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Integrated Molecular Analysis of Papillary Renal Cell Carcinoma and Precursor Lesions Unfolds Evolutionary Process from Kidney Progenitor-Like Cells

  • Rola M. Saleeb
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

    Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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  • Mina Farag
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
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  • Qiang Ding
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
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  • Michelle Downes
    Affiliations
    Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

    Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada
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  • Georg Bjarnason
    Affiliations
    Division of Medical Oncology and Hematology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada
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  • Fadi Brimo
    Affiliations
    Department of Pathology, McGill University Health Center, Montreal, Quebec, Canada
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  • Pamela Plant
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
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  • Fabio Rotondo
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
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  • Zsuzsanna Lichner
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
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  • Antonio Finelli
    Affiliations
    Division of Urology, Department of Surgery, University Health Network, Toronto, Ontario, Canada
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  • George M. Yousef
    Correspondence
    Address correspondence to George M. Yousef, M.D., Ph.D., Paediatric Laboratory Medicine, Hospital for Sick Children, 555 University Ave, Room 3227, Toronto, ON M5G 1X8, Canada.
    Affiliations
    Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

    Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

    Paediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada
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Open ArchivePublished:August 02, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.07.002
      Papillary renal cell carcinoma (PRCC) is the most common type of RCC in end-stage kidney disease (ESKD). Papillary adenoma (PA) is a small benign lesion morphologically similar to PRCC and is suggested to be its precursor. PA is also prevalent in ESKD. The evolution of PAs to PRCCs and their relationship to ESKD are poorly understood. A total of 140 PAs, normal kidneys, ESKDs, and PRCCs were analyzed. Previously described markers of renal tubular progenitor cells were analyzed using immunohistochemistry and quantified with digital analysis. Progenitor cells were significantly increased in ESKD (P < 0.0001) and PAs (P = 0.02) in comparison with the normal kidney. Pathway analysis using global miRNA and chromosomal copy number variations revealed a common developmental theme between PA and the PRCCs. Whole exome sequencing showed a KMT2C-specific pathogenic mutation among all PAs and PRCCs. KMT2C is a chromosome 7 epigenetic regulator implicated in development and oncogenesis. Collectively, results show possible connection of PRCCs to PA and the progenitor-like cell population, which are increased in response to renal tubular injury. In addition, each PRCC histologic subtype had its own set of mutational changes, indicating divergence from a common precursor. The study reports previously unknown biological aspects of PRCC development and could influence current surveillance criteria and early detection strategies of PRCC tumors.
      Papillary renal cell carcinoma (PRCC) is a cancer that is suggested to arise from the renal tubules, and it represents the second most common type of renal cell carcinoma (RCC) in adults or the most common type of non–clear cell RCCs.
      • Chevarie-Davis M.
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      Morphologic and immunohistochemical spectrum of papillary renal cell carcinoma: study including 132 cases with pure type 1 and type 2 morphology as well.
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      Morphologic typing of papillary renal cell carcinoma: comparison of growth kinetics and patient survival in 66 cases.
      Papillary adenoma (PA) is a benign kidney tumor with similar morphology to PRCC; however, it is deemed benign on the basis of the size cutoff of ≤15 mm, by the last World Health Organization urological classification (ie, tumors that are ≤15 mm are labeled as a benign PA, whereas tumors >15 mm are called PRCC, which is considered malignant).
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      WHO Classifications of Tumours of the Urinary System and Male Genital Organs.
      Until recently, that cutoff was 5 mm; however, the change in the cutoff decision was based on the clinical behavior rather than the biological nature of these lesions.
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      WHO Classifications of Tumours of the Urinary System and Male Genital Organs.
      PA is suggested to be the precursor lesion to PRCC on the basis of earlier studies of whole chromosomal copy number abnormalities, yet this remains to be validated by more comprehensive genomic analysis methods.
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      Gains of chromosomes 7, 17, 12, 16, and 20 and loss of Y occur early in the evolution of papillary renal cell neoplasia: a fluorescent in situ hybridization study.
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      In addition, both PA and PRCC show similar immunohistochemical (IHC) staining patterns.
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      Renal papillary adenoma: a putative precursor of papillary renal cell carcinoma.
      Incidental PAs are reported in 7% of nephrectomy specimens and 7% to 40% of autopsy studies,
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      Renal papillary adenoma: a putative precursor of papillary renal cell carcinoma.
      and they are more frequent in resected kidneys accompanying PRCC, more so than other RCC subtypes.
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      Renal papillary adenoma: a putative precursor of papillary renal cell carcinoma.
      PRCC was recognized as having two morphologic subtypes,
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      Papillary Renal Cell Carcinoma: A Clinicopathologic and Immunohistochemical Study of 105 Tumors.
      PRCC type 1 (PRCC1) and PRCC type 2 (PRCC2). We and others have shown the subtypes to be distinct molecularly and prognostically,
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      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      with PRCC2 associated with a worse prognosis.
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      Morphologic typing of papillary renal cell carcinoma: comparison of growth kinetics and patient survival in 66 cases.
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      Papillary renal cell carcinoma: prognostic value of morphological subtypes in a clinicopathologic study of 43 cases.
      Recently, we uncovered another proposed subtype of PRCC on the basis of the combined morphology and molecular makeup of these tumors: it is designated PRCC type 3 (PRCC3) and constitutes 35% of PRCC cases.
      • Saleeb R.M.
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      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      A previously reported entity (oncocytic low-grade PRCC) was further characterized, and a new classification of PRCC tumors into four biologically distinct subtypes was proposed
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
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      • Plant P.
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      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      (Table 1). The relationship between PA and these different PRCC subtypes remains unclear. Also, the molecular mechanisms by which PAs can progress to PRCC are poorly understood.
      Table 1Summary of Morphologic and IHC Characteristics of the Proposed New PRCC Classification
      FeaturePRCC1PRCC2PRCC3PRCC4/OLG
      Cytoplasmic quantityScant, occasionally moderateAbundantModerateAbundant
      Cytoplasmic colorBasophilic, eosinophilic, or clearingEosinophilic or clearingEasonophilic or clearingOncocytic or easinophilic
      Cell sizeSmall to intermediateLargeIntermediateLarge
      Nucleolar prominence at 10×Inconspicuous, rarely prominentProminentOften prominentInconspicuous, rarely prominent
      Nuclear pseudostratificationAbsentMostly present, occasionally absentMostly absent, occasionally presentAbsent; linear; nuclei arranged away from base of the cells
      Nuclear sizeSmallLargeSmall to intermediateIntermediate
      ISUP nucleolar grade1 to 2; focal, 3Mostly 3Mostly 31 to 2
      ABCC2 IHCNegativeStrong diffuse positiveWeaker patchy positiveStrong diffuse positive
      CA9 IHCNegativePositive Golgi pattern (perinuclear dot)NegativeNegative
      GATA3 IHCNegativeNegativeNegativePositive
      ABCC2, ATP binding cassette subfamily C member 2; CA9, carbonic anhydrase 9; GATA3, GATA binding protein 3; IHC, immunohistochemistry; ISUP, The International Society of Urological Pathology; OLG, oncocytic low grade.
      Both PA and PRCC are known to be the most common lesions in end-stage kidney disease (ESKD; approximately 70% of ESKD tumors are PRCCs).
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      Hes et al
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      End-stage kidney disease: gains of chromosomes 7 and 17 and loss of Y chromosome in non-neoplastic tissue.
      revealed chromosomal changes in end-stage kidney's nonneoplastic tissue, similar to PRCC1. Woldu et al
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      Renal insufficiency is associated with an increased risk of papillary renal cell carcinoma histology.
      attributed these findings to resident kidney stem cells that are potentially up-regulated with kidney damage. Endorsing this, Lindgren et al
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      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      isolated a population of renal tubular cells with enhanced stem cell/progenitor cell properties and demonstrated significant similarities at the transcriptomic level between a renal tubular progenitor cell population and both the PA and PRCC. These cells were overexpressing several markers, including B-cell lymphoma 2 (BCL2) and cytokeratin 7 (CK7).
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      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      CK7 is known to stain PRCC lesions, whereas BCL2 is an antiapoptotic marker.
      In our previous analysis, we identified pathways enriched in PRCC subtypes that correspond to normal kidney developmental pathways, such as WNT signaling, transforming growth factor (TGF)-β, and fibroblast growth factor (FGF) pathways.
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
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      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
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      Of interest, these pathways are also reported in colon cancer, which is thought to arise from crypt colonic stem (progenitor) cells and has colonic adenoma as its known precursor lesion.
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      Signaling cross-talk between TGF-beta/BMP and other pathways.
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      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
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      Colon cancer stem cells.
      Understanding initiating events that drive PRCC oncogenesis could potentially have a significant impact on PRCC prevention and management. PA could represent the link between end-stage kidney and PRCC. In this study, we aim to uncover the sequence of biological events implicated in the transformation of the normal kidney to ESKD, to PA, and eventually to the different subtypes of PRCCs. We also attempt to explore the possible connection between the renal tubular progenitor cells and PRCC.

      Materials and Methods

      Sample Collection

      Research ethics board approvals were obtained from the corresponding institutions. Cases were collected from three Canadian Institutions: St. Michael's Hospital, Sunnybrook Health Sciences Centre, and McGill University Health Center. The latter cohort was detailed in previous publications.
      • Chevarie-Davis M.
      • Riazalhosseini Y.
      • Arseneault M.
      • Aprikian A.
      • Kassouf W.
      • Tanguay S.
      • Latour M.
      • Brimo F.
      Morphologic and immunohistochemical spectrum of papillary renal cell carcinoma: study including 132 cases with pure type 1 and type 2 morphology as well.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Samples included normal kidney (n = 5), ESKD (n = 7), PA (n = 20), and PRCC (n = 108). Formalin-fixed, paraffin-embedded tissue blocks were collected for analysis. A formalin-fixed, paraffin-embedded tissue microarray was prepared from the McGill University Health Center cases, as previously described.
      • Chevarie-Davis M.
      • Riazalhosseini Y.
      • Arseneault M.
      • Aprikian A.
      • Kassouf W.
      • Tanguay S.
      • Latour M.
      • Brimo F.
      Morphologic and immunohistochemical spectrum of papillary renal cell carcinoma: study including 132 cases with pure type 1 and type 2 morphology as well.
      • Karamchandani J.R.
      • Gabril M.Y.
      • Ibrahim R.
      • Scorilas A.
      • Filter E.
      • Finelli A.
      • Lee J.Y.
      • Ordon M.
      • Pasic M.
      • Romaschin A.D.
      • Yousef G.M.
      Profilin-1 expression is associated with high grade and stage and decreased disease-free survival in renal cell carcinoma.

      Nucleic Acid Extraction

      Ten shaves of pure lesional (or normal) areas (10 μm thick) were selected for processing. Shaves were obtained from different areas of the lesion to account for heterogeneity. DNA was isolated using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX), according to the manufacturer's protocol. Total RNA was isolated from the sections using the miRNeasy kit (Qiagen, Mississauga, ON, Canada), as previously described.
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      • Ordon M.
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      • Jewett M.A.
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      Low expression of miR-126 is a prognostic marker for metastatic clear cell renal cell carcinoma.
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      miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in renal cell carcinoma.
      DNA and RNA concentration and purity were measured spectrophotometrically (Nanodrop 2000; Thermo Fisher Scientific, Waltham, MA).
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      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      • Wala S.J.
      • Karamchandani J.R.
      • Saleeb R.
      • Evans A.
      • Ding Q.
      • Ibrahim R.
      • Jewett M.
      • Pasic M.
      • Finelli A.
      • Pace K.
      • Lianidou E.
      • Yousef G.M.
      An integrated genomic analysis of papillary renal cell carcinoma type 1 uncovers the role of focal adhesion and extracellular matrix pathways.

      Chromosomal CNV Assessment

      The nCounter Human Karyotype panel (NanoString Technologies, Inc., Seattle, WA) was used to assess chromosomal copy number variations (CNVs) using 338 probes spanning all 24 chromosomes. DNA input used was optimized at 300 ng per sample. A total of 21 samples underwent the CNV analysis: 3 normal kidneys, 3 ESKD, 3 PA, as well as the 12 PRCC samples (4 PRCC1, 4 PRCC2, and 4 PRCC3) from our previous analysis.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Samples were normalized with the invariant probes method.

      miRNA Expression Analysis

      NanoString Human miRNA version 3 hybridization platform (NanoString Technologies, Inc.) was used to assess miRNA expression profile (800 miRNAs), as previously described.
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      • Hood L.
      Direct multiplexed measurement of gene expression with color-coded probe pairs.
      Twelve samples were analyzed: three normal kidney, three PA, as well as the six PRCC samples obtained from our previous analysis (two PRCC1, one PRCC3, and three PRCC2).
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      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      The normalization method adopted was a geometric mean of the top 100 most highly expressed miRNAs.
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      Whole Exome Sequencing

      Whole exome sequencing of 12 samples (3 PA, 3 PRCC1, 3 PRCC2, and 3 PRCC3) was performed using the Illumina (San Diego, CA) NextSeq platform. The TruSeq exome kit (Illumina) was used for library preparation and enrichment. The NextSeq 500/550 high-output kit version 2, 150 cycles (Illumina), was used for the sequencing on high output run, generating 400 million reads for three samples.
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      • Li M.
      • Hakonarson H.
      ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.
      • Patil V.
      • Pal J.
      • Somasundaram K.
      Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing.
      Alternate variant frequencies of <10% were eliminated. Because there was no germline mutational comparison, the variants with Single Nucleotide Polymorphism Database (dbSNP) identifications (IDs) were eliminated for the final analysis, and the variants were analyzed closely with Catalog of Somatic Mutations in Cancer (COSMIC) IDs (IDs in cancer).
      • Patil V.
      • Pal J.
      • Somasundaram K.
      Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing.
      • Wang S.R.
      • Malik S.
      • Tan I.B.
      • Chan Y.S.
      • Hoi Q.
      • Ow J.L.
      • He C.Z.
      • Ching C.E.
      • Poh D.Y.S.
      • Seah H.M.
      • Cheung K.H.T.
      • Perumal D.
      • Devasia A.G.
      • Pan L.
      • Ang S.
      • Lee S.E.
      • Ten R.
      • Chua C.
      • Tan D.S.W.
      • Qu J.Z.Z.
      • Bylstra Y.M.
      • Lim L.
      • Lezhava A.
      • Ng P.C.
      • Wong C.W.
      • Lim T.
      • Tan P.
      Technical validation of a next-generation sequencing assay for detecting actionable mutations in patients with gastrointestinal cancer.
      • Hartmaier R.J.
      • Charo J.
      • Fabrizio D.
      • Goldberg M.E.
      • Albacker L.A.
      • Pao W.
      • Chmielecki J.
      Genomic analysis of 63,220 tumors reveals insights into tumor uniqueness and targeted cancer immunotherapy strategies.

      Bioinformatics and Gene Set Enrichment Analysis

      To analyze the DNA CNV results, unsupervised clustering analysis was performed using the NanoString nSolver software version 3.0 (NanoString Technologies, Inc.), hierarchical clustering module, and the hierarchical clustering image viewer on GenePattern (Broad Institute, Cambridge, MA). Supervised clustering analysis (GenePattern Comparative Marker Selection module) was performed to compare all studied groups (ESKD, PA, PRCC1, PRCC2, and PRCC3) with the normal kidney to delineate the most significant chromosomal regions harboring gains
      • Kuehn H.
      • Liberzon A.
      • Reich M.
      • Mesirov J.P.
      Using GenePattern for gene expression analysis.
      using the GenePattern Bioinformatics software package version 3.9.9 (Broad Institute). Enriched chromosomal regions selected were subsequently analyzed for surrounding enriched genes (Atlas of Genetics). The specific enriched genes for each tissue group were further explored for the implicated molecular pathways of statistical significance using the Reactome Pathway database and Ingenuity Pathway Analysis software version 2.4 (Qiagen, Mississauga, Canada).
      • Butz H.
      • Szabó P.M.
      • Nofech-Mozes R.
      • Rotondo F.
      • Kovacs K.
      • Mirham L.
      • Girgis H.
      • Boles D.
      • Patocs A.
      • Yousef G.M.
      Integrative bioinformatics analysis reveals new prognostic biomarkers of clear cell renal cell carcinoma.
      • Butz H.
      • Szabó P.
      • Khella H.
      • Nofech-Mozes R.
      • Patocs A.
      • Yousef G.
      miRNA-target network reveals miR-124 as a key miRNA contributing to clear cell renal cell carcinoma aggressive behaviour by targeting CAV1 and FLOT1.
      • Croft D.
      • O'Kelly G.
      • Wu G.
      • Haw R.
      • Gillespie M.
      • Matthews L.
      • Caudy M.
      • Garapati P.
      • Gopinath G.
      • Jassal B.
      • Jupe S.
      • Kalatskaya I.
      • MayMahajan S.
      • May B.
      • Ndegwa N.
      • Schmidt E.
      • Shamovsky V.
      • Yung C.
      • Birney E.
      • Hermjakob H.
      • D'Eustachio P.
      • Stein L.
      Reactome: a database of reactions, pathways and biological processes.
      For miRNA analysis, the Comparative Marker Selection module was used to detect the significantly up-regulated miRNAs in the PA versus normal. Enriched miRNAs were selected for target prediction using DIANA-miRPath. Corresponding genes were analyzed with the Reactome database to depict the top pathways implicated in the transformation of normal kidney to PA.
      Genes with single-nucleotide and indel variants selected as pathogenic from the whole exome sequencing analysis were further studied for molecular pathways through the Reactome database.

      Immunohistochemistry

      IHC was performed using a Ventana automated system and Ventana Iview DAB Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ). IHC staining of ATP binding cassette subfamily C member 2 (ABCC2) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) was performed by the streptavidin-biotin-peroxidase complex protocol using an ABCC2-specific mouse monoclonal antibody (Monosan, Uden, the Netherlands; catalog number MON9026; dilution 1:200) and an LGR5-specific mouse monoclonal antibody (dilution 1:100). A standard previously published protocol was used.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.

      IHC Scoring, Quantitative Image Analysis, and Statistical Analysis

      Slides stained with BCL2, CK7, and LGR5 were scanned and analyzed with the Aperio ImageScope analysis software version 12.3.2.8013 (Leica Biosystems, Wetzlar, Germany). The algorithm used (nsr) combines staining intensity and percentage to provide a quantitative measurement of strong positive staining cells.
      β-Catenin–stained tissue slides, as well as the PRCC tissue microarray stained slides, were assessed manually using a combined intensity and percentage scoring via two independent pathologists (R.M.S. and G.M.Y.; each category has a score from 1 to 3, and then both scores are added together).
      • Gabril M.
      • Girgis H.
      • Scorilas A.
      • Rotondo F.
      • Wala S.
      • Bjarnason G.A.
      • Ding Q.
      • Evans A.
      • Tawedrous E.
      • Pasic M.
      • Finelli A.
      • Al-Haddad S.
      • Yousef G.M.
      S100A11 is a potential prognostic marker for clear cell renal cell carcinoma.
      • Fitzgibbons P.L.
      • Goldsmith J.D.
      • Souers R.J.
      • Fatheree L.A.
      • Volmar K.E.
      • Stuart L.N.
      • Nowak J.A.
      • Astles J.R.
      • Nakhleh R.E.
      Analytic validation of immunohistochemical assays: a comparison of laboratory practices before and after introduction of an evidence-based guideline.

      Results

      PA Shares Several Chromosomal Aberrations with End-Stage Kidney Disease

      Copy number aberrations of 338 chromosomal regions were compared between normal kidney, ESKD, and PA. The PA group showed gains in multiple chromosomes similar to the end-stage kidney samples (Figure 1A). When compared with PRCCs, PA samples clustered in the same arm with the ESKD (Figure 1B), whereas PRCC subtypes clustered in a distinct arm. Specific analysis of the significantly enriched chromosomal regions revealed a 22% overlap between PA and ESKD (Supplemental Figure S1).
      Figure thumbnail gr1
      Figure 1Copy number variation analysis showing similarities between the papillary adenomas (PAs) and end-stage kidney disease (ESKD). A: Combined analysis of the 338 chromosomal (Chr) regions shows similar gains between the PA and the ESKD groups. Each column represents the combined aberrations of three cases. B: Hierarchical clustering analysis showing PA cases to cluster with ESKD, but distinct from PRCC. Consistent with previous report, PRCC1 formed a unique distinct cluster, whereas PRCC2 and PRCC3 showed occasional overlap.

      PA Shares Gains in Six Chromosomal Regions of Biological Significance with PRCC Subtypes

      Differential expression analysis with the comparative marker selection module (selecting enriched chromosomal regions in comparison to the normal kidney) revealed six regions that consistently harbored gains among PA and all PRCC subtypes (chromosome 18:9402604, chromosome 17:16355177, chromosome 7:27529574, chromosome 10:117416534, chromosome 17:8081210, and chromosome 7:127701508) (Figure 2). Pathway analysis of the genes located on these regions (Reactome pathway database) revealed significant key pathways that have previously been shown to be enriched in the different PRCC subtypes. The SMAD2/3 down-regulation and TGF-β pathway was previously noted in PRCC3 (P = 4.29 × 105 and P = 0.004, respectively).
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      NOTCH signaling, which has been reported in PRCC1 by transcriptomic analysis, was also among the significant pathways (P = 0.003).
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      FGF signaling, which has been correlated before to PRCC2 was likewise enriched (P = 0.003).
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Other significant pathways were mitogen-activated protein kinase (MAPK) activation (P = 0.004) and tumor necrosis factor signaling and death receptor signaling (P = 0.002 and P = 0.003, respectively). The later pathways are consistent with what has been reported in the literature regarding antiapoptotic pathways being enriched in papillary tumors.
      • Lindgren D.
      • Boström A.-K.
      • Nilsson K.
      • Hansson J.
      • Sjölund J.
      • Möller C.
      • Jirström K.
      • Nilsson E.
      • Landberg G.
      • Axelson H.
      • Johansson M.E.
      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      Figure thumbnail gr2
      Figure 2Copy number variation analysis shows unanimous gains in six genomic regions in papillary adenoma (PA) and PRCC subtypes. Venn diagram showing the number of significant chromosomal (Chr) gains in each group, with an overlap of six regions common to all groups. Inset: The unanimous gains in all groups. Regions highlighted in red were also enriched in the end-stage kidney disease group.
      Consistent with the previous results, analysis of the same six regions using the Ingenuity Pathway Analysis software revealed cell death and development and embryonic development as the top molecular pathways implicated. Other enriched pathways included cell to cell signaling and interactions. Integrating these pathways demonstrated some key molecules at the center of the interactions as BCL2 (we further examined BCL2 expression as a marker of renal tubular progenitor cells, as detailed below), nuclear receptor corepressor 1 (NCOR1; implicated in the TGF-β that is related to PRCC3) (Supplemental Figure S2), as well as glycogen synthase kinase-3 β (involved in β-catenin activation, which was demonstrated to be up-regulated in PA, as detailed below) (Supplemental Figure S3).

      Pathways Shared between PA, PRCC, and ESKD

      Of interest, two of the six chromosomal regions described above were also shared with ESKD. Both were located at chromosome 7, which frequently holds aberrations in PRCCs
      • Jones T.D.
      • Eble J.N.
      • Wang M.
      • MacLennan G.T.
      • Delahunt B.
      • Brunelli M.
      • Martignoni G.
      • Lopez-Beltran A.
      • Bonsib S.M.
      • Ulbright T.M.
      • Zhang S.
      • Nigro K.
      • Cheng L.
      Molecular genetic evidence for the independent origin of multifocal papillary tumors in patients with papillary renal cell carcinomas.
      • Brunelli M.
      Gains of chromosomes 7, 17, 12, 16, and 20 and loss of Y occur early in the evolution of papillary renal cell neoplasia: a fluorescent in situ hybridization study.
      (chromosome 7:27529574, chromosome 7:127701508). Reactome pathway analysis for genes located in these regions exhibit regulation of gene expression in progenitor cells (PAX4 gene) as the top pathway (P = 0.01). Also, among the enriched pathways are DNA damage/telomere abnormalities and tumor necrosis factor/death receptor signaling.
      Performing Ingenuity Pathway Analysis on these two chromosomal regions revealed almost identical molecular themes: cell cycle, cellular development, cellular growth and proliferation, and organismal injury. Key molecules at the center of this interaction were myelocytomatosis (MYC), MAPK1, and breast cancer 1 (BRCA1) (Supplemental Figure S4), as well as the HOXA genes implicated in embryonic development.
      • Pezzani L.
      • Milani D.
      • Manzoni F.
      • Baccarin M.
      • Silipigni R.
      • Guerneri S.
      • Esposito S.
      HOXA genes cluster: clinical implications of the smallest deletion.
      The telomere extension by telomerase showed as a significant canonical pathway for both the six and two region analysis (P = 0.02 and P = 0.009, respectively). Taken together, these results indicate that DNA damage from kidney injury together with abnormalities in cell division and disruption of apoptosis are at the core of progression of papillary lesions from the end-stage kidney to PRCC. In addition, the data point toward progenitor cell implication in the PRCC progression process, as detailed further below.

      Tracing Kidney Progenitor Cells Using IHC Reveals Their Enrichment in Kidney Injury and PA

      To test our hypothesis that PRCCs arise from a population of renal tubular regenerative/progenitor cells that are enriched in the end-stage kidney, the expression of previously reported renal stem cell/progenitor cell markers (BCL2 and CK7)
      • Lindgren D.
      • Boström A.-K.
      • Nilsson K.
      • Hansson J.
      • Sjölund J.
      • Möller C.
      • Jirström K.
      • Nilsson E.
      • Landberg G.
      • Axelson H.
      • Johansson M.E.
      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      was first compared between normal kidney, end-stage kidney, and PA. The assessment revealed enhanced renal tubular staining of both markers in ESKD (Figure 3, A–D) and diffused strong staining in PA (Figure 3E) compared with the normal kidney. A population of small-sized budding regenerating tubules present in areas of kidney injury that strongly stained with these markers was particularly noted, and these tubules were always increased in number around areas containing PA (Figure 3, F and G).
      Figure thumbnail gr3
      Figure 3Immunohistochemical staining of reported renal tubular progenitor cell markers B-cell lymphoma 2 (BCL2) and cytokeratin 7 (CK7). A and C: Representative photomicrographs showing a small number of tubular epithelial cells staining for these markers in the normal kidney. B and D: In the end-stage kidney, almost all renal tubular cells show strong positive staining for these markers. E: Papillary adenoma staining diffusely positive for BCL2. F: Budding regenerative tubules staining strongly positive for BCL2. G: Budding tubules staining strongly positive for CK7 (red arrows) next to a papillary adenoma (black arrow), which is also staining strong diffuse positive for the marker. Scale bars = 200 μm.
      Aperio image analysis software was used to quantify strong BCL2 and CK7 staining cells in normal kidney, ESKD, and PA. Compared with normal kidney, there was a significant increase in that tracked cell population in ESKD (BCL2, P < 0.0001; CK7, P < 0.0001) and PA (BCL2, P = 0.02; CK7, P = 0.07). CK7 and BCL2 staining was also increased significantly in the budding/atrophic tubules (BCL2, P <0.0001; CK7, P = 0.04) and in area surrounding PA (BCL2, P = 0.005; CK7, P = 0.36) compared with the normal kidney (Figure 4, A and B). Our results indicate an increase in a kidney progenitor cell population in kidney injury, which likely causes the budding regenerative tubules, then PA, and eventually PRCC.
      Figure thumbnail gr4
      Figure 4Immunohistochemical staining of reported renal tubular progenitor cell markers BCL2 and CK7. A: Digital image analysis for BCL2-stained slides from five different kidney conditions shows a significant increase of the BCL2-positive cell population (progenitor cells) in end-stage kidney disease (ESKD) as well as in papillary adenoma (PA), its surrounding background, and the budding surrounding tubules. B: Digital image analysis for strong CK7-stained progenitor cells shows a significant increase of the cell population in the ESKD as well as in the PA, its surrounding background, and the budding surrounding tubules. *P < 0.05, **P < 0.01, and ****P < 0.0001. NSR, a score that assesses the percentage of strong positive-stained cells.
      Next, another stem cell marker, LGR5, was assessed to further validate that the renal tubular cells staining for BCL2 and CK7 are a progenitor cell population. LGR5 is known to contribute to kidney development and marks epithelial stem cells of adult organs that use WNT signaling as a regenerative pathway analogous to the kidneys.
      • Curcio C.
      • Lanzini M.
      • Calienno R.
      • Mastropasqua R.
      • Marchini G.
      The expression of LGR5 in healthy human stem cell niches and its modulation in inflamed conditions.
      • Baker A.-M.
      • Graham T.A.
      • Elia G.
      • Wright N.A.
      • Rodriguez-Justo M.
      Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.
      • Barker N.
      • Rookmaaker M.B.
      • Kujala P.
      • Ng A.
      • Leushacke M.
      • Snippert H.
      • van de Wetering M.
      • Tan S.
      • Van Es J.H.
      • Huch M.
      • Poulsom R.
      • Verhaar M.C.
      • Peters P.J.
      • Clevers H.
      Article Lgr5 + ve stem/progenitor cells contribute to nephron formation during kidney development.
      ESKD and areas of kidney injury exhibited enhanced LGR5 immunostaining compared with the normal kidney (P = 0.0002) (Figure 5, A–C). When costaining LGR5 and BCL2 on the same slides, LGR5-stained areas corresponded to the areas that stained strongly for BCL2 (Figure 5, D–I). Taken together, these data provide evidence that a renal tubular progenitor/stem cell population is enhanced in kidney injury and can cause PA; consequently, PRCC is the most prevalent RCC in end-stage kidney disease.
      • Inoue T.
      • Matsuura K.
      • Yoshimoto T.
      • Nguyen L.T.
      • Tsukamoto Y.
      • Nakada C.
      • Hijiya N.
      • Narimatsu T.
      • Nomura T.
      • Sato F.
      • Nagashima Y.
      • Kashima K.
      • Hatakeyama S.
      • Ohyama C.
      • Numakura K.
      • Habuchi T.
      • Nakagawa M.
      • Seto M.
      • Mimata H.
      • Moriyama M.
      Genomic profiling of renal cell carcinoma in patients with end-stage renal disease.
      Figure thumbnail gr5
      Figure 5Immunostaining for leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5; an epithelial stem cell marker). A: Normal kidney showing markedly less staining. B: Staining is significantly increased in end-stage kidney disease (ESKD). C: Quantitative image analysis showing significantly less staining with LGR5 in the normal kidney than in the ESKD. DI: LGR5 and BCL2 costaining; LGR5 is shown in green, and BCL2 is shown in brown. D and E: Sections from normal kidney showing weak blush separate staining for both markers. FI: Sections from end-stage kidney confirming LGR5 stain the same cell population as BCL2 in regenerative tubules. Both markers show increased intensified costaining. ***P < 0.001. Scale bars: 200 μm (A and B); 50 μm (DI). NSR, a score that assesses the percentage of strong positive-stained cells.

      Exome Sequencing Ascertains the Connection between PA and PRCC Subtypes

      Whole exome sequencing of PA, PRCC1, PRCC2, and PRCC3 was performed (Supplemental Figure S5). There was considerable overlap between PRCC and PA, with approximately 50% of the PA mutations common among all PRCCs (Figure 6), further confirming our hypothesis of a common precursor origin to all PRCC subtypes that start with PA.
      Figure thumbnail gr6
      Figure 6Whole exome sequencing reveals connections between papillary adenoma (PA) and all the PRCC subtypes (P). Venn diagram showing the considerable overlap among the PRCC subtypes in regard to their PA shared mutational signature. Approximately 50% of the variants shared with the PA group are common to all subtypes, also showing that the PAs are equally related to all PRCCs.
      Next, possible germline variants were excluded by eliminating those that were previously reported in germline settings (had a dbSNP ID). Subsequently, only variant mutations with a projected functional significance on the corresponding protein were selected, excluding potentially nonfunctional variants.
      Illumina Variant Interpreter informatics software version Beta was used to assess the tumors' mutational signatures. These signatures vary between tumors and are thought to carry imprints of previous mutagenic exposures.
      • Greenman C.
      • Stephens P.J.
      • Smith R.
      • Dalgliesh G.L.
      • Hunter C.
      • Bignell G.R.
      • et al.
      UKPMC Funders Group patterns of somatic mutation in human cancer genomes.
      All papillary lesions (PA and PRCC subtypes) showed near identical somatic mutational signatures, with >90% of the mutations conferring to signatures 1, 12, 5, and 20
      • Letouzé E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.
      • Tubacher E.
      • Bayard Q.
      • Bacq D.
      • Meyer V.
      • Semhoun J.
      • Bioulac-sage P.
      • Prévôt S.
      • Azoulay D.
      • Paradis V.
      • Imbeaud S.
      • Deleuze J.
      • Zucman-rossi J.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver.
      • Alexandrov L.B.
      • Stratton M.R.
      Mutational signatures: the patterns of somatic mutations hidden in cancer genomes.
      (Supplemental Figure S6). Signatures 1 and 5 are tumor age-related signatures that are unanimously found in all cancers. Signature 20 is associated with mismatch repair aberrations or prolonged exposure to mutagens. It was consistently around approximately 15% of the mutations in all papillary cases. Signature 12, however, is still of unknown association and has been previously reported only in liver cancer.
      • Letouzé E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.
      • Tubacher E.
      • Bayard Q.
      • Bacq D.
      • Meyer V.
      • Semhoun J.
      • Bioulac-sage P.
      • Prévôt S.
      • Azoulay D.
      • Paradis V.
      • Imbeaud S.
      • Deleuze J.
      • Zucman-rossi J.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver.
      This unique combination of mutational signatures in PRCCs has not been previously reported (Supplemental Figure S6).

      Exome Sequencing Reveals Epigenetic Regulators as Potential Tumor Triggers

      A total of eight significant somatic gene mutations were unanimously present in all papillary lesions (PA and all PRCC subtypes). These mutations corresponded to the genes lysine methyl transferase 2 C (KMT2C), dual specificity phosphatase 5 (DUSP5), UGGT2, LIMD1, OR9G1, CACNA1B, FRG1B, and PRAMEF6 (Table 2). Of these, chromosome 7 KMT2C frameshift insertion (G>G/GT; 15195071; alternate variant frequency, approximately 20%) was the only variant reported previously in cancer in the COSMIC database
      • Patil V.
      • Pal J.
      • Somasundaram K.
      Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing.
      • Furney S.J.
      • Turajlic S.
      • Fenwick K.
      • Lambros M.B.
      • Mackay A.
      • Ricken G.
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      • Kozarewa I.
      • Hakas J.
      • Zvelebil M.
      • Lord C.J.
      • Ashworth A.
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      • Herlyn M.
      • Murata H.
      • Marais R.
      Genomic characterisation of acral melanoma cell lines.
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      • Chang K.
      • Dinh H.H.
      • Drummond J.A.
      • et al.
      Cancer Genome Atlas Network
      Comprehensive molecular characterization of human colon and rectal cancer.
      (Supplemental Figure S7). This gene is reported to act as an epigenetic regulator and a possible tumor suppressor gene.
      • Liu L.
      • Kimball S.
      • Liu H.
      • Holowatyj A.
      • Yang Z.-Q.
      Genetic alterations of histone lysine methyltransferases and their significance in breast cancer.
      Another unanimous interesting gene variant is the chromosome 10 DUSP5, with a possible role in oncogenesis (GC>GC/AT; 112266822; alternate variant frequency, approximately 90%),
      • Ueda K.
      • Arakawa H.
      • Nakamura Y.
      Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53.
      which is also reported to interplay with many of our significant signaling pathways.
      • Osaki L.H.
      • Gama P.
      MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation.
      • Bikkavilli R.K.
      • Feigin M.E.
      • Malbon C.C.
      p38 Mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3.
      Pathway enrichment of the genes common to all papillary lesions shows that they all share embryonic developmental pathways (P = 0.008), chromatin organization pathways (P = 0.02), and MAPK signaling pathways (P = 0.01).
      Table 2Mutations: SNVs and Indels That Are Common to all Papillary Lesions (PA, PRCC1, PRCC2, and PRCC3)
      GeneVariantChromosomeCoordinateTypeConsequence
      KMT2CG>G/GT7151945071InsertionFrameshift variant
      UGGT2TTT>TTT/CTC1396540141MNPSplice region variant
      DUSP5GC>GC/AT10112266822MNPMissense variant
      LIMD1GCA>GCA/TGC345677637MNPSplice region variant
      OR9G1AC>AC/GT1156468047MNPMissense variant
      CACNA1BTGGTGAG---------------- (3584)9140773610Deletion
      FRG1BG>G/A2029623254SNVSplice region variant
      Indel, insertion/deletion; MNP, multinucleotide pleomorphism; PA, papillary adenoma; SNV, single-nucleotide variant.

      A Unique Set of Mutations and Pathways Specific to Each PRCC Subtype, Denoting Their Unique Identities

      Genes were additionally assessed with somatic mutations that are exclusive or common to each of the specific PRCC subtypes. Exclusive for the PRCC2 group, a unanimous mutation was detected in the chromosome 2 FN1 (fibronectin 1) gene (GC>GC/AA; 216299550). In previous reports, FN1 correlated with aggressive RCC (particularly PRCC) and thyroid cancers.
      • Sponziello M.
      • Rosignolo F.
      • Celano M.
      • Maggisano V.
      • Pecce V.
      • De Rose R.F.
      • Lombardo G.E.
      • Durante C.
      • Filetti S.
      • Damante G.
      • Russo D.
      • Bulotta S.
      Fibronectin-1 expression is increased in aggressive thyroid cancer and favors the migration and invasion of cancer cells.
      • Waalkes S.
      • Atschekzei F.
      • Kramer M.W.
      • Hennenlotter J.
      • Vetter G.
      • Becker J.U.
      • Stenzl A.
      • Merseburger A.S.
      • Schrader A.J.
      • Kuczyk M.A.
      • Serth J.
      Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer.
      Also, fibronectin matrix formation projected as significantly enriched in PRCC2 (P = 0.02). A consistent deletion in the group of protocadherin γ subfamily genes (PCDHG 5q31.3) in most PRCC2 and PRCC3 cases was also discovered. To confirm this finding, The Cancer Genome Atlas PRCC cohort was assessed for similar deletions. Twenty-five cases with deletions in different members of the PCDHG family, all of which had a morphology corresponding to a higher-grade PRCC (PRCC2 or PRCC3), were detected. In comparison, none of the PRCC1 cases had deletions in the PCDHG family. Of interest, the FGFR4 gene (5q35.2), which lies close to the PCDHG locus consistently, harbored a homozygous single-nucleotide polymorphism (SNP; A>G/G; 176523597) across all PRCC2 cases. However, whether FGFR4 was a germline mutation given that it had a dbSNP ID could not be excluded. We have repeatedly identified FGF signaling to be activated in PRCC2
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      (Table 3).
      Table 3Genes Common or Exclusive to Each PRCC Group
      GeneVariantChromosomeTypeCoordinateConsequence variantdbSNP IDCOSMIC IDOther groups
      PRCC1
      NOTCH1 (2/3 and 1/3)G>G/A

      G>G/A
      9SNV

      SNV
      139399892

      139400340
      Missense

      Splice
      No

      No
      No

      No
      None
      NOTCH2 (1/3)T>T/C1SNV120572612Splice acceptorNoNoNone
      RASA4 (3/3)A>A/G7SNV102234975MissenseNoNoPRCC2 and PRCC3 (1/3 and 2/3)
      PRCC2
      PCDHG genes (2/3)Deletion 7 and 20 genes5DeletionN/AN/ANoNoPRCC3 (2/3)
      FN1 (3/3)GC>GC/AA2MNP216299550SpliceNoNoNone
      FGFR4 (2/3)G>G/A5SNV176520243MissenseYesYesPRCC3 (1/3)
      PRCC3
      NCOR2 (2/3)T>T/TGCCG12Insertion124824739FrameshiftNoYesPRCC2 (1/3)
      MET pathway-related genes among all PRCCs
      HGFT>T/C7SNV81346685SpliceYesNoAll PRCC1

      All PRCC2

      All PRCC3

      All PA
      ITGB1C>C/A10SNV33190567SpliceYesNoAll PRCC1

      All PRCC2

      All PRCC3

      No PA
      LRRK2G>G/A12SNV40619082MissenseYesNoAll PRCC1

      No PRCC2

      1 PRCC3

      All PA
      GGA2G>G/C16SNV23521643SpliceYesNoAll PRCC1

      All PRCC2

      All PRCC3

      All PA
      For dbSNP ID, reported germline mutations; for COSMIC ID, mutations reported in cancer.
      COSMIC, Catalog of Somatic Mutations in Cancer; dbSNP, Single Nucleotide Polymorphism Database; ID, identification; MET, mesenchymal–epithelial transition; MNP, multinucleotide pleomorphism; N/A, not applicable; PA, papillary adenoma; SNV, single-nucleotide variant.
      The PRCC3 group had several consistently enriched immune signaling pathways, including programmed cell death protein 1 (PD-1) signaling (P = 1.11 × 1017), cytokine signaling (P = 1.26 × 107), and immune system (P = 0.009). Tumor microenvironment immune dysregulation occurs secondarily to abnormalities in the TGF-β signaling pathway.
      • Papageorgis P.
      • Stylianopoulos T.
      Role of TGFB in regulation of the tumor microenvironment and drug delivery (review).
      In addition, two of three PRCC3 cases had a frameshift insertion in NCOR2 (also implicated in the TGF-β pathway), and one of three PRCC2 (T>T/TGCCG; 124824739) versus no cases in the PRCC1. An enrichment in the TGF-β and immune-related pathways in PRCC3 has been previously noted
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      (Table 3). Consistent with these findings, the PRCC3 group harbored an inverse translocation (15:41853734:76553740) among all its samples that hold TGF-β pathway gene SMAD3. In comparison, the PRCC1 and PRCC2 groups carried a unanimous inverse translocation (16:14580876:33950176), which holds many of the ABC transporter family genes.
      The NOTCH1 or NOTCH2 gene was exclusively mutated in PRCC1 group. The NOTCH pathway is known to interact with the WNT pathway that is activated in PRCC1.
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Wang R.
      • Sun Q.
      • Wang P.
      • Liu M.
      • Xiong S.
      • Luo J.
      • Huang H.
      • Du Q.
      • Geller D.A.
      • Cheng B.
      Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.
      In addition, the PRCC1 subtype showed enrichment in the RAS/MAPK signaling (P = 0.01). The RASA4 gene showed multiple SNPs among PRCC1 cases and was less common among other subtypes. The RAS/MAPK interacts with the NOTCH,
      • Mittal S.
      • Subramanyam D.
      • Dey D.
      • Kumar R.V.
      • Rangarajan A.
      Cooperation of Notch and Ras/MAPK signaling pathways in human breast carcinogenesis.
      β-catenin/Wnt, and TGF-β pathways, the last two particularly in colon cancer
      • Zeller E.
      • Hammer K.
      • Kirschnick M.
      • Braeuning A.
      Mechanisms of RAS/??-catenin interactions.
      • Cheruku H.R.
      • Mohamedali A.
      • Cantor D.I.
      • Tan S.H.
      • Nice E.C.
      • Baker M.S.
      Transforming growth factor-β, MAPK and Wnt signaling interactions in colorectal cancer.
      (Table 3).
      Other genes that were commonly mutated among the papillary lesions, however, had dbSNP IDs (so they cannot be confirmed as somatic mutations) and were HGF, ITGB1, and GGA2, all of which are implicated in the mesenchymal–epithelial transition (MET) pathway. The MET pathway has been repeatedly reported to be activated in PRCC.
      Cancer Genome Atlas Research Network
      Comprehensive molecular characterization of papillary renal cell carcinoma.
      • Albiges L.
      • Guegan J.
      • Le Formal A.
      • Verkarre V.
      • Rioux-Leclercq N.
      • Sibony M.
      • Bernhard J.C.
      • Camparo P.
      • Merabet Z.
      • Molinie V.
      • Allory Y.
      • Orear C.
      • Couvé S.
      • Gad S.
      • Patard J.J.
      • Escudier B.
      MET is a potential target across all papillary renal cell carcinomas: result from a large molecular study of pRCC with CGH array and matching gene expression array.
      PRCC3 had the highest prevalence of homozygous SNPs in these genes (three of three in HGF, three of three in ITGB1, and two of three in GGA2), followed by PRCC1 (two of three in HGF, two of three in GGA2, and none in ITGB1), and PRCC2 (two of three in HGF, two of three in ITGB1, and one of three in GGA2). No actual MET gene mutations were discovered in our cohort (Table 3).

      miRNA Pathway Analysis

      Cases of normal kidney and PA were selected for pathway analysis using their miRNA global expression profiles. After identifying a subset of miRNAs that were differentially expressed in adenomas compared with the normal kidney, target prediction, followed by pathway analysis, was performed to identify the top molecular pathways implicated in PA. Pathways enriched in PA pointed toward DNA damage (SUMOylation DNA damage), compromise of the p53 DNA damage checkpoint (p53 checkpoint damage), and initiation of the antiapoptotic and cell cycle pathways (G0 and early G1 cell cycle) (Supplemental Table S1). This suggests DNA damage and chromosomal instability as early events and is consistent with the CNV and mutational analyses. It also correlates with what is known about PRCC being the most prevalent RCC in end-stage kidney disease (49% to 75%).
      • Inoue T.
      • Matsuura K.
      • Yoshimoto T.
      • Nguyen L.T.
      • Tsukamoto Y.
      • Nakada C.
      • Hijiya N.
      • Narimatsu T.
      • Nomura T.
      • Sato F.
      • Nagashima Y.
      • Kashima K.
      • Hatakeyama S.
      • Ohyama C.
      • Numakura K.
      • Habuchi T.
      • Nakagawa M.
      • Seto M.
      • Mimata H.
      • Moriyama M.
      Genomic profiling of renal cell carcinoma in patients with end-stage renal disease.
      Of interest, there were many enriched pathways indicating induction of β-catenin. β-Catenin is known to induce the WNT signaling pathway, which we have previously reported to be enriched in PRCC.
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      It is also known to be the pathway activated in colonic adenomas, which is a known precursor lesion to colon cancer.
      • Kobayashi M.
      • Honma T.
      • Matsuda Y.
      • Suzuki Y.
      • Narisawa R.
      • Ajioka Y.
      • Asakura H.
      Nuclear translocation of beta-catenin in colorectal cancer.
      To validate the induction of the β-catenin pathway in PA, 16 PA cases were selected for IHC β-catenin staining. Normal β-catenin IHC staining is membranous; both internalization of staining and loss of membranous staining are associated with activation of β-catenin pathway.
      • Kobayashi M.
      • Honma T.
      • Matsuda Y.
      • Suzuki Y.
      • Narisawa R.
      • Ajioka Y.
      • Asakura H.
      Nuclear translocation of beta-catenin in colorectal cancer.
      • Demunter A.
      • Libbrecht L.
      • Degreef H.
      • De Wolf-Peeters C.
      • van den Oord J.J.
      Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations.
      A total of 15 of 16 PA cases showed abnormal β-catenin staining, validating our results obtained through Gene Set Enrichment Analysis (Figure 7, A–C). Similarly, there was aberrant staining (complete loss) in six of seven PRCC cases of the different subtypes (Supplemental Figure S8).
      Figure thumbnail gr7
      Figure 7β-Catenin shows abnormal staining patterns in papillary adenoma (PA) lesions. Two staining patterns are reported to be associated with β-catenin activation, loss of membranous expression, and diffuse cytoplasmic and nuclear (internalization) staining. A: PA with loss of membranous β-catenin expression (right panel); the surrounding renal tubules from the same case show normal membranous expression (left panel). This pattern is associated in the literature with its activation. B: A small PA exhibiting diffuse cytoplasmic and nuclear (internalization) staining of β-catenin, which is another pattern associated with β-catenin activation. C: A large PA showing partial cytoplasmic and nuclear staining with β-catenin (red arrow), also indicating at least a partial activation of the pathway. Scale bars = 200 μm.

      The Evolution of PRCC into Different Subtypes Starts Early in the Disease Process

      Tumors that arise from normal progenitor cells as colonic adenocarcinoma tend to change their expression patterns of progenitor cell marker after tumor formation, depending on the cellular hierarchy of the tumor.
      • Baker A.-M.
      • Graham T.A.
      • Elia G.
      • Wright N.A.
      • Rodriguez-Justo M.
      Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.
      Generally, the LGR5 progenitor cell marker is associated with a worse prognosis in such tumors.
      • Baker A.-M.
      • Graham T.A.
      • Elia G.
      • Wright N.A.
      • Rodriguez-Justo M.
      Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.
      • Sun B.
      • Ye X.
      • Li Y.
      • Zhang W.
      Lgr5 is a potential prognostic marker in patients with cervical carcinoma.
      • de Sousa e Melo F.
      • Kurtova A.V.
      • Harnoss J.M.
      • Kljavin N.
      • Hoeck J.D.
      • Hung J.
      • Anderson J.E.
      • Storm E.E.
      • Modrusan Z.
      • Koeppen H.
      • Dijkgraaf G.J.P.
      • Piskol R.
      • de Sauvage F.J.
      A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer.
      • Michelotti G.
      • Jiang X.
      • Sosa J.A.
      • Diehl A.M.
      • Henderson B.
      LGR5 is associated with tumor aggressiveness in papillary thyroid cancer.
      To gain a better understanding of the evolution of PRCCs, three renal tubular progenitor cell markers were assessed: BCL2, CK7, and LGR5 in PA and PRCC. The PRCC1 and PRCC3 groups showed a significantly increased expression of the BCL2 and CK7 markers than the PRCC2 group. Conversely, the more aggressive PRCC2 subtype showed significantly increased staining with the LGR5 marker (Supplemental Figure S9A). The data are also consistent with what is known about the expression of CK7 and its tendency to stain more PRCC1 tumors.
      • Chevarie-Davis M.
      • Riazalhosseini Y.
      • Arseneault M.
      • Aprikian A.
      • Kassouf W.
      • Tanguay S.
      • Latour M.
      • Brimo F.
      Morphologic and immunohistochemical spectrum of papillary renal cell carcinoma: study including 132 cases with pure type 1 and type 2 morphology as well.
      Subsequently, the expression of these markers was examined in PA. The PA cases were divided on the basis of their ABCC2 staining. The PA1 cases were the cases that showed complete absence of ABCC2 stain. The PA2 cases were the adenomas with diffuse strong ABCC2 staining analogous to the background renal tubules. The PA3 cases were cases with weaker ABCC2 staining. Samples that had diffuse strong ABCC2 expression (analogous to the PRCC2 from our previous data
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      ) showed a trend toward lower BCL2 expression but more LGR5 expression (a pattern that is comparable to PRCC2), whereas those with absent or patchy ABCC2 expression (similar to PRCC1 and PRCC3, respectively
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      ) showed higher BCL2 and lower LGR5 expression. However, the CK7 showed comparable expression among all groups (Supplemental Figure S9B). The results indicate a tendency toward subtype differentiation early on in the course of tumor development.

      A Proposed Model of PRCC Evolution Based on Integrated miRNA, CNV, and Mutational Analysis

      Combining together all enriched pathways obtained from ESKD, PA, and the three PRCC subtypes, we propose an interconnected PRCC evolution model with distinct events occurring at each tumor stage (Figure 8).
      • Guo X.
      • Wang X.-F.
      Signaling cross-talk between TGF-beta/BMP and other pathways.
      • Katoh M.
      • Katoh M.
      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
      • Bikkavilli R.K.
      • Feigin M.E.
      • Malbon C.C.
      p38 Mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3.
      • Wang R.
      • Sun Q.
      • Wang P.
      • Liu M.
      • Xiong S.
      • Luo J.
      • Huang H.
      • Du Q.
      • Geller D.A.
      • Cheng B.
      Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.
      • Cheruku H.R.
      • Mohamedali A.
      • Cantor D.I.
      • Tan S.H.
      • Nice E.C.
      • Baker M.S.
      Transforming growth factor-β, MAPK and Wnt signaling interactions in colorectal cancer.
      • Monga S.P.S.
      • Mars W.M.
      • Pediaditakis P.
      • Bell A.
      • Mulé K.
      • Bowen W.C.
      • Wang X.
      • Zarnegar R.
      • Michalopoulos G.K.
      Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes.
      • Mah K.M.
      • Houston D.W.
      • Weiner J.A.
      The γ-protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane.
      • Wilson S.I.
      • Rydström A.
      • Trimborn T.
      • Willert K.
      • Nusse R.
      • Jessell T.M.
      • Edlund T.
      The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.
      • Turner N.
      • Grose R.
      Fibroblast growth factor signalling: from development to cancer.
      • Xu Q.
      • Krause M.
      • Samoylenko A.
      • Vainio S.
      Wnt signaling in renal cell carcinoma.
      • Kim K.H.
      • Seol H.J.
      • Kim E.H.
      • Rheey J.
      • Jin H.J.
      • Lee Y.
      • Joo K.M.
      • Lee J.
      • Nam D.
      Wnt/β-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells.
      • Liu Y.
      • Wang T.
      • Yan J.
      • Jiagbogu N.
      • Heideman D.A.M.
      • Canfield A.E.
      • Alexander M.Y.
      HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.
      • Zhou D.
      • Tan R.J.
      • Fu H.
      • Liu Y.
      Wnt/β-catenin signaling in kidney injury and repair: a double-edged sword.
      Initiating the PA stage requires DNA damage, disruption of the p53 checkpoint, and turning on cell cycle propagation events, such as MAPK, MET signaling, NOTCH, and β-catenin activation.
      • Osaki L.H.
      • Gama P.
      MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation.
      • Liu Y.
      • Wang T.
      • Yan J.
      • Jiagbogu N.
      • Heideman D.A.M.
      • Canfield A.E.
      • Alexander M.Y.
      HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.
      • Hayward P.
      • Kalmar T.
      • Martinez Arias A.
      Wnt/Notch signalling and information processing during development.
      • Li B.
      • Jia Z.
      • Wang T.
      • Wang W.
      • Zhang C.
      • Chen P.
      • Ma K.
      • Zhou C.
      Interaction of Wnt/??-catenin and notch signaling in the early stage of cardiac differentiation of P19CL6 cells.
      • Prakash S.
      • Swaminathan U.
      • Nagamalini B.R.
      • Krishnamurthy A.B.
      Beta-catenin in disease.
      • Boivin F.J.
      • Sarin S.
      • Dabas P.
      • Karolak M.
      • Oxburgh L.
      • Bridgewater D.
      Stromal β-catenin overexpression contributes to the pathogenesis of renal dysplasia.
      Many of these events are enriched in end-stage kidney disease. These pathways interact with the WNT, TGF-β, and FGF pathways to further develop into different PRCC subtypes.
      • Katoh M.
      • Katoh M.
      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
      • Tiong K.H.
      • Mah L.Y.
      • Leong C.-O.
      Functional roles of fibroblast growth factor receptors (FGFRs) signaling in human cancers.
      • Wheler J.J.
      • Atkins J.T.
      • Janku F.
      • Moulder S.L.
      • Stephens P.J.
      • Yelensky R.
      • Valero V.
      • Miller V.
      • Kurzrock R.
      • Meric-Bernstam F.
      Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors.
      • Akhmetshina A.
      • Palumbo K.
      • Dees C.
      • Bergmann C.
      • Venalis P.
      • Zerr P.
      • Horn A.
      • Kireva T.
      • Beyer C.
      • Zwerina J.
      • Schneider H.
      • Sadowski A.
      • Riener M.-O.
      • MacDougald O.A.
      • Distler O.
      • Schett G.
      • Distler J.H.W.
      Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.
      Connecting all stages is an embryonic developmental theme (identified consistently by all molecular modalities) that points toward a progenitor cell origin for PRCC.
      Figure thumbnail gr8
      Figure 8Model proposed for PRCC evolution. A simplified illustration showing the connection between the pathways uncovered by the current study (copy number variation, miRNA, and mutational analysis) at the different tumor development stages. Genes showing mutations at each stage are depicted in red, next to its associated molecular pathway. Vertical dashed lines are the transition lines between normal kidney, papillary adenoma, and PRCC. Solid blue arrows show the resulting pathways when normal P53 and apoptosis are lost. Dotted blue arrows indicate connections between the pathways as depicted by the literature. The illustration shows a model in which PRCC tumors of different subtypes divert from a common precursor.
      • Guo X.
      • Wang X.-F.
      Signaling cross-talk between TGF-beta/BMP and other pathways.
      • Katoh M.
      • Katoh M.
      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
      • Bikkavilli R.K.
      • Feigin M.E.
      • Malbon C.C.
      p38 Mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3.
      • Wang R.
      • Sun Q.
      • Wang P.
      • Liu M.
      • Xiong S.
      • Luo J.
      • Huang H.
      • Du Q.
      • Geller D.A.
      • Cheng B.
      Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.
      • Cheruku H.R.
      • Mohamedali A.
      • Cantor D.I.
      • Tan S.H.
      • Nice E.C.
      • Baker M.S.
      Transforming growth factor-β, MAPK and Wnt signaling interactions in colorectal cancer.
      • Monga S.P.S.
      • Mars W.M.
      • Pediaditakis P.
      • Bell A.
      • Mulé K.
      • Bowen W.C.
      • Wang X.
      • Zarnegar R.
      • Michalopoulos G.K.
      Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes.
      • Mah K.M.
      • Houston D.W.
      • Weiner J.A.
      The γ-protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane.
      • Wilson S.I.
      • Rydström A.
      • Trimborn T.
      • Willert K.
      • Nusse R.
      • Jessell T.M.
      • Edlund T.
      The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.
      • Turner N.
      • Grose R.
      Fibroblast growth factor signalling: from development to cancer.
      • Xu Q.
      • Krause M.
      • Samoylenko A.
      • Vainio S.
      Wnt signaling in renal cell carcinoma.
      • Kim K.H.
      • Seol H.J.
      • Kim E.H.
      • Rheey J.
      • Jin H.J.
      • Lee Y.
      • Joo K.M.
      • Lee J.
      • Nam D.
      Wnt/β-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells.
      • Liu Y.
      • Wang T.
      • Yan J.
      • Jiagbogu N.
      • Heideman D.A.M.
      • Canfield A.E.
      • Alexander M.Y.
      HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.
      • Zhou D.
      • Tan R.J.
      • Fu H.
      • Liu Y.
      Wnt/β-catenin signaling in kidney injury and repair: a double-edged sword.
      APC, adenomatous polyposis coli; Chr, chromosome; FGF, fibroblast growth factor; GK3β, glycogen synthase kinase-3 β; MAPK, mitogen-activated protein kinase; MET, mesenchymal–epithelial transition; TGF-β, transforming growth factor-β.

      Discussion

      PRCC and PA lesions are both common in ESKD.
      • Hora M.
      • Hes O.
      • Reischig T.
      • Urge T.
      • Klecka J.
      • Ferda J.
      • Michal M.
      • Eret V.
      Tumours in end-stage kidney.
      • Saleeb R.
      • Faragalla H.
      • Yousef G.M.
      • Stewart R.
      • Streutker C.J.
      Malignancies arising in allograft kidneys, with a first reported translocation RCC post-transplantation: a case series.
      • Klatte T.
      • Marberger M.
      Renal cell carcinoma of native kidneys in renal transplant patients.
      PA accompanies approximately 73% of RCCs in ESKD.
      • Denton M.D.
      • Magee C.C.
      • Ovuworie C.
      • Mauiyyedi S.
      • Pascual M.
      • Colvin R.B.
      • Cosimi A.B.
      • Tolkoff-Rubin N.
      Prevalence of renal cell carcinoma in patients with ESRD pre-transplantation: a pathologic analysis.
      The prevalence of one PRCC subtype or the other in ESKD is not clear from the literature reports. Some authors have reported similarities between chromosomal aberrations that occur in ESKD to PRCCs.
      • Hes O.
      • Sima R.
      • Nemcova J.
      • Hora M.
      • Bulimbasic S.
      • Kazakov D.V.
      • Urge T.
      • Reischig T.
      • Dvorak M.
      • Michal M.
      End-stage kidney disease: gains of chromosomes 7 and 17 and loss of Y chromosome in non-neoplastic tissue.
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      Renal cell carcinoma of native kidneys in renal transplant patients.
      Others have gone further to suggest that PRCCs are transcriptomically related to a regenerative/progenitor cell population in the renal tubules.
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      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      The biological and clinical differences between PRCC subtypes have previously been analyzed,
      • Saleeb R.M.
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      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Saleeb R.M.
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      • Farag M.
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      • Rotondo F.
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      • Wala S.
      • Plant P.
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      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      and a third PRCC subtype has been identified.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
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      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Pathways that were uncovered in all subtypes were feeding back to the embryonic developmental theme.
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      • Bell A.
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      • Michalopoulos G.K.
      Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes.
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      • Trimborn T.
      • Willert K.
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      • Jessell T.M.
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      The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.
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      • Kim E.H.
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      • Jin H.J.
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      Wnt/β-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells.
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      Interaction of Wnt/??-catenin and notch signaling in the early stage of cardiac differentiation of P19CL6 cells.
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      Wnt signaling in stem and cancer stem cells.
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      • Clevers H.
      Wnt signaling, Lgr5, and stem cells in the intestine and skin.
      These pathways (WNT, TGF-β, and FGF) are implicated in renal development
      • Takasato M.
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      • Chuva de Sousa Lopes S.M.
      • Little M.H.
      Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.
      and are also shown to be enriched secondarily to kidney injury (WNT, TGF-β).
      • Zhou D.
      • Tan R.J.
      • Fu H.
      • Liu Y.
      Wnt/β-catenin signaling in kidney injury and repair: a double-edged sword.
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      Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.
      • Brunskill E.W.
      • Potter S.S.
      Changes in the gene expression programs of renal mesangial cells during diabetic nephropathy.
      Of interest, these pathways interact in colonic adenocarcinoma, which is believed to arise from intestinal crypt regenerative/stem cells.
      • Abdul Khalek F.J.
      • Gallicano G.I.
      • Mishra L.
      Colon cancer stem cells.
      • Barker N.
      • van Es J.H.
      • Kuipers J.
      • Kujala P.
      • van den Born M.
      • Cozijnsen M.
      • Haegebarth A.
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      • Begthel H.
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      • Clevers H.
      Identification of stem cells in small intestine and colon by marker gene Lgr5.
      • Prange W.
      • Breuhahn K.
      • Fischer F.
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      • Pietsch T.
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      • Eilers R.
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      Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes.
      Hence, in this study, we tested the hypothesis that PRCC arises from kidney progenitor cells through studying the proposed precursor lesions: ESKD and PA.
      Our data showed that ESKD and PA share a significant number of chromosomal aberrations. To trace the hypothesized progenitor cell population, staining was performed with markers that were proposed to stain renal tubular progenitor cells (BCL2 and CK7),
      • Lindgren D.
      • Boström A.-K.
      • Nilsson K.
      • Hansson J.
      • Sjölund J.
      • Möller C.
      • Jirström K.
      • Nilsson E.
      • Landberg G.
      • Axelson H.
      • Johansson M.E.
      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      as well as another established stem cell marker (LGR5). LGR5 stains epithelial stem cells in organs that use the WNT pathway in regeneration (such as the kidney).
      • Curcio C.
      • Lanzini M.
      • Calienno R.
      • Mastropasqua R.
      • Marchini G.
      The expression of LGR5 in healthy human stem cell niches and its modulation in inflamed conditions.
      • Baker A.-M.
      • Graham T.A.
      • Elia G.
      • Wright N.A.
      • Rodriguez-Justo M.
      Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.
      • Zhou D.
      • Tan R.J.
      • Fu H.
      • Liu Y.
      Wnt/β-catenin signaling in kidney injury and repair: a double-edged sword.
      • Barker N.
      • van Es J.H.
      • Kuipers J.
      • Kujala P.
      • van den Born M.
      • Cozijnsen M.
      • Haegebarth A.
      • Korving J.
      • Begthel H.
      • Peters P.J.
      • Clevers H.
      Identification of stem cells in small intestine and colon by marker gene Lgr5.
      All three markers were significantly increased in ESKD compared with the normal kidney. LGR5 highlighted the same population of cells as CK7 and BCL2. Furthermore, the BCL2 and CK7 were also overexpressed in PA and the budding regenerative tubules that are commonly seen in injured kidney. BCL2 is an antiapoptotic marker; Lindgren et al
      • Lindgren D.
      • Boström A.-K.
      • Nilsson K.
      • Hansson J.
      • Sjölund J.
      • Möller C.
      • Jirström K.
      • Nilsson E.
      • Landberg G.
      • Axelson H.
      • Johansson M.E.
      Isolation and characterization of progenitor-like cells from human renal proximal tubules.
      found the apoptotic pathways to be down-regulated in kidney progenitor cells. BCL2 has also been shown to be crucial for tubule formation and repair after kidney injury.
      • Veis D.J.
      • Sorenson C.M.
      • Shutter J.R.
      • Korsmeyer S.J.
      Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.
      It was similarly noted that the antiapoptotic pathways were activated in ESKD (by miRNA pathway enrichment analysis). When analyzing chromosomal aberrations that were common between PA and PRCC, BCL2 was a central molecule in the interaction network.
      Considerable overlap was demonstrated between the PA and the three PRCC subtypes by CNVs, SNP, and indel genomic analysis, supporting our hypothesis that they arise from the same precursor lesion. PA and PRCCs shared aberrations in six chromosomal regions carrying genes involved in pathways that are significantly related to all papillary lesions (TGF-β, NOTCH, FGF, MAPK, and cell death/apoptosis).
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
      • Downes M.R.
      • Pace K.
      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Genes that held significant SNP and indel variants among all groups also shared a common theme of embryonic development, differentiation, chromosomal organization, and MAPK signaling pathways.
      To detect possible early driver genes at the essence of PRCC pathogenesis, significant somatic gene mutations that were common to PA and all PRCC subtypes were filtered. Eight genes were detected with specific variant mutations common to all lesions. Of these, the KMT2C frameshift insertion was previously reported in cancer.
      • Liu L.
      • Kimball S.
      • Liu H.
      • Holowatyj A.
      • Yang Z.-Q.
      Genetic alterations of histone lysine methyltransferases and their significance in breast cancer.
      KMT2C is a histone lysine methyl transferase, which induces methylation of histone lysine residue, thus causing chromatin organizational epigenetic changes.
      • Hyun K.
      • Jeon J.
      • Park K.
      • Kim J.
      Writing, erasing and reading histone lysine methylations.
      KMT2C is among the most frequently reported mutated genes in breast, prostate, bladder, and renal cancers,
      • Liu L.
      • Kimball S.
      • Liu H.
      • Holowatyj A.
      • Yang Z.-Q.
      Genetic alterations of histone lysine methyltransferases and their significance in breast cancer.
      • Rao R.C.
      • Dou Y.
      Hijacked in cancer: the MLL/KMT2 family of methyltransferases.
      • Humphrey P.A.
      • Moch H.
      • Cubilla A.L.
      • Ulbright T.M.
      • Reuter V.E.
      The 2016 WHO classification of tumours of the urinary system and male genital organs—part B: prostate and bladder tumours.
      and also one of the most common genes carrying mutations in The Cancer Genome Atlas PRCC cohort. It is reported to act as a tumor suppressor (through regulation of tumor enhancer genes), and although its mutations are commonly heterozygous (as in our tumors), some reported that they could reach full penetrance if accompanied by TP53 disruption and other oncogene activation.
      • Rao R.C.
      • Dou Y.
      Hijacked in cancer: the MLL/KMT2 family of methyltransferases.
      KMT2C naturally plays a role in cell development and differentiation; mice deficient in both gene alleles have developmental defects.
      • Rao R.C.
      • Dou Y.
      Hijacked in cancer: the MLL/KMT2 family of methyltransferases.
      Also, KMT2C is implicated in development through regulation of HOXA genes.
      • Wang P.
      • Lin C.
      • Smith E.R.
      • Guo H.
      • Sanderson B.W.
      • Wu M.
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      • Alexander T.
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      • Ge K.
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      Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II.
      The HOXA locus on chromosome 7 was one of two regions found to harbor gains across all papillary lesions and ESKDs. The DUSP5 was another gene of potential significance common to all papillary lesions. It is reported to be a direct target of p53 and, hence, acts as a tumor suppressor gene in cancer.
      • Ueda K.
      • Arakawa H.
      • Nakamura Y.
      Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53.
      • Osaki L.H.
      • Gama P.
      MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation.
      The miRNA pathway analysis of PA showed p53 checkpoint damage, which corresponds to DUSP5 activity, and may also be synergistic to the KMT2C mutational effect. Both genes are possible drivers of PRCC formation; however, the results need to be confirmed in larger cohorts and with mechanistic studies.
      The mutational analysis exhibited different sets of prevalent mutations among each subtype, confirming their unique identities. PRCC1 had more frequent mutations in the NOTCH1 and NOTCH2 genes. The NOTCH pathway interacts with the WNT pathway and the MET pathway, all of which were reported specifically in PRCC1.
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
      • Ding Q.
      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
      • Rompré-brodeur A.
      • Rotondo F.
      • Beharry V.
      • Wala S.
      • Plant P.
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      • Evans A.
      • Bjarnason G.
      • Bartlett J.M.
      • Yousef G.M.
      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      Cancer Genome Atlas Research Network
      Comprehensive molecular characterization of papillary renal cell carcinoma.
      PRCC2 had more prevalent mutations in FGFR4 and FN1 and common deletions in the PCDHG family genes. FGF was also enriched in PRCC2 (by miRNA and CNV analysis). It is a pathway that interacts with WNT signaling in kidney development.
      • Saleeb R.M.
      • Plant P.
      • Tawedrous E.
      • Krizova A.
      • Brimo F.
      • Evans A.J.
      • Wala S.J.
      • Bartlett J.
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      • Boles D.
      • Rotando F.
      • Farag M.
      • Yousef G.M.
      Integrated phenotypic/genotypic analysis of papillary renal cell carcinoma subtypes: identification of prognostic markers, cancer-related pathways, and implications for therapy.
      Tumors that have both WNT and FGF tend to have aggressive behavior.
      • Katoh M.
      • Katoh M.
      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
      • Katoh M.
      • Katoh M.
      WNT signaling pathway and stem cell signaling network.
      The PCDHG gene family members are located on chromosome 5q31 near the FGF1 gene and not far from FGFR4. In addition, the PCDH gene mutations are thought to enhance the release of β-catenin and WNT signaling, relating them to PA.
      • Mah K.M.
      • Houston D.W.
      • Weiner J.A.
      The γ-protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane.
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      Epigenetic silencing of protocadherin 10 in colorectal cancer (review).
      The FN1 gene correlates with aggressive forms of RCC (such as PRCC2).
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      PRCC3 harbors mutations in the NCOR genes that relate to the TGF-β pathway. Immune-related pathways, such as PD-1 signaling, were also implicated in PRCC3, comparable to our previous analysis.
      • Saleeb R.M.
      • Brimo F.
      • Farag M.
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      • Evans A.
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      Towards biological subtyping of papillary renal cell carcinoma with clinical implications through histological, immunohistochemical and molecular analysis.
      TGF-β pathways are recognized to affect the tumor immune microenvironment.
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      Role of TGFB in regulation of the tumor microenvironment and drug delivery (review).
      Integrating all of the molecular pathways uncovered at each potential stage, we propose an interconnected model of a sequence of events underlying PRCC development (Figure 8).
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      • Wang X.-F.
      Signaling cross-talk between TGF-beta/BMP and other pathways.
      • Katoh M.
      • Katoh M.
      Cross-talk of WNT and FGF signaling pathways at GSK3B to regulate B-catenin and SNAIL signaling cascades.
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      Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.
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      • Cantor D.I.
      • Tan S.H.
      • Nice E.C.
      • Baker M.S.
      Transforming growth factor-β, MAPK and Wnt signaling interactions in colorectal cancer.
      • Monga S.P.S.
      • Mars W.M.
      • Pediaditakis P.
      • Bell A.
      • Mulé K.
      • Bowen W.C.
      • Wang X.
      • Zarnegar R.
      • Michalopoulos G.K.
      Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes.
      • Mah K.M.
      • Houston D.W.
      • Weiner J.A.
      The γ-protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane.
      • Wilson S.I.
      • Rydström A.
      • Trimborn T.
      • Willert K.
      • Nusse R.
      • Jessell T.M.
      • Edlund T.
      The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.
      • Turner N.
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      Fibroblast growth factor signalling: from development to cancer.
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      HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.
      • Zhou D.
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      • Liu Y.
      Wnt/β-catenin signaling in kidney injury and repair: a double-edged sword.
      In that model, activation of β-catenin was at the core of early PRCC development (PA). The activation was validated further with the identification of irregular β-catenin expression among PA lesions. Both abnormal patterns of β-catenin staining in our cohort were associated in the literature with its activation.
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      Nuclear translocation of beta-catenin in colorectal cancer.
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      Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations.
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      Of interest, our results point to potential differentiation of PA toward the different PRCC subtypes, indicating that the evolution and differentiation of PRCCs might be an early event in the disease course. In this study, the PA cases selected for molecular analysis were all <0.5 cm (the old cutoff for PA versus the current 1.5 cm). The rationale was to capture the tumor precursor events as early as possible in the disease process before they evolve and diverge into different PRCC subtypes. Prospective molecular studies of PAs with different size cutoffs are warranted.
      Proper detection of PA lesions has a potential clinical significance regarding the early prevention of PRCC. In colon cancer, the early management of colonic adenomas has dramatically reduced the incidence of colonic adenocarcinoma. Hence, our enhanced understanding of the renal precursor lesions in the era of advanced imaging and localized ablation techniques may also reduce the incidence of PRCC.
      In conclusion, we provide evidence that PRCC potentially originates from kidney tubular regenerative/progenitor cells and elaborate on the possible evolution of PRCC from early preneoplastic lesions into the different neoplastic subtypes. We uncover potential tumor driver genes that have not been reported before in PRCC. We further validate and expand on the biological differences between the fully developed PRCC subtypes, which can guide their prospective management. The limitations of our study include the limited number of samples analyzed with whole exome sequencing (n = 12) and the heterogenic nature of the PRCC biology. This study provides additional evidence as to the pathogenesis of PRCC; however, some of the findings need to be validated by additional studies.

      Acknowledgments

      We thank Bhooma Thiruvahindrapuram, Gaganjot Kuar (bioinformatic group), and Sergio Pereira (next-generation sequencing facility manager) (The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON, Canada) for assistance with the bioinformatics analysis and technology assistance.

      Supplemental Data

      • Supplemental Figure S2

        Ingenuity Pathway Analysis of the six regions highlights interactions for cell death and development, embryonic development, and cell to cell signaling, with BCL2 and NCOR1 genes at the core of the interactions. Among these interactions, the genes highlighted in yellow are part of the two regions that are also shared by the end-stage kidney disease.

      • Supplemental Figure S3

        Ingenuity Pathway Analysis of the genes of the six regions common to papillary adenoma and PRCC subtypes. Glycogen synthase kinase-3 β (GSK3β) and AKT are at the center of the projected interactions. Molecules in yellow are also part of the two regions shared by the end-stage kidney disease.

      • Supplemental Figure S4

        Ingenuity Pathway Analysis of the two chromosome 7 regions that show gains in end-stage kidney disease (ESKD), and all papillary groups show cell cycle, cell development, and cell injury pathways. The projected interactions highlight the embryonic development components homeobox A (HOXA), DNA repair breast cancer 1 (BRCA1), and mitogen-activated protein kinase (MAPK) molecules at the core of the interactions. PA, papillary adenoma.

      • Supplemental Figure S5

        Whole exome sequencing and number of single-nucleotide variants (SNVs) and insertion/deletion (indel) variants present in each papillary group. Data are expressed as means ± SD. PA, papillary adenoma.

      • Supplemental Figure S6

        Whole exome sequencing reveals connections between papillary adenoma (PA) and all the PRCC subtypes. All papillary lesions harbor the same set of mutational signatures (signatures 1, 5, 12, and 20). A: PA. B: PRCC1. C: PRCC2. D: PRCC3.

      • Supplemental Figure S7

        Whole exome sequencing reveals connections between papillary adenoma (PA) and all the PRCC subtypes. Epigenetic regulator KMT2C gene carries the same mutational frameshift insertion in all papillary lesions. Different groups of papillary lesions are as follows: PA (A), PRCC1 (B), PRCC2 (C), and PRCC3 (D).

      • Supplemental Figure S9

        Progenitor cell markers [B-cell lymphoma 2 (BCL2), cytokeratin 7 (CK7), and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)] in the three PRCC subtypes and papillary adenomas indicate that the differentiation starts early on in the disease course. A: The BCL2 and CK7 markers show highest level of staining in the PRCC1, followed by the PRCC3 and PRCC2 groups, whereas LGR5 shows the exact reverse, with strongest staining (expression) in the PRCC2, followed by the PRCC3 and PRCC1. This is consistent with what is known regarding the association of LGR5 expression with aggressive tumors and CK7 higher expression in PRCC1. B: The papillary adenoma (PA) cohort was divided using our previously published immunohistochemistry (IHC) panel

        • Saleeb R.M.
        • Brimo F.
        • Farag M.
        • Rompré-brodeur A.
        • Rotondo F.
        • Beharry V.
        • Wala S.
        • Plant P.
        • Downes M.R.
        • Pace K.
        • Evans A.
        • Bjarnason G.
        • Bartlett J.M.
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        into PA1, PA2, and PA3, analogous to the PRCC1, PRCC2, and PRCC3 subtypes, respectively. The staining patterns of the different PA subgroups match their related PRCC group on both BCL2 and LGR5 stains. The CK7 did not project significant differences in staining between the groups. The results indicate that the differentiation into different subtypes starts at the stage of PA. Data are expressed as means ± SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

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