Novel multiplex stool-based assay for the detection of early-stage colon cancer in a Chinese population

Table 1 Primers for pyrosequencing of three DNA methylation biomarkers

Target gene	Primer	Sequence	Size (bp)	Target DMS
PAX8	Forward	5’-GGGGGTTAGGGGATTTTGATTATA-3’	166	chr2:114035984; 114035988; 114035995; 114035998; 114036006
	Reverse	Biotin-5’-ATCTCATACCCTTCTCCTAAATTTATAC-3’
	Sequencing	5’-ATGGAGTTGTGAGGT-3’
RASSF1	Forward	5’-TTTATTTATTGGGTGGGGTAGGA-3’	141	chr3:50378714; 50378718
	Reverse	Biotin-5’-CCTCAAAATCACCATCCAACCTCTAC-3’
	Sequencing	5’-GGGAGATAGGTTAGTAGTTTTA-3’
SFRP2	Forward	5’-GATTAGGGATAATTAGGTAAAAGGAGTT-3’	166	chr4:154711281; 154711305
	Reverse	Biotin-5’-ATTCATCCCCTACCTACCAAAAAACACC-3’
	Sequencing	5’-AGTTAGAGATATTAGATTTTAGG-3’

PAX8: Paired box 8; RASSF1: Ras-association domain family 1; SFRP2: Secreted frizzled-related protein 2; DMS: Differentially methylated site.

Table 2 Clinicopathological features of the early-stage colon cancer patients enrolled in this study, n (%)

Variable	Total (n = 125)	Training set (n = 75)	Test set (n = 50)
Age (yr)	69 (range, 48-94)	68 (range, 51-94)	70 (range, 48-88)
Sex
Male	66 (52.8)	39 (52.0)	27 (54.0)
Female	59 (47.2)	36 (48.0)	23 (46.0)
Serum CEA (≥ 5 ng/mL)
Positive	37 (29.6)	21 (28.0)	16 (32.0)
Negative	88 (70.4)	54 (72.0)	34 (68.0)
FIT (≥ 200 ng/mL)
Positive	78 (62.4)	48 (64.0)	30 (60.0)
Negative	47 (37.6)	27 (36.0)	20 (40.0)
Tumor site
Proximal	64 (51.2)	36 (48.0)	28 (56.0)
Distal	61 (48.8)	39 (52.0)	22 (44.0)
TNM stage
I	47 (37.6)	30 (40.0)	17 (34.0)
II	78 (62.4)	45 (60.0)	33 (66.0)
T stage
T1-3	71 (56.8)	44 (58.7)	27 (54.0)
T4	54 (43.2)	31 (41.3)	23 (46.0)
Histological differentiation
Well/moderately	98 (78.4)	60 (80.0)	38 (76.0)
Poorly	27 (21.6)	15 (20.0)	12 (24.0)

CEA: Carcinoembryonic antigen; FIT: Fecal immunochemical test; TNM: Tumor-node-metastasis.

Table 3 Diagnostic sensitivity, specificity and area under the receiver operating characteristic curve for various indexes

Study population	Index	Sensitivity (%)	Specificity (%)	AUC (95%CI)
Training set	Serum CEA	28.0	100.0	0.640 (0.551-0.729)
(n = 150 subjects)	FIT	64.0	96.0	0.800 (0.726-0.874)
	PAX8_P4	76.0	77.3	0.810 (0.741-0.880)
	RASSF1_P1	50.7	93.3	0.782 (0.709-0.854)
	SFRP2_P1	50.7	86.7	0.697 (0.612-0.782)
	sDNA panel	82.7	77.3	0.866 (0.810-0.923)
	sDNA panel + FIT	86.7	86.7	0.924 (0.881-0.966)
Test set	Serum CEA	32.0	100.0	0.660 (0.552-0.768)
(n = 100 subjects)	FIT	60.0	98.0	0.790 (0.697-0.883)
	sDNA panel	76.0	84.0	0.864 (0.795-0.933)
	sDNA panel + FIT	82.0	96.0	0.909 (0.850-0.967)
Combined set	Serum CEA	29.6	100.0	0.648 (0.580-0.716)
(n = 250 subjects)	FIT	62.4	96.8	0.796 (0.738-0.854)
	sDNA panel	75.2	84.0	0.859 (0.815-0.904)
	sDNA panel + FIT	80.0	93.6	0.918 (0.884-0.952)
Subgroup for stage I	FIT	63.8	96.8	0.803 (0.716-0.891)
(n = 172 subjects)	sDNA panel + FIT	74.5	93.6	0.891 (0.832-0.950)
Subgroup for stage II	FIT	61.5	96.8	0.792 (0.721-0.863)
(n = 203 subjects)	sDNA panel + FIT	83.3	93.6	0.934 (0.899-0.970)

AUC: Area under the receiver operating characteristic curve; CI: Confidence interval; CEA: Carcinoembryonic antigen; FIT: Fecal immunochemical test; sDNA: Stool DNA.