Vivien Moris
email: morisvivien@gmail.com

Pierre Bensa²

Brice Gerenton
email: brice.gerenton@gmail.com

Philippe Rizzi
email: philipperizzi@gmail.com

Sarah Cristofari

Narcisse Zwetyenga
email: nzwetyenga@gmail.com

David Guilier

Pierre Bensa
email: pierre.bensa@orange.fr

Sarra Cristofari
email: sarra.cristofari@aphp.fr

David Guillier
email: docteurguillierdavid@gmail.com

The Cervicofacial lift under Pure local anaesthesia diminishes the incidence of postoperative haematoma

2

Abstract

The cervicofacial lift is a frequently performed procedure in plastic surgery. It is the reference technique for facial rejuvenation and restoration of the oval form of the face and it is essential to treat excess skin. One of the most frequent complications of this procedure is the formation of haematomas.

The aim of this article is to analyze the incidence of bleeding in the standard cervicofacial lift carried out under pure local anaesthesia. The entire operation, including liposuction and tightening of the SMAS, can be done under local anaesthesia. A total of 1500 patients, who have undergone cervicofacial lift under pure local anaesthesia between November 1995 and January 2016, were included in the study. The incidence of early bleeding (in the first 15 days following the operation) was recorded and analyzed. Pre-intra-and postoperative monitoring of the arterial blood pressure revealed stable pressure in the peri-operative period. The mean difference in blood pressure when comparing the intraoperative to the postoperative periods was 7 mmHg for the systolic BP [range from 3 -25] and 4 mmHg [range from 2 -12] for the diastolic BP, with lower mean values in the postoperative period. We identified nine bleeding events in 1500 patients (0.6%).

The cervicofacial lift under pure local anaesthesia does not radically modify the surgical technique. It allows better control of arterial blood pressure of patients throughout the surgical procedure and avoids fluctuations in blood pressure, which is one of the main causes of bleeding and haematoma formation. The infiltration of xylocaine adrenaline combined with the absence of hypotensive general anaesthesia diminishes the incidence of haematoma and ensures an early return home for the patients.

Introduction

The cervicofacial lift is a frequent and useful intervention. Despite the popularity of new techniques for rejuvenation of the face, such as Botox, fillers, lipofilling (1) and suspension sutures, the "traditional" cervicofacial lift is the only technique available to plastic surgeons to remove the inevitable excess skin that appears with age (2).

The standard cervicofacial lift consists of tightening the skin and the superficial musculoaponeurotic system (SMAS). This procedure on the muscle and skin can durably eliminate wrinkles in the lower third of the face and the neck. The excess skin which arises from the tightening of the SMAS is removed by surgery. Liposuction is generally performed in the area under the chin and the neck to treat the unattractive "double-chin". In most cases, this operation is carried out under general anaesthesia, less commonly under sedation combined with local anaesthesia and exceptionally under pure local anaesthesia without using any systemic muscle relaxants or anxiolytic drugs. One of the most feared complications is the onset of postoperative haematoma as it starves the skin of oxygen and can potentially cause skin necrosis. The onset of haematoma after a cervicofacial lift correlates with the patient's pre-intra-and postoperative arterial blood pressure [START_REF] Ramanadham | Evolution of hypertension management in face lifting in 1089 patients: optimizing safety and outcomes[END_REF][START_REF] Kleinberger | What is the best method for minimizing the risk of hematoma formation after rhytidectomy? The Laryngoscope[END_REF].

The aim of this article was to analyze the incidence of bleeding following a standard cervicofacial lift (5-7) carried out under pure local anaesthesia.

Materials and Methods

Patients:

From November 1995 to January 2016, 1500 patients who underwent a cervicofacial lift under pure local anaesthesia were included.

Inclusion:

The indication for a cervicofacial lift with tightening of the SMAS under local anaesthesia was proposed by the surgeon following two consultations. The shape and volume of the face as well as the quality of the skin (tension and elasticity) were taken into consideration.

Exclusion:

Smokers > 5 cigarettes a day were excluded, as were persons with skin disorders (Xeroderma pigmentosum, sclerodermia, lupus…) or with body dysmorphia disorder. Associated surgery of the face was also excluded (mask lift, forehead lift, rhinoplasty). Only blepharoplasties associated with the cervicofacial lift were not excluded. Patients treated with anticoagulants (oral or injectable) or antiplatelets (aspirin, plavix) were excluded.

Principal judgement criterion:

The onset of an early bleeding event (in the 15 days following surgery) was recorded and analysed. Bleeding events were defined as a postoperative haematoma or active bleeding requiring draining (out-patient or operating room).

Pre-, intra-, and postoperative arterial blood pressure was continuously monitored (every 4 min). Surveillance was started 30 min before the start of the local anaesthesia and continued for 2 hours after the end of the surgery.

Secondary judgement criterion:

Local complications were looked for: infection of the surgical site, wound dehiscence, facial paralysis and an asymmetric result.

Pain was evaluated using a Visual Analogue Scale and postoperative nausea was documented.

Surgical procedure

All patients underwent the same surgical procedure, a cervicofacial lift with a SMAS flap. No patients were given medication before the operation. . Neither anxiolytics, nor analgesics, nor anti-hypertensives were administered. A simple analgesic treatment with paracetamol and codeine was prescribed in the postoperative period. The first dose was taken 2 hours after the intervention before the patient returned home.

Local anaesthesia:

Local anaesthesia was administered once the preoperative marking had been done with patients standing or sitting. (Figure 1)

Patients were positioned in supine position with legs downwards to diminish arterial blood pressure in the head and neck. The injection sites were swabbed with chlorhexidine before local anaesthesia was injected.

The preparation for the local anaesthesia was a modification of the Klein formula (8):

-250 cc of injectable NaCl 0.9% -80 ml of xylocaine : 10mg/ml with adrenaline : 0.005mg/ml -10 ml of naropeine 7.5 mg/ml

Local anaesthesia was started with a syringe of 1cc and a 30 gauge needle and injections into key points: under the chin and around both ear lobes. These are the only three painful injections for the patient. Incisions were made at these key points using a N° 15 scalpel blade, and a Klein cannula was inserted. The diluted anaesthesia solution was thus infiltrated gently and atraumatically via a blunt-tipped cannula 100 mm in length and 1.25 mm in diameter manufactured by the French Aesthetic group. The solution was injected abundantly under the skin in the cheeks and in the pre-auricular, cervical and retro-auricular areas.

This first stage of the anaesthesia takes an average of 15 minutes. (Figures 2,3,4)

The solution is left to take effect for 10 minutes before the incision (time necessary for the adrenaline to create a bloodless field). In the meantime, the table is prepared and the 2 nd skin prep for the incision sites is done. The principal advantage of this technique is that patients can cooperate by raising their head, thus making it easier to place surgical drapes in aseptic conditions. Liposuction of the cervical area and under the chin is carried out via an incision under the chin and using a N°4 cannula. Liposuction extracts the excess fat and prepares the dissection plane by creating dissection tunnels using the cannula. . (Figure 5)

A traditional lift with tightening of the SMAS was carried out using partial SMASectomy.

The creation of a subcutaneous plane was facilitated by the hydrodissection and liposuction.

The incision starts in the scalp behind the hairline and continues downwards passing in front of the ear so that the scar will be on the interior side of the tragus, it then passes under the lobe and continues behind the ear where it ends. The skin is lifted using skin hooks and undermining is first achieved by using a cold N° 15 scalpel blade and then using Metzenbaum scissors to cut along the infiltration tunnels but keeping a distance of 3 cm from the lip commissures. Haemostasis is achieved step-by-step using bipolar coagulation forceps.

Halstead forceps are used to pinch and pull the SMAS so as to identify useful traction vectors.

The SMAS is elevated by two forceps and incised between them using a cold blade (Figure 6). This manoeuvre makes it possible to incise the SMAS without damaging the branches of the facial nerve. The sub-aponeurotic elevation is achieved using a swab held between forceps and completed with a chisel (Figure 78). The SMAS flap is pulled tight and sutured in the proximal position using 3-0 Vicryl sutures and respecting the lines of traction identified earlier.

The skin flaps are repositioned and the line of the skin resection is determined using a marker clamp. The skin is resected using a cold blade and iridectomy scissors starting with the retroauricular cervical flap (Figures 9 and10). The subcutaneous suture using 3-0 Vicryl starts behind the ear on a Redon 6 French drain which will be left free in the dressing. This drain will be removed one day after the surgery. The pre-auricular skin resection is then done.

Particular attention must be paid to the resection around the lobe, which has to be done under tension to obtain a natural-looking result.

The skin is sutured using Indermil-type glue. Neither running nor simple interrupted sutures should be used for the skin (Figures 11 and12). Vaseline is applied to the wounds, which are then covered with compresses dampened with anaesthetic solution and held in place with 10cm-wide Velpeau bands. It is essential to check the position of the lobes once the dressing is in place to avoid necrosis of the lobe due to incorrect positioning of the dressing.

Results:

Patients characteristics (Table 1):

There were 1350 women and 150 men with a mean age of 58 years [40 -69] (Table 1). It was the first surgery in 1394 patients, the second in 123 patients and the third in 50 patients. There were 510 smokers, who reduced the number of cigarettes or stopped smoking before the operation (maximum 5 cigarettes a day, 4 weeks before and 4 weeks after the intervention).

Comorbidities included 311 patients with treated hypertension measured at > 130/80 during the anaesthesia consultation, and 120 patients with treated diabetes with HbA1c < 8%.

The mean duration to perform the local anaesthesia was 15 minutes.

The mean duration of the surgery excluding the time of anaesthesia was 2 hours.

Principal Judgement Criterion:

In the 1500 patients, there were nine bleeding events (0.6%). All nine of these patients suffered from hypertension, measured at >150/90 during the anaesthesia consultation despite dual antihypertensive therapy.

The mean systolic blood pressure was 123 mmHg [78 -169] in the preoperative stage, 147 mmHg [88 -178] during the procedure and it stabilized at 142 mmHg [85 -175] after surgery in the postoperative stage (figure 13).

Mean variations in blood pressure from the preoperative to the intraoperative stage were 22 mmHg [4 -45] for systolic BP and 8 mmHg [4 -15] for diastolic BP. The mean variations in blood pressure between the intraoperative and postoperative period were 7 mmHg for the systolic BP [3 -25] and 4 mmHg [2 -12] for the diastolic BP (Figure 14).

Secondary Judgement Criterion (Table 2):

Three infections requiring drainage occurred and were managed at the patients' bedside. No antibiotics were prescribed. Twenty patients presented wound dehiscence requiring skin care for > 15 days.

Ten patients presented skin necrosis requiring skin care for > 15 days despite early treatment with alpha-blockers. There were no cases of facial paralysis (mouth or forehead), but 148 cases of hypoesthesia around the pre-tragal and peri-lobular scar (10% of patients). There were no cases of numbness in the wider auricular area. Two men developed keloid scars, in the pre-tragal and retro-auricular area, and were treated with scar excision associated with intralesional steroids. Pain VAS was evaluated immediately after and the day after the operation: maximum pain was reported at the start of the infiltration and assessed at 3/10 in the pre-tragal and retro-auricular area. The postoperative VAS at D1 was 2/10; patients complained more about a feeling of tension in the face than about pain itself. Concerning imperfections in the result: 10 female patients underwent a supplementary lift at 1 year. These women were all aged over 60 and had loose skin.

general anaesthesia (weaning off drugs, effort of breathing through a tube). We found a very low proportion of bleeding events (0.6%) in this series.

The lowest systolic blood pressure value was 78 mmHg in preoperative stage. This result is considered normal as it was measured after 10 min rest in a decubitus dorsal position. The patient was a female that had a BMI=18kg/m2 and was known to have low blood pressure on a daily basis. The highest systolic blood pressure value was 169 mmHg in preoperative stage. This is the measurement of an overweight male patient.

A high systolic blood pressure can cause multiple risks in this surgery:

The diffusion of adrenaline in the systemic blood circulation is related to blood pressure. A patient with hypertension can have adrenaline systemic rush which can cause multiple rhythmic and ischemic heart diseases. On the other hand, the risk of hematoma is higher with an elevated systolic blood pressure. In our series, all patients with a hematoma had a preoperative uncontrolled hypertension.

For these reasons, the patient selection is very important in cosmetic surgery especially in elective facial surgery, giving the intrinsic risk factor of hematoma during the facelift procedure. Therefore, we now only accept patients with a systolic blood pressure below 140 mmHg.

This result is lower than figures reported in the literature, with proportions ranging from 3% in patients without hypertension to 12,9% in those with hypertension (3;11). In 1976, Baker et al demonstrated that postoperative hypertension is the main etiologic factor in hematoma formation. Over a 30-year period, the incidence of hematoma requiring surgical evacuation decreased from 8.7% to 3.97% after the initiation of a strict perioperative blood pressure control regimen [START_REF] Baker | Reducing the incidence of hematoma requiring surgical evacuation following male rhytidectomy: a 30-year review of 985 cases[END_REF] . The appearance of a haematoma correlates with postoperative peak blood pressure. Certain authors have proposed using anti-hypertensive agents like Clonidine or beta-blockers (Atenolol) to control arterial blood pressure [START_REF] Ramanadham | Evolution of hypertension management in face lifting in 1089 patients: optimizing safety and outcomes[END_REF][START_REF] Moreira | Atenolol prevents the formation of expansive hematoma after rhytidoplasty[END_REF]. The adrenaline injected in the local anaesthesia solution is slowly resorbed and the vasoconstrictive action continues for 4 to 10 hours (8). This long latency reduces bleeding during the peri-operative period, when variations in blood pressure are the most frequent. The early return home to a stress-free, welcoming environment reduces the risk of late rebound hypertension (>10 hours), which is sometimes observed when nurses do their night ward rounds. A massive haematoma can be life-threatening by obstructing the patient's upper airways.

The presence of even a small haematoma can contribute to partial or total necrosis of the skin flap. The skin may also change colour through the fixation of haemoglobin in the subcutaneous tissue. Resorption of a haematoma can leave residual changes of shape and skin texture. All of these phenomena have an impact on the aesthetic result.

Some authors have proposed inserting drains to prevent the development of haematomas.

Perkins et al tried to evaluate the rate of hematoma and seroma formation in a series of rhytidectomies performed with and without the use of intrawound vacuum drains. A retrospective chart review was conducted on 222 consecutive face-lifts performed by a single surgeon using an identical surgical technique before and after the use of vacuum wound drains. The review identified hematomas in 8% of surgeries without vacuum drains and 7% with vacuum drains. The decrease in hematoma formation did not reach statistical significance [START_REF] Perkins | Prevention of seromas and hematomas after face-lift surgery with the use of postoperative vacuum drains[END_REF]. This practice has been frequently called into question in recent studies which have shown that drains do not prevent or reduce the risk of haematoma [START_REF] Jones | The efficacy of surgical drainage in cervicofacial rhytidectomy: a prospective, randomized, controlled trial[END_REF]. The presence of a drain can lead to complications by facilitating the bacterial contamination of surgical wounds, and can also lead to discomfort and anxiety in patients.

We used a drain without suction in our procedure. It only stayed one day. This was supposed to be useful to drain a hematoma if a mechanical drainage was performed as well. In real terms, it appeared to be useless, since hematoma had to be evacuated during a surgical procedure. Therefore, drainage in this context is more an old habit (supposedly, relieving the "stress" of the surgeon) rather than a useful procedure. Regarding the results of our study, drainage won't be used during this surgery anymore. Beside being inefficient to avoid hematoma, it can cause bacterial contamination and patient discomfort.

The use of fibrin-based biological glue was tested to reduce the surgical dead space by sticking the skin flap to deeper tissues, and thereby reducing the risk of haematoma and seroma. Studies have not clearly demonstrated the efficacy of this treatment [START_REF] Killion | A systematic examination of the effect of tissue glues on rhytidectomy complications[END_REF], which carries a risk of viral transmission as the fibrin is a blood product.

Grover et al determined the parameters which were significantly associated with formation of haematoma in a consecutive series of facelifts performed by two high-volume operators. The records of 1078 patients who underwent a facelift between 1994 and 1999 were reviewed: 45 haematomas occurred (4.2%). Analysis revealed significant associations between haematoma formation and anterior platysmaplasty (P= 0.009), systolic pressure (P= 0.02), gender (P= 0.03), aspirin or non-steroidal anti-inflammatory intake (P= 0.04) and smoking (P= 0.049). Preoperative hypertension was found to be significantly associated with haematoma following rhytidectomy. This is consistent with the findings of Straith et al who reported a series of 500 facelifts in which 8 patients suffered haematomas that required surgical evacuation [START_REF] Grover | The prevention of haematoma following rhytidectomy: a review of 1078 consecutive facelifts[END_REF].

Our study reviewed 1500 facelifts with SMASectomy : dissection, resection, elevation and suture under tension of the SMAS flap. We did not perform anterior platysmaplasty (18), which has been correlated with a higher risk of haematoma [START_REF] Grover | The prevention of haematoma following rhytidectomy: a review of 1078 consecutive facelifts[END_REF]. The SMAS flap was sutured with Vicryl 3-0 and no purse-string reinforcement was done [START_REF] Van Der Lei | The purse-string reinforced SMASectomy short scar facelift[END_REF]. We only used glue for the skin closure. We are not convinced that glue closures can cause wound dehiscence because the real strength on skin closure comes from sub-cutaneous suture.

When hematoma was developped, the wound was partially re-openned to evacuate the blood and therefore caused wound dehiscence.

Our surgical technique was a limited skin dissection without additional steps such as submandibular gland resection or platysmoplasty. This type of surgical procedure is possible under local anaesthesia.

The reduction of the number of steps in our surgical technique to only SMASectomy rhytidectomy and cervical liposuction can explain the very low frequency of hematoma in this cohort.

Our philosophy concerning the prevention of haematomas following a cervicofacial lift is to avoid drugs side effects as much as possible. For our team, the procedure is straightforward and quick, and does not require any modification of the patient's usual treatment. Giving patients new drugs, such as premedication, modifies their basal physiological status, and in particular can affect their blood pressure. Preventive intra-and postoperative antihypertensive treatments are not used to avoid rebound hypertension caused by the inevitable withdrawal of these drugs. The simplification of medical treatments, the non-use of fibrin-based biological glue and the absence of general anesthesia make the procedure far simpler.

Patients therefore undergo surgery with their basal physiological blood pressure and with as little pre-intra-and postoperative variation as possible. Patients can return home on the same day free from the side effects of anaesthesia. Convalescence is easier and faster.

Conclusion

Cervicofacial lift under local anaesthesia does not radically modify the surgical technique. It enables better control of a patient's arterial blood pressure throughout the surgical procedure and avoids the variations in blood pressure which can cause haematoma. The cooperation of the patient facilitates changes of position and diminishes the risk of contamination induced by intubation. Moreover, the absence of an endotracheal tube makes the surgery easier. Finally, the absence of multiple general anaesthesia agents: hypnotics, morphine and neuromuscular blocking agents, means that patients can return home quickly and safely thus accelerating convalescence.

There is no Conflict of Interest or Funding for this work.

Discussion:

The principal advantage of local anaesthesia as mentioned above is the cooperation of the patient when the sterile drapes are installed. Patients can also turn their heads to facilitate skin dissection and excision. The absence of intubation also simplifies these procedures and reduces the risk of contamination. In addition, as there is no rigidity between the neck and chin caused by the intubation, the surgery is more precise, notably regarding liposuction under the chin and tightening of the cervical SMAS, and the absence of septic adhesive bandage on the face and neck reduces the risk of contamination of the surgical wound.

Infiltration using a blunt cannula is painless and atraumatic, and the adrenaline gives a bloodless operating field, thus facilitating the dissection [START_REF] Klein | Tumescent technique for local anesthesia[END_REF][START_REF] Noël | Tumescent local anesthesia[END_REF]. The liposuction phase creates tunnels, which facilitate the subcutaneous dissection.

The most common complication following facelift surgery is haematoma, and the most critical risk factor that causes haematoma is elevated systolic blood pressure. In general, systolic blood pressure of <150 mm Hg and diastolic blood pressure of >65 mm Hg are recommended. The possible causes of perioperative hypertension are anxiety, epinephrine, pain, postoperative nausea and vomiting [START_REF] Chung | Perioperative Hypertension Management during Facelift under Local Anesthesia with Intravenous Hypnotics[END_REF]. Another major advantage of local anaesthesia techniques is the stability of blood pressure during and after the procedure. Indeed, the drugs used in general anaesthesia systematically reduce arterial blood pressure, which is maintained at around 90 mmHg. Under local anaesthesia, the patient's blood pressure is maintained at normal levels (generally between 120 and 140 mmHg). Haemostasis is easier to maintain at blood pressure levels close to the patient's usual arterial blood pressure. Moreover, following local anaesthesia there is no rebound hypertension, which is seen when patients wake up after


Maxime Vanmechelen

Diether Lambrechts

Thomas Van Brussel

Annelies Verbiest

Gabrielle Couchy

Patrick Schöffski

Herlinde Dumez

Philip R Debruyne

Evelyne Lerut

Jean-Pascal Machiels

Vincent Richard

Maarten Albersen

Vincent Verschaeve

Stéphane Oudard

Arnaud Méjean

Pascal Wolter

Jessica Zucman-Rossi

Benoit Beuselinck
email: benoit.beuselinck@uzleuven.be

Fibroblast Growth Factor Receptor-2 polymorphism rs2981582 is correlated with progression-free survival and overall survival in patients with metastatic clear-cell renal cell carcinoma treated with sunitinib

Keywords: clear-cell renal cell carcinoma, sunitinib, outcome, single nucleotide polymorphisms, fibroblast growth factor receptor MICRO-

We describe a potential biomarker associated with progression-free survival and overall survival on sunitinib in metastatic clear-cell renal cell carcinoma. rs2981582 is a polymorphism in the fibroblastgrowth-factor-receptor-2. In our series of 154 patients treated with sunitinib, the TT-variant, present in 13% of the patients, was associated with shorter progression-free survival and overall survival.

CLINICAL PRACTICE POINTS

Biomarkers predicting outcome on vascular-endothelial-growth-factor-receptor tyrosine kinase inhibitors (VEGFR-TKIs) in metastatic clear-cell renal cell carcinoma are lacking. We have found rs2981582, a polymorphism in the fibroblast-growth-factor-receptor-2 (FGFR2), to be a potential biomarker associated with progression-free survival and overall survival on the VEGFR-TKI sunitinib in metastatic clear-cell renal cell carcinoma. In our series of 154 patients, TT-variant carriers had a poorer outcome compared to CT/CC-carriers: median progression-free survival was 8 versus 15 months (p=0.0007) and median overall survival 22 versus 33 months (p=0.04), respectively. Moreover, median shrinkage of selected tumor target lesions during treatment with sunitinib was -16% versus -31% (p=0.002). On multivariate analysis, rs2981582 remained an independent predictor of progression-free survival and overall survival. Previously, the same impact was shown in metastatic clear-cell renal cell carcinoma patients treated with the VEGFR-TKI pazopanib. TT-variant carriers might have increased angiogenesis through the FGFR2-pathway, leading to escape of the tumor when treated with sunitinib or pazopanib. These findings, when validated, might have a clinical impact in future: they could be used for patient counselling on prognosis and might also explain the efficacy of FGFR-blockers in m-ccRCC.

3

INTRODUCTION

Clear-cell renal cell carcinoma (ccRCC) is characterized by ubiquitous loss of a functional Von Hippel Lindau (VHL) protein, caused by mutation, promotor hypermethylation or loss of heterozygosity [START_REF] Sato | Integrated molecular analysis of clear-cell renal cell carcinoma[END_REF][START_REF] Genome | Comprehensive molecular characterization of clear cell renal cell carcinoma[END_REF]. This results in an increase in hypoxia inducible factor (HIF) [START_REF] Fakih | Intramedullary spinal cord metastasis (ISCM) in renal cell carcinoma: a series of six cases[END_REF] and, among other effects, subsequent activation of vascular endothelial growth factor (VEGF) dependent angiogenesis. Targeted therapies directed against the VEGF-pathway are the current standard of care as first-line treatment of m-ccRCC patients [START_REF] Motzer | Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma[END_REF][START_REF] Motzer | Pazopanib versus sunitinib in metastatic renal-cell carcinoma[END_REF].

Apart from bevacizumab, which is an anti-VEGF antibody, these therapies are tyrosine kinase inhibitors (TKIs) such as sunitinib, pazopanib, axitinib, cabozantinib or sorafenib, inhibiting VEGF-receptors (VEGFR) and other molecular targets. Sunitinib and pazopanib are most often used in first-line therapy [START_REF] Motzer | Sunitinib versus interferon alfa in metastatic renal-cell carcinoma[END_REF][START_REF] Sternberg | Pazopanib in locally advanced or metastatic renal cell carcinoma: results of a randomized phase III trial[END_REF]. Clinical responses are highly variable and even patients who initially respond well will ultimately develop secondary resistance [START_REF] Mckay | Impact of Bone and Liver Metastases on Patients with Renal Cell Carcinoma Treated with Targeted Therapy[END_REF]. Unfortunately, there are no validated predictive biomarkers for response or resistance in m-ccRCC patients treated with VEGFR-TKIs.

The VEGF-dependent pro-angiogenetic pathway targeted by these therapies has been the object of several studies searching for predictive biomarkers. VHL-mutations are not correlated with efficacy [START_REF] Kim | Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review[END_REF]. On a transcriptomic level, upregulation of angiogenesis-related genes has been associated with better response to VEGFR-TKIs [START_REF] You | Predictive role of tissue-based molecular markers in patients treated with sunitinib for metastatic renal cell carcinoma[END_REF][START_REF] Garcia-Donas | Prospective study assessing hypoxia-related proteins as markers for the outcome of treatment with sunitinib in advanced clear-cell renal cell carcinoma[END_REF][START_REF] Beuselinck | Pro-angiogenic gene expression is associated with better outcome on sunitinib in metastatic clear-cell renal cell carcinoma[END_REF][START_REF] Beuselinck | Molecular subtypes of clear cell renal cell carcinoma are associated with sunitinib response in the metastatic setting. Clinical cancer research : an official journal of the American Association for[END_REF]. Finally, several studies have linked SNPs in genes encoding proteins in the VEGF-pathway with outcome in m-ccRCC patients treated with sunitinib [START_REF] Garcia-Donas | Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with first-line sunitinib: a multicentre, observational, prospective study[END_REF][START_REF] Dornbusch | Evaluation of polymorphisms in angiogenesis-related genes as predictive and prognostic markers for sunitinib-treated metastatic renal cell carcinoma patients[END_REF][START_REF] Beuselinck | Validation of VEGFR1 rs9582036 as predictive biomarker in metastatic clear-cell renal cell carcinoma patients treated with sunitinib[END_REF][START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF]. However, validation of these findings in independent patient series has been challenging [START_REF] Liu | Meta-analysis on the association of VEGFR1 genetic variants with sunitinib outcome in metastatic renal cell carcinoma patients[END_REF][START_REF] Motzer | Investigation of novel circulating proteins, germ line single-nucleotide polymorphisms, and molecular tumor markers as potential efficacy biomarkers of first-line sunitinib therapy for advanced renal cell carcinoma[END_REF].

Activation of VEGF-independent neo-angiogenesis, for instance through the FGFR-pathway, is suggested as one of the putative mechanisms of resistance to VEGF-directed therapy [START_REF] Casanovas | Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors[END_REF]. When the VEGFdependent pro-angiogenetic pathway is blocked by VEGFR-TKIs, neo-angiogenesis and tumor growth could continue through the FGFR-pathway. Therefore, FGFR-blockers such as dovitinib and lenvatinib have been tested in m-ccRCC [START_REF] Motzer | Dovitinib versus sorafenib for third-line targeted treatment of patients with metastatic renal cell carcinoma: an open-label, randomised phase 3 trial[END_REF][START_REF] Motzer | Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial[END_REF]. The TT-variant of SNP rs2981582 C>T in FGFR2 has been associated with increased FGFR2 gene expression in breast cancer cell lines [START_REF] Meyer | Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer[END_REF].

The possible impact of SNP rs2981582 in FGFR2 on outcome in m-RCC patients treated with VEGFR-TKIs was previously shown in patients treated with pazopanib. In 380 patients treated in first-line with pazopanib in three studies, among them the pazopanib pivotal trial (7), Xu et al. showed the negative impact of the TT-variant in rs2981582 on PFS (p=0.053) [START_REF] Xu | Pazopanib efficacy in renal cell carcinoma: evidence for predictive genetic markers in angiogenesis-related and exposure-related genes[END_REF]. In 241 patients included in the pazopanib pivotal trial, rs2981582 was associated with mOS (p=0.008; HR 1.40; 95%CI 1.09-1.81) [START_REF] Xu | IL8 polymorphisms and overall survival in pazopanib-or sunitinib-treated patients with renal cell carcinoma[END_REF], favoring

MATERIALS AND METHODS

For this retrospective study, germ-line DNA samples were collected in the "CIT-rein" kidney tumor bank (frozen normal kidney tissue) in patients treated at the University Hospitals Leuven (peripheral blood samples) and in patients included in the Belgian multicentric METASUN study (peripheral blood samples).

The French-Belgian multicentric CIT-rein kidney tumor bank contains frozen kidney tumor samples collected at 20 academic hospitals in Belgium and France. Eligible patients could have received cytokines as systemic treatment for kidney tumors before starting sunitinib as a monotherapy. Patients who received previous treatment with any other targeted therapy before starting sunitinib were excluded. The study was approved by the medical ethics review boards of all participating institutions, and signed informed consent was obtained from all patients. DNA was isolated at INSERM U1162 in Paris, France, from fresh frozen normal kidney tissue sampled in the nephrectomy specimen using the Qiaquick extraction kit (Qiagen, Valencia, CA, USA) and quantified by fluorometry (Fluoroskan Thermo Labsystems, Cergy-Pontoise, France). DNA was isolated from peripheral blood at the Vesalius Research Center in Leuven, Belgium, with the Qiagen DNA kit (Qiagen, Valencia, CA, USA) and final DNA concentration quantified with Nanodrop (Nanodrop, Wilmington, DE, USA). High-throughput SNP genotyping was performed at the Vesalius Research Center in Leuven, Belgium, using the Sequenom MassArray platform (Sequenom, San Diego, CA, USA) [START_REF] Reumers | Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing[END_REF]. Genotyping analysis was performed by investigators blinded for the clinical data.

All patients were treated in routine clinical practice. The treating oncologist could change treatment approach concerning drug schedule, dose-reduction policy, and timing of radiological assessments in accordance with current local practice guidelines. CT thorax-abdomen was performed in most cases every two cycles of sunitinib. All the patients started their sunitinib therapy at the standard sunitinib dose of 50 mg/day four weeks on two weeks off. Commonly used prognostic factors were assessed: sarcomatoid dedifferentiation, presence of bone metastases and the variables included in the IMDC score (International Metastatic Renal Cell Carcinoma Database Consortium): baseline neutrophil count, baseline platelets and hemoglobin, calcium, time between initial diagnosis and start of systemic therapy and Karnofsky performance status [START_REF] Heng | Prognostic factors for overall survival in patients with metastatic renal cell carcinoma treated with vascular endothelial growth factor-targeted agents: results from a large, multicenter study[END_REF]. Primary kidney tumors were also classified according to the molecular ccrcc1-4 classification as described previously [START_REF] Beuselinck | Molecular subtypes of clear cell renal cell carcinoma are associated with sunitinib response in the metastatic setting. Clinical cancer research : an official journal of the American Association for[END_REF]. This expression-profile based classification has a prognostic value in patients treated with metastasectomy (28) and a predictive value in patients treated with sunitinib [START_REF] Beuselinck | Molecular subtypes of clear cell renal cell carcinoma are associated with sunitinib response in the metastatic setting. Clinical cancer research : an official journal of the American Association for[END_REF] or pazopanib [START_REF] Verbiest | Molecular Subtypes of Clear Cell Renal Cell Carcinoma Are Associated With Outcome During Pazopanib Therapy in the Metastatic Setting[END_REF].

Clinical data were collected at 19 different sites in France and Belgium. The main objective of the study was to investigate the impact of rs2981582 on outcome in m-ccRCC patients treated with sunitinib and to investigate whether this impact would be prognostic or predictive.

The primary endpoints of the study were PFS, RR and tumor shrinkage. The secondary endpoint was OS.

In fact, OS can be influenced by sequential therapies administered after first-line sunitinib, particularly immune checkpoint inhibitors, that have an activity mechanism thought to be independent of angiogenesis.

We defined PFS as the interval between the first day on treatment with sunitinib and the date of radiological progressive disease or death. Patients who had not progressed at database closure were censored at last follow-up. OS was defined as the interval between the first day on sunitinib and the date of death or last date of follow-up. Objective response was assessed by treating doctors using Response Evaluation Criteria in Solid Tumors (RECIST). We studied not only the impact of RECIST-categories (complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD)), but also the precise percentage of RECIST tumor shrinkage compared to baseline, whenever available. The precise percentage of tumor shrinkage can give additional and more precise information compared to the RECIST categories CR-PR-SD-PD. On one hand, although the difference between a SD with 29% of tumor shrinkage and a PR with 31% of shrinkage is not an important difference in shrinkage, patients are classified in another response category. On the other hand, two patients with tumor shrinkage of 35% and 95% will both be classified in the PR-group, but the response has been more important in the latter case.

The impact of rs2981582 was studied in a discovery and a validation cohort. The discovery cohort was composed of the 88 patients included in our previous publication [START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF], in which we reported the impact of SNPs in several genes such as VEGFR3, ABCB1, NR1/3, NR1/2, PDGFRA and FGFR2. However, in this previous publication, concerning FGFR2, we only reported outcome in CC-carriers (n=23) compared to TTcarriers (n=12), because we aimed to replicate data presented in 2011 in an abstract by Xu et al. comparing OS in CC-versus TT-carriers treated with pazopanib. Outcome for CT-carriers was not reported in this previous study. Now, we aimed to study more in detail the impact of the three different genotype combinations (CC, CT and TT) on outcome in our series of patients genotyped in 2011. The validation cohort was composed of new patient samples genotyped from 2013 on.

All patient characteristics were tested in univariate fashion to study the association with mPFS and mOS using Kaplan-Meier estimates and in a multivariate model using Cox proportional hazards. Fisher exact test was used to compare percentages and student's t-test was applied for comparison of tumor shrinkage between carriers of different genotypes. All variables that did correlate with PFS and OS on univariate analysis with a p-value of <0.2 were included in the multivariate analysis. Results with a p-value of <0.05 were considered as significant in the univariate and multivariate analyses. Statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) and XLSTAT software (Addinsoft, Paris, France).

RESULTS

Included patients

We included 154 patients who started sunitinib between November 2005 and July 2016 and closed the follow-up database in September 2017. This series included the 35 patients assessed in the project published earlier [START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF]. Table 1 shows the clinical characteristics of patients included in this project. Mean age at diagnosis was 59 years (range 30-80) with a male predominance (71%). The majority of patients were of Caucasian origin. 55% had Fuhrman grade IV ccRCCs on the initial nephrectomy specimen or biopsy. According to IMDC prognostic criteria, 15% of patients were categorized into the favorable risk group, 61% had intermediate and 24% poor risk. In 85 patients, the primary tumor was classified according to the ccrcc1-4 classification. At the time of final analysis, 121 (79%) patients had reached progression and 108 (70%) had died. The median follow-up was 47.5 months (range 2.0 -239.0 months) after the start of sunitinib. The global mPFS was 13 months and mOS 30 months. Best RECIST response assessment was available in 147 patients. 11/147 (7%) patients had a CR, 60/147 (41%) patients a PR, 53/147 (36%) SD and 23/147 (16%) PD as best response. In 6 patients, there was a clinical benefit, but response assessment was poorly defined in the medical records, and as a consequence, it was unclear whether the best response was either PR or SD in these 6 patients. One patient died after one month of treatment with sunitinib. These results are comparable to phase III and expanded access response data [START_REF] Motzer | Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma[END_REF][START_REF] Gore | Final results from the large sunitinib global expanded-access trial in metastatic renal cell carcinoma[END_REF] 1).

Validation cohort

The 1).

Total cohort

In the total cohort, mPFS was 8 and 15 months for TT-and CT/CC-carriers, respectively (p=0.0007). mOS was 22 and 33 months for the TT-and CT/CC-carriers, respectively (p=0.04) (Figure 1). PR rate was 37% in patients with the TT-genotype compared to 50% in patients with the CT/CC-genotype. This difference was not significant. Complete responses (n=11) were only noticed in the CT/CC-genotype subgroup.

Median tumor shrinkage was -16% for patients with the TT-genotype versus -31% for patients with the CT/CC-genotype (p=0.002) (Figure 2).

When comparing the three different genotypes separately, mPFS was 8, 15 and 14 months for patients with the TT-, CT-and CC-genotype, respectively (p=0.005). The curves of CT-and CC-carriers were overlapping (Figure 3). PR rate was 35%, 51% and 48%, for TT-, CT and CC-carriers, respectively, however, these differences were not significantly different. PD as best response was observed in 21%, 13% and 16% of the patients, respectively. The median percentage of tumor shrinkage was -16%, -35% and -28% for patients with the TT-, CT-and CC-genotype, respectively (p=0.09). mOS was 22, 35 and 19 months, respectively (p=0.04). The OS-curve of the CC-carriers lied in between the curves of the CTcarriers and TT-carriers (Figure 3).

Multivariate analysis

In the multivariate analysis, we included the commonly used prognostic markers that were significant on univariate analysis (the presence of bone metastases, baseline neutrophil count, baseline platelet count, sarcomatoid dedifferentiation, Karnofsky Performance Status, baseline hemoglobin levels, baseline lactate dehydrogenase activity and time between nephrectomy to systemic therapy <12 months (Table 2)). Table 1 shows that all baseline patient characteristics usually associated with prognosis, including IMDC score, were well balanced between TT-and CT/CC-carriers, except baseline LDH (above 1.5*upper limit of normal: 21% in TT-versus 4% in CT/CC-carriers; p=0.02). However, median baseline LDH-levels were identical in TT-and CT/CC-carriers (232,0 versus 247.5 U/L, p=0.8). In the multivariate analysis, rs2981582 remained as independently associated with PFS with a HR of 2.858 (p<0.0001; 95%CI 1.659-4.923) and with OS with a HR of 1.795 (p=0.049; 95%CI 1.003-3.212) (Table 3). LDH levels were not associated with PFS (HR 0.677 (95%CI 0.240-1.910); p= 0.46) nor OS (HR 0.531 (95%CI 0.181-1.562); p=0.25). Supplementary Figure 1 shows that the negative impact of the TT-variant on PFS can be observed in all IMDC risk group patients. Thus, the poor outcome for TT-patients seems not to be driven by the higher frequency of elevated LDH nor by IMDC risk stratification.

Supplementary internal validation

As an additional internal validation of our results, we analyzed the impact of rs2981582 in the subgroup of patients treated at Belgian (n=102) and at French sites (n=52) patients included in this study. An identical significant impact on mPFS was observed in both subgroups (Supplementary Figure 2).

DISCUSSION

The aim of this study was to investigate the impact of SNP rs2981582 in FGFR2 in patients with m-ccRCC treated with sunitinib as first-line VEGFR-targeted therapy.

In this series of 154 patients, we found a statistically and clinically significant impact of the TT-variant on outcome. Compared to patients with the CT/CC-genotype, TT patients had significantly poorer mPFS and mOS and less important tumor shrinkage on sunitinib. rs2981582 remained as an independent predictor of mPFS and mOS on multivariate analysis. The impact of rs2981582 was stronger on PFS than on OS, but we have to consider the impact of subsequent therapy lines, among them immune checkpoint inhibitors, on OS.

Considering merely the correlation with mPFS and mOS, it is impossible to differentiate if the impact of rs2981582 is prognostic or predictive. The impact of rs2981582 would be prognostic, if a longer mPFS and mOS are the consequence of a more indolent disease in CC/CT-carriers and a more aggressive disease in TT-carriers. The impact of rs2981582 would be predictive, if a longer mPFS and mOS are the result of an improved efficacy of sunitinib in CC/CT-carriers compared to TT-carriers. In the latter case, the polymorphism should also be strongly correlated to tumor shrinkage. Based on our findings, although rs2981582 was correlated to median tumor shrinkage, we still cannot state that rs2981582 is a predictive biomarker for response on sunitinib. Indeed, even in TT-carriers, partial responses have been noticed in our patient series.

Similar data in literature are scarce. rs2981582 in FGFR2 was previously found to be associated with treatment outcome in m-ccRCC patients treated in first-line with pazopanib. rs2981582 was associated with mPFS (p=0.053) [START_REF] Xu | Pazopanib efficacy in renal cell carcinoma: evidence for predictive genetic markers in angiogenesis-related and exposure-related genes[END_REF] and with mOS (p=0.008; HR 1.40; 95%CI 1.09-1.81) [START_REF] Xu | IL8 polymorphisms and overall survival in pazopanib-or sunitinib-treated patients with renal cell carcinoma[END_REF], favoring CT/CCcarriers.

The validation of findings on the prognostic or predictive value of specific SNPs in m-ccRCCs treated with VEGFR-TKIs has been challenging [START_REF] Beuselinck | Kidney cancer: Single nucleotide polymorphisms in mRCC-is their time up?[END_REF]. The most concordant results were found in SNPs in the efflux pump ABCB1 [START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF][START_REF] Diekstra | CYP3A5 and ABCB1 Polymorphisms as Predictors for Sunitinib Outcome in Metastatic Renal Cell Carcinoma[END_REF][START_REF] Van Der Veldt | Genetic polymorphisms associated with a prolonged progression-free survival in patients with metastatic renal cell cancer treated with sunitinib. Clinical cancer research : an official journal of the[END_REF][START_REF] Chu | Association of ABCB1 and FLT3 Polymorphisms with Toxicities and Survival in Asian Patients Receiving Sunitinib for Renal Cell Carcinoma[END_REF][START_REF] Beuselinck | Efflux pump ABCB1 single nucleotide polymorphisms and dose reductions in patients with metastatic renal cell carcinoma treated with sunitinib[END_REF] and in interleukin-8 [START_REF] Xu | IL8 polymorphisms and overall survival in pazopanib-or sunitinib-treated patients with renal cell carcinoma[END_REF][START_REF] Motzer | Germline Genetic Biomarkers of Sunitinib Efficacy in Advanced Renal Cell Carcinoma: Results From the RENAL EFFECT Trial[END_REF]. Findings on the impact of SNPs in VEGFR1 (rs9582036) [START_REF] Beuselinck | Validation of VEGFR1 rs9582036 as predictive biomarker in metastatic clear-cell renal cell carcinoma patients treated with sunitinib[END_REF] and VEGFR3 (rs307826) [START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF][START_REF] Garcia-Donas | Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with first-line sunitinib: a multicentre, observational, prospective study[END_REF], although similarly shown in independent series, were not confirmed in other patient cohorts [START_REF] Liu | Meta-analysis on the association of VEGFR1 genetic variants with sunitinib outcome in metastatic renal cell carcinoma patients[END_REF][START_REF] Motzer | Investigation of novel circulating proteins, germ line single-nucleotide polymorphisms, and molecular tumor markers as potential efficacy biomarkers of first-line sunitinib therapy for advanced renal cell carcinoma[END_REF]. However, findings concerning the impact of rs2981582 are now coherent in 154 patients treated with sunitinib and 380 with pazopanib, totalizing 534 patients. This is an argument in favor of the robustness of these findings, which now should be validated in further independent patient series.

FGFR2 amplifications and mutations have been described in multiple cancer types [START_REF] Brooks | Molecular pathways: fibroblast growth factor signaling: a new therapeutic opportunity in cancer. Clinical cancer research : an official journal of the[END_REF]. However, FGFR2 mutations are rare in ccRCCs ( 2) and data on FGFR2 amplification are scarce. FGFR2 is located on chromosome 10q26 and encodes a receptor tyrosine kinase that is involved in multiple processes like cell growth, invasiveness, mortality and VEGF-independent angiogenesis [START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF]. FGFR2 amplifications have been reported in up to 10% of gastric cancers, most of which are diffuse-type with relatively poor prognosis [START_REF] Kunii | FGFR2amplified gastric cancer cell lines require FGFR2 and Erbb3 signaling for growth and survival[END_REF]. In a series of 125 patients with invasive ductal breast carcinoma, a significant association between cytoplasmic FGFR2 expression levels and tumor size was shown. Higher expression levels of FGFR2

were associated with lower OS and disease-free survival [START_REF] Sun | Increased expression of fibroblastic growth factor receptor 2 is correlated with poor prognosis in patients with breast cancer[END_REF]. Finally, the association with rs2981582 and breast cancer susceptibility is another argument in favor of a (patho)physiologic impact of this polymorphism. In a meta-analysis, FGFR2 was confirmed as a breast cancer susceptibility gene, and various variants of FGFR2 are significantly associated with breast cancer risk. For rs2981582, 39 studies for a total of 93.000 patients and 107.000 controls were evaluated. The corresponding odds ratio (OR) for developing breast cancer in heterozygous individuals was 1.21 whereas homozygous individuals (TT) carried an OR of 1.48 compared to people carrying the wild type (CC) (p<0.001) [START_REF] Cui | Variants of FGFR2 and their associations with breast cancer risk: a HUGE systematic review and meta-analysis[END_REF].

The TT-polymorphism in rs2981582 906C>T leads to increased transcription and expression of FGFR2 [START_REF] Meyer | Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer[END_REF] and thus possibly to increased VEGF-independent angiogenesis. When VEGF inhibitors successfully block angiogenetic pathways that rely on VEGF, other pro-angiogenic factors and pathways, such as the FGF-FGFR-axis, can be activated and be responsible for further vessel growth and disease progression (kinase switch theory). The result is a stimulation of endothelial cell-, fibroblast-and tumor cell growth and function. Unfortunately, FGFR2 mRNA-expression data were not available. However, most probably, it will not be FGFR2-expression in the primary kidney tumor, but in metastases resisting to sunitinib which could be correlated to the FGFR2-genotype. Unfortunately, tissue samples of metastases resisting to systemic therapy are usually only rarely available.

FGFR inhibitors such as lenvatinib and dovitinib, have been tested in m-ccRCC in clinical studies.

Lenvatinib is a TKI targeting FGFR1,2,3,4 and VEGFR. Lenvatinib was tested in a phase II trial in patients progressing on a previous VEGF-targeted therapy. Patients received lenvatinib and the mammalian target of rapamycin (mTOR) inhibitor everolimus or single agent treatment with these two agents. Lenvatinib plus everolimus or lenvatinib alone resulted in a PFS benefit compared to everolimus in monotherapy. The RR was 43% in patients receiving lenvatinib plus everolimus, compared to 6% in patients receiving everolimus in monotherapy. This RR with lenvatinib was higher than the RR usually seen in second line VEGFR-TKIs [START_REF] Motzer | Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial[END_REF][START_REF] Sacre | Prognostic factors in second-line targeted therapy for metastatic clear-cell renal cell carcinoma after progression on an anti-vascular endothelial growth factor receptor tyrosine kinase inhibitor[END_REF]. Dovitinib is a TKI targeting, besides the VEGFR, also FGFR1 and FGFR3. Dovitinib was tested in a phase III study as a third-line therapy in m-ccRCC patients treated in first-line with VEGF-targeted therapy and in second-line with everolimus. Patients were randomized between dovitinib and sorafenib.

Surprisingly, mPFS and mOS were similar in both treatment arms and the study was considered negative.

The results of this study have challenged the hypothesis that resistance to anti-VEGF-TKIs is mainly due to FGFR activation [START_REF] Motzer | Dovitinib versus sorafenib for third-line targeted treatment of patients with metastatic renal cell carcinoma: an open-label, randomised phase 3 trial[END_REF]. Possibly, the difference in efficacy between lenvatinib and dovitinib can be explained by a larger FGFR-inhibition by lenvatinib.

Our pharmacogenomics study has several potential limitations. First, it was a retrospective, uncontrolled analysis of patients treated in several centers without a central protocol dictating the treatment schedule and dose modifications or the timing of radiological assessments. Secondly, because our patients were mainly Caucasian, the relevance of these polymorphisms needs to be assessed in other ethnic groups because of possible genetic heterogeneity. Finally, at this moment these findings cannot be used for patient selection for treatment with VEGFR-TKIs. However, these results provide further evidence that FGFR2 is involved in resistance to VEGFR-TKIs in m-ccRCC patients.

CONCLUSIONS

Polymorphism rs2981582 in FGFR2 is correlated to outcome in m-ccRCC patients treated with sunitinib.

The TT-genotype is associated with poorer PFS, poorer OS and reduced target lesion shrinkage during treatment compared to the CC-and CT-genotype. Prospective validation of this SNP is now waranted.

FIGURE 1 :FIGURE 2 :

 12 FIGURE 1: KAPLAN-MEIER ESTIMATES SHOWING THE IMPACT OF rs2981582 ON PROGRESSION-FREE SURVIVAL AND OVERALL SURVIVAL IN THE DISCOVERY COHORT, THE VALIDATION COHORT AND THE TOTAL PATIENT SERIES.

FIGURE 3 :

 3 FIGURE 3: KAPLAN-MEIER ESTIMATES SHOWING THE IMPACT OF THE THREE GENOTYPES (CC, CT AND TT) OF rs2981582 ON PROGRESSION-FREE SURVIVAL AND OVERALL SURVIVAL

  validation cohort was composed of 67 new patients, genotyped from 2013 on: 7 TT-carriers, 38 CTcarriers and 22 CC-carriers. mPFS was 6, 12, and 12 months, respectively, in TT-, CT-and CC-carriers (p=0.06). Again, Kaplan-Meier curves for PFS were overlapping in CC-and CT-carriers. When CT-and CC-carriers were pooled, mPFS was 6 versus 12 months for TT-and CT/CC-carriers (p=0.02; HR 0.2363

	95%CI 0.07142-0.7819). mOS was 13 versus 34 months the TT-and CT/CC-carriers (p=0.03; HR 0.2444
	95%CI 0.06865-0.8703)(Figure

TABLE 1 : PATIENT CHARACTERISTICS AT DIAGNOSIS AND AT THE START OF SUNITINIB TREATMENT AND BASELINE CLINICAL AND BIOCHEMICAL PARAMETERS ASSOCIATED WITH PFS AND OS

 1

	AT INITIAL DIAGNOSIS	TOTAL (=154)	CC+CT (n=134)	TT (n=20)	p-value
	Male		71% (109/154)	73% (98/134)	55% (11/20)	0.12
	Age (mean, range)		59 (30-80)	59 (35-78)	62 (30-80)
	Ethnic origin	Caucasian	90% (139/154)	91% (122/134)	85% (17/20)	0.42
		Unknown	10% (15/154)	9% (12/134)	15% (3/20)	0.42
	M1 (synchronous metastases)	57% (85/150)	56% (73/131)	63% (12/19)	0.63
	Fuhrman	Grade 4	55% (82/148)	56% (72/128)	50% (10/20)	0.64
	Sarcomatoid	≥25%	4% (5/138)	3% (4/120)	6% (1/18)	0.51
	dedifferentiation
	AT THE START OF SUNITINIB
	Karnofsky	≤ 70	16% (25/153)	17% (23/133)	10% (2/20)	0.53
	Performance Status
	Neutrophils	>4.500/mm³	46% (70/151)	44 % (58/131)	60% (12/20)	0.23
	Platelets	>400.000/mm³	18% (27/153)	18% (24/133)	15% (3/20)	1.00
	Hemoglobin	<13 g/dl (men)) Low (<11.5 g/dl (women) or	40% (61/153)	38% (51/133)	50% (10/20)	0.34
	LDH	>1.5ULN	6% (9/149)	4% (5/130)	21% (4/19)	0.02
	Corrected Calcium	>10 mg/dl	10% (11/112)	11% (11/98)	0% (0/14)	0.35
	Time from nephrectomy to systemic treatment	<12 months	65% (100/153)	63% (84/133)	80% (16/20)	0.21
	Immunotherapy before sunitinib	18% (27/153)	19% (25/134)	11% (2/19)	0.53
	Site of metastasis	Lung Liver	75% (116/154) 20% (31/154)	76% (102/134) 19% (26/134)	70% (14/20) 25% (5/20)	0.58 0.56
		Bone	36% (55/154)	36 % (48/134)	35% (7/20)	1.00
		Brain	8% (13/154)	9% (12/134)	5% (1/20)	1.00
		Ccrcc1	35% (30/85)	36% (26/73)	33% (4/12)	0.88
	Molecular ccrcc1-4	Ccrcc2	46% (39/85)	44% (32/73)	58% (7/12)	0.35
	classification	Ccrcc3	4% (3/85)	4% (3/73)	0% (0/12)	0.47
		Ccrcc4	15% (13/85)	16% (12/73)	8% (1/12)	0.47
		Favorable	15% (22/150)	15% (20/132)	11% (2/18)	1.00
	IMDC prognosis	Intermediate	61% (92/150)	61% (80/132)	67% (12/18)	0.80
		Poor	24% (36/150)	24% (32/132)	22% (4/18)	1.00

TABLE 2 : UNIVARIATE ANALYSIS -ASSOCIATION BETWEEN SNP AND OUTCOME

 2 In univariate analysis, median PFS and median OS were estimated by Kaplan-Meier and p-values are derived from a log-rank test. SNP: single nucleotide polymorphism. PFS: progression free survival. OS: overall survival. FGFR2: fibroblast growth factor receptor 2. LDH: lactate dehydrogenase activity. ULN: upper limit of normal.

	VARIABLES		Number	Median PFS	p-value	Median	p-value
			of	(months)		OS
			patients			(months)
	FGFR2 rs2981582 906C>T	No	134	15	0.0007	33	0.04
		Yes	20	8		22
	Bone metastases	No	99	14	0.098	34	0.01
		Yes	55	11		19
	Neutrophil count >4.500/mm³	No	139	14	0.0001	31	<0.0001
		Yes	13	3		6
	Platelet count >400.000/mm³	No	133	14	0.01	34	0.0002
		Yes	20	7		13.5
	Sarcomatoid dedifferentiation ≥25%	No	133	14	0.0006	30	0.008
		Yes	5	2		14
	Karnofsky Performance Status ≤70	No	128	14	0.007	31	0.006
		Yes	25	8		14
	Hemoglobin low	No	70	16	0.097	35	0.18
	<11.5 g/dl women or <13 g/dl men	Yes	83	11		23
	LDH >1.5 ULN	No	140	14	0.11	31	0.15
		Yes	9	10		22
	Corrected Calcium >10mg/dl	No	137	13	0.46	30	0.48
		Yes	11	21		29
	Time from nephrectomy to	No	53	15	0.08	42	0.01
	systemic treatment <12m	Yes	100	12		27

TABLE 3 : MULTIVARIATE ANALYSIS FOR PFS AND OS

 3

	Variable

CT/CC-carriers. We previously published the impact of rs2981582 on outcome in m-RCC patients treated with sunitinib. We compared outcome in 23 patients with the CC-genotype and 12 with the TT-genotype. mPFS was 14 versus 7.5 months, respectively (p=0.012), but no impact on OS was shown[START_REF] Beuselinck | Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib[END_REF]. Outcome of the CT-carries was not studied, because our analysis at that moment was based on an abstract of Xu et al. comparing OS in CC-versus TT-carriers treated with pazopanib.The aim of the present study was to validate the impact of SNP rs2981582 in a larger series of m-ccRCC patients treated with sunitinib as first-line VEGF-targeted therapy and to study more in detail the impact of the three different genotype combinations (CC, CT and TT) on outcome.

GRANT SUPPORT