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Background: DNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastatic ALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identify ALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring. Methods: Longitudinal plasma samples (n = 79) of 21 ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15 ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies. Results: We devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles of ALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g., PCDH10, TBX2, CDO1, and HOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman, ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rank p = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression. Conclusion: We demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the leading cause of cancer-related death in most western countries in both, males and females, accounting for roughly 20–25% of all cancer deaths. For choosing the most appropriate therapy regimen a definite diagnosis is a prerequisite. However, histological characterization of bronchoscopic biopsies particularly with low tumor cell content is often challenging. Therefore, this study aims at (a) determining the value of DNA methylation analysis applied to specimens obtained by bronchoscopic biopsy for the diagnosis of lung cancer and (b) at comparing aberrantly CpG loci identified in bronchoscopic biopsy with those identified by analyzing surgical specimens. Results: We report the HumanMethylation450-based DNA methylation analysis of paired samples of bronchoscopic biopsy specimens either from the tumor side or from the contralateral tumor-free bronchus in 37 patients with definite lung cancer diagnosis and 18 patients with suspicious diagnosis. A differential DNA methylation analysis between both biopsy sites of patients with definite diagnosis identified 1303 loci. Even those samples were separated by the set of 1303 loci in which histopathological analysis could not unambiguously define the dignity. Further differential DNA methylation analyses distinguished between SCLC and NSCLC. We validated our results in an independent cohort of 40 primary lung cancers obtained by open surgical resection and their corresponding controls from the same patient as well as in publically available DNA methylation data from a TCGA cohort which could also be classified with high accuracy. Conclusions: Considering that the prognosis correlates with tumor stage at time of diagnosis, early detection of lung cancer is vital and DNA methylation analysis might add valuable information to reliably characterize lung cancer even in histologically ambiguous sample material. [ABSTRACT FROM AUTHOR]

Hwang et al. recently showed that VGF substantially contributes to the resistance of human lung cancer cells towards epidermal growth factor receptor kinase inhibitors. This was further linked to enhanced epithelial-mesenchymal transition. Here, we demonstrate that VGF is epigenetically modified in non-small cell lung cancer tissues compared to corresponding tumor-free lung tissues from the same donors by using methylome bead chip analyses. These epigenetic modifications trigger an increased transcription of the VGF gene within the tumors, which then leads to an increased expression of the protein, facilitating epithelial-mesenchymal transition and the resistance to kinase inhibitors. These results should be taken into account in the design of novel therapeutic and diagnostic approaches. [ABSTRACT FROM AUTHOR]

Targeting checkpoint inhibitors using monoclonal antibodies results in significantly better outcome of cancer patients compared to conventional chemotherapy. However, the current companion diagnostics to predict response is so far suboptimal, since they base on more or less reliable immunohistochemical approaches. In order to overcome these limitations, we analyzed epigenetic modifications of PDCD1 (PD1), CD274 (PD-L1), and CTLA4 in NSCLC tissues from 39 patients. Results were correlated with transcriptome data. Significant differences in the CpG-methylation patterns between tumor tissues and matched controls were observed for CTLA4 and PDCD1 (PD1) showing a decreased methylation of these genes compared to matched tumor-free tissues from the same patients. Results were confirmed by bisulfide sequencing in an independent validation cohort. Hypomethylation also resulted in increased expression of these genes as shown by transcriptome data. These epigenetic pathways as a hallmark of NSCLC might be useful to generate more precise diagnostic approaches in the future. [ABSTRACT FROM AUTHOR]

Recently, it was shown that the epigenetic age of non-small cell lung cancer (NSCLC) tissues is different from the chronological age of patients. Here, we demonstrate that Regucalcin and Survivin, molecules which are known to be involved in the process of aging and overcoming aging, are epigenetically modified in NSCLC tissues compared to corresponding tumor-free tissues from the same donors by using methylome bead chip and corresponding transcriptome analyses. A high expression of Survivin on the RNA level was negatively correlated with patients' survival in adenocarcinomas while a high Regucalcin expression was correlated positively. In stage 1 adenocarcinomas, this separation is even sharper for both genes. Within these, adenocarcinomas, smokers with low expression of Survivin show a better outcome, while the high expression of Regucalcin seems to be protective in never smokers. On the protein level, these molecules were detected by immunohistochemistry using tissue microarrays. Since Survivin can be secreted and we observed a high abundance of the protein also in the adjacent immune cells of the tumor microenvironment, an effect on benign cells can be assumed. These findings show that epigenetic re-programming of Survivin and Regucalcin in non-small cell lung cancer leads to enhanced expression of Survivin and reduced expression of Regucalcin, with a possible role of both molecules as predictive markers. [ABSTRACT FROM AUTHOR]