Comparison of real-time PCR and nCounter NanoString techniques to validate copy number alterations in oral cancer

Copy number alterations (CNAs) are imperative in determining the patient's prognostic and predictive status. Real-time polymerase chain reaction (PCR) is extensively used to validate the results of global genomic profiling methods. However, nCounter NanoString is yet to be thoroughly assessed and compared for copy number analysis. For the first time, we have comprehensively compared the aforementioned techniques in 119 oral cancer samples to evaluate 24 genes. Spearman's rank correlation and Cohen's Kappa score were calculated to identify concurrence. Kaplan-Meier curves with the Log-rank test were used to identify genes related to disease prognosis, including recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS). Spearman's rank correlation ranged from r = 0.188 to 0.517, and Cohen's kappa score showed a moderate to substantial agreement. Additionally, in real-time PCR, ISG15 was associated with a better prognosis for RFS, DSS and OS, along with ATM, CASP4, and CYB5A with poor RFS. However, in nCounter NanoString, ISG15 was associated with poor prognosis for RFS, DSS, and OS, while CDK11A showed poor prognosis for RFS. Real-time PCR remains a robust method to validate the genomic biomarkers. However, these observations should be rigorously validated by conducting additional, well-designed, independent studies.