CCR-16-2267 3003..3011 Cancer Therapy: Clinical A Phase II Trial of Dovitinib in BCG-Unresponsive Urothelial Carcinoma with FGFR3 Mutations or Overexpression: Hoosier Cancer Research Network Trial HCRN 12-157 Noah M. Hahn1,2,3, Trinity J. Bivalacqua1,2,3, Ashley E. Ross1,2,3, George J. Netto1,2,3, Alex Baras1,2,3, Jong Chul Park1, Carolyn Chapman1, Timothy A. Masterson4, Michael O. Koch4, Richard Bihrle4, Richard S. Foster4,Thomas A. Gardner4, Liang Cheng5, David R. Jones6, Kyle McElyea5, George E. Sandusky5, Timothy Breen7, Ziyue Liu8, Costantine Albany9, Marietta L. Moore9, Rhoda L. Loman9, Angela Reed9, Scott A.Turner10, Francine B. De Abreu10, Torrey Gallagher10, Gregory J. Tsongalis10, Elizabeth R. Plimack11, Richard E. Greenberg12, and Daniel M. Geynisman11 Abstract Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non– muscle-invasive urothelial carcinoma of the bladder (NMIUC) population. Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intrave- sical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHCþ) or FGFR3 mutations (Mutþ) assessed in a CLIA-licensed labora- tory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHCþ Mut�, 3 IHCþ Mutþ). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carci- noma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 þ CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3–4 event. Six-month CR rate was 8% (0% in IHCþ Mut�; 33% in IHCþ Mutþ). The primary endpoint was not met. Pharmacodynamically active (94– 5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment. Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003–11. �2016 AACR. Introduction Urothelial carcinoma of the bladder is the fifth most common human cancer diagnosis. In 2016, more than 76,000 individuals are expected to be diagnosed with urothelial carcinoma and more than 16,000 patients to die from their disease (1). Most new urothelial carcinoma cases (�50,000 patients) are non–muscle- invasive at diagnosis with disease limited to the mucosal epithe- lium (Ta/Tis) and immediate connective tissue layer beneath the 1Johns Hopkins University Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland. 2The Johns Hopkins Greenberg Bladder Cancer Insti- tute, Baltimore, Maryland. 3The James Buchanan Brady Urological Institute, Baltimore, Maryland. 4Department of Urology, Indiana University Simon Cancer Center, Indianapolis, Indiana. 5Department of Pathology and Labo- ratory Medicine, Indiana University Simon Cancer Center, Indianapolis, Indi- ana. 6Department of Clinical Pharmacology, Indiana University Simon Cancer Center, Indianapolis, Indiana. 7Hoosier Cancer Research Network, Indiana- polis, Indiana. 8Indiana University Department of Biostatistics, Schools of Public Health and Medicine, Indianapolis, Indiana. 9Division of Hematology and Oncology, Indiana University Simon Cancer Center, Indianapolis, Indiana. 10Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, New Hampshire. 11Department of Hematol- ogy and Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania. 12Department of Urology, Fox Chase Cancer Center, Philadelphia, Pennsylvania. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Prior presentation: Presented in part as a poster at the 2016 American Society of Clinical Oncology Annual Meeting, Chicago, IL. Study ClinicalTrials.gov Identifier: NCT01732107. Corresponding Author: Noah M. Hahn, Department of Oncology and Urology, The Bunting-Blaustein Cancer Research Building 1, Room 1M42, 1650 Orleans Street, Baltimore, MD 21287. Phone: 443-287-0553; Fax: 410-614-8397; E-mail: nhahn4@jhmi.edu doi: 10.1158/1078-0432.CCR-16-2267 �2016 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 3003 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ mucosa (T1; ref. 2). The clinical course of non–muscle-invasive urothelial carcinoma of the bladder (NMIUC) is dominated by frequent recurrences requiring surveillance (with cystoscopy, bladder biopsy, urine cytology, etc.). The need for long-term invasive monitoring and treatment has significant cost and mor- bidity for patients with urothelial carcinoma. Compared with other malignancies, urothelial carcinoma ranks highest in lifetime per patient costs with an average cost from diagnosis to death of $96,500 per patient (3). Standard therapy for high-risk patients with NMIUC includes transurethral resection of bladder tumor (TURBT) augmented by intravesical administration of Bacillus Calmette-Guerin (BCG), an attenuated bovine mycoplasma–derived agent. Two meta- analyses of randomized trials of TURBT plus BCG versus TURBT alone demonstrated a reduction in 12-month tumor recurrence rate from 56% to 29% (P < 0.001) and a reduction in progression to muscle-invasive stages from 13.8% to 9.8% (P ¼ 0.001) in association with BCG therapy (4, 5). While BCG therapy is successful at preventing early tumor recurrences, most patients do not maintain sustained remissions. With 5-year follow-up, recurrent bladder tumors requiring repetitive TURBT and further cystoscopic surveillance are observed in 40% to 66% of patients (6, 7). For post-BCG tumor recurrences, BCG-unresponsive dis- ease is defined by any of the following features: recurrent NMIUC after 2 prior adequate BCG regimens, recurrent T1 disease at the initial 3-month posttreatment TURBT, recurrent NMIUC within 6 months of last BCG administration, and NMIUC involving the prostatic urethra (8). Transient remissions are often observed with additional intravesical therapy approaches; however, only 10% to 15% of patients remain recurrence-free at 1 year (9, 10). Thus, cystectomy is considered a standard treatment in BCG-unrespon- sive patients (11). A need clearly exists to explore the clinical efficacy of novel agents in this high-risk NMIUC population. Across multiple cancer types, the critical role of angiogenesis in tumor migration, proliferation, and metastasis is well established with VEGF and VEGFR serving as key mediators (12, 13). In urothelial carcinoma, associations between increased tumor VEGF expression and high-grade disease, advanced stage, and poor prognosis have been observed (14–16). Initial phase II trials in metastatic patients with urothelial carcinoma combining che- motherapy with the anti-VEGFR2 monoclonal antibody bevaci- zumab have demonstrated promising overall survival outcomes compared with historical controls with a definitive phase III trial of chemotherapy with or without bevacizumab completed and data maturing (17, 18). In addition to VEGFR, FGFR3 has been implicated as a critical facilitator of urothelial carcinoma carcinogenesis, particularly in NMIUC (19, 20). FGFR3 mutations or overexpression promote FGFR dimerization and constitutive activation of downstream signaling pathways in the absence of ligand in up to 80% of low- grade NMIUC tumors (21). These mutations result in a hyper- plastic phenotype dominated by frequent tumor recurrences with infrequent progression to muscle-invasive stages. While FGFR3 mutations are highly associated with low-grade NMIUC, over- expression of FGFR3 has been observed in up to 42% of high- grade muscle-invasive urothelial carcinoma tumors (22). Further- more, either an FGFR3 mutation or overexpression of the FGFR3 protein in the absence of mutation has been observed in 54% of muscle-invasive urothelial carcinoma tumors (22). Thus, while FGFR3 mutations likely are an early event in the tumorigenesis of low-grade noninvasive urothelial carcinoma tumors, alterations of FGFR3 may still play a role in the continued proliferation of high-grade urothelial carcinoma. Dovitinib is an oral tyrosine kinase inhibitor of FGFR1-3, VEGFR1-3, PDGFRb, c-Kit, RET, TrkA, CSF-1R, and FLT3 which has demonstrated a tolerable safety profile in single agent and combination regimens (23). Increasing evidence demonstrates that FGFR1 is a crucial mediator of tumor angiogenesis (24). In preclinical tumor models, blockade of the FGF pathway has proven to be an effective method of overcoming resistance to VEGFR inhibitors (25). Given the previously described impor- tance of VEGF in urothelial carcinoma progression and the frequent FGFR3 aberrations in NMIUC, we conducted a multi- site pilot trial in patients with BCG-unresponsive NMIUC har- boring FGFR3 gene alterations to evaluate the clinical and biologic outcomes of oral dovitinib therapy. Materials and Methods Study design A single-arm, nonrandomized, multicenter phase II study (NCT01732107) was conducted between 3 sites: Indiana Univer- sity Simon Cancer Center (Indianapolis, IN), Fox Chase Cancer Center (Philadelphia, PA), and Johns Hopkins Sidney Kimmel Comprehensive Cancer Center (Baltimore, MD). Standard of care and correlative biospecimens were collected pre- and posttreat- ment from all patients. The study was approved by the institu- tional review boards of each site. Patients Key eligibility criteria included: histologically confirmed Ta, T1, or Tis stage NMIUC assessed by TURBT performed within 42 days of registration; somatic tumor mutations in FGFR3 exons 7, 10, or 15 (S373C, G372C, Y375C, G382R, K652E, K652Q, K652T, K652M, A393E, S249C, and R248C) or tumor overexpression of phosphorylated (pFGFR3) by immunohistochemistry (IHC) Translational Relevance This trial reports the toxicity, pharmacodynamics, and clin- ical efficacy profiles of the oral FGFR1-3 and VEGFR1-3 multi- targeted tyrosine kinase inhibitor, dovitinib, in a pilot phase II investigation in patients with Bacillus Calmette-Guerin (BCG)-unresponsive non–muscle-invasive urothelial carcino- ma of the bladder (NMIUC) with tumors harboring FGFR3 alterations. In addition to demonstrating reductions in post- treatment pFGFR3, confirmed biologically active dovitinib concentrations were observed in the bladder urothelium. This trial is the first NMIUC study to require genomic testing as an eligibility requirement and to demonstrate successful achieve- ment of therapeutic urothelial tissue concentrations of sys- temically administered targeted therapies. Thus, it greatly expands the potential therapeutic approaches to treat this high-risk population. Lack of clinical efficacy was hampered by frequent drug toxicity and a paucity of patients harboring FGFR3 mutations. Additional FGFR3 targeting approaches in molecularly enriched urothelial carcinoma populations are ongoing and clearly worthy of further study, including in patients with NMIUC. Hahn et al. Clin Cancer Res; 23(12) June 15, 2017 Clinical Cancer Research3004 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ defined as 1þ or greater tumor pFGFR3 staining; recurrent NMIUC despite at least 2 prior intravesical treatment regimens (no limit), one of which must have been BCG; patients medically unfit for or refusing cystectomy; age >18 years; ECOG perfor- mance status 0–2; adequate hematologic and liver function; creatinine clearance >30 mL/min by modified Cockcroft–Gault equation; and documented, written informed consent. Major exclusion criteria included: evidence of muscle-invasive or met- astatic disease on pre-study screening tests; concurrent upper tract urothelial carcinoma; prior VEGFR or FGFR-targeted therapy. Treatment Patients were treated with dovitinib 500 mg by oral admin- istration once per day for 5 consecutive days followed by 2 days off each week. A cycle was defined as 4 weeks of therapy. No maximum number of treatment cycles was stipulated. Dose reductions to 400 and 300 mg were permitted in the event of treatment-related toxicity. Because of drug–drug interactions, full dose anti-coagulation with warfarin was not allowed, however, use of low-molecular weight heparin at full-dose was permitted. Usage of anti-emetic and colony-stimulating growth factor medications was at the discretion of the treating physician. Disease evaluations At baseline, the absence of metastatic disease was confirmed by abdomen and pelvis CT scan and chest X-ray or CT scan. Adequate cardiac function was confirmed by echocardiogram and electro- cardiogram assessments. History and physical examination find- ings, vital signs, baseline symptoms, and laboratory assessments were performed within 14 days of registration. Exams, vital signs, toxicity evaluations (per CTCAE v4.0), and laboratory assess- ments were performed biweekly for the first 2 cycles of treatment and every 4 weeks thereafter. All patients were evaluated with urine cytology and cystoscopy every 3 months during the first year and per the treating physi- cian's discretion thereafter. TURBT's were required at 3- and 6- month posttreatment with only for cause TURBT's thereafter. At each TURBT, biopsy tissue was obtained from all previous and new tumor sites, the bladder dome, anterior bladder wall, left lateral bladder wall, right lateral bladder wall, and the bladder trigone. Patients with any NMIUC at the 6-month evaluation or beyond were considered relapses as were patients with carcinoma in situ (CIS) at the 3-month evaluation. Patients with papillary- only disease at the 3-month cystoscopy/TURBT who declined further dovitinib therapy were classified as relapsed. Testing of urine for evidence of relapse by FISH was allowed but not required. The same was true for the use of blue light cystoscopy. An isolated FISH-positive urine finding was not classified as a relapse event. Complete response (CR) was defined as no evi- dence of any remaining urothelial carcinoma tumors of any T stage (including Tis) as assessed by cystoscopic examination and urine cytology. In addition to these criteria, the 6-month CR rate required no evidence of tumor within the 6-month posttreatment TURBT biopsies. The 1-year relapse-free survival rate was defined as the proportion of patients treated with dovitinib with no evidence of any urothelial carcinoma at 12 months of follow up. Patients with any evidence of muscle-invasive tumors (T2 or above) or metastatic disease in follow-up were considered as progressive disease. At the time of all TURBTs or cystectomy, tumor samples were sent for standard-of-care diagnostic evaluation and complete pathologic staging information was recorded. Samples from the same blocks were cut and archived for correlative studies. Resolution of any treatment-related toxicities was confirmed 30 days after administration of a patient's last dovitinib dose. Patients were not followed for long-term overall survival outcomes. FGFR3 mutation analysis At baseline, 5 individual 5-mm-thick slides were cut from the patient's representative TURBT block with the highest grade tumor and greatest volume of tumor present. In slides with less than 40% tumor cells present, macrodissection was performed to ensure maximum tumor cell DNA content. Also, at baseline, a 30-mL urine sample was obtained from all patients and centrifuged at 3,500 rpm for 10 minutes. The resulting super- natant and cell pellet were transferred into separate cryovials and stored at �70�C until analyzed. Slides and urine cell pellets were shipped to the laboratory for Clinical Genomics and Advanced Technology (CGAT) at the Dartmouth Hitchcock Medical Center. Tumor and urine cell pellet DNA extraction was performed per manufacturer's specification [Qiagen Pure- gene (tissue) and Qiagen DNeasy (cell pellet)]. FGFR3 muta- tional status was determined using a custom designed SNaP- shot assay (ThermoFisher Scientific) for all common mutations in FGFR3 coding exons including exons 7, 10, and 15. The presence or absence of specific FGFR3 mutations (S373C, G372C, Y375C, G382R, K652E, K652Q, K652T, K652M, A393E, S249C, and R248C) was communicated to HCRN within 14 days of specimen receipt. Phosphorylated FGFR3 IHC analysis Simultaneously at baseline, 5 individual 5-mm-thick slides from the patient's tumor and a single hematoxylin and eosin (H&E) slide from the same block were shipped to the IUSCC Immunohistochemistry Core Laboratory for FGFR3 IHC anal- ysis. Slides were heated to 60�C for 15 minutes. Slides were deparaffinized and rehydrated sequentially with xylene (5 minutes � 2), 100% ethyl alcohol solution (2 minutes � 2), and 95% ethyl alcohol solution (2 minutes � 2) on a Sakura linear stainer. Antigen retrieval utilized PT Link (PT10030, Dako) in conjunction with EnVision FLEX High pH target retrieval solution (K8000, Dako). Cycles began at 85�C and were heated to 100�C for 20 minutes followed by cooling back to 85�C and placement in wash buffer (K8002, Dako). Baseline phosphorylated FGFR3 staining for trial eligibility evaluation was performed on a Dako Autostainer platform utilizing the sc- 33041 anti-FGFR3 (phospho Y724) antibody (Santa Cruz Biotechnology). The sc-33041 pFGFR3 antibody was optimized to a 1:100 dilution for 30 minutes prior to the conduct of this trial utilizing 15 breast cancer cases as positive controls. Fol- lowing pFGFR3 staining, slides were dehydrated sequentially with 95% ethyl alcohol solution (2 minutes � 1), 100% ethyl alcohol solutions (3 minutes � 2), and xylene (5 minutes � 2) followed by coverslipping. The immunostained slides were evaluated by 2 different pathologists. Areas within the tumor were scored as follows: 0 ¼ negative, 1þ ¼ mild staining, 2þ ¼ moderate staining, 3þ ¼ strong staining. Both positive and negative controls were run in addition to the samples. Because a Dovitinib in BCG-Unresponsive NMIUC with FGFR3 Alterations www.aacrjournals.org Clin Cancer Res; 23(12) June 15, 2017 3005 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ clinically relevant cutoff for pFGFR3 IHC intensity had not previously been established, tumors with any staining intensity (1þ or greater) were considered pFGFR3-overexpressing. Dur- ing the conduct of the trial, improved commercial pFGFR3 antibodies became available. The correlative pre- and posttreat- ment pFGFR3 analyses were, therefore, performed utilizing the ab155960 anti-FGFR3 (phospho Y724) antibody (Abcam). The ab155960 evaluation scheme was validated across 19 different individual cases of bladder cancer for antibody specificity. Triplicate runs of this validation scheme showed that low strainers (1þ), moderate strainers (2þ), and strong strainers (3þ) were replicated across all runs. High, medium, and low staining positive controls were identified and used across all runs. All other antibody optimization procedures mirrored those of the sc-33041 antibody with the exception that the ab155960 antibody was optimized with to a 1:25 dilution for 40 minutes. The sc-33041 antibody continued to be utilized for eligibility determination throughout the entire conduct of the study. Aperio's ScanScope CS whole slide digital imaging sys- tem (Leica Biosystems) was used for baseline and posttreat- ment pFGFR3 pathology imaging. The system imaged all slides at 20�. The scan time ranged from 1 1/2 minutes to a maximum time of 2 1/4 minutes. The whole images were housed and stored in their Spectrum software system and images were shot from the whole slides. Quantification of pFGFR3 staining was performed on the HALO image analysis platform (Indica Labs). An algorithm was designed on the basis of pattern recognition that quantified tumor cells within pFGFR3-positive areas (tumor) and pFGFR3-negative areas (invasive margin). HALO's classifier package performed image analysis based on RGB (red, green, blue) spectra which was used to detect cells positively expressing pFGFR3 against negative expressing counterstained hematoxylin cells. The algorithm calculated the classified area (mm2) and percentage of tumor expression (% positive cells/% of all nucleated cells) using the HALO classifier package. The total percentage of positive expression in each group was averaged and SD was calculated. Further analysis was per- formed on 3 hotspots on each tissue via HALO's area quanti- fication package. An algorithm was designed to quantify pos- itive pFGFR3-expressing tumor cells in weak, moderate, and high positivity values. An average of hotspots for the tissues collected at day 1 was calculated along with SD. These data were compared with an average of hotspots of the tissues collected between cycle 3 days 26 and 30 for their total positivity and according to weak, moderate, high, and total expression. Dovitinib pharmacokinetic tissue analysis At the 3-month posttreatment disease assessment, a bladder biopsy of tumor or normal appearing urothelium was obtained for pharmacokinetic analysis to confirm achievement of bio- logically active dovitinib tissue concentrations via oral drug administration. The pharmacokinetic biopsy sample was flash- frozen, stored in liquid nitrogen, and shipped to the IUSCC Clinical Pharmacology Analytical Core (CPAC) for analyses. Tissue samples were homogenized in PBS, internal standard (sorafenib) was added to each sample, the samples were extracted with ethyl acetate, and injected into an HPLC-MS/ MS (API 4000; AB Sciex). Plasma was used for the matrix of the standard samples to estimate tissue concentrations. The lower limit of quantification was 8 ng/sample. For ease of comparison, tissue concentrations (ng/g) were converted to the nanomolar concentrations (assuming 1-g tissue is equiva- lent to 1-mL water). Statistical considerations The primary endpoint of the trial was 6-month CR rate. With a 6-month CR rate of clinical interest of �25%, a sample size of 20 patients provided an 80% power to exclude a lower bound of �10% utilizing a one-sided 90% confidence interval (CI) of Agresti–Couli type. With an estimated FGFR3 mutation or overexpression present in 40% of BCG-unresponsive tumors, screening of 50 patients' tumors was estimated to enroll the required 20 patients on dovitinib therapy. An evaluation of early stopping was planned at the first 10 patients completing 3-month assessment for progression to T2 or greater stages, whose objective was to stop the study if the likelihood of progression rate was more than 20%. A rule was chosen that the study should be terminated if 5 or more progressions were observed of 10 patients, which is the minimal number that leads to a 90% Agresti–Coull CI with a lower bound above 20%. Rates of CR, progressive disease, and treatment-related toxicity were summarized by 95% CIs. Associations between pre- and posttreatment pFGFR3 IHC staining intensity were compared by paired t testing with significance set at P < 0.05. Results Patients Between November 2013 and October 2014, 17 patients were screened and 13 patients were enrolled. Fifteen patients (88%) had sufficient tumor tissue for FGFR3 mutation testing. Two patients with tumors demonstrating no FGFR3 mutations were considered screen failures after the study amendment capping the enrollment of FGFR3 mutation–negative patients was in place. Further accrual was stopped because of cessation of clinical development of dovitinib. Patient demographics are summarized in Table 1 and included: median age 70 years (range, 57–78 years), 85% male, and 85% Caucasian. Baseline TURBT tumor stages were: CIS, 3 patients; Ta or T1, 8 patients; and Ta or T1 with concurrent CIS, 2 patients. Patients had received a median of 3 prior intravesical regimens (range, 2–6) with all patients having received at least 2 prior BCG induction courses. The median time from last intravesical therapy was 6 months (range, 1–33). Tumor FGFR3 mutations were detected in 3 patients (18% of screened patients) with a concordant urine FGFR3 mutation detected in 1 of the 3 patients. Dovitinib treatment Patients received a median of 4 cycles of dovitinib treatment (range, 1–19). Ten patients (77%) required dovitinib dose reduc- tions. Two patients (15%) discontinued dovitinib treatment prematurely and did not undergo planned 3-month posttreat- ment disease evaluations. Reasons for discontinuation included: physician discretion discontinuation of treatment due to a trau- matic intracranial hemorrhage sustained in a ground-level fall unrelated to study treatment (1 patient) and patient choice to withdraw from study (1 patient). In addition, dovitinib therapy was discontinued per treating physician's discretion in a single patient after 19 cycles after the patient revealed a prior history of retinal detachment unknown to the treating team at study enrollment. Hahn et al. Clin Cancer Res; 23(12) June 15, 2017 Clinical Cancer Research3006 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ Toxicity Dovitinib therapy was associated with frequent toxicity. All 13 patients (100%) experienced at least 1 grade 3 or 4 event. Treatment-related grade 4 hypertriglyceridemia was observed in 1 patient (8%). Treatment-related grade 3 events included fatigue, elevated g-glutamyl transferase (GGT), and elevated lipase in 2 patients (15%) each as well as headache, hypertriglyceridemia, stomatitis, and rash in 1 patient (8%) each. One patient (8%) suffered a subdural intracranial hemorrhage that did not require operative intervention in association with a ground-level fall on an ice-covered winter sidewalk that was not deemed treatment- related. All grade 3–4 events and other toxicities occurring in more than 20% of patients are summarized in Table 2. Complete all- grade toxicity is included in Supplementary Table S1. Tumor response Antitumor responses to dovitinib treatment were infrequent. Of the 13 patients enrolled, a pathologic CR was observed in 1 patient (8%). Nonresponse was observed in 11 patients (85%) and progression to muscle-invasive stage occurred in 1 patient (8%). The single patient with CR did harbor an FGFR3 S249C mutation. Thus, the pathologic CR rate amongst FGFR3 mutþ patients was 33% (1 of 3) as summarized in Table 3. The patient remains in a CR at 19þ months of follow-up. Eight patients (62%) underwent cystectomy per the discretion of their physician at any time point following completion of study therapy with a wide variety of pathologic stages ranging from pT0N0 to pNþ disease (Supplementary Table S2). Dovitinib pharmacokinetic tissue analysis Fresh tumor or adjacent normal urothelium biopsy tissue was available for dovitinib pharmacokinetic analysis from 9 of the 11 patients who underwent posttreatment disease evaluations. As shown in Fig. 1, dovitinib was detectable at pharmacologically active levels in all patients examined with tissue concentrations ranging from 94 to 5,813 nmol/L. IHC analysis of dovitinib treatment on pFGFR3 All baseline slides for eligibility determination demonstrated positive pFGFR3 staining as assessed by the sc-33041 pFGFR3 antibody. Staining intensities according to the use of the sc- 33041 and ab155960 pFGFR3 antibodies showed significant heterogeneity (Supplementary Table S3). Pre- and post-dovi- tinib treatment slides were available from 9 patients including 8 tumor pairs. Utilizing the quantitative Halo Classifier imaging platform, reductions in averaged pFGFR3 staining area from 41.2 to 31.3 mm2 were observed following dovitinib treatment. As depicted in Fig. 2, this posttreatment reduction in mean pFGFR3 staining area showed a strong trend but did not reach statistical significance (P ¼ 0.08). Marked reductions in pFGFR3 staining were observed in 4 of 9 patients, of which one reduction is demonstrated in Fig. 3. Discussion Until the recent FDA approval of the immunotherapy agent atezolizumab in metastatic urothelial carcinoma patients, near- ly a quarter century had passed without any significant advances in systemic therapy for urothelial carcinoma (26). While the approval of atezolizumab is encouraging, it is impor- tant to note that only a small subset of patients derive benefit. Thus, additional novel approaches to treat urothelial carcinoma are clearly needed. In particular, innovative strategies for the two thirds of patients with urothelial carcinoma initially pre- senting with NMIUC are paramount. Given the established relevance of VEGFR in urothelial carcinoma cancer invasion and metastases and the striking frequency of FGFR3 aberrations in low-grade NMIUC, we postulated that an FGFR3/VEGFR2- directed approach with dovitinib would prove both feasible and beneficial in patients with BCG-unresponsive NMIUC with tumors harboring FGFR3 alterations. Our study failed to demonstrate significant clinical activity with dovitinib therapy in the enrolled study population. Lim- itations in the enrollment criteria for the study population likely played a major factor in the absent antitumor activity observed. At the time the study was designed, the relative importance of FGFR3 mutations versus gene fusions versus overexpression was unknown. Furthermore, clinically relevant cutoffs for pFGFR3 IHC staining had not been established and available commercial pFGFR3 antibodies were limited. There- fore, even though robust methodology was developed prior to study initiation to optimize pFGFR3 antibody procedures, our trial allowed patients with any degree of pFGFR3 IHC staining at baseline to enroll. This allowed for an early influx of IHCþ Mut� patients. As demonstrated by the frequent heterogeneity that was observed in baseline IHC intensities according to the pFGFR3 antibody utilized, this likely resulted in a less Table 1. Baseline patient and tumor characteristics Patient Gender Age, y Race T-stage Prior regimens Time from last therapy, mo Tumor FGFR3 mutation Urine FGFR3 mutation pFGFR3 IHC intensity 1 F 77 C T1 þ CIS BCG � 2, Gem 58.3 G382R None 3þ 2 M 67 C CIS BCG � 2, MMC 8.3 None None 3þ 3 M 71 C Ta BCG � 2 6.3 None None 3þ 4 M 64 C Ta BCG � 2, MMC 23.7 None None 3þ 5 M 75 C Ta BCG � 4, MMC, Val 5.6 S249C S249C 3þ 6 M 70 AA T1 BCG � 4, MMC 6.2 None None 3þ 7 M 69 U CIS BCG � 2, Val 3.7 None None 3þ 8 M 78 C Ta BCG � 2 5.7 None None 3þ 9 M 57 C Ta BCG � 2, MMC 1.4 None None 3þ 10 M 71 C T1 BCG � 2 3.7 None None 3þ 11 F 67 C CIS BCG � 2, MMC, Val 15 None None 3þ 12 M 77 C T1 þ CIS BCG � 3, Val 24.2 None None 3þ 13 M 57 C Ta BCG � 2 33.1 S249C NE 2þ Abbreviations: AA, African-American; C, Caucasian, F, female; Gem, gemcitabine; M, male; MMC, mitomycin C; NE, not evaluable; U, unknown, Val, valrubicin. Dovitinib in BCG-Unresponsive NMIUC with FGFR3 Alterations www.aacrjournals.org Clin Cancer Res; 23(12) June 15, 2017 3007 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ biologically enriched population than intended. An amend- ment to cap the number of IHCþ Mut� patient enrollment at 10 patients was instituted; however, the trial was closed after enrolling only 3 Mutþ patients. In recent trial reports of other FGFR3 inhibitors (JNJ-42756493, BGJ398, AZD4547) in met- astatic patients with urothelial carcinoma, it now appears clear that activating FGFR3 mutations or fusions are required for tumor responses (27–29). In trials of these agents mandating either FGFR3 mutation or fusions, reduction of tumor size was observed in 50% to 60% of metastatic patients with urothelial carcinoma (27, 29). Interestingly, in a prior report of dovitinib in metastatic patients with urothelial carcinoma, no responses were observed among 12 patients with FGFR3 mutations (30). It is not clear whether differences in FGFR3 mutation testing methodology or individual drug FGFR3-binding site properties explain discordant clinical activity. An observed CR 1 of the 3 Mutþ patients treated with dovitinib in our trial is consistent with the more recent FGFR3 inhibitor results. With only 3 Mutþ patients enrolled, it is impossible for our study to provide any meaningful CIs around the true CR rate. However, it is encouraging that the single CR patient has demonstrated a sustained remission out to 19þ months. Furthermore, a strong trend in decreased posttreatment pFGFR3 staining was observed regardless of FGFR3 mutation status. In addition to patient selection limitations, the high rate of treatment-related toxicity led to frequent dose reductions including 2 of the 3 FGFR3 Mutþ patients discontinuing dovi- tinib early. These dose modifications led to reduced dovitinib dose intensity in most patients and may have compromised antitumor effects. For future trials, particularly in the NMIUC population, our study provides a good example of the need to have a drug that is not only effective but also tolerable at therapeutic doses to impart true benefit. Specifically, as in the case of dovitinib, the acceptance of relatively high rates of chronic toxicity in heavily pretreated metastatic solid tumor phase I trials may be greater than in NMIUC, given that NMIUC can be cured with cystectomy (31). In future design of NMIUC trials, particular attention to high rates of acute or chronic grade 1–2 toxicities is warranted particularly if a drug will require chronic or lifelong administration to prevent tumor recurrence. In addition, perioperative complication rates from patients who proceed to posttreatment cystectomies are of critical importance in NMIUC trials, particularly when agents with known effects on bleeding and wound-healing such as FGFR or VEGFR inhibitors Table 2. All grade 3–4 adverse events and other adverse events occurring in more than 20% of patients Adverse event Grade 1 Grade 2 Grade 3 Grade 4 Constitutional Fatigue 5 (39%) 4 (31%) 2 (15%) 0 (0%) Pain 6 (46%) 6 (46%) 0 (0%) 0 (0% Fall 0 (0%) 0 (0% 1 (8%) 0 (0%) Other constitutional 3 (23%) 2 (15%) 0 (0%) 0 (0%) Vascular Hypertension 0 (0%) 2 (15%) 2 (15%) 0 (0%) Headache 5 (39%) 1 (8%) 1 (8%) 0 (0%) Intracranial hemorrhage 0 (0%) 0 (0% 1 (8%) 0 (0%) Gastrointestinal GERD 2 (15%) 3 (23%) 1 (8%) 0 (0%) Constipation 2 (15%) 2 (15%) 0 (0%) 0 (0%) Diarrhea 8 (62%) 2 (15%) 0 (0%) 0 (0%) Anorexia 4 (31%) 1 (8%) 0 (0%) 0 (0%) Weight loss 4 (31%) 0 (0%) 0 (0%) 0 (0%) Dysgeusia 5 (39%) 2 (15%) 0 (0%) 0 (0%) Nausea/Emesis 6 (46%) 0 (0%) 0 (0%) 0 (0%) Emesis 4 (31%) 0 (0%) 0 (0%) 0 (0%) Other gastrointestinal 2 (15%) 3 (23%) 0 (0%) 0 (0%) Skin Stomatitis 0 (0%) 0 (0%) 1 (8%) 0 (0%) Rash 4 (31%) 1 (8%) 1 (8%) 0 (0%) Hand–foot syndrome 2 (15%) 1 (8%) 0 (0%) 0 (0%) Dry mouth 4 (31%) 0 (0%) 0 (0%) 0 (0%) Other skin 6 (46%) 2 (15%) 0 (0%) 0 (0%) Genitourinary Bladder spasms 0 (0%) 3 (23%) 0 (0%) 0 (0%) Other urinary 7 (54%) 1 (8%) 0 (0%) 0 (0%) Infection Fever 4 (31%) 0 (0%) 0 (0%) 0 (0%) Infection 0 (0%) 8 (62%) 0 (0%) 0 (0%) Pulmonary Hoarseness 3 (23%) 0 (0%) 0 (0%) 0 (0%) Other pulmonary 4 (31%) 1 (8%) 0 (0%) 0 (0%) Musculoskeletal Arthralgia/Myalgia 4 (31%) 2 (15%) 0 (0%) 0 (0%) Metabolic Hypertriglyceridemia 1 (8%) 2 (15%) 1 (8%) 1 (8%) Elevated alkaline phosphatase 2 (15%) 1 (8%) 0 (0%) 0 (0%) Elevated GGT 0 (0%) 1 (8%) 2 (15%) 0 (0%) Hypoalbuminemia 2 (15%) 1 (8%) 0 (0%) 0 (0%) Elevated lipase 0 (0%) 0 (0%) 2 (15%) 0 (0%) Other metabolic 6 (46%) 0 (0%) 0 (0%) 0 (0%) Hematologic Anemia 4 (31%) 0 (0%) 0 (0%) 0 (0%) Table 3. Tumor response to dovitinib treatment Patient Baseline T-stage Tumor FGFR3 mutations Duration of treatment, mo Posttreatment T-stage Response category 1 T1 þ CIS G382R 0.8 NE NR 2 CIS None 2.7 CIS NR 3 Ta None 4.4 Ta NR 4 Ta None 2.8 Ta NR 5 Ta S249C 0.7 NE NR 6 T1 None 2.7 T1 NR 7 CIS None 5.3 CIS NR 8 Ta None 3.3 T1 NR 9 Ta None 3 Ta NR 10 T1 None 2.7 T2 PD 11 CIS None 4.4 CIS NR 12 T1 þ CIS None 3.2 T1 NR 13 Ta S249C 17.5 T0 CR Abbreviations: NE, not evaluable; NR, nonresponder; PD, progressive disease. Hahn et al. Clin Cancer Res; 23(12) June 15, 2017 Clinical Cancer Research3008 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ are studied. While no life-threatening perioperative complica- tions were observed in our trial, our sample size is insufficient to discount the possibility of such risks. Despite the absent clinical activity, our study establishes several innovative principles in the design of NMIUC trials that should facilitate improved future clinical trial designs in this population. First, our study demonstrated the feasibility of tumor genomic testing as an eligibility requirement in the NMIUC population in a multisite setting. In fact, of the 17 patients screened, 15 (88%) had sufficient tumor available for FGFR3 mutation testing. While investigation of oral kinase inhibitors in patients with NMIUC has been pursued by other investigators, to our knowledge, our trial is the first to be undertaken in a molecularly enriched NMIUC population (32). With the establishment of intrinsic basal and luminal tumor subtypes from analysis of The Cancer Genome Atlas (TCGA) urothelial carcinoma samples, we expect an increased need for future urothelial carcinoma clinical trials to target specific genomically defined patient subsets (33). Our study demonstrates that, despite the small tumor samples obtained from standard-of-care TURBT specimens, enrichment of NMIUC patient subsets based on molecular testing is pos- sible and should be pursued if scientific hypotheses warrant it. In addition, our results establish the frequency of FGFR3 mutations in the BCG-unresponsive NMIUC population at 18% (3 of 17 patients), a previously unknown benchmark. Our a priori design assumption that the FGFR3 mutation rate in patients with BCG-unresponsive NMIUC would fall somewhere between the reported rates in low-grade NMIUC (65%) and muscle-invasive urothelial carcinoma (15%) 41.3 28.0 9.4 3.8 31.3 24.3 6.3 0.7 0 5 10 15 20 25 30 35 40 45 Total Weak Moderate Strong Baseline Pos�reatment P er ce nt p FG FR 3 po si tiv ity b y IH C pFGFR3 IHC Intensity group Figure 2. Pre- and post-dovitinib pFGFR3 IHC results. 1603 159 5813 812 726 1135 94 2115 2483 1 10 100 1,000 10,000 2 3 6 7 8 10 11 12 13L og 10 D ov iti ni b tis su e co nc en tr at io n (n m ol /L ) Patient study number Dovitinib IC50 = 10 nmol/L Figure 1. Posttreatment dovitinib tissue concentration. Dovitinib in BCG-Unresponsive NMIUC with FGFR3 Alterations www.aacrjournals.org Clin Cancer Res; 23(12) June 15, 2017 3009 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ patients proved incorrect. Our results suggest that BCG-unre- sponsive NMIUC more closely resembles muscle-invasive and metastatic urothelial carcinoma than a low-grade NMIUC pre- decessor tumor. The lower rate of FGFR3 mutations observed in the BCG-unresponsive NMIUC population has implications on future sample size considerations of FGFR3-targeting trials in this population. Importantly, our trial showed that oral administration of dovitinib unanimously achieved pharmacologically active urothelial tissue concentrations. This finding suggests that lack of clinical activity was related to drug toxicity and study population design issues rather than drug delivery failure. These results support further investigation of systemically administered agents in the NMIUC population. A caveat, how- ever, is the fact that the urothelial tissue bioavailability is not usually investigated or provided in preclinical testing data provided in investigator brochures of most novel cancer drugs. A high intact urinary excretion of drug can be reassuring of adequate urothelial tumor drug concentration exposure. How- ever, if urothelial tissue concentrations are critical in the deci- sion process to assess the effectiveness of systemic versus intravesical routes of drug administration, development of clinical pharmacology assays to measure urothelial tissue drug concentrations are strongly recommended. Finally, our study demonstrates the importance of multispeci- alty investigator engagement in the conduct of early-stage urothe- lial carcinoma trials. At each participating center, a urologist, medical oncologist, and pathologist were identified to serve as local champions for the trial. While multidisciplinary teams in varying forms are often utilized in the administration of neoad- juvant cisplatin-based chemotherapy for muscle-invasive urothe- lial carcinoma, our study highlights the importance of also developing highly functional cross-discipline research collabora- tions in NMIUC patients. The need for urothelial carcinoma multispecialty research infrastructure is increasing in parallel with the rapid expansion of clinical trials being conducted in the muscle-invasive adjuvant, neoadjuvant, and BCG-unresponsive NMIUC populations. In summary, our study firmly establishes that pFGFR3 IHC alone should not be used as a solitary qualifying criteria for enrollment in future urothelial carcinoma trials of FGFR3 kinase inhibitors. In addition, the unfavorable toxicity profile of dovi- tinib precludes further development in the NMIUC population. However, antitumor activity consistent with other reports in FGFR3 Mutþ patients was observed further implying FGFR3 as a viable therapeutic target in urothelial carcinoma across all stages including NMIUC. The demonstration that genomic testing as an eligibility requirement in NMIUC patients is feasible and the detection of pharmacologically active dovitinib urothelial tissue concentrations by oral drug administration are novel findings with implications for future NMIUC trial designs. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Authors' Contributions Conception and design: N.M. Hahn, M.O. Koch, R.S. Foster, Z. Liu, G.J. Tsongalis Development of methodology: N.M. Hahn, G.J. Netto, D.R. Jones, G.E. San- dusky, T. Breen, Z. Liu, S.A. Turner, F.B. de Abreu, G.J. Tsongalis, R.E. Greenberg Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.M. Hahn, T.J. Bivalacqua, A.E. Ross, G.J. Netto, A.S. Baras, J.C. Park, C. Chapman, T.A. Masterson, M.O. Koch, R. Bihrle, R.S. Foster, T.A. Gardner, L. Cheng, T. Breen, C. Albany, M.L. Moore, A. Reed, F.B. de Abreu, T.L. Gallagher, G.J. Tsongalis, E.R. Plimack, R.E. Greenberg Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.M. Hahn, T.J. Bivalacqua, G.J. Netto, J.C. Park, R.S. Foster, L. Cheng, D.R. Jones, K. McElyea, G.E. Sandusky, T. Breen, Z. Liu, C. Albany, S.A. Turner, F.B. de Abreu, E.R. Plimack Writing, review, and/or revision of the manuscript: N.M. Hahn, T.J. Bivalac- qua, A.E. Ross, G.J. Netto, R.S. Foster, A.S. Baras, T.A. Gardner, L. Cheng, D.R. Jones, G.E. Sandusky, Z. Liu, C. Albany, F.B. de Abreu, G.J. Tsongalis, E.R. Plimack, R.E. Greenberg, D.M. Geynisman Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N.M. Hahn, A.S. Baras, K. McElyea, S.A. Turner, G.J. Tsongalis Study supervision: N.M. Hahn, T.A. Masterson, R.A. Loman, R.E. Greenberg Grant Support This study was supported by grant funding from investigator-initiated funds from Novartis and NCI Cancer Center support grant funding (P30CA006973). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 13, 2016; revised November 7, 2016; accepted Novem- ber 8, 2016; published OnlineFirst December 8, 2016. A B Figure 3. Dovitinib pFGFR3 IHC pathology samples. A, Patient 3 – Baseline. B, Patient 3 – Posttreatment cycle 3 day 26. Hahn et al. Clin Cancer Res; 23(12) June 15, 2017 Clinical Cancer Research3010 on April 5, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2016; DOI: 10.1158/1078-0432.CCR-16-2267 http://clincancerres.aacrjournals.org/ References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7–30. 2. Ries L, Harkins D, Krapcho M, Mariotto A, Miller B, Feuer E, et al. SEER cancer statistics review, 1975–2003. 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Bivalacqua, Ashley E. Ross, et al. Cancer Research Network Trial HCRN 12-157 Mutations or Overexpression: HoosierFGFR3Carcinoma with A Phase II Trial of Dovitinib in BCG-Unresponsive Urothelial Updated version 10.1158/1078-0432.CCR-16-2267doi: Access the most recent version of this article at: Material Supplementary http://clincancerres.aacrjournals.org/content/suppl/2017/06/21/1078-0432.CCR-16-2267.DC1 Access the most recent supplemental material at: Cited articles http://clincancerres.aacrjournals.org/content/23/12/3003.full#ref-list-1 This article cites 28 articles, 6 of which you can access for free at: E-mail alerts related to this article or journal.Sign up to receive free email-alerts Subscriptions Reprints and .pubs@aacr.org To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Permissions Rightslink site. 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