Abstract 5249: Intratumor heterogeneity and the detection of potential driver mutations in cfDNA in a NSCLC cohort using UltraSEEKTM
Authors: Jamal-Hanjani Mariam , De Bruin Elza , Wilson Gareth , McGranahan Nicholas , Rowan Andrew , Mosko Michael , Metzler Heath , Nygren Anders , Swanton Charles 
Published: August 2015

Abstract
Introduction
Increasing evidence supports the heterogeneous clonal composition and branched evolution of non-small cell lung cancer (NSCLC) tumors. Circulating biomarkers, such as circulating cell-free tumor DNA (cfDNA), may potentially be used to track the evolution of such tumors in order to detect minimal residual disease, monitor treatment response, detect the emergence of resistance, and subsequent disease relapse.
Experimental procedures
In a cohort of 14 primary NSCLC tumors, DNA was extracted from fresh frozen tissue and multi-region whole-exome sequencing (mean depth of 219x) was performed on a total of 44 tumor regions. Single nucleotide variants, indels and copy number data were identified using VarScan2 (v2.3.6). Non-silent mutations were validated using ultra-deep amplicon sequencing (mean depth 1220x). The subclonal composition of each tumor was used to construct phylogenetic trees. Candidate driver genes were selected by referencing large-scale lung cancer or pan-cancer sequencing studies, and the COSMIC gene census. To validate the identified driver mutations and to further screen all tumor regions for additional low-level heterogeneity, we developed patient-specific UltraSEEK panels. The UltraSEEK technology utilizes mutation-specific single base extension and MassARRAY® MALDI-TOF Mass Spectrometry to identify low frequency mutated alleles down to 0.1%. This method was used to determine if potential driver mutations could be detected in cfDNA extracted at the time of diagnosis in a subset of patients.
Summary of data
Intratumor heterogeneity was evident in all tumors, with a median of 25% heterogeneous mutations (range 5-61%). Phylogenetic tree analysis revealed branched tumor evolution, with mutations in potential driver genes occurring both early (trunk) and late (branch). The UltraSEEK method identified truncal and branch mutations in tumor DNA, as well as truncal mutations in the EGFR, TP53, BRAF, RB1 and MLL2 genes in two out of six patients. These mutations are predicted to have deleterious functional consequences and may therefore play a role in disease pathogenesis.
Conclusions
Spatial and temporal heterogeneity of driver mutations was evident in all NSCLC tumors. The use of liquid biopsies, such as cfDNA, has the potential to aid early detection and guide treatment initiation in cancer patients. They may also avoid the need for repeated tissue sampling, and be of value in cases where disease sites, such as brain or bone, are not easily accessible to biopsy.
Citation Format: Mariam Jamal-Hanjani, Elza De Bruin, Gareth Wilson, Nicholas McGranahan, Andrew Rowan, Michael Mosko, Heath Metzler, Anders Nygren, Charles Swanton. Intratumor heterogeneity and the detection of potential driver mutations in cfDNA in a NSCLC cohort using UltraSEEKTM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2015-5249