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Next-Generation Sequencing Reveals Potential Predictive Biomarkers and Targets of Therapy for Urothelial Carcinoma in Situ of the Urinary Bladder

Open AccessPublished:November 14, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.10.004
      Bacillus Calmette-Guérin instillation after removal of the tumor is the first line of treatment for urothelial carcinoma in situ (CIS), the precursor lesion of most muscle-invasive bladder cancers. Bacillus Calmette-Guérin therapy fails in >50% of cases, and second-line radical cystectomy is associated with overtreatment and drastic lifestyle consequences. Given the need for alternative bladder-preserving therapies, we identified genomic alterations (GAs) in urothelial CIS having the potential to predict response to targeted therapies. Laser-capture microdissection was applied to isolate 30 samples (25 CIS and 5 muscle controls) from 26 fresh-frozen cystectomy specimens. Targeted next-generation sequencing of 31 genes was performed. The panel comprised genes frequently affected in muscle-invasive bladder cancer of nonpapillary origin, focusing on potentially actionable GAs described to predict response to approved targeted therapies or drugs that are in registered clinical trials. Of CIS patients, 92% harbored at least one potentially actionable GA, which was identified in TP53/cell cycle pathway–related genes (eg, TP53 and MDM2) in 72%, genes encoding chromatin-modifying proteins (eg, ARID1A and KDM6A) in 68%, DNA damage repair genes (eg, BRCA2 and ATM) in 60%, and phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes (eg, ERBB2 and FGFR1) in 36% of the cases. These data might help guide the selection of targeted therapies to be investigated in future clinical CIS trials, and they may provide a basis for future mechanistic studies of urothelial CIS pathogenesis.
      With an estimated number of 380,000 new cases and 150,000 deaths per year worldwide, bladder cancer is the most common malignancy of the urinary tract.
      • Ferlay J.
      • Shin H.-R.
      • Bray F.
      • Forman D.
      • Mathers C.
      • Parkin D.M.
      Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.
      More than 90% of all bladder tumors diagnosed in Europe and North America are urothelial carcinomas.
      • Knowles M.A.
      • Hurst C.D.
      Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.
      Most bladder cancers present as non–muscle-invasive, low-grade papillary carcinomas, characterized by FGFR3 gene alterations and an excellent prognosis (5-year survival of approximately 90%). These tumors infrequently progress to muscle-invasive disease but frequently recur. In contrast, muscle-invasive bladder cancer (MIBC) is associated with an unfavorable prognosis (5-year survival <50%) and a high risk of progression to metastasis.
      • Knowles M.A.
      • Hurst C.D.
      Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.
      Most MIBC cases arise via carcinoma in situ (CIS), a flat high-grade lesion associated with TP53 mutations. CIS is found in approximately 10% of all patients diagnosed with non–muscle-invasive bladder cancer (NMIBC).
      • Kirkali Z.
      • Chan T.
      • Manoharan M.
      • Algaba F.
      • Busch C.
      • Cheng L.
      • Kiemeney L.
      • Kriegmair M.
      • Montironi R.
      • Murphy W.M.
      • Sesterhenn I.A.
      • Tachibana M.
      • Weider J.
      Bladder cancer: epidemiology, staging and grading, and diagnosis.
      The fraction of primary (isolated) CIS is estimated to be 3% of all patients diagnosed with bladder cancer.
      • Casey R.G.
      • Catto J.W.F.
      • Cheng L.
      • Cookson M.S.
      • Herr H.
      • Shariat S.
      • Witjes J.A.
      • Black P.C.
      Diagnosis and management of urothelial carcinoma in situ of the lower urinary tract: a systematic review.
      It is estimated that, without any treatment, 40% to 60% of CIS patients develop muscle invasion within 5 years after diagnosis.
      • Sylvester R.J.
      • van der Meijden A.
      • Witjes J.A.
      • Jakse G.
      • Nonomura N.
      • Cheng C.
      • Torres A.
      • Watson R.
      • Kurth K.H.
      High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder.
      Currently, therapy for bladder cancer, including CIS, is mainly based on non-specific treatments [ie, chemotherapy or bacillus Calmette-Guérin (BCG) immunotherapy in addition to surgery], depending on the risk estimation for the disease diagnosed.
      • Babjuk M.
      • Burger M.
      • Zigeuner R.
      • Shariat S.F.
      • van Rhijn B.W.G.
      • Compérat E.
      • Sylvester R.J.
      • Kaasinen E.
      • Böhle A.
      • Palou Redorta J.
      • Rouprêt M.
      EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013.
      ,
      • Alfred Witjes J.
      • Lebret T.
      • Compérat E.M.
      • Cowan N.C.
      • De Santis M.
      • Bruins H.M.
      • Hernández V.
      • Espinós E.L.
      • Dunn J.
      • Rouanne M.
      • Neuzillet Y.
      • Veskimäe E.
      • van der Heijden A.G.
      • Gakis G.
      • Ribal M.J.
      Updated 2016 EAU guidelines on muscle-invasive and metastatic bladder cancer.
      Recently, the approval of immune checkpoint inhibitors by the US Food and Drug Administration and the European Medicines Agency has opened new therapeutic options for patients with advanced bladder cancer.
      • Bellmunt J.
      • Powles T.
      • Vogelzang N.J.
      A review on the evolution of PD-1/PD-L1 immunotherapy for bladder cancer: the future is now.
      For urothelial bladder cancer, immune checkpoint inhibitor drugs have been approved for the treatment of patients with metastatic bladder cancer as second-line therapy in the post-platinum setting or for cisplatin-ineligible individuals in first line if patients exhibit a combined positive score >10%.
      • Tripathi A.
      • Plimack E.R.
      Immunotherapy for urothelial carcinoma: current evidence and future directions.
      Although new-generation immunotherapies have been shown to result in durable responses and seem to be relatively well tolerated, most patients do not respond because of primary or acquired resistance mechanisms.
      • Sharma P.
      • Hu-Lieskovan S.
      • Wargo J.A.
      • Ribas A.
      Primary, adaptive, and acquired resistance to cancer immunotherapy.
      For CIS patients, intravesical BCG-based immunotherapy after transurethral resection of tumor cells remains the standard of care, as recommended by the European Association of Urology and the American Urological Association.
      • Babjuk M.
      • Burger M.
      • Zigeuner R.
      • Shariat S.F.
      • van Rhijn B.W.G.
      • Compérat E.
      • Sylvester R.J.
      • Kaasinen E.
      • Böhle A.
      • Palou Redorta J.
      • Rouprêt M.
      EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013.
      ,
      • Hall M.C.
      • Chang S.S.
      • Dalbagni G.
      • Pruthi R.S.
      • Seigne J.D.
      • Skinner E.C.
      • Wolf J.S.
      • Schellhammer P.F.
      Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update.
      However, it is estimated that BCG therapy fails in >50% of cases,
      • Sylvester R.J.
      • van der Meijden A.
      • Witjes J.A.
      • Jakse G.
      • Nonomura N.
      • Cheng C.
      • Torres A.
      • Watson R.
      • Kurth K.H.
      High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder.
      caused by cessation of treatment because of toxicity or resistance to BCG immunotherapy.
      • Tang D.H.
      • Chang S.S.
      Management of carcinoma in situ of the bladder: best practice and recent developments.
      Radical cystectomy is recommended as second-line treatment in case of BCG failure.
      • Babjuk M.
      • Burger M.
      • Zigeuner R.
      • Shariat S.F.
      • van Rhijn B.W.G.
      • Compérat E.
      • Sylvester R.J.
      • Kaasinen E.
      • Böhle A.
      • Palou Redorta J.
      • Rouprêt M.
      EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013.
      ,
      • Hall M.C.
      • Chang S.S.
      • Dalbagni G.
      • Pruthi R.S.
      • Seigne J.D.
      • Skinner E.C.
      • Wolf J.S.
      • Schellhammer P.F.
      Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update.
      Although early radical cystectomy is associated with an excellent tumor-specific survival,
      • Herr H.W.
      • Sogani P.C.
      Does early cystectomy improve the survival of patients with high risk superficial bladder tumors?.
      the rate of overtreatment is assumed to be up to 50%
      • Sylvester R.J.
      • van der Meijden A.
      • Witjes J.A.
      • Jakse G.
      • Nonomura N.
      • Cheng C.
      • Torres A.
      • Watson R.
      • Kurth K.H.
      High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder.
      and radical cystectomy is a morbid surgery significantly impacting quality of life.
      • Nykopp T.K.
      • Batista da Costa J.
      • Mannas M.
      Black PC: Current clinical trials in non-muscle invasive bladder cancer.
      Taken together, there is large need for alternative bladder-preserving therapies for urothelial CIS, reflected by the wide landscape of active clinical trials in high-grade NMIBC.
      • Nykopp T.K.
      • Batista da Costa J.
      • Mannas M.
      Black PC: Current clinical trials in non-muscle invasive bladder cancer.
      In recent years, advances in next-generation sequencing (NGS) technologies have led to substantial progress in the molecular characterization of several diseases, including bladder cancer, and thereby the identification of potential biomarkers and candidate therapeutic targets.
      TCGA Research Network
      Comprehensive molecular characterization of urothelial bladder carcinoma.
      • Ross J.S.
      • Wang K.
      • Al-Rohil R.N.
      • Nazeer T.
      • Sheehan C.E.
      • Otto G.A.
      • He J.
      • Palmer G.
      • Yelensky R.
      • Lipson D.
      • Ali S.
      • Balasubramanian S.
      • Curran J.A.
      • Garcia L.
      • Mahoney K.
      • Downing S.R.
      • Hawryluk M.
      • Miller V.A.
      • Stephens P.J.
      Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy.
      • Kim P.H.
      • Cha E.K.
      • Sfakianos J.P.
      • Iyer G.
      • Zabor E.C.
      • Scott S.N.
      • Ostrovnaya I.
      • Ramirez R.
      • Sun A.
      • Shah R.
      • Yee A.M.
      • Reuter V.E.
      • Bajorin D.F.
      • Rosenberg J.E.
      • Schultz N.
      • Berger M.F.
      • Al-Ahmadie H.A.
      • Solit D.B.
      • Bochner B.H.
      Genomic predictors of survival in patients with high-grade urothelial carcinoma of the bladder.
      • Hedegaard J.
      • Lamy P.
      • Nordentoft I.
      • Algaba F.
      • Høyer S.
      • Ulhøi B.P.
      • et al.
      Comprehensive transcriptional analysis of early-stage urothelial carcinoma.
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      Although most of the work focused on advanced bladder cancer,
      TCGA Research Network
      Comprehensive molecular characterization of urothelial bladder carcinoma.
      • Ross J.S.
      • Wang K.
      • Al-Rohil R.N.
      • Nazeer T.
      • Sheehan C.E.
      • Otto G.A.
      • He J.
      • Palmer G.
      • Yelensky R.
      • Lipson D.
      • Ali S.
      • Balasubramanian S.
      • Curran J.A.
      • Garcia L.
      • Mahoney K.
      • Downing S.R.
      • Hawryluk M.
      • Miller V.A.
      • Stephens P.J.
      Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy.
      • Kim P.H.
      • Cha E.K.
      • Sfakianos J.P.
      • Iyer G.
      • Zabor E.C.
      • Scott S.N.
      • Ostrovnaya I.
      • Ramirez R.
      • Sun A.
      • Shah R.
      • Yee A.M.
      • Reuter V.E.
      • Bajorin D.F.
      • Rosenberg J.E.
      • Schultz N.
      • Berger M.F.
      • Al-Ahmadie H.A.
      • Solit D.B.
      • Bochner B.H.
      Genomic predictors of survival in patients with high-grade urothelial carcinoma of the bladder.
      ,
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      fewer advances have been made regarding NMIBC.
      • Hedegaard J.
      • Lamy P.
      • Nordentoft I.
      • Algaba F.
      • Høyer S.
      • Ulhøi B.P.
      • et al.
      Comprehensive transcriptional analysis of early-stage urothelial carcinoma.
      ,
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      Especially the genomic alterations (GAs) underlying the preinvasive CIS, the precursor lesion of fatal MIBC, have been characterized insufficiently so far.
      • Rentsch C.A.
      • Müller D.C.
      • Ruiz C.
      • Bubendorf L.
      Comprehensive molecular characterization of urothelial bladder carcinoma: a step closer to clinical translation?.
      In this study, laser-capture microdissection (LCM) and targeted NGS were combined to identify therapeutically actionable GAs [mutations and copy number variations (CNVs)] in a cohort of fresh-frozen CIS.

      Materials and Methods

      Patients and Sample Preparation

      From 26 cryopreserved cystectomy specimens, 30 samples (25 urothelial CIS and 5 smooth muscle control specimens) were isolated by LCM. The 26 patients were treated by cystectomy at the University Hospital RWTH Aachen (Aachen, Germany) between 2005 and 2011 and were retrospectively included in the present study. The study was conducted at the University Hospital RWTH Aachen in accordance with local institutional review board–approved protocols of the Medical Faculty of the RWTH Aachen University (approval number EK 291/16) and the principles expressed in the Declaration of Helsinki. The clinicopathologic parameters for the 26 cystectomy cases are summarized in Table 1 and Supplemental Table S1. The pathologic diagnosis of each urothelial CIS case as well as the absence of cancer cells in smooth muscle controls (distant from tumor areas) were confirmed on hematoxylin and eosin–stained slides by an experienced uropathologist (R.K.) before LCM-based isolation of cells. Moreover, great care has been taken to isolate mere CIS regions without any contact to invasive areas. Figure 1 shows a hematoxylin and eosin–stained tissue section of an exemplary cryopreserved CIS sample that is part of our CIS cohort.
      Table 1Clinicopathologic Data of the CIS Patients Analyzed in This Study
      Sample IDCystectomySexAge, yearsTumor gradepTpNpMPretreatment
      CIS011M82G3pT310No
      CIS022F66G3pT40NANo
      MU052F66G3pT40NANo
      CIS033M73G3pT3a0NANo
      CIS044M43G3pT20NANo
      CIS055M75G3pT10NANo
      CIS066M69G3pT41NANo
      CIS077M62G3pT3a2NANo
      CIS088F77G3pT3a1NANo
      CIS099M81G3pT3b0NANo
      CIS1010M72G3pT2a0NAYes
      CIS1111M50G3pT201No
      CIS1312F70G3pT3a1NANo
      CIS1413M77G3pT20NAYes
      CIS1514F70G3pT2aNANAYes
      CIS1615M66G3pT3b0NANo
      CIS1716M76G3pT3a0NANo
      CIS1817M76G3pT3a0NANo
      MU0418F62G3pT20NANo
      CIS2019M79G3pT32NANo
      CIS2120F75G3pT2a0NANo
      MU0120F75G3pT2a0NANo
      CIS2221M61G2 high gradepT1b0NANo
      CIS2322M57G3pTis0NANo
      MU0322M57G3pTis0NANo
      CIS2423M63G2 high gradepT10NANo
      CIS2524M69G3pT3aNANAYes
      CIS2625M65G3pT1a0NAYes
      MU0225M65G3pT1a0NAYes
      CIS2726M60G3pT41NANo
      CIS, carcinoma in situ; F, female; ID, identification; M, male; MU, smooth muscle sample; NA, not available; pM, distant metastasis status; pN, regional lymph node status; pT, primary tumor classification.
      Figure thumbnail gr1
      Figure 1Hematoxylin and eosin–stained tissue section of an exemplary cryopreserved carcinoma in situ (CIS) sample used for laser-capture microdissection. A: Overview of a specimen with maintained luminal urothelium, edematous stroma, and lymphofollicular infiltrates. No invasive tumor is seen in proximity, and detrusor muscle is present. The boxed area is shown at higher magnification in B. B: CIS of the urothelium, best maintained in a fold but also in adjacent flat urothelium. Accompanying mild chronic edematous cystitis in the adjacent stroma. Scale bars: 5 mm (A); 250 μm (B).
      For LCM of CIS (and smooth muscle) cells from the cryoconserved cystectomy specimens, ethanol-fixed, methylene blue–stained tissue sections (10 μm thick) were prepared on nuclease- and nucleic acid–free membrane slides (Zeiss, Oberkochen, Germany) to isolate an area of 6 to 10 × 106 μm2 per case using the PALM MicroBeam IP 230VZ (Zeiss) and adhesive caps (Zeiss). Control hematoxylin and eosin slides were prepared after every 10 sections to ensure the histology on the sample slides. DNA was extracted using the QIAamp DNA Micro kit (Qiagen, Hilden, Germany) and was quantified by a Qubit fluorometric assay (Thermo Fisher Scientific, Waltham, MA). To assess the quality of isolated DNA, a real-time quantitative PCR–based method using the TruSeq FFPE DNA Library Prep QC kit (Illumina, San Diego, CA) was applied.

      Library Preparation and Targeted NGS

      Genomic target regions for NGS analysis were selected as follows: to identify common GAs (mutations and CNVs) potentially inherent in CIS, publicly available sequencing data for MIBC of nonpapillary origin of The Cancer Genome Atlas network,
      TCGA Research Network
      Comprehensive molecular characterization of urothelial bladder carcinoma.
      accessible via the cBio cancer genomics portal version 3.1.0,
      • Cerami E.
      • Gao J.
      • Dogrusoz U.
      • Gross B.E.
      • Sumer S.O.
      • Aksoy B.A.
      • Jacobsen A.
      • Byrne C.J.
      • Heuer M.L.
      • Larsson E.
      • Antipin Y.
      • Reva B.
      • Goldberg A.P.
      • Sander C.
      • Schultz N.
      The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
      were used. Of these data, potentially druggable GAs in genes impacting the selection of targeted anticancer therapies on the market or that are in registered clinical trials were further studied (Supplemental Table S2). Libraries were generated applying the TruSeq Costum Amplicon Low Input kit (Illumina), according to the manufacturer's instructions. In brief, at least 10 ng of high-quality DNA and 20 ng of medium-quality DNA (as determined by TruSeq FFPE DNA Library Prep QC kit) were used for hybridizing the custom target gene oligonucleotide pools. To improve the validity of the sequencing data, a dual-strand sequencing protocol making use of two sets of custom target gene oligonucleotide pools (named CATA and CATB) was applied, enabling sequence analysis of both complementary DNA strands. Subsequent to hybridization and an extension-ligation step, target regions were amplified using unique index adapter combinations (Illumina). After cleanup, library quality control was assessed by bioanalyzer analysis (Agilent Technologies, Santa Clara, CA). Finally, normalized libraries were sequenced on a NextSeq 500 platform (Illumina) using a NextSeq 500/550 Mid Output kit (2 × 150 cycles; Illumina). For single-nucleotide variant analysis, bam-file generation was performed with the DNA amplicon module version 2.0.0 (Basespace; Illumina). Single-nucleotide variant analysis was conducted using Sequence Pilot Software version 4.4 (SeqNext module; JSI Medical Systems, Ettenheim, Germany). For all analyzed samples, region of interest coverage was >96% at 300×. Missense and silent variants with an allele frequency >2% in the normal population [according to 1000 Genomes (http://www.internationalgenome.org, last accessed September 2, 2019) or dbSNP version 153 (https://www.ncbi.nlm.nih.gov/snp, last accessed September 27, 2019)] and non-splicing–relevant silent and intronic variants in general were considered nonpathogenic. In addition, all untranslated region and missense variants annotated as benign or likely benign in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar, last accessed September 27, 2019) were also excluded. The TERT gene promoter was checked for the two known recurrent mutations: c.-124C>T and c.-146C>T. The cutoff for the variant allele frequency was set to 5%. As reference genome for panel design, hg19 was used, whereas hg38 served as reference genome for mutation annotation. For CNV analysis, a recently developed in-house algorithm (validated using three NGS panels; n = 144 samples), based on an exponential growth model for amplification of PCR products, was applied. For every gene analyzed for CNVs, at least 10 amplicons were included in the panel design. Coverage data were normalized; and amplicon clustering, based on the PCR efficiency among all samples, was performed (for each amplicon). Genes were considered harboring a CNV if at least 30% of the amplicons of a gene were recognized as outliers in most of the (five) response models built within a cluster. The underlying reference genome for the panel design was hg19.
      The raw sequencing data of this study are deposited in the National Center for Biotechnology Information Sequence Read Archive version 2.15.0 (https://www.ncbi.nlm.nih.gov/sra; accession number PRJNA551281).

      Fluorescence in Situ Hybridization

      To validate NGS-based detection of MDM2 gene amplification, fluorescence in situ hybridization was performed on corresponding formalin-fixed, paraffin-embedded tissue samples using the ZytoLight SPEC MDM2/CEN 12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany). The test is based on the use of two fluorescently labeled oligonucleotide probes: a MDM2-specific probe (green fluorochrome ZyGreen) and a probe targeting the centromeric region of chromosome 12 (orange fluorochrome ZyOrange) on which the MDM2 locus is located. The test was performed according to the manufacturer's instructions. In brief, deparaffinized and rehydrated tissue sections were immersed in pretreatment buffer for 20 minutes at 98°C, followed by enzymatic pepsin digestion for 10 minutes at 37°C. Subsequently, the dual-color probe was applied to the tissue sections, followed by DNA denaturation at 75°C and a hybridization step at 37°C overnight. After stringent washing and DAPI counterstaining, hybridized probes were visualized by fluorescence microscopy (Axiovert S135 microscope; Zeiss). A minimum of 50 nuclei per case was analyzed, and the mean ratio of green (MDM2) over orange (CEN 12) hybridization signals was calculated. A case showing a ratio of >2.0 was considered MDM2-amplified.

      Results

      The NGS tissue cohort comprised 25 fresh-frozen urothelial CIS samples from 25 cystectomy specimens of 5 female and 20 male patients with a mean age of 69 years (range, 43 to 82 years). For control purposes, five normal smooth muscle samples were also included in the cohort. Tumor multifocality was observed in 96% (24/25) of cystectomies, and 80% (20/25) of patients exhibited muscle-invasive disease. Of the patients, 80% (20/25) did not receive pretreatment before cystectomy (Table 1 and Supplemental Table S1).
      Mutations and CNVs in 31 genes (Supplemental Table S2) were analyzed. In total, 108 GAs (mutations and CNVs), including multiple aberrations per gene, were identified in 25 LCM-isolated CIS samples, with an average of 4.3 alterations per sample (range, 0 to 13) (Supplemental Tables S3 and S4). Affected genes per CIS case are summarized in Figure 2. With 52% (13/25) of affected cases, TERT gene promoter mutations were the most frequent GAs detected (Figure 2). Of CIS specimens, 72% (18/25) harbored at least one GA in genes of the TP53/cell cycle pathway (TP53, MDM2, CCND1, CDKN1A, CDKN2A, CCNE1, and FBXW7). Genes encoding chromatin-modifying proteins (ARID1A, KDM6A, CREBBP, and EP300) were impacted with a similar frequency of 68% (17/25) of cases. Moreover, DNA damage response genes (BRCA2, ATM, BRCA1, and ERCC2) exhibited variants in 60% (15/25) and phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes (ERBB2, FGFR1, PIK3CA, EGFR, RAF1, and NF1) exhibited variants in 36% (9/25) of CIS patients. The validity of the CNV data was approved in fluorescence in situ hybridization experiments exemplarily performed for MDM2 (Supplemental Figure S1).
      Figure thumbnail gr2
      Figure 2Mutations and copy number variations identified in a cohort of 25 laser-capture microdissected, fresh-frozen urothelial carcinoma in situ (CIS) samples. Analyzed genes without detected alterations are not shown.
      Of all GAs (mutations and CNVs), 86% (93/108) were considered potentially actionable, including variants of yet unknown significance in genes with predictive potential (Supplemental Tables S3 and S4). On average, 3.7 potentially actionable GAs were detected per patient (range, 0 to 11); and 92% (23/25) of CIS cases harbored at least one GA in genes potentially impacting the selection of targeted therapies. Frequently altered genes found in CIS that were considered potentially actionable and potential related therapeutic options are summarized in Figure 3 and are listed in detail in Supplemental Table S5.
      • Liu Y.
      • Kwiatkowski D.J.
      Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target.
      • Moser R.
      • Xu C.
      • Kao M.
      • Annis J.
      • Lerma L.A.
      • Schaupp C.M.
      • Gurley K.E.
      • Jang I.S.
      • Biktasova A.
      • Yarbrough W.G.
      • Margolin A.A.
      • Grandori C.
      • Kemp C.J.
      • Méndez E.
      Functional kinomics identifies candidate therapeutic targets in head and neck cancer.
      • Shen J.
      • Peng Y.
      • Wei L.
      • Zhang W.
      • Yang L.
      • Lan L.
      • Kapoor P.
      • Ju Z.
      • Mo Q.
      • Shih I.-M.
      • Uray I.P.
      • Wu X.
      • Brown P.H.
      • Shen X.
      • Mills G.B.
      • Peng G.
      ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors.
      • Williamson C.T.
      • Miller R.
      • Pemberton H.N.
      • Jones S.E.
      • Campbell J.
      • Konde A.
      • Badham N.
      • Rafiq R.
      • Brough R.
      • Gulati A.
      • Ryan C.J.
      • Francis J.
      • Vermulen P.B.
      • Reynolds A.R.
      • Reaper P.M.
      • Pollard J.R.
      • Ashworth A.
      • Lord C.J.
      ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.
      • Samartzis E.P.
      • Gutsche K.
      • Dedes K.J.
      • Fink D.
      • Stucki M.
      • Imesch P.
      Loss of ARID1A expression sensitizes cancer cells to PI3K- and AKT-inhibition.
      • Shen J.
      • Ju Z.
      • Zhao W.
      • Wang L.
      • Peng Y.
      • Ge Z.
      • Nagel Z.D.
      • Zou J.
      • Wang C.
      • Kapoor P.
      • Ma X.
      • Ma D.
      • Liang J.
      • Song S.
      • Liu J.
      • Samson L.D.
      • Ajani J.A.
      • Li G.-M.
      • Liang H.
      • Shen X.
      • Mills G.B.
      • Peng G.
      ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade.
      • Bitler B.G.
      • Aird K.M.
      • Garipov A.
      • Li H.
      • Amatangelo M.
      • Kossenkov A.V.
      • Schultz D.C.
      • Liu Q.
      • Shih I.-M.
      • Conejo-Garcia J.R.
      • Speicher D.W.
      • Zhang R.
      Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.
      • Farmer H.
      • McCabe N.
      • Lord C.J.
      • Tutt A.N.J.
      • Johnson D.A.
      • Richardson T.B.
      • Santarosa M.
      • Dillon K.J.
      • Hickson I.
      • Knights C.
      • Martin N.M.B.
      • Jackson S.P.
      • Smith G.C.M.
      • Ashworth A.
      Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.
      • Teo M.Y.
      • Bambury R.M.
      • Zabor E.C.
      • Jordan E.
      • Al-Ahmadie H.
      • Boyd M.E.
      • Bouvier N.
      • Mullane S.A.
      • Cha E.K.
      • Roper N.
      • Ostrovnaya I.
      • Hyman D.M.
      • Bochner B.H.
      • Arcila M.E.
      • Solit D.B.
      • Berger M.F.
      • Bajorin D.F.
      • Bellmunt J.
      • Iyer G.
      • Rosenberg J.E.
      DNA damage response and repair gene alterations are associated with improved survival in patients with platinum-treated advanced urothelial carcinoma.
      • McCabe N.
      • Turner N.C.
      • Lord C.J.
      • Kluzek K.
      • Bialkowska A.
      • Swift S.
      • Giavara S.
      • O'Connor M.J.
      • Tutt A.N.
      • Zdzienicka M.Z.
      • Smith G.C.M.
      • Ashworth A.
      Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.
      • Liu Q.
      • Gheorghiu L.
      • Drumm M.
      • Clayman R.
      • Eidelman A.
      • Wszolek M.F.
      • Olumi A.
      • Feldman A.
      • Wang M.
      • Marcar L.
      • Citrin D.E.
      • Wu C.-L.
      • Benes C.H.
      • Efstathiou J.A.
      • Willers H.
      PARP-1 inhibition with or without ionizing radiation confers reactive oxygen species-mediated cytotoxicity preferentially to cancer cells with mutant TP53.
      • Ler L.D.
      • Ghosh S.
      • Chai X.
      • Thike A.A.
      • Heng H.L.
      • Siew E.Y.
      • Dey S.
      • Koh L.K.
      • Lim J.Q.
      • Lim W.K.
      • Myint S.S.
      • Loh J.L.
      • Ong P.
      • Sam X.X.
      • Huang D.
      • Lim T.
      • Tan P.H.
      • Nagarajan S.
      • Cheng C.W.S.
      • Ho H.
      • Ng L.G.
      • Yuen J.
      • Lin P.-H.
      • Chuang C.-K.
      • Chang Y.-H.
      • Weng W.-H.
      • Rozen S.G.
      • Tan P.
      • Creasy C.L.
      • Pang S.-T.
      • McCabe M.T.
      • Poon S.L.
      • Teh B.T.
      Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2.
      • Andersen C.
      • Asmar F.
      • Klausen T.
      • Hasselbalch H.
      • Gronbaek K.
      Somatic mutations of the CREBBP and EP300 genes affect response to histone deacetylase inhibition in malignant DLBCL clones.
      • Grivas P.
      • Mortazavi A.
      • Picus J.
      • Hahn N.M.
      • Milowsky M.I.
      • Hart L.L.
      • Alva A.
      • Bellmunt J.
      • Pal S.K.
      • Bambury R.M.
      • O'Donnell P.H.
      • Gupta S.
      • Guancial E.A.
      • Sonpavde G.P.
      • Faltaos D.
      • Potvin D.
      • Christensen J.G.
      • Chao R.C.
      • Rosenberg J.E.
      Mocetinostat for patients with previously treated, locally advanced/metastatic urothelial carcinoma and inactivating alterations of acetyltransferase genes.
      • Ogiwara H.
      • Sasaki M.
      • Mitachi T.
      • Oike T.
      • Higuchi S.
      • Tominaga Y.
      • Kohno T.
      Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality via apoptotic cell death due to abrogation of MYC expression.
      • Barth I.
      • Schneider U.
      • Grimm T.
      • Karl A.
      • Horst D.
      • Gaisa N.T.
      • Knüchel R.
      • Garczyk S.
      Progression of urothelial carcinoma in situ of the urinary bladder: a switch from luminal to basal phenotype and related therapeutic implications.
      • Hayashi T.
      • Seiler R.
      • Oo H.Z.
      • Jäger W.
      • Moskalev I.
      • Awrey S.
      • Dejima T.
      • Todenhöfer T.
      • Li N.
      • Fazli L.
      • Matsubara A.
      • Black P.C.
      Targeting HER2 with T-DM1, an antibody cytotoxic drug conjugate, is effective in HER2 over expressing bladder cancer.
      • Her N.-G.
      • Oh J.-W.
      • Oh Y.J.
      • Han S.
      • Cho H.J.
      • Lee Y.
      • Ryu G.H.
      • Nam D.-H.
      Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells.
      • Chen L.
      • Pastorino F.
      • Berry P.
      • Bonner J.
      • Kirk C.
      • Wood K.M.
      • Thomas H.D.
      • Zhao Y.
      • Daga A.
      • Veal G.J.
      • Lunec J.
      • Newell D.R.
      • Ponzoni M.
      • Tweddle D.A.
      Preclinical evaluation of the first intravenous small molecule MDM2 antagonist alone and in combination with temozolomide in neuroblastoma.
      • Canon J.
      • Osgood T.
      • Olson S.H.
      • Saiki A.Y.
      • Robertson R.
      • Yu D.
      • Eksterowicz J.
      • Ye Q.
      • Jin L.
      • Chen A.
      • Zhou J.
      • Cordover D.
      • Kaufman S.
      • Kendall R.
      • Oliner J.D.
      • Coxon A.
      • Radinsky R.
      The MDM2 inhibitor AMG 232 demonstrates robust antitumor efficacy and potentiates the activity of p53-inducing cytotoxic agents.
      • Gong X.
      • Litchfield L.M.
      • Webster Y.
      • Chio L.-C.
      • Wong S.S.
      • Stewart T.R.
      • Dowless M.
      • Dempsey J.
      • Zeng Y.
      • Torres R.
      • Boehnke K.
      • Mur C.
      • Marugán C.
      • Baquero C.
      • Yu C.
      • Bray S.M.
      • Wulur I.H.
      • Bi C.
      • Chu S.
      • Qian H.-R.
      • Iversen P.W.
      • Merzoug F.F.
      • Ye X.S.
      • Reinhard C.
      • De Dios A.
      • Du J.
      • Caldwell C.W.
      • Lallena M.J.
      • Beckmann R.P.
      • Buchanan S.G.
      Genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4 and CDK6 inhibitor abemaciclib.
      • Pan Q.
      • Sathe A.
      • Black P.C.
      • Goebell P.J.
      • Kamat A.M.
      • Schmitz-Draeger B.
      • Nawroth R.
      CDK4/6 inhibitors in cancer therapy: a novel treatement strategy for bladder cancer.
      • Rubio C.
      • Martínez-Fernández M.
      • Segovia C.
      • Lodewijk I.
      • Suarez-Cabrera C.
      • Segrelles C.
      • López-Calderón F.
      • Munera-Maravilla E.
      • Santos M.
      • Bernardini A.
      • García-Escudero R.
      • Lorz C.
      • Gómez-Rodriguez M.J.
      • de Velasco G.
      • Otero I.
      • Villacampa F.
      • Guerrero-Ramos F.
      • Ruiz S.
      • de la Rosa F.
      • Domínguez-Rodríguez S.
      • Real F.X.
      • Malats N.
      • Castellano D.
      • Dueñas M.
      • Paramio J.M.
      CDK4/6 inhibitor as a novel therapeutic approach for advanced bladder cancer independently of RB1 status.
      • Zeng S.-X.
      • Zhu Y.
      • Ma A.-H.
      • Yu W.
      • Zhang H.
      • Lin T.-Y.
      • Shi W.
      • Tepper C.G.
      • Henderson P.T.
      • Airhart S.
      • Guo J.-M.
      • Xu C.-L.
      • deVere White R.W.
      • Pan C.-X.
      The phosphatidylinositol 3-kinase pathway as a potential therapeutic target in bladder cancer.
      • Ross R.L.
      • McPherson H.R.
      • Kettlewell L.
      • Shnyder S.D.
      • Hurst C.D.
      • Alder O.
      • Knowles M.A.
      PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.
      • Tadesse S.
      • Caldon E.C.
      • Tilley W.
      • Wang S.
      Cyclin-dependent kinase 2 inhibitors in cancer therapy: an update.
      • Karkera J.D.
      • Cardona G.M.
      • Bell K.
      • Gaffney D.
      • Portale J.C.
      • Santiago-Walker A.
      • Moy C.H.
      • King P.
      • Sharp M.
      • Bahleda R.
      • Luo F.R.
      • Alvarez J.D.
      • Lorenzi M.V.
      • Platero S.J.
      Oncogenic characterization and pharmacologic sensitivity of activating fibroblast growth factor receptor (FGFR) genetic alterations to the selective FGFR inhibitor erdafitinib.
      • Grünewald S.
      • Politz O.
      • Bender S.
      • Héroult M.
      • Lustig K.
      • Thuss U.
      • Kneip C.
      • Kopitz C.
      • Zopf D.
      • Collin M.P.
      • Boemer U.
      • Ince S.
      • Ellinghaus P.
      • Mumberg D.
      • Hess-Stumpp H.
      • Ziegelbauer K.
      Rogaratinib: a potent and selective pan-FGFR inhibitor with broad antitumor activity in FGFR-overexpressing preclinical cancer models.
      • Nogova L.
      • Sequist L.V.
      • Perez Garcia J.M.
      • Andre F.
      • Delord J.-P.
      • Hidalgo M.
      • Schellens J.H.M.
      • Cassier P.A.
      • Camidge D.R.
      • Schuler M.
      • Vaishampayan U.
      • Burris H.
      • Tian G.G.
      • Campone M.
      • Wainberg Z.A.
      • Lim W.-T.
      • LoRusso P.
      • Shapiro G.I.
      • Parker K.
      • Chen X.
      • Choudhury S.
      • Ringeisen F.
      • Graus-Porta D.
      • Porter D.
      • Isaacs R.
      • Buettner R.
      • Wolf J.
      Evaluation of BGJ398, a fibroblast growth factor receptor 1-3 kinase inhibitor, in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors: results of a global phase I, dose-escalation and dose-expansion study.
      • Cocco E.
      • Lopez S.
      • Black J.
      • Bellone S.
      • Bonazzoli E.
      • Predolini F.
      • Ferrari F.
      • Schwab C.L.
      • Menderes G.
      • Zammataro L.
      • Buza N.
      • Hui P.
      • Wong S.
      • Zhao S.
      • Bai Y.
      • Rimm D.L.
      • Ratner E.
      • Litkouhi B.
      • Silasi D.-A.
      • Azodi M.
      • Schwartz P.E.
      • Santin A.D.
      Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.
      • Konecny G.E.
      • Winterhoff B.
      • Kolarova T.
      • Qi J.
      • Manivong K.
      • Dering J.
      • Yang G.
      • Chalukya M.
      • Wang H.-J.
      • Anderson L.
      • Kalli K.R.
      • Finn R.S.
      • Ginther C.
      • Jones S.
      • Velculescu V.E.
      • Riehle D.
      • Cliby W.A.
      • Randolph S.
      • Koehler M.
      • Hartmann L.C.
      • Slamon D.J.
      Expression of p16 and retinoblastoma determines response to CDK4/6 inhibition in ovarian cancer.
      • Sathe A.
      • Koshy N.
      • Schmid S.C.
      • Thalgott M.
      • Schwarzenböck S.M.
      • Krause B.J.
      • Holm P.S.
      • Gschwend J.E.
      • Retz M.
      • Nawroth R.
      CDK4/6 inhibition controls proliferation of bladder cancer and transcription of RB1.
      • Rose T.L.
      • Chism D.D.
      • Alva A.S.
      • Deal A.M.
      • Maygarden S.J.
      • Whang Y.E.
      • Kardos J.
      • Drier A.
      • Basch E.
      • Godley P.A.
      • Dunn M.W.
      • Kim W.Y.
      • Milowsky M.I.
      Phase II trial of palbociclib in patients with metastatic urothelial cancer after failure of first-line chemotherapy.
      • Rebouissou S.
      • Bernard-Pierrot I.
      • de Reyniès A.
      • Lepage M.-L.
      • Krucker C.
      • Chapeaublanc E.
      • Hérault A.
      • Kamoun A.
      • Caillault A.
      • Letouzé E.
      • Elarouci N.
      • Neuzillet Y.
      • Denoux Y.
      • Molinié V.
      • Vordos D.
      • Laplanche A.
      • Maillé P.
      • Soyeux P.
      • Ofualuka K.
      • Reyal F.
      • Biton A.
      • Sibony M.
      • Paoletti X.
      • Southgate J.
      • Benhamou S.
      • Lebret T.
      • Allory Y.
      • Radvanyi F.
      EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype.
      • Siddiqui M.R.
      • Railkar R.
      • Sanford T.
      • Crooks D.R.
      • Eckhaus M.A.
      • Haines D.
      • Choyke P.L.
      • Kobayashi H.
      • Agarwal P.K.
      Targeting epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) expressing bladder cancer using combination photoimmunotherapy (PIT).
      • Powles T.
      • Huddart R.A.
      • Elliott T.
      • Sarker S.-J.
      • Ackerman C.
      • Jones R.
      • Hussain S.
      • Crabb S.
      • Jagdev S.
      • Chester J.
      • Hilman S.
      • Beresford M.
      • Macdonald G.
      • Santhanam S.
      • Frew J.A.
      • Stockdale A.
      • Hughes S.
      • Berney D.
      • Chowdhury S.
      Phase III, double-blind, randomized trial that compared maintenance lapatinib versus placebo after first-line chemotherapy in patients with human epidermal growth factor receptor 1/2–positive metastatic bladder cancer.
      • Autenrieth M.E.
      • Seidl C.
      • Bruchertseifer F.
      • Horn T.
      • Kurtz F.
      • Feuerecker B.
      • D'Alessandria C.
      • Pfob C.
      • Nekolla S.
      • Apostolidis C.
      • Mirzadeh S.
      • Gschwend J.E.
      • Schwaiger M.
      • Scheidhauer K.
      • Morgenstern A.
      Treatment of carcinoma in situ of the urinary bladder with an alpha-emitter immunoconjugate targeting the epidermal growth factor receptor: a pilot study.
      • Mao J.-H.
      • Kim I.-J.
      • Wu D.
      • Climent J.
      • Kang H.C.
      • DelRosario R.
      • Balmain A.
      FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression.
      • Caenepeel S.
      • Brown S.P.
      • Belmontes B.
      • Moody G.
      • Keegan K.S.
      • Chui D.
      • et al.
      AMG 176, a selective MCL1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapies.
      • Kotschy A.
      • Szlavik Z.
      • Murray J.
      • Davidson J.
      • Maragno A.L.
      • Le Toumelin-Braizat G.
      • et al.
      The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.
      • Knievel J.
      • Schulz W.
      • Greife A.
      • Hader C.
      • Lübke T.
      • Schmitz I.
      • Albers P.
      • Niegisch G.
      Multiple mechanisms mediate resistance to sorafenib in urothelial cancer.
      • Necchi A.
      • Lo Vullo S.
      • Raggi D.
      • Perrone F.
      • Giannatempo P.
      • Calareso G.
      • Togliardi E.
      • Nicolai N.
      • Piva L.
      • Biasoni D.
      • Catanzaro M.
      • Torelli T.
      • Stagni S.
      • Colecchia M.
      • Busico A.
      • Pennati M.
      • Zaffaroni N.
      • Mariani L.
      • Salvioni R.
      Neoadjuvant sorafenib, gemcitabine, and cisplatin administration preceding cystectomy in patients with muscle-invasive urothelial bladder carcinoma: an open-label, single-arm, single-center, phase 2 study.
      • Johannessen C.M.
      • Reczek E.E.
      • James M.F.
      • Brems H.
      • Legius E.
      • Cichowski K.
      The NF1 tumor suppressor critically regulates TSC2 and mTOR.
      • Marjon K.
      • Cameron M.J.
      • Quang P.
      • Clasquin M.F.
      • Mandley E.
      • Kunii K.
      • McVay M.
      • Choe S.
      • Kernytsky A.
      • Gross S.
      • Konteatis Z.
      • Murtie J.
      • Blake M.L.
      • Travins J.
      • Dorsch M.
      • Biller S.A.
      • Marks K.M.
      MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis.
      Figure thumbnail gr3
      Figure 3Potentially actionable genomic carcinoma in situ (CIS) alterations and suggested targeted drugs. Top: Impacted pathways and frequency of CIS cases harboring at least one potentially actionable genomic alteration in the respective pathway. The three most frequently affected genes of each pathway are depicted. Bottom: Potential targeted therapies. CDK4/6i, cyclin dependent kinase 4/6 inhibitor; CHEKi, checkpoint kinase inhibitor; CREBBPi, CREB binding protein inhibitor; DDR, DNA damage response; EP300, E1A binding protein p300; ERBB2, erb-b2 receptor tyrosine kinase 2; EZH2i, enhancer of zeste 2 polycomb repressive complex 2 subunit inhibitor; FGFRi, fibroblast growth factor receptor inhibitor; MAPK, mitogen-activated protein kinase; MDM2i, MDM2 proto-oncogene inhibitor; PARPi, poly (ADP-ribose) polymerase inhibitor; PI3K, phosphatidylinositol 3-kinase; PI3Ki, phosphatidylinositol 3-kinase inhibitor.

      Discussion

      In contrast to MIBC, the molecular characterization of NMIBC lacks behind.
      • Rentsch C.A.
      • Müller D.C.
      • Ruiz C.
      • Bubendorf L.
      Comprehensive molecular characterization of urothelial bladder carcinoma: a step closer to clinical translation?.
      ,
      • Sjödahl G.
      • Jackson C.L.
      • Bartlett J.M.S.
      • Siemens D.R.
      • Berman D.M.
      Molecular profiling in muscle-invasive bladder cancer: more than the sum of its parts.
      Although the number of studies addressing this lack of knowledge is increasing,
      • Hedegaard J.
      • Lamy P.
      • Nordentoft I.
      • Algaba F.
      • Høyer S.
      • Ulhøi B.P.
      • et al.
      Comprehensive transcriptional analysis of early-stage urothelial carcinoma.
      ,
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      ,
      • Hurst C.D.
      • Alder O.
      • Platt F.M.
      • Droop A.
      • Stead L.F.
      • Burns J.E.
      • Burghel G.J.
      • Jain S.
      • Klimczak L.J.
      • Lindsay H.
      • Roulson J.-A.
      • Taylor C.F.
      • Thygesen H.
      • Cameron A.J.
      • Ridley A.J.
      • Mott H.R.
      • Gordenin D.A.
      • Knowles M.A.
      Genomic subtypes of non-invasive bladder cancer with distinct metabolic profile and female gender bias in KDM6A mutation frequency.
      • Scott S.N.
      • Ostrovnaya I.
      • Lin C.M.
      • Bouvier N.
      • Bochner B.H.
      • Iyer G.
      • Solit D.
      • Berger M.F.
      • Lin O.
      Next-generation sequencing of urine specimens: a novel platform for genomic analysis in patients with non-muscle-invasive urothelial carcinoma treated with bacille Calmette-Guérin.
      • Nassar A.H.
      • Umeton R.
      • Kim J.
      • Lundgren K.
      • Harshman L.
      • Van Allen E.M.
      • Preston M.
      • Dong F.
      • Bellmunt J.
      • Mouw K.W.
      • Choueiri T.K.
      • Sonpavde G.
      • Kwiatkowski D.J.
      Mutational analysis of 472 urothelial carcinoma across grades and anatomic sites.
      most work studying NMIBC by NGS spared aggressive CIS lesions, probably because of difficulties related to limited availability of tissue material.
      • Rentsch C.A.
      • Müller D.C.
      • Ruiz C.
      • Bubendorf L.
      Comprehensive molecular characterization of urothelial bladder carcinoma: a step closer to clinical translation?.
      ,
      • Hurst C.D.
      • Knowles M.A.
      Mutational landscape of non-muscle-invasive bladder cancer.
      To the best of our knowledge, the present work studied the currently largest cohort of urothelial CIS samples of the urinary bladder using an NGS-based approach to identify GAs with the potential to guide the investigation of targeted therapies in future clinical trials.
      With a frequency of 52%, TERT gene promoter mutations were the most common genetic alterations in our CIS cohort; that is comparable to mutational rates in CIS observed in earlier studies.
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      ,
      • Kinde I.
      • Munari E.
      • Faraj S.F.
      • Hruban R.H.
      • Schoenberg M.
      • Bivalacqua T.
      • Allaf M.
      • Springer S.
      • Wang Y.
      • Diaz L.A.
      • Kinzler K.W.
      • Vogelstein B.
      • Papadopoulos N.
      • Netto G.J.
      TERT promoter mutations occur early in urothelial neoplasia and are biomarkers of early disease and disease recurrence in urine.
      TERT promoter mutations detected in the present study were confined to two hot spot positions recently described in bladder cancer
      • Hurst C.D.
      • Platt F.M.
      • Knowles M.A.
      Comprehensive mutation analysis of the TERT promoter in bladder cancer and detection of mutations in voided urine.
      (namely, to the positions -124 and -146 bp upstream of the ATG translation start codon in 77% and 23% of TERT-mutated cases, respectively).
      In accordance with the proposed model of a distinct development of papillary and flat urothelial lesions,
      • Knowles M.A.
      • Hurst C.D.
      Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.
      CIS cases in our study were characterized by frequent TP53 mutations (44%), but complete absence of activating FGFR3 alterations. Deletions in the CDKN2A gene locus were detected at obviously lower frequency in our cohort (4%) compared with MIBC cases of nonpapillary origin (31%),
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      which is in accordance with observations made in two recent NMIBC studies.
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      ,
      • Scott S.N.
      • Ostrovnaya I.
      • Lin C.M.
      • Bouvier N.
      • Bochner B.H.
      • Iyer G.
      • Solit D.
      • Berger M.F.
      • Lin O.
      Next-generation sequencing of urine specimens: a novel platform for genomic analysis in patients with non-muscle-invasive urothelial carcinoma treated with bacille Calmette-Guérin.
      The observed discrepancy might be attributable to a lower sensitivity of our amplicon-based CNV detection method compared with the array-based technology applied in the MIBC The Cancer Genome Atlas study
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      or might be related to CIS biology.
      In addition to frequent aberrations in the TP53/cell cycle pathway (72%), genes encoding chromatin-modifying proteins (ARID1A, KDM6A, CREBBP, and EP300) were found to be altered at a similar rate (68%), followed by aberrations in DNA damage repair–associated genes (BRCA2, ATM, BRCA1, and ERCC2) in 60% of the analyzed CIS samples. A high prevalence of alterations in chromatin-modifying and DNA damage pathway genes in high-grade NMIBC
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      and MIBC
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      has been noted before.
      More important, 92% of CIS patients harbored at least one GA potentially predicting response to targeted therapy. Of these, TP53 was the most frequently mutated gene analyzed in this study. Although p53 itself is not a therapeutic target, recent data suggest that TP53-mutated cancer cells, showing a defective G1 checkpoint, rely on an intact G2-M cell cycle checkpoint to ensure cell cycle arrest and repair in case of DNA damage.
      • Moser R.
      • Xu C.
      • Kao M.
      • Annis J.
      • Lerma L.A.
      • Schaupp C.M.
      • Gurley K.E.
      • Jang I.S.
      • Biktasova A.
      • Yarbrough W.G.
      • Margolin A.A.
      • Grandori C.
      • Kemp C.J.
      • Méndez E.
      Functional kinomics identifies candidate therapeutic targets in head and neck cancer.
      Inhibition of either of the two kinases WEE1 G2 checkpoint kinase (WEE1) or checkpoint kinase 1 (CHEK1), regulating G2-M checkpoint activation, resulted in synthetic lethality in TP53-mutant cells. More important, combination of a WEE1/CHEK1 inhibitor with a DNA-damaging drug enhanced cancer cell death.
      • Moser R.
      • Xu C.
      • Kao M.
      • Annis J.
      • Lerma L.A.
      • Schaupp C.M.
      • Gurley K.E.
      • Jang I.S.
      • Biktasova A.
      • Yarbrough W.G.
      • Margolin A.A.
      • Grandori C.
      • Kemp C.J.
      • Méndez E.
      Functional kinomics identifies candidate therapeutic targets in head and neck cancer.
      In bladder cancer, a similar reliance on an intact G2-M checkpoint was noted, especially in TP53/CDKN1A double-mutant cells.
      • Liu Y.
      • Kwiatkowski D.J.
      Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target.
      WEE1/CHEK1 inhibitors are currently investigated in clinical trials in solid tumors showing cell cycle/DNA repair defects (including TP53 mutations) as monotherapy or in combination with DNA-damage inducing drugs, such as US Food and Drug Administration–approved poly (ADP-ribose) polymerase (PARP) inhibitors (https://clinicaltrials.gov; NCT02797964 and NCT03330847).
      Alterations in ARID1A have been observed in 36% of CIS cases, comprising a high portion of samples harboring inactivating truncating mutations and gene deletions (78%). AT-rich interaction domain 1A (ARID1A) is a component of switch/sucrose non-fermentable chromatin remodeling complexes, potential tumor suppressors that are mutated in approximately 20% across human cancers.
      • Kadoch C.
      • Hargreaves D.C.
      • Hodges C.
      • Elias L.
      • Ho L.
      • Ranish J.
      • Crabtree G.R.
      Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.
      Inactivation of switch/sucrose non-fermentable complexes, including ARID1A deficiency, has been proposed to be a predictive biomarker for a plethora of targeted therapies in cancer.
      • Shen J.
      • Peng Y.
      • Wei L.
      • Zhang W.
      • Yang L.
      • Lan L.
      • Kapoor P.
      • Ju Z.
      • Mo Q.
      • Shih I.-M.
      • Uray I.P.
      • Wu X.
      • Brown P.H.
      • Shen X.
      • Mills G.B.
      • Peng G.
      ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors.
      • Williamson C.T.
      • Miller R.
      • Pemberton H.N.
      • Jones S.E.
      • Campbell J.
      • Konde A.
      • Badham N.
      • Rafiq R.
      • Brough R.
      • Gulati A.
      • Ryan C.J.
      • Francis J.
      • Vermulen P.B.
      • Reynolds A.R.
      • Reaper P.M.
      • Pollard J.R.
      • Ashworth A.
      • Lord C.J.
      ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.
      • Samartzis E.P.
      • Gutsche K.
      • Dedes K.J.
      • Fink D.
      • Stucki M.
      • Imesch P.
      Loss of ARID1A expression sensitizes cancer cells to PI3K- and AKT-inhibition.
      ,
      • Bitler B.G.
      • Aird K.M.
      • Garipov A.
      • Li H.
      • Amatangelo M.
      • Kossenkov A.V.
      • Schultz D.C.
      • Liu Q.
      • Shih I.-M.
      • Conejo-Garcia J.R.
      • Speicher D.W.
      • Zhang R.
      Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.
      For example, it has been observed that ARID1A loss of function disabled cell cycle checkpoint activation and thus proper DNA repair, sensitizing cells to PARP inhibitors.
      • Shen J.
      • Peng Y.
      • Wei L.
      • Zhang W.
      • Yang L.
      • Lan L.
      • Kapoor P.
      • Ju Z.
      • Mo Q.
      • Shih I.-M.
      • Uray I.P.
      • Wu X.
      • Brown P.H.
      • Shen X.
      • Mills G.B.
      • Peng G.
      ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors.
      Other data suggested that ARID1A deficiency might impair DNA topoisomerase II alpha function, resulting in an accumulation of DNA lesions and thus enhanced reliance on ATR serine/threonine kinase activity for checkpoint activation and repair.
      • Williamson C.T.
      • Miller R.
      • Pemberton H.N.
      • Jones S.E.
      • Campbell J.
      • Konde A.
      • Badham N.
      • Rafiq R.
      • Brough R.
      • Gulati A.
      • Ryan C.J.
      • Francis J.
      • Vermulen P.B.
      • Reynolds A.R.
      • Reaper P.M.
      • Pollard J.R.
      • Ashworth A.
      • Lord C.J.
      ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.
      ,
      • Dykhuizen E.C.
      • Hargreaves D.C.
      • Miller E.L.
      • Cui K.
      • Korshunov A.
      • Kool M.
      • Pfister S.
      • Cho Y.-J.
      • Zhao K.
      • Crabtree G.R.
      BAF complexes facilitate decatenation of DNA by topoisomerase IIα.
      The authors demonstrated that application of ATR serine/threonine kinase inhibitors, currently tested in clinical trials in advanced bladder cancer and other solid tumors (https://clinicaltrials.gov; NCT03682289 and NCT02567409), might be a rational therapy for ARID1A-deficient carcinomas.
      • Williamson C.T.
      • Miller R.
      • Pemberton H.N.
      • Jones S.E.
      • Campbell J.
      • Konde A.
      • Badham N.
      • Rafiq R.
      • Brough R.
      • Gulati A.
      • Ryan C.J.
      • Francis J.
      • Vermulen P.B.
      • Reynolds A.R.
      • Reaper P.M.
      • Pollard J.R.
      • Ashworth A.
      • Lord C.J.
      ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.
      In addition to ARID1A alterations potentially enhancing response to PARP inhibitors, 56% of CIS samples in this study exhibited at least one GA in the DNA damage repair genes BRCA1, BRCA2, and ATM, including pathogenic missense variants, gene deletions, and missense mutations of unknown significance. Inactivation of homologous recombination–associated genes is known to sensitize cancer cells to PARP inhibition.
      • McCabe N.
      • Turner N.C.
      • Lord C.J.
      • Kluzek K.
      • Bialkowska A.
      • Swift S.
      • Giavara S.
      • O'Connor M.J.
      • Tutt A.N.
      • Zdzienicka M.Z.
      • Smith G.C.M.
      • Ashworth A.
      Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.
      The potential benefit of PARP inhibitors is currently studied in patients with advanced bladder cancer (https://clinicaltrials.gov; NCT03375307 and NCT03397394). Moreover, alterations in DNA damage response and repair-related genes, including BRCA1, BRCA2, ATM, and ERCC2 (impacted in 60% of samples analyzed in this study), have been associated with improved response to platinum chemotherapy in advanced urothelial carcinoma.
      • Teo M.Y.
      • Bambury R.M.
      • Zabor E.C.
      • Jordan E.
      • Al-Ahmadie H.
      • Boyd M.E.
      • Bouvier N.
      • Mullane S.A.
      • Cha E.K.
      • Roper N.
      • Ostrovnaya I.
      • Hyman D.M.
      • Bochner B.H.
      • Arcila M.E.
      • Solit D.B.
      • Berger M.F.
      • Bajorin D.F.
      • Bellmunt J.
      • Iyer G.
      • Rosenberg J.E.
      DNA damage response and repair gene alterations are associated with improved survival in patients with platinum-treated advanced urothelial carcinoma.
      Cisplatin-based combination therapies, currently studied in high-risk NMIBC (https://clinicaltrials.gov; NCT02202772), might represent a bladder-preserving treatment option for a subgroup of CIS patients.
      Other genes encoding chromatin-modifying proteins have been found to be frequently altered in our CIS cohort (namely, KDM6A in 24% of cases as well as CREBBP and EP300 in 20% of samples each). Concerning KDM6A, encoding a histone lysine demethylase,
      • Schulz W.A.
      • Lang A.
      • Koch J.
      • Greife A.
      The histone demethylase UTX/KDM6A in cancer: progress and puzzles.
      most altered cases (83%) harbored inactivating truncating mutations, as noted recently in NMIBC.
      • Pietzak E.J.
      • Bagrodia A.
      • Cha E.K.
      • Drill E.N.
      • Iyer G.
      • Isharwal S.
      • Ostrovnaya I.
      • Baez P.
      • Li Q.
      • Berger M.F.
      • Zehir A.
      • Schultz N.
      • Rosenberg J.E.
      • Bajorin D.F.
      • Dalbagni G.
      • Al-Ahmadie H.
      • Solit D.B.
      • Bochner B.H.
      Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
      ,
      • Hurst C.D.
      • Alder O.
      • Platt F.M.
      • Droop A.
      • Stead L.F.
      • Burns J.E.
      • Burghel G.J.
      • Jain S.
      • Klimczak L.J.
      • Lindsay H.
      • Roulson J.-A.
      • Taylor C.F.
      • Thygesen H.
      • Cameron A.J.
      • Ridley A.J.
      • Mott H.R.
      • Gordenin D.A.
      • Knowles M.A.
      Genomic subtypes of non-invasive bladder cancer with distinct metabolic profile and female gender bias in KDM6A mutation frequency.
      More important, preclinical data showed that lysine demethylase 6A deficiency sensitized bladder cancer cells to inhibition of enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), antagonizing lysine demethylase 6A activity
      • Ler L.D.
      • Ghosh S.
      • Chai X.
      • Thike A.A.
      • Heng H.L.
      • Siew E.Y.
      • Dey S.
      • Koh L.K.
      • Lim J.Q.
      • Lim W.K.
      • Myint S.S.
      • Loh J.L.
      • Ong P.
      • Sam X.X.
      • Huang D.
      • Lim T.
      • Tan P.H.
      • Nagarajan S.
      • Cheng C.W.S.
      • Ho H.
      • Ng L.G.
      • Yuen J.
      • Lin P.-H.
      • Chuang C.-K.
      • Chang Y.-H.
      • Weng W.-H.
      • Rozen S.G.
      • Tan P.
      • Creasy C.L.
      • Pang S.-T.
      • McCabe M.T.
      • Poon S.L.
      • Teh B.T.
      Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2.
      ; and a scheduled clinical trial (https://clinicaltrials.gov; NCT03854474) in advanced bladder cancer now aims to study the potential benefit of combined pembrolizumab and tazemetostat (EZH2 inhibitor) therapy. Inactivating truncating mutations or gene deletions in either CREBBP or EP300, encoding two homologous histone acetyltransferases,
      • Attar N.
      • Kurdistani S.K.
      Exploitation of EP300 and CREBBP lysine acetyltransferases by cancer.
      were detected in 20% of CIS cases in this study. Although recent clinical data do not support the use of histone deacetylase inhibitors for treatment of CREBBP/EP300-mutated bladder cancers,
      • Grivas P.
      • Mortazavi A.
      • Picus J.
      • Hahn N.M.
      • Milowsky M.I.
      • Hart L.L.
      • Alva A.
      • Bellmunt J.
      • Pal S.K.
      • Bambury R.M.
      • O'Donnell P.H.
      • Gupta S.
      • Guancial E.A.
      • Sonpavde G.P.
      • Faltaos D.
      • Potvin D.
      • Christensen J.G.
      • Chao R.C.
      • Rosenberg J.E.
      Mocetinostat for patients with previously treated, locally advanced/metastatic urothelial carcinoma and inactivating alterations of acetyltransferase genes.
      a synthetic lethal relationship between E1A binding protein p300 (EP300) and CREB binding protein (CREBBP) has been described in cancer cells
      • Ogiwara H.
      • Sasaki M.
      • Mitachi T.
      • Oike T.
      • Higuchi S.
      • Tominaga Y.
      • Kohno T.
      Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality via apoptotic cell death due to abrogation of MYC expression.
      and might have potential for bladder cancer therapy as EP300/CREBBP bromodomain inhibitors have recently entered clinical trials (https://clinicaltrials.gov; NCT03568656).
      Another important finding of this study is the presence of alterations in known therapeutic drug target genes of the phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway [namely, ERBB2 (20%), FGFR1 and PIK3CA (both 12%), and EGFR and RAF1 (both 4%)]. The ERBB2 locus was altered by amplification (4%) and missense mutations in the extracellular domain (16%), including the pathogenic activating S310F mutation
      • Greulich H.
      • Kaplan B.
      • Mertins P.
      • Chen T.-H.
      • Tanaka K.E.
      • Yun C.-H.
      • Zhang X.
      • Lee S.-H.
      • Cho J.
      • Ambrogio L.
      • Liao R.
      • Imielinski M.
      • Banerji S.
      • Berger A.H.
      • Lawrence M.S.
      • Zhang J.
      • Pho N.H.
      • Walker S.R.
      • Winckler W.
      • Getz G.
      • Frank D.
      • Hahn W.C.
      • Eck M.J.
      • Mani D.R.
      • Jaffe J.D.
      • Carr S.A.
      • Wong K.-K.
      • Meyerson M.
      Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.
      and a variant of unknown significance in two cases each. ERBB2 amplification is a relatively rare event in CIS,
      • Barth I.
      • Schneider U.
      • Grimm T.
      • Karl A.
      • Horst D.
      • Gaisa N.T.
      • Knüchel R.
      • Garczyk S.
      Progression of urothelial carcinoma in situ of the urinary bladder: a switch from luminal to basal phenotype and related therapeutic implications.
      and variants impacting amino acid position S310 (S310F/Y) are the most frequent missense mutations found in ERBB2 in bladder cancer.
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      Although ERBB2-targeted therapies are US Food and Drug Administration–approved for ERBB2-positive breast and gastric cancer,
      • Meric-Bernstam F.
      • Johnson A.M.
      • Dumbrava E.E.I.
      • Raghav K.
      • Balaji K.
      • Bhatt M.
      • Murthy R.K.
      • Rodon J.
      • Piha-Paul S.A.
      Advances in HER2-targeted therapy: novel agents and opportunities beyond breast and gastric cancer.
      results of previous clinical trials in advanced bladder, using drugs inhibiting ERBB2 signaling (trastuzumab and lapatinib), are inconsistent.
      • Oudard S.
      • Culine S.
      • Vano Y.
      • Goldwasser F.
      • Théodore C.
      • Nguyen T.
      • Voog E.
      • Banu E.
      • Vieillefond A.
      • Priou F.
      • Deplanque G.
      • Gravis G.
      • Ravaud A.
      • Vannetzel J.M.
      • Machiels J.-P.
      • Muracciole X.
      • Pichon M.-F.
      • Bay J.-O.
      • Elaidi R.
      • Teghom C.
      • Radvanyi F.
      • Beuzeboc P.
      Multicentre randomised phase II trial of gemcitabine+platinum, with or without trastuzumab, in advanced or metastatic urothelial carcinoma overexpressing Her2.
      • Wülfing C.
      • Machiels J.-P.H.
      • Richel D.J.
      • Grimm M.-O.
      • Treiber U.
      • De Groot M.R.
      • Beuzeboc P.
      • Parikh R.
      • Pétavy F.
      • El-Hariry I.A.
      A single-arm, multicenter, open-label phase 2 study of lapatinib as the second-line treatment of patients with locally advanced or metastatic transitional cell carcinoma.
      • Hainsworth J.D.
      • Meric-Bernstam F.
      • Swanton C.
      • Hurwitz H.
      • Spigel D.R.
      • Sweeney C.
      • Burris H.A.
      • Bose R.
      • Yoo B.
      • Stein A.
      • Beattie M.
      • Kurzrock R.
      Targeted therapy for advanced solid tumors on the basis of molecular profiles: results from MyPathway, an open-label, phase IIa multiple basket study.
      More important, we have recently demonstrated moderate to strong positivity for ERBB2 in 65% of CIS samples and only weak expression in adjacent normal urothelium.
      • Barth I.
      • Schneider U.
      • Grimm T.
      • Karl A.
      • Horst D.
      • Gaisa N.T.
      • Knüchel R.
      • Garczyk S.
      Progression of urothelial carcinoma in situ of the urinary bladder: a switch from luminal to basal phenotype and related therapeutic implications.
      Thus, as the driver status of ERBB2 in bladder cancer is unclear,
      • Kiss B.
      • Wyatt A.W.
      • Douglas J.
      • Skuginna V.
      • Mo F.
      • Anderson S.
      • Rotzer D.
      • Fleischmann A.
      • Genitsch V.
      • Hayashi T.
      • Neuenschwander M.
      • Buerki C.
      • Davicioni E.
      • Collins C.
      • Thalmann G.N.
      • Black P.C.
      • Seiler R.
      Her2 alterations in muscle-invasive bladder cancer: patient selection beyond protein expression for targeted therapy.
      antibody-drug conjugates, such as trastuzumab emtansine (T-DM1), are suggested as a rational therapy for urothelial CIS, supported by preclinical data in bladder cancer.
      • Hayashi T.
      • Seiler R.
      • Oo H.Z.
      • Jäger W.
      • Moskalev I.
      • Awrey S.
      • Dejima T.
      • Todenhöfer T.
      • Li N.
      • Fazli L.
      • Matsubara A.
      • Black P.C.
      Targeting HER2 with T-DM1, an antibody cytotoxic drug conjugate, is effective in HER2 over expressing bladder cancer.
      Clinical trials in late-stage bladder cancer investigating T-DM1 are underway, but results are not available yet (https://clinicaltrials.gov; eg, NCT02999672).
      Although this work comprises the currently largest cohort of urothelial CIS samples analyzed by an NGS-based approach, it is limited to carefully mapped frozen cystectomy samples to find unequivocal and sufficient material for analysis. Larger-scale studies are needed to validate these findings and may partially be possible by targeted analysis of formalin-fixed transurethral resection samples, for which we have an extended data base.
      • Barth I.
      • Schneider U.
      • Grimm T.
      • Karl A.
      • Horst D.
      • Gaisa N.T.
      • Knüchel R.
      • Garczyk S.
      Progression of urothelial carcinoma in situ of the urinary bladder: a switch from luminal to basal phenotype and related therapeutic implications.
      Also, this sequencing study was focused on a specific gene set; and it is meaningful to perform more comprehensive analyses of this entity in upcoming studies. More important, because of our primary motive of finding therapy targets, matched control samples were not analyzed for each CIS case and a certain discrimination of germline and somatic variants was thus not possible.
      In summary, targeted NGS of fresh-frozen urothelial CIS samples identified at least one potentially actionable alteration in most CIS cases. These data should help in guiding the selection of targeted intravesical/systemic therapies that should be investigated in future clinical trials.

      Acknowledgments

      We thank Angela Maurer (University Hospital Rheinisch-Westfälische Technische Hochschule Aachen) for supporting the assessment of the next-generation sequencing data.

      Author Contributions

      S.G. and R.K. conceptualized the study; S.G., N.O.-B., U.S., and K.G. designed the methods; S.G., U.S., and I.L. performed the experiments; N.O.-B., K.G., and S.G. analyzed the results; K.G. and N.O.-B. developed the in-house software for CNV analysis; S.G. wrote the manuscript; and E.L., N.O.-B., R.K., N.T.G., I.L., A.H., K.G., U.S., and K.L.-D. edited the manuscript; N.T.G., K.L.-D., and A.H. provided resources; S.G. and R.K. supervised the study; S.G. acquired funding.

      Supplemental Data

      • Supplemental Figure S1

        Detection of MDM2 gene amplification by fluorescence in situ hybridization. Confirmation of MDM2 gene locus amplification in the cases carcinoma in situ (CIS) 03 (A) and CIS09 (B). No MDM2 amplification is detected in the control samples CIS04 (C) and CIS05 (D). A representative image for each case is depicted. Green signals correspond to the MDM2 locus on chromosome 12, whereas orange signals correspond to the centromeric region of chromosome 12. Original magnification, ×1000 (AD).

      References

        • Ferlay J.
        • Shin H.-R.
        • Bray F.
        • Forman D.
        • Mathers C.
        • Parkin D.M.
        Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.
        Int J Cancer. 2010; 127: 2893-2917
        • Knowles M.A.
        • Hurst C.D.
        Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.
        Nat Rev Cancer. 2015; 15: 25-41
        • Kirkali Z.
        • Chan T.
        • Manoharan M.
        • Algaba F.
        • Busch C.
        • Cheng L.
        • Kiemeney L.
        • Kriegmair M.
        • Montironi R.
        • Murphy W.M.
        • Sesterhenn I.A.
        • Tachibana M.
        • Weider J.
        Bladder cancer: epidemiology, staging and grading, and diagnosis.
        Urology. 2005; 66: 4-34
        • Casey R.G.
        • Catto J.W.F.
        • Cheng L.
        • Cookson M.S.
        • Herr H.
        • Shariat S.
        • Witjes J.A.
        • Black P.C.
        Diagnosis and management of urothelial carcinoma in situ of the lower urinary tract: a systematic review.
        Eur Urol. 2015; 67: 876-888
        • Sylvester R.J.
        • van der Meijden A.
        • Witjes J.A.
        • Jakse G.
        • Nonomura N.
        • Cheng C.
        • Torres A.
        • Watson R.
        • Kurth K.H.
        High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder.
        Urology. 2005; 66: 90-107
        • Babjuk M.
        • Burger M.
        • Zigeuner R.
        • Shariat S.F.
        • van Rhijn B.W.G.
        • Compérat E.
        • Sylvester R.J.
        • Kaasinen E.
        • Böhle A.
        • Palou Redorta J.
        • Rouprêt M.
        EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013.
        Eur Urol. 2013; 64: 639-653
        • Alfred Witjes J.
        • Lebret T.
        • Compérat E.M.
        • Cowan N.C.
        • De Santis M.
        • Bruins H.M.
        • Hernández V.
        • Espinós E.L.
        • Dunn J.
        • Rouanne M.
        • Neuzillet Y.
        • Veskimäe E.
        • van der Heijden A.G.
        • Gakis G.
        • Ribal M.J.
        Updated 2016 EAU guidelines on muscle-invasive and metastatic bladder cancer.
        Eur Urol. 2017; 71: 462-475
        • Bellmunt J.
        • Powles T.
        • Vogelzang N.J.
        A review on the evolution of PD-1/PD-L1 immunotherapy for bladder cancer: the future is now.
        Cancer Treat Rev. 2017; 54: 58-67
        • Tripathi A.
        • Plimack E.R.
        Immunotherapy for urothelial carcinoma: current evidence and future directions.
        Curr Urol Rep. 2018; 19: 109
        • Sharma P.
        • Hu-Lieskovan S.
        • Wargo J.A.
        • Ribas A.
        Primary, adaptive, and acquired resistance to cancer immunotherapy.
        Cell. 2017; 168: 707-723
        • Hall M.C.
        • Chang S.S.
        • Dalbagni G.
        • Pruthi R.S.
        • Seigne J.D.
        • Skinner E.C.
        • Wolf J.S.
        • Schellhammer P.F.
        Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update.
        J Urol. 2007; 178: 2314-2330
        • Tang D.H.
        • Chang S.S.
        Management of carcinoma in situ of the bladder: best practice and recent developments.
        Ther Adv Urol. 2015; 7: 351-364
        • Herr H.W.
        • Sogani P.C.
        Does early cystectomy improve the survival of patients with high risk superficial bladder tumors?.
        J Urol. 2001; 166: 1296-1299
        • Nykopp T.K.
        • Batista da Costa J.
        • Mannas M.
        Black PC: Current clinical trials in non-muscle invasive bladder cancer.
        Curr Urol Rep. 2018; 19: 101
        • TCGA Research Network
        Comprehensive molecular characterization of urothelial bladder carcinoma.
        Nature. 2014; 507: 315-322
        • Ross J.S.
        • Wang K.
        • Al-Rohil R.N.
        • Nazeer T.
        • Sheehan C.E.
        • Otto G.A.
        • He J.
        • Palmer G.
        • Yelensky R.
        • Lipson D.
        • Ali S.
        • Balasubramanian S.
        • Curran J.A.
        • Garcia L.
        • Mahoney K.
        • Downing S.R.
        • Hawryluk M.
        • Miller V.A.
        • Stephens P.J.
        Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy.
        Mod Pathol. 2014; 27: 271-280
        • Kim P.H.
        • Cha E.K.
        • Sfakianos J.P.
        • Iyer G.
        • Zabor E.C.
        • Scott S.N.
        • Ostrovnaya I.
        • Ramirez R.
        • Sun A.
        • Shah R.
        • Yee A.M.
        • Reuter V.E.
        • Bajorin D.F.
        • Rosenberg J.E.
        • Schultz N.
        • Berger M.F.
        • Al-Ahmadie H.A.
        • Solit D.B.
        • Bochner B.H.
        Genomic predictors of survival in patients with high-grade urothelial carcinoma of the bladder.
        Eur Urol. 2015; 67: 198-201
        • Hedegaard J.
        • Lamy P.
        • Nordentoft I.
        • Algaba F.
        • Høyer S.
        • Ulhøi B.P.
        • et al.
        Comprehensive transcriptional analysis of early-stage urothelial carcinoma.
        Cancer Cell. 2016; 30: 27-42
        • Pietzak E.J.
        • Bagrodia A.
        • Cha E.K.
        • Drill E.N.
        • Iyer G.
        • Isharwal S.
        • Ostrovnaya I.
        • Baez P.
        • Li Q.
        • Berger M.F.
        • Zehir A.
        • Schultz N.
        • Rosenberg J.E.
        • Bajorin D.F.
        • Dalbagni G.
        • Al-Ahmadie H.
        • Solit D.B.
        • Bochner B.H.
        Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets.
        Eur Urol. 2017; 72: 952-959
        • Robertson A.G.
        • Kim J.
        • Al-Ahmadie H.
        • Bellmunt J.
        • Guo G.
        • Cherniack A.D.
        • et al.
        Comprehensive molecular characterization of muscle-invasive bladder cancer.
        Cell. 2017; 171: 540-556.e25
        • Rentsch C.A.
        • Müller D.C.
        • Ruiz C.
        • Bubendorf L.
        Comprehensive molecular characterization of urothelial bladder carcinoma: a step closer to clinical translation?.
        Eur Urol. 2017; 72: 960-961
        • Cerami E.
        • Gao J.
        • Dogrusoz U.
        • Gross B.E.
        • Sumer S.O.
        • Aksoy B.A.
        • Jacobsen A.
        • Byrne C.J.
        • Heuer M.L.
        • Larsson E.
        • Antipin Y.
        • Reva B.
        • Goldberg A.P.
        • Sander C.
        • Schultz N.
        The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
        Cancer Discov. 2012; 2: 401-404
        • Liu Y.
        • Kwiatkowski D.J.
        Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target.
        Mol Cancer Ther. 2015; 14: 174-182
        • Moser R.
        • Xu C.
        • Kao M.
        • Annis J.
        • Lerma L.A.
        • Schaupp C.M.
        • Gurley K.E.
        • Jang I.S.
        • Biktasova A.
        • Yarbrough W.G.
        • Margolin A.A.
        • Grandori C.
        • Kemp C.J.
        • Méndez E.
        Functional kinomics identifies candidate therapeutic targets in head and neck cancer.
        Clin Cancer Res. 2014; 20: 4274-4288
        • Shen J.
        • Peng Y.
        • Wei L.
        • Zhang W.
        • Yang L.
        • Lan L.
        • Kapoor P.
        • Ju Z.
        • Mo Q.
        • Shih I.-M.
        • Uray I.P.
        • Wu X.
        • Brown P.H.
        • Shen X.
        • Mills G.B.
        • Peng G.
        ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors.
        Cancer Discov. 2015; 5: 752-767
        • Williamson C.T.
        • Miller R.
        • Pemberton H.N.
        • Jones S.E.
        • Campbell J.
        • Konde A.
        • Badham N.
        • Rafiq R.
        • Brough R.
        • Gulati A.
        • Ryan C.J.
        • Francis J.
        • Vermulen P.B.
        • Reynolds A.R.
        • Reaper P.M.
        • Pollard J.R.
        • Ashworth A.
        • Lord C.J.
        ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.
        Nat Commun. 2016; 7: 13837
        • Samartzis E.P.
        • Gutsche K.
        • Dedes K.J.
        • Fink D.
        • Stucki M.
        • Imesch P.
        Loss of ARID1A expression sensitizes cancer cells to PI3K- and AKT-inhibition.
        Oncotarget. 2014; 5: 5295-5303
        • Shen J.
        • Ju Z.
        • Zhao W.
        • Wang L.
        • Peng Y.
        • Ge Z.
        • Nagel Z.D.
        • Zou J.
        • Wang C.
        • Kapoor P.
        • Ma X.
        • Ma D.
        • Liang J.
        • Song S.
        • Liu J.
        • Samson L.D.
        • Ajani J.A.
        • Li G.-M.
        • Liang H.
        • Shen X.
        • Mills G.B.
        • Peng G.
        ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade.
        Nat Med. 2018; 24: 556-562
        • Bitler B.G.
        • Aird K.M.
        • Garipov A.
        • Li H.
        • Amatangelo M.
        • Kossenkov A.V.
        • Schultz D.C.
        • Liu Q.
        • Shih I.-M.
        • Conejo-Garcia J.R.
        • Speicher D.W.
        • Zhang R.
        Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers.
        Nat Med. 2015; 21: 231-238
        • Farmer H.
        • McCabe N.
        • Lord C.J.
        • Tutt A.N.J.
        • Johnson D.A.
        • Richardson T.B.
        • Santarosa M.
        • Dillon K.J.
        • Hickson I.
        • Knights C.
        • Martin N.M.B.
        • Jackson S.P.
        • Smith G.C.M.
        • Ashworth A.
        Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.
        Nature. 2005; 434: 917-921
        • Teo M.Y.
        • Bambury R.M.
        • Zabor E.C.
        • Jordan E.
        • Al-Ahmadie H.
        • Boyd M.E.
        • Bouvier N.
        • Mullane S.A.
        • Cha E.K.
        • Roper N.
        • Ostrovnaya I.
        • Hyman D.M.
        • Bochner B.H.
        • Arcila M.E.
        • Solit D.B.
        • Berger M.F.
        • Bajorin D.F.
        • Bellmunt J.
        • Iyer G.
        • Rosenberg J.E.
        DNA damage response and repair gene alterations are associated with improved survival in patients with platinum-treated advanced urothelial carcinoma.
        Clin Cancer Res. 2017; 23: 3610-3618
        • McCabe N.
        • Turner N.C.
        • Lord C.J.
        • Kluzek K.
        • Bialkowska A.
        • Swift S.
        • Giavara S.
        • O'Connor M.J.
        • Tutt A.N.
        • Zdzienicka M.Z.
        • Smith G.C.M.
        • Ashworth A.
        Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.
        Cancer Res. 2006; 66: 8109-8115
        • Liu Q.
        • Gheorghiu L.
        • Drumm M.
        • Clayman R.
        • Eidelman A.
        • Wszolek M.F.
        • Olumi A.
        • Feldman A.
        • Wang M.
        • Marcar L.
        • Citrin D.E.
        • Wu C.-L.
        • Benes C.H.
        • Efstathiou J.A.
        • Willers H.
        PARP-1 inhibition with or without ionizing radiation confers reactive oxygen species-mediated cytotoxicity preferentially to cancer cells with mutant TP53.
        Oncogene. 2018; 37: 2793-2805
        • Ler L.D.
        • Ghosh S.
        • Chai X.
        • Thike A.A.
        • Heng H.L.
        • Siew E.Y.
        • Dey S.
        • Koh L.K.
        • Lim J.Q.
        • Lim W.K.
        • Myint S.S.
        • Loh J.L.
        • Ong P.
        • Sam X.X.
        • Huang D.
        • Lim T.
        • Tan P.H.
        • Nagarajan S.
        • Cheng C.W.S.
        • Ho H.
        • Ng L.G.
        • Yuen J.
        • Lin P.-H.
        • Chuang C.-K.
        • Chang Y.-H.
        • Weng W.-H.
        • Rozen S.G.
        • Tan P.
        • Creasy C.L.
        • Pang S.-T.
        • McCabe M.T.
        • Poon S.L.
        • Teh B.T.
        Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2.
        Sci Transl Med. 2017; 9: eaai8312
        • Andersen C.
        • Asmar F.
        • Klausen T.
        • Hasselbalch H.
        • Gronbaek K.
        Somatic mutations of the CREBBP and EP300 genes affect response to histone deacetylase inhibition in malignant DLBCL clones.
        Leuk Res Rep. 2012; 8: 1-3
        • Grivas P.
        • Mortazavi A.
        • Picus J.
        • Hahn N.M.
        • Milowsky M.I.
        • Hart L.L.
        • Alva A.
        • Bellmunt J.
        • Pal S.K.
        • Bambury R.M.
        • O'Donnell P.H.
        • Gupta S.
        • Guancial E.A.
        • Sonpavde G.P.
        • Faltaos D.
        • Potvin D.
        • Christensen J.G.
        • Chao R.C.
        • Rosenberg J.E.
        Mocetinostat for patients with previously treated, locally advanced/metastatic urothelial carcinoma and inactivating alterations of acetyltransferase genes.
        Cancer. 2019; 125: 533-540
        • Ogiwara H.
        • Sasaki M.
        • Mitachi T.
        • Oike T.
        • Higuchi S.
        • Tominaga Y.
        • Kohno T.
        Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality via apoptotic cell death due to abrogation of MYC expression.
        Cancer Discov. 2015; 6: 430-445
        • Barth I.
        • Schneider U.
        • Grimm T.
        • Karl A.
        • Horst D.
        • Gaisa N.T.
        • Knüchel R.
        • Garczyk S.
        Progression of urothelial carcinoma in situ of the urinary bladder: a switch from luminal to basal phenotype and related therapeutic implications.
        Virchows Arch. 2018; 472: 749-758
        • Hayashi T.
        • Seiler R.
        • Oo H.Z.
        • Jäger W.
        • Moskalev I.
        • Awrey S.
        • Dejima T.
        • Todenhöfer T.
        • Li N.
        • Fazli L.
        • Matsubara A.
        • Black P.C.
        Targeting HER2 with T-DM1, an antibody cytotoxic drug conjugate, is effective in HER2 over expressing bladder cancer.
        J Urol. 2015; 194: 1120-1131
        • Her N.-G.
        • Oh J.-W.
        • Oh Y.J.
        • Han S.
        • Cho H.J.
        • Lee Y.
        • Ryu G.H.
        • Nam D.-H.
        Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells.
        Cell Death Dis. 2018; 9: 792
        • Chen L.
        • Pastorino F.
        • Berry P.
        • Bonner J.
        • Kirk C.
        • Wood K.M.
        • Thomas H.D.
        • Zhao Y.
        • Daga A.
        • Veal G.J.
        • Lunec J.
        • Newell D.R.
        • Ponzoni M.
        • Tweddle D.A.
        Preclinical evaluation of the first intravenous small molecule MDM2 antagonist alone and in combination with temozolomide in neuroblastoma.
        Int J Cancer. 2019; 144: 3146-3159
        • Canon J.
        • Osgood T.
        • Olson S.H.
        • Saiki A.Y.
        • Robertson R.
        • Yu D.
        • Eksterowicz J.
        • Ye Q.
        • Jin L.
        • Chen A.
        • Zhou J.
        • Cordover D.
        • Kaufman S.
        • Kendall R.
        • Oliner J.D.
        • Coxon A.
        • Radinsky R.
        The MDM2 inhibitor AMG 232 demonstrates robust antitumor efficacy and potentiates the activity of p53-inducing cytotoxic agents.
        Mol Cancer Ther. 2015; 14: 649-658
        • Gong X.
        • Litchfield L.M.
        • Webster Y.
        • Chio L.-C.
        • Wong S.S.
        • Stewart T.R.
        • Dowless M.
        • Dempsey J.
        • Zeng Y.
        • Torres R.
        • Boehnke K.
        • Mur C.
        • Marugán C.
        • Baquero C.
        • Yu C.
        • Bray S.M.
        • Wulur I.H.
        • Bi C.
        • Chu S.
        • Qian H.-R.
        • Iversen P.W.
        • Merzoug F.F.
        • Ye X.S.
        • Reinhard C.
        • De Dios A.
        • Du J.
        • Caldwell C.W.
        • Lallena M.J.
        • Beckmann R.P.
        • Buchanan S.G.
        Genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4 and CDK6 inhibitor abemaciclib.
        Cancer Cell. 2017; 32: 761-776.e6
        • Pan Q.
        • Sathe A.
        • Black P.C.
        • Goebell P.J.
        • Kamat A.M.
        • Schmitz-Draeger B.
        • Nawroth R.
        CDK4/6 inhibitors in cancer therapy: a novel treatement strategy for bladder cancer.
        Bladder Cancer. 2017; 3: 79-88
        • Rubio C.
        • Martínez-Fernández M.
        • Segovia C.
        • Lodewijk I.
        • Suarez-Cabrera C.
        • Segrelles C.
        • López-Calderón F.
        • Munera-Maravilla E.
        • Santos M.
        • Bernardini A.
        • García-Escudero R.
        • Lorz C.
        • Gómez-Rodriguez M.J.
        • de Velasco G.
        • Otero I.
        • Villacampa F.
        • Guerrero-Ramos F.
        • Ruiz S.
        • de la Rosa F.
        • Domínguez-Rodríguez S.
        • Real F.X.
        • Malats N.
        • Castellano D.
        • Dueñas M.
        • Paramio J.M.
        CDK4/6 inhibitor as a novel therapeutic approach for advanced bladder cancer independently of RB1 status.
        Clin Cancer Res. 2019; 25: 390-402
        • Zeng S.-X.
        • Zhu Y.
        • Ma A.-H.
        • Yu W.
        • Zhang H.
        • Lin T.-Y.
        • Shi W.
        • Tepper C.G.
        • Henderson P.T.
        • Airhart S.
        • Guo J.-M.
        • Xu C.-L.
        • deVere White R.W.
        • Pan C.-X.
        The phosphatidylinositol 3-kinase pathway as a potential therapeutic target in bladder cancer.
        Clin Cancer Res. 2017; 23: 6580-6591
        • Ross R.L.
        • McPherson H.R.
        • Kettlewell L.
        • Shnyder S.D.
        • Hurst C.D.
        • Alder O.
        • Knowles M.A.
        PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.
        BMC Cancer. 2016; 16: 553
        • Tadesse S.
        • Caldon E.C.
        • Tilley W.
        • Wang S.
        Cyclin-dependent kinase 2 inhibitors in cancer therapy: an update.
        J Med Chem. 2019; 62: 4233-4251
        • Karkera J.D.
        • Cardona G.M.
        • Bell K.
        • Gaffney D.
        • Portale J.C.
        • Santiago-Walker A.
        • Moy C.H.
        • King P.
        • Sharp M.
        • Bahleda R.
        • Luo F.R.
        • Alvarez J.D.
        • Lorenzi M.V.
        • Platero S.J.
        Oncogenic characterization and pharmacologic sensitivity of activating fibroblast growth factor receptor (FGFR) genetic alterations to the selective FGFR inhibitor erdafitinib.
        Mol Cancer Ther. 2017; 16: 1717-1726
        • Grünewald S.
        • Politz O.
        • Bender S.
        • Héroult M.
        • Lustig K.
        • Thuss U.
        • Kneip C.
        • Kopitz C.
        • Zopf D.
        • Collin M.P.
        • Boemer U.
        • Ince S.
        • Ellinghaus P.
        • Mumberg D.
        • Hess-Stumpp H.
        • Ziegelbauer K.
        Rogaratinib: a potent and selective pan-FGFR inhibitor with broad antitumor activity in FGFR-overexpressing preclinical cancer models.
        Int J Cancer. 2019; 145: 1346-1357
        • Nogova L.
        • Sequist L.V.
        • Perez Garcia J.M.
        • Andre F.
        • Delord J.-P.
        • Hidalgo M.
        • Schellens J.H.M.
        • Cassier P.A.
        • Camidge D.R.
        • Schuler M.
        • Vaishampayan U.
        • Burris H.
        • Tian G.G.
        • Campone M.
        • Wainberg Z.A.
        • Lim W.-T.
        • LoRusso P.
        • Shapiro G.I.
        • Parker K.
        • Chen X.
        • Choudhury S.
        • Ringeisen F.
        • Graus-Porta D.
        • Porter D.
        • Isaacs R.
        • Buettner R.
        • Wolf J.
        Evaluation of BGJ398, a fibroblast growth factor receptor 1-3 kinase inhibitor, in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors: results of a global phase I, dose-escalation and dose-expansion study.
        J Clin Oncol. 2017; 35: 157-165
        • Cocco E.
        • Lopez S.
        • Black J.
        • Bellone S.
        • Bonazzoli E.
        • Predolini F.
        • Ferrari F.
        • Schwab C.L.
        • Menderes G.
        • Zammataro L.
        • Buza N.
        • Hui P.
        • Wong S.
        • Zhao S.
        • Bai Y.
        • Rimm D.L.
        • Ratner E.
        • Litkouhi B.
        • Silasi D.-A.
        • Azodi M.
        • Schwartz P.E.
        • Santin A.D.
        Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.
        Br J Cancer. 2016; 115: 303-311
        • Konecny G.E.
        • Winterhoff B.
        • Kolarova T.
        • Qi J.
        • Manivong K.
        • Dering J.
        • Yang G.
        • Chalukya M.
        • Wang H.-J.
        • Anderson L.
        • Kalli K.R.
        • Finn R.S.
        • Ginther C.
        • Jones S.
        • Velculescu V.E.
        • Riehle D.
        • Cliby W.A.
        • Randolph S.
        • Koehler M.
        • Hartmann L.C.
        • Slamon D.J.
        Expression of p16 and retinoblastoma determines response to CDK4/6 inhibition in ovarian cancer.
        Clin Cancer Res. 2011; 17: 1591-1602
        • Sathe A.
        • Koshy N.
        • Schmid S.C.
        • Thalgott M.
        • Schwarzenböck S.M.
        • Krause B.J.
        • Holm P.S.
        • Gschwend J.E.
        • Retz M.
        • Nawroth R.
        CDK4/6 inhibition controls proliferation of bladder cancer and transcription of RB1.
        J Urol. 2016; 195: 771-779
        • Rose T.L.
        • Chism D.D.
        • Alva A.S.
        • Deal A.M.
        • Maygarden S.J.
        • Whang Y.E.
        • Kardos J.
        • Drier A.
        • Basch E.
        • Godley P.A.
        • Dunn M.W.
        • Kim W.Y.
        • Milowsky M.I.
        Phase II trial of palbociclib in patients with metastatic urothelial cancer after failure of first-line chemotherapy.
        Br J Cancer. 2018; 119: 801-807
        • Rebouissou S.
        • Bernard-Pierrot I.
        • de Reyniès A.
        • Lepage M.-L.
        • Krucker C.
        • Chapeaublanc E.
        • Hérault A.
        • Kamoun A.
        • Caillault A.
        • Letouzé E.
        • Elarouci N.
        • Neuzillet Y.
        • Denoux Y.
        • Molinié V.
        • Vordos D.
        • Laplanche A.
        • Maillé P.
        • Soyeux P.
        • Ofualuka K.
        • Reyal F.
        • Biton A.
        • Sibony M.
        • Paoletti X.
        • Southgate J.
        • Benhamou S.
        • Lebret T.
        • Allory Y.
        • Radvanyi F.
        EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype.
        Sci Transl Med. 2014; 6: 244ra91
        • Siddiqui M.R.
        • Railkar R.
        • Sanford T.
        • Crooks D.R.
        • Eckhaus M.A.
        • Haines D.
        • Choyke P.L.
        • Kobayashi H.
        • Agarwal P.K.
        Targeting epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) expressing bladder cancer using combination photoimmunotherapy (PIT).
        Sci Rep. 2019; 9: 2084
        • Powles T.
        • Huddart R.A.
        • Elliott T.
        • Sarker S.-J.
        • Ackerman C.
        • Jones R.
        • Hussain S.
        • Crabb S.
        • Jagdev S.
        • Chester J.
        • Hilman S.
        • Beresford M.
        • Macdonald G.
        • Santhanam S.
        • Frew J.A.
        • Stockdale A.
        • Hughes S.
        • Berney D.
        • Chowdhury S.
        Phase III, double-blind, randomized trial that compared maintenance lapatinib versus placebo after first-line chemotherapy in patients with human epidermal growth factor receptor 1/2–positive metastatic bladder cancer.
        J Clin Oncol. 2017; 35: 48-55
        • Autenrieth M.E.
        • Seidl C.
        • Bruchertseifer F.
        • Horn T.
        • Kurtz F.
        • Feuerecker B.
        • D'Alessandria C.
        • Pfob C.
        • Nekolla S.
        • Apostolidis C.
        • Mirzadeh S.
        • Gschwend J.E.
        • Schwaiger M.
        • Scheidhauer K.
        • Morgenstern A.
        Treatment of carcinoma in situ of the urinary bladder with an alpha-emitter immunoconjugate targeting the epidermal growth factor receptor: a pilot study.
        Eur J Nucl Med Mol Imaging. 2018; 45: 1364-1371
        • Mao J.-H.
        • Kim I.-J.
        • Wu D.
        • Climent J.
        • Kang H.C.
        • DelRosario R.
        • Balmain A.
        FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression.
        Science. 2008; 321: 1499-1502
        • Caenepeel S.
        • Brown S.P.
        • Belmontes B.
        • Moody G.
        • Keegan K.S.
        • Chui D.
        • et al.
        AMG 176, a selective MCL1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapies.
        Cancer Discov. 2018; 8: 1582-1597
        • Kotschy A.
        • Szlavik Z.
        • Murray J.
        • Davidson J.
        • Maragno A.L.
        • Le Toumelin-Braizat G.
        • et al.
        The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.
        Nature. 2016; 538: 477-482
        • Knievel J.
        • Schulz W.
        • Greife A.
        • Hader C.
        • Lübke T.
        • Schmitz I.
        • Albers P.
        • Niegisch G.
        Multiple mechanisms mediate resistance to sorafenib in urothelial cancer.
        Int J Mol Sci. 2014; 15: 20500-20517
        • Necchi A.
        • Lo Vullo S.
        • Raggi D.
        • Perrone F.
        • Giannatempo P.
        • Calareso G.
        • Togliardi E.
        • Nicolai N.
        • Piva L.
        • Biasoni D.
        • Catanzaro M.
        • Torelli T.
        • Stagni S.
        • Colecchia M.
        • Busico A.
        • Pennati M.
        • Zaffaroni N.
        • Mariani L.
        • Salvioni R.
        Neoadjuvant sorafenib, gemcitabine, and cisplatin administration preceding cystectomy in patients with muscle-invasive urothelial bladder carcinoma: an open-label, single-arm, single-center, phase 2 study.
        Urol Oncol. 2018; 36: 8.e1-8.e8
        • Johannessen C.M.
        • Reczek E.E.
        • James M.F.
        • Brems H.
        • Legius E.
        • Cichowski K.
        The NF1 tumor suppressor critically regulates TSC2 and mTOR.
        Proc Natl Acad Sci U S A. 2005; 102: 8573-8578
        • Marjon K.
        • Cameron M.J.
        • Quang P.
        • Clasquin M.F.
        • Mandley E.
        • Kunii K.
        • McVay M.
        • Choe S.
        • Kernytsky A.
        • Gross S.
        • Konteatis Z.
        • Murtie J.
        • Blake M.L.
        • Travins J.
        • Dorsch M.
        • Biller S.A.
        • Marks K.M.
        MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis.
        Cell Rep. 2016; 15: 574-587
        • Sjödahl G.
        • Jackson C.L.
        • Bartlett J.M.S.
        • Siemens D.R.
        • Berman D.M.
        Molecular profiling in muscle-invasive bladder cancer: more than the sum of its parts.
        J Pathol. 2019; 247: 563-573
        • Hurst C.D.
        • Alder O.
        • Platt F.M.
        • Droop A.
        • Stead L.F.
        • Burns J.E.
        • Burghel G.J.
        • Jain S.
        • Klimczak L.J.
        • Lindsay H.
        • Roulson J.-A.
        • Taylor C.F.
        • Thygesen H.
        • Cameron A.J.
        • Ridley A.J.
        • Mott H.R.
        • Gordenin D.A.
        • Knowles M.A.
        Genomic subtypes of non-invasive bladder cancer with distinct metabolic profile and female gender bias in KDM6A mutation frequency.
        Cancer Cell. 2017; 32: 701-715.e7
        • Scott S.N.
        • Ostrovnaya I.
        • Lin C.M.
        • Bouvier N.
        • Bochner B.H.
        • Iyer G.
        • Solit D.
        • Berger M.F.
        • Lin O.
        Next-generation sequencing of urine specimens: a novel platform for genomic analysis in patients with non-muscle-invasive urothelial carcinoma treated with bacille Calmette-Guérin.
        Cancer Cytopathol. 2017; 125: 416-426
        • Nassar A.H.
        • Umeton R.
        • Kim J.
        • Lundgren K.
        • Harshman L.
        • Van Allen E.M.
        • Preston M.
        • Dong F.
        • Bellmunt J.
        • Mouw K.W.
        • Choueiri T.K.
        • Sonpavde G.
        • Kwiatkowski D.J.
        Mutational analysis of 472 urothelial carcinoma across grades and anatomic sites.
        Clin Cancer Res. 2019; 25: 2458-2470
        • Hurst C.D.
        • Knowles M.A.
        Mutational landscape of non-muscle-invasive bladder cancer.
        Urol Oncol. 2018; ([Epub ahead of print] doi:10.1016/j.urolonc.2018.10.015)
        • Kinde I.
        • Munari E.
        • Faraj S.F.
        • Hruban R.H.
        • Schoenberg M.
        • Bivalacqua T.
        • Allaf M.
        • Springer S.
        • Wang Y.
        • Diaz L.A.
        • Kinzler K.W.
        • Vogelstein B.
        • Papadopoulos N.
        • Netto G.J.
        TERT promoter mutations occur early in urothelial neoplasia and are biomarkers of early disease and disease recurrence in urine.
        Cancer Res. 2013; 73: 7162-7167
        • Hurst C.D.
        • Platt F.M.
        • Knowles M.A.
        Comprehensive mutation analysis of the TERT promoter in bladder cancer and detection of mutations in voided urine.
        Eur Urol. 2014; 65: 367-369
        • Kadoch C.
        • Hargreaves D.C.
        • Hodges C.
        • Elias L.
        • Ho L.
        • Ranish J.
        • Crabtree G.R.
        Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.
        Nat Genet. 2013; 45: 592-601
        • Dykhuizen E.C.
        • Hargreaves D.C.
        • Miller E.L.
        • Cui K.
        • Korshunov A.
        • Kool M.
        • Pfister S.
        • Cho Y.-J.
        • Zhao K.
        • Crabtree G.R.
        BAF complexes facilitate decatenation of DNA by topoisomerase IIα.
        Nature. 2013; 497: 624-627
        • Schulz W.A.
        • Lang A.
        • Koch J.
        • Greife A.
        The histone demethylase UTX/KDM6A in cancer: progress and puzzles.
        Int J Cancer. 2019; 145: 614-620
        • Attar N.
        • Kurdistani S.K.
        Exploitation of EP300 and CREBBP lysine acetyltransferases by cancer.
        Cold Spring Harb Perspect Med. 2017; 7: a026534
        • Greulich H.
        • Kaplan B.
        • Mertins P.
        • Chen T.-H.
        • Tanaka K.E.
        • Yun C.-H.
        • Zhang X.
        • Lee S.-H.
        • Cho J.
        • Ambrogio L.
        • Liao R.
        • Imielinski M.
        • Banerji S.
        • Berger A.H.
        • Lawrence M.S.
        • Zhang J.
        • Pho N.H.
        • Walker S.R.
        • Winckler W.
        • Getz G.
        • Frank D.
        • Hahn W.C.
        • Eck M.J.
        • Mani D.R.
        • Jaffe J.D.
        • Carr S.A.
        • Wong K.-K.
        • Meyerson M.
        Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.
        Proc Natl Acad Sci U S A. 2012; 109: 14476-14481
        • Meric-Bernstam F.
        • Johnson A.M.
        • Dumbrava E.E.I.
        • Raghav K.
        • Balaji K.
        • Bhatt M.
        • Murthy R.K.
        • Rodon J.
        • Piha-Paul S.A.
        Advances in HER2-targeted therapy: novel agents and opportunities beyond breast and gastric cancer.
        Clin Cancer Res. 2019; 25: 2033-2041
        • Oudard S.
        • Culine S.
        • Vano Y.
        • Goldwasser F.
        • Théodore C.
        • Nguyen T.
        • Voog E.
        • Banu E.
        • Vieillefond A.
        • Priou F.
        • Deplanque G.
        • Gravis G.
        • Ravaud A.
        • Vannetzel J.M.
        • Machiels J.-P.
        • Muracciole X.
        • Pichon M.-F.
        • Bay J.-O.
        • Elaidi R.
        • Teghom C.
        • Radvanyi F.
        • Beuzeboc P.
        Multicentre randomised phase II trial of gemcitabine+platinum, with or without trastuzumab, in advanced or metastatic urothelial carcinoma overexpressing Her2.
        Eur J Cancer. 2015; 51: 45-54
        • Wülfing C.
        • Machiels J.-P.H.
        • Richel D.J.
        • Grimm M.-O.
        • Treiber U.
        • De Groot M.R.
        • Beuzeboc P.
        • Parikh R.
        • Pétavy F.
        • El-Hariry I.A.
        A single-arm, multicenter, open-label phase 2 study of lapatinib as the second-line treatment of patients with locally advanced or metastatic transitional cell carcinoma.
        Cancer. 2009; 115: 2881-2890
        • Hainsworth J.D.
        • Meric-Bernstam F.
        • Swanton C.
        • Hurwitz H.
        • Spigel D.R.
        • Sweeney C.
        • Burris H.A.
        • Bose R.
        • Yoo B.
        • Stein A.
        • Beattie M.
        • Kurzrock R.
        Targeted therapy for advanced solid tumors on the basis of molecular profiles: results from MyPathway, an open-label, phase IIa multiple basket study.
        J Clin Oncol. 2018; 36: 536-542
        • Kiss B.
        • Wyatt A.W.
        • Douglas J.
        • Skuginna V.
        • Mo F.
        • Anderson S.
        • Rotzer D.
        • Fleischmann A.
        • Genitsch V.
        • Hayashi T.
        • Neuenschwander M.
        • Buerki C.
        • Davicioni E.
        • Collins C.
        • Thalmann G.N.
        • Black P.C.
        • Seiler R.
        Her2 alterations in muscle-invasive bladder cancer: patient selection beyond protein expression for targeted therapy.
        Sci Rep. 2017; 7: 42713