Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

Table 1 Circulating tumor cells in pancreatic cancer

Ref.	Patient characteristics	Number of patients with PCa	Controls	Enrichment/isolation method	Detection method	Detection rate
Funaki et al[28], 1996	Pre- or post-treatment	9	NA	None	RT-PCR: CEA mRNA	33.3% of PCa
Funaki et al[29], 1998	Preoperative	3	NA	None	RT-PCR: CEA-mRNA
Miyazono et al[30], 1999	Intra-operative	21	15 HV	Density gradient enrichment	RT-PCR: CEA-mRNA	61.9% of PCa
Chausovsky et al[33], 1999	Metastatic PCa	28	22 BD	Density gradient enrichment	RT-PCR: CK20 mRNA	78.6% of PCa
Z'graggen et al[124], 2001	All stages, preoperative	105	66 HV	Density gradient enrichment	ICC: CK-AE1/AE3	Sensitivity: 26%, specificity: 96%
Lukyanchuk et al[34], 2003	NA	11	18 HV	Density gradient enrichment	RT-PCR: CK-20 and PSCA	CK-20: 19 of 47 (40.4%), PSCA: 22 of 47 (46.8%)
Clarke et al[37], 2003	NA	11	23 HV	Density gradient enrichment	RT-PCR: EGFR mRNA	18% of PCa, 0.0% of HV
Mataki et al[32], 2004	All stages	20	15 HV, 15 BD	Density gradient enrichment	RT-PCR: CEA mRNA	Sensitivity: 75.0%, specificity: 94.6%
Zhang et al[35], 2005	Stages II and III	40	5 BD, 5 HV	Not available	RT-PCR: CK20 mRNA	57.5% of PCa
Soeth et al[36], 2005	Preoperative	154	NA	Density gradient enrichment	RT-PCR: CK20 mRNA	33.8% of PCa
Ishizone et al[38], 2006	All stages	55	10 CP, 70 HV	Density gradient enrichment	RT-PCR: a4GnT	76.4% of PCa, 40.0% of CP, 17.1% of HV
Hoffmann et al[39], 2007	All stages	37	16 CP, 15 BD	Density gradient enrichment	RT-PCR: CK-19 mRNA	64% of PCa
Kurihara et al[41], 2008	Stages II, III and IV	26	11 CP, 10 HV	CELLSEARCH®		42% of PCa
Zhou et al[40], 2011	All stages	25	15 BD	Immunomagnetic separation	RT-PCR: C-MET, h-TERT, CK20, and CEA	Sensitivity: 100%, specificity: 93.3%
Khoja et al[125], 2012	Stages III and IV	54 PCa	NA	Size-based selection	RT-PCR: EpCAM, CK, vimentin, and CEA	ISET 49/54 (93%) vs CELLSEARCH® 21/54 (40%)
de Albuquerque et al[126], 2012	Stages III and IV	34	40 HV	Immunomagnetic separation	RT-PCR: KRT19, MUC1, EpCAM, CEACAM5, and BIRC5	Sensitivity: 47.1%, specificity: 100%
Kamande et al[127], 2013	All stages	12	5 HV	Microfluidic; ICC	DAPI+, CD45-, CK+	100% of PCa
Bidard et al[42], 2013	Locally advanced PCa	79	NA	CELLSEARCH®		11% of PCa
Iwanicki-Caron et al[43], 2013	All stages	40	NA	Size-based selection	Cell size and cytopathologic criteria	Sensitivity: 55.5%, specificity: 100%, accuracy: 70%
Bobek et al[45], 2014	All stages	24	NA	Size-based selection	DAPI, CK, CEA, vimentin IHC	66.7% of PCa
Sheng et al[48], 2014	Metastatic PCa	18	NA	Multifluidic, "GEM"Chip		94.4% of PCa
Rhim et al[47], 2014	All stages	11	19 HV	Geometrically enhanced differential immunocapture	ICC for DAPI, CD45, CK, and PDX-1	73% of PCa
Catenacci et al[128], 2015	Stages II, III and IV	18	NA	Immune-magnetic separation	CD45-negative and positive for CK8, -18, and/or -19 and DAPI	118.4 ± 36.8 CTCs/7.5 mL PVB, compared with a mean of 0.8 ± 0.4 CTCs/7.5 mL PB
Earl et al[81], 2015	All stages	35	NA	CELLSEARCH®		20% of PCa
Cauley et al[44], 2015	All stages	105	9 HV	Size-based selection	Cytomorphologic criteria	48.6% of PCa
Kulemann et al[46], 2015	Preoperative	11	9 HV	Size-based selection: ScreenCell	Cytologic and detection of KRAS mutation	75% of early PCa, 71.4% of advanced PCa
Zhang et al[129], 2015	All stages	32	30 HV	ICC and FISH	DAPI+, CD45-, and CK+, or CEP8 > 2+	Sensitivity: 63.6%, specificity: 94.4%
Bissolati et al[130], 2015	Intra-operative	20	NA	CELLSEARCH®		PVB: 40%, PB: 20%
Zhang et al[131], 2015		15	15 HV	Immunomagnetic separation	BC-15 aptamer or anti-CK staining	73.3% of PCa

BD: Benign disease; CEA: Carcinoembryonic antigen; CK: Cytokeratin; CP: Chronic pancreatitis; CTC: Circulating tumor cell; DAPI: 4′,6′-diamidino-2-phenylindole; EpCAM: Epithelial cellular adhesion molecule; FISH: Fluorescence in situ hybridization; HV: Healthy volunteer; ICC: Immunocytochemistry; IHC: Immunohistochemistry; NA: Not applicable; PB: Peripheral blood; PCa: Pancreatic cancer; PVB: Portal venous blood; RT-PCR: Reverse transcription polymerase chain reaction.

Table 2 Circulating cell-free DNA in pancreatic cancer

Ref.	Patients	Controls	Sample	Method	Target candidate	Findings
Shapiro et al[64], 1983	201 MD	185 BD	Serum	Radioimmunoassay	Circulating DNA levels	Serum DNA concentration is markedly elevated in 90% of patients with PCa as compared with HV
Yamada et al[72], 1998	21 PCa	-	Plasma	Mutant allele-specific amplification method	K-ras mutation	In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment
Giacona et al[132], 1998	3 PCa	3 HV	Plasma	Gel electrophoresis and measuring the variation in length by electron microscopy	Length	There are significant differences in non-cell-associated DNA in plasma between patients with PCa and HV
Theodor et al[73], 1999	20 PCa	6 CP, 5 HV	Serum	PCR	K-ras mutation	K-ras gene mutations at codon 12 were detected in the sera of 14 of 20 patients with PCa and in none of the 6 patients with CP, or in the 5 HVs
Castells et al[74], 1999	44 PCa	37 CP	Plasma	Restriction fragment length polymorphism-PCR and single-strand conformation polymorphism techniques	K-ras mutation	Plasma K-ras analysis is a highly specific, low-sensitivity approach that has diagnostic and prognostic clinical implications in patients with PCa
Zambon et al[75], 2000	29 PCa	12 HV	Serum	ME-PCR	K-ras mutation	K-ras was amplifiable in 2 patients with PCa (6.9%), and K-ras was not amplifiable in any of the 12 serum samples obtained from HVs
Maire et al[76], 2002	47 PCa	31 CP	Serum	PCR and allele-specific amplification	KRAS2 mutations	KRAS2 mutations were found in 22 patients (47%) with PCa and in 4 controls with CP (13%) (P < 0.002)
Melnikov et al[65], 2009	30 PCa	30 HV	Plasma	Multiplexed array-mediated analysis of DNA methylation	Methylation	Differential methylation profiling of plasma DNA can detect PCa with 76% sensitivity and 59% specificity
Liggett et al[66], 2010	30 PCa	30 CP, 30 HV	Plasma	Microarray-mediated methylation analysis	Methylation	Methylation analysis achieved 81.7% sensitivity and 78% specificity (P < 0.01) in the detection of CP (HV vs CP) and 91.2% sensitivity and 90.8% specificity (P < 0.01) in the differential detection of PCa (PCa vs CP)
Chen et al[79], 2010	91 PCa	-	Plasma	Direct sequencing	K-ras	K-ras codon 12 mutations were found in 30 of 91(33%) plasma DNA samples and significantly reflected the clinical parameters, including TNM tumor staging and liver metastasis, and independent predict shorter survival time
Wu et al[80], 2014	24 PCa	25 HV	Plasma	COLD-PCR combined with an unlabeled-probe HRM approach	K-ras	KRAS mutations were identified in 26 of 36 PCa cases (72.2%), but none were detected in the disease control and/or healthy group
Earl et al[81], 2015	31 PCa	-	Plasma	Digital PCR	KRAS	KRAS mutant cfDNA was detected in 26% of patients at all stages, which correlated strongly with OS, 60 d for KRAS mutation-positive vs 772 days for KRAS mutation-negative patients
Zill et al[85], 2015	26 PCa	-	Plasma	Sequenced on an Illumina Hi-Seq 2500	KRAS, TP53, APC, FBXW7, and SMAD4	The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across 5 informative genes
Singh et al[133], 2015			Plasma		Levels of ctDNA and K-ras mutation	Higher levels of plasma DNA were significantly associated with lower OS and advanced stage. However, k-ras mutation did not correlate with any of the clinicopathological parameters or survival
Kinugasa et al[82], 2015	141 PCa	20 CP, 20 HV	Serum	Digital PCR	G12V, G12D, and G12R in codon 12 of K-ras gene	K-ras mutation rate in ctDNA was 62.6%. The survival of patients with K-ras mutations in ctDNA was significantly shorter than that of patients without mutations
Sausen et al[83], 2015	77 PCa	-	Plasma	Next-generation sequencing		The 43% of patients with localized disease had detectable ctDNA at diagnosis. Detection of ctDNA after resection predicts clinical relapse and poor outcome, with recurrence by ctDNA detected 6.5 months earlier than with CT imaging
Takai et al[84], 2015	259 PCa	-	Plasma	Picoliter-droplet digital PCR and targeted deep sequencing	KRAS mutation	KRAS mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA

BD: Benign disease; cfDNA: Cell-free DNA; COLD-PCR: Co-amplification at lower denaturation-temperature PCR; CP: Chronic pancreatitis; CT: Computed tomography; ctDNA: Circulating tumor DNA; HV: Healthy volunteer; ICC: Immunocytochemistry; PCa: Pancreatic cancer; MD: Malignant disease; OS: Overall survival; PCR: Polymerase chain reaction; ME-PCR: Mutant-enriched PCR.

Table 3 Circulating noncoding RNA in pancreatic cancer

Ref.	Sample	Candidate target	Potential value
		miRNA
Wang et al[102], 2009	Plasma	miR-210 (↑), miR-21 (↑), miR-155 (↑), miR-196a (↑)	D
Ho et al[134], 2010	Plasma	miR-210 (↑)	D
Li et al[135], 2010	Plasma	miR-200a (↑), miR-200b (↑)	D
Ali et al[136], 2010	Plasma	miR-21 (↑)	D/P
Kong et al[105], 2011	Serum	miR-196a (↑)	D/S/P
LaConti et al[137], 2011	Serum	miR-155 (↑)	D
Morimura et al[138], 2011	Plasma	miR-18a (↑)	D
Liu et al[139], 2012	Serum	miR-16 (↑), miR-196a (↑)	D
Liu et al[140], 2012	Serum	miR-20a (↑), miR-21 (↑), miR-24 (↑), miR-25 (↑),	D/P
		miR-99a (↑), miR-185 (↑), miR-191 (↑)
Li et al[141], 2012	Serum	miR-1290 (↑)	D
Wang et al[142], 2013	Whole blood	miR-27a-3p (↑)	D
Kawaguchi et al[143], 2013	Plasma	miR-221 (↑), miR-375 (↓)	D/S
Zhao et al[144], 2013	Serum	miR-192 (↑)	D
Li et al[141], 2013	Serum	miR-1290 (↑)	D
Wang et al[145], 2013	Serum	miR-21 (↑)	T/P
Carlsen et al[146], 2013	Plasma	miR-375 (↑)	D
Que et al[147], 2013	Serum	miR-17-5p (↑), miR-21 (↑), miR-155 (↓), miR-196a (↓)	D/S
Schultz et al[103], 2014	Whole blood	Multigene index	D
Gao et al[148], 2014	Plasma	miR-16 (↑)	D
Chen et al[149], 2014	Plasma	miR-182 (↑)	D/S/P
Ganepola et al[150], 2015	Plasma	miR-22 (↑), miR-642b (↑), miR-885-5p (↑)	D
Abue et al[151], 2015	Plasma	miR-21 (↑), miR-483-3p (↑)	D/S/P
Slater et al[152], 2015	Serum	miR-196a (↑), miR-196b (↑)	D
Kojima et al[104], 2015	Serum	Multigene index	D
Xu et al[153], 2015	Plasma	miR-486-5p (↑), miR-938 (↑)	D
Madhavan et al[154], 2015	Serum	miR-1246 (↑), miR-3976 (↑), miR-4306 (↑), miR-4644 (↑)	D
Komatsu et al[123], 2015	Plasma	miR-223 (↑)	D/P
Miyamae et al[106], 2015	Plasma	miR-744 (↑)	D/S/P/T
		Other noncoding RNAs
Wang et al[117], 2015	Plasma	HOTTIP-005 (↑), RP11-567G11.1 (↑)	D/P
Baraniskin et al[155], 2013	Plasma/serum	U2 snRNA (↑)	D

D: Diagnostic marker; miRNA: MicroRNA; P: Prognostic marker; S: Staging marker; T: Treatment marker.