Array real-time PCR The array RT-PCR measurements for selected transcript panels were performed on independent http://www.selleckchem.com/products/Trichostatin-A.html biopsy specimens. According to the lowest standard deviation of ��CT values, 18S ribosomal RNA was chosen as a reference among the seven housekeeping genes placed on the array real-time PCR plate. PCA figure shows that normal, adenoma and CRC biopsy samples are classified into three distinct groups (Figure 1C). Discriminant analysis of 11 markers on independent RT-PCR samples showed correct classification for 95.6% of the original grouped cases, and 94.1% of the cross-validated cases (Table 4). When only 2 sample groups were compared, discriminatory power of the gene panel is also proved to be considerably high during the ROC curve analysis of CRC and normal samples (sensitivity: 100%, specificity: 100%).

The adenoma and healthy samples could be clearly separated by 95.8% sensitivity and 95.0% specificity values. In case of adenoma vs. CRC comparison, the ROC curve analysis showed separation with 95.8% sensitivity and specificity. Discrimination between high-grade dysplastic adenoma and early CRC samples The set of 11 classifiers could classify the 24 high-grade dysplastic adenoma and the 24 early CRC (stage Dukes A or B) samples analyzed on microarrays by 83.3% specificity and 100% sensitivity (Figure 3A). This marker set was also suitable for discrimination between high-grade dysplastic adenoma (n=11) and early cancer (n=10) samples in real-time PCR analysis. The hierarchical cluster diagram of the real-time PCR samples represents that all the 10 CRC samples were correctly classified, and 3 of the 11 adenoma samples were misclustered (Figure 3C).

These samples were adenoma 6, adenoma 10 and adenoma 11 biopsy samples. However samples 6 and 11 were found to be misclassified as during a patient follow up they were rediagnosed as in situ carcinoma (Figure 3D, E). Application of ROC statistic showed even higher differentiation since 100% sensitivity and 90.9% specificity observed in the comparison of samples. Red highlight refers to 6 and 11 adenoma samples which were above or near to the threshold. Green highlight refers to adenoma 10 samples which were clustered with CRC samples but ROC statistic shows clear separation from that group (Figure 3B, D). After patient follow the aforementioned samples transferred into CRC group and new multiple logistic regression was applied.

Comparison of 9 high-grade dyslpalstic adenoma 12 and early cancer resulted 100% sensitivity and 100% specificity (Figure 3D), thereby optimize sensitivity (100%) and specificity Cilengitide (90.9%) of original sample classification (Figure 3B). Discussion In this study a characteristic transcript set was determined which is specific for the colorectal dysplasia-carcinoma transition using whole genomic microarray in 53 biopsy samples.