Evaluating plasma and tissue biopsy for DNA methylation markers in early colorectal cancer detection: a systematic review

SI no.	Title, Year	Reference no.	Sample type	Population		Age range (years)	CRC stage		DNA methylation marker
SI no.	Title, Year	Reference no.	Sample type	Male	Female	Age range (years)	0–I	II	DNA methylation marker
1	Identifying potential DNA methylation markers in early-stage CRC, 2020	[10]	FFPE tissue; Blood plasma	37	14	34–77	36	6	SFMBT2, VAV3-AS1, ZNF132, KCNQ5, ITGA4, THBD, FBN1, EVC, C9orf50, TWIST1, ZNF304
2	Blood leukocytes methylation levels analysis indicate methylated plasma test is a promising tool for CRC early detection, 2011	[11]	Blood plasma	55	36	22–89	13	31	SEPT9, SDC2
3	A novel cfDNA methylation-based model improves the early detection of CRC, 2021	[12]	Tissue DNA samples; plasma cfDNA	287	202	18–89	66	86	SEPT9, BCAT1, IKZF1, cg10673833
4	Evaluation of epigenetic methylation biomarkers for the detection of CRC using ddPCR, 2023	[13]	FFPE tissue	57	48	-	24	35	BCAT1, GATA5, IKZF1 (V1), IKZF1 (V2), IRF4, ITGA4, HIC1, NPY, SDC2, SEPT9, WIF1
5	Combined SEPT9 and BMP3 methylation in plasma for CRC early detection and screening in a Brazilian population, 2023	[14]	Blood plasma	11	32	57.1 (mean)	9	9	SEPT9, BMP3
6	CRC detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study, 2023	[3]	Blood plasma	32	40	69.8 (mean)	0	11	AGBL4, ALX4, BCAT1, BMP3, FLI1, GRIA4, IKZF1, NDRG4, NPTX2, PRIMA1, RARB, SEPT9, SDC2, SFRP1, SFRP2, SLC8A1, TWIST1, VIM, WNT5A, ZNF331
7	Combining methylated SEPTIN9 and RNF180 plasma markers for diagnosis and early detection of gastric cancer, 2023	[15]	Blood plasma	384	176	<65, >65	94	53	SEPT9, RNF180
8	cfDNA methylation profiles enable early detection of colorectal and gastric cancer, 2016	[16]	Blood plasma	28	42	25–89	10	25	SEPT9, ATXN1, PCDH10, MYO1G, NGFR, IKZF1, ITGA4
9	Circulating-tumor DNA methylation of HAND1 gene: a promising biomarker in early detection of CRC, 2017	[17]	Blood plasma	18	12	31–68	-	9	HAND1, SEPT9

1 cfDNA, cell-free DNA; CRC, colorectal cancer; ddPCR, droplet digital PCR; FFPE, formalin-fixed paraffin-embedded.

Flow chart of study selection process. CRC, colorectal cancer; PMC, PubMed Center.

Summary of study samples demographic profile based on sex and early CRC stage. CRC, colorectal cancer.

Percentages of identified CRC DNA methylation markers from the list of eligible research articles. CRC, colorectal cancer.

Table 2.

Methods used for the identification of CRC DNA methylation markers

SI No.	DOI	Sample type	Extraction & isolation method	Quantification of methylation method	Markers identified
1	10.1016/j.ygeno.2020.06.007	FFPE tissue; Blood plasma	AllPrep DNA/RNA FFPE Kit (QIAGEN)	RT-PCR	SFMBT2, VAV3-AS1, ZNF132, KCNQ5, ITGA4, THBD, FBN1, EVC, C9orf50, TWIST1, ZNF304
2	10.7150/jca.57114	Blood plasma	QIAamp DNA Blood Mini Kit (QIAGEN)	Bisulfite Sequencing and PCR Assays	SEPT9, SDC2
3	10.1002/1878-0261.12942	Tissue samples; plasma	MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher Scientific)	Bisulfite conversion and targeted sequencing	SEPT9, BCAT1, IKZF1, cg10673833
4	10.1038/s41598-023-35631-5	FFPE tissue	QIAamp DNA Kit (QIAGEN)	ddPCR using Bio-Rad QX200 system	BCAT1, GATA5, IKZF1 (V1), IKZF1 (V2), IRF4, ITGA4, HIC1, NPY, SDC2, SEPT9, WIF1
5	10.1002/cam4.6224	Blood plasma	QIAmp circulating Nucleic Acid Kit (QIAGEN)	ddPCR	SEPT9, BMP3
6	10.1038/s41416-023-02337-4	Blood Plasma	MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher Scientific)	MSP with Zymo Lightning Conversion Reagent	AGBL4, ALX4, BCAT1, BMP3, FLI1, GRIA4, IKZF1, NDRG4, NPTX2, PRIMA1, RARB, SEPT9, SDC2, SFRP1, SFRP2, SLC8A1, TWIST1, VIM, WNT5A, ZNF331
7	10.1002/cac2.12478	Blood plasma	QIAamp Circulating Nucleic Acid Kit (QIAGEN)	qPCR	SEPTIN9, RNF180
8	10.62347/TPTQ3682	Blood plasma	MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher Scientific)	MSP with Zymo Lightning Conversion Reagent	SEPT9, ATXN1, PCDH10, MYO1G, NGFR, IKZF1, ITGA4
9	10.1186/s12920-024-01893-9	Blood plasma	AddPrep Genomic DNA Extraction Kit (Addbio)	qMS-PCR	HAND1, SEPT9

1 CRC, colorectal cancer; ddPCR, droplet digital PCR; FFPE, formalin-fixed paraffin-embedded; mPCR, multiplex PCR; MSP, methylation-specific PCR; qMS-PCR, quantitative methylation-specific PCR; qPCR, quantitative PCR; RT-PCR, real-time PCR.

Table 3.

Sensitivity (%) and specificity (%) of each identified DNA methylation marker

SI No.	DOI	Sample type	Markers identified	Sensitivity (%)	Specificity (%)
1	10.1016/j.ygeno.2020.06.007	FFPE tissue; Blood plasma	SFMBT2	92.20	94.60
			VAV3-AS1	92.20	89.20
			ZNF132	86.30	94.60
			KCNQ5	90.20	89.20
			ITGA4	90.20	94.60
			THBD	86.30	94.60
			FBN1	86.30	91.90
			EVC	84.30	86.50
			C9orf50	94.10	86.50
			TWIST1	72.60	100.0
			ZNF304	66.70	100.0
2	10.7150/jca.57114	Blood plasma	SEPT9	75.80	94.70
2	10.7150/jca.57114	Blood plasma	SDC2	60.40	86.80
3	10.1002/1878-0261.12942	Tissue DNA samples; plasma cfDNA	SEPT9	48.20	91.50
			BCAT1/IKZF1	66.00	95.00
			cg10673833	89.70	86.80
4	10.1038/s41598-023-35631-5	FFPE tissue	BCAT1	73.30–75.20	92.40–94.30
			GATA5	73.30–74.30	91.40–92.40
			IKZF1 (V1)	75.20	93.30
			IKZF1 (V2)	70.50	95.20
			IRF2	81.90–82.90	90.5–91.40
			ITGA4	82.90	88.60–89.50
			HIC1	43.80	78.10
			NPY	80.00	90.50
			SDC2	75.20–76.20	94.30–95.20
			SEPT9	70.50–72.40	88.60–90.50
			WIF1	65.70	96.20
5	10.1002/cam4.6224	Blood plasma	SEPT9	50.00	90.00
5	10.1002/cam4.6224	Blood plasma	BMP3	40.00	90.00
6	10.1038/s41416-023-02337-4	Blood plasma	AGBL4	40.30	77.50
			ALX4	-	-
			BCAT1	43.10	64.80
			BMP3	41.70	76.10
			FLI1	38.90	76.10
			GRIA4	-	-
			IKZF1	43.10	78.90
			NDRG4	62.50	47.90
			NPTX2	48.60	74.60
			PRIMA1	-	-
			RARB	-	-
			SEPT9	48.60	69.00
			SDC2	41.70	70.40
			SFRP1	40.30	77.50
			SFRP2	31.90	85.90
			SLC8A1	45.80	73.20
			TWIST1	-	-
			VIM	45.80	70.40
			WNT5A	51.40	60.60
			ZNF331	50.00	66.20
7	10.1002/cac2.12478	Blood plasma	SEPT9	40.00	96.00
7	10.1002/cac2.12478	Blood plasma	RNF180	46.20	87.30
8	10.62347/TPTQ3682	Blood plasma	SEPT9	68.00	80.00
			ATXN1	-	-
			PCDH10	-	-
			MYO1G	-	-
			NGFR	-	-
			IKZF1	-	-
			ITGA4	-	-
9	10.1186/s12920-024-01893-9	Blood plasma	HAND1	93.33	80.00
9	10.1186/s12920-024-01893-9	Blood plasma	SEPT9	66.67	86.67

1 cfDNA, cell-free DNA; FFPE, formalin-fixed paraffin-embedded.

Summary of **(A)** risk of bias and **(B)** applicability concerns of the included papers after QUADAS-2 assessment. QUADAS-2, Quality Assessment of Diagnostic Accuracy Studies-2.

Table 4.

Quality assessment ratings using the QUADAS-2 scale for reviewed studies (n = 9)

Signaling questions	Patient selection					Index test				Reference standard				Flow and timing
Signaling questions	Q1	Q2	Q3	Risk of bias	Applicability concerns	Q4	Q5	Risk of bias	Applicability concerns	Q6	Q7	Risk of bias	Applicability concerns	Q8	Q9	Risk of bias
(1) Zhang et al. [16]	Y	N	N	High	Low	N	U	Low	Unclear	U	Y	Unclear	High	Y	N	High
(2) Chen et al. [17]	N	N	Y	Low	High	Y	Y	Unclear	Low	Y	N	Low	Low	N	Y	High
(3) Wu et al. [18]	U	Y	U	Unclear	Low	N	N	High	Low	Y	N	Low	Low	Y	N	Low
(4) Petit et al. [19]	N	N	Y	High	Low	Y	U	Low	High	N	Y	High	Low	N	Y	High
(5) Lima et al. [6]	Y	Y	N	High	High	Y	Y	Low	Low	Y	Y	Low	Unclear	U	Y	Unclear
(6) Brenne et al. [3]	N	Y	U	Low	Unclear	N	Y	Unclear	Low	N	Y	High	Low	Y	U	Low
(7) Nie et al. [20]	U	N	Y	Unclear	Low	N	U	High	Unclear	Y	N	Low	High	N	Y	High
(8) Lei [21]	N	Y	N	Low	High	Y	N	Low	High	Y	U	Low	Low	Y	N	Low
(9) Shavali et al. [22]	Y	N	N	High	Low	N	U	Unclear	Low	U	N	Unclear	Low	Y	N	Low

1 N, no; Y, yes; U, unclear.

1 QUADAS-2, Quality Assessment of Diagnostic Accuracy Studies-2.

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DOI: https://doi.org/10.2478/abm-2025-0029 | Journal eISSN: 1875-855X | Journal ISSN: 1905-7415

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Language: English

Page range: 266 - 280

Published on: Oct 31, 2025

Published by: Chulalongkorn University

In partnership with: Paradigm Publishing Services

Keywords:

blood plasma,

colorectal cancer,

DNA methylation markers,

Related subjects:

Assistive professions, nursing,

Basic medical science,

Basic medical science, other,

Clinical medicine,

Clinical medicine, other

© 2025 Hans Ezekiel T. Olorosisimo, Charlemagne G. Sumperos, Aeryll Lesley A. Adviento, Angel Ann T. Lachica, John Louie A. Cabalteja, An Gheline R. Ubiña, Josiah T. Valentin, Pamela Rose Bremner, published by Chulalongkorn University
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 19 (2025): Issue 5 (October 2025)

Evaluating plasma and tissue biopsy for DNA methylation markers in early colorectal cancer detection: a systematic review

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