A Comprehensive Evaluation of Circulating Tumor DNA and Circulating Tumor Cells as a Predictive Marker in Lung Cancer

Lung cancer is not only amongst the top 3 most common cancer type in both men and women, but also the leading cause of cancer mortality in Singapore and the United States. Platinum-based chemotherapy is currently the mainstay in drug treatment for lung cancer patients although it only relieves disease-related symptoms temporarily. Recently, the use of molecularly targeted therapy has led to improved survival in subsets of patients. In particular, the presence of EGFR or ALK mutations from lung biopsies predicts improved response rate and progression-free survival from the use of tyrosine kinase inhibitors or crizotinib respectively. Importantly, EGFR mutations are more common in tumors of Asian patients (about 35%) versus non-Asians (about 10%).

The main cause of mortality in patients with solid cancers is metastases from dissemination via blood circulation. The ability to detect the presence of tumor cells in circulation even before overt metastases occur has evolved rapidly in recent years. The prognostic importance of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in several cancers has been found and this technique is widely recognized to be of major clinical and biologic importance. This study evaluates the use of ctDNA and CTCs in predicting disease activity and drug response in lung cancer patients and serves to complement existing methods to achieve a non-invasive and accurate means to guide treatment decisions.

Indeed, the use of ctDNA and CTCs as a marker for risk assessment and treatment monitoring is advantageous as it is accessible may be monitored on a regular basis on a non-invasive basis. This will better tailor our surveillance programmes and treatment to optimize disease outcomes.

The Investigators have developed an EGFR mutation detection kit that detects the 3 main EGFR mutations at ultrasensitive levels. The Investigators have applied this to a variety of clinical materials including formalin-fixed paraffin embedded DNA, pleural effusions, circulating DNA as well as circulating tumor cells and found that the kits can pick up these mutations with high sensitivity and specificity.

A microsieve membrane filter device to effectively isolate circulating tumor cells from whole blood sample has been developed by the Institute of Bioengineering and Nanotechnology. The device exploits the size difference between cancer cells and blood cells to achieve the cancer cell capture on a microsieve filter with > 85% recovery rate from 4.5-ml whole blood within 10 min, demonstrated with cancer cells spiked into whole blood samples. The device contains a densely packed pore array (> 8000 pores/mm^2) with an optimized pore diameter of 10 µm. Fluid regulation and cell counting are achieved by a simple vacuum pumping and laboratory fluorescence microscope. The captured cells are stained with anti-EpCAM-PE for CTCs, anti-CD45-FITC for white blood cells, and 4',6-diamidino-2-phenylindole (DAPI) for cell nuclei. The CTCs are identified with anti-EpCAM positive, anti-CD45 negative and DAPI positive, characterized using a fluorescence microscope.