Hadrontherapy Interactions in Molecular and Cellular Biology.

The resistance of cancer cells to radiotherapy is a major issue in the curative treatment of cancer patients. This resistance can be intrinsic or acquired after irradiation and has various definitions, depending on the endpoint that is chosen in assessing the response to radiation. This phenomenon might be strengthened by the radiosensitivity of surrounding healthy tissues. Sensitive organs near the tumor that is to be treated can be affected by direct irradiation or experience nontargeted reactions, leading to early or late effects that disrupt the quality of life of patients. For several decades, new modalities of irradiation that involve accelerated particles have been available, such as proton therapy and carbon therapy, raising the possibility of specifically targeting the tumor volume. The goal of this review is to examine the up-to-date radiobiological and clinical aspects of hadrontherapy, a discipline that is maturing, with promising applications. We first describe the physical and biological advantages of particles and their application in cancer treatment. The contribution of the microenvironment and surrounding healthy tissues to tumor radioresistance is then discussed, in relation to imaging and accurate visualization of potentially resistant hypoxic areas using dedicated markers, to identify patients and tumors that could benefit from hadrontherapy over conventional irradiation. Finally, we consider combined treatment strategies to improve the particle therapy of radioresistant cancers.

Differential normal skin transcriptomic response in total body irradiated mice exposed to scattered versus scanned proton beams.

Proton therapy allows to avoid excess radiation dose on normal tissues. However, there are some limitations. Indeed, passive delivery of proton beams results in an increase in the lateral dose upstream of the tumor and active scanning leads to strong differences in dose delivery. This study aims to assess possible differences in the transcriptomic response of skin in C57BL/6 mice after TBI irradiation by active or passive proton beams at the dose of 6 Gy compared to unirradiated mice. In that purpose, total RNA was extracted from skin samples 3 months after irradiation and RNA-Seq was performed. Results showed that active and passive delivery lead to completely different transcription profiles. Indeed, 140 and 167 genes were differentially expressed after active and passive scanning compared to unirradiated, respectively, with only one common gene corresponding to RIKEN cDNA 9930021J03. Moreover, protein-protein interactions performed by STRING analysis showed that 31 and 25 genes are functionally related after active and passive delivery, respectively, with no common gene between both types of proton delivery. Analysis showed that active scanning led to the regulation of genes involved in skin development which was not the case with passive delivery. Moreover, 14 ncRNA were differentially regulated after active scanning against none for passive delivery. Active scanning led to 49 potential mRNA-ncRNA pairs with one ncRNA mainly involved, Gm44383 which is a miRNA. The 43 genes potentially regulated by the miRNA Gm44393 confirmed an important role of active scanning on skin keratin pathway. Our results demonstrated that there are differences in skin gene expression still 3 months after proton irradiation versus unirradiated mouse skin. And strong differences do exist in late skin gene expression between scattered or scanned proton beams. Further investigations are strongly needed to understand this discrepancy and to improve treatments by proton therapy.

Outcome beyond third-line chemotherapy for metastatic triple-negative breast cancer in the French ESME program.

PURPOSE: Among metastatic breast cancer (MBC) patients, those with a triple-negative breast cancer phenotype (mTNBC) have the worst prognosis, but the benefit of chemotherapy beyond second line on outcome remains uncertain. The purpose of this study was to identify predictive factors of outcome after third- or fourth-line chemotherapy. METHODS: The ESME-MBC database is a French prospective real-life cohort with homogeneous data collection, including patients who initiated first-line treatment for MBC (2008-2016) in 18 cancer centers. After selection of mTNBC cases, we searched for independent predictive factors (Cox proportional-hazards regression models) for overall survival (OS) on third- and fourth-line chemotherapy (OS3, OS4). We built prognostic nomograms based on the main prognostic factors identified. RESULTS: Of the 22,266 MBC cases in the ESME cohort, 2903 were mTNBC, 1074 (37%) and 598 (20%) of which had received at least 3 or 4 lines of chemotherapy. PFS after first- and second-line chemotherapy (PFS1, PFS2) and number of metastatic sites ≥3 at baseline were identified by multivariate analysis as prognostic factors for both OS3 (HR = 0.76 95%CI[0.66-0.88], HR = 0.55 95%CI[0.46-0.65], HR = 1.36 95%CI[1.14-1.62], respectively), and OS4 (HR = 0.76 95%CI[0.63-0.91], HR = 0.56 95%CI[0.45-0.7], HR = 1.37 95%CI[1.07-1.74]), respectively. In addition, metastasis-free interval was identified as a prognostic factor for OS3 (p = 0.01), while PFS3 influenced OS4 (HR = 0.75 95%CI[0.57-0.98]). Nomograms predicting OS3 and OS4 achieved a C-index of 0.62 and 0.61, respectively. CONCLUSION: The duration of each previous PFS is a major prognostic factor for OS in mTNBC patients receiving third- or fourth-line chemotherapy. The clinical utility of nomograms including this information was not demonstrated.

Detection of extracellular 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker of oxidative damage in X-irradiated fibroblast cultures: optimization of analytical procedure.

We have developed a simple methodology, based on single-step solid-phase extraction followed by isocratic high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD), to determine extracellular 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in culture supernatants of normal human dermal fibroblasts. A standard addition method, using externally added 8-oxodG (0.5 and 1 pmol) was employed to eliminate matrix effects arising from the chemically complex, protein-rich medium. Secondly, applying this procedure to X-ray irradiated fibroblasts, we report a significant twofold increase in the levels of 8-oxodG at the radiobiologically relevant dose of 6 Gy. This suggests that extracellular 8-oxodG might be a useful biomarker for oxidative stress following moderate doses of X-irradiation.

Biological Effects of Scattered Versus Scanned Proton Beams on Normal Tissues in Total Body Irradiated Mice: Survival, Genotoxicity, Oxidative Stress and Inflammation.

Side effects of proton therapy are poorly studied. Moreover, the differences in the method of dose delivery on normal tissues are not taken into account when proton beams are scanned instead of being scattered. We proposed here to study the effects of both modalities of proton beam delivery on blood; skin; lung and heart in a murine model. In that purpose; C57BL/6 mice were total body irradiated by 190.6 MeV proton beams either by Double Scattering (DS) or by Pencil Beam Scanning (PBS) in the plateau phase before the Bragg Peak. Mouse survival was evaluated. Blood and organs were removed three months after irradiation. Biomarkers of genotoxicity; oxidative stress and inflammation were measured. Proton irradiation was shown to increase lymphocyte micronucleus frequency; lung superoxide dismutase activity; erythrocyte and skin glutathione peroxidase activity; erythrocyte catalase activity; lung; heart and skin oxidized glutathione level; erythrocyte and lung lipid peroxidation and erythrocyte protein carbonylation even 3 months post-irradiation. When comparing both methods of proton beam delivery; mouse survival was not different. However, PBS significantly increased lymphocyte micronucleus frequency; erythrocyte glutathione peroxidase activity and heart oxidized glutathione level compared to DS. These results point out the necessity to take into account the way of delivering dose in PT as it could influence late side effects.

The Possibility of Using Genotoxicity, Oxidative Stress and Inflammation Blood Biomarkers to Predict the Occurrence of Late Cutaneous Side Effects after Radiotherapy.

Despite the progresses performed in the field of radiotherapy, toxicity to the healthy tissues remains a major limiting factor. The aim of this work was to highlight blood biomarkers whose variations could predict the occurrence of late cutaneous side effects. Two groups of nine patients treated for Merkel Cell Carcinoma (MCC) were established according to the grade of late skin toxicity after adjuvant irradiation for MCC: grade 0, 1 or 2 and grade 3 or 4 of RTOG (Radiation Therapy Oncology Group)/EORTC (European Organization for Research and Treatment of Cancer). To try to discriminate these 2 groups, biomarkers of interest were measured on the different blood compartments after ex vivo irradiation. In lymphocytes, cell cycle, apoptosis and genotoxicity were studied. Oxidative stress was evaluated by the determination of the erythrocyte antioxidant capacity (superoxide dismutase, catalase, glutathione peroxidase, reduced and oxidized glutathione) as well as degradation products (protein carbonylation, lipid peroxidation). Inflammation was assessed in the plasma by the measurement of 14 cytokines. The most radiosensitive patients presented a decrease in apoptosis, micronucleus frequency, antioxidant enzyme activities, glutathione and carbonyls; and an increase in TNF-a (Tumor Necrosis Factor a), IL-8 (Interleukin 8) and TGF-ß1 (Transforming Growth Factor ß1) levels. These findings have to be confirmed on a higher number of patients and before radiotherapy and could allow to predict the occurrence of late skin side effects after radiotherapy.

[Cardiac complications of breast radiation therapy]. / Complications cardiaques de la radiothérapie mammaire.

Adjuvant radiation therapy in breast cancer is a standard of care, either post-lumpectomy or in case of lymph node involvement. Internal mammary chain (IMC) is more and more included in the clinical target volume, because it increases overall survival. This increase must be weighed against cardiac complications in left breast cancer. Intensity modulated radiation therapy (IMRT) is used in this indication in order to better cover target volumes, but tends to increase irradiated healthy volumes, including the heart. The average cardiac dose is higher with IMRT, while it is also predictive of cardiovascular events in patients treated in 3D. This article aims to make an inventory of the IMC irradiations, as well as a review of the mechanisms of radiation-induced cardiac toxicity and ways to diagnose it early. Cooperation between medical oncologists, radiotherapy oncologists and cardiologists is needed to better support patients.

The ongoing French metastatic breast cancer (MBC) cohort: the example-based methodology of the Epidemiological Strategy and Medical Economics (ESME).

PURPOSE: The currently ongoing Epidemiological Strategy and Medical Economics (ESME) research programme aims at centralising real-life data on oncology care for epidemiological research purposes. We draw on results from the metastatic breast cancer (MBC) cohort to illustrate the methodology used for data collection in the ESME research programme. PARTICIPANTS: All consecutive ≥18 years patients with MBC treatment initiated between 2008 and 2014 in one of the 18 French Comprehensive Cancer Centres were selected. Diagnostic, therapeutic and follow-up data (demographics, primary tumour, metastatic disease, treatment patterns and vital status) were collected through the course of the disease. Data collection is updated annually. FINDING TO DATE: With a recruitment target of 30 000 patients with MBC by 2019, we currently screened a total of 45 329 patients, and >16 700 patients with a metastatic disease treatment initiated after 2008 have been selected. 20.7% of patients had an hormone receptor (HR)-negative MBC, 73.7% had a HER2-negative MBC and 13.9% were classified as triple-negative BC (ie, HER2 and HR status both negative). Median follow-up duration from MBC diagnosis was 48.55 months for the whole cohort. FUTURE PLANS: These real-world data will help standardise the management of MBC and improve patient care. A dozen of ancillary research projects have been conducted and some of them are already accepted for publication or ready to be issued. The ESME research programme is expanding to ovarian cancer and advanced/metastatic lung cancer. Our ultimate goal is to achieve a continuous link to the data of the cohort to the French national Health Data System for centralising data on healthcare reimbursement (drugs, medical procedures), inpatient/outpatient stays and visits in primary/secondary care settings. TRIAL REGISTRATION NUMBER: NCT03275311; Pre-results.

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and alpha-tocopherol.

PURPOSE: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of alpha-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation. MATERIALS AND METHODS: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 - 21 days). RESULTS: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively. CONCLUSION: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.

Modulation of DNA damage by pentoxifylline and alpha-tocopherol in skin fibroblasts exposed to Gamma rays.

Previous in vivo studies showed the combination pentoxifylline (PTX) and alpha-tocopherol was highly efficient in reducing late radiation-induced skin damage. The present work aimed at investigating the molecular and cellular mechanisms involved in the effects of this combination. Primary cultures of confluent dermal fibroblasts were gamma-irradiated in the presence of PTX and trolox (Tx), the water-soluble analogue of alpha-tocopherol. Drugs were added either before or after radiation exposure and were maintained over time. Their antioxidant capacity and their effect on radiation-induced ROS production was assessed together with cell viability and clonogenicity. DNA damage formation was assessed by the alkaline comet assay and by the micronucleus (MN) test. Cell cycle distribution was also determined. The combination of PTX/ Tx was shown to reduce both immediate and late ROS formation observed in cells after irradiation. Surprisingly, decrease in DNA strand breaks measured by the comet assay was observed any time drugs were added. In addition, the micronucleus test revealed that for cells irradiated with 10 Gy, a late significant increase in MN formation occurred. The combination of PTX/Tx was shown to be antioxidant and to decrease radiation-induced ROS production. The observed effects on DNA damage at any time the drugs were added suggest that PTX/Tx could interfere with the DNA repair process.

Differential Radiosensitivity of Uveal Melanoma Cell Lines After X-rays or Carbon Ions Radiation.

PURPOSE: We compared the radiosensitivity of uveal melanoma (UM) cell lines after x-ray or carbon-ions radiation (C-ions). METHODS: We characterized the radiosensitivity toward x-rays and C-ions of UM cell lines: 92.1, MEL270, SP6.5, MKT-BR, µ2, and TP17. Normal choroidal melanocytes and the retinal pigment epithelial cell line ARPE19 were used as controls for normal cells. X-rays were delivered with an energy of 6 MV at a dose rate of 2 Gy/min. X-rays served as a reference for Relative Biological Effectiveness (RBE) evaluation. Radiation with C-ions was delivered at 75 MeV/u (34 keV/µm) at a dose rate of 2 Gy/min. After single-doses (0-8 Gy) of medical x-rays (6 MV) or C-ions (33 keV/µm), cells sensitivity was measured using standard colony formation assay, and cell growth was examined by counting the cell colonies. The effect of x-rays or C-ions on the expression and activation of ERK1/2 was evaluated by Western Blot. RESULTS: C-ions presented with regard to the x-rays a RBE of 1.9 to 2.5 at 10% of UM cells survival. The x-ray sensitivity of UM cells was neither influenced by the synchronization of cells in phase G0/G1 of the cell cycle nor by the level of oxygenation. X-ray and C-ions radiation had the same effects on cell cycle leading to a mitotic catastrophe that appeared earlier after C-ions than x-ray treatment. However, C-ions radiation induced a sustained inhibition of ERK1/2 activation compared to the transitory induction of that signalization pathway after x-ray radiation. CONCLUSIONS: This in vitro study shows that C-ions had a better biological effectiveness than x-rays leading to a sustained inhibition of the ERK1/2 pathway.

Dramatic increase in oxidative stress in carbon-irradiated normal human skin fibroblasts.

Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D0 (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D(10%) (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D(0%) (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this discrepancy between the two types of radiation should be investigated.