Abstract POSTER-CTRL-1203: Circulating TP53 mutations as a biomarker of high-grade serous cancer
Authors: Christie Elizabeth , Hunter Tane , Doyle Maria ,   Australian Ovarian Cancer Study, Bowtell David 
Published: August 2015

Abstract
Women who carry a germline BRCA1 or BRCA2 mutation are at high risk of developing ovarian cancer in their lifetime. While risk can be reduced by prophylactic bilateral salpingo-oopherectomy (RRSBO), for those high-risk women who delay, or choose not to undergo RRSBO, current surveillance options have not shown sufficient specificity or sensitivity to prevent an advanced stage diagnosis (Buys et al (2011) JAMA). As such there is a need to identify novel, sensitive biomarkers to employ in this at-risk population. We recently found that BRCA1/2 mutation carriers predominantly develop high-grade serous cancers (HGSC) (Alsop et al (2012) J Clin Oncol), therefore, identifying a biomarker of HGSC will be useful for screening BRCA1/2 carriers. Mutation in TP53 represents the ideal biomarker of HGSC; mutation occurs early in tumour development (Crum et al (2007) Clin Med Res), it is present in almost 100% of tumours (Ahmed, et al (2010) J Pathol, TCGA (2011) Nature), and pathogenic mutations can be readily distinguished from passengers (Olivier et al (2010) Cold Spring Harb Perspect Biol). Tumour specific TP53 mutations have been identified in the circulating DNA of late stage ovarian cancer patients by Sanger sequencing (Swisher et al (2005) Am J Obstet Gynecol). Next generation sequencing (NGS) provides increased sensitivity for identification of rare mutant alleles (Flaherty et al (2011) Nucl Acids Res) and has been used to monitor disease burden in advanced stage cases (Murtaza et al (2013) Nature).
Supported by a Marsha Rivkin Challenge Grant, we explored the feasibility of detecting free circulating mutant TP53 DNA in plasma from HGSC patients, using multiplex PCR to amplify TP53 DNA from the circulating DNA followed by ultra-deep NGS. The main challenge has been in developing bioinformatics filtering processes to increase specificity and sensitivity. We utilised multiple variant callers to identify low frequency, high quality, pathogenic variants that are then compared to the IARC TP53 database and COSMIC. Variants called in our sequence data that are absent from these databases are flagged as lower confidence calls. Consistent sequencing artefacts were removed from consideration. High confidence variants were tested for their presence in multiple amplicons, to generate a final list of variants ranked in terms of confidence based on multiple lines of bioinformatics evidence. Using this methodology, we have been able to detect tumour specific TP53 mutations in circulating DNA of 40% of predominately late stage cases. In those cases where the mutation was not called in the circulating DNA, the mutant allele frequency was less than 0.1% and could not be distinguished from the background errors associated with NGS. The extremely low frequency of mutations in the plasma of women with late stage HGSC, against a background of normal TP53 sequences, currently hampers immediate adoption of circulating TP53 mutations as an early detection biomarker. Our work provides proof of principle for the approach, however, we need to develop additional strategies to push detection into a range that is useful for screening at risk women.
Citation Format: Elizabeth Christie, Tane Hunter, Maria Doyle, Australian Ovarian Cancer Study, David Bowtell. Circulating TP53 mutations as a biomarker of high-grade serous cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1203.