Abstract P3-06-42: Detection of PIK3CA mutations in cell-free plasma DNA from women with early breast cancer
Authors: Hoskins Kent F , Sidani Amer , Jones Tiffany , Green Stefan , Emmadi Rajyasree , Chenn Anjen 
Published: May 2015

Abstract
Background
Activating mutations in the PIK3CA gene represent the most common molecular aberration in luminal breast cancers. Three hot-spot mutations (E542K, E545K, H1047R) account for approximately 70% of the PIK3CA mutations identified. The presence of a mutation is likely to become an important predictive biomarker to select patients for treatment with PIK3CA inhibitors. Tumor-derived DNA can be detected in the systemic circulation in the form of cell-free plasma DNA (cfpDNA), and the ability to interrogate tumor-derived DNA from an easily acquired biospecimen like cfpDNA has practical advantages over analysis of tissue specimens. Furthermore, cfpDNA can be monitored serially and could provide a dynamic measure of tumor progression and response to therapy. Droplet digital PCR (ddPCR) is a high-throughput technique that provides highly precise and sensitive detection of rare alleles in samples with ultra-low abundance of target DNA.
Methods
A nested case-control study was conducted with plasma samples collected as part of a study to identify blood-based biomarkers. Blood was collected from women scheduled for a breast biopsy at one of seven participating mammography units. All blood samples were collected prior to the biopsy procedure and were processed using a standard protocol. Cases are women diagnosed with invasive ductal carcinoma, stage I-III, that is positive for the estrogen receptor (ER+) and negative for HER2/neu (0 or 1+ by IHC). Controls are women with a benign, non-proliferative lesion, matched by collection site, year of collection and age. cfpDNA was isolated using a commercial kit (Macherey-Nagel, Bethlehem, PA). DNA was extracted from FFPE biopsy specimens by standard techniques. Central pathologic review confirmed at least 10% tumor cellularity in biopsy specimens from cases. A ddPCR assay was developed on a BioRad QX100 Droplet Digital PCR instrument using standard TaqManR assays with probe pairs designed to detect the 3 PIK3CA hotspot mutations and corresponding wild type (wt) alleles. The assay was validated using DNA from four cell lines, each positive for one of the hotspot mutations or wt at all 3 loci (Horizon Diagnostics, Cambridge, UK). ddPCR was performed on cfpDNA and DNA extracted from FFPE biopsy specimens in cases and controls. Next generation DNA sequencing (NGS) of exons 9 and 20 of PIK3CA was performed on biopsy specimens from cases using an Ion Torrent Personal Genome Sequencer.
Results
Validation of the ddPCR assay with serial dilutions of cell line DNA demonstrated the assay is highly sensitive with mutant alleles detected at an allele frequency down to 25% when input DNA template is as low as 2 ng. With DNA template input of 20 ng (typical recovery of cfpDNA), mutations can be detected down to an allele frequency of 0.78%. We will present data on the rate of hotspot mutation detection with the ddPCR assay performed on cfpDNA and FFPE samples in cases (n=80) and controls (n=80), and we will report performance characteristics of the assay. Mutation status of tumors determined by NGS will serve as the gold standard.
Conclusion
We have developed a ddPCR assay to detect PIK3CA hotspot mutations in cfpDNA, and will report the performance characteristics of the assay in 80 cases of untreated, ER+ early breast cancer.
Citation Format: Kent F Hoskins, Amer Sidani, Tiffany Jones, Stefan Green, Rajyasree Emmadi, Anjen Chenn. Detection of PIK3CA mutations in cell-free plasma DNA from women with early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-42.