In spite of the encouraging findings from the National Lung Screening Trial, there is still an ongoing debate on the cost-benefit profile of low-dose computed tomography for lung cancer screening. Consequently, development of complementary biomarkers with less invasive and higher accuracy for lung cancer detection will alleviate this important clinical requirement. To conquer this requirement, we tried to develop a non-invasive, high-throughput DNA methylation-based screening test using sputum to screen subjects with lung cancer burden.
We have previously reported a unique and highly sensitive assay that uses a single-step bisulfite modification of DNA, followed by fluorescence-based PCR to measure DNA methylation (fluoresence-HiSA) to analyze methylation of 8 loci in 4 genes in sputum specimens. In this study, we developed a new assay by using Luminex® technology (Luminex-Hi-SA) which can anticipate methylation status in 52 CpG sites in the 8 loci. By using two procedures, we analyzed 207 sputum samples from patients intended for resection of lung tumors, and subjects without any lesion detected by high dose computed tomography. All sputum specimens were collected before surgical resection, with complete clinical annotation, including 89 adenocarcinoma, 25 squamous cell carcinoma, 3 small cell carcinoma, 4 large cell carcinoma, 13 metastatic lung carcinoma, 5 other malignant carcinoma, and 68 subjects without any malignant lesions.
Our assays successfully identified two or more methylated markers in sputum collected on replicate analysis from 58.3% of malignant patients and 14.7% of subjects without any malignant lesions (AUC = 0.75317) by fluoresence-HiSA. To our surprise, Luminex-Hi-SA improved the accuracy for the detection of subjects with lung cancer burden (AUC=0.94399).
Our novel, non-invasive, DNA methylation-based screening test using sputum by using Luminex® technology can robustly detect a variety of lung cancers. Our modified DNA methylation assay for sputum provides an effective and promising tool for the non-invasive screening for lung cancers, highlighting its clinical utility in reducing the mortality and morbidity associated with lung cancers.
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All authors have declared no conflicts of interest.