Liquid biopsy leads to a paradigm shift in the treatment of pancreatic cancer

Table 1 Liquid biopsy in the early diagnosis of pancreatic cancer

Ref.	Journal	No. of patients	Biomarker	Method	Main findings
Ankeny et al[76], 2016	Br J Cancer	72	CTC	Microfluidic NanoVelcro CTC chip	Detection rate of PDAC: 54/72 (sensitivity = 75.0%, specificity = 96.4%)
Rhim et al[77], 2014	Gastroenterology	51	CTC	Microfluidic geometrically enhanced differential immunocapture	CTC (≥ 3) in 33% of patients with cystic lesions and no clinical diagnosis of cancer, 73% with PDAC, and 0% of controls
Xu et al[78], 2017	Int J Mol Sci	40	CTC	NE-iFISH	The positive rate of diagnosis of PDAC is nearly 97% in combination with CA19-9
Poruk et al[79], 2017	Clin Cancer Res	60	CTC	ISET method & immunofluorescence	The positive rate is 12% in stageⅠPDAC
Gao et al[80], 2016	J Exp Clin Cancer Res	25	CTC	SE-iFISH platform	Sensitivity of 88 % and specificity of 90 % in PDAC
Kulemann et al[81], 2015	Pancreas	11	CTC	ScreenCell; Cyto kit	No difference in the rate of CTC detection between early-stage and advanced-stage diseases (P = 0.71)
Melo et al[33], 2015	Nature	56	Exosome	FACS analysis	The sensitivity and specificity of GPC1+ circulating exosomes in diagnosing PDAC were both 100%
Buscail et al[82], 2019	Cancers	30	Exosome and CTC	FACS, Cellsearch and RosetteSepTM	Combining quantification of GPC1-positive exosomes and CTC detection identified all the PDAC patients, showed a negative predictive value of 100%, and an overall diagnostic accuracy of 91%
Lai et al[83], 2017	Cancer Lett	40	Exosome and miRNA	LC-MS & RT-qPCR	High levels of exosomal miR-10b, miR-21, miR-30c, and miR-181a and low levels of miR-let7a differentiated PDAC from healthy and chronic pancreatitis samples
Liang et al[84], 2017	Nat Biomed Eng	23	EV	nPES assay	Pre-therapy EphA2-EV blood levels accurately distinguished stage I/II pancreatic cancer patients from NC (AUC = 0.96) and pancreatitis patients (AUC = 0.93)
Que et al[85], 2013	World J Surg Oncol	49	miRNA	RT-PCR	Serum exosomal miR-17-5p was higher in PDAC patients than in non–PDAC patients and healthy participants
Cote et al[86], 2014	Am J Gastroenterol	215	miRNA	RT-PCR	Increased expression of miRNA-10b, -155, and -106b in plasma appears highly accurate in diagnosing PDAC
Ouyang et al[87], 2015	Oncogene	42	miRNA	RT-PCR	Plasma miR-10b levels significantly increased in comparison with normal controls
Slater et al[88], 2014	Transl Oncol	59	miRNA	Real-time PCR	A combination test of miRNA-196a and miRNA-196b, whose expression is upregulated from the PanIN state, can identify patients with PanIN 2/3
Madhavan et al[89], 2015	Int J Cancer	220	Exosome and miRNA	miRNeasyMinikit, RT-PCR, qRT-PCR and flow cytometry	The selected miR-1246, miR-4644, miR-3976 and miR-4306 were significantly upregulated in 83% of PDAC serum-exosomes, but rarely in control groups

AUC: Area under the curve; CA19-9: Carbohydrate antigen 19-9; CTC: Circulating tumor cell; EphA2: Ephrin type-A receptor 2; EV: Extracellular vesicles; FACS: Fluorescence-activated cell sorting; ISET: Isolation by size of epithelial tumor cells; LC-MS: Liquid chromatography-tandemmass spectrometry; miRNA: microRNA; nPES: Nanoplasmon-enhanced scattering; NC: Normal healthy control; NE-iFISH: Negative enrichment immunofluorescence and in situ hybridization of chromosome 8; PDAC: Pancreatic ductal adenocarcinoma; RT-qPCR: Quantitative reverse transcription polymerase chain reaction; SE-iFISH: Subtraction enrichment and immunostaining-fluorescence in situ hybridization.

Table 2 Liquid biopsy in the predicting prognosis of pancreatic cancer

Ref.	Journal	year	No. of patients	Biomarker	Method	Main findings
Singh et al[91], 2015	Cancer Invest	2015		cfDNA		Higher level of plasma DNA (> 62 ng/mL) was found to associate significantly with lower overall survival time (P = 0.002), presence of vascular encasement (P = 0.030) and metastasis (P = 0.001)
Lapin et al[92], 2018	J Transl Med	2018	61	cfDNA	2100 Bioanalyzer	Pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, P = 0.005) and OS (HR = 2.236, P = 0.028)
Wang et al[93], 2021	Pancreas	2021	97	cfDNA	PCR	The 1- and 5-year survivals for those with high cfDNA were poorer; 70.2% and 21.2%, respectively, as compared with 93.4% and 23.7% for those with low cfDNA level
Castells et al[94], 1999	J Clin Oncol	1999	47	ctDNA	PCR-RFLP and SSCP	Plasma KRAS mutations were identified as the only independent prognostic factor (odds ratio, 1.51; 95%CI: 1.02 to 2.23)
Ako et al[95], 2017	Pancreatology	2017	40	ctDNA	ddPCR	KRAS mutation at G12V in the plasma or serum conferred a significantly poorer prognosis than without the mutation (P < 0.01)
Hadano et al[96], 2016	Br J Cancer	2016	105	ctDNA	ddPCR	Patients who were preoperative ctDNA+ had a significantly poorer prognosis with respect to OS (P < 0.0001)
Nakano et al[97], 2018	Br J Cancer	2018	45	ctDNA	PNA directed, PCR clamping	There were no significant differences in DFS and OS between patients with and without KRAS mutations from preoperative serum
Watanabe et al[98], 2019	PLoS One	2019	78	ctDNA	ddPCR	No effect of the presence of KRAS-mutated ctDNA before surgery on RFS (median: 16.9 mo vs 32.4 mo) was observed
Bernard et al[99], 2019	Gastroenterology	2019	34	ctDNA and exosome DNA	ddPCR	Increased exosome DNA levels after neoadjuvant therapy were significantly associated with disease progression (P = 0.003)
Kinugasa et al[100], 2015	Cancer	2015	75	ctDNA	ddPCR	KRAS mutations in plasma correlated with poor OS (P = 0.002)
Tjensvoll et al[101], 2016	Mol Oncol	2016	14	ctDNA	PNA clamp PCR	Kaplan-Meier survival analyses indicated that patients with a positive ctDNA before or after initiation of chemotherapy had shorter PFS and OS
Chen et al[102], 2010	Eur J Surg Oncol	2010	91	ctDNA	Direct sequencing	KRAS codon 12 mutation from plasma DNA was an independent negative prognostic factor (HR, 7.39; 95%CI: 3.69-14.89)
Sausen et al[103], 2015	Nat Commun	2015	101	ctDNA	Next-generation sequencing and digital PCR	ctDNA was an independent prognostic marker of OS in advanced disease, with OS of 6.5 mo vs 19.0 mo for ctDNA-positive and negative patients, respectively
Khoja et al[105], 2012	Br J Cancer	2012	54	CTC	Cellsearch and ISET	The PFS and OS for patients without vs those with CTCswas 140 d vs 94 d (P = 0.13) and 164 d vs 127 d (P = 0.26), respectively
Earl et al[106], 2015	BMC Cancer	2015	45	CTC	Cellsearch	A Cox regression analysis showed a significant difference in OS for CTC positive vs negative patients with a HR of 3.0 (P = 0.023)
Zhang et al[107], 2015	Int J Cancer	2015	61	CTC	The EpCAM-independent method	CTCs positive pancreatic cancer patients exhibit a worse (P = 0.0458) survival rate
Okubo et al[108], 2017	Eur J Surg Oncol	2017	65	CTC	Cellsearch	A multivariate analysis identified the presence or absence of CTCs as an independent prognostic factor (P = 0.049)
Ankeny et al[76], 2016	Br J Cancer	2016	100	CTC	Microfluidic NanoVelcro CTC chip	A cut-off of 3 CTCs in 4 mL venous blood was able to discriminate between local/regional and metastatic disease (AUROC = 0.885; 95%CI: 0.800-0.969; and P < 0.001)
Chang et al[109], 2016	Clin Chem	2016	63	CTM	anti-EpCAM conjugated supported lipid bilayer-coated microfluidic chips	CTM was an independent prognostic factor of OS and PFS (P 0.0001 and P = 0.003, respectively)
Bidard et al[110], 2013	Ann Oncol	2013	79	CTC	Cellsearch	CTC positivity was associated with poor tumor differentiation (P = 0.04), and with shorter OS in multivariable analysis (P = 0.01)
Kurihara et al[111], 2008	J Hepatobiliary Pancreat Surg	2008	47	CTC	Cellsearch	MST of the CTC-positive and -negative patients were 110.5 and 375.8 d (P < 0.001)
de Albuquerque et al[112], 2008	Oncology	2012	74	CTC		Median PFS time was 66.0 d for patients with baseline CTC positivity and 138.0 days for CTC-negative patients (P = 0.01)
Kulemann et al[81], 2015	Pancreas	2015	21	CTC	ScreenCell	The presence of CTC did not adversely affect MST: 16 mo in CTC-positive (n = 18) vs 10 mo in CTC-negative (n = 3) patients
Li et al[116], 2018	Cell Physiol Biochem	2018	73	miRNA	Arraystar Human miRCURYTM LNA Array	Multivariate analyses showed that exosomal miR-222 was independent risk factors for PDAC survival (P = 0.046)
Wang et al[117], 2018	Cancer Res	2018	50	miRNA	qRT-PCR	Exosomal miR-301a-3p overexpression predicted late TNM stage and poor survival in human PDAC (P = 0.0182)
Frampton et al[118], 2018	Oncotarget	2018	43	GPC1+ circulating exosomes	ELISA	Patients with high crExos GPC1 levels have significantly larger PDACs (> 4 cm; P = 0.012)
Costa-Silva et al[119], 2015	Nat Cell Biol	2015	55	Exosome	ELISA	Increased levels of MIF in exosomes isolated from patients with PDAC with progression of disease post-diagnosis compared with PDAC patients with no evidence of disease five years post-diagnosis (P < 0.01) and with healthy controls (P < 0.01), but not patients with liver metastasis

AUROC: Area under the curve; cfDNA: Cell-free DNA; ctDNA: Circulating tumor DNA; CTC: Circulating tumor cell; CTM: Circulating tumor microemboli; DFS: Disease-free survival; ddPCR: Droplet digital polymerase chain reaction; ELISA: Enzyme-linked immuno-sorbent assay; EPCAM: Epithelial cell adhesion molecule; ISET: Isolation by size of epithelial tumor cells; HR: Hazard ratio; MST: Median survival time; MIF: Macrophage migration inhibitory factor; miRNA: microRNA; OS: Overall survival; PFS: Progression-free survival; PCR: Polymerase chain reaction; PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism; PNA: Peptide nucleic acid; PDAC: Pancreatic ductal adenocarcinoma; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; RFS: Recurrence-free survival; SSCP: Single-strand conformation polymorphism.

Table 3 Liquid biopsy in recurrence monitoring of pancreatic cancer

Ref.	Journal	No. of patients	Biomarker	Method	Main findings
Nakano et al[97], 2018	Br J Cancer	45	ctDNA	PNA-directed PCR clamping	Multivariate analysis revealed that KRAS mutations in postoperative serum are an independent prognostic factor for DFS (P = 0.027). Furthermore, the change from not detecting mutant KRAS in preoperative to mutant KRAS in postoperative cfDNA was an independent prognostic factor for OS (P = 0.004)
Hussung et al[120], 2021	BMC Cancer	25	ctDNA	ddPCR, PCR	An increased KRAS mutated ctDNA during adjuvant chemotherapy and follow-up was a highly predictive dynamic marker of early relapse and poor OS
Watanabe et al[98], 2019	PLoS One	78	ctDNA	ddPCR	Detection of mutant KRAS on postoperative ctDNA was associated with OS regardless of recurrence (P = 0.005)
Groot et al[121], 2019	Clin Cancer Res	59	ctDNA	ddPCR	ctDNA detected during follow-up predicted clinical recurrence (sensitivity 90%, specificity 88%) with a median lead time of 84 d
Sausen et al[103], 2015	Nat Commun	20 (surgery group)	ctDNA	Next-generation sequencing and digital PCR	Patients with detectable ctDNA after surgical resection (n = 10) were more likely to relapse and die from disease compared with those with undetectable ctDNA (P = 0.0199)
Jiang et al[122], 2020	Front Oncol	27	ctDNA	Next-generation sequencing	Patients with ctDNA-positive status postoperatively had a markedly reduced DFS compared to those with ctDNA-negative status (P = 0.019)
Kim et al[123], 2018	Clin Chem	106	ctDNA	ddPCR	Patients who had increased KRAS MAF values at 6 mo had a shorter OS (P = 0.036) than those who had decreased values
Yamaguchi et al[124], 2021	Ann Surg Oncol	97	ctDNA	ddPCR	The multivariate analysis showed that the presence of preoperative ctDNA was associated with poorer OS (P = 0.008) and that postoperative ctDNA was not associated with either RFS or OS
Guo et al[125], 2020	Br J Cancer	113 and 44 (discovery and validation cohorts)	ctDNA	ddPCR	Survival analysis showed that plasma KRAS mutations, especially KRAS G12D mutation, had significant association with OS and RFS of resectable PDAC. Plasma KRAS G12D mutation showed a strong correlation with early distant metastasis
Lee et al[126], 2019	Ann Oncol	42	ctDNA	PCR-based-SafeSeqS assays	Preoperative ctDNA detection was associated with inferior RFS (P = 0.002) and OS (P = 0.015). Detectable ctDNA following curative intent resection was associated with inferior RFS (P < 0.0001) and OS (P = 0.003)
Pietrasz et al[127], 2017	Clin Cancer Res	31	ctDNA	Next-generation sequencing	The presence of ctDNA was associated with a shorter DFS (4.6 mo vs 17.6 mo; P = 0.03) and shorter OS (19.3 mo vs.32.2 mo; P = 0.027)
Okada et al[128], 2020	J Gastroenterol	66 (surgery group)	ctDNA	Digital PCR	Patients with preoperative ctDNA MAF > 0.45% exhibited significantly shorter disease-free survival than those with lower MAF (HR 3.179, 95%CI: 1.025-9.859; P = 0.0452)
Park et al[129], 2021	Sci Rep	40	CTC	CD-PRIM kit	On multivariable logistic regression analysis, CTC positivity was an independent risk factor for early recurrence (P = 0.027) and systemic recurrence (P = 0.033)
Allenson et al[130], 2017	Ann Oncol	142 and 121 (discovery and validation cohort)	Exosome and ctDNA	Electron microscopy, flow cytometry and particle analysis and ddPCR	Higher exosome KRAS MAFs were associated with decreased disease-free survival in patients with localized disease (P = 0.031)
Takahasi et al[131], 2018	J Hepatobiliary Pancreat Sci	50	miRNA	qRT-PCR	In cox proportional hazards model analysis, exosomal miR-451a showed significance to OS and DFS (P = 0.001, P = 0.004)
Kawamura et al[132], 2019	J Hepatobiliary Pancreat Sci	55	miRNA	qRT-PCR	miR-4525, miR-451a, and miR-21 from portal vein can be utilized for the evaluation of pancreatic cancer recurrence (P = 0.002, 0.001 and 0.002, respectively)

ctDNA: Circulating tumor DNA; cfDNA: Cell-free survival; CTC: Circulating tumor cell; DFS: Disease-free survival; ddPCR: Droplet digital polymerase chain reaction; HR: Hazard ratio; MAF: Mutant allele frequency; miRNA: microRNA; OS: Overall survival; PNA: Peptide nucleic acid; PCR: Polymerase chain reaction; PDAC: Pancreatic ductal adenocarcinoma; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; RFS: Recurrence-free survival; SafeSeqS: Safe-sequencing system.

Table 4 Liquid biopsy in the therapeutic effect monitoring of pancreatic cancer

Ref.	Journal	No. of patients	Biomarker	Method	Main findings
Del Re et al[133], 2017	Sci Rep	27	ctDNA	ddPCR	There was a statistically significant difference in PFS and OS in patients with increase vs stability/reduction of ctDNA in the sample collected at day 15 (P = 0.03 and P = 0.009, respectively)
Kruger et al[134], 2018	Ann Oncol	54	ctDNA	BEAMing	An increase in ctDNA at day 14 correlated with disease progression on subsequent imaging with a sensitivity of 83% and specificity of 100%
Watanabe et al[98], 2019	PLoS One	39	ctDNA	ddPCR	The emergence of KRAS ctDNA in longitudinal tests was associated with prognosis (P < 0.005)
Wei et al[135], 2019	Cancer Lett	13 (chemotherapy group)	CTC	Vimentin or EpCAM immobilized microfluidic chip	In patients exhibiting a response, their CTC counts decreased or remained the same, except for one case
Okubo et al[108], 2017	Eur J Surg Oncol	65	CTC	Cellsearch	The overall survival rate was significantly lower in patients with than in those without CTCs even after chemotherapy and chemoradiotherapy (P = 0.045)
Xu et al[78], 2017	Int J Mol Sci	83	CTC	NE-iFISH	The proportion of triploid CTC detected by the NE-iFISH was significantly decreased after chemotherapy (P < 0.001)
Tian et al[136], 2016	Oncol Lett	17	microRNA	RT-qPCR	Significant upregulation of serum miRNAs (miR-21, miR-210, miR-221 and miR-7), at earlier time points (3-6 wk) was observed in non-responders of chemotherapy compared to responders
Bernard et al[99], 2019	Gastroenterology	104 (chemotherapy group)	exosome and ctDNA	ddPCR	In the longitudinal analysis in chemotherapy group, a MAF peak above 1% in exosome DNA was significantly associated with radiologic progression (P = 0.0003)
An et al[137], 2017	J Proteome Res	10	exosome	iTRAQ	They analyzed exosomes before treatment, after one cycle of induction gemcitabine-based chemotherapy, and at 3 wk after starting chemoradiation therapy and compared these samples to serum derived from healthy volunteers. They identified eight proteins that changed during a course of therapy in all patients
Liang et al[84], 2017	Nat Biomed Eng	23 (neoadjuvant chemotherapy group)	EV	nPES	EphA2-EVs were also informative in detecting early responses to neoadjuvant therapy (P < 0.05)
Yin et al[138], 2021	Clin Cancer Res	36	somatic mutations, CTCs, and ctDNA	Next-generation sequencing & ISET	Somatic mutations, CTCs, and ctDNA existed even in patients with PDAC with pathologic complete response to NAT, which could possibly predict early recurrence and reduced survival
Poruk et al[140], 2016	Ann Surg	50	CTC	ISET	The detection of CTCs expressing both vimentin and cytokeratin was predictive of recurrence (P = 0.01)
Gemenetzis et al[139], 2018	Ann Surg	57	CTC	ISET	Patients who received neoadjuvant chemotherapy had significantly lower total CTCs (tCTCs, P = 0.007), eCTCs (P = 0.007), and mCTCs (P = 0.034), compared with untreated patients eligible for upfront resection

ctDNA: Circulating tumor DNA; CTC: Circulating tumor cell; CR: Complete response; ddPCR: Droplet digital polymerase chain reaction; eCTCs: Epithelial circulating tumor cell; EpCAM: Epithelial cell adhesion molecule; EphA2: Ephrin type-A receptor 2; EV: Extracellular vesicles; iTRAQ: Isobaric tag for relative and absolute quantitation; ISET: Isolation by size of epithelial tumor cells; MAF: Mutant allele frequency; mCTCs: Mesenchymal circulating tumor cell; nPES: Nanoplasm-enhanced scattering; NAT: Neoadjuvant chemotherapy; NE-iFISH: Negative enrichment immunofluorescence and in situ hybridization of chromosome 8; OS: Overall survival; PFS: Progression-free survival; RT-qPCR: Quantitative reverse transcription polymerase chain reaction; tCTC: Total circulating tumor cell.