European Journal of Cancer (2013) 49, 2161–2169

A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

journa l homepag e : www.e j cancer . com

avb3 Integrin and Fibroblast growth factor receptor 1(FGFR1): Prognostic factors in a phase I–II clinical trialassociating continuous administration of Tipifarnibwith radiotherapy for patients with newly diagnosed glioblastoma

Anne Ducassou a, Emmanuelle Uro-Coste b,c, Pierre Verrelle d, Thomas Filleron e,Alexandra Benouaich-Amiel f, Vincent Lubrano g,c, Jean-Christophe Sol g,c,Marie-Bernadette Delisle b,c, Gilles Favre h,c, Solea Ken a, Anne Laprie a,c,Peter De Porre i, Christine Toulas h, Muriel Poublanc e,Elizabeth Cohen-Jonathan Moyal a,c,h,⇑

a Institut Claudius Regaud, Departement de Radiotherapie, Toulouse F-31000, Franceb CHU de Toulouse, Service d’Anatomie Pathologique, Hopital de Rangueil, Toulouse F-31000, Francec Universite Paul Sabatier, Toulouse F-31000, Franced Centre Jean Perrin, Departement de Radiotherapie, Clermont-Ferrand 63000, Francee Institut Claudius Regaud, Bureau des Etudes Cliniques, Toulouse F-31000, Francef Institut Claudius Regaud, Departement d’Oncologie Medicale, Toulouse F-31000, Franceg CHU de Toulouse, Service de Neurochirurgie, Hopital de Rangueil, Toulouse F-31000, Franceh INSERM, Unite 1037, CRCT, Toulouse F-31000, Francei Janssen Research & Development, Turnhoutseweg 23, Beerse B-2340, Belgium

Available online 6 April 2013

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KEYWORDS

GlioblastomaFarnesyltransferaseFGFR1ILKIntegrinsRadiotherapyPhase II

59-8049/$ - see front matter

tp://dx.doi.org/10.1016/j.ejca.

Corresponding author: AddrPont St Pierre, 31052 TouloE-mail address: moyal.eliza

� 2013 E

2013.02.0

ess: INSEuse, Fra

beth@cla

Abstract Background: Based on our previous results showing the involvement of the farnesy-lated form of RhoB in glioblastoma radioresistance, we designed a phase II trial associatingthe farnesyltransferase inhibitor Tipifarnib with radiotherapy in patients with glioblastomaand studied the prognostic values of the proteins which we have previously shown control thispathway.Patients and methods: Patients were treated with 200 mg Tipifarnib (recommended dose (RD))given continuously during radiotherapy. Twenty-seven patients were included in the phase IIwhose primary end-point was time to progression (TTP). Overall survival (OS) and biomarkeranalysis were secondary end-points. Expressions of avb3, avb5 integrins, FAK, ILK,

lsevier Ltd. All rights reserved.

33

RM UMR 1037, CRCT Toulouse, Departement de Radiotherapie, Institut Claudius Regaud, 20–24 ruence. Tel.: +33 5 61 42 41 78; fax: +33 5 61 42 46 43.udiusregaud.fr (E. Cohen-Jonathan Moyal).

2162 A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169

fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) werestudied by immuno-histochemistry in the tumour of the nine patients treated at the RD duringthe previously performed phase I and on those of the phase II patients. We evaluated the cor-relation of the expressions of these proteins with the clinical outcome.Results: For the phase II patients median TTP was 23.1 weeks (95%CI = [15.4; 28.2]) whilethe median OS was 80.3 weeks (95%CI = [57.8; 102.7]). In the pooled phase I and II popula-tion, median OS was 60.4w (95%CI = [47.3; 97.6]) while median TTP was 18.1w(95%CI = [16.9; 25.6]). FGFR1 over-expression (HR = 4.65; 95%CI = [1.02; 21.21],p = 0.047) was correlated with shorter TTP while FGFR1 (HR = 4.1 (95% CI = [1.09–15.4]; p = 0.036)) and avb3 (HR = 10.38 (95%CI = [2.70; 39.87], p = 0.001)) over-expressionswere associated with reduced OS.Conclusion: Association of 200 mg Tipifarnib with radiotherapy shows promising OS but noincrease in TTP compared to historical data. FGFR1 and avb3 integrin are independent badprognostic factors of OS and TTP.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Despite post-operative combination of radiotherapyand temozolomide,1 the prognosis of the patients witha glioblastoma (GBM) remains poor because of a localrecurrence mainly due to resistance of the GBM cellsto radiotherapy.

Our team has been involved for several years in thedeciphering of molecular pathways involved in the regu-lation of tumour micro-environment and radioresis-tance. We have shown that fibroblast growth factor 2(FGF)-2 controls radioresistance through RhoB, whosefarnesylated form modulates GBM cell radioresistancein vitro but also controls hypoxia in vitro and in vivo inGBM models.2–5 Furthermore inhibition of farnesyla-tion of RhoB led to radiosensitisation, vascularisationnormalisation and oxygenation in GBM xenografts.3,6

We recently demonstrated that upstream of RhoB,avb3 and avb5 integrins control GBM radioresistancevia the integrin linked kinase (ILK) and RhoB.7 More-over, we showed in vitro and in vivo that these integrinscontrol a major factor of radio and chemoresistance,hypoxia, inhibition of this pathway leading to oxygena-tion and normalisation of the vascularisation.8,9

Because of the pivotal role of the farnesylated form ofRhoB in the GBM radioresistance, we developed aphase I/II trial combining the farnesyltransferase inhib-itor (FTI) Tipifarnib in continuous administration withradiotherapy in patients with GBM, allowing us todefine the recommended dose (RD) of 100 mg bid withencouraging results at this treatment dose.10 In this arti-cle, we report the results from the bicentric, open-label,single arm, phase II trial of 100 mg bid Tipifarnib givenconcurrently with conformational radiotherapy inadults with newly diagnosed GBM. Moreover, due toour previous results, we studied on the pre-treatmenttumour specimens from the patients enrolled in thephase I/II study, the expression of the proteins we previ-ously demonstrated to be involved in RhoB-mediatedGBM radioresistance and hypoxia.

2. Patients and methods

2.1. Patient eligibility

This phase I/II trial was approved by the local ethicscommittee (n�1-02-54), the institutional review board,and by the French Agency for Health Products(n�030201). All patients provided a written informedconsent. Thirteen patients were included in the phase Icomponent of this clinical trial.10 The phase II startedon December 2005 in the two French comprehensivecancer centres Claudius Regaud Institute in Toulouseand Jean Perrin Institute in Clermont Ferrand.

Patients with newly diagnosed, histologically con-firmed GBM multiforme (Grade IV astrocytoma)according to the World Health Organization criteria,11

previously untreated except for biopsy or surgery, wereeligible. No prior chemotherapy or prior cranial irradia-tion was allowed. Other inclusion criteria including age,biology and imaging criteria were described in the phaseI related article.10

2.1.1. Treatment plan

According to the result of the phase I,10 Tipifarnib((R115777; Zarnestra), Johnson & Johnson Pharmaceu-tical Research and Development, Beerse, Belgium), apotent and selective non-peptidomimetic FTI was orallyadministered daily at the RD of 100 mg bid every 12 hstarting one week before and until the end of the radio-therapy, continuously 7 days/week. No temozolomidetreatment was administered because the initiation dateof this phase I–II study was anterior to the date at whichTemozolomide became part of the GBM standardtreatment.

Radiotherapy was administered with a total dose of60 Gy in 2 Gy daily fractions delivered 5 d per week over6 weeks.12 No dose reduction was allowed.

At progression, all patients were treated withTemozolomide.

Table 1Immunohistochemical specifications of the antibodies used.

Antibody (Company) Clone Dilution Time ofincubation

Antigen retrieval

FAK (Cell Signaling Technology) 3285 1:100 1 h Incubation with a EDTA buffer (pH 9.0)for 40 min at 96 �C

ILK (Santa Cruz Biotechnology) 65-1 1:100 1 h Incubation with a EDTA buffer (pH 9.0)for 40 min at 96 �C

avb3 Integrin (Millipore-Chemicon) MAB 2008 cloneSAP

1:50 1 h Incubation with a citrate buffer (pH 6.0) for40 min at 96 �C

avb5 Integrin (Abcam) Ab15459 1:50 1 h Incubation with a citrate buffer (pH 6.0) for40 min at 96 �C

Fibroblast growth factor 2 (FGF2)(Calbiochem)

Ab3 3H3 1:200 Overnight 4 �C Incubation with a EDTA buffer (pH 9.0)for 40 min at 96 �C

Fibroblast growth factor receptor 1 (FGFR1)(Thermo Scientific)

M19B2 1:100 1 h Incubation with a citrate buffer (pH 6.0) for40 min at 96 �C

Table 2Patient characteristics of the phase II.

Characteristic N (%)

A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169 2163

All patients had a pre-treatment MRI (Siemens Mag-netom Avanto 1.5 T, Erlangen, Germany) within 7 dafter inclusion.

GenderMale 18 (66.7)Female 9 (33.3)

Age (years)Median (range) 59 (38–79)

Performance status (WHO)0 16 (18.5)1 10 (37)2 1 (3.7)

SurgeryBiopsy 5 (18.5)Partial resection 5 (18.5)Gross resection 17 (63)

RPA classesIII 2 (7.4)IV 20 (74.1)V 5 (18.5)

2.1.2. Biomarker study

This analysis was conducted at the pathology depart-ment of the University Hospital of Toulouse, France.

Paraffin blocks of the tumours from the patientsenrolled in the phase I and treated at the RD (N = 9)and from the patients enrolled in the phase II (N = 27)were collected, and immuno-stainings for FAK, ILK,avb3 and avb5 integrins, FGF2 and fibroblast growthfactor receptor 1 (FGFR1) were performed on 4-lm sec-tions (Table 1). Second anti-bodies were then used andantibody–antigen complexes were visualised with adetection kit (Dako K0675).

Two pathologists blinded to patient outcomes scoredeach sample first independently and then together.Results for each observer were averaged to obtain thefinal immunoreactive score (IRS), combining both infor-mation on percentage of labelled cells and intensity ofstaining as previously published.13

The methylation status of the methyl-guanine methyltransferase (MGMT) was also determined on paraffinblocks when available. The methylation-specific poly-merase chain reaction (MS-PCR) was used.14,15

2.1.3. Study end-points

The primary objective of this trial was to evaluate thetime to progression (TTP). Secondary objectives wereoverall survival (OS), objective response rate and thecorrelation between protein expression in the tumourand TTP or OS. A safety analysis was also conducted.

MRI was performed every 2 months over a period of6 months, and then every 3 months until progression.Tumour size was measured according to a volumetricanalysis by using the Sysiphe-Neuroimaging SoftwareToolbox,16 on three dimensional T1 + gadolinium andFlair sequences. Responses were characterised asfollows: complete response: disappearance of all

enhancing lesions evaluated by MRI, patient neurologi-cally stable or improved; partial response: evidence ofP50% reduction in tumour volume on T1 gado, patientneurologically stable or improved; progressive disease:25% increase of tumour volume on T1 gado or Flairor appearance of new lesions, patient neurologicallyworse; stable disease: all other situations. The cut-offdate was August 2010.

2.1.4. Statistical analysisWith a median TTP of 15w and a target of 30w, using

a 5% significance level and 80% power, 24 patients wererequired.17 Assuming a 10% drop-out rate, 27 patientswere included in the phase II.

Categorical variables were reported by frequenciesand percentages, continuous variables were presentedby median and range. TTP and OS were estimated byKaplan–Meier method. Univariate analyses were per-formed using the log-rank test. A minimum p-valueapproach was used to dichotomise proteins referringto the TTP. Multivariate analyses were performed using

Table 3Toxicities during the phase II study, according to the CTCAEv3.

G0(N; %)

G1(N;%)

G2(N; %)

G3(N;%)

G4(N;%)

Allergy/Immunology 26;96.3

1; 3.7 0; 0 0; 0 0; 0

Auditory/Ear 25;92.6

1; 3.7 1; 3.7 0; 0 0; 0

Blood/Bone Marrow 21;77.8

6;22.2

0; 0 0; 0 0; 0

Cardiac (general,Arrhythmia)

25;92.6

0; 0 2; 7.4 0; 0 0; 0

Constitutional symptoms 11;40.7

7;25.9

8; 29.6 1; 3.7 0; 0

Dermatology/Skin 13;48.1

8;29.6

5; 18.5 1; 3.7 0; 0

Gastrointestinal 13;48.1

6;22.2

6; 22.2 2; 7.4 0; 0

Haemorrhage/Bleeding 24;88.9

2; 7.4 1; 3.7 0; 0 0; 0

Infection 26;96.3

0; 0.0 1; 3.7 0; 0 0; 0

Lymphatic 26;96.3

1; 3.7 0; 0 0; 0 0; 0

Metabolic/Laboratory 19;70.4

7;25.9

0; 0 1; 3.7 0; 0

Musculoskeletal 23;85.2

2; 7.4 2; 7.4 0; 0 0; 0

Neurology 4; 14.8 5;18.5

16;59.3

2; 7.4 0; 0

Ocular/Visual 19;70.4

4;14.8

4; 14.8 0; 0 0; 0

Pain 11;40.7

3;11.1

11;40.7

2; 7.4 0; 0

Pulmonary/Upperrespiratory

25;92.6

1; 3.7 1; 3.7 0; 0 0; 0

Renal/Genitourinary 25;92.6

1; 3.7 1; 3.7 0; 0 0; 0

Vascular 26;96.3

1; 3.7 0; 0 0; 0 0; 0

Fig. 1. Time to progression and overall survival of the 27 patientstreated for glioblastoma (GBM) multiforme with radiotherapy andTipifarnib in the phase II study.

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Cox model. Variables with p < 0.15 in univariateanalysis were included in multivariate models. Ap-value < 0.05 was considered statistically significant inmultivariate analysis. Correlations between two contin-uous variables were investigated using Spearman’scoefficients.

3. Results

3.1. Phase II analysis

3.1.1. Demographic data

Twenty-seven patients were enrolled into the phase IIstudy between December 2005 and January 2009(Table 2).

3.1.2. Treatment

Median time of radiation treatment was 6.4w(range = (5.6–7.3)). All the patients received the fullcourse of radiotherapy without any interruption. Threepatients interrupted the Tipifarnib, one during 9 dbecause of a grade 3 cutaneous rash, the second one dur-ing one day and the last one during 3 d, due to a urinaryinfection with elevation of serum creatinin.

3.1.3. Safety

Toxicity was modest (Table 3). The majority of toxicevents were grade 1 or 2. Five serious adverse events(18.5%) were observed, and only one, a cutaneous rash,was a grade 3 and imputable to Tipifarnib. The otherserious adverse events were neurologic (seizure, speechimpairment) and vascular (1 thrombosis) not related toTipifarnib.

3.1.4. Response rate and survival

After a median follow up of 80w (range = (21.3;180.1)), 26 patients had progressed. Median TTP was23.1w (95%CI = [15.4; 28.2]); 33.33% (95%CI = [16.77;50.86]) of the patients had no progression at 6 monthsand 7.41% (95%CI = [1.30; 21.03]) at 1 year (Fig. 1).The best response was stable disease in 14 patients(51.9%).

At last follow-up, 21 patients had died and the OSwas 74% at 1 year (95%CI = [53.19; 86.70]) and26.59% at 2 years (95%CI = [10.47; 45.96]) (Fig. 1).The median OS was 80.3w (95%CI = [57.8; 102.7]).

3.2. Biological marker studies performed on the phase I

and phase II patients

Among the 36 patients treated at the RD during thephase I–II trial, the expression of the following six pro-teins were studied on available samples of 35 patients:ILK (N = 34), FGFR1 (N = 33), FGF2 (N = 35),avb3 (N = 35), avb5 (N = 33) and FAK (N = 32),.Moreover, only 14/35 samples were available for evalu-

Fig. 2. Expression of FAK (A), avb3 integrin (B), avb5 integrin (C) anendothelial cells in the invasion front of patients with glioblastoma (GBM).D) Magnification, �100

Table 4Immuno-Reactive Score (IRS) of the tumour samples of the patientsenrolled in the phases I and II.

Markers (N, number of available samples) Immunoreactivescore: N

ILK Tumour (32) Median 4Range (0; 12)

ILK Endothelial cells (34) Median 12Range (0; 12)

Fibroblast growth factor receptor-1 (FGFR-1)tumour (33)

Median 6Range (0; 9)

FGFR-1 Endothelial cells (31) Median 6Range (0; 12)

Fibroblast growth factor 2 (FGF2) tumour (35) Median 6Range (0; 12)

avb3 Integrin Tumour (35) Media 4Range (0; 12)

avb3 Integrin endothelial cells (35) Median 8Range (0; 12)

avb5 Integrin tumour (32) Median 5Range (1; 12)

avb5 Integrin endothelial cells (33) Median 8Range (0; 12)

FAK Tumour (32) Median 2Range (0; 12)

FAK Endothelial cells (31) Median 8Range (0; 12)

A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169 2165

ating MGMT status. Among them, two were methylated(14.3%).

ILK, FAK, avb3 and avb5 integrin staining were pre-dominantly detected in endothelial cells, but were alsopresent in tumour cells (Table 4). As shown in Fig. 2,FAK (Fig. 2A), avb3 integrin (Fig. 2B) and avb5 inte-grin (Fig. 2C) were often observed in invasion fronts.FGF2 and FGFR1 (Fig. 2D) were predominantlyobserved in tumour cells. No correlation between pro-tein expression in endothelial cells and clinical outcomewas observed. avb3 Integrin expression in tumour cellswas statistically correlated with age (p = 0.0146).

ILK expression was statistically linked with avb3integrin expression (rho = 0.49; p = 0.0047), whereasFGF2 expression was inversely correlated to FAKexpression (rho = 0.3911; p = 0.0269).

3.3. Correlation between protein expressions in tumour

cells and clinical outcome

3.3.1. Time to progression

Thirty-three patients progressed during follow-up.The median TTP was 18.1w (95%CI = [16.9; 25.6]). In

d fibroblast growth factor receptor 1 (FGFR1) (D) in tumour andImmunohistochemistry was performed as described in ‘Section 2’. (A–

Table 5Univariate analysis (Cox analysis): time to progression (TTP) and overall survival (OS) for the patients of the phases I and II according tobiological markers (Prog = Progression).

N Time to progression Overall survival

Prog. Median 95%CI p Death Median 95%CI p

Age 0.1072 0.2628660 years 21 20 16.9 [14.4; 23.8] 16 74.7 [42.8; 107.3]>60 years 14 13 26.7 [17.1; 42] 13 52.4 [46.6; 80.3]

Gender 0.4731 0.6090Male 22 22 17.1 [14.4; 28.4] 18 64 [43.9; 102.7]Female 13 11 25.3 [16.3; 26.7] 11 57.9 [46.6; 101.3]

PS 0.7326 0.35120 22 21 23.1 [16.3; 28.3] 16 65 [44; 107.3]1/2 13 12 17.1 [14.1; 34.3] 13 52.4 [43.8; 101.3]

Surgery 0.1267 0.0024Biopsy 17 10 17 [12.8; 25.4] 11 46.6 [21.3; 52.4]Large 7 2 23.9 [16.3; 25.6] 7 74.7 [57.9; 131.9]

Fibroblast growth factor 2 (FGF2) 0.1692 0.8182<8 22 22 18.1 [14.4; 26.7] 18 72.8 [46.6; 101.3]P8 13 11 17.1 [16.3; 42] 11 52.4 [38; 103.3]

avb3 integrin 0.9221 0.0014<4 15 14 17.1 [14.4; 25.6] 10 102.7 [38; NR]P4 20 19 23.1 [16.3; 34.3] 19 47.3 [38.7; 60.4]

avb5 integrin 0.7943 0.4260<6 16 14 16.8 [14.1; 25.4] 13 60 [42.8; 131.8]P6 16 16 25.3 [16.3; 30.3] 13 59.4 [45.2; 97.6]

ILK 0.0772 0.0284<6 20 18 23.1 [15.4; 37.1] 15 97.6 [44; 131.8]P6 12 12 16.9 [13.6; 25.3] 11 48 [42.8; 74.7]

FAK 0.0488 0.0066<4 19 18 18.1 [13.6; 25.4] 17 48.0 [38.7; 64]P4 13 12 28.3 [15.4; 43.3] 9 97.6 [60.4; NR]

Fibroblast growth factor receptor 1 (FGFR1) 0.0748 0.1104<4 7 6 18.1 [13.6; 92.4] 5 102.7 [19.9; NR]P4 26 25 17.1 [15.4; 25.6] 22 57.9 [46.6; 80.3]

Table 6Multivariate analysis (Cox analysis): Time to progression (TTP) and overall survival (OS) for the patients of the phases I and II according tosurgical treatment, age and biological markers. The hazard ratios (HR) are presented with 95% confidence interval.

IRS Time to progression Overall survival

HR 95%CI p HR 95%CI p

SurgeryBiopsy 2.66 [0.85; 8.34] 0.093 3.98 [1.37; 11.56] 0.011Large surgery 1 1

ILK tumour cells<6 1 1P6 1.46 [0.61; 3.52] 0.396 0.69 [0.22; 2.17] 0.530

Fibroblast growth factor receptor 1 (FGFR1) tumour cells<4 1 1P4 4.65 [1.02; 21.21] 0.047 4.10 [1.09; 15.40] 0.036

avb3 Integrin tumour cells<4 NA 1P4 NA 10.38 [2.70; 39.87] 0.001

FAK tumour cells<4 0.96 [0.33; 2.80] 0.947 2.63 [0.90; 7.69] 0.077P4 1 1

Age at inclusion660 years 3.04 [1.0; 8.82] 0.041 NA>60 years 1 NA

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Fig. 3. Overall survival in univariate analysis of patients in phase I and II according to: (A) ILK expression in tumour cells (ILK low: IRS < 6 ;ILK high: IRS P 6); (B) FAK expression in tumour cells (FAK low: IRS < 4; FAK high: IRS P 4); (C) avb3 integrin expression in tumour cells(avb3 low: IRS < 4; avb3 high: IRS P 4); (D) fibroblast growth factor receptor 1 (FGFR1) expression in tumour cells (FGFR1 low: IRS < 4;FGFR1 high: IRS P 4).

A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169 2167

univariate analysis (Table 5), no correlation was foundbetween TTP and age, gender, PS or type of surgery.

Patients with ILK over-expression had a tendency tohave a shorter TTP (p = 0.0772) with a median TTP of23.1w when IRS < 6 and 16.9w when IRS P 6. FAKover-expression was significantly associated with alonger TTP (p = 0.0488). Although FGFR1 expressionwas not statistically correlated with TTP, patients withover-expression of FGFR1 seemed to be associated witha poorer TTP at 6 months, 42.9% when IRS < 4 and30.8% when IRS P 4.

In multivariate analysis (Table 6), the only factorsindependently associated with TTP were the age andthe FGFR1 over-expression.

3.4. Overall survival

Twenty-nine patients were dead at last follow-up. Themedian OS was 60.4w (95%CI = [47.3; 97.6]). In univar-iate analysis (Table 5), no correlation was identifiedbetween OS and age, gender or performance status.Patients who underwent a biopsy have a significantlypoorer survival compared to patients treated by grossresection (p = 0.002).

No significant correlation was found betweenFGFR1 expression and OS, but patients with FGFR1over-expression tend to have a poorer survival (medianOS = 57.9w) compared to those with lower expression(median OS = 102.7ws). avb3 Integrin over-expressionwas highly significantly associated with OS, since the2-year OS was 45.8% when IRS < 4 and 10% whenIRS P 4 (p = 0.0014). ILK over-expression was signifi-cantly associated with a poorer OS (p = 0.0284)(Fig. 3A). avb3 Integrin over-expression was statisticallyassociated with an increased risk of death (p = 0.0014),median OS were 102.7w (IRS < 4) and 47.3w (IRS P 4)(Fig. 3C). FAK over-expression was correlated with alower risk of death (p = 0.0066) (Fig. 3B). The medianOS were 48w for patients with FAK lower-expressionand 97.6w for patients who over-expressed FAK.

In the multivariate analysis (Table 6), patients whounderwent a biopsy were associated with a significantincreased risk of death compared to the patients whounderwent a surgical resection. The adjusted HR ofOS of patients with a FGFR1 over-expression(IRS P 4) compared with patients with low expressionof FGFR1 (IRS < 4) was 4.1 (95% CI, 1.09–15.4;p = 0.036) (Fig. 3D). avb3 Over-expression appeared

2168 A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169

significantly associated with an increased risk of death,the adjusted HR was estimated to be 10.38 (95%CI[2.70; 39.87], p = 0.001) (Fig. 3C). These results clearlyshow that the FGFR1 and avb3 integrin over-expres-sion in tumour cells were closely associated with worsesurvival rates in patients with a GBM.

4. Discussion

We confirm in this phase II study that continuousadministration of Tipifarnib with radiotherapy is safeat the dose of 100 mg bid. We obtained encouragingresults with a 18.5 month median OS compared to his-torical data of 14.6 months for the patients treated withthe Stupp protocol. However, at relapse, all patientswere treated with Temozolomide and 14 patientsreceived Bevacizumab. TTP was shorter in our studycompared to the historical results.1 Two hundred milli-grams per day of Tipifarnib (versus the usually RD of300 mg bid) even though given continuously during7 weeks may be not able to inhibit the farnesylation ofRhoB in the tumour, or inhibition of farnesylation alonemay be not sufficient to radiosensitise GBM. Trials asso-ciating radiotherapy with at least Temozolomide andTipifarnib in continuous administration should be per-formed. A phase I trial has shown an acceptable toler-ance of 300 mg bid Tipifarnib given concurrently withradiotherapy with or without Temozolomide, but noresults of survival were shown.18 Association of Tipifar-nib with radiotherapy has been studied in a paediatricphase I trial. The maximum tolerated dose was125 mg/m2 bid with 1-year survival and progression-freesurvival rates of 36.4% and 9.4%, respectively.19

Our biological analysis shows that avb3 integrin andFGFR1 are bad prognostic factors of OS and thatFGFR1 is a bad prognostic factor of TTP. We and oth-ers have shown that avb3 integrin controls GBM andendothelial cells’ radioresistance.7,20–22 We have shownthat this integrin controls GBM radioresistance viaILK and RhoB, but also controls hypoxia.7,9 In ourtrial, Tipifarnib was only given during radiotherapywithout any adjuvant treatment. avb3 Integrin beingalso involved in invasion and GBM angiogenesis, inhibi-tion of this pathway with Tipifarnib during radiother-apy may only act on the radiosensitisation but not onthe invasion, which may explain why avb3 integrinexpression appears as a bad prognostic factor of OS.Moreover, we show a correlation between avb3 integrinand ILK expressions, and that ILK expression, that wehave previously shown to control GBM radioresistancedownstream of avb3 integrin,7 is also associated with ashorter survival. These results confirm the importanceof this pathway in GBM radioresistance and aggressive-ness, and suggest that blocking farnesylation alone isnot sufficient to control GBM radioresistance or thatthese factors are prognostic markers independently of

farnesylation inhibition. avb3 Integrin has been shownto be a prognostic marker in other tumours such as cer-vix carcinoma and melanoma23,24 and a marker ofchemo-radiosensitivity in lung cancer.25 Our presentstudy is, to our knowledge, the first one which describesan impact of the avb3 integrin overexpression on sur-vival in patients with GBM and confirms the importanceto target this pathway in GBM treatment. Clinical trialsare currently developed associating Cilengitide, a spe-cific avb3 and avb5 integrin inhibitor, with Temozolo-mide and radiotherapy in patients with GBM.26

Because we have previously shown that FGF-2 con-trols radioresistance via RhoB,5,27 we studied FGF-2and FGFR1 expressions in the tumour of the patientsaccrued in our trial. We did not find a correlation betweenFGF-2 expression and clinical outcome as previouslyshown,28,29 however, we report that FGFR1 overexpres-sion in GBM tumour cells was correlated with a poor OSand a shorter TTP. No study has yet shown an impact ofFGFR1 overexpression on survival or TTP.30,31 In lungcancer, FGFR1 has been shown to confer a bad prognosisonly in univariate analysis.32 Our study confirms our lab-oratory results showing that FGFR1 controls GBMradioresistance while pharmacologic inhibition ofFGFR1 induces in vitro and in vivo radiosensitisation inGBM model.33 Our results strongly suggest that besidesthe avb3 integrin pathway, the FGFR pathway is also acrucial one to target in the treatment of GBM patientsin the aim to radiosensitise this tumour.

In conclusion, we report and confirm the safety of thecontinuous administration of 100 mg bid Tipifarnibwith radiotherapy with encouraging results for OS.However, because of modest effect on TTP, clinical trialsshould be performed associating Tipifanib with Tem-ozolomide and radiotherapy. Moreover, we show theimportance of the expression of avb3 integrin andFGFR1 as bad prognostic factors of OS and of TTPfor patients with GBM, confirming their involvementin GBM radioresistance and aggressiveness. After con-firming these results in a larger population, the studyof the expression of these receptors could be performedin patients enrolled in clinical trials associating inhibi-tors of these proteins with standard radio-chemother-apy, in order to better select patients who couldbenefit from these treatments.

Conflict of interest statement

Elizabeth Cohen-Jonathan Moyal has served on paidadvisory board for Merck KGaA. The other authorshave declared no conflict of interest.

Acknowledgement

This work was supported by a grant from Janssen-Cilag.

A. Ducassou et al. / European Journal of Cancer 49 (2013) 2161–2169 2169

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