Aberrantly methylated circulating DNA as a tool for diagnostics and evaluation of therapeutic effect in lung cancer

Ponomaryeva A.A.1, Rykova Е.2, Cherdyntseva N.V.1, Skvortsova T.2, Dobrodeev A.Yu.1, Zav’yalov A.1, Tuzikov S.1, Vlassov V.2, Laktionov P.2

A. Ponomaryova1, Е. Rykova2, N. Cherdyntseva1, T. Skvortsova2, А. Dobrodeev1, A. Zav’yalov1, S. Tuzikov1, V. Vlassov2, P. Laktionov2

1Cancer Research Institute of SB of the RAMS, Tomsk, Russia

2Institute of Chemical Biology and Fundamental Medicine of SB of the RAS, Novosibirsk, Russia


Introduction: Cancer cell-specific aberrantly methylated DNA was found in blood plasma from cancer patients, indicating that cell-free DNA circulating in blood (cirDNA) is a convenient source of tumor-associated DNA markers for the minimally invasive diagnostic tests. Aim: Estimation of the diagnostic and prognostic significance of methylation changes in RARB2, RASSF1A tumor suppressor genes detected in the cirDNA from lung cancer patients before and after combined treatment. Methods: Blood samples were taken from 33 healthy subjects (HS) and 60 patients with non-small cell lung cancer (NSCLC) before treatment, after neoadjuvant chemotherapy, surgery and during post-treatment follow-up. CirDNA was extracted from blood plasma and cell-surface-bound cirDNA (csb-cirDNA) fraction which was obtained by successive treatment of blood cells with PBS/EDTA and trypsin solutions. Concentration of methylated and unmethylated RASSF1A and RARB2 tumor suppressor genes circulating in blood was quantified by methylation-specific PCR and methylation index (IM) was calculated as IM = 100 х [copy number of methylated /(copy number of methylated + unmethylated gene)] for plasma cirDNA and csb-cirDNA. Results: Methylation index for RASSF1A, RARB2 genes was elevated 2–3 fold in plasma cirDNA and csb-cirDNA from lung cancer patients versus HS (Mann — Whitney U-test, p<0.05). Exceeding of at least one of RASSF1A or RARB2 IM values over the selected cut-offs discriminates NSCLC patients from healthy subjects with 90% sensitivity and 82% specificity when plasma cirDNA and csb-cirDNA were analyzed. The mean RARВ2 IM in csb-cirDNA was higher for stage III patients compared with stage I–II patients (p < 0.05). In post-surgery period most patients had more pronounced decrease of RASSF1A, RARB2 IM in both plasma cirDNA and csb-cirDNA compared with IM values detected before treatment and after chemotherapy. The increased level of methylation markers was found in blood of patients with recurrent NSCLC after treatment. Conclusions: Detection of RARB2, RASSF1A methylation changes in csb-cirDNA and plasma cirDNA of NSCLC patients’ blood appeared to be more informative in comparison with analysis of plasma cirDNA only. The obtained data provides evidence that RARB2, RASSF1A IM analysis in the cirDNA is valuable for lung cancer diagnostics, evaluation of cancer treatment efficiency and post-treatment monitoring.

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