Al 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 samplesThe set of 11 classifiers could U 90152 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. RedTesting of the identified marker set with 11 classificatory genes on independent samplesAdditional microarrays. Principal component analysis of microarray data from independent biopsy samples resulted in distinct clusters of normal, adenoma and CRC cases with small overlaps between the diagnostic groups (Figure 1B). In discriminant analysis 93.6 of the original samples and 91.5 of crossvalidated samples were correctly classified (Table 4). In paired comparison, according to the discriminatory set with 11 classifiers, the independent CRC and normal samples could be clearly separated. The sensitivity was 100 , the specificity was 100 . Using the discriminatory panel, independent adenoma andMicroarray ?original sample set (53) Log2FC (AD vs. N) Log2FC (CRC vs. N) 24.9 4.5 4.7 6.6 4.2 20.9 4.1 3.7 1.4 3.3 3.2 4.0 6.3 5.7 3.9 6.3 4.7 8.4 5.1 4.1 20.5 1.4 1.9 20.4 1.5 3.8 5.3 4.2 9.7 0.1 4.0 3.0 1.4 4.1 9.0 1.4 1.7 5.1 3.4 4.8 3.9 2.2 2.0 2.5 3.0 1.5 4.4 1.1 2.2 6.1 3.9 1.5 25.4 25.1 0.2 Log2FC (CRC vs. AD) Log2FC (AD vs. N) Log2FC (CRC vs. N) Log2FC (CRC vs. AD) 26.3 3.4 3.3 5.2 0.2 5.0 4.6 2.5 3.4 4.6 8.2 Microarray ?independent sample 1379592 set (94) RT-PCR independent sample set (68) Log2FC (AD vs. N) 25.8 1.7 1.0 2.2 2.4 20.04 1.1 1.0 1.4 1.8 1.8 Log2FC (CRC vs. N) 24.1 4.7 3.3 4.4 4.5 2.7 4.6 3.4 6.0 6.3 3.2 Log2FC (CRC vs. AD) 1.7 3.0 2.3 2.2 2.1 2.7 3.5 2.4 4.6 4.5 1.Table 3. The set of 11 discriminatory transcripts.MedChemExpress ADX48621 Affymetrix IDGene SymbolGene name207504_atCAcarbonic anhydrase VII39402_atIL1Binterleukin 1, beta212657_s_atIL1RNinterleukin 1 receptor antagonist202859_x_atILinterleukin218469_atGREMgremlin204470_atCXCLche.Al 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 samplesThe 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. RedTesting of the identified marker set with 11 classificatory genes on independent samplesAdditional microarrays. Principal component analysis of microarray data from independent biopsy samples resulted in distinct clusters of normal, adenoma and CRC cases with small overlaps between the diagnostic groups (Figure 1B). In discriminant analysis 93.6 of the original samples and 91.5 of crossvalidated samples were correctly classified (Table 4). In paired comparison, according to the discriminatory set with 11 classifiers, the independent CRC and normal samples could be clearly separated. The sensitivity was 100 , the specificity was 100 . Using the discriminatory panel, independent adenoma andMicroarray ?original sample set (53) Log2FC (AD vs. N) Log2FC (CRC vs. N) 24.9 4.5 4.7 6.6 4.2 20.9 4.1 3.7 1.4 3.3 3.2 4.0 6.3 5.7 3.9 6.3 4.7 8.4 5.1 4.1 20.5 1.4 1.9 20.4 1.5 3.8 5.3 4.2 9.7 0.1 4.0 3.0 1.4 4.1 9.0 1.4 1.7 5.1 3.4 4.8 3.9 2.2 2.0 2.5 3.0 1.5 4.4 1.1 2.2 6.1 3.9 1.5 25.4 25.1 0.2 Log2FC (CRC vs. AD) Log2FC (AD vs. N) Log2FC (CRC vs. N) Log2FC (CRC vs. AD) 26.3 3.4 3.3 5.2 0.2 5.0 4.6 2.5 3.4 4.6 8.2 Microarray ?independent sample 1379592 set (94) RT-PCR independent sample set (68) Log2FC (AD vs. N) 25.8 1.7 1.0 2.2 2.4 20.04 1.1 1.0 1.4 1.8 1.8 Log2FC (CRC vs. N) 24.1 4.7 3.3 4.4 4.5 2.7 4.6 3.4 6.0 6.3 3.2 Log2FC (CRC vs. AD) 1.7 3.0 2.3 2.2 2.1 2.7 3.5 2.4 4.6 4.5 1.Table 3. The set of 11 discriminatory transcripts.Affymetrix IDGene SymbolGene name207504_atCAcarbonic anhydrase VII39402_atIL1Binterleukin 1, beta212657_s_atIL1RNinterleukin 1 receptor antagonist202859_x_atILinterleukin218469_atGREMgremlin204470_atCXCLche.
