Abstract 3662: Cell-free DNA sequencing demonstrates persistence of nucleic acid-based therapy—opportunities for detection and monitoring
Authors: Sikora Marcin , Artyomenko Alexander , Strauss James , Lanman Richard B. , Raymond Victoria M. , Odegaard Justin 
Published: August 2018

Introduction: Cell-free circulating tumor DNA (cfDNA) is utilized for genomic alteration detection to inform therapy selection and is an emerging noninvasive disease monitoring tool. Nucleic acid-based therapies (NATs) directly or indirectly manipulate DNA or mRNA transcript levels for therapeutic effect. cfDNA is in routine clinical use and NATs are in an expanding number of clinical trials, yet little is known about potential interactions of these technologies including the need for cfDNA next-generation sequencing (NGS) to recognize this source of exogenous DNA.

Methods: A 62-year-old male with weight loss and abdominal pain was found to have a tumor in the head of the pancreas and a metastatic liver lesion. Fine-needle aspirate of the tumor confirmed mucinous adenocarcinoma of the pancreas but sample was insufficient for molecular testing. cfDNA NGS was performed (Guardant Health, CA) to identify genomic alterations (e.g., BRCA, EGFR alterations, ERBB2 amplification).

Results: cfDNA NGS revealed dramatic over-representation (41% of all reads) of TP53-derived sequences with atypical insert sizes and distribution, read start/stop positions, and post-clipping read lengths. Atypical sequences comprised only TP53 coding sequence, without introns. Alignment and reconstruction assembled the atypical reads into a 6003 bp circular construct with the complete TP53 coding sequence inserted into a synthetic expression vector. Consultation with the treating physician revealed that patient progressed on FOLFIRINOX and was subsequently enrolled on a clinical trial utilizing a liposome-encapsulated TP53 expression vector with gemcitabine and nab-paclitaxel. His first experimental treatment was four days prior to cfDNA sample collection. Importantly, over-representation of this synthetic construct obscured native TP53 sequences, resulting in a 47% increase in predicted assay limit of detection despite standard performance metrics being within normal ranges. The atypical features of the expression vector reads make it possible to automate detection and quantification, even at significantly lower concentration than described here. The expression vector bears no human genome homology, allowing read identification via mapping. TP53 intron “deletions” were detected by a novel deletion detection algorithm. Finally, insert length and read start-stop distributions are markedly dissimilar between native and synthetic sequences

Conclusions: NATs generate unique and previously unappreciated interference with cfDNA sequencing which can be identified and filtered using automatable and generalizable bioinformatics techniques. cfDNA sequencing identified an NAT construct, four days after administration, demonstrating that the ability to identify and quantify NATs may enable noninvasive assessment of NAT delivery efficacy and/or pharmacologic persistence.

Citation Format: Marcin Sikora, Alexander Artyomenko, James Strauss, Richard B. Lanman, Victoria M. Raymond, Justin Odegaard. Cell-free DNA sequencing demonstrates persistence of nucleic acid-based therapy—opportunities for detection and monitoring [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 3662.