Comparison of Mutated KRAS and Methylated HOXA9 Tumor-Specific DNA in Advanced Lung Adenocarcinoma

Abstract

Circulating tumor DNA (ctDNA) has been suggested as a biomarker in non-small cell lung cancer. The optimal target for measuring ctDNA has not yet been established. This study aimed to investigate methylated Homeobox A9 (meth-HOXA9) as an approach to detect ctDNA in advanced lung adenocarcinoma and compare it with mutated Kirsten rat sarcoma viral oncogene homolog (mut-KRAS) in order to determine the mutual agreement. DNA was purified from formalin-fixed, paraffin-embedded non-malignant lung tissue and lung adenocarcinoma tissue, and plasma from healthy donors and lung adenocarcinoma patients, respectively. KRAS mutations in tumor tissue were identified by next-generation sequencing and quantified in tumor and plasma by droplet digital polymerase chain reaction (ddPCR). The meth-HOXA9 analysis was based on bisulfite-converted DNA from tumor and plasma and quantified by ddPCR. Samples consisted of 20 archival non-malignant lung tissues, 48 advanced lung adenocarcinomas with matched plasma samples, and 100 plasma samples from healthy donors. A KRAS mutation was found in the tumor in 34/48 (70.8%) adenocarcinoma patients. All tumors were positive for meth-HOXA9, while none of the non-malignant lung tissues were. Meth-HOXA9 was detected in 36/48 (75%) of plasma samples, and the median level was 0.7% (range of 0–46.6%, n = 48). Mut-KRAS was detected in 29/34 (85.3%) of the plasma samples, and the median level was 1.2% (range of 0–46.1%, n = 34). There was a good correlation between meth-HOXA9 and mut-KRAS in plasma (Spearman’s rho 0.83, p < 0.001). Meth-HOXA9 is present in tissue from incurable lung adenocarcinoma but not in non-malignant lung tissue. It may be used as an approach for detecting ctDNA. The results demonstrated a high agreement between meth-HOXA9 and mut-KRAS in patients with advanced lung adenocarcinoma.

Methylated Circulating Tumor DNA in Blood as a Tool for Diagnosing Lung Cancer: A Systematic Review and Meta-Analysis

“…Gao, 2015 [39], analyzed all markers on both plasma and serum, and they reported higher sensitivity in the serum cohort (RASSF1A 43.1% versus 52.5% and APC 24.1% versus 42.5% for plasma versus serum, respectively). The main method applied by the studies was quantitative methylation-specific PCR (QMSP, 27/33); three studies used digital PCR [46,50,56] and two studies used sequencing-based assays [55,59]. One study employed a method where the target DNA was increased by PCR and then quantified by Surface Enhanced Raman Spectroscopy [49].…”

Prognostic Impact of Circulating Methylated Homeobox A9 DNA in Patients Undergoing Treatment for Recurrent Ovarian Cancer

“…Details on DNA isolation and meth- HOXA9 analysis have been described previously [ 26 , 27 ] and are available in the Supplementary Materials [ 28 , 29 ], with details on primers and probes described in Table S1 . In short, ctDNA was extracted from 4 mL plasma, and bisulfite converted following droplet digital PCR (ddPCR) analysis using an in-house designed methylation-specific assay ( HOXA9 ) and a control assay (Albumin) described in the reference [ 29 ].…”

Early ctDNA response to chemotherapy. A potential surrogate marker for overall survival

“…Measurement of the tumour-specific, methylated neuropeptide Y-gene (meth-NPY) was applied in CRC, and the hypermethylated HOXA9 gene (meth-HOXA9) was used in ovarian and lung cancer. The details on the measurements are available in the supplementary material and have also been described previously [14,15]. In short, it involves bisulphite conversion followed by droplet digital PCR (ddPCR) analysis.…”