Improved Detection of Circulating Epithelial Cells in Patients with Intraductal Papillary Mucinous Neoplasms

Abstract

Background: Recent work has demonstrated early shedding of circulating epithelial cells (CECs) from premalignant intraductal papillary mucinous neoplasms (IPMNs). However, the potential use of CECs as a "liquid biopsy" for patients with IPMNs has been limited by antigen dependence of CEC isolation devices and the lack of robust detection biomarkers across CEC phenotypes.

Materials and methods: We utilized a negative depletion microfluidic platform to purify CECs from contaminating leukocytes and coupled this platform with immunofluorescence, RNA in situ hybridization, and RNA sequencing (RNA-seq) detection and enumeration.

Results: Using established protein (EpCAM, cytokeratins) and novel noncoding RNA (HSATII, cytokeratins) biomarkers, we detected CECs in 88% of patients bearing IPMN lesions. RNA-seq analysis for MUC genes confirm the likely origin of these CECs from pancreatic lesions.

Conclusion: Our findings increase the sensitivity of detection of these cells and therefore could have clinical implications for cancer risk stratification.

Implications for practice: This work describes a high-sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at high risk of malignant transformation. Further research efforts are underway to define the transcriptional programs that might allow discrimination between circulating cells released from tumors that will become malignant and cells released from tumors that will not. After further refinement, this combination of technologies could be deployed for monitoring and early detection of patients at high risk for developing new or recurrent pancreatic malignancies.

Keywords: Circulating epithelial cells; Early detection; Pancreatic cancer.

Figure 1.

Experimental rationale and schema. (A): Representative RNA in situ hybridization images of resected primary intraductal papillary mucinous neoplasm specimens showing HSATII expression (red) and control immune cell IHC markers (CD8 left, CD163 right; both brown). (B): Schematic of workflow used for CEC isolation and enumeration. Erythrocytes and platelets are first separated by hydrodynamic sorting; then magnetic separation of bead‐bound leukocytes takes place after passage through curved channels to orient the nucleated cells in a single‐file line. Erythrocytes are shown in red, leukocytes coated with magnetic beads in blue, and circulating epithelial cells in yellow. Abbreviations: CEC, circulating epithelial cell; RBC, red blood cell; WBC, white blood cell.

Figure 2.

Detection of CECs in patient whole blood using the circulating tumor cell iChip followed by a combination of immunofluorescent cell staining, in situ hybridization (ISH), and RNA sequencing. (A): Representative immunofluorescent image of a CK+/EpCAM+/CD45− CEC and a leukocyte. (B): Quantification of CECs per mL detected using IF. (C): Representative RNA‐ISH image of a HSATII+/keratin+ CEC. (D): Quantification of CECs per mL detected using RNA‐ISH. p values are reported for a two‐tailed nonparametric Mann‐Whitney test, and the box plot shows data quartiles (25%, median, 75%). (E): Heat map showing expression of differentially expressed mucin genes (p < .05) expressed at high levels in CECs isolated from patients with IPMNs and PDACs but not in cells isolated from HDs. IPMN1 denotes low risk and IPMN2 denotes high risk for pancreatic cancer development. Expression in log10 scale is shown. Abbreviations: CEC, circulating epithelial cell; CK, cytokeratin; HD, healthy donor; IF, immunofluorescent staining; IPMN, intraductal papillary mucinous neoplasm; PDAC, pancreatic ductal adenocarcinoma; RPM, reads per million.