Molecular analysis of circulating cell-free DNA in lung cancer
thesisposted on 06.03.2017, 11:24 by Ricky Mark Trigg
Two-thirds of patients with non-small cell lung cancer (NSCLC) are diagnosed with incurable disease, reflecting the need for early detection. However, research in this area has been hampered by the inability to identify individuals with early-stage tumours or preneoplastic lesions that may progress to malignancy. In this thesis, the Kras+/LSL-G12D mouse was used to model lung preneoplasia, permitting analysis of circulating-free DNA (cfDNA) in comparison with tumour burden. Using end-point polymerase chain reaction (PCR), tumour-derived cfDNA (ctDNA) was detected in 3/7 Kras+/LSL-G12D mice with lung preneoplasia but no evidence of malignant transformation. Moreover, total levels of cfDNA were elevated in Kras+/LSL-G12D mice (mean, 66.6 ng/mL) relative to Kras+/+ mice (mean, 2.5 ng/mL) (P = 0.006). To permit a quantitative analysis of cfDNA, digital and quantitative real-time PCR strategies were then developed to detect KrasLSL-G12D and KrasLox-G12D. Using these assays, analysis of the sensitivity of ctDNA in Kras+/LSL-G12D mice may inform its diagnostic potential in human NSCLC. In a separate project, somatic copy number alteration (SCNA) in cfDNA was investigated in 143 patients with advanced NSCLC. Analysis of SCNA in five commonly amplified genes (EGFR, FGFR1, HER2, MET and PIK3CA) by qPCR and ddPCR identified gene amplification in 18 patients. Next-generation sequencing identified mutations in 28 patients, of which only 2 patients also had evidence of SCNA. Interestingly, patients with SCNA in cfDNA had significantly poorer overall survival (OS) than patients without (HR, 1.78; 95% CI, 1.05 – 3.02; P = 0.03). This significance was improved further when SCNA and mutation data were combined; patients with one or both types of alteration had significantly poorer progression-free survival (PFS) (HR, 1.50; 95% CI, 1.01 – 2.21; P = 0.02) and OS (HR, 1.88; 95% CI, 1.24 – 2.84; P = 0.003) than patients without. This is the first time SCNA in cfDNA has demonstrated predictive potential in any solid cancer type.