Abstract 2582: A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay
Authors: He Yuting , Wongchenko Matthew , Skoletsky Joel , Burns Christine , Li Yali , Maness Paula , Kim Doris , Lipson Doron , Stephens Philip , Miller Vincent , Ross Jeffrey , Gendreau Steven , Sun James 
Published: August 2018

Abstract
Introduction Patients with PI3K/Akt-pathway activation may be sensitive to selective Akt-inhibitors that are currently under development. We have developed a novel next-generation sequencing (NGS)-based composite biomarker assay that identifies patients with PI3K/Akt-pathway activated tumors by identifying activating PIK3CA and AKT1 alterations, and inactivating alterations in PTEN. This assay was analytically validated, and applied to triple-negative breast cancer (TNBC) patients in the LOTUS trial (NCT02162719), a placebo-controlled phase II clinical trial to assess the safety and efficacy of adding ipatasertib to paclitaxel treatment in patients with metastatic TNBC (Kim et al., 2017).
Methods DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability, and tumor mutational burden. The assay further evaluated the PI3K/Akt-pathway activation biomarker status that consists of six features: 1-2) AKT1 and PIK3CA activating mutations, 3) PTEN homozygous deletion, 4) PTEN heterozygous deletion (HE), 5) PTEN dominant negative mutations, and 6) bi-allelically inactivated (BI) PTEN mutations defined as mutation plus loss of heterozygosity (LOH). We evaluated the limit of detection (LoD) and the precision of the biomarker for two novel genomic features: HE and BI, with the other four features previously validated.
Results Analytical validation of novel biomarker features: The LoD of detecting PTEN HE and BI was determined to be 30%, the lowest tumor content at which the features can be detected at 90% probability. In the precision study, 100% (81/81) agreement was achieved across different replicates within the same sequencer run and across different sequencer runs for biomarker positive samples, demonstrating high reproducibility in calling PTEN HE and BI.
Conclusions We developed and analytically validated an NGS-based assay that identifies complex and novel genomic alterations (heterozygous deletion and bi-allelic inactivation) in PTEN that is part of a composite PI3K/Akt-pathway activation biomarker. This assay identified patients that appeared to derive greater benefit in the Phase II LOTUS study as compared to using PTEN IHC to only identify patients with PTEN protein loss (Kim et al., 2017). This assay could be generalized to identify other biomarkers with similar types of genetic alterations. It also demonstrates that NGS-based CGP can broaden the intent to treat population to be more specifically related to the mechanism of action of a drug, while also being more selective to patients with potential to respond.
Citation Format: Yuting He, Matthew Wongchenko, Joel Skoletsky, Christine Burns, Yali Li, Paula Maness, Doris Kim, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Steven Gendreau, James Sun. A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2582.