Voxel-wise analysis: A powerful tool to predict radio-induced toxicity and potentially perform personalised planning in radiotherapy.

Dose - volume histograms have been historically used to study the relationship between the planned radiation dose and healthy tissue damage. However, this approach considers neither spatial information nor heterogenous radiosensitivity within organs at risk, depending on the tissue. Recently, voxel-wise analyses have emerged in the literature as powerful tools to fully exploit three-dimensional information from the planned dose distribution. They allow to identify anatomical subregions of one or several organs in which the irradiation dose is associated with a given toxicity. These methods rely on an accurate anatomical alignment, usually obtained by means of a non-rigid registration. Once the different anatomies are spatially normalised, correlations between the three-dimensional dose and a given toxicity can be explored voxel-wise. Parametric or non-parametric statistical tests can be performed on every voxel to identify the voxels in which the dose is significantly different between patients presenting or not toxicity. Several anatomical subregions associated with genitourinary, gastrointestinal, cardiac, pulmonary or haematological toxicity have already been identified in the literature for prostate, head and neck or thorax irradiation. Voxel-wise analysis appears therefore first particularly interesting to increase toxicity prediction capability by identifying specific subregions in the organs at risk whose irradiation is highly predictive of specific toxicity. The second interest is potentially to decrease the radio-induced toxicity by limiting the dose in the predictive subregions, while not decreasing the dose in the target volume. Limitations of the approach have been pointed out.

[Fabien Arcelin (1876-1942), or how to become a radiation oncologist when born an archeologist]. / Fabien Arcelin (1876­1942), ou comment on devient radiothérapeute quand on naît archéologue.

Fabien Arcelin, physician working in Lyon, pioneer of radiology and radiotherapy, was both mentored by Destot (one of the fathers of the French radiology for his thesis of medicine) and by his own father Adrien Arcelin (one of the two codiscoverers of the prehistorical site of the Roche-de-Solutré in Burgundy) for archeological works. Hence, radiologist in Lyon during the week and archeologist during the weekend, Fabien Arcelin made significant advances in both radiation research and archeology. He was notably the discoverer of the first tombs of Aurignacian men. In radiology, he examined about 2000 radiographic heart views with the Destot's orthodiagraph to assess the cardiothoracic ratio and wrote a reference book about the use of X-rays to detect kidney stones. He made the first survey about the secondary effects of radiotherapy, important database for pointing out individual radiosensitivity. In parallel to the radiotherapy trials of Regaud at Curie institute (Paris), he performed the first series of anticancer treatments in Lyon.

[Étienne Destot (1864-1918) or the other father of French radiology]. / Étienne Destot (1864­1918) ou l'autre père de la radiologie française.

Étienne Destot is a French physician from Burgundy who benefited, during his studies in Lyon, from the quality of teaching of the best specialists of the time: Augagneur for hygiene, Testut for anatomy, Ollier for surgery, Lépine for the medical applications of electricity and the Lumière brothers for the technological development. During its experiments, he met Despeignes, the first radiation oncologist, Regaud pioneer of radiobiology and Bouchacourt who pointed out individual radiosensitivity. Less than two months after the X-rays discovery by Roentgen, he produced one of the first French radiographic views that were at the origin of our current knowledge in bone and cartilage anatomy and traumatology. He funded the first department of radiology in France in a former library of the major hospital of Lyon, where he made a number of original advances. It appears obvious that, while Antoine Beclère was the great organizer of the French radiology, Destot was its pathfinder. Destot was at the origin of several technological advances that gave stereoscopy, internal organs imaging and quantification of the heart-thorax ratio. By contrast, he was not convinced of the therapeutic properties of X-rays even if he contributed to the technological development of X-ray tubes. Victim of radiations, exhausted, Destot died on December 1918, by helping the Great War victims. His name is written in a war tribute monument in Arc-et-Senans (Burgundy).

[Radiation-induced bronchiolitis obliterans with organizing pneumonia]. / Complications pulmonaires de la radiothérapie après cancer du sein : penser à la BOOP.

Bronchiolitis obliterans with organizing pneumonia is an inflammatory reaction that can occur as a consequence of various pulmonary affections. Radiotherapy is not the sole and systematic cause of bronchiolitis obliterans with organizing pneumonia. Radiation-induced should not be confused with post-radiation, dose-dependent, inflammatory pulmonary fibrosis, which is non-immunological and located within the irradiation field. The role of immunity, local inflammation and individual radiosensitivity in bronchiolitis obliterans with organizing pneumonia is not well defined. Bronchiolitis obliterans with organizing pneumonia represents 1% of irradiated patients with breast cancer. It results in fever (flu-like symptoms), a rather dry cough and dyspnea. In the post-radiation context, bronchiolitis obliterans with organizing pneumonia may be diagnosed several months and up to a year after breast irradiation. The treatment consists of prolonged steroids or immunosuppressants, which do not prevent chronicity in 15% of patients and death in up to 5% of cases, the remaining 80% of patients healing without sequelae.

[1896: How nascent radiotherapy gathered Roentgen, Pasteur and the Lumière brothers]. / 1896 : comment la radiothérapie naissante a pu réunir Roentgen, Pasteur et les frères Lumière.

On the 4th July, 1896, in his medical office of Les Échelles (Savoie, France), Victor Despeignes performed the very first radiotherapy against cancer, documented by indisputable proofs. However, the intellectual and practical approach that leads to this first radiotherapy may appear unexpected to date. Indeed, it is likely that the treatment that Despeignes applied to his neighbour, who did not suffer from stomach cancer, was born with the indirect support of the Lumière brothers, on the basis of a biological theory (the parasitory theory of cancer) that is irrelevant today, with an experimental protocol based on experiments performed on tuberculosis et in a bifractionated model linked by the constraints of a district medical doctor.

[The enigma of the biological interpretation of the linear-quadratic model finally resolved? A summary for non-mathematicians]. / L'énigme de l'interprétation biologique du modèle linéaire-quadratique enfin résolue ? Une synthèse pour les non-mathématiciens.

The linear-quadratic (LQ) model is the only mathematical formula linking cellular survival and radiation dose that is sufficiently consensual to help radiation oncologists and radiobiologists in describing the radiation-induced events. However, this formula proposed in the 1970s and α and ß parameters on which it is based remained without relevant biological meaning. From a collection of cutaneous fibroblasts with different radiosensitivity, built over 12 years by more than 50 French radiation oncologists, we recently pointed out that the ATM protein, major actor of the radiation response, diffuses from the cytoplasm to the nucleus after irradiation. The evidence of this nuclear shuttling of ATM allowed us to provide a biological interpretation of the LQ model in its mathematical features, validated by a hundred of radiosensitive cases. A mechanistic explanation of the radiosensitivity of syndromes caused by the mutation of cytoplasmic proteins and of the hypersensitivity to low-dose phenomenon has been proposed, as well. In this review, we present our resolution of the LQ model in the most didactic way.

[Repeated radiation dose effect and DNA repair: Importance of the individual factor and the time interval between the doses]. / Effets de répétitions de doses d'irradiation et réparation de l'ADN : importance du facteur individuel et de l'intervalle de temps entre les doses.

The dose fractionation effect is a recurrent question of radiation biology research that remains unsolved since no model predicts the clinical effect only with the cumulated dose and the radiobiology of irradiated tissues. Such an important question is differentially answered in radioprotection, radiotherapy, radiology or epidemiology. A better understanding of the molecular response to radiation makes possible today a novel approach to identify the parameters that condition the fractionation effect. Particularly, the time between doses appears to be a key factor since it will permit, or not, the repair of certain radiation-induced DNA damages whose repair rates are of the order of seconds, minutes or hours: the fractionation effect will therefore vary according to the functionality of the different repair pathways, whatever for tumor or normal tissues.

[Léon Bouchacourt (1865-1949): How an obstetrician pointed out individual radiosensitivity]. / Léon Bouchacourt (1865-1949) : comment un obstétricien mit en évidence la radiosensibilité individuelle.

Léon Bouchacourt (1865-1949) was a misknown pioneer of radiology and radiotherapy from Lyon, France. While he was resident in obstetrics in Hôpitaux de Paris from 1892 to 1898, he met Charcot, the future polar explorer, and wrote the first thesis dissertation about X-rays. He invented a new radiology technique for cavitary organs such as vagina, rectum and mouth, the endodiascopy, which permitted him to perform the first pelvimetry and dental radiographies in France. While he undertook the first trials of contact-radiotherapy, he was confronted with radiation-induced reactions. In 1911, he wrote the first paper about individual radiosensitivity. During the First World War, he commanded one of the radiology vehicles, he met Irène Curie and developed his « radiological helmet ¼, which will hold his name and became essential for interventional radiology. After the war, with Béclère and ten others, Léon Bouchacourt funded the French Radiology Society. He ended his career by thinking about public health and the different aspects of the duties of radiologists.

[Management of craniofacial type 1 neurofibromatosis]. / Prise en charge des atteintes craniofaciales de la neurofibromatose de type 1.

Type I neurofibromatosis (NF) is the most common autosomal dominant disease. It concerns one in 3000 births, the penetrance is close to 100% and 50% of new cases are de novo mutations (17q11.2 chromosome 17 location). Cranio-maxillofacial region is concerned in 10% of the cases, in different forms: molluscum neurofibroma, plexiform neurofibroma, cranio-orbital neurofibroma, parotido-jugal neurofibroma, cervical neurofibroma. These lesions have different prognosis depending on the craniofacial localization: ocular functional risk, upper airway compressive risk, nerve compression risk, aesthetic and social impact. The maxillofacial surgeon in charge of patients with type I NF should follow the patient from the diagnosis and organize the different surgical times in order to take care about the different issues: vital, functional and aesthetic. We describe the treatment of facial localizations of type 1 NF as it is done at the University Hospital of Lyon and at the Rhône-Alpes-Auvergne neurofibromatosis reference center.

[Victor Despeignes (1866-1937): how a hygienist became the first radiation oncologist]. / Victor Despeignes (1866-1937) : comment un hygiéniste devint le premier radiothérapeute de l'Histoire.

Between the 4th and 23rd July 1896, Victor Despeignes has performed the first anticancer radiotherapy that has been validated by undeniable publications and practical facts. However, the events and the intellectual approach that led him to perform this unique treatment are misknown. This unpublished biography endeavours to better understand the work of Victor Despeignes who was, first of all, an hygienist physician. The conjunction of Pasteur's ideas, the discovery of X-rays and a probable technical help of the Lumière brothers led him to irradiate a cancerous tumour. The life of Victor Despeignes can be divided in three parts; the Lyon period (1866-1894) during which he became physician and researcher in parasitology, working on the quality of tap water and tuberculosis; the period spent between 1894 and 1907 in Buis-les-Baronnies (Drôme) and thereafter in Les Échelles (Savoie) during which he was a simple district physician and performed the first radiotherapy; the Chambery period (1907-1937) during which he was the Director of the Town Hygiene and provided a major action for public health.

[Claudius Regaud (1870-1940): A pioneer of radiobiology and radiotherapy]. / Claudius Regaud (1870-1940) : relecture des archives d'un pionnier de la radiobiologie et de la radiothérapie.

Born in 1870, Claudius Regaud was a pioneer of radiobiology and radiotherapy. As histologist, he developed a new staining technique that allowed him to describe in detail all the reproduction system of a number of animal models. As radiobiologist, he contradicted the interpretations of Tribondeau and Bergonié about relationships between cell proliferation and radiosensitivity. In 1908, he suggested that chromatin was the main target of radiation. As physician, he defined the first bases of anti-cancer radiation treatments and treated patients suffering from incurable cancer from 1911. As military doctor, he organized war hospitals by creating multidisciplinary teams for the surgery of hurts. Organizer, he was one of the founders of the League against Cancer. As radiotherapist and brachytherapist, he contributed to make Institut Curie an international reference center for research and teaching, with nearly a thousand treated patients. As globe-trotter, he was at the origin of the creation of numerous worldwide radiotherapy and radiobiology centers. He died in December 1940 and let an impressive but still misknown scientific heritage. A re-reading of the familial archives and the Regaud Fund of Institut Curie is the occasion to remind the contribution of Regaud.

[Radiation biology: major advances and perspectives for radiotherapy]. / Biologie des radiations: avancées majeures et perspectives pour la radiothérapie.

At the beginning of the 21st century, radiation biology is at a major turning point in its history. It must meet the expectations of the radiation oncologists, radiologists and the general public, but its purpose remains the same: to understand the molecular, cellular and tissue levels of lethal and carcinogenic effects of ionizing radiation in order to better protect healthy tissues and to develop treatments more effective against tumours. Four major aspects of radiobiology that marked this decade will be discussed: technological developments, the importance of signalling and repair of radiation-induced deoxyribonucleic acid (DNA) damage, the impact of individual factor in the response to radiation and the contribution of radiobiology to better choose innovative therapies such as protontherapy or stereotactic body radiation therapy (SBRT). A translational radiobiology should emerge with the help of radiotherapists and radiation physicists and by facilitating access to the new radio and/or chemotherapy modalities.

[Radiobiological features of anti-cancer treatments involving synchrotron radiation: outcome and perspectives]. / Aspects radiobiologiques des traitements anticancéreux par rayonnement synchrotron: bilan et perspectives.

Each technological development of radiotherapy is an example of interaction between physicians and physicists. In the past, it was the case for the first X-rays generators, betatrons and particle accelerators. To date, this is the case for Cyberknives and intensity modulation radiotherapy. In the future, this will be the case for proton- and hadron-therapy. However, in a general tendency of favouring higher radiation energies, leaving the 250kV orthovoltage irradiators and preferring accelerators delivering some tens MeV to reach the deepest tumours, how to consider the anti-cancer applications of synchrotron radiation that provides X-rays in the 10-100keV "only"? Since the first approaches developed in the USA in seventies until the last preclinical trials performed at the European Synchrotron Radiation Facility of Grenoble, the radiobiological features of the chemoradiotherapy involving synchrotron radiation will be described and analysed throughout a transversal view considering physicochemical bases, biomolecular and cellular mechanisms and results from the preclinical trials in order to provide a general outcome and some eventual transfer perspectives.

[Very low-dose hyper-radiosensitivity: impact for radiotherapy of micrometastases]. / Hyperradiosensibilité aux très faibles doses: impact en radiothérapie des micrométastases.

Radiobiologists have pointed out a novel radiobiological phenomenon observed in many tumor and normal cell lines: hyper-radiosensitivity to very low-dose (HRS) followed by induced radioresistance (IRR) after a threshold dose of 0.1-0.3 Gy that depends on the cell line. Radioresistance at high dose (i.e. higher than 0.5 Gy) and metastatic potential of tumor cells are likely major factors of failure in radiotherapy. A careful review of literature suggests that: 1) radiotherapy does not increase the metastatic potential of tumor cells; 2) radioresistance at high dose and metastatic potential are not related. However, inside a given tumor cell line, highly metastatic clones may elicit more cells showing HRS or are more radiosensitive at high dose than poorly metastatic ones. Recent data obtained from molecular techniques (comet and immunofluorescence assays) applied to single cells irradiated at very low radiation doses (1-100 mGy) suggest that DNA single-strand breaks (SSB) and double-strand breaks (DSB) may be the key-lesions responsible for the HRS phenomenon. These data suggest that the HRS phenomenon may find application in radiotherapy for micrometastasis. These early disseminated and probably unvascularised cells may escape the influence of high-dose chemotherapy after excision of the primary tumor. Considering the link between metastatic potential and HRS, we have previously proposed to apply very low-dose total body irradiation (TBI) at M(0) stage that may prevent the development of micrometastases. Literature data suggest that the smallest radiation dose that can produce HRS without increasing the risk of cancer may be in the milliGrays range.

[Intrinsic radiosensitivity and DNA double-strand breaks in human cells]. / Radiosensibilité intrinsèque et cassures double-brin de l'ADN dans les cellules humaines.

Among the large spectrum of DNA damage induced by radiation, DNA double-strand breaks (DSBs) are considered, to date, as the key-lesions responsible for the cell killing. However, although it was always intuitive to radiobiologists, such a conclusion has only been reached after technical developments and conceptual advances and remains consensual rather than demonstrated formally. In this article, we have reviewed the results that have lead to the conclusion that the assessment of successful DSB repair can be the basis of reliable assays predictive of the clinical response to radiotherapy and some chemotherapeutic treatments. We have discussed a number of technical artifacts, the biases due to the extrapolation of data obtained in yeast and rodent model systems to the human situation and the variety of phenotypes observed in human cells and in particular: 1) the most recent techniques developed, based on immunofluorescence, which have revolutionized our understanding of the molecular events occurring early after irradiation but have also raised the crucial questions about the choice of techniques to assess DSB repair and their specificity for different steps of the repair process; 2) While the homologous recombination repair pathway is predominant in yeasts, its importance in human cells appears less obvious, and raises the problem that the existence of randomized repair events may produce many more errors in human cells than in small genome organisms; 3) the impairment of DSB repair is observed in a plethora of genetic diseases, leading to radiosensitivity, immunodeficiency and sometimes cancer-proneness, but the low frequency and the pleiotropism of such diseases makes difficult the development of a single predictive assay. Therefore, although complete DSB repair appears to be crucial for cell survival, further research is still needed to provide innovative techniques fro measuring repair which can be successfully transferred to the clinic and used to ensure the avoidance of deleterious side-effects to cancer therapies.

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum.

XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.

The tumor suppressor activity induced by adenovirus-mediated BRCA1 overexpression is not restricted to breast cancers.

The BRCA1 (breast cancer 1) breast cancer susceptibility gene is recognized as responsible for most familial breast and ovarian cancers and is suggested to be a tissue-specific tumor suppressor gene. In this report, we investigated the tissue specificity of tumor inhibitory activities induced by a recombinant adenovirus coding for wild-type BRCA1 (wtAdBRCA1). We demonstrated a pronounced in vitro antiproliferative effect on H1299 lung and HT29 colon cells upon infection with AdBRCA1. We describe a prolonged G1 cell cycle arrest associated with a decrease in the hyperphosphorylated form of Rb, suggesting that the Rb/E2F pathway is implicated in BRCA1-induced cell growth arrest. We also observed a significant antitumor effect in these pre-established subcutaneous tumors after in situ delivery of AdBRCA1, although these two tumors do not express wt p53, and also estrogen alpha and beta, progesterone and androgen receptors. Moreover, BRCA1 can induce a strong prolonged cell cycle arrest and apoptotic cell death but no significant antiangiogenic effect in H1299 tumors. Finally, our data indicate that intratumor administration of wtAdBRCA1 significantly inhibits growth of lung and colon steroid hormone-independent tumors.

BRCA1 carries tumor suppressor activity distinct from that of p53 and p21.

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.

The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway.

Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB.

A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells.

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.