EpCAM-based Flow Cytometric Detection of Circulating Tumor Cells in Gallbladder Carcinoma Cases

Abstract

Purpose: Liquid biopsy has entered the arena of cancer diagnostics in the past decade and detection of circulating tumor cells (CTC) is one diagnostic component. CTCs in gallbladder cancer (GBC) have hitherto not been comprehensively analysed. Methods and Results: The current study focused on the diagnostic role of CTCs in 27 cases of treatment-naive GBC and 6 normal controls as well as 6 cases of cholecystitis. An EasySep kit featuring negative immunomagnetic bead separation and flow cytometric detection of EpCAM positive and CD45 negative cells revealed CTCs in 25 of the 27 cases. At a cut-off point of ≥1, the CTC count discriminated GBC from controls with a sensitivity, specificity and diagnostic accuracy of 92.6%, 91.7% and 92.3%, respectively. CTC levels in turn correlated significantly with clinico-pathological parameters of cases in terms of known prognostic indicators, with significant diagnostic potential at a cut-off point of >4, to discriminate disease stage I and II vs. III and IV GBC. With a cut-off of >3, the CTC count discriminated tumor stages I and II vs. III and IV and at >6 CTCs could discriminate metastatic vs. non metastatic GBCs with a sensitivity, specificity and diagnostic accuracy of 55. 6%, 100.0% and 85.2, respectively. A review of CTC in pancreatico-biliary malignancies is included. Conclusion: Detection and quantification of CTCs may serve as a non-invasive biomarker for GBC diagnosis in correlation with radiological studies.

Keywords: Circulating tumor cells; gall bladder cancer; liquid biopsy; EpCAM.

Figure 1

Plot 1, R1 drawn on Forward scatter vs. Side scatter plot to exclude debris (exclusion gate/NOT gate); Plot 2, R2 drawn on Forward scatter vs. 7-AAD to include only viable events which are 7-AAD positive; Plot 3, shows cells in G1 gate = R2 AND NOT R1 on CD45 FITC vs. EpCAM PE, R4 is drawn on bright CD45 positive cells (lymphocyte CD45+, EpCAM –ve) and R3 is drawn around EpCAM + CD45 –ve event (CTCs); Plot 4, is gated on G2=R4 AND R2 AND NOT R1. This plot gives lowest cut-off based on size for lymphocyte on which a further region R5 was made; Plot 5, is gated on G3=R3 AND R2 AND NOT R1. This plot shows CTCs on Forward scatter vs. Side scatter on which further region R6 is created which is exactly same as R5 on X-axis, considering that CTCS would not be smaller than lymphocytes; Plot 6, is gated on G4= R6 AND R3 AND R2 AND NOT R1. This plot shows CTC count after strictly excluding debris, dead cells and events which are smaller in size than lymphocytes. a)shows 18 CTC in Stage IV GBC b) shows 1 CTC in Stage I c) shows No CTC in normal healthy control.