PMCID: PMC4636050.-- et al. ; Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis.We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC. ; E.B. is a Rosetrees Trust fellow; M.J.H. has a Cancer Research UK fellowship; N.Mu. received funding from the Rosetrees Trust; M.G. is funded by the UK Medical Research Council; I.V. is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal; R.C.R. and D.M.R. are partly funded by the Cambridge Biomedical Research Centre and Cancer Research UK Cancer Centre; P.V.L. is a postdoctoral researcher of the Research Foundation—Flanders (FWO); S.M.J. is a Wellcome Senior Fellow in Clinical Science; and C.S. is a senior Cancer Research UK clinical research fellow and is funded by Cancer Research UK, the Rosetrees Trust, European Union Framework Programme 7 (projects PREDICT and RESPONSIFY, ID:259303), the Prostate Cancer Foundation, the European Research Council and the Breast Cancer Research Foundation. This research is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. ; Peer Reviewed