1. 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
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2. 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.
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3. 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
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4. 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.
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5. Pulsed Electric Field Treatment
Bertil R.R. Persson, Lund University, 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
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6. 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.
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7. Exponential Growth of N32 Tumours implanted in the back leg of Rats
Bertil R.R. Persson, Lund University, Lund , Sweden
Exponential Growth of N32 Tumours implanted in the back leg of Rats
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8. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden
Radiation therapy prior to High Voltage Pulse treatment. Average of 5 rats.
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9. High Voltage pulses prior to Radiation Therapy. Average of 4 rats.
Bertil R.R. Persson, Lund University, Lund , Sweden
High Voltage pulses prior to Radiation Therapy. Average of 4 rats.
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10. Tumour growth rate “TGR”
Bertil R.R. Persson, Lund University, 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
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11. Mean tumour growth rate
Bertil R.R. Persson, Lund University, Lund , Sweden
Mean tumour growth rate
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12. Specific Therapeutic Effect “STE”
Bertil R.R. Persson, Lund University, 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
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13. Specific therapeutic effect "STE"
Bertil R.R. Persson, Lund University, 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.
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14. Specific Therapeutic Effect & Therapeutic Enhancement Ratio
Bertil R.R. Persson, Lund University, Lund , Sweden
Specific Therapeutic Effect & Therapeutic Enhancement Ratio
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15. The therapeutic enhancement ratio “TER”
Bertil R.R. Persson, Lund University, 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
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16. Therapeutic Enhancement Ratio “TER”
Bertil R.R. Persson, Lund University, 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 .
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17. Specific Therapeutic Effect & Therapeutic Enhancement Ratio
Bertil R.R. Persson, Lund University, Lund , Sweden
Specific Therapeutic Effect & Therapeutic Enhancement Ratio
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18. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden
Survival after Combined treatment radiation prior/after High Voltage Pulses
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19. Treatment data and results
Bertil R.R. Persson, Lund University, 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=
p= 0.031
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20. 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.
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21. EP+RT; 2 hrs after treatment
Bertil R.R. Persson, Lund University, 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
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22. EP+RT; 2 hrs post treatment.
Bertil R.R. Persson, Lund University, 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.
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23. EP+RT; 36 hrs post treatment.
Bertil R.R. Persson, Lund University, 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.
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24. TGR Summary of 4 equal experimental series
Bertil R.R. Persson, Lund University, Lund , Sweden
TGR Summary of 4 equal experimental series
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25. Specific Therapeutic Effect: STE = (TGRCtrl-TGRExp)/TGRCtrl
Bertil R.R. Persson, Lund University, Lund , Sweden
Specific Therapeutic Effect: STE = (TGRCtrl-TGRExp)/TGRCtrl
Summary of 4 eqivalent experimental series
Tumour Enhancement Ratio = 0.95 0.29
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26. DISCUSSION AND CONCLUSIONS
Bertil R.R. Persson, Lund University, 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.
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27. Reactive Oxygen Species (”ROS”) and Free Radicals
Bertil R.R. Persson, Lund University, 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, ).
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 .
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28. 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.
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29. 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 % of the glioma bearing mice was cured.”
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30. PEF + Radiation+Immunisation
Bertil R.R. Persson, Lund University, Lund , Sweden
PEF + Radiation+Immunisation
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31. Lymphocyte Proliferation after PEF & Radiation treatment
Bertil R.R. Persson, Lund University, 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
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32. Lymphocyte Proliferation after IF, PEF and Radiation treatment
Bertil R.R. Persson, Lund University, 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
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33. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden
SLUT
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34. Lymphocyte Proliferation after IF, PEF and Radiation treatment
Bertil R.R. Persson, Lund University, Lund , Sweden
Lymphocyte Proliferation after IF, PEF and Radiation treatment
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35. Bertil R.R. Persson, Lund University, 22185 Lund , Sweden
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