly lower for the same tumor volumes. This
difference is caused by both different equations for the treatment volume
and on different BLM doses for different tumor volumes in the ESOPE
protocol (1000 IU/cm3 for tumor volumes < 0.5 cm3, 500 IU/ cm3 for volumes between 0.5 and 1.0 cm3 and 250 IU/cm3 for volumes > 1.0 cm3)
(100). In this thesis the same dose (1000 IU/cm3) was used for all tumor
volumes. The tumor volume equation used in the ESOPE protocol is
V=ab2/6 (V=treatment volume, a=the longest tumor diameter,
b=perpendicular diameter). With IV BLM administration the recommended dose in the ESOPE protocol is 15 000 IU/m2 surface area.
ECT in non-melanoma skin cancer

Although the skin is easily accessible for ECT relatively few studies of
treatment of primary skin cancer have been published. In Table 3 four
studies with more than two patients and a median follow-up of at least 5
months are summarized (102-105). BCC was the most common tumor
type. However, in one study CSCC and Bowens disease were also treated.
The complete response rate (CRR) varied between 78 and 100 %. In three
studies IT BLM was used. Because of differences in calculating the treatment volume the doses used were lower than in the patients treated in this
thesis. In the study by Salwa et al three elderly patients with peri-ocular
BCC was treated with complete responses in a follow-up period between 5
and 8 months (104). ECT with intravenous BLM has been tried successfully in three patients with Gorlins-Goltz syndrome showing a CR in 86/99
tumors (106). It has also been tried successfully in treatment of Kaposis
sarcoma and recurrent Merkel cell carcinomas (93, 107, 108).
40
FREDRIK LANDSTRM
Table 3. Studies of ECT treatment in primary NMSC. In the first study there were
54 tumors treated, in the other studies there were only one tumor per patient (102105).
Study
Glass, 1996
Rodgriguez-C.,
2001
Gargiulo, 2010
Salwa, 2014
Pats
Histology
Treatment
Follow-up
(months)
CRR
(%)
20
BCC
IT BLM
18
98.1
BCC
IT BLM
8.6
77.8
15
BCC, SCC,
BD
IV BLM
13
80
BCC
IT BLM
100
BCC, Basal Cell Cacinoma; BD, Bowens Disease; BLM, Bleomycin; CRR, Complete Respoense Rate; SCC, SquamousCell Carcinoma
ECT in head and neck cancer

As mentioned before, there are at present no commercially available flexible applicators thereby limiting the use of ECT in the treatment of head
and neck cancer. With bendable electrode applicators like the ones used in
this thesis access to tumors in the whole oral cavity and the oropharynx
can be achieved.
In the first clinical ECT trial published in 1993 by Belehradek et al was
40 cutaneous HNSCC metastases in eight patients was treated with ECT
using IV BLM. A CR was achieved in 57% of the tumors with a 36-day
(12-250 days) mean follow-up period (89).
As with NMSC there have been few studies investigating the potentially
curative aspects of ECT in head and neck cancer. In Table 4, three studies
are summarized. In 1998, Panje et al treated 10 patients with either recurrent or primary T1-T4 head and neck cancer RT (109). There was a CR in
5/10 patients with a mean follow-up time of 4 months. These patients
were then followed and 4 additional patients were treated. The long-term
FREDRIK LANDSTRM
41
follow-up (median 31.5 months) was reported in 2001 with CR in 6/14

patients (110). One case of osteomyelitis and one pharyngocutaneous
fistula was reported. Panje et al reported two cases of ECT of sinonasal
cancer where one patient had a CR 20 months after treatment (111). In
2003 Burian et al reported treatment of 12 patients with T1-T2 oral cavity
and oropharyngeal cancer (112). The treatment area was resected after
four weeks with viable cancer in 10/12 patients. In 2014 Campana et al
reported the retrospective outcome in 12 patients with cancer in the oral
cavity and oropharynx. The CRR was 58.3% and the reported side effects
were mycositis in 8/12 patients and ulcerations in 7/12 patients (113).
Table 4. Studies of ECT treatment of primary and recurrent head and neck cancer
(110, 112, 113).
Study
Pats
Histology
Treatment Follow-up
(months)
CRR
(%)
Allegretti, 2001
14
HNSCC, AC,
ACC
IT BLM
31.5
42.9
Burian, 2003
12
HNSCC
IT BLM
83.3
Campana, 2014
12
HNSCC
IT/IVBLM
14
58.3
AC, Adenocarcinoma; ACC, Adenoidcystic Cancer; HNSCC, Head and neck Squamous
Cell Carcinoma)
From treatment to healing

After ECT with IT BLM the treated tissues go through three distinct phases with different lengths depending on the tissue treated.
1. A phase of swelling that starts after treatment and continue for
days. This phase is more evident in the oral cavity than in the skin.
2. A phase of demarcation and necrosis. In the treated area a distinct
border between viable hyper-vascularized tissue and a central necrosis develops. If the necrosis is not resected it will be discharged
42
FREDRIK LANDSTRM
within a few weeks. In the skin an ulceration develop followed by a

crust formation covering the treatment area for several weeks to
months (Pictures 1A and 1B).
3. A phase of healing with minimal scarring which lasts a few weeks
in the oral cavity and a few months in the skin (Pictures 1C and
1D).
1A
1B
1C
1D
Figure 17. Necrosis, four weeks after ECT in a patient with ECT for a T2-tongue
cancer (A). Ulceration, ten weeks after ECT in a patient with a recurrent cutaneous SCC in the cheek (B). Healing, six weeks after ECT (same patient as in 1A)
(C). Healing, two years after ECT (same patient as in 1B) (D) .
FREDRIK LANDSTRM
43
The EU-CCBE-2003 and EU-HNBE-2003 trials

In 2003 Genetronics, a subsidiary of Inovio Biomedical Corporation (San
Diego, CA, USA), started two clinical phase II multicentre trials of ECT in
Europe with the MedPulser system; the EU-CCBE-2003 trial (ClinicalTrials.gov Identifier: NCT00198276) for treatment of cutaneous and subcutaneous cancer and the EU-HNBE-2003 trial (NCT00198263) for
treatment of T1 and T2 head and neck cancer. The Centre for Head and
Neck Oncology in rebro was the only centre in Sweden participating in
these studies. Since these were phase II trials there were no control groups
and no randomization for comparison to established treatments.
We received approvals from the Regional Board for Ethical Evaluation
(decisions 2004: M-296 and 2005:005) and the Medical Products Agency,
both in Uppsala, Sweden for enrolment of 10 patients to each trial. In
March of 2005 we received approval to enrol 20 additional patients to the
EU-HNBE-2003 trial. The primary and secondary outcome measures,
planned enrolment, our enrolment and time frame of both studies are
shown in Table 5. Beside the inclusion and exclusion criteria, Inovio had
no influence over the patient selection; the company also had no part in
the treatment of the patients or in the data collection process.
In July of 2007 Inovio stopped the enrolment in both studies due to an
increased mortality in the ECT-arm in two other studies comparing ECT
to surgery in treatment of primary and recurrent cancer in the oral cavity
and the palatine tonsils (HNBE-03-01, NCT00198315) and in the base of
tongue, lateral pharynx wall, hypopharynx and larynx (HNBE-03-02,
NCT00198328). To our knowledge, no publication of these studies has
been published. We applied for and were granted full permission by Inovio
to publish our results without any interference from the company in the
follow-up or in writing and submission of the manuscripts.
However, we did not have any access to the data from other participating centres but there have, to our knowledge, not been any other publication publications from these aborted studies. We continued to follow all
patients for 24 months and the head and neck patients from our county
for 60 months; patients referred to us for treatment from other counties
were referred back and followed at the referring departments after 24
month. All the follow-up data in all patients with head and neck cancer
treated at our centre is reported to our local database.
Papers I, II, III and IV of this thesis is based on the two and five year
follow-up data from the 26 patients treated in the framework of these
trials.
44
FREDRIK LANDSTRM
Table 5. Outcome, enrolment and original time frame of the EU-CCBE-2003 and
EU-HNBE-2003 ECT trials.
EU-CCBE-2003
EU-HNBE-2003
Primary outcome
Local tumor recurrence in Local tumor recurrence

6 months after ECT
in 8 months after ECT
Secondary outcomes
Pharmacoeconomic factors
MedPulser system performance
Adverse events in 6
months after ECT
Enrolment
95 patients
Pharmacoeconomic
factors
MedPulser system performance
Adverse events in 4
months after ECT
Evaluation of organ
function and QoL 4,8
and 12 months after
ECT
88 patients
Start
January 2004
February 2004
Completion
September 2008
September 2008
The MedPulser system

The MedPulser system was used in the treatment of all patients in this
thesis. It was an EP system intended for clinical use designed and manufactured by Genetronics. It consisted of two main components; the MedPulser Instrument and the MedPulser Applicator (Figure 18). The instrument
produced a cycle of six 100 s square-wave pulses with the field strength
1100 V/cm. The applicators were hexagonal array needle electrodes with a
diameter of 0.5 cm or 1 cm and with needle lengths adjustable to 3 cm
(Figure 19). The needle angle relative the handle was adjustable between
0 and 90. All patients in this thesis were treated with this system. The
development of the MedPulser system has since been discontinued and it
has never been commercially available.
FREDRIK LANDSTRM
45
Figure 18. The Medpulser instrument with applicator attached.
Figure 19. The Medpulser applicator.
46
FREDRIK LANDSTRM
The present investigation

Aims
The overall aims of this thesis
To investigate if ECT is an efficacious and safe curative treatment
in head and neck cancer and non-melanoma skin cancer in the
head and neck area.
To assess the functional and quality of life outcome in patients
with head and neck cancer treated with ECT.
To investigate if ECT has selective effects in vitro on survival in different cell-types.
Specific aims for each paper

I
Primary outcome: The two-year local tumor control rate and treatment
safety in 6 patients with NMSC that treated with ECT with IT administered BLM.
Secondary outcome: The two-year survival and functional outcome after
ECT.
II
safety in 15 patients with oral tongue cancer that were treated with ECT
with IT administered BLM.
Secondary outcome: The two-year survival rate and functional outcome
after treatment with ECT.
III
safety in 5 patients with cancer in the oral cavity and the oropharynx that
were treated with ECT with IT administered BLM.
Secondary outcome: The two-year survival rate and functional outcome
after treatment with ECT.
FREDRIK LANDSTRM
47
IV
Primary outcome: The five-year local tumor control rate and treatment
safety in nineteen patients with head and neck cancer that were treated
with ECT with a curative intent. Quality of life (QoL) outcome assessed at
baseline and after twelve months with pairwise comparison of subgroups
stratified for adjuvant RT, tumor location and smoking status.
Secondary outcome: The five-year survival rate after treatment with ECT.
V
Assessment of cell survival in vitro after ECT with BLM in human tongue
cancer cells, fibroblasts and endothelial cells in order to investigate possible cell-type survival selectivity. Under the null hypothesis the mean survival was the same in all cell-types.
Materials
Study group
Paper I
Six patients (3 men, 3 women; mean age 76.5) who were diagnosed with
non-melanoma skin cancer in the head and neck area were enrolled in the
study. The inclusion and exclusion criteria are presented in Table 6. There
were three BCC and three SCC tumors. Three of the tumors were recurrences previously treated with surgery. CT scans were performed for correct TNM classification, except in one patient with a low-aggressive BCC
of the temporal region. Histological type, localization, and T classification
are shown in Table 7. Four of the tumors could be described as complicated by proximity to the buccal and zygomatic branches of the facial nerve
and the parotid duct (patient 2); proximity to the floor of the external
meatus of the auditory canal, with risk of extensive spread (Patient 3);
growth involving the medial rectus muscle of the eye (Patient 5); or proximity to the orbit (Patient 6). All patients gave their written informed consent for treatment with ECT after receiving detailed information about the
treatment alternatives, ECT or surgery. The treatment intention was curative in all patients.
48
FREDRIK LANDSTRM
Table 6. Inclusion and exclusion criteria for the patients treated with ECT in Papers I, II, III and IV.
Inclusion criteria
Exclusion criteria
Biopsy verified CSCC, BCC, melanoma, > 50% suspected encasement of major
Merkel cell carcinoma or cutaneous
blood vessel
lymphoma (Paper I)
Biopsy verified cancer in the oral cavity, Tumor bone invasion
pharynx, larynx or salivary glands (Papers II, III, IV)
Tumors with volumes requiring BLM
dose < 80 U
Complete tumor and margin accessibility with BLM and electrodes
18 years or older
Contraceptive methods for men and
women with childbearing potential
Baseline performance status: ECOG 02*
Life expectancy of at least 6 months
Patients must sign a written Informed
Consent
Hypersensitivity to BLM
Lifetime BLM dose > 400 U
Patients unsuitable for general anesthesia
Emphysema/pulmonary fibrosis
Patients with pacemakers that cannot
be turned off
History of uncontrolled cardiac arrhythmia
Pregnancy or nursing
*Grade 0: Fully active, able to carry on all pre-disease performance without restriction.
Grade 1: Restricted in physically strenuous activity but ambulatory and able to
carry out work of a light or sedentary nature, e.g., light housework, office work.
Grade 2: Ambulatory and capable of all self-care, but unable to carry out any
work activities. Up and about more than 50% of waking hours.
Papers II, IV
Fifteen patients (8 males, 7 females; mean age 54.3 years) diagnosed with
T1 or T2 cancers of the oral tongue were enrolled in the study. The inclusion and exclusion criteria are presented in Table 6. The tumors were
SCCs in 14 patients and an adenocarcinoma in 1 patient (Table 8). To
determine the correct TNM classification a CT scan of the neck and
measurement and digital palpation of the tongue tumor under anaesthesia
were performed for all patients. All patients gave their written informed
FREDRIK LANDSTRM
49
consent for treatment with ECT after detailed information about the
treatment alternatives, ECT or laser resection of the tumor, the usual
treatment in our department. The treatment intention was curative in all
patients.
Papers III, IV
Five patients (all male; mean age 63.8 years) with cancer in the head and
neck region were enrolled in the study (Table 9). The inclusion and exclusion criteria are presented in Table 6. Four patients had primary T2 SCC
in the oral cavity or the oropharynx and one patient had a metastasis of
renal clear cell carcinoma in the right masseter muscle. A CT-scan of the
neck and thorax and digital palpation with measurement of the tumor,
when needed under general anaesthesia, was performed on all patients to
determine the correct TNM classification. All patients gave their written
informed consent for treatment with ECT after detailed information about
the treatment alternatives, ECT and the standard treatment options in our
department (Table 9). In one patient with generalized metastatic disease
(Patient 1) the treatment intention was palliative. In the other four patients
the treatment intention was curative.
Cells
Paper V
Four different human cell-types were chosen for the investigation, endothelial cells, fibroblasts (FB), and two different HNSCC cell lines. Endothelial cells were selected to investigate a possible endothelial basis for the
anti-vascular effects of ECT seen in vivo (46, 47) and fibroblasts because
of their important role in wound healing after treatment. HNSCC was
chosen because it is the most common type of head and neck cancer.
Human umbilical vein endothelial cells (HUVEC) were prepared from
umbilical cords of healthy infants as previously described (114). Normal
human fibroblasts (FB) were isolated by explanting pieces of dermis obtained from elective abdominal or chest surgery. The tissue was removed
using standard surgical procedures. Two human tongue squamous cell
carcinoma cell lines CAL 27 (CRL-2095) (115, 116) and SCC-4 (CRL1624) (117, 118) were obtained from American Type Culture Collection
(Manassas, VA).
50
FREDRIK LANDSTRM
Methods
ECT protocol (Papers I, II, III and IV)
All patients were treated under general anaesthesia with muscle relaxation,
using mivacurium chloride to reduce muscle contractions. The tumor was
measured, and the treatment volume was calculated with the formula:
0.5(a + 1)(b + 1)2, where a = longest tumor diameter and b = maximum
perpendicular diameter.
The tumor, including a 1.0-cm margin of tissue with a normal macroscopic appearance, was then infiltrated as uniformly as possible with bleomycin sulfate (Baxter, Halle, Germany) (4,000 IU/mL) using a fanning
technique. The dose was 1,000 IU per cm3 tumor volume (0.25 mL/cm3).
Ten minutes after the injection, electroporation was performed using a
0.5- or 1.0-cm hexagonal array needle applicator (Genetronics). The applicator was connected to the Medpulser electroporation instrument (Genetronics), which delivered a pulsed electrical field (6 x 100 s square-wave
pulses, 1100 V/cm).
The tumor was treated in overlapping sequences beginning at the margin and ending in the centre of the tumor. The two patients in whom the
eyes were involved were treated in cooperation with an ophthalmic surgeon. Except for control biopsies 8 weeks after treatment and removal of
obviously necrotic tissue at the revisits in some of the patients treated in
the oral cavity, no tissue was resected. Instead, we left the treated area
intact to follow the secondary healing process.
Additional treatment (Papers I, II, III and IV)

RT after ECT was performed using the same criteria as after surgery based
on TNM- stage and estimated depth of tumour infiltration 5 mm. Neck
dissections was performed concurrently with ECT in the patients with
neck node disease at the time inclusion and subsequently in patients that
developed regional recurrences. Two patients, one with a persistent tumor
(pat 3, paper I) and one with a recurrent tumor (patient 5, paper I) after
ECT underwent salvage surgery and RT. No primary reconstruction was
performed but reconstructive surgery was performed subsequently in two
patients that developed osteoradionecrosis (ORN) (patients 2 and 4, paper
III) and in one patient that developed a fistula (patient 3, paper III).
FREDRIK LANDSTRM
51
Assessment of local control (Papers I, II, III, IV)

Biopsies of the treatment area were carried out in all patients 8 weeks after
ECT. To ensure that the biopsies were representative they were performed
in general anaesthesia after careful palpation and inspection of the treatment area in all patients with head and neck cancer and in three of the
patients with NMSC. In the three remaining patients with NMSC the biopsies were performed in local anaesthesia. The number of biopsies, in
each patient, varied between one and five. The patients were then followed
clinically every third to fourth month using inspection and palpation.
The patients with NMSC that did not develop recurrences were not followed beyond two years. The patients with head and neck cancer living in
our county (rebro ln) were followed for five years. Patients referred to
us from other counties were referred back for follow-up after two years. If
no recurrence had occurred in five years the patients were declared cancer
free. The follow-up data was collected from our local register at the Head
and Neck Oncology Center in rebro.
Assessment of treatment safety (Papers I, II and III)

All treatment malfunctions of the Medpulser system was recorded as well
as all serious adverse events (SAE), defined as treatment-related events
resulting in death, which was life threatening, required inpatient hospitalization or resulted in persistent or significant disability/incapacity.
Assessment of functional outcome (Paper I)

The patient treated for a tumor in the vicinity of the facial nerve was examined clinically every third to fourth month after ECT including tests of
all four facial nerve branches. The two patients that were treated for periocular tumors were assesses by an ophthalmic surgeon after treatment.
Assessment of functional outcome (Papers II and III)

Assessment of eating in public, understandability of speech and normalcy
of diet were carried out before and 12 months after treatment using the
validated Performance Status Scale for Head and Neck Cancer (PSS-HN
scale) (Appendix I)(119). In paper II a voice recording of the patients reading a standard text was carried out between 12 and 15 months after
treatment. The voice recordings were assessed by a phoniatrician who
were not involved in the treatment.
52
FREDRIK LANDSTRM
Assessment of Quality of life (Paper IV)

The validated European Organisation for Research and Treatment of
Cancer Head & Neck 35 (EORTC H&N 35) questionnaire was used for
QoL assessment at baseline and 12 months after ECT(Appendix II)(120,
121). The questionnaire is specific for patients with head and neck cancer
and consists of seven multiple-question subscales for assessment of pain,
swallowing, senses (taste/smell), speech, social eating, social contact, and
sexuality. It also consists of eleven single-item scales assessing problems
associated with teeth, mouth-opening, xerostomia, sticky saliva, coughing,
feeling ill, need for painkillers, need for nutritional supplements, need for
feeding tube, weight loss and weight gain. The smoking status at the time
of treatment was collected from the patients journals.
ECT protocol (Paper V)

Fifteen mg (equalling 15 000 IU) of bleomycin (Baxter, Halle, Germany)
was dissolved in 1.5 ml sterile 0.9% sodium chloride to a stock solution of
10 mg/ml.
For each cell type, 100.000 well-suspended cells in 200 L of serum-free
cell culture medium were added to electroporation cuvettes with a 2 mm
electrode gap (Bio-Rad, Hercules, CA, USA). BLM was then added to final
concentrations of 1, 10, 100 and 1000 g/ml.
Electroporation was performed using the Gene Pulser Xcell (BioRad, Hercules, CA, USA). 8 square-wave pulses, each with duration of 0.1
ms and a 0.1 s interval between pulses were used, corresponding to clinical
ECT parameters (51). The electrical field strengths were 500, 1000, 1200
and 1500 V/cm. 1.8 ml of cell culture medium was then added to the cuvette. After mixing, the electroporated cells were seeded into a black 96well plate in quadruplicates.
To separate the effect of electroporation itself from the effect of ECT
we also exposed each cell type to EP only. As controls, quadruplicates of
each cell-type were added to the cuvette and mixed without BLM and then
seeded in a 96-well plate without being electroporated. To account for the
background fluorescence (blank) we also added wells with only cell culture medium.
Assessment of cell survival (Paper V)

The survival of all cell types was assessed 24 (day 1), 48 (day 2), 72 (day
3) and 96 hours (day 4) after EP/ECT using both the protease-based Mul-
FREDRIK LANDSTRM
53
tiTox-Fluor cytotoxicity assessment assay (Promega, Madison, WI,

USA) and a crystal violet assay (Sigma-Aldrich Sweden AB, Stockholm,
Sweden). All fluorometric readings were done using the FLUOstar Optima microplate reader (BMG Labtech, Ortenberg, Germany).
Statistical methods
Paper IV
The EORTC H&N35 QoL questionnaire produces both ordinal and nominal (dichotomous) raw data that were standardized to scores between 0
and 100 by linear transformations according to the EORTC manual where
a higher score indicates worse symptoms (122). The Wilcoxon signed rank
test was used for paired ordinal data (pain, swallowing, senses, speech,
social eating, social contact, sexuality, teeth, mouth-opening, dry mouth,
sticky saliva, coughing and feeling ill) and McNemars test for the paired
nominal data (need for painkillers, nutritional supplements, feeding tube,
weight loss and weight gain). For subgroup comparisons the Mann Whitney U-test was used for the ordinal data and Fishers exact test for nominal
data. The = 0.05 level was chosen for statistical significance for all the
conducted tests. SPSS software (Armonk, NY, USA) was used for all statistical tests.
Paper V
To accept or reject the null hypotheses we were only concerned with the
survival after 96 hours. We used the data obtained from the MultitoxFluor assay because of its very good linear relationship between the fluorescence and the number of cells alive. The fluorescence values of the
blanks were subtracted from the data. In order to compare the between
group survival independently from cell-type specific factors, for example
different proliferation rates, the survival data was then normalized (% of
control group from the same cell-type) prior to statistical analysis. The
data were tested for normality and equal variance using the Shapiro-Wilk
and Levernes tests respectively. Welchs F-test was used to establish if
there were any statistically significant within-group differences. In those
cases we performed the Games-Howell post-hoc test for between-group
comparisons. = 0.01 was chosen as the level of statistical significance.
SPSS (Armonk, NY, USA) software was used in all statistical tests. To
measure the effect size of the statistically significant survival differences
54
FREDRIK LANDSTRM
Cohens d formula with pairwise pooled standard deviations to account

for unequal variances in the different cell groups was used.
Results
Paper I
Primary outcome: The outcome is summarized in Table 7. There was a
persistent tumor in the external meatus after two ECT-treatments requiring extensive salvage surgery and radiotherapy (Patient 3). There were no
other local recurrences recorded in the two-year follow-up period and no
other patient received adjuvant treatment. The median follow-up time was
24 months. The patient with a metatypical BCC (Patient 5) had a local
recurrence 2.5 years after ECT treated with salvage surgery (exenteration
of the orbit) and RT.
There was one SAE not reported in the original paper (97); the patient
with a BCC in the temporal area (Patient 1) had an episode of epileptic
seizure 6 months after treatment. EEG and a CT-scan were normal and
the patient did not have any additional seizures in the follow-up period.
There was one adverse event, a treatment-related perforation of the nasal
septum cartilage. There were no recorded malfunctions of the Medpulser
system.
Secondary outcome: All patients were alive and tumour-free two years
after treatment (Table 7). In the patient with a recurring SCC tumor in the
cheek, no sign of facial nerve paralysis or damage to the parotid duct was
recorded, even though these structures were located in the ECT treatment
area. The two patients with tumors in the medial canthus experienced a
period of greater tear fluid but no visual impairment. Within 2 months,
the function of the eyelids and the tear apparatus was normal in both
patients.
FREDRIK LANDSTRM
55
Table 7. Demographics, tumor characteristics, additional treatment and outcome

after ECT in six patients with NMSC.
Pat Sex Age
Tumor
type
Tumor
size
Localization Recurrence Additional

treatment
temporal
region
65
BCC Glas
25x25x5
IA
81
SCC
28x35x15 cheek
SCC
external
20x15x10
meatus
10x10x5
Follow- Tumor
up
status
(months)
None
24
NED
None
24
NED
Surgery
+RT
PT
70
66
SCC
None
24
NED
None
24
NED
medial
canthus
None
24
NED
vestibule
82
medial
Metatypic
15x15x10
canthus
BCC
95
BCC Glas
18x12x5
II
BCC, Basal Cell Carcinoma; NED, No Evidence of Disease; PT, Persistant Tumor,
RT, Radiotherapy, SCC, Squamous Cell Carcinoma.
Pat 3 was treated with ECT two times.
Paper II
Primary outcome: The outcome is summarized in Table 8. The local tumor control rate in the two-year follow-up period was 100% based on 12
surviving patients. All biopsies two months after ECT were negative for
cancer and there were no clinical signs of recurrence subsequently. Ten
patients received adjuvant RT and five patients were treated with ECT
alone. The median follow-up time was 24 months.
There were no recorded malfunctions of the Medpulser system. There
was one recorded serious adverse event; the first patient treated was readmitted because of treatment-related pain.
Secondary outcome: The overall survival in the two-year follow-up period
was 80 % (12/15) and the disease specific survival was 85.7 % (12/14)
(Table 8). Two patients died from regional recurrences (RR) and one patient died from a gastrointestinal bleeding, all without signs of local recurrence. The functional outcome was very good (Table 9). The only notable
differences were that after treatment two patients needed liquid assistance
when eating a normal diet and one patient that could eat all meats before
56
FREDRIK LANDSTRM
treatment could only eat soft foods afterwards (Table 9). All three had
been treated with adjuvant RT.
The five patients treated with ECT alone had the highest scores in all
three parameters (eating in public, understandability of speech and normalcy of diet) before as well as after treatment (Table 9). In the voice recordings all 12 surviving patients had normal speech intelligibility, although five had minor articulation indistinctiveness but with no impact on
the ability to produce any speech sound. The overall communication ability was normal for all patients.
Table 8. Demographics, tumor characteristics, treatment date, additional treatment and clinical outcome after ECT in 15 patients with oral tongue cancer.
Sex Age
M
M
F
F
F
F
M
M
M
F
F
M
M
F
M
Type
Tumor
size(mm)
Treatment Neck
Radiodate
dissection therapy
30 T2N0M0 SCC
45 T2N0M0 SCC
74 T1N0M0 SCC
65 T2N0M0 SCC
71 T2N0M0 SCC
61 T1N0M0 SCC
60 T2N0M0 SCC
71 T2N0M0 SCC
20 T2N2aM0 SCC
47 T1N2bM0 SCC
63 T2N0M0 SCC
45 T2N0M0 AC
64 T2N0M0 SCC
53 T2N0M0 SCC
46 T2NOMO SCC
25x25x5
25x2x10
17x17x5
22x14x3
35x3x15
20x18x3
40x20x20
35x30x20
25x25x15
18x15x5
30x25x10
22x18x5
27x20x3
30x19x12
22x17x1
02-feb-05
07-apr-05
14-apr-05
06-dec-05
01-dec-05
19-jan-06
24-jan-06
04-apr-06
18-apr-06
15-jun-06
20-jul-06
14-nov-06
27-dec-06
01-mar-07
22-mar-07
TNM
No
Ipsilateral
Bilateral
No
Ipsilateral
No
Ipsilateral
No
Ipsilateral
Ipsilateral
No
No
No
No
No
T+N
T+N
T+N(ipsilat)
No
T+N
No
T+N
T+N
T+N
N
T+N
No
No
T+N
No
Last
follow-up
(month)
Tumor
status
tongue
Tumor
status
overall
24
24
24
24
8
24
24
8
24
1
24
24
24
24
24
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
NED
RR
RR
NED
DWD
NED
NED
DWOD
NED
DWD
NED
NED
NED
NED
NED
SCC= Squamous cell carcinoma, AC=Adenocarcinoma T=Tongue, N=Neck,

NED = No evidence of disease,
RR = Regional recurrence DWD = Dead with disease, DWOD = Dead without disease
FREDRIK LANDSTRM
57
Table 9. PSS-HN assessments before and 12 months after ECT in patients with
oral tongue cancer.
Pat No
Eating in public
Understandability of speech
Normalcy of diet
Before
100
After
100
Before
100
After
100
Before
100
After
100
2
3
100
75
100
75
100
100
100
100
100
80
100
50
4
6
100
100
100
100
100
100
100
100
100
100
100
100
7
9
50
100
75
100
100
100
75
100
50
100
90
90
11
12
100
100
100
100
100
100
100
100
100
100
90
100
13
14
100
100
100
100
100
100
100
100
100
100
100
100
15
100
100
100
100
100
100
PSS-HN= Performance Status Scale for Head and Neck cancer patients
Eating in public: 100=no restriction, 75=no restriction of place but restriction of
diet in public, 50=eats only at selected places with selected people. Understandability of speech: 100=always understandable, 75=occasional repetition necessary.
Normalcy of diet: 100=no restriction,
90=full diet(liquid assist), 80=all meat, 50=soft, chewable foods
Paper III
Primary outcome: The outcome is summarized in Table 10. The local
tumor control rate in the two-year follow-up period was 100% based on 4
surviving patients. All biopsies at 2 months after ECT were negative for
cancer and there were no clinical signs of recurrence subsequently. Three
of the five patients received adjuvant RT and one patient were treated
with ECT alone. One patient had been treated with palliative RT before
ECT (Patient 1). The median follow-up time was 24 months.
There was one recorded malfunction of the Medpulser system; the
treatment, however, could be completed successfully. There were four
SAEs recorded in the follow-up period; two cases of ORN, one buccal
fistula and one nearly lethal bleeding. The three patients that developed
58
FREDRIK LANDSTRM
ORN or fistulas were treated with secondary reconstructive surgery (one

free flap, two loco-regional flaps).
Secondary outcome: Four of the five patients were alive and tumourfree overall at 24 months (80%) (Table 10). The functional outcome was
poor. Three of the four surviving patients scored lower in all three parameters (eating in public, understandability of speech, normalcy of diet) after
12 months than before treatment (Table 11). Two of the patients still had
feeding tubes one year after treatment.
Table 10. Demographics, tumor characteristics, adjuvant treatment, and two-year

clinical outcome in 5 patients treated with ECT for cancer in the head and neck
area.
Sex
Age
Localization
59
65
59
Masseter
muscle
Floor of
mouth
Bucca
78
58
Floor of
mouth
Base of
tongue
Histology
TNM
Clear cell TXNXM1

carcinoma
SCC
T2N0M0
SCC
T2N0M0
SCC
T2N0M0
SCC
T2N0M0
Tumor
size (mm)
RT
Follow-up Tumor status

(months)
Local Overall
43 26
26
30 30
25
20 20
5
25 25
5
25 25
8
Before
NED
DWD
P+N
24
NED
NED
P+N
24
NED
NED
P+N
24
NED
NED
No
24
NED
NED
DWD, dead with disease; M, male; N, neck; NED, no evidence of disease; P, primary tumor; RT, radiotherapy; SCC, squamous cell carcinoma
FREDRIK LANDSTRM
59
Table 11. PSS-HN assessments in four patients with cancer in the oral cavity and
oropharynx before and 12 months after ECT.
Pat No
Eating in public
Understandability of speech
Normalcy of diet
Before
100
After
0
Before
100
After
50
Before
100
After
0
3
4
100
100
100
75
100
100
100
50
100
100
100
30
100
25
75
50
80
20
Eating in public: 100 = no restriction, 75 = no restriction of place but restriction of

diet in public, 25= eats only at home in presence of selected individuals, 0 = always
eats alone. Understandability of speech: 100 = always understandable, 75 = occasional repetition necessary, 50 = usually understandable; face-to-face contact necessary. Normalcy of diet: 100 = no restriction, 80 = all meat, 30 = pureed foods (in
blender), 20 = warm liquids, 0 = non-oral feeding (tube-fed). PSS-HN, Performance Status Scale for Head and Neck Cancer
Paper IV
Primary outcome: The outcome is summarized in Table 12. The local
control rate in the five-year follow-up period was 100% based on 12 surviving patients. Six of these patients had received adjuvant RT and six
were treated with ECT alone. The median follow-up time was 58 months.
There was one additional SAEs recorded in addition to the five previously reported (pain, ORN, fistula and bleeding) (98, 123) (Table 12). The
patient treated for a tongue base cancer (Patient 19) developed severe
problems with aspiration and was eventually offered a laryngectomy,
which the patient declined.
The QoL data (median values) at baseline and 12 months after ECT are
shown in Table 13. The data was based on the 18 patients that choose to
participate at baseline and 15 patients a year after treatment. By then three
patients had died and one patient chose not to participate. There was a
statistically significant increase in problems with senses (p=0.006), speech
(p=0.01), opening mouth (p=0.02) and mouth dryness (p=0.005) and a
decrease in problems with social contact after treatment for the whole
group (p=0.042) (Table 13). There were no statistically significant differ-
60
FREDRIK LANDSTRM
ences for the subgroup that were treated with only ECT (6 patients before
and 5 patients after) (Table 13).
Three pair of subgroups was compared; patients treated with ECT
alone (ECT) vs. patients treated with adjuvant RT (ECT+RT), patients
with oral tongue cancer (Tongue) vs. patients with oral cavity cancer in
other sites (Non-tongue) and patients that smoked during treatment
(Smokers) vs. patients that didnt (Non-smokers). Twelve months after
ECT there were statistically significant unfavourable outcomes regarding
swallowing (p=0.01) and xerostomia (p=0.04) in the ECT+RT group
compared to the ECT group. When comparing tumor location, the nontongue cancer patients had a statistically significantly worse outcome than
the tongue cancer patients after ECT regarding need for painkillers
(p=0.03). Smokers had significantly worse speech problems (p= 0.008)
than the non-smoking group.
Secondary outcome: The overall five-year survival was 63.1% (12/19)
and the tumour-specific five-year survival was 75% (12/16) (Table 12).
Two patients had already died of RR at the two-year follow-up and two
more patients died of RR subsequently. Three patients died of intercurrent
disease in the follow-up period. There were no signs of local recurrence in
the seven patients that died during follow-up.
FREDRIK LANDSTRM
61
Table 12. Demographics, smoking, tumor characteristics, serious adverse events

and five-year outcome in 19 head and neck cancer patients treated with ECT.
62
FREDRIK LANDSTRM
Table 13. The EORTC H&N 35 quality of life data (median and range) for all
patients at baseline and one year after ECT. The p-values are the outcome of the
Wilcoxon signed rank test and McNemars test. There were significantly increased
problems with senses, speech, opening mouth and xerostomia and a significant
decrease in problems with social contact. The group sizes are shown at the bottom
of the table.
QoL items
Baseline
12 months
p-value
Swallowing
0 (0-41.7)
8.3 (0-58.3)
0.17
Senses (taste, smell)
0 (0-66.6)
16.7 (0-33.3)
0.006
Speech
0 (0-44.4)
11.1 (0-55.5)
0.01
Social eating
8.3 (0-66.6)
8.3 (0-75)
0.066
Social contact
0 (0-66.6)
0 (0-33.3)
0.042
Sexuality
0 (0-66.6)
0 (0-100)
0.67
Teeth
0 (0-33.3)
0 (0-33.3)
0.56
Opening mouth
0 (0-33.3)
0 (0-66.6)
0.02
33.3 (0-66.6)
66.6 (33.3-100)
0.005
Sticky saliva
0 (0-100)
33.3 (0-100)
0.13
Coughing
0 (0-66.6)
0 (0-33.3)
0.56
Felt ill
0 (0-33.3)
0 (0-66.6)
0.16
Painkillers
0 (0-100)
0 (0-100)
Nutritional supplements
Xerostomia
0 (0-100)
0 (0-100)
0.62
Feeding tube
0 (0-0)
0 (0-100)
Weight loss
0 (0-100)
0 (0-100)
Weight gain
0 (0-100)
0 (0-100)
0.51
n=18
n=15
Paper V
The difference in survival day 4 after ECT between the fibroblasts and the
other cell types (SCC-4, CAL-27 and the HUVEC) was statistically significant at the = 0.01level (Figure 20 and Table 14). The null hypothesis
FREDRIK LANDSTRM
63
was therefore rejected for the fibroblasts. Cohens d for the differences
varied between 18.7 and 42.6 indicating that the survival differences were
large. For the HUVEC cells, on the other hand, the null hypothesis was
accepted. When compared to the SCC cell lines there were no statistically
significant differences in survival after ECT. There were, however, statistically significant survival differences between the HUVEC cells and all the
other cell types day 1 after ECT. Cohens d values between 9.4 and 14.8
again indicated that these differences were also large (Figure 21 and Table
15).
Figure 20. Mean relative survival SEM day 1 to 4 for all cell types after ECT
with low dose (10 g/ml) and high dose (100 g/ml) BLM exposure. Cell survival
was assessed with the MultiTox-Fluor assay. The data is based on four independent experiments.
64
FREDRIK LANDSTRM
Table 14. Result of Welchs F-test for all cell types 4 days after EP alone, ECT
with 10 g/ml BLM (ECT L) and 100 g/ml (ECT H) (A). The between cell type
comparison with the Games-Howell post-hoc test for the significant results from
Welchs test (ECT L and ECT-H) and Cohens d value for these statistically significant differences showing large differences (B). The data was based on four independent experiments. (FB, Fibroblasts; HUVEC, Human umbilical vein endothelial
cells; CAL-27 and SCC-4 are two human SCC cell lines).
A
Groups
F-statistic Welchs test
p-value
EP
4.79
0.048
ECT L
343.1
< 0.001
ECT H
610.4
< 0.001
B
Cell type
ECT L
ECT H
Games-Howell
Cohen's d
Games-Howell
Cohen's d
FB - HUVEC
p < 0.001
21.8
p < 0.001
34.8
FB - CAL-27
p < 0.001
24.5
p < 0.001
42.6
FB - SSC-4
p < 0.001
21.8
p < 0.001
18.8
HUVEC - CAL-27
p = 0.20
NS
p = 0.88
NS
HUVEC - SCC-4
p = 0.28
NS
p = 0.09
NS
CAL-27 - SCC-4
p = 0.02
NS
p = 0.08
NS
FREDRIK LANDSTRM
65
Figure 21. Mean relative survival SEM for all cell types day 1 to 4 after EP
alone. The cell viability was assessed with the MultiTox-Fluor assay. The data was
based on four independent experiments. (FB, Fibroblasts; HUVEC, Human umbilical vein endothelial cells; CAL-27 and SCC-4 are two human SCC cell lines).
Table 15. Result of Welchs F-test for all cell types day 1 and 2 after EP alone (A).
The results from the between group comparison with the Games-Howell post-hoc
test for the significant results from Welch test (EP day 1 and day 2) and Cohens d
value for these statistically significant differences showing large differences (B).
The data was based on four independent experiments. (NS=not statistically significant)
A
Groups
F-statistic Welchs test
p-value
EP day 1
115.2
< 0.001
EP day 2
136.8
< 0.001
66
FREDRIK LANDSTRM
Cell type
EP day 1
EP day 2
Games-Howell
Cohen's d
Games-Howell
Cohen's d
HUVEC - FB
p = 0.001
13.4
p < 0.001
19.8
HUVEC -CAL-27
p = 0.001
11.2
p = 0.001
14.7
HUVEC -SCC4
p = 0.007
9.4
p = 0.016
NS
FREDRIK LANDSTRM
67
Discussion
For patients with HNSCC or NMSC both the disease and the treatment
can cause significant functional impairment and unwelcome changes in
physical appearance. In the last decades there has been a tremendous progress in the knowledge about the molecular basis of cancer as well as in
diagnostics and treatment (68, 76). Improvements in diagnostic radiology
like PET-CT and diffusion MRI could lead to earlier detection of both
primary tumors and recurrent disease. Intensity modulated RT, functional
neck dissections, transoral robotic surgery and free flap reconstruction
have decreased the treatment related morbidity.
However, much remain to be achieved. For example, while the survival
in oropharyngeal cancer patients has increased significantly in the last
decades in Sweden the survival rate in patients with tongue cancer has
increased only modestly (82). Despite an increased knowledge about the
molecular basis of HNSCC and NMSC, surgery and RT remain the cornerstones of treatment. There is, however, a need for more safe and efficacious treatment modalities since field cancerization and cancer stem cells
increases the risk of second primary tumours and recurrence in previously
treated sites (84, 86).
Methodological limitations and strengths

Papers I, II, III and IV
The study design (phase II trial) did not include control groups treated
with the standard modalities (surgery and RT). The study groups consisted
of 6, 15 and 5 patients, respectively and there were four different cancer
types investigated. Obviously, the group sizes, the different tumors involved and the lack of control groups limits the conclusions that can be
made. For instance, no direct comparison to surgery and RT could be
made. In Paper IV the subgroups assessed for QoL were too small to find
all potential significant outcome differences. The significant differences
found, on the other hand, were probably large enough to be clinically
relevant.
One consequence of treatment with ECT is the lack of a histological
specimen. Therefore, potentially important prognostic factors like the
depth of infiltration and perineural invasion cannot be adequately determined. It can have potential consequences for the results presented in this
study since the depth of infiltration was estimated, not measured, leading
to potential misclassification and bias in selection of patients for adjuvant
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FREDRIK LANDSTRM
RT. The adjuvant RT treatment also limits the assessment of efficacy to

the patients treated with only ECT. The strengths of these studies were the
curative intention, the standardized treatment and follow-up protocols and the
length of the follow-up periods (24 and 60 months). No patient was lost to follow
up. Also, there have been no selective reporting; the outcome in all treated
patients was reported. This was also one of the reasons reason for including Papers I and II in two meta-analyses of ECT efficacy (96, 99).
Paper V
There were only three cells groups investigated (SCC, fibroblasts and endothelial cells). There were two SCC cell lines and only one fibroblast and
one endothelial cell line. The outcome might be different if other cell lines
had been used. The investigation ended 4 days after ECT. Perhaps the
long-term outcome would have been different if the experiment had continued since the BLM mediated mitotic cell death is a slow process.
The strengths of the study were that two independent methods for cell
survival assessment and a conservative statistical test (Welchs F test) were
used. Also, a conservative significance level was used ( 0.01). Cohens d
was calculated for the significant survival differences also indicating the
size of these differences.
Efficacy of ECT
The previously reported efficacy of ECT (89-92) has been confirmed by
this thesis. In Paper I there was a CR in 4/6 patients with NMSC two
years after mono modality treatment with ECT (Table 7) (97). Moreover,
ECT was an organ sparing treatment in two patients with periocular BCC
(Paper I). However, in one of these patients the tumor recurred 2.5 years
after ECT and was treated with exenteration of the orbit and RT. The
tumor (metatypical BCC) had a long history of recurrence and in this context a delay before exenteration of 2.5 years could actually be considered a
success. Salwa et al have reported CR in 3 patients with periocular BCC
using the ESOPE protocol. However, the follow-up period in that study
was only 5-8 months (104).
In paper IV the, to our knowledge, largest case series (19 patients) with
the longest follow-up period (five years) of curative ECT in head and neck
cancer was reported. Six patients, five with oral tongue cancer and one
with tongue base cancer, were cured by ECT alone (Table 12). Four of
these patients had T2 tumors and the histopathology was HNSCC in five
patients and an adenocarcinoma in one patient. In addition, six patients
FREDRIK LANDSTRM
69
were cured by ECT and adjuvant RT and none of the nineteen patients
had any local recurrences (Table 12). The adjuvant dose was 57.8 Gy,
which was lower than the dose for macroscopic cancer (68-70 Gy). Therefore, the five-year local control in these patients was probably a combined
effect of ECT and RT. There is a need for further investigation since ECT
in combined modality treatment has not been extensively reported (124,
125).
The tumor specific five-year survival reported in paper IV was 75% and
the over-all survival was 63.1%. This is in agreement with previously reported five-year survival in patients with T1 and T2 tongue cancer treated
with surgery and/or RT (62-83.5 %) (126-128).
The outcome in Paper IV (100 % CRR) compares favourably to the
outcome reported in the previously largest studies of head and neck cancer
treated with curative ECT. In 2001 Allegretti et al reported a series of 14
patients with T1-T4 head and neck cancer treated with ECT with IT BLM.
A 42.9% CRR was reported in the median 31.5 month follow-up period
(110). In 2014 Campana et al reported a 58.3 % CRR in a retrospective
study of 12 patients with oral cavity or oropharyngeal cancer (113). The
median follow-up period in that study was 14 months. An important reason for the better outcome seen in our study (Paper IV) could be that the
BLM dose was higher due to differences in calculation of the treatment
volume; a 1 cm margin was included in the formula used in this thesis (see
Methods). The difference in outcome could probably also be explained by
the more advanced tumors (T3, T4) included in Allegrettis study compared to our study of only T1 and T2 tumors (Paper IV).
In 2014 Mali et al published two papers on the efficacy of ECT (96,
99). The first was a meta-analysis of treatment efficacy related to tumor
type and the administration route of the drug used. Papers I and II in this
thesis were included. IT administration was significantly more effective
than IV administration. No differences, however, could be found between
cisplatin and BLM (96). The other study was a meta-analysis investigating
the relationship between tumor size and ECT efficacy; Paper I in this thesis
was included (97). There was a statistically significant lower response rate
for tumors 3 cm. If anything, the results of the study suggests that the
ESOPE guidelines that recommends lower doses of BLM in larger tumors
(250 IU/cm3) should be re-evaluated (100).
Eight of 26 patients treated in this thesis had tumors 3 cm (Papers IIV). The local control was 100% in these patients but only two were
treated with ECT alone. Considering the very good long-term results re70
FREDRIK LANDSTRM
ported in this thesis the BLM dose used (1000 IU/cm3 tumor volume) and
the calculation of the treatment volume should be further evaluated especially in ECT with curative intention.
Only one patient in this thesis was treated with palliative intention; the
patient with a metastasis of renal clear cell carcinoma in the masseter
muscle (Paper III). That patient was successfully treated with ECT after
palliative RT had failed.
Although it is somewhat rewarding to experience that a method under
investigation actually seems to work it is just as important to analyse why
and when it doesnt. In Paper I a patient with CSCC in the external meatus had a persistent tumor after two ECT treatments and was eventually
treated with salvage surgery and RT (Table 7). We know that the stratum
corneum have a very low conductivity resulting in very high electrical
fields in EP. The result could be that the electrical fields in the underlying
cartilage are too weak to cause EP. Whatever the reason, treatment in the
external meatus cannot be recommended.
Safety of ECT
This thesis has also confirmed ECT as a safe treatment with some exceptions. The EP system (Medpulser) that was used was reliable; all treatments could be completed successfully. Treatment of patients with NMSC
(Paper I) and oral tongue cancer (Paper II) was safe with two SAEs in 21
patients (pain and an epileptic seizure) (97, 98). However, in the five patients with cancer in the floor of mouth, bucca and base of tongue there
were five SAEs; two ORN, a mucocutaneous fistula, a nearly fatal bleeding and aspiration (Paper III). Both cases of ORN and the fistula required
secondary reconstructive surgery. ORN is, of course, caused by RT and
this study (Paper III) is obviously very small but none of the 10 patients
with oral tongue cancer that received the same RT dose developed ORN
(Paper II).
Furthermore, in a retrospective study of 189 patients treated with RT
there were no reported cases of ORN in patients that had received the
lowest doses, 60-65 Gy (129). The patients in this thesis were treated with
an even lower dose 57.8 Gy (Paper II-IV). This might suggest that ECT
can be a risk factor for ORN in patients that will receive RT, especially in
proximity to the mandible (Paper III). ECT with BLM has a welldocumented vascular disrupting effect that may have a role in the pathogenesis (46, 47). There is limited reporting of complications with curative
ECT in head and neck cancer. Allegretti et al reported a pharyngocutaeous
FREDRIK LANDSTRM
71
fistula and Campana reported mycositis and ulceration after treatment

(110, 113).
Functional and quality of life outcome after ECT

The patient with a recurring CSCC in the proximity of the facial nerve and
parotid duct was successfully treated with ECT (Paper I). There were no
signs of facial nerve injury or parotid swelling in the follow-up period.
Surgery would almost certainly have led to facial nerve injury.
The patients treated for periocular BCC experienced increased tear fluid
a few weeks after treatment but no visual impairment (Paper I). The functional outcome was also very good one year after ECT in the patients with
tongue cancer reported in Paper II (Table 9) (98). The patients treated
with ECT alone had maximal scores in all three PSS-HN parameters (eating in public, understandability of speech and normalcy of diet) both before and after treatment (Paper II). In the patients with cancer in the floor
of mouth, bucca and tongue base reported in Paper III the functional outcome was poor (Table 11). The PSS-HN assessments were worse in all
parameters one year after treatment in three of the four surviving patients.
The QoL assessments in the head and neck cancer patients showed a
statistically significant increase in problems with sense (taste, smell),
speech, mouth opening and xerostomia one year after treatment (Paper
IV) (Table 13). These differences were probably caused by RT, not ECT in
itself. When comparing three pair of patient subgroups there were a significantly worse outcomes in swallowing in the patients treated with ECT
and RT compared to the patients treated with ECT alone, in speech problems in patients that smoked during treatment and in need for painkillers
in the patients with oral cavity cancer other than tongue cancer (Table
13). Considering the small group sizes these significant differences probably have to be clinically significant. However, because of the overlap in
smoking habits, tumor location and RT exposition in these small groups
the results should be with interpreted with caution.
In summary, while ECT seems to be a safe, efficacious treatment with
very good functional outcome in NMSC (Paper I) and oral tongue cancer
(Paper II) it seems to be less safe in the floor of mouth, bucca and base of
tongue (Paper III). The functional outcome was also poor in these patients
(Paper III). One conclusion drawn is that for these patients, ECT was not
the preferred treatment. With surgical resection and primary reconstruction, these patients in all probability would have had a better outcome.
The long-term results in the patients with oral tongue cancer make a
72
FREDRIK LANDSTRM
strong case for further investigation of ECT as a curative treatment in T1

and T2 primary tongue cancer (Paper IV).
Selectivity of ECT
One of the most important features of ECT is its proposed selectivity. This
is especially important in head and neck cancer where sparing of normal
tissue often means spared function. There are many theoretical reasons for
selectivity. The cell radius is important. If we recall the equation for induced membrane potential the Vrev needed for EP is inversely proportional
to the cell radius. Since an axon is one tenth of the usual cell radius, EP of
the axon would require an electrical field 10 times higher than the cancer
cells. This is one selective mechanisms of ECT and could probably explain
the lack of facial nerve damage in the patient treated for a CSCC in the
cheek (Paper I).
Also, the mitotic cell death seen with BLM is selective towards nondividing cells (29). Proliferative cells also have lower membrane potentials
and are therefore easier to electroporated (Figure 4) (9). Tumor tissue has
more irregular vasculature and the antivascular effects of ECT could possibly have a selective effect on tumors with high metabolism (50). There is
also some evidence that the immune system could be involved in the effect
of ECT (57, 59).
In paper V the possibility of a selective effect of ECT on cell survival between different cell-types was investigated (Figures19 and 20, Tables 14
and 15). Two significant selective effects were found. The survival of the
fibroblasts was significantly higher than the survival of SCC cells and endothelial cells four days after ECT (Figure 19, Table 14). The survival of
the endothelial cells was significantly lower than in the fibroblasts and the
SCC cells one day after EP alone (Figure 20, Table 15). This suggests that
the endothelial cells could have important roles in both the reversible
vascular lock effect of EP and the irreversible vascular disrupting
effect of ECT (47, 48, 50). There was no difference in survival between the
endothelial cells and the cancer cells after ECT. Endothelial cells also seem
to play an important supporting and promoting role for cancer stem cells
(88). Hypothetically, ECT could therefore possibly have effects, both direct and indirect, on cancer stem cells. The results presented in Paper V
leads to a more complete understanding of ECT and its effect in different
cell types. More research is needed to confirm or reject these results in
other cell-types. This will hopefully lead to more selective ECT protocols
in the future.
FREDRIK LANDSTRM
73
Costs
The patients in this thesis were all treated in the same setting, under general anaesthesia and admitted in our ward. Because of the few patients
treated and the many locales involved the only group that could be used in
comparisons were the 15 patients with oral tongue cancer.
The costs can be divided into treatment time in the operating room,
admission, number of revisits and the cost for the materials and the pharmacological cost. The treatment time for ECT in tongue cancer is similar
to surgery with monopolar diathermia, currently the usual treatment in
our department. The admission time was, at least for the first patients,
longer than in patients treated with surgery, probably due to our inexperience with this modality.
In the last patients treated the admission times were about the same as
for surgery, two to four days. For patients with tongue cancer treated with
surgery there is usually only one revisit in the first month after treatment.
In the ECT patients, however, there were approximately two to three revisits in the same period. Again, this was done, in part, to study the treatment effect but also to remove necrotic tissue in many of the patients.
The cost of BLM and the needle electrodes for the Cliniporator system
currently used is approximately 5 000 SEK per treatment which is a little
more than the cost of surgery with monopolar diathermia per treatment. It
is fairly obvious that the cost-benefit for treating uncomplicated NMSC
with ECT, usually done in a out-patient setting, is not feasible. However,
with the 5000 Hz electroporators available today there is possible to treat
NMSC in local anaesthesia thereby reducing the costs considerably.
Treatment recommendations
So where, if at all, does ECT fit into the armamentarium in the treatment
of head and neck cancer? New modalities must be compared to surgery
and RT, preferably in randomized control trials, and proven to be at least
as safe and efficient to become first line treatments. The situation is different in recurrent head and neck cancer where additional RT or surgery may
not be possible or would result in very significant morbidity. In these cases
alternative modalities can be especially important. These alternative
treatments must of course have been proven to be reliable, safe and efficient. However, the level of evidence needed to become a first line treatment could take years in multicentre studies and this should not exclude
patients from safe and efficient treatments, especially when surgery and
RT are no longer useful options.
74
FREDRIK LANDSTRM
To put ECT in a useful context the author has made a list of when and
where ECT could, should and should not be used. This is, of course, a
somewhat subjective list based in part on evidence, available treatment
alternatives, side effects and the subjective opinion of the author. In all
cases there is an underlying assumption that the tumor with adequate
margins is fully accessible to the drug and the electrodes.
When to recommend ECT as a first line of treatment:
In palliative treatment of multiple cutaneous metastases with diameters < 3 cm using the ESOPE protocol (protocol 1). There are numerous studies showing the efficacy of ECT in this setting and
there are no good treatment alternatives (130, 131).
In palliative treatment of recurrent NMSC and HNSCC after multimodality treatment where additional surgery is not feasible. If the
tumor diameter > 3 cm, the ECT protocol in this thesis should be
used (protocol 2). If tumor diameter < 3 cm protocol 1 should be
used.(99, 132, 133).
When to recommend ECT as an alternative to surgery:
In primary NMSC with protocol 2 as an organ and functionsparing treatment, especially in BCC in the proximity of the orbit
where the alternative is exenteration (with eye muscle involvement
but without involving the globe) or in proximity of the facial nerve
(97, 104).
In other primary/recurrent NMSC with protocol 2 in areas with
high risk of recurrence (mask areas of the face) or with illdefined tumor borders.
In primary T1 and T2 oral tongue cancer with ill-defined borders
in patients previously treated with RT. Protocol 2 should be used.
FREDRIK LANDSTRM
75
When not to recommend ECT:

In tumors with bone invasion.
In well-defined NMSC not in the proximity of the facial nerve or
the orbit.
In tumors in the floor of mouth, bucca and base of tongue especially if the patient will receive or have previously received RT.
In tumors involving the cartilaginous external meatus.
Future perspectives
There are physical limitations to ECT treatment in head and neck cancer.
The drug must be reliably administered and the tumour area has to be
accessible with electrodes (39). With the limited access of the currently
available applicators this excludes treatment of tumours in the oropharynx, the hypopharynx and the larynx. Instead treatment is currently limited to the more accessible parts of the oral cavity. A reason for the lack of
suitable applicators could be that, so far, mostly medical oncologists have
been involved in the research, development and treatment. The technology
has, basically, been developed for palliative treatment of skin metastasis.
This is not a critique of those dedicated oncologists but of head and
neck surgeons that so far has shown little interest in the technique and its
applications. With more surgeons involved the curative potential of ECT
could be investigated more fully and the demand for a wider variety of
applicators would increase.
If ECT will have a future as a treatment modality in primary head and
neck cancer depends on not yet conducted studies comparing its efficacy
and functional outcome to surgery and RT. Based on the results in this
thesis the following future studies are proposed:
A randomized controlled trial of ECT vs surgery in primary T1 and
T2 tongue cancer.
A randomized controlled trial of ECT vs surgery with adjuvant RT
in advanced stage tongue cancer (T3, T4).
A randomized controlled trial of ECT vs surgery in parotid cancer
without facial nerve paralysis.
76
FREDRIK LANDSTRM
The long-term data presented in this thesis could hopefully increase the
interest in ECT as a curative treatment modality and facilitate the realisation of these and many more studies.
FREDRIK LANDSTRM
77
Conclusions
ECT seems to be an efficacious and safe curative treatment for
non-melanoma skin cancer in the head and neck area where it can
be an organ and function-sparing treatment.
ECT seems to be an efficacious and safe curative treatment with a
very good functional outcome in oral tongue cancer where it
should be further investigated as a potential primary treatment.
ECT with adjuvant RT seems to be an efficacious treatment of oral
cavity cancer in the floor of mouth and bucca. In these localizations, however, the safety and functional outcome can be poor.
There is evidence of survival selectivity in different cell types after
ECT in vitro. Fibroblasts seem to have a significantly increased
survival when compared to human SCC cells and endothelial cells.
In addition, endothelial cells seem to have a significant survival decrease after EP alone.
78
FREDRIK LANDSTRM
Acknowledgements
My sincere gratitude to all of you that have helped me with this thesis. I
would especially like to thank the following:
Professor Claes Mller for guiding me through this thesis like a
partner, not a parent. Thank you!
Lennart Lfgren, for introducing me to ECT.
Johan Reizenstein for your constructive skepticism.
Mathias von Beckerath, for your contributions to this thesis and
for being a friend.
Hans Gustafsson, for always telling the painful truth.
Mikael Ivarsson and Anita Koskela, for all your support and patience in the laboratory.
Stefan Kristiansson, for your words of encouragement.
Gun-Britt Adamsson for all your help with the data collection. You
will be missed.
To all my colleagues in the ENT department, without your support
this thesis would not have been possible.
To all colleagues and nurses involved in the treatment and followup of the patients in this thesis.
To all my colleagues in the Head and Neck Oncology Centre.
To all the patients that chose to participate in these studies. Without you there will be no progress.
FREDRIK LANDSTRM
79
My parents Gunnar and Marie, for being my role models.

My sister Cecilia, for all your good advice.
Anna, for being in my life. I love you.
Noah, Gabriella, Elias and Mikaela, you make everything worthwhile. Thank you for your patience during the last month. Now I
want to spend my time with you again!
80
FREDRIK LANDSTRM
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Appendix 1
PERFORMANCE STATUS SCALE FOR HEAD & NECK

CANCER PATIENTS - PSS-HN
Suggestions for Administration
These performance scales may be rated by health professionals (e.g., physicians, nurses, nutritionists) or
other personnel (e.g., clerks, data managers). Ratings are determined through use of an unstructured
interview format.
Normalcy of Diet
Begin by asking the patient what kinds of foods (s)he has been eating. Ask what foods are difficult to eat.
Based on the patient's response, choose an item at the low end of the scale. Move up the scale giving
examples of foods in each category and asking the patient if (s)he is eating those food items. Even if the
patient says that (s)he eats everything, inquire about specific items beginning with 50, soft chewable foods
and moving upwards. Stop at the item at, and above which the patient cannot eat. The patient then receives
the score below that. If the patient indicates that (s)he is eating a full diet, also inquire whether (s)he needs
to drink more liquids than usual with meals; eating a full diet with intake of extra fluids is scored 90. If the
patient can take foods orally, but is also using a feeding tube, score based on solid food.
Public Eating
Score the Public Eating scale by asking the patient where (s)he eats (in a restaurant, at home, at
friends/relatives' homes, etc.) and with whom (s)he eats (always alone, with family/friends, etc). Ask patient
if (s)he chooses different foods (softer, less messy, etc.) when eating with others. When was the last time the
patient ate in a restaurant, cafeteria, MacDonald's, picnic, family reunion? Choose the score beside the
description that best fits the patient. A patient on a restricted diet, (e.g., tube feeding, pureed foods) who
does not eat in public but will join others in a public eating setting should be rated 75. Score 999 for
inpatients.
Understandability of Speech
This scale is scored based on the interviewer's ability to understand the patient during conversation (in this
case, based on conversation about patient's diet and social activities). Choose the score beside the
description that best fits the patient. See if you can understand the patient if you are looking away while
(s)he is talking.
Special Considerations for Inpatients: Administration of the PSS-HN varies somewhat for inpatients. Score
the Normalcy of Diet and Understandability of Speech Scale as indicated. The Eating in Public Scale is not
applicable as inpatients generally have little opportunity to eat with others or leave their hospital rooms.
Inpatients receive a score of 999 on the Eating in Public Scale.
PERFORMANCE STATUS SCALE FOR

HEAD AND NECK CANCER PATIENTS: PSS-HN
Patient Name ____________________
ID# /__/__/ / / / /__/__/__/
Date /__/__/__/__/__/__/
NORMALCY OF DIET /__/__/__/
PUBLIC EATING /__/__/__/
100
Full diet (no restrictions)
100
90
Full diet (liquid assist)
No restriction of place, food or

companion (eats out at any
opportunity)
80
All meat
75
70
Raw carrots, celery
60
Dry bread and crackers
No restriction of place, but restricts

diet when in public (eats anywhere,
but may limit intake to less "messy"
foods (e.g., liquids)
50
Soft chewable foods (e.g., macaroni,

canned/soft fruits, cooked vegetables,
fish, hamburger, small pieces of meat)
50
Eats only in presence of selected

persons in selected places
25
Eats only at home in presence of

selected persons
40
Soft foods requiring no chewing

(e.g., mashed potatoes, apple
sauce, pudding)
30
Pureed foods (in blender)
20
Warm liquids
10
Cold liquids
Non-oral feeding (tube fed)
0
999
Always eats alone

Inpatient
UNDERSTANDABILITY OF SPEECH /__/__/__/

100
Always understandable
75
Understandable most of the time;

occasional repetition necessary
50
Usually understandable; face-to-face

contact necessary
25
Difficult to understand
Never understandable; may use written

communication
List MA, Ritter-Sterr C, Lansky SB. A Performance Status Scale for Head and Neck Cancer Patients.
Cancer. 66:564-569, 1990
Appendix 2
Publications in the series

rebro Studies in Medicine
1.
Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term

sequelae from closed head injury.
2.
Jansson, Kjell (2004). Intraperitoneal Microdialysis.

Technique and Results.
3.
Windahl, Torgny (2004). Clinical aspects of laser treatment of

lichen sclerosus and squamous cell carcinoma of the penis.
4.
Carlsson, Per-Inge (2004). Hearing impairment and deafness.

Genetic and environmental factors interactions consequences.
A clinical audiological approach.
5.
Wgster, Dick (2005). CXCL16 and CD137 in Atherosclerosis.
6.
Jatta, Ken (2006). Inflammation in Atherosclerosis.
7.
Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on

Malignant Diseases in Childhood.
8.
Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis

and Mycoplasma genitalium by genetic and serological methods.
9.
Norn, Torbjrn (2006). Clostridium difficile, epidemiology and

antibiotic resistance.
10. Anderzn Carlsson, Agneta (2007). Children with Cancer Focusing

on their Fear and on how their Fear is Handled.
11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in
vascular smooth muscle cells.
12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry
in children.
13. Eliasson, Henrik (2008). Tularemia epidemiological, clinical and
diagnostic aspects.
14. Walldn, Jakob (2008). The influence of opioids on gastric function:
experimental and clinical studies.
15. Andrn, Ove (2008). Natural history and prognostic factors in
localized prostate cancer.
16. Svantesson, Mia (2008). Postpone death? Nurse-physician
perspectives and ethics rounds.
17. Bjrk, Tabita (2008). Measuring Eating Disorder Outcome

Definitions, dropouts and patients perspectives.
18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery.
19. Parihar, Vishal Singh (2008). Human Listeriosis Sources and Routes.
20. Berglund, Carolina (2008). Molecular Epidemiology of MethicillinResistant Staphylococcus aureus. Epidemiological aspects of MRSA
and the dissemination in the community and in hospitals.
21. Nilsagrd, Ylva (2008). Walking ability, balance and accidental falls in
persons with Multiple Sclerosis.
22. Johansson, Ann-Christin (2008). Psychosocial factors in patients
with lumbar disc herniation: Enhancing postoperative outcome by
the identification of predictive factors and optimised physiotherapy.
23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco
products.
24. Hahn-Strmberg, Victoria (2008). Cell adhesion proteins in different
invasive patterns of colon carcinoma: A morphometric and molecular
genetic study.
25. Bttiger, Anna (2008). Genetic Variation in the Folate Receptor-
and Methylenetetrahydrofolate Reductase Genes as Determinants
of Plasma Homocysteine Concentrations.
26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence,
treatment seeking behaviour, experiences and perceptions among
persons with and without urinary leakage.
27. Elfstrm, Peter (2009). Associated disorders in celiac disease.
28. Skrberg, Kurt (2009). Anabolic-androgenic steroid users in treatment:
Social background, drug use patterns and criminality.
29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment
Prevalence, Measurement and Long-term Outcome.
30. Vumma, Ravi (2009). Functional Characterization of Tyrosine
and Tryptophan Transport in Fibroblasts from Healthy Controls,
Patients with Schizophrenia and Bipolar Disorder.
31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis
from a virulence and immunogenic perspective that includes variations
in novel vaccine antigens.
32. Allvin, Rene (2009). Postoperative Recovery. Development of a

Multi-Dimensional Questionnaire for Assessment of Recovery.
33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia
with Special Reference to Folate and Fibrinolysis.
34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide
study of assiciated morbidity and mortality.
35. Sderqvist, Fredrik (2009). Health symptoms and potential effects on
the blood-brain and blood-cerebrospinal fluid barriers associated with
use of wireless telephones.
36. Neander, Kerstin (2009). Indispensable Interaction. Parents perspectives
on parentchild interaction interventions and beneficial meetings.
37. Ekwall, Eva (2009). Womens Experiences of Gynecological Cancer
and Interaction with the Health Care System through Different Phases
of the Disease.
38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance
in Neisseria meningitidis phenotypic and genotypic characteristics.
39. Hammer, Ann (2010). Forced use on arm function after stroke.
Clinically rated and self-reported outcome and measurement during
the sub-acute phase.
40. Westman, Anders (2010). Musculoskeletal pain in primary health
care: A biopsychosocial perspective for assessment and treatment.
41. Gustafsson, Sanna Aila (2010). The importance of being thin
Perceived expectations from self and others and the effect on
self-evaluation in girls with disordered eating.
42. Johansson, Bengt (2010). Long-term outcome research on PDR
brachytherapy with focus on breast, base of tongue and lip cancer.
43. Tina, Elisabet (2010). Biological markers in breast cancer and acute
leukaemia with focus on drug resistance.
44. Overmeer, Thomas (2010). Implementing psychosocial factors in
physical therapy treatment for patients with musculoskeletal pain
in primary care.
45. Prenkert, Malin (2010). On mechanisms of drug resistance in
acute myloid leukemia.
46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal

Sphincters in Obese Patients.
47. Josefson, Anna (2010). Nickel allergy and hand eczema
epidemiological aspects.
48. Almon, Ricardo (2010). Lactase Persistence and Lactase NonPersistence. Prevalence, influence on body fat, body height, and
relation to the metabolic syndrome.
49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis.
50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of
Cystic Fibrosis.
51. Franzn, Karin (2011). Interventions for Urinary Incontinence in
Women. Survey and effects on population and patient level.
52. Loiske, Karin (2011). Echocardiographic measurements of the heart.
With focus on the right ventricle.
53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of
Staphylococcus epidermidis isolated from prosthetic joint infections.
54. Eriksson Crommert, Martin (2011). On the role of transversus
abdominis in trunk motor control.
55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal
Sphincters.
56. Hollndare, Fredrik (2011). Managing Depression via the Internet
self-report measures, treatment & relapse prevention.
57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding
Properties in Neuropsychiatric Disorders using the Fibroblast Cell
Model.
58. Vidlund, Mrten (2011). Glutamate for Metabolic Intervention in
Coronary Surgery with special reference to the GLUTAMICS-trial.
59. Zakrisson, Ann-Britt (2011). Management of patients with Chronic
Obstructive Pulmonary Disease in Primary Health Care.
A study of a nurse-led multidisciplinary programme of pulmonary
rehabilitation.
60. Lindgren, Rickard (2011). Aspects of anastomotic leakage, anorectal
function and defunctioning stoma in Low Anterior Resection of the
rectum for cancer.
61. Karlsson, Christina (2011). Biomarkers in non-small cell lung

c arcinoma. Methodological aspects and influence of gender,
histology and smoking habits on estrogen receptor and epidermal
growth factor family receptor signalling.
62. Varelogianni, Georgia (2011). Chloride Transport and Inflammation
in Cystic Fibrosis Airways.
63. Makdoumi, Karim (2011). Ultraviolet Light A (UVA) Photoactivation
of Riboflavin as a Potential Therapy for Infectious Keratitis.
64. Nordin Olsson, Inger (2012). Rational drug treatment in the elderly:
To treat or not to treat.
65. Fadl, Helena (2012). Gestational diabetes mellitus in Sweden:
screening, outcomes, and consequences.
66. Essving, Per (2012). Local Infiltration Analgesia in Knee
Arthroplasty.
67. Thuresson, Marie (2012). The Initial Phase of an Acute Coronary
Syndrome. Symptoms, patients response to symptoms and
opportunity to reduce time to seek care and to increase ambulance use.
68. Mrild, Karl (2012). Risk Factors and Associated Disorders of
C eliac Disease.
69. Fant, Federica (2012). Optimization of the Perioperative Anaesthetic
Care for Prostate Cancer Surgery. Clinical studies on Pain, Stress
Response and Immunomodulation.
70. Almroth, Henrik (2012). Atrial Fibrillation: Inflammatory and
pharmacological studies.
71. Elmabsout, Ali Ateia (2012). CYP26B1 as regulator of retinoic acid
in vascular cells and atherosclerotic lesions.
72. Stenberg, Reidun (2012). Dietary antibodies and gluten related
seromarkers in children and young adults with cerebral palsy.
73. Skeppner, Elisabeth (2012). Penile Carcinoma: From First Symptom
to Sexual Function and Life Satisfaction. Following Organ-Sparing
Laser Treatment.
74. Carlsson, Jessica (2012). Identification of miRNA expression
profiles for diagnosis and prognosis of prostate cancer.
75. Gustavsson, Anders (2012): Therapy in Inflammatory Bowel Disease.
76. Paulson Karlsson, Gunilla (2012): Anorexia nervosa treatment

expectations, outcome and satisfaction.
77. Larzon, Thomas (2012): Aspects of endovascular treatment of
abdominal aortic aneurysms.
78. Magnusson, Niklas (2012): Postoperative aspects of inguinal hernia
surgery pain and recurrences.
79. Khalili, Payam (2012): Risk factors for cardiovascular events and
incident hospital-treated diabetes in the population.
80. Gabrielson, Marike (2013): The mitochondrial protein SLC25A43
and its possible role in HER2-positive breast cancer.
81. Falck, Eva (2013): Genomic and genetic alterations in endometrial
adenocarcinoma.
82. Svensson, Maria A (2013): Assessing the ERG rearrangement for
clinical use in patients with prostate cancer.
83. Lnn, Johanna (2013): The role of periodontitis and hepatocyte
growth factor in systemic inflammation.
84. Kumawat, Ashok Kumar (2013): Adaptive Immune Responses in the
Intestinal Mucosa of Microscopic Colitis Patients.
85. Nordenskjld, Axel (2013): Electroconvulsive therapy for
depression.
86. Davidsson, Sabina (2013): Infection induced chronic inflammation
and its association with prostate cancer initiation and progression.
87. Johansson, Benny (2013): No touch vein harvesting technique in
coronary by-pass surgery. Impact on patency rate, development
of atherosclerosis, left ventricular function and clinical outcome
during 16 years follow-up.
88. Sahdo, Berolla (2013): Inflammasomes: defense guardians in
host-microbe interactions.
89. Hrer, Tal (2013): Early detection of major surgical postoperative
complications evaluated by microdialysis.
90. Malakkaran Lindqvist, Breezy (2013): Biological signature of HER2positive breast cancer.
91. Lidn, Mats (2013): The stack mode review of volumetric datasets
applications for urinary stone disease.
92. Emilsson, Louise (2013): Cardiac Complications in Celiac Disease.
93. Dreifaldt, Mats (2013): Conduits in coronary artery bypass grafting
surgery: Saphenous vein, radial and internal thoracic arteries.
94. Perniola, Andrea (2013): A new technique for postoperative pain
management with local anaesthetic after abdominal hysterectomy.
95. Ahlstrand, Erik (2013): Coagulase-negative Staphylococci in
Hematological Malignancy.
96. Sundh, Josefin (2013): Quality of life, mortality and exacerbations
in COPD.
97. Skoog, Per (2013): On the metabolic consequences of abdominal
compartment syndrome.
98. Palmetun Ekbck, Maria (2013): Hirsutism and Quality of Life with
Aspects on Social Support, Anxiety and Depression.
99. Hussain, Rashida (2013): Cell Responses in Infected and Cystic
Fibrosis Respiratory Epithelium.
100. Farkas, Sanja (2014): DNA methylation in the placenta and in cancer
with special reference to folate transporting genes.
101. Jildenstl, Pether (2014): Influence of depth of anaesthesia on postoperative cognitive dysfunction (POCD) and inflammatory marker.
102. Sderstrm, Ulf (2014): Type 1 diabetes in children with non-Swedish
background epidemiology and clinical outcome
103. Wilhelmsson Gstas, Mona (2014): Psychotherapy patients in
mental health care: Attachment styles, interpersonal problems and
therapy experiences
104. Jarl, Gustav (2014): The Orthotics and Prosthetics Users Survey:
Translation and validity evidence for the Swedish version
105. Demirel, Isak (2014): Uropathogenic Escherichia coli, multidrugresistance and induction of host defense mechanisms
106. Mohseni, Shahin (2014): The role of -blockade and anticoagulation therapy in traumatic brain injury
107. Bai, Vladimir T. (2014): Molecular mechanisms mediating

development of pulmonary cachexia in COPD
108. Kirrander, Peter (2014): Penile Cancer: Studies on Prognostic Factors
109. Trs, Bianca (2014): Genome-based characterization of Neisseria
meningitidis with focus on the emergent serogroup Y disease
110. von Beckerath, Mathias (2014): Photodynamic therapy in the Head
and Neck
111. Waldenborg, Micael (2014): Echocardiographic measurements at
Takotsubo cardiomyopathy - transient left ventricular dysfunction.
112. Lillsunde Larsson, Gabriella (2014): Characterization of HPV-induced
vaginal and vulvar carcinoma.
113. Palm, Eleonor (2015): Inflammatory responses of gingival fibroblasts in
the interaction with the periodontal pathogen Porphyromonas gingivlis.
114. Sundin, Johanna (2015): Microbe-Host Interactions in Post-infectious
Irritable Bowel Syndrome.
115. Olsson, Lovisa (2015): Subjectiv well-being in old age and its association
with biochemical and genetic biomarkers and with physical activity.
116. Klarstrm Engstrm, Kristin (2015): Platelets as immune cells in sensing
bacterial infection.
117. Landstrm, Fredrik (2015): Curative Electrochemotherapy in the Head
and Neck Area

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Curative Electrochemotherapy in the Head and Neck Area

To Noah, Gabriella, Elias and Mikaela

rebro Studies in Medicine 117

Curative Electrochemotherapy in the Head and Neck Area

Fredrik Landstrm, 2015

Basal Cell Carcinoma

Curative Electrochemotherapy in the Head and Neck Area

II. Electroporation therapy for T1 and T2 oral tongue cancer.

III. Electrochemotherapy possible benefits and limitations to its use in

V. Electrochemotherapy evidence for cell-type selectivity in vitro.

Curative Electrochemotherapy in the Head and Neck Area

The cell membrane

Curative Electrochemotherapy in the Head and Neck Area

Examples of common phospholipids in human cell membranes are shown

Figure 2. Chemical structure of the common phospholipids phosphatidylserine,

Curative Electrochemotherapy in the Head and Neck Area

Membrane proteins can somewhat oversimplified be divided into three

Curative Electrochemotherapy in the Head and Neck Area

The membrane potential

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area

Figure 5. The induced membrane potential Vm by an external electrical field E in

Curative Electrochemotherapy in the Head and Neck Area

noseconds, to formation of perforating hydrophilic pores filled with

Figure 6. Electroporation hypothesis. Changes in the PLM induced by an electrical

This leads to a drastic increase in the transmembrane transport of ions and

Curative Electrochemotherapy in the Head and Neck Area

Figure 7. Summary of different EP applications in relation to electrical field

Curative Electrochemotherapy in the Head and Neck Area

Also, recently the investigation of very short (nanosecond) pulsed electrical

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area

Human tongue SCC

Chinese hamster fibroblast

Human endothelial cell

SCC, Squamous cell carcinoma

BLM can be inactivated by the intracellular enzyme bleomycin hydrolase;

Curative Electrochemotherapy in the Head and Neck Area

BLM and hence a low-permeant rather than non-permeant drug (Figure

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area

polarity was sequentially changed counter-clockwise in a pairwise 2x2

Effects of ECT on the muscle

Curative Electrochemotherapy in the Head and Neck Area

Effects of ECT on the skin

Effects of ECT on the vascular system

Curative Electrochemotherapy in the Head and Neck Area

oxygen, the antivascular mechanisms can also, theoretically, have negative

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area

Effects of ECT on the heart

Effects of ECT on the nervous system

Effects of ECT on the immune system

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area

The treated tumors

Curative Electrochemotherapy in the Head and Neck Area

Cutaneous squamous cell carcinoma (CSCC) is the third most common

Curative Electrochemotherapy in the Head and Neck Area

Head and neck squamous cell carcinoma

Curative Electrochemotherapy in the Head and Neck Area

Alcohol and tobacco consumption, especially smoking, have historically

Curative Electrochemotherapy in the Head and Neck Area

Curative Electrochemotherapy in the Head and Neck Area