translating the therapeutic potential of azd4547 in fgfr1...

Cancer Therapy: Preclinical

Translating the Therapeutic Potential of AZD4547 in FGFR1-Amplified Non–Small Cell Lung Cancer through the Use ofPatient-Derived Tumor Xenograft Models

Jingchuan Zhang1, Lin Zhang1, Xinying Su1, Ming Li1, Liang Xie1, Florian Malchers3, ShuQiong Fan1,XiaoLu Yin1, YanPing Xu1, Kunji Liu1, Zhengwei Dong1, Guanshan Zhu1, Ziliang Qian1, Lili Tang1, Ping Zhan1,Qunsheng Ji1, Elaine Kilgour2, Paul D. Smith2, A. Nigel Brooks2, Roman K. Thomas3,4, and Paul R. Gavine1

AbstractPurpose: To investigate the incidence of FGFR1 amplification in Chinese non–small cell lung cancer

(NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor

receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC

FGFR1–amplified patient-derived tumor xenograft (PDTX) models.

Experimental Design: A range of assays was used to assess the translational relevance of FGFR1

amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacody-

namic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n ¼ 127), and,

importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547.

Results: The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of

squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective

profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified

FGFR1 (GI50¼ 0.003–0.111 mmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of

five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in

vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level.

Conclusions: This study provides novel epidemiologic data through identification of FGFR1 gene

amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the

clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX

models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the

translational science presented here provides a strong rationale for investigation of AZD4547 as a

therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1. Clin

Cancer Res; 18(24); 6658–67. �2012 AACR.

IntroductionDespite significant geographical variations, the overall

global lung cancer incidence rate remains the highestamongst all cancer types (1). Disease mortality is of equalconcern with average 5-year survival rates of about 15% inthe United States (2). Non–small cell lung cancer (NSCLC)

accounts for about 85% of lung cancer cases and includesthe major histologic subtypes of adenocarcinoma, large cellcarcinoma, and squamous cell carcinoma(SCC). Regardlessof histology, treatment regimens have been dominatedlargely by platinum-based chemotherapeutics which typi-cally prolong median progression-free survival for less than1 year (3).However, recent advances inmolecularly targetedtreatment options, including EGFR inhibitors for EGFRmutation–positive tumors andanaplastic lymphomakinaseinhibitors for EML4-ALK fusion–positive tumors, have pro-vided significant improvements in survival (4–6). Unfortu-nately, these genetic events are rare and limited almostexclusively to the adenocarcinomas of "never smoked"patients and thus, SCC, primarily a smokers’ disease (7),still represents a disease of highunmet needboth in termsoftractable genetic targets and more effective therapies.

Further molecular segmentation has been suggestedrecently through the discovery of genetic amplification ofthe fibroblast growth factor receptor 1 (FGFR1) gene insquamous cell lung clinical samples (8, 9). FGFR pathway

Authors' Affiliations: 1Innovation Center China, AstraZeneca R&D,Shanghai, China; 2AstraZeneca R&D, Alderley Park, United Kingdom; andDepartments of 3Translational Genomics and 4Pathology, University ofCologne, Cologne, Germany

Note: Presented in part at the AACR Annual Meeting 2012 by P. R. Gavine(Poster number 1917 – Translating the therapeutic potential of AZD4547 inFGFR1-amplified non–small cell lung cancer through the use of primarylung explant models).

Corresponding Author: Paul R. Gavine, AstraZeneca Innovation CenterChina, Building 7, 898 Halei Road, Zhangjiang Hi-Tech Park, Shanghai,201203, PR China. Phone: 0086-15801994860; Fax: 0086-2161097700;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-2694

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(24) December 15, 20126658

on June 13, 2018. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 18, 2012; DOI: 10.1158/1078-0432.CCR-12-2694





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signaling normally contributes to the physiologic processesof tissue repair, hematopoiesis, angiogenesis, and embry-onic development; however, FGFs and FGFRs have emergedas driving oncogenes within a significant proportion ofhuman tumors. Deregulation of FGFR signaling throughmultiple mechanisms (including gene amplification) hasbeen documented within clinical samples of breast (10),multiple myeloma (11), noninvasive bladder (12), endo-metrial (13), gastric (14), and prostate cancers (15). Prece-dent for the success of targeting tumor-amplified tyrosinekinases as a therapeutic strategy has been shown clinicallyusing agents such as trastuzumab [ERBB2 in breast cancer(ref. 16)] and cetuximab [EGFR in colorectal cancer(ref. 17)]. We have previously characterized the in vitro andin vivo activity of the novel, potent, and selective small-molecule FGFR inhibitor, AZD4547 (18).Although lung cancer incidence and mortality rates

continue to decline in the West, the same does not holdtrue for China and Japan, where the principle risk factors ofsmoking, air pollution, and an ageing population contrib-ute to increasing incidence (19). Indeed, geographicaldifferences also appear to exist in disease histology witha higher prevalence of adenocarcinoma in Japan than inmost Western countries (20, 21). Tumor genetics alsodiffer significantly with region, with a higher incidence ofactivating EGFR mutations in Chinese NSCLCs than in theWest, and conversely a lower incidence of KRas mutation(20, 22).Accordingly, in this study, we aimed to investigate the

incidence of FGFR1 amplification in a Chinese NSCLCpatient population. AZD4547 was initially used to screena large panel of lung cancer cell lines and displayed potentantiproliferative activity in 2 cell lines containing amplifiedFGFR1. More importantly, we established translational

significance by showing potent AZD4547 antitumor effica-cy and pharmacodynamic activity in a panel of FGFR1-amplified squamous lung patient-derived tumor xenograft(PDTX) models.

Materials and MethodsAZD4547

Synthesis of N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5-diemthylpiperazin-1-yl)benzamide(AZD4547, AstraZeneca) has been described previously(18). The free base of AZD4547 (molecular weight ¼463.6) was used in all preclinical studies. For in vitrostudies, AZD4547 was prepared as a 10 mmol/L stocksolution and diluted in the relevant assay media. For in vivostudies, AZD4547 was formulated in a 1% (v/v) solution ofpolyoxyethylenesorbitan monooleate (Tween 80) in deio-nizedwater. Animals were given AZD4547 or vehicle controlonce daily (qd) by oral gavage.

Cell cultureCell lines were obtained from the American Type Culture

Collection (ATCC), the German Resource Centre for Bio-logical Material (DSMZ), or from internal collections asdescribed previously (23). DMS114 and NCI-H1975 celllines were routinely grown in RPMI-1640 supplementedwith 10% (v/v) fetal calf serum (Biochrom AG) and 2mmol/L L-glutamine (Invitrogen). Cell lines were geneti-cally tested and authenticated using the StemElite IDSystemKit (Promega) and were not cultured for more than 6months before conducting the work described here.

In vitro antiproliferative cell panel screeningCell line screeningwas conducted as described previously

(23). Viability was determined after 96 hours by measuringcellular ATP content (CellTiter-Glo, Promega). Half-maxi-mal inhibitory concentrations (GI50) were determined withthe statistical data analysis software "R" with the package"IC50."

FGFR1 FISHThe FGFR1 FISH probe was generated internally by

directly labeling BAC (CTD-2288L6) DNA with SpectrumRed (Vysis, Cat # 30-803400). The CEP8 Spectrum Greenprobe (Vysis, Cat #32-132008) for the centromeric regionofchromosome 8 was used as internal control. FISH assayswere conducted on 4-mm dewaxed and dehydrated form-aldehyde-fixed, paraffin-embedded (FFPE) sections. TheSpotLight Tissue pretreatment Kit (Invitrogen, Cat #00-8401) was used for pretreatment (boiled in reagent 1 for�15 minutes then coated with reagent 2 for �10 minutes,minor time adjustments were made for individual sam-ples). Sections and probes were co-denatured at 80�C for5 minutes and then hybridized at 37�C for 48 hours.After a quick post-wash-off process (0.3%NP40/1xSSC at75.5�C for 5 minutes, twice in 2�SSC at room temper-ature for 2 minutes), sections were finally mounted with0.3 mg/mL 40,6-diamidino-2-phenylindole (DAPI; Vector,

Translational RelevanceDeregulated fibroblast growth factor receptor (FGFR)

expression plays an important role in driving manycancers. In this report, we focus on squamous cellcarcinoma, where there exists a desperate need for effec-tive treatment therapies, and identify FGFR1 gene ampli-fication in a cohort of Chinese patients with non–smallcell lung cancer (NSCLC). Through the use of high-throughput cell line screening, we confirm the abilityof the novel FGFR inhibitor, AZD4547, to modulateFGFR signaling and inhibit tumor cell proliferation onlyin lung tumor cell lines harboring amplified FGFR1.Importantly, we extend the clinical significance of thesefindings to show tumor stasis or regression effects in apanel of FGFR1-amplified squamous lung patient–derived tumor xenograft models (PDTX), but not innonamplified models. Thus, our findings provide astrong rationale for the investigation of AZD4547 as anovel therapeutic option for patients with squamousNSCLCs harboring FGFR1 amplification.

AZD4547 Is Active in FGFR1-Amplified Squamous NSCLC PDTX Models

www.aacrjournals.org Clin Cancer Res; 18(24) December 15, 2012 6659




Cat #H-1200), and stored at 4�C avoiding light for atleast 30 minutes before scoring. FGFR1 gene and CEP8signals were observed using a fluorescence microscopeequipped with the appropriate filters allowing visualiza-tion of the intense red FGFR1 gene signals, the intensegreen chromosome 8 centromere signals, and the bluecounterstained nuclei. Enumeration of the FGFR1 geneand chromosome 8 was conducted by microscopic exam-ination of 50 tumor nuclei, which yielded a ratio ofFGFR1 to CEP8. Tumors with FGFR1 to CEP8 ratio �2or presence of �10% gene cluster were defined as ampli-fied (AMP).

Analysis of FGFR1 mRNA expressionRNAsamples fromPDTXmodelswere reverse transcribed

to cDNA using High Capacity RNA-to-cDNA master mix(Applied Biosystems). FGFR1 mRNA expression was thendetermined by quantitative PCR (qPCR) assay using the ABI7900HTplatform.About 50ngRNA inputwas used for eachqPCR reaction, and the gene expression results were nor-malized using glyceraldehyde 3-phosphate dehydrogenase(GAPDH) levels.

Protein expression analysisCell lines were treated with AZD4547 or dimethyl sulf-

oxide (DMSO) control for 2 hours at 37�C. Frozen tumorfragments were lysed in 1� cell lysis buffer (Cell SignalingTechnologies) containing phosphatase and protease inhi-bitors (Sigma) using a Fast Prep Homogenizer (MP Biome-dicals). Immunoprecipitation studies were conducted bypreclearing with protein G sepharose beads (Invitrogen) for2 hours at 4�C. Cleared supernatants, containing beads andprimary antibody, were then gently rocked overnight at4�C, followed by centrifugation, washing in fresh lysisbuffer and transfer directly to into SDS loading buffer.Western blotting was conducted using standard SDS-PAGEprocedures and antibody incubation conducted overnightat 4�C. Antibodies were obtained from the followingsources: FGFR1 (Epitomics), pFGFR (Y653/Y654) (CST),pFRS2 (Tyr436) (R&DSystems), pErk1/2 (Thr202/Tyr204),and GAPDH (Cell Signaling Technologies). Secondaryantibodies were applied and immunoreactive proteinsvisualized using "SuperSignal West Dura" Chemilumines-cence substrate according to the manufacturer’s instruc-tions (Pierce).

ImmunohistochemistryAntigen retrieval was conducted on FFPE tissues for 5

minutes in pH 6 retrieval buffer (S1699; Dako) followed bywashing in running tapwater for 5minutes. Sections (3mm)were rinsed in TBS with Tween (TBST) and incubated withendogenous peroxidase block on a LabVision autostainerfor 10 minutes. Slides were washed twice in TBST andthen incubated with primary antibody (FGFR1, Epitomics2144-1, 1:50; pERK, Thr202/Tyr204, CST4376, 1:50; pS6Ser240/244, CST2215, 1:100; CC3, CST9661, 1:100,respectively) for 60 minutes at room temperature andfinally washed twice in TBST. The appropriate Envision

or biotinylated goat anti-rabbit immunoglobulin second-ary antibodies were used (DAKO E0432 or Vector Labo-ratories, Inc) and staining was detected using diamino-benzidine (K3468; Dako). For Ki67 immunohistochemical(IHC) analysis, ARK kit (DAKO K3954) was used toprevent cross-reaction with mouse tissue. Sections wereincubated with biotinylated primary antibody (Ki67,DAKO M7240; 1: 100) for 15 minutes at room tempera-ture and then washed twice in TBST. Following 15 minutesstreptavidin–peroxidase treatment and washing in TBST,sections were developed and counterstained as describedabove.

For baseline expression or modulation detection, IHCscoring of FGFR1, phospho-S6, and phospho-Erk was cal-culated according to the following formula: scoring ¼ 0 �[% cells with no staining (0)]þ 1� [% cells staining faint tobarely visible (1þ)] þ 2 � [% cells staining weak tomoderately (2þ)] þ 3 � [% cells staining strongly (3þ)].This method combines positive intensity and percentagetumor cell staining and was determined by 2 separatepathologists usingmicroscopy. Quantification of Ki67- andCC3-positive signals was conducted using the Ariol system(Genetix).

In vivo efficacy studies using lung PDTX mouse modelsApanel of PDTXmousemodels was established as part of

a previous study using patient NSCLC tissues acquiredlocally during resection (manuscript submitted to EuropeanJournal of Cardio-Thoracic Surgery). Prior written informedconsent was obtained from all patients, and the studyprotocol was approved by the local hospital ethics com-mittee. Eight- to 10-week-old female nude (nu/nu) mice(Vital River) were used for in vivo studies. All experimentsusing immunodeficientmicewere carried out in accordancewith the guidelines approved by Institutional AnimalCare and Use Committees (IACUC). PDTX mouse modelswere established by directly implanting fresh surgical tumortissue into immunodeficient mice. Briefly, PDTX tissuefragments (�15 mm3) were implanted subcutaneouslyvia Trocar needle into female nude mice. Tumor-bearingmice with a tumor size range of 100 to 200 mm3 wererandomly divided into vehicle control or AZD4547treatment groups (8 animals per group). Animals weretreated orally by gavage needle. Subcutaneous tumors innude mice and mice body weight were measured twiceweekly. Tumor volumes were calculated by measuring 2perpendicular diameterswith calipers [formula:V¼ (length�width2)/2]. Percentage tumor growth inhibition [%TGI¼1� [change of tumor volume in treatment group/change oftumor volume in control group) � 100] was used for theevaluation of antitumor efficacy. For tumor regression, inwhich the tumor volume after treatment was smaller thanthe tumor volume at the beginning, the following equationwas used: T/T0 (%) ¼ 100 � DT/T0, DT is change of tumorvolume in treatment group. Statistical significance wasevaluated using a one-tailed, two-sample t test. P < 0.05was considered statistically significant. Tumor volume wascalculated as described previously (24).

Zhang et al.

Clin Cancer Res; 18(24) December 15, 2012 Clinical Cancer Research6660




ResultsPotent in vitro AZD4547 antiproliferative activitycorrelates with FGFR1 gene amplification andpharmacodynamic activity in a lung cancer cell linepanel screenTo characterize antiproliferative sensitivity to AZD4547,

an in vitro screenwas conductedusing a comprehensive lungtumor cell line panel (23, 25). Cellular proliferation wasassessed using a standard metabolism-based proliferationassay and GI50 values determined (Fig. 1A). Of 78 cell lines,only 2 (DMS114 and NCI-H1581) displayed a profoundsensitivity to AZD4547,withGI50 values of 0.111 and 0.003mmol/L, respectively. The DMS114 (small cell) and NCI-H1581 (large cell) lung cancer lines are well established andhavebeenpreviously shown toharbor amplifiedFGFR1 (8).

GI50 values for the remaining cell lines were all >4 mmol/L.An in vivo antitumor efficacy studyofmice harboringH1581xenografts treated daily with 12.5 mg/kg AZD4547 resultedin tumor regressions (Supplementary Fig. S1).

To assess pharmacodynamic modulation of FGFR signal-ing using AZD4547 in vitro, one amplified (DMS114) andone nonamplified control cell line (NCI-H1975—EGFRL858R/T790M)were chosen for further study. FISH stainingand further in vitro antiproliferative analysis confirmedFGFR1 amplification and AZD4547 sensitivity in theDMS114 line, but not in NCI-H1975 (Supplementary Fig.S2 and Supplementary Table S1). Pharmacodynamic mod-ulation of FGFR signaling within these lines was assessedfollowing a 2-hour incubation with AZD4547 and subse-quent Western blotting with anti-sera raised against phos-pho-FRS2(Y436) and phospho-Erk1/2(T202/Y204), bothwell-established downstream markers of FGFR signaling.Clear, titratable inhibition of both p-FRS2 and p-ERK1/2was observed in the DMS114 cell line, but not within NCI-H1975 (Fig. 1B). Thus, due to a proliferative dependence onFGFR1 gene amplification and signaling within theDMS114 cell line, strong pathway inhibition usingAZD4547 led to potent antiproliferative activity which wasnot observed in the nonaddicted NCI-H1975 cell line.

FGFR1 gene amplification is a frequent occurrence inChinese NSCLC patient samples

To establish the incidence of FGFR1 amplification inChineseNSCLCpatient samples, 127 tumor fragmentswereobtained locally at surgery and FFPE sections processed forFISH analysis using a specific FGFR1 gene probe. A total of11 of 127NSCLC samples (8.7%)were confirmed as FGFR1gene–amplified (defined as an FGFR1/CEP8 gene proberatio of �2 or cluster signals in �10% of tumorcells; Table 1). Of the 11 FGFR1-amplified tumor samples,6 of 48 were of squamous origin (12.5%) and 5 of 76 wereidentified as adenocarcinoma (7%).

Statistical analysis of patient clinicopathologic para-meters showed clear associations of FGFR1 amplificationwithmale gender and "ever smoker" status (P¼ 0.0046 and0.0022, respectively, c2 likelihood ratio test; Table 2). Torule out any potential confounding effect of gender, themale-only sampleswere further analyzed for smoking statusand FGFR1 amplification correlation. Despite not reachingstatistical significance, the data did suggest a correlative

Figure 1. Potent in vitro AZD4547 antiproliferative activity correlates withFGFR1 gene amplification and pharmacodynamic activity in a lungcancer cell line panel screen. A,GI50 values (y-axis) of AZD4547 across 78lung cancer cell lines (x-axis). Only DMS114 and NCI-1581 lines areFGFR1-amplified (copy number � 4). Cell line NCI-H1975 (used in Bbelow) is marked with an asterisk. B, AZD4547 treatment inhibits FGFRsignaling through p-FRS2 and p-Erk1/2 in DMS114, but not NCI-H1975.Cell lines were incubated for 2 hours with the stated concentrations ofAZD4547 and then lysed and immunoblotted for the proteins indicated.

Table 1. Incidence of FGFR1 gene amplification in Chinese NSCLC patient samples

NSCLC tumor histology

All (n ¼ 127) SCC (n ¼ 48) AC (n ¼ 76) AC/SCC (n ¼ 3)

FGFR1 amplification incidence [no. of samples (%)] 11 (8.7) 6 (12.5) 5 (7.0) 0 (0)

NOTE:One hundredand twenty-seven patientNSCLC tumor fragmentswere obtained locally at surgery and FFPE sectionsprocessedfor FISH analysis using a specific FGFR1 gene probe. FGFR1 FISH amplification incidence is shown here within SCC andadenocarcinoma (AC). Three samples were defined as mixed morphology (AC/SCC). FGFR1 amplification (FISH 6) was defined asan FGFR1/CEP8 gene probe ratio of �2 or cluster signals in �10% of tumor cells.






trend between smoking status and FGFR1 amplification (P¼ 0.0573, c2 likelihood ratio test). No correlations werefound between FGFR1 amplification and patient age, tumorgrade, or tumor stage. At the time of analysis, survival datawas too immature to be able to determine the effect ofFGFR1 amplification on patient survival.

AZD4547 treatment results in pharmacodynamicmodulation of FGFR signaling and potent antitumoractivity in FGFR1-amplified squamous PDTX models

To test preclinically the hypothesis that use of a selectiveFGFR pharmacologic inhibitor could offer therapeutic ben-efit to patients with NSCLCs harboring FGFR1-amplifiedtumors, we established a panel of primary SCC xenograftmodels derived directly from patient tumormaterial. Tissuesections from these models were characterized using hema-toxylin and eosin (H&E) staining and FGFR1 FISH and IHCanalysis. Of these, 5 models (L123, LC038, LC026, L121,and L133) were identified as FGFR1-amplified (FISH score6) and (with the exception of model L133) high-levelFGFR1 protein expression confirmed by IHC (denoted as"þþþ"; Fig. 2A). Model LC036 served as a negative controlwith normal FGFR1 gene copy number and undetectableprotein expression. Allmodels testedwild-type for EGFR,K-Ras and negative for the EML4-ALK gene fusion (data notshown).

AZD4547 antitumor efficacy was tested first using theFGFR1-amplified model, L121. Tumor-bearing mice wererandomly grouped and dosed orally, once daily with vehi-cle, 3.125, 6.25, or 12.5 mg/kg AZD4547 for a period of 15days. Potent tumor regressions were observed in the 6.25and 12.5 mg/kg treatment groups (TGI¼ 159% and 190%,respectively; P� 0.0001), whereas tumor stasis followed byslow regrowth was observed in the 3.125 mg/kg treatmentgroup (TGI¼84%,P¼0.0019; Fig. 2B). To confirmdirect in

vivo target engagement and modulation of tumor FGFRsignaling by AZD4547, a separate study was used usingL121 tumor–bearingmice, treatedwith a single dose of 12.5mg/kg AZD4547. Tumors were excised at 0, 1, 2, 4, 8, 24,and 48 hours post-dose and processed for both IHC andWestern blot analysis. Clear dynamic modulation of phos-pho-FGFR1was detected by immunoprecipitation/Westernblot analysis of L121 tumor lysates following AZD4547dosing, with maximal inhibition occurring around 4 hoursand signal recovery between 24 and 48 hours (Fig. 2C).Similarly, markers of downstream FGFR signaling, phos-pho-S6 and phospho-Erk, also showed significant modu-lation at 8 hours post-dose as determined by quantified IHCstaining of L121 tumor sections (55% and 73% reductionsin p-S6 and p-Erk staining, respectively, both P � 0.05; Fig.2D). Phenotypic markers were also assessed in this studyand included Ki67 and cleaved caspase 3 (CC3)—prolifer-ative and apoptotic markers, respectively. At 24 hours post-dose, significant induction of CC3 was observed (48% ofcells IHC-positive, P � 0.05), whereas at 48 hours, Ki67staining was dramatically reduced in response to AZD4547dosing (72% of cells IHC-negative, P � 0.05). Dynamicmodulation of these markers over multiple timepoints isshown in Supplementary Table S2.

Next, we assessed the antitumor efficacy of AZD4547 in 4additional FGFR1-amplified PDTX models (L123, LC038,LC026, and L133) and used model LC036 to test the nullhypothesis. Following implantation and establishment oftumor fragments in nude mice, randomized groups weredosed orally with AZD4547, once daily at either 12.5 or 25mg/kg for 2 to 3 weeks. In 2 of the 4 FGFR1-amplifiedmodels, potent tumor regressions were observed (TGI ¼199% and 134% in models L123 and LC026, respectively;both P � 0.0001), whereas model LC038 displayed sus-tained tumor stasis (TGI ¼ 94%; P � 0.0001; Fig. 3).

Table 2. Correlation analyses of FGFR1 amplification and clinicopathologic parameters

FGFR1 status

Parameter Value Nonamplified n (%) Amplified n (%) Pa

Gender Male 81 (88) 11 (12) 0.0046Female 38 (100) 0 (0)

Stage 1,2 79 (92) 7 (2) 0.56503,4 68 (88) 7 (12)

T 1,2 33 (92) 3 (8) 0.86243,4 68 (91) 7 (9)

N 0 67 (94) 4 (6) 0.10611,2 34 (85) 6 (15)

Histology SCC 42 (88) 6 (12) 0.2655AC 71 (93) 5 (7)

"Ever smoker" Yes 53 (84) 10 (16) 0.0022No 63 (98) 1 (2)

NOTE:Patient clinicopathologic informationwasobtained for 127ChineseNSCLC tumor specimensandanalyzed for correlationswithFGFR1 amplification status.aSignificance was determined using the c2 likelihood ratio test (P � 0.05).

Zhang et al.





Interestingly, despite being characterized as FGFR1-ampli-fied using FISH analysis, model L133 displayed relativelypoor antitumor efficacy in response to AZD4547 treatment(TGI ¼ 55% at 25 mg/kg AZD4547, P ¼ 0.01), likely aconsequence of very low FGFR1 protein expression. Impor-tantly, minimal (nonsignificant) efficacy was observed inthe negative control model, LC036 (TGI ¼ 18%; P ¼ 0.25)confirming the previously documented in vivo FGFR selec-tivity of AZD4547 (18). In all of the antitumor efficacystudies presented here, treatment with AZD4547 was welltolerated and did not result in significant body weight loss.

AZD4547 antitumor efficacy correlates well with FGFR1FISH score and protein expression level

To further inform our understanding of the relationshipbetween FGFR1 gene and protein level with AZD4547antitumor efficacy, Western blotting of PDTX model lysateswas conductedusinganFGFR1-specific antisera and thedatacombined with FISH, IHC, and efficacy data (Fig. 4). Twofurther FGFR1 nonamplified models (LC011 and LC022,FISH score 4 andFISHscore 5, respectively)were included inthis analysis. Notably, AZD4547 treatment at either 12.5 or25 mg/kg gave potent tumor stasis or regression effects in 4

Figure 2. AZD4547 treatment results in pharmacodynamic modulation of FGFR signaling and tumor regression in an FGFR1-amplified patient-derivedsquamous cell lung cancer model. A, characterization of 5 FGFR1-amplified squamous cell lung cancer models (L123, LC038, LC026, L121, and L133) andone nonamplified squamous cell lung cancer model (LC036) using morphologic, IHC, and FISH staining techniques. FGFR1 IHC score and FGFR1amplification status are embeddedwithin the images ("AMP" denotes gene amplified). For FISH images, FGFR1 gene probe signals appear red,CEP8 signalsare green, and DAPI-counterstained nuclei appear blue. Scale bars represent 50 mm for H&E/IHC images and 30 mm for FISH images. All images within eachrow are to the same scale. B, AZD4547 was administered by oral gavage once (qd) daily to nu/nu mice bearing established PDTX model (L121) xenograftfragments at the doses indicated. Tumor volumes are plotted against time. C, immunoprecipitation (IP)/Western blot (WB) analysis (IP: total FGFR1,WB: p-FGFR) showing modulation of p-FGFR1 within L121 tumor lysates following a single dose of 12.5 mg/kg AZD4547. Three animals were used for eachtimepoint and individual lanes represent tumor lysate from one animal. D, IHC analysis of L121 tumor sections showing modulation of FGFR-downstreamsignaling (p-Erk and p-S6) and proliferative (Ki67) and apoptotic markers (CC3). p-Erk and p-S6 analysis was conducted at 8 hours following a singledoseof 12.5mg/kgAZD4547,whereasCC3andKi67datawere collected at 24and48hours post-dose, respectively. QuantifiedHscoresweredetermined foreach group (n ¼ 3 animals/group) and statistical significance established for each marker using a one-tailed t test (P � 0.05; data not shown). The scale barrepresents 50 mm and all images are to the same scale.






of 5 of the FGFR1-amplifiedmodels (94%–199% TGI). The3 remaining nonamplified models (LC036, LC011, andLC022) displayed inferior antitumor efficacy ranging from18% to 69% TGI using 25 mg/kg AZD4547. This trend inAZD4547 antitumor response based on gene amplificationwas also observed when using FGFR1 gene copy number(GCN) analysis. Potent stasis and regression effects wereonly observed in those models with FGFR1 GCN>6.

Similarly, an analysis of FGFR1 protein expression usingboth IHC and Western blotting confirmed that the modelsin which AZD4547 induced tumor stasis or regression allshowed strong FGFR1 protein expression (intense bandsonWestern blot or IHC score 3þ). Model L133 is of interestas despite being FGFR1 gene amplified (FISH score 6,GCN¼ 6, IHC score 0–1), it displayed relatively low FGFR1protein expression and consequently inferior sensitivity to

Figure 3. AZD4547 displayspotent antitumor efficacy in 3 of4 additional FGFR1-amplifiedpatient-derived squamous lungcancer models but is inactive in anFGFR1 nonamplified controlmodel (LC036). AZD4547 wasadministered by oral gavage once(qd) daily to nu/nu mice bearingestablished patient-derived humanlung tumor xenograft fragments atthe doses indicated. Tumorvolumes are plotted against time.

Figure 4. AZD4547 antitumorefficacy correlateswell with FGFR1gene andprotein expression levels.Model summary table displayingFGFR1 FISH score, gene copynumber (GCN), IHC score, proteinexpression by Western blotting,and antitumor efficacy in responseto 2 to 3 weeks, once daily oralAZD4547 treatment (25 or12 mg/kg). Western blot datawere obtained using FGFR1 andGAPDH antisera on fresh tumorfragment lysates. P values werecalculated using a one-tailedt test. �, AZD4547 dosed at12.5 mg/kg/qd.

Zhang et al.





AZD4547 compared with the other amplifiedmodels (55%TGI at 25mg/kg,P¼0.01). Subsequent analysis using qPCRrevealed that model L133 FGFR1 messenger RNA (mRNA)levels were barely detectable in comparison to high-levelmRNA expression in the other FGFR1 gene–amplifiedmod-els (Supplementary Fig. S3).Thus, AZD4547 inducedpotent tumor stasis or regression

in 4 of the 5 FGFR1 gene–amplified squamous lung PDTXmodels tested here, whereas in nonamplified models, thebest response observed was partial tumor growth inhibi-tion. AZD4547 antitumor efficacy correlates well withFGFR1 gene amplification (FISH score 6) and consequenthigh-level protein expression (IHC 3þ).

DiscussionThis report describes the incidence of FGFR1 amplifica-

tion in Chinese NSCLCs, and importantly, through ourobservations of potent antitumor efficacy in multiplepatient-derived lung cancer xenograft models harboringFGFR1 gene amplification, provides strong translationalsupport for the clinical use of AZD4547 in patients withtumors bearing amplification of FGFR1.In light of previous reports highlighting geographical

differences in tumor molecular genetics (20, 22), theobserved enrichment of FGFR1 amplification incidencewithin our Chinese squamous NSCLC samples (12.5%) isnotable from a molecular epidemiology perspective and isbroadly consistent with previously published data onWest-ern NSCLC cohorts (8, 9). Our Chinese data confirmed thewell-known correlation of smoking status ("ever-smokers")with SCCs (P ¼ 0.0026, Fisher exact test; SupplementaryTable S3). No correlations were found between FGFR1amplification and any other clinicopathologic parameters.Interestingly, although lower than squamous, we alsoobserved FGFR1 amplification within our Chinese adeno-carcinoma samples (7%). Because of the novelty of thisfinding, we confirmed the classification of these samplesthrough independent review. This has potential importancedue to the higher incidence of lung adenocarcinoma inAsian populations and also the high smoking prevalenceamongst Asian males. Accordingly, these data warrant fur-ther investigation and confirmation in a larger patientcohort.We note that previous studies have identified FGFR1-

containing amplicons of differing sizes (133–208 kbp;refs. 8, 26), thus raising the possibility that genes otherthan FGFR1 could be driving or contributing to tumorigen-esis. Published data using short hairpin RNA (shRNA) in an8p11-amplified lung cell line did not support tumorigenicinvolvement of the WHSC1L1 and FLJ43582 genes whileconfirming the role of FGFR1 in cell viability (8). While insome cases (e.g., such as case L133 reported here) genesother than FGFR1 (e.g., WHSC1L1; ref. 27) may be thefunctional target of the 8p amplicon, the profound antitu-mor efficacy observed only in the FGFR1-amplified primarymodels elicited by the selective FGFR inhibitor, AZD4547,supports our conclusion that proliferation within these

models (and the patient tumors from which they werederived) is driven primarily by FGFR1 amplification andsignaling.

From a molecular segmentation perspective, our datahighlight the significance of FGFR1 amplification as a noveltherapeutic target. Characterization of our Chinese NSCLCsamples revealed a higher incidence of EGFR mutation(36.0%) and lower incidence of KRas mutation (4.4%)thanWesternNSCLCs (17% and 22%, respectively; ref. 20),but more importantly, our data suggest that these geneticaberrations are almost entirely mutually exclusive withFGFR1 amplification (Supplementary Table S4). This find-ing extended to our lung PDTX models (data not shown)and has clear implications for clinical trial design andpatient stratification.

Cell line–derived xenografts have proven use as modelsfor pharmacologic studies and as efficacymodels in cases ofoncogene addiction.However, the use of cell line xenograftsis limited by the lack of molecular and cellular heteroge-neity. PDTX models offer the promise of better diseasemodels through increased diversity of molecular lesionsand the preservation of 3-dimensional tumor stromal cellcomponents and interactions (28). Importantly, amplifica-tion of the FGFR1 gene was maintained between the PDTXmodels used herein and the original patient tumor samplesfrom which they were derived (Zhang JC, manuscript sub-mitted). Furthermore, all studies were conducted usingmodels which had undergone fewer than 8 serial in vivofragment passages, thus maintaining distinct regions oftumor heterogeneity and tumor/stromal architecture.Accordingly, we believe that the AZD4547 antitumor effi-cacy data presented here have strong translationalsignificance.

Within FGFR1-amplified PDTX models, once daily, oraldosing of AZD4547 resulted in either tumor stasis or rapidtumor regressions. Antitumor activity correlated with inhi-bition of tumor phospho-FGFR1 levels and downstreamsignaling through p-Erk and p-S6. Furthermore, antitumorefficacy correlated with inhibition of tumor Ki67 stainingand induction of apoptosis. Althoughnot specifically exam-ined here, previous in vitro mechanistic studies usingAZD4547 led us to speculate that the tumor cell apoptosisobserved in model L121 is likely a consequence ofAZD4547-induced BIM expression (18). With regard toinhibition of tumor angiogenesis, previous experience withAZD4547 leads us to conclude that such effects are unlikelyto result from AZD4547 treatment. Published preclinicaldata using AZD4547 at efficacious dose levels have failed toshow any effects on tumor angiogenesis as measured byCD31 IHC staining (18). Furthermore, we did not observein vivo inhibition of kinase insert domain receptor (KDR)signaling, nor AZD4547 efficacy in models which areknown to be sensitive to anti-KDR agents. Importantly, in7 of 8 models, we were able to show a good correlationbetween FGFR1 gene level, protein expression, and efficacyin response to AZD4547. Specifically, only those modelswith FISH score 6 (GCN � 10) and corresponding high-level protein expression (IHC 3þ) showed tumor stasis or






regression in response to AZD4547. In contrast, modelL133 did not respond despite a FISH score of 6, but notablybore relatively low protein expression by IHC (score 0–1)and Western blotting. Further analysis using qPCR con-firmed near undetectable levels of FGFR1 mRNA expres-sion, relative to the other FGFR1-amplified models. Tumorheterogeneity is an unlikely explanation for this finding, asmultiple FISH analyses of this model detected FGFR1amplification throughout the entirety of each tumor section(data not shown).Hence, other genes (e.g.,WHSC1L1)maybe driving tumorigenesis in this model. Overall, this PDTXmodel dataset supports the potential for the use of FISH toselect FGFR1 gene–amplified patients likely to benefit fromtreatment with the FGFR inhibitor AZD4547 and highlightsthat assessment of FGFR1 protein expression by IHC couldprovide an additional approach.

To conclude, our data highlight the occurrence of FGFR1gene amplification in a cohort of Chinese NSCLC andenrichment within SCC. Moreover, our data suggest acorrelative trend between smoking status and FGFR1 ampli-fication. Furthermore, we show the ability of the novel andselective FGFR inhibitor, AZD4547, to drive tumor regres-sions in patient-derived models of FGFR1-amplified squa-mous cell lung disease where there is currently high unmetmedical need. Taken together, these data support furtherinvestigation of AZD4547 as a targeted therapeutic optionfor patients with NSCLCs with tumors harboring geneticamplification of FGFR1. AZD4547 is currently being eval-uated in phase I/II clinical trials.

Disclosure of Potential Conflicts of InterestP.D. Smith has ownership interest (including patents) in AstraZeneca. R.

K. Thomas reports the following potential sources of conflict of interest:consulting and lecture fees (Sanofi-Aventis, Merck KGaA, Bayer, Lilly, Roche,Boehringer Ingelheim, Johnson & Johnson, AstraZeneca, Atlas-Biolabs,

Daiichi-Sankyo, Blackfield); research support (AstraZeneca, Merck, EOS).He is also a founder and shareholder of Blackfield, a company involved incancer genome services and cancer genomics–based drug discovery. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Zhang, K. Liu, Q. Ji, E. Kilgour, P.D. Smith, A.N.Brooks, R.K. Thomas, P.R. GavineDevelopment of methodology: J. Zhang, L. Zhang, X. Su, M. Li, Y. Xu, K.Liu, G. Zhu, L. Tang, P.R. GavineAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J. Zhang, L. Zhang, X. Su,M. Li, F.Malchers, Y. Xu,K. Liu, Z. Dong, L. Tang, R.K. ThomasAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): J. Zhang, L. Zhang, X. Su, F. Malchers, X.Yin, Z. Dong, Z. Qian, L. Tang, P. Zhan, E. Kilgour, P.D. Smith, R.K. Thomas,P.R. GavineWriting, review, and/or revision of the manuscript: J. Zhang, X. Su, X.Yin, Z. Dong, G. Zhu, E. Kilgour, P.D. Smith, A.N. Brooks, R.K. Thomas, P.R.GavineAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Zhang, X. Su, S. Fan, X. YinStudy supervision: P.D. Smith, A.N. Brooks, P.R. GavineIHC studies: Y. Xu

Grant SupportThe majority of this research was conducted and funded by AstraZeneca.

R.K. Thomas is supported by the State of Nordrhein-Westfalen through thePerMed initiative (grant z1104me004a/005-1111-0025), by the GermanMinistry of Science and Education (BMBF) as part of the NGFNplus program(grant 01GS08100), by the Deutsche Forschungsgemeinschaft (DFG)through SFB832 (TP6) and TH1386/3-1, by the EU-Framework ProgrammeCURELUNG (HEALTH-F2-2010-258677), by a Stand Up To Cancer Inno-vative Research Grant, a Program of the Entertainment Industry Foundation(SU2C-AACR-IR60109), by the Behrens-Weise Foundation, and by an anon-ymous foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 15, 2012; revised September 27, 2012; accepted October1, 2012; published OnlineFirst October 18, 2012.

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Correction

Correction: Translating the TherapeuticPotential of AZD4547 in FGFR1-AmplifiedNon–Small Cell Lung Cancer through the Useof Patient-Derived Tumor Xenograft Models

In this article (Clin Cancer Res 2012;18:6658–67), which was published in theDecember 15, 2012, issue of Clinical Cancer Research (1), Dr. Jakob Sch€ottle hasbeen added as an author.

The authors should be ordered as follows:

Zhang J, Zhang L, Su X, LiM, Xie L,Malchers F, Fan S, Yin X, Xu Y, Liu K, Dong Z,ZhuG,Qian Z, Tang L, Sch€ottle J, Zhan P, Ji Q, Kilgour E, Smith PD, Brooks AN,Thomas RK, and Gavine PR

The authors regret this error.

Reference1. Zhang J, Zhang L, Su X, Li M, Xie L, Malchers F, et al. Translating the therapeutic potential of

AZD4547 in FGFR1-amplified non–small cell lung cancer through the use of patient-derivedtumor xenograft models. Clin Cancer Res 2012;18:6658–67.

Published OnlineFirst May 24, 2013.doi: 10.1158/1078-0432.CCR-13-1265�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(13) July 1, 20133714

2012;18:6658-6667. Published OnlineFirst October 18, 2012.Clin Cancer Res Jingchuan Zhang, Lin Zhang, Xinying Su, et al. Patient-Derived Tumor Xenograft Models

Small Cell Lung Cancer through the Use of−-Amplified NonFGFR1Translating the Therapeutic Potential of AZD4547 in

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