Molecular characterization of circulating tumor cells from pancreatic cancer patients by single-cell quantitative reverse transcription PCR

Abstract

Pancreatic cancer is a highly lethal disease, which kills approximately 70 000 people per year in Europe alone. The median survival after the time of diagnosis is five to six months for patients with advanced cancer, making pancreatic cancer one of the deadliest of all solid cancers. The disease has few early symptoms, therefore, most patients are diagnosed at advanced stages. The poor prognosis is related to late detection and early dissemination of the disease. The median age of diagnosis is 73 years for pancreatic cancer. The treatment options for pancreatic cancer are rather limited, and there is a great need for more effective treatment and new biomarkers for monitoring of the disease. Circulating tumor cells (CTCs) are promising new biomarkers suggested to be of clinical relevance also in pancreatic cancer. CTCs are cells in the blood stream that are shred from the primary tumor or metastasis. These cells could offer new insight into treatment effects, monitoring of disease progression and tumor cells genotypes. Some of the CTCs contribute to the formation of distant metastasis. CTC examination is considered a minimal invasive liquid biopsy of the cancer, because the CTCs seem to represent the population of cancer cells present in solid tumors. Enrichment and detection of CTCs have been investigated for several epithelial cancers with great success. However, just a few studies have been published for pancreatic cancer. The aim of this study was to perform molecular characterization of single CTCs. The CTCs were isolated from blood samples from pancreatic cancer patients and analyzed by quantitative reverse-transcription PCR (RT-qPCR). The pancreatic cancer patients that were recruited for this thesis were part of an ongoing trial at Stavanger University Hospital and Haukeland University Hospital, called PACT-ACT (PAncreatic Cancer Treatment And Circulating Tumor Cells). Tumor cells were enriched from the whole blood by density gradient separation, followed by immunomagnetic enrichment. The single CTCs were identified using immunofluorescent staining of EpCAM and CD45. Isolation of single cells was performed using micromanipulation. The single cells were lysed, reverse-transcribed and pre-amplified, followed by qPCR for 11 specific mRNAs. The method for enrichment and characterization of CTCs in this study was optimized and validated through several experiments. The tumor cells were successfully separated from the leukocytes by the molecular characterization. Blood samples from patients were analyzed and we were able to enrich, detect, isolate and perform molecular characterization of single CTCs in peripheral blood samples from patients with pancreatic cancer. However, further optimization of the method should be implemented to enhance the RNA quality of the single cells. The overall impression of the research on the field is promising, and CTC detection is expected to have future clinical utility for pancreatic cancer patients, as well as for other solid cancers.