Role of MLH1 methylation in esophageal cancer carcinogenesis and its clinical significance

Figures & data

Table 1 The basic characteristics of all eligible studies

Study	Year	Country	Ethnicity	Method	Carcinoma		Normal			Precarcinoma		NOS
					M+	Samples	M+	Samples	Source	M+	Samples
Eads et al^Citation33	2001	USA	Caucasian	qMSP	2	22	0	31	A	1	19	7
Nie et al^Citation34	2002	China	Asian	MSP	5	21	0	25	A	2	13	7
Geddert et al^Citation35	2004	Germany	Caucasian	MSP	7	50	0	50	A	NA	NA	8
Tzao et al^Citation36	2005	China	Asian	MSP	37	60	0	20	A	NA	NA	7
Clement et al^Citation37	2006	Switzerland	Caucasian	MS-DBA	1	27	0	16	H	NA	NA	7
Guo et al^Citation38	2006	China	Asian	MSP	16	69	1	17	H	8	60	7
Ishii et al^Citation39	2007	Japan	Asian	COBRA	6	56	1 0	56 42	A H	0	21	8
Wang et al^Citation40	2008	China	Asian	MSP	4	125	0 0	125 10	A H	NA	NA	8
Liao et al^Citation41	2009	China	Asian	MSP	22	105	2	105	A	NA	NA	7
Moriichi et al^Citation42	2009	Japan	Asian	MSP	NA	NA	0	21	NA	7	83	7
Vasavi et al^Citation43	2010	India	African	MSRE	33	50	NA	NA	NA	NA	NA	8
Lu et al^Citation44	2011	China	Asian	MSP	4	120	0	120	A	NA	NA	8
Ling et al^Citation45	2011	China	Asian	qMSP	102	235	21	235	A	NA	NA	8
Wang et al^Citation46	2011	China	Asian	MSRE	1	13	0	55	H	0	21	7
Chen et al^Citation47	2012	China	Asian	MSP	11	257	0	257	A	NA	NA	8
Su et al^Citation22	2014	China	Asian	MSP	9	51	6	51	A	NA	NA	8
Fukui et al^Citation48	2016	Japan	Asian	MS-HRM	8	10	NA	NA	NA	29	128	8
Guilleret et al^Citation49	2016	Switzerland	Caucasian	MLM	17	26	0	8	H	NA	NA	7
Wu et al^Citation50	2017	China	Asian	MSP	53	87	NA	NA	NA	NA	NA	8

Abbreviations: A, autologous (control samples from the same patients); COBRA, combined bisulfite restriction analysis; H, heterogeneous (control samples from other individuals); M+, positive for MLH1 methylation test; MLH1, mutL homolog-1; MLM, methylation ligation-dependent macroarray; MS-DBA, methylation-sensitive dot-blot assay; MS-HRM, methylation-sensitive high-resolution melting analyses; MSP, methylation-specific polymerase chain reaction; MSRE, methylation-sensitive restriction endonuclease; NA, not available; NOS, Newcastle–Ottawa Scale; qMSP, quantitative methylation-specific polymerase chain reaction.

Figure 1 Flow diagram of the selection process for this meta-analysis.

Table 2 Subgroup analyses of MLH1 promoter methylation in esophageal cancer

Subgroup	Case		Control		Pooled OR (95% CI)	P-value	Heterogeneity
	M+	Total	M+	Total			I^2 (%)	P-value
Ethnicity
Caucasian	27	125	0	105	11.90 (2.75–51.51)	<0.01	0	0.62
Asian	217	1,112	31	1,118	8.15 (5.75–12.08)	<0.01	16.2	0.29
Histology
ESCC	224	1,162	31	1,168	8.32 (5.64–12.29)	<0.01	10.6	0.34
EAC	20	75	0	55	9.98 (1.83–54.32)	<0.01	0	0.45
Control source
Autologous	209	1,102	30	1,075	8.28 (5.58–12.29)	<0.01	15.9	0.29
Heterogeneous	28	290	1	140	6.54 (2.12–11.69)	<0.01	0	0.55
Methods
MSP	115	858	9	780	8.61 (4.64–15.97)	<0.01	0	0.88
No MSP	129	379	22	443	8.25 (5.12–13.30)	<0.01	34.7	0.14
Sample size
<60	48	266	7	334	5.59 (2.74–11.39)	<0.01	17	0.3
≥60	196	971	24	889	9.74 (6.21–15.27)	<0.01	0	0.75
Published year
<2010	100	535	4	497	12.74 (5.84–27.78)	<0.01	0	0.89
≥2010	144	702	27	726	6.99 (4.52–10.82)	<0.01	42.9	0.12

Abbreviations: EAC, esophageal adenocarcinoma; ESCC, esophageal squamous cell carcinoma; M+, positive for MLH1 methylation test; MLH1, mutL homolog-1; MSP, methylation-specific polymerase chain reaction; OR, odds ratio.

Figure 2 Pooled forest plot of MLH1 methylation frequency during the carcinogenesis of esophageal cancer.

Notes: (A) Carcinoma versus healthy controls: OR=8.40, 95% CI =5.75–12.28. (B) Carcinoma versus precancerous lesions: OR=3.08; 95% CI =1.64–5.92. (C) Precancerous lesions versus healthy controls: OR=3.75; 95% CI =1.08–13.02.
Abbreviations: MLH1, mutL homolog-1; OR, odds ratio.

Figure 3 Sensitivity analysis of pooled ORs for the association between MLH1 methylation and esophageal cancer.

Abbreviations: MLH1, mutL homolog-1; OR, odds ratio.

Figure 4 Begg’s funnel plots and Egger’s test of publication bias for MLH1 methylation during the carcinogenesis of esophageal cancer.

Notes: (A) Carcinoma versus healthy controls: Begg’s test: P=0.69; Egger’s test: P=0.29. (B) Carcinoma versus precancerous lesions: Begg’s test: P=0.42; Egger’s test: P=0.52. (C) Precancerous lesions versus healthy controls: Begg’s test: P=0.81; Egger’s test: P=0.53.
Abbreviations: EC, esophageal cancer; MLH1, mutL homolog-1; NC, normal control; PC, precancerous lesions.

Table 3 Association between MLH1 promoter methylation and clinicopathological features of esophageal cancer patients

Characteristics	No	Case/control	Pooled OR (95% CI)	P-value	Heterogeneity
					I^2%	P-value
Age	4	Older/younger	1.79 (1.20–2.66)	<0.01	0	0.4
Gender	4	Male/female	1.12 (0.61–2.05)	0.71	0	0.99
Smoking behavior	3	Yes/no	0.90 (0.46–1.74)	0.75	0	0.78
Alcohol consumption	3	Yes/no	0.81 (0.42–1.57)	0.54	0	0.73
Differentiation grade	4	Poor/well and moderate	1.45 (0.92–2.28)	0.11	26.7	0.25
Location	4	Up and middle/down	0.87 (0.58–1.31)	0.5	0	0.9
T stage	6	T3+4/T1+2	3.7 (2.37–5.77)	<0.01	78.8	<0.01
Lymph node metastasis	6	Yes/no	2.65 (1.81–3.88)	<0.01	73.2	<0.01
Distant metastasis	4	Yes/no	7.60 (1.23–47.19)	0.03	63.6	0.04
Clinical stage	4	III + IV/I + II	4.46 (2.88–6.91)	<0.01	88.9	<0.01

Abbreviations: MLH1, mutL homolog-1; No, number of studies; OR, odds ratio.

Figure 5 Forest plot for pooled HR and the corresponding 95% CI of MLH1 methylation for OS of EC patients.

Abbreviations: EC, esophageal cancer; HR, hazard ratio; MLH1, mutL homolog-1; OR, odds ratio; OS, overall survival.

Figure 6 SROC plots of methylated MLH1 for the diagnosis of esophageal cancer.

Abbreviations: MLH1, mutL homolog-1; SENS, sensitivity; SPEC, specificity; SROC, summary of receiver operating characteristic.

Figure 7 Fagan plot analysis to evaluate the diagnostic power of methylated MLH1 for esophageal cancer.

Notes: (A) The posttest probability was 89% at a pretest probability of 25%. (B) The posttest probability was 96% at a pretest probability of 50%. (C) The posttest probability was 99% at a pretest probability of 75%.
Abbreviations: LR, likelihood ratio; MLH1, mutL homolog-1.

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