Endoscopic Ultrasound-Guided Acquisition of Portal Venous Circulating Tumor Cells as a Potential Diagnostic and Prognostic Tool for Pancreatic Cancer

Abstract

Background: Circulating tumor cells (CTCs) were a promising liquid biopsy for pancreatic cancer (PC) but circulate in low counts in peripheral blood. We evaluated the diagnostic and prognostic values of portal vein (PoV) CTCs in PC patients.

Methods: PoV was aspirated under EUS guidance from 40 patients with suspected pancreaticobiliary cancers. Epithelial-mesenchymal-transition-related subtypes of CTCs were identified via immunofluorescence using EpCAM and Twist antibodies. The diagnostic and prognostic performance of PoV CTCs was investigated by receiver-operating characteristic (AUC) curve and Kaplan-Meier survival analysis.

Results: In total, 40 patients including 31 with PC, 4 with non-pancreatic periampullary cancer and 5 with benign pancreatic diseases (BPD) were enrolled. CTCs were detected more in PoV compared with peripheral blood. PoV CTC numbers in BPD patients were lower than in PC patients. The number of PoV CTCs, especially mesenchymal-CTCs (M-CTCs), was positively correlated with the tumor burden, instead of epithelial-CTCs (E-CTCs). The combination of PoV CTC numbers and CA19-9 demonstrated better diagnostic efficiency (AUC value 0.987) than either alone in differentiating PC with BPD. Moreover, the diagnostic efficacy of PoV CTCs and M-CTCs were obviously better than that of E-CTCs and CA19-9 in distinguishing early and late stage PC. Lastly, high PoV CTC and M-CTC numbers were both associated with shorter overall survival.

Conclusion: Acquisition of the PoV samples in PC patients via EUS-guided procedures has been proved safe and feasible. PoV CTCs, especially M-CTCs, have great potentials in diagnosing and predicting the prognosis of PC, especially in combination with CA19-9.

Keywords: circulating tumor cell; diagnosis; endoscopic ultrasound; portal vein blood; prognosis.

Conflict of interest statement

The authors report no conflicts of interest in this work.

© 2021 Zhang et al.

Figures

Figure 1

EUS-guided access of the portal vein. (A) Pancreatic mass was hypoechoic with poorly defined boundaries. (B) EUS-FNA to obtain histological cytology and histological examination. (C) The main portal vein was identified under EUS guidance with Doppler wave verification. (D) Puncture the primary vein with a needle for portal venous blood acquisition.

Figure 2

Flowchart of the protocol for CTC detection in our study. The peripheral and portal vein blood were collected from each patient. Red blood Cells (RBCs) in the samples were lysed, and residual cell pellets were resuspended. Samples were labeled with anti-leukocyte-specific antibodies (CD45) monoclonal antibody coated magnetic beads for 20 min, followed by separation of the beads using a gradient centrifugation liquid and magnetic stand. The resulting solution with any CTCs was smeared on a single slide, fixed, and dried for CTC EMT phenotype detection by immunofluorescence.

Figure 3

Flowchart of the enrolled patients.

Figure 4

Verification and detection of EMT phenotype in PC cell lines and portal vein CTCs from PC patients. (A) EMT phenotype of this platform was verified in representative “epithelial” (Bxpc3, sw1990) and “mesenchymal” (Miapaca-2, Panc-1) PC cell lines. The cells were analyzed using a 200X× objective. (B) Representative images of CTCs in portal vein blood from a patient with PC. Leukocytes were stained for CD45 (red fluorescence). CTCs were stained for epithelial markers (EpCAM, Orange fluorescence) and mesenchymal markers (Twist, green fluorescence). The cells were analyzed using a 400× objective.

Figure 5

Circulating tumor cells (CTCs) in portal vein blood (PoV) constitute a potential biomarker for PC. (A) CTC numbers in PoV were significantly higher than in peripheral blood (median, 10 vs 6 cell/5mL of blood, p<0.0001); (B) Comparison of PoV CTC numbers in patients with PC, non-PC and BPD. PC, pancreatic cancer; non-PC, non-pancreatic periampullary cancer; BPD, benign pancreatic diseases. (C) CTC numbers among patients with BPD, and PC patients at different stages. (D) The diagnostic performance of PoV CTC numbers, PV CTC numbers, CA19-9, and combined PoV CTC numbers with CA19-9 in differentiating PC and BPD was shown by ROC curves.

Figure 6

Correlation of the number of CTCs, M-CTCs and E-CTCs in PoV and the number of CTCs in PV with the clinical features of PC patients. (A) The comparison of PoV CTC numbers detected using Twist and EpCAM antibodies. (B–E) Correlation of the numbers of CTCs (B) M-CTCs (C) E-CTCs (D) in portal vein blood and the numbers of CTCs in peripheral vein blood (E) with lymph node invasion, distant metastasis and PC stages. (F and G) ROC curves of CTCs, M-CTCs, E-CTCs in portal vein blood, CTCs in peripheral blood and serum CA19-9 to distinguish PC patients between stage I–II and III–IV.

Figure 7

Overall survival (OS) in patients with different levels of CTCs and M-CTCs in PoV. (A) Kaplan–Meier survival analysis showed that patients with lower CTC (CTCs<10/5 mL) had better OS compared with higher CTC (CTCs ≥10/5mL) (p=0.004). (B) OS in patients with lower M-CTC (M-CTCs <8/5mL) was superior to those with higher M-CTC (M-CTCs ≥8/5mL) (p=0.007). (C) The hazard ratio (HR) with 95% CI of the CTCs and M-CTCs parameters and clinicopathological characteristics for OS in PC patients based on COX regression analysis.