Abstract

BackgroundMost cancer deaths are caused by metastases, resulting from circulating tumor cells (CTC) that detach from the primary cancer and survive in distant organs. The aim of the present study was to develop a CTC gene signature and to assess its prognostic relevance after surgery for pancreatic ductal adenocarcinoma (PDAC).MethodsNegative depletion fluorescence activated cell sorting (FACS) was developed and validated with spiking experiments using cancer cell lines in whole human blood samples. This FACS-based method was used to enrich for CTC from the blood of 10 patients who underwent surgery for PDAC. Total RNA was isolated from 4 subgroup samples, i.e. CTC, haematological cells (G), original tumour (T), and non-tumoural pancreatic control tissue (P). After RNA quality control, samples of 6 patients were eligible for further analysis. Whole genome microarray analysis was performed after double linear amplification of RNA. ‘Ingenuity Pathway Analysis’ software and AmiGO were used for functional data analyses. A CTC gene signature was developed and validated with the nCounter system on expression data of 78 primary PDAC using Cox regression analysis for disease-free (DFS) and overall survival (OS).ResultsUsing stringent statistical analysis, we retained 8,152 genes to compare expression profiles of CTC vs. other subgroups, and found 1,059 genes to be differentially expressed. The pathway with the highest expression ratio in CTC was p38 mitogen-activated protein kinase (p38 MAPK) signaling, known to be involved in cancer cell migration. In the p38 MAPK pathway, TGF-β1, cPLA2, and MAX were significantly upregulated. In addition, 9 other genes associated with both p38 MAPK signaling and cell motility were overexpressed in CTC. High co-expression of TGF-β1 and our cell motility panel (≥ 4 out of 9 genes for DFS and ≥ 6 out of 9 genes for OS) in primary PDAC was identified as an independent predictor of DFS (p=0.041, HR (95% CI) = 1.885 (1.025 – 3.559)) and OS (p=0.047, HR (95% CI) = 1.366 (1.004 – 1.861)).ConclusionsPancreatic CTC isolated from blood samples using FACS-based negative depletion, express a cell motility gene signature. Expression of this newly defined cell motility gene signature in the primary tumour can predict survival of patients undergoing surgical resection for pancreatic cancer.Trial RegistrationClinical trials.gov NCT00495924

Highlights

  • Most cancer deaths are caused by metastases, resulting from circulating tumor cells (CTC) that detach from the primary cancer and survive in distant organs

  • Since distant organ metastases are the end-results of circulating tumor cells (CTC), molecular characterization of CTC is promising in the quest for effective therapeutic agents and novel prognostic markers [2]

  • Patients and tissues Fresh tumour (T) and surrounding non-tumoural control (P) tissue samples were obtained from surgical specimens from 10 patients (M/F: 5/5; median age: 61,0 (50 – 74) years) who had undergone surgical resection for pancreatic ductal adenocarcinoma (PDAC) (Table 1)

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Summary

Introduction

Most cancer deaths are caused by metastases, resulting from circulating tumor cells (CTC) that detach from the primary cancer and survive in distant organs. Current cancer treatments are mainly based on primary tumour characterization instead of on characterization of metastases or cancer cells in the blood circulation. Since distant organ metastases are the end-results of circulating tumor cells (CTC), molecular characterization of CTC is promising in the quest for effective therapeutic agents and novel prognostic markers [2]. Identification and isolation of CTC using predefined marker expression has failed to identify certain cancer cell phenotypes, and their genotypes. Identify and enable reliable and reproducible molecular characterization of CTC without a priori selection could provide new insights into cancer research and improve management of the disease

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