Curative Electrochemotherapy in the Head and Neck Area

To Noah, Gabriella, Elias and Mikaela

Örebro Studies in Medicine 117

FREDRIK LANDSTRÖM

Title: Curative Electrochemotherapy in the Head and Neck Area. Publisher: Örebro University 2015 www.oru.se/publikationer-avhandlingar

Print: Örebro University, Repro 02/2015

ISSN 1652-4063 ISBN 978-91-7529-061-4 Abstract

Fredrik Landström (2015): Curative Electrochemotherapy in the Head and Neck Area. Örebro 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 non- melanoma 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 tumor- specific 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 Landström, School of Medicine, Örebro University, SE-701 82 Örebro, Sweden, [email protected]

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 Basal Cell Carcinoma BLM Bleomycin CAL-27 Squamous Cell CER Cytotoxicity Enhancement Ratio CR Complete Response CRR Complete Response Rate CRT Controlled Randomized Trial CSC Cancer Stem Cells CSCC Cutaneous Squamous Cell Carcinoma CT Computed Tomography CUP Cancer of Unknown Primary CV Crystal Violet staining method Da Dalton

ΔVm Induced membrane potential DWD Dead With Disease DWOD Dead Without evidence of Disease ECOG Eastern Cooperative Oncology Group ECT Electrochemotherapy EGFR Epidermal Growth Factor Receptor EORTC European Organisation for Research and Treatment of Cancer E Electric Field Strength EP Electroporation EPT Electroporation Therapy ESOPE European Standard Operation Procedures for Electrochemotherapy FB Fibroblast FOM Floor of Mouth G Electric Conductance Gy Gray HNC Head and Neck Cancer HNSCC Head and Neck Squamous Cell Carcinoma HPV Human Papilloma Virus HUVEC Human Umbilical Vein Endothelial Cells I Electric Current IGRT Image Guided RadioTherapy IMRT Intensity Modulated RadioTherapy

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 9

IRE Irreversible Electroporation IT Intratumoral IU International Units IV Intravenous NED No Evidence of Disease NMSC Non Melanoma Skin Cancer ORN Osteoradionecrosis PBL Phospholipid Bilayer PDT Photodynamic Therapy PSSHN Performance Status Scale for Head and Neck cancer QLQ-H&N35 Quality of Life Questionnaire-Head and Neck 35 RR Regional Recurrence RT Radiotherapy SC Stratum Corneum SCC Squamous Cell Carcinoma SCC-4 Squamous Cell Carcinoma Cell Line SEM Standard Error of Mean TNM Tumor, Node, Metastasis classification of malignant tumors U Units

Vrev Threshold for Reversible Electroporation

Virrev Threshold for Irreversible Electroporation

Vm Resting Membrane Potential VMAT VoluMetric Arc Therapy

10 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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. Landström FJ, Nilsson CO, Crafoord S, Reizenstein JA, Adamsson GB, Löfgren LA. Dermatol Surg. 2010 Aug; 36(8): 1245-50.

II. Electroporation therapy for T1 and T2 oral tongue cancer. Landström FJ, Nilsson CO, Reizenstein JA, Nordqvist K, Adamsson GB, Löfgren AL. Acta Otolaryngol. 2011 Jun; 131(6): 660-4.

III. Electrochemotherapy – possible benefits and limitations to its use in the head and neck region. Landström FJ, Nilsson CO, Reizenstein JA, Adamsson GB, Löfgren AL, Möller C. Acta Otolaryngol. 2015; 135: 90–95

IV. Long-term follow-up in patients treated with curative electrochemotherapy. Landström FJ, Reizenstein JA, Adamsson GB, Löfgren AL, von Beckerath M, Möller C. Submitted to Acta Otolaryngologica.

V. Electrochemotherapy – evidence for cell-type selectivity in vitro. Landström FJ, Ivarsson M, Koskela A, Magnuson A, von Beckerath M, Möller C. Submitted to Bioelectrochemistry.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 deform- ability 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).

14 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 Cl- ions 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). Prolif- erating cells, both tumor and non-tumor, seems to have a lower Vm than non-proliferating cells (Figure 4) (9).

16 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 insula- tor 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 ΔVm when an external electrical field E is applied can be described by the equation: ΔVm = f E r cos θ, where f is a factor related to the form of the cell, r is the cell radius and θ 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 17

Figure 5. The induced membrane potential ΔVm by an external electrical field E in a cell with radius r and angle θ causes hyperpolarization in the anode pole and depolarization in the cathode pole of the cell. ΔVm varies between a maximal value for θ = 0 and 0 for θ = 90°. The degree of permeabilization is bigger in the depolar- ized pole allowing for transport of larger molecules like DNA (13, 14).

The induced potential ΔVm is superimposed on the resting membrane potential Vm. When a certain threshold Vrev for ΔVm + 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 ΔVm and Vm have the same vectorial direction. At the cathode side these directions are opposite requiring a higher ΔVm 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 µm, this could be compared to an axon with a typical diameter of 0.5 µm 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 rear- rangements of the membrane phospholipid molecules leading, within na-

18 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 ΔVm + 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 homeo- stasis (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).

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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 µs, 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).

20 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 µs 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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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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 non- permeant 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 CER SCC-25 Human tongue SCC 400 DC3-F Chinese hamster fibroblast 300-700 HMEC-1 Human endothelial cell 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

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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, doxoru- bicin, 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 µs 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 dis- tributed 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

24 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 im- mediately 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 se- quence 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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Effects of ECT on the skin The skin consists of three layers, the epidermis, the dermis and the subcu- tis. 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, gen- erates 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 cap- turing 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 12- 48 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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).

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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 thor- oughly 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 µs, 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. Further- more, 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 immuno-

30 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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-Dupré-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 that´s 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 ele- vated 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 fre- quently 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 35 24 59 Mouth, unspecified location 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 HPV- induced cancer is different from alcohol and tobacco-induced cancer; it involves inactivation of the tumor suppressor genes TP53 and the reti- noblastoma 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 histological- ly 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 in-

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 preser- vation. 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 office- based 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 Swe- den 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 deter- mines 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 inhibi- tion 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 perfu- sion and oxygenation. The perimeter of the tumor often has a better per- fusion 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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, Saar- brücken, 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, Kaposi’s 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. Kaposi’s 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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=πab2/6 (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 Bowen’s 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 Kaposi’s sarcoma and recurrent Merkel cell carcinomas (93, 107, 108).

40 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 (102- 105).

Study Pats Histology Treatment Follow-up CRR (months) (%)

20 BCC IT BLM 18 98.1 Glass, 1996

Rodgriguez-C., 9 BCC IT BLM 8.6 77.8 2001 BCC, SCC, 15 IV BLM 13 80 Gargiulo, 2010 BD

3 BCC IT BLM 5 100 Salwa, 2014

BCC, Basal Cell Cacinoma; BD, Bowen’s Disease; BLM, Bleomycin; CRR, Com- plete 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 CRR (months) (%)

Allegretti, 2001 14 HNSCC, AC, IT BLM 31.5 42.9 ACC

Burian, 2003 12 HNSCC IT BLM 1 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 (Clinical- Trials.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 Örebro 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 out- Pharmacoeconomic fac- Pharmacoeconomic comes tors factors MedPulser system per- MedPulser system performance formance Adverse events in 6 Adverse events in 4 months after ECT months after ECT Evaluation of organ function and QoL 4,8 and 12 months after ECT Enrolment 95 patients 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 manufac- tured by Genetronics. It consisted of two main components; the MedPuls- er Instrument and the MedPulser Applicator (Figure 18). The instrument produced a cycle of six 100 µs 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 45

Figure 18. The Medpulser instrument with applicator attached.

Figure 19. The Medpulser applicator.

46 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 Primary outcome: The two-year local tumor control rate and treatment 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 Primary outcome: The two-year local tumor control rate and treatment 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 complicat- ed 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

Table 6. Inclusion and exclusion criteria for the patients treated with ECT in Pa- pers 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 (Pa- pers II, III, IV) Tumors with volumes requiring BLM Hypersensitivity to BLM dose < 80 U Complete tumor and margin accessibil- Lifetime BLM dose > 400 U ity with BLM and electrodes 18 years or older Patients unsuitable for general anesthesia Contraceptive methods for men and Emphysema/pulmonary fibrosis women with childbearing potential Baseline performance status: ECOG 0- Patients with pacemakers that cannot 2* be turned off Life expectancy of at least 6 months History of uncontrolled cardiac ar- rhythmia Patients must sign a written Informed Pregnancy or nursing Consent *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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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. Endo- thelial 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 (CRL- 1624) (117, 118) were obtained from American Type Culture Collection (Manassas, VA).

50 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 (Gen- etronics), which delivered a pulsed electrical field (6 x 100 μs 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 (Örebro län) 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 Örebro.

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 hospitali- zation 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 ex- amined 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 read- ing 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 patient’s 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 μL 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 μg/ml. Electroporation was performed using the Gene Pulser Xcell ™ (Bio- Rad, 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 96- well 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 Opti- ma™ 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 McNemar’s test for the paired nominal data (need for painkillers, nutritional supplements, feeding tube, weight loss and weight gain). For subgroup comparisons the Mann Whit- ney U-test was used for the ordinal data and Fishers exact test for nominal data. The α = 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 Multitox- Fluor 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 Leverne´s tests respectively. Welch’s 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. α = 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

Cohen´s 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 55

Table 7. Demographics, tumor characteristics, additional treatment and outcome after ECT in six patients with NMSC.

Pat Sex Age Tumor Tumor Localization Recurrence Additional Follow- Tumor type size treatment up status (months)

BCC Glas temporal 1 M 65 25x25x5 0 None 24 NED IA region 2 M 81 SCC 28x35x15 cheek 2 None 24 NED external Surgery 3 M 70 SCC 20x15x10 1 3 PT meatus +RT 4 F 66 SCC 10x10x5 vestibule 0 None 24 NED Metatypic medial 5 F 82 15x15x10 7 None 24 NED BCC canthus BCC Glas medial 6 F 95 18x12x5 0 None 24 NED II canthus 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 read- mitted 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 TNM Type Tumor Treatment Neck Radio- Last Tumor Tumor size(mm) date dissection therapy follow-up status status (month) tongue overall

M 30 T2N0M0 SCC 25x25x5 02-feb-05 No T+N 24 NED NED

M 45 T2N0M0 SCC 25x2x10 07-apr-05 Ipsilateral T+N 24 NED RR

F 74 T1N0M0 SCC 17x17x5 14-apr-05 Bilateral T+N(ipsilat) 24 NED RR

F 65 T2N0M0 SCC 22x14x3 06-dec-05 No No 24 NED NED

F 71 T2N0M0 SCC 35x3x15 01-dec-05 Ipsilateral T+N 8 NED DWD

F 61 T1N0M0 SCC 20x18x3 19-jan-06 No No 24 NED NED

M 60 T2N0M0 SCC 40x20x20 24-jan-06 Ipsilateral T+N 24 NED NED

M 71 T2N0M0 SCC 35x30x20 04-apr-06 No T+N 8 NED DWOD

M 20 T2N2aM0 SCC 25x25x15 18-apr-06 Ipsilateral T+N 24 NED NED

F 47 T1N2bM0 SCC 18x15x5 15-jun-06 Ipsilateral N 1 NED DWD

F 63 T2N0M0 SCC 30x25x10 20-jul-06 No T+N 24 NED NED

M 45 T2N0M0 AC 22x18x5 14-nov-06 No No 24 NED NED

M 64 T2N0M0 SCC 27x20x3 27-dec-06 No No 24 NED NED

F 53 T2N0M0 SCC 30x19x12 01-mar-07 No T+N 24 NED NED

M 46 T2NOMO SCC 22x17x1 22-mar-07 No No 24 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

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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 After Before After Before After 1 100 100 100 100 100 100 2 100 100 100 100 100 100 3 75 75 100 100 80 50 4 100 100 100 100 100 100 6 100 100 100 100 100 100 7 50 75 100 75 50 90 9 100 100 100 100 100 90 11 100 100 100 100 100 90 12 100 100 100 100 100 100 13 100 100 100 100 100 100 14 100 100 100 100 100 100 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. Understandabil- ity 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 tumour- free 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 Histology TNM Tumor RT Follow-up Tumor status size (mm) (months) Local Overall

M 59 Masseter Clear cell TXNXM1 43 × 26 × Before 4 NED DWD muscle carcinoma 26 M 65 Floor of SCC T2N0M0 30 × 30 × P + N 24 NED NED mouth 25 M 59 Bucca SCC T2N0M0 20 × 20 × P + N 24 NED NED 5 M 78 Floor of SCC T2N0M0 25 × 25 × P + N 24 NED NED mouth 5 M 58 Base of SCC T2N0M0 25 × 25 × No 24 NED NED tongue 8 DWD, dead with disease; M, male; N, neck; NED, no evidence of disease; P, primary tumor; RT, radiotherapy; SCC, squamous cell carcinoma

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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 After Before After Before After 2 100 0 100 50 100 0 3 100 100 100 100 100 100 4 100 75 100 50 100 30 6 100 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, Perfor- mance 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 didn’t (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 non- tongue 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.

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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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 McNemar’s 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 Xerostomia 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) 1 Nutritional supplements 0 (0-100) 0 (0-100) 0.62 Feeding tube 0 (0-0) 0 (0-100) 1 Weight loss 0 (0-100) 0 (0-100) 1 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 α = 0.01level (Figure 20 and Table 14). The null hypothesis

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 63 was therefore rejected for the fibroblasts. Cohen´s 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. Cohen´s 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 µg/ml) and high dose (100 µg/ml) BLM exposure. Cell survival was assessed with the MultiTox-Fluor assay. The data is based on four independent experiments.

64 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

Table 14. Result of Welch´s F-test for all cell types 4 days after EP alone, ECT with 10 μg/ml BLM (ECT L) and 100 μg/ml (ECT H) (A). The between cell type comparison with the Games-Howell post-hoc test for the significant results from Welch’s test (ECT L and ECT-H) and Cohen´s 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).

Groups F-statistic Welch´s test p-value

EP 4.79 0.048 ECT L 343.1 < 0.001 ECT H 610.4 < 0.001

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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 Welch´s 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 Cohen´s d value for these statistically significant differences showing large differences (B). The data was based on four independent experiments. (NS=not statistically significant)

Groups F-statistic Welch’s test p-value

EP day 1 115.2 < 0.001 EP day 2 136.8 < 0.001

66 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 cor- nerstones 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

68 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 (Welch´s F test) were used. Also, a conservative significance level was used (α ≤ 0.01). Cohen’s 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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). There- fore, 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 Allegretti’s 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 I- IV). The local control was 100% in these patients but only two were treated with ECT alone. Considering the very good long-term results re-

70 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 doesn’t. 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 excep- tions. 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 well- documented 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 non- dividing 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 inexperi- ence 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 diam- eters < 3 cm using the ESOPE protocol (protocol 1). There are nu- merous 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 function- sparing 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 ill- defined 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

The long-term data presented in this thesis could hopefully increase the interest in ECT as a curative treatment modality and facilitate the realisa- tion of these and many more studies.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 localiza- tions, 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 LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 Möller for guiding me through this thesis like a partner, not a parent. Thank you!

• Lennart Löfgren, 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 follow- up 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. With- out you there will be no progress.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 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 worth- while. Thank you for your patience during the last month. Now I want to spend my time with you again!

80 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area References 1. Mir LM, Orlowski S, Belehradek J, Jr., Paoletti C. Electrochemother- apy potentiation of antitumour effect of bleomycin by local electric pulses. Eur J Cancer. 1991;27(1):68-72. 2. Neumann E, Kakorin S, Toensing K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochemistry and bioenergetics (Lausanne, Switzerland). 1999;48(1):3-16. 3. Alberts B JA, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell. New York, NY: Garland Publishing; 2007. 4. Sheriff DS, Ali EF. Perspective on plasma membrane cholesterol ef- flux and spermatozoal function. Journal of human reproductive sciences. 2010;3(2):68-75. 5. van Uitert I, Le Gac S, van den Berg A. The influence of different membrane components on the electrical stability of bilayer lipid membranes. Biochimica et biophysica acta. 2010;1798(1):21-31. 6. Akamatsu Y. Reminiscence of our research on membrane phospholipids in mammalian cells by using the novel technology. Proceedings of the Japan Academy Series B, Physical and biological sciences. 2012;88(10):536-53. 7. Day RE, Kitchen P, Owen DS, Bland C, Marshall L, Conner AC, et al. Human aquaporins: regulators of transcellular water flow. Bio- chimica et biophysica acta. 2014;1840(5):1492-506. 8. Tekle E, Wolfe MD, Oubrahim H, Chock PB. Phagocytic clearance of electric field induced 'apoptosis-mimetic' cells. Biochemical and biophysical research communications. 2008;376(2):256-60. 9. Yang M, Brackenbury WJ. Membrane potential and cancer progression. Frontiers in physiology. 2013;4:185. 10. Levin M. Molecular bioelectricity in developmental biology: new tools and recent discoveries: control of cell behavior and pattern formation by transmembrane potential gradients. BioEssays : news and reviews in molecular, cellular and developmental biology. 2012;34(3):205-17.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 81

11. Chen C, Smye SW, Robinson MP, Evans JA. Membrane electroporation theories: a review. Medical & biological engineering & computing. 2006;44(1-2):5-14. 12. Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta physiologica Scandinavica. 2003;177(4):437-47. 13. Burgain-Chain A SD. DNA Electrotransfer: An Effective Tool for Gene Therapy. In: Martin F, editor. Gene Therapy-Tools and Poten- tial Applications 2013. 14. Tekle E, Astumian RD, Chock PB. Electro-permeabilization of cell membranes: effect of the resting membrane potential. Biochem Bio- phys Res Commun. 1990;172(1):282-7. 15. Zimmermann U, Pilwat G, Riemann F. Dielectric breakdown of cell membranes. Biophysical journal. 1974;14(11):881-99. 16. Gabriel B, Teissie J. Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electroper- meabilized cell membrane. Biophysical journal. 1997;73(5):2630-7. 17. Saulis G SR. Comparison of electroporation threshold for different cell lines in vitro. Acta Physica Polonica. 2009;115(6):1056-8. 18. Teissie J, Rols MP. An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. Bio- physical journal. 1993;65(1):409-13. 19. Bockmann RA, de Groot BL, Kakorin S, Neumann E, Grubmuller H. Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophysical journal. 2008;95(4):1837-50. 20. Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA. Calcium in cell injury and death. Annual review of pathology. 2006;1:405-34. 21. Deipolyi AR, Golberg A, Yarmush ML, Arellano RS, Oklu R. Irre- versible electroporation: evolution of a laboratory technique in interventional oncology. Diagnostic and interventional radiology (Ankara, Turkey). 2014;20(2):147-54.

82 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

22. Weaver JC, Smith KC, Esser AT, Son RS, Gowrishankar TR. A brief overview of electroporation pulse strength-duration space: a region where additional intracellular effects are expected. Bioelectrochemis- try. 2012;87:236-43. 23. Gehl J, Skovsgaard T, Mir LM. Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs. Anti- cancer drugs. 1998;9(4):319-25. 24. Narayanan G, Froud T, Suthar R, Barbery K. Irreversible electroporation of hepatic malignancy. Seminars in interventional radiology. 2013;30(1):67-73. 25. Hofmann GA, Dev SB, Nanda GS, Rabussay D. Electroporation therapy of solid tumors. Critical reviews in therapeutic drug carrier systems. 1999;16(6):523-69. 26. Orlowski S, Belehradek J, Jr., Paoletti C, Mir LM. Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs. Biochemical pharmacology. 1988;37(24):4727-33. 27. Mir LM, Tounekti O, Orlowski S. Bleomycin: revival of an old drug. General pharmacology. 1996;27(5):745-8. 28. Gothelf A, Mir LM, Gehl J. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer treatment reviews. 2003;29(5):371-87. 29. Tounekti O, Pron G, Belehradek J, Jr., Mir LM. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Cancer research. 1993;53(22):5462-9. 30. Okada H, Mak TW. Pathways of apoptotic and non-apoptotic death in tumour cells. Nature reviews Cancer. 2004;4(8):592-603. 31. Pron G, Mahrour N, Orlowski S, Tounekti O, Poddevin B, Belehrad- ek J, Jr., et al. Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism. Biochemical pharmacology. 1999;57(1):45-56. 32. Roy SN, Horwitz SB. Characterization of the association of radio- labeled bleomycin A2 with HeLa cells. Cancer research. 1984;44(4):1541-6.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 83

33. Cemazar M, Parkins CS, Holder AL, Chaplin DJ, Tozer GM, Sersa G. Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy. British journal of cancer. 2001;84(4):565-70. 34. Hofmann GA, Dev SB, Dimmer S, Nanda GS. Electroporation therapy: a new approach for the treatment of head and neck cancer. IEEE transactions on bio-medical engineering. 1999;46(6):752-9. 35. Poddevin B, Orlowski S, Belehradek J, Jr., Mir LM. Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. Biochemical pharmacology. 1991;42 Suppl:S67-75. 36. Mir L. Bases and rationale of the electrchemotherapy. EJC Supple- ments. 2006;4:38-44. 37. Soultati A, Mountzios G, Avgerinou C, Papaxoinis G, Pectasides D, Dimopoulos MA, et al. Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications. Cancer treatment reviews. 2012;38(5):473-83. 38. Kotnik T, Macek-Lebar A, Miklavcic D, Mir LM. Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent. BioTechniques. 2000;28(5):921-6. 39. Miklavcic D, Corovic S, Pucihar G, Pavselj N. Importance of tumour coverage by sufficiently hihg local electric field for effective electrochemotherapy. EJC Supplements. 2006;4:45-51. 40. Gilbert RA, Jaroszeski MJ, Heller R. Novel electrode designs for electrochemotherapy. Biochimica et biophysica acta. 1997;1334(1):9- 14. 41. Corovic S, Lackovic I, Sustaric P, Sustar T, Rodic T, Miklavcic D. Modeling of electric field distribution in tissues during electroporation. Biomedical engineering online. 2013;12:16. 42. Heller R, Coppola D, Pottinger C, Gilbert R, Jaroszeski MJ. Effect of electrochemotherapy on muscle and skin. Technology in cancer research & treatment. 2002;1(5):385-92.

84 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

43. Miklavcic D, Pucihar G, Pavlovec M, Ribaric S, Mali M, Macek- Lebar A, et al. The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy. Bioelectrochemistry (Amsterdam, Neth- erlands). 2005;65(2):121-8. 44. Prausnitz MR, Bose VG, Langer R, Weaver JC. Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery. Proceedings of the National Academy of Sciences of the United States of America. 1993;90(22):10504-8. 45. Gothelf A, Mahmood F, Dagnaes-Hansen F, Gehl J. Efficacy of transgene expression in porcine skin as a function of electrode choice. Bioelectrochemistry (Amsterdam, Netherlands). 2011;82(2):95-102. 46. Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochimica et biophysica acta. 2002;1569(1-3):51-8. 47. Sersa G, Cemazar M, Miklavcic D, Chaplin DJ. Tumor blood flow modifying effect of electrochemotherapy with bleomycin. Anticancer Res. 1999;19(5b):4017-22. 48. Sersa G, Jarm T, Kotnik T, Coer A, Podkrajsek M, Sentjurc M, et al. Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma. British journal of cancer. 2008;98(2):388-98. 49. Domenge C, Orlowski S, Luboinski B, De Baere T, Schwaab G, Belehradek J, Jr., et al. Antitumor electrochemotherapy: new advances in the clinical protocol. Cancer. 1996;77(5):956-63. 50. Jarm T, Cemazar M, Miklavcic D, Sersa G. Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases. Expert review of anticancer therapy. 2010;10(5):729-46. 51. Gehl J, Geertsen PF. Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy. Melanoma research. 2000;10(6):585-9. 52. Al-Khadra A, Nikolski V, Efimov IR. The role of electroporation in defibrillation. Circulation research. 2000;87(9):797-804.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 85

53. Mali B, Jarm T, Corovic S, Paulin-Kosir MS, Cemazar M, Sersa G, et al. The effect of electroporation pulses on functioning of the heart. Medical & biological engineering & computing. 2008;46(8):745-57. 54. Tsourtos G, Spong J, Stough C. The effects of electro-convulsive therapy on the speed of information processing in major depression. Journal of affective disorders. 2007;103(1-3):263-6. 55. Schoellnast H, Monette S, Ezell PC, Maybody M, Erinjeri JP, Stub- blefield MD, et al. The delayed effects of irreversible electroporation ablation on nerves. European radiology. 2013;23(2):375-80. 56. Reisner AD. Possible mechanisms for delayed neurological damage in lightning and electrical injury. Brain injury. 2013;27(5):565-9. 57. Mir LM, Orlowski S, Poddevin B, Belehradek J, Jr. Electrochemo- therapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses. European cytokine network. 1992;3(3):331-4. 58. Miyazaki S, Gunji Y, Matsubara H, Shimada H, Uesato M, Suzuki T, et al. Possible involvement of antitumor immunity in the eradication of colon 26 induced by low-voltage electrochemotherapy with bleomycin. Surgery today. 2003;33(1):39-44. 59. Hadrup SR, Gehl J, Sorensen RB, Geertsen PF, Straten PT, Andersen MH. Persistence of survivin specific T cells for seven years in a melanoma patient during complete remission. Cancer biology & therapy. 2006;5(5):480-2. 60. Basal Cell Carcinoma in Sweden 2004–2008 Socialstyrelsen, 2009. 61. Scrivener Y, Grosshans E, Cribier B. Variations of basal cell carcinomas according to gender, age, location and histopathological subtype. The British journal of dermatology. 2002;147(1):41-7. 62. Kraft S, Granter SR. Molecular pathology of skin neoplasms of the head and neck. Archives of pathology & laboratory medicine. 2014;138(6):759-87. 63. Arnon O, Rapini RP, Mamelak AJ, Goldberg LH. Mohs micrographic surgery: current techniques. The Israel Medical Association journal : IMAJ. 2010;12(7):431-5.

86 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

64. Roenigk RK, Ratz JL, Bailin PL, Wheeland RG. Trends in the presentation and treatment of basal cell carcinomas. The Journal of dermatologic surgery and oncology. 1986;12(8):860-5. 65. Seccia V, Muscatello L, Dallan I, Bajraktari A, Briganti T, Ursino S, et al. Electrochemotherapy and its controversial results in patients with head and neck cancer. Anticancer research. 2014;34(2):967-72. 66. Ratushny V, Gober MD, Hick R, Ridky TW, Seykora JT. From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma. The Journal of clinical investigation. 2012;122(2):464-72. 67. Rowe DE, Carroll RJ, Day CL, Jr. Prognostic factors for local recurrence, metastasis, and survival rates in squamous cell carcinoma of the skin, ear, and lip. Implications for treatment modality selection. Journal of the American Academy of Dermatology. 1992;26(6):976- 90. 68. Parikh SA, Patel VA, Ratner D. Advances in the management of cutaneous squamous cell carcinoma. F1000prime reports. 2014;6:70. 69. Leibovitch I, Huilgol SC, Selva D, Hill D, Richards S, Paver R. Cuta- neous squamous cell carcinoma treated with Mohs micrographic surgery in Australia I. Experience over 10 years. Journal of the American Academy of Dermatology. 2005;53(2):253-60. 70. Kwan W, Wilson D, Moravan V. Radiotherapy for locally advanced basal cell and squamous cell carcinomas of the skin. International journal of radiation oncology, biology, physics. 2004;60(2):406-11. 71. Guibert M, David I, Vergez S, Rives M, Filleron T, Bonnet J, et al. Brachytherapy in lip carcinoma: long-term results. International journal of radiation oncology, biology, physics. 2011;81(5):e839-43. 72. Willey A, Mehta S, Lee PK. Reduction in the incidence of squamous cell carcinoma in solid organ transplant recipients treated with cyclic photodynamic therapy. Dermatologic surgery : official publication for American Society for Dermatologic Surgery [et al]. 2010;36(5):652-8.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 87

73. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer Journal international du cancer. 2015;136(5):E359-86. 74. Franceschi S, Bidoli E, Herrero R, Munoz N. Comparison of cancers of the oral cavity and pharynx worldwide: etiological clues. Oral oncology. 2000;36(1):106-15. 75. Ramqvist T, Dalianis T. Oropharyngeal cancer epidemic and human papillomavirus. Emerging infectious diseases. 2010;16(11):1671-7. 76. Machiels JP, Lambrecht M, Hanin FX, Duprez T, Gregoire V, Schmitz S, et al. Advances in the management of squamous cell carcinoma of the head and neck. F1000prime reports. 2014;6:44. 77. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer. 1953;6(5):963-8. 78. van Oijen MG, Slootweg PJ. Oral field cancerization: carcinogen- induced independent events or micrometastatic deposits? Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Soci- ety of Preventive Oncology. 2000;9(3):249-56. 79. Ritchie KE, Nor JE. Perivascular stem cell niche in head and neck cancer. Cancer letters. 2013;338(1):41-6. 80. Sparano A, Weinstein G, Chalian A, Yodul M, Weber R. Multivari- ate predictors of occult neck metastasis in early oral tongue cancer. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery. 2004;131(4):472-6. 81. Fukano H, Matsuura H, Hasegawa Y, Nakamura S. Depth of invasion as a predictive factor for cervical lymph node metastasis in tongue carcinoma. Head & neck. 1997;19(3):205-10. 82. Hammarstedt L, Lu Y, Marklund L, Dalianis T, Munck-Wikland E, Ye W. Differential survival trends for patients with tonsillar, base of tongue and tongue cancer in Sweden. Oral oncology. 2011;47(7):636-41.

88 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

83. Nordgren M, Hammerlid E, Bjordal K, Ahlner-Elmqvist M, Boysen M, Jannert M. Quality of life in oral carcinoma: a 5-year prospective study. Head & neck. 2008;30(4):461-70. 84. Day GL, Blot WJ. Second primary tumors in patients with oral cancer. Cancer. 1992;70(1):14-9. 85. Hasina R, Lingen MW. Angiogenesis in oral cancer. Journal of dental education. 2001;65(11):1282-90. 86. Krishnamurthy S, Nor JE. Head and neck cancer stem cells. Journal of dental research. 2012;91(4):334-40. 87. Lin Q, Yun Z. Impact of the hypoxic tumor microenvironment on the regulation of cancer stem cell characteristics. Cancer biology & therapy. 2010;9(12):949-56. 88. Krishnamurthy S, Dong Z, Vodopyanov D, Imai A, Helman JI, Prince ME, et al. Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells. Cancer research. 2010;70(23):9969-78. 89. Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek J, Jr., Mir LM. Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer. 1993;72(12):3694-700. 90. Heller R, Jaroszeski MJ, Glass LF, Messina JL, Rapaport DP, DeCon- ti RC, et al. Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer. 1996;77(5):964- 71. 91. Gaudy C, Richard MA, Folchetti G, Bonerandi JJ, Grob JJ. Random- ized controlled study of electrochemotherapy in the local treatment of skin metastases of melanoma. Journal of cutaneous medicine and surgery. 2006;10(3):115-21. 92. Bloom DC, Goldfarb PM. The role of intratumour therapy with electroporation and bleomycin in the management of advanced squamous cell carcinoma of the head and neck. European journal of surgical oncology : the journal of the European Society of Surgical On- cology and the British Association of Surgical Oncology. 2005;31(9):1029-35.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 89

93. Di Monta G, Caraco C, Benedetto L, La Padula S, Marone U, Torne- sello ML, et al. Electrochemotherapy as "new standard of care" treatment for cutaneous Kaposi's sarcoma. European journal of surgical oncology : the journal of the European Society of Surgical On- cology and the British Association of Surgical Oncology. 2014;40(1):61-6. 94. Rebersek M, Cufer T, Cemazar M, Kranjc S, Sersa G. Electrochemo- therapy with cisplatin of cutaneous tumor lesions in breast cancer. Anti-cancer drugs. 2004;15(6):593-7. 95. Snoj M, Cemazar M, Srnovrsnik T, Kosir SP, Sersa G. Limb sparing treatment of bleeding melanoma recurrence by electrochemotherapy. Tumori. 2009;95(3):398-402. 96. Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology. 2013;39(1):4-16. 97. Landstrom FJ, Nilsson CO, Crafoord S, Reizenstein JA, Adamsson GB, Lofgren LA. Electroporation therapy of skin cancer in the head and neck area. Dermatologic surgery : official publication for Ameri- can Society for Dermatologic Surgery [et al]. 2010;36(8):1245-50. 98. Landstrom FJ, Nilsson CO, Reizenstein JA, Nordqvist K, Adamsson GB, Lofgren AL. Electroporation therapy for T1 and T2 oral tongue cancer. Acta oto-laryngologica. 2011;131(6):660-4. 99. Mali B, Miklavcic D, Campana LG, Cemazar M, Sersa G, Snoj M, et al. Tumor size and effectiveness of electrochemotherapy. Radiology and oncology. 2013;47(1):32-41. 100. Mir LM, Gehl J, Sersa G, Collins CG, Garbay J-R, Billard V, et al. Standard operating procedures of the electrochemotherapy: Instruc- tions for the use of bleomycin and cisplatin administered either systemically or locally and electric pulses delivered by the Cliniporator by means of inasive or noninvasive electrodes. European Journal of Cancer Supplements. 2006(4):14-25.

90 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

101. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy – An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. EJC Supplements.4(11):3-13. 102. Glass LF, Jaroszeski M, Gilbert R, Reintgen DS, Heller R. Intrale- sional bleomycin-mediated electrochemotherapy in 20 patients with basal cell carcinoma. Journal of the American Academy of Dermatol- ogy. 1997;37(4):596-9. 103. Rodriguez-Cuevas S, Barroso-Bravo S, Almanza-Estrada J, Cristobal- Martinez L, Gonzalez-Rodriguez E. Electrochemotherapy in primary and metastatic skin tumors: phase II trial using intralesional bleomycin. Archives of medical research. 2001;32(4):273-6. 104. Salwa SP, Bourke MG, Forde PF, O'Shaughnessy M, O'Sullivan ST, Kelly EJ, et al. Electrochemotherapy for the treatment of ocular basal cell carcinoma; a novel adjunct in the disease management. Journal of plastic, reconstructive & aesthetic surgery : JPRAS. 2014;67(3):403- 6. 105. Gargiulo M, Moio M, Monda G, Parascandolo S, Cubicciotti G. Electrochemotherapy: actual considerations and clinical experience in head and neck cancers. Annals of surgery. 2010;251(4):773. 106. Kis E, Baltas E, Kinyo A, Varga E, Nagy N, Gyulai R, et al. Success- ful treatment of multiple basaliomas with bleomycin-based electrochemotherapy: a case series of three patients with Gorlin-Goltz syndrome. Acta dermato-venereologica. 2012;92(6):648-51. 107. Scelsi D, Mevio N, Bertino G, Occhini A, Brazzelli V, Morbini P, et al. Electrochemotherapy as a new therapeutic strategy in advanced Merkel cell carcinoma of head and neck region. Radiology and oncology. 2013;47(4):366-9. 108. Curatolo P, Quaglino P, Marenco F, Mancini M, Nardo T, Mortera C, et al. Electrochemotherapy in the treatment of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial. Annals of surgical oncology. 2012;19(1):192-8.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 91

109. Panje WR, Hier MP, Garman GR, Harrell E, Goldman A, Bloch I. Electroporation therapy of head and neck cancer. The Annals of otology, rhinology, and laryngology. 1998;107(9 Pt 1):779-85. 110. Allegretti JP, Panje WR. Electroporation therapy for head and neck cancer including carotid artery involvement. The Laryngoscope. 2001;111(1):52-6. 111. Panje WR, Sadeghi N. Endoscopic and electroporation therapy of paranasal sinus tumors. American journal of rhinology. 2000;14(3):187-91. 112. Burian M, Formanek M, Regele H. Electroporation therapy in head and neck cancer. Acta Otolaryngol. 2003;123(2):264-8. 113. Campana LG, Mali B, Sersa G, Valpione S, Giorgi CA, Strojan P, et al. Electrochemotherapy in non-melanoma head and neck cancers: a retrospective analysis of the treated cases. The British journal of oral & maxillofacial surgery. 2014;52(10):957-64. 114. Kallman J, Schollin J, Hakansson S, Andersson A, Kihlstrom E. Ad- herence of group B streptococci to human endothelial cells in vitro. APMIS : acta pathologica, microbiologica, et immunologica Scandi- navica. 1993;101(5):403-8. 115. Jiang L, Ji N, Zhou Y, Li J, Liu X, Wang Z, et al. CAL 27 is an oral adenosquamous carcinoma cell line. Oral oncology. 2009;45(11):e204-7. 116. Gioanni J, Fischel JL, Lambert JC, Demard F, Mazeau C, Zanghellini E, et al. Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment. European journal of cancer & clinical oncology. 1988;24(9):1445-55. 117. Rheinwald JG, Beckett MA. Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes. Cell. 1980;22(2 Pt 2):629-32. 118. Rheinwald JG, Beckett MA. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer research. 1981;41(5):1657-63.

92 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

119. List MA, D'Antonio LL, Cella DF, Siston A, Mumby P, Haraf D, et al. The Performance Status Scale for Head and Neck Cancer Patients and the Functional Assessment of Cancer Therapy-Head and Neck Scale. A study of utility and validity. Cancer. 1996;77(11):2294-301. 120. Bjordal K, Hammerlid E, Ahlner-Elmqvist M, de Graeff A, Boysen M, Evensen JF, et al. Quality of life in head and neck cancer patients: validation of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-H&N35. Journal of clinical oncology : official journal of the American Society of Clinical Oncol- ogy. 1999;17(3):1008-19. 121. Aaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, et al. The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. Journal of the National Cancer Institute. 1993;85(5):365-76. 122. Fayers PM, Aaronson NK, K. B, M. G, D. C, A. B. The EORTC QLQ-C30 Scoring Manual (3rd Edition). Brussels: European Organi- sation for Research and Treatment of Cancer. 2001. 123. Landstrom FJ, Reizenstein JA, CO SN, Beckerath MV, Lofgren AL, Adamsson GB, et al. Electrochemotherapy - possible benefits and limitations to its use in the head and neck region. Acta otolaryngologica. 2015;135(1):90-5. 124. Raeisi E, Aghamiri SM, Bandi A, Rahmatpour N, Firoozabadi SM, Kafi-Abad SA, et al. The antitumor efficiency of combined electrochemotherapy and single dose irradiation on a breast cancer tumor model. Radiology and oncology. 2012;46(3):226-32. 125. Snoj M, Rudolf Z, Cemazar M, Jancar B, Sersa G. Successful sphinc- ter-saving treatment of anorectal malignant melanoma with electrochemotherapy, local excision and adjuvant brachytherapy. Anti- cancer drugs. 2005;16(3):345-8. 126. Rusthoven K, Ballonoff A, Raben D, Chen C. Poor prognosis in patients with stage I and II oral tongue squamous cell carcinoma. Can- cer. 2008;112(2):345-51.

FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area 93

127. Mitchell R, Crighton LE. The management of patients with carcinoma of the tongue. The British journal of oral & maxillofacial surgery. 1993;31(5):304-8. 128. O'Brien CJ, Lahr CJ, Soong SJ, Gandour MJ, Jones JM, Urist MM, et al. Surgical treatment of early-stage carcinoma of the oral tongue-- wound adjuvant treatment be beneficial? Head & neck surgery. 1986;8(6):401-8. 129. Glanzmann C, Gratz KW. Radionecrosis of the mandibula: a retrospective analysis of the incidence and risk factors. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. 1995;36(2):94-100. 130. Wong CY, Helm MA, Kalb RE, Helm TN, Zeitouni NC. The presentation, pathology, and current management strategies of cutaneous metastasis. North American journal of medical sciences. 2013;5(9):499-504. 131. Mevio N, Bertino G, Occhini A, Scelsi D, Tagliabue M, Mura F, et al. Electrochemotherapy for the treatment of recurrent head and neck cancers: preliminary results. Tumori. 2012;98(3):308-13. 132. Macri GF, Greco A, Gallo A, Fusconi M, Marinelli C, de Vincentiis M. Use of electrochemotherapy in a case of neck skin metastasis of oral squamous cell carcinoma: case report and considerations. Head & neck. 2014;36(9):E86-90. 133. Campana LG, Bertino G, Rossi CR, Occhini A, Rossi M, Valpione S, et al. The value of electrochemotherapy in the treatment of peristo- mal tumors. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Associa- tion of Surgical Oncology. 2014;40(3):260-2.

94 FREDRIK LANDSTRÖM Curative Electrochemotherapy in the Head and Neck Area

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 No restriction of place, food or companion (eats out at any 90 Full diet (liquid assist) opportunity)

80 All meat 75 No restriction of place, but restricts diet when in public (eats anywhere, 70 Raw carrots, celery but may limit intake to less "messy" foods (e.g., liquids) 60 Dry bread and crackers 50 Eats only in presence of selected 50 Soft chewable foods (e.g., macaroni, persons in selected places canned/soft fruits, cooked vegetables, fish, hamburger, small pieces of meat) 25 Eats only at home in presence of selected persons 40 Soft foods requiring no chewing (e.g., mashed potatoes, apple 0 Always eats alone sauce, pudding) 999 Inpatient 30 Pureed foods (in blender)

20 Warm liquids UNDERSTANDABILITY OF SPEECH /__/__/__/

10 Cold liquids 100 Always understandable 0 Non-oral feeding (tube fed) 75 Understandable most of the time; occasional repetition necessary

50 Usually understandable; face-to-face contact necessary

25 Difficult to understand

0 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 Örebro 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. Wågsäter, 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. Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance. 10. Anderzén 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. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies. 15. Andrén, Ove (2008). Natural history and prognostic factors in localized prostate cancer. 16. Svantesson, Mia (2008). Postpone death? Nurse-physician perspectives and ethics rounds. 17. Björk, 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 Methicillin- Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals. 21. Nilsagård, 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-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study. 25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α 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. Elfström, Peter (2009). Associated disorders in celiac disease. 28. Skårberg, 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, Renée (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. Söderqvist, 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 parent–child interaction interventions and beneficial meetings. 37. Ekwall, Eva (2009). Women’s 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 Non- Persistence. 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. Franzén, 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. Holländare, 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, Mårten (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 carcinoma. 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. Mårild, Karl (2012). Risk Factors and Associated Disorders of Celiac 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. Lönn, 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. Nordenskjöld, 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. Hörer, Tal (2013): Early detection of major surgical postoperative complications evaluated by microdialysis. 90. Malakkaran Lindqvist, Breezy (2013): Biological signature of HER2- positive breast cancer. 91. Lidén, 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 Ekbäck, 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. Jildenstål, Pether (2014): Influence of depth of anaesthesia on postoperative cognitive dysfunction (POCD) and inflammatory marker. 102. Söderström, Ulf (2014): Type 1 diabetes in children with non-Swedish background – epidemiology and clinical outcome 103. Wilhelmsson Göstas, 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, multidrug- resistance and induction of host defense mechanisms 106. Mohseni, Shahin (2014): The role of ß-blockade and anticoagula- tion therapy in traumatic brain injury 107. Bašić, Vladimir T. (2014): Molecular mechanisms mediating development of pulmonary cachexia in COPD 108. Kirrander, Peter (2014): Penile Cancer: Studies on Prognostic Factors 109. Törös, 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. Klarström Engström, Kristin (2015): Platelets as immune cells in sensing bacterial infection. 117. Landström, Fredrik (2015): Curative Electrochemotherapy in the Head and Neck Area