Development of Circulating Tumour Cell Molecular Diagnostics Using a Novel Microfluidic Device

For Aim 1, two 5ml pre-treatment blood and corresponding tumor samples will be obtained from NSCLC patients at the National University Health System. Sampling will be organized to avoid the blood samples being the first sample taken after skin puncture to minimize contamination with skin epithelial cells. For the first blood sample, CTCs will be isolated and retrieved using the NUS developed CTC bio-chip according to methods described previously.21 From second blood sample, CTCs will be isolated, fixed on the chip and stained for EpCAM, CD45 and DAPI to assess for cell purity and quantity. DNA will be extracted from the retrieved CTCs and tumour samples, and analyzed exon 19 deletion, L858R and T790M mutations by digital PCR on the Fluidigm Biomark according to methods described previously.23 EGFR mutation status in blood and tumour samples will then be compared for their concordance. For Aim 2, patients with NSCLC being treated with gefitnib will be approached. Two 5ml blood samples will be obtained pre-treatment (baseline) and then every 4 weeks of treatment (one cycle of gefitinib) from NSCLC patients at the National University Health System. CTCs will also be obtained and analysed from patients on another protocol receiving gefitinib/ hydroxychloroquine. (B/08/196. A phase II with a lead in phase I study to examine the tolerability, safety profile and efficacy of Hydroxychloroquine and Gefitinib in advanced Non-Small Cell Lung Cancer.) In this study, CTCs are already being collected. Hence the investigators intend to use samples from B/08/196 for CTC analysis using the CTC biochip platform. For each timepoint, CTCs will be isolated, retrieved and analyzed for EGFR mutation status as described above. Clinical response will be determined using the RECIST criteria.34 Associations between pre-treatment EGFR mutation type quantities and best clinical response will be assessed by Fisher's exact test. Association between trends in EGFR mutation type quantities and tumour size over all treatment cycles will be assessed by observation as performed in the study by Maheswaran et al.9 The investigators plan to analyze 30 patients over one year for both aims of this study. Some cases will have relevant samples (serial blood samples and corresponding tumour) for both aims. The sample size is based on that of the study by Maheshwaran et al.9 and also the likely volume of suitable subjects during 1 year at NUHS. Relevant data for sample size estimation is otherwise lacking. In particular, the frequency of T790M mutations in a relevant patient population is lacking, highlighting the lack of adequate analytical systems for its assessment such as the one proposed in this study.