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1
DNA DAMAGE AND CELLULAR RESPONSE ALONG AND AROUND THE BRAGG
CURVE OF HEAVY IONS (DNA-BRAGG).
Spoke persons: Dr. G. Schettino1 ([email protected])
Collaborators: Prof. K.M. Prise1, Dr. F.J. Currell
1, Dr Pankaj Chuadhary
1, Mr Thomas Marshall
1, Dr.
L. Manti2, Dr. G.A.P. Cirrone
3,
1CCRCB, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT7 9BL, UK
2Università di Napoli “Federico II”, Scienze Fisiche, Monte S. Angelo, 80126, Napoli, Italy
3LNS-INFN, Via S. Sofia, Catania, Italy
Introduction.
The main goal of radiotherapy is the localized delivery of radiation dose to a tumour whilst
minimizing damaging effects to the surrounding healthy tissues. Ion beams represent, in theory, the
most effective type of radiation due to their physical dose deposition pattern [1] and have been used
for radiotherapy for some years [2], [3] with protons used in many facilities worldwide while carbon
ions have been exploited in Japan and Germany with impressive results [4], [5]. Despite these results,
there are still uncertainties on the biological effects caused by heavy ions especially related to non-
lethal and late effects. Although it has been proved that the relative biological effectiveness (RBE) for
cell killing is higher for heavy ions compared to low LET radiation, the same could be true for late and
non-lethal effects which occur in the plateau region of the ion trajectory where, by definition, healthy
tissues are exposed to low but not negligible doses. It is therefore essential to develop a rigorous theory
of ion radiation action at the cellular and molecular level to further improve tumour hadron therapy. To
date, radiotherapy studies investigating the biological response of different types of charged particles
have focused mainly on the cell killing effect on tumour cells or tissues at the Bragg peak [6].
However, damage caused at the beam entrance, beyond the Bragg peak and indeed in the immediate
proximity of the ion path is unavoidable and needs to be quantified. This damage is likely to be sub-
lethal and occurring in healthy tissues. Ultimately, it is normal tissue effects, including risks of
secondary cancers, which will determine the treatment outcome. Using a variety of approaches, the
present proposal aims to investigate in depth the damage and cellular response caused by heavy ion
exposure along the ion path and in its proximity.
The aim of these experiments is to investigate in detail the damage caused in live cells by
therapeutically relevant ion beams (i.e. protons and carbon ions) across and around the Bragg peak.
Our central hypothesis is that damage and cellular response will vary greatly along and around
the ion path and be related to both the physical and “biological” dose. The biological impact of
physical parameters such as dose deposition profiles, size of SOBP, LET, dose rate and fractionation
schedule needs to be further investigated and included in existing models to design optimal cancer
treatment strategies. Additionally, we anticipate DNA damage caused by ion fragmentation, secondary
electrons and bystander signals to have non-negligible effects with a contribution to cancer treatment
plans which has still to be fully investigated. Finally, it is crucial that biological investigations are not
just limited to cell killing but extended to other end points (i.e. chromosomal aberrations, senescence,
invasion and epigenetic changes in general) to clearly define the biological consequences of ion beam
exposures. The experimental data, together with computer modelling, will improve our understanding
of the basic ion-biological sample interactions and knowledge of the associated risks providing critical
information to improve the development of biological models predictive for the therapeutic use of ion
beams.
Scientific proposal.
The experiments proposed are aimed to address three linked key questions:
Q.1. How does DNA damage and cell response vary across the Bragg curve?
Using normal (human fibroblasts AG01522) and cancer (glioblastoma U87) cell lines, we propose
to perform immunofluorescence (i.e., γ-H2AX, 53BP1), cell survival and chromosome aberration
assays to investigate DNA damage induction/.repair and cell response as a function of the cell position
2
along the ion beam path. The onset of premature cellular senescence will also be evaluated as a sub-
lethal stress response. The data will delineate the biological Bragg curve for the selected end points
and highlight the potential of ion beams to induce effects other than tumour cell killing. The effect of
dose, dose rate, size of SOBO, fractionation and ion mass will also be investigated. The data will help
in predicting tumour control and healthy tissue risks in hadron therapy settings offering experimental
evidence to optimize existing and future treatment plans.
Q.2. How does track structure relate to cellular effects?
The approach described in Q.1. will be extended to investigate the relationship between spatio-
temporal ionization pattern and biological effects caused by ion irradiation. Amount, pattern and repair
dynamic of DNA double strand breaks (dsb) as detected by visualizing γ-H2AX, 53BP1 and related
DNA repair proteins foci using advanced imaging analysis, will be correlated to cellular response
(lethal and non-lethal effects) and physical parameters (LET, dose rate, SOBP size, fractionation
schedule) for samples placed along the primary ion path as well as in its immediate proximity. This
study will help to correlate biological effectiveness of the above mentioned end points to the unique
ionization pattern produced by high LET radiation.
Q.3. What is the contribution of bystander signals following heavy ion traversals?
In parallel to the direct irradiation effect, we are also planning to investigate the bystander
response of cells that do not receive any physical dose but share the same medium as the irradiated
samples. The bystander response (in terms of DNA damage repair, cell survival and chromosome
aberrations) will be characterised as a function of dose and ion species. We are also aiming to
investigate the LET dependence by relating the bystander response to specific parts of the Bragg curve
used for the direct irradiation. This will provide clues on the bystander triggering mechanisms while
the use of scavengers will help us in delineating the main components of the bystander signal. Finally,
comparison of the bystander response with data from direct exposure will offer some indication of the
relevance of bystander signals for the clinical use of heavy ions [7].
In order to perform the above mentioned experiments, we envision using a mixture of commercially
available and specifically designed/manufactured experimental set-ups (new and existing) to take full
advantages of the available LSN facilities.
Team expertise and previous data.
We have previously studied the effect of protons on both in vitro cell lines [8], [9] and 3D tissue
models [10]. These studies were performed using 3.5 MeV protons and microbeam facilities to
determine both the direct effect of proton traversals as well as the bystander response using cell
survival and micronuclei assays. The data highlight that precise and specifically designed experimental
set ups are able to detect biological responses which differ significantly from those determined
theoretically or by using average dose distributions. Since 2008, we are also part of the ACE
(Antiproton Cell Experiment) collaboration to evaluate the possible use and advantages of antiprotons
in radiotherapy. The experiments performed at CERN are aimed to quantify the direct, bystander and
secondary particle (i.e. from annihilation events) component of the damage induced in live cells. Data
so far indicate a qualitative as well as quantitative difference in the DNA damage caused in the Bragg
peak compared to the plateau region where also significant sub-lethal damage was detected (i.e.
micronucleus induction). Preliminary investigations of sub-lethal damage (chromosome aberrations)
from discrete positions of the plateau region of energetic carbon ions have also been performed [11]
using the LNS facilities. The data highlight the expertise of the collaboration, the feasibility of the
studies and how increase of biological effectiveness for end points other than cell killing may not
coincide with dose deposition as described by the physical Bragg curve. Finally, our team has also
considerable expertise in designing and performing biological experiments at overseas facilities having
ongoing collaborations with CERN and UK synchrotron facilities (Diamond Light Source).
Outcomes from our previous experimental sessions at the INFN-LNS are highlighted below.
3
Briefly, in our preliminary test (2011) we have successfully characterized a spread out Bragg peak
(SOBP) configuration for 62 MeV/u carbon ions with help from Dr. P. Cirrone’s group and performed
biological investigations at both the centre of the spread out and in the plateau region using normal
human fibroblast cells (AG01522). Cell survival data (RBE10% (Plateau) = 2.3, RBE10% (SOBP) = 4.2)
are in agreement with published data confirming more complex lesions being produced in the SOBP
region. These results have provided the basis for our DNA damage/repair investigations which
highlighted differences in repair kinetic as a function of position along the beam path at which the
samples were exposed. In all cases (spread out and plateau exposures), significant residual damage was
detected at 24 hrs post irradiation for carbon ions contrary to what observed with protons and
conventional 225 kVp X-ray exposures. This highlighted the appropriateness of the method and the
possibility of correlating DNA damage/repair with late effects. Bystander and chromosome aberration
preliminary experiments were also performed to validate and optimise the set up. These data have been
used to secure substantial funds (£500000 MRC project specifically aimed to these investigations has
been sponsored with start on 1st April 2012), have provided substantial data for a PhD thesis (Dr. J.
Kavanagh successfully graduated in Feb 2012) and have been the centre of oral presentations at 2
international meetings (Nano-IBCT, Caen Oct2011 and ICTR-PHE, Geneva Feb2012). One
manuscript has been published in IOP Journal of Physics: Conference Series and a second one is under
review in Nature Scientific Report.
Experimental sessions in 2012-2013 were aimed to investigate the variation in the biological
effectives (RBE) in normal and cancerous cells along clinically relevant proton beams. Due to the
limited beam time available, we concentrated our efforts on proton beams using the clinical setup
available at CATANA. Detailed report attached.
Beam time request.
Experiment investigations in 2013-2014 will concentrate on DNA repair, bystander signalling
and out-of-field effects. More data will also be needed to complete the preliminary senescence studies
since the effectiveness with which this sublethal effect is induced does vary along the Bragg curve, and
particularly along the SOBP. Chromosome aberration induction, as a biomarker of cytogenetic damage
and cancer risk will also be investigated. Cell proliferation studies will be complemented by invasion
and migration investigations to link radiation response to metastasis and epigenetic changes induced
by radiation treatments. We are also planning to address the impact of fractionation on the variation of
the biological effectiveness across the Bragg curve. Despite fractionation being a standard approach in
radiotherapy, there is a significant lack of radiobiological investigation with fractionated ion exposures
and new data are required to optimise clinical trials.
In order to perform the described experiments in suitable replicates to assure statistical robust
data sets, we estimate the need of 4 experimental sessions using the proton clinical setup of the
CATANA beamline. Each session should consist of 5 BTUs (Beam Time Units) of 8 hrs each. The
length of each session is critical to allow for equipment set-up and accurate dosimetry (~1 BTU),
reasonable number of sample handling and in particularly allowing fractionated exposures with ~ 24 hr
gap between irradiations as for clinical practise. Total BTU = 20. Additionally, access to local
biological facilities 2-3 days pre- and post-irradiation will be necessary for sample handling and set-
up. The experiments will be performed using proton beams from the LNS cyclotron of energy ~62
MeV. Beam diameter and dose rate are not critical parameters and previous set-ups used at LNS for
radiobiology experiments (i.e. ~2x2 cm2, ~1 Gy/min or ~ 1-10 nA) would be adequate.
References.
1. Orecchia, R., A. Zurlo, A. Loasses, et al., Particle beam therapy (hadrontherapy): basis for interest and clinical
experience. European Journal of Cancer, 1998. 34(4): p. 459-68.
2. Hug, E.B. and J.D. Slater, Proton radiation therapy for chordomas and chondrosarcomas of the skull base. Neurosurgery
Clinics of North America, 2000. 11(4): p. 627-38.
3. Miyamoto, T., N. Yamamoto, H. Nishimura, et al., Carbon ion radiotherapy for stage I non-small cell lung cancer.
Radiotherapy & Oncology, 2003. 66(2): p. 127-40.
4
4. Jereczek-Fossa, B.A., M. Krengli, and R. Orecchia, Particle beam radiotherapy for head and neck tumors:
radiobiological basis and clinical experience. Head Neck, 2006. 28(8): p. 750-60.
5. Kamada, T., H. Tsujii, H. Tsuji, et al., Efficacy and safety of carbon ion radiotherapy in bone and soft tissue sarcomas.
Journal of Clinical Oncology, 2002. 20(22): p. 4466-71.
6. Furusawa, Y., K. Fukutsu, M. Aoki, et al., Inactivation of aerobic and hypoxic cells from three different cell lines by
accelerated (3)He-, (12)C- and (20)Ne-ion beams. Radiation Research, 2000. 154(5): p. 485-96.
7. Prise, K. M. and J. M. O'Sullivan, Radiation-induced bystander signalling in cancer therapy. Nature Review Cancer,
2009. 9(5): p. 351-60.
8. Schettino, G., M. Folkard, K.M. Prise, et al., Low-dose hypersensitivity in Chinese hamster V79 cells targeted with
counted protons using a charged-particle microbeam. Radiation Research, 2001. 156: p. 526-534.
9. Prise, K.M., M. Folkard, A.M. Malcomson, et al., Single ion actions: the induction of micronuclei in V79 cells exposed
to individual protons. Advances in Space Research, 2000. 25(10): p. 2095-2101.
10. Schettino, G., G.W. Johnson, S.A. Marino, and D.J. Brenner, Development of a method for assessing non-targeted
radiation damage in an artificial 3D human skin model. International Journal of Radiation Biology, 2010.
11. Manti, L., M. Durante, G. Grossi, et al., Chromosome aberrations in human lymphocytes from the plateau region of the
bragg curve for carbon-ion beam. Nuclear Instruments and Methods in Physics Research B, 2007. 259: p. 884-888.
5
STATUS REPORT
Project name: DNA-BRAGG
Spokesperson: Dr. Giuseppe Schettino, Dr. Lorenzo Manti
Collaborators: Prof. Kevin Prise, Dr. Fred Currell, Dr Pankaj Chaudhary, Mr Thomas
Marshall, Dr. Lorenzo Manti, Dr Pablo Cirrone
Beam time requested: 8 BTU with carbon ions (62 MeV/u) + 8 BTU with protons (62 MeV)
Beam time allocated: 8 BTU with protons (62 MeV) in 3 experimental sessions (2 BTU in
Dec 2012, 3 BTU in Feb 2013, 3 BTU in June 2013)
Due to the significant cut in the beam time requested, our experimental efforts have concentrated on
the investigation of the variation in biological effectiveness (RBE) along monochromatic and
modulated proton beams. This was done for normal (AG01522) and cancerous (U87) cell lines with
different intrinsic radio-sensitivity. As suggested by the advisory committee, experiments were
designed in suitable replicated to allow for a robust statistical analysis and reduce the uncertainties.
Extensive dosimetry and Monte Carlo simulations were also planned to provide physical parameters to
which report the biological responses. As data from the experimental session in February 2013 are still
been analysed and experimental session scheduled for June 2013 still to be completed, this report will
focus on data from previous experimental sessions (May 2012 and December 2012).
Experiments were performed using proton beams of 62 MeV in the CATANA facility and aimed to
measure the biological effectiveness in terms of lethal cellular damage (cell death as for lack of
clonogenicity) in U87 cells (human tumour glioblastoma) and normal human fibroblasts (AG01522).
According to the proposed experimental plan, we assessed the biological outcome at several position
along a monochromatic (Pristine Bragg Peak) beam and a modulated (Spread Out Bragg Peak) using
clinical configurations. Characterization of the modulated proton beam was performed in collaboration
with Dr. Cirrone’s group and represented a training opportunity for a 1st year PhD student (Mr Thomas
Marshall). All experimental sessions were very successful with 6 different depth positions investigated
for the Pristine and Spread Out configuration. Samples were exposed to five doses (range 0-6 Gy as for
clinical interest) for each position and experiments repeated in duplicate and when possible triplicate
according to standard radiobiology protocols. Over 100 samples were irradiated per session. Through
collaboration with the local group (Dr. Pablo Cirrone) and supported by Monte Carlo simulations,
sample positioning was achieved with resolution <50 microns which greatly reduced the dose
uncertainties. Data have then been analyzed to calculate the Relative Biological Effectiveness (RBE,
critical parameter in radiotherapy with ion beams) as a function of depth along the proton beam and
compared to X-ray exposures which were performed following the same protocol in our Institution.
RBE values have been found to vary significantly only towards the end of the Bragg peak and
especially for high expected survival fractions (i.e. low doses). The data are currently being analysed
in terms of biologically effective dose for a typical clinical dose delivery to assess the potential impact
of such variation compared to the commonly used fixed RBE value of 1.1. Small differences are also
observed as a function of intrinsic radiosensitivity confirming that RBE variation as a function of LET
becomes more critical with increasing cellular radio-sensitivity. No statistical significant differences
between the RBE measured for the Pristine peaks and the distal part of a SOBP have been observed.
This supports the hypothesis that the biological response of a modulated ion beam can be predicted by
convoluting the response to a monochromatic beam and considering the physical characteristics of the
beam modulation (i.e. energy spread and weight factor). This is currently under investigation using the
data collected and simulation outcome from Geant4 modelling. Data from the Pristine configuration
are also being analysed as a function of the LET (and cell line characteristics) with the intent of
providing reference data for existing models (such as the Local Effect Model) and a new simulation
module for the Geant4 code (collaboration with Dr.Cirrone).
Figure 1. Dose distributions (Markus Chamber) and relative LET values (Geant4 simulations)
for Pristine and SOBP configurations used for the experiments. Sample positioning have been
verified using Gafchromic film and comparing the qualitative dose profile obtained with the
quantitative measurements of the Markus Chamber.
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10
Relative Dose
Pristine Dose & LET Profile in Water
Dose - Markus Chamber
Sample Positions
LET - Geant4
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10
Relative Dose
SOBP Dose & LET Profile in Water
Dose
Sample Positions
LET -
Dose distributions (Markus Chamber) and relative LET values (Geant4 simulations)
for Pristine and SOBP configurations used for the experiments. Sample positioning have been
film and comparing the qualitative dose profile obtained with the
quantitative measurements of the Markus Chamber.
0
5
10
15
20
25
30
15 20 25 30
LET (keV/µm)
Depth (mm)
Pristine Dose & LET Profile in Water
Markus Chamber
Sample Positions
0
5
10
15
20
25
30
10 15 20 25 30Depth (mm)
SOBP Dose & LET Profile in Water
Dose - Markus Chamber
Sample Positions
- Geant4
6
Dose distributions (Markus Chamber) and relative LET values (Geant4 simulations)
for Pristine and SOBP configurations used for the experiments. Sample positioning have been
film and comparing the qualitative dose profile obtained with the
LET (keV/µm)
10
15
20
25
30
LET (keV/µm)
7
Figure 2. RBE values as a function of depth and survival level for Pristine and SOBP
configurations for both cell lines used (AG01522 and U87).
Part of the above data and model have been presented (oral communication) at the European
Meeting for Radiation Research (ERRS 2012, Vietri sul Mare - Italy, 15-19 October 2012) and
submitted as part of an abstract for the Nano-IBCT conference (20-24 May 2013, Poland) and the
Radiation Research Society annual meeting (15-19 September 2012, New Orleans - USA). Data
collected so far at the INFN-LNS have been included in a manuscript been published in IOP
Journal of Physics: Conference Series (Manti, L., Campajola L., Perozziello F. M., Kavanagh J. N.
and Schettino G. (2012). "Development of a low-energy particle irradiation facility for the study
of the biological effectiveness of the ion track end." Journal of Physics: Conference Series
373(1)), a second one currently under review in Nature Scientific Reports and a third one been
drafted (aimed for International Journal of Radiation Oncology by summertime).
Pristine Bragg SOB
8
Preliminary DNA damage and bystander investigations have also been performed. Figure 3
reports the repair kinetics for DNA damage induced by direct exposure and bystander signalling
(~2 cm away from beam). Samples were placed at two different depths (entrance and peak of a
monochromatic 62 MeV proton beam) to address the LET contribution. No significant difference in
the repair of directly induced DNA lesion has been observed and future experiments will aim to
study the repair kinetic beyond the Bragg peak (distal end) where the LET increases to ~25
keV/µm. Other DNA repair markers will also be used to investigate pathways activated as a
function of DNA damage complexity. Small but significant amount of DNA damage has also been
observed in bystander cells with suggestion of LET dependency on the repair efficiency as
indicated by the slightly higher number of foci persisting at 24hr post exposure in samples around
the Bragg peak position (Bystander P5). Future beam times will be used to further investigate the
bystander contribution and its spatial correlation.
Figure 3. DNA repair dynamics (γ-H2AX assay) in directly exposed and bystander AG01522
cells. Samples were positioned at the entrance (P1, LET ~2 keV/µm) and at the peak position
(P5, LET ~11 keV/µm) of a monochromatic 62 MeV proton beam.
9
Figure 4. DNA damage induction/repair assessed through monitoring γ-H2AX (Green) and
ATM (Red) foci in time post exposure. Pictures relative to 1 Gy of 62 MeV protons (Pristine
configuration) at the entrance and peak depth positions.
SIPS (Stress-Induced Premature Senescence) was studied in normal human endothelial cells
by means of the expression of senescence-associated β-galactosidase. Accumulation in healthy
tissues of prematurely senescing cells may lead to organ and/or tissue malfunction and
degeneration. Furthermore, endothelial cells from tumour vasculature entering senescence may
secrete factors increasing tumour cells’ proliferation (Senescence-Associated Secretory
Phenotype). Cells were exposed in five positions along the SOBP Bragg curve (Fig. 1), positions
being labelled as P1, P2, P3, P4 and P6, covering almost the whole particle beam range. Cells
were assayed at 3 time points post-irradiation (Fig. 5). Results show that protons are effective at
inducing SIPS. At the entrance (P1), a relatively low dose (0.5 Gy) induced a significantly higher
proportion of senescing cells both acutely (day 2) and as a delayed response (day 27) compared
to replicative physiological senescence (control). Along the SOBP (P2, P3 and P4, proximal,
middle and distal parts, respectively), a fractionation typical dose (2 Gy) elicits a rather
inhomogeneous response: On proximal position, protons were quite effective at causing an acute
SIPS, whereas the proportion of senescing cells observed from those irradiated at middle- or
distal-SOBP positions increased steadily with time post-irradiation. Interestingly, a significant
persistent induction of SIPS was observed also for cells irradiated just beyond the SOBP (P6).
ENTRANCE PEAK
2
4
hrs
0.5
hrs
10
Control
P1 0,5 Gy
P2 2 Gy
P3 2 Gy
P4 2 Gy
P6 0,5 Gy
P6 2Gy
Percentage of senescent cells (%)
0
20
40
60
80
Day 2
Day 12
Day 27
Figure 5. Induction of premature senescence in normal human endothelial cell as a function
of position along a SOBP proton beam. P1-P6 positions refers to Figure 1
Beam time request.
Experiment investigations in 2013-2014 will concentrate on DNA repair, bystander
signalling and out-of-field effects. More data will also be needed to complete the preliminary
senescence studies since the effectiveness with which this sublethal effect is induced does vary
along the Bragg curve, and particularly along the SOBP. Chromosome aberration induction, as a
biomarker of cytogenetic damage and cancer risk will also be investigated. Cell proliferation
studies will be complemented by invasion and migration investigations to link radiation response
to metastasis and epigenetic changes induced by radiation treatments. We are also planning to
address the impact of fractionation on the variation of the biological effectiveness across the
Bragg curve. Despite fractionation being a standard approach in radiotherapy, there is a
significant lack of radiobiological investigation with fractionated ion exposures and new data are
required to optimise clinical trials.
In order to perform the described experiments in suitable replicates to assure statistical
robust data sets, we estimate the need of 4 experimental sessions using the proton clinical setup
of the CATANA beamline. Each session should consist of 5 BTUs (Beam Time Units) of 8 hrs each.
The length of each session is critical to allow for equipment set-up and accurate dosimetry (~1
BTU), reasonable number of sample handling and in particularly allowing fractionated exposures
with ~ 24 hr gap between irradiations as for clinical practise. Total BTU = 20. Additionally, access
to local biological facilities 2-3 days pre- and post-irradiation will be necessary for sample
handling and set-up. The experiments will be performed using proton beams from the LNS
cyclotron of energy ~62 MeV. Beam diameter and dose rate are not critical parameters and
previous set-ups used at LNS for radiobiology experiments (i.e. ~2x2 cm2, ~1 Gy/min or ~ 1-10
nA) would be adequate.