Guillaume Chassagnon
email: guillaume.chassagnon@aphp.fr

Katharina Martini

Philippe Giraud

Marie-Pierre Revel

Radiological assessment after stereotactic body radiation of lung tumours Évaluation radiologique après la radiothérapie stéréotaxique des tumeurs pulmonaires

Keywords: Radiosurgery, Lung neoplasms, Multidetector computed tomography, Positron emission tomography computed tomography

The increasing use of stereotactic body radiation therapy for lung tumours comes along with new posttherapeutic imaging findings that should be known by physicians involved in patient follow-up.

Radiation-induced lung injury is much more frequent than after conventional radiation therapy, it can also be delayed and has a different radiological presentation. Radiation-induced lung injury after stereotactic body radiation therapy involves the lung parenchyma surrounding the target tumour and appears as a dynamic process continuing for years after completion of the treatment. Thus, the radiological pattern and the severity of radiation-induced lung injury are prone to changes during follow-up, which can make it difficult to differentiate from local recurrence. Contrary to radiationinduced lung injury, local recurrence after stereotactic body radiation therapy is rare. Other complications mainly depend on tumour location and include airway complications, rib fractures and organizing pneumonia. The aim of this article is to provide a wide overview of radiological changes occurring after SBRT for lung tumours. Awareness of changes following stereotactic body radiation therapy should help avoiding unnecessary interventions for pseudo tumoral presentations.

Résumé

L'utilisation plus fréquente de l'irradiation stéréotaxique pour le traitement des tumeurs pulmonaires s'accompagne de nouveaux aspects en imagerie qui devraient être connus des médecins impliqués dans le suivi de ces patients. La pneumopathie radique y est plus fréquente qu'après une radiothérapie classique, elle peut également être plus tardive et s'associe à une présentation radiologique différente.

La pneumopathie radique après une radiothérapie en conditions stéréotaxiques est localisée au parenchyme entourant la tumeur traitée et se présente comme un processus dynamique pouvant se modifier jusqu'à plusieurs années après la fin du traitement. Ainsi, l'aspect radiologique et la sévérité de la pneumopathie radique ont tendance à se modifier au cours du suivi, ce qui rend difficile le diagnostic différentiel avec une récidive locale. Contrairement à la pneumopathie radique, la récidive locale est rare après la radiothérapie en conditions stéréotaxiques. Les autres complications dépendent principalement de la localisation de la tumeur et incluent les atteintes des voies aériennes, les fractures costales et la pneumopathie organisée. L'objectif de cet article est de présenter une large revue des modifications radiologiques après la radiothérapie en conditions stéréotaxiques des tumeurs pulmonaires. La connaissance des aspects post-thérapeutiques peut permettre d'éviter des examens ou des interventions inutiles en présentation pseudo tumorale.

Mots clé

Radiochirurgie ; Néoplasies pulmonaires ; scanographie multidétecteurs ; tomographie par émission de positron couplée à la tomodensitométrie

Introduction

Technological advances in image guidance and treatment planning have made it possible to develop stereotactic body radiation therapy, which consists of delivering a very high radiation dose to the target tumour while restricting the inclusion of normal tissues [START_REF] Martin | Stereotactic body radiotherapy: a review[END_REF].

Stereotactic body radiation therapy has gradually become a standard treatment for patients with early stage non-small cell lung cancer non eligible for surgical treatment or oligometastatic status.

Studies have shown that stereotactic body radiation therapy allows 3-year local control rates higher than 90% for early stage non-small cell lung cancer and ranging from 60 % to 91% for lung metastases [START_REF] Senthi | Patterns of disease recurrence after stereotactic ablative radiotherapy for early stage non-small-cell lung cancer: a retrospective analysis[END_REF][START_REF] Timmerman | Stereotactic body radiation therapy for inoperable early stage lung cancer[END_REF][START_REF] Chang | Stereotactic ablative radiotherapy versus lobectomy for operable stage I non-small-cell lung cancer: a pooled analysis of two randomised trials[END_REF][START_REF] Ricco | Lung metastases treated with stereotactic body radiotherapy: the RSSearch® patient Registry's experience[END_REF][START_REF] Okunieff | Stereotactic body radiation therapy (SBRT) for lung metastases[END_REF][START_REF] Alongi | Review and uses of stereotactic body radiation therapy for oligometastases[END_REF]. Additionally, a pooled analysis of two randomized trials has shown that stereotactic body radiation therapy could be an option for treating operable stage I non-small cell lung cancer [START_REF] Chang | Stereotactic ablative radiotherapy versus lobectomy for operable stage I non-small-cell lung cancer: a pooled analysis of two randomised trials[END_REF].

The increased use of stereotactic body radiation therapy for lung tumours comes along with new post-therapeutic imaging features that should be known by physicians involved in patient follow-up.

The main difference between stereotactic body radiation therapy and conventionally fractionated radiotherapy relates to radiation-induced lung injury that is much more frequent in the former case, can be delayed and has a different radiological presentation. Post-treatment changes may wrongly be attributed to local recurrence.

Therefore, the aim of this article is to provide a wide overview of radiological changes occurring after stereotactic body radiation therapy for lung tumours.

Radiation-induced lung injury

Radiation-induced lung injury is related to an oxidative damage caused by the formation of free radicals. The early stage is characterized by an exudative phase with damages of the alveolar epithelial and vascular endothelial cells causing the release of inflammatory mediators [START_REF] Huang | The cellular and molecular mechanism of radiation-induced lung injury[END_REF][START_REF] Ghaye | Imaging after radiation therapy of thoracic tumors[END_REF]. This phase is followed by an organizing phase with an accumulation of blood exudate and inflammatory cells at the site of tissue injury. If the exacerbated inflammatory response persists, it leads to lung tissue damage with progressive development of fibrosis at the late stage.

Risk factors for radiation-induced lung injury include a higher radiation dose, higher proportion of tissues submitted to a high radiation dose, and a small number of fractions [START_REF] Ghaye | Imaging after radiation therapy of thoracic tumors[END_REF]. This explains the higher proportion of radiation-induced lung injury after stereotactic body radiation therapy as compared to conventional radiotherapy. Indeed, the concept of stereotactic body radiation therapy is to safely deliver a high dose with a hypofractionated schedule to a small radiation field, minimizing the exposition of normal tissue.

Radiation-induced lung injury is usually divided in two stages: an early stage, up to the 6 th month after treatment also named transient radiation pneumonitis and a late stage also named as chronic radiation fibrosis [START_REF] Ghaye | Imaging after radiation therapy of thoracic tumors[END_REF].

In the context of conventional radiotherapy, transient radiation pneumonitis usually occurs within the first 3 months after the completion of treatment. If inflammation persists beyond the 6 th month, fibrotic remodelling occurs to form chronic radiation fibrosis. Lesions are usually considered to be stable after the 24t h month and associate architectural distortion, volume loss and presence of traction bronchiectasis. It is noteworthy that most transient radiation pneumonitis resolve without any sequelae whereas chronic radiation fibrosis is not always preceded by a transient radiation pneumonitis phase.

Conversely to conventional radiotherapy, radiation-induced lung injury occurs in most patients after stereotactic body radiation therapy. Because of the small size of the radiation field, radiationinduced lung injury remains localized to the close surrounding of the tumour and is usually asymptomatic. Indeed, only 9% of acute stereotactic body radiation therapy-related radiation-induced lung injuries are symptomatic [START_REF] Zhao | Simple factors associated with radiation-induced lung toxicity after stereotactic body radiation therapy of the thorax: a pooled analysis of 88 studies[END_REF], compared to 13 to 37% after conventional conformational radiotherapy. In both situations, symptoms may include dyspnoea, cough, fever and pleuritis. Differential diagnosis with chronic obstructive pulmonary disease exacerbation can be clinically difficult. A good response to steroid therapy is observed in about 80% of cases. Risk factors for symptomatic stereotactic body radiation therapy-related radiation-induced lung injury are older age, larger tumour size and more advanced stage (IB versus IA non-small cell lung cancer) [START_REF] Zhao | Simple factors associated with radiation-induced lung toxicity after stereotactic body radiation therapy of the thorax: a pooled analysis of 88 studies[END_REF]. In a pooled analysis of 88 studies, Zhao et al. found no significant correlation between dosimetric factors and the rate of symptomatic forms [START_REF] Zhao | Simple factors associated with radiation-induced lung toxicity after stereotactic body radiation therapy of the thorax: a pooled analysis of 88 studies[END_REF]. On the other hand, Okubo et al. reported that subclinical interstitial lung disease was a risk factor for symptomatic radiation pneumonitis [START_REF] Okubo | Predicting risk factors for radiation pneumonitis after stereotactic body radiation therapy for primary or metastatic lung tumours[END_REF].

The median time interval from the completion of stereotactic body radiation therapy to the occurrence of radiation-induced lung injury is approximately 17 weeks, but in 25% of cases, the first radiation-induced lung changes occur more than 1 year after stereotactic body radiation therapy [START_REF] Dahele | Radiological changes after stereotactic radiotherapy for stage I lung cancer[END_REF]. This time interval is longer than that for conventional radiation therapy in which changes usually appear within 4 weeks [START_REF] Febbo | Stereotactic body radiation therapy for early-stage nonsmall cell lung cancer: a primer for radiologists[END_REF].

At the early stage after stereotactic body radiation therapy, there is no evidence of increased density in the lung parenchyma surrounding the treated lesion in about 21% to 46% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF]. This include cases where tumour is stable, regressive or resolving, or cases with fibrosis in the position of the original tumour, not larger than the original tumour. In the other patients, stereotactic body radiation therapy-induced lung changes are usually categorized in four general patterns (Figure 1) that have been defined as follows by Palma et al. [START_REF] Palma | Radiological and Clinical pneumonitis after stereotactic lung radiotherapy: a matched analysis of three-dimensional conformal and volumetric-modulated arc therapy techniques[END_REF]:

•
diffuse consolidation corresponding to a consolidation larger than 5 cm in largest dimension and being more extended than the aerated lung in the involved region; this pattern is found in 14% to 38% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF];

• patchy consolidation corresponding to a consolidation measuring less than 5 cm in largest dimension and/or being less extensive than the aerated lung in the involved region; this pattern is found in 8% to 33% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF];

• diffuse ground glass opacity corresponding to ground glass opacity larger than 5 cm, without consolidation. The involved region contains more ground glass opacity than normal lung. This pattern is found in 4% to 12% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF];

• patchy ground glass opacity corresponding to a ground glass opacity area measuring less than 5 cm, without consolidation, and/or involved region containing less ground glass opacity than normal lung; this pattern is also found in 4% to 12% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF].

At the late stage, after 6 month from completion of stereotactic body radiation therapy, only 1 to 20% of patients do not present radiation fibrosis [START_REF] Dahele | Radiological changes after stereotactic radiotherapy for stage I lung cancer[END_REF][START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF]. In the other patients, three patterns are used to describe radiation pneumonitis [START_REF] Koenig | Radiation injury of the lung after three-dimensional conformal radiation therapy[END_REF] (Figure 2):

• modified conventional pattern is the most frequently encountered pattern (46% to 71% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF]). It corresponds to a consolidation associated with volume loss and bronchiectasis.

Its presentation is close to that observed in conventional radiation fibrosis but is less extensive.

Consolidation may be more extensive than the initial tumour and may be associated with ground glass opacity;

• scar-like fibrosis patterns is found in 11% to 22% of patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF]. CT images show linear opacities in the region of the treated tumour, associated with volume loss;

• mass-like fibrosis pattern presents as a focal consolidation larger than the treated tumour. This cofounding aspect is found in 7% to 20% of patients and the main differential diagnosis is local recurrence [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF]. The frequency of this pattern explains the difficulty of radiological follow-up of patients treated with stereotactic body irradiation.

Interestingly, Dahele et al. demonstrated that post-stereotactic body radiation therapy radiation fibrosis is a dynamic process continuing for years and that its pattern and severity are prone to changes during follow-up (Figure 3) [START_REF] Dahele | Radiological changes after stereotactic radiotherapy for stage I lung cancer[END_REF]. In their cohort of 61 patients, the incidence of radiation fibrosis at 6, 12, 24, and 36 months was 56%, 73%, 87%, and 99%, respectively. Therefore, the first CT changes developed more than 1 year after treatment in 27% of cases. They also showed that the proportion of mass-like pattern tends to increase up to 2 years after stereotactic body radiation therapy whereas the proportions of patients with no radiation fibrosis or a modified conventional pattern decrease over time. Similarly, the maximum severity of the radiation fibrosis was usually observed around 2 years from completion of stereotactic body radiation therapy and then decreased in intensity. In 47% of cases, the morphology or severity of the radiation fibrosis continued to evolve more than 2 years after stereotactic body radiation therapy. This dynamic process of the radiation fibrosis makes it difficult to distinguish post-stereotactic body radiation therapy changes from tumour recurrence.

Tumour recurrence

Stereotactic body radiation therapy has been reported to have an efficacy comparable to that of surgery for the treatment of early stage non-small cell lung cancer [START_REF] Nguyen | Can stereotactic fractionated radiation therapy become the standard of care for early stage non-small cell lung carcinoma[END_REF][START_REF] Chang | Stereotactic ablative radiotherapy versus lobectomy for operable stage I non-small-cell lung cancer: a pooled analysis of two randomised trials[END_REF]. Recurrences are usually classified as local when occurring at the site or adjacent to the treated tumour, regional when occurring in a regional lymph node (ipsilateral hilar or mediastinal lymph lymphadenopathy), or distant when occurring at any other site. In a cohort of 676 patients with stage I-II non-small cell lung cancer treated with stereotactic body irradiation, Senthi et al. reported 5-year local and regional control rates of 90% and 87%, respectively [START_REF] Senthi | Patterns of disease recurrence after stereotactic ablative radiotherapy for early stage non-small-cell lung cancer: a retrospective analysis[END_REF]. In their study, most recurrences occurred distantly. The 5-year distant control rate was 80%. These rates are close to those observed after surgery [START_REF] Nguyen | Can stereotactic fractionated radiation therapy become the standard of care for early stage non-small cell lung carcinoma[END_REF][START_REF] Robinson | Patterns of failure after stereotactic body radiation therapy or lobar resection for clinical stage I non-small-cell lung cancer[END_REF]. Median time to local recurrence was 13 months but local recurrence occurred up to 5 years following the treatment. Thus, local recurrence is uncommon after stereotactic body radiation therapy whereas radiation-induced lung injury is common, but there is an overlap between the median time to local recurrences and the peak of radiation-induced lung injury.

In non-small cell lung cancer, risk factors for local recurrence include larger size, squamous cell histology, low biologically effective dose and altered stereotactic body radiation therapy dose schedule [START_REF] Febbo | Stereotactic body radiation therapy for early-stage nonsmall cell lung cancer: a primer for radiologists[END_REF][START_REF] Loganadane | Stereotactic ablative radiotherapy for early stage non-small cell lung cancer: A critical literature review of predictive factors of relapse[END_REF][START_REF] Woody | A histologic basis for the efficacy of SBRT to the lung[END_REF]. In patients treated for lung metastasis, risk factor for progression are larger size, more than three lung metastases and presence extrathoracic metastases at the time of stereotactic body radiation therapy [START_REF] Borm | Prognostic factors in stereotactic body radiotherapy of lung metastases[END_REF].

While regional and distant recurrence are usually not difficult to diagnose, diagnosing local recurrence is challenging due to the overlap of the radiological appearance with that of radiationinduced lung injury (Figures 4 and5). However, distinguishing between the two entities is of great importance in patients who may be amenable to salvage therapy.

The challenge of post-stereotactic body radiation therapy follow-up

As it is the case after targeted therapies or radiofrequency ablation, the use of response evaluation criteria in solid tumors (RECIST) for post-stereotactic body radiation therapy follow-up suffers from several limitations [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF][START_REF] Mattonen | Pulmonary imaging after stereotactic radiotherapy-does RECIST still apply?[END_REF]. Indeed, local response may be difficult to assess because size increase is not necessarily associated with local recurrence. In a cohort of 22 patients, Mattonen et al. reported a RECIST overall accuracy of 52% for predicting tumour recurrence at 2 to 5 months post-stereotactic body radiation therapy with a false positive rate of 72.7% and a false negative rate of 25% [START_REF] Mattonen | Early prediction of tumor recurrence based on CT texture changes after stereotactic ablative radiotherapy (SABR) for lung cancer[END_REF]. Thus, new criteria are required to more accurately evaluate tumour response.

A list of high-risk features has been proposed for the detection of local recurrence on CT imaging. This list was initially created based on a systematic review of the literature and then validated and further enriched in a cohort of 36 patients [START_REF] Huang | Radiographic changes after lung stereotactic ablative radiotherapy (SABR) -Can we distinguish recurrence from fibrosis? A systematic review of the literature[END_REF][START_REF] Huang | High-risk CT features for detection of local recurrence after stereotactic ablative radiotherapy for lung cancer[END_REF]. The high-risk features include:

• enlarging opacity;

• enlargement observed after 12 months;

• continuous enlargement on consecutive CT scans;

• craniocaudal growth of at least 5mm or 20%;

• bulging margin;

• linear margin disappearance;

• and disappearance of air bronchogram.

Huang et al. showed that all these features were significantly associated with local recurrence (p < 0.01), and that the odds of recurrence increased 4-fold for each additional detected high-risk feature [START_REF] Huang | High-risk CT features for detection of local recurrence after stereotactic ablative radiotherapy for lung cancer[END_REF]. They also found that the presence of at least three high-risk features had a sensitivity and specificity superior to 90% for recurrence. In another series of 39 patients, Peulen et al. found that a cut-off value of at least four high-risk features gave the best result with a sensitivity of 92% and a specificity of 85% [START_REF] Peulen | Validation of high-risk computed tomography features for detection of local recurrence after stereotactic body radiation therapy for early-stage non-small cell lung cancer[END_REF]. They also reported that the occurrence of an isolated pleural effusion without any additional high-risk feature should not considered as a sign of recurrence.

The role of positron emission tomography-CT (PET-CT) to differentiate between radiationinduced lung injury and local recurrence has also been evaluated. PET-CT response following stereotactic body radiation therapy may differ from response after conventional radiotherapy where standard uptake value usually decreases and returns to background in case of successful treatment.

Studies have shown that persistent increased metabolic activity on PET-CT, even 1 to 2 years after stereotactic body radiation therapy, may not always represent persisting or recurrent disease [START_REF] Hoopes | FDG-PET and stereotactic body radiotherapy (SBRT) for stage I non-small-cell lung cancer[END_REF][START_REF] Matsuo | Characterization of FDG-PET images after stereotactic body radiation therapy for lung cancer[END_REF].

Increased metabolic activity, usually observed in case of local recurrence, can also be related to radiation-induced lung injury. Ishimori et al. reported that standard uptake value increased in four out of nine patients who underwent PET-CT before and after stereotactic body radiation therapy [START_REF] Ishimori | 18 F)-FDG and ( 11 C)-methionine PET for evaluation of treatment response of lung cancer after stereotactic radiotherapy[END_REF]. In two patients, standard uptake value increased was observed more than 3 months following stereotactic body radiation therapy. Vahdat et al. reported a mean standard uptake value of 2 (range: 1.5 to 2.8) for controlled tumours at 18 to 24 months in a series of 21 patients with stage IA non-small cell lung cancer [START_REF] Vahdat | CyberKnife radiosurgery for inoperable stage IA non-small cell lung cancer: ( 18 F)-fluorodeoxyglucose positron emission tomography/computed tomography serial tumor response assessment[END_REF], however standard uptake values over 5 were observed in patients without local recurrence [START_REF] Hoopes | FDG-PET and stereotactic body radiotherapy (SBRT) for stage I non-small-cell lung cancer[END_REF]. Thus, PET-CT is not currently recommended as part of systematic follow-up procedure but when disease recurrence, including local recurrence is suspected.

A post-stereotactic body radiation therapy follow-up scheme combining high-risk features and PET-CT has been proposed by Huang et al. for patients who are candidates for salvage treatment [START_REF] Huang | High-risk CT features for detection of local recurrence after stereotactic ablative radiotherapy for lung cancer[END_REF].

Patients are categorized as having a low-(no high-risk feature), intermediate-(one or two high-risk features), or high-risk (three or more high-risk features) of recurrence depending on the number of high-risk features on follow-up CT scans. For intermediate-risk patients, a PET-CT is recommended.

If maximum standard uptake value is less than 5, close CT or PET-CT should be performed and then biopsy should be considered if results are non-reassuring. In intermediate-risk patients with maximum standard uptake value greater than 5.0 or exceeding pretreatment maximum standard uptake value and in high-risk patients, additional interventions including biopsy, resection or non-surgical salvage are recommended.

Ronden et al. investigated the incidence and patterns of change of HRFs on follow-up CT-scans

in a series of 88 patients (747 CTs) without local recurrence [START_REF] Ronden | Incidence of high-risk radiologic features in patients without local recurrence after stereotactic ablative radiation therapy for early-stage non-small cell lung cancer[END_REF]. They found that a majority of patients (53% according to at least three of the five readers) without local recurrence had at least two high-risk features, while about one fourth (23% according to at least three of the five readers) had at least three high-risk features. An enlarging opacity and an enlargement after 12 months were the two most frequent high-risk features. They had both a maximum incidence of 23.4% at 3 years poststereotactic body radiation therapy. Only two patients (2.3%) did not have an enlarging opacity on any follow-up scan. These authors proposed to exclude an enlargement after 12 months as an independent high-risk feature. Despite the high frequency of cases with at least two and at least three high-risk features, the authors found that only six patients underwent a PET-CT for a suspected local recurrence and only one patient underwent a transthoracic biopsy. These results underline the importance of radiological expertise and suggest that the previously mentioned recommendations should probably be reconsidered.

Other approaches to differentiate between radiation-induced lung injury and local recurrence based on magnetic resonance imaging (MRI) and radiomics have been evaluated. Using diffusionweighted MRI to assess tumour response in 15 patients with early stage non-small cell lung cancer, Shintani et al. showed that the median apparent diffusion coefficient values at 3 and 6 months after stereotactic body radiation therapy were significantly lower in patients with local recurrence whereas no significant difference was observed in the maximum standard uptake value at any time point [START_REF] Shintani | Assessment of treatment response after lung stereotactic body radiotherapy using diffusion weighted magnetic resonance imaging and positron emission tomography: A pilot study[END_REF].

The apparent diffusion coefficient value is a quantitative parameter reflecting water mobility in the microenvironment. In case of successful treatment, the apparent diffusion coefficient is expected to increase due to the increase in extracellular space associated with cell death. However, the authors did not observe significant differences between apparent diffusion coefficient values after 6 months ( 9month and 12 months) in patients with and without local recurrence and there was an overlap between the measured values. Radiomics is a field of medical imaging aiming to extract features (e.g. image characteristics invisible to the human eye) from medical images to create image-driven biomarkers [START_REF] Nishino | State of the art: Response assessment in lung cancer in the era of genomic medicine[END_REF][START_REF] Afshar | From handcrafted to deeplearning-based cancer radiomics: Challenges and opportunities[END_REF]. The objective is to select imaging features of various complexity and to combine them using machine learning methods in order to establish correlations with clinical outcomes such as the presence of local recurrence. Several studies have evaluated radiomics on PET and CT images in the context of stereotactic body radiation therapy either to predict outcome on pretreatment images [START_REF] Bousabarah | Radiomic analysis of planning computed tomograms for predicting radiation-induced lung injury and outcome in lung cancer patients treated with robotic stereotactic body radiation therapy[END_REF][START_REF] Starkov | The use of texture-based radiomics CT analysis to predict outcomes in early-stage non-small cell lung cancer treated with stereotactic ablative radiotherapy[END_REF][START_REF] Huynh | CT-based radiomic analysis of stereotactic body radiation therapy patients with lung cancer[END_REF][START_REF] Li | Unsupervised machine learning of radiomic features for predicting treatment response and overall survival of early stage non-small cell lung cancer patients treated with stereotactic body radiation therapy[END_REF] or to differentiate between radiation-induced lung injury and local recurrence [START_REF] Mattonen | Early prediction of tumor recurrence based on CT texture changes after stereotactic ablative radiotherapy (SABR) for lung cancer[END_REF][START_REF] Mattonen | Detection of local cancer recurrence after stereotactic ablative radiotherapy (SABR) for Lung cancer: physician performance versus radiomic assessment[END_REF]. Comparing the performance of a radiomics model to those of six physicians for detecting local recurrence, Mattonen et al. found that radiomics can detect early changes associated with local recurrence [START_REF] Mattonen | Detection of local cancer recurrence after stereotactic ablative radiotherapy (SABR) for Lung cancer: physician performance versus radiomic assessment[END_REF].

Their algorithm had a false positive and a false negative rates of 24% and 23%, respectively.

Complications after SBRT

Stereotactic body radiation therapy is associated with a lower complication rate than that reported for conventional radiotherapy. Indeed, even though stereotactic body radiation therapy-related radiation pneumonitis and radiation fibrosis are being observed in the vast majority of patients, only a few are symptomatic and should thus be considered as true complications. Other complications include:

Radiation-induced organizing pneumonia

Organizing pneumonia can present with symptoms close to symptomatic radiation pneumonitis, including dyspnoea, cough and fever. Whereas lesions are limited to the irradiated area in radiation pneumonitis, lung infiltrates in organizing pneumonia are observed outside the radiation field and frequently migrate (Figure 6). The mechanism of radiation-induced organizing pneumonia remains unknown but lymphocyte-mediated hypersensitivity reactions and induced immune disorders have been suggested [START_REF] Ghaye | Imaging after radiation therapy of thoracic tumors[END_REF]. In a cohort of 189 patients, Murai et al. reported an incidence of symptomatic stereotactic body radiation therapy-related organized pneumonia of 4% and 5% at 1 and 2 years after treatment, respectively [START_REF] Murai | Organizing pneumonia after stereotactic ablative radiotherapy of the lung[END_REF]. Ochiai et al. reported a time interval from the completion of stereotactic body radiation therapy to the occurrence of organizing pneumonia of 6 to 18 months [START_REF] Ochiai | Radiationinduced organizing pneumonia after stereotactic body radiotherapy for lung tumor[END_REF]. Symptoms and lesions can rapidly improve under steroid therapy, but up to 20% to 44% of patients relapse.

Complications observed for central tumours

Central tumours are defined as tumours located within 2 cm of any mediastinal critical structure, including the bronchial tree, oesophagus, heart, brachial plexus, major vessels, spinal cord, phrenic nerve, and recurrent laryngeal nerve [START_REF] Chang | Stereotactic ablative radiotherapy for centrally located early stage non-small-cell lung cancer: what we have learned[END_REF]. Central tumours are associated to a higher risk of complications [START_REF] Kang | Complications from stereotactic body radiotherapy for lung cancer[END_REF].

In case of central tumour abutting the trachea or a proximal bronchus, it is possible to observe at the early stage a bronchial thickening of the adjacent airway with or without stenosis [START_REF] Febbo | Stereotactic body radiation therapy for early-stage nonsmall cell lung cancer: a primer for radiologists[END_REF][START_REF] Kang | Complications from stereotactic body radiotherapy for lung cancer[END_REF]. At the late stage, a stricture can persist. This can cause obstruction or even atelectasis. More rarely, bronchial necrosis with tracheobronchial fistula may occur. Recurrent pneumonitis in patients who underwent stereotactic body radiation therapy for central lung tumour is suggestive of the diagnosis [START_REF] Febbo | Stereotactic body radiation therapy for early-stage nonsmall cell lung cancer: a primer for radiologists[END_REF].

When the treated tumour is close to the oesophagus, oesophagitis, stricture, perforation, and/or tracheoesophageal fistula can be encountered [START_REF] Kang | Complications from stereotactic body radiotherapy for lung cancer[END_REF].

Vascular complications such as aortitis or fatal haemoptysis complicating radiation-induced injury to the central pulmonary arteries are also possible but have been rarely reported [START_REF] Kang | Complications from stereotactic body radiotherapy for lung cancer[END_REF].

Complications observed for peripheral tumours

In patients treated for subpleural tumours, rib fractures may occur (Figure 7). This delayed complication usually occurs 16 to 48 months after completion of stereotactic body radiation therapy with an incidence of 16 to 24%. One third of patients are symptomatic [START_REF] Nambu | Rib fracture after stereotactic radiotherapy for primary lung cancer: prevalence, degree of clinical symptoms, and risk factors[END_REF][START_REF] Aoki | Radiation-induced rib fracture after stereotactic body radiotherapy with a total dose of 54-56 Gy given in 9-7 fractions for patients with peripheral lung tumor: impact of maximum dose and fraction size[END_REF]. These patients can also present focal pleural thickening [START_REF] Febbo | Stereotactic body radiation therapy for early-stage nonsmall cell lung cancer: a primer for radiologists[END_REF].

Follow-up recommendations

The currently recommended algorithm for imaging follow-up after stereotactic body radiation therapy for non-small cell lung cancer mainly follows follow-up recommendations after lung surgery [START_REF] Huang | Follow-up of patients after stereotactic radiation for lung cancer: a primer for the nonradiation oncologist[END_REF][START_REF] Jaklitsch | The American Association for Thoracic Surgery guidelines for lung cancer screening using low-dose computed tomography scans for lung cancer survivors and other high-risk groups[END_REF][START_REF]NCCN clinical practice guidelines in oncology -non-small cell lung cancer n[END_REF].

It includes a chest CT every 3 to 6 months during the first year, then a chest CT every 6 months for 3 years and an annual chest CT from the 4 th after stereotactic body radiation therapy. Contrast medium injection is optional the first 2 years and then unenhanced chest CT is recommended. Importantly, PET-CT is not recommended as part of the systematic follow-up.

The presence of high-risk features on CT images can help detecting local recurrence but is not specific. In the setting of suspected recurrence, discussion in a multidisciplinary team is recommended to decide on expert opinion whether closer follow-up, PET-CT or biopsy should be performed.

Conclusion

Stereotactic body radiation therapy is an increasingly popular method for the management of primary and secondary lung tumours. Knowing specificities of radiological assessment after stereotactic body radiation therapy is important, especially being able to recognize radiation-induced lung injury as a possible aetiology of lung changes in the radiation field, even several years after the completion of treatment. Despite the published high-risk CT features and other approaches based on radiomics and lung MRI, differentiating between radiation-induced lung injury and local recurrence remains difficult.

It is important to know that radiation-induced lung injury may mimic local recurrence and is more frequent than the later. Increased knowledge of post-stereotactic body radiation therapy imaging changes and multidisciplinary team discussions should help avoiding unnecessary examinations or interventions. Twenty months after the completion of stereotactic body radiation therapy, a pulmonary CT angiography was performed for suspicion of pulmonary embolism due to chest pain. The CT angiography was negative for pulmonary embolism but showed stereotactic body radiation therapyinduced rib fractures (arrows).

Figure 1 .Figure 2 .

 12 Figure 1. Radiological patterns of transient radiation pneumonitis after stereotactic body radiation therapy. a) No evidence of increasing density. b) Diffuse consolidation larger than 5 cm in largest dimension. c) Patchy consolidation. d) Diffuse ground glass opacity larger than 5 cm, without consolidation. e) Patchy ground glass opacity

Figure 3 .

 3 Figure 3. Changes in pattern and severity of radiation-induced lung injury after stereotactic body radiation therapy. a) Baseline chest CT shows a small nodule in the left lower lobe. b) At 6 months, no radiation pneumonitis is seen. c-g: Radiation-induced lung injury first manifests at 12 months (c), withan increasing severity from the 12 th to the 36 th month (d: 18 months; e: 24 months; f: 30 months; g: 36 months). On chest CT scan performed at 36 months, a mass-like fibrosis larger than the treated lesion is seen (g).

Figure 4 .

 4 Figure 4. Post-stereotactic body radiation therapy radiation-induced lung injury mimicking local recurrence. a) Baseline chest CT shows a nodule in the right lower lobe. b) 6 months post-stereotactic body radiation therapy chest CT. c) 12 months post-stereotactic body radiation therapy chest CT. d)At 18 months after completion of stereotactic body radiation therapy, a mass larger than the treated lesion is seen. Therefore, a PET-CT is performed and shows only a mild ( 18 F)-fluorodeoxyglucose uptake (maximum standard uptake value: 2). e) 19 months post stereotactic body radiation therapy chest CT. f) As the mass continued to enlarge on the chest CT performed at 24 months, the patient underwent a transthoracic biopsy that showed no tumoral cells, only inflammatory changes. g) 25 months post-stereotactic body radiation therapy chest CT.

Figure 5 .

 5 Figure 5. Recurrence of a lung adenocarcinoma after stereotactic body radiation therapy. a,b) Baseline chest CT (a) and PET-CT (b) show a hypermetabolic lung nodule in the right lower lobe (maximum standard uptake value: 4). c)Follow-up chest CT performed 18 months after completion of the treatment (shows a mass-like consolidation in the radiation field. d,e) PET-CT examination shows increased fluorodeoxyglucose uptake of the lung mass (maximum standard uptake value: 7, on d)higher than that of the treated nodule on the baseline examination, as well as an area of increased metabolic activity in the liver (e). This was suggestive of local and distant recurrence.

Figure 6 .

 6 Figure 6. Stereotactic body radiation therapy-induced organizing pneumonia. A 65-year-old patient with a right lower lobe adenocarcinoma treated by stereotactic body irradiation. a) baseline exam. b) Follow-up examination at 12 months after completion of stereotactic body radiation therapy showing

Figure 7 .

 7 Figure 7. Stereotactic body radiation therapy-induced rib fracture. a) CT angiography in an 80-year old patient with stage I peripheral lung adenocarcinoma treated by stereotactic body irradiation. b)

Author contributions

Conceptualization: GC, KM, PG, MPR. Writing -original draft: GC -review and editing: KM, PG, MPR.


Georges Hayek
email: georges.hayek@aphp.fr

Bruno Kastler

Interventional radiology for treatment of bone metastases Apport de la radiologie interventionnelle dans le traitement des métastases osseuses

Keywords: Interventional radiology, bone metastasis, image-guided tumour ablation Radiologie interventionnelle, métastases osseuses, ablation tumorale guidée par l'imagerie

Therapeutic advances in oncology have led to longer survival in many forms of cancer, including those complicated by bone metastases. When a bone metastasis is painful or when there is a risk of fracture, interventional radiology procedures can be carried out for pain control and/or stabilization.

All of these techniques can be performed under local anaesthesia. Cementoplasty and vertebroplasty

Résumé

Les progrès thérapeutiques en oncologie ont entraîné une survie plus longue dans de nombreuses formes de cancer, y compris celles compliquées par des métastases osseuses. Lorsqu'une métastase osseuse est douloureuse ou lorsqu'il existe un risque de fracture, des procédures de radiologie interventionnelle peuvent être réalisées pour le contrôle de la douleur et/ou la stabilisation de la fracture. Toutes ces techniques peuvent être accomplies sous anesthésie locale. La cimentoplastie et la vertébroplastie sont des procédures de stabilisation consistant en l'injection percutanée de ciment acrylique dans une lésion osseuse lytique. L'effet sur la douleur peut s'expliquer par la consolidation de l'os affaibli, fracturé ou préfracturé, mais aussi dans une moindre mesure par les effets toxiques, chimiques et thermiques du ciment. Les techniques d'ablation tumorale comprennent l'alcoolisation ou l'ablation thermique (par la chaleur avec radiofréquence et micro-ondes, ou froid par cryoablation).

L'ablation thermique percutanée des tumeurs osseuses est le plus souvent réalisée en tant que technique palliative entraînant une réduction significative et durable des symptômes. L'ablation par radiofréquence consiste à placer des aiguilles à travers lesquelles passe un courant électrique. L'ablation par micro-ondes agit en provoquant des vibrations à très haute fréquence des molécules d'eau. La cryoablation libère de l'argon à l'extrémité d'une aiguille, formant une « boule de glace » détruisant efficacement les cellules tumorales. Chacune de ces techniques peut être combinée à la radiothérapie, ou effectuée avant ou après l'irradiation. Enfin, l'embolisation tumorale peut avoir pour objectif de contrôler la douleur ou de préparer une intervention chirurgicale pour réduire le risque d'hémorragie peropératoire.

Introduction

Therapeutic advances in oncology have led to longer survival in many forms of cancer, including those complicated by bone metastases. Some cancers that used to have a very short survival have developed into chronic diseases, in which bone metastases can occur. Systemic treatments will help control the global disease. When there is a painful bone metastasis or when there is a risk of fracture, interventional radiology procedures can be carried out for analgesic purposes and/or to stabilize the lesion. Depending on the case, they may replace or be combined with radiation therapy and/or surgery. In rare cases, they may even be performed for curative purposes in oligometastatic patients with lesions smaller than 3 to 4 cm in order to control the disease.

These acts of interventional bone radiology can be classified into consolidation techniques (vertebroplasty, cementoplasty, percutaneous screw placement, etc.) and tumour destruction techniques using thermal (radiofrequency ablation, microwave ablation and cryoablation), chemical (alcohol) and embolization methods.

Stabilization techniques

Cementoplasty and vertebroplasty

Cementoplasty and vertebroplasty (i.e. cementoplasty of a vertebra) consist in the percutaneous injection of acrylic cement into a lytic bone lesion. The initial aim is analgesic by the effect of consolidation of a weakened piece of bone. These techniques derive from those initially developed in 1987 by Galibert and Deramond in Amiens on seven angiomatous vertebrae [START_REF] Galibert | Preliminary note on the treatment of vertebral angioma by percutaneous acrylic vertebroplasty[END_REF]. The indications then extended to metastatic vertebrae, myeloma lesions and osteoporotic fractures.

They consist in injecting a biocompatible cement into the bone, under x-ray and/or CT control.

The most commonly used cement is polymethylmethacrylate (PMMA). The liquid phase (monomer) and the PMMA powder are mixed once the needle is positioned in the bone lesion. As soon as the consistency of the cement slightly thickens, the cement can be carefully injected. After approximately 8 to 10 minutes, the cement hardens during the polymerization phase. This is accompanied by an exothermic reaction with temperature peaks of up to 75°C in the centre of the treated bone.

The analgesic effect is mainly attributed to the consolidation and stabilization of the pathological bone. Some authors believe that thermal elevation during polymerization may play an accessory analgesic role by destroying nociceptors. Once solidified, PMMA cement has perfect stability and excellent compressive strength. These characteristics explain why cementoplasty finds excellent indications in load-bearing bone areas subjected mainly to compressive forces such as vertebral bodies (if no vertebral instability) and the acetabulum. On the other hand, bone areas where shear forces predominate or long bones such as ribs are not suitable for simple cementoplasty.

Cementoplasty and vertebroplasty are performed according to the teams either in a digital interventional radiology room, in an angiography room equipped with cone beam CT or on an interventional CT with or without fluoroscopy. They can be performed under local or general anaesthesia depending on the clinical context, the general condition of the patient and the number of sites to be treated. Local anaesthesia is carried out from the skin to the bone cortex using a mixture of anaesthetic combining xylocaine for rapid action but short duration of action (about 2 hours) and ropivacaine for a delayed effect (after about 30 minutes) and much longer duration of action (about 6 hours). A small intraosseous anaesthesia by nerve block is associated with it after the needle has been inserted.

At the lumbar level, the needle is placed by transpedicular or lateropedicular route when the pedicle is very thin. At the thoracic level, the intercostotransverse route is preferred. A single needle is usually sufficient to fill the vertebral body. Sometimes a bipedicular approach is performed immediately, or at a later stage in the case of suboptimal filling with the first needle in place. The cervical spine is approached anterolaterally, between the large vessels of the neck and the tracheooesophageal axis but can also be approached posteriorly under CT guidance. Once the needle is in place in the bony part, a coaxial biopsy is performed if necessary.

Depending on the size of the lesion, 1 to several cm 3 of cement are slowly injected under fluoroscopic control or under CT scan control (iterative injection of small quantities of cement with immediate control by repeating a CT acquisition centred on the bone element to be treated). Lysis of the cortex and in particular of the posterior wall of the vertebral body is not a contraindication but requires particular caution when injecting the cement.

Apart from infections linked to the procedure, which are very rare, complications may arise during the positioning of the needle or during the injection of the cement. Cement leakage in the perivertebral veins, in the soft tissues or in the intervertebral discs is almost always asymptomatic.

Cement leakage in the spinal canal is most often well tolerated if it is of small volume (very rare spinal cord compression). Foraminal leakage may be responsible for neurological complications (radiculalgia), most of the time regressing spontaneously or after infiltration. Pulmonary embolism by cement is almost always asymptomatic if it is not massive.

The decision to perform the procedure is taken in a multidisciplinary board to evaluate the respective place of surgery, radiation therapy, medical treatments and their possible combination, taking into account the general condition of the patient, his life expectancy, the local or general extent of the disease and the number of spinal levels needing to be treated.

The analgesic effect of cementoplasty can be explained by the consolidation of weakened, fractured or prefractured bone, but also to a lesser extent by the toxic, chemical and thermal effect of the cement. The sedation of the pain generally appears immediately and makes it possible to stop major analgesics whereas radiation therapy may require one or two weeks to be effective.

We recommend combining cementing with thermal ablation, especially in oligometastatic lesions, in order to avoid possible tumour dissemination through the drainage veins (especially at the vertebral level) inherent to any cement injection. If analgesic radiation therapy is planned, it may be carried out before or after the cementoplasty according to the team's habits. On the other hand, if stereotactic radiation therapy for curative purposes is planned, it must be carried out before the cementoplasty so as not to disturb the calculation of the dose of radiation to be delivered.

The lesions for which vertebroplasty can be proposed are:

• asymptomatic osteolytic lesions at risk of fracture in an area where there are compressive forces (vertebra, pelvis, sternum, etc.) (2) (fig. 1); • painful osteolytic lesions with prefracture or fracture in an area where there are compressive forces (vertebra, pelvis, sternum, etc.) (fig. 2 and3); in the case of unstable lesions, surgery will be recommended instead.

A simple injection of cement is not possible in sclerotic metastases. Thermal ablation is a good alternative (see below).

Percutaneous fixation by internal cemented screw

Fixation by internal cemented screws is a technique that stabilizes a bone metastasis through the placement of a perforated metal screw combined with the injection of cement through the screw.

Treatment may be performed for pain palliation or prevention or in the context of an impending pathologic fracture. The addition of metal elements to the cementoplasty provides greater strength.

The procedure is minimally invasive and is performed under fluoroscopic or CT guidance.

Tumour destruction techniques

Intratumoral nerve blocks

Prior to ablation treatment, a percutaneous nerve block test injection of a mixture of anaesthetics (two thirds ropivacaine-one third xylocaine) may be performed. Several milliliters of this mixture are instilled in the tumour and the test block effect is sometimes prolonged by a palliative therapeutic effect of several days to a few weeks. This injection is both a diagnostic test on the origin of the pain and allows the patient to wait serenely for the heavier ablative treatment.

Alcoholization

Introduced into a tumour, alcohol causes desiccation and tissue destruction. Using pure alcohol, it was proposed for analgesic use in refractory extraspinal lesions before the advent of thermoablation techniques (3). It is a painful procedure requiring an analgesic intratumoral block to be performed prior to the instillation of alcohol.

Radiofrequency ablation, microwave ablation or cryoablation

Percutaneous thermal ablation (radiofrequency ablation, microwave ablation or cryoablation) of bone tumours is most often performed as a palliative measure. Numerous studies have shown that percutaneous ablation of painful bone metastases results in a significant and lasting reduction in symptoms [START_REF] Deib | Percutaneous microwave ablation ablation and cementoplasty: clinical utility in the treatment of painful extraspinal osseous metastatic disease and myeloma[END_REF]. It is therefore an alternative to radiation therapy and long-term morphine treatments.

It can be performed on bone metastases that has already been treated by radiation therapy but remain painful or as a first-line treatment in the case of painful bone metastasis resistant to analgesics before radiation therapy. Percutaneous ablation can also be performed for curative purposes in patients with a slowly progressing cancer with few bone lesions, provided the lesions are small (less than 4 cm)

(5,6) (fig. 4).

Whether palliative or curative, percutaneous ablation should be followed by an injection of cement when the treated metastasis has a lytic component and is located on a load-bearing bone. The purpose of this consolidating cementoplasty is to compensate for the additional fracture risk caused by bone destruction following the percutaneous ablation.

Radiofrequency ablation consists of placing under CT guidance, an electrode in the tumour to deliver a sinusoidal current of 400 to 500 kHz [START_REF] Alemann | Treatment of painful extraspinal bone metastases with percutaneous bipolar radiofrequency ablation under local anesthesia: feasibility and efficacy in twenty-eight cases[END_REF]. The regions through which the radiofrequency ablation current travels are subjected to ion agitation which induces tissue heating by friction between particles. The aim is to expose the tumour cells to a temperature above 60 °C (threshold for tissue necrosis). Its effectiveness is well established [START_REF] Dupuy | Percutaneous radiofrequency ablation ablation of painful osseous metastases: a multicenter American College of Radiology Imaging Network trial[END_REF]. The more recently introduced microwave ablation acts by causing very high frequency vibrations of water molecules. It is much more rapid in action (and therefore less painful) and allows treating larger lesions [START_REF] Kastler | Microwave ablation thermal ablation of spinal metastatic bone tumors[END_REF][START_REF] Kastler | Analgesic effects of microwave ablation ablation of bone and soft tissue tumors under local anesthesia[END_REF]. Unlike radiofrequency ablation, it is not sensitive to the conductive properties of the tissue and is not hindered by the vaporization phenomena inherent to any tissue heating. Similarly, cryoablation requires the placement under CT guidance of one or more needles within the tumour [START_REF] Mcmenomy | Percutaneous cryoablation of musculoskeletal oligometastatic disease for complete remission[END_REF]. Its principle is based on the Joule-Thompson effect: a rapid decompression of argon gas at the tip of the needle allows reaching a temperature of -180 °C in a few seconds. It has the advantage of generally seeing the size of the 'ice ball'-induced lesion.

In the treatment of bulky bone lesions refractory to pain medication, the goal of thermal ablation is analgesic, i.e. to improve the quality of life of the patients and not to destroy the entire tumour. It will target the edges between the tumour and bone structures in an attempt to destroy the nerve endings of the endosteum that are probably involved in the genesis of the pain.

When there are nerve structures, such as the spinal cord or motor nerves in close proximity, the interventional radiologist must be precautious because they are very sensitive to heat (above 45 °C or below -5 °C). Consequently, the procedure must be extremely controlled for spinal tumours. A tumour of the vertebral body could be treated by thermal ablation provided that the posterior vertebral wall is intact and that it is at a distance of over 1 cm from the spinal cord. Thermocouples can be used in these cases to protect fragile structures [START_REF] Kastler | Feasibility of real-time intraprocedural temperature control during bone metastasis thermal microwave ablation ablation: a bicentric retrospective study[END_REF]. For bone localisations close to neurological structures, cryoablation seems more suitable than radiofrequency ablation. Indeed, the zone of ice that develops around the tip of the needle is sometimes visible under CT scan during the procedure ('ice ball') and can therefore be monitored precisely.

Cold being less painful than heat, cryoablation is better tolerated under local anaesthesia than thermal ablation by radiofrequency or microwave ablation. On the other hand, after cryoablation a prolonged increase in pain can be observed in the postoperative period, up to one week, related to the inflammation in the periphery of the treated zone.

Contrary to simple cementoplasty, sclerotic bone lesions are accessible to thermal ablation.

Embolization

It can be used presurgically in the case of hypervascular bone metastases (e.g. kidney cancer metastases) to devascularize the tumour and reduce the risk of bleeding during the operation. It can be performed independently for both palliative and analgesic purposes (i.e. reduction of tumour volume decreasing the invasion into surrounding soft tissue and periosteal 'stretching').

A large retrospective study of 309 embolization procedures for painful bone metastases due to kidney, thyroid, breast and lung cancers in 243 patients showed a decrease in pain of more than 50% in 97% of patients and a decrease in the use of pain medication for an average duration of 8.1 months (12).

Conclusion

Percutaneous interventional radiology offers a range of minimally invasive analgesic and consolidation techniques that can be performed under local anaesthesia. In patients with bone metastases that are refractory to medical pain therapy, it allows rapid and significant pain control, thus

Fig. 1 .Fig. 2 .

 12 Fig. 1. T6 vertebroplasty under exclusive CT guidance and local anesthesia in a very painful vertebral compression fracture in a 56-year-old man with multiple bone myeloma. a: sagittal reconstruction of the spinal CT scan showing a pathological vertebral compression fracture of T6 (arrow) secondary to extensive lysis of the vertebral body; b: axial section of the CT through T6 showing a large lytic lesion of the vertebral body of T6 (arrow); c: sagittal reconstruction of the post-vertebroplasty control CT; d: axial section of the post-vertebroplasty CT showing a good filling of the osteolytic lesion by the cement (arrow) without extravertebral leakage.

Fig. 3 .

 3 Fig. 3. Cementoplasty of a metastatic lytic lesion of a urothelial cancer reaching the posterior wall of the left acetabulum under exclusive CT guidance and local anaesthesia. a: axial section of the CT through the acetabulum showing the lytic lesion of the posterior wall (arrow), patient is in prone position; b: axial section of the CT showing the needle in place in the lesion; c: post-procedure control showing the filling of the lesion with cement (arrow).

Fig. 4 .

 4 Fig. 4. Microwave ablation therapy of a small sclerotic metastasis of L3 with curative aim in a 80 year-old man with lung cancer. a: axial section of the CT scan showing the small metastatic sclerotic lesion located in the vertebral body of L3 (arrow); b: axial section CT showing the microwave ablation probe in place in the osteocondensing lesion; c: sagittal T2 STIR-weighted image on the postprocedure control MRI. The lesion appears in low signal on T2 STIR. The high T2 STIR signal on the periphery of the tumour destruction zone is the expected appearance of a successful ablation; d: axial T1weighted with fat suppression and postcontrast injection image on the postprocedure control MRI. The lesion does not take up the contrast agent, whereas the periphery enhances after contrast injection (arrow) and along the tumour ablation trajectory (arrowhead).

cm), a curative effect can be expected depending on their location.


L Abi Nassif

Stéphane Rioual

W Farah

M Fauchon

Y Toueix

Claire Hellio

Marie Abboud

Benoit Lescop

L Abi Nassif

Electrophoretic deposition of zinc alginate coatings on stainless steel for marine antifouling applications

Keywords: alginate, biofouling, zinc, Stainless steel, bacteria, microalgae

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Marine biofouling can be defined as the undesirable settlement and growth of organisms on biotic and abiotic immersed surfaces in seawater such as ships, oil and gas platforms, pilings, mariculture facilities, buoys and pontoon [START_REF] Yebra | Antifouling technology-past, present and future steps towards efficient and environmentally friendly antifouling coatings[END_REF]. Due to the numerous impacted applications, it leads to a significant economic cost [START_REF] Schultz | Economic impact of biofouling on a naval surface ship[END_REF]. This process is caused by the attachment of organic molecules and organisms such as bacteria, microalgae, macroalgae, or invertebrates (barnacles, mussels, ascidians…) on a wide range of surfaces and can be therefore strongly limited by the use of anti-biofouling coatings. Tributyltin (TBT) based coatings were proven highly toxic towards non-target organisms and are now banned since several years [START_REF] Yebra | Antifouling technology-past, present and future steps towards efficient and environmentally friendly antifouling coatings[END_REF]. Coatings containing antimicrobial agents such as copper, silver, zinc oxide under different forms (ions, nanoparticles…) have been proposed as substitute to TBT-based paints. However, the toxicity of copper and silver ions is now well-established [START_REF] Nys | The impact and control of biofouling in marine finfish aquaculture[END_REF][START_REF] Srinivasan | Managing the Use of Copper-Based Antifouling Paints[END_REF] while the one of nanoparticles is still debated [START_REF] Pandiarajan | Properties, synthesis and toxicity of silver nanoparticles[END_REF]. Within this context, the development of biocompatible coatings is a priority. Recently, our group has developed bio-based alginate hydrogels [START_REF] Abi Nassif | Development of alginate hydrogels active against adhesion of microalgae[END_REF][START_REF] Abi Nassif | Reduction of potential ennoblement of stainless steel in natural seawater by an ecofriendly biopolymer[END_REF]. These previous studies have shown that a mixed Zinc / Calcium alginate protects the surfaces by blocking the adhesion of microalgae together with a low toxicity, a property that is essential for environmental friendly applications. Steel metal surfaces were protected against biofouling by placing active hydrogel cubes of few cm 3 in their surrounding environment [START_REF] Abi Nassif | Reduction of potential ennoblement of stainless steel in natural seawater by an ecofriendly biopolymer[END_REF]. The present work aims at investigating the antifouling property of coatings produced by active alginates.

Coating of surfaces can be performed by numerous techniques such as sol-gel, plasma spray, or electrophoretic deposition (EPD). EPD is a versatile and inexpensive technique with advantages such as the need for a simple apparatus, short processing time and minor restrictions on substrate shape [START_REF] Avcu | Electrophoretic deposition of chitosan-based composite coatings for biomedical applications: A review[END_REF][START_REF] Singh | Electrophoretic deposition of bioactive glass composite coating on biomaterials and electrochemical behavior study: A review[END_REF]. This method consists of applying a potential difference between an electrode and the conductive substrate. Charged particles or polymer chains in aqueous solution then move towards the substrate under the influence of the electrical field, resulting in coatings between 0.1 to 100 µm thick [START_REF] Boccaccini | Electrophoretic deposition of biomaterials[END_REF]. Being an anionic polysaccharide, alginate was successfully deposited by EPD [START_REF] Yokoyama | Formation of optically anisotropic alginic acid gels under DC electric fields[END_REF].

Within the present study, we propose to evaluate the antifouling property of Zinc/Calcium alginate coatings produced by EPD on 316L stainless steel (316L SS). Based on our knowledge, this is the first attempt to report the effect of zinc alginate electrophoretic coating against marine microorganisms (bacteria and microalgae). The coatings were analyzed by Grazing incidence X-ray diffraction, electron microscopy SEM and by X-ray Photoelectron Spectroscopy. Their antifouling properties were tested on microorganisms, which can be considered as pioneer in marine biofilm formation: four bacteria (Halomonas aquamarina, Vibrio aesturianus, Vibrio harveyi and Pseudoalteromonas elyakovii) and two microalgae strains (Halomphora coffeaformis and Cylindrothecca closterium). Copper / Calcium alginate coatings were also produced to highlight the effects of several ions on the growing of marine microorganisms.

Materials and methods

Material preparation

316L is a well-known austenitic stainless steel containing a high proportion of Cr (about 16 % atomic), Ni (10 %) and Mo (2 %) and a very low proportion of carbon (< 0.04 %). It has excellent corrosion resistance and good mechanical properties. The 316L samples were designed with a circular shape of 13 mm diameter and a thickness of 1 mm. They were treated with silicon carbide paper and cleaned using acetone. Moreover, to improve the adherence of the alginate coating, the roughness of samples was increased by immersion in a H3PO4 solution (C = 1 mol/L) during 10 min. A solution of sodium alginate (Sigma Aldrich 71238, M/G ratio: 0.85) at a concentration of 10 g/L was prepared under by magnetic stirring during 5 hours. The stainless steel and a counter electrode (graphite) were immersed in the sodium alginate solution. The distance between the two electrodes was set at 20 mm and an electrical potential of 5 V was applied. Then, the incorporation of cations such as Ca 2+ , Cu 2+ , Zn 2+ into the coating was performed via an ion exchange mechanism: the substitution of hydrogen ions in the alginate matrix by cations [START_REF] Germanos | Diffusion and agglomeration of iron oxide nanoparticles in magnetic calcium alginate beads initiated by copper sorption[END_REF]. This was achieved by immersing the materials in different solutions: CaCl2 (0.36 mol.L -1 ), CuSO4 (0.1 mol.L -1 ), ZnC4H6O4 (0.1 mol.L -1 ) for pure coatings and 0.27 mol.L -1 (calcium) and 0.05 mol.L -1 (copper / zinc) for mixed-alginate coatings respectively.

Physicochemical characterization of the coating

The analysis of the morphology of the hydrogels and their chemical composition were performed using field emission SEM (Scanning Electron Microscopy) coupled with EDX (Energy Dispersive X-ray analysis). The chemical composition of the surface was characterized by XPS using an apparatus consisting of an Al Ka X-ray source (Thermo VG) and a cylindrical mirror analyzer (RIBER). The adventitious C(1s) peak was used for the binding energy calibration at 284.8 eV. The crystalline structure of the coated samples was analyzed by grazing incidence angle X-ray diffraction (GIXRD) with an Empyrean PANalytical apparatus using the CuKa radiation (1.5408 Å) with an incidence angle of 0.5°.

The main interest of the GIXRD measurements is its high sensitivity to the surface of the material and therefore to the coating.

Biological assays

For the assessment of bioactivity against microfouling organisms, both the inhibition of cell adhesion and growth were evaluated [START_REF] Trepos | Antifouling Compounds from the Sub-Arctic Ascidian Synoicum pulmonaria: Synoxazolidinones A and C, Pulmonarins A and B, and Synthetic Analogues[END_REF]. All the strains were purchased from culture collection: American Type Culture Collection (ATCC) and Algobank (AC) respectively for bacteria and microalgae. The strains used for the experimental work are four bacteria (Halomonas aquamarina ATCC 14400, Vibrio aestuarianus ATCC 35048, Vibrio harveyi ATCC 14126 and Pseudoalteromonas elyakovii ATCC 700519) and two microalgae (Halamphora coffeaeformis AC713 and Cylindrotheca closterium AC170) model species commonly used in biofouling studies [START_REF] Moodie | Design and biological evaluation of antifouling dihydrostilbene[END_REF].

The algal strains were maintained and grown at 20°C in F/2 medium, under a light exposure regime of 12 hours light (irradiance: 140 μmol.m -2 .s -1 ) / 12 hours darkness. Prior to experiments, microalgal stock solutions at the concentration of 0.1 mg/L chlorophyll a were prepared [START_REF] Trepos | Antifouling Compounds from the Sub-Arctic Ascidian Synoicum pulmonaria: Synoxazolidinones A and C, Pulmonarins A and B, and Synthetic Analogues[END_REF]. For the assay of hydrogel coating, experiments were run in 6 replicates. 1 mL of the microalgal stock solution (0.1 mg/L chlorophyll a) was transferred to the wells of transparent 24 Iwaki well containing the material to test. The plates were then incubated for 5 days at 20°C under constant light exposure of 12 hours light/darkness (irradiance: 140 μmol.m -2 .s -1 ). Microalgal growth inhibition was measured according to standard protocols [START_REF] Trepos | Antifouling Compounds from the Sub-Arctic Ascidian Synoicum pulmonaria: Synoxazolidinones A and C, Pulmonarins A and B, and Synthetic Analogues[END_REF][START_REF] Moodie | Design and biological evaluation of antifouling dihydrostilbene[END_REF]. Microalgal growth was estimated by chlorophyll a quantification using a fluorometric method (excitation at 485 nm and an emission at 645 nm). Results are expressed as percentage of inhibition compared to a control (uncoated steel).

Prior to experiments, bacteria were pre-grown in sterile Marine Bacterial Medium (MBM) (0.5% peptone (Neutralised Bacteriological Peptone, Oxoid Ltd, Basingstoke, UK) in sterile filtered (Whatman 1001-270, pore size 11 μm) natural seawater, at 20 °C [START_REF] Bovio | The Sponge-Associated Fungus Eurotium chevalieri MUT 2316 and its Bioactive Molecules: Potential Applications in the Field of Antifouling[END_REF]. For the assay of hydrogel coating, experiments were run in 6 replicates. 1 mL of the bacterial stock solution (2×10 8 colony forming units/mL) was transferred to the wells of transparent 24 Iwaki well containing the material to test [START_REF] Thabard | Sargassum polyceratium (Phaeophyceae, Fucaceae) surface molecule activity towards fouling organisms and embryonic development of benthic species[END_REF]. The plates were then incubated for 72 hours at 20°C. Bacterial growth inhibition was measured according to standard protocols [START_REF] Bovio | The Sponge-Associated Fungus Eurotium chevalieri MUT 2316 and its Bioactive Molecules: Potential Applications in the Field of Antifouling[END_REF]. Bacterial growth was monitored spectroscopically (Tecan Infinite M200) at 630 nm [START_REF] Trepos | Evaluation of cationic micropeptides derived from the innate immune system as inhibitors of marine biofouling[END_REF]. Results are expressed as percentage of inhibition compared to a control (uncoated steel).

Results and discussion

Physical characterization of the coatings

Following the procedure presented in the materials and methods section, 316L SS samples were coated with alginic acid using the EPD. This process is effective due to the dissociation of sodium alginate in anionic Alg -species: NaAlg Na + + Alg -. At the anode (stainless steel electrode), water molecules are decomposed (2 H2O O2 + 4 H + + 4 e -), which lowers the pH. The positive potential at the stainless steel electrode attracts the Alg - species, which react with hydrogen to form the alginic acid gel Alg(H) on the stainless steel surface. Figure 1 displays the morphology of the surface. As shown in Fig. 1(a), despite of the use of a mixture of ethanol / water (2:3) (v/v), bubbles formation associated with H2 production during the EPD process is evident. In this case, a constant potential of 5 V was applied during 10 s between the two electrodes. In order to improve the quality of the coating, the deposition process was carried out by two 5 V pulses for 5 s. Figure 2(a) presents the GIXRD pattern of the coated sample. The three peaks corresponding to iron in fcc austenite phase without any evidence of martensite phase are observed in agreement with Öztürk et al [START_REF] Öztürk | Microstructural and mechanical characterization of nitrogen ion implanted layer on 316L stainless steel[END_REF]. Additional peaks at 13.3° and 21.0°, attributed to the alginate [START_REF] Dong | Alginate/gelatin blend films and their properties for drug controlled release[END_REF], prove the presence of the alginate coating on stainless steel. The thickness of the coating was estimated by peeling a coating and by obtaining a SEM image. Figure 2(b) shows this SEM image: the coating is a dense layer 2.9 µm thick. As a result, the surface is completely and uniformly covered. [START_REF] Hryniewicz | Electrochemical and XPS studies of AISI 316L stainless steel after electropolishing in a magnetic field[END_REF] in accordance with the composition of steel. For surfaces coated with alginic acid (Alg(H)), carbon and oxygen atoms concentrations increase due to the presence of hydroxyl and carboxyl groups in the alginate coating matrix. The presence of sodium can be noted due to the use of sodium alginate in the initial solution.

Immersion of the Alg(H) coating in solutions containing copper, zinc or calcium ions then leads to a change in its composition. Indeed, as shown in Table 1, the presence of these ions in the coating is clearly demonstrated. This result can be explained by the ion exchange process that occurs between H + and Na + of Alg(H) and the cations present in the solution. As this Table shows, the presence of chloride in Alg(Ca) is also observed due to the use of CaCl2 in the solution [START_REF] Abi Nassif | Reduction of potential ennoblement of stainless steel in natural seawater by an ecofriendly biopolymer[END_REF]. The ionic states of the various elements on the surface were determined by XPS.

Figure 3 shows the survey spectra of uncovered and alginate-covered 316L SS up to a binding energy of 1150 eV. The uncovered steel spectrum is dominated by carbon, oxygen, iron and chromium species in accordance with the previous XPS data [START_REF] Hryniewicz | Electrochemical and XPS studies of AISI 316L stainless steel after electropolishing in a magnetic field[END_REF] and with our EDX data. The Fe(2p) and Cr(2p) spectra (not shown here) are dominated by structures at 710.0 (Fe 2p3/2), 724.1 (Fe 2p1/2), 575.6 (Cr 2p3/2) and 585.6 eV (Cr 2p1/2). These positions prove the existence of oxidized species at the surface [START_REF] He | Corrosion of stainless steel 316L in simulated formation water environment with CO2-H2S-Cl[END_REF] to form a passive layer. It should be noted that small contributions of the metallic species Fe 0 , Cr 0 are also reported indicating the thinness of the oxide layer. As reported in Figure 3, the XPS spectra measured on alginate coatings differ greatly from those obtained on steel. The total disappearance of the iron and chromium peaks confirms the complete covering of the surface. The baseline observed in the uncovered surface is classical for iron material [START_REF] Bhargava | Characterization of the ''native'' surface thin film on pure polycrystalline iron: A high resolution XPS and TEM study[END_REF], but the baseline of the covered surface spectra is different. This observation is consistent with the total masking of the 316L SS surface.

Structures associated to the Na(1s), Cu(2p) or Zn(2p) atoms at high binding energy demonstrate the presence of these species in the coatings according to our EDX data. iron and chromium oxides [START_REF] Tardio | Comparative study of the native oxide on 316L stainless steel by XPS and ToF-SIMS[END_REF], while the high energy part is related to hydroxide and carbonates species present at the surface [START_REF] Tardio | Comparative study of the native oxide on 316L stainless steel by XPS and ToF-SIMS[END_REF]. For alginate-covered steels, no oxide-associated peaks are observed and only one peak at about 532 eV is observed. This peak can be attributed to the chemical groups of alginate such as the C -OH groups of alcohol, C -O -C groups of acetal and hemiacetal and carboxylate groups. The peak shift with the cation with the alginate is explained by the interaction of the cations with the functional groups of the alginate [START_REF] Germanos | Diffusion and agglomeration of iron oxide nanoparticles in magnetic calcium alginate beads initiated by copper sorption[END_REF][START_REF] Deschatre | Characterization of exopolysaccharides after sorption of silver ions in aqueous solution[END_REF]. The existence of the well-known shake-up satellites proves that copper ions are in the Cu 2+ form [START_REF] Biesinger | Resolving surface chemical states in XPS analysis of first row transition metals, oxides and hydroxides: Sc, Ti, V, Cu and Zn[END_REF], as usually observed in biomaterials [START_REF] Sun | Enhanced Cu(II) and Cr(VI) biosorption capacity on poly(ethylenimine) grafted aerobic granular sludge[END_REF]. These positions can be related to the interaction between Cu 2+ ions and hydroxyls [START_REF] Biesinger | Resolving surface chemical states in XPS analysis of first row transition metals, oxides and hydroxides: Sc, Ti, V, Cu and Zn[END_REF]. The high FWHM value (full width at half maximum) of the 2p peaks can be explained by the complexity of the chemical environment such as oxygen or carboxylate species [START_REF] Cano | An XPS study of copper corrosion originated by formic acid vapour at 40% and 80% relative humidity[END_REF]. The Zn(2p3/2) spectrum of the zinc alginate coating, shown in Fig. 4(c), has a symmetrical peak at 1022.6 eV. As the position of this peak was not sufficient to determine the chemical environment, the Zn(LMM) Auger spectrum was measured. It is shown in Fig. 4(d). The position of the Zn Auger peak is at 986.8 eV. Note that a shoulder at 990.1 eV can be attributed to the Na(KLL) Auger peak. The estimated α parameter value of 2009.4 eV is consistent with the Zn 2+ bonding with hydroxyl and/or carboxylate species [START_REF] Diler | Initial formation of corrosion products on pure zinc and MgZn2 examinated by XPS[END_REF]. Thus, the XPS data prove the existence of Cu 2+ and Zn 2+ ions at the coating surface.

Biological assays of the materials

When microorganisms attach to surfaces, they form a biofilm surrounded by a complex polymer matrix. This fixed and communal form of life offers many advantages, including protection against toxic agents. Thus most microbial consortia in the natural environment are found in the form of biofilms. Communication between cells plays an important role in synchronizing processes within the biofilm and is done through the quorum sensing (QS) system. QS can modulate the regulation of gene expression ([29]Lami 2019). It is important to note as well that it was proven that marine bacteria and microalgae can interact via quorum sensing molecules ([30] Joint et al., 2002). In order to develop new antifouling solutions, specific experiments can be carried out to determine the mode of action of molecules on the quorum sensing. However, this is not possible when testing new surfaces.

For this reason, we have chosen to evaluate the effect of surfaces on cell adhesion and growth.

These biological phases occur before the formation of biofilms, and it is therefore relevant to develop innovative surfaces able to control or inhibit them.

The biofilm formation is initially governed by the adhesion of microorganisms on surfaces, with a dominance of bacteria and microalgae. Consequently, the influence of alginate coatings on the growth of pioneer marine microalgae and bacteria was evaluated. To decrease the toxicity of the materials against the organisms, copper/zinc quantity was reduced with respect to alginates studied in the previous part. Calcium ions were also integrated in the alginates to enhance the insolubility property of the coating and to eventually enhance antifouling property [START_REF] Abi Nassif | Development of alginate hydrogels active against adhesion of microalgae[END_REF]. The calcium and copper/zinc concentrations were set at 0.27 and 0.05 mol.L -1 respectively. These coatings are named as CaZn and CaCu. The influence of these coatings compared to a pure calcium alginate (Ca) is investigated to highlight the role of different ions in reducing the growth of bacteria/microalgae.

Figure 5 presents the growth inhibition of four marine bacteria (Fig. 5(a-d)) and two microalgae (Fig. 5(e,f)) when considering CaZn, CaCu and Ca coatings. Uncoated 316L SS samples were used as control. As observed in Fig. 5(a-d), the calcium alginate coatings leads only to a weak effect against bacteria with less than 15 % of growth inhibition. Fig. 5(a,b,d)

show that the inclusion of zinc and copper in alginate leads to a bacteria growth inhibition between 50 and 70 %. No major difference are observed between CaZn and CaCu alginates.

Copper and zinc coatings are therefore effective against marine bacteria, as demonstrated previously on microalgae [START_REF] Abi Nassif | Development of alginate hydrogels active against adhesion of microalgae[END_REF]. In contrast to these results, for Vibrio harveyi in Fig. 5(c), the growth inhibition with CaZn alginate is negligible and close to the control and calcium coating data. In this case, the CaCu coating is definitely more efficient. This effect can be explained by the fact that many Vibrio harveyi harboured antimicrobial resistant genes and thus can be found to be resistant to specific antifouling coatings [START_REF] Deng | Horizontal gene transfer contributes to virulence and antibiotic resistance of Vibrio harveyi 345 based on complete genome sequence analysis[END_REF]. After successively assessing the influence of coatings against bacteria development, two microalgae strains (Cylindrothecca closterium and Halomphora coffeaformis) were studied. Firstly, Fig. 5(e) presents the assays performed on Cylindrothecca closterium. The influence of calcium alginate on the inhibition is negligible or maybe slightly negative: the coating could enhance alga growth [START_REF] He | Mg 2+ /Ca 2+ promotes the adhesion of marine bacteria and algae and enhances following biofilm formation in artificial seawater[END_REF][START_REF] Patrauchan | Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp[END_REF]. The behaviors of CaCu and CaZn coatings are similar to those observed with bacteria (Halomonas aquamarina, Vibrio aesturianus, Pseudoalteromonas elyakovii) with a high growth reduction (about 75 %) and no major difference between copper and zinc action. Similar results were previously obtained using hydrogels [START_REF] Abi Nassif | Development of alginate hydrogels active against adhesion of microalgae[END_REF]. Indeed, the reduction of growth was observed to depend only on the availability of copper and zinc ions in the alginate material, independently of calcium ions. CaZn and CaZn coatings lead to a strong reduction of algal growth (more than 85 %), a value definitely higher than for Cylindrothecca closterium. In this case, the calcium-alginate coating presents a non-negligible inhibition effect (Fig. 5f). This result is in agreement with our previous study performed with bulk hydrogels [START_REF] Abi Nassif | Development of alginate hydrogels active against adhesion of microalgae[END_REF]. The high efficiency of CaZn and CaCu alginate may therefore be explained by the synergical effect of both Cu(Zn) and Ca ions in the inhibition process.

The calcium-zinc alginate coatings present a clear growth inhibition effect on all tested bacteria and microalgae. The role of the copper and zinc ionic species seems to be nearly identical in the reduction of growing process. Only results achieved on Vibrio harveyi show a different sensitivity to zinc and copper ions. Concerning the role of calcium ions, almost no influence of calcium ions on the inhibition is observed, excepted for the microalga Cylindrothecca closterium. The anti-bacterial and microalgal efficiency of the coating is ascribed to the release of ions in its surrounding environment. The similar sensitivity to the bacteria / microalgae to the zinc or copper alginate coatings can be explained by the equivalent quantity of metal ions (Cu 2+ , Zn 2+ ) in the material [START_REF] Stanic | Synthesis, characterization and antimicrobial activity of copper and zincdoped hydroxyapatite nanopowders[END_REF] as shown by our EDX data.

Stanic et al [START_REF] Stanic | Synthesis, characterization and antimicrobial activity of copper and zincdoped hydroxyapatite nanopowders[END_REF] have explained the antimicrobial activity of the polymer including metal ions (Cu 2+ , Zn 2+ …) by the following mechanism: ions form strong bonds with thiol, imidazole, amino and carboxyl groups of microorganism membrane proteins, causing structural changes of the membrane. The membranes exhibit a significant increase in permeability, leaving the microorganism cells incapable of properly regulating transport through the plasma membrane.

This leads to the death of the cells. The difference observed on Vibrio harveyi between CaCu and CaZn can be related to the efficiency of the copper and zinc ions to bond with the different groups or to resistance effects.

Therefore, our results showed the effectiveness of zinc alginate coating against marine fouling. One of the interests of the coating is that it is based on a natural, abundant and cheap product: alginate. This biosourced material is then not toxic against marine microoragnisms.

In addition, the EPD technique requires only a low-voltage power supply and does not require any thermal annealing. As a result, the price of the coating is greatly reduced to few cents.

Conclusion

This study highlights the feasibility of producing a uniform alginate coating containing zinc and calcium ions by electrophoretic deposition on stainless steel. Its characterization demonstrates the presence of ions in their 2+ state into the coating initiated by exchange process between the initial alginic acid coating and cations present in solutions. Biological assays have shown an effective growth inhibition of bacteria and microalgae. This result is consistent with previous studies performed with bulk alginate and is explained by the release of ions from the coating. Therefore, the proposed method should be considered as a promising antifouling technique due to its low price, easy to develop and environmentally friendly. For further development, corrosion protection and the service lifetime of coatings must be addressed.

  Fig. 1(b) shows the strong reduction in bubbles quantity by the pulses.

Figure 1 .

 1 Figure 1. SEM images of the alginate coating layer without (a) and with (b) pulses with a voltage of 5V.

Figure 2 .

 2 Figure 2. Grazing incidence angle (0.5°) X-ray diffractogram of 316L SS covered by alginate (a) and SEM micrograph of peeling alginate coating layer (b).

Figure 3 .

 3 Figure 3. Full XPS spectra of uncovered and alginate-covered 316L SS.

Figure 4 (

 4 Figure 4(a) shows the O(1s) spectra of the various alginate coatings and steel. For steel, it presents two broad structures at 529 and 532 eV. The low energy part is attributed to

Figure 4 .

 4 Figure 4. O(1s), Cu(2p), Zn(2p) XPS and Zn(LMM) Auger spectra of uncovered and alginate-covered 316L SS.

Fig. 4 (

 4 Fig. 4(b,c) presents Cu and Zn spectra of the alginate coatings. In Fig. 4(b), the Cu(2p) spectrum shows peaks corresponding to the 2p3/2 (934.5 eV), 2p1/2 (953.6 eV) and associated satellites (943.6 / 962.7 eV). The existence of the well-known shake-up satellites proves that

[Figure 5 .

 5 Figure 5. Evaluation of growth inhibition of four bacteria: Halomonas aquamarina (a), Vibrio aesturianus (b), Vibrio harveyi (c), Pseudoalteromonas elyakovii (d) and two microalgae strains: Cylindrothecca closterium (e) and Halomphora coffeaformis (f).

Figure 5 (

 5 Figure 5(f) presents the results using the microalga Halomphora coffeaformis. Both

Table 1

 1 presents the chemical composition of 316L SS covered and not covered by Alg(H) obtained from EDX experiments. The uncovered surface is composed of mainly carbon, iron, chromium and nickel

Table 1 .

 1 EDX data showing the atomic percentage of each element for the different samples

		C	O	Fe	Cr Ni Mo Si	Al Na Ca Cu Zn Cl
	316L SS	39.2	4.6	37.6 9.8 4.6 1.9 1.0 1.3	-	-	-	-	-
	Alg (H)	73.3	14.1	7.9	2.1 0.9 0.4 0.2	-	1.1	-	-	-	-
	Alg (Ca) 67.4	24.3	3.7	1.2 0.3 0.4 0.2	-	-	1.9	-	-	0.6
	Alg (Cu) 66.0	28.4	2.0	0.7 0.2 0.4 0.1	-	-	-	2.2	-	-
	Alg (Zn) 71.3	19.5	4.5	1.3 0.4 0.5 0.2	-	-	-	-	2.3	-

Acknowledgments

This work is supported by the European Union through the European Regional Development Fund (ERDF), the Ministry of Higher Education and Research, the Région Bretagne, the Conseil départemental du Finistère and Brest Métropole Océane, through the CPER Project 2015-2020 MATECOM. One of the authors (LAN) acknowledges the 'Agence