Circulating and disseminated tumour cells as potential biomarkers for treatment response and disease progression in patients with locally advanced and/or metastatic pancreatic cancer

Abstract

Introduction: Pancreatic cancer is the fourth most common cause of cancer related deaths in the Western countries. The poor prognosis of pancreatic cancer patients is often associated with early dissemination of the disease, late detection due to unspecific symptoms and chemotherapy resistance. There are two ways for tumour cells to enter the blood circulation, either by passive shedding of tumour cells from the primary tumour or by an active process called epithelial-to-mesenchymal transition (EMT). Circulating tumour cells (CTCs) that are resident in the bone marrow (BM) are called disseminated tumour cells (DTCs).

Around 85% of pancreatic cancers harbour point mutations in the KRAS gene, and these mutations represent highly tumour-specific traits that might be applied as surrogate markers for tumour cell detection. Hence, we wanted to use both KRAS mutations and four mRNAs as surrogate markers for CTC/DTC detection in blood (PB) and BM from patients with locally advanced and/or metastatic pancreatic cancer before and during chemotherapy.

Patients and methods: Six metastatic pancreatic cancer patients were included in the study and samples from nine healthy individuals constituted the control group. In the first part of the study we compared different strategies for enrichment of CTCs/DTCs, a manually prepared and a commercially RBC lysis buffer versus the LymphoprepTM protocol. A sensitivity analysis was performed to determine the detection limit of each mRNA marker with regard to the lowest amount of tumour cells detectable with RT-qPCR. The four mRNAs were also evaluated in six pancreatic tumour samples. Following tumour cell enrichment by Lymphoprep, CTCs and DTCs were detected indirectly using the epithelial-specific surrogate mRNAs CK8, CK19, EpCAM and CEACAM5, as well as KRAS mutations by real-time PCR.

Results: All the mRNA markers were highly expressed in the six pancreatic tumour samples compared to PB and BM samples from nine healthy individuals emphasizing their use as surrogate markers for CTC/DTC detection. Furthermore, our preliminary data show that we detect CTCs and DTCs in PB and BM samples obtained before treatment in 5/6 and 5/5 patients, respectively. Repeated blood sampling from three patients and BM samples from one patient, also confirmed the presence of CTCs and DTCs after initiation of the treatment. KRAS mutations were detected in CTCs and DTCs from 1/6 patients. Conclusion: We detect CTCs and DTCs in PB and BM samples obtained both before and during gemcitabine treatment of metastatic pancreatic cancer patients with mRNA quantification and KRAS mutation detection by real-time PCR. However, inclusion of more patients is required to conclude on the clinical value of these data.