Abstract C204: Characterization of TP53 mutations by DNA and RNA sequencing of platin-resistant ovarian cancer FFPE biopsies
Authors: Haynes Brian C. , Ilsley Diane , Fahey Marie , Latham Gary J. , Somers Elizabeth B. , O'Shannessy Daniel J. 
Published: May 2016

Introduction: TP53 is the most frequently mutated gene across all cancers at approximately 50% and in cancers such as high grade serous ovarian up to 96%. In addition to the high incidence of DNA mutations at the gene locus, the TP53 pathway is also dysregulated through alternative mechanisms which involve other members of the regulatory cascade such as overexpression of Mdm2 or Mdmx.

In contrast to other tumor-suppressors, TP53 possesses a spectrum of mutations including both loss and gain of function. Some mutations such as E177R result in a partial loss of TP53 function, eliminating the apoptotic fate while retaining senescence and cell-cycle arrest. Despite the centrality of TP53 in cancer, its prognostic significance remains unclear and the development of targeted therapies is ongoing. Characterizing TP53 mutation status in conjunction with transcriptional profiling of tumor biopsies will enable a more comprehensive understanding of the functional consequences of different classes of TP53 mutations and aid in distinguishing partial from total abrogation of TP53 pathway function.

Asuragen has developed sensitive assays and algorithms to characterize DNA mutations and RNA expression profiles from the most challenging poor quality clinical samples. In order to enable a more complete understanding of the functional consequences of TP53 mutations (or lack thereof), we present an application of these approaches to characterize TP53 function in FFPE ovarian cancer biopsies through an integrated analysis of DNA and RNA sequencing.

Methods: Primary FFPE tumor biopsies were collected from a cohort of 200 paclitaxel/carboplatin resistant ovarian cancer patients. DNA from isolated tumor biopsies and matched normal specimens was profiled by the Quantidex™ NGS TP53 Assay (Asuragen, Inc.) and library analysis was performed using the Quantidex™ Reporter. TP53 mutation status was independently assessed by the AmpliSeq™ Cancer Hotspot Panel (Thermo Fisher Scientific). Whole transcriptome RNA-Seq was performed on isolated total RNA material. Gene and isoform expression quantitation as well as SNV and indel calling was performed on the RNA-Seq libraries.

Results: TP53 mutation status for Quantidex TP53 and AmpliSeq assays was found to be concordant for regions of TP53 covered by both assays. The Quantidex TP53 Assay has more complete coverage of COSMIC mutations, 99% compared to 68% for AmpliSeq. Many TP53 positives were detected by the Quantidex Assay but negative by AmpliSeq due to coverage differences. The remaining discordant calls in commonly covered regions were assessed with matched RNA-Seq data. The comparison with RNA-Seq, led to the observation of specific cases of allele specific preferential expression of TP53 gain of function mutations. RNA-Seq expression profiles were also evaluated leading to the identification of gene signatures that associate with different TP53 mutation classes.

Conclusions: TP53 profiling through orthogonal DNA-Seq assays and matched RNA-Seq has enabled the identification of a set of high confidence associations between TP53 mutations and RNA expression signatures in ovarian cancer. These results point to a path for clinical assays in which DNA mutations and pathway signatures are integrated in support of diagnostic and precision medicine applications.

Citation Format: Brian C. Haynes, Diane Ilsley, Marie Fahey, Gary J. Latham, Elizabeth B. Somers, Daniel J. O'Shannessy. Characterization of TP53 mutations by DNA and RNA sequencing of platin-resistant ovarian cancer FFPE biopsies. [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 C204.