Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Radiation Therapy combined with Pulsed Electric Field - A new efficient tumor treatment modality Bertil R. Persson1, Catrin Bauréus Koch1,2, Gustav Grafström1, Per Engström1, Crister Ceberg1, Henrietta Nittby2, Bengt Widegren3, Leif G. Salford2, 1Medical Radiation Research, Lund, Sweden, 2Dept of Neurosurgery, Lund, Sweden, 3Dept of Tumor Immunology, Lund, Sweden 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Aim of the study Study the therapeutic effect of treatment subcutaneously implanted glioma N32 tumours on male rats of the Fischer-344 strain on the thigh with Pulsed Electric Fields (PEF) in combination with Radiation therapy. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden E X P E R I M E N T A L S E T - U P N32 GLIOMA TUMOUR Steel plates connected to exponential high voltage pulse supply CONNECTION TO EP MODULE SLIDE CALIPER 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Treatment procedure After 4 weeks, a solid tumour has grown to a size of 1 cm located directly under the skin. The fur over the tumour was shaven and the tumour was fixed in position between two plate electrodes. Electrocardial paste was used to achieve good electric conductivity between tissue and electrodes. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Pulsed Electric Field Treatment Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Pulsed Electric Field Treatment Electric pulses were delivered through two flat electrodes sized 2x2 cm and mounted on a slide calliper to measure the distance. 16 pulses with a field strength of 1400 V/cm and a time constant of 1.0 ms were delivered at approximately one pulse per second. All animals were treated during four consecutive days. Animals were sacrificed when tumour volume reached 5 cm3 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Radiation Therapy Radiation therapy was performed with 60Co-gamma radiation focused on the tumour. The total absorbed radiation dose given to the tumours was 20 Gy, given in 4 fractions of 5 Gy each day in four consecutive days. Radiation treatment was conducted before the treatments with high voltage pulses (X+EP), or the reverse (EP+X) the pulsation of the tumor took place 4 to 5 minutes before radiation treatment. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Exponential Growth of N32 Tumours implanted in the back leg of Rats Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Exponential Growth of N32 Tumours implanted in the back leg of Rats 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Radiation therapy prior to High Voltage Pulse treatment. Average of 5 rats. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

High Voltage pulses prior to Radiation Therapy. Average of 4 rats. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden High Voltage pulses prior to Radiation Therapy. Average of 4 rats. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Tumour growth rate “TGR” Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Tumour growth rate “TGR” TGR is estimated from the tumour volume measurements of each tumour fitted to a model of exponential growth. The tumour volume growth rate is thus defined according to the following equation. where TV Tumour volume TGR Tumour growth rate constant day-1 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Mean tumour growth rate Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Mean tumour growth rate 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Specific Therapeutic Effect “STE” Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Specific Therapeutic Effect “STE” The average of the individual Tumour growth rate constant in the group of exposed rats. day-1 The average of the individual Tumour growth rate constant in the group of control rats. day-1 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Specific therapeutic effect "STE" Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Specific therapeutic effect "STE"   The STE is equal to 0 when the average of tumour growth rate constant of the exposed group, is equal to the average of the tumour growth rate constant of the control. The STE is equal to 1 when the average tumour growth rate constant of the exposed group, is equal to 0, which means arrested tumour growth. The STE is larger than 1 when the average tumour growth rate constant of the exposed group, is less than 0, which means a declining tumour volume. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Specific Therapeutic Effect & Therapeutic Enhancement Ratio Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Specific Therapeutic Effect & Therapeutic Enhancement Ratio 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

The therapeutic enhancement ratio “TER” Bertil R.R. Persson, Lund University, 22185 Lund , Sweden The therapeutic enhancement ratio “TER” TER of the combined treatments is the ratio of the specific therapeutic effect “STE” of the experimental combination of Electrical pulses and radiation and the specific therapeutic effect the hypothetical independent combination of the two agents. where the hypothetical therapeutic effect by independent (additive) action of ionizing radiation and Electrical pulses is given by 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Therapeutic Enhancement Ratio “TER” Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Therapeutic Enhancement Ratio “TER” TER is a measure of any synergistic or diminishing effect obtained in the combination of the two agents. TER > 1 may due to synergistic interaction of sublethal lesions induced by both agents to produce more lethal events. TER< 1 may due to “overkilling” that reduce the effect compared to the additive action . 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Specific Therapeutic Effect & Therapeutic Enhancement Ratio Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Specific Therapeutic Effect & Therapeutic Enhancement Ratio 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Survival after Combined treatment radiation prior/after High Voltage Pulses 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Treatment data and results Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Treatment data and results Type of Treatment Survival time (days) excl. cures TGD (%) of non-cured Tot N Number of cures (CR>80 d post treatm) Fisher exact p two-tailed Exp. vs. Contr. Exp. vs. RT. Control 41.5  1.1   8 RT 50  1.1 21 5 EP 70  14.6 69 4 1 p= 0.33 p= 0.44 EP+RT 98.3  0.7 137 9 6 p= 0.0090 p= 0.031 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Control Lower quarter of picture 1s dominated by spontaneous, amorphous necrosis bordered by a dark blue convoluted band of vital tumor, the tumor rim sharply demarcated from subcutaneous tissue and above that skin with hair follicles. 20. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

EP+RT; 2 hrs after treatment Bertil R.R. Persson, Lund University, 22185 Lund , Sweden EP+RT; 2 hrs after treatment The tumor rim between old, deep amorphous necrosis and skin consists of vital tumor cells and contains d. multiple areas of small fresh hemorrhages and e. small areas of loose tissue due to edema. 20 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

EP+RT; 2 hrs post treatment. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden EP+RT; 2 hrs post treatment. EP+RT; 2 hrs post treatment. Endothelium of some major vessels with continuously stained and apparently intact vessel walls. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

EP+RT; 36 hrs post treatment. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden EP+RT; 36 hrs post treatment. 36 hrs post combined treatment with E-pulses and Radiation. Arrows show examples of staining of fragmented endothelial linings of blood vessels. None of the major vessels appears intact. The upper third of the picture is dominated by spontaneous amorphous necrosis. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

TGR Summary of 4 equal experimental series Bertil R.R. Persson, Lund University, 22185 Lund , Sweden TGR Summary of 4 equal experimental series 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Specific Therapeutic Effect: STE = (TGRCtrl-TGRExp)/TGRCtrl Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Specific Therapeutic Effect: STE = (TGRCtrl-TGRExp)/TGRCtrl Summary of 4 eqivalent experimental series Tumour Enhancement Ratio = 0.95 0.29 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

DISCUSSION AND CONCLUSIONS Bertil R.R. Persson, Lund University, 22185 Lund , Sweden DISCUSSION AND CONCLUSIONS Pulsed Electric Fields (1400 V/cm) of millisecond duration has similar effects on regression of glioma tumour implanted on the flank of Fischer 344 rats as konventional radiation therapy (Radiomimic effect) Pulsed Electric Fields (1400 V/cm) of ms duration combined with radiation therapy has an astonishing enhancément of the therapeutic effect with high fraction of complete remissions. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Reactive Oxygen Species (”ROS”) and Free Radicals Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Reactive Oxygen Species (”ROS”) and Free Radicals Reactive Oxygen Species (”ROS”) are induced by applying high voltage electrical pulses in vivo ROS are associated with cell-damaging action. (Gabriel,B. and Teissié, J. Eur. J. Biochem. 221, 25-33 1994). Oxidative stress is one of several factors known to i induce programmed cell death, i.e. apoptosis {BRIELMEIER1998}. By combining Electical impulse treatment and radiation therapy ROS react with the radiation generated free radicals that enhance cell destruction . 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Vascular Effects Impaired oxygen supply due to extensive vascular damage or ischemia also cause apoptotic tumour cell death. Sersa and colleagues have reported on marked and long lasting (> 24 hrs) decrease in tumour blood flow after application of high-voltage electric pulses (RAMIREZ1998}{SERSA1999}. Repeated treatment with pulsed electric fields may act as a continuous ischemia or vascular occlusion, and effectively cause tumour cell death {CHAPLIN1994}{DENEKAMP1983}{PARKINS1994}. Tumours generally have a chaotic and badly structured microvasculature compared to normal tissue. This characteristic may render tumours especially sensitive to the effects of electric pulses {SERSA1999}. Pulsed Electric Fields appear to cause an immediate vascular contraction, which results from nervous reflexes and local myogenic spasm {GUYTON1996}{TORTORA1996}. This vascular spasm is transient but may last considerably longer for tumour tissue than for normal tissue. 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Local tumor irradiation augments the antitumour effect of cytokine producing autologous cancer cell vaccines in a murine glioma model ( Katalin Lumniczky et al. Budapest, Hungary 2002) ”Strong synergism was observed by combining cytokine vaccination (GM-CSF, IL-4, IL-12) with local tumor irradiation 6 Gy: about 80-100% of the glioma bearing mice was cured.” 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

PEF + Radiation+Immunisation Bertil R.R. Persson, Lund University, 22185 Lund , Sweden PEF + Radiation+Immunisation 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Lymphocyte Proliferation after PEF & Radiation treatment Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Lymphocyte Proliferation after PEF & Radiation treatment   Controls EPsqu EPexp Rad EPexp +Rad 50k cells 173  311 14  40 8  181 -9  185 37  1 15 k Cell 447  1001 97  268 79  657 -40  588 636  729 450 k Cells 558  915 -25  427 -70  1453 140  1940 4086  3555 AVERAGE 383  185 29  62 6  181 31  244 1587  874 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Lymphocyte Proliferation after IF, PEF and Radiation treatment Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Lymphocyte Proliferation after IF, PEF and Radiation treatment   IF EPsq +IF EPexp+ IF Rad+IF EPexp +Rad+IF 50k cells 1110  1188 96  64 419  587 104  147 19  18 15 k Cell 909  708 331  409 351  506 896  997 45  49 450 k Cells 2280  3923 2076  2604 80  269 1935  2743 88  147 AVERAGE 1433  515 834  434 283  103 978  415 51  28 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden SLUT 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Lymphocyte Proliferation after IF, PEF and Radiation treatment Bertil R.R. Persson, Lund University, 22185 Lund , Sweden Lymphocyte Proliferation after IF, PEF and Radiation treatment 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se

Bertil R.R. Persson, Lund University, 22185 Lund , Sweden 2002-05-03 SNOG Bertil.Persson@radfys.lu.se Bertil.Persson@radfys.lu.se