aENEA Radiation Sources Laboratory, Research Center Frascati, Rome, Italyb Istituto Superiore di Sanità - Technology and Health Department, Viale Regina Elena 299, 00161 Roma, Italy

c ENEA C.R. Casaccia, UT. BIORAD Radiobiology, Via Anguillarese, 301, 00123, Roma, Italyd INFN-Sezione di Roma1, Gr.coll.Sanità

M. Vadruccia, A. Ampollinia, P. Anellob, M. Balduzzic, G. Bazzanoa, G. Espositob,d, C. Maricnoc, C. Patronoc, L. Picardia, C. Ronsivallea, C. Snelsc, M. A. Tabocchini b,d, A. Testac

Radiobiology Experiments for Characterization of the low-energy TOP-IMPLART Proton Beam

ABSTARCT: The TOP (Oncological Therapy with Protons)-IMPLART (Intensity Modulated Proton Linear Accelerator for RadioTherapy) [1] accelerator is a facility for proton-therapy under construction in ENEA C. R. Frascati Radiation Sources Laboratory in collaboration with ISS and IFO. It is a completely linear pulsed accelerator consisting of a 7 MeV injector followed by a high frequency booster up to 150 MeV. A vertical beam extraction-line at the exit of injector has been devoted to in-vitro radiobiology experiments for the characterization of the low-energy proton beam.Irradiation experiments were carried out on V79 and CHO Chinese hamster cells. Both cell lines have been widely used in radiobiology studies with different radiation qualities – including by the authors [2, 3] - thus allowing the evaluation and the comparison of results. In order to carry out the biological characterization of the facility, the radiation responses of V79 and CHO cells have been evaluated in terms of clonogenic survival. Moreover, a protocol of Micronuclei Assay for the in situ evaluation of radiation induced DNA damage has been adapted to the specific exposure conditions of the vertical proton beam.Cells were plated in stainless steel Petri dishes, especially designed for charged particle irradiation [1], and grown attached to a 60 μm mylar foil representing the base of the dish.The irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range 0.5-8 Gy.The dose-response curves - characterized by an initial shoulder followed by a straight portion - were well fitted by a linear-quadratic function.The obtained results were found in good agreement with literature data [3, 4]. [1] C. Ronsivalle et al., The TOP IMPLART Project, Eur. Phys. J Plus (2011) 126, number 7, 68; [2] Belli et al, NIM 1987; [3] Belli et al, IJRB 1998; [4] JT Tang'et al, BJC 1997.TOP – IMPLART PROJECTThe TOP-IMPLART project, developed by ENEA, the Italian National Institute of Health (ISS) and Regina Elena National Cancer Institute IFO, Rome, is devoted to the realization of a new proton therapy center to be sited in Rome (Italy). The project is based on a 230 MEV proton accelerator consisting of a sequence of linear accelerators. The low-energy segment (up to 35 MeV) is composed by a commercial 7MeV proton Linac (sequence of a source, a RFQ and a DTL operating at the frequency of 425 MHz) produced by AccSys-Hitachi, followed by 4 SCDTL-modules added to leading the proton energy up to 11.6, 18, 27 and 35MeV.The TOP-IMPLART facility is designed for fully active scanning 4D (intensity, energy and transversal position x-y) IMPT protocols. Peculiar characteristics of the system are: modularity, pulsed operation naturally suited to IMPT, fast energy variation, high quality beam.

Vertical Beam Line 7 MeV Protons Dedicated to In vitro Radiobiology

70 cm long - 2 mm aluminium collimator - 2

m gold scatterer spreading uniformly the beam on an area of 13 mm diameter – 50 m

Kapton vacuum/air window degrading the

beam energy to 6.2 MeV

SCDTL-1

RADIOBIOLOGICAL EXPERIMENTS

The first radiobiological experimental campaign was carried out to characterize the proton beam. To this purpose, cell killing experiments and micronuclei (MN) assay were conducted at the vertical transport line on V79 and CHO cells. Irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range 0.5-8 Gy.

0.01

0.1

1

0 2 4 6 8 10

SF 1

SF 2

SF 3

SF

Su

rviv

ing

Fra

ctio

n

Dose / Gy

CHO TOP protons (3 exps)

0.01

0.1

1

0 2 4 6 8 10

SF 1SF 2SF 3SF 4SF

Su

rviv

ing

Fra

ctio

n

Dose / Gy

V79 TOP protons (4 exps)

STAINLESS STEEL CELLS SAMPLE HOLDER ASSEMBLAGE

mylar foil

protons

cell monolayer

beam

Myl

arCe

lls

Dose-response curves for clonogenic survival in V79 and CHO cells

The dose-response curves for clonogenic survival

were found to be characterized by an initial

shoulder (more pronunced in V79 than in CHO

cells) followed by a straight portion, that can be

well fitted by a linear-quadratic function of the

dose.

= 0.208±0.016= 0.020±0.003

= 0.186±0.019= 0.090±0.003

In situ MN assay

~ 104 CHO cellsseeded on the irradiation dish(60 μm mylar)

• fresh medium containing Cytochalasin-b (1.5 μg/ml) added•20-24 h incubation (37°C, 5% CO2) •Hypotonic shock and fixing•Staining with 5% Giemsa

24h 5 MeVproton

irradiation

All the steps of experimental protocol used for MN assay have been adapted to the specific exposure conditions of the TOP-IMPLART vertical proton beam.

MN

40 X

After the staining the mylar membrane is mounted on a glass

slide for microscope analysis

Up to now layout of the TOP – IMPLART facility

The results on V79 cells, widely used in

hadrontherapy experiments, were found in good

agreement with the previous data obtained at LNL

[3]. Also the data obtained using CHO cells were

consistent with literature results [4].