Prof Thomas Aparicio
email: thomas.aparicio@aphp.fr

Magali Svrcek

Julie Henriques

Pauline Afchain

Astrid Lièvre

David Tougeron

Johan Gagniere

Eric Terrebonne

Guillaume Piessen

Jean-Louis Legoux

Jean- Louis Legoux

Cédric Lecaille

Marc Pocard

Jean-Marc Gornet

Aziz Zaanan

Sandrine Lavau-Denes

Thierry Lecomte

David Deutsch

Dewi Vernerey

Pierre Laurent Puig

Panel gene profiling of small bowel adenocarcinoma, results from the NADEGE prospective cohort

Keywords: small intestine adenocarcinoma, genomic profiling, cohort study, Crohn's disease, Lynch syndrome, MMR status Association des Gastro-Entérologues-Oncologues AJCC: American Joint Committee on Cancer deficient mismatch repair DNA: desoxyribonucleic acid GERCOR: Groupe Coopérateur Multidisciplinaire en Oncologie microsatellite instability MMR: mismatch repair

à la 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.

BACKGROUND

Small bowel adenocarcinoma (SBA) is a rare tumour of poor prognosis [START_REF] Aparicio | Small bowel adenocarcinoma: epidemiology, risk factors, diagnosis and treatment[END_REF]. Nevertheless, it is the first aetiology of small bowel cancer in France [START_REF] Bouvier | Trends in incidence of small bowel cancer according to histology: a population-based study[END_REF] and second aetiology in the USA [START_REF] Pan | Epidemiology of cancer of the small intestine[END_REF]. Concordant findings report an increasing incidence of SBA [START_REF] Bouvier | Trends in incidence of small bowel cancer according to histology: a population-based study[END_REF][START_REF] Legué | Trends in incidence, treatment and survival of small bowel adenocarcinomas between 1999 and 2013: a population-based study in The Netherlands[END_REF][START_REF] Faivre | Incidence, prevalence and survival of patients with rare epithelial digestive cancers diagnosed in Europe in 1995-2002[END_REF].

Few studies have investigated the molecular phenotype of SBA. A previous study reports that the genomic profile of SBA is closer to colon adenocarcinoma rather than gastric adenocarcinoma [START_REF] Haan | Small bowel adenocarcinoma copy number profiles are more closely related to colorectal than to gastric cancers[END_REF]. Recently, a large genomic analysis mainly on stage IV tumours has reported a distinct profile of SBA compared to gastric or colon adenocarcinoma [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF]. Indeed, if RAS mutation prevalence is similar to colon cancer, APC mutations are much less frequent, BRAF rarely involved V600E point mutations and ERBB2 mutations or microsatellite instabilities (MSI) are more frequent than in colon cancer [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF][START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF][START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF][START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF]. A prognostic value had been inconsistently associated with ERBB2 mutations [START_REF] Adam | DNA Sequencing of Small Bowel Adenocarcinomas Identifies Targetable Recurrent Mutations in the ERBB2 Signaling Pathway[END_REF], MSI [START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF] or TP53 mutations [START_REF] Alvi | Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility[END_REF]. Some differences of genetic profile were reported according to the small bowel segment. Indeed, several studies found that ERBB2 mutations were more frequent in duodenum [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF][START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF][START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF], but conflicting results are reported for other genetic alterations according to localisation across the studies. The limits of most studies are the small number of patients and the lack of clinical data or prognosis evaluation.

The NADEGE cohort has enrolled prospectively consecutive patients with all stages of SBA during a four-year period in France. Clinical tumour characteristics differ according to sporadic SBA or secondary to a predisposing disease. Crohn disease was significantly associated with younger age, poor differentiation, and ileum location, whereas Lynch syndrome was associated with younger age, poor differentiation, an early stage, and duodenum location.

Tumour grade and stage were the main prognostic factors [START_REF] Aparicio | Small bowel adenocarcinoma: Results from a nationwide prospective ARCAD-NADEGE cohort study of 347 patients[END_REF]. The BIONADEGE study is an ancillary study of the NADEGE cohort aimed to assess the genomic profile according to a predisposing disease for SBA, to SBA localisation or stage and assess the prognostic value of these genomic alterations.

PATIENTS AND METHODS

Study population

The NADEGE cohort has recruited 347 patients in 74 participating PharMingen, Le Pont de Claix, France). The Bond Polymer Refine Detection kit (Leica) was used as the detection system. Immunostaining of MLH1, MSH2, MSH6 and PMS2 in tumour cells was evaluated as positive or negative as assessed in TMA. Tumours were considered negative when there was a complete absence of nuclear staining of neoplastic cells in the presence of an internal positive control assessed in a whole slide. All the tumour with a negative staining of one of the MMR protein were considered as dMMR.

Molecular analysis

The same paraffin blocks were used for DNA extractions and for IHC analyses. DNA was extracted from formalin-fixed, paraffin-embedded neoplastic tissue that had been macrodissected with reference to the Hematoxylin and Eosin stained section.

Next generation sequencing (NGS) investigates the presence of 739 hotspot somatic mutations in 46 genes involved in carcinogenesis using cancer hotspot panel from Thermofisher (Table S1). DNA extraction, NGS sequencing and mutation calling were performed as described previously [START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF].

Statistical analysis

Descriptive analysis of the initial tumour stage (reference) and variables measured at baseline was performed. Categorical variables were summarised as frequencies and percentages and continuous variables as medians and ranges. The comparison of gene alteration frequencies according the sub-group of patients was assessed with the χ² test or Fisher's exact test, as appropriate, for categorical variables.

Patients with metastatic disease were defined as those who had metastasis at the time of the inclusion and those who developed additional metastatic recurrence tumours during follow-up.

Therefore, some patients in this trial were analysed twice: first, as cases with localized tumours, and second, as cases with metastases.

Overall survival (OS) was defined as the time from diagnosis of a primary tumour (localized tumour) or of metastasis (synchronous or metachronous) until death due to any cause.

Patients who were still alive at the last follow-up were censored. Patients with synchronous resected metastasis were excluded from the analysis of metastatic patient subgroup in order to assess OS of patients with unresectable metastases.

The survival curves for OS were estimated by the Kaplan-Meier and were compared using the log-rank test. The follow-up time was assessed by the reverse Kaplan-Meier method. The medians and 95% confidence intervals (95% CIs) were calculated and 3-year rates with 95%CI were also provided.

The hazard ratios (HRs) and their 95% CIs were estimated with the Cox proportional hazard model. Univariate analysis was performed to determine baseline characteristics associated with OS for patients with mutational status available. All variables with p values of <0.1 were included in multivariate analysis. The correlations between variables were assessed and proportional hazard assumptions were examined graphically by log-minus-log plots of survival.

All statistical analyses were conducted with a two-sided alpha significance level of 5% using SAS 9.3 software (SAS institute Inc., Cary, NC, USA). As the analyses were exploratory, p values were not adjusted for multiple testing.

RESULTS

Patient and tumour characteristics

Among the 347 patients included in the analysis of clinical NADEGE dataset, 196 tumour blocks were collected for immunohistochemistry and molecular analysis. The quantity or quality of extracted DNA could not allow molecular analysis in 71 tumours. Finally, the mutation status was obtained for 125 patients (Figure 1). Patient characteristics are presented in table 1. The clinical and tumour characteristics were comparable in the patients from the whole NADEGE [START_REF] Aparicio | Small bowel adenocarcinoma: Results from a nationwide prospective ARCAD-NADEGE cohort study of 347 patients[END_REF] and the BIONADEGE cohorts except for metastatic stage at diagnosis underrepresented in the BIONADEGE cohort (36% in NADEGE vs 18% in BIONADEGE, p<0.0001).

The gene mutation frequency according to tumour stage and primary are presented in table 2.

The detail of raw NGS data are presented in the table S1. Overall, at least one genomic alteration was observed in 90.4% of tumours. There is no difference into the frequency of at least one genomic alteration according to the tumour stage: 89.2% and 95.4% for localized or metastatic tumour at diagnosis, respectively. There was no difference into the frequency of at least one genomic alteration according to primary tumour site: 92.0%, 82.1% and 95.4% for duodenum, jejunum or ileum, respectively. Overall, the number of mutations observed per tumour was 0 in 9.6%, 1 in 32.0%, 2 in 26.4% and >3 in 32.0% of the patients. The proportion of tumours with >3 mutations were also similar according to stage: 30.4% and 40.9% for localized and metastatic tumours at diagnosis, respectively and according to primary: 33.3%, 21.4% and 40.9% for duodenum, jejunum or ileum, respectively. The most frequent genomic alteration observed were: KRAS (44.0%), TP53 (38.4%), PIK3CA (20.0%), APC (18.4%), SMAD4 (14.4%) and ERBB2 (7.2%). A KRAS mutation was more frequent in metastatic tumours at diagnosis than in localized tumours (72.7% vs 38.2%, p=0.003). A BRAF mutation was observed in 5 (4%) cases and among them there is only one V600E mutation. There was no significant difference of mutation rate according to primary location for the most frequently altered genes.

The comparison of gene mutation frequency between patients with Lynch syndrome and those with no predisposing disease revealed different profiles (Table 3). There is a trend for less frequent KRAS mutations in Lynch syndrome and more frequent TP53 and PIK3CA mutations in Crohn's disease compare to no predisposing disease. No APC mutation was observed in any Crohn's disease. There is a trend of more frequent ERBB2 mutations in Lynch syndrome compare to no predisposing disease. Moreover, no ERBB2 mutation was observed in Crohn's disease. Several rare mutations are more frequent in tumour with Lynch syndrome than in no predisposing syndrome such as ATM, FGFR3 and FGFR1 gene mutations. IDH1 mutations are more frequent in tumours with Crohn's disease than in no predisposing disease.

Results according to MMR status

MMR status was determined with immunohistochemical (IHC) analysis of MMR proteins in 180 patients. A deficient MMR (dMMR) tumour was observed in 50 (28%) patients. A negative staining was observed for both MLH1 and PMS2 in 21 (42%), MSH2 and MSH6 in 18 (36%), PMS2 with MLH1 inconclusive test in 4 (8%), MSH6 with inconclusive MSH2 in 2 (4%), PMS2 alone in 2 (4%), MSH6 alone in 2 (4%) and MSH2 with inconclusive MSH6 in one (2%).

The comparisons of patient and tumour characteristics according to MMR status are given in table [START_REF] Legué | Trends in incidence, treatment and survival of small bowel adenocarcinomas between 1999 and 2013: a population-based study in The Netherlands[END_REF]. The dMMR tumours were associated with a younger age, a less metastatic stage at diagnosis, less KRAS mutations but more SMARCB1 mutations. There is also a trend for less TP53 mutations and more ERBB2 mutations.

Survival analysis

The median follow-up was 56 months (95% confidence interval (CI) [47-63]). The 3-years OS of the 196 patients with block available was 64.4% (95%CI 56.6% -71.1%) and 71.7% (95%CI 61.9% -79.4%) for the 125 patients with mutation status available.

Survival analysis in the 125 patients with mutational status available

Univariate analysis was performed in the sub-group of patients with mutation statuses available to assess the prognostic factors for OS including clinical parameters and the gene mutation with a frequency over 10% (Table 5). No genomic alteration was associated with OS (Table 5). In the multivariate analysis including stage, Lynch syndrome and tumour differentiation, only poor tumour differentiation remained associated with higher risk of death

Survival analysis in the 180 patients with MMR status available

The 3-years OS rate was 79.9% (95%CI 64.7 -89.1) for patients with dMMR tumours and 58.5 months (95%CI 48.5 -67.1) for patients with pMMR tumours. There is a trend for better survival in patients with dMMR tumours (HR=0.59; 95%CI [0.32-1.06], p=0.0765) (Figure S1).

In the subgroup of patients with localized tumours the 3-years OS were 82.9% (95%CI 67.2 -91.5) and 72.5% (95%CI 60.2 -81.6) for patients with dMMR and pMMR respectively (logrank p=0.3957; HR=0.74 [0.36-1.50], p=0.3976). Due to the small number of patients with a dMMR metastatic tumour (n=2) the comparison of median OS according to MMR status was not reported.

DISCUSSION

Our study on a large number of patients with SBA revealed different tumour mutation profiles according to predisposing diseases (Crohn's disease or Lynch syndrome compared to tumour without predisposing disease) or an MMR status.

Our results are concordant with the genomic alteration profile reported in three previous studies that reported a mutation rate for KRAS from 43.4% to 53.6%, TP53 from 41% to 58.4%, PIK3CA from 9% to 18.4%, APC from 13.2% to 26.8%, SMAD4 from 9.6% to 17.4% and ERBB2 from 8.4% to 14% [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF][START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF][START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF]. Moreover, we found that KRAS mutations as pMMR status were associated with metastasis. This is the first report showing that a genomic alteration is associated with advanced stage in SBA to our best knowledge.

We found no significant association with one of the mutations observed in more than 5% of the tumour and primary tumour site. Two previous studies have reported an association with ERBB2 mutation and duodenal location [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF][START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF]. In our study as in the Härinnen et al study [START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF] the ERBB2 mutation rate was higher in tumour of the proximal small bowel without reaching significance. Some rare mutations seem to have a different distribution according to the small bowel segment. IDH1 mutations were only reported in the ileum tumour which may be explained by the association with Crohn's disease that was mainly associated with ileum tumours in the NADEGE cohort [START_REF] Aparicio | Small bowel adenocarcinoma: Results from a nationwide prospective ARCAD-NADEGE cohort study of 347 patients[END_REF]. FBXW7 mutation was predominantly observed in the jejunum tumour. This result is concordant with the Schrock et al results that have reported a trend of more FBXW7 mutation in non-duodenal SBA [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF].

The genomic alteration profile was different according to predisposing diseases or the MMR status. Crohn's disease was associated with tumour genomic alterations of IDH1. Moreover, a trend for more frequent KDR mutations but no APC mutation was observed in Crohn's disease compare to no predisposing disease. IDH1 mutations and also high mutation rate of TP53 were already reported associated with Crohn's disease in colorectal cancer [START_REF] Yaeger | Genomic Alterations Observed in Colitis-Associated Cancers Are Distinct From Those Found in Sporadic Colorectal Cancers and Vary by Type of Inflammatory Bowel Disease[END_REF]. A recent publication reported an association of IDH1 and SMAD4 mutations with Crohn's disease in SBA [START_REF] Liao | Clinicopathological and Molecular Characterisation of Crohn's Disease-associated Small Bowel Adenocarcinomas[END_REF]. We did not find any association of SMAD4 mutation and Crohn's disease in our study. Tumour KDR gene alteration, coding for VEGFR2, has not been previously reported to be associated to Crohn's disease. The lack of APC mutation in SBA associated with inflammatory bowel disease was already reported by Schrock et al [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF]. A lower frequency of APC mutation in colorectal cancer associated with inflammatory bowel disease as compared to sporadic colorectal cancer was also reported [START_REF] Yaeger | Genomic Alterations Observed in Colitis-Associated Cancers Are Distinct From Those Found in Sporadic Colorectal Cancers and Vary by Type of Inflammatory Bowel Disease[END_REF]. No ERBB2 mutation was observed in tumour associated with Crohn's disease in our study as it was previously observed in the Schrock study [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF]. Altogether ours and previous results support the hypothesis that the SBA associated with Crohn's disease has a different carcinogenesis from sporadic cancer as it is observed in colorectal cancer [START_REF] Ullman | Intestinal inflammation and cancer[END_REF].

In SBA associated with Lynch syndrome, there is a trend of less KRAS mutations and more ERBB2 mutations compared to tumours without predisposing diseases. Other rare mutations such as ATM, FGFR3 and FGFR1 are associated with Lynch syndrome in our study. The risk of developing a cancer for patient with Lynch syndrome if they had an ATM mutant allele is a matter of debate [START_REF] Jones | ATM polymorphism and hereditary nonpolyposis colorectal cancer (HNPCC) age of onset (United States)[END_REF]. We could not determine in our study if the ATM mutation was inherited or acquired. FGFR3 R248C hotspot mutation has already been associated to the Lynch syndrome in upper tract urothelial carcinoma [START_REF] Donahu | Genomic Characterization of Upper-Tract Urothelial Carcinoma in Patients With Lynch Syndrome[END_REF] but not with SBA until our report.

We found some specificity in the subgroup of dMMR tumours compared to pMMR tumours.

Patients with dMMR tumours are younger than patients with pMMR tumours. This is the inverse result that it is observed in colorectal cancer [START_REF] Aparicio | High prevalence of deficient mismatch repair phenotype and the V600E BRAF mutation in elderly patients with colorectal cancer[END_REF]. That may be explained by the fact that in our study the proportion of Lynch syndrome among dMMR tumour reach 34%. Nevertheless, as it is observed in colorectal cancer, the dMMR tumours are rarely metastatic at diagnosis. In our study, KRAS mutations are less frequent in dMMR tumours compared to pMMR tumours. This has not been previously reported in SBA and deserves a confirmatory study. There is also a trend for less TP53 alterations but more ERBB2 alterations in dMMR tumours than in pMMR tumours. The association of ERBB2 mutations and dMMR has previously been reported [START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF].

We report a higher frequency of SMARCB1 mutations in dMMR tumours, SMARCB1 has already been described in dMMR colorectal tumours [START_REF] Kondelin | Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer[END_REF].

We did not find any association between genomic alteration and prognosis. One previous study reports a poor prognosis associated with a genomic alteration of the ERBB signalling cascade [START_REF] Adam | DNA Sequencing of Small Bowel Adenocarcinomas Identifies Targetable Recurrent Mutations in the ERBB2 Signaling Pathway[END_REF] but ERBB2 mutations solely had no prognostic value. The dMMR phenotype was already reported as good prognostic factor for disease free-survival in one study [START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF]. In our study as in a previous one [START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF] there is a trend for better prognosis in patients with a dMMR tumour. The prognostic effect of dMMR phenotypes seems restricted to patients with localized and resected tumours. In patients with metastatic tumours the MMR status seems to have no effect. It must be pointed out that no patient with a dMMR tumour received immunotherapy. TP53 mutations were reported associated with poor survival in a previous study [START_REF] Alvi | Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility[END_REF] but had no significant prognostic value in our study either in localized tumour or metastatic tumour like in another previous study [START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF]. KRAS mutations were reported as a poor prognostic predictor in the subgroup of patients with a pT1-T3 tumour (21) but also associated with a better survival in patients with metastatic tumour [START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF]. In our study there was no significant effect of KRAS mutation but a trend of a worst prognosis in localized tumours and a better prognosis in metastatic tumours. It has been previously reported that KRAS mutations were associated with a poor OS in colorectal stage III pMMR tumours [START_REF] Taieb | Prognostic Value of BRAF and KRAS Mutations in MSI and MSS Stage III Colon Cancer[END_REF]. The prognostic value of KRAS mutations deserves further evaluation in SBA. A BRAF mutation was only observed in 4% of the tumours in our study. In previous studies the frequency of BRAF mutations range from 1% to 11% [START_REF] Schrock | Genomic Profiling of Small-Bowel Adenocarcinoma[END_REF][START_REF] Hänninen | Exome-wide somatic mutation characterization of small bowel adenocarcinoma[END_REF][START_REF] Laforest | ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma[END_REF][START_REF] Jun | Clinicopathologic and prognostic associations of KRAS and BRAF mutations in small intestinal adenocarcinoma[END_REF]. As in the previous studies the majority of BRAF mutations reported in our study were not the V600E mutation. No prognostic value of BRAF mutations was reported in SBA. It must be pointed out that in metastatic colorectal cancer non-V600E BRAF mutations are not associated to a poor prognosis in contrast to the V600E BRAF mutations [START_REF] Jones | Non-V600 BRAF Mutations Define a Clinically Distinct Molecular Subtype of Metastatic Colorectal Cancer[END_REF].

Several genomic alterations reported in our study may be targeted. It has recently been reported that a treatment with immune checkpoint inhibitor gives a prolonged survival in patients with metastatic dMMR SBA [START_REF] Marabelle | Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study[END_REF]. Preclinical data suggest that ERBB2 inhibitors reduce tumour growth of ERBB2 mutated tumours [START_REF] Adam | DNA Sequencing of Small Bowel Adenocarcinomas Identifies Targetable Recurrent Mutations in the ERBB2 Signaling Pathway[END_REF]. Thus, ERBB2 inhibition deserve clinical evaluation in patients with ERBB2 mutated SBA. Other gene alterations of PTEN, PI3KCA or PTEN may be considered for targeted treatment [START_REF] Mateo | A framework to rank genomic alterations as targets for cancer precision medicine: the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)[END_REF]. Some rare mutations deserve also further evaluation. Signal of efficacy have been reported with PARP inhibitors in patient with ATM deficiency [START_REF] Jette | ATM-Deficient Cancers Provide New Opportunities for Precision Oncology[END_REF]. IDH1 inhibitions have shown efficacy in cholangiocarcinoma [START_REF] Lowery | Safety and activity of ivosidenib in patients with IDH1-mutant advanced cholangiocarcinoma: a phase 1 study[END_REF].

IDH1 mutations should be screened in patient with SBA associated with Crohn's disease and IDH1 inhibitors need evaluation in those patients.

Our study had some limitations: first, even if this study is one of the largest genomic profiling of SBA, the sample size does not allow an accurate evaluation of rare mutation impact.

Secondly, the gene panel used is limited but contains the most frequently altered genes in SBA. Thirdly, the constitutional gene mutations were not assessed in case of Lynch syndrome in our study. Fourth, we did not performed MSI testing nevertheless a previous study has reported no discordance between MMR IHC and MSI testing [START_REF] Aparicio | Small bowel adenocarcinoma phenotyping, a clinicobiological prognostic study[END_REF]. Finally, we assume that our results are exploratory and should be taken with caution for the rare mutations as we did not perform a Bonferroni correction in our analysis. Moreover, the clinical characteristics were comparable in the NADEGE [START_REF] Aparicio | Small bowel adenocarcinoma: Results from a nationwide prospective ARCAD-NADEGE cohort study of 347 patients[END_REF] and BIONADEGE cohorts except for metastatic stage at diagnostic underrepresented in the BIONADEGE cohort due to missing tumour samples suitable for genomic analysis. Thus, our results in metastatic tumours are limited. Additional studies pooling several databases are needed to specify the association of genomic profile, clinical data and prognosis.

In conclusion, our study shows that there are different genomic alteration profiles in SBA that depends on the existence, or lack thereof, of a predisposing disease. This advocates to analyse separately sporadic SBA and those related to predisposing disease in future studies.

With caution due to sample size, genomic alteration had no prognostic impact except a trend for a favourable prognosis associated with dMMR phenotypes in localized tumour.

Nevertheless, some genomic alterations may be targeted. A compilation of worldwide experiences for off label targeted therapy is urgently needed for this orphan disease.

Previous presentation: This work has been presented at the 2019 ASCO meeting

  French institutions from January 2009 to December 2012. All consecutive stage I-IV patients with histologically proven, newly diagnosed or with recurrent SBA (local or distant) were enrolled into the NADEGE cohort. Ampullary and non-adenocarcinoma tumours were excluded. TNM staging was done according to the criteria of AJCC and UICC (7th UICC TNM Staging System) performed at diagnosis by computed tomography (CT) scan and/or magnetic resonance imaging. The following clinical data were prospectively collected: demographics, cancer treatment history, tumour stage, lymph node invasion, tumour differentiation, initial treatment, and survival. The predisposing disease or genetic syndrome was assessed by investigator declaration. The tumour blocks of either tumour biopsy from primary or metastasis or tumour surgical resection were collected. Immunohistochemistry Tissue microarrays (TMA) were constructed from 0.6-mm diameter tissue cores obtained from formalin fixed paraffin embedded tumor specimens. Hematoxylin and Eosin staining was performed on each TMA slide to confirm the presence of tumor tissue. The expression of MLH1, MSH2, MSH6, and PMS2 was assessed as previously described (9). Briefly, 4 μm sections were cut onto silane-treated Super Frost slides (CML, Nemours, France) and left to dry at 37°C overnight. The slides were deparaffinised in xylene and rehydrated in pure ethanol. Endogenous peroxidase was blocked using 3% hydrogen peroxide in methanol for 30 min. Before immunostaining, antigen retrieval was performed by immersing sections in citrate buffer (pH 6.0). Sections were then incubated for 15 minutes at room temperature with antibodies to MLH1 (dilution 1/70, clone G168-728, Pharmingen, San Diego, CA), MSH2 (dilution 1/100, clone FE11, Calbiochem, Oncogene Research Products, Cambridge, MA), MSH6 (dilution 1/100, clone 44, Becton Dickinson, Lexington, NC), PMS2 (clone A16-4, 1:150 dilution, BD

(

  HR=2.48; 95%CI [1.19-5.21]; p=0.0159). There is trend for a better prognosis associated with early stage (p=0.0774) and Lynch syndrome (p=0.0648). The results of univariate analysis according to localized or metastatic tumour are given in table S2. In the sub-group of 102 patients with localized and resected tumour, no genomic alteration was associated with OS. There is a trend for a worst 3 years OS in patients with tumour KRAS mutation (63.3% [95%CI 43.0 -78.1] versus 82.0% [95%CI 69.1 -89.9], p=0.3551). In the subgroup of 31 patients with metastatic disease (unresectable synchronous metastasis and metachronous metastasis) the median OS was 22.6 months (95%CI 12.7-59.7). No genomic alteration was associated with OS. Median OS was 32.3 months (95%CI 12.5 -59.7) and 21.0 months (95%CI 8.1 -36) in patients with mutated and wild type tumour KRAS, respectively (p=0.5235). The median OS was 27.3 (95%CI 9.1 -59.7) and 16.2 (95%CI 3.9 -not assessable) in patients with mutated and wild type tumour TP53, respectively (p=0.9123).

Figure 1 :

 1 Figure 1: Flow chart

Table 1 :

 1 Patient's characteristics

		Overall population in Nadege analysis
		347 patients
		196 patients with block analysed
		(180 patients with conclusive results for MMR)
	Accepted Article	125 patients with conclusive results for mutation analysis (121 patients with conclusive results for MMR) 102 localised resected patients 22 metastatic patients Stage at diagnosis progression Resection 24 patients with metastatic 1 patient M0 but resection unknown
		7 unresectable patients	15 resected patients

Table 4 :

 4 Patients and tumour characteristics according to MMR status

		pMMR
		tumours	dMMR tumours
		n=130
	Characteristics	(72.2%)	n=50 (27.8%)	P value
	Sex: Men	67 (51.5%)	28 (56.0%)
	Women	63 (48.5%)	22 (44.0%)	0.5912
	Age (median)	64	58	0.1760
	Primary: Duodenum	83 (64.3%)	33 (66.0%)
	Jejunum	22 (17.0%)	11 (22.0%)
	Ileum	24 (18.6%)	6 (12.0%)	0.4889
	Stage at diagnosis	n=129	n=49

Accepted Article

Acknowledgments

BIONADEGE was granted by INCa and sponsor by Assistance Publique Hôpitaux de Paris (Délégation à la Recherche Clinique). ARCAD-NADEGE cohort was granted by A.R.C.A.D. and sponsored by GERCOR. The authors thank to all investigators: Pauline Afchain (Paris), Thomas Aparicio (Paris), Jean-Baptiste Bachet (Paris), Nathalie Bonichon-Lamichhane (Bordeaux), Laurent Cany (Périgueux), François Caroli-Bosc (Angers), Christos Christidis (Paris), Philippe Collin (Reims), Romain Coriat (Paris), David Deutsch (Bobigny), Joëlle Egreteau (Lorient), Pierre-Luc Etienne (Saint Brieuc), Francine Fein (Besançon), Johan Gagnière (Clermont-Ferrand), Claire Garnier-Tixidre (Grenoble), Vincenzo Giardina (Bezier), Jean-Marc Gornet (Paris), Victoire Granger (Grenoble), Rosine Guimbaud (Toulouse), Kamran Imani (Tarbes), Sandrine Lavau-Denes (Limoges), Cédric Lecaille (Bordeaux), Thierry Lecomte (Tours), Jean-Louis Legoux (Orléans), Astrid Lièvre (Rennes), Catherine Ligeza-Poisson (Saint Nazaire), Christophe Locher (Meaux), Catherine Lombard-Bohas (Lyon), May Mabro (Suresnes), Sylvain Manfredi (Dijon), André Mathieu (Narbonne), Jérôme Meunier (Orléans), Drifa Moussata (Lyon), Suzanne Nguyen (Beauvais), Arnaud Patenotte (Semur en Auxois), Hervé Perrier (Marseille), Guillaume Piessen (Lille), Marc Pocard (Lariboisière), Fabienne Portales (Montpellier), Corinne Sarda (Castre), Eric Terrebone (Pessac), David Tougeron (Poitiers), Aziz Zaanan (Paris). The authors also thank Sabine Helfen and Zahia ben Abdesselam from the Clinical Research Unit and Clinical Research Centre, Avicenne Hospital, Bobigny as study manager, Mourad Benallaoua from Gastroenterology department for operational assistance, Alexandra Patry for technical assistance for tumour micro-array and Elsa Benamouzig for English editing.

Funding

This work was supported by a grant from INCa (Programme Hospitalier de Recherche Translationelle Cancer, PRTK14 N°091) and a grant n° NA 2009 from the A.R.CA.D. foundation.

Data availability statement:

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions:

Study design: TA, MS, JH, DV and PLP.

Data acquisition: TA, MA, PA, AL, DT, JG, ET, GP, JLL, CL, MP, JMG, AZ, SLD, TL, DD and PLP.

Statistical analysis: JH and DV.

Manuscript preparation: TA, MS, JH, DV and PLP.

Manuscript review: all authors

Conflict of interest statement

The authors declare no conflict of interest for this publication.


Mohamed Sahnoune

Aurélie Taguet

Belkacem Otazaghine

Mustapha Kaci

José-Marie Lopez-Cuesta

Fire Retardancy Effect of Phosphorus-Modified Halloysite on Polyamide-11 Nanocomposites

Halloysite nanotubes (HNTs) were successfully incorporated as flame retardants in polyamide-11 (PA11) after their modification with methyl phosphonic acid. Fourier transform infrared spectroscopy, thermal gravimetric analysis (TGA) and pyrolysis-gas chromatography-mass spectrometry were used to evidence the functionalization of the clay. Raw and modified HNTs were then incorporated by melt mixing in PA11 at 20 wt%. Compositions containing both ammonium polyphosphate (APP) and HNTs were also prepared. TGA and pyrolysis combustion flow calorimeter exhibited enhancement in thermal stability upon incorporation of both raw and modified halloysite nanotubes while APP causes degradation at lower temperature. Cone calorimeter data showed that modified halloysite acts by forming an insulating barrier during the combustion, which limits heat and mass transfers. Moreover, elemental analysis of sample residues after cone test evidenced that a part of the phosphorus of the modified halloysite was transferred to the gaseous phase. These results suggest the full potential of halloysite as fire retardant agent for polyam-ides.

INTRODUCTION

Polyamide-11 (PA11) is a biocompatible commercial aliphatic polymer with excellent characteristics such as good oil resistance or low coefficient of friction and has the advantage to be less hydro-philic than the commonly used polyamide-6 and 6,6 [1,2]. Therefore, PA11 is widely used as an engineering polymer in a large range of industrial fields from automotive to offshore applications including food packaging and sport equipments. Unfortunately, like all the polyamides, the fire behavior of PA11 is limited and applications in electric industries or in the building sector require the use of fire retardants. Several approaches to improve fire retardancy of polyamides can be envisaged, including the use of flame retardants additives [3,4], grafting fire retardant groups on the polymer chain [5] or copolymerization with fire retardant monomers [START_REF] Shalaby | [END_REF]. Nevertheless, most of current fire retardants cause destabilization of polyamides (discoloration and reduction in melt viscosity) because of sensitivity to acids, even at low concentrations [3]. Moreover, phenomena of migration of the additives during processing, water conditioning or ageing of the polymer are liable to entail loss of fire retardancy because of the undergone hydrolysis. Hence, nanocomposites technology has emerged as a route to improve the flammability of polyamides, not only with the use of nanoparticles alone, such as nanoclays [7][8][9], but more efficiently through synergies with flame retardants [10][11][12][13].

Halloysite nanotubes (HNTs) are minerals of kaolinite group with the chemical composition Al 2 Si 2 O 5 (OH) 4 .nH 2 O, which exhibit many advantages such as biocompatibility, thermal stability and reinforcing effect [14]. The literature reports numerous polymer/halloysite nanocomposite systems, leading to enhancements in mechanical properties, improvement of thermal stability, nucleating effect and also flame retardancy [15][16][17][18]. Indeed, it has been reported that halloysite, alone or in synergy with usual fire retardant, can enhance fire behavior of various polymers such as polypropylene [19][20][21], polyamide-6 [22], polylactic acid (PLA) [23], poly(butylene succinate) [24] or linear low-density polyethylene [25] through thermal insulation barriers that can be developed at the surface of the composite during the burning. It has been shown that this barrier reduces the mass loss rate, limits the heat release rate (HRR) and extends the time to flameout [22]. The encapsulation into the HNTs lumen of products resulting from the thermal degradation of the polymer is also known to contribute to the fire behavior enhancement of the nanocomposites [26]. Many factors have to be examined while considering nanoparticles as fire retardant, such as aspect ratio, shape or amount that can influence the barrier effect. To improve the flame retardant effect of halloysite, some authors have retained the strategy of chemical modification of the clay. For example, Li et al. [27] grafted maleic anhydride and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) at the surface of HNTs before adding it at 5 wt% in PLA. They reported that the improved fire retardancy was due to a slow release of DOPO derivations and a subsequent flame inhibition, together with secondary barrier effect. Marney et al. [28] intercalated halloysite with phenyl phosphonic acid, leading to an enhanced fire behavior of PA6/halloysite nanocomposites. Halloysite has unique tubular structure with aluminol groups at the inner surface and silanol ones at the outer. This difference of composition was used in this study to modify selectively inner surface of halloysite with methyl phosphonic acid (MPA). Indeed, the reactivity between HNTs aluminol groups and phosphonic functions allows the selective grafting of inner surface of HNTs [29,30]. In a recent article [31], organophosphorous flame retardant dimethyl methylphosphonate (DMMP) was loaded into HNTs lumen using vacuum (leading to HNT-D). A 20 wt% of HNT-D was added into UV-curable epoxy resin and a better flame retardancy was measured. The authors revealed that the flame retardant mechanism consisted in two steps: a first DMMP gasification followed by a polyphosphoric-HNTs carbon layer generation on the condensed phase. To the best of our knowledge, there are no other articles dealing with the effect of the chemical modification of HNTs on the flame retardancy of polymers, especially polyamides.

Correspondence to: A. Taguet; e-mail: aurelie.taguet@mines-ales.fr Therefore, the main objective of this paper is to investigate the potential of halloysite, after its functionalization with MPA, as a fire retardant agent for PA11. Halloysite was used alone or in combination with a standard phosphorous fire retardant. The incorporation of the clay in PA11 was performed by melt compounding at 20 wt%. Moreover, the results with the modified halloysite were compared to those of neat PA11 and PA11 containing raw halloysite and ammonium polyphosphate (APP).

EXPERIMENTAL Materials

PA11 (Rilsan ® LMNO) was kindly supplied by Arkema. This grade is free of any stabilizers or additives. Its mean molecular weight Mn = 22,000 g/mol and average molar weight = 51,000 g/mol with a density of 1.02 g/cm 3 . Raw halloysite (referenced as Hal) derived from Djebel Debbagh deposit (Guelma, eastern region of Algeria). Halloysite particles having an average diameter of 25 μm and a surface area of 51.4 m 2 /g. This halloysite was characterized in details previously in [32]. MPA was purchased from ABCR. Less-hygroscopic APP Exolit 423 was kindly supplied from Clariant. Ethanol and acetone were purchased from Sigma Aldrich and Panreac, respectively. Before using, HNTs and polymer pellets were dried under vacuum at 80 C for 24 h and overnight, respectively, to remove moisture traces.

Halloysite Functionalization

The halloysite functionalization consisted of a reaction between halloysite and MPA. A 10 g of halloysite, 1 g of MPA and 100 ml of an ethanol/water (90/10 w/w) solution were introduced into a 250 mL flask fitted with a condenser. The mixture was then stirred and heated at solvent reflux for 15 h. The mixture was next centrifuged (speed: 5,000 rpm) to eliminate the liquid phase and washed three times with acetone. Finally, the obtained halloysite (referenced as Hal-P) was dried under vacuum.

Samples Preparation

Various compositions containing 20 wt% of Hal, Hal-P, APP, or a mixture of APP and Hal or Hal-P were prepared and Table 1 summarizes the studied compositions. The samples were prepared by melt mixing under vacuum in a semi-industrial twin screw extruder (BC 21 Clextral) at a screw speed of 250 rpm with a screw diameter (D) of 25 mm and length to diameter ratio (L/D) of 48. The temperature profile was 140 C/250 C/245 C/235 C/230 C from hopper to die. After pelletizing, the nanocomposites granules were dried under vacuum at 80 C overnight and then injection molded into standard test specimens for fire testing (plates of 100 × 100 × 4 mm 3 ) using an injection molding machine KM50-180X (Krauss Maffei). The temperature profile setting ranged from 220 C to 225 C and the mold temperature was kept at 40 C. Holding pressure and screw rotation speed were 135 bar and 100 rpm, respectively, with a throughput of 50 cm 3 /s.

Characterization

Fourier Transform Infrared Spectroscopy.

The surface modification of halloysite after functionalization was investigated by Fourier transform infrared (FTIR) spectroscopy. FTIR was performed on a spectrometer IFS 66 (Bruker) on KBr pellets (1:99 ratio) and spectra were obtained by collecting 64 scans between 400 and 4,000 cm -1 with a resolution of 2 cm -1 .

Pyrolysis-Gas Chromatography-Mass Spectrometry.

A Pyroprobe 5000 pyrolyzer (CDS analytical) was used to flash pyrolyze the samples in a helium environment. This pyrolyzer is equipped with an electrically heated platinum filament. One coil probe enables the pyrolysis of samples (<1 mg) placed in a quartz tube between two pieces of rockwool. The sample was heated at 900 C. The temperature was held for 15 s, and then the gases were drawn to the gas chromatograph for 5 min. The pyroprobe 5000 is interfaced to a 450-GC chromatograph (Varian) by means of a chamber heated at 270 C. In the oven, the initial temperature of 70 C was raised to 250 C at 10 C/min. The column is a Varian Vf-5 ms capillary column (30 m × 0.25 mm; thickness = 0.25 μm) and helium (1 L/min) was used as the carrier gas, a split ratio was set to 1:50. The gases were introduced from the GC transfer line to the ion trap analyzer of the 240-MS mass spectrometer (Varian) through the direct-coupled capillary column.

X-Ray Diffraction.

The crystalline structure of the specimens was examined using wide angle XRD measurements. An X-ray diffractometer (XRD) Bruker D8 (Bruker, USA) equipped with a Cu-Kα radiation source (λ = 1.5418 A ) and operated at 30 kV and 40 mA, was used for the investigations in the interval range 2ϑ from 5 to 70 with a scanning step of 0.007 .

Thermogravimetric Analysis.

The thermal stability of the halloysites and the nanocomposite samples was studied by thermal gravimetric analysis (TGA). The analyses were performed by a thermogravimetric analyzer (Setsys Evolution, Setaram). In order to remove all the physisorbed water of the halloysites, an isothermal step (10 min, 110 C) was performed before starting the analysis and then the samples were heated to 900 C at a heating rate of 10 C/min, while the nanocomposite samples were heated to 600 C at 10 C/min. Measurements were carried out under air or nitrogen atmospheres with a flow rate of 20 mL/min on samples of ~20 mg.

Transmission Electron Microscopy.

Transmission electron microscopy (TEM) was conducted to evaluate the degree of dispersion of both raw and modified HNTs in the PA11 matrix. Micrographs were obtained with a JEOL 1200EX TEM (JEOL) operating at an accelerating voltage of 100 kV. The TEM samples were embedded in a LR white resin and ultrathin-sectioned at 70 nm using a Leica EM UC7 ultra-microtome with a diatome diamond knife Ultra 45 (Nissei Sangyo). Sections were collected on 300 mesh copper grids coated with carbon film. vibration of alkyl groups inside the confined spaces of halloysite lumen [29,30]. Pyrolysis-gas chromatography-mass spectrometry (Py-GC/ MS) was also used to evidence the functionalization and the obtained results are presented in Fig. 2. The first observation after halloysite modification concerns the main observed peak at 1.5 min in Hal that is assigned to a sulfonate fraction resulting from a pollution due to the raw nature of the used HNTs. We

Scanning Electron Microscopy.

Scanning electron microscopy (SEM) was conducted under high vacuum with an environmental SEM Quanta 200 FEG (FEI Company) operating at 12.5 kV. It was first used to investigate the morphology of the PA11/HNTs samples. Injection molded test specimens were cryo-fractured and coated with a thin carbon layer. The fracture surfaces were observed in the back-scattered electrons mode. Moreover, SEM was used to observe the residues collected after cone calorimeter test for PA11/H and PA11/HP.

Pyrolysis Combustion Flow Calorimeter.

Pyrolysis combustion flow calorimeter (PCFC) has been used to study the fire behavior of mg-sized samples. PCFC analysis was carried out according to the method A (anaerobic pyrolysis) of the ASTM D7309 using a Fire Testing Technology (FTT) apparatus with a heating rate of 1 C/s, the maximum pyrolysis temperature was 750 C and the combustion temperature was 900 C (corresponding to complete combustion). The flow in combustor was a mixture of O 2 /N 2 20/80 at 100 cm 3 .min -1 and the sample weight was 0.5-2 mg.

Cone Calorimeter.

The fire behavior of the studied compositions was mainly studied using a FTT cone calorimeter. The measurements were carried out according to ISO 5660-1 test procedure at incident heat flux of 50 kW/m 2 and the ignition of the flame was initiated using a spark igniter. The cone data reported here are averages of at least three replicates reproducible with variation <10%. The fire property parameters considered were HRR, peak of HRR (PHRR), total heat released (THR), maximum average rate of heat emission (MAHRE), and time to ignition (TTI).

Elemental Analysis.

The phosphorus content was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Measurements were carried out by SGS Multilab. Modified halloysite (Hal-P) and the residue after cone calorimeter test of PA11/HP were analyzed. Hal-P contains 16 wt% of water whereas the residue of PA11/HP is in the dehydrated form. Hence, water was removed in the calculation of the final phosphorus content of Hal-P and the dry mass of phosphorus (wt% of P) is given in the Results and Discussion part.

RESULTS AND DISCUSSION

Characterization of the Functionalization

The purpose of the functionalization is to confer a better flame retardancy action through the modification of halloysite using a phosphorous compound, the MPA. The functionalization is assumed to be performed on the inner surface of HNTs, since phosphonic compounds can only react with aluminol groups present at the inner surface [29]. Modified HNTs (Hal-P) were obtained after several steps of centrifugation and washing in order to eliminate all the MPA molecules that were not covalently attached to the aluminol groups of the HNTs. Figure 1 shows FTIR spectra of Hal, Hal-P and MPA. A weak band can be observed at 1,320 cm -1 for Hal-P (Fig. 1b). This additional band is attributed to the alkyl group of the MPA [33]. It is difficult to observe new bands, even in the transmission mode, because of the localization of the MPA at the inner surface. Indeed, the peak absorbance shifts toward lower values due to the more constricted observe the appearance of a new peak at 2.1 min corresponding to another sulfur species, which can be the result of a combination between the decomposition products of MPA and the sulfur fraction of raw halloysite during the pyrolysis step.

The XRD patterns of the samples (Fig. 3) confirm the presence of halloysite (basal reflection at 2ϑ = 12 ) in its tubular form (very intense reflection at about 20 ) with presence of some other mineral impurities like quartz and calcite [34]. As both XRD spectra of Hal and Hal-P matches, it is shown that MPA does not change the crystalline structure of the nanotubes, as the peaks remain unchanged even after functionalization. This proves that MPA is not intercalated but either encapsulated or grafted onto the lumen surface or grafted at the edge of the nanotubes [28,35].

TGA curves of the clay samples are displayed in Fig. 4. Experiments were conducted under air because halloysite modified with a phosphonic acid molecule has a tendency to react with nitrogen. As evidenced, both halloysites show a first step of thermal decomposition up to 230 C due to the release of the remaining physisorbed water (surface and interlayer) [START_REF] Belkassa | Colloids Surfaces A Physicochem[END_REF]. The main mass loss corresponding to the structural dehydroxylation of the hydroxyl groups of the surface of HNTs occurs between 400 C and 570 C [START_REF] Belkassa | Colloids Surfaces A Physicochem[END_REF]. Hal-P features a slightly more pronounced loss, which was attributed to the decomposition of the grafted organic part. This slight difference is due to the non-complete degradation of the MPA molecules that have lost only around 37% of their weight at 900 C, under air.

Hence, this additional mass loss for Hal-P compared with Hal does not allow calculating accurately a grafting rate of MPA.

Morphology

The dispersion of both raw and modified HNTs was first evaluated by TEM. The pictures (Fig. 5) reveal the individual dispersion of the two HNTs at the nanoscale in the PA11 matrix. Furthermore, SEM micrographs of the fractured surface of PA11/H and PA11/HP nanocomposites are presented in Fig. 6. The images confirm the homogeneous and uniform dispersion of both Hal and Hal-P in the PA11 matrix. However, some small bundles of HNTs are also visible but they are less than 15 μm in size. They correspond in fact to the micronic particles resulting from the comminution process of halloysite, which does not allow a complete release of nanotubes to be performed, especially at high filler content that is 20 wt%.

The morphological investigations through TEM and SEM indicate clearly that halloysite modification through MPA did not influence the state of dispersion of the clay in the matrix when incorporated at 20 wt%.

Thermal Stability of the Nanocomposites

The thermal and fire behavior of PA11 containing 20 wt% of Hal and Hal-P was compared with those containing 20 wt% of APP and a mixture between APP and Hal or Hal-P. The thermal stability of the different compositions was investigated mainly by TGA. TGA and differential thermogravimetric (DTG) curves obtained under nitrogen and air atmospheres are shown in Figs. 7 and8, respectively.

At first, in non-oxidant atmosphere a decrease in PA11 thermal stability in presence of APP is noticed. Thus, the temperature of the maximum rate of degradation, corresponding to the peak of the DTG curves, is reduced by 60 C compared with neat PA11, whereas halloysite improves thermal stability by 15 C. Intermediate results are observed when APP is partially substituted by HNTs but with a better effect in the presence of modified halloysite.

Under oxidative atmosphere, the same trends are observed as APP causes earlier degradation (-72 C) while halloysite enhances thermal stability of PA11 matrix. Nevertheless, the improvement of thermal stability is only significant in presence of modified halloysite (+14 C for the temperature of maximum degradation rate).

The early degradation of the systems containing APP (whatever the atmosphere) is due to release of water from APP and to the formation of phosphoric acid that causes PA11 hot hydrolysis and advanced thermal decomposition [37]. Conversely, it is suggested that the enhancement of the thermal stability in presence of halloysite is due to its high specific surface area, which restricts the motion of macromolecular species when the polymer decomposes. Moreover, its tubular structure with large lumen volume may entrap some degradation products, resulting in an effective delay in mass transfer of volatile decomposition products [19].

PCFC measurements under inert and oxidative atmospheres are shown in Fig. 9a andb, respectively.

Under anerobic atmosphere (nitrogen), a significant influence on degradation in presence of APP is observed, as the temperature of the pHRR decreases by 40 C in comparison to neat PA11. In addition, a higher value of pHRR is noticed (667 W/g for neat PA11 to 1060 W/g for PA/AP), whereas, incorporation of raw or As the pHRR of PA11/HP is almost the same whatever the atmosphere, it means that Hal-P may limit oxygen diffusion through the nanocomposite during thermal degradation. Moreover, a reduction in pHRR when 5 wt% of APP is substituted by Hal-P is also observed, in comparison with APP alone. Since it has been shown that PCFC test does not take into account physical effects [38,39], we can argue that the decrease in pHRR with incorporation of Hal-P can be also due to the substitution of a fraction of the organic polymer matrix by halloysite or catalytic effects able to modify the degradation pathway of the polymer [40].

Cone Calorimeter

Fire behavior of the different compositions was mainly studied by cone calorimeter and the heat release curves at 50 kW/m 2 heat flux are illustrated in Fig. 10. The cone calorimeter collected data are listed in Table 2.

From Fig. 10, it can be seen that most of the HRR curves are very sharp, indicating intense burning and strong heat release, except for PA11/HP, when the incorporation of 20 wt% of modified halloysite to the matrix leads to a flat HRR curve with a time to flame out much longer than that of neat PA11. This plateau of HRR, corresponding to a thick charring behavior [41], is ascribed to the formation of a strong protective char layer that lasts longer before breaking during the combustion. The presence of 20 wt% of APP enhances pHRR by 9% while all the other compositions lead to a decrease in pHRR in comparison to the neat PA11 with the better results for PA11/HP (-57%).

The cone calorimetric data (Table 2) confirm that the flammability of PA11/HP was decreased in comparison to the other compositions, as both the time of flameout (TOF) and the MAHRE of the formulation are all optimized compared to those of neat PA11. The addition of any flame retardant systems (APP, HNTs or both) decreases the TTI in Table 2 because the release of water and phosphoric acid (in the case of APP) involves hydrolysis of the PA11. The observation of a slightly lower char yield than the original mass of HNTs incorporated into the composites is anticipated, as HNTs are expected to lose their associated water, and undergo de-hydroxylation [42]. However, a higher amount of residue is found for PA11/HP in comparison to PA11/H, which can be ascribed to an incomplete pyrolysis due to char promotion through catalytic effects in presence of Hal-P (images of the residue are available in Supporting Information).

From the results, it can be considered that APP does not improve fire retardancy of PA11. This is probably due, in our case to the amount of APP that was not optimized to reach the best flame retardant effect for PA11. Moreover, the stability of the protective char layer formed by APP is limited because of both PA11 hydrolysis due to water release and the formation of phosphoric acid during APP degradation [11]. Indeed, it is known that APP acts in the condensed phase via a protective char during combustion [43]. In the case of Hal-P, a cohesive protective char layer is formed during the combustion, which does not break down and forms an insulating layer between the melted polymer and the flame zone. This acts to reduce the heat and mass transfer between the gaseous (flame zone) and condensed phase (melted polymer) [22], thereby slowing down the pyrolysis process and consequently lowering the heat release rate (pHRR).

The substitution of 5 wt% of APP by HNTs, raw or modified, results in an improvement of the fire reaction in comparison to neat PA11 and PA11/AP due to an additive effect of halloysite. It is particularly evident for PA11/AP/HP (containing 5 wt% of Hal-P) in comparison with PA11/AP/H and PA11/AP. Hence, a reinforcing effect of the charred structure promoted by APP owing to the presence of HNTs could be considered. Moreover, reactions between halloysite and APP degradation products to form aluminophosphates that will stabilize the char layer at high temperature can be assumed [24,44].

When analyzing the results it appears that the MPA functionalization exerts a critical influence on the fire behavior. This is consistent with the literature where it was found that phenyl phosphonic acid modified halloysite can disrupt both the pyrolysis and combustion process in PA6/halloysite nanocomposites [28].

Residue Analysis

Some analyses were undertaken on the cone calorimeter residues. First, elemental analysis was carried out to quantify both the amount of phosphorus present on Hal-P and remaining in the residue of the samples containing the modified halloysite. The results (Table 3) indicate that Hal-P contains 2.06 wt% of phosphorus, whereas the residue after cone calorimeter test of PA11/HP contains 1.86 wt%. This difference can be due to the transfer of some phosphorus content in the gaseous phase during the combustion, which can suppose an effect of Hal-P in the gaseous phase.

The residues for PA11/H and PA11/HP were also collected at the middle-time of the cone calorimeter test with degraded and non-degraded parts. The SEM micrographs of these samples noted PA11/H-middle and PA11/HP-middle, respectively are given in Fig. 11. For PA11/H-middle, a clearly straight limit between the burned material (at the top) and the unspoiled barrier. This strong barrier due to the formation of aluminum phosphate does not break during the combustion and prevents heat and mass transfer between gaseous and condensed phases.

These results suggest the full potential of halloysite after its functionalization to become an effective and environment-friendly fire retardant agent.

one is visible, meaning that the pyrolysis front advances straight. For the PA11/HP-middle sample, the microstructure is highly different as different zones can be noticed (at least three zones pointed by three arrows). We assume that the phosphorous compound grafted on Hal-P reacts with the PA11 beside, to form a more cohesive residue that impedes the pyrolysis to progress straight ahead. As seen by the microstructure of the upper burned part of the PA11/HP-middle sample, it is considered that phosphorus also reacts with aluminum leading to an aluminum phosphate (AlPO 3 ) [45].

CONCLUSIONS

Halloysite modification with methyl phosphonic acid was successfully performed and FTIR, TGA, and Py-GC/MS analyses evidenced that functionalization. The influence of the halloysite modification on thermal stability and fire behavior of PA11 nanocomposites was then investigated in comparison to those of composites filled with raw halloysite, APP and mixed compositions. Halloysite, especially the modified one, improves thermal stability of the PA11 in both inert and oxidative atmospheres while APP induces thermal degradation. Moreover, compared with APP that releases phosphoric acid at temperatures <300 C, the phosphonic acid grafted on the HNTs allows PA11 flammability to be improved by the formation of an insulating and cohesive char

FIG. 1 .

 1 FIG. 1. FTIR spectra of Hal, Hal-P and MPA (a). The region between 1,300 and 1,330 cm -1 (b).

FIG. 4 .

 4 FIG. 4. TGA curves of Hal and Hal-P under air.

FIG. 5 .

 5 FIG. 5. TEM pictures of PA11/H and PA11/HP.

FIG. 8 .

 8 FIG. 8. TGA (a) and DTG (b) curves of the different compositions obtained at 10 C/min under air. [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 9 .

 9 FIG. 9. PCFC curves under nitrogen (a) and air (b) of the different compositions. [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 1 .

 1 Code and composition of the samples

	Samples	PA11 (wt%)	APP (wt%)	Hal (wt%)	Hal-P (wt%)
	PA11	100	0	0	0
	PA11/AP	80	20	0	0
	PA11/H	80	0	20	0
	PA11/HP	80	0	0	20
	PA11/AP/H	80	15	5	0
	PA11/AP/HP	80	15	0	5

TABLE 2 .

 2 Cone calorimeter data at heat flux of 50 kW/m 2

		TTI (s)	TOF (s)	pHHR (kW/m 2 )	Time pHRR (s)	MAHRE (kW/m 2 )	THR (MJ/m 2 )	Residue (%)
	PA11	88 AE 2	440 AE 29	977 AE 30	190 AE 5	440 AE 10	152 AE 2	0
	PA11/AP	52 AE 3	543 AE 10	1067 AE 65	195 AE 5	452 AE 21	138 AE 2	15.9
	PA11/H	65 AE 7	543 AE 57	816 AE 90	255 AE 10	365 AE 33	139 AE 3	15.2
	PA11/HP	53 AE 5	680 AE 2	416 AE 3	255 AE 90	288 AE 5	142 AE 2	17.7
	PA11/AP/H	44 AE 12	609 AE 48	825 AE 23	130 AE 5	411 AE 21	135 AE 1	17.4
	PA11/AP/HP	48 AE 7	550 AE 21	726 AE 32	152 AE 8	404 AE 4	132 AE 2	14.8

TABLE 3 .

 3 wt% of P obtained by elemental analysis (ICP-AES)

	Samples	Results from ICP-AES (mg/kg)	wt% of P
	Hal-P	20587	2.06
	Residue of PA11/HP	18621	1.86

ACKNOWLEDGMENTS

The authors are grateful to EGIDE for its financial support through the Tassili program 13MDU891, to Jean-Jacques Flat from Arkema, and Soalka for supplying PA11 and raw Algerian halloysite, respectively. The authors are also thankful to Benjamin Gallard and Loic Dumazert of C2MA for their help during samples elaboration and characterization, respectively.