title: Gene-expression analysis of a colorectal cancer-specific discriminatory transcript set on formalin-fixed, paraffin-embedded (FFPE) tissue samples.
abstract: BACKGROUND
  A recently published transcript set is suitable for gene expression-based discrimination of normal colonic and colorectal cancer (CRC) biopsy samples. Our aim was to test the discriminatory power of the CRC-specific transcript set on independent biopsies and on formalin-fixed, paraffin-embedded (FFPE) tissue samples.
  
  
  METHODS
  Total RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) from fresh frozen biopsies stored in RNALater (CRC (n = 15) and healthy colonic (n = 15)), furthermore from FFPE specimens including CRC (n = 15) and normal adjacent tissue (NAT) (n = 15) specimens next to the tumor. After quality and quantity measurements, gene expression analysis of a colorectal cancer-specific marker set with 11 genes (CA7, COL12A1, CXCL1, CXCL2, CHI3L1, GREM1, IL1B, IL1RN, IL8, MMP3, SLC5A7) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler480 System (Roche). In situ hybridization for two selected transcripts (CA7, CXCL1) was performed on NAT (n = 3), adenoma (n = 3) and CRC (n = 3) FFPE samples.
  
  
  RESULTS
  Although analytical parameters of automatically isolated RNA samples showed differences between fresh frozen biopsy and FFPE samples, both quantity and the quality enabled their application in gene expression analyses. CRC and normal fresh frozen biopsy samples could be distinguished with 93.3% sensitivity and 86.7% specificity and FFPE samples with 96.7 and 70.0%, respectively. In situ hybridization could confirm the upregulation of CXCL1 and downregulation of CA7 in colorectal adenomas and tumors compared to healthy controls.
  
  
  CONCLUSIONS
  According to our results, gene expression analysis of the analyzed colorectal cancer-specific marker set can also be performed from FFPE tissue material. With the addition of an automated workflow, this marker set may enhance the objective classification of colorectal neoplasias in the routine procedure in the future.
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. alexandra.kalmar@gmail.com.|Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. alexandra.kalmar@gmail.com.|2nd Department of Medicine Semmelweis University, Szentkirályi str. 46., 1088, Budapest, Hungary. alexandra.kalmar@gmail.com.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kalmár
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. wwbarna@yahoo.com.
authors.forename: Barnabás
authors.initials: B
authors.lastname: Wichmann
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. orsg1@yahoo.com.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Galamb
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. spisak_sandor@yahoo.com.
authors.forename: Sándor
authors.initials: S
authors.lastname: Spisák
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. drtothkinga@yahoo.com.
authors.forename: Kinga
authors.initials: K
authors.lastname: Tóth
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. leiszterkata@gmail.com.
authors.forename: Katalin
authors.initials: K
authors.lastname: Leiszter
authors.affiliation: Bioneer A/S, Hørsholm, Denmark. bsn@bioneer.dk.
authors.forename: Boye Schnack
authors.initials: BS
authors.lastname: Nielsen
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. bartak.barbara@gmail.com.
authors.forename: Barbara Kinga
authors.initials: BK
authors.lastname: Barták
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. tulassay.zsolt@med.semmelweis-univ.hu.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary. molnar.bela1@med.semmelweis-univ.hu.
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
mesh_terms: D019540:Area Under Curve; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics*; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization; D016612:Paraffin Embedding; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde