Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer.
ABSTRACT Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
- SourceAvailable from: Lukas C Heukamp[show abstract] [hide abstract]
ABSTRACT: In cancer, genetically activated proto-oncogenes often induce "upstream" dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce "downstream" dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.Proceedings of the National Academy of Sciences 10/2009; 106(43):18351-6. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Lung cancer is the leading cause of cancer mortality worldwide, yet there exists a limited view of the genetic lesions driving this disease. In this study, an integrated high-resolution survey of regional amplifications and deletions, coupled with gene-expression profiling of non-small-cell lung cancer subtypes, adenocarcinoma and squamous-cell carcinoma (SCC), identified 93 focal copy-number alterations, of which 21 span <0.5 megabases and contain a median of five genes. Whereas all known lung cancer genes/loci are contained in the dataset, most of these recurrent copy-number alterations are previously uncharacterized and include high-amplitude amplifications and homozygous deletions. Notably, despite their distinct histopathological phenotypes, adenocarcinoma and SCC genomic profiles showed a nearly complete overlap, with only one clear SCC-specific amplicon. Among the few genes residing within this amplicon and showing consistent overexpression in SCC is p63, a known regulator of squamous-cell differentiation. Furthermore, intersection with the published pancreatic cancer comparative genomic hybridization dataset yielded, among others, two focal amplicons on 8p12 and 20q11 common to both cancer types. Integrated DNA-RNA analyses identified WHSC1L1 and TPX2 as two candidates likely targeted for amplification in both pancreatic ductal adenocarcinoma and non-small-cell lung cancer.Proceedings of the National Academy of Sciences 07/2005; 102(27):9625-30. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.Nature 04/2007; 446(7132):153-8. · 38.60 Impact Factor
, 62ra93 (2010);
2 Sci Transl Med
, et al.Jonathan Weiss
Tractable FGFR1 Dependency in Squamous Cell Lung Cancer
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Frequent and Focal FGFR1 Amplification Associates with
Therapeutically Tractable FGFR1 Dependency in
Squamous Cell Lung Cancer
Jonathan Weiss,1* Martin L. Sos,1*†Danila Seidel,1,2* Martin Peifer,1Thomas Zander,3
Johannes M. Heuckmann,1Roland T. Ullrich,1Roopika Menon,4Sebastian Maier,4Alex Soltermann,5
Holger Moch,5Patrick Wagener,6Florian Fischer,1Stefanie Heynck,1Mirjam Koker,1Jakob Schöttle,1
Frauke Leenders,1,2Franziska Gabler,1,2Ines Dabow,1,2Silvia Querings,1Lukas C. Heukamp,7
Hyatt Balke-Want,1Sascha Ansén,3Daniel Rauh,8Ingelore Baessmann,9Janine Altmüller,9
Zoe Wainer,10Matthew Conron,10Gavin Wright,10Prudence Russell,11Ben Solomon,12
Elisabeth Brambilla,13,14Christian Brambilla,13,14Philippe Lorimier,13Steinar Sollberg,15
Odd Terje Brustugun,16,17Walburga Engel-Riedel,18Corinna Ludwig,18Iver Petersen,19Jörg Sänger,20
Daniëlle Heideman,24Federico Cappuzzo,26Claudia Ligorio,27Stefania Damiani,27Michael Hallek,3,28
Rameen Beroukhim,29,30,31,32William Pao,33Bert Klebl,34Matthias Baumann,34Reinhard Buettner,7
(Published 15 December 2010; Volume 2 Issue 62 62ra93)
Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adeno-
carcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor
(EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks ther-
genetic alterations thatwere therapeuticallyamenableand thenperformed high-resolution genecopynumber analy-
ses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung
cancer (n = 155),but notin other lung cancer subtypes,and,byfluorescence in situ hybridization, confirmed the pres-
based screening with the FGFR inhibitor PD173074in a large (n = 83) panel of lung cancer cell lines,we demonstrated
that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified
sion of an FGFR1-resistant allele (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cyto-
toxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage
in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth
and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
for cancer treatment. For examples, the ERBB2 amplification in breast
(1), and KIT or PDGFRA (platelet-derived growth factor receptor A)
mutations in gastrointestinal stromal tumors lead to sensitivity to the
KIT/ABL/PDGFR inhibitor imatinib (2). In lung adenocarcinoma, pa-
tients with EGFR-mutant tumors (3–5) experience tumor shrinkage
and prolonged progression-free survival when treated with epidermal
growth factor receptor (EGFR) inhibitors (6). Furthermore, EML4-
ALK gene fusion–positive lung cancers can be effectively treated with
anaplastic lymphoma kinase (ALK) inhibitors (7, 8).
However, these alterations almost exclusively occur in the rare
adenocarcinomas of patients who never smoked, but are uncommon
alterations in squamous cell lung cancer (10), no therapeutically tract-
able targets have so far been identified. Thus, therapeutic options for
squamous cell lung cancer patients remain scarce, because molecularly
in squamous cell lung cancer patients.
mens using Affymetrix 6.0 SNP (single-nucleotide polymorphism)
arrays, which yielded high-resolution genomic profiles (median inter-
marker distance <1 kb). To separate driver lesions from random noise,
we applied the GISTIC algorithm (13, 14). We identified 25 significant
www.ScienceTranslationalMedicine.org 15 December 2010Vol 2 Issue 62 62ra93
on December 16, 2010
SOX2 on chromosome 3q26.33 (Fig. 1A and table S1) (10) and 26 sig-
nificant deletions (fig. S1 and table S1). The second most significant
amplified sample (Fig. 1A). This region spanned 133 kb (table S1) and
was amplified at high amplitude (four or more copies) in 15 of 155
(9.7%) squamous cell lung cancer specimens (Fig. 1A). Notably, 11
of the tumors with FGFR1 amplification were from smokers, whereas
of the 15 tumors with amplified amounts of FGFR1 also harbored a
mutation in TP53 (table S2). Moreover, patientswho had tumors with
FGFR1 amplification [copy number > 9 in fluorescence in situ hybrid-
(copy number = 2 in FISH analysis) (fig. S2). We next analyzed copy
number alterations in lung adenocarcinoma specimens (n = 77) and
found no significant (q > 0.25) amplification (four or more copies; 1.3%)
at 8p12 (Fig. 1B).
Finally, we analyzed a publicly available lung cancer SNP array data
FGFR1 was amplified in 6 of 581 (1%) nonsquamous cell lung cancers
(Fig. 1C). Thus, FGFR1 amplification is significantly enriched in squa-
set (P = 0.03) (table S3) and when compared to a published data set of
nonsquamous cell lung cancer (P < 0.0001) (Fig. 1C). FISH using an
8p12-specific probe on an independent set of 153 squamous cell lung
cancers confirmed the presence of frequent high-level amplification of
FGFR1 in 34 of 153 (22%) patients (Fig. 1D and table S4), 27 of whom
were current smokers and none of whom were nonsmokers. We note
that FISH is not sensitive to the admixture of nontumoral cells; thus,
focal amplification of FGFR1 is likely to be more frequent in squamous
sequenced the FGFR1 gene in 94 squamous cell lung cancers and 94
adenocarcinomas and found one mutation (FGFR1P578H) in the ade-
nocarcinoma cohort, indicating that FGFR1 mutations might play only
a minor role and might not drive alterations in the pathogenesis of
lung cancer (16).
Next, we performed high-throughput cell line screening (17, 18)
to determine the activity of the non–isoform-specific FGFR inhibitor
PD173074 (19) in a collection of 83 lung cancer cell lines (table S5)
concentration (GI50values) below 1.0 mM (Fig. 2A); remarkably, three
8p12 by6.0SNP arrayanalysis(Fig.2B),suggestingthat FGFR1 ampli-
fications are significantly (P = 0.0002) associated with FGFR inhibitor
activity (Fig. 2A). As expected, FGFR1-amplified cells expressed higher
amounts of total FGFR1 protein (fig. S3). One (H520) of the three
from a squamous cell lung cancer patient (table S5). We next tested
whether amplification of FGFR1 could be linked with sensitivity to
FGFR inhibition in an unbiased fashion. Application of a K-nearest
neighbor–based analysis, followed by leave-one-out cross-validation
(17), revealed FGFR1 amplification to be the only genetic predictor of
PD173074 sensitivity that retained significance following Bonferroni-
based multiple testing correction (P < 0.05; table S6). Previous studies
indicated that expression of FGFR ligands might contribute to the sen-
sitivity to FGFR inhibitors in lung cancer (21). We did not observe
elevated amounts of FGF2 in the FGFR1-amplified cell lines (fig. S4A),
nor did we observe a difference in the expression of FGFR ligands be-
tween patients harboring FGFR1 amplification and those without
robust phosphorylation of FGFR, suggesting ligand-independent acti-
vation, which was further enhanced upon addition of exogenous FGF2
or FGF9 (fig. S4C), compatible with paracrine activation of FGFR1 in
FGFR1-amplified cells. We next measured induction of apoptosis in
FGFR1-amplified cells after treatment with PD173074 and found a sig-
the group of sensitive cells (Fig. 2C and table S7). Furthermore, FGFR
inhibition led to decreased colony formation of FGFR1-amplified but
not of EGFR-mutant cells in soft agar (Fig. 2D), further enforcing the
notion that amplification of FGFR1 drives proliferation of these lung
cancer cell lines. Treatment with PD173074 reduced the amounts of
phosphorylated FGFR1 (fig. S5) and of the adaptor molecule FRS2 in
EGFR-mutant cell line HCC827 (Fig. 2E). We also observed inhibition
of phosphorylation of extracellular signal–regulated kinase (ERK) but
To validate FGFR1 as the critical target of PD173074 in FGFR1-
amplified lung cancer cells, we ectopically expressed the V561M
mutation (22) at the gatekeeper position of FGFR1 (FGFR1V561M),
1Max Planck Institute for Neurological Research, Klaus-Joachim-Zülch Laboratories of the Max
PlanckSociety andtheMedical Facultyof the UniversityofCologne,50931 Cologne,Germany.
2Laboratory of Translational Cancer Genomics, Center of Integrated Oncology Köln-Bonn,
Pathology, Comprehensive Cancer Center, University Hospital Tübingen, 72076 Tübingen,
Germany.5Institute for Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland.
6Department of Surgery, Weill Medical College, Cornell University, New York, NY 10065, USA.
7Institute of Pathology,Universityof Bonn, 53123 Bonn,Germany.8ChemicalGenomicsCenter,
St. Vincent’s Hospital, Melbourne, 3065 Victoria, Australia.12Department of Haematology and
Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, 3002 Victoria, Australia.
13Department of Pathology, Université Joseph Fourier, 38041 Grenoble, France.14Institut Albert
Bonniot INSERM U823, Université Joseph Fourier, 38042 Grenoble, France.15Department of
Thoracic Surgery, Rikshospitalet, Oslo University Hospital, 0027 Oslo, Norway.16Department of
Radiation Biology, Norwegian Radium Hospital, N-0310 Oslo, Norway.17Department of Oncol-
ogy, Radiumhospitalet, Oslo University Hospital, N-0310 Oslo, Norway.18Thoracic Surgery,
Lungenklinik Merheim,Klinikender StadtKöln gGmbH,51109 Cologne,Germany.19Institute of
for Pathology Bad Berka, 99438 Bad Berka, Germany.21Department for Internal Medicine II,
University Clinic Jena, Friedrich-Schiller University, 07740 Jena, Germany.22Department of
Pulmonary Diseases, University Medical Centre Groningen, 9713 GZ Groningen, Netherlands.
23Department of Pathology, University Medical Centre Groningen, 9713 GZ Groningen,
Netherlands.24Department of Pathology, VU University Medical Center Amsterdam, 1007 MB
Amsterdam, Netherlands.25Department of Pulmonary Diseases, VU University Medical Center
Amsterdam, 1007 MB Amsterdam, Netherlands.26Department of Medical Oncology, Ospedale
Civile, 57100 Livorno, Italy.27Department of Haematology and Oncologic Science, University
Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Medicine,Brigham andWomen’s Hospital, Boston, MA02115, USA.31Departmentof Medicine,
Center GmbH, 44227 Dortmund, Germany.35Department of Pathology, Hospital Merheim,
Kliniken der Stadt Köln gGmbH, 51109 Cologne, Germany.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
(M.L.S.); email@example.com (R.K.T.)
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on December 16, 2010
hinge region of the kinase (23) (fig. S6).
Expression of FGFR1V561Min FGFR1-
amplified lung cancer cells abolished
PD173074-mediated cytotoxicity and de-
phosphorylation of FGFR (Fig. 3A),
consistent with the notion that FGFR1 is
the critical target of PD173074 in FGFR1-
amplified lung cancer cells. Furthermore,
in a panel of 105 biochemically screened
kinases,FGFR1wasoneofonly two kinases
strongly inhibited by PD173074 (table
S8), recapitulating previous studies (22).
The high analytical resolution of the
6.0 SNP arrays, together with the large
size of our data set, limited the number
of candidate genes in the 8p12 amplicon
to only two genes, FGFR1 and FLJ43582.
in lung cancer applying lower-resolution
techniques suggested WHSC1L1 to be
the relevant oncogene in the 8p12 ampli-
con (24). To test whether genes other
than FGFR1 drive tumorigenesis in the
8p12-amplified tumors, we silenced the
constructs in the 8p12-amplified lung
cellular viability (fig. S7), silencing of
FGFR1 strongly reduced the viability of
the FGFR1-amplified lung cancer cells
(Fig. 3B). In light of the focality of the
8p12 amplicon (including FGFR1 and
FLJ43582) and the lack of effect of shRNA-
mediated knockdown of either FLJ43582
or WHSC1L1 in FGFR1-amplified cells,
our data suggest that FGFR1 is the rele-
vant target in these cells. Notably, the cell
line H1703, which bears a copy number
gain at 8p12 and that had been reported
trast, H1703 cells depend on PDGFRA
for their survival (25) because of amplifi-
is primarily FGFR1 and its amplification
induces FGFR1 dependency.
Finally, treatment with PD173074
(100 mg/kg, twice a day) resulted in tu-
mor shrinkage in mice engrafted with
FGFR1-amplified cells (Fig. 3C). This re-
duction in tumor size was paralleled by
reduction in the amounts of phospho-
ERK but not of phospho-AKT in immu-
Fig. 1. FGFR1 is amplified in squamous cell lung cancer (SQLC). (A) Left panel: Significant (14) [FDR
(false discovery rate) value; x axis] amplifications across all chromosomes (y axis) in SQLC (n = 155)
as assessed by GISTIC. Right panel: Copy number alterations (blue, deletion; white, copy number–
neutral; red, amplification) at chromosome 8 (y axis) across all SQLC samples (x axis). Samples are
ordered according to focal amplification of FGFR1. (B) Significant (G score; y axis) copy number
changes in adenocarcinoma (AC; n = 77) (black line) and SQLC (red dotted line) at chromosome 8.
The q value for the presence of 8p12 amplification is 8.82 × 10−28for SQLC and greater than 0.25 for
adenocarcinoma. The chromosomal positions of FGFR1 (8p12) and MYC are highlighted (black ar-
rows). (C) Frequency of FGFR1 amplification (% of samples ≥ copy number 4; y axis) in non-SQLC from
a published data set (14), adenocarcinoma, and SQLC. P values indicate statistical significance. (D) FISH
analysis (green, control; red, FGFR1) of 153 SQLC samples (FGFR1-HA: copy number >9; FGFR1-LA: copy
number >2 and <9; FGFR1-N: copy number 2). Presented are example images from the three different
FGFR1 amplification groups.
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on December 16, 2010
nohistochemical analyses of explanted tumors, validating our in vitro
findings that MAPK signaling is the key pathway engaged by ampli-
fied FGFR1 (fig. S9A). Treatment at 50 mg/kg twice a day resulted in
only a minimal exposure when compared to the gavage of 100 mg/kg
twice a day because of the short half-life of the compound in vivo
on VEGFR2 (vascular endothelial growth factor receptor 2), the ob-
In contrast, xenografted EGFR-mutant H1975 cells did not show signs
of regression upon PD173074 treatment (fig. S9C). Thus, FGFR1
amplification leads to FGFR1 dependency in vivo.
Here, we have identified frequent high-level amplification of FGFR1 in
squamous cell lung cancer of smokers; this amplification sensitizes the
tumors to FGFR1 inhibition.Previous studies in lung cancer cohorts of
mixed subtypes and low technological resolution (24, 28) or small size
(10) have reported occasional amplification of the 8p locus in lung
the high prevalence of this amplicon in squamous cell lung cancer
(~10%) in comparison to other lung cancer subtypes (1%). Given the
prevalence of this amplification is likely to still be substantively under-
amplification is one of the hallmark alterations in squamous cell lung
cancer,similar toamplification of SOX2. These two alterations were al-
most completely mutually exclusive (table S9), suggesting an epistatic
Fig. 2. FGFR1 amplifications are associated with FGFR inhibitor activity. (A)
GI50values (y axis) of PD173074 across 83 lung cancer cell lines (x axis).
FGFR1-amplified (copy number ≥4) cell lines are marked with asterisks. (B)
Copy number alterations (x axis; blue, deletion; white, copy number 2; red,
amplification) on chromosome 8 with a zoom in on 8p12 (FGFR1 locus is
between PD173074 at 1 mM and DMSO control after 72 hours; y axis) across
24 cell lines (x axis; asterisks denote FGFR1 amplification copy number ≥4)
as measured by flow cytometry (after annexin V/PI staining). (D) FGFR1-
amplified cell lines were plated in soft agar and treated with either DMSO
(control) or decreasing concentrations of PD173074. (E) Phosphorylation of
FGFR and of downstream molecules in FGFR1-amplified (H1581 and H520)
and in FGFR1 wild-type (EGFR-mutant) cells (HCC827) after treatment with
PD173074 as assessed by immunoblotting.
Fig. 3. FGFR1-amplified cells are dependent on FGFR1 in vitro and in vivo.
of FGFR1-amplified cells expressing wild-type (wt)ormutant(V561M) FGFR1
treated with PD173074 [0.5 mM (white bars) and 1.0 mM (gray bars)]. Right
panel: Phosphorylation of FGFR in the FGFR1V561Mand FGFR1wtcells de-
tected by immunoblotting. (B) Left panel: Viability (PD173074 treatment as
compared to DMSO control; y axis) of H1581 cells after transduction with
control shRNA or shRNA targeting FGFR1. Right panel: Silencing of FGFR1
y axis), tumor volume was measured over time (x axis).
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on December 16, 2010
of apoptosis. Thus, FGFR1 amplification represents an opportunity for
targeted therapy in squamous cell lung cancer. We therefore suggest
that FGFR1 inhibitors, which are currently in clinical testing in tumor
types bearing genetic alterations in FGFR genes (29–31), should be
MATERIALS AND METHODS
The tumor specimens analyzed in this study have been collected under
local Institutional Review Board approval. All patients gave written
informed consent. Genomic DNA was hybridized to Affymetrix 6.0
sities were normalized and modeled with a Gaussian mixture model.
Background-corrected intensities were normalized across all arrays of
one batch by quantile normalization. Raw copy numbers were cal-
culated by dividing the normalized tumor-derived signal intensities
ized in the same batch. Raw copy number data were segmented by
circular binary segmentation and visualized in the integrated genome
viewer (IGV) (32). GISTIC was performed as described previously
was performed on whole-genome amplified DNA of primary tumors.
Cell lines were sequenced with complementary DNA (cDNA). All raw
Tissue microarray construction
Tissue microarray slides were obtained from formalin-fixed, paraffin-
embedded lung squamous cell carcinoma samples. The tissue microar-
rays contained samples of a total of 172 patients from the University
Hospital Zurich; each of these samples was present in duplicate cores,
each core 0.6 mm in diameter (33). A second tissue microarray of 22
patients from Weill Cornell Medical Center was obtained, with each
sample present in triplicate cores, each core 0.6 mm in diameter. Sub-
sequently, 153 samples were used for FISH analysis.
After RNA isolation, biotin-labeled complementary RNA (cRNA)
preparation was performed with Epicentre TargetAmp Kit (Epicentre
Biotechnologies) and Biotin-16-UTP (10 mM; Roche Molecular Bio-
chemicals) or Illumina TotalPrep RNA Amplification Kit (Ambion).
Biotin-labeled cRNA (1.5 mg) was hybridized to Sentrix whole-genome
bead chips WG6 version 2 (Illumina) and scanned on the Illumina
BeadStation 500X. For data collection, we used Illumina BeadStudio
188.8.131.52 software. Gene pattern analysis platform (34) was used to visu-
alize the normalized data.
FGFR1 amplification FISH assay
A FISH assay was used to detect the FGFR1 amplification at the chro-
mosomal level on the tissue microarrays. We performed fluorescence
is located on a stable region of chromosome 8p23.2 and selected on the
basis of SNP array analysis. Only samples where the control bacterial
FGFR1 locus spanning 8p11.23 to 8p11.22. We used the digoxigenin-
labeled BAC clones CTD 2523O9, which produces a green signal, as
reference probe. The target probe was labeled with biotin to produce
a red signal with RP11-148D21 BAC clones (Invitrogen). Deparaffin-
ized sections were pretreated with a 100 mM tris and 50 mM EDTA
5 minand immediatelyplaced onice.Subsequently,thetissuesections
and FGFR1 FISH probes were co-denatured at 94°C for 3 min and hy-
with streptavidin–Alexa 594 conjugates (dilution 1:200) and antibodies
to digoxigenin–fluorescein isothiocyanate (FITC) (dilution, 1:200).
Slides were then counterstained with 4′,6-diamidino-2-phenylindole
(DAPI) and mounted. The samples were analyzed under a 63× oil im-
appropriate filters, a charge-coupled device camera, and the FISH im-
aging and capturing software Metafer 4 (Metasystems). The evaluation
of the tests was done independently by three experienced evaluators
thresholds for assigning a sample to the FGFR1 “high-amplification”
group were a copy number of nine. All samples that had a copy num-
ber below nine and above two were assigned to the group of “low-
Cell lines and reagents
the German Resource Centre for Biological Material (DSMZ), or from
Cell line screening
ious concentrations of PD173074. Viability was determined after 96
hours by measuring cellular adenosine triphosphate (ATP) content
(CellTiter-Glo, Promega). Half-maximal inhibitory concentrations
(GI50) were determined with the statistical data analysis software “R”
with the package “ic50.”
For determination of apoptosis, cells were seeded in six-well plates,
incubated for 24 hours, treated with either DMSO (control) or 1.0 mM
PD173074 for 72 hours, and stained with annexin V and propidium
iodide (PI). Finally, the cells were analyzed on a FACSCanto flow cy-
tometer (BD Biosciences). The difference between the relative percent-
ageof annexinV/PI–positive cells treated with DMSOand cellstreated
with PD173074 was determined (induction of apoptosis rate).
Lentiviral RNA interference and retroviral expression
The V561M mutation was introduced into FGFR1 cloned in pBABE-
Puro by site-directed mutagenesis. Replication-incompetent retro-
viruses were produced by cotransfection with the pCL-ampho plasmid
in human embryonickidney (HEK) 293T cells.Hairpin shRNA target-
in table S10. Replication-incompetent lentiviruses were produced from
pLKO.1-Puro–based vectors by cotransfection with D8.9 and pMGD2
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on December 16, 2010
selected with puromycin (1.5 mg/ml), and 5 days after selection, cells
were counted with trypan blue.
The following antibodies were used for immunoblotting: b-actin (MP
Bioscience); phospho-FGFR (Tyr653, Tyr654), phospho-FRS2 (Tyr196),
phospho-AKT (Ser473), phospho-S6, S6, AKT, phospho-ERK, and
ERK (Cell Signaling Technology); total FGFR1 (Santa Cruz Bio-
technology);and horseradish peroxidase(HRP)–conjugated antibodies
to rabbit and mouse (Millipore).
Soft agar assay
Cells were suspended in growth media containing 10% fetal calf serum
medium (10% FCS; 1.0% agar). Growth medium containing indicated
compound concentrations was added on top. Colonies were analyzed
with the Scanalyzer imaging system (LemnaTec).
Xenograft mouse models
All animal procedures were approved by the local animal protection
PD173074 (15 mg/ml for 50 mg/kg or 30 mg/ml for 100 mg/kg sched-
ule) dissolved in vehicle (sodium lactate) or vehicle detergent alone.
Tumor size was monitored by measuring perpendicular diameters
as described previously (17). For the determination of tumor growth
under treatment with PD173074, each experiment presented in the
figures compromises the measurement of five different tumors.
tests. Prediction of compound activity was performed with the KNN
Fig. S1. Significant deletions are observed in squamous cell lung cancer.
Fig. S2. FGFR1 amplification has no significant impact on overall survival of SQLC patients.
Fig. S3. FGFR1 amplification correlates with FGFR1 protein expression.
Fig. S4. Expression of FGFR ligands does not correlate with FGFR1 amplification status.
Fig. S5. Treatment of FGFR1-amplified cell line H520 with PD173074 leads to dephos-
phorylation of FGFR1 as measured by immunoprecipitation.
Fig. S6. PD173074 binds inside the ATP-binding pocket of FGFR1.
Fig. S7. Knockdown of genes adjacent to FGFR1 on 8p12 does not affect cell viability.
Fig. S8. PD173074 is not active in the PDGFRA- and FGFR1-amplified cell line H1703.
Fig. S9. PD173074 shows antitumor activity in vivo.
Table S1. Significant amplifications and deletions are noted in a subset of 155 SQLC samples.
Table S2. Clinical features and co-occurrent mutations of FGFR1-amplified SQLC samples.
Table S3. Significant amplifications and deletions are noted in a subset of 77 adenocarcinoma
Table S4. FGFR1 amplification is detected using FISH on tumor microarrays.
Table S5. GI50values are not associated with mutation status across the lung cancer cell line panel.
Table S6. KNN algorithm–based scoring predicts PD173074 sensitivity.
Table S7. PD173074 induces apoptosis in FGFR1-amplified cell lines.
Table S8. PD173074 has specific activity against two kinases.
Table S9. FGFR1 and SOX2 amplification in squamous cell lung carcinoma.
Table S10. Sequences of all shRNA constructs that were used in the study.
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C. Reinhardt and A. Ullrich for discussion. Funding: This work was supported by the Deutsche
of the NGFNplus program (grants 01GS08100 to R.K.T. and 01GS08101 to J. Wolf and P.N.); the
Max Planck Society (M.I.F.A.NEUR8061 to R.K.T.); the Deutsche Forschungsgemeinschaft (DFG)
through SFB (TP6 to R.K.T. and R.T.U.; TP5to L.C.H. and R. Buettner); the Ministry for Innovation,
Science, Research and Technology of the State of Nordrhein-Westfalen (MIWT, 4000-12 09 to
R.K.T. and B.K.); and an anonymous foundation to R.K.T. E.B. and C.B. were supported by the
Scholarship. G.W. was supported by the Australasian Society of Cardiac and Thoracic Surgeons
Foundation Grant, Peter MacCallum Foundation Grant, and a private research donation from
family and friends of former patients. Author contributions: J. Weiss and M.L.S. designed and
performed the experiments, analyzed the data, and wrote the manuscript. D.S. designed the
S.M., F.F., S.H., M.K., J. Schöttle, F.G., I.D., S.Q., L.C.H., H.B.-W., I.B., and J.A. performed the
experiments and discussed the data. A.S., H.M., P.W., S.A., Z.W., M.C., G.W., P.R., B.S., E.B., C.B.,
R. Beroukhim, W.P., B.K., M.B., R. Buettner, K.E., E. Stoelben, J. Wolf, and P.N. contributed critical
tumor specimens and contributed to the discussion of the data.S.P.reviewed tumor histology,
analyzed the FISH data, and wrote the manuscript. R.K.T. conceived the project, designed the
experiments, analyzed the data, and wrote the manuscript. Competing interests: R.K.T. re-
ringer, AstraZeneca, and ATLAS Biolabs, as well as research support from Novartis and
AstraZeneca. J. Wolf is a member of advisory boards of Roche, AstraZeneca, Novartis, Amgen,
receives consulting fees for Novartis Institutes for BioMedical Research. W.P. receives consulting
fees from Molecular MD, AstraZeneca, BMS, and Symphony Evolution. H.G. received research
support from Eli Lilly, Roche, and Boehringer Ingelheim through the University Medical Center
Groningen. The other authors declare that they have no competing interests. Accession
numbers: All raw data are publicly available (GEO; GSE25016).
Submitted 6 July 2010
Accepted 24 November 2010
Published 15 December 2010
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