Molecular Detection of EMT Markers in Circulating Tumor Cells from Metastatic Non-Small Cell Lung Cancer Patients: Potential Role in Clinical Practice

Figure 4

Reliability assessment of the RT-PCR assay. (a) Comparison between RT-PCR assay and control methods for cell counts. The reliability of RT-PCR method was assessed by Bland & Altman plot. At the different experimental points, the differences between methods were within mean ± 1.96 SD; therefore, the RT-PCR assay was interchangeable with both the qIF and CF assays. (b) Evaluation of the linearity of RT-PCR assay. A significant correlation between the number of spiked A549^EMT cells and levels of mRNA expression was found for all target genes, mainly for the expression of vimentin (r²: 0.68), Snail2 (r²: 0.72), and Twist1 (r²: 0.69). (r correlation coefficient for other target genes: Snail1 (r²: 0.68), Twist2 (r²: 0.68), ZEB1 (r²: 0.57), and ZEB2 (r²: 0.37)). (c) ROC curves and cutoff values for EMT-target genes (VIM: >0.678; sens 83.3, spec 73.6, likelihood 3.17, AUC 0.837, ; Twist1: >0.551; sens 100, spec 83.3, likelihood 6.00, AUC 1.00, ; Twist2: >0.551, sens 100, spec 83.3, likelihood 6.00, AUC 1.00, ; Snail1: >0.718, sens 74.0, spec 83.3, likelihood 4.44, AUC 0.77, ; Snail2: >0.559, sens 96.0, spec 83.3, likelihood 5.76, AUC 0.993, ; ZEB1: >0.765, sens 72.0, spec 83.3, likelihood 4.32, AUC 0.736, ; ZEB2: > 0.88, sens 92.0, spec 83.3, likelihood 5.52, AUC 0.923, ).

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