Multiplex PCR-Based Detection of Circulating Tumor Cells in Lung Cancer Patients Using CK19, PTHrP, and LUNX Specific Primers

Micrometastatic disease is the primary cause of cancer recurrence and is mostly missed using current imaging approaches. The combined detection of cytokeratin 19 (CK19), parathyroid hormone-related protein (PTHrP), and lung-specific X protein (LUNX) messenger RNAs (mRNAs) on 125 peripheral blood samples from cancer patients revealed strong correlation with disease stage and presence of distant metastases. This method serves as a complementary tool for patient monitoring and detection of systemic disease. Introduction: The aim of this study was to develop a multiplex polymerase chain reaction (PCR)-based method for detection of circulating tumor cells in peripheral blood of lung cancer (LC) patients. Patients and Methods: Peripheral blood was collected from 71 healthy donors and 125 LC patients at different pathological stages. Samples were analyzed using multiplex PCR, and specific primers for CK19, PTHrP, and LUNX mRNA. The sensitivity of our method was set at 10 LC cells (A549 cells) in 3 mL of peripheral blood of healthy donors using spiking experiments. Results: The detection rates in LC patients for CK19, PTHrP, and LUNX were 45.6%, 64.8%, and 28%, and in healthy individuals were 7%, 7%, and 5.6%, respectively. Overall, our method produced 77.8% positive detections for at least 1 molecular marker. Twenty-eight (22.2%) were negative for expression of all markers, 39 (31.2%) were positive for expression of 1 marker, 42 (33.6%) were positive for expression of 2 markers, and 17 (13.6%) were positive for expression of all 3 markers. Detection of CK19 mRNA expression positively correlated with LC stage and distant metastases. PTHrP mRNA detection correlated positively with LC stage, presence of bone metastasis, and squamous cell carcinoma, and LUNX mRNA detection correlated with lymph node involvement. Combined detection of 2 or 3 markers was significantly correlated with metastatic disease, and negative detection of all 3 molecular markers was correlated with early stage nonmetastatic disease. Conclusion: Multiple PCR-based detection of CK19, PTHrP, and LUNX mRNA expression provides useful information for disease stage and dissemination in LC patients.