Gene-expression analysis of a colorectal cancer-specific discriminatory transcript set on formalin-fixed, paraffin-embedded (FFPE) tissue samples

Alexandra Kalmár, Barnabás Wichmann, Orsolya Galamb, Sándor Spisák, Kinga Tóth, Katalin Leiszter, Boye Schnack Nielsen, Barbara Kinga Barták, Zsolt Tulassay, Béla Molnár

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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 LightCycler®480 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. Conclusion: 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.

Original languageEnglish
Article number126
JournalDiagnostic Pathology
Volume10
Issue number1
DOIs
Publication statusPublished - Jul 25 2015

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Keywords

  • Colorectal cancer
  • Formalin-fixed
  • Fresh frozen
  • Gene expression
  • In situ hybridization
  • Paraffin-embedded
  • qRT-PCR

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

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