Cell-free DNA liquid biopsy for early detection of gastrointestinal cancers: A systematic review

Table 1 Characteristics of included studies

Ref.	Year	Country	Mono/multicentric	Type of cancer	Total number of patients in validation cohort	Type of groups analyzed
Li et al[13]	2020	China	Monocentric	PDAC	208	Cancer vs healthy
Chen et al[14]	2020	China	Monocentric	Gastric, esophagus, colorectal, lung or liver	418	Cancer diagnosed vs healthy; Pre-diagnosed patients vs healthy
Guler et al[18]	2020	United States	Multicentric	PDAC	228	Cancer vs healthy
Junca et al[12]	2020	France	NA	Colorectal	130	Cancer vs healthy vs advanced-adenoma vs non-advanced adenoma and/or hyperplastic polyp(s)
Tao et al[15]	2020	China	NA	HCC	175	HBV-related HCC vs cancer-free HBV patients
Cristiano et al[19]	2019	United States	Multicentric	Breast, colorectal, lung, ovarian, pancreatic, gastric, bile duct	423	Cancer vs healthy
Li et al[17]	2019	China	Monocentric	Colorectal	140	Cancer vs healthy
Qu et al[16]	2019	China	Multicentric	HCC	331	HBsAg1 positive without cancer based on screening with serum AFP and ultrasonography
Cai et al[11]	2019	China	Multicentric	HCC	1194	Cancer vs healthy vs 392 LC/HB vs BLL
Wan et al[9]	2019	United States	Multicentric	Colorectal	817	Cancer vs healthy
Jensen et al[20]	2019	Denmark	NA	Colorectal	234	Cancer vs healthy
Nunes et al[21]	2018	Portugal	NA	Breast, colorectal, lung	356	Cancer vs healthy
Perrone et al[22]	2014	Italy	Monocentric	Colorectal	170	Cancer vs healthy vs premalignant lesion (adenoma/hyperplasia)

PDAC: Pancreatic ductal adenocarcinoma; HCC: Hepatocellular carcinoma; LC/HB: Liver cirrhosis/hepatitis B; BLL: Benign liver lesions; HBV: Hepatitis B virus; AFP: Alpha-fetoprotein.

Table 2 Number of patients in each group

Ref.	Total patients in validation cohort	Nbr patient cancer group	Nbr patient healthy group	Nbr patient additional group 1	Nbr patient in aditionnal group 2
Li et al[13]	208	101	107	-	-
Chen et al[14]	418	113	2071	98 pre-diagnosed patients	-
Guler et al[18]	228	23	205	-	-
Junca et al[12]	130	20	40	39 advance adenoma	31 non-advance adenoma
Tao et al[15]	175	89	86	-	-
Cristiano et al[19]	423	208	215	-	-
Li et al[17]	140	74	66	-	-
Qu et al[16]	331	-	-	HBsAg (+)	-
Cai et al[11]	1194	809	256	129 LC/CHB	-
Wan et al[9]	817	546	271	-	-
Jensen et al[20]	234	143	91	-	-
Nunes et al[21]	356	253	103	-	-
Perrone et al[22]	170	34	63	73 adenoma/hyperplasia	-

LC: Liver cirrhosis, CHB: Chronic hepatitis B virus infection. 90 GC patients without surgery and 110 who had undergone surgery.

Table 3 Risk of bias of included studies, determined using the ROBINS-I tool (2016)

Ref.		Entry	Judgement	Support for judgement
Li et al[13]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Chen et al[14]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Guler et al[18]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Junca et al[12]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analysesconsistent with a priori plan
Tao et al[15]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants in the supplementary materials
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants in the supplementary materials
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Cristiano et al[19]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Li et al[17]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Qu et al[16]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Low risk	Pre-registered protocol available (NCC201709011)
Cai et al[11]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Wan et al[9]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Jensen et al[20]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Nunes et al[21]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	No information	No information about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	No information	No information about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan
Perrone et al[22]	A	Bias due to confounding	Low risk	No confounding factors
	B	Bias in selection of participants into the study	Low risk	Information provided about the start of follow up and intervention for the participants
	C	Bias in classification of interventions	Low risk	Information provided about the start of follow up and intervention for the participants
	D	Bias due to deviationsfrom intended interventions	Low risk	No deviations from the planned interventions
	E	Bias due to missing data	Low risk	All data were reported
	F	Bias in measurement of outcomes	Low risk	Comparable methods of outcome assessment in the groups, intervention received in each group unlikely to influence the outcome measure, any error in measuring the outcome is unrelated to intervention
	G	Bias in selection of the reported result	Moderate risk	No pre-registered protocol available; outcome measurements and analyses consistent with a priori plan

Table 4 Details of extraction and sequencing methods used in each of the included studies

Ref.	Source of cfDNA	Focus in cfDNA	Extraction method (used kit)	Sequencing method	Sequencing method details
Li et al[13]	Plasma	Methylated markers	QIAamp Circulating Nucleic Acid Kit (Qiagen, 55114)	NGS	Illumina HiSeq 2000 platform
Chen et al[14]	Plasma	Cancer-specific methylation signatures	QIAamp Circulating Nucleic Acid kit (Qiagen, 55114)	NGS	APA Library Quantification Kit for Illumina (KK4844) and sequenced on an Illumina NextSeq 500
Guler et al[18]	Plasma	5hmC modifications	QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germantown, MD)	NGS	NextSeq550 instrument with version 2 reagent chemistry (Illumina, San Diego, CA).
Junca et al[12]	Plasma	KRAS and BRAF mutational status	QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany)	RT-PCR	Q24 PyroMark system (Qiagen, Hilden, Germany)
Tao et al[15]	Plasma	Somatic copy number aberration	QIAamp CirculatingNucleic Acid Kit (Qiagen)	NGS	Next generation sequencing (Illumina)
Cristiano et al[19]	Plasma	Fragmentation size	Qiagen Circulating Nucleic Acids Kit (Qiagen GmbH)	NGS	NEBNext DNA Library Prep Kit for Illumina
Li et al[17]	Plasma	Aberrant DNA hypermethylation of CpGislands	DNeasy Blood & TissueKit (Qiagen)	NGS	Methylated CpG tandem ampli-fication and sequencing
Qu et al[16]	Plasma	Specific mutations	ARCHITECT i2000SR Chemical luminescence immunity analyzer	NGS	Next generation sequencing
Cai et al[11]	Plasma	5hmC modifications	NA	NGS	5hmC-Seal
Wan et al[9]	Plasma	cfDNA mutations patterns	MagMAX cfDNA Isolation Kit	NGS	Illumina NovaSeq 6000 Sequencing System
Jensen et al[20]	Plasma	Tumour-specific DNA methylation	Gentra Puregene Tissue Kit (Qiagen)	DD-PCR	Bisulfite sequencing and methylation-specific droplet digital PCR
Nunes et al[21]	Plasma	Aberrant DNA methylation	QIAamp MinElute ccfDNA (Qiagen, Hilden, Germany)	qMSP	qMSP
Perrone et al[22]	Plasma	KRAS mutated cfDNA	Qiamp DNA Blood Extraction Kit (Qiagen)	RT-PCR	RT-PCR

NGS: Next-generation sequencing; RT-PCR: Real-time polymerase chain reaction; qMSP: Multiplex methylation-specific polymerase chain reaction.

Table 5 Sensibility and sensitivity of included studies

	Ref.	Group of validation cohorts	Sensitivity	Specificity	Positive predictive value	Negative predictive value	AUC
PDCA	Li et al[13]	Cancer vs healthy	93.2	95.2	NA	NA	0.943
	Chen et al[14]	Cancer vs healthy	NA	NA	NA	NA	0.921
HCC	Guler et al[18]	HBV-related HCC vs cancer-free HBV group 1	18	97.4	NA	NA	0.92
		HBV-related HCC vs cancer-free HBV group 2	29	95.6	NA	NA	0.81
	Junca et al[12]	HCC vs cancer-free HBV	100	94	17	100	NA
	Tao et al[15]	HCC vs healthy	82.7	76.4	NA	NA	0.884
		HCC vs high risk (HBV and cirrhosis)	82.7	67.4	NA	NA	0.846
Various cancer types	Cristiano et al[19]	Pre-diagnosis vs healthy	84.9	96.1	NA	NA	NA
		Post-diagnosis vs healthy	87.5	96.1
	Li et al[17]	All cancer vs healthy	80	95	NA	NA	0.94
			73	98
		Gastric cancer vs healthy	81	95
			81	98
		Colorectal cancer vs healthy	81	95
			70	98
		Bile duct cancer vs healthy	88	95
			81	98
		Pancreatic cancer vs healthy	71	95
			65	98
	Qu et al[16]	All cancer vs healthy	74.2	73.5	87.1	52.1	NA
		Colorectal cancer vs healthy	78.4	69.9	48.3	90
Colorectal	Cai et al[11]	Cancer/adenoma vs healthy	16.9	100	100	59.2	NA
	Wan et al[9]	Cancer vs healthy	74	90	NA	NA	0.887
	Jensen et al[20]	Cancer vs healthy	85	85	NA	Na	0.92
	Nunes et al[21]	Cancer vs healthy	85	99	NA	NA	NA
	Perrone et al[22]	Cancer vs healthy	NA	NA	NA	NA	0.709
		Adenomas vs healthy	NA	NA	NA	NA	0.535

HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; PDAC: Pancreatic ductal adenocarcinoma.