Hypermethylation of multiple tumor-related genes associated with DMNT3b up-regulation served as a biomarker for early diagnosis of esophageal squamous cell carcinoma

Figures & data

Figure 1 Representative results of MSP analysis of the methylated form of 12 tumor-related genes. M, markers; N, negative control (water blank); P, positive control (universal methylated DNA); T, tumor samples. Samples were scored as methylated when there was a clearly visible band with the methylated primers.

Figure 2 mRNA expression of DNMTs in patients with ESCC (n = 46). (A) Real-time reverse transcription-PCR results for DNMTs. The black bars correspond to non-tumor tissues (NT), and the white bars correspond to ESCC. The means of expression levels of DNMTs are depicted relative to ACTB housekeeping gene expression. Asterisk indicates statistical significance after multiple testing (p < 0.017). (B) DNMT1, 3a and 3b mRNA were expressed in a coordinate manner in the same tumor tissues. “+” indicates elevated mRNA expression as opposed to “−” which indicates a not elevated result. Numbers above the bars indicate the percentage in the total concordant group (+/+ and −/−) and non-concordant group (+/− and −/+). For the association between categories, p = 0.051 for DNMT1/3b, p < 0.001 for DNMT1/3a and p = 0.008 for DNMT3a/3b.

Figure 3 Representative immunohistochemical staining results for DNMT3b protein in two patients with ESCC. Strong nuclear expression was seen in the layer of epithelium in ESCC tissues (A and C). Negative staining was observed in paired non-tumor tissues (B and D). (Original magnification ×400 in A–D).

Figure 4 Hypermethylation of cell-free serum DNA at five gene promoter regions in ESCC patients (n = 45) and healthy individuals (n = 15). S, ESCC samples; HI, healthy individuals samples; WBC DNA isolated from the WBCs of healthy volunteers served as a negative control and SssI treated fully methylated DNA served as positive control. NIM was color-scaled between white (representing no methylation detected) and black (indicating that ≥10% of bisulfite-converted input copies were methylated). Asterisk indicates NIM values less than 0.5% but greater than 0.

Table 1 Frequency of promoter methylation for individual gene in ESCC patients (n = 47)

Genes	Frequency of methylation (%)		p value
	ESCC	NT
p16	44.7%	21.3%	0.027
p14	14.9%	0.0%	0.012
DAPK	46.8%	12.8%	0.001*
RAR-β	46.8%	12.8%	0.001*
CDH1	42.6%	21.3%	0.046
APC	12.8%	4.3%	0.267
TIMP3	14.9%	2.1%	0.059
GSTP1	0.0%	0.0%	N/A
MGMT	12.8%	4.3%	0.267
ADAM23	0.0%	0.0%	N/A
RASSF1A	14.9%	4.3%	0.158
FHIT	6.4%	2.1%	0.617

NT, non-tumor tissue; N/A, not available value;

* statistical significance after multiple testing (p < 0.0042).

Table 2 Correlation between DNA hypermethylation and clinico-pathological parameters of ESCC patients (n = 47)

	Frequency of methylation (%)
	p16	p14	DAPK	RAR-β	CDH1	APC	TIMP3	MGMT	RASSF1A	FHIT	≥2 genes
Gender
male	43.2	16.2	40.5	48.6	43.2	13.5	16.2	16.2	8.1	8.1	73.0
female	50.0	10.0	70.0	40.0	40.0	10.0	10.0	0.0	40.0	0.0	80.0
p value	0.734	1	0.154	0.73	1	1	1	0.317	0.029	1	1
Age group (year)
<60	36.0	20.0	48.0	32.0	40.0	12.0	16.0	12.0	16.0	4.0	72.0
<60	54.5	9.1	45.5	63.6	45.5	13.6	13.6	13.6	13.6	9.1	77.3
p value	0.248	0.423	1	0.042	0.773	1	1	1	1	0.593	0.747
Size (cm) *
<5	40.0	16.0	64.0	48.0	52.0	12.0	12.0	8.0	20.0	8.0	84.0
≥5	52.4	9.5	23.8	47.6	28.6	14.3	19.0	19.0	9.5	0.0	61.9
p value	0.553	0.673	0.009	1	0.14	1	0.686	0.39	0.428	0.493	0.107
Location of tumor *
Upper	50.0	33.3	66.7	50.0	33.3	16.7	16.7	16.7	0.0	0.0	50.0
Middle	36.0	8.0	44.0	48.0	40.0	16.0	12.0	12.0	16.0	4.0	76.0
Lower	60.0	13.3	40.0	46.7	46.7	6.7	20.0	13.3	20.0	6.7	80.0
p value	0.384	0.242	0.591	1	0.841	0.704	0.851	1	0.734	1	0.458
Differentiation *
Poorly	50.0	0.0	44.4	44.4	50.0	5.6	22.2	0.0	33.3	11.1	83.3
Moderately	52.4	23.8	52.4	52.4	33.3	14.3	9.5	28.6	4.8	0.0	76.2
Well	14.3	14.3	28.6	42.9	42.9	28.6	14.3	0.0	0.0	0.0	42.9
p value	0.212	0.087	0.57	0.924	0.565	0.285	0.647	0.019	0.035	0.432	0.105
pT category *
1–2	54.5	18.2	63.6	54.5	45.5	18.2	27.3	0.0	36.4	9.1	90.9
3–4	42.9	11.4	40.0	45.7	40.0	11.4	11.4	17.1	8.6	2.9	68.6
p value	0.73	0.619	0.298	0.734	1	0.619	0.333	0.311	0.046	0.425	0.241
pN category *
0	45.8	8.3	41.7	37.5	37.5	16.7	16.7	8.3	16.7	8.3	66.7
1–2	45.5	18.2	50.0	59.1	45.5	9.1	13.6	18.2	13.6	0.0	81.8
p value	1	0.405	0.768	0.237	0.765	0.667	1	0.405	1	0.49	0.321
Smoking *
Smoker	46.9	12.5	40.6	43.8	40.6	9.4	18.8	18.8	12.5	6.3	75.0
Non-smoker	42.9	14.3	57.1	57.1	42.9	21.4	7.1	0.0	21.4	0.0	71.4
p value	1	1	0.349	0.525	1	0.35	0.413	0.157	0.658	1	1
Alcohol *
Drinker	42.9	14.3	39.3	46.4	39.3	17.9	17.9	21.4	14.3	7.1	75.0
Non-drinker	50.0	11.1	55.6	50.0	44.4	5.6	11.1	0.0	16.7	0.0	72.2
p value	0.746	1	0.367	1	0.767	0.38	0.688	0.068	1	0.513	1

* Clinical data was not available for one patient.

Table 3 Nuclear immunoreactivity of DNMT3b in ESCC patients (n = 44)

	DNMT3b (%)					p value
	−	+	++	+++	++++
ESCC	27.3	18.2	15.9	27.3	11.4	p < 0.001
NT	56.8	36.4	4.5	2.3	0.0

NT, non-tumor tissue. The proportions of positive cells were evaluated using the scoring system to determine as negative (−) to extremely strong positive (++++) staining.

Table 4 Correlation between DNA hypermethylation and mRNA expression of DNMT3b in ESCC (n = 46)

	DNMT3b mRNA expression (%)		p value
	Upregulated	Not upregulated
p16
methylated	90.0	10.0	0.022
unmethylated	57.7	42.3
p14
methylated	57.1	42.9	0.385
unmethylated	74.4	25.6
DAPK
methylated	77.3	22.7	0.521
unmethylated	66.7	33.3
RAR-β
methylated	66.7	33.3	0.527
unmethylated	76.0	24.0
CDH1
methylated	73.7	26.3	1
unmethylated	70.4	29.6
APC
methylated	83.3	16.7	0.659
unmethylated	70.0	30.0
TIMP3
methylated	83.3	16.7	0.659
unmethylated	70.0	30.0
MGMT
methylated	66.7	33.3	1
unmethylated	72.5	27.5
RASSF1A
methylated	66.7	33.3	1
unmethylated	72.5	27.5
FHIT
methylated	50.0	50.0	0.49
unmethylated	72.7	27.3
More genes methylation
≥2 genes	82.4	17.6	0.021
<2 genes	41.7	58.3

Table 5 Diagnostic information of serum DNA methylation at five genes

Gene	AUC (95%, CI)	Sensitivity (%)	Specificity (%)
p16	0.533 (0.369–0.698)	6.7	100
DAPK	0.800 (0.673–0.927)	73.3	86.7
RAR-β	0.567 (0.405–0.728)	26.7	86.7
CDH1	0.822 (0.689–0.955)	84.4	80.0
RASSF1A	0.778 (0.655–0.901)	62.2	93.3
Any gene	0.733 (0.561–0.906)	100.0	46.7
Two or more genes	0.911 (0.839–0.983)	82.2	100.0

CI, confidence interval.