DNA methylation: its role in transcriptional regulation and association with lung cancer

Figures & data

Figure 1 Bar chart demonstrating the distribution of the most important groups describing the function of the genes with abnormal DNA methylation in lung cancer tissue.

Notes: Gene ontology was performed based on biological function using The Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 (http://david.abcc.ncifcrf.gov/).

Table 1 List of recent (post-2012) studies identifying abnormal DNA methylation patterns in human lung cancer tissue

Publication	Gene (genomic region)	Methylation	Methodology	Sample size	Comments
Bediaga et al^Citation33	hsa-miR-43a	Hyper 83%	Pyrosequencing	39 NSCLC patients (T/N)	DNA methylation abnormalities contribute little to the deregulation of the miRNAs tested
	hsa-miR-9.2	Hyper 45%
	hsa-miR-9.3	Hyper 46%
	hsa-miR-27b	Hypo 51%
	hsa-miR-182	Hypo 31%
	hsa-miR-24.1	Hypo 39%
	hsa-miR-23b	Hypo 28%
	hsa-miR-200b	Hypo 23%
	has-miR-486	Hypo 44%
	hsa-miR-338	Hypo 74%
	LINE-1	Hypo 77%
Bradly et al^Citation77	MGMT, DAPK, RASSF1, CDH1, LE17-3-a, NORE1, PTEN p16	No age-related differences	Pyrosequencing	198 NSCLC patients	Patients ≥60 years with p 16 hypermethylation had shortened time to recurrence and a shortened survival time
		Hyper
Bruno et al^Citation110	WT1	Mean percentage of methylation tumors: 16.2±3.4 normal adjacent tissue: 5.6±l.7	Pyrosequencing	16 NSCLC patients	ACs present greater WT1 methylation than SCCs As the smoking habit increases, methylation decreases
Carvalho et al^Citation111	CDKN2A	Hyper	MethylCap-seq Validation: bisulfite sequencing, MSP	7 NSCLC patients (T/N) validation: 96 NSCLC patients (T/N)	Statistically significant differences were observed for each of the five loci/genes between lung cancer samples and controls
	chr2:119,331,343 to 119,331,692	Hyper
	chr2:119,328,097 to 119,328,400	Hyper			The methylation status of the chromosomic region chr2:l19,331,343 to 119,331,692 could distinguish between AC and SCC
	RASSFI	Hyper
	ZIC4	Hyper
Cortes-Sempere et al^Citation92	IGFBP3	Methylated in resistant to cisplatin patients	Bisulfite, MSP	61 NSCLC patients (T)	The authors propose a test that could predict resistance to cisplatin therapy, with an accuracy and precision of 0.84 and 0.9, respectively
de Fraipont et al^Citation88	RASSF1, DAPK1		MSP, pyrosequencing	208 NSCLC samples (T)	RASSF 1A methylation is related to poor prognosis of early-stage NSCLC and longer disease-free survival after paclitaxel-based neoadjuvant chemotherapy
					RASSF 1 and DAPK 1 methylation, along with tumor stage, can define three subgroups with strikingly different prognosis
Dietrich et al^Citation84	PITX2, SH0X2	Mean percent of methylation for SHOX was 41.8% and for PITX2 was 13.1%	qMSP	465 (SHOX2) and 445 (PITX2) NSCLC patients (T)	High methylation of SHOX2 and PITX2 was a predictor of progression-free survival
Do et al^Citation68	ATM &RCAI MLHI XPC ERCCI MGMT NEILI RAD23B	Frequency of methylation: No methylation detected No methylation detected No methylation detected No methylation detected 2% 13% 42% 2%	MS-HRM. External validation using the TCGA database. Validation: Big Dye terminator v3.1	56 NSCLC samples (T)	Promoter methylation frequency of ATM, BRCA1, MLH 1, and XPC is likely to be overestimated in the literature
Geng et al^Citation71	NEUROG2, NID2, RASSFIA, APC and HOXC9		MSP	15 healthy volunteers, 103 stage 1 NSCLC tumors (T), and 26 noncancerous controls (N)	This five-gene panel with sensitivity of 91.26% with specificity of 84.62% could detect stage 1 NSCLC The methylation status of NEUROG2 could distinguish AC from SCC
Zhao et al^Citation70	MGMT	Median percent methylation in tumors was 26.1% and in normal adjacent tissue 2.4%	MSP, qMSP	1,160 NSCLC tissues and 970 controls	This meta-analysis identified a strong association between methylation of MGMT gene and NSCLC
Gu et al^Citation74	P16^INK4A	Median percent methylation in tumors was 44% and in normal adjacent tissue 15%	MSP, qMSP	2,652 NSCLC patients with 5,1 75 samples (T/N)	Strong and significant correlation between tumor tissue and autologous samples of P161NK4A promoter methylation
Guo et al^Citation75	APC	Hypermethylated in cancer	MSP, qMSP	2,259 NSCLC tumor samples and 1,039 controls	This meta-analysis showed that APC methylation was strongly associated with NSCLC, especially AC
Harada et al^Citation78	&RCAI	Methylation in 13 of 70 patients (18.6%)	MSP	70 patients with curatively resected stage 1 NSCLC (T/N)	Recurrence-free survival rate in patients with BRCAI methylation was lower than in those without BRCAI methylation
Heller et al^Citation72	miR- 193a miR-9-3	Mean percent methylation in tumors was 33% and in normal adjacent tissue 21 % Mean percent methylation in tumors was 25% and in normal adjacent tissue 1 1 %	MS-HRM, BGS	101 patients (T/N) with stage l-III NSCLC	76% of the patients were methylated for miR-9-3 and/or miR-193a Poor survival of patients with miR-9-3-methylated compared to miR-9-3-unmethylated SCC
Heller et al^Citation79	HOXA2 PRDM14	Frequency of methylation 78% 72%	Methylated DNA immunoprecipitation, microarray, MS-HRM. Validation: BGS	101 stage 1, II, or III NSCLC patients (T/N)	HOXA2 and HOXAIO methylation was associated with better survival in SCC
	EVXI	68%			G ATA3 was most frequently methylated in AC, while SFMBT2 in SCCs
	PCD HA 12	62%
	TAL1	61%
	IRX2	56%
	POU3F4	55%
	GATA3	46%			POU3F4 methylation was associated with sex
	SFMBT2	45%
	HOXA10	40%			G ATA3 methylation was observed more frequently in late stages
	DMRTA2	39%
	SHOX2	39%
Hochhauser et al^Citation95	MGMT	1.35%	qMSP	Among 740 patients screened, only 10 NSCLC cases were positive for MGMT methylation	Aim of this clinical trial was to investigate whether response to temozolomide can be predicted by MGMT methylation. Low number of cases led to uninterpretable results
Hua et al^Citation73	RARβ	Hypermethylated in cancer	MSP	1,347 NSCLC tissue samples and 1,137 autologous controls	This meta-analysis showed that RARp promoter methylation was higher in tumor tissues compared to control
Huang et al^Citation112	1,413 genes		Mehylation profiles from Gene Expression Omnibus with the accession number GSE 16559	57 NSCLC patients and 52 control samples	20 clusters of dysfunctional genes in NSCLC were identified based on methylation, miRNA, and mRNA differences
Jia et al^Citation113	DACT2	Mehylation found in 41 % of tumor and 0% of normal samples	MSP, bisulfite sequencing (BSSQ)	106 NSCLC samples and 4 tissue samples from patients without cancer	DACT2 promoter methylation observed in lung cancer
Ko et al^Citation89		Frequency of mehylation	MSP	328 FFPEs from patients with resected NSCLC (T)	Hypermethylation of RASSF1A was associated with high risk of recurrence in node-negative stage 1 and II NSCLCs
	BAX	67%
	GSTP1	11%
	HOXA11	72%
	Integrin a 4	28%
	MyoD1	59%
	RARβ2	34%
	RASSFIA	52%
	SOCS-1	42%
Koga et al^Citation80	CHFR	Frequency of methylation: 10%	MSP	165 ACs (T)	CHFR hypermethylation was correlated with poor prognosis and lymphatic vessel invasion
Kontic et al^Citation144	RASSFIA CDHI3	Median (IQR) methylation change between tumors and normal adjacent tissue 0.91 (−0.15, 11.7) 2.43 (−1.41, 6.9)	Pyrosequencing	65 NSCLC patients (T/N) and 51 blood samples	Higher methylation of RASSFIA, CDH13, and DAPK genes in lung tumors compared to normal lung Histology and sex were associated with CDH 13 methylation
	MGMT ESRI DAPK	0.12 (−0.27, 0.95) 0.28 (−0.67, 1.46) 0.87 (−0.97, 2.26)			Stage was associated with methylation of MGMT
Lee et al^Citation114	TMEFF2	Methylation found in 52.5% of tumor samples	MSP	Tumor and corresponding nonmalignant lung tissue specimens (n=l 39)	TMEFF2 methylation was more frequent in ACs without EGFR mutation
Lee et al^Citation90	RASSFIA	Methylation found in 44.7% of tumor and 2.6% of normal samples	Pyrosequencing, MSP	206 NSCLC tumor and 40 adjacent normal samples	RASSF 1A methylation was more frequent in ever- smokers and tumors with TP53 mutation Strong methylation was an unfavorable prognostic factor for stage 1 and SCC patients
Lee et al^Citation115	Wifl	Methylation found in 47.5% of tumor and 20.9% of normal tissues	MSP	139 NSCLC patients (T/N)	Wif I is hypermethylated in tumors compared to normal adjacent tissue Wifl methylation is associated with unfavorable prognosis in ACs patients with EGFR mutation
Li et al^Citation116			DNA Methylation data from NCBI GEO: accession number GSE32867	28 ACs and 27 adjacent normal samples	Identification of 108 differentially methylated genes in tumor compared to normal tissues, involved in cell development, immune response, and apoptosis pathways
Li et al^Citation94	miR-503	Methylation found in 80% of tumor and 20% of normal samples	Bisulfite Restriction Analysis (COBRA), Bisulfite-treated DNA sequencing assays (BSP)	65 NSCLC patients (T/N)	miR-503 is methylated in tumors compared to normal samples
Liu et al^Citation117	ANKRD18A	Methylation found in 68.4% of tumor and 0% of normal samples	M ethylation-specific PCR, bisulfite-sequencing PCR	38 NSCLC tumors and 20 normal samples	ANKRD 18A hypermethylation was associated with lung cancer
Liu et al^Citation118	ANKRDI8B	Methylation found in 53.1% of tumor and 0% of normal samples	Methyl ation-specific PCR and BGS	98 NSCLC patients (10 adjacent normal samples)	ANKDI8B exhibited higher methylation in tumor compared to normal samples ANKD18B hypermethylation was associated with poor differentiation and earlier stage tumors
Liu et al^Citation119	ALX4	Methylation found in 55% of tumor and 0% of normal samples	MSP and BGS	98 NSCLC patients (20 adjacent normal samples)	Tumor suppressor ALX4 is downregulated in NSCLC due to DNA methylation
Liu et al^Citation120	LHX6	Methylation found in 56% of tumor and 0% of normal samples	MSP and BGS	93 primary NSCLC tumor and 20 normal samples	Tumor suppressor LHX6 is downregulated in NSCLC due to DNA methylation
Lokk et al^Citation87	Whole genome	Differential methylation of clustered genes	InfiniumH Human- M ethylation27 RevB BeadChips (Illumina Inc.). Validation: Sanger Sequencing	48 patients with stage 1 NSCLC and 18 matching cancer-free lung samples	DXS9879E (LAGE3), RTELI and MTM 1 hypermethylation, and SCUBE3, SYT2, KCNC3, KCNC4, GRIK3, CRB 1, SOCS2, ACTAI, ZNF660, MDFI, ALDH 1 A3, and SRD5A2 hypomethylation were associated with poor survival
Lu et al^Citation121	IncRNA MEG3	Methylation found in 68% of normal and 96% of tumor samples	Bisulfite sequencing	44 patients (T/N)	Donwregulation of IncRNA MEG3 observed in NSCLC is partly due to promoter hypermethylation
Meng et al^Citation122	miR-31		TCGA data set (Illumina Infinium Human DNA Methylation 450 beadchip data)	1 77 lung AC samples containing both methylation and miR-3 1 expression from the 233-patient set of the databank	DNA promoter methylation leads to repression of miR-3 1 expression
Morân et al^Citation91	CALCA, MMP-2	Hyper	Illumina GoldenGate Methylation bead array, validation with qMSP (only for RASSFI and pi6)	46 NSCLC samples (T/N)	Hypermethylation of CALCA and MMP-2 was associated with a poor clinical outcome
	RASSF1	Hyper			Hypermethylation of RASSF 1 was a protective variable
	CALCA, HOXB2, IRF7, MOS, MTIA, MYCL2, p16, RARA, RASSFI, SEP15, SOX17 and SPARC	Hyper			SCC was associated with hypermethylation of these 12 genes Global hypermethylation was associated with bad prognosis in stage III A NSCLC patients
	SFN (14-3-3 sigma)	Hypo			Well-differentiated tumors showed hypermethylation of IRF5 and NPR2
	ZMYNDIO IRF5, NRP2	Hypo Hyper
Na et al^Citation85	DKK1	Methylation found in 48.9% of tumors and 22.3% of normal samples	MSP	139 NSCLC patients (T/N)	DKK1 methylation was more frequent in patients with stage 1 NSCLC
					DKK 1 methylation was associated with better survival of males, ever-smokers and AC patients without EGFR mutation
Nadal et al^Citation82	MicroRNA-34b/c	More than 5% methylation found in 42% of tumor and 10% of normal samples	Melting-curve analysis. Validation: BGS	140 early-stage lung ACs and 10 nonmalignant samples	Methylation of microRNA-34b/c predicts poor disease-free survival in early-stage ACs
Nelson et al^Citation67	HOXA9 SOXI DDRI	Methylation fold change in cancer: Hyper (10.3 fold) Hyper (5.9-fold) Hypo (8.1 -fold)	Illumina GoldenGate arrays, validation: bisulfite pyrosequencing	47 NSCLC patients. Validation set: 99 NSCLC patients (T/N)	The identified genes consistently have altered methylation in lung tumors (no difference between SCC and ACs)
Ko et al^Citation89	SLC5A8	Hypermethylated in cancer	Restriction landmark genomic scanning. Validation: bisulfite- modified sequencing and qMSP	23 NSCLC patients (T/N)	Hypermethylation of SLC5A8 promoter, which is mainly observed in tumor samples, seems to affect the expression of SLC5A8
Ramnath et al^Citation86	CfP 24A1	More than 10% methylation found in 35.5% of tumor samples	Bisulfite DNA pyrosequencing	90 surgically resected lung ACs	Patients with unmethylated CYP24AI had worse disease-free and overall survival
Sandoval et al^Citation123	HISTIH4F, PCDHGB6, NPBWR1, ALX1, and HOXA9		DNA methylation microarray. Validation: pyrosequencing	Array: 444 stage 1 NSCLC patients. Validation: 143 stage 1 NSCLC patients	A signature based on the number of hypermethylated events distinguished stage 1 NSCLC patients with high and low risk of relapse
Sato et al^Citation124	ADCY5, EVX1, GFRA1, PDE9A, and TBX20	Hyper	Single-CpG resolution Infinium array. Validation: pyrosequencing	145 AC patients (T/N) and 36 normal tissue from patients without any primary lung tumor	Methylation of these five genes in tumor samples was correlated with less mRNA production and tumor aggressiveness
Sato et al^Citation125	Genome wide		Single-CpG resolution Infinium array. Validation: pyrosequencing	139 NSCLC paired samples and 36 normal samples from patients without any cancer previously tested. Validation: 50 paired samples	DNA methylation profiles related to carcinogenetic factors such as smoking and chronic obstructive pulmonary disease appear to be established in noncancerous lung tissue from patients with ACs and may determine the aggressiveness of the tumors
Scesnaite et al^Citation126		Frequency of methylation	MSP	212 NSCLC patients	Hypermethylation of p 16 was predominant in men and SCCs
	p16, RARB, RASSF 1	20%-30%			RARB hypermethylation was more frequent in females and ACs
	MGMT, DAPKI	15%-20%
Shi et al^Citation127	Cos-trans-methylation quantitative trait loci in the genome		Illumina Infinium HumanMethylation450 Bead Chip, methylation quantitative trait loci analyses	244 stage l-IIIA NSCLC patients (T/N)	Identification of 34,304 cis- and 585 transmethylation quantitative trait loci
Shinjo et al^Citation128	CCNA1, ACAN, GFRA1, EDARADD, MGC45800, and p16		Genome-wide DNA methylation microarray analysis. Validation: pyrosequencing	41 AC patients. Validation: 128 AC patients (T/N)	The study reveals six newly identified CIMP (CpG island methylator phenotype) markers that may be useful in the accurate and practical epigenomic classifications of lung cancer
Selamat et al^Citation129	ABCA3	Hyper	Illumina Infinium HumanMethylation27 platform	59 AC patients (T/N)	The hypermethylated cluster was associated with smoking status and KRAS mutations in AC
	SOX17 TMEM204 FAM83A AGR2, KRT8, SFN LGALS4	Hyper Hyper Hypo Hypo Hyper in smokers compared to nonsmokers
Suzuki et al^Citation130		Frequency of methylation	Pyrosequencing	56 NSCLC patients (T/N)	LINE-1 methylation was lower in SCC than ACs
	LINE-1	55% (hypo)
	SLIT2	64% (hyper)			SLIT2 methylation was higher in COPD when compared with non-COPD cases
	MAL	46% (hyper)			MAL hypermethylation was higher in men, smokers and patients without EGFR mutations
	IGFBP7	54% (hyper)
Tan et al^Citation131	PRDM5	Hypermethylated in cancer	MSP	30 SCC patients (T/N)	PRDM5 methylation was correlated with tumor differentiation and lymph node metastasis
Tang et al^Citation132	PCDH10	Methylation found in 50% of tumors and 0% of normal samples	MSP	40 NSCLC patients (T/N)	PCDH 10 promoter methylation was related to smoking
Tekpli et al^Citation133	CYPIA1	High methylation in never- smokers	Pyrosequencing	120 early-stage NSCLC patients (T/N)	Low CYPIAI enhancer methylation in histologically normal lung was associated with high CYPIAI mRNA levels and smoking-induced genetic alterations
Tennis et al^Citation134	Wnt7a	Hypermethylated in cancer	Pyrosequencing	82 NSCLC patients (T/N)	The Wnt7a promoter is hypermethylated in cancer resulting in decreased activity
Tessema et al^Citation135	TOX2	Found methylated in 28% (TOX2), 5% (TOX) and 58% (TOX3) of tumor and in 0% of normal samples	MSP, bisulfite sequencing	190 primary NSCLC tumors (and some adjacent normal samples)	Prevalence for TOX3 methylation among lung cancer patients was greater in SCC (79%) compared to ACs (56%)
	TOX TOX3 TOX4	No methylation detected
Walter et al^Citation136	ERBB2 ZEB2	Hypermethylated in cancer	qMSP (with pre-amp) and pyrosequencing	42 late-stage NSCLC (T) 60 late-stage NSCLC (T)	ERBB2 and ZEB2 methylation status was strongly associated with an epithelial phenotype in patients who fail frontline chemotherapy
Wilkerson et al^Citation137	Genome wide		Methylation-SNP analysis (DNA digested by methylation-sensitive Ηρall to Hpall-undigested DNA by Affymetrix 250K Sty microarrays)	Published cohorts (n=504) Validation cohort (n=116)	ACs are classified in Magnoid, Squamoid, and Bronchioid. The main methylation alteration detected was genome-wide hypermethylation in Magnoid
Wu et al^Citation81	TFP1-2	Found methylated in 27.1% of the samples	MSP	133 non-metastatic NSCLC patients (T)	Methylated TFPI-2 was associated with poor survival
Yin et al^Citation138	SOX17	Methylated in 60.2% of the tumor samples	MSP	88 primary NSCLC samples	SOX 1 7 methylation was associated with sex and poor tumor differentiation
Zhao et al^Citation139	RARβ	Methylation found in 57.5% of tumor and 17.5% of normal samples	MSP	80 NSCLC patients (T/N)	RARp methylation was more frequently found in tumors (more closely related to SCC) compared to normal adjacent tissue
Zhao et al^Citation70	MYF6, S1X6, BCL2, and RARB	Hyper	MSP	101 stage 1 NSCLC patients and 30 patients with noncancerous lung diseases	A panel of four genes (MYF6, SIX6, BCL2, and RARB) has a sensitivity of 93.07% and a specificity of 83.33% in the diagnosis of stage 1 NSCLC Co-methylation of SIX6 and SOXI correlated with SCC Co-methylation of BCL2, RARB, and SIX6, but not the methylation of either single gene, was associated with smoking
	ALXI, HPPI, OC2, PDGFRA, and SMPD3				No difference in methylation of ALXI, HPPI, OC2, PDGFRA, and SMPD3 between tumor and normal samples
Zhu et al^Citation93	SFRP5		MSP	155 patients with stage IIIB-1 V NSCLC who received EGFR-TKI therapy (T)	Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy (P=0.002), independent of EGFR genotype
Cao et al^Citation83	miR-886-3p	Hypermethylated in cancer	Bisulfite sequencing (BSP) and methylation-speciflc PCR (MSP)	33 SCLC patients	miR-886 promoter methylation was correlated with shorter overall survival of SCLC patients
Kalari et al^Citation140	Genome wide		MIRA technique Validation: COBRA and bisulfite genome sequencing	18 SCLC tumor and 5 normal samples	This study identified 73 gene targets that are methylated in >77% of primary SCLC tumors
Tanaka et al^Citation141	miR-34a miR-34b/c	Methylation found in 15% of SCLC and 28% of NSCLC Methylation found in 67% of SCLC and 26% of NSCLC	MSP	12 SCLC, 47 NSCLC resected tumors, and 15 malignant pleural effusions in SCLC patients	The frequency of miR-34b/c methylation was higher in SCLC than in NSCLC

Abbreviations: AC, adenocarcinoma; BGS, bisulfite genomic sequencing; BSP, bisulfite sequencing PCR; COBRA, combined bisulfite restriction analysis; COPD, chronic obstruction pulmonary disease; FFPE, formalin fixed paraffin embedded; GEO, Gene Expression Omnibus; IQR, interquartile range; lncRNA, long non coding RNA; mRNA, messenger RNA; MS-HRM, methylation-sensitive high-resolution melting; MSP, methylation specific PCR; NCBI, National Center for Biotechnology Information; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; qMSP, quantitative methylation-specific PCR; SCC, squamous cell carcinoma; SCLC, small-cell lung cancer; T, tumor-only samples; T/N, tumor-normal pairs.

Table 2 List of recent (post-2012) studies involving detection of DNA methylation in body fluids from patients with lung cancer

Publication	Gene (genomic region)	Sample size	Methodology	Tissue/body fluid	Comments
van der Drift et al^Citation99	RASSFIA	129 lung cancer patients and 28 controls	qMSP	Bronchial washings	RASSFIA hypermethylation in 50% of central tumors and 31% of peripheral tumors RASSFIA methylation detected in 4/17 patients with nondiagnostic bronchoscopy When combined with KRAS mutation, methylation RASSF 1A provided an additional diagnostic value of 29%
Dietrich et al^Citation100	SHOX2	125 lung cancer patients and 125 controls	HeavyMethyl qPCR	Bronchial aspirates	Evaluation of SHOX2 methylation in a case-control study conferred 96% specificity and 78% sensitivity
Nikolaidis et al^Citation60	pi6, RASSFI, TERT, WTI	Training set: 194 cases/213 controls Validation set: 139 cases/109 controls	qMSP	Bronchial lavage	Methylation panel showed 82% sensitivity, 91% specificity, and 85.9% diagnostic accuracy
Darwiche et al^Citation101	SH0X2	154 lung cancer patients (lymph node aspirates)	qMSP	EBUS-TBNA	94% sensitivity and 98% specificity for detection of malignant lymph nodes by SHOX2 methylation in EBUS-TBNA Negative predictive value increases from 80% to 99% when SHOX2 assessment is used in combination with pathological examination
Shin et al^Citation142	MAGE A3, pi6	65 lung cancer cases, 68 benign lung diseases, 30 healthy individuals	Nested-MSP	Sputum (induced)	MAGE A3 and p 16 methylation was more frequent in the sputum of lung cancer than in benign lung disease and healthy controls Methylation of MAGE A3 and p 16 was higher in MAGE AI-A6- positive group when compared to the negative group
Hubers et al^Citation102	RASSFI A, APC, CYGB	Training set: 98 cases/90 controls Validation set: 60 cases/445 controls	qMSP	Sputum (spontaneous)	RASSF 1A best of the three in discriminating lung cancer cases in both sets (sensitivity 41 %-52% and specificity 94%-96%) Assessing RASSF 1A improved the sensitivity of cytological evaluation, from 22% to 52% without diminishing the specificity (94%)
Leng et al^Citation103	Overall, 3 1 candidate genes evaluated CO panel: MGMT, DAPK, PAX5b, Dali, PCDH20, Jph3, Kifla NM panel: DAPK, PAX5b, PAX5a, Dali, GATA5, SULF2, CXCLI4	CO cohort:64 lung cancer cases/64 controls NM cohort: 40 stage 1 asymptomatic lung cancer cases/90 controls	MSP	Sputum (spontaneous)	Seven-gene panels improved the classification accuracy obtained with covariates alone (CO cohort: 62%—71 % and NM cohort: 58%-77%) NM cohort: methylation of five or more genes in the seven-gene panel conferred a 22-fold increase in risk for lung cancer
Liu et al^Citation118	ANKRD18B	Sputum: 85 lung cancer cases Plasma: 65 lung cancer cases 10 controls (tissue)	MSP and BGS	Sputum Plasma	ANKRD 18B aberrant methylation detected in the sputum of 69.7% (23/33) of the patients who showed tissue methylation ANKRD 18B aberrant methylation detected in the plasma of 72.7% (24/33) of the patients who showed tissue methylation ANKRD 18B hypermethylation is cancer-specific (not found in control samples)
Balgkouranidou et al^Citation105	BRMSI	Training set: 57 stage l-lll NSCLC patients (tumor tissue, adjacent normal and corresponding plasma - for 48 patients) Validation set: 74 stage IV NSCLC patients cell-free DNA Control group: 24 healthy donors cell-free DNA	MSP tissue samples qMSP - cell-free DNA samples	Tissue (fresh frozen) Plasma	BRMSI promoter was methylated both in NSCLC primary tissues (59.6%) and in corresponding cell-free DNA (47.9%) but not in cell-free DNA from healthy individuals (0%) Stage l-lll NSCLC patients with BRMSI methylation in cell-free DNA had lower disease-free interval and overall survival In stage IV patients, methylation of BRMSI in cell-free DNA was associated with a lower progression-free and overall survival
Kontic et al^Citation144	RASSFIA, CDH 13, MGMT, ESRI, DAPK	Primary tumor tissue and nonmalignant lung tissue from 65 NSCLC patients and corresponding blood samples (from 51 cases)	Pyrosequencing	Tissue (fresh frozen)	DAPK, RASSF 1 A, and CDH 13 were significantly hypermethylated in lung tumors compared with matched normal adjacent tissue CDH 13 hypermethylation was observed more frequently in women and adenocarcinomas
				Whole blood	Stage IV patients were more likely to have MGMT hypermethylation ESR1 showed higher methylation levels in blood from patients with hypermethylation in their tumor samples
Lee et al^Citation114	TMEFF2	139 NSCLS T/N	Nested – MSP	Tissue (fresh frozen)	TMEFF2 hypermethylation was found in 52.5% (73/139) of tumors and 15.8% (22/139) of adjacent normal adjacent tissue TMEFF2 methylation was significantly lower in adenocarcinomas with EGFR mutations
		316 NSCLC patients and 50 healthy donors		Serum	Detection of TEMFF2 methylation in serum showed 9.2% sensitivity and 100% specificity
Lietal^Citation106	RASFFI, RARβ	56 lung cancer patients and 52 controls	MSP	Peripheral blood	RASSF 1 methylation was observed in 85.71% (48/56) of lung cancer patients’ blood RARβ methylation was found in 80.36% (45/56) of lung cancer patients’ blood Methylation of both genes was observed in 75% (42/56) of lung cancer patients’ blood Control samples did not showed methylation neither in RASSF 1 nor in RARp
Ponomaryova et al^Citation107	RASFFI, RARβ	60 untreated NSCLC patients (blood samples prior to treatment, after neoadjuvant treatment and after surgery) 32 healthy volunteers (age comparable)	qMSP	Blood (plasma and cellular fraction)	RASFF1 and RARβ methylation showed 85% sensitivity and 75% specificity (81% accuracy) in circulating DNA (free and cell surface-bound combined analysis) RASFF 1 and RARβ methylation levels in cirDNA decreased after chemotherapy, reaching healthy subject levels after tumor resection Tumor relapses were observed in all NSCLC patients showing increased methylation levels of either gene 9 months after surgery
Powrozek et al^Citation104	SEPTIN9	70 lung cancer patients	Abbott mSEPT9 detection Kit	Serum	Aberrant promoter methylation of SEPTIN9 detected in 44.3% of lung cancer patients, providing 92.3% specificity
		100 healthy individuals	(real-time PCR)		SEPTIN9 methylation more frequently observed in NSCLC than SCLC patients (53% vs 26%)
Tan et al^Citation143	CDKN2A, RASFFI A, FHIT	200 primary lung cancer patients 200 healthy individuals	qMSP (SYBR green)	Peripheral blood	Methylation levels of CDKN2A, RASFF1 A, and FHIT were significantly higher in lung cancer cases when compared to controls
lise et al^Citation109	SHOX2	Pleural effusions from 1617 patients	qMSP	Pleural effusion	SHOX2 methylation detected not only malignant pleural effusions from lung cancer patients but also metastases of other malignant tumors Best diagnostic accuracy was achieved when cytological evaluation was combined with SHOX2 methylation (58% sensitivity and 96.2% specificity)

Abbreviations: BGS, bisulfite genomic sequencing; CO, Colorado; EBUS-TNA, endobronchial ultrasound with transbronchial needle aspiration; NM, New Mexico; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; qMSP, quantitative methylation-specific PCR; qPCR, quantitative PCR; T/N, tumor -normal pairs.

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