We report here a novel, high analytical sensitivity multiplexed-qPCR assay for monitoring human and mouse-specific circulating cell-free DNA (cfDNA) using only 15 microliters of blood harvested from patient-derived xenograft (PDX) mice. PDX tumors maintained in murine models are commonly employed to study the biology and drug sensitivity of patient tumors. Our multiplexed qPCR assay incorporates both human and mouse multi-copy PCR amplifications targets in a single qPCR reaction. The multiplex also includes an internal synthetic DNA positive control to monitor accuracy of PCR amplification. All three targets are quantitated by monitoring three fluorophores in a single qPCR reaction. In PDX mice, the implanted human tumor sheds DNA as circulating tumor DNA (human ctDNA). Select metastatic tumors also shed human circulating tumor cells (CTCs) into the mouse’s blood that also can be detected with our assay. Our qPCR assay utilizes an Alu retrotransposon sequence, present at thousands of copies per human genome, to detect and quantitate human ctDNA, and a mouse specific multi-copy target to detect mouse cfDNA in plasma harvested from PDX mice. These high copy, repetitive elements are ideally suited for qPCR targets to quantitate circulating-tumor DNA, which is predominately in low abundance. The low detection limits of the assay (below 1pg/µL of human DNA extracted from mouse plasma) allows for longitudinal studies of mice, as the volume of blood required is minimal (as low as 15 µL) and can be repeatedly collected from live mice without the need for a terminal bleed. With the assay quantitating both human ctDNA and mouse cfDNA, normalization of the amount of tumor DNA can be calculated, allowing for more reliable tracking of the tumor growth in the mouse model. Furthermore, CTC DNA had higher measurable concentrations in the peripheral blood of both invasive TNBC PDX models compared to cfDNA concentrations. We have tested this assay successfully in mice that contain various tumor burdens (CRC, prostrate, SCLC, and TNBC). Data will be presented to show the relative amounts of human DNA present in these mice. The assay also accurately predicted the success or failure of the initial tumor engraftment of a patient tumor in a mouse—this can be used to reduce cost by eliminating unsuccessful grafts, without waiting for months to observe if the tumor grew or not. This innovative assay provides a relatively quick, economic, and easy method to monitor ongoing response to therapy.

Citation Format: Sudhir K. Sinha, Hiromi Brown, Andrew Loftus, Margarite D. Matossian, Bridgette M. Collins-Burow, Matthew E. Burow. Development of a qPCR cfDNA analysis method for the assessment of tumor burden and metastatic status in live PDX mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1077.