M.D Jebrane Bouaoud
email: jebrane.bouaoud@gmail.com

Guillaume Beinse

Nicolas Epaillard

Melika Amor-Sehlil

François Bidault

Isabelle Brocheriou

Geneviève Hervé

Jean-Philippe Spano

François Janot

Pascaline Boudou-Rouquette

Pascaline Boudou- Rouquette

Mourad Benassarou

Thomas Schouman

Patrick Goudot

Gabriel Malouf

François Goldwasser

Chloe Bertolus

Lack of efficacy of neoadjuvant chemotherapy in adult patients with maxillo-facial high-grade osteosarcomas: A French experience in two reference centers

Keywords: head and neck cancer, osteosarcoma, neoadjuvant chemotherapy

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

INTRODUCTION

Osteosarcomas (OS) are malignant neoplasms, locally aggressive and usually affecting long extremity bones of adolescents and young adults [START_REF] Luetke | Osteosarcoma treatment -where do we stand? A state of the art review[END_REF][START_REF] Bielack | Advances in the management of osteosarcoma[END_REF]. Maxillo-facial OS (MFOS) are rare, representing less than ten percent of all OS [START_REF] Guadagnolo | Osteosarcoma of the jaw/craniofacial region: outcomes after multimodality treatment[END_REF] and typically occur in the third or fourth decade of life [START_REF] Jasnau | Craniofacial osteosarcoma Experience of the cooperative German-Austrian-Swiss osteosarcoma study group[END_REF]. In this localization, metastases occur less frequently than in long bones osteosarcomas [START_REF] Laskar | Osteosarcoma of the head and neck region: lessons learned from a single-institution experience of 50 patients[END_REF]. Indeed, recurrence and evolution are mainly localized to the primary site [START_REF] Ketabchi | Sarcomas of the head and neck: a 10-year retrospective of 25 patients to evaluate treatment modalities, function and survival[END_REF]. Because of its scarcity and subsequent lack of data [START_REF] Thariat | Osteosarcomas of the mandible: are they different from other tumor sites?[END_REF], MFOS management is mostly based on long extremity OS guidelines.

Multimodal management is recognized as the standard of care and has been demonstrated to improve outcomes in long extremity OS [START_REF] Durfee | Review of Osteosarcoma and Current Management[END_REF][START_REF] Whelan | Survival from highgrade localised extremity osteosarcoma: combined results and prognostic factors from three European Osteosarcoma Intergroup randomised controlled trials[END_REF]. Indeed, compared to surgery alone, overall survival was increased with adjuvant multi-agent chemotherapy in long extremity OS [START_REF] The | Bone sarcomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF].

Neoadjuvant chemotherapy (neo-CT) has been developed for the treatment of long bones OS for several reasons: [START_REF] Luetke | Osteosarcoma treatment -where do we stand? A state of the art review[END_REF] to choose postoperative adjuvant CT based on the response of the primary tumor to preoperative CT [START_REF] Rosen | Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy[END_REF][START_REF] Link | The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity[END_REF] and [START_REF] Bielack | Advances in the management of osteosarcoma[END_REF] to allow more time to design endoprosthetic devices for limb-salvage procedures [START_REF] Isakoff | Osteosarcoma: Current Treatment and a Collaborative Pathway to Success[END_REF]. A good histological response to neo-CT is defined by more than 90% of necrosis on the surgical specimen. In the neoadjuvant setting, pathological response has been reported to be significantly associated with better overall survival [START_REF] Marina | Biology and therapeutic advances for pediatric osteosarcoma[END_REF], leading to establish the threshold of >90% as a predictive marker for good overall survival. Although there is a strong correlation between the degree of necrosis and survival [START_REF] Provisor | Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the Children's Cancer Group[END_REF][START_REF] Bacci | Prognostic significance of serum lactate dehydrogenase in osteosarcoma of the extremity: experience at Rizzoli on 1421 patients treated over the last 30 years[END_REF], the criteria "good histological response to neo-CT" for extremity OS, is limited to 50% of large series in literature [START_REF] Friebele | Osteosarcoma: A Meta-Analysis and Review of the Literature[END_REF]. Moreover, considering multimodal management, neo-CT is not proven to add survival benefit [START_REF] Gerrand | UK guidelines for the management of bone sarcomas[END_REF][START_REF] Mirabello | Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the Surveillance, Epidemiology, and End Results Program[END_REF] and thus despite intensified treatment [START_REF] Meyers | Intensification of preoperative chemotherapy for osteogenic sarcoma: results of the Memorial Sloan-Kettering (T12) protocol[END_REF].

In MFOS, less than 30% of patients achieve good histological response after neo-CT [START_REF] Boon | Impact of chemotherapy on the outcome of osteosarcoma of the head and neck in adults[END_REF]. Moreover, no significant benefit on overall survival was found in large series [START_REF] Thariat | Osteosarcomas of the mandible: multidisciplinary management of a rare tumor of the young adult a cooperative study of the GSF-GETO, Rare Cancer Network, GETTEC/REFCOR and SFCE[END_REF][START_REF] Society Of Otolaryngology-Head | Osteogenic sarcoma of the mandible and maxilla: a Canadian review (1980-2000)[END_REF]. On the other hand, because of surgical constrains relative to maxillo-facial anatomy [START_REF] Nthumba | Osteosarcoma of the jaws: a review of literature and a case report on synchronous multicentric osteosarcomas[END_REF], the achievement of complete resection is a critical therapeutic parameter in this field [START_REF] Jasnau | Craniofacial osteosarcoma Experience of the cooperative German-Austrian-Swiss osteosarcoma study group[END_REF][START_REF] Thariat | Osteosarcomas of the mandible: multidisciplinary management of a rare tumor of the young adult a cooperative study of the GSF-GETO, Rare Cancer Network, GETTEC/REFCOR and SFCE[END_REF][START_REF] Laskar | Osteosarcoma of the head and neck region: lessons learned from a single-institution experience of 50 patients[END_REF][START_REF] Granados-Garcia | Free osseous and soft tissue surgical margins as prognostic factors in mandibular osteosarcoma[END_REF][START_REF] Patel | Improved outcomes in patients with osteogenic sarcoma of the head and neck[END_REF]. Therefore, neo-CT appears as a potentially detrimental strategy, because of the potential lack of efficacy, as well as the delay to 3 surgical resection. Indeed, the risk of local evolution during neo-CT, whose effectiveness is not proven, is not consistent with the therapeutic challenges of MFOS.

In this orphan situation, the safety of neo-CT in MFOS in term of local control needs to be established. Our aim is to report the histological and radiological local response rates to neo-CT in a series of MFOS treated since 2001 in two MFOS referral centers in France.

PATIENTS and METHODS

Population included in the analysis

All consecutive patients treated and followed for a MFOS between 2001 and 2016 in two referral centers in France (la Pitié-Salpêtrière Hospital, APHP, RESAP, Paris; Gustave Roussy Cancer Campus, Villejuif) were screened for inclusion in this cohort.

We included all adult patients treated for a histologically proven MFOS, with at least surgery and/or chemotherapy (with or without metastasis).

Collection of data

Collected data included: baseline patient (clinical and demographic data) and tumor characteristics (pathological and radiological data), chemotherapy regimens, surgical characteristics, outcomes (first date of relapse or progression, date of death or date of last news). Resection margins were considered as clear (R0) or not (microscopically involved (R1) or macroscopically intralesional (R2)).

All available radiological exams (CT-scan, Magnetic resonance imaging (MRI)) in each center were independently reviewed by two expert radiologists in order to collect tumor size before and after neo-CT. Radiological responses were assessed according to change in maximal tumor diameter (Recist v1.1 criterion).

Tumor histological response to neo-CT was collected from pathology reports, as Pathologists routinely evaluate it using the Rosen's criteria to assess the percent of necrotic tumor cells in the surgical specimen after neo-CT (good response if > 90% of necrosis on the surgical specimen) [START_REF] Rosen | Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy[END_REF][START_REF] Huvos | Primary osteogenic sarcoma: pathologic aspects in 20 patients after treatment with chemotherapy en bloc resection, and prosthetic bone replacement[END_REF] This study was approved by Institutional review board and done in accordance with the Helsinki declaration. Written consent was not required from patients because of the retrospective noninterventional design, consistently with French standard regulations. Collection of data and analysis was in accordance with guidelines of the French national committee for protection of personal data (CNIL).

Objective and end points

The primary objective of this study was to describe the pathological and radiological responses to neo-CT.

Secondary outcomes of interest were disease free survival (DFS), defined by the duration between surgery and first relapse (or death) or censoring by the date of last news alive, and correlation between tumor responses and surgical margins.

Statistical analyses

The retrospective design of this study and the scarcity of the disease prevented us to calculate a population to confirm statistical hypotheses. Binary data were described by ratios. Quantitative data were described using median and range. Correlations between binary variables were assessed using Fisher exact test. Because of the limited number of patients included, correlations between binary and quantitative variables were assessed using non-parametric Wilcoxon test. Follow-up was estimated using the reverse Kaplan-Meier estimation method (1-KM). Survival curves were performed using the Kaplan-Meier method and analyzed using two-sided log-rank test. Exploratory survival analysis was performed with Cox logistic regression model. Proportional hazards assumption was tested for each analysis. Survivals are considered from the date of first treatment (surgery, or first neo-CT infusion), to the date of event of interest, censored by the date of last news. Significance was defined by p<0.05. All analyses were performed using R software version 3.3.3.

RESULTS

Patients and tumors characteristics

Forty patients were screened for the inclusion (Figure 1). Thirty-five high grade MFOS were included for the final analysis (n=5 excluded: other diagnosis (n=4) and missing data (n=1; histological report unavailable).

Patient and tumor characteristics (n=35) are presented in Table 1 and were consistent with literature [START_REF] Van Den Berg | Osteosarcoma: A Comparison of Jaw versus Nonjaw Localizations and Review of the Literature[END_REF]. Briefly, median age was 36.8 years old (range 18.5-84.4). All patients had good general condition, 64% were male. The delay between first symptoms and diagnosis was extremely variable, with a median time to diagnosis of 15 weeks (range 5-108). The first symptom was an evolving mass syndrome in 83% of cases (Table 1). All tumors were developed from mandibular bone (60%), or maxillary bone (40%). Most of tumors (75%) had local development corresponding to stage IIA/B of AJCC staging system for long bone sarcoma. Four patients had metastasis at diagnosis (skin: n=1; lung: n=3) and three had radiological suspicion of lymph nodes metastasis (not confirmed histologically).

Median initial tumor size was 40 mm (range 15-99). All MFOS were high grade tumors. The main histological subtypes were chondroblastic (37%), osteoblastic (31%) or undifferentiated (14%).

Twenty-nine percent of tumors had a mixed histological type.

Therapeutic management

Twenty-four (77.4%) patients received neo-CT. Regimens were based on Adriamycine, Platine Ifosfamide and high-dose methotrexate. Regimens heterogeneity prevented any further analyses for correlations with resection margins or survival.

Four patients (n=3 localized disease and n=1 with lung metastasis) did not undergo surgical resection because of locally advanced/unresectable disease at initial diagnosis (n=1) or after neo-CT (n=3). Surgical resection of tumor was performed for 31 (89%) patients (Figure 1). Among these, 10 (32%) did not receive previous neo-CT. For the 21 patients operated after neo-CT, the median delay between the first cycle of neo-CT and surgery was 14 weeks (range 5-40) and 8 (38%) had R1 resection margins. Overall, 27/31 (87%) patients had available data for surgical resection margins, 17 (63%) were clear (R0), 10 (37%) were marginal R1 and no intralesional R2 margins was observed.

Adjuvant CT was administered to 16/27 patients.

No patient received RT prior to surgery. Adjuvant RT was realized for 8 patients with positive surgical margins.

Radiological response to neo-CT

Among patients who underwent surgical resection after neo-CT (n=21), responses distributions were eight (44%) progressions (tumor size increase > 20%), ten (66%) stable diseases, while none had significant radiological tumor shrinkage when using RECIST1.1 criterion (decrease > 30% in tumor maximal diameter) (Figure 2). Median tumor size change from the baseline was + 4 millimeters [-10; +20] representing a median of +12.8% of size increase. (Table 2).

The three patients who received neo-CT (n=1 and n=2 having received 6 and 4 cycles respectively) but were not resected thereafter had not radiological examinations available. They were in clinical progression. They were resectable at initial diagnosis and became unresectable after neo-CT due to the large progression of the tumor. Overall, 16/24 patients (66.7%) had an increase in tumor size or were in clinical progression during the neo-CT.

Histological response to neo-CT

Among patients who underwent surgical resection after neo-CT, 20 had histological data available.

One out of 20 (5%) patient experienced a 100% tumor necrosis and was the only one responder (necrosis >90%) to neo-CT (Figure 3). Regarding others, tumor necrosis was ≤30% for 14 patients (70%), 31-60% for three patients (15%) and 61-90% for two patients (10%).

Survival and prognostic factors

Median follow-up was 43.6 months (95%CI [32.1; 63.7]; range [1.0 -160.7]). We observed 8 deaths during the period of follow-up. One patient died of tumor bleeding prior to any treatment, one patient treated by surgery alone died of pulmonary infection in the post-surgical period. The six others had received neo-CT and died after tumor relapses either locally (3) or secondary to metastatic disease progression (3). Among patients who were resected for the primary tumor (n=31/35), 8 (26%) relapsed, with a 3years DFS of 76% (95% confidence interval [60%; 95%]). Among the 8 patients who relapsed, 5 have died at the date of last news. The 3 other patients were still alive after long follow-up (34 months, 40 months, 160 months) R0 resections were significantly associated with a better DFS compare to R1 resections (log-rank test, p=0.004) (Figure 4), with a 3-years DFS of 100% versus 37.5% respectively. Other factors significantly associated with poorer DFS were the median initial and pre-operative tumor sizes (Table 3).

Higher initial and post neo-CT tumor size were significantly associated with R1 margins (supplementary table 1).

DISCUSSION

This study is the first to report the radiological and pathological response rates after neo-CT for a recent cohort of MFOS. During fifteen years period, 35 patients with a confirmed diagnosis of MFOS were treated in two reference centers. Among this cohort of patients, 11 patients were operated before any CT and 24 received neo-CT followed by surgery for 21 of them. The clinical tumor response rate was 4% (1/24 receiving neo-CT) and three patients (12.5%) initially resectable became unresectable due to clinical tumor progression (n=2 maxillary and n=1 mandible MFOS). The radiological response was 0% (RECIST v1.1 criterion). Radiological/clinical reviews revealed that 66.7% of patients experienced a tumor size increase during neo-CT. The pathological response was 5% (1/20 with available data) and necrosis rates were ≤ 30% for 14 (70%) patients. These data are particularly important given that tumor size and R0 surgical resection were significantly associated with DFS.

MFOS represents fewer than ten percent of all osteosarcomas and have a predominantly local development [START_REF] O'neill | Head and neck sarcomas: epidemiology, pathology, and management[END_REF][START_REF] Sarkar | Pathological and clinical features of primary osseous tumours of the jaw[END_REF]. The scarcity of these tumors, the lack of knowledge and the heterogeneity of reported cohorts explain the lack of evidence-based treatment guidelines for the optimal management of these tumors [START_REF] Tudor-Green | Current update on the diagnosis and management of head and neck hard tissue sarcomas[END_REF][START_REF] Thiele | Interdisciplinary combined treatment of craniofacial osteosarcoma with neoadjuvant and adjuvant chemotherapy and excision of the tumour: a retrospective study[END_REF].

The benefit of neo-CT in the treatment of MFOS is not widely accepted in literature [START_REF] Baumhoer | Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry[END_REF]. Nonsignificant or contradictory results [START_REF] Thariat | Osteosarcomas of the mandible: multidisciplinary management of a rare tumor of the young adult a cooperative study of the GSF-GETO, Rare Cancer Network, GETTEC/REFCOR and SFCE[END_REF][START_REF] Patel | Improved outcomes in patients with osteogenic sarcoma of the head and neck[END_REF][START_REF] Kassir | Osteosarcoma of the head and neck: meta-analysis of nonrandomized studies[END_REF][START_REF] Granowski-Lecornu | Osteosarcoma of the jaws: factors influencing prognosis[END_REF][START_REF] Mücke | Effect of neoadjuvant treatment in the management of osteosarcomas of the head and neck[END_REF] are issues from retrospective studies that included heterogeneous patients before 2000, with few of them receiving neo-CT [START_REF] Granados-Garcia | Free osseous and soft tissue surgical margins as prognostic factors in mandibular osteosarcoma[END_REF][START_REF] Thiele | Interdisciplinary combined treatment of craniofacial osteosarcoma with neoadjuvant and adjuvant chemotherapy and excision of the tumour: a retrospective study[END_REF]. A recent large series has reported a limited pathological response rate to neo-CT (27%) [START_REF] Thariat | Osteosarcomas of the mandible: are they different from other tumor sites?[END_REF].

Regarding adjuvant chemotherapy, the limited number of patients in our cohort precluded relevant statistical analyses comparing patients who received adjuvant chemotherapy versus patients who did not. In 2016 and 2017, Yiming Chen et al., have reported a series of n=160 head and neck osteosarcoma and affirmed that adjuvant chemotherapy improves overall survival. They found that the overall survival was significantly better with adjuvant chemotherapy among various treatment plans and that primary surgery alone vs. primary surgery and chemotherapy group showed borderline significance [START_REF] Chen | Osteosarcoma of head and neck: A retrospective study on prognostic factors from a single institute database[END_REF][START_REF] Chen | Chemotherapy in head and neck osteosarcoma: Adjuvant chemotherapy improves overall survival[END_REF].

The role of surgery, and particularly the achievement of R0 resection margins, has been largely reviewed and reported to be associated with better outcomes [START_REF] Jasnau | Craniofacial osteosarcoma Experience of the cooperative German-Austrian-Swiss osteosarcoma study group[END_REF][START_REF] Laskar | Osteosarcoma of the head and neck region: lessons learned from a single-institution experience of 50 patients[END_REF][START_REF] Granados-Garcia | Free osseous and soft tissue surgical margins as prognostic factors in mandibular osteosarcoma[END_REF][START_REF] Kassir | Osteosarcoma of the head and neck: meta-analysis of nonrandomized studies[END_REF][START_REF] Mücke | Outcome in adult patients with head and neck sarcomas--a 10-year analysis[END_REF][START_REF] Kämmerer | Clinical, therapeutic and prognostic features of osteosarcoma of the jaws -experience of 36 cases[END_REF][START_REF] Chen | Osteosarcoma of the Mandible: A Site-Specific Study on Survival and Prognostic Factors[END_REF][START_REF] Jeong | Osteosarcoma of the jaws in Koreans: analysis of 26 cases[END_REF]. Local failures are the main causes of death in MFOS compared to other sites [START_REF] Thariat | Osteosarcomas of the mandible: are they different from other tumor sites?[END_REF]. The therapeutic issues and the operability of MFOS depend on tumor parameters such as size and volume [START_REF] Lee | Characteristics and prognostic factors of osteosarcoma of the jaws: a retrospective cohort study[END_REF][START_REF] Gadwal | Primary osteosarcoma of the head and neck in pediatric patients: a clinicopathologic study of 22 cases with a review of the literature[END_REF]. It is particularly true for posterior tumors, close to the skull base. Thus, the delay until surgery and the subsequent increase in tumors size could be detrimental as suggested by the death of two patients with clinical progression during neo-CT (which were initially resectable), and by the correlation between tumor size and R1 resection. As per routine practice in our centers, some teams focused on early tumor response evaluation to discontinue neo-CT, in order to perform radical tumor resection (R0) during the window of therapeutic opportunity for surgery [START_REF] Kimura | Conventional osteosarcoma of the mandible successfully treated with radical surgery and adjuvant chemotherapy after responding poorly to neoadjuvant chemotherapy: a case report[END_REF].

In line with the aforementioned therapeutic issues, our results highlight a major concern: the overall lack of efficacy of neo-CT could lead to a delay until surgery, during which tumor can grow beyond the theoretical limits of complete resection.

Why MFOS appeared so resistant to neo-CT and different from other locations remained a partially unsettled question [START_REF] Ferrari | Osteosarcoma of the Jaw: Classification, Diagnosis and Treatment[END_REF]. Some large series clearly underlined biologic differences between MFOS and OS of other localization [START_REF] Baumhoer | Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry[END_REF] reflecting the molecular heterogeneity of human osteosarcoma [START_REF] Penel | Presentation and outcome of frequent and rare sarcoma histologic subtypes: A study of 10,262 patients with localized visceral/soft tissue sarcoma managed in reference centers[END_REF]. For example, recent molecular characterization study allowed the identification of a new-subtype of mandibular osteosarcoma with RASAL1/MDM2 amplification [START_REF] Guérin | A new subtype of high-grade mandibular osteosarcoma with RASAL1/MDM2 amplification[END_REF].

More generally the chemoresistance in OS appears to be mediated by numerous molecular mechanisms which include decreased intracellular drug accumulation, drug inactivation, enhanced DNA repair, perturbations in signal transduction pathways, apoptosis-and autophagy-related chemoresistance, microRNA (miRNA) dysregulation, cancer stem cell (CSC)-mediated drug resistance and Interaction of OS cells and the micro-environment [START_REF] He | Molecular mechanisms of chemoresistance in osteosarcoma (Review)[END_REF][START_REF] Li | Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment[END_REF][START_REF] Abarrategi | Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and Targeted Therapies[END_REF][START_REF] Lindsey | Osteosarcoma Overview[END_REF][START_REF] Bhuvaneshwar | Genome sequencing analysis of blood cells identifies germline haplotypes strongly associated with drug resistance in osteosarcoma patients[END_REF] (Supplementary Table 3).

Although identified, the precise role of each of these mechanisms of chemoresistance remains unclear.

To illustrate it, we can mention the autophagy and apoptosis processes, which have been already referred to as a double-edged sword. On one hand, they promote osteosarcoma cells survival, while in other circumstances, they can lead to tumor cell death. Furthermore, there is a close interplay between autophagy and apoptosis during OS cells development, progression and response to therapy (role of the PI3K and Akt regulators).

Furthermore, recent studies have highlighted the importance of OS-CSCs, which have been associated with chemoresistance, relapse, and metastasis events [START_REF] Abarrategi | Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and Targeted Therapies[END_REF]. However, almost all the current studies on the mechanisms of OS-CSCs related chemoresistance are in their infancy and better understanding would help provide better targets for therapies. Some studies have highlighted other biological processes which have been already reported as implicated in the chemoresistance of other cancers as the epithelial-mesenchymal transition (EMT) biological process (Visfatin, an EMT-related transcription factors, is involved in the cisplatin resistance of osteosarcoma cells via upregulation of Snail and Zeb1 and cPLA2a, cytosolic phospholipase A2, could promote OS cell invasion) [START_REF] Wang | Visfatin is involved in the cisplatin resistance of osteosarcoma cells via upregulation of Snail and Zeb1[END_REF][START_REF] Pang | cPLA2a correlates with metastasis and poor prognosis of osteosarcoma by facilitating epithelial-mesenchymal transition[END_REF]. More recently Bhuvaneshwar et al., have reported intronic and intergenic hotspot regions from 26 genes significantly associated with resistance to cisplatin, doxorubicin, and methotrexate, in children with osteosarcoma [START_REF] Bhuvaneshwar | Genome sequencing analysis of blood cells identifies germline haplotypes strongly associated with drug resistance in osteosarcoma patients[END_REF]. Among significant results were mutations in genes belonging to AKR enzyme family (AKRD1), the cell-cell adhesion biological process (genes of the cadherin family CDH13, CDH9 and PKHD1 resulting in a phenotype called "cell adhesion-mediated drug resistance," or CAM-DR) and the PI3K pathways.

Molecular studies could help to anticipate the chemoresistance. Indeed, a molecular classification of OS had identified a 45-gene signature that could predict with 100% accuracy the chemoresponse of osteosarcoma patients prior to the initiation of treatment [START_REF] Lau | Molecular classification of osteosarcoma[END_REF]. This support the fact that neo-CT should not be generalized but prescribed in a subset of patients with high level of expected chemosensitivity. This could explain why the only patient of our cohort who experienced a tumor response after neo-CT had no any viable tumor cell on the surgical specimen.

The identification of biomarkers could allow to detect tumor onset, progression and response to therapy for OS [START_REF] Kimura | Conventional osteosarcoma of the mandible successfully treated with radical surgery and adjuvant chemotherapy after responding poorly to neoadjuvant chemotherapy: a case report[END_REF][START_REF] Raimondi | Circulating biomarkers in osteosarcoma: new translational tools for diagnosis and treatment[END_REF]. By predicting response to therapy, these biomarkers, as well as the immunohistochemical analysis of the microenvironment may represent novel tools for therapeutic stratification [START_REF] Gomez-Brouchet | CD163-positive tumor-associated macrophages and CD8-positive cytotoxic lymphocytes are powerful diagnostic markers for the therapeutic stratification of osteosarcoma patients: An immunohistochemical analysis of the biopsies fromthe French OS2006 phase 3 trial[END_REF].

To identify new molecular targets and develop new drugs, further studies are required to a better understanding of the molecular pathogenesis Osteosarcoma [START_REF] Chiappetta | Whole-exome analysis in osteosarcoma to identify a personalized therapy[END_REF]. Recent genome-wide sequencing analyzes have demonstrated that Osteosarcomas are genetically complex and heterogeneous (intraand intertumoral) [START_REF] Baumhoer | Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry[END_REF]. Structural and numerical alterations (somatic copy number alterations) are much more common than recurrent point mutations. Regarding Cancer-causing genes, also called driver genes or drivers, numerous somatic mutations have been identified by next-generation sequencing of Osteosarcomas [START_REF] Rickel | Molecular genetics of osteosarcoma[END_REF] (Supplementary Table 4). The most common driver genes associated with osteosarcomas development are TP53 and RB1. TP53 and RB1 mutations have been identified as causative driver genes in almost 50% of cases. Frequent alterations in PTEN and PI3K/mTOR signaling pathways have also been reported. Furthermore, about 90% of all osteosarcomas appear to have mutations in BRCA-associated genes and genomically show a striking similarity to BRCA1 / 2-mutated tumors (so-called 'BRCAness') [START_REF] Baumhoer | Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry[END_REF][START_REF] Rickel | Molecular genetics of osteosarcoma[END_REF]. It suggests that a high percentage of OS tumors may be HRR-deficient (homologous recombination repair defect) and therefore be vulnerable to additional DNA damage caused by double-strand breaks. At all, like a double-edged sword, all these alterations confer a growth advantage, but also creates vulnerabilities in osteosarcoma cells. This give us opportunities to test targeted therapies targeting the different genes and pathways involved [START_REF] Brown | Biology of Bone Sarcomas and New Therapeutic Developments[END_REF][START_REF] Wedekind | Immunotherapy for osteosarcoma: Where do we go from here?[END_REF] (Supplementary table 5). Some recent studies support for the potential uses of immunotherapy, including monoclonal antibodies, immunomodulators, Adoptive T-cell therapy, vaccine therapy, Immunologic checkpoint blockade and oncolytic virotherapy for the eradication of OS cells [START_REF] Wedekind | Immunotherapy for osteosarcoma: Where do we go from here?[END_REF][START_REF] Wan | Strategies and developments of immunotherapies in osteosarcoma[END_REF] (Supplementary table 5 and6). At all, combination strategies are probably necessary to achieve meaningful and durable responses to therapies for osteosarcoma, especially immunotherapies. Indeed, as seen with conventional CT, tumors development involves multiple pathways to resist to therapies. The recent development developments in genomics, therapeutics and imaging technologies will allow the early detection of the genomic risk of sarcomas for each patient and may participate to better personalized management.

Despite the inherent limitations due to our retrospective data collection, and the relatively limited number of patients analyzed, which precluded further analysis of overall survival, our results were consistent with other series [START_REF] Baumhoer | Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry[END_REF]. Indeed, our results suggest that MFOS should not be managed as OS of other localization. The natural history of MFOS is distinctive from other sites [START_REF] Peng | Head and neck sarcomas: analysis of the SEER database[END_REF]. Regarding demographic pattern, the mean age of diagnosis of MFOS is 30 years of age while children and adolescents are most often affected for other sites [START_REF] Chen | Osteosarcoma of head and neck: A retrospective study on prognostic factors from a single institute database[END_REF]. Regarding embryologic development, head and neck bones are structurally quite different in origin from the body. Furthermore, when compared to long bones OS, MFOS showed no clinical, radiological and histological chemosensitivity [START_REF] Bacci | Prognostic factors for osteosarcoma of the extremity treated with neoadjuvant chemotherapy: 15-year experience in 789 patients treated at a single institution[END_REF]. Finally, this disease seems to harbor a different evolution, characterized by a local invasion more than lung metastasis, which suggest the need to a better local control rather than to eradicate micrometastasis.

Conclusion

MFOS is a rare disease. This large retrospective cohort of MFOS indicates the lack of benefit and potentially deleterious effects of neo-CT. We suggest privileging primary surgery in initially localized resectable MFOS. The benefit of adjuvant chemotherapy should be prospectively studied.

Neo-CT could benefit only for a limited group of patients with high predisposition of chemosensitivity, on the basis of molecular analysis. Collaborative and large high-throughput genomic analysis are warranted to better characterize MFOS, and to allow the emergence of predictive biomarkers, as well as the development of targeted therapies.

Figure legends and tables.

  Figure legends and tables.

Figure 1 .Table 1 .

 11 Figure 1. Flow-chart: patient selection and treatments.

Figure 2 .

 2 Figure 2. Radiological response to neoadjuvant chemotherapy for patients who underwent

Figure 3 .

 3 Figure 3. Histological response to neoadjuvant chemotherapy for the n=18 patients who

Figure 4 .

 4 Figure 4. Disease free survival according to surgical margins

Table 3 .

 3 Disease free survival following surgical resection according to patients' characteristics. Patients included: n=31 (in 21 patients who underwent chemotherapy -as assessed by radiological reviewing. Maximal diameter was taken into account. Pathological response as assessed by tumor necrosis on surgical specimen).

Table 2 . Radiological and pathological response to neoadjuvant chemotherapy.

 2

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Supplementary Table

 1. Patients' characteristics associated with positive surgical resection margins (n=27).

Supplementary table 2.

Differences between treatments of patients with MFOS according to their metastatic status. (MFOS: maxillofacial osteosarcoma; (M-) patients with localized/non-metastatic MFOS and (M+) with metastases; CT: chemotherapy; NA: Not applicable).

Supplementary table 3. Molecular mechanisms of chemoresistance in OS (adapted from


Deyang Zhao

Yantao Wang

Frederic Delbecq

Christophe Len
email: christophe.len@chimieparistech.psl.eu

Continuous flow conversion of alkyl levulinates into γ-valerolactone in the presence of Ru/C as catalyst

Keywords: Continuous flow, alkyl levulinates, γ-valerolactone,5%Ru/C

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

Introduction

The rapid developments in industry and transportation all over the world have led to a drastic increase in the demand for fuels. The gradual depletion of fossil fuel resources has stimulated the global efforts to search for alternative sources. Biomass is an abundant and renewable alternative resource that is the best candidate to replace fossil fuels for the sustainable production of energy [1][2][3][4][5], as well as chemicals [6][7][8][9][10][11].

The development of efficient processes to convert biomass resource into liquid fuels and valuable chemicals is a key research area in the next few decades [12][13][14][15][16][17][18][19]. Recent advances in the field of catalytic valorization of lignocellulose for the sustainable production of bulk chemicals and fuel additives have been reported [20].

γ-Valerolactone (GVL) as one of the most appealing molecules shows a very promising versatile intermediate for production of fuel additives, a renewable solvent, food ingredient, liquid fuel, and ideal precursor for the production of more valuable chemicals [21,22] in a wide range of applications (e.g., cutting oils and brake fluids) [23]. Due to very high interest towards GVL, its efficient production is currently a topic of intensive research.

The production of GVL from biomass has received extensive research interests.

Starting from cellulose and hemicellulose [19], successive hydrolysis, dehydration/rehydration and then hydrogenation furnished GVL. The use of intermediates such as carbohydrate [24,25], 5-hydroxymethylfurfural and furfuryl alcohol (FA) [26] was also developed efficiently for the production of GVL (Scheme 1). The hydrogenation of levulinic acid (LA) to GVL using heterogeneous and homogeneous catalysts in combination with molecular hydrogen has been studied extensively [27,28]. Unfortunately, the synthesis of LA in acidic aqueous media has got some drawbacks: production of a large amount of waste, a high cost for mineral acid recovery, humin formation and a rather difficult separation of the highly polar molecule LA [29,30]. Comparatively, the hydrogenation of alkyl levulinates (AL) to GVL is more attractive because AL was obtained in better yield with a more efficient separation process. Thus, it is meaningful to establish a new integrated route to produce GVL from hemicellulose via successive formation of xylose, furfural, FA and AL. Scheme 1. Production of biomass-derived GVL from cellulose (in green) and hemicellulose (in blue) via AL as a common intermediate.

Molecular hydrogen (H2

) is widely used as the reductant for catalytic transformation of LA or AL to GVL whether in batch or continuous flow [27,28,31].

Recently, heterogeneous catalytic transfer hydrogenation (CTH) of bio-based compound in the presence of hydrogen donors (e.g. 2-PrOH) has emerged [32]. CTH is more attractive for techno-economic point of view but also because the interphase transfer is improved (solid/liquid for ROH vs. solid/liquid/gas for H2). Furthermore it offers a simple, efficient and safe option for the valorization of biomass derived molecules using abundant and inexpensive alcohols as hydrogen source and solvent as compared to H2 [33][34][35]. Various metal catalysts have been demonstrated to have good activities for the conversion of AL and LA to GVL using CTH process. Kobayashi et al. reported a CTH process for the conversion of cellulose to sugar alcohols over supported Ru/C as the catalyst and 2-PrOH as the hydrogen donor [36].

Batch microwave-assisted hydrogenation as alternative technology was studied for the conversion of LA and AL to GVL through catalysts screening experiments (Pt/C, Pd/C, Rh/C, Cu/C, Ni/C and Ru/C) and ruthenium exhibited the highest activity [37].

Heterogeneous Zr-based catalysts, such as ZrO2 [38], hydrated zirconia (ZrO(OH)2•xH2O) [39] and Zr-beta [40] having amphoteric nature permitted to produce GVL in good productivity.

Continuous flow chemistry as alternative technology offers significant processing advantages including improved thermal management, mixing control, application to a wider range of reaction conditions, scalability, energy efficiency, waste reduction, safety, use of heterogeneous catalysis, multistep synthesis and much more [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55]. In the context to develop new green process, few works on the production of GVL in continuous flow have been reported [31,42]. Starting from LA, the use of heterogeneous 5% Ru/C in the presence of sulfonated phosphine as ligand and hydrogen under 10 bar at 140 °C permitted the production of GVL in continuous flow with a high productivity (0.8 mol g -1 h -1 ) [31]. In parallel, our group described a high GVL productivity via CTH with Zr-based catalyst (0.0923 mol g -1 h -1 ) starting from methyl levulinate (ML) in the presence of functional metal organic framework at elevated temperature (240 o C) in continuous flow [42]. To date, an outstanding of the continuous flow catalytic transfer hydrogen process to produce GVL from AL is requested. In this context, the present work tested some commercial catalyst: 5%Ru/C, 5%Pd/C, 10%Pt/C and 5% Ru/Al2O3 for a continuous flow conversion of biosourced ML to GVL using CTH process.

Materials and methods

Materials

Methanol (MeOH, ≥ 99.9%), propan-1-ol (1-PrOH, ≥ 99.5%), propan-2-ol (2-PrOH, ≥ 99.5%), ML (99%), GVL (99 %) and n-decane (≥ 99%) were purchased from Sigma-Aldrich and further used without purification. ML feedstocks of 0.2, 0.4 and 0.8 M were prepared by dissolving 2.602 g, 5.204 g and 10.40 g ML in propan-2-ol (100 mL) in a volumetric flask using n-decane (5.0mg/mL) as external standard. The commercial 5%Ru/C, 5%Pd/C, 10%Pt/C and 5% Ru/Al2O3 were purchased from Sigma-Aldrich and further used without purification.

General procedure for the synthesis of GVL in continuous flow experiment

The experiments were carried out in a high-temperature high-pressure H-Cube Pro TM Flow Reactor ThalesNano TM , Hungary (Figure S1), connected to a HPLC pump to supply a continuous feed of ML (0.2-0.8 M) in 2-PrOH,MeOH or 1-PrOH. A 70 mm CatCart cartridge (0.88 mL empty volume) was packed with 330 mg catalyst by applying vacuum suction at the bottom of the cartridge. The total flow through volume (including feed, reactor and product sections) was 6 mL. First, pure 2-PrOH, MeOH or 1-PrOH was pumped through the system and then the feed was changed to the ML feedstock. The flow was continued until the temperature (100-150 °C) and hydrodynamic pressurization (0-50 bar) of the reactor module were reached. Then, in function of the flow rate (0.2-0.8 mL min -1 ), the reaction proceeded during a certain time (12-50 min) before collecting the first sample (time zero). Further samples were collected after regular time intervals.

Identical procedure was done starting from ethyl levulinate (EL) and butyl levulinate (BL).

Product analysis

GC-FID analysis was performed on a gas chromatograph (HP, 14009 Arcade, New York, United States) coupled with a FID detector equipped with a Supelco 2-8047-U capillary column (15 m x 0.25 mm i.d. and 0.25 µm film thickness, Alltech Part No.31163-01). H2 was used as carrier gas at 1 mL min -1 flow rate. The samples were injected directly onto the column using septum-equipped programmable injector (SPI) system. The temperature of the injector was set 250 °C and the oven started at 70 °C, held for 1 min, raised to 250 °C at a rate of 20 °C min -1 , held for 10 min. The respectively. The response factors (RF) of ML and GVL relative to a fixed concentration (5.0 mg/mL) of n-decane as internal standard were experimentally determined at 3.62 and 3.01, respectively. The linear regression coefficients of the calibrations were high in all cases (r 2 > 0.99). The RF for MHP, EHP and BHP were calculated according to the Effective Carbon Number (ECN) method [START_REF]Modern practice of gas chromatography[END_REF], based on the RF and ECN factor of ML (3.75), EL (4.75), BL (6.75) and the ECN factor of MHP (4), EHP (5), BHP (7). The yield and conversion in continuous flow were calculated according to eqs. ( 1) -( 2):

(1)

% = × 100 ! (2) " # % = $ %&' '() - '&() + %&' '()
× 100 ! constant during the experiments (no evaporation loss). All results (conversion and yield) are expressed as molar percentages. The metal-based catalyst productivity (, -. / , 0 12 ℎ 12 ) was calculated using eq. ( 3):

(3 12 × 0.06 × 4 4 #0 × 0 0 5 , 0 ,0 /

) # 4 " 5 , / 12 ℎ 12 = $6 ! #0 , ,

Results and discussion

Catalyst activity screening experiments

Continuous flow chemistry generally offers a better control and allows a higher flexibility in reaction conditions, which typically require optimization to improve the conversion and selectivity. 2-PrOH has been reported as an active hydrogen donor, which could provide good conversion and product selectivity when compared with other primary alcohols such as methanol, ethanol, 1-propanol [START_REF] Kuwahara | [END_REF]58]. Thus, 2-PrOH was selected as hydrogen donor for CTH of ML under continuous flow conditions.

Starting from ML (0.2 M) in 2-PrOH at 130 °C, four commercial catalysts 5%Pd/C, 5%Ru/C, 5%Ru/Al2O3 and 10%Pt/C were tested for the two successive steps:

hydrogenation and lactonization aiming to reach high GVL yields (Fig. 1). The blank experiment (activated carbon as catalyst) did not give any ML conversion whatever the residence time (0-120 min). However, when the tested noble metals were supported on activated carbon GVL was obtained in moderate to good yield. In our hands, the highest GVL yield (89%) was achieved in the presence of 5%Pd/C with 10% higher than that with 5% Ru/C. Nevertheless, 5% Ru/C was used for the next step of our optimization since this catalyst permitted to have the highest GVL productivity (0.2 mol g -1 h -1 ). This result was in accordance with that reported in the literature [59].

Effect of reaction temperature for the production of GVL

In a first step, the effect of reaction temperature (100-150 °C) was studied using ML (0.2 M) at 20 bar in a continuous flow system (0.4 mL min -1 ) (Fig. 2). Whatever the temperature, ML furnished two main compounds: GVL and MHP (Fig. S2) and unidentified side compounds with a full conversion. With the increase of reaction temperature from 100 °C to 150 °C, the GVL yield and productivity (Pr) increase from 30% to 85% yield and from 0.084% to 0.254%, respectively. It was obvious that the MHP yield decrease from 60% to 8% with the increase of temperature because the lactonization of MHP to GVL was favored at higher temperature [60]. Whatever the temperature used, the amount of side products was almost similar (3%). Among them, isopropyl 4-hydroxypentanoate obtained by transesterification between ML and 2-PrOH was identified (Fig. S3). Besides, the 5%Ru/C catalyst showed an excellent catalytic stability at the temperatures investigated. Due to the maximum temperature limitation of H-Cube Pro TM Flow Reactor, higher reaction temperature than 150ºC

was not available.

Effect of reaction pressure for the production of GVL

The effect of hydrodynamic pressure (20, 40, 60 bar) at 150 °C was also investigated for 120 min, which corresponded to the processing capacity 58 mL (120 min x 0.4 mL min -1 + 10 mL) in continuous flow (Fig. 3). Whatever the pressure used, full conversion of ML was obtained. The yield of GVL was improved ca. 7% with the increase of reaction pressure from 20 bar to 40 bar and the yield of MHP decreased 5%. With a higher pressure (60 bar vs 40 bar), no apparently changes of GVL and MHP yields were observed. From the point of energy conservation, the pressurization was set as 40 bar for the following optimization reactions.

Effect of flow rate for the production of GVL

processing capacity (10 mL to 58 mL) (Fig. 4). Whatever the flow rate used, ML was fully converted. With a low flow rate (0.2 mL min -1 ), the highest GVL yield was achieved (95% yield) with only 5% MHP. As expected, the decrease of residence time implying by the increase of the flow rate afforded a lower GVL yield (87-92% vs 95%) and a higher MHP yield (6-8% vs 2%). It was obvious that a lower flow rate offered a longer residence time, which favored the reactant contact with the Ru catalyst. In term of productivity, a short residence time with 0.8 mL min -1 flow rate gave a better result compared with a long residence time obtained with 0.2 mL min -1 flow rate (0.551 vs 0.131). Nevertheless, the presence of MHP as side product was a limiting step of the process and required more energy for the work-up and purification. Therefore, the flow rate of 0.2 mL min -1 with only 2-3 % of MHP was choose for the next step of the optimization.

Effect of the concentration of ML for the production of GVL

With the perspectives to obtain a higher productivity and to have a solvent economy, variation of ML concentration was studied. In the present work, ML feeding rate was kept constant (0.16 mmol min -1 ) and ML concentration was increased while flow rate was decreased (Fig. 5). Although the ML feeding rate was constant the conversion of ML, yields of GVL and MHP were different between 0.2 mL min -1 and 0.4-0.8 mL min -1 . With a low flow rate (0.2 mL min -1 ), the conversion of ML was lower than that obtained with a flow rate of 0.4-0.8 mL min -1 (90% vs 100%). Similar observations were observed with the GVL yields (80% vs 87-90%) and the MHP yields (6% vs 9%). Whatever the flow rate, these experiments showed that the target GVL and the acyclic MHP were obtained as the main chemicals and as the minor chemicals, respectively. The best results obtained using 0.8 mL min -1 and ML (0.2 M) can be explained by a short residence time and low concentration of starting material. The reactant adsorption on catalyst surface as well as the desorption of the target product were more efficient and favor high mass transfer rate. With ML concentration (0.2 M and 0.4 M), almost the same GVL productivity (ca. 0.55) because similar ML conversion and GVL yield were achieved. From the point of saving solvent, higher feedstock concentration could be a better choice. ML feeding rate was increased to 0.32 mmol min -1 (ML 0.4 M and flow rate 0.8 mL min -1 ). The conversion of ML (98%) and the yield of GVL (85%) with ca.11% MHP yield were obtained and the productivity (0.99 mol g -1 h -1 ) was remarkably superior to several systems in previous report (Table 1). Selected works describing the production of GVL from LA or AL in the presence of hydrogen source and 5% Ru/C as catalyst in either batch reactor with or without microwave radiation or continuous flow reactor were described (Table 1). CTH using formic acid and 2-PrOH gave not good productivity even the yield of GVL can reach 93% yield (Table 1, entries 1 and 2). It was noteworthy that the use of high concentrated solution of ML (1.4 M vs 0.43 M) and higher pressure of H2 (30 bar vs 12 bar) permitted to have a better productivity (2.25 mol g -1 h -1 vs 1.12 mol g -1 h -1 )

(Table 1, entries 3 and 4). Due to practical considerations, microwave-assisted hydrogenation of LA and AL was studied in batch reactor and not in continuous flow reactor. In this context, the use of 2-PrOH and LA as well as ML permitted to produce GVL with a good productivity (> 2 mol g -1 h -1 ) (Table 1, entries 5 and 6).

Unfortunately, when the alkyl chain was higher than one carbon atom the productivity decreased drastically (Table 1, entries 7 and 8). Starting from LA in the presence of 2-BuOH the microwave-assisted organic synthesis did not furnished GVL with good yield but the productivity was significant (1.92 mol g -1 h -1 ) (Table 1, entry 9). In accordance with literature, microwave-assisted reaction permitted to have a short reaction time (30 minutes).

The main problem of this process was the formation of side compounds, which limit their further utilization. For continuous flow as alternative technology, the production of GVL was studied using hydrogenation and CTH processes. Whatever the pressure used, LA furnished GVL in good to excellent yields with a productivity lower than 0.9 mol g -1 h -1 (Table 1, entries [10][11][12]. Starting from AL such as BL in the presence of primary alcohol

(1-BuOH) as hydrogen donor, the productivity decreased ten times (0.085 mol g -1 h -1 vs 0.832 mol g -1 h -1 ) (table 1, entry 13). Our optimized process (ML (0.4 M), 5%Ru/C (330 mg), 2-PrOH, 150 °C, 40 bar, 0.8 mL min -1 ) furnished the target lactone in 83% yield with a productivity of 0.99 mol g -1 h -1 , which is the best reported in the literature for a continuous flow process.

Table 1 5%Ru/C as catalysts reported for the conversion of LA and AL to GVL. [62]

[60]

[37]

[37]

[37]

[37]

Effect of the nature of the alkyl levulinate (AL) for the production of GVL

Among the different results obtained with 5% Ru/C as catalyst, our optimized conditions (ML (0.4 M), 5% Ru/C, 2-PrOH, 150 °C, 40 bar, 0.8 mL min -1 ) was selected and variation of the nature of the alcohol was investigated (Table 2). With the increase of carbon atoms (from C1 to C4) (Table 2, entries 1-3), the yield of GVL decreased from 85% to 31% while the intermediate AHP yield increased from 13% to 63%. As a consequence, the productivity decreased from 0.99 to 0.25 mol g -1 h -1 . In accordance with the literature, the subsequent reduction of the ketone to the corresponding alcohol and the lactonization were facilitated with an AL having a little molecular weight [60]. When the carboxylic acid LA was used as starting material, GVL was obtained in 59% with a low conversion (67%) but the productivity was similar to that starting from EL (ca. 0.5 mol g -1 h -1 ) (Table 2, entry 4). b AHP for alkyl 4-hydroxypentanoate.

c 4-hydroxypentanoic acid.

Stability of the catalyst for the production of GVL

Further catalyst stability experiments were performed using our optimized conditions. The conversion remained stable over 9 hours at least, the yields of GVL and MHP were ca.83% and 10 %, respectively (Fig. 6). In our lab-scale process, the stability of 5% Ru/C catalyst was good.

Conclusion

This works reports a new efficient process using commercial heterogeneous catalyst 5% Ru/C for the production of γ-valerolactone (GVL) in continuous flow from either levulinic acid (LA) or alkyl levulinates (AL). After the investigation of methyl levulinate (ML) experiment parameters (reaction temperature, pressure, flow rate, starting material concentration), the optimization condition was at ML (0.4 M) in 2-PrOH, 5% Ru/C(330mg), 150 °C, 40 bar, flow rate=0.8 mL min -1 . Feedstock were fully converted with 85% GVL yield, and 13% methyl 4-hydroxypentanoate (MHP) yield, with the highest productivity is 0.99 mol g -1 h -1 . Effect of the nature of the AL experiments showed the lactonization were facilitated with shorter AL alkyl chains.

Finally 5%Ru/C shows good stability in 9 h cycle experiments.

  ionization mode was FID (70 eV, 300 μA, 250 °C). The identification of the compounds was performed by comparison of the retention times with pure standards and by GC/MS analysis. The solvent delay time was 0.90 min. The retention times of GVL, ML, EL, BL, methyl 4-hydroxypentanoate (MHP), ethyl 4-hydroxypentanoate (EHP), butanol 4-hydroxypentanoate (BHP) and LA, n-decane were 3.58 min, 3.39 min, 3.92 min, 5.21 min, 3.69 min, 4.16 min, 5.44 min, 4.98 min, 2.30 min.

  Mass spectra were record on a Hewlett-Packart HP 5973 mass spectrometer via a GC/MS coupling with a Hewlett-Packart HP 6890 chromatograph equipped with a capillary column HP-5MS (50 m x 0.25 mm i.d. and 0.25 µm film thickness, Alltech Part No.31163-01). lonisation was performed by electronic impact (EL, 70 eV). Mass spectra are reported as m/z (% of relative intensity). Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded using a Bruker AC 300 (300 MHz) or a Bruker AC 400 (400 MHz). The chemical shifts are expressed in parts per million (ppm) referenced to residual chloroform (7.26 ppm). Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were recorded using a Bruker AC 300 (75 MHz) or a Bruker AC 400 (100 MHz). The chemical shifts are expressed in parts per million (ppm) relative to the centre line of the triplet at 77.16 ppm for CDCl3

Fig. 1 .

 1 Fig. 1. Effect of catalysts and residence time on (a) the GVL yields and (b) on the

Fig. 2 .

 2 Fig. 2. Effect of reaction temperature on GVL productivity and products yield in

Fig. 3 .

 3 Fig. 3. Effect of hydrodynamic pressure on GVL productivity and products yield and

Fig. 4 .

 4 Fig. 4. Effect of flow rate on GVL productivity and products yield in continuous flow

Fig. 5 .

 5 Fig.5. Effect of flow rate and substrate concentration with a constant ML feeding rate

  in batch reactor. b microwave-assisted reaction (300 W) in batch reactor. c conventional heating in continuous flow reactor. d residence time/min.

Fig. 6 .

 6 Fig. 6. Operational stability of the catalyst 5% Ru/C in continuous flow alcoholysis of

Table 2

 2 Effect of AL molecular weight as starting material for the production of GVL. a Reaction conditions: ML (0.4 M), 5% Ru/C (330 mg), 2-PrOH, 150 °C, 40 bar, 0.8 mL min -1 .

	Entry AL	Conversion	GVL yield	AHP yield	Productivity
			(%)	(%)	(%) b	(mol g -1 h -1 )
	1	ML	99	85	13	0.99
	2	EL	95	64	31	0.55
	3	BL	94	31	63	0.25
	4	LA	67	59	6 c	0.50

a

Acknowledgements D.Z. and Y.W. would like to thank the China Scholarship Council (CSC) for the financial support.

Appendix A. Supplementary data

The Supporting Information is available: