NCI-EDRN Validation of Salivary Oral Cancer Biomarkers

Objectives: We have shown previously a panel of 7 salivary mRNA biomarkers for oral cancer detection. We aimed to validate these salivary markers in a phase 2 biomarker study which was conducted in a blinded manner simultaneously by the Biomarker Reference Laboratory (BRL) of the NCI's Early Detection Research Network (EDRN) and our laboratory.

Methods: Anonymised aliquots of saliva samples (n=64) were distributed to two different individuals from each laboratory. Total RNA was isolated semi-automatically (KINGFISHER, Thermo Scientific) and the expression of these biomarkers were quantified by a novel multiplex RT-PCR preamplification and subsequent singleplex real-time PCR. All reactions were loaded automatically (BioMek 3000) in 96-well plates. Data (Ct numbers and relative copy numbers) were then reported to and analyzed by statisticians at EDRN Data Management and Coordinating Center.

Results: 1. Highly comparable results were generated in both labs (Table). 2. Three genes for EDRN-BRL and all 7 from our lab, were elevated in oral cancer samples significantly (p<0.05). 3. Area under the curve (AUC) of 0.86 and 0.85 were achieved by EDRN-BRL and our lab, respectively (7 marker combined).

Conclusion: The predictive power and standardized measurement of these salivary oral cancer biomarkers were highly reproducible. Importantly, they were independently validated by NCI's EDRN. A second validation (n=100) is currently underway prior to a phase 3 multi-center validation. 

Genes	Area under ROC curve		Wilcoxon P-value		False Discovery Rate
	EDRN	Wong	EDRN	Wong	EDRN	Wong
IL8	0.775	0.798	<0.001	<0.001	0.091	<0.001
IL1B	0.677	0.717	0.024	0.005	0.056	0.006
H3F3A	0.635	0.676	0.088	0.023	0.002	0.017
OAZ1	0.652	0.676	0.053	0.023	0.084	0.017
S100P	0.628	0.685	0.055	0.017	0.068	0.018
SAT	0.709	0.721	0.007	0.004	0.026	0.008
DUSP1	0.594	0.665	0.144	0.034	0.129	0.021

Supported by PHS NIH Grant R01 DE15970 (D.T.W.) and NIH/National Cancer Institute 5U24 CA08636607 (D.C.).