Regulation Of Tumor Suppressor Genes Research Articles

Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases.

A New Approach to Evaluating Aberrant DNA Methylation Profiles in Hepatocellular Carcinoma as Potential Biomarkers

Hypermethylation of CpG islands in the promoter region of tumor suppressor genes (TSGs) and their subsequent silencing is thought to be one of the main mechanisms of carcinogenesis. MBD2b enrichment coupled with a NimbleGen array was applied to examine the genome-wide CpG island methylation profile of hepatocellular carcinoma (HCC). Hypermethylated DNA of 58 pairs of HCC and adjacent tissue samples was enriched and hybridized in the same array. Aberrant hypermethylated peaks of HCC and adjacent tissues were screened and annotated after data processing using NimbleScan2.5 and our newly developed Weighting and Scoring (WAS) method, respectively. Validation using bisulfite sequencing of randomly selected ANKRD45, APC, CDX1, HOXD3, PTGER and TUBB6 genes demonstrated significant hypermethylation modification in HCC samples, consistent with the array data.

Polymorphisms in CARS are associated with gastric cancer risk: a two-stage case-control study in the Chinese population.

The cysteinyl transfer RNA synthetase gene (CARS) is located on chromosome band 11p15.5, which is an important tumor-suppressor gene region. Mutations in CARS have been identified in many kinds of cancers; however, evidence for a relationship between genetic variants in CARS and gastric cancer at the population level is still lacking. Thus, we explored the association of variants in CARS with gastric cancer using a two-stage case-control strategy in Chinese. We undertook a two-stage case-control study to investigate the association between polymorphisms in CARS and risk of gastric cancer with use of an Illumina Infinium® BeadChip and an ABI 7900 system. Four single nucleotide polymorphisms (SNPs) were significantly associated with gastric cancer risk in both the discovery stage and the validation stage after adjustment for age and sex. In addition, the combined results of the two stages showed these SNPs were related to gastric cancer risk (P falsediscoveryrate≤0.001 for rs384,490, rs729662, rs2071101, and rs7394702). In silico analyses revealed that rs384490 and rs7394702 could affect transcription factor response elements or DNA methylation of CARS, and rs729662 was associated with the prognosis of gastric cancer. Additionally, expression quantitative trait loci analysis showed rs384490 and rs729662 might alter expression of CARS-related genes. The potential functional SNPs in CARS might influence the biological functions of CARS or CARS-related genes and ultimately modify the occurrence and development of gastric cancer in Chinese. Further large-scale population-based studies or biological functional assays are warranted to validate our findings.

Promoter Hypermethylation Analysis of the Tumor Suppressor Genes RASSF1A and RASSF2A in Iranian Endometrial Carcinoma Patients

Introduction: The endometrial cancer (EC) is the seventh most common malignancies worldwide among females with good prognosis in early stages of the disease. The CpG Island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we investigated the methylation pattern in promoter region of RASSF1A and RASSF2A genes in Endometrial cancer patients in Iranian women to identify correlations among promoter hypermethylation, disease risk and clinicopathological parameters. Methods: 28 patients and 22 healthy controls were studied. Isolation of genomic DNA from FFPE and peripheral blood was performed and Methylation-Specific PCR (MSP) was applied for analysis of the promoter CpG methylation status of RASSF1A and RASSF2A genes in the studied population. Results: A significant difference was found among the study groups and the presence of promoter CpG hypermethylation status in the RASSF1A (P = 0.0321) and RASSF2A (P = 0.0003) genes. RASSF1A, and RASSF2A gene promoter methylations were present in 53.57% and 42.85% of EC samples when compared to those in the controls with 31.81% and 9.09% respectively. Furthermore, methylation status between tissue and blood samples of RASSF1A, and RASSF2A genes was not significant (P = 0.49 and 0.09 respectively). Our results indicated a corollation between ages, menosososal state and tumor grade with RASSF1A, and RASSF2A promoter methylation. Conclusions: In our study, Hypermethylation of both RASSF1A and RASSF2A genes are important events in carcinogenesis of endometrial cancer. Epigenetic alternations may have diagnostic value for early diagnosis and better clicinal management of susceptibility to endometrial malignancies.

Repression of DOK7 mediated by DNMT3A promotes the proliferation and invasion of KYSE410 and TE-12 ESCC cells

Increasing evidence shows that aberrant epigenetic regulation of tumor suppressor genes is a contributing factor to their altered expression in esophageal squamous cell carcinoma (ESCC). In the current study, we investigate the role of DOK7 in ESCC cells. We found that enforced expression of DOK7 inhibited the proliferation and invasion of ESCC cells. We also found that treatment of ESCC cells with the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-azadC), induced the demethylation of DOK7 in promoter and DOK7 expression. Moreover, silencing DNMT3A decreased methylation of DOK7 and increased DOK7 expression, followed by repressing the proliferation and invasion of ESCC cells. Collectively, our data indicated that silencing DNMT3A inhibits proliferation and invasion in ESCC cells by inducing demethylation of DOK7.

Synonymous Somatic Variants in Human Cancer Are Not Infamous: A Plea for Full Disclosure in Databases and Publications.

Single-nucleotide variants (SNVs) are the most frequent genetic changes found in human cancer. Most driver alterations are missense and nonsense variants localized in the coding region of cancer genes. Unbiased cancer genome sequencing shows that synonymous SNVs (sSNVs) can be found clustered in the coding regions of several cancer oncogenes or tumor suppressor genes suggesting purifying selection. sSNVs are currently underestimated, as they are usually discarded during analysis. Furthermore, several public databases do not display sSNVs, which can lead to analytical bias and the false assumption that this mutational event is uncommon. Recent progress in our understanding of the deleterious consequences of these sSNVs for RNA stability and protein translation shows that they can act as strong drivers of cancer, as demonstrated for several cancer genes such as TP53 or BCL2L12. It is therefore essential that sSNVs be properly reported and analyzed in order to provide an accurate picture of the genetic landscape of the cancer genome.

Genistein and Trichostatin A Induction of Estrogen Receptor Alpha Gene Expression, Apoptosis and Cell Growth Inhibition in Hepatocellular Carcinoma HepG 2 Cells

Epigenetic changes such as DNA methylation and histone acetylation play important roles in determining gene expression. Hypermethylation of CpG islands of the promoter region of tumor suppressor genes can greatly influence carcinogenesis through transcriptional silencing. Acetylation of lysine in histone tails causes relaxation of chromatin, which facilitates gene transcription, while deacetylation is associated with condensed chromatin resulting in gene silencing. DNA demethylating agents such as genistein (GE) and histone deacetylase inhibitors (HDACIs) such as trichostatin A (TSA) may strongly reactivate silenced genes and exposure to these two agents in combination is reported to enhance estrogen receptor alpha (ERα) reactivation and induction of apoptosis. The present study was designed to evaluate the effect of these compounds on ERα gene expression, cell viability and apoptosis in hepatocellular carcinoma (HCC) Hep G2 cells. GE exerted biphasic effects; it stimulated cell growth at a low concentration (1 μM) but inhibitory influence was noted with high concentrations (10, 20 and 40 μM). In contrast, TSA demonstrated inhibitory effects on growth at all of concentrations tested. Furthermore, GE and GE/TSA significantly induced apoptosis at all concentrations, but TSA only after 72 h. GE induced ERα re-expression and this was maximal in combined treatment groups treated with GE/TSA for 72 h.Discussion:Our finding clearly indicates that GE and TSA have an inhibitory cell growth, induce apoptosis and reactivate the ERα gene expression.Conclusion:GE and TSA can significantly inhibit the growth of HCC cells and play a significant role in apoptosis and reactivation of ERα gene.

Gene Expression Array Analysis to Identify Candidate Tumor Suppressor Genes in Melanoma.

Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma.

Signalling pathways in UHRF1-dependent regulation of tumor suppressor genes in cancer.

Epigenetic silencing of tumor suppressor genes (TSGs) through DNA methylation and histone changes is a main hallmark of cancer. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a potent oncogene overexpressed in various solid and haematological tumors and its high expression levels are associated with decreased expression of several TSGs including p16 INK4A, BRCA1, PPARG and KiSS1. Using its several functional domains, UHRF1 creates a strong coordinated dialogue between DNA methylation and histone post-translation modification changes causing the epigenetic silencing of TSGs which allows cancer cells to escape apoptosis. To ensure the silencing of TSGs during cell division, UHRF1 recruits several enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 to the right place at the right moment. Several in vitro and in vivo works have reported the direct implication of the epigenetic player UHRF1 in tumorigenesis through the repression of TSGs expression and suggested UHRF1 as a promising target for cancer treatment. This review describes the molecular mechanisms underlying UHRF1 regulation in cancer and discusses its importance as a therapeutic target to induce the reactivation of TSGs and subsequent apoptosis.

Anti-leukemic activity of DNA methyltransferase inhibitor procaine targeted on human leukaemia cells

AbstractChromatin remodeling in DNA is fundamental to gene expression, DNA replication and repair processes. Methylation of promoter regions of tumor-suppressor genes and histone deacetylation leads to gene silencing and transcriptionally repressive chromatin. For the past few decades DNA methylation agents became very attractive as the targets for cancer therapy. The purpose of this work was to examine the effects of DNMT inhibitor procaine on growth inhibition, apoptosis and differentiation of human leukaemia cells. The changes in expression of genes, proteins and histone modifications caused by procaine were evaluated under different treatments. We demonstrated that procaine arrests growth of human leukaemia cells and in combination with all-trans retinoic acid (ATRA) induces cancer cell differentiation. Procaine causes reduction of expression of DNA methyltransferases as well. The treatment of human leukaemia cells with procaine increase the expression of molecules associated with differentiation (CD11b, E-cadherin, G-CSF) and apoptosis (PPARγ). Moreover, the examined DNMT inhibitor enhances certain gene transcription activation via chromatin remodelling – the changes in histone H3K4(Me)3 and H3K9Ac/S10P modifications were detected. Our results suggest, that DNMT inhibitor – procaine, can be used for further investigations on epigenetic differentiation therapy of leukaemia cells especially when used in combination with retinoic acid.

Methylation of CDX2 as a Predictor in Poor Clinical Outcome of Patients with Colorectal Cancer

Increased DNA methylation within the promoter regions of tumor suppressor genes has been associated with gene silencing in various cancers. CDX2, a caudal-related homeobox transcription factor, represents a key tumor suppressor in colorectal cancer. However, the pathological role of its promoter methylation has not yet been well defined in colorectal cancer. We measured CDX2 promoter methylation by methylation-specific PCR (MSP) and CDX2 mRNA levels by real-time quantitative reverse transcription-PCR in both tumor and normal control tissues collected from 108 colorectal cancer patients and then performed correlation analyses. We also determined whether CDX2 promoter methylation status was associated with any clinicopathological features and prognosis in these patients. CDX2 was expressed in all normal mucosa, but only in 38 (35.2%) tumor tissues. CDX2 promoter methylation was detected in 43 (39.8%) tumor samples, but in none of the normal tissue specimens. DNA methylation correlated with decreased mRNA level in these tumors (p < 0.05). The methylation of the CDX2 promoter was associated with enhanced lymph node metastases and shorter survival time (p = 0.0273), but was independent of tumor stage, tumor differentiation, gender, and age. Enhanced CDX2 promoter methylation is associated with gene silencing in a subgroup of colorectal cancer patients with lymph node metastasis and shorter survival times.

Molecular alterations in the carcinogenesis and progression of hepatocellular carcinoma: Tumor factors and background liver factors.

Although hepatocellular carcinoma (HCC) is associated with poor prognosis worldwide, the molecular mechanisms underlying the carcinogenesis and progression of this disease remain unclear. Several tumor characteristics have previously been demonstrated to be prognostic factors of survival following hepatic resection, or the recurrence of HCC or other types of cancer. Comparisons of normal tissues and HCC tumor tissues have revealed the presence of numerous molecular alterations in HCC, including genetic and epigenetic mechanisms, particularly mutations in certain genes and DNA methylation in the promoter regions of tumor-suppressor genes. A number of studies have previously used array analysis to detect variations in the expression levels of cancer-associated genes and microRNAs, and in DNA methylation. However, an investigation of HCC tumor tissues may not determine the effect of noncancerous liver tissues (background liver) in patients with HCC. As HCC may recur multicentrically following resection, a damaged or chronically diseased HCC background liver may be considered as a pre-cancerous organ. Therefore, the influence of the background liver on HCC requires further study. Detailed studies regarding the background liver may be essential for the improved understanding of the carcinogenesis and progression of this malignancy; however only a few studies have investigated the microenvironment of the HCC background liver. The present review discusses prior molecular studies of hepatocarcinogenesis that focus on HCC and background liver tissues.

Zinc enhances CDKN2A, pRb1 expression and regulates functional apoptosis via upregulation of p53 and p21 expression in human breast cancer MCF-7 cell.

Zinc (Zn) is an essential trace elements, its deficiency is associated with increased incidence of human breast cancer. We aimed to study the effect of Zn on human breast cancer MCF-7 cells cultured in Zn depleted and Zn adequate medium. We found increased cancer cell growth in zinc depleted condition, further Zn supplementation inhibits the viability of breast cancer MCF-7 cell cultured in Zn deficient condition and the IC25, IC50 value for Zn is 6.2μM, 15μM, respectively after 48h. Zn markedly induced apoptosis through the characteristic apoptotic morphological changes and DNA fragmentation after 48h. In addition, Zn deficient cells significantly triggered intracellular ROS level and develop oxidative stress induced DNA damage; it was confirmed by elevated expression of CYP1A, GPX, GSK3β and TNF-α gene. Zinc depleted MCF-7 cells expressed significantly (p≤0.001) decreased levels of CDKN2A, pRb1, p53 and increased the level of mdm2 expression. Zn supplementation (IC50=15μM), increased significantly CDKN2A, pRB1 & p53 and markedly reduced mdm2 expression; also protein expression levels of CDKN2A and pRb1 was significantly increased. In addition, intrinsic apoptotic pathway related genes such as Bax, caspase-3, 8, 9 & p21 expression was enhanced and finally induced cell apoptosis. In conclusion, physiological level of zinc is important to prevent DNA damage and MCF-7 cell proliferation via regulation of tumor suppressor gene.

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

Association of aberrant methylation at promoter regions of tumor suppressor genes with placental pathologies.

The resemblance between invasive behavior of cancer cells and placental trophoblasts and the role of aberrant epigenetic regulation in cancer development is well known. We analyzed the role of promoter region CpG-methylation and H3K9/27me3 of tumor suppressor genes in normal and pathological pregnancies and utilized their CpG-methylation data to search for fetal DNA epigenetic marker in maternal blood. CpG and H3K9/27-methylation associated decreased expression of RASSF1A and APC and increased expression of P16, RB1 and PRKCDBP was observed with advancing normal gestation. Gestational trophoblastic diseases and preeclampsia revealed gene-specific epigenetic deregulation of candidate tumor suppressor genes. Furthermore, APC and PRKCDBP showed the potential to act as fetal DNA epigenetic markers, similar to RASSF1A. Deregulation of methylation of tumor suppressor genes contributes to the development of preeclampsia and gestational trophoblastic diseases. APC and PRKCDBP may act as fetal DNA epigenetic markers for prenatal diagnosis.

Methylation of CpG islands in the promoter regions of P16, hMLH1 and CDH1 genes in patients with gastric cancer.

e15531Background: Aberrant methylation of CpG islands in the promoter regions of tumor suppressor genes has been demonstrated to be one of the mechanisms for tumor development. In this study, we an...

Rutin attenuates high cholesterol diet‐induced hepatotoxicity in rat model via regulation of tumor suppressor genes; p53 and caspase‐3 and P21

Hypercholesterolemia‐induced oxidative stress has been implicated in the pathogenesis of various disease including cardiovascular disorders and non‐alcoholic fatty liver disease (NAFLD). Tumor suppressor genes such as p53 play a central role in NAFLD. p53 activation facilitates apoptosis, oxidative stress, steatosis and injury. Levels of p21 often determine the cellular response to different stress induced by drug and others. Caspase‐3‐mediated cleavage of p21. This study was designed to investigate the potential protective effects of dietary antioxidant rutin (RT) combination with high cholesterol diet (HCD) induced hepatotoxicity in male Wistar albino rats. Twenty four male Wistar rats were used in the present study. Animals were divided into four groups; (1) control, (2) RT, (3) HCD and (4) HCD+RT. Animals received freshly prepared experimental diets for 6 consecutive weeks. In plasma, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were estimated. In hepatic tissue, gene expressions of p53 and caspase‐3 and P21 were measured using real time PCR. Rats fed with HCD for 6 weeks had significant increase in plasma ALT and AST levels compared to control and HCD+RT groups. In the current study, HCD increase p53 and caspase‐3 mRNA expression and reduced the mRNA expression P21. Rutin in combination with HCD induced a significant protective effect against the alteration in p53 and caspase‐3 and P21 genes expression. In conclusion, our findings demonstrated that rutin could attenuate the hepatotoxicity induced by HCD in rat model via regulation of tumor suppressor genes.

Meta-analysis of the Association between RASSF1A Gene Promoter Methylation and Non-small Cell Lung Cancer

背景与目的肿瘤发生、发展过程中，抑癌基因启动子区域CpG岛异常甲基化起着重要的作用。已有研究显示RAS相关区域家族1A(Ras association domain family 1A, RASSF1A)基因，作为一个抑癌基因其启动子区域甲基化与非小细胞肺癌(non-small cell lung cancer, NSCLC)的发生发展密切相关。在NSCLC患者癌组织中RASSF1A基因启动子往往表现为异常高甲基化。本研究采用meta分析的方法探讨RASSF1A基因启动子甲基化与NSCLC发生之间的关系。方法通过检索Medline、EMBASE、CNKI和万方数据库，按照已拟定的纳入与剔除标准筛选收集公开发表的关于RASSF1A基因启动子甲基化与NSCLC相关性的研究。以比值比(odds ratio, OR)和95%置信区间(confidence interval, CI)为效应指标，分析RASSF1A基因启动子甲基化与NSCLC的关系。结果共有23篇文献纳入本研究，RASSF1A基因启动子甲基化率在NSCLC患者肺部组织和对照组中分别为41.50%(95%CI: 34%-49%)和5.58%(95%CI: 2%-9%)，meta分析显示肿瘤组织中的甲基化率高于对照组(OR=8.72, 95%CI:4.88-15.58, P < 0.05)；亚组分析显示:肿瘤组织中的甲基化频率高于血浆(OR=10.99, 95%CI:2.48-48.68)和正常对照组织(OR=8.74, 95%CI: 4.39-17.41)。结论NSCLC患者肺癌组织中RASSF1A基因启动子甲基化率高于对照组，组织中RASSF1A基因启动子甲基化率对肺癌的发生更具影响，RASSSF1A甲基化可能与肺癌的发生存在相关并可作为肺癌诊断的潜在标志物。

The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

Post-translational modifications of chromatin components are significantly involved in the regulation of tumor suppressor gene and oncogene expression. Connective tissue growth factor (CTGF) is an epigenetically regulated growth factor with functions in angiogenesis and cell-matrix interactions and plays a pivotal role in hepatocellular carcinoma(HCC). The pharmacologic inhibition of histone and protein deacetylases represents a new approach to interfere with pathways of apoptosis and angiogenesis. We investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on human HCC cell lines HepG2 (p53wt) and Hep3B (p53null) and in a subcutaneous xenograft model and explored the influence on angiogenesis. Specimens were characterized by quantitative real-time PCR. Protein was separated for western blotting against CTGF, VEGF, VEGF receptor-1 (VEGFR-1/FLT-1), VEGF receptor-2 (VEGFR-2/KDR), MAPK and phospho-MAPK. Invivo, HepG2 cells were xenografted to NMRI mice and treated with daily i.p. injections of 10mg/kg panobinostat. After 1, 7 and 28days, real-time PCR was performed. Immunohistochemistry and western blotting were examined after 28days. An increased significant expression of CTGF was only seen after 24h treatment with 0.1µM panobinostat in HepG2 cells and Hep3B cells, whereas after 72h treatment CTGF expression clearly decreased. In the xenografts, treatment with panobinostat showed a minimal CTGF expression after 1day and 4weeks, respectively. Invitro as well as invivo, VEGF was not affected by panobinostat treatment at any time. In conclusion, panobinostat influences extracellular signaling cascades via CTGF-dependent pathways.

Assessment of methylation status of locoregional lymph nodes in lung cancer using EBUS-NA.

Hypermethylation of the promoter region of tumor suppressor genes is associated with carcinogenesis in lung cancer (LC). Endobronchial ultrasound with needle aspiration (EBUS-NA) is a semi-invasive method for obtaining cell blocks from lymph nodes, which can be used for epigenetic analyses. To establish the relationship between methylation status of p16, DAPK, RASSF1a, APC and CDH13 genes in lymph nodes sampled by EBUS-NA, tumor staging and prognosis. Methylation status of DAPK, p16, RASSF1a, APC and CDH13 genes was assessed in EBUS-NA cell blocks from LC patients and related to stage and survival. Eighty-five consecutive patients [mean age 67 (SD 8)] were included. Methylation of ≥1 gene was found in 43 malignant nodes (67 %). A higher prevalence of RASSF1a methylation was observed in small cell lung cancer patients [9/10 (90 %) vs. 15/53 (28 %); p < 0.001 χ(2) test]. Methylation of APC and/or p16 was related to advanced staging in non-small cell lung cancer (NSCLC) [15/29 (52 %) vs. 6/24 (25 %), p = 0.048, χ(2) test]. Patients with NSCLC showing methylation of APC and/or p16 had also lower 6-month survival (p = 0.019, log rank test), which persisted after adjustment for age and subtyping (HR = 6, 95 % CI [1.8-19.5], p = 0.003, Cox regression). Epigenetic analyses are feasible in EBUS-NA cell blocks and may identify methylation patterns associated with worse prognosis. Methylation of p16 and APC genes in NSCLC patients was associated with advanced staging and lower 6-month survival.