Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood

Int J Oncol. 2010 Aug;37(2):519-25. doi: 10.3892/ijo_00000701.

Abstract

Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the controls. Our data confirm the extreme sensitivity of RT-PCR, making this technique able to detect minimal amounts of mRNA expressed in a non-tissue-specific manner. Moreover, DGED remains a powerful tool to identify candidate epithelial markers in blood, such as colon related mRNAs. However, to date, none of these qualified as tumour markers.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor / analysis*
  • Biomarkers, Tumor / genetics
  • Carcinoma / blood
  • Carcinoma / diagnosis
  • Carcinoma / genetics*
  • Case-Control Studies
  • Colorectal Neoplasms / blood
  • Colorectal Neoplasms / diagnosis
  • Colorectal Neoplasms / genetics*
  • Female
  • Gene Expression Profiling / instrumentation
  • Gene Expression Profiling / methods*
  • Genetic Association Studies
  • Humans
  • Image Processing, Computer-Assisted / methods
  • Male
  • Middle Aged
  • Neoplastic Cells, Circulating / chemistry
  • Neoplastic Cells, Circulating / metabolism
  • Neoplastic Cells, Circulating / pathology
  • RNA, Messenger / analysis
  • Reverse Transcriptase Polymerase Chain Reaction* / methods
  • Signal Processing, Computer-Assisted*

Substances

  • Biomarkers, Tumor
  • RNA, Messenger