Combined multimodal ctDNA analysis and radiological imaging for tumor surveillance in Non-small cell lung cancer

Background: Radiology is the current standard for monitoringtreatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Noninvasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility. Methods: We analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. Results: A 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observedbetween baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT)-monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay ((meth)cfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in (meth)cfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status. Conclusion: Taken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring

Automated summary

In this study, we analyzed plasma and tumor samples from lung cancer patients using next-generation sequencing, radiological imaging, and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. The results showed a 62% concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing in baseline liquid and tissue biopsies from stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial ctDNA mutation analysis revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, ctDNA methylation assay showed a high area under the curve (AUC) in early and late stage cases. The decrease in ctDNA and (meth)cfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status.