Mohamad-Ali Fawal

Thomas Jungas

Alice Davy
email: alice.davy@univ-tlse3.fr

Inhibition of DHFR targets the self-renewing potential of brain tumor initiating cells

Keywords:

Brain tumors are a heterogeneous group of benign and malignant tumors arising from the brain parenchyma and its surrounding structures, with in general a poor clinical outcome due to high recurrence. One of the underlying causes for this somber prognostic is the presence of brain tumor initiating cells (BTIC) endowed with self-renewal potential, multi-lineage differentiation and resistance to treatment. One promising therapeutic avenue for brain tumors is targeting BTIC self-renewal potential and forcing their differentiation. A compelling candidate is onecarbon metabolism shown to play a key role in maintaining stem cell self-renewal in several lineages. Here, we focus on dihydrofolate reductase (DHFR), a key enzyme in one-carbon metabolism, and demonstrate this enzyme's overexpression in several human brain tumors and its expression in human BTIC. We show that DHFR inhibition, either by Methotrexate (MTX) or EphB activation with synthetic ligands, reduces the tumorigenic potential of 4 human BTIC lines, by reducing their self-renewal capacities both in vitro and in a cerebral organoid glioma (GLICO) model. Our data indicate that driving BTIC differentiation by inhibiting DHFR may provide a new therapeutic approach to treating highly refractory aggressive tumors.

Introduction

Brain tumors (BTs) are often deadly, and when not, impact dramatically on the patients' quality of life. While there have been significant advances in surgical and imaging techniques, BTs treatments are still inefficient, most likely due to extensive infiltration of tumor cells into normal brain parenchyma, their cellular heterogeneity and relapse [START_REF] Lapointe | Primary brain tumours in adults[END_REF]. The complexity of malignant gliomas has led to many interpretations, one of which is the brain tumor initiating cells (BTIC) hypothesis. The central idea of this hypothesis is that very few renewable and multipotent BTIC propagate and sustain the full composition of tumor mass cell types. Such cells have been identified and isolated from different grades (low and high) and types of brain cancer, including gliomas and medulloblastomas [START_REF] Vora | Culture and isolation of brain tumor initiating cells[END_REF]. BITC exhibit self-renewal, high proliferative capacity and multi-lineage differentiation potential in vitro. They also initiate tumors that phenocopy the parent tumor in immunocompromised mice [START_REF] Laks | Neurosphere formation is an independent predictor of clinical outcome in malignant glioma[END_REF]. It is, therefore, essential to develop new therapies able to target this subpopulation and to improve the prognosis of glioma patients.

One-carbon metabolism is a metabolic pathway that plays an essential role in nucleic acid synthesis [START_REF] Ducker | One-carbon metabolism in health and disease[END_REF]. Because inhibition of this pathway blocks cell proliferation, pharmacological inhibitors, such as antifolate compounds (e.g. methotrexate (MTX)), are widely used for chemotherapy in a variety of cancers including brain and spinal cord tumors [START_REF] Newman | One-carbon metabolism in cancer[END_REF][START_REF] Vezmar | Biochemical and clinical aspects of methotrexate neurotoxicity[END_REF]. One major drawback of these treatments, nevertheless, is the high toxicity associated with inhibition of one-carbon metabolism in healthy tissues. In addition, cancer cells acquire resistance to antifolate agents by over expressing dihydrofolate reductase (DHFR) [START_REF] Bertino | Resistance mechanisms to methotrexate in tumors[END_REF] as well as other key enzymes of the pathway [START_REF] Ducker | One-carbon metabolism in health and disease[END_REF]. Besides its well-known role in cell proliferation, more recent studies have uncovered a role for one-carbon metabolism in maintaining stem cell pluripotency, a function linked to methylation reactions via the methionine cycle [START_REF] Shiraki | Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells[END_REF][START_REF] Shyh-Chang | Influence of threonine metabolism on Sadenosylmethionine and histone methylation[END_REF]. We have shown recently that activation of EphB receptors with synthetic ephrinB1 ligands decreases DHFR expression as well as other one-carbon metabolism enzymes, although to a lesser extent. This inhibition of one-carbon metabolism decreases the self-renewal of embryonic neural stem cells (NSC) and locks them into a differentiation-ready state through epigenetic remodeling of H3K4me3 on key progenitor genes [START_REF] Fawal | Cross talk between one-carbon metabolism, Eph signaling, and histone methylation promotes neural stem cell differentiation[END_REF]. Taken together, these studies indicate that modulation of one-carbon metabolism could be a valuable approach to targeting BTIC self-renewal and forcing their differentiation.

Here we present evidence that DHFR inhibition, either by MTX treatment or EphB receptor activation, leads to reduction of BTIC selfrenewal potential by forcing their differentiation. Furthermore, EphB receptor activation is sufficient to prevent MTX-induced increase in DHFR protein level. Finally, we use iPSC-derived human cerebral organoids as 3D grafting platforms to test the invasiveness potential of BTIC. We show that MTX treatment prior to co-culture has long term consequences on the capacity of BTIC to invade and proliferate within human tissues. Our findings indicate that BTIC ability to generate tumor mass (proliferative potential) and daughter BTIC (self-renewal potential) is uncoupled and this offers new strategies to target the latter in glioma.

Materials and methods

Culture and maintenance of glioma cell lines

Glioma cell lines "BTIC" were a gift from Samuel Weiss' lab. They were derived from tumor samples obtained from the Tumor Tissue Bank at Foothills Hospital, Calgary, Alberta, Canada and cultured under neural stem cell promoting conditions as described previously [START_REF] Kelly | Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens[END_REF]. All BTIC lines are wild type for IDH [START_REF] Cusulin | Precursor states of brain tumor initiating cell lines are predictive of survival in xenografts and associated with glioblastoma subtypes[END_REF]. BTIC were cultured with growing medium [DMEM/F12 medium containing 0.6% glucose (Cat#UG3050, Euromedex), 5 mM HEPES (Cat#H3375, Sigma), 1 mM putrescine (Cat#P5780, Sigma), 5 ng/ml basic fibroblast growth factor 2 (FGF-2) (Cat#F029, Sigma), 20 ng/ml epidermal growth factor (EGF) (Cat#E9644, Sigma), 10 ng/ml insulin-transferrin-sodium selenite supplement (Cat#I1844-1VL, Sigma) and 2% B27 supplement (Cat#17504-044,Invitrogen)] in a 5% CO 2 incubator at 37 • C. Fresh medium was added every 2-3 days to the culture and passage was realized once a week using Accutase® (Cat#A6964, Sigma).

hiPSC culture and cerebral organoid generation

Human iPSC cell line (LUMC0004iCTRL10) was provided by the LUMC hiPSC core facility and grown on Matrigel (Corning #354277) with mTeSR-1(StemCell™ Technologies).

The generation of hiPSC derived cerebral organoids was performed according to the unguided protocol described previously (Lancaster, Nature 2013) using the commercially available reagents from Stem-Cell™ Technologies (#08570 and #08571).

Briefly, confluent hiPSCs were detached using GCDR (#07174, StemCell™ Technologies) and plated in Ultra low-attachment 96-well plates at 9000 cells per well with Embryoid Body Formation Medium supplemented with 10 μM ROCKi (Y27642, StemCell™ Technologies). This step corresponds to 0 day in vitro (DIV). Embryoid bodies were fed on day 2 and 4 with fresh medium without ROCKi, before being transferred on day 5 to a 48-wells ultra-low attachment plate with Induction Medium. At 7 DIV each embryoid body was embedded in 15 μl Matrigel droplets and grown for 3 days in Expansion Medium. In vitro Organoids were transferred to 6-well plates on day 10 and placed under orbital agitation and in Organoid Maturation Medium until use for co-culture with BTIC. Medium was refreshed every 3-4 days.

Co-culture of BTIC and cerebral organoids

To generate BTIC that stably express GFP, BTIC were transfected with pCDNA-GFP plasmid and grown under puromycine selection in growing medium. For co-culture experiments, 30 DIV cerebral organoids were transferred to a 24-well plate (5 organoids per well). Excess medium was removed, and 10,000 stable GFP-expressing BTIC were plated in each organoid-containing well (10,000 BTIC/2 mL of growing media per well). Plates were incubated at 37 • C and tumor-bearing organoids were maintained on an orbital shaker for up to 14 days at 37 • C.

Stimulation of Eph forward signaling

Prior to stimulation, BTIC spheres were dissociated with Accutase® (Cat#A6964, Sigma), and aliquots of 10 5 cells were placed in growing medium and then stimulated with 1 μg/ml eB1-Fc (Cat#473-EB, R&S Systems) pre-clustered with 0.1 μg/ml anti-human IgG (Cat#G-101-C-ABS, R&D Systems). Cells were stimulated for 30min and 72 h and lysed in protein lysis buffer for Western blot analysis.

Cell proliferation assay

Spheres were dissociated with Accutase® (Cat#A6964, Sigma), and cells were seeded in triplicate in a 6-well plate at 10 4 cell/ml density. Cell number was determined using Cellometer™ (Cat#1001201, Nexcelom) according to the manufacturer's instructions.

Sphere forming assay

Spheres were dissociated with Accutase® (Cat#A6964, Sigma), and about one hundred cells were seeded in a 96-well plate, and the total number of spheres was counted 2 weeks after incubation. eB1-Fc (Cat#473-EB, R&S Systems) and Methotrexate (Cat#M8407, Sigma) were added to the assay culture medium upon seeding to a final concentration of 1 μg/ml and 2 μM, respectively. Sphere-Forming Unit (SFU) was calculated: SFU = (number of spheres counted/number of input cells) * 100.

Quantitative PCR

Total RNA was extracted from cell pellets 72 h post-culture using TRIreagent (Cat#TR118, MRC) according to the manufacturer's instructions. 1 μg RNA was used for reverse transcription. Genomic DNA was degraded with 1 μl DNase (Cat#M6101, Promega) for 20 min at 37 • C in 20 μl RNase/DNase-free water (Cat#W4502, Bio-RAD), and the reaction was stopped by adding 1 μl stop solution under heat inactivation at 65 • C for 10 min. 2 μl dNTPs (10 mM; Promega) and 2 μl oligodTs (100 mM, idtDNA) were added for 5 min at 65 • C, then 8 μl of 5 × buffer, 2 μl RNasin (Cat#N2511, Roche), and 4 μl of 100 mM DTT (Cat#P1171, Promega) were added for 2 min at 42 • C. The mix was divided into equal volumes in a reverse-transcriptase-negative control tube with addition of 1 μl water, and in a reverse-transcriptase-positive tube with 1 μl superscript enzyme and placed at 42 • C for 1 h. The reaction was stopped at 70 • C for 15 min, and cDNAs were diluted (10-, 100-, and 1000-fold) and processed for quantitative PCR in triplicate for each dilution. 10 μl diluted cDNA was mixed with 10 μl premix Evagreen (Cat# 1725204, Bio-RAD) containing 1 μM of each primer, and the PCR program was run for 40 cycles on a CFX96 BioRad Realtime system (Cat#185-5096, Bio-RAD). mRNA expression level relative to control genes (S16, GAPDH and TBP) were calculated using the 2-ΔΔCts method. Primers are listed in Table 1.

Immunofluorescence and flow cytometry

For immunofluorescence, cells were grown and treated as described earlier [START_REF] Fawal | Cross talk between one-carbon metabolism, Eph signaling, and histone methylation promotes neural stem cell differentiation[END_REF]. Floating spheres were collected and fixed at room temperature with 4% paraformaldehyde (Cat#15710, Fisher Scientific) for 30 min, then permeabilized and blocked with PBTA solution (0.5% BSA (Cat#04100811C, Euromedex), 0.5% FBS (Cat#10500064, Thermo-Fisher), and 0.1% Triton X-100 (Cat#T9284, Sigma) in PBS (Cat#D1408, Sigma) for 30 min. Primary and secondary antibodies diluted in PBTA were incubated overnight at 4 • C and 2 h at room temperature, respectively. Spheres were collected with centrifugation at 1200 rpm for 10 min at each step. Spheres were finally mounted on glass slides (Superfrost) (Cat#LR90SF03, ThermoFisher) with coverslips, using mounting medium [4.8% wt/vol Mowiol (Cat#81381, Sigma) and 12% wt/vol glycerol (Cat#G9012, Sigma) in 50 mM Tris pH 8.5

(Cat#26-128-3094-B, Euromedex)].
Microscope acquisitions were performed on an SP5 DM600B confocal microscope (40 × /1.3 APO; 20 × /0.7 APO, PMT; Leica Biosystems). Observation was performed using Type-F mineral oil (1153859; Leica Biosystems).

Protein extraction and western blot

For protein extraction, spheres were pelleted by centrifugation at 1500 rpm for 10 min and washed twice with ice-cold PBS (Cat#D1408, Sigma) solution. Whole cell extracts were obtained by re-suspending cell pellets in the following ice-cold lysis buffer (150 mM NaCl (Cat#1112-A, Euromedex), 50 mM Tris-HCl pH 7.4 (Cat#EU0011, Euromedex), 0.5 mM EDTA (Cat#EU0007, Euromedex), 2 mM Na3VO4 (Cat#S6508, Sigma), 1% Nonidet P-40 (NP-40) (Cat#N6507, Sigma), 0.5 mM EGTA (Cat#E4378, Sigma) and 0.1 mM PMSF (Cat#78830, Sigma)) supplemented by protease inhibitors (Cat#11836170001, Roche) for 1 h on ice. Protein lysates were then vortexed, sonicated with Biorupter® Sonicator for 10 cycles (Cat#B01020001, Diagenode), and centrifuged at 13,000 rpm for 10 min. Cleared lysates were used for Western blot analyses. Samples were denatured by boiling in loading buffer (4 × 100 mM Tris-HCL, pH 6.8, 8% SDS (Cat#EU0660, Euromedex), 40% glycerol (Cat#G9012, Sigma), 4% β-mercaptoethanol (Cat#63689, Sigma), and bromophenol blue (Cat#B0126, Sigma)) before loading and electrophoresis on an 8 or 16% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membrane (Cat#GE10600002, GE Healthcare), blocked for 30 min and incubated with primary antibody in 5% nonfat dry milk in TBS-T [20 mM Tris base (Cat#26-128-3094-B, Euromedex), 150 mM NaCl (Cat#S9888, Sigma), and 0.05% Tween 20 (Cat#2001-A, Euromedex) adjusted to pH 7.6 with 1 M HCl (Cat#30721, Sigma)] overnight at 4 • C. Milk was replaced with 5% BSA (Cat#04100811C, Euromedex) for detection of phosphorylated epitopes. Western blots are representative of at least three independent experiments.

DHFR catalytic assay

DHFR activity, measured as folate reductase, was determined by the dihydrofolate reductase assay kit according to the manufacturer's instructions. Briefly, cells were pelleted by centrifugation at 1500 rpm for 10 min and washed twice with ice-cold PBS solution (Cat#D1408, Sigma). Cell pellets were re-suspended in 300 μl assay buffer supplemented with protease inhibitors (Cat#11836170001, Roche) and lysed mechanically using glass beads (Cat#G8772, Sigma). Lysates were precleared by centrifugation at 13,000 rpm for 10 min. The supernatant was used immediately for enzyme assay after determining protein concentration.

Statistical analyses

For experiments involving a single pair of conditions, statistical significance between sets of data containing more than two samples, one-way or two-way analysis of variance with either Bonferroni, or Fisher's LSD multiple comparison post-test was used, as indicated, to determine adjusted p-values with Prism5 (GraphPad software). Sample sizes of sufficient power were chosen on the basis of similar published research and were confirmed statistically by appropriate tests. Statistically significant differences are reported at: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Results

Eph:ephrin and DHFR expression in human brain tumors

In recent years, high throughput whole genome analyses of large sets of human gliomas have generated massive quantities of data that can be used to assess gene expression and correlation between genes.

Taking advantage of these expression datasets, through GlioVis portal [START_REF] Bowman | GlioVis data portal for visualization and analysis of brain tumor expression datasets[END_REF], we explored DHFR as well as Eph:ephrin B expression in human BTs. In the Rembrandt and Gravendeel datasets, as expected, DHFR expression was consistently upregulated in all BTs analyzed, regardless of histology (Fig. 1A and B and Fig. S1A), and was associated with poorer prognosis (Fig. 1C). Since our previous findings in embryonic NSC established a link between DHFR and Eph:ephrinB [START_REF] Fawal | Cross talk between one-carbon metabolism, Eph signaling, and histone methylation promotes neural stem cell differentiation[END_REF], we surveyed all class B ephrins and their cognate Eph receptors. Amongst all genes analyzed, in the Rembrandt dataset, EFNB1 and EphB2 exhibited an increased expression in glioblastoma (GBM) samples, while EFNB3, EphB6 and EphA4 exhibited a decreased expression in these tumors (Fig. 1D, G and Fig. S1B). To validate these differential expression patterns and test whether they correlate with tumor grade, we analyzed both Gravendeel and Rembrandt datasets. This analysis showed that EFNB1 expression was upregulated in high grade glioblastomas in the Rembrandt dataset (Figs. 1E andS1C), however, no significant changes were observed in the Gravendeel dataset (Fig. S1D). Expression of EphB2 was upregulated in high grade glioblastomas (GBM) in both datasets (Fig. 1H andS1C, D). EFNB2 expression was increased in high grade GBM, while expression of EphB1 was decreased in high grade compared to low grade GBM (Figs. S1C andD). Expression of EphA4 was not significantly changed in high grade GBM in the Rembrandt dataset but was increased in the Gravendeel dataset (Figs. S1C andD). Changes in the expression of EFNB3, EphB3 and EphB6 were not statistically significant in either dataset (Figs. S1B,C,D). Lastly, high expression of both EFNB1 and EphB2 correlated with poorer prognosis (Fig. 1F,I). These data highlight the correlation between high DHFR levels and BTs and reveal the complexity of Eph:ephrin B expression in these tumors. Furthermore, these data identify DHFR and Eph:ephrin B as a potential targets for intervention in GBM.

Characterization of Eph:ephrin/DHFR axis in BTIC lines

To explore the putative role of DHFR in GBM BTIC self-renewal, we used 4 different human-derived glioma cell lines. The 4 glioma-derived BTIC lines established from different patients exhibited distinct morphological features when grown in culture: 12 M formed large spheres with rough edges, 25 M formed smooth large spheres and both 50 EF and 53 M formed small smooth spheres (Fig. 2A). To further characterize these BTIC lines, we analyzed cell composition of these spheres at different passages by fluorescence activated cell sorting (FACS) using progenitor (Nestin) and differentiated cell markers (TUJ1: neurons, O4: oligodendrocytes, GFAP: astrocytes). While the proportion of progenitor-like cells in these BTIC lines ranged between 50% and 80%, the proportion of the differentiated cell types varied between these different cell lines (Fig. 2B). When we used the sphere formation assay as an indicator of these different lines self-renewal capacities, we observed no correlation with the progenitor-like cell numbers observed above (Fig. 2C). This indicates that not all progenitor-like cells identified with the Nestin marker are able to generate tumor-like spheres. Thus, we chose the sphere forming assay approach to assess self-renewal potential of BTIC lines in this study. We then characterized the proliferation potential of the 4 different lines (Fig. 2D). Unexpectedly, the highly proliferative cell lines (12 M, 25 M) had similar self-renewal potential compared to the less proliferating cell line 53 M, indicating that proliferation rate does not directly correlate with the self-renewing capacities in these cell lines. Then, we profiled DHFR expression and activity levels. While 50 EF had slightly lower DHFR protein levels (Fig. 2F), there was no significant difference in DHFR activity among these different cell lines (Fig. 2G). Altogether these results indicate that DHFR is expressed at varying levels in BTIC, and that changes in its expression or activity do not appear to correlate with their proliferative or selfrenewing potential.

We have previously shown that inhibition of DHFR, either pharmacologically (MTX) or transcriptionally by activation of EphB pathway with ephrinB1 recombinant protein (eB1-Fc), led to NSC depletion [START_REF] Fawal | Cross talk between one-carbon metabolism, Eph signaling, and histone methylation promotes neural stem cell differentiation[END_REF]. Given the similarities between NSC and BTIC, we wanted to explore the effects of DHFR inhibition on BTIC self-renewal. We first profiled Eph and ephrinB expression in these glioma cell lines. By qRT-PCR, we quantified mRNA levels of EFNB1, EFNB2, EphB1, EphB2 and EphA4. Amongst the 4 cell lines, 25 M had the lowest Eph:ephrin expression, while 50 EF had the highest. The lines 12 M and 53 M had average Eph:ephrin expression levels (Fig. 3A). Taken together these data show that Eph:ephrin expression is detected at varying levels in all 4 BTIC lines, with no obvious correlation between their proliferation and self-renewing potential. In all BTIC lines, eB1-Fc treatment was successful in activating the EphB pathway, as evidenced by the increased EphB1/2 phosphorylation 30 min post-treatment (Fig. 3B). In addition, similarly to what we previously observed in NSC, activation of EphB receptors led to decreased DHFR protein levels 72 h post-treatment in all BTIC lines (Fig. 3C). However, MTX treatment led to increased levels of DHFR protein which is consistent with one of the mechanisms of MTX resistance in cancer cells [START_REF] Bertino | Resistance mechanisms to methotrexate in tumors[END_REF]. Since Eph:ephrin signaling inhibits DHFR at the transcriptional level [START_REF] Fawal | Cross talk between one-carbon metabolism, Eph signaling, and histone methylation promotes neural stem cell differentiation[END_REF], we tested whether activation of Eph signaling could prevent MTX-induced DHFR overexpression. Following MTX treatment, all cell lines responded with increased DHFR expression 72 h post-treatment (Fig. 3C). However, though cells treated with both eB1-Fc and MTX showed increased levels of DHFR, it was reduced compared to cells treated with MTX alone (Fig. 3C).

Altogether, these in vitro data reveal that the molecular interaction between Eph signaling and DHFR that we previously described in mouse NSC, is conserved in human BTIC lines. Furthermore, the pharmacologically-induced increase of DHFR levels that is at the base of MTX resistance can be inhibited at the transcriptional level by activating EphB signaling.

Inhibition of DHFR reduces the self-renewing capacities of BTIC

Since we demonstrated that the molecular pathway linking Eph: ephrin to DHFR is conserved in GBM-derived cell lines, we tested whether, as in NSC, inhibition of DHFR activity in these cells can also drive their differentiation. First, we compared the consequence of MTX and eB1-Fc treatment on DHFR activity. In all BTIC lines, both treatments led to a reduction in DHFR activity after 72 h compared to untreated cells (Fig. 4A). Using the sphere forming assay, we addressed whether MTX and eB1-Fc treatments could reduce the self-renewing potential of BTIC. Compared to untreated cells, all glioma cell lines showed reduced self-renewal following both treatments (Fig. 4B), yet MTX was more efficient than eB1-Fc in 2 BTIC lines (25 M and 53 M). Furthermore, both eB1-Fc and MTX treatment decreased the progenitor Fig. 2. DHFR is expressed and active in BTIC and activity does not correlate with proliferative potential (A) Representative light microscopy images of glioma cell lines spheres. Each insert shows an enlargement of selected area. (B) Quantification by FACS analysis of BTIC lines after 7d culture under normal culture conditions and stained with Nestin, TUJ1, O4 and GFAP antibodies. (C) Spheres were cultured for 7 days under stem culture conditions. Spheres were then dissociated, seeded into different wells (10 2 cells/ml) and cultured under stem culture conditions for 1 week, and the number of spheres were counted. Sphere-Forming Unit (SFU) is calculated according to the following formula: SFU = (number of spheres counted/number of input cells) * 100. (G) DHFR enzymatic activity was measured in cell lysates of different BTIC lines. Statistical analysis was performed using one-way ANOVA test followed by the Bonferroni method (C, D, E and G). fraction, as evidenced by the SOX2 staining (Fig. 4C) and both treatments increased the differentiated cell population (Fig. S2A-C) in all BTIC lines. However, while MTX treatment inhibited the increase in cell numbers in all BTIC lines, eB1-Fc treatment had no effect on cellular proliferation (Fig. S3A-D). Lastly, we tested for a potential synergistic effect between MTX and eB1-Fc treatments. Simultaneous treatment with both MTX and eB1-Fc did not further decrease DHFR activity compared to MTX treatment alone in any of the glioma cell lines (Fig. S4A), and no significant difference was observed in the self-renewal potential when compared to MTX treatment alone, with the exception of 25 M (Fig. S4B). Since this decrease in self-renewal potential was not correlated with a further decrease in DHFR activity in 25 M, we hypothesize that it is due to "non-DHFR" ephrin signaling downstream targets. Taken together, these data indicate that the decrease in progenitor pool following DHFR inhibition is not coupled to a decrease in cell proliferation. In addition, no synergistic or additive effect of both treatments was observed on DHFR activity or BTIC self-renewal in tested conditions.

Co-culture with human cerebral organoids reveals long term consequences of DHFR inhibition

BTIC properties are highly influenced by the tumor microenvironment, the tumor host-cell interactions and the ability to grow and invade the complex 3D extracellular matrix. Recently, iPSC derived human brain organoids have been shown to be a powerful tool for recreating the putative initiating events and observing a process normally visible in human patients [START_REF] Ogawa | Glioblastoma model using human cerebral organoids[END_REF][START_REF] Linkous | Modeling patient-derived glioblastoma with cerebral organoids[END_REF]. To test the long term consequences of DHFR inhibition on BTIC properties, we adapted an established protocol to generate human iPSC-derived cerebral organoids. We generated stably GFP-expressing BTIC lines that were treated for 7 days with MTX prior to co-culture with human cerebral organoids. These cell lines were then co-cultured with 30 day-old in vitro human cerebral organoids for an additional 14 days, in the absence of drug (Fig. 5A). Co-cultured non-treated BTIC lines were able to graft onto human organoids and form macroscopically visible GFP tumor masses (Fig. S4A). MTX pre-treatment reduced the capacity of these cell lines to form these macroscopically visible GFP tumor masses (Fig. S5A). Furthermore, sectioning of organoids showed that while non-treated BTIC were able to form tumor masses and invade the surrounding tissues, no such behavior was observed in MTX treated cells (Fig. 5B). These data indicate that MTX pre-treatment has long term consequences on BTIC, efficiently decreasing their tumorigenic and invasion potential.

Discussion

Two of the major clinical challenges facing contemporary cancer therapies are resistance and cancer relapse, which is attributed to a small population of tumor cells with high self-renewal potential, called BITC. Targeting the self-renewing capacity of BTIC has been at the center of current day therapies [START_REF] Annett | Targeting cancer stem cells in the clinic: current status and perspectives[END_REF]. Among the several approaches that target these tumor initiating cells, inducing their differentiation seems to be a very promising strategy [START_REF] Cruz | Solid tumor differentiation therapy -is it possible?[END_REF]. Our analysis of glioma databases revealed that DHFR is highly expressed in gliomas and we show that it is also expressed in BTIC lines. In our study, DHFR expression was not measured separately in BTIC and differentiated cells, yet, recent findings in the developing neocortex have revealed that DHFR expression is elevated in NSC and strongly reduced in differentiated neurons [START_REF] Telley | Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex[END_REF]. Inhibition of DHFR activity, either transcriptionally (e.g. EphB activation) or pharmacologically (e.g. MTX treatment), decreases the self-renewal capacities of both NSC and BTIC and promotes their differentiation. Unexpectedly, while MTX treatment greatly reduces BTIC cell numbers in culture, eB1-Fc treatment has no significant effect. These data point out that cell proliferation and self-renewing potential are uncoupled in glioma-derived BTIC. The reduced cell number following MTX treatment could be due to non-DHFR mediated effects. Indeed, it has been recently shown that MTX shares structural similarities with known histone deacetylase inhibitors (e.g. butyrate). Other than its effect on histone methylation through inhibition of the 1C folate pathway, MTX could also increase histones acetylation in patient-derived osteosarcoma cell lines [START_REF] Sramek | Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation[END_REF][START_REF] Yang | Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate[END_REF]. Hence, MTX treatment could have a broader role than eB1-Fc treatment as an epigenetic modifier. The role of epigenetic changes in carcinogenesis has gained a great deal of interest in the last few years, and multiple studies have addressed the epigenetic changes in glioblastoma [START_REF] Lin | Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene[END_REF][START_REF] Romani | Epigenetic targeting of glioblastoma[END_REF]. It would, therefore, be of great interest to identify the epigenetic changes induced following both MTX and eB1-Fc treatments and to cross check them with those identified in glioma, in order to improve personalized therapies. Whether the long term effect of MTX treatment that we report here is due to epigenetic changes will have to be studied further. EphB activation yielded results similar to MTX treatment in reducing the self-renewal potential of BTIC and in promoting cell differentiation, without the cytotoxic effects associated with the latter. Since MTX has been associated with neurotoxicity and long term neurocognitive impairment [START_REF] Vezmar | Biochemical and clinical aspects of methotrexate neurotoxicity[END_REF][START_REF] Sandberg | Methotrexate administration directly into the fourth ventricle in children with malignant fourth ventricular brain tumors: a pilot clinical trial[END_REF][START_REF] Krull | Chemotherapy pharmacodynamics and neuroimaging and neurocognitive outcomes in long-term survivors of childhood acute lymphoblastic leukemia[END_REF], our data indicate that EphB activation could serve as a potential alternative to MTX treatment, specifically during pregnancy. Accumulating evidence supports the role of tyrosine kinases in glioma proliferation and invasion [START_REF] Pearson | Targeting cellular pathways in glioblastoma multiforme[END_REF][START_REF] Carrasco-García | Role of receptor tyrosine kinases and their ligands in glioblastoma[END_REF]. Numerous studies have analyzed the Eph family of tyrosine kinases and their ligands, the ephrins, in glioma and have associated their altered expression with glioma histological grade, their growth, angiogenesis, invasion and adverse outcome [START_REF] Day | Eph receptors as therapeutic targets in glioblastoma[END_REF][START_REF] Ferluga | Ephs and Ephrins in malignant gliomas[END_REF][START_REF] Nakada | Role of Eph/ephrin tyrosine kinase in malignant glioma[END_REF]. While the role of Eph:ephrin signaling in glioma invasion is well characterized, its role in the initiation and progression of gliomas remains overlooked. This is partly due to the complexity of Eph: ephrins interactions. For instance, Eph:ephrin A family members have been shown to have somewhat perplexing dual roles, with both tumor suppressor and tumor promoting functions being described [START_REF] Nakada | Role of Eph/ephrin tyrosine kinase in malignant glioma[END_REF][START_REF] Ferluga | Simultaneous targeting of Eph receptors in glioblastoma[END_REF]. Recent findings have pointed out the importance of EphA2 and EphA3 in glioma tumor initiating cells in the context of recurrent glioblastoma [START_REF] Qazi | Cotargeting ephrin receptor tyrosine kinases A2 and A3 in cancer stem cells reduces growth of recurrent glioblastoma[END_REF]. It would therefore be interesting to test for a potential link with DHFR. Although it is clear that Eph:ephrin B signaling has functional roles in gliomas [START_REF] Nakada | Role of Eph/ephrin tyrosine kinase in malignant glioma[END_REF][START_REF] Krusche | EphrinB2 drives perivascular invasion and proliferation of glioblastoma stem-like cells[END_REF], these members are less characterized. It is Fig. 4. Inhibition of DHFR activity reduces BTIC self-renewal and the number of Sox2+ cells (A) BTIC cells treated with either with eB1-Fc or MTX for 7 days were lysed and DHFR activity was measured. (B) Spheres were dissociated and incubated for 7 days with either MTX or eB1-Fc. Spheres were then dissociated, seeded into different wells (10 2 cells/ ml), cultured under stem culture conditions for 1 week and the number of spheres were counted. Sphere-Forming Unit (SFU) is calculated according to the following formula: SFU = (number of spheres counted/number of input cells) * 100. (C) Representative confocal immunofluorescence images of spheres treated with either MTX, eB1-Fc for 7d and stained with SOX2 (red) and DraQ5 (DNA stain, blue).

Scale bars represent 100 μm. Statistical analysis was performed using one-way ANOVA test followed by the Bonferroni method (A and B). believed that Eph:ephrin B's activity might limit the proliferative potential of neural stem cells during brain tumor development. For instance, ephrinB1 is essential for the self-renewal of NSC and for maintaining them within the stem cell niche [START_REF] Arvanitis | Ephrin B1 maintains apical adhesion of neural progenitors[END_REF][START_REF] Arvanitis | Ephrin-B1 reverse signaling controls a posttranscriptional feedback mechanism via miR-124[END_REF] and ephrinB2 expression in blood vessels induces NSC quiescence [START_REF] Ottone | Direct cell-cell contact with the vascular niche maintains quiescent neural stem cells[END_REF]. Besides influencing neural stem cells in the adult brain, EphB2 signaling also regulates niche cell plasticity and plays a critical role in stem cell niche maintenance and self-renewal [START_REF] Nomura | EphB signaling controls lineage plasticity of adult neural stem cell niche cells[END_REF].

In conclusion, we have shown that EphB activation reduces the level of DHFR and that it targets specifically the self-renewing potential of BTIC without affecting their proliferation potential. Future studies will determine the benefit and the long term effects of combining EphB activation with treatments targeting different pathways such as cellular proliferation on glioma cells, and will open new perspectives in administrating these drugs in clinical practice.

Fig. 1 .

 1 Fig. 1. Eph:ephrin and DHFR expression in human glioblastomas (A, B) DHFR log2 mRNA expression as a function of brain tumor type (A), and GBM histological grade (B) (Rembrandt database). (C) Kaplan Meier survival analysis in human glioblastoma samples expressing high or low levels of DHFR (Rembrandt database). (D, E) EFNB1 log2 mRNA expression as a function of brain tumor type (D), and GBM histological grade (E) (Rembrandt database). (F) Kaplan Meier survival analysis in human glioblastoma samples expressing high or low levels of EFNB1 (Rembrandt database). (G, H) EphB2 log2 mRNA expression as a function of brain tumor type (G), and GBM histological grade (H) (Rembrandt database). (I) Kaplan Meier survival analysis in human glioblastoma samples expressing high or low levels of EphB2 (Rembrandt database). Statistical analysis was performed using Tukey's Honest significant difference (A, B, D, E, G and H) and Log-rank/Wilcoxon test (C, F and I).

  Fig.2. DHFR is expressed and active in BTIC and activity does not correlate with proliferative potential (A) Representative light microscopy images of glioma cell lines spheres. Each insert shows an enlargement of selected area. (B) Quantification by FACS analysis of BTIC lines after 7d culture under normal culture conditions and stained with Nestin, TUJ1, O4 and GFAP antibodies. (C) Spheres were cultured for 7 days under stem culture conditions. Spheres were then dissociated, seeded into different wells (10 2 cells/ml) and cultured under stem culture conditions for 1 week, and the number of spheres were counted. Sphere-Forming Unit (SFU) is calculated according to the following formula: SFU = (number of spheres counted/number of input cells) * 100. (D) Cell numbers quantification after 4, 8, 12 and 16 days of 4 different glioma cell lines: 12 M, 25 M, 50 EF and 53 M. (E) qRT-PCR analysis of BTIC lines. DHFR mRNA expression relative to control S16 mRNA is shown. (F) BTIC cell lysates were collected and analyzed by Western blot with the indicated antibodies. (G) DHFR enzymatic activity was measured in cell lysates of different BTIC lines. Statistical analysis was performed using one-way ANOVA test followed by the Bonferroni method (C, D, E and G).

Fig. 3 .

 3 Fig. 3. BITCs express EphR whose activation leads to inhibition of DHFR (A) qRT-PCR analysis of cultured BTIC lines. Relative mRNA expression relative to control S16 mRNA is shown for the indicated genes. (B) BTIC cell pellets were collected 30min postdissociation and incubation with IgG (control) or eB1-Fc, lysed and analyzed by Western blot with the indicated antibodies. (C) BTIC cell pellets were collected 72 h postdissociation and incubation with either IgG (control), eB1-Fc, MTX or both eB1-FC/MTX, lysed and analyzed by Western blot with the indicated antibodies. Two different exposures of the DHFR signal are shown (++ indicates higher exposure time).

Table 1

 1 Primer sequence used for quantitative RT-PCR analysis.

	mRNA Transcript	Forward primer (5 ′ -3 ′ )	Reverse primer (5 ′ -3 ′ )
	DHFR	CATGGTTGGTTCGCTAAACTG	GAGGTTGTGGTCATTCTCTGGAAATA
	S16	AGGAGCGATTTGCTGGTGTGG	GCTACCAGGGCCTTTGAGATGG
	GAPDH	TGGATGCAGGGATGATGTTC	ATTCAACGGCACAGTCAAGG
	TBP	GAGCTGTGATGTGAAGTTTCT	TCTGGGTTTGATCATTCTGTAG
	Ephrin-B1	CGTGTTGGTCACCTGCAATAG	CAGGCTTCCATTGGATGTTGA
	Ephrin-B2	TGTCTGCTTGGTCTTTATCAACC	AAGGACTGGTACTATACCCACAG
	Eph-B1	GGTTTCCCACGGCATCTC	AGAGGAGGGAAAAGGACCAGG
	Eph-B2	GTCCATCTGTCCCGTCCTC	AAAAGGGCTTGGGAGATTCAT
	Eph-A4	CGTTATGTGGGAAGTGATGTCATA	TTCCTCAATGGCTTTAATCACATCT

Acknowledgments

We are grateful to S. Weiss for sharing the BTIC lines. We thank P. Belenguer's team for sharing reagents and C. Monod for her help with editing the manuscript. We acknowledge core support from the Imagery Platform of Toulouse. This work was funded by ARC (Association pour la Recherche contre le Cancer; PJA 20131200200), FRM (Fondation pour le Recherche Médicale; DEQ20180339174) and ANR (Agence Nationale de la Recherche; ANR-15-CE13-0010-01). M.A. Fawal was supported by an ARC fellowship. Core funding was from the Université Paul Sabatier and the CNRS.

Authors contribution

M.A.F. and A.D. conceptualized the study, M.A.F. designed, performed and analyzed experiments, and co-wrote the manuscript. T.J. assisted and performed FACS analysis. All authors contributed to the editing of the manuscript.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.canlet.2021.01.026.


G Klausner

I Troussier

Sarah Kreps

Emmanuelle Fabiano

O Laccourreye

Professeur Philippe Giraud
email: philippe.giraud@aphp.fr

Impact du curage ganglionnaire cervical dans les cancers de l'oropharynx N2-3 traités par chimio-radiothérapie exclusive : étude observationnelle en vie réelle

Keywords: Oropharyngeal Neoplasms, Neck Dissection, induction chemotherapy, definitive chemo-radiotherapy

Titre courant : Impact du curage ganglionnaire cervical not statistically different between the two groups. The median follow-up was 60. 1 [3.2-119] months. Incidence of neck failure was higher in patients without ND (p=0.015). The neck failure rate at 5 years was 19. ], p=0.015) without ND versus 0% following ND. All lymph node failures occurred in the planned target volume at 70 Gy.

Upfront ND suggested a decrease in the mean dose received by the homolateral parotid gland (p=0.01), mandible (p=0.02), and thyroid gland (p=0.02).

Acute toxicity of CRT after ND suggested a reduction in grade ≥ 3 adverse events (p=0.04), early discontinuation of concomitant chemotherapy (p=0.009) and feeding tube-dependence (p=0.008) in univariate analysis. During follow-up, there was no difference between the two groups in terms of xerostomia, dysgeusia, dysphagia or gastrostomy dependence in univariate analysis.

Conclusion

ND prior to definitive CRT in N2-3 oropharyngeal SCC was associated with high neck control without additional mid and long-term morbidity.

Résumé

Objectif de l'étude

Évaluer l'efficacité en termes d'échec ganglionnaire du curage ganglionnaire cervical (CGC) initial dans les carcinomes épidermoïdes de l'oropharynx (CEO) N2-3 traités par chimio-radiothérapie (CRT) exclusive, ainsi que l'impact dosimétrique et la tolérance aigüe et tardive de cette approche.

Matériel et méthode

Résultats

Deux groupes étaient constitués : 47 (62,7%) patients sans CGC initial, et 28 (37,3%) patients avec un CGC avant la CRT. Les caractéristiques initiales des patients n'étaient pas statistiquement différentes entre les deux groupes. Le recul médian était de 60,1 [START_REF] Hamoir | Radical neck dissection: is it still indicated?[END_REF] mois. L'incidence des échecs ganglionnaires était plus élevée chez les patients n'ayant pas eu de CGC (p=0,015). Le taux d'échec ganglionnaire à 5 ans était de 19,8% (IC95% [START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF][START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF]) en l'absence de CGC contre aucune rechute (0%) après CGC. Tous les échecs ganglionnaires se trouvaient dans le volume cible planifié à 70 Gy.

Le CGC suggérait une diminution de la dose moyenne reçue par la parotide homolatérale (p=0,01), la mandibule (p=0,02) et la thyroïde (p=0,02). La tolérance aiguë de la CRT après CGC suggérait une réduction des événements indésirables de grade ≥ 3 (p=0,04), de l'arrêt précoce de la chimiothérapie concomitante (p=0,009) et du recours à une gastrostomie (p=0,008) en analyse univariée. Au cours du suivi, il n'existait aucune différence entre les deux groupes en termes de xérostomie, de dysgueusie, de dysphagie ou de dépendance à la gastrostomie en analyse univariée.

Conclusion

Le CGC avant la CRT exclusive dans les CEO N2-3 était associé à un très bon contrôle ganglionnaire sans morbidité supplémentaire à moyen et long terme.

Mots-clés : cancer de l'oropharynx ; curage ganglionnaire cervical ; chimiothérapie d'induction ; chimio-radiothérapie exclusive

Abstract

Purpose

To assess the efficacy in terms of neck failure of an initial neck dissection (ND) before definitive chemo-radiotherapy (CRT) in N2-3 oropharyngeal squamous-cell carcinomas (SCC), as well as the dosimetric impact and the acute and delayed morbidity of this approach.

Materials and methods

All patients consecutively treated between 2009 and 2018 with definitive CRT using intensity-modulated conformal radiotherapy (IMRT) for a histologically proven N2-3 oropharyngeal SCC were retrospectively included. The therapeutic approach consisted of induction chemotherapy, followed by cisplatine-based CRT preceded or not by ND. ND was discussed on a case-by-case basis in a dedicated multidisciplinary tumor board for patients with a dissociated response to induction chemotherapy, defined as a better response on the primary than on the node. CRT without ND was systematically performed in case of a major lymph node response to induction chemotherapy (decrease in size of 90% or more). IMRT using a simultaneous-

Results

Two groups were constituted: 47 (62.7%) patients without an initial ND, and 28 (37.3%) patients with a ND prior to definitive CRT. Initial patient characteristics were

Introduction

Les cancers de l'oropharynx sont dans 95% des cas des carcinomes épidermoïdes dont la présentation au diagnostic est fréquemment une masse cervicale indolore [START_REF] Chung | Pattern of lymph node metastasis in hypopharyngeal squamous cell carcinoma and indications for level VI lymph node dissection[END_REF]. Historiquement, les rechutes ganglionnaires des carcinomes épidermoïdes de l'oropharynx pouvaient survenir dans 30% des cas environ après chimio-radiothérapie (CRT) [START_REF] Peters | Neck surgery in patients with primary oropharyngeal cancer treated by radiotherapy[END_REF]. Ainsi, selon les équipes, un curage ganglionnaire cervical peut être pratiqué comme traitement de première intention des carcinomes épidermoïdes de la tête et du cou (CETC) localement avancés, indépendamment des caractéristiques de la tumeur primitive, d'autant que les progrès des techniques chirurgicales, avec notamment la réalisation d'un curage ganglionnaire radical modifié, ont considérablement réduit la morbidité de ce geste [START_REF] Hamoir | Radical neck dissection: is it still indicated?[END_REF]. Toutefois, un essai randomisé de phase III a récemment démontré que l'imagerie fonctionnelle par tomographie à émission de positons couplée au [18F] flurodéoxyglucoce (TEP-FDG) était efficiente pour évaluer la réponse thérapeutique dans les stratégies basées sur la CRT première dans les CETC N2-3 [START_REF] Mehanna | PET-CT Surveillance versus Neck Dissection in Advanced Head and Neck Cancer[END_REF]. Entre temps, la radiothérapie conformationelle avec modulation d'intensité (RCMI) est devenue la technique recommandée pour l'irradiation des CETC depuis qu'une diminution significative des effets secondaires à moyen et long terme comme la xérostomie, la dysphagie ou la dysphonie ait été démontrée grâce à cette technique avec un bénéfice direct sur la qualité de vie des patients [START_REF] Vergeer | Intensity-modulated radiotherapy reduces radiation-induced morbidity and improves health-related quality of life: results of a nonrandomized prospective study using a standardized follow-up program[END_REF][START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF].

Ainsi, la controverse demeure donc quant à la meilleure approche thérapeutique pour les CETC localement avancés avec atteinte ganglionnaire N2-3 [START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF]. D'un côté, la stratégie consistant en une chimiothérapie d'induction suivie d'une RCMI en association avec le cetuximab ne semble pas supérieure à la CRT exclusive à base de sel de platine [START_REF] Geoffrois | Induction Chemotherapy Followed by Cetuximab Radiotherapy Is Not Superior to Concurrent Chemoradiotherapy for Head and Neck Carcinomas: Results of the GORTEC 2007-02 Phase III Randomized Trial[END_REF] Les analyses statistiques ont été conduites à l'aide du logiciel R (version 3.6.0).

Une p-value < 0,005 était définie comme significative et une p-value entre 0,005 et 0,05 était considérée comme suggestive [START_REF] Ioannidis | The Proposal to Lower P Value Thresholds to .005[END_REF]. patients. Le nombre médian de ganglions retrouvés sur l'examen anatomopathologique de la pièce opératoire était de 30 . Onze patients (soit 39,3% des patients opérés) avaient une adénopathie cervicale en rupture capsulaire.

Résultats

En ce qui concerne la chimiothérapie, 45 (60%) patients ont reçu du cisplatine en induction, 26 (57,8%) d'entre eux ont pu recevoir une CRT à base de cisplatine, 13 (28,9%) à base de carboplatine et 6 (13,3%) à base de cetuximab, sans différence entre les deux groupes (p=0,9). La dose cumulée moyenne de cisplatine était de 239 (± 122) mg/m 2 après curage ganglionnaire initial et de 269 (± 122) mg/m 2 en l'absence de chirurgie, sans différence entre les deux groupes (p=0,4). Le recul médian entre la fin de la CRT et la date des dernières nouvelles était de 60,1 [START_REF] Hamoir | Radical neck dissection: is it still indicated?[END_REF] Malgré tout, les échecs ganglionnaires observés avec une CRT concomitante en RCMI étaient concordants avec les séries historiques ayant évaluées la réponse ganglionnaire à une radiothérapie exclusive. Les adénopathies métastatiques semblaient déjà le siège de zones radio-résistantes, surtout en cas de volume hypoxique important ou de tumeur supérieure à 3 cm [START_REF] Wolf | Effectiveness of salvage neck dissection for advanced regional metastases when induction chemotherapy and radiation are used for organ preservation[END_REF][START_REF] Strasser | Management implications of evaluating the N2 and N3 neck after organ preservation therapy[END_REF]. L'intérêt du curage ganglionnaire premier est de retirer ces zones à haut risque d'échec thérapeutique potentiel, de maximiser le contrôle ganglionnaire tout en réduisant significativement la dose délivrée au niveau de cou et des organes sains. Par ailleurs, les progrès chirurgicaux ont permis de commencer la CRT moins de sept semaines après le curage ganglionnaire, sans compromettre l'efficacité thérapeutique. En effet, allonger ce délai au-delà de sept semaines après la chirurgie était associé à une moins bonne SG selon Graboyes et al [START_REF] Graboyes | Effect of time to initiation of postoperative radiation therapy on survival in surgically managed head and neck cancer[END_REF]. D'ailleurs, la SG observée dans notre cohorte était particulièrement favorable. L'hypothèse probable serait la localisation oropharyngée de la tumeur primitive associée à plus de 60% à HPV. Des études prospectives ont permis d'établir que le statut HPV positif étaient à la fois prédictif de la chimioradiosensibilité de la maladie dans son ensemble et pronostic d'une meilleur SG [START_REF] Mak | Treatment response in the neck: p16+ versus p16-oropharyngeal cancer[END_REF][START_REF] Lee | Predicting response to radical (chemo)radiotherapy with circulating HPV DNA in locally advanced head and neck squamous carcinoma[END_REF].

  Tous les patients consécutivement traités entre 2009 et 2018 par CRT exclusive à l'aide d'une radiothérapie conformationelle avec modulation d'intensité (RCMI) pour un CEO N2-3 selon la 7 ème édition de la classification TNM AJCC/UICC étaient rétrospectivement inclus. La stratégie thérapeutique consistait en une chimiothérapie d'induction, suivie d'une CRT exclusive à base de cisplatine précédée ou non d'un CGC. Le CGC était discuté au cas par cas en réunion de concertation multidisciplinaire dédiée chez les patients présentant une réponse dissociée à la chimiothérapie d'induction, à savoir une réponse de meilleure qualité sur le primitif que sur le N. Une CRT sans CGC était systématiquement réalisée en cas de réponse ganglionnaire majeure à la chimiothérapie d'induction (diminution en taille de 90% ou plus). La RCMI délivrait en boost intégré une dose de 70 Gy en 35 fractions sur les volumes tumoraux macroscopiques, 63 Gy sur les niveaux à risque intermédiaire ou en rupture capsulaire et 54 Gy sur les aires ganglionnaires prophylactiques.

  integrated boost delivered 70 Gy in 35 fractions on macroscopic tumor volumes, 63 Gy on intermediate-risk levels or extra-nodal extension and 54 Gy on prophylactic lymph node areas.

  . D'un autre côté, les stratégies d'intensification reposant sur une CRT accélérée ne semblent pas supérieures à la CRT en étalement conventionnel. Les essais GORTEC 96-01 et 99-02 concluaient à un contrôle local et ganglionnaire insuffisant malgré l'intensification des traitements, cause majeure de décès dans les stades ganglionnaires avancés, notamment N3 [9]. Dans ce contexte où les échecs ganglionnaires restent un défi majeur, et les traitements de rattrapage très morbides, la question d'un curage ganglionnaire cervical avant la CRT reste d'actualité. D'ailleurs, les indications d'une approche multimodale associant un curage ganglionnaire initial suivie d'une CRT en RCMI ne sont pas à ce jour homogènes ; l'efficacité et la morbidité de cette stratégie méritent donc d'être évaluées à l'ère de la RCMI. L'objectif principal de cette étude était donc d'évaluer l'efficacité en termes d'échec ganglionnaire du curage ganglionnaire cervical initial dans les carcinomes épidermoïdes de l'oropharynx N2-3 traités par CRT exclusive en RCMI. Les objectifs secondaires étaient la survie globale, l'impact dosimétrique et la tolérance aigüe et tardive de cette stratégie thérapeutique. Matériel et méthodes Tous les patients adultes consécutivement traités entre 2009 et 2018 par CRT exclusive à l'aide d'une RCMI dans notre institution pour un carcinome épidermoïde de l'oropharynx N2-3 prouvé histologiquement ont été rétrospectivement inclus. Le stade TNM était défini selon la 7 ème édition de la classification TNM AJCC/UICC. Le statut Human Papilloma Virus (HPV) était défini selon les résultats de l'hybridation in reçue dans le site de la récidive. Les données collectées ont été extraites lors de la relecture individuelle des dossiers médicaux informatisés ; les patients ayant signés un consentement lors de l'admission dans notre institution et conforme à la Commission Nationale de l'Informatique et des Libertés (CNIL). L'étude a été rédigée conformément aux recommandations des rapports STROBE [12]. Analyses statistiques Une rechute locale était définie par la persistance ou la résurgence de la maladie au niveau de la tumeur primitive, et un échec ganglionnaire par la persistance ou la résurgence de la maladie au niveau des aires ganglionnaires cervicales. Une rechute métastatique était définie par l'apparition de métastase(s) en dehors des aires ganglionnaires de drainage. La survie globale (SG) était le temps entre la fin des traitements et le décès. Les patients étaient censurés à la date des dernières nouvelles. Les variables quantitatives ont été décrites par leur médiane, minimum et maximum ou leur moyenne et écart-type, et les variables qualitatives par leur fréquence et leur pourcentage. Les comparaisons des caractéristiques cliniques des patients ont été réalisées, pour les variables continues, à l'aide d'un test t de Student ou d'un test de Mann-Whitney Wilcoxon ; pour les variables catégorielles, les tableaux de contingence et leur test du Chi-2 ou le test exact de Fisher ont été utilisés. Les corrélations entre chaque paire de variables ont été estimées par le coefficient de corrélation de Spearman (rho). Un rho de Spearman au-dessus d'un seuil de 0,7 ou inférieur à -0,7 a été considéré comme une forte corrélation entre les variables. Les événements carcinologiques dont l'échec ganglionnaire et la SG ont été analysés avec la méthode de Kaplan-Meier. Les comparaisons des courbes d'échec ganglionnaire et de SG entre deux groupes ont été réalisées par le test du log-Rank. Les variables incluses dans l'analyse multivariée étaient associées à la SG avec une p-value < 0,1 en analyse univariée. Les analyses univariées et multivariées ont été réalisées à l'aide du modèle de Cox à risque proportionnel pour obtenir les hazard-ratio (HR) et leur intervalle de confiance à 95% (IC95%) respectif.

  mois.Contrôle local et ganglionnaireLe contrôle local de la tumeur primitive à 5 ans était de 88,4% (IC95% [76,9-100]) après curage ganglionnaire initial contre 91,4% (IC95% [83,2-99,8]) en l'absence de geste chirurgical, sans différence significative en termes de survie sans échec local entre les deux groupes (p=0,9). L'incidence des échecs ganglionnaires était plus élevée chez les patients n'ayant pas eu de curage ganglionnaire avant la CRT (p=0,015) (Figure1). A 5 ans, le taux de rechute ganglionnaire était de 19,8% (IC95%[START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF][START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF]) chez les patients non opérés contre aucune rechute (0%) après curage ganglionnaire initial. Les neufs échecs ganglionnaires sont survenus dans le volume cible planifié à 70 Gy (PTV 70 Gy), dont six dans le CTV N 70 Gy et trois dans le CTV N 63 Gy.Prise en charge des échecs ganglionnairesSur les neufs patients en échec ganglionnaire, trois patients sont décédés de leur poursuite évolutive ganglionnaire, et six patients ont eu une chirurgie de rattrapage, consistant en un curage ganglionnaire radical modifié pour quatre d'entre eux et en une excision sélective pour les deux autres. Le temps médian entre la fin de la CRT et la chirurgie de rattrapage était de 6 mois[START_REF] Hamoir | Radical neck dissection: is it still indicated?[END_REF][START_REF] Mehanna | PET-CT Surveillance versus Neck Dissection in Advanced Head and Neck Cancer[END_REF][START_REF] Vergeer | Intensity-modulated radiotherapy reduces radiation-induced morbidity and improves health-related quality of life: results of a nonrandomized prospective study using a standardized follow-up program[END_REF][START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF][START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF][START_REF] Geoffrois | Induction Chemotherapy Followed by Cetuximab Radiotherapy Is Not Superior to Concurrent Chemoradiotherapy for Head and Neck Carcinomas: Results of the GORTEC 2007-02 Phase III Randomized Trial[END_REF][START_REF] Tao | Very accelerated radiotherapy or concurrent chemoradiotherapy for N3 head and neck squamous cell carcinoma: Pooled analysis of two GORTEC randomized trials[END_REF][START_REF] Maingon | Chapitre commun à la prise en charge et à la procédure de préparation des traitements des cancers de la tête et du cou[END_REF][START_REF] Blanchard | Guidelines update: Post-treatment follow-up of adult head and neck squamous cell carcinoma: Screening for metastasis and metachronous esophageal and bronchial locations[END_REF][START_REF] Cuschieri | The STROBE guidelines[END_REF][START_REF] Ioannidis | The Proposal to Lower P Value Thresholds to .005[END_REF][START_REF] Wolf | Effectiveness of salvage neck dissection for advanced regional metastases when induction chemotherapy and radiation are used for organ preservation[END_REF][START_REF] Strasser | Management implications of evaluating the N2 and N3 neck after organ preservation therapy[END_REF][START_REF] Vergeer | Intensity-modulated radiotherapy reduces radiation-induced morbidity and improves health-related quality of life: results of a nonrandomized prospective study using a standardized follow-up program[END_REF]. Les effets indésirables survenus dans les suites de la chirurgie de rattrapage et persistants au cours du suivi ont été un déficit moteur complet de l'élévation de l'épaule et une dysphonie pour un patient après excision sélective. En termes d'efficacité, un patient a présenté une deuxième récidive ganglionnaire cervicale homolatérale ayant causée son décès trois ans après l'excision sélective de rattrapage, trois patients sont décédés des suites de métastases à distance, et les deux autres patients étaient en rémission clinique et radiologique.Survie globaleLa SG à 1 an, 2 ans et 5 ans était respectivement de 94,7% (IC95% [89,7-99,9]), 89% (IC95% [82,1-96,5]) et de 80,4% (IC95% [71,[START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF]). En analyse univariée, les facteurs suivant étaient associés à un pronostic défavorable en termes de SG (p<0,1) : le statut HPV négatif (p=0,01), le nombre de paquets-années consommés (p=0,002), l'absence de curage ganglionnaire cervical initial (p=0,06) et le volume du CTV T 70 Gy (p=0,01). Le statut HPV et la consommation de tabac présentaient une corrélation négative significative (p<0,001). En analyse multivariée, les facteurs pronostics suivant étaient suggestifs d'une SG défavorable (p<0,05) : le volume du CTV T 70 Gy (HR=1,02, IC95% [1-1,04], p=0,04), le statut HPV négatif (HR=3,25, IC95% [1,2-9,1], p=0,025) mais pas la réalisation d'un curage ganglionnaire initial (p=0,12). Impact dosimétrique Le volume cible clinique de la tumeur primitive (CTV T) traité à 70 Gy ne présentait pas de différence statistique selon que le patient ait eu un curage (35 ± 27 cc) ou non (39 ± 24 cc) (p=0,5). Les conséquences attendues du curage ganglionnaire cervical initial sur la planification de la radiothérapie étaient la nette diminution du volume cible clinique ganglionnaire (CTV N) traité à 70 Gy (9 (± 27) cc) comparé aux adénopathies en place (53 (± 41) cc) (p<0,001) ainsi que du volume du CTV N recevant 63 Gy (113 (± 59) cc versus 147 (± 67,5) cc, p=0,03). A noter, les volumes des CTV T et CTV N 70 Gy n'étaient pas statistiquement corrélés (p=0,37). Les comparaisons des doses moyennes reçues lors de la CRT définitive par les principaux organes à risque ont été résumées dans le tableau 2. Le curage ganglionnaire cervical initial permettait de diminuer la dose moyenne délivrée par la RCMI à la glande parotide homolatérale (p=0,01), à la mandibule (p=0,01) ainsi qu'à la thyroïde (p=0,02). Tolérance Effets indésirables aigus de la chirurgie Les suites du curage ganglionnaire ont été compliquées pour quatre patients par une hémorragie du site opératoire, une pneumopathie d'inhalation, un lymphoedème compressif et une paralysie faciale périphérique. Le délai médian entre la chirurgie et le premier jour de la RCMI était de 41 [28-68] jours. Effets indésirables aigus de la CRT La tolérance aigüe de la CRT avait tendance à être meilleure chez les patients ayant eu un curage ganglionnaire (tableau 3) en termes d'effets indésirables de grade ≥ 3 (p=0,04), d'arrêt prématuré de la chimiothérapie concomitante (p=0,009), d'interruption de la radiothérapie pendant quatre jours ou plus (p=0,06) et de recours à une gastrostomie (p=0,008) en analyse univariée. Cependant, aucune corrélation forte n'a été retrouvée entre les volumes du CTV N ou du CTV T 70 Gy et la survenue d'effets indésirables de grade ≥ 3 ou d'interruption de la radiothérapie pendant quatre jours ou plus. Effets secondaires tardifs La xérostomie tous grades confondus était la complication la plus fréquemment rencontrée avec une incidence de 86,5% (IC95% [77-93]) à 6 mois, 77,8% (IC95% [67-87]) à 12 mois, 59,4% (IC95% [46-72]) à 24 mois et 37,2% (IC95% [23-53]) à 5ans. Les taux de xérostomie tous grades confondus ne différaient pas significativement selon que les patients aient eu un curage cervical ou non (Figure1A). L'incidence de la xérostomie de grade ≥ 2 était de 50% (IC95% [38-62]) à 6 mois, 23,6% (IC95% [14-25]) à 12 mois, 18,8% (IC95% [10-30]) à 24 mois et 4,7% (IC95% [0,5-16]) à 5 ans sans aucune différence en fonction de la stratégie thérapeutique.La deuxième complication la plus fréquente était la dysgueusie avec une incidence tous grades confondus de 29,7% (IC95%) à 6 mois, 19,4% (IC95%), 12,5% (IC95%[START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF][START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF][START_REF] Geoffrois | Induction Chemotherapy Followed by Cetuximab Radiotherapy Is Not Superior to Concurrent Chemoradiotherapy for Head and Neck Carcinomas: Results of the GORTEC 2007-02 Phase III Randomized Trial[END_REF][START_REF] Tao | Very accelerated radiotherapy or concurrent chemoradiotherapy for N3 head and neck squamous cell carcinoma: Pooled analysis of two GORTEC randomized trials[END_REF][START_REF] Maingon | Chapitre commun à la prise en charge et à la procédure de préparation des traitements des cancers de la tête et du cou[END_REF][START_REF] Blanchard | Guidelines update: Post-treatment follow-up of adult head and neck squamous cell carcinoma: Screening for metastasis and metachronous esophageal and bronchial locations[END_REF][START_REF] Cuschieri | The STROBE guidelines[END_REF][START_REF] Ioannidis | The Proposal to Lower P Value Thresholds to .005[END_REF][START_REF] Wolf | Effectiveness of salvage neck dissection for advanced regional metastases when induction chemotherapy and radiation are used for organ preservation[END_REF][START_REF] Strasser | Management implications of evaluating the N2 and N3 neck after organ preservation therapy[END_REF][START_REF] Graboyes | Effect of time to initiation of postoperative radiation therapy on survival in surgically managed head and neck cancer[END_REF][START_REF] Mak | Treatment response in the neck: p16+ versus p16-oropharyngeal cancer[END_REF][START_REF] Lee | Predicting response to radical (chemo)radiotherapy with circulating HPV DNA in locally advanced head and neck squamous carcinoma[END_REF][START_REF] Prades | Planned neck dissection before combined chemoradiation for pyriform sinus carcinoma[END_REF][START_REF] Seidler | Dual-Energy CT Texture Analysis With Machine Learning for Impact du curage ganglionnaire cervical, G. Klausner 24 the Evaluation and Characterization of Cervical Lymphadenopathy[END_REF][21][22][23]) et de 11,6% (IC95%) sans aucune différence en fonction de la stratégie thérapeutique. L'incidence de la dysphagie tous grades confondus était de 28,4% (IC95%[19- 40]) à 6 mois, 16,7% (IC95% [9-27]) à 12 mois, 6,3% (IC95%[START_REF] Peters | Neck surgery in patients with primary oropharyngeal cancer treated by radiotherapy[END_REF][START_REF] Hamoir | Radical neck dissection: is it still indicated?[END_REF][START_REF] Mehanna | PET-CT Surveillance versus Neck Dissection in Advanced Head and Neck Cancer[END_REF][START_REF] Vergeer | Intensity-modulated radiotherapy reduces radiation-induced morbidity and improves health-related quality of life: results of a nonrandomized prospective study using a standardized follow-up program[END_REF][START_REF] Nutting | Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial[END_REF][START_REF] Klausner | Neck management in head and neck squamous cell carcinomas: where do we stand?[END_REF][START_REF] Geoffrois | Induction Chemotherapy Followed by Cetuximab Radiotherapy Is Not Superior to Concurrent Chemoradiotherapy for Head and Neck Carcinomas: Results of the GORTEC 2007-02 Phase III Randomized Trial[END_REF][START_REF] Tao | Very accelerated radiotherapy or concurrent chemoradiotherapy for N3 head and neck squamous cell carcinoma: Pooled analysis of two GORTEC randomized trials[END_REF][START_REF] Maingon | Chapitre commun à la prise en charge et à la procédure de préparation des traitements des cancers de la tête et du cou[END_REF][START_REF] Blanchard | Guidelines update: Post-treatment follow-up of adult head and neck squamous cell carcinoma: Screening for metastasis and metachronous esophageal and bronchial locations[END_REF][START_REF] Cuschieri | The STROBE guidelines[END_REF][START_REF] Ioannidis | The Proposal to Lower P Value Thresholds to .005[END_REF][START_REF] Wolf | Effectiveness of salvage neck dissection for advanced regional metastases when induction chemotherapy and radiation are used for organ preservation[END_REF][START_REF] Strasser | Management implications of evaluating the N2 and N3 neck after organ preservation therapy[END_REF][START_REF] Graboyes | Effect of time to initiation of postoperative radiation therapy on survival in surgically managed head and neck cancer[END_REF]) à 24 mois et 11,6% (IC95% [4-25]) à 5 ans. Les taux de dysphagie ne différaient pas significativement chez les patients opérés ou non (Figure1B). L'incidence de la dysphagie de grade ≥ 2 était de 14,9% (IC95%[START_REF] Geoffrois | Induction Chemotherapy Followed by Cetuximab Radiotherapy Is Not Superior to Concurrent Chemoradiotherapy for Head and Neck Carcinomas: Results of the GORTEC 2007-02 Phase III Randomized Trial[END_REF][START_REF] Tao | Very accelerated radiotherapy or concurrent chemoradiotherapy for N3 head and neck squamous cell carcinoma: Pooled analysis of two GORTEC randomized trials[END_REF][START_REF] Maingon | Chapitre commun à la prise en charge et à la procédure de préparation des traitements des cancers de la tête et du cou[END_REF][START_REF] Blanchard | Guidelines update: Post-treatment follow-up of adult head and neck squamous cell carcinoma: Screening for metastasis and metachronous esophageal and bronchial locations[END_REF][START_REF] Cuschieri | The STROBE guidelines[END_REF][START_REF] Ioannidis | The Proposal to Lower P Value Thresholds to .005[END_REF][START_REF] Wolf | Effectiveness of salvage neck dissection for advanced regional metastases when induction chemotherapy and radiation are used for organ preservation[END_REF][START_REF] Strasser | Management implications of evaluating the N2 and N3 neck after organ preservation therapy[END_REF][START_REF] Graboyes | Effect of time to initiation of postoperative radiation therapy on survival in surgically managed head and neck cancer[END_REF][START_REF] Mak | Treatment response in the neck: p16+ versus p16-oropharyngeal cancer[END_REF][START_REF] Lee | Predicting response to radical (chemo)radiotherapy with circulating HPV DNA in locally advanced head and neck squamous carcinoma[END_REF][START_REF] Prades | Planned neck dissection before combined chemoradiation for pyriform sinus carcinoma[END_REF][START_REF] Seidler | Dual-Energy CT Texture Analysis With Machine Learning for Impact du curage ganglionnaire cervical, G. Klausner 24 the Evaluation and Characterization of Cervical Lymphadenopathy[END_REF][21][22][23][24][25]) à 6 mois, 9,7% (IC95% [4-19]) à 12 mois, 6,3% (IC95% [2-15]) à 24 mois et 4,7% (IC95% [0,5-16]) à 5 ans sans aucune différence en fonction de la stratégie thérapeutique. La dépendance à la gastrostomie définie par la nécessité d'une alimentation par voie entérale totale ou partielle était estimée à 12,2% (IC95% [6-22]) à 6 mois, 4,2% (IC95% [1-12]) à 12 mois et 3,1% (IC95% [0,4-11]) à 24 mois, sans aucune différence en rapport avec le statut chirurgical. Discussion L'objectif de cette étude était d'évaluer l'efficacité en termes d'échec ganglionnaire du curage ganglionnaire cervical initial dans les carcinomes épidermoïdes de l'oropharynx N2-3 traités par CRT exclusive, ainsi que la SG, l'impact dosimétrique, et la morbidité de cette approche. Avec un recul médian de 5 ans, l'incidence des échecs ganglionnaires était plus élevée chez les patients n'ayant pas eu de curage ganglionnaire avant la CRT. De plus, les échecs ganglionnaires de la CRT exclusive se trouvaient majoritairement dans le CTV N 70 Gy. Par ailleurs, les patients ayant eu un évidement cervical initial avant la CRT présentaient des résultats plus favorables à la fois en termes de planification dosimétrique qu'en termes de tolérance aiguë de la CRT, sans morbidité supplémentaire au cours du suivi ni d'avantage en SG. En effet, au-delà de l'exérèse de l'adénopathie tumorale, la réalisation d'un curage ganglionnaire initial permettait d'adapter la dose délivrée lors de la CRT exclusive en fonction des résultats anatomopathologiques, et de réduire ainsi les volumes cervicaux irradiés à 70 Gy et à 63 Gy. Ainsi la dose moyenne reçue par les principaux organes à risque, notamment la glande parotide homolatérale, la mandibule et la thyroïde était plus basse après un curage initial dont les conséquences cliniques et sur la qualité de vie des patients seraient à évaluer prospectivement. A ce jour, aucune étude prospective ne porte sur la question d'une dose et de volumes plus faibles guidés par les résultats anatomopathologiques de l'évidement cervical initial pour réduire les effets secondaires aigus et tardifs à l'ère de la RCMI. La principale limite, inhérente à la nature rétrospective de notre étude, était probablement un biais d'indication ayant conduit à opérer les adénopathies carcinologiquement résécables et à récuser les adénopathies non résécables et les patients non opérables. Plus précisément, une adénopathie est généralement considérée comme non résécable en cas de continuum avec la tumeur primitive, de localisation rétropharyngée ou en base de crâne, d'extension péri-carotidienne supérieure à 180° ou d'envahissement cutané. Une autre limite de notre étude était le recueil rétrospectif des effets secondaires à moyen et longs termes tant de la CRT que de la chirurgie. Bien que les toxicités potentielles aient été systématiquement recherchées lors de la surveillance, l'absence de mesure du flux salivaire et de questionnaires dédiés à l'évaluation de la xérostomie, de la dysphagie ou des séquelles d'un curage ganglionnaire cervical impose de confirmer prospectivement l'absence de différence retrouvée en termes de morbidité entre les deux stratégies thérapeutiques d'autant que la qualité de vie des patients n'a pas pu être évaluée.

Tableau 1 .

 1 Actuellement, aucun facteur clinique ou radiologique ne permet de prédire à lui seul le succès de la chimiothérapie d'induction ou de la CRT exclusive. Après échec de la chimiothérapie d'induction (progression ou stabilité), l'évolution naturelle de la maladie se traduit par un moins bon contrôle local et ganglionnaire. Paradoxalement, les taux d'échec métastatique à distance ne sont pas significativement plus élevés. Il devient donc indispensable d'identifier des facteurs prédictifs de réponse à la chimiothérapie d'induction ou à la CRT exclusive. Certaines séries décrivaient comme moins radiosensibles des adénopathies nécrotiques ou supérieures à 3 cm ou de primitif hypopharyngée, sans que ces caractéristiques n'aient été confirmées prospectivement [19]. La radiomique et les algorithmes d'intelligence artificielle tels que les apprentissages neuronaux (deep learning) pourraient être des outils pour catégoriser en amont les adénopathies susceptibles d'être chimio-radiosensibles ou au contraire susceptibles d'échec thérapeutique [20]. En cas de maladie ganglionnaire à priori chimio-radiosensible, une CRT première pourrait être privilégiée. Inversement, un traitement complémentaire, local et/ou systémique serait à envisager pour des maladies ganglionnaires peu radiosensibles, de moins bon pronostic, dont un échec à la CRT première est attendu. Dans ce contexte, une chimiothérapie d'induction plutôt Impact du curage ganglionnaire cervical, G. Klausner 20 qu'une chimiothérapie adjuvante après une CRT exclusive dont la tolérance est souvent délicate sera discutée pour minimiser le risque d'évolution métastatique à distance. En parallèle, la question du curage ganglionnaire cervical dont la morbidité apparait faible se discute pour maximiser le contrôle ganglionnaire notamment en cas de maladie stable ou de réponse partielle de mauvaise qualité à la chimiothérapie d'induction. Conclusion L'impact du curage ganglionnaire cervical dans les carcinomes épidermoïdes de l'oropharynx N2-3 assure un très bon contrôle ganglionnaire sans morbidité supplémentaire à moyen et long terme. UP-FRONT NECK (NCT02918955), essai randomisé de phase III comparant la CRT exclusive avec ou sans curage ganglionnaire cervical initial dans les CETC localement avancés, est en cours et les résultats de la réponse à cette question sont attendus courant 2022. Conflit d'intérêt : aucun. Caractéristiques des 75 patients traités pour un carcinome épidermoïde de l'oropharynx N2-3 selon si une chimio-radiothérapie ou un curage ganglionnaire ait été réalisé en premier.

Figure 1 .Figure 2 .

 12 Figure 1. Courbes Kaplan-Meier de l'incidence cumulée de l'échec

  58%) patients ayant eu ensuite un curage ganglionnaire avant la CRT contre 15 (42%) patients une CRT exclusive (p<0,001). Deux patients ayant présentés une réponse < 50% sur le T et le N ont également eu un curage ganglionnaire avant CRT. Au total, 47 (62,7%) patients ont reçu une CRT définitive sans geste chirurgical et 28 (37,3%) patients ont eu un curage ganglionnaire cervical avant la CRT définitive. Les caractéristiques cliniques initiales des patients ne présentaient pas de différence statistiquement significative selon qu'un évidement ganglionnaire ait été réalisé ou non (tableau 1). Au diagnostic, 23 (31%) patients avaient une adénopathie au diagnostic non résécable d'emblée sans différence significative entre les deux groupes (p=0,41). Sept patients ont présenté une réponse à la chimiothérapie d'induction ayant permis la réalisation d'un curage ganglionnaire avant la CRT. Le geste chirurgical était un curage ganglionnaire radical modifié pour 23 (82,1%) patients et radical pour 5 (17,9%)

	Population et traitements
	Entre 2009 et 2018, 75 patients ont été traités par RCMI pour un carcinome
	épidermoïde de l'oropharynx N2-3. Au diagnostic, le performance status OMS était de
	0 pour 65 (86,7%) patients et de 1 pour 10 (13,3%) patients. Les caractéristiques
	initiales des patients inclus ont été détaillées dans le tableau 1.
	Comme traitement de première intention, 69 (92%) patients ont reçu une
	chimiothérapie d'induction, cinq patients un curage ganglionnaire cervical et un patient
	une CRT exclusive. Après chimiothérapie d'induction, 11 (16%) des patients
	présentaient une réponse ganglionnaire majeure et ont reçu une CRT sans curage
	ganglionnaire préalable. Une réponse dissociée à la chimiothérapie était observée
	chez 36 (48%) patients, 21 (

  Tolérance aiguë de la chimio-radiothérapie des 75 patients traités pour un carcinome épidermoïde de l'oropharynx N2-3 selon si une chimioradiothérapie ou un curage ganglionnaire ait été réalisé en premier.

	Tableau 3. Complications aigües	Chimio-radiothérapie	Curage ganglionnaire	p-value
					0,57
		(n = 47)		(n = 28)
	Médiane [min-max]	63,5 [42,7-81,3]	61 [42,7-81,3]	64,5 [47-75,9]
	Effets secondaires de grade ≥ 3				0,04
	Sexe				0,75
	Oui	21 (44,7%)	6 (21,4%)
	Homme	62 (82,7%)	38 (80,85%)	24 (85,7%)
	Non	26 (55,3%)	22 (78,6%)
	Femme	13 (17,3%)	9 (19,15%)	4 (14,3%)
	Interruption de la radiothérapie ≥ 4 jours				0,06
	Localisation				0,42
	Oui	14 (29,8%)	3 (10,7%)
	Amygdale	39 (52%)	27 (57,5%)	12 (42,9%)
	Non	33 (70,2%)	25 (89,3%)
	Base de langue	20 (26,7%)	12 (25,5%)	8 (28,5%)
	Arrêt prématuré de la chimiothérapie concomitante			0,009
	Sillon amygdaloglosse	9 (12%)	4 (8,5%)	5 (17,9%)
	Oui	22 (46,8%)	5 (17,9%)
	Vallécule	4 (5,3%)	2 (4,25%)	2 (7,1%)
	Non	25 (53,2%)	23 (82,1%)
	Paroi pharyngée	3 (4%)	2 (4,25%)	1 (3,6%)
	Recours à une gastrostomie				0,008
	Stade T				0,07
	Oui	36 (76,6%)	13 (46,4%)
	T1	8 (10,7%)	2 (4,3%)	6 (21,4%)
	Non	11 (23,4%)	15 (53,6%)
	T2	19 (25,3%)	11 (23,4%)	8 (28,6%)
	% de perte de poids au terme des traitements			0,98
	T3	7 (9,3%)	4 (8,5%)	3 (10,7%)
	Médiane [min ; max]	-8,2 [-18,3 ; +3,3]	-6,4 [-18,2 ; +1,4]
	T4	41 (54,7%)	30 (63,8%)	11 (39,3%)
	Durée totale de l'ensemble des traitements, mois			0,05
	Stade N				0,37
	Médiane [min ; max]	4,7 [3,3 ; 6]	5,3 [3,3 ; 6,1]
	N2a	5 (6,7%)	2 (4,3%)	3 (10,7%)
	N2b	45 (60%)	27 (57,5%)	18 (64,3%)
	N2c	18 (21%)	14 (29,8%)	4 (14,3%)
	N3	7 (9,3%)	4 (8,5%)	3 (10,7%)
	Adénopathie résécable au diagnostic				0,41
	Oui	52 (69%)	21 (75%)	31 (66%)

situ et/ou de la Polymerase Chain Reaction (PCR) HPV. Les critères d'exclusion étaient un traitement par radiothérapie conformationnelle en trois dimensions et la présence de métastase(s) au diagnostic. Tous les traitements ont été validés dans une réunion de concertation multidisciplinaire dédiée. La stratégie thérapeutique de notre institution pour les carcinomes épidermoïdes de l'oropharynx localement avancés consistait en une chimiothérapie d'induction à base cisplatine, de docétaxel et de 5fluorouracile (TPF) en l'absence de contre-indication, suivie d'une CRT exclusive à base de cisplatine précédée ou non d'un curage ganglionnaire cervical. Celui-ci était discuté au cas par cas, en réunion de concertation multidisciplinaire dédiée, chez les patients présentant une réponse dissociée à la chimiothérapie d'induction, à savoir une réponse de meilleure qualité au niveau de la tumeur primitive que sur l'adénopathie. Une CRT sans geste chirurgical préalable était systématiquement réalisée en cas de réponse ganglionnaire majeure à la chimiothérapie d'induction (diminution en taille de 90% ou plus de l'adénopathie). La CRT exclusive en RCMI délivrait une dose de 70 Gy en 35 fractions de 2 Gy sur les volumes tumoraux initiaux oropharyngés et ganglionnaires macroscopiques, 63 Gy en fraction de 1,8 Gy sur les aires ganglionnaires à risque intermédiaire (aires contenant au moins une adénopathie en place ou ayant contenues, après curage ganglionnaire, une adénopathie en rupture capsulaire sur l'examen anatomopathologique), et 54 Gy en fraction de 1,6 Gy sur les aires ganglionnaires prophylactiques selon une technique de boost intégré [10]. La surveillance des patients débutait 3 mois après la fin des traitements en alternance entre les services d'oncologie-radiothérapie et de chirurgie cervico-faciale et suivait les recommandations de la Société Française d'Otorhinolaryngologie (SFORL) [11]. En cas de d'échec ganglionnaire, une TEP-FDG était réalisée et était fusionnée au scanner de planification dosimétrique initial afin de préciser la localisation et la dose