Precise Enumeration Of Cell-Free Dna From Patient-Derived Cell Lines

Cancer cells secrete fragments of their genetic material in the form of cell-free DNA (cfDNA) producing a potential biomarker for following cell growth and drug response. While cfDNA has been popularized as a liquid biopsy analyte for monitoring disease status in patients with metastases, we have also applied cfDNA detection in vitro for following the establishment of patient-derived organoid cancer cell lines. Because the diversity of tumor subtypes in many cancers requires extensive optimization, the combinations of growth factors or other supplements can vary with each culture, even for the same cancer type. This challenging task is complicated further by contamination with normal cells. Historically, determining which condition is optimal for enriching for tumor cell growth required sacrificing at least some portion of the culture for a phenotypic analysis, adding cost and time to replenish the culture. As an alternative, we have developed a robust, sensitive, and patient-specific assay that can be rapidly applied as new culture conditions are optimized, with a priori knowledge of the clonal mutations present in the tumor sample. For a given panel of point mutations, multiplex PCR primers were generated using the IonTorrect AmpliSeq Design tool, creating locus-specific primers flanking each site by approximately 60bp on each side. To the 5’ end of each forward primer, a 7-base degenerate unique molecular identifier (UMI) was synthesized, to which an additional 32-base sequence complimentary to the forward Illumina Nextera adaptor was further added. To the 3’ end of the reverse primer, a 34-base sequence complimentary to the reverse Illumina Nextera adaptor was added. Once the patient-specific primer set is obtained, the total time from sample to result was less than two days. For each patient-specific primer set, the forward primers were pooled and used to tag DNA extracted from the cell culture supernatant of patient-derived cell lines with the adaptor and UMI sequence in a single hybridization and elongation cycle of PCR. Primers were blocked with terminal transferase and removed by spin-column, and the tagged product was amplified in 30 linear PCR cycles with the reverse primer pool. Following purification and size selection, the tagged and partial-adaptor-ligated library was amplified in 35 cycles of PCR with barcoded Illumina adaptors. Libraries were quantified, pooled, and sequenced on a MiSeq. Upon analysis of sequencing data, including the identification of unique template molecules, each template sample produced a number of starting molecules of DNA. With this critical data, we can determine which conditions successfully enriched for the growth of tumor cells and their subsequent release of cfDNA. Citation Format: Sean Hennigan, Shana Trostel, Adam G. Sowalsky. Precise enumeration of cell-free DNA from patient-derived cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-275. doi:10.1158/1538-7445.AM2017-LB-275