Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma Priya Chudasama1, Marcus Renner2, Melanie Straub3, Sadaf S

Published OnlineFirst August 17, 2016; DOI: 10.1158/1078-0432.CCR-16-0860

Personalized Medicine and Imaging Clinical Cancer Research Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma Priya Chudasama1, Marcus Renner2, Melanie Straub3, Sadaf S. Mughal4, Barbara Hutter4, Zeynep Kosaloglu5, Ron Schweßinger4, Matthias Schefﬂer6, Ingo Alldinger7, Simon Schimmack7, Thorsten Persigehl8, Carsten Kobe9, Dirk Jager€ 5,10, Christof von Kalle1,11,12,13, Peter Schirmacher2,12, Marie-Kristin Beckhaus14, Stephan Wolf12,14, Christoph Heining1,11, Stefan Groschel€ 1,11,Jurgen€ Wolf6, Benedikt Brors4,12, Wilko Weichert3,15, Hanno Glimm1,11,12, Claudia Scholl1,12, Gunhild Mechtersheimer2, Katja Specht3,15, and Stefan Frohling€ 1,11,12

Abstract

Purpose: Altered FGFR1 signaling has emerged as a therapeutic Results: Increased FGFR1 copy number was detected in 74 target in epithelial malignancies. In contrast, the role of FGFR1 in of 190 (38.9%; cohort 1), 13 of 79 (16.5%; cohort 2), and 80 soft-tissue sarcoma (STS) has not been established. Prompted by of 254 (31.5%; cohort 3) patients. FGFR1 overexpression the detection and subsequent therapeutic inhibition of amplified occurred in 16 of 79 (20.2%, cohort 2) and 39 of 254 FGFR1 in a patient with metastatic leiomyosarcoma, we investi- (15.4%; cohort 3) patients. Targeting of FGFR1 by RNA gated the oncogenic properties of FGFR1 and its potential as a interference and small-molecule inhibitors (PD173074, drug target in patients with STS. AZD4547, BGJ398) revealed that the requirement for FGFR1 Experimental Design: The frequency of FGFR1 amplification signaling in STS cells is dictated by FGFR1 expression levels, and overexpression, as assessed by FISH, microarray-based com- and identified the MAPK–ERK1/2 axis as critical FGFR1 effec- parative genomic hybridization and mRNA expression profiling, tor pathway. SNP array profiling, and RNA sequencing, was determined in Conclusions: These data identify FGFR1 as a driver gene in three patient cohorts. The sensitivity of STS cell lines with or multiple STS subtypes and support FGFR1 inhibition, guided without FGFR1 alterations to genetic and pharmacologic FGFR1 by patient selection according to the FGFR1 expression and inhibition and the signaling pathways engaged by FGFR1 were monitoring of MAPK–ERK1/2 signaling, as a therapeutic investigated using viability assays, colony formation assays, and option in this challenging group of diseases. Clin Cancer Res; biochemical analysis. 23(4); 962–73. 2016 AACR.

Introduction characterized by remarkable histologic diversity, comprising more than 50 subtypes, and a variable clinical course, ranging Soft-tissue sarcomas (STS) are mesenchymal malignancies that from indolent to highly invasive and metastatic, thereby posing originate from connective tissues at all anatomic sites and account substantial diagnostic and therapeutic challenges (1). Numerous for 1% of all cancers, with approximately 11,930 and 28,000 clinical trials have shown that in advanced-stage STS, conven- newly diagnosed cases per year in the United States and the tional chemotherapy may provide symptom palliation and delay European Union, respectively (1–3). Soft-tissue sarcomas are disease progression but does not prolong survival, which typically

1Department of Translational Oncology, National Center for Tumor Diseases many. 13DKFZ-Heidelberg Center for Personalized Oncology (HIPO), Heidelberg, (NCT) Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany. 14Genomics and Proteomics Core Facility, DKFZ, Heidelberg, Ger- Germany. 2Department of General Pathology, Institute of Pathology, Heidelberg many. 15German Cancer Consortium, Munich, Germany. University Hospital, Heidelberg, Germany. 3Institute of Pathology, Technische Universitat€ Munchen,€ Munich, Germany. 4Division Applied Bioinformatics, DKFZ Note: Supplementary data for this article are available at Clinical Cancer and NCT Heidelberg, Heidelberg, Germany. 5Clinical Cooperation Unit Applied Research Online (http://clincancerres.aacrjournals.org/). Tumor Immunity, DKFZ and NCT Heidelberg, Heidelberg, Germany. 6Depart- M. Renner and M. Straub equally contributed to this article. ment of Internal Medicine I, Center for Integrated Oncology, Cologne University Hospital, Cologne, Germany. 7Department of General, Visceral and Transplan- Corresponding Author: Stefan Frohling,€ National Center for Tumor Dis- tation Surgery, Heidelberg University Hospital, Heidelberg, Germany. 8Depart- eases (NCT), Im Neuenheimer Feld 460, Heidelberg 69120, Germany. ment of Radiology, Cologne University Hospital, Cologne, Germany. 9Depart- Phone: 49-6221-56-35212; Fax: 49-6221-56-5389; E-mail: ment of Nuclear Medicine, Cologne University Hospital, Cologne, Germany. [email protected] 10Department of Internal Medicine VI, Heidelberg University Hospital, Heidel- doi: 10.1158/1078-0432.CCR-16-0860 berg, Germany. 11Section for Personalized Oncology, Heidelberg University Hospital, Heidelberg, Germany. 12German Cancer Consortium, Heidelberg, Ger- 2016 American Association for Cancer Research.

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FGFR1 Is a Therapeutic Target in Soft-Tissue Sarcoma

the potential of FGFR1 to serve as a biomarker for FGFR Translational Relevance inhibitor therapy in STS. We identify FGFR1 amplification and Soft-tissue sarcomas (STS) are highly diverse and clinically overexpression as frequent events in multiple STS subtypes, challenging malignancies. In the setting of metastatic STS, demonstrate that STS cells harboring FGFR1 amplification and systemic therapy using a limited spectrum of conventional overexpression are highly sensitive to genetic and pharmaco- cytotoxic drugs may provide symptom palliation and prevent logic FGFR1 inhibition, and delineate the MAPK–ERK1/2 sig- rapid disease progression, but does not prolong survival in the naling axis as critical FGFR1 effector pathway in STS cells. majority of cases. Hence, there is an unmet need for novel, molecularly targeted STS therapies. Alterations of the FGFR1 receptor tyrosine kinase have been identified as oncogenic Materials and Methods drivers in various cancers, and small-molecule FGFR inhibitors Patient samples are being evaluated as anticancer drugs; however, the role of The frequency of FGFR1 alterations was determined in two FGFR1 in STS has not been established. Here, we report that retrospective patient cohorts (Supplementary Tables S1–S3). the FGFR1 gene is recurrently amplified and overexpressed in Cohort 1 comprised 190 patients with high-grade STS [alveolar multiple STS subtypes, and that increased FGFR1 expression, soft-part sarcoma, epithelioid sarcoma (ES), angiosarcoma (AS), alone and in the context of FGFR1 amplification, imparts undifferentiated pleomorphic sarcoma (UPS), leiomyosarcomas sensitivity to FGFR inhibitors. These findings provide a ratio- (LMS), synovial sarcoma (SS)] who underwent biopsy or surgical nale for molecularly stratified clinical trials of FGFR inhibitors resection of their tumors at the Klinikum rechts der Isar, Technical in patients with STS. University Munich, Munich, Germany, between 1995 and 2015. Cohort 2 comprised specimens from 79 patients with untreated high-grade STS [UPS, LMS, SS, malignant peripheral nerve sheath tumor (MPNST), dedifferentiated liposarcoma (DDLS), myxofi- ranges from 11 to 15 months after the development of distant brosarcoma (MFS), myxoid liposarcoma (MLS), and pleomor- metastases (1, 4–7). Thus, there is an urgent need for more phic liposarcoma (PLS)] that were collected at the Institute effective and well-tolerated therapies. of Pathology, Heidelberg University Hospital, Heidelberg, FGFR1–4 are receptor tyrosine kinases (RTK) that are activated Germany, between 1989 and 2008. Tumors were categorized upon heparin-mediated binding to their cognate ligands, which according to current World Health Organization criteria (1), and triggers dimerization, transphosphorylation of the activation grading was performed using the French Federation Nationale des loop, and phosphorylation of tyrosines in the cytoplasmic tail. Centres de Lutte Contre le Cancer system. Cases of MLS, SS, and Subsequently, PLCG1 and the docking protein FRS2 are DDLS were also verified by detection of the FUS–DDIT3 and recruited, which in turn stimulate the PLCG1–PKC, PI3K–AKT, SS18–SSX fusion genes and MDM2 amplification, respectively. and MAPK–ERK1/2 pathways (8). FGFR signaling is tightly All tissue specimens were independently reviewed by two board- involved in cell survival, proliferation, and differentiation and certified pathologists. is also known to regulate the development of organ systems, angiogenesis, and wound repair (8–11). Apart from these phys- Whole-exome sequencing iologic functions, aberrant FGFR signaling resulting from muta- DNA from tumor tissue and peripheral blood was isolated tion, amplification, translocation, and overexpression of FGFR using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen), fol- family members has been implicated in several epithelial and lowed by quality control using gel electrophoresis and a Bioa- hematologic malignancies (10, 12, 13). The "druggability" of nalyzer 2100 system (Agilent). Exome capturing was performed FGFR family members has established them as promising targets using SureSelect Human All Exon V5þUTRs in-solution capture for therapeutic intervention. As a consequence, several broad- reagents (Agilent). Briefly, 1.5-mg genomic DNA were fragmen- spectrum RTK inhibitors, such as dovitinib, ponatinib, and tedto150–200 bp (paired-end) insert size with a Covaris S2 nintedanib, as well as FGFR-selective inhibitors, for example device, and 250 ng of Illumina adapter-containing libraries AZD4547, BGJ398, and PD173074, are either approved for were hybridized with exome baits at 65C for 16 hours. Paired- clinical use or are currently being investigated in the clinical end sequencing (101 bp) was carried out with a HiSeq 2500 trials for FGFR-driven malignancies (14). instrument (Illumina) in rapid mode. Reads were mapped to TheroleofFGFR1 amplification as an oncogenic driver and the 1000 Genomes Phase II assembly of the human reference potential drug target has been extensively studied in epithelial genome (NCBI build 37.1) using BWA (version 0.6.2) with malignancies such as breast and squamous cell lung cancer default parameters and maximum insert size set to 1,000 bp. (10, 15). In contrast, little is known about the relevance of BAM files were sorted with SAMtools (version 0.1.19; ref. 18), deregulated FGFR signaling in adult STS. Missiaglia and col- and duplicates were marked with Picard tools (version 1.90). leagues detected FGFR1 amplification and concomitant over- Average target coverage was 129 for the tumor and 122 for expression in a subset of embryonal and alveolar rhabdomyo- the control. In both, more than 80% of the targets had coverage sarcoma (16), and Ishibe and colleagues studied the growth- of at least 50. Copy number variants (CNV) were analyzed by promoting effect of ligand-induced FGFR signaling in synovial read depth plots and an in-house pipeline using the VarScan2 sarcoma cell lines (17). However, systematic studies of large copynumber and copyCaller modules (19). Regions were fil- patient cohorts encompassing multiple STS subtypes comple- tered for unmappable genomic stretches, merged by requiring mented by functional studies are lacking. In this study, at least 70 markers per called copy number event, and anno- prompted by the detection, and subsequent therapeutic target- tated with RefSeq genes using BEDTools (20). Genotyping data ing, of amplified FGFR1 in a patient with metastatic leiomyo- were deposited in the European Genome-phenome Archive sarcoma within a clinical sequencing program, we investigated under accession No. EGAS00001001844.

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Microarray-based comparative genomic hybridization and body: anti-FGFR1 (D8E4; Cell Signaling Technology). As neg- mRNA expression profiling ative control, the primary antibody was substituted by normal Genomic DNA from primary human STS samples was isolated rabbit IgG (Santa Cruz Biotechnology). All control stainings using the Allprep DNA/RNA/Protein Mini Kit (Qiagen), fol- were negative. lowed by precipitation with ethanol and 5 mol/L ammonium m acetate. Tumor DNA (1.5 g) or gender-matched control DNA Cell culture were labeled with the Genomic DNA ULS Labeling Kit (Agilent). HS-SY-II, SW982, and 1273/99 cells were provided by Wolf- fi A NanoDrop instrument (Thermo Scienti c) was used to mea- gang Hartmann (Munster€ University Hospital, Munster,€ Ger- > sure the degree of labeling, and only samples with values 2% many). SK-LMS-1 cells were a gift from Sebastian Bauer (Essen were used for hybridization. Microarray processing, including University Hospital, Essen, Germany). SKUT1 and SKUT1B were hybridization, washing, and scanning steps, was carried out purchased from the ATCC. Cell line identity and purity were according to the manufacturer's recommendations (Agilent). In verified using the Multiplex Cell Authentication and Contami- brief, paired tumor and gender-matched reference samples were nation Tests (Multiplexion). All cell lines were regularly tested for hybridized on a Human Genome CGH Microarray 244A in a mycoplasma contamination using the Venor GeM Mycoplasma SureHyb Chamber (Agilent) and incubated in a hybridization Detection Kit (Minerva). Cell lines were cultured as follows: HS- oven (Agilent) at 65 C and 20 rotations per minute for 40 hours. SY-II in DMEM (Life Technologies), 10% FBS (Biochrom), 0.5% For washing of microarrays, procedure A was chosen in an sodium pyruvate; 1273/99 in Ham F12 (Life Technologies), 10% ozone-controlled environment. Finally, slides were scanned with FBS; SW982 in DMEM, 10% FBS; SKLMS-1 in RPMI1640 (Life an Agilent High-Resolution C Scanner with the following set- Technologies), 15% FBS; SKUT1 and SKUT1B in MEM (Life m tings: resolution, 5 m; Tiff, 16 bit; red and green PMT, 100%; Technologies), 10% FBS. All media were supplemented with < > XDR, No XDR . Data extraction was done using Feature Extrac- 1% penicillin/streptomycin and 1% L-glutamine (Biochrom). tion Software v10 (Agilent Technologies). R package "DNAcopy" PD173074, BGJ398, and AZD4547 were obtained from Selleck. was used for normalization of profiles, and outliers were smoothed. R package "CGHcall" was used for calling of copy Quantitative RT-PCR number profiles. Gene expression profiling was performed using Total RNA was isolated using the RNeasy Mini Kit (Qiagen) HumanHT-12 v3 Expression BeadChip technology (Illumina) as and reverse-transcribed using the High-Capacity cDNA Reverse described previously (21), and the dataset is available at NCBI's Transcription Kit (Applied Biosystems). Real-time RT-PCR Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; reactions using KiCqStart SYBR Green Primers (Sigma) and accession No. GSE52392). LightCycler 480 SYBR Green I Master reagents (Roche) were performed to quantify FGFR1 expression relative to endoge- Tissue microarray construction nous PBGD. Formalin-fixed, paraffin-embedded STS samples were assem- bled into a tissue microarray (TMA) using a Tissue Microarrayer (Beecher Instruments) with a core size of 0.6 mm. Three to six RNA interference areas containing vital tumor tissue were marked by two pathol- Short hairpin RNA (shRNA) sequences targeting FGFR1 ogists and selected for TMA generation. (TRCN0000312574, TRCN0000312572) and a nontargeting control sequence (SHC002) from the TRC-Hs 1.0 (Human) shRNA FISH Library were cloned into the pLKO.1puro lentiviral shRNA vector. – Cell lines were trypsinized, resuspended in phosphate-buff- VSV-G pseudotyped lentiviral particles were produced by ered formalin, precipitated with FBS, and embedded in paraffin. cotransfection of 293T cells with pLKO.1 constructs and the Sections (3–4 mm) from paraffin blocks or TMA were depar- compatible packaging plasmids pSPAX2 and pMD2.G. Cell lines m affinized with xylol, treated with 0.005% pepsin for 25 minutes, were incubated with lentiviral supernantants in presence of 8 g/ and hybridized overnight at 37C with the ZytoLight SPEC mL polybrene (Millipore) for 48 hours, and stably transduced – m FGFR1/CEN8 Dual Color Probe (ZytoVision). Denaturation cells were selected with puromycin (1 5 g/mL) for 5 days. of the probe at 85C for 4 minutes was performed using a ThermoBrite System (Leica). Posthybridization saline-sodium Western blotting citrate washing steps were performed at 37C, and slides were Western blotting was performed as described previously (23). stained with 40,6-diamidino-2-phenylindole before analysis. Briefly, whole-cell protein extracts were prepared using RIPA cell FGFR1 signals were analyzed using an Axioplan 2 fluorescent lysis buffer, and 40 mg of protein were subjected to SDS-PAGE microscope (Zeiss) and 40 and 63 objectives, and images and transferred to nitrocellulose membranes (Whatman). Mem- were taken using a Leica Application Suite V3.8. Changes in branes were blocked with 5% BSA or low-fat milk and incubated FGFR1 copy number were categorized according to the criteria with the following antibodies: anti-ß-actin (Millipore); anti- proposed by Schildhaus and colleagues (22). FGFR1, anti-phospho-PLCG1, anti-PLCG1, anti-phospho-AKT (S473), anti-AKT, anti-phospho-ERK (T202/Y204), anti-ERK IHC (Cell Signaling Technology). To investigate the effects of phar- Tissue sections were deparaffinized and rehydrated with a macologic FGFR1 inhibition on FGFR1-mediated signaling, STS graded ethanol series. Heat-based antigen retrieval was carried cell lines were subjected to serum starvation for four hours, out in citrate buffer (DAKO), and immunohistochemical stain- treatment with 2.5 mmol/L AZD4547 for 4 hours, and incuba- ing was performed using the SignalStain Boost IHC Detection tion with 20 ng/mL recombinant human basic FGF (Peprotech) Reagent (Cell Signaling Technology) in accordance with the for 15 minutes prior to the preparation of protein extracts and manufacturer's instructions and the following primary anti- Western blotting.

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FGFR1 Is a Therapeutic Target in Soft-Tissue Sarcoma

Cell viability and proliferation assays PDGFR, and FGFR inhibitor nintedanib in combination with The effects of FGFR1 knockdown or pharmacologic FGFR1 everolimus (ClinicalTrials.gov Identifier NCT01349296). Sur- inhibition on cell viability and proliferation were determined prisingly, a CT scan performed 6 weeks after discontinuation of using the CellTiter96AQueousOne Solution Proliferation Assay BGJ398 and before treatment with nintedanib and everolimus (Promega). Cells (SK-LMS-1, 1 103; SW982, SKUT1, SKUT1B, was initiated which showed that several metastases had 1.5 103; HS-SY-II; 6 103, 1273/99, 5 103) were seeded in decreased in size, whereas others continued to grow, indicating 96-well plates in triplicates, and absorbance at 490 nm was a delayed response to FGFR inhibition (Fig. 1D; Supplementary measured on five consecutive days or after 72 hours. Table S4). Subsequent on-protocol treatment with nintedanib and everolimus resulted in rapid clinical improvement but was Anchorage independence assays discontinued by the patient after 1 month, necessitating her The effect of FGFR1 knockdown on anchorage-independent withdrawal from the trial. An 18F-fluorodeoxyglucose (FDG) growth was determined as described previously (24). Cells (SK- positron emission tomography scan 2 months after the last LMS-1, 5 103; SW982, 1 104; HS-SY-II and 1273/99, 2 104; intake of study medication demonstrated near-physiologic SKUT1 and SKUT1B, 1.5 103) were seeded in 35-mm plates in FDG uptake (Fig. 1E), again indicating prolonged efficacy, and triplicates, and colony formation was analyzed after 2 (SK-LMS-1) the patient was started on continuous low-dose trofosfamide or 4 (SW982, HS-SY-II, 1273/99) weeks by staining with 0.005% in combination with celecoxib. Her condition remained crystal violet for 1 hour. stable for 8 months when a gradual increase in metabolic activity of the liver metastases was accompanied by slow Statistical analysis clinical deterioration, and the patient was lost to follow-up Experiments were performed at least three times in triplicates; 14 months after initiation of FGFR-directed therapy. This unless otherwise indicated, one representative experiment is demonstration of FGFR1 amplification and overexpression in shown. Error bars represent SDs of triplicate measurements. leiomyosarcoma, a common STS subtype and, in particular, Computations were performed using GraphPad Prism. the sustained clinical benefit achieved with short courses of FGFR inhibition prompted us to systematically investigate the Study approval frequency and functional implications of FGFR alterations in Tumor tissue and a matched normal blood sample for whole- adult STS. exome sequencing were obtained following written informed consent under an Institutional review board–approved protocol Recurrent amplification and overexpression of FGFR1 in covering all aspects relevant to clinical cancer genome sequencing. multiple STS subtypes This study was conducted in accordance with the Declaration of To assess the frequency of FGFR1 alterations broadly across STS Helsinki. subtypes, we first analyzed FGFR1 DNA copy number status by FISH in a cohort of 190 patients with high-grade STS (cohort 1; Table 1 and Supplementary Tables S1 and S2). In each case, 60 Results tumor cell nuclei were analyzed, and the results were evaluated Amplification of FGFR1 in metastatic leiomyosarcoma according to a standardized scoring system that was developed by To identify therapeutically tractable genetic alterations in a 23- Schildhaus and colleagues to reproducibly identify high- and low- year-old woman with metastatic retroperitoneal leiomyosar- level FGFR1 amplifications in patients with non–small cell lung coma who had progressed on prior treatment with doxorubi- cancer (22). An important feature of this system is the application cin/ifosfamide and trabectedin, we performed whole-exome of multiple complementary criteria to capture the highly hetero- sequencing (WES) of tumor and matched normal tissue within geneous patterns of FGFR1 gene copy number. Specifically, high- NCT MASTER (Molecularly Aided Stratification for Tumor Erad- level amplification is defined as FGFR1/CEN8 ratio 2, FGFR1 ication Research), an Institutional review board–approved clin- copy number per nucleus 6, or percentage of tumor cells con- ical sequencing program for younger patients with advanced- taining 15 gene copies 10%. Low-level amplification is stage cancer across all histologies (25). DNA copy number defined as percentage of tumor cells containing 5 gene copies analysis showed high-level amplification of a region on the 50%. Sixty-one of 190 cases (32.1%) showed >2 CEN8 signals short arm of chromosome 8, which included the FGFR1 locus due to polysomy, which may in rare cases result in an FGFR1/ (Fig. 1A). This finding was confirmed by FISH using probes CEN8 ratio <1 despite an increase in absolute FGFR1 copy number for FGFR1 and the centromere of chromosome 8 as control compared with normal tissue. These alterations were classified as (Fig. 1B), which yielded an FGFR1/CEN8 ratio 2 indicative of polysomy/copy number gain. In addition, FGFR1 amplification high-level amplification according to the criteria developed by was detected in 13 of 190 cases (6.8%). STS subtypes affected by Schildhaus and colleagues (22). The amplification correlated FGFR1 copy number alterations in this cohort included ES, AS, with high expression of FGFR1 protein in the tumor but not the UPS, and LMS. surrounding normal tissue (Fig. 1C). To further investigate FGFR1 alterations using genome-wide On the basis of these results, the patient was enrolled in a approaches, we next analyzed FGFR1 DNA copy number status phase I clinical trial of the pan-FGFR inhibitor BGJ398 (Clin- and mRNA expression by array-based comparative genomic icalTrials.gov Identifier NCT01004224). Staging after 2 months hybridization and mRNA expression profiling in our previously of treatment showed a 24% increase in the sum of the diameters described cohort of 79 patients with untreated high-grade STS of target lesions, consistent with progressive disease according (cohort 2; Supplementary Tables S1 and S3; ref. 21). Copy to response evaluation criteria in solid tumors. Treatment, number analysis revealed that FGFR1 was gained or amplified therefore, had to be discontinued, and the patient consented in 13 of 79 cases (16.5%; Fig. 2A, left). To determine whether these to participate in another phase I trial of the pan-VEGFR, genomic changes translated into elevated FGFR1 expression, we

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A 4 1 3 5 7 9 11 13 15 17 19 21 X

Segment mean –2

–4 BC

D Baseline Follow-up 1 Follow-up 2 E Mesentery (T02) Baseline: 29.3 mm Follow-up 1: 34.7 mm (+18.6%) Follow-up 2: 20.4 mm (–41.4%)

Liver (T04) Baseline: 14.4 mm Follow-up 1: 19.6 mm (+36.5%) Follow-up 2: 16.3 mm (–17.0%)

Lung (T06) Baseline: 13.6 mm Follow-up 1: 16.7 mm (+22.2%) Follow-up 2: 19.4 mm (+16.6%)

Lymph node (T07) Baseline: 17.8 mm Follow-up 1: 21.6 mm (+21.8%) Follow-up 2: 18.5 mm (–14.2%)

Figure 1. Amplification of FGFR1 in a patient with metastatic leiomyosarcoma. A, CNV plot showing focal amplification of chromosome 8p (arrow). B, Representative FISH signal pattern showing high-level amplification of FGFR1 (green, arrows) relative to the centromere of chromosome 8 (red). Original magnification, 40. C, Photomicrographs showing high expression of FGFR1 in a liver metastasis (left and bottom right) but not the surrounding normal tissue (left and top right). Scale bars, 500 mm (left) and 100 mm (right). D, Computed tomography scans performed at baseline, after 2 months of BGJ398 treatment (follow-up 1), and 6 weeks after discontinuation of BGJ398 treatment (follow-up 2). Shown are the results for four of seven target lesions. E, FDG positron emission tomography scan showing near-physiologic FDG uptake 2 months after discontinuation of nintedanib and everolimus treatment. Lesions with slightly increased metabolic activity are indicated (arrows).

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Table 1. FGFR1 copy number alterations detected by FISH in STS Cohort 1 a Disomy Polysomy/gain Amplification (No. of cases, %) Subtype No. of cases (No. of cases, %) (No. of cases, %) Ib IIb IIIb IVc Alveolar soft-part sarcoma 10 10 (100) Epithelioid sarcoma 11 8 (72.7) 3 (27.3) Angiosarcoma 29 24 (82.8) 4 (13.8) 1 (3.4) Undifferentiated pleomorphic sarcoma 91 28 (30.8) 49 (53.8) 6 (6.6) 3 (3.3) 2 (2.2) Leiomyosarcoma 10 4 (40) 5 (50) 1 (10) Synovial sarcoma 39 39 (100) Total 190 113 (59.5) 61 (32.1) 8 (4.2) 3 (1.6) 2 (1.1) aI, FGFR1/CEN8 ratio 2; II, average number of FGFR1 signals per tumor cell 6; III, percentage of tumor cells containing 15 FGFR1 signals or large clusters 10%; IV, percentage of tumor cells containing 5 FGFR1 signals 50%. bHigh-level amplification. cLow-level amplification.

defined the mean FGFR1 mRNA level across all samples plus was supported further by our observation that 92 of 254 cases one SD as threshold for overexpression. According to this cutoff, (36.2%) in the TCGA cohort showed copy number gain or 16 of 79 cases (20.2%) showed FGFR1 overexpression. Among amplification of FRS2, of which 43 (16.9%) showed FRS2 over- these were five cases (31.3%) with FGFR1 gain/amplification, expression (Supplementary Fig. S3). FRS2 encodes an FGFR whereas 11 cases (68.7%) were diploid for the FGFR1 locus adaptor protein that induces downstream MAPK–ERK1/2 signal- (Fig. 2A, left). ing and has been implicated in high-grade liposarcoma as well as To validate these findings, we used the cBioPortal for Cancer other cancers (31, 32). Genomics (26, 27) to query The Cancer Genome Atlas (TCGA) cohort of 254 STS cases (cohort 3; Supplementary Table S1) for FGFR1 essentiality in human STS cells changes in FGFR1 DNA copy number and mRNA expression, as To determine the functional impact of FGFR1 alterations in determined by SNP array analysis and RNA sequencing, respec- STS, we first screened a panel of human STS cell lines for FGFR1 tively (Fig. 2A, right). Here, gene copy numbers were classified amplification (Fig. 3A) and overexpression (Fig. 3B and C) using using the GISTIC algorithm as (i) deep loss or homozygous FISH, quantitative RT-PCR, and immunoblotting. HS-SY-II SS deletion, (ii) shallow loss or heterozygous deletion, (iii) diploid, cells demonstrated high-level amplification of the FGFR1 locus, (iv) low-level gain, and (v) high-level amplification (28). Accord- which resulted in high FGFR1 mRNA and protein expression. ing to these categories, the FGFR1 locus was gained in 69 of 254 SKUT1B uterine LMS cells, on the other hand, showed high FGFR1 cases (27.1%) and amplified in 11 of 254 cases (4.3%). Analysis of mRNA and protein levels in the absence of genomic FGFR1 FGFR1 mRNA levels using the cutoff described above showed that amplification, indicating upregulation of FGFR1 through a tran- 39 of 254 cases (15.4%) overexpress FGFR1 (Fig. 2A, right). scriptional mechanism. The cell lines SKUT1, of which SKUT1B is Interestingly, high-level amplification or gain of the FGFR1 locus a substrain, 1273/99 (SS), SK-LMS-1 (LMS), and SW982 (spindle were present in five (12.8%) and 17 (43.6%) of 39 FGFR1-over- cell sarcoma, not otherwise specified) showed moderate FGFR1 expressing cases, respectively, whereas 14 (35.9%) and three mRNA and protein expression and no FGFR1 amplification. (7.7%) samples were diploid or characterized by heterozygous To investigate whether FGFR1 amplification and/or overexpres- FGFR1 loss. Thus, findings from the high-grade STS and TCGA sion confer FGFR1 dependence, we next performed FGFR1 knock- cohorts indicate that elevated FGFR1 expression can also occur in down using lentivirally delivered shRNA constructs (Fig. 4A). In the absence of increased FGFR1 copy number. the high FGFR1-expressing cell lines HS-SY-II and SKUT1B, sup- Analysis of clinical data from cohorts 2 and 3 revealed FGFR1 pression of FGFR1 resulted in strong inhibition of cell prolifer- copy number gain and overexpression in seven different STS ation and survival, whereas cell lines with moderate FGFR1 subtypes, including UPS, LMS, DDLS, PLS, MFS, SS, and MPNST expression, namely SW982, SK-LMS-1, and SKUT1, were affected (Fig. 2B and Supplementary Fig. S1). Furthermore, three cases in to a lesser extent (Fig. 4B). Likewise, FGFR1 depletion abrogated the TCGA cohort showed FGFR1 mutations, namely N546K, anchorage-independent growth of HS-SY-II and SKUT1B cells H166Y, and E804K in an MPNST, LMS, and UPS case, respectively and had a moderate effect in SW982, SK-LMS-1, and SKUT1 cells (Fig. 2A, right, red dots). Among these, N546K, which has been (Fig. 4C). 1273/99 cells were unresponsive to FGFR1 knockdown. reported to occur in glioblastoma, pilocytic astrocytoma, and These results indicate that the degree of dependence on oncogenic papillary glioneuronal tumors, affects the kinase domain critical FGFR1 signaling of STS cells is primarily determined by FGFR1 for aberrant FGFR1 signaling (29). expression levels. In addition to STS patient samples, we analyzed FGFR1 expression in 1,036 human cell lines, representing 37 cancer types, using Sensitivity of human STS cells to selective FGFR inhibitors the Cancer Cell Line Encyclopedia database (30). Ranking of cell To test whether the effects of shRNA-mediated FGFR1 suppres- lines according to FGFR1 levels derived from gene-centric, robust sion could be phenocopied by pharmacologic inhibition multiarray average-normalized mRNA expression data identified of FGFR1, we incubated STS cell lines with increasing concentra- STS cell lines (n ¼ 21) among the top FGFR1 expressers, only tions of the selective small-molecule FGFR inhibitors PD173074, slightly surpassed by chondrosarcoma, mesothelioma, and kid- AZD4547, and BGJ398 (33–35). In line with the knockdown ney cancer (Fig. 2C and Supplementary Fig. S2). approach, high FGFR1-expressing HS-SY-II and SKUT1B Together, these data suggested a pathogenic role for deregulated cells exhibited profound sensitivity to all three inhibitors, with FGFR1 signaling in a relevant proportion of STS patients. The AZD4547 having the strongest effect, whereas SW982, SK-LMS-1, potential importance of the FGFR pathway for STS pathogenesis SKUT1, and 1273/99 showed intermediate to no sensitivity to

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A High-grade sarcoma cohort TCGA sarcoma cohort n n 8 ( = 79) 1616 ( = 254)

7 1515

6 1414

5 1313

4 1212

3 1111

FGFR1 Expression (log2) 2 1010

99 FGFR1 expression (RNA Seq V2 RSEM, log2) 0 Hetdel/ Diploid Gain/ Deep Shallow Diploid Gain Amplification Homodel Amplification deletion deletion

B FGFR1 DNA Copy number status FGFR1 DNA Copy number status LMS MPNST 6% SS 8% SS 10% DDLS 19% 31% MPNST 37% MFS UPS 13% 6%

LMS PLS UPS DDLS 19% 18% 20% 13%

High-grade sarcoma cohort TCGA Sarcoma cohort

C FGFR1-Entrez ID: 2260 mRNA Expression level (RMA, log2) mRNA

Figure 2. Recurrent ampliﬁcation and overexpression of FGFR1 in multiple STS subtypes. A, FGFR1 mRNA expression according to FGFR1 DNA copy number in a cohort of 79 patients with untreated high-grade STS (left) and the TCGA sarcoma cohort (right). Dots represent individual patients. Red dots in the right indicate patients harboring FGFR1 mutations. Dashed lines indicate thresholds for FGFR1 overexpression. Hetdel, heterozygous deletion; Homodel, homozygous deletion. B, Distribution of histologic subtypes among patients with FGFR1 overexpression in the high-grade STS cohort (left) and the TCGA sarcoma cohort (right). C, Ranking of cancer cell lines (n ¼ 1,036) of different histologies from the Cancer Cell Line Encyclopedia according to normalized FGFR1 mRNA expression.

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FGFR1 Is a Therapeutic Target in Soft-Tissue Sarcoma

A HS-SY-II 1273/99 SK-LMS-1

SW982 SKUT1 SKUT1B Figure 3. Amplification and overexpression of FGFR1 in STS cell lines. A, Representative FISH signal pattern showing high-level amplification of FGFR1 (green, arrow) relative to the centromere of chromosome 8 (red) in HS-SY-II cells. Nonamplified 1273/99, SW982, SKUT1, and SKUT1B cells harbor two FGFR1 signals on average. B, FGFR1 mRNA expression of STS cell lines. C, FGFR1 protein expression of STS cell lines. Multiple bands B C represent different isoforms and/or 1.2 posttranslational modifications of FGFR1. 1.0 HS-SY-II 1273/99 SK-LMS-1 SW982 SKUT1 SKUT1B 0.8

0.6 FGFR1 0.4

0.2 β-Actin Relative FGFR1 expression 0.0 9 9 1B -SY-II MS-1 -L SW982 SKUT1 HS 1273/ K SKUT S

FGFR inhibitors (Fig. 5A). These results further support the MAPK–ERK1/2signalingaxisascriticalFGFR1effectorpathway conclusion that FGFR1 expression levels are a potential biomarker whose suppression dictates the sensitivity of FGFR1-driven STS of sensitivity to FGFR pathway inhibition in STS patients that cells to FGFR-targeted therapy. might be of value for enrichment of future clinical trial populations. Discussion FGFR1-driven PI3K–AKT, PLCG1, and MAPK–ERK1/2 signaling In this study, we establish FGFR1 amplification and overexpres- in human STS cells sion as recurrent events in nine different categories of STS, Under physiologic conditions, ligand-mediated FGFR1 acti- including subtypes with specific genetic alterations and relatively vation leads to recruitment of the adaptor protein FRS2, which few chromosomal changes, such as SS and DDLS, as well as induces MAPK–ERK1/2 and PI3K–AKT signaling (36). Further- subtypes that are characterized by complex karyotypes and lack more, FGFR1 stimulates the PLCG1–PKC pathway in an FRS2- pathognomonic genetic alterations, such as UPS, MPNST, LMS, independent manner (10, 14). To investigate the signaling PLS, and MFS. Furthermore, we demonstrate that high FGFR1 events triggered by FGFR1 in STS cells, we subjected STS cell expression confers sensitivity to genetic or pharmacologic FGFR lines to serum starvation and incubated them with the FGFR inhibition, and delineate the MAPK–ERK1/2 signaling axis as inhibitor AZD4547 with or without addition of FGF (Fig. 5B). critical FGFR1 effector pathway whose suppression dictates the In HS-SY-II, SW982, SK-LMS-1, SKUT1, and SKUT1B cells, sensitivity of high FGFR1-expressing STS cells to FGFR blockade. AZD4547 treatment resulted in reduced basal and ligand-stim- The identification of altered FGFR1 signaling as pharmacolog- ulated phosphorylation of AKT, PLCG1, and ERK1/2. In con- ically tractable event in a sizeable proportion of STS patients trast, 1273/99 cells showed only marginal dephosphorylation indicates that genomics-driven, individualized treatment of ERK1/2 in response to AZD4547, whereas AKT and PLCG1 approaches may represent an alternative or adjunct to conven- phosphorylation responded to FGFR blockade. Together with tional chemotherapy across different histologies. Thus far, the the observation that viability and proliferation of 1273/99 cells genomic landscape of STS remains incompletely characterized, were not affected by shRNA-mediated or pharmacologic FGFR1 and as a consequence, targets for molecularly directed therapies inhibition (Figs. 4C and 5A), the latter result points to the remain elusive in the majority of cases, with CDK4 and MDM2 in

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A HS-SY-II SW982 1273/99 _ _ _ NTC shRNA-1 shRNA-2 NTC shRNA-1 shRNA-2 NTC shRNA-1 shRNA-2

FGFR1 100 KDa

50 KDa β-Actin

SK-LMS-1 SKUT1 SKUT1B _ _ _ shRNA-1 shRNA-2 NTC shRNA-1 shRNA-2 NTC shRNA-1 shRNA-2 NTC

FGFR1 100 KDa

50 KDa β-Actin

B HS-SY-II SW982 1273/99 2.5 shRNA-1 3.0 shRNA-1 2.5 shRNA-1 shRNA-2 2.5 shRNA-2 shRNA-2 2.0 NTC NTC 2.0 NTC 2.0 1.5 1.5 1.5 1.0 1.0 Figure 4. 1.0 FGFR1 essentiality in STS cell lines. A, 0.5 0.5 0.5 Knockdown of FGFR1 in STS cell lines. Relative proliferation Relative proliferation 0.0 0.0 Relative proliferation 0.0 –, untransduced; shRNA-1 and shRNA- 12345 12345 123452, FGFR1-speciﬁc shRNAs; NTC, Day Day Day nontargeting control shRNA. B, Effect SK-LMS-1 SKUT1 SKUT1B of FGFR1 knockdown on viability and 2.0 shRNA-1 2.5 shRNA-1 2.0 shRNA-1 proliferation of STS cell lines. Error shRNA-2 shRNA-2 shRNA-2 bars, SDs of triplicate measurements. NTC 2.0 NTC NTC 1.5 1.5 C, Effect of FGFR1 knockdown on 1.5 anchorage-independent growth of 1.0 1.0 1.0 STS cell lines. Error bars, SD of 0.5 0.5 triplicate measurements. 0.5 Relative proliferation Relative proliferation 0.0 Relative proliferation 0.0 0.0 12345 12345 12345 Day Day Day

C 80 HS-SY-II 200 SW982 200 1273/99

60 150 150

40 100 100

20 50 50 No. of colonies No. of colonies 0 No. of colonies 0 0 1 2 - C 1 2 C 1 -2 C A- T NT NA- NT N hRNA hRNA- hR hRNA- hRNA s s s s shRN s

200 SK-LMS-1 250 SKUT1 250 SKUT1B

150 200 200 150 150 100 100 100 50 50 50 No. of colonies No. of colonies 0 0 No. of colonies 0 1 2 C 1 -2 C 1 -2 C A NA- NT NA- NT N NT R R R h hRNA- h hRNA hRNA- s s s s s sh

well-differentiated liposarcoma and DDLS and mutant PIK3CA in bitors (41–43). Our findings indicate that comprehensive geno- MLS being notable exceptions (37–40). However, the examples of mic profiling will facilitate the identification of additional ther- dermatofibrosarcoma protuberans, inflammatory myofibroblas- apeutic targets in STS, including more common subtypes such as tic tumor, and perivascular epithelioid cell tumor illustrate the DDLS, PLS, UPS, and LMS, and aid in identifying patients who are potential of genetic studies to inform the treatment of STS, as these most likely to benefit from a particular therapy. For example, there rare diseases are driven by mutations in signaling pathways are thus far no molecular determinants of efficacy in patients (COL1A1-PDGF fusion, ALK fusions, and TSC1/2 mutations, receiving pazopanib, the only targeted drug approved to treat STS respectively) that can be targeted by small-molecule kinase inhi- (44, 45). Given that pazopanib has activity against FGFR family

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FGFR1 Is a Therapeutic Target in Soft-Tissue Sarcoma

A 1.2 1.2 1.2 1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4 Cell viability Cell viability Cell viability 0.2 0.2 0.2

0.0 0.0 0.0 9 5 9 9 8 6 5 O 9 8 6 19 39 78 56 12 1 39 7 12 00 19 39 7 5 1 3 62 15 3 62 1 312 625 1,2502,5005,000 1,2502,5 5,000 1,2502,5005,000 DMSO DMSO DMS PD173074 (nmol/L) BGJ398 (nmol/L) AZD4547 (nmol/L) HS-SY-II SW982 1273/99 SK-LMS-1 SKUT1 SKUT1B B HS-SY-II SW982 1273/99 AZD4547 − + − + − + − + − + − + FGF −− ++ −− ++ −− ++ KDa p-PLCG1 (Y783) 130

t-PLCG1 130

p-AKT (S473) 60

t-AKT 60

p-ERK (T202/Y204) 42/44

t-ERK 42/44

SK-LMS-1 1TUKS B1TUKS AZD4547 − + − + − + − + − + − + FGF −− ++ −− ++ −− ++ KDa p-PLCG1 (Y783) 130

t-PLCG1 130

p-AKT (S473) 60

t-AKT 60

p-ERK (T202/Y204) 42/44

t-ERK 42/44

Figure 5. Sensitivity of STS cell lines to selective FGFR inhibitors. A, Effects of different FGFR inhibitors on viability and proliferation of STS cell lines. Error bars, SDs of triplicate measurements. B, Effects of AZD4547 and FGF on components of the PLCG1, PI3K–AKT, and MAPK–ERK1/2 signaling pathways in STS cell lines.

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members, it appears warranted to analyze the outcome of pazo- Authors' Contributions panib-treated STS patients according to their tumors' FGFR1 Conception and design: P. Chudasama, C. von Kalle, J. Wolf, H. Glimm, status. C. Scholl, G. Mechtersheimer, K. Specht, S. Frohling€ Similar to other malignancies driven by aberrant signaling of Development of methodology: P. Chudasama, M. Straub, B. Hutter, R. Schweßinger, K. Specht RTK such as FGFR family members (46, 47), STS cells with fi Acquisition of data (provided animals, acquired and managed patients, FGFR1 ampli cation and/or overexpression showed reduced provided facilities, etc.): P. Chudasama, M. Renner, M. Straub, M. Scheffler, viability and proliferation as well as impaired colony formation I. Alldinger, S. Schimmack, T. Persigehl, C. Kobe, P. Schirmacher, C. Heining, in response to knockdown and/or pharmacologic inhibition of S. Groschel,€ J. Wolf, G. Mechtersheimer, K. Specht, S. Frohling,€ M.-K. Beckhaus, FGFR1. Of note, the "addiction" of STS cells to FGFR1-mediated S. Wolf signaling appears to be determined by FGFR1 expression in a Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Chudasama, M. Straub, S.S. Mughal, B. Hutter, dose-dependent manner, with cell lines characterized by mod- Z. Kosaloglu, M. Scheffler, T. Persigehl, C. Kobe, D. J€ager, J. Wolf, B. Brors, erate FGFR1 expression showing intermediate sensitivity to W. Weichert, C. Scholl, G. Mechtersheimer, K. Specht FGFR1 blockade. Thus, assessment of FGFR1 mRNA and/or Writing, review, and/or revision of the manuscript: P. Chudasama, M. Renner, protein expression, rather than DNA copy number, may be best B. Hutter, M. Scheffler, I. Alldinger, S. Schimmack, T. Persigehl, C. Kobe, D. J€ager, suited to prospectively identify STS patients likely to respond to P. Schirmacher, S. Groschel,€ J. Wolf, B. Brors, H. Glimm, C. Scholl, G. Mechter- € FGFR1-targeted therapy, as has recently been reported for lung sheimer, S. Frohling Administrative, technical, or material support (i.e., reporting or organizing cancer and head and neck squamous cell carcinoma (48, 49). data, constructing databases): M. Renner, T. Persigehl, P. Schirmacher, Our signaling studies also indicate that suppression of STS W. Weichert, G. Mechtersheimer growth by FGFR blockade will require inhibition of MAPK– Study supervision: P. Chudasama, C. von Kalle, C. Heining, K. Specht, ERK1/2 signaling. This suggests that analysis of on-treatment S. Frohling€ biopsies by, for example, immunohistochemical assessment of fi phosphorylated ERK may enable early identi cation of patients Acknowledgments – at risk for resistance to FGFR inhibition due to persistent MAPK The authors thank the DKFZ-HIPO and NCT POP Sample Processing ERK1/2 activity. Such resistance may appear only subsequent to Laboratory, the DKFZ Genomics and Proteomics Core Facility, and the treatment or may be an intrinsic property of the tumor. For DKFZ-HIPO Data Management Group for technical support and expertise. We example, collateral mechanisms of MAPK–ERK1/2 activation also thank Katja Beck, Daniela Richter, Karolin Willmund, Roland Eils, and may be "hard-wired" into the genomes of STS patients, under- Peter Lichter for infrastructure and program development within DKFZ-HIPO scoring the need for comprehensive genomic studies to identify and NCT POP, and Axel Ullrich for support. Tissue samples were provided by the NCT Heidelberg Tissue Bank in accordance with its regulations and additional drivers of sarcomagenesis and targets for combina- after approval by the Ethics Committee of Heidelberg University and by the torial treatment approaches (50). Institute of Pathology, Technische Universit€at Munchen,€ in accordance with its In summary, our findings identify FGFR1 as an oncogenic ethical regulations. driver in multiple STS subtypes and suggest that FGFR1 inhibition, guided by patient selection according to FGFR1 expres- Grant Support sion and monitoring of MAPK–ERK1/2 signaling, may broaden This work was supported by grant H021 from DKFZ-HIPO and the the therapeutic armamentarium against STS, a hypothesis that NCT Precision Oncology Program (POP) and by The Wilhelm Sander can readily be tested in molecularly stratified clinical trials. Foundation. The costs of publication of this article were defrayed in part by the Disclosure of Potential Conflicts of Interest payment of page charges. This article must therefore be hereby marked advertisement M. Scheffler reports receiving speakers bureau honoraria from and is a in accordance with 18 U.S.C. Section 1734 solely to indicate consultant/advisory board member for Boehringer Ingelheim. J. Wolf reports this fact. receiving speakers bureau honoraria from and is a consultant/advisory board member for Novartis. No potential conflicts of interest were disclosed by the Received April 6, 2016; revised July 12, 2016; accepted July 28, 2016; other authors. published OnlineFirst August 17, 2016.

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Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma

Priya Chudasama, Marcus Renner, Melanie Straub, et al.

Clin Cancer Res 2017;23:962-973. Published OnlineFirst August 17, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-0860

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