Acquisition of Portal Venous Circulating Tumor Cells From Patients With Pancreaticobiliary Cancers by Endoscopic Ultrasound

Abstract

Background & aims: Tumor cells circulate in low numbers in peripheral blood; their detection is used predominantly in metastatic disease. We evaluated the feasibility and safety of sampling portal venous blood via endoscopic ultrasound (EUS) to count portal venous circulating tumor cells (CTCs), compared with paired peripheral CTCs, in patients with pancreaticobiliary cancers (PBCs).

Methods: In a single-center cohort study, we evaluated 18 patients with suspected PBCs. Under EUS guidance, a 19-gauge EUS fine needle was advanced transhepatically into the portal vein and as many as four 7.5-mL aliquots of blood were aspirated. Paired peripheral blood samples were obtained. Epithelial-derived CTCs were sorted magnetically based on expression of epithelial cell adhesion molecules; only those with a proper morphology and found to be CD45 negative and positive for cytokeratins 8, 18, and/or 19 and 4',6-diamidino-2-phenylindole were considered to be CTCs. For 5 samples, CTCs also were isolated by flow cytometry and based on CD45 depletion. ImageStream was used to determine the relative protein levels of P16, SMAD4, and P53. DNA was extracted from CTCs for sequencing of select KRAS codons.

Results: There were no complications from portal vein blood acquisition. We detected CTCs in portal vein samples from all 18 patients (100%) vs peripheral blood samples from only 4 patients (22.2%). Patients with confirmed PBCs had a mean of 118.4 ± 36.8 CTCs/7.5 mL portal vein blood, compared with a mean of 0.8 ± 0.4 CTCs/7.5 mL peripheral blood (P < .01). The 9 patients with nonmetastatic, resectable, or borderline-resectable PBCs had a mean of 83.2 CTCs/7.5 mL portal vein blood (median, 62.0 CTCs/7.5 mL portal vein blood). In a selected patient, portal vein CTCs were found to carry the same mutations as those detected in a metastatic lymph node and expressed similar levels of P16, SMAD4, and P53 proteins.

Conclusions: It is feasible and safe to collect portal venous blood from patients undergoing EUS. We identified CTCs in all portal vein blood samples from patients with PBCs, but less than 25% of peripheral blood samples. Portal vein CTCs can be used for molecular characterization of PBCs and share features of metastatic tissue. This technique might be used to study the pathogenesis and progression of PBCs, as well as a diagnostic or prognostic tool to stratify risk of cancer recurrence or developing metastases.

Keywords: Endoscopic Ultrasound; EpCAM; Pancreatic Cancer; Portal Venous Blood.

Figure 1

EUS-guided access of the portal vein. (A) The portal vein is identified under EUS guidance with Doppler wave verification. (B) EUS-guided, transhepatic, FNA puncture of the portal vein with a 19G EUS FNA needle for portal venous blood acquisition. PV, portal vein.

Figure 2

CTC enumeration from peripheral blood and portal venous blood. (A) Summary table of CTC enumeration per 7.5 mL of peripheral and portal venous blood by malignancy type. (B) Representative immunofluorescence staining of a single CTC and a CTC cluster captured by CellSearch enumeration (DAPI, magenta; CK, green) (C) Vertical scatter plot with the means ± SEM for CTCs enumerated from the peripheral blood and portal venous blood in patients with confirmed PBCs. Bar indicates a statistically significant difference using paired t test. (D) Box and whisker plot with individual points for CTC enumeration per 7.5 mL of portal venous blood (PVB) in all patients with confirmed PBCs and the subset of patients with borderline-resectable or resected PBCs vs unresectable PBCs. IPMN, intraductal papillary mucinous neoplasm.

Figure 3

Tumor-suppressor genes, p16/CDKN2A, TP53, and SMAD4 protein expression in AGS control cells and portal venous CTCs. (A) Flow cytometry of CD45-depleted portal venous blood gated by ImageStream for EpCAM+, DAPI+ CTCs. Representative immunofluorescent staining of the proteins in (B) individual AGS control cells and (C) portal venous CTCs during EpCAM+ flow cytometry via ImageStream technology. (D) Representative immunofluorescent staining of the proteins in CTC clusters identified in portal venous blood. (E) Median intensity for immunofluorescence of CTC p53, smad4, p16 proteins relative to AGS control cell line normalized to mean cell size (mean aspect ratio of the bright field). (F) Immunohistochemistry confirmation of positive smad4, loss of p16, and heterogeneous p53 expression in a metastatic lymph node relative to AGS control cells and positive control tissues (40× magnification; scale bars: 50 µm).

Figure 4

Confirmation of CTNNB1 mutation detected in CTC DNA. Top 2 sequences show reference protein (top row) and complementary DNA (cDNA) (second row) sequences as G (glycine) and GGA, respectively. Bottom 2 sequencing traces show forward GAA (third row) and reverse TTC (bottom row) sequencing of portal venous CTCs isolated from the patient consistent with G34E missense mutation as found in a metastatic lymph node. Red boxes highlight the sequencing location of the CTNNB1 gene with G>A nucleotide change resulting in a G34E mutation.