MMP-1 and Pro-MMP-10 As Potential Urinary Pharmacodynamic Biomarkers of FGFR3-Targeted Therapy in Patients with Bladder Cancer

Published OnlineFirst October 17, 2014; DOI: 10.1158/1078-0432.CCR-13-3336

Clinical Cancer Personalized Medicine and Imaging Research

MMP-1 and Pro-MMP-10 as Potential Urinary Pharmacodynamic Biomarkers of FGFR3-Targeted Therapy in Patients with Bladder Cancer

Xiangnan Du1, Benjamin C. Lin2, Qian-Rena Wang1, Hao Li1, Ellen Ingalla1, Janet Tien1, Isabelle Rooney3, Avi Ashkenazi4, Elicia Penuel2, and Jing Qing1

Abstract Purpose: The aim of this study was to identify noninvasive pharmacodynamic biomarkers of FGFR3- targeted therapies in bladder cancer to facilitate the clinical development of experimental agent targeting FGFR3. Experimental Design: Potential soluble pharmacodynamic biomarkers of FGFR3 were identified using a combination of transcriptional profiling and biochemical analyses in preclinical models. Two matrix metalloproteinases (MMP), MMP-1 and MMP-10, were selected for further studies in human bladder cancer xenograft models treated with a specific anti-FGFR3 monoclonal antibody, R3Mab. Serum and urinary levels of MMP-1 and MMP-10 were determined in healthy donors and patients with bladder cancer. The modulation of MMP-1 and MMP-10 by R3Mab in patients with bladder cancer was further evaluated in a phase I dose-escalation study. Results: MMP-1 and MMP-10 mRNA and protein were downmodulated by FGFR3 shRNA and R3Mab in bladder cancer cell lines. FGFR3 signaling promoted the expression and secretion of MMP-1 and pro-MMP- 10 in a MEK-dependent fashion. In bladder cancer xenograft models, R3Mab substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, both MMP-1 and pro-MMP-10 were elevated in the urine of patients with advanced bladder cancer. In a phase I dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in patients with bladder cancer. Conclusion: These findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for R3Mab in patients with bladder cancer. Clin Cancer Res; 20(24); 6324–35. 2014 AACR.

Introduction localized and distant metastatic bladder cancer is approx- Bladder cancer is the fifth most common cancer world- imately 35% and 5% to 6%, respectively (2). Thus, novel wide, with an estimated 386,330 new cases and 150,200 therapeutic agents are urgently needed to improve patient deaths in 2008 (1). Despite radical cystectomy and intensive outcome. chemotherapy, the 5-year survival rate for patients with FGFR3 belongs to a family of four structurally and functionally related single-transmembrane receptor tyrosine kinases, which transduce signals from many of the 22 1Translational Oncology, Genentech, Inc., South San Francisco, California. identified FGF polypeptides in humans (3–5). Upon 2Oncology Biomarker Development, Genentech, Inc., South San Fran- cisco, California. 3Oncology Clinical Science ECD, Genentech, Inc., South canonical FGF binding in a ternary complex with heparin San Francisco, California. 4Research Oncology, Genentech, Inc., South sulfate proteoglycans, FGFR3 undergoes dimerization, San Francisco, California. autophosphorylation, and activation. This triggers the Note: Supplementary data for this article are available at Clinical Cancer recruitment of adaptor proteins, such as FGFR substrate Research Online (http://clincancerres.aacrjournals.org/). 2a (FRS2a), leading to activation of several downstream X. Du and B.C. Lin contributed equally to this article. signaling cascades, including the Ras–Raf–MAPK, Corresponding Authors: Elicia Penuel, ES Oncology Biomarker Devel- PI3K–Akt–mTOR, and phospholipase C g (PLCg)path- opment, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. ways. Dysregulation and activation of FGFR3 through a Phone: 650-467-7214; Fax: 650-467-5470; E-mail: epenuel@gene.com; variety of mechanisms has been identified in a wide and Jing Qing, Department of Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Phone: 650-467-8266; spectrum of human cancers, including multiple myeloma E-mail: [email protected] (6–8), squamous cell carcinoma of the lung (9, 10), doi: 10.1158/1078-0432.CCR-13-3336 breast cancer (11, 12), glioblastoma (13, 14), testicular 2014 American Association for Cancer Research. cancer (15), and bladder cancer (16).

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While the advances in developing FGFR3-targeted ther- Translational Relevance apeutic agents are encouraging, identification and valida- Dysregulation of FGFR3 has been implicated in the tion of accessible, mechanism-based biomarkers to gauge pathogenesis of bladder cancer, and several investiga- targeted pharmacodynamic modulation is urgently need- tional agents targeting FGFR3 have been evaluated in ed to facilitate clinical investigation. In the current study, early-phase clinical studies. To facilitate the clinical combining transcriptional profiling and biochemical development of FGFR3-targeted therapy, we sought to approaches, we identified MMP-1 and pro-MMP-10 as identify potential noninvasive pharmacodynamic bio- bona fide FGFR3 downstream targets and the urinary level markers to gauge drug activities. Combining transcrip- of these metalloproteinases could serve as potential easily tional profiling and biochemical analyses, we identified accessible pharmacodynamic biomarkers for FGFR3-tar- and confirmed two matrix metalloproteinases, MMP-1 geted therapy in bladder cancer. and MMP-10, as downstream targets of FGFR3. Inhibit- ing FGFR3 function with a humanized monoclonal Materials and Methods antibody, R3Mab, led to a significant reduction of Cell culture, siRNA transfection, and reagents MMP-1 and MMP-10 in xenograft models in conjunc- Bladder cancer cell lines RT112 which contain FGFR3- tion with attenuated tumor growth. Importantly, in a TACC3 fusion endogenously (21) was purchased from the phase I dose-escalation trial, R3Mab caused an acute German Collection of Microorganisms and Cell Cultures reduction of urinary MMP-1 and pro-MMP-10 levels in (DSMZ, Germany). RT112 cells stably expressing doxycy- patients with advanced bladder cancer. These data war- cline-inducible shRNAs targeting FGFR3 or enhanced GFP rant further investigation in larger clinical studies using (EGFP) were described in our previous study (29). Bladder these potentially actionable noninvasive pharmacody- cancer cell line UMUC-14 which contains FGFR3S249C was namic biomarkers to stratify patients with elevated obtained as described (29). Bladder cancer cell line SW780 MMP-1 and pro-MMP-10 and potentially to monitor which contains FGFR3-BAIAP2L1 fusion (21) was pur- therapeutic response. chased from ATCC. All cells are stored at early passages in a central cell bank at Genentech. Cell lines were authenti- cated by short tandem repeat and genotyped upon reexpan- sions, and experiments were carried out with low-passage Specifically, accumulating genetic and functional evi- cultures of these stocks. The cells were maintained with dence has established FGFR3 as a potential oncogenic driver DMEM supplemented with 10% FBS (Sigma), 100 U/mL and therapeutic target in bladder cancer. About 60% to 70% penicillin, 0.1 mg/mL streptomycin, and L-glutamine under of non–muscle-invasive low-grade papillary and 11% to conditions of 5% CO2 at 37 C. 16% of muscle-invasive bladder tumors contain somatic Rapamycin and PI3K inhibitor LY294002 were obtained activating mutations in FGFR3 (17–20), the majority of from Cell Signaling Technology. A potent and selective which are missense substitutions in the extracellular or MEK1/2 inhibitor PD0325901 (Pfizer) was purchased from juxtamembrane domains of FGFR3 that lead to ligand- Synthesis Med Chem. independent receptor dimerization and activation. Besides All RNA interference experiments were carried out with activating mutations, gene fusion between FGFR3 and ON-TARGETplus siRNAs (25 nmol/L, Dharmacon). Cells TACC3 or BAIAP2L1 has been recently identified in bladder were transfected with Lipofectamine RNAiMax (Invitro- cancer cell lines as well as primary tumors, resulting in gen), and RNA or conditioned medium was collected at aberrant regulation and activation of FGFR3 (21, 22). In 48 or 72 hours after transfection. addition, high levels of wild-type FGFR3 is found in more than 40% to 50% of muscle-invasive and metastatic bladder Gene expression array and quantitative reverse cancers (19, 23). Moreover, expression of activated FGFR3 transcriptase-PCR analyses of mRNA expression level transforms NIH-3T3 cells and hematopoietic cells in culture The microarray studies with RT112 cells expressing doxy- and confers tumorigenicity in vivo (24–26). Importantly, cycline-inducible shRNAs targeting FGFR3 or EGFP were several preclinical studies using genetic and/or pharmaco- conducted as described (36), and can be accessed at the logic approaches have demonstrated that inhibition of Gene Expression Omnibus database under the accession FGFR3 in bladder cancer suppresses cell proliferation in number GSE41035. To detect transcripts of MMP-1 and culture and tumor growth in xenograft and orthotopic MMP-10, quantitative reverse transcriptase (qRT)-PCR was models (23, 27–31). Collectively, these data suggest that performed with predesigned TaqMan gene expression FGFR3 could be an oncogenic driver in a subset of bladder assays (Applied Biosystems). All reactions were performed cancers and hence that targeting this receptor may be at least in triplicates. The relative amount of all mRNAs was therapeutically beneficial. Indeed, several small-molecule calculated using the comparative Ct method after normal- inhibitors of FGFR have entered into clinical investigations ization to human RPL19. for cancer therapy (32–34). A specific human monoclonal anti-FGFR3 antibody we developed (designated R3Mab Protein analyses or MFGR1877S) has been evaluated in a phase I clinical Cells were treated as described in the figure legends. For trial (35). total cell lysates, cells were washed twice with ice-cold PBS

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and extracted in RIPA buffer (Millipore) supplemented with C.B-17 SCID mice, 6 to 10 weeks of age, were purchased phosphatase inhibitor cocktail PhosSTOP and Complete from Charles River Laboratory. Female athymic nude mice protease inhibitor cocktail (Roche Applied Science). For were obtained from Harlan Sprague Dawley. Mice were analysis of protein expression in tumor xenografts, lysate maintained under specific pathogen-free conditions. from tumor tissues was extracted with lysis buffer Female C.B-17 SCID or athymic nude mice were inoculated [consisting of 150 mmol/L sodium chloride, 20 mmol/L subcutaneously with 7 106 RT112 cells in Hank balanced Tris (pH 7.5), 2 mmol/L EDTA, 1% Triton X-100, 10 salt solution (HBSS)/Matrigel (1:1 v/v, BD Biosciences) or mmol/L sodium fluoride, supplemented with protease inhi- 5 106 UMUC-14 cells in HBSS (0.1 to 0.2 mL/mouse), bitors and phosphatase inhibitors] by pulverizing the fro- respectively. Mice with tumors reaching a mean volume of zen tissues using the FastPrep-24 homogenizer as described 150 to 200 mm3 were randomly grouped into groups of 12, by the manufacturer (MP Biomedicals). and were treated with vehicle (PBS) or R3Mab (0.3–100 mg/ Protein lysates were analyzed by SDS-PAGE and Western kg) via weekly intraperitoneal injections. Body weights and blot analysis. The primary blotting antibody for total FRS2 caliper measurements were taken twice weekly. The tumor (sc-8318) was purchased from Santa Cruz Biotechnology. volume (mm3) was calculated using the following formula: 2 The following primary antibodies were purchased from length width 0.5. Log2 (tumor volume) growth traces Cell Signaling Technology: pFRS2 Y196 (#3864), pMAPK were fitted to each treatment group with restricted cubic (#9101), total MAPK (#4695), pAKT (#9271), and total splines for the fixed time effect in each group. Fitting was AKT (#9272). Anti-MMP-1 (MAB901) and anti-MMP-10 done as previously described (37) via a linear mixed effects (MAB910) were purchased from R&D Systems. The blots model, using the R package "nlme," version 3.1–108 in R were visualized using a chemiluminescent substrate (ECL version 2.15.2 (R Development Core Team 2012; R Foun- Plus, Amersham Pharmacia Biotech). dation for Statistical Computing). Plotting was performed using Excel 14.3.2 (Microsoft Corporation). Analyses of secreted MMP-1, pro-MMP-10, and MMP-10 To analyze MMP-1 and MMP-10 levels in tumor tissues in conditioned medium and serum, mice with tumors reaching a mean volume of Secreted MMPs were measured with the following ELISA 150 to 200 mm3 were randomly grouped into cohorts of 10, Kits according to the manufacturers’ instructions: Total and were treated with a control human IgG1 or R3Mab human MMP-1 protein (ELH-MMP1) and total human (30 mg/kg) on days 1, 4, and 7. Tumors and mouse serum MMP-10 (ELH-MMP10-001) ELISA kits were purchased were collected at indicated time points and kept frozen. from RayBiotech, and human pro-MMP-10 ELISA kit Human MMP-1 and pro-MMP-10 level in mouse serum (DM100) was purchased from R&D Systems. were determined using the ELISA assays as described above. To collect conditioned medium: RT112 and UMUC-14 cells were grown to 90% confluence in DMEM containing Clinical study design 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and The phase I MFG4991g trial (35) was an open-label dose- 100 mg/mL streptomycin. To evaluate the effects of FGFR3 escalation study to evaluate the safety, tolerability, and knockdown on MMP-1 and MMP-10 protein expression, pharmacokinetics of R3Mab (designated MFGR1877S) in cells were cultured in the presence or absence of doxycycline patients with solid tumors. The trial consisted of a dose (1 mg/mL) for the indicated time, and conditioned medium escalation of single-agent R3Mab administered intrave- was collected. To study the effect of R3Mab on MMP-1 and nously at 2, 4, 8, 15, and 30 mg/kg once every 28 days. MMP-10 expression, 106 cells per dish were seeded onto 10 Serum and urine samples were collected at designated time cm dishes in complete growth medium and allowed to points, frozen in liquid nitrogen, and shipped to Genen- attach overnight. Then, serum-free DMEM containing tech, Inc. for measurement of pharmacodynamic biomar- R3Mab (15 mg/mL) or a control antibody was applied, and kers of R3Mab activity. supernatants were collected at the indicated time points. The conditioned medium was concentrated 10-fold using ELISA analyses of MMPs in human serum and urine Amicon Ultra Centrifugal filters (Millipore) for ELISA anal- The levels of a panel of MMPs were measured by ELISA in ysis, and the corresponding cells were lysed in the RIPA serum and urine samples from normal healthy volunteers, buffer (50 mmol/L pH 7.4 Tris-Cl, 1% NP40, 0.25% sodium procured bladder cancer patients, and patients enrolled in deoxycholate) with complete protease inhibitor cocktail the phase I MFG4991g clinical trial. To this end, the human (Roche Applied Science). MMP-1 and MMP-10 amounts MMP 3-Plex (i.e., MMP-1, MMP-3, and MMP-9; Mesoscale were normalized to total cellular proteins. Discovery) and human pro-MMP-10 immunoassay (R&D Systems) kits were utilized according to the manufacturer’s Xenograft studies and collection of serum for ELISA instructions. For the human pro-MMP-10 immunoassay analyses kit, serum samples were diluted 2-fold in the appropriate All animal studies were conducted in accordance with the assay diluent. For the human MMP 3-Plex kit, serum sam- Guide for the Care and Use of Laboratory Animals, pub- ples were diluted 10-fold. For all MMP kits, urine was used lished by the NIH (NIH publication 8523, revised 1985). undiluted. As a reference for quantification, a standard The Institutional Animal Care and Use Committee at Gen- curve was used for each kit according to the manufacturer’s entech reviewed and approved all animal protocols. Female directions. Mann–Whitney tests were used for comparison

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between two groups. A value of P < 0.05 was considered these genes was also verified in UMUC-14 bladder cancer statistically significant. cells subjected to siRNA-mediated FGFR3 knockdown (Sup- plementary Fig. S2A), or treated with a specific anti-FGFR3 Results human monoclonal antibody, R3Mab (Supplementary Fig. FGFR3 knockdown inhibits MMP-1 and MMP-10 S2B). Together, these data suggest that FGFR3 signaling expression and secretion promotes transcription of MMP-1 and MMP-10 in bladder Using doxycycline-inducible shRNA targeting FGFR3, we cancer cells. previously determined the transcriptional profile of RT112- Next, we examined the effect of FGFR3 knockdown on the derived cell lines that express endogenous or depleted levels expression and secretion of MMP-1 and MMP-10 proteins. of FGFR3, using microarray analysis (36). To facilitate RT112 cells were treated with or without doxycycline for 72 identification of serum or urine pharmacodynamic markers hours to deplete FGFR3 protein, and the conditioned media of FGFR3-targeted therapeutics, we focused on secreted were collected at specific time points thereafter. FGFR3 molecules that were specifically modulated by FGFR3 deple- shRNAs substantially diminished the secretion of MMP-1 tion. Comparing independently established stable cell lines and pro-MMP-10 as early as 24 hours after induction and transduced with a doxycycline-inducible control shRNA the decrease was sustained at 48 and 72 hours, whereas the versus three independent FGFR3 shRNAs, we found that control shRNA had no effect (Fig. 1B). MMP-10 was down- two matrix metalloproteinases, MMP-1 and MMP-10, were regulated in a similar manner by FGFR3 shRNAs (Supple- among the genes showing the greatest decline upon FGFR3 mentary Fig. S3). Consistently, pretreatment with R3Mab knockdown (Supplementary Table S1). The transcripts were also blocked the production and secretion of MMP-1, pro- downregulated by about 16-fold and 4-fold for MMP-1 and MMP-10, and MMP-10 proteins into conditioned media by MMP-10, respectively (Supplementary Fig. S1). These both RT112 and UMUC-14 cells (Fig. 1C and Supplemen- microarray results were further confirmed using qRT-PCR tary Fig. S4). Together, these results suggest that FGFR3 analysis of the mRNA abundance of these two genes signaling promotes the expression and secretion of MMP-1 (Fig. 1A). In addition, the FGFR3-dependent regulation of and MMP-10 proteins by these bladder cancer cell lines. As

A 1 1.4 1.2 0.8 1 0.6 0.8 0.4 0.6 0.4 (+Dox/ − Dox) (+Dox/ − Dox) 0.2 0.2 0 0 Relative MMP-1 mRNA Figure 1. Inhibition of FGFR3 Ctrl R3 R3 R3 Relative MMP-10 mRNA Ctrl R3 R3 R3 reduces the mRNA and protein shRNA shRNA2 shRNA4 shRNA6 shRNA shRNA2 shRNA4 shRNA6 expression of MMP-1 and MMP- 10 in RT112 bladder cancer cells. B No Dox Dox No Dox Dox A, doxycycline-induced 500 350 knockdown of FGFR3 reduces 300 mRNA levels of MMP-1 and MMP- 400 10. B, doxycycline-induced 250 knockdown of FGFR3 reduces 300 200 total MMP-1 and pro-MMP-10 150 secreted in the conditioned 200

Total MMP-1 100 medium. C, a speciﬁc FGFR3- pro-MMP-10 100 blocking antibody, R3Mab, 50 (ng/mg cellular protein) (ng/mg cellular (ng/mg cellular protein) (ng/mg cellular reduces total MMP-1, pro-MMP- 0 0 10, and total MMP-10 secreted in h h24 72 h48 24 h 48 h 72 h 24 h 48 h 72 h h h24 h48 h72 h24 h48 h72 h24 72 h48 the conditioned medium by RT112 Ctrl shRNA R3 shRNA2 R3 shRNA4 Ctrl shRNA R3 shRNA2 R3 shRNA4 cells. C 120 600 35 Ctrl Ab Ctrl Ab Ctrl Ab 30 100 R3Mab 500 R3Mab R3Mab 25 80 400 20 60 300 15 40 200 10 pro-MMP-10 Total MMP-1 20 100 Total MMP-10 5 (ng/mg cellular protein) (ng/mg cellular (ng/mg cellular protein) (ng/mg cellular 0 protein) (ng/mg cellular 0 0 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

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the ELISA detection of pro-MMP-10 is more sensitive than tion of its corresponding target, as assessed by the phos- that of MMP-10, and both pro- and enzymes show similar phorylation of AKT or MAPK, respectively (Fig. 2C). While modulation by FGFR3 inhibition, we focused on pro-MMP- PI3K inhibition elicited minimal effect on the expression 10 protein levels in subsequent studies. and secretion of MMP-1 and MMP-10 proteins, MEK inhibition significantly diminished both basal and FGF1- FGFR3 signaling promotes MMP-1 and MMP-10 induced expression of these two proteins (Fig. 2D). Thus, expression mainly through MEK FGFR3 promotes MMP-1 and MMP-10 expression in RT112 To study the molecular mechanisms underlying FGFR3 bladder cancer cells mainly through the MEK-MAPK axis. regulation of MMP-1 and MMP-10 expression, we activated FGFR3 signaling in bladder cancer cells with FGF1. We first R3Mab blocks FGFR3 signaling and reduces human analyzed the mRNA levels of the two MMPs. FGF1 treatment MMP-1 and MMP-10 protein levels in both bladder of RT112 cells induces robust FGFR3 activation in a dose- tumor xenograft tissues and mouse serum and time-dependent fashion (36), and higher mRNA levels To evaluate whether MMP-1 and MMP-10 could serve of both MMP-1 and MMP-10 were detected after 6 or as potential pharmacodynamic markers for FGFR3-tar- 12-hour incubation (Fig. 2A). Consistent with these geted therapeutics, we studied the effect of R3Mab on the changes, secreted MMP-1 and pro-MMP-10 proteins expression and secretion of these enzymes in vivo.Two detected in conditioned media were higher after a 24-hour R3Mab-responsive tumor xenograft models, RT112 and incubation with FGF1 (Fig. 2B), and accumulated further at UMUC-14, were selected for this study. Weekly intraper- 48 and 72 hours (data not shown). itoneal administration of R3Mab suppressed xenograft To determine whether the induction of MMP-1 and growth of these models in a dose-dependent manner MMP-10 by FGF1 depends on specific FGFR3 signaling (Fig. 3A and Supplementary Fig. S5). First, we evaluated mediators, we blocked the two major signaling branches FGFR3 signaling and MMP-1 and MMP-10 protein levels downstream of FGFR3 using the PI3K inhibitor Ly294002 in tumor xenograft tissues. Compared with the control and the MEK1/2 inhibitor PD325901. In RT112 bladder antibody, R3Mab markedly reduced the phosphorylation cancer cells, each inhibitor blocked FGF1-induced activa- and activation of FRS2 and MAPK, two of the essential

PD901 Ly294 A 8 6 C No FGF1 No FGF1 (nmol/L) (µmol/L) +FGF1 +FGF1 6 DMSO 10 100 2 20 4 4 FGF1 + −− + − + − − ++ 2 pAKT mRNA level mRNA level 2 Figure 2. FGF1 signaling stimulates Relative MMP-1 Relative MMP-10 MMP-1 and MMP-10 expression 0 0 AKT 6 h 12 h 6 h 12 h through MEK. A, FGF1 induces MMP-1 and MMP-10 mRNA B No FGF1 No FGF1 pMAPK expression in RT112 cells. B, FGF1 +FGF1 +FGF1 treatment stimulates MMP-1 and 60 300 MAPK pro-MMP-10 protein expression 250 and secretion into conditioned 200 40 Actin medium by RT112 cells. C, pharmacologic inhibition of FGF1 150 signaling in RT112 cells. RT112 20 100 cells were treated with PD325901 Pro-MMP-10 Total MMP-1 50 (PD901) and Ly294002 (Ly294) at (ng/mg cellular protein) (ng/mg cellular protein) 0 0 indicated concentrations for 3 hours, followed by FGF1 (25 ng/mL) stimulation for 10 D 450 40 minutes. Cell lysates were No FGF1 No FGF1 subjected to Western blot +FGF1 analysis. D, MEK inhibitor, but +FGF1 30 300 not PI3K inhibitor, blocked 20 FGF1-induced expression and

MMP-1 secretion of MMP-1 and 150

pro-MMP-10 pro-MMP-10 by RT112 cells. 10 (ng/mg cellular protein) (ng/mg cellular protein) 0 0

DMSO DMSO

µ Ly294 2 mol/L µ µ PD901 10 nmol/L Ly294 20 mol/L Ly294 2 mol/L PD901 10 nmol/L Ly294 20 µmol/L PD901 100 nmol/L PD901 100 nmol/L

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A B Ctrl Ab R3Mab Vehicle R3Mab, 1 mg/kg R3Mab, 3 mg/kg Animal ID 223 238 246 210 220 226 R3Mab, 10 mg/kg 242 249 202 208 R3Mab, 30 mg/kg R3Mab, 100 mg/kg pFRS2 Figure 3. R3Mab attenuates RT112 1,400 ) 3 tumor growth, blocks FGFR3 1,200 Total FRS2 signaling and reduces human 1,000 MMP-1 and MMP-10 levels in pMAPK xenograft tumor tissues and 800 mouse serum. A, effect of R3Mab 600 Total MAPK on pre-established RT112 bladder 400 xenograft tumor growth. R3Mab Total MMP-1 was administered intraperitoneally 200

n ¼ Fitted tumor volume (mm weekly (arrows). 12 per group. 0 Total MMP-10 B, effect of R3Mab on FGFR3 2520151050 signaling and MMP levels in tumor R3Mab Actin xenograft tissues. Mice with pre- Day established RT112 tumors were grouped into cohorts of 5 for treatment with either a control C Day 4 Day 6 Day 10 antibody (Ctrl Ab) or R3Mab 8 8 12 10 P < 0.0001 (30 mg/kg). Tumors were 6 P < 0.0001 6 P < 0.0001 harvested at day 7, and lysates 8 extracted from xenograft tumor 4 4 6 tissues were subjected to Western 4

2 2 MMP-1 (ng/mL) MMP-1 (ng/mL)

MMP-1 (ng/mL) 2 blot analysis. C and D, the kinetics 0 of R3Mab effect on serum levels 0 0 Ctrl Ab R3Mab Ctrl Ab R3Mab of human MMP-1 (C) and Ctrl Ab R3Mab pro-MMP-10 (D) in mice bearing D n ¼ RT112 xenograft tumors. 10 15.0 20 20 per group. 12.5 P < 0.0001 P < 0.0005 P < 0.0001 15 15 10.0 7.5 10 10 5.0 5 5 MMP-10 (ng/mL) MMP-10 (ng/mL) 2.5 MMP-10 (ng/mL) 0.0 0 0 Ctrl Ab R3Mab Ctrl Ab R3Mab Ctrl Ab R3Mab

downstream mediators of FGFR3 signaling (Fig. 3B). To assess whether the modulation of MMPs by R3Mab Concomitantly, both total MMP-1 and MMP-10 protein correlates with efficacy, we also analyzed pro-MMP-10 levels in the tumor lysates were decreased significantly in levels in three xenograft models whose growth is not affect- response to R3Mab (Fig. 3B). ed by R3Mab, namely HT1376, UMUC-4, and SW780 We next evaluated the effect of R3Mab on human (Supplementary Fig. S8A and data not shown). Levels of MMP-1 and MMP-10 protein levels in the circulating pro-MMP-10 were too low to be detected in serum from blood from xenograft models expressing human bladder mice bearing UMUC-4 or HT1376 xenograft tumors (data tumors. Nude mice with pre-established RT112 tumors not shown). While pro-MMP-10 was easily detected in mice were treated with a low or a high dose of R3Mab (3 or with SW780 tumors, levels of pro-MMP-10 were not affect- 30 mg/kg, respectively), and mouse serum was collected ed upon R3Mab treatment as observed in responsive mod- at 48 or 96 hours after treatment. Human pro-MMP-10 els (Supplementary Fig. S8B). protein levels in mouse serum were reduced in a dose- Together, these data demonstrated that R3Mab-based dependent manner in R3Mab-treated group at 48 and 96 inhibition of FGFR3 signaling and tumor growth occurs in hours, and the effect was more pronounced in the high- conjunction with a substantial and sustained reduction of dose group at 96 hours (Supplementary Fig. S6). We MMP-1 and MMP-10 protein expression and secretion, extended the analysis to days 4, 6, and 10 after treatment. suggesting that these two metalloproteinases could be Compared with the control antibody, R3Mab substan- explored as potential pharmacodynamic markers of FGFR3 tially and significantly suppressed the serum levels of pathway inhibition. human MMP-1 and pro-MMP-10 throughout the time course (Fig. 3C and D). A similar reduction of serum MMP-1 and pro-MMP-10 proteins are elevated in the levels of these two proteins was induced by R3Mab urine of bladder cancer patients treatment of mice bearing UMUC-14 tumors (Supple- Next, we evaluated MMP-1 and pro-MMP-10 protein mentary Fig. S7A and S7B). levels in urine and serum specimens from healthy donors

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and patients with bladder cancer (acquired from commer- (n ¼ 15; Fig. 4A). Similarly, elevated levels of pro-MMP- cial sources). In urine samples from healthy donors (n ¼ 10 were observed in urine samples from patients with 25), MMP-1 was mostly undetectable or below the lower bladder cancer relative to patients with other solid tumors limit of quantitation (LLOQ), but pro-MMP-10 was con- (Fig. 4A). In agreement with observations in the commer- sistently detected above the LLOQ, albeit within a low and cially procured specimens, there was no difference in serum narrow range (Fig. 4A). In contrast, both MMP-1 and pro- MMP-1 and pro-MMP-10 levels between patients with MMP-10 proteins were detected at significantly higher levels bladder cancer or other solid tumors (Fig. 4B). Together, in urine from patients with bladder cancer (n ¼ 35; Fig. 4A). these data demonstrate that these two metalloproteinases A different pattern was observed in serum: both metallo- are significantly elevated in bladder cancer patient–derived proteinases were detected above the LLOQ in all serum urine relative to analogous samples from healthy donors or samples from the same patients; however, there was no from patients with several other solid tumors. However, we difference in levels detected in healthy donors versus did not observe a correlation between baseline MMP levels patients with bladder cancer (Fig. 4B). and either FGFR3 protein expression level (as measured As R3Mab has been evaluated in a phase I dose-escalation retrospectively by immunohistochemistry) or clinical clinical trial (MFG4991g) conducted in relapsed or refrac- response in the MFG4991g phase I trial (38). tory, locally advanced, or metastatic solid tumors (Supple- mentary Table S2; ref. 35), analogous measurements of R3Mab reduces urinary MMP-1 and pro-MMP-10 levels MMP1 and pro-MMP-10 were made in clinical pretreatment in patients with bladder cancer urine and serum specimens (n ¼ 26). All patients with To examine whether MMP-1 and/or pro-MMP-10 could bladder cancer (n ¼ 10) enrolled in this study have relapsed be used as pharmacodynamic biomarkers, patterns of or refractory metastatic lesions, and 8 of 10 patients dis- their expression were also evaluated in urine samples from played significantly higher urinary MMP-1 levels, with only the MFG4991g phase I clinical trial collected predose, or at two patients having MMP-1 below the LLOQ. In contrast, cycle 1, day 2 (C1D2), cycle 1, day 4 or 5 (C1D4D5) and, urinary MMP-1 was undetectable or below the LLOQ in a in some cases, at later time points depending on how long majority of samples from patients with cancer with other the patient stayed on study and on availability of samples solid tumors, including colorectal, squamous cell carcino- (Fig. 5). In all bladder cancer patients (based on 8 evaluable ma of the head and neck, ovarian, thyroid, esophageal, urine sample pairs), exposure to R3Mab reduced MMP-1 adrenal, adenoid cystic carcinoma, or carcinoid tumor levels by >50% (Fig. 5A and B). Assessment of

A Urine B Serum ** **

1,000 Figure 4. 100 1,000 MMP-1 and pro-MMP-10 100 are elevated in bladder cancer 10 10 (BLC) patient urine but not in 1 serum. A, levels of MMP-1 and 1 0.1 pro-MMP-10 in commercially 0.1 procured urine samples from 0.01 0.01 patients with bladder cancer 0.001 0.001 n ¼ Total MMP-1 (ng/mL) ( 35) relative to healthy donors 0.0001 Total MMP-1 (ng/mL) 0.0001 (n ¼ 25), or from the MFG4991g Healthy BLC Non-BLC BLC Health BCL Non-BCL BCL phase I clinical trial, including n = 5 n = 35 n = 16 n = 10 n = 3 n = 23 n = 15 n = 10 patients with bladder cancer n ¼ Procured samples Ph1 clinical samples Procured samples Ph1 clinical samples ( 10) and patients with other solid tumor types (non-BLC, n ¼ 16). Plotted n values are *** * representative of only those 1,000 1,000 samples that were detectable 100 100 (>LOD). The dotted line represents the LLOQ. B, levels of MMP-1 and 10 10 pro-MMP-10 in serum are 1 1 equivalent in healthy donors, 0.1 0.1 bladder cancer patients, and in 0.01 0.01 non–bladder cancer patients. 0.001 Note: only 5 healthy donor serum pro-MMP10 (ng/mL) 0.001 pro-MMP10 (ng/mL) samples were evaluated. 0.0001 0.0001 Healthy BLC Non-BLC BLC Healthy BLC Non-BLC BLC n = 25 n = 35 n = 8 n = 8 n = 5 n = 35 n = 16 n = 10

Procured samples Ph1 clinical samples Procured samples Ph1 clinical samples

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Urinary Pharmacodynamic Markers for R3Mab in Bladder Cancer

ACBladder cancer Non–bladder cancer Bladder cancer Non–bladder cancer 10 10

1 1

0.1 0.1 Figure 5. MMP-1 and pro-MMP-10 are reduced upon exposure to 0.01 0.01 R3Mab in patients with bladder MMP-1 (ng/mL) cancer in the MFG4991g phase I 0.001 0.001 pro-MMP-10 (ng/mL) study. A, the overall urinary levels of MMP-1 are reduced by R3Mab 0.0001 0.0001 e e 5 e 2 5 se 2 5 s 2 5 s D in patients with bladder cancer but o o D D os o 4 1D 4D 4 C1D2 d C1D D C D ed C1 re – red 1 r 1D Pred P not in non bladder cancer P C P C C1D4D C1 patients. B, relative levels of MMP- 1 compared with predose levels. C, the overall urinary levels of pro- B Bladder cancer Non–bladder cancer D Bladder cancer Non–bladder cancer MMP-10 are reduced by R3Mab in 150 500 patients with bladder cancer but 400 300 not in non–bladder cancer 200 patients. D, relative levels of pro- 100 150 MMP-10 compared with predose levels. C1D2, cycle 1 day 2; 100 C1D4D5, cycle 1, day 4 or day 5. 50 50 MMP-1 (% predose) pro-MMP-10 (% predose) 0 0 e 2 5 e 2 5 e 2 5 e 2 5 s D D D D D D 1 4 1 4D 1 4 1 do C dos C D dos C dos C re 1D re 1 e P C P C Pr C1D Pre C1D4D pharmacodynamic modulation in non–bladder cancer study (Fig. 6A). Similar to pro-MMP-10 and MMP-1 data, patients was hampered by the limited number (3/16) of no significant difference was detected in serum (Fig. 6B). samples having sufficient MMP-1 levels at baseline. While Pharmacodynamic monitoring of these MMPs showed that most samples were detectable, only 3 were above the lowest in most but not all cases MMP-3 and MMP-9 were down- level of quantitation in the assay. However, in the cases in modulated by R3Mab similarly to pro-MMP-10 and MMP-1 which MMP-1 could be quantified, MMP-1 levels were in patient samples where all four MMPs were detectable reduced by >50% (Fig. 5A and B). (Fig. 6C). Together, these data suggested that anti-FGFR3 Unlike MMP-1, all urine samples had detectable pro- therapy specifically downmodulated urinary, but not MMP-10 levels for analysis. Pro-MMP-10 was reduced by serum, MMP levels in patients with bladder cancer. It is >50% in half of the patients with bladder cancer treated with worth noting that similar to observations at baseline, no R3Mab (4/8 evaluable pairs). In contrast, there was no correlation between pharmacodynamics measures and reduction in pro-MMP-10 in urine samples from patients either FGFR3 expression or clinical response was observed with non–bladder cancer (Fig. 5C and D). All of the patients (38). with bladder cancer that showed reduced levels of pro- MMP-10 were treated at the highest dose (30 mg/kg). However, two other patients in the highest dose cohort did Discussion not show modulation of pro-MMP-10 levels. In contrast, Bladder cancer is the second most common genitourinary MMP-1 was downmodulated upon antibody treatment in tract malignancy, accounting for more than 73,000 new most dose cohorts including the 8, 15, and 30 mg/kg dose cases each year and about 15,000 deaths in 2012 in the cohorts. United States alone (2). The receptor tyrosine kinase, A number of MMPs have been postulated as potential FGFR3, has been postulated as an important oncogenic diagnostic and/or prognostic biomarkers in bladder cancer, driver and potential therapeutic target in this disease based but the role for MMPs as pharmacodynamic biomarkers has on multiple lines of evidence, including a high frequency of largely been unexplored (39). To extend our preliminary activating mutations (17–20), aberrant regulation and acti- observations, additional analysis was performed to evaluate vation of FGFR3 by gene fusion with TACC3 or BAIAP2L1 two other MMPs, MMP-3, and MMP-9, which also harbor a (21, 22), overexpression of FGFR3 protein in tumor tissues group 1 promoter element similar to MMP-1 and MMP-10 (19, 23), and a large body of preclinical loss-of-function (40). Statistical analysis (Mann–Whitney test) indicated studies (23, 27–31). Recent progress toward clinical inves- that MMP-3 (P ¼ 0.0251), but not MMP-9 was significantly tigation of experimental agents targeting FGFR3 has made it higher in urine from patients with bladder cancer relative to imperative to identify accessible pharmacodynamic bio- non–bladder cancer patients in the MFG4991g phase I markers to gauge target modulation and facilitate clinical

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Du et al.

A Urine C Predose C1D2 C1D4/5 BLC 150.00 10,000 Non-BLC 100.00 1,000

100 50.00 Figure 6. Levels and modulation of 10 0.00 MMP-3 and MMP-9 by R3Mab in 1 MMP1 values (% predose) 116 117 121 122 123 126 127 the MFG4991g phase I study. 150.00 0.1 A, urinary MMP-3 is elevated in patients with bladder cancer

MMP values (ng/mL) 100.00 0.01 relative to non–bladder cancer 0.001 patients in the MFG4991g phase I 50.00 – MMP-3 MMP-9 study. On the basis of Mann Whitney test, P ¼ 0.0251 for 0.00 pro MMP10 (% predonse) P 116 117 121 122 123 126 127 MMP-3; > 0.05 for MMP-9. B, no 150.00 corresponding difference of these B Serum MMPs was detected in serum. C, samples (n ¼ 7) with detectable BLC 100.00 levels of all four MMPs showed 1,000 Non-BLC variable modulation in response to 50.00 R3Mab administration. C1D2,

MMP3 (% predose) cycle 1 day 2; C1D4D5, cycle 1, 0.00 100 116 117 121 122 123 126 127 day 4 or day 5. 150.00

10 100.00

MMP values (ng/mL) 50.00 1 MMP9 (% predose) 0.00 MMP-3 MMP-9 116 117 121 122 123 126 127

studies. Here, we report that FGFR3 stimulates MMP-1 and in the conditioned medium. This inhibitory effect is mainly pro-MMP-10 expression and secretion in human bladder mediated through the FGFR3–MEK–MAPK pathway, but cancer cells through the MEK–MAPK pathway. Blocking not the FGFR3-PI3K axis. These results extend earlier studies FGFR3 signaling by a specific anti-FGFR3 human mono- by others showing that the expression of several MMP genes clonal antibody, R3Mab, inhibited human tumor xenograft is regulated by some growth factors and cytokines in bladder growth in association with a marked reduction in circulat- cancer. For example, basic FGF is able to stimulate the ing human MMP-1 and pro-MMP-10 levels. Moreover, expression of MMP-2 and MMP-9 in HT1376 cells (41), patients with bladder cancer had an increased level of and EGF elicits the rapid induction of MMP-1 and MMP-10 urinary MMP-1 and pro-MMP-10 proteins. Importantly, in mRNA in T24 bladder cancer cells through the Stat3/acti- a phase I dose-escalation study of R3Mab, inhibition of vator protein-1 (AP-1) family of transcription factors (42). FGFR3 in patients with relapsed or refractory metastatic Importantly, in the current study, by selectively blocking bladder cancer resulted in an acute reduction of urinary FGFR3 function with R3Mab (i.e., a specific anti-FGFR3 MMP-1 and pro-MMP-10 levels. These results unveil an monoclonal antibody), we were able to clearly demonstrate important role for FGFR3 in regulating MMP-1 and pro- that MMP-1 and pro-MMP-10 are bona fide downstream MMP-10 expression and secretion in bladder cancer, and targets of FGFR3 signaling in bladder cancer. It is worth support further evaluation of these metalloproteinases as noting that the current study does not exclude the possi- potential pharmacodynamic markers in future FGFR3-tar- bility that other signaling pathways may also regulate these geted clinical studies. two MMPs through MAPK or alternative mechanisms in In our unbiased expression analysis of genes encoding bladder cancers. secreted molecules, we found that upon FGFR3 knockdown To assess whether MMP-1 and pro-MMP-10 could serve in RT112 bladder cancer cells, MMP-1, and pro-MMP-10 as accessible pharmacodynamic biomarkers for FGFR3-tar- were among the most prominently downregulated genes. geted therapy, we have evaluated the serum and urinary Our studies further demonstrate that ligand-induced FGFR3 levels of these two proteins in healthy subjects and in activation promotes both mRNA and protein expression of patients with bladder cancer, as well as in patients with these two metalloproteinases, and acute pharmacologic various solid cancers enrolled in the MFG4991g phase I inhibition of canonical FGFR3 signaling markedly study of R3Mab. Compared with healthy controls and diminishes the level of secreted MMP-1 and pro-MMP-10 patients with non–bladder cancer malignancies, patients

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Urinary Pharmacodynamic Markers for R3Mab in Bladder Cancer

with advanced bladder cancer showed significantly elevated decreased transcription of these genes. Indeed, in 4T1 urine levels of MMP-1 and pro-MMP-10 protein. In con- breast cancer cells, the pan-FGFR inhibitor TKI258 sup- trast, the concentration of these two proteins in circulating presses the transcription of MMP-1, -3, -9, and -10 simul- blood displayed no difference amongst the different groups. taneously (49). This observation is in agreement with several published The matrix metalloproteinases degrade extracellular studies. MMP-1 and MMP-10 mRNA levels have been matrix components and activate growth factors, contribut- reported to be significantly higher in bladder cancers com- ing to tumor growth, progression, and metastasis (50). pared with normal urothelial tissues (43, 44), and MMP-1 Several MMPs have been postulated to be diagnostic or and pro-MMP-10 immunoreactivity is evidently stronger in prognostic markers in bladder cancer (39, 50). Our current tumor cells of both superficial and muscle-invasive bladder study demonstrates that FGFR3 signaling directly regulates cancers (45, 46). In addition, patients with bladder cancer MMP-1 and pro-MMP-10 expression and secretion in blad- with an increased urinary MMP-1 protein level are more der cancer through the MEK–MAPK pathway. Urinary likely to have an aggressive tumor of advanced stage and MMP-1 and pro-MMP-10 levels were reduced substantially grade (47, 48). Although published data on urinary pro- by R3Mab treatment in both preclinical models and MMP-10 in patients with bladder cancer is more limited, a patients with bladder cancer. This is the first study, to our recent study demonstrates that a higher urinary pro-MMP- knowledge, that identifies urinary MMP-1 and pro-MMP-10 10 concentration could serve as a potential diagnostic as potential pharmacodynamic biomarkers. While we biomarker when used in combination with a panel of six observed clear pharmacodynamic modulation in response other analytes (44). Taken together, the differential expres- to R3Mab administration, there did not appear to be a sion pattern between patients with bladder cancer and correlation between modulation and dose or FGFR3 expres- healthy subjects, the relative ease in acquisition of void sion level or clinical benefit (as assessed by time on study or urine samples, and the robust detection of urinary MMP-1 disease progression). The lack of correlation could be due to and pro-MMP-10 levels suggest that these two metallopro- small sample size and/or a change in FGFR3 expression teinases could serve as useful and clinically relevant non- levels during disease progression or prior drug treatment. invasive biomarkers. Although this phase I study was not powered to assess One important finding of our current study is that in predictive biomarkers, the data presented here warrant preclinical bladder cancer xenograft models, R3Mab treat- further investigation in larger studies using these potentially ment inhibited FGFR3 signaling, attenuated tumor growth, actionable pharmacodynamic biomarkers that can be used and reduced MMP-1 and pro-MMP-10 protein expression in in the clinic in a noninvasive manner to stratify patients tumor lysates as well as in mouse serum. Consistent with with elevated MMP-1 and pro-MMP-10 profiles and poten- these preclinical studies, our phase I study with 26 patients tially monitor therapeutic response. demonstrated that administration of R3Mab led to an acute reduction of urinary MMP-1 by more than 50% in 8 of 8 Disclosure of Potential Conflicts of Interest evaluable patients with bladder cancer. The evaluation in J. Qing, X. Du, B.C. Lin, Q.-R. Wang, H. Li, E. Ingalla, J. Tien, I. Rooney, non–bladder cancer patients was hampered by the fact that A. Ashkenazi, and E. Penuel are employees of Genentech. No other potential very few patients had measureable levels of MMP-1 at conflicts of interest were disclosed. baseline. In the only 3 patients (2 colorectal and 1 head and neck SCC) that were evaluable, MMP-1 was also Authors' Contributions markedly reduced. It is noteworthy that although a mini- Conception and design: B.C. Lin, A. Ashkenazi, E. Penuel, J. Qing Development of methodology: X. Du, B.C. Lin mum threshold level of MMP-1 is required for monitoring Acquisition of data (provided animals, acquired and managed patients, pharmacodynamic modulation, the extent of downmodu- provided facilities, etc.): X. Du, B.C. Lin, Q.-R. Wang, H. Li, E. Ingalla, J. Tien, I. Rooney lation was not correlated with the baseline values. A similar Analysis and interpretation of data (e.g., statistical analysis, biosta- effect was observed for pro-MMP-10, as R3Mab resulted in a tistics, computational analysis): X. Du, B.C. Lin, Q.-R. Wang, H. Li, decrease of urinary pro-MMP-10, albeit in only 4 of 8 E. Ingalla, J. Tien, A. Ashkenazi, E. Penuel, J. Qing Writing, review, and/or revision of the manuscript: B.C. Lin, J. Tien, patients with bladder cancer. Interestingly, no modulation I. Rooney, A. Ashkenazi, E. Penuel, J. Qing of pro-MMP-10 was detected in non–bladder cancer Administrative, technical, or material support (i.e., reporting or orga- patients, suggesting that pro-MMP-10 might be more spe- nizing data, constructing databases): B.C. Lin, J. Tien Study supervision: J. Tien, I. Rooney, J. Qing cific for bladder cancer. Additional MMPs were monitored in the clinical study. Similar to MMP-1 and pro-MMP-10, urinary MMP-3, and Acknowledgments The authors thank Zora Modrusan for microarray profiling and Jinfeng to a lesser extent, MMP-9, were also elevated in bladder Liu for the informatics analysis of the array data. The authors also thank Mark relative to non–bladder cancer patients. In a majority of Davis and Genentech’s FGFR3 development team for designing and con- patients, there was a similar pattern of downmodulation ducting the phase I clinical trial of R3Mab. The costs of publication of this article were defrayed in part by the of these MMPs by R3Mab despite some noticeable differ- payment of page charges. This article must therefore be hereby marked ences. This observation could be partially due to the fact advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate that these MMPs all have a proximal AP-1 binding site this fact. in their promoters (40); conceivably, R3Mab-mediated Received December 11, 2013; revised September 18, 2014; accepted inhibition of the MEK-MAPK-AP-1 axis may result in October 2, 2014; published OnlineFirst October 17, 2014.

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MMP-1 and Pro-MMP-10 as Potential Urinary Pharmacodynamic Biomarkers of FGFR3-Targeted Therapy in Patients with Bladder Cancer

Xiangnan Du, Benjamin C. Lin, Qian-Rena Wang, et al.

Clin Cancer Res 2014;20:6324-6335. Published OnlineFirst October 17, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-3336

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