Michael H. Holzscheiter 1 SPSC Meeting October 26
Status Report on AD-4/ACEStatus Report on AD-4/ACEAntiproton Cell ExperimentAntiproton Cell Experiment
The Biological Effectiveness of Antiproton Annihilation
Nzhde Agazaryan1, Niels Bassler2, Gerd Beyer3, John J. DeMarco1, Michael Doser4,
Dragan Hajdukovic5, Michael H. Holzscheiter6, Toshiyasu Ichioka2, Keisuke S. Iwamoto1, Sandra Kovacevic5, Helge V. Knudsen2, Rolf
Landua4, Carl J. Maggiore6, William H. McBride1, Søren Pape Møller2, Jorgen
Petersen7, Vesna Popovic5, James B. Smathers1, Lloyd D. Skarsgard8, Timothy D.
Solberg1, Ulrik I. Uggerhøj2, Sanja Vranjes9, H. Rodney Withers1, Michelle Wong8,
Bradly G. Wouters10
1 UCLA Medical School, 2 University of Aarhus, 3 Geneva University Hospital4 CERN, 5 University of Montenegro, 6 PBar Labs, LLC,
7 Aarhus University Hospital, 8 British Columbia Cancer Research Centre9 Vinca Institute Belgrade 10University of Maastricht
Michael H. Holzscheiter 2 SPSC Meeting October 26
Antiprotons deliver a higher biological dose for an Antiprotons deliver a higher biological dose for an equal effect in the entrance channel than protonsequal effect in the entrance channel than protons(and possibly heavy ions).(and possibly heavy ions).
The damage outside the beam path due to long and The damage outside the beam path due to long and medium range annihilation products is small and medium range annihilation products is small and does not significantly effect treatment planning.does not significantly effect treatment planning.
Antiprotons offer the possibility of real time imaging Antiprotons offer the possibility of real time imaging using high energy gammas and pions, even at low using high energy gammas and pions, even at low (pre-therapeutical) beam intensity.(pre-therapeutical) beam intensity.
Antiproton Therapy is based on three claims which need
proof:
Michael H. Holzscheiter 3 SPSC Meeting October 26
•We have measured cell survival in the peak and plateau We have measured cell survival in the peak and plateau regions of an antiproton beam stopped in a biological regions of an antiproton beam stopped in a biological medium.medium.
•Extracting the relative doses which produce equivalentExtracting the relative doses which produce equivalent cell kill in the peak and the plateau region we can define cell kill in the peak and the plateau region we can define
the BEDR (Biological Effective Dose Ratio) as the ratio of the BEDR (Biological Effective Dose Ratio) as the ratio of these doses. (We only need to know the relative dose).these doses. (We only need to know the relative dose).
Results from the 2003 run period
• We can compare these results to the same experiment We can compare these results to the same experiment using a proton beam of comparable energyusing a proton beam of comparable energy..
Michael H. Holzscheiter 4 SPSC Meeting October 26
• Irradiate sample tube with living cells suspended in gel.
• Slice sample tube in <1 mm slices and determine survival fraction for each slice.
Repeat for varying (peak) doses.
Biological Analysis Technique
Michael H. Holzscheiter 5 SPSC Meeting October 26
Calculate “plateau” survival using slices 1 – 4.
Determine “peak” survival from slice 8 and 9.
Plot “peak” and “plateau” survival vs. relative dose (Plateau dose, particle fluence, etc.) and extract the Biological Effective Dose Ratio (BEDR).
Biological Analysis Technique
Dose (arb. units)
Michael H. Holzscheiter 6 SPSC Meeting October 26
Cell Survival Measurements
Axial tube
Radial tubes
Location of Bragg Peak
Michael H. Holzscheiter 7 SPSC Meeting October 26
0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25
Tube B 1 Gy
Tube D 2.7 Gy
Tube F 4.6 Gy
Tube J 13.6 Gy
Sur
viva
l Fra
ctio
n
Depth in Sample [mm]
Data points inserted based on aminimum count of 1 surviving cell and known plating efficiency.
Cell Survival Measurements
3.8 Gy
8.9 Gy
15.5 Gy
40.0 Gy
Michael H. Holzscheiter 8 SPSC Meeting October 26
Plateau average (slices 1,2)
Broad peak average (slices 8,8’,9,9’)
Narrow peak average (slices 8’,9)
0 2 4 6 8 10 12 14 16 18 20 22 24
0.01
0.1
1
B - 1Gy E - 1Gy C - 2Gy D - 3Gy F - 5Gy J - 25 GyS
urvi
ving
Fra
ctio
n
Depth (mm)
Michael H. Holzscheiter 9 SPSC Meeting October 26
0 5 10 15 20 25 3010-2
10-1
100
BEDR(20%S)=9.2 - Broad peakBEDR(20%S)=9.8 - Narrow peak
Plateau Broad peak average Narrow peak average
Su
rviv
ing
Fra
ctio
n
Particle Fluence (arb. units)
BEDR AnalysisBEDR Analysis
Dose (arb. units) 0 5 10 15 20 25 3010 -2
10 -1
10 0
Plateau (CERN 2) Plateau (CERN 1) Peak (CERN 2) Peak (CERN 1)
Particle Fluence (arb. units)
Su
rviv
ing
Fra
ctio
n
Comparison toComparison toJune 7, 2003June 7, 2003
Dose (arb. units)
Michael H. Holzscheiter 10 SPSC Meeting October 26
0 5 10 15 20 25 3010
-2
10 -1
100
BEDR(20%S)=9.2 - Broad peakBEDR(20%S)=9.8 - Narrow peak
Plateau Broad peak average Narrow peak average
Sur
vivi
ng F
ract
ion
Particle Fluence (arb. units)
CERN (50 MeV Antiprotons)CERN (50 MeV Antiprotons)
Dose (arb. units)
TRIUMF TRIUMF (reanalyzed for 50 MeV Protons)(reanalyzed for 50 MeV Protons)
Michael H. Holzscheiter 11 SPSC Meeting October 26
The method works very well. The method works very well.
We are able to measure the survival response of V79-WNRE cells in the We are able to measure the survival response of V79-WNRE cells in the plateau and peak regions of a SOBP antiproton peak. plateau and peak regions of a SOBP antiproton peak.
In the early test experiment we obtained good data at 3 different doses in the In the early test experiment we obtained good data at 3 different doses in the plateau, and complete data at one dose in the peak. plateau, and complete data at one dose in the peak.
In the September run we obtained complete survival curves for 5 different In the September run we obtained complete survival curves for 5 different doses (in 6 measurements). The sensitivity in axial direction is high enough doses (in 6 measurements). The sensitivity in axial direction is high enough to detect the dose modulation due to the degrader used.to detect the dose modulation due to the degrader used.
An analysis of the data for the BEDR gives a result which is significantly An analysis of the data for the BEDR gives a result which is significantly higher than the value for protons (obtained at slightly higher energy and higher than the value for protons (obtained at slightly higher energy and using a different degrader) . using a different degrader) .
We observe only negligible cell kill outside of the beam in either the radial or We observe only negligible cell kill outside of the beam in either the radial or axial (beyond the peak) position at even the highest dose. This means not axial (beyond the peak) position at even the highest dose. This means not only that there is no significant spread of dose outside the beam due to the only that there is no significant spread of dose outside the beam due to the annihilation event but also no significant pion contamination in the beam.annihilation event but also no significant pion contamination in the beam.
Summary at end of 2003
Michael H. Holzscheiter 12 SPSC Meeting October 26
2004 Run Cycle
The BEDR enhancement has been proven to be The BEDR enhancement has been proven to be significant.significant.
NEXT STEPS:NEXT STEPS:
Detailed studies of the peripheral damage due to the medium andDetailed studies of the peripheral damage due to the medium and long range products from the antiproton annihilation. long range products from the antiproton annihilation.
Clonogenic studies may not be the best approach Clonogenic studies may not be the best approach search for alternative assays. search for alternative assays.
Increased efforts on dosimetry in the periphery to the beam Increased efforts on dosimetry in the periphery to the beam
Systematic studies to find faster (and more automated) methods Systematic studies to find faster (and more automated) methods to extract biological data.to extract biological data.
Preparatory studies towards real time imaging.Preparatory studies towards real time imaging.
Michael H. Holzscheiter 13 SPSC Meeting October 26
Evidence of LOW Peripheral Damage
0 5 10 15 20
J - 25 Gy
Depth (mm)
Distal
Peripheral Damage Study
0.001
0.01
0.1
1
10
0 2 4 6 8 10 12 14 16 18
Depth in Gel [mm]
Su
rviv
al F
ractio
n
20 Gy
5 Gy
< 2
mm
At the highest doses we can seea small effect outside the Braggpeak up to 1 – 2 mm distance
Need more sensitive assay Clonogenic may not be best DNA damage is detectable
Radial
0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25 30
Depth [mm]
Su
rviv
al
4.6 GY
13.8 GY
21
Sam
ple
Tub
e
Michael H. Holzscheiter 14 SPSC Meeting October 26
The COMET Assay
The comet assay is a gel electrophoresis method used to visualize and measure DNA strand breaks in individual cells using microscopy:
cell with DNA fragmentation
cell with major DNA fragmentation
cell without DNA fragmentation
Automated Analysis on individual cells
Statistical accuracy through analysisof > 100 cells per sample
Michael H. Holzscheiter 15 SPSC Meeting October 26
The COMET Assay – Early Results
0 5 10 15 20 25 30 35 400.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Tai
l Mom
ent
Depth in Gelatin [mm]
Cell sample irradiated with 15 Gy
Slices (0.5 mm and 1 mm) forplateaupeakperipheral (distal)
COMET Assay performed by the Inst. for Occup. Medicine, Zagreb
No detectable damage above control sample
Michael H. Holzscheiter 16 SPSC Meeting October 26
Peripheral Damage – Neutron Dosimetry
Placement of 6Li and 7Li TLD chipsat 30 to 120 mm from annihilation volume
Total dose in Peak ~ 7 GyDose seen at 20 mm < 2 cGy
Additional dose in 6Li fromthermal neutrons only
Additional measurements toestablish neutron spectrum
in preparation
Michael H. Holzscheiter 17 SPSC Meeting October 26
Peripheral Damage – Neutron Dosimetry
Bubble Technology Industries (BTI) Neutron Dosimeters
Superheated freon bubblets in gelatin undergo phase transitions when hit by neutrons
Michael H. Holzscheiter 18 SPSC Meeting October 26
Peripheral Damage – Neutron Dosimetry
Detectors are re-usable Detectors are energy dependent
Michael H. Holzscheiter 19 SPSC Meeting October 26
Monte Carlo Simulations
Original GEANT4 did not properly describe antiproton annihilation!
No ions produced above ‘sNewest version of GEANT4 with addition of (unofficial) modules
now produces ionsBut still no annihilation on periphery includedResults are still questionable – need benchmarks to test code
Michael H. Holzscheiter 20 SPSC Meeting October 26
Real Time Imaging Tests
Antiprotons stop in target:Disc of 1.5 cm diameter and 2 mm thicknessSet-up 1 cm slit using 10 cm thick led blocksImage seen if slit is in line of sight with sourceBackground only if slit points away from source
Michael H. Holzscheiter 21 SPSC Meeting October 26
Future Directions
Finish Laying the Foundations (2004/2006) Finalize Clonogenic Assay Studies Intensify Peripheral Damage Studies First Demonstration of Real Time Imaging of shaped targets
Source of Pbars: AD (3 – 5 x 107/85 seconds, T = 100 – 500 ns)
BEDR Measurements on antiprotons using pristine peakhigher beam energy needed (100 MeV)will allow better comparison to existing proton and heavy ion dataperform direct comparison measurement with heavy ions
Up to now we have not seen out of beam effectstill searching for more sensitive assaycan do better with tighter beam focus (DEM line
completion)complete neutron dosimetry
Initial Demonstration only established detector capabilityhigh resolution imaging at low intensity will need small focusand would be much easier with slow extraction (detector pile-
up)
Michael H. Holzscheiter 22 SPSC Meeting October 26
Future Directions
Finish Laying the Foundations (2004/2006) Finalize Clonogenic Assay Studies Intensify Peripheral Damage Studies First Demonstration of Real Time Imaging of shaped targets
Source of Pbars: AD (3 – 5 x 107/85 seconds, T = 100 – 500 ns)
Moving Forward: R&D towards final certification (2006 +)Development of beam delivery and energy modulation
~ 1 mm focus, scanning possibility (Complete DEM line)Real time imaging of shaped target
Implement semi-slow extraction (106 – 107/second)?
Comparison with protons and heavy ions (2005)
Michael H. Holzscheiter 23 SPSC Meeting October 26
Hardware needed:
Excitation sextupoles: 2 XRC available in dispersion free regions (sections 16 and 41)Electrostatic septum: not available in ADMagnetic septum: SM5306 is available
More detailed design studyBeam lifetime measurements at 300 MeV/cCommissioning of this option
(Semi-)Slow Extraction?
Michael H. Holzscheiter 24 SPSC Meeting October 26
Future Directions
Finish Laying the Foundations (2004/2006) Finalize Clonogenic Assay Studies Intensify Peripheral Damage Studies First Demonstration of Real Time Imaging of shaped targets
Source of Pbars: AD (3 – 5 x 107/85 seconds, T = 100 – 500 ns)
Moving Forward: R&D towards certification of method (2006 +)Development of beam delivery and energy modulation
~ 1 mm focus, scanning possibility (Complete DEM line)Real time imaging of shaped target
Implement semi-slow extraction (106 – 107/second)?Initial in vivo testing? 4 x 108 pbars deliver 1 Gy to 1 cc tumor (10 shots or 15 minutes) Possibilities to increase intensity per shot exist
Need studies on life-time and radiation protection issues
Comparison with protons and heavy ions (2005)
Michael H. Holzscheiter 25 SPSC Meeting October 26
Mode of Operation
Biological Measurements require long beam timesIrradiation of 4-5 cell samples at biological relevant
dose levels requires 24 hours of beam time. Time window between sample preparation and
analysisis maximum 72 hours.
Logistics is difficult as several teams need to be working in concert
….and have low repetition rate Cell preparation + analysis typically takes 4+ weeks
Continue with few long run periods (4 x 24 hours)
Michael H. Holzscheiter 26 SPSC Meeting October 26
Mode of Operation
‘Physics’ studies (dosimetry, imaging, beam delivery)
are possible with shorter shifts (8 hours)can be done by separate small sub-teamsand can be performed back-to-back
This would be best if 8 hour shifts could be taken one week (5 shifts) at a time
Michael H. Holzscheiter 27 SPSC Meeting October 26
Mode of Operation
Date Time Scheduled Topics Comments
May 21 8 hours Beam Development Cancelled due to PS delay
June 11 8 hours Focussing tests/Dosimetry Cancelled due to AD/PS Problems
June 28 16 hours Dosimetry using TLD’s Significant time lost to AD problem
July 2 8 hours Alanin tests Found misalignment of beam line
July 19 24 hours Peripheral damage studies Cancelled to PS problem (septum)
August 6 8 hours 6Li, 7Li dosimetry First smooth run of the year
August 23 24 hours Alternative assay studies Initial studies of COMET
August 27 8 hours Dosimetry Peripheral neutron dose
September 10 8 hours Dosimetry 2nd run on neutron dose
September 20 24 hours Biological studies Cancelled due to collaboration timing
September 24 8 hours Neutron Bubble Spectrometer Fast neutron spectrum
October 15 8 hours Imaging tests First high energy gamma detection
October 25 24 hours Peripheral damage studies COMET and clonogenic assays