Selvaraju Veeriah

Tracking the Evolution of Non–Small-Cell Lung Cancer

BACKGROUND Among patients with non‐small‐cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early‐stage NSCLC. METHODS In this prospective cohort study, we performed multiregion whole‐exome sequencing on 100 early‐stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence‐free survival. RESULTS We observed widespread intratumor heterogeneity for both somatic copy‐number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy‐number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy‐number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10‐4), which remained significant in multivariate analysis. CONCLUSIONS Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601.)

Tracking genomic cancer evolution for precision medicine: the lung TRACERx study.

TRACERx, a prospective study of patients with primary non-small cell lung cancer, aims to map the genomic landscape of lung cancer by tracking clonal heterogeneity and tumour evolution from diagnosis to relapse.

Expansion of airway basal epithelial cells from primary human non-small cell lung cancer tumors: 3T3+Y for NSCLC cell culture

Pre‐clinical non‐small cell lung cancer (NSCLC) models are poorly representative of the considerable inter‐ and intra‐tumor heterogeneity of the disease in patients. Primary cell‐based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3‐J2 feeder cell culture in the presence of Y‐27632, a RHO‐associated protein kinase (ROCK) inhibitor, in what are known as “conditional reprograming conditions” (CRC) or 3T3u2009+u2009Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.

Abstract LB-A03: Allele specific HLA loss and immune escape in lung cancer evolution

Cancer cells adopt a variety of mechanisms to evade the immune system and avoid T-cell recognition. Disruption of human leukocyte antigen (HLA), which may lead to reduced neoantigen presentation, has been proposed as an immune escape strategy in many cancers, including lung cancer. Mutations in HLA class I genes are infrequent in early stage non-small cell lung cancers (NSCLC), suggesting alternative mechanisms of HLA disruption may be common. To date, the polymorphic nature of the locus has precluded copy number analysis and exploration of HLA loss. To investigate the prevalence and importance of HLA disruption, we present LOHHLA (Loss Of Heterozygosity in Human Leukocyte Antigen), a computational tool to determine HLA allele-specific copy number from sequencing data. Building upon previous work imputing HLA haplotypes from sequencing data (Shukla, 2015; Szolek, 2014) and utilizing previously published datasets (Jamal-Hanjani, 2017; Brastianos, 2015), we endeavored to address the prevalence and timing of HLA LOH in lung cancer and its impact on tumor evolution and neoantigen presentation. Using LOHHLA, we find HLA LOH occurs in 40% of NSCLCs and is associated with a high subclonal neoantigen burden, APOBEC mediated-mutagenesis, upregulation of cytolytic activity and PD-L1 positivity. The focal nature of these alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggest that HLA LOH is an immune escape mechanism, selected later in NSCLC tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for immunotherapeutic approaches targeting neoantigens. Citation Format: Nicholas McGranahan, Rachel Rosenthal, Crispin T. Hiley, Andrew J. Rowan, Thomas B.K. Watkins, Gareth A. Wilson, Nicolai J. Birkbak, Selvaraju Veeriah, Peter Van Loo, Javier Herrero, Charles Swanton. Allele specific HLA loss and immune escape in lung cancer evolution [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A03.