Subtyping Lung Cancer Using DNA Methylation in Liquid Biopsies

Abstract

Background: Lung cancer (LCa) is the most frequently diagnosed and lethal cancer worldwide. Histopathological subtyping, which has important therapeutic and prognostic implications, requires material collection through invasive procedures, which might be insufficient to enable definitive diagnosis. Aberrant DNA methylation is an early event in carcinogenesis, detectable in circulating cell-free DNA (ccfDNA). Herein, we aimed to assess methylation of selected genes in ccfDNA from LCa patients and determine its accuracy for tumor subtyping. Methods: Methylation levels of APC, HOXA9, RARβ2, and RASSF1A were assessed in three independent study groups (study group #1: 152 tissue samples; study group #2: 129 plasma samples; study group #3: 28 benign lesions of lung) using quantitative methylation-specific PCR. Associations between gene promoter methylation levels and LCa subtypes were evaluated using non-parametric tests. Receiver operating characteristic (ROC) curve analysis was performed. Results: In study group #2, HOXA9 and RASSF1A displayed higher methylation levels in small-cell lung cancer (SCLC) than in non-small-cell lung cancer (NSCLC). HOXA9 displayed high sensitivity (63.8%), whereas RASSF1A disclosed high specificity (96.2%) for SCLC detection in ccfDNA. Furthermore, HOXA9 methylation levels showed to be higher in squamous cell carcinoma in comparison with adenocarcinoma in study group #1. Conclusions: Methylation level assessments in ccfDNA may provide a minimally invasive procedure for LCa subtyping, complementing standard diagnostic procedures.

Methylation in HOX Clusters and Its Applications in Cancer Therapy

“…The methylation profile of HOXA9 has also been proposed as a reliable biomarker to identify resistance to cisplatin-based therapy in bladder cancer [34], and as a tool for testicular germ cell tumors subtyping [100]. In addition, the methylation status of HOXA9 was considered relevant for subtyping lung cancer using liquid biopsies [110] or for its early detection in circulating cell-free DNA [58]. HOXA9 hypermethylation was also found to be a tool to identify advanced neck squamous cell carcinomas favoring tumor progression and metastasis [57], predict survival in breast cancer patients, together with HOXA10 hypermethylation [111], and detect early onset of endometrial cancer [58].…”

Epigenetic Research in Stem Cell Bioengineering—Anti-Cancer Therapy, Regenerative and Reconstructive Medicine in Human Clinical Trials

“…Methylation levels in adenomatous polyposis coli (APC), homeobox gene A9 (HOXA9), retinoic acid receptor beta 2 (RARβ2), and Ras association domain-containing protein 1 (RASSF1A) were assessed as a novel non-invasive method for lung cancer subtyping. Higher methylation patterns of HOXA9 and RASSF1A were detected in small-cell lung cancer rather than non-small-cell lung cancer [97]. Quantitative panel assessment of two-gene promoter methylation patterns (singleplex-miR-34b/c, miR-193b and multiplex-APC, GSTP1, RARβ2, respectively), in tissue biopsies and urine of prostate cancer patients demonstrated higher methylation levels of both panels compared to controls [98].…”

Hypermethylation of tumor necrosis factor decoy receptor gene in non‑small lung cancer

“…However, tissue biopsy is inconvenient and traumatic. Liquid biopsy, such as circulating tumor cells, circulating tumor DNA and exosomes can provide more comprehensive disease information (42,43). Furthermore, investigating the methylation levels of TNFRSF10C and TNFRSF10D genes of patients' blood samples, and combining data from both tissues and blood may allow their development as novel diagnostic biomarkers of lung cancer.…”