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

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.