Effectiveness of circulating tumor DNA for detection of KRAS gene mutations in colorectal cancer patients: a meta-analysis

Figures & data

Figure 1 A flow chart showing the studies included in this meta-analysis.

Table 1 Characteristics of eligible studies

First author	Year	Country	Patients	TNM	Tissue	Blood	Collection	Method	Codon	Sensitivity	Specificity	TP	FN	FP	TN
Bettegowda et al^Citation16	2014	Multi	206	Advanced	NA	Plasma	NA	dPCR	12, 13	0.872	0.992	68	10	1	127
Danese et al^Citation17	2015	Italy	85	I–IV	Frozen	Plasma	Before	ARMS-qPCR	12, 13	0.815	0.931	22	5	4	54
Kim et al^Citation18	2015	Korea	65	Advanced	FFPE	Serum	NA	RFLP-PCR	12, 13	0.581	0.765	18	13	8	26
Kopreski et al^Citation19	2000	USA	135	NA	FFPE	Plasma	Before	RFLP-PCR	12	0.829	0.930	29	6	7	93
Kuo et al^Citation20	2014	China	52	I–IV	NA	Plasma	NA	PNA-PCR	12, 13	1.000	0.703	15	0	11	26
Lefebure et al^Citation21	2010	France	24	IV	NA	Serum	NA	Real-time PCR	12, 13	0.500	1.000	7	7	0	10
Lin et al^Citation22	2014	China	133	I–IV	Frozen	Plasma	Before	MassARRAY	12, 13	0.456	1.000	41	49	0	43
Liu et al^Citation23	2012	China	62	NA	Frozen	Plasma	Before	Nested cold-PCR	12	0.750	0.920	9	3	4	46
Miyano et al^Citation24	2012	Japan	42	I–IV	FFPE	Plasma	Before	PNA-PCR	12, 13	0.615	0.931	8	5	2	27
Morgan et al^Citation25	2012	USA	71	IV	FFPE	Plasma	Before	ARMS-qPCR	12, 13	0.313	0.949	10	22	2	37
Morgan et al^Citation25	2012	USA	71	IV	FFPE	Serum	Before	ARMS-qPCR	12, 13	0.281	1.000	9	23	0	39
Mulcahy et al^Citation26	2000	Switzerland	14	NA	NA	Plasma	Before	MASA-PCR	12	0.857	1.000	6	1	0	7
Perrone et al^Citation27	2014	Italy	12	NA	FFPE	Plasma	Before	ME-PCR	12, 13	0.000	1.000	0	5	0	7
Pu et al^Citation28	2013	China	115	I–IV	FFPE	Serum	Before	Nested PCR	12, 13	0.243	0.949	9	28	4	74
Ryan et al^Citation29	2003	the Netherlands	78	I–IV	FFPE	Serum	Before	ME-PCR	12, 13	0.756	0.973	31	10	1	36
Sakai et al^Citation30	2015	Japan	15	NA	FFPE	Plasma	NA	NGS	12, 13	0.714	1.000	5	2	0	8
Sefrioui et al^Citation31	2015	France	34	IV	FFPE	Plasma	Before	dPCR	12, 13	0.688	1.000	11	5	0	18
Spindler et al^Citation32	2015	Denmark	211	IV	FFPE	Plasma	Before	ARMS-qPCR	12, 13	0.800	0.958	112	28	3	68
Taly et al^Citation33	2013	France	50	IV	Frozen	Plasma	NA	qPCR	12, 13	0.789	0.935	15	4	2	29
Thierry et al^Citation15	2014	France	95	IV	NA	Plasma	NA	qPCR	12, 13	0.923	0.982	36	3	1	55
Xu et al^Citation34	2014	China	242	IV	FFPE	Plasma	Before	PNA-PCR	12, 13	0.547	0.904	64	53	12	113

Abbreviations: TNM, tumor–node–metastasis; TP, true positive; FN, false negative; FP, false positive; TN, true negative; NA, not applicable; dPCR, digital polymerase chain reaction; ARMS-qPCR, allele refractory mutation system – quantitative polymerase chain reaction; FFPE, formalin-fixed and paraffin-embedded; RFLP-PCR, restriction fragment length polymorphism – polymerase chain reaction; PNA-PCR, peptide nucleic acid-mediated polymerase chain reaction; PCR, polymerase chain reaction; MASA-PCR, mutant specific alleles – polymerase chain reaction; ME-PCR, mutant-enriched polymerase chain reaction, NGS, next-generation sequencing; qPCR, quantitative polymerase chain reaction.

Figure 2 The Deeks regression line showing the publication bias of studies.

Abbreviations: ESS, effective sample size; DOR, diagnostic odds ratio.

Table 2 QUADAS-2 score of eligible studies

First author	Risk of bias				Application concerns
	Patient selection	Index test	Reference standard	Flow and timing	Patient selection	Index test	Reference standard
Bettegowda et al^Citation16	L	U	U	L	L	L	L
Danese et al^Citation17	L	U	U	L	L	L	L
Kim et al^Citation18	L	U	U	L	L	L	L
Kopreski et al^Citation19	L	U	U	L	L	L	L
Kuo et al^Citation20	L	U	U	L	L	L	L
Lefebure et al^Citation21	L	U	U	L	L	L	L
Lin et al^Citation22	L	L	L	L	L	L	L
Liu et al^Citation23	L	U	U	L	L	L	L
Miyano et al^Citation24	L	U	U	L	L	L	L
Morgan et al^Citation25	L	L	L	L	L	L	L
Morgan et al^Citation25	L	L	L	L	L	L	L
Mulcahy et al^Citation26	L	U	U	L	L	L	L
Perrone et al^Citation27	L	U	U	L	L	L	L
Pu et al^Citation28	L	U	U	L	L	L	L
Ryan et al^Citation29	L	U	U	L	L	L	L
Sakai et al^Citation30	L	U	U	L	L	L	L
Sefrioui et al^Citation31	L	H	L	L	L	L	L
Spindler et al^Citation32	L	L	L	L	L	L	L
Taly et al^Citation33	L	U	U	L	L	L	L
Thierry et al^Citation15	L	U	U	L	L	L	L
Xu et al^Citation34	L	L	L	L	L	L	L

Abbreviations: L, low; U, unclear; H, high; QUADAS-2, quality assessment of studies of diagnostic accuracy included in systematic reviews-2.

Figure 3 The SROC curve of ctDNA for detection of KRAS gene mutations.

Abbreviations: SROC, summary receiver operating characteristic; AUC, area under the curve.

Figure 4 Fagan’s nomogram of ctDNA for detection of KRAS gene mutations.

Abbreviation: LR, likelihood ratio.

Figure 5 LR scattergram of ctDNA for detection of KRAS gene mutations.

Abbreviations: LR, likelihood ratio; NLR, negative likelihood ratio; PLR, positive likelihood ratio; CI, confidence interval; LUQ, left upper quadrant; LRP, likelihood ratio for positive results; RUQ, right upper quadrant; LLQ, left lower quadrant; RLQ, right lower quadrant; LRN, likelihood ratio for negative results.

Table 3 Meta-analysis of subgroup

Subgroup analyses	n	Sensitivity	Specificity	AUROC	DOR	PLR	NLR
All	21	0.67 (0.55–0.78)	0.96 (0.93–0.98)	0.95 (0.92–0.96)	53.95 (26.24–110.92)	18.33 (10.14–33.16)	0.34 (0.24–0.49)
TNM stage
Advanced	10	0.66 (0.50–0.79)	0.97 (0.93–0.99)	0.95 (0.93–0.97)	71.52 (21.18–241.57)	24.88 (9.36–66.17)	0.35 (0.22–0.54)
I–IV	6	0.73 (0.40–0.92)	0.93 (0.86–0.97)	0.94 (0.92–0.96)	37.80 (14.18–100.79)	11.02 (6.18–19.66)	0.29 (0.11–0.77)
Storage of tissue
FFPE	12	0.55 (0.40–0.69)	0.96 (0.92–0.98)	0.93 (0.90–0.95)	29.19 (12.47–68.33)	13.63 (6.88–27.01)	0.47 (0.34–0.65)
Frozen	4	0.70 (0.51–0.84)	0.95 (0.88–0.98)	0.94 (0.91–0.96)	48.17 (18.44–125.87)	15.07 (6.29–36.09)	0.31 (0.18–0.54)
Format of blood
Plasma	16	0.74 (0.60–0.84)	0.97 (0.93–0.98)	0.96 (0.94–0.97)	77.45 (39.03–153.72)	20.93 (11.58–37.82)	0.27 (0.17–0.43)
Serum	5	0.47 (0.29–0.66)	0.96 (0.84–0.99)	0.83 (0.79–0.86)	22.16 (4.30–114.30)	12.25 (2.69–55.84)	0.55 (0.39–0.79)
Detection method
ARMS-qPCR	4	0.58 (0.30–0.81)	0.96 (0.91–0.98)	0.96 (0.94–0.97)	34.90 (11.17–109.07)	15.38 (6.96–34.01)	0.44 (0.23–0.85)
Detection site
Codon 12	21	0.67 (0.55–0.78)	0.96 (0.93–0.98)	0.95 (0.92–0.96)	53.95 (26.24–110.92)	18.33 (10.14–33.16)	0.34 (0.24–0.49)
Codon 12, 13	18	0.65 (0.50–0.77)	0.97 (0.93–0.99)	0.95 (0.92–0.96)	55.56 (23.97–128.81)	20.26 (9.97–41.18)	0.37 (0.25–0.54)

Note: Data presented as n (95% confidence interval).

Abbreviations: AUROC, area under the ROC curve; DOR, diagnostic odds ratio; PLR, positive likelihood ratio; NLR, negative likelihood ratio; TNM, tumor–node–metastasis; FFPE, formalin-fixed and paraffin-embedded; ARMS-qPCR, allele refractory mutation system – quantitative polymerase chain reaction.

BettegowdaCSausenMLearyRJDetection of circulating tumor DNA in early- and late-stage human malignanciesSci Transl Med20146224224ra24

 PubMed Web of Science ®Google Scholar

DaneseEMinicozziAMBenatiMComparison of genetic and epigenetic alterations of primary tumors and matched plasma samples in patients with colorectal cancerPLoS One2015105e012641725946211

 PubMedGoogle Scholar

KimSTChangWJJinLSungJSChoiYJKimYHCan serum be used for analyzing the KRAS mutation status in patients with advanced colorectal cancer?Cancer Res Treat201547479680325687873

 PubMedGoogle Scholar

KopreskiMSBenkoFABorysDJKhanAMcGarrityTJGockeCDSomatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNAJ Natl Cancer Inst2000921191892310841827

 PubMedGoogle Scholar

KuoYBChenJSFanCWLiYSChanECComparison of KRAS mutation analysis of primary tumors and matched circulating cell-free DNA in plasmas of patients with colorectal cancerClin Chim Acta201443328428924685572

 PubMed Web of Science ®Google Scholar

LefebureBCharbonnierFDi FioreFPrognostic value of circulating mutant DNA in unresectable metastatic colorectal cancerAnn Surg2010251227528020010083

 PubMed Web of Science ®Google Scholar

LinJKLinPCLinCHClinical relevance of alterations in quantity and quality of plasma DNA in colorectal cancer patients: based on the mutation spectra detected in primary tumorsAnn Surg Oncol201421suppl 4S680S68624841357

 PubMed Web of Science ®Google Scholar

LiuPLiangHXueLPotential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/Taq-Man(®) -MGB probe genotyping methodExp Ther Med20124110911223060932

 PubMed Web of Science ®Google Scholar

MiyanoSHanazawaKKitabatakeTFujisawaMKojimaKDetecting KRAS mutations in peripheral blood of colorectal cancer patients by peptide nucleic acid clamp PCRExp Ther Med20124579079423226727

 PubMed Web of Science ®Google Scholar

MorganSRWhiteleyJDonaldEComparison of KRAS mutation assessment in tumor DNA and circulating free DNA in plasma and serum samplesClin Med Insights Pathol20125152222661904

 PubMedGoogle Scholar

MulcahyHELyauteyJLederreyCPlasma DNA K-ras mutations in patients with gastrointestinal malignanciesAnn N Y Acad Sci2000906252810818591

 PubMed Web of Science ®Google Scholar

PerroneFLampisABertanCCirculating free DNA in a screening program for early colorectal cancer detectionTumori2014100211512124852853

 PubMed Web of Science ®Google Scholar

PuXPanZHuangYComparison of KRAS/BRAF mutations between primary tumors and serum in colorectal cancer: biological and clinical implicationsOncol Lett20135124925423255930

 PubMedGoogle Scholar

RyanBMLefortFMcManusRA prospective study of circulating mutant KRAS2 in the serum of patients with colorectal neoplasia: strong prognostic indicator in postoperative follow upGut200352110110812477769

 PubMed Web of Science ®Google Scholar

SakaiKTsurutaniJYamanakaTExtended RAS and BRAF mutation analysis using next-generation sequencingPLoS One2015105e012189125954997

 PubMedGoogle Scholar

SefriouiDSarafan-VasseurNBeaussireLClinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancerDig Liver Dis2015471088489026160500

 PubMedGoogle Scholar

SpindlerKLPallisgaardNAndersenRFBrandslundIJakobsenACirculating free DNA as biomarker and source for mutation detection in metastatic colorectal cancerPLoS One2015104e010824725875772

 PubMed Web of Science ®Google Scholar

TalyVPekinDBenhaimLMultiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patientsClin Chem201359121722173123938455

 PubMed Web of Science ®Google Scholar

ThierryARMouliereFEl MessaoudiSClinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNANat Med201420443043524658074

 PubMed Web of Science ®Google Scholar

XuJMLiuXJGeFJKRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancerJ Exp Clin Cancer Res20143310425491325

 PubMed Web of Science ®Google Scholar