The Impact of Clonal Composition on the Efficacy of Radiation Therapy in Lung Adenocarcinoma

Abstract

Lung adenocarcinomas (LUAD), although classified unitarily, are composed of cancers with substantial intertumoral genetic diversity. This variation in LUAD has led to the development of genetic markers that have successfully guided drug therapies. Despite impressive initial clinical responses, there is variability in the extent of response even within homogeneous baskets and resistance to targeted and genotoxic therapies ultimately ensues. The extent of these failures is attributed to the topographic differences in genomes within tumors, or intratumoral heterogeneity, remains poorly studied which represents a major obstacle to tumor eradication. Tumors can have genetically distinct subclones that compete for space and resources and differentially resist therapy. Herein, we seek to elucidate subclone architecture in the most common type of lung cancer for the purpose of guiding radiotherapeutic strategies. We identified 405 candidate BRAF variants by analyzing targeted and genome-wide screen data from a collection of 48,397 tumors representing 35 cancers deposited in COSMIC. We selected 28 BRAF variants by random sampling and 7 variants considering local mutational density, evolutionary conservation and prior knowledge. We used site-directed mutagenesis to generate mutant clones and transferred alleles into lentiviral vectors. We generated 74 expression constructs adding wild-type and vector controls and representing biological replicates then stably-expressed each variant in bronchial epithelial cells (BEAS-2B). Total mRNA gene expression was assayed using RNAseq. Gene level copy number and mutational data were combined with estimates of the sample purity to infer the cancer cell fraction, or the proportion of cancer cells with the single nucleotide variant (CCFSNV) as follows: CCFSNV = (VAF*(2+(ploidyCNV-2)*CCFCNV))/purity. We identified BRAF driver mutations as predominately clonal in some cancers (melanoma) and subclonal in others (LUAD). Clonality was associated with the prevalence of V600 mutations and its selective amplification, a frequent occurrence in melanoma. We modelled the propagation and selection of tumors containing distinct categories of BRAF mutations to estimate their evolutionary trajectories. Hyperactivating BRAF mutant cells rapidly swept to clonality, resulting in a significant reduction of genetic diversity in the affected tumors. Mutations with differential activation of the Braf signaling pathway conferred a “softer” clonal sweep or remained subclonal maintaining tumor genetic diversity. Subclones containing BRAF variants had significant therapeutic implications for both targeted inhibitors of BRAF and/or MEK and genotoxic stress, conferring attenuated responses to both therapies. Our study uncovers patterns of distinct BRAF clonal evolutionary dynamics and nominates new radiotherapeutic strategies based on both the type of mutation and its subclone composition.