Dynamic Changes in Circulating Methylated Markers in Response to Antitumor Therapy of Rectal Cancer.

BACKGROUND: Rectal cancer (RC) occupies a leading position in the structure of oncological morbidity and mortality. Aberrant methylation of tumor-suppressor genes and hypomethylation of retrotransposons were shown to be detectable in cell-free DNA, circulating in the blood (cfDNA) of cancer patients, indicating the possibility to use them as diagnostic and prognosis markers. PURPOSE: Evaluation of the changes in the methylation level of LINE-1 elements and SEPTIN9 and IKZF1 genes in the cell-surface-bound cfDNA (csb-cfDNA) from the blood of RC patients after antitumor therapy at a long-term follow-up. METHODS: Blood samples were obtained from RC patients (n = 25) before treatment, after preoperative chemotherapy (3 courses according to the XELOX scheme), 10-15 days after surgery, and every 3 months during 12 months of dynamic observation. The methylation level of LINE-1, SEPTIN9, and IKZF1 in the csb-cfDNA was evaluated by quantitative methyl-specific PCR. RESULTS: The LINE-1 methylation level in the csb-cfDNA increased 1.6 times in RC patients after chemotherapy and 3 times after tumor resection versus methylation level before therapy. The SEPTIN9 gene methylation level in the csb-cfDNA decreased by 1.7 times in RC patients after chemotherapy and by 2.3 times after tumor resection compared with the values before the treatment. The IKZF1 gene methylation level decreased by 2 times in RC patients after combined therapy. Notably, all patients with relapses (n = 5) showed an increase in methylation level for the SEPTIN9 and IKZF1 genes and a decrease of methylation level for the LINE-1 elements by 2 times or more in comparison with the level 10-15 days after surgery. There were no changes in the circulating SEPTIN9, IKZF1, and LINE-1 methylation levels during the 12-month follow-up period after the combined therapy of RC patients (n = 20) without relapses. CONCLUSION: The results indicate that SEPTIN9, IKZF1, and LINE-1 methylation levels in the csb-cfDNA are potential markers of the effectiveness of antitumor therapy and early detection of relapse in RC patients.

Genomic and Transcriptomic Research in the Discovery and Application of Colorectal Cancer Circulating Markers.

Colorectal cancer (CRC) is the most frequently occurring malignancy in the world. However, the mortality from CRC can be reduced through early diagnostics, selection of the most effective treatment, observation of the therapy success, and the earliest possible diagnosis of recurrences. A comprehensive analysis of genetic and epigenetic factors contributing to the CRC development is needed to refine diagnostic, therapeutic, and preventive strategies and to ensure appropriate decision making in managing specific CRC cases. The liquid biopsy approach utilizing circulating markers has demonstrated its good performance as a tool to detect the changes in the molecular pathways associated with various cancers. In this review, we attempted to brief the main tendencies in the development of circulating DNA and RNA-based markers in CRC such as cancer-associated DNA mutations, DNA methylation changes, and non-coding RNA expression shifts. Attention is devoted to the existing circulating nucleic acid-based CRC markers, the possibility of their application in clinical practice today, and their future improvement. Approaches to the discovery and verification of new markers are described, and the existing problems and potential solutions for them are highlighted.

Potentialities of aberrantly methylated circulating DNA for diagnostics and post-treatment follow-up of lung cancer patients.

To date, aberrant DNA methylation has been shown to be one of the most common and early causes of malignant cell transformation and tumors of different localizations, including lung cancer. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA circulating in the blood (cirDNA) is a convenient tumor-associated DNA marker that can be used as a minimally invasive diagnostic test. In the current study, we investigated the methylation status in blood samples of 32 healthy donors and 60 lung cancer patients before and after treatment with neoadjuvant chemotherapy followed by total tumor resection. Using quantitative methylation-specific PCR, we found that the index of methylation (IM), calculated as IM = 100 × [copy number of methylated/(copy number of methylated + unmethylated gene)], for the RASSF1A and RARB2 genes in the cirDNA isolated from blood plasma and cell-surface-bound cirDNA was elevated 2- to 3-fold in lung cancer patients compared with healthy donors. Random forest classification tree model based on these variables combined (RARB2 and RASSF1A IM in both plasma and cell-surface-bound cirDNA) lead to NSCLC patients' and healthy subjects' differentiation with 87% sensitivity and 75% specificity. An association of increased IM values with an advanced stage of non-small-cell lung cancer was found for RARB2 but not for RASSF1A. Chemotherapy and total tumor resection resulted in a significant decrease in the IM for RARB2 and RASSF1A, in both cirDNA fractions, comparable to the IM level of healthy subjects. Importantly, a rise in the IM for RARB2 was detected in patients within the follow-up period, which manifested in disease relapse at 9 months, confirmed with instrumental and pathologic methods. Our data indicate that quantitative analysis of the methylation status of the RARB2 and RASSF1A tumor suppressor genes in both cirDNA fractions is a useful tool for lung cancer diagnostics, evaluation of cancer treatment efficiency and post-treatment monitoring.

RARß2 gene methylation level in the circulating DNA from blood of patients with lung cancer.

Alterations in the patterns of DNA methylation are among the earliest and most common events in tumorigenesis. Epigenetic changes were shown to be detectable in DNA, circulating in blood (cirDNA) of cancer patients, indicating the resources to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. RARß2 methylation level was significantly increased in plasma cirDNA and cell surface-bound cirDNA (csb-cirDNA) from patients with non-small cell lung cancer compared with healthy individuals (7620 and 1083 copies/ml in the csb fractions, 3589 and 1068 copies/ml in the blood plasma; P=0.003 and 0.001). The cell-bound-to-cell-free RARß2 methylation ratio was found to be elevated in patients with non-small cell lung cancer compared with control (2.12 and 1.01, respectively; P=0.023). RARß2 methylation level in csb-cirDNA and plasma cirDNA was higher in stage III patients compared with stage I-II patients (P=0.02 and 0.03). In the subgroup of patients with squamous cell carcinoma, RARß2 methylation level in the cbs-cirDNA was higher compared with patients with adenocarcinoma (P=0.04). Epigenetic alterations of tumor suppressor gene RARß2 in the total cirDNA (plasma cirDNA and csb-cirDNA) were found to be associated with lung cancer progression. The data obtained indicate that cirDNA-based testing provides a valuable source for subsequent verification of methylated DNA markers for lung cancer diagnostics and prognosis.