Validation of Reference Genes for Oral Cancer Detection Panels in a Prospective Blinded Cohort

Jack L Martin,Ramona Natacha Pena I Subirà

doi:10.1371/journal.pone.0158462

Abstract

BackgroundReference genes are needed as internal controls to determine relative expression for clinical application of gene expression panels. Candidate constitutively expressed genes must be validated as suitable reference genes in each body fluid and disease entity. Prior studies have predominantly validated oral squamous cell carcinoma associated messenger RNAs (mRNAs) based on quantitative polymerase chain reaction (qPCR) quantification cycle (Cq) values without adjustment for housekeeping genes.MethodsOne hundred sixty eight patients had saliva collected before clinically driven biopsy of oral lesions suspicious for cancer. Seven potential housekeeping mRNAs and six pre-specified oral cancer associated mRNAs were measured with qPCR by personnel blinded to tissue diagnosis. Housekeeping gene stability was determined with the NormFinder program in a training set of 12 randomly selected cancer and 24 control patients. Genes with stability indices <0.02 were then tested in the validation set consisting of the remaining cancer and control patients and were further validated by the geNorm program. Cancer gene delta Cqs were compared in case and control patients after subtracting the geometric mean of the reference gene raw Cqs.ResultsB2M and UBC had stability indices >0.02 in the training set and were not further tested. MT-ATP6, RPL30, RPL37A, RPLP0 and RPS17 all had stability indices <0.02 in the training set and in the verification set. The geNorm M values were all ≤1.10. All six pre-specified cancer genes (IL8, IL1, SAT, OAZ1, DUSP1 and S100P) were up-regulated in cancer versus control patients with from nearly twofold to over threefold higher levels (p<0.01 for all based on delta Cq values).ConclusionsFive reference genes are validated for use in oral cancer salivary gene expression panels. Six pre-specified oral carcinoma associated genes are demonstrated to be highly significantly up-regulated in cancer patients based on delta Cq values. These cancer and reference genes are suitable for inclusion in gene expression panels for research and clinical applications.Trial RegistrationClinicalTrials.gov NCT01587573

Highlights

Five reference genes are validated for use in oral cancer salivary gene expression panels
Significantly up-regulated in cancer patients based on delta Cq values. These cancer and reference genes are suitable for inclusion in gene expression panels for research and clinical applications
[2] A group of messenger RNAs (mRNAs) discovered to be up-regulated in the presence of oral squamous cell cancer have been confirmed in multiethnic cohorts and independently validated by the National cancer Institute–Early Detection Research Network. [3,4,5,6] Prior studies predominantly compared quantitative polymerase chain reaction (qPCR) raw cycle threshold (Cq) values in oral cancer and control subjects without adjustment for rigorously selected internal reference genes. [3,6,7]

Summary

Introduction

Salivary contains a broad range of biomarkers and has gained increasing interest as a readily accessible body fluid for disease detection and surveillance. [1] The salivary transcriptome has been demonstrated to change in the presence of systemic malignancy with disease specific footprints. [2] A group of mRNAs discovered to be up-regulated in the presence of oral squamous cell cancer have been confirmed in multiethnic cohorts and independently validated by the National cancer Institute–Early Detection Research Network. [3,4,5,6] Prior studies predominantly compared qPCR raw cycle threshold (Cq) values in oral cancer and control subjects without adjustment for rigorously selected internal reference genes. [3,6,7]For clinical applications reference genes are needed to serve as an internal control for gene expression assays. [8,9] Robust reference genes that are stable between samples are necessary to detect subtle changes in gene expression and to correct for variability between assays performed at different times. [9,10] Constitutively expressed potential reference genes must be validated for each specific disease entity and in the body fluid or tissue of interest. [10,11] For, example, analysis by our group of patient level data from prior studies has demonstrated that commonly used reference genes can often show differences in expression in the saliva of cancer and control patients. [12] The goal of the present study is to validate reference genes suitable for salivary gene expression assays in an intended use population of patients with lesions suspicious for the presence of oral squamous cell carcinoma. [3,4,5,6] Prior studies predominantly compared qPCR raw cycle threshold (Cq) values in oral cancer and control subjects without adjustment for rigorously selected internal reference genes. [9,10] Constitutively expressed potential reference genes must be validated for each specific disease entity and in the body fluid or tissue of interest. [12] The goal of the present study is to validate reference genes suitable for salivary gene expression assays in an intended use population of patients with lesions suspicious for the presence of oral squamous cell carcinoma. Prior studies have predominantly validated oral squamous cell carcinoma associated messenger RNAs (mRNAs) based on quantitative polymerase chain reaction (qPCR) quantification cycle (Cq) values without adjustment for housekeeping genes

Objectives

Methods

Discussion

Conclusion