Abstract 3506: mRNA biomarkers of colorectal cancer development tested on biopsy, fresh frozen and formalin-fixed, paraffin embedded (FFPE) specimens in an automated workflow.
Authors: Molnar Bela , Kalmar Alexandra , Wichmann Barnabás , Galamb Orsolya , Spisak Sandor , Sipos Ferenc , Tóth Kinga , Leiszter Katalin , Patai Arpad V. , Schöller Andrea , Tulassay Zsolt 
Published: August 2014

Introduction: The early molecular detection of the colorectal dysplasia-carcinoma transition may enhance the classification of colonic tissue samples. Recently, a colorectal cancer-specific transcript set including COL12A1, CXCL2, CA7, Il1B, MMP3, IL8 was identified on colonic biopsy samples using whole genomic microarrays (WG MA).

Aims: Our aim was to evaluate the applicability of these markers on independent biopsy and also on fresh frozen and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from healthy and CRC patients by expression arrays and RT-PCR. Furthermore, automated RNA isolation was introduced and evaluated for increased sample number in one run.

Materials and methods: Total RNA from 3 normal and 3 CRC FFPE specimens were analyzed by using Affymetrix U133Plus 2.0 WG expression arrays. Total RNA was isolated from 30 biopsy samples stored in RNALater (15 CRC, 15 normal), 20 fresh frozen surgically removed colonic tissues (10 CRC, 10 adjacent normal) and 60 FFPE (30 CRC, 30 adjacent normal) samples with the automated MagNA Pure 96 Cellular RNA Large volume kit and reverse transcription was done using Transcriptor First Strand cDNA Synthesis Kit (Roche). Gene expression analysis was performed with real-time PCR using RealTime ready assays and the LightCycler 480 system (Roche).

Results: The FFPE specimens could be clustered correctly in 100% by the results of the microarray analysis. According to the gene expression levels of the set of 11 transcripts, the biopsy samples could be distinguished by 93,3% sensitivity and 93,3% specificity by RT-PCR. The discriminatory power of the marker set was proved to be high also on fresh frozen and FFPE samples (sens:&gt;90 %, spec: &gt;80 %). The automated RNA isolation technique has not influenced the classification power of the markers, but decreased the workaround time by 100% (4 hours to 2 hours).

Conclusion: The analyzed set of markers in an automated environment could correctly characterize the healthy and the tumorous colonic tissue samples on biopsy, fresh frozen and also on FFPE tissue samples by MAs and RT-PCR. On the basis of these results, these markers can enhance the automated, early and differential detection of colorectal cancer.

Citation Format: Bela Molnar, Alexandra Kalmar, Barnabás Wichmann, Orsolya Galamb, Sandor Spisak, Ferenc Sipos, Kinga Tóth, Katalin Leiszter, Arpad V. Patai, Andrea Schöller, Zsolt Tulassay. mRNA biomarkers of colorectal cancer development tested on biopsy, fresh frozen and formalin-fixed, paraffin embedded (FFPE) specimens in an automated workflow. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3506. doi:10.1158/1538-7445.AM2013-3506