SDC2 and TFPI2 Methylation in Stool Samples as an Integrated Biomarker for Early Detection of Colorectal Cancer

Figures & data

Table 1 Patient Characteristics of Stool Samples Used in This Study

Clinicopathological Characteristics	Healthy (n=53)	Ade (n=16)	CRC (n=61)	GC (n=37)	Others (n=13)
Sex (%)
Male	27	11	38	22	10
Female	26	5	23	15	3
Age, mean (range)	48.9 (20–77)	61.1 (45–82)	58.6 (31–81)	59.1 (25–77)	55.7 (39–73)
Location
Left from Colerectal		13	52
Right from Colerectal		3	9
Size
≥ 3 cm (n=32)			32
< 3 cm (n=29)			29
Stage
I			8	10
II			18	7
III			25	8
IV			10	12

Table 2 Other Clinicopathological Characteristics of Ade

Clinicopathological Characteristics	Numbers
Size
≥ 1 cm	8
< 1 cm	8
Villous component
Yes	6
No	10
Dysplasia
Well differentiated	11
Poorly differentiated	5

Table 3 Primers and Probes Used in This Study

	Name	Sequence 5ʹ–3’	Description	Amplicons Size
SDC2	SDC2 F	CGAGTTTGAGTCGTAATCGTTGC	SDC2 forward primer	188bp
	SDC2 R	TCCGCCGACACGCAAACCACCAAACC	SDC2 reverse primer
	SDC2 P	AACAAAACGAAACCTCCTACCCAAC	SDC2 probe
TFPI2	TFPI2 F	CGCGGAGATTTTTTGT	TFPI2 forward primer	164bp
	TFPI2 R	AACAAACATCGTCGCAAACCTC	TFPI2 reverse primer
	TFPI2 P	ATAAAACCCGACAAAATCCG	TFPI2 probe
ACTB	ACTB F	TAGGTTAGACGGGGGATATGT	ACTB forward primer	90bp
	ACTB R	CACAATAAATCTAAACAAACTCC	ACTV reverse primer
	ACTB P	TCCCAAAACCCCAACACACT	ACTB probe

Figure 1 SDC2 promoter methylation level in subgroups of patients with CRC, stratified based on gender, age and other criteria (UALCAN). (A) Boxplot showing relative promoter methylation level of SDC2 in normal and CRC samples. (B) Boxplot showing relative promoter methylation level of SDC2 in normal individuals of either gender or male or female CRC patients. (C) Boxplot showing relative promoter methylation level of SDC2 in normal individuals of any age or in CRC patients aged 21–40, 41–60, 61–80, or 81–100 yr. (D) Boxplot showing relative promoter methylation level of SDC2 in normal individuals of either race or in CRC patients of Caucasian, African-American or Asian. (E) Boxplot showing relative promoter methylation level of SDC2 in normal individuals of either weight or in CRC patients with normal weight, extreme weight, obese or overweight. (F) Boxplot showing relative promoter methylation level of SDC2 in normal individuals or in CRC patients in stages 1, 2, 3 or 4 tumors. (G) Boxplot showing relative promoter level of SDC2 in normal individuals of either histology or adenocarcinoma or mucinous adenocarcinoma CRC patients. (H) Boxplot showing relative promoter methylation level of SDC2 in normal individuals of any nodal metastasis status or in CRC patients in N0, N1, N2. ***p< 0.001.

Figure 2 TFPI2 promoter methylation level in subgroups of patients with CRC, stratified based on gender, age and other criteria (UALCAN). (A) Boxplot showing relative promoter methylation level of TFPI2 in normal and CRC samples. (B) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals of either gender or male or female CRC patients. (C) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals of any age or in CRC patients aged 21–40, 41–60, 61–80, or 81–100 yr. (D) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals of either race or in CRC patients of Caucasian, African-American or Asian. (E) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals of either weight or in CRC patients with normal weight, extreme weight, obese or overweight. (F) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals or in CRC patients in stages 1, 2, 3 or 4 tumors. (G) Boxplot showing relative promoter level of TFPI2 in normal individuals of either histology or adenocarcinoma or mucinous adenocarcinoma CRC patients. (H) Boxplot showing relative promoter methylation level of TFPI2 in normal individuals of any nodal metastasis status or in CRC patients in N0, N1, N2. ***p < 0.001.

Figure 3 Heatmap for ACTB, SDC2 and TFPI2 in CRC. (A) The heatmap for ACTB in TCGA-CRC using MethSurv. (B) The heatmap for SDC2 in TCGA-CRC using MethSurv. (C) The heatmap for TFPI2 in TCGA-CRC using MethSurv.

Figure 4 Methylation levels of CpG citesof ACTB, SDC2 and TFPI2 in CRC and Normal. (A) Methylation levels throughout the region around ACTB gene between normal tissues and CRC tumors are compared. (B) Methylation levels throughout the region around SDC2 gene between normal tissues and CRC tumors are compared. (C) Methylation levels throughout the region around TFPI2 gene between normal tissues and CRC tumors are compared. Green probes indicate CpG islands.

Figure 5 Kaplan-Meier survival curves comparing the high and low methylation levels of SDC2 and TFPI2 in CRC patients (UCSC-XENA). (A) The overall survival curve for CRC patients with high or low methylation level of SDC2. (B) The disease-specific survival curve for CRC patients with high or low methylation level of SDC2. (C) The overall survival curve for CRC patients with high or low methylation level of TFPI2. (D) The disease survival curve for CRC patients with high or low methylation level of TFPI2.

Figure 6 Genes differentially expressed in correlation with SDC2 methylation in CRC (LinkedOmics). (A) A Pearson test was used to analyze correlations between SDC2 methylation and genes differentially expressed in CRC. (B and C) Heat maps showing genes positively and negatively correlated with SDC2 methylation in CRC (TOP 50). Red indicates positively correlated genes and green indicates negatively correlated genes.

Figure 7 Genes differentially expressed in correlation with TFPI2 methylation in CRC (LinkedOmics). (A) A Pearson test was used to analyze correlations between TFPI2 methylation and genes differentially expressed in CRC. (B and C) Heat maps showing genes positively and negatively correlated with TFPI2 methylation in CRC (TOP 50). Red indicates positively correlated genes and green indicates negatively correlated genes.

Figure 8 Significantly enriched GO annotations and KEGG pathways about SDC2 methylation in CRC. (A) Cellular components. (B) Biological processes. (C) Molecular functions. (D) KEGG pathway analysis.

Figure 9 Significantly enriched GO annotations and KEGG pathways about TFPI2 methylation in CRC. (A) Cellular components. (B) Biological processes. (C) Molecular functions. (D) KEGG pathway analysis.

Table 4 Positive Detection Rates and Youden Indexes for Integration of Methylated SDC2 and TFPI2 in Detecting Normal, Ade and CRC in Stool Samples. N/A, Not Applicable

Gene	Group	Subject Number	Positive Detection Rate (%)	Youden Index (%)	Specificity (%)	p value
SDC2	Normal	53	1.9 (1/53)	N/A
	Ade	16	68.8 (11/16)	66.9	98.1	<o.oo1*
	CRC	61	77.0 (47/61)	75.1		<o.oo1^#
TFPI2	Normal	53	5.7 (3/53)	N/A
	Ade	16	75.0 (12/16)	69.3	94.3	<o.oo1*
	CRC	61	90.2 (55/61)	84.5		<o.oo1^#
SDC2+ TFPI2	Normal	53	5.7 (3/53)	N/A
	Ade	16	81.3 (13/16)	75.6	94.3	<o.oo1*
	CRC	61	93.4 (57/61)	87.7		<o.oo1^#

Notes: *p value between Ade and Normal. ^#p value between CRC and Normal.

Table 5 Correlation Between Methylation Detection Rate and the Clinicopathologic Features in CRC Stool Samples

Clinicopathological Parameters	mSDC2 Rate (%)	p value	mTFPI2 Rate (%)	p value	Total Positive Detection Rate (%)	p value
Sex
Male (n=38)	73.7 (28/38)	0.422	89.5 (34/38)	1	94.7 (36/38)	1
Female (n=23)	82.6 (19/23)		91.3 (21/23)		91.3 (21/23)
Age
≥ 60 (n=27)	70.4 (19/27)	0.269	62.2 (23/27)	0.465	88.9 (24/27)	0.447
< 60 (n=34)	82.4 (28/34)		94.1 (32/34)		97.1 (33/34)
Location
Left (n=52)	80.8 (42/52)	0.218	92.3 (48/52)	0.456	94.2 (49/52)	1
Right (n=9)	55.6 (5/9)		77.8 ((7/9)		88.9 (8/9)
Size
≥ 3 cm (n=32)	84.4 (27/32)	0.153	96.9 (31/32)	0.156	96.9 (31/32)	0.535
< 3 cm (n=29)	69.0 (20/29)		82.8 (24/29)		89.7 (26/29)
Clinical stage
I–II (n=26)	76.9 (20/26)	0.984	88.5 (23/26)	1	92.3 (24/26)	1
III–IV (n=35)	77.1 (27/35)		91.4 (32/35)		94.3 (33/35)
Symptom status
Symptomatic (n=36)	83.3 (30/36)	0.085	88.9 (32/36)	0.177	97.2 (35/36)	0.074
Asymptomatic (n=25)	64.0 (16/25)		72.0 (18/25)		80.0 (20/25)

Notes: mSDC2 rate refers to detection rate of methylated SDC2; mTFPI2 rate refers to detection rate of methylated TFPI2; total positive rate refers to integrated detection rate of methylated SDC2 and TFPI2.

Table 6 Correlation Between Methylation Detection Rate and the Clinicopathologic Features in Ade Stool Samples

Clinicopathological Parameters	mSDC2 Rate (%)	p value	mTFPI2 Rate (%)	p value	Total Positive Detection Rate (%)	p value
Sex
Male (n=11)	63.6 (7/11)	1	63.6 (711)	0.245	72.7 (8/11)	0.509
Female (n=5)	80.0 (4/5)		100.0 (5/5)		100.0 (5/5)
Age
≥ 60 (n=9)	77.8 (7/9)	0.596	66.7 (6/9)	0.585	66.7 (7/9)	1
< 60 (n=7)	57.1 (4/7)		85.7 (6/7)		85.7 (6/7)
Location
Left (n=13)	76.9 (10/13)	0.214	76.9 (10/13)	1	84.6 (11/13)	0.489
Right (n=3)	33.3 (1/3)		66.7 (2/3)		66.7 (2/3)
Size
≥ 1 cm (n=8)	87.5 (7/8)	0.282	75.0(6/8)	1	87.5 (7/8)	1
< 3 cm (n=8)	50.0 (4/8)		75.0 (6/8)		75.0 (6/8)
Villous component
Yes (n=6)	100.0 (6/6)	0.093	83.3 (5/6)	1	100.0 (6/6)	0.25
No (n=10)	50.0 (5/10)		70.0 (7/10)		70.0 (7/10)
Dysplasia
Well differentiated (n=11)	72.7 (8/11)	1	72.7 (8/11)	1	81.8 (9/11)	1
Poorly differentiated (n=5)	60.0 (3/5)		80.0 (4/5)		80.0 (4/5)

Notes: mSDC2 rate refers to detection rate of methylated SDC2; mTFPI2 rate refers to detection rate of methylated TFPI2; total positive rate refers to integrated detection rate of methylated SDC2 and TFPI2.

Table 7 Comparison of Methylation Detection of CRC, Gastric Cancer and Other Gastrointestinal Tumors

Cancer Types	mSDC2 Rate (%)	p value	mSDC2 Rate (%)	p value	Total Positive Rate(%)	p value
CRC (61)	80.3 (49/61)		90.2 (55/61)		93.4 (57/61)
GC (N=37)	10.8 (4/37)	<0.001	29.7 (11/37)	<0.001	35.1 (13/37)	<0.001
Others (n=12)	0 (0/13)		21.4 (3/13)		21.4 (3/13)

Notes: mSDC2 rate refers to detection rate of methylated SDC2; mTFPI2 rate refers to detection rate of methylated TFPI2; total positive rate refers to integrated detection rate of methylated SDC2 and TFPI2. Others refers to other gastrointestinal tumors.

Table 8 Comparison of Integrated Detection of Methylated SDC2 and TFPI2 to Other Screening Methods in CRC

Method	Methylation Detection (+)	Methylation Detection (-)	p value	Sensitivity (%)
FOBT
FOBT+	44	1	<0.001	73.7 (45/61)
FOBT-	13	3
AFP
AFP+ (>8.78 ng/mL)	4	1	<0.001	8.2 (5/61)
AFP- (≤8.78 ng/mL)	53	3
CEA
CEA+ (>5 ng/mL)	3	1	<0.001	65.6 (4/61)
CEA- (≤5 ng/mL)	54	3
CA125
CA125+ (>35 U/mL)	9	0	<0.001	14.8 (9/61)
CA125- (≤35 U/mL)	48	4
CA153
CA153+ (>28 U/mL)	1	0	<0.001	1.6 (1/61)
CA153- (≤28 U/mL)	56	4
CA199
CA199+ (>37 U/mL)	7	1	<0.001	13.1 (8/61)
CA199- (≤37 U/mL)	50	3

Notes: Methylation detection (+) refers to positive detection of integrated methylated SDC2 and TFPI2; methylation detection (-) refers to negative detection of integrated methylated SDC2 and TFPI2. Chi-square test was used in that comparison.