Circulating and stool nucleic acid analysis for colorectal cancer diagnosis

Table 1 Summary of studies that have tested DNA integrity and genetic alteration markers in blood samples in colorectal cancer

Study	Biomarker(s)	Methods	Case analyzed	Main results
Flamini et al[17], 2006	Serum cfDNACEAcfDNA and CEA	qRT PCR	CRC: 75HD: 75	Sensitivity	Specificity
				81%	73%
				39%	97%
				88%	71%
Frattini et al[51], 2006	Plasma cfDNA	DNA Dipstick Kit	CRC: 70HD: 20	CRC (T0) mean value DNA: 495.7 ng/mL
				CRC (FU at 4 mo) mean value DNA: 170.6 ng/mL
				CRC (FU at 10 mo) mean value DNA: 240.9 ng/mL
				CRC (FU at 10 mo)(DFP) mean value DNA: 136.2 ng/mL
				CRC (FU at 10 mo)(RP) mean value DNA: 694.4 ng/mL
				HD: mean value DNA 10.3 ng/mL
				Altered in only 37% of CRC patients
Umetani et al[61], 2006	Plasma cfDNASerum cfDNA	ALU qRT PCR	Breast, Colorectal, Thyroid Cancer: 22Thyroid A: 2	Mean ± SD value plasma cfDNA: 180 ± 150 pg/μL
				Mean ± SD value serum cfDNA: 970 ± 730 pg/μL
Umetani et al[64], 2006	Absolute serum DNAIntegrity serum DNA Alu repeats (2 sites)	ALU qRT PCR	CRC: 32HD: 51	CRC (I-II) mean absolute value DNA: 1.63 ng/μL
				CRC (I-II) mean integrity DNA: 0.22 ng/μL
				CRC (III-IV) mean absolute DNA: 1.73 ng/μL
				CRC (III-IV) mean integrity DNA: 0.22 ng/μL
Boni et al[52], 2007	Plasma cfDNACEA	qRT PCR	CRC: 67HD: 67	CRC: Mean value DNA 57.93 ng/mL
				HD: Mean value DNA 0.85 ng/mL
				Evaluated in only 47% of patients
Danese et al[65], 2010	Serum cfDNACEA	RT-PCR	CRC: 118P: 49HD: 26	Sensitivity	Specificity
				83%	92%
				36%	100%
Mead et al[31], 2011	Plasma cfDNA (4 DNA markers) CEA	PCRELISA	CRC: 24P: 26HD: 35	AUC: cfDNA: 0.81
				AUC (CEA and cfDNA): 0.855
				P: Sensitivity: 83%; Specificity 72%
Czeiger et al[66], 2011	Serum cfDNA	Fluorometric Assay	CRC: 38HD: 34	Sensitivity	Specificity
				42%	94%
da Silva Filho et al[67], 2013	Serum cfDNAAlu repeats (2 sites)	qPCR	CRC not operated on: 27CRC operated on: 33HD: 30	CRC not operated on: 0.08-62.10 pg/μL
				CRC operated on: 0.01-186.7 pg/μL
				HD: 0.01-26.11 pg/μL
				Mean value range (according to different Alu analysis)

cfDNA: Cell-free circulating DNA; HD: Healthy donors; A: Adenomas; P: Polyps; T₀: Time 0; FU: Follow up; DFP: Disease free patients; RP: Recurrence patients; AUC: Area under the receiver-operating characteristic (ROC) curve; qRT PCR: Quantitative real time polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay.

Table 2 Summary of studies that have tested DNA integrity and genetic alteration markers in stool samples

Study	Biomarker(s)	Methods	Assay for Long DNA	Case analyzed	Sensitivity	Specificity
Ahlquist et al[88], 2000	Stool DNA integrity	L-DNA (4 sites)	Hybrid	CRC: 22	91%	93%
	KRAS, TP53, APC	Mutation analysis	Capture	A ≥ 1 cm: 11	82%	NA
	BAT 26	MSI	PCR	HD: 28
Tagore et al[89], 2003	Stool DNA integrity	L-DNA (6 sites)	Hybrid	CRC: 52	64%	96%
	KRAS, TP53, APC	Mutation analysis	Capture	AA: 28
	BAT 26	MSI	PCR	HD: 212	57%	NA
Calistri et al[90], 2003	Stool DNA integrity	L-DNA (8 sites)	PCR	CRC: 56	62%	97%
	KRAS, TP53, APC	Mutation analysis		HD: 38
	D2S123, D5S346, D17S250
	BAT 25, BAT 26	MSI
Boyton et al[91], 2003	Stool DNA integrity	L-DNA (6 sites)	Hybrid	CRC: 27	56%	97%
			Capture	HD: 77
			PCR
Calistri et al[92], 2004	Stool DNA integrity	FL-DNA (8 sites)	Capillary	CRC: 85	76%	93%
			Electrophoresis	HD: 59
Whitney et al[93], 2004	Stool DNA integrity	L-DNA (4 sites)	Magnetic	CRC: 86	70%	96%
	KRAS, TP53, APC	Mutation analysis	Bead-Based	HD: 100
	BAT 26	MSI	Sequence-Specific Purification
			Capture
Imperiale et al[94], 2004	Stool DNA integrity	L-DNA (4 sites)	Hybrid Capture	CRC: 31	52%	95%
	KRAS, TP53, APC	Mutation analysis	PCR	AA: 407	15%	NA
	BAT 26	MSI		P: 648	8%	NA
				HD: 1423
Kutzner et al[95], 2005	Stool DNA integrity	L-DNA (4 sites)	Hybrid	CRC: 57	65%	91%
	APC	Mutation analysis	Capture	HD: 44
	BAT 26	MSI	PCR
Zou et al[96], 2006	Stool DNA integrity	L-DNA: Alu-assay (2 sites)	RT PCR	CRC: 18	44%	100%
				HD: 20
Itzkowitz et al[97], 2007	Stool DNA integrity	L-DNA (4 sites Locus D, Locus Y)	RT PCR	CRC: 40	88%	82%
	Vimentin	Methylation analysis		HD: 122
Abbaszadegan et al[98], 2007	Stool DNA integrity	L-DNA: (1476 bp fragments)	PCR	CRC: 25	64%	95%
	p16	Methylation analysis		HD: 20
	BAT 26	MSI
Ahlquist et al[99], 2008	Stool DNA integrity	L-DNA (4 sites)	PCR	CRC: 12	25%	96%
	KRAS, APC, TP53	Mutations Analysis		A ≥ 1 cm: 135	17%	NA
	BAT 26	MSI		A < 1 cm: 469
	Stool DNA test 1			P: 341	4%	NA
				HD: 1473	5%	NA
Itzkowitz et al[47], 2008	Stool DNA integrity	L-DNA ( 4 sites, Locus D, Locus Y)	RT PCR	CRC: 82	83%	82%
	Vimentin	Methylation Analysis		HD: 363
Calistri et al[100], 2009	Stool DNA integrity	FL-DNA (8 sites)	Capillary	CRC: 100	79%	89%
			Electrophoresis	HD: 100
Calistri et al[101], 2010	Stool DNA integrity	FL-DNA (8 sites)	Capillary	CRC: 26	Cancer risk prediction with markers combination
	iFOBT	iFOBT	Electrophoresis	A HR: 264
				A LR: 54
				HD: 216
Kalimutho et al[102], 2011	Stool DNA integrity	L-DNA (4 sites)	QdHPLC	CRC: 28	86%	81%
	Calprotectin	ELISA		A: 69	17%	NA
				HD: 95	72%	75%
Ahlquist et al[103], 2012	Stool DNA integrity	L-DNA	QuARTS	CRC: 252	85%	89%
	KRAS	Mutations Analysis		A ≥ 1 cm: 133	63%	NA
	NDRG4, BMP3, vimentin,	Methylation Analysis		A < 1 cm: 94	54%	NA
	TFP12/α-actine			HD: 293

L-DNA: Long-DNA; FL-DNA: Fluorescence long DNA; CRC: Colorectal cancer; A: Adenoma; AA: Advanced Adenoma; A HR: Adenoma high risk; A LR: Adenoma low risk; HD: Healthy donors; HP: Hyperplasic polyps; NA: Not available; RT-PCR: Real time polymerase chain reaction; QdHPLC: Quantitative denaturing high performance liquid chromatografy; QuARTS: Quantitative allele specific real-time target and signal amplification; MSI: Microsatellite instability.