Lucia M. Valastro

A 62 MeV proton beam for the treatment of ocular melanoma at Laboratori Nazionali del Sud-INFN (CATANIA)

At the INFN Laboratori Nazionali del Sud in Catania (Italy) the first Italian protontherapy facility, named CATANA (Centre di AdroTerapia e Applicazioni Nucleari Avanzate) has been realized in collaboration with the University of Catania. It is based on the use of the 62 MeV proton beam delivered by the K = 800 superconducting cyclotron installed and working at LNS since 1995. The facility is mainly devoted to the treatment of ocular diseases like uveal melanoma. A beam treatment line in air has been realized together with a dedicated positioning patient system. The facility is in operation since the beginning of 2002 and 52 patients have been successfully treated up to now. The main features of CATANA together with the clinical and dosimetric features will be extensively reported; particularly, will be described the proton beam line, that has been entirely realized at LNS, with all its elements, the experimental transversal and depth dose distributions of the 62 MeV proton beam obtained for a final collimator of 25 mm diameter and the experimental depth dose distributions of a modulated proton beam obtained for the same final collimator. Finally, the clinical results over one year of treatments, describing the features of the treated diseases will be reported.

Radiobiological analysis of human melanoma cells on the 62 MeV CATANA proton beam

Purpose: To measure the ability of protons and γ-rays to effect cell viability and cell survival of human HTB140 melanoma cells. Materials and methods: Exponentially growing HTB140 cells were irradiated close to the Bragg peak maximum of the 62 MeV protons or with 60Co γ-rays with single doses, ranging from 8 – 24 Gy. Cell viability using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay was evaluated at 6 h, 24 h, 48 h or 7 days after irradiation and clonogenic survival was assessed at 7 days after irradiation. Cell cycle phase redistribution and the level of apoptosis were evaluated at 6 h and 48 h after irradiation. Results: The study of cell viability as a function of time (cell survival progression) and cell survival, using a clonal assay, demonstrated the considerably stronger inactivation effect of protons compared to γ-rays with a relative biological effectiveness (RBE) of ∼1.64. Cell cycle phase distribution and apoptosis levels with time enabled us to investigate the development and the character of the damage induced by irradiation. Due to the high radio-resistance of HTB140 cells, cell cycle phase redistribution exhibited only a modest cell accumulation in G2/M phase. Protons but not γ-rays induced apoptosis. Conclusions: It appears that protons reduce the number of HTB140 cells by apoptosis as well as by severe DNA damage, while γ-rays eliminate viable cells primarily by the production of irreparable DNA damage. Protons have an increased RBE relative to γ-rays.

Response of a radioresistant human melanoma cell line along the proton spread-out Bragg peak.

Purpose: To analyse changes of cell inactivation and proliferation under therapeutic irradiation conditions along the proton spread out Bragg peak (SOBP) with particular emphasis on its distal declining edge. Materials and methods: HTB140 cells were irradiated at four positions: plateau, middle, distal end and distal declining edge of the 62 MeV proton SOBP. Doses ranged from 2–16 Gy. They were normalised in the middle of SOBP and delivered following the axial physical dose profile. Survival, proliferation and cell cycle were assessed seven days after irradiation. Results: Moving from proximal to distal irradiation position surviving fractions at 2 Gy (SF2) decreased from 0.88–0.59. Increased radiosensitivity of the cells was noticed for the doses below 4 Gy, resulting in two gradients of cell inactivation, stronger for lower and weaker for higher doses. Relative biological effectiveness (RBE) increased from 1.68–2.84 at the distal end of SOBP. A further rise of RBE reaching 7.14 was at its distal declining edge. Following the axial physical dose profile of SOBP the strongest inactivation was attained at its distal end and was comparable to that at its declining edge. Conclusions: Survival data confirmed very high radioresistance of HTB140 cells. An effect similar to low-dose hyper radiosensitivity (HRS) was observed for order of magnitude larger doses. Better response of cells to protons than to γ-rays was illustrated by rather high RBE. Strong killing ability at the SOBP distal declining edge was the consequence of increasing proton linear energy transfer.

Response of a Human Melanoma Cell Line to Low and High Ionizing Radiation

Abstract:  Effects of single irradiation with gamma rays and protons on human HTB140 melanoma cell growth were compared. Exponentially growing cells were irradiated close to the Bragg peak maximum of the unmodulated 62 MeV protons, as well as with 60Co gamma rays. Applied doses ranged from 8 to 24 Gy. Viability of cells and proliferation capacity were assessed 7 days after irradiation. Induction of apoptosis and cell cycle phase redistribution were observed 6 and 48 h after irradiation. Significant inhibitory effects of both irradiation qualities were detected 7 days after irradiation. Important reduction of HTB140 cell viability was observed after irradiation with protons. Almost linear and highly significant (P < 0.001) decrease of cell proliferation was observed 7 days after irradiation with gamma rays and protons, as compared to nonirradiated controls. Protons induced apoptosis, both 6 and 48 h after irradiation. With the increase of post‐irradiation incubation time, number of apoptotic cells decreased. Exposure of HTB140 cells to gamma rays did not provoke apoptotic cell death. Important number of cells in G1‐S phase, detected by the cell cycle phase redistribution analyses, suggested high metabolic activity of irradiated melanoma cells within the first 48 h. Both irradiation qualities caused modest G2‐M arrest 6 and 48 h after irradiation, thus supporting results that illustrated high radioresistance of HTB140 cells.

Assessment of the inhibitory effects of different radiation qualities or chemotherapeutic agents on a human melanoma cell line

The correlation between time dependent viabilities, after applying two radiation qualities and two alkylating agents on HTB140 melanoma cells, has been studied. Irradiations were performed with gamma-rays and 62 MeV protons, close to the Bragg peak maximum, delivering doses of 8-24 Gy. Treatments with fotemustine (FM) and dacarbazine (DTIC) were carried out with concentrations of 0.05-2mM. High radio-resistance of HTB140 cells revealed by a clonogenic assay was confirmed by microtetrasolium and sulforhodamine B, through the surviving fraction at 2 Gy (SF2), being 0.961-0.956 for gamma-rays and 0.931-0.887 for protons. A better efficiency of protons was illustrated by relative biological effectiveness at 2 Gy (RBE), ranging from 1.69 to 1.89. A kinetic study of concentration dependent cytotoxicity indicated that the best effect of the drugs, estimated as the concentration that produces 50% of growth inhibition (IC(50)), was obtained at 48 h, having values of 76 microM for DTIC and 145 microM for FM. The cytostatic ability of the drugs pointed out that the presence of DTIC at 24h, compared to FM, was insufficient to produce an effect. Protons and FM demonstrated their pro apoptotic capacity. Cross-resistance between treatments applied to the HTB140 cells was observed, protons being the most efficient, while DTIC, FM and gamma-rays demonstrated a lower level of cell inactivation.

Preliminary investigation on the use of the MOSFET dosimeter in proton beams

Metal Oxide Semiconductor (MOS) device structures can be used to measure ionizing radiation through the mechanism of hole trapping in the oxide layer leading to changing of electrical characteristic of the device. They are a new type of direct reading semiconductor dosimeters. Due to their extremely small physical size, ability to permanently store the accumulated dose, dose-rate independence and their ease of use make them very promising for in vivo dosimetry. They are attractive for dosimetry in small radiation fields used in modern radiation oncology modalities, as conformal radiotherapy, IMRT, stereotactic radiotherapy/radiosurgery and proton therapy. Preliminary results on the use of commercial MOSFET dosimeters (TN-502RD, Thomson & Nielsen Electronics Ltd, Canada) irradiated on therapeutic 62 MeV proton beams are presented. Linearity with absorbed dose, sensibility and energy dependence were investigated. Moreover, the possibility to use of MOSFET dosimeters in order to measure the Output Factors (OF) for very small irradiation fields was verified. The comparison of OF obtained using MOSFETs and other dosimetry systems is reported.

Viability of a human melanoma cell after single and combined treatment with fotemustine, dacarbazine, and proton irradiation.

Abstract:  Viability of human HTB140 melanoma cells after being exposed to fotemustine (FM) and dacarbazine (DTIC) as well as to proton irradiation was studied. Effects of 100 and 250 μM drugs were assessed after incubation of 6, 24, 48, 72, and 96 h. Irradiations were performed with 62 MeV therapeutic protons, delivering to the cell monolayer single doses of 2, 4, 8, 12, and 16 Gy. Viability was evaluated 7 days after irradiation. Inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB) assays. Combined effects of each drug and protons, were carried out using the same drug concentrations. Proton doses applied were those used in therapy, that is, 12 and 16 Gy. With the increase of drug concentration or irradiation dose, level of cell inactivation reached approximately 60%, 48 h after drug treatment or 7 days after irradiation at 16 Gy. Considering the rate of drug concentrations used, as well as the level of doses applied, it appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents tested. The combined treatment with FM or DTIC and protons did not show significant changes of cell viability as compared to the effects of single agents. Since the time point for measuring cumulative effects of drug and irradiation was 48 h post irradiation, it seems that the obtained level of viability could be attributed primarily to the effects of drugs.

Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation

BackgroundConsidering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed.MethodsCells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 μM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time.ResultsSingle proton irradiations have reduced the number of cells to ~50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA.ConclusionThe obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

Neuro-Radiosurgery Treatments: MRI Brain Tumor Seeded Image Segmentation Based on a Cellular Automata Model

Gross Tumor Volume (GTV) segmentation on medical images is an open issue in neuro-radiosurgery. Magnetic Resonance Imaging (MRI) is the most prominent modality in radiation therapy for soft-tissue anatomical districts. Gamma Knife stereotactic neuro-radiosurgery is a mini-invasive technique used to deal with inaccessible or insufficiently treated tumors. During the planning phase, the GTV is usually contoured by radiation oncologists using a manual segmentation procedure on MR images. This methodology is certainly time-consuming and operator-dependent. Delineation result repeatability, in terms of both intra- and inter-operator reliability, is only obtained by using computer-assisted approaches. In this paper a novel semi-automatic segmentation method, based on Cellular Automata, is proposed. The developed approach allows for the GTV segmentation and computes the lesion volume to be treated. The method was evaluated on 10 brain cancers, using both area-based and distance-based metrics.

Fully Automatic Multispectral MR Image Segmentation of Prostate Gland Based on the Fuzzy C-Means Clustering Algorithm

Prostate imaging is a very critical issue in the clinical practice, especially for diagnosis, therapy, and staging of prostate cancer. Magnetic Resonance Imaging (MRI) can provide both morphologic and complementary functional information of tumor region. Manual detection and segmentation of prostate gland and carcinoma on multispectral MRI data is not easily practicable in the clinical routine because of the long times required by experienced radiologists to analyze several types of imaging data. In this paper, a fully automatic image segmentation method, exploiting an unsupervised Fuzzy C-Means (FCM) clustering technique for multispectral T1-weighted and T2-weighted MRI data processing, is proposed. This approach enables prostate segmentation and automatic gland volume calculation. Segmentation trials have been performed on a dataset composed of 7 patients affected by prostate cancer, using both area-based and distance-based metrics for its evaluation. The achieved experimental results are encouraging, showing good segmentation accuracy.