title: Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing.
abstract: OBJECTIVE
  A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set).
  
  
  METHODS
  Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq®Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor).
  
  
  RESULTS
  The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R²=.95 and R²=.94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial-mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-β1and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets.
  
  
  CONCLUSIONS
  This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|b Department of Medicine , Haukeland University Hospital , Bergen , Norway.
authors.forename: Oystein S
authors.initials: OS
authors.lastname: Eikrem
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|b Department of Medicine , Haukeland University Hospital , Bergen , Norway.
authors.forename: Philipp
authors.initials: P
authors.lastname: Strauss
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|c Department of Urology , Haukeland University Hospital , Bergen , Norway.
authors.forename: Christian
authors.initials: C
authors.lastname: Beisland
authors.affiliation: d Spheromics , Kontiolahti , Finland.|e Institute for Molecular Medicine Finland (FIMM), University of Helsinki , Helsinki , Finland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Scherer
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.
authors.forename: Lea
authors.initials: L
authors.lastname: Landolt
authors.affiliation: f Department of Cancer Research and Molecular Medicine , Norwegian University of Science and Technology , Trondheim , Norway.
authors.forename: Arnar
authors.initials: A
authors.lastname: Flatberg
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|g Department of Pathology , Haukeland University Hospital , Bergen , Norway.
authors.forename: Sabine
authors.initials: S
authors.lastname: Leh
authors.affiliation: f Department of Cancer Research and Molecular Medicine , Norwegian University of Science and Technology , Trondheim , Norway.
authors.forename: Vidar
authors.initials: V
authors.lastname: Beisvag
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.
authors.forename: Trude
authors.initials: T
authors.lastname: Skogstrand
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|c Department of Urology , Haukeland University Hospital , Bergen , Norway.
authors.forename: Karin
authors.initials: K
authors.lastname: Hjelle
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.
authors.forename: Anjana
authors.initials: A
authors.lastname: Shresta
authors.affiliation: a Department of Clinical Medicine , University of Bergen , Bergen , Norway.|b Department of Medicine , Haukeland University Hospital , Bergen , Norway.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Marti
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics; D002097:C-Reactive Protein / Q000235:genetics; D000071231:Carbonic Anhydrase IX / Q000235:genetics*; D002292:Carcinoma, Renal Cell / Q000175:diagnosis / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000235:genetics*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D007680:Kidney Neoplasms / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D015398:Signal Transduction / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000235:genetics; D058949:Uromodulin / Q000235:genetics; D000099153:Pentraxins
chemical_list: D014408:Biomarkers, Tumor; D002097:C-Reactive Protein; D000071231:Carbonic Anhydrase IX; D015815:Cell Adhesion Molecules; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D053773:Transforming Growth Factor beta1; D058949:Uromodulin; D000099153:Pentraxins; C072273:TNFAIP6 protein, human; C064045:delta protein
keywords: CA9; PNCK; TNFAIP6; clear cell renal cell carcinoma; formalin-fixed and paraffin-embedded biopsies; high-throughput RNA sequencing; transcriptome