Combined detection of plasma GATA5 and SFRP2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas

Table 1 Summary of primer sequences and annealing temperatures used for methylation-specific polymerase assays

Gene	Primer	Sequence (5'-3')	Annealing temperature (°C)	Ref.
GATA5	MF	TTAGAAATCGAGGAAATCGC	54
	MR	GTAAACCCCCTCGTTACGTA
	UF	TGTTTAGAAATTGAGGAAATTGT	48
	UR	CCCATAAACCCCCTCATTACATA
SFRP2	MF	TTTTTGTAGGGGCGTTTTTATAAC	58	[21]
	MR	TATCGATATACTCCCCAATACCG
	UF	AGATTTTTGTAGGGGTGTTTTTATAAT	54
	UR	ACCTATCAATATACTCCCCAATACCA
ITGA4	MF	TAGAGTTATTTCGCGTTTTGCG	56	[22]
	MR	CTTCGAATACTCGCGCTACTT
	UF	GTTTAGAGTTATTTTGTGTTTTGTG	50
	UR	AAAACTTCAAATACTCACACTACT

M: Methylated; U: Unmethylated; F: Forward; R: Reverse.

Table 2 Clinicopathological features and DNA hypermethylation in plasma samples of 57 colorectal cancer patients and 30 patients with adenomas

Parameters	n	GATA5			SFRP2			ITGA4
		M	U	P value	M	U	P value	M	U	P value
Colorectal cancer
Gender
Male	34	20	14	0.627	21	13	0.174	10	24	0.157
Female	23	15	8		10	13		11	12
Age, yr
≤ 60	28	16	12	0.516	16	12	0.681	8	20	0.203
> 60	29	19	10		15	14		13	16
Tumor size, cm
< 5	42	22	20	0.019	21	21	0.266	13	29	0.123
≥ 5	15	13	2		10	5		8	7
Differentiation
Well	6	2	4	0.038	2	4	0.012	0	6	0.007
Moderately	36	20	16		16	20		11	25
Poorly	15	13	2		13	2		10	5
TNM stage
I-II	33	15	18	0.008	14	19	0.034	8	25	0.021
III-IV	24	20	4		17	7		13	11
Lymph node metastasis
N0	33	15	18	0.008	14	19	0.034	8	25	0.021
N1–3	24	20	4		17	7		13	11
Distant metastasis
M0	53	31	22	0.151	27	26	0.168	19	34	0.620
M1	4	4	0		4	0		2	2
Location
Colon	19	10	9	0.336	12	7	0.347	6	13	0.560
Rectum	38	25	13		19	19		15	23
Adenomas
Tumor size, cm
≤ 1	14	5	9	0.431	5	9	0.201	4	10	1.000
> 1	16	8	8		7	9		5	11
Tumor number
1	26	13	13	0.113	12	14	0.13	8	18	1.000
≥ 2	4	0	4		0	4		1	3
Intraepithelial neoplasia
Low	24	11	13	0.673	8	16	0.184	6	18	0.329
High	6	2	4		4	2		3	3

Table 3 Comparison of predictive ability of GATA5, SFRP2, ITGA4 methylation, when used alone or combined, in colorectal cancer and adenomas

	Sensitivity (95%CI)	Specificity (95%CI)	Odds ratio (95%CI)	P value
Adenomas
GATA5	43.33% (25.46%-62.57%)	78.72% (64.34%-89.30%)	2.83 (1.04-7.73)	0.039
SFRP2	40.00% (22.66%-59.40%)	72.34% (57.36%-84.38%)	1.74 (0.66-4.60)	0.259
ITGA4	30.00% (14.37%-49.40%)	80.85% (66.74%-90.85%)	1.81 (0.62-5.26)	0.273
GATA5 or SFRP2	63.33% (43.86%-80.07%)	65.96% (50.69%-79.14%)	3.35 (1.29-8.71)	0.012
GATA5 or ITGA4	60.00% (40.60%-77.34%)	65.96% (50.69%-79.14%)	2.91 (1.13-7.50)	0.025
SFRP2 or ITGA4	55.67% (37.43%-74.53%)	61.70% (46.38%-75.49%)	1.48 (0.92-2.39)	0.114
GATA5 or SFRP2 or ITGA4	70.00% (50.60%-85.27%)	55.32% (40.12%-69.83%)	1.57 (1.06-2.33)	0.030
GATA5 and SFRP2	26.77% (12.28%-45.89%)	91.49% (79.62%-90.00%)	3.13 (1.03-9.51)	0.032
GATA5 and ITGA4	10.00% (2.11%-26.53%)	82.46% (70.09%-91.25%)	0.41 (0.10-1.64)	0.231
SFRP2 and ITGA4	13.33% (3.76%-30.72%)	85.11% (71.69%-93.80%)	0.88 (0.23-3.31)	1.000
GATA5 and SFRP2 and ITGA4	6.67% (0.82%-22.07%)	93.62% (82.46%-98.66%)	1.04 (0.19-5.89)	1.000
Colorectal cancer
GATA5	61.40% (47.57%-74.00%)	78.72% (64.34%-89.30%)	5.89 (2.44-14.18)	< 0.01
SFRP2	54.39% (40.66%-67.64%)	72.34% (57.36%-84.38%)	3.12 (1.37-7.12)	< 0.01
ITGA4	36.84% (24.45%-50.66%)	80.85% (66.74%-90.85%)	2.46 (1.00-6.09)	0.048
GATA5 or SFRP2	73.68% (60.34%-84.46%)	65.96% (50.69%-79.14%)	5.43 (2.33-12.61)	< 0.01
GATA5 or ITGA4	73.68% (60.34%-84.46%)	65.96% (50.69%-79.14%)	5.43 (2.33-12.61)	< 0.01
SFRP2 or ITGA4	70.18% (56.60%-81.57%)	61.70% (46.38%-75.49%)	3.79 (1.67-8.59)	< 0.01
GATA5 or SFRP2 or ITGA4	80.70% (68.09%-89.95%)	55.32% (40.12%-69.83%)	5.18 (2.16-12.41)	< 0.01
GATA5 and SFRP2	42.86% (29.71%-50.00%)	91.49% (79.62%-90.00%)	8.06 (2.54-25.5)	< 0.01
GATA5 and ITGA4	6.52% (1.37%-17.90%)	82.46% (70.09%-91.25%)	0.33 (0.08-1.27)	0.094
SFRP2 and ITGA4	21.05% (11.38%-33.89%)	85.11% (71.69%-93.80%)	1.52 (0.55-4.25)	0.419
GATA5 and SFRP2 and ITGA4	15.79% (7.48%-27.87%)	93.62% (82.46%-98.66%)	2.75 (0.7-10.82)	0.217