Development of a serum miRNA panel for detection of early stage non-small cell lung cancer

Abstract

Minimally invasive testing for early detection of lung cancer to improve patient survival is a major unmet clinical need. This study aimed to develop and validate a serum multi-microRNA (multimiR) panel as a minimally invasive test for early detection of nonsmall cell lung cancer (NSCLC) regardless of smoking status, gender, and ethnicity. Our study included 744 NSCLC cases and 944 matched controls, including smokers and nonsmokers, male and female, with Asian and Caucasian subjects. Using RT-qPCR and a tightly controlled workflow, we quantified the absolute expression of 520 circulating microRNAs (miRNAs) in a Chinese cohort of 180 early stage NSCLC cases and 216 healthy controls (male smokers). Candidate biomarkers were verified in two case-control cohorts of 432 Chinese and 218 Caucasians, respectively (including females and nonsmokers). A multimiR panel for NSCLC detection was developed using a twofold cross-validation and validated in three additional Asian cohorts comprising 642 subjects. We discovered 35 candidate miRNA biomarkers, verified 22 of them, and developed a five-miR panel that detected NSCLC with area under curve (AUC) of 0.936-0.984 in the discovery and verification cohorts. The panel was validated in three independent cohorts with AUCs of 0.973, 0.916, and 0.917. The sensitivity of five-miR test was 81.3% for all stages, 82.9% for stages I and II, and 83.0% for stage I NSCLC, when the specificity is at 90.7%. We developed a minimally invasive five-miR serum test for detecting early stage NSCLC and validated its performance in multiple patient cohorts independent of smoking status, gender, and ethnicity.

Keywords: blood biomarker; early detection; microRNA.

Fig. 1.

Candidate biomarkers segregate NSCLC cases from noncancer controls. Heatmap of normalized miRNA expression levels following unsupervised hierarchical clustering of all 424 subjects in the discovery cohort using 272 expressed miRNAs (Upper) and 35 candidate biomarkers (Lower).The horizontal axis represents the NSCLC status for the samples, with black color for NSCLC subjects and white color for non-NSCLC control subjects. The heatmap shows relative miRNA expression level with red indicating higher expression and green indicate lower expression.

Fig. 2.

Biomarkers verified in cohorts including subjects with different ethnicities, gender, and smoking status. (A) Comparison of biomarker fold changes between female and male subjects and between smokers and nonsmokers in Verification cohorts 1 (Asian) and 2 (Caucasian). (B) Heatmap of normalized miRNA expression levels following unsupervised hierarchical clustering of all 1,070 subjects in the combined cohort (Discovery, Validation 1 and 2) using 22 validated biomarkers.

Fig. 3.

Development of five-miR biomarker panel for NSCLC detection. (A) Boxplots of AUC values calculated from 200 rounds of the twofold cross-validation procedure for biomarker panels comprising two to eight miRNAs. Box represents the 25th, 50th, and 75th percentiles of AUC values calculated for the detection of NSCLC by the biomarker panels containing miRNAs from 2 to 8. (B) ROC curves of NSCLC detection sensitivity and specificity for the five-miR biomarker panel in Discovery and Verification Cohorts 1–2. (C) Table of miRNAs included in the final five-miR panel showing their expression in NSCLC and coefficients used for constructing the panel.

Fig. 4.

Validation of five-miR biomarker panel for detection of all stages of NSCLC. (A) ROC curves of NSCLC detection sensitivity and specificity for the five-miR panel in Validation Cohorts 3–5. (B) Sample scores calculated from the five-miR panel prediction model for every subject in each case-control cohort, classified by cancer stage where available. (C) AUC for the five-miR biomarker panel in all of the cohorts of this study. The AUCs were calculated for all stage cancers, early stage cancers (stages I and II), and stage I cancers.