Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 25:10:126.
doi: 10.1186/s13000-015-0363-4.

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

Alexandra Kalmár  1   2   3 , Barnabás Wichmann  4 , Orsolya Galamb  5 , Sándor Spisák  6 , Kinga Tóth  7 , Katalin Leiszter  8 , Boye Schnack Nielsen  9 , Barbara Kinga Barták  10 , Zsolt Tulassay  11 , Béla Molnár  12
Affiliations

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

Alexandra Kalmár et al. Diagn Pathol. .

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.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Analytical parameters of the automatically isolated RNA samples. a RNA yield (μg RNA); b) OD260/280; c) OD260/230 and d) RNA integrity number (RIN) of normal and tumor fresh frozen biopsy and FFPE samples. Individual quality or quantity measurements are represented by dots on the boxplots, while boxes indicate median and standard deviation of the data
Fig. 2
Fig. 2
Heat maps of real-time PCR data representing gene expression alteration of the 11 analyzed transcripts in (a) fresh frozen biopsyand (b) FFPE samples. Color scale encodes relative overexpression (red) to underexpression (green)
Fig. 3
Fig. 3
Discriminant analysis of (a) fresh frozen biopsy and (b) FFPE samples on the basis of gene expression levels of 11 transcripts. The table contains predicted group membership data on the original grouped cases and on the cross validated samples
Fig. 4
Fig. 4
Interactive dot diagram of (a) fresh frozen biopsy and (b) FFPE samples according to the multiple logistic regression equation. On the basis of the previously published transcript set, a multiple logistic regression equation was applied to the present study’s results. The results can be visualized on interactive dotblots, normal and tumor groups are displayed as dots in two separate groups and the horizontal line indicates the cut off point with the best sensitivity and specificity results. The sensitivity and specificity values are calculated by the algorithm, that can be seen beside the dotblots
Fig. 5
Fig. 5
In situ hybridization for CA7 and CXCL1 mRNAs. Paraffin sections from normal colon (a-d), colon adenoma (e-h) and colon cancer (i-l) were stained with LNA probes for CXCL1 mRNA (a,e,i), CA7 mRNA (b,f,j), a generic unspecific sequence, scramble (c,g,k), and a positive control probe, miR-126 (d,h,l). The CXCL1 ISH signal is seen in a population of macrophage-like cells located in the cancer stroma (i, arrows) just below the luminal ulceration (indicated by U), whereas no CXCL7 ISH signal is detected in the normal colon mucosa and in the adenoma (a,e). The CA7 ISH signal is seen in a subset of epithelial cells in normal colon mucosa (b, arrows), whereas no ISH signal is detected in the colon adenoma and cancer tissue (f,j). miR-126 ISH signal is prevalent in endothelial cells (arrows in d,h,l), and only background staining is seen with scramble probe (c,g,k). Tissue sections were counter stained with Nuclear Fast Red. 50 μm bar is representative for all images
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Huerta S. Recent advances in the molecular diagnosis and prognosis of colorectal cancer. Expert review of molecular diagnostics. 2008;8:277–88. - PubMed
    1. Galamb O, Wichmann B, Sipos F, Spisak S, Krenacs T, Toth K, et al. Dysplasia-carcinoma transition specific transcripts in colonic biopsy samples. PLoS One. 2012;7:e48547. doi: 10.1371/journal.pone.0048547. - DOI - PMC - PubMed
    1. Ribeiro-Silva A, Zhang H, Jeffrey SS. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol. 2007;8:118. doi: 10.1186/1471-2199-8-118. - DOI - PMC - PubMed
    1. Budczies J, Weichert W, Noske A, Muller BM, Weller C, Wittenberger T, et al. Genome-wide gene expression profiling of formalin-fixed paraffin-embedded breast cancer core biopsies using microarrays. J Histochem Cytochem . 2011;59:146–57. doi: 10.1369/jhc.2010.956607. - DOI - PMC - PubMed
    1. Chung JY, Braunschweig T, Hewitt SM. Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue. Diagn Mol Pathol. 2006;15:229–36. doi: 10.1097/01.pdm.0000213468.91139.2d. - DOI - PubMed

Publication types