1

DNA DAMAGE AND CELLULAR RESPONSE ALONG AND AROUND THE BRAGG

CURVE OF HEAVY IONS (DNA-BRAGG).

Spoke persons: Dr. G. Schettino1 (g.schettino@qub.ac.uk)

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.