Abstract 1155: A discriminatory set of transcripts, suitable for differentiation of normal, colorectal cancer and adenoma cases from both fresh frozen biopsy and formalin-fixed, paraffin-embedded (FFPE) tissue samples - potential biomarkers of colorectal dyplasia-carcinoma transition
Authors: Alexandra Kalmar, Orsolya Galamb, Ferenc Sipos, Barnabás Wichmann, Sándor Spisák, Tibor Krenács, Kinga Tóth, Katalin Leiszter, Zsolt Tulassay, Béla Molnár
Journal: Cancer Research (2012)

Abstract Background and aims: Though histopathology is still the gold standard for differentiating stages of colorectal disease progression, the cut slide may represent only a fragment of the whole sample and disease features, such as dysplastic areas of flat adenomas, may remain hidden due to inappropriate sampling or tissue orientation. The early molecular detection of the colorectal dysplasia-carcinoma transition may enhance the strenght of diagnosis from colonic biopsies. Using high-throughput microarray-based biomarker screening low cost diagnostic array real-time PCR panels may be developed. We aimed to determine molecular markers for enhancing the differentiation of high-grade dysplasia from colorectal carcinoma (CRC) and to analyze the applicability of FFPE tissue samples. Material and Methods: Discriminatory transcript set was identified using HGU133plus2 microarrays (Affymetrix Inc.) on 53 biopsy samples (22 CRC, 20 adenoma, 11 normal). For testing the classificatory power of the discriminatory genes, 94 independent biopsies (27 CRC, 29 adenoma, 38 normal) were analyzed on microarrays. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma, 20 normal) using LightCycler®480 system (Roche) and RealTime ready assays. Reverse transcription was done using Transcriptor First Strand cDNA Synthesis Kit (Roche). For testing the applicability of FFPE tissues, total RNA was isolated from 10um-thick slides of 5 CRC and 2 adjacent normal samples using High Pure RNA Paraffin Kit (Roche). The same cDNA synthesis and real-time PCR conditions were applied as in case of fresh frozen samples. Results: A set of 11 transcripts was determined which could correctly discriminate between not only the normal, adenoma and CRC samples, but between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent fresh frozen biopsy samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples were correctly classified in discriminant analysis. According to the RT-PCR results for the set of 11 markers, tumorous and normal FFPE tissue samples could be distinguished by 100% sensitivity and specificity. Conclusion: The identified transcripts could correctly characterize the dysplasia-carcinoma transition in colonic tissue samples, also on a large independent sample set and on FFPE tissue samples. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic panels can become part of the automated routine procedure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1155. doi:1538-7445.AM2012-1155