Abstract 3976: “Fit-for-purpose” PIK3CA assays for patient stratification in breast cancer clinical trials
Authors: Gong Hua C. , Manjeshwar Sharmila , Abraham Violet , Rivera Shawn 
Published: August 2019

Abstract
Background: Prospective clinical trials stratifying by PIK3CA genotype is necessary to determine if the mutation predicts for increased sensitivity to agents targeting the phosphoinositide 3-kinase (PI3K) pathway. It is crucial to select appropriate assays to optimize operational, scientific and timeline requirements for patient enrollment in a real world setting. Here we report application of three different PIK3CA mutation test methods during the course of a PI3K TKI development.
Methods: We customized Sanger sequencing, cobas® assay and a proprietary investigational use only (IUO) assay to detect point mutations in PIK3CA in order to accommodate the development needs at the different stages of clinical development of PIK3CA TKIs. We performed method comparison studies to determine the most cost effective and “fit-for-purpose” assay for clinical trial testing. Specimen requirements, assay performance characteristics, turnaround-time (TAT) and valid result rates were assessed to select the best test method for patient stratification to meet the requirements of each stage of drug development.
Results: PIK3CA test results can be obtained from a single, 5μm FFPE section with ≥0.1% and ≥10% tumor in the IUO and cobas® tests respectively. Sanger Sequencing requires longer processing time and ≥30% tumor content. The cobas® assays were validated in FFPE samples and can reliably detect PIK3CA mutations in exons 9 and 20 with ≥5% mutation level. The method comparison study showed that the concordance rate between Sanger sequencing and cobas® PIK3CA assay is 86% among the 360 samples tested while it is 96% between the cobas® assay and the IUO assay among 395 samples tested. The IUO assay is slightly more sensitive, and calls out the exact mutation while the cobas® assay may not. The Sanger sequencing demonstrated the lowest sensitivity and required more DNA input, which resulted in more samples failing to yield a valid result. The cobas® assay is a more user-friendly assay allowing better laboratory efficiency and TAT. Patients selected using the IUO assay showed a median progression-free survival nearly twice as long among those receiving a PI3K TKI compared to placebo.
Conclusions: The most important factor in selecting a testing platform for clinical trial patient stratification is the assay performance characteristics. We employed the Sanger sequencing assay in the initial trial testing, then switched to the cobas® assay, and finally to the IUO assay to meet the requirements as the drug progressed through different stages of clinical development. The IUO assay has the performance characteristics that meet the regulatory requirements and a validated clinical utility in selecting patients for PI3K TKI treatment, thus on its way to a companion diagnostic submission.
Citation Format: Hua C. Gong, Sharmila Manjeshwar, Violet Abraham, Shawn Rivera. “Fit-for-purpose” PIK3CA assays for patient stratification in breast cancer clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3976.