Abstract LB-A05: Profiling cell free DNA in breast cancer and non-small cell lung cancer using broad NGS assessment
Authors: Solovieff Nadia , Hims Matt , Leary Rebecca , Chiang Derek , Germa Caroline , Massacesi Cristian , Hirawat Samit , Scherer Stefan J. , Morrissey Michael , Winckler Wendy , di Tomaso Emmanuelle 
Published: May 2016

Abstract
Introduction: Cell free DNA (cfDNA) has become a promising approach for non-invasive assessment of the tumor genome. Many cfDNA assays target hotspot alterations in a focused set of genes, but do not provide a broad characterization of the cancer. We have developed and optimized a large next generation sequencing (NGS) panel covering the coding regions of over 500 genes. Using this panel, we sequenced cell free DNA from plasma and matched tumor DNA in patients with early stage breast cancer, late stage breast cancer and late stage lung cancer.
Methods: Plasma was collected from patients with cancer using a double spin protocol and, when available, matched archival tumor tissue (representing different time interval with blood collection) was obtained. Next generation sequencing libraries were generated from cell free DNA isolated from 70 plasma samples and genomic DNA from 58 matched tumor samples. The NGS libraries were enriched for the gene panel of interest and were sequenced to a targeted depth of 1,000X for plasma and 300X for matched tumors. We optimized parameters of standard bioinformatics tools to robustly call low allelic fraction events, detecting single nucleotide variants down to 1%, as well as indels and copy number alterations.
Results: We identified 8 PIK3CA hotspot alterations in plasma from late stage breast and lung cancers, in addition to many alterations across driver genes such as AKT1, EGFR, IDH2, NRAS, PTEN and TP53. In plasma samples from patients with late stage breast cancer, we found 4 ESR1 mutations exclusive to the plasma samples, of which 3 are known resistance mutations to endocrine therapy. Copy number alterations in EGFR, CCND1 and KRAS were also identified in patient plasma. When comparing the number of alterations across tumor stages, we found that late stage breast (mean = 12.5 variants) and lung cancers (mean = 12.5 variants) had a larger number of alterations present in plasma than early stage breast cancers (mean = 4.5 variants). We compared somatic mutations calls in plasma and matched tumor samples and found a concordance of 53%-67% at the variant level across patients with late stage cancers (N = 37 pairs). Higher variant level concordance was observed among plasma-tumor pairs collected less than a year apart (N = 11 pairs; 76%-84%) versus more than 5 years apart (N = 8 pairs; 41%-50%).
Conclusion: We have developed and optimized a 500+ gene panel for direct sequencing of cfDNA, and we demonstrate that this broad assessment of circulating tumor DNA can be used for non-invasive characterization of the cancer genome landscape. The number of alterations identified in patient plasma is consistent with higher levels of ctDNA being present in late stage disease than in early stage disease. The time dependent degree of concordance between plasma and tumor collection suggests that cell free DNA assays may provide a more accurate characterization of the current tumor mutational landscape than an archival tumor sample. The identification of plasma specific ESR1 alterations highlights the importance of cfDNA in the context of identifying mechanisms of resistance, particularly for metastatic disease when tumor tissue collection may not be feasible. In addition, a broad NGS panel provides the opportunity to identify lesions unevaluated by targeted assays and to discover resistance mutations.
Citation Format: Nadia Solovieff, Matt Hims, Rebecca Leary, Derek Chiang, Caroline Germa, Cristian Massacesi, Samit Hirawat, Stefan J. Scherer, Michael Morrissey, Wendy Winckler, Emmanuelle di Tomaso. Profiling cell free DNA in breast cancer and non-small cell lung cancer using broad NGS assessment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A05.