Abstract 5110: Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications
Authors: O'Rourke Dennis J. , Wang Danyi , Feng Zheng 
Published: June 2022

Abstract
Background: Cerebrospinal fluid (CSF) has been recognized as an important biofluid for biomarker analysis of central nervous system (CNS) tumors due to the proximity to the disease/tumor site as compared to serum or plasma. CNS tumors have significantly enriched tumor-derived cfDNA/circulating tumor DNA (ctDNA) in CSF, however, the absolute quantities are low. Therefore, highly sensitive targeted methods such as next generation sequencing or digital droplet PCR are required to efficiently detect mutations. Recent studies have suggested the feasibility of detecting H3K27M in CSF and the potential of these mutations as biomarkers in CNS malignancies. We report the establishment of a protocol for CSF collection, cfDNA extraction and proof of concept study for the analysis of H3K27 mutations as potential clinical biomarkers for CNS tumors.
Methods: CSF was collected from healthy donors (n=10) via lumbar puncture. cfDNA was isolated from approximately 2-3mL CSF from each donor using the Qiagen Circulating Nucleic Acid kit and quantified using the Qubit High Sensitivity DNA kit. Five normal donors were first tested for cfDNA recovery from CSF and then the remaining five were pooled and tested via a spike-in recovery experiment of KRAS G12D mutation ddPCR detection assay. Primer/probes specific to wildtype H3F3A/HIST1H3B and mutant to K27M were designed for ddPCR testing with cfDNA from the CSF of CNS tumor patients (n=10).
Results: cfDNA was successfully isolated from CSF of five healthy samples. The mean cfDNA concentration was 7.6ng DNA per mL CSF with a range of 3.89-11.27 ng/mL CSF. The low yield of cfDNA further confirm the need for an extremely sensitive detection method for biomarkers in CSF. Synthetic DNA for the KRAS G12D mutation was spiked into a pool of healthy volunteer CSF at quantities as low as 1fg and then measured successfully downstream in ddPCR, illustrating the ability of this workflow to recover and measure very low levels the mutant DNA. The 1fg spike in amount yielded an average of 0.77copies/uL. Multiple ddPCR assays were designed for the HIST1H3B assay but after several iterations, no successful assay could be established for this mutation: a result consistent with other reported findings. However, an assay to measure H3F3A p.K27M was identified and is currently being optimized.
Conclusion: Collectively, we demonstrated the use of CSF as a matrix for biomarker testing in a clinical setting. Using commercial kits, ctDNA can be extracted from CSF and used downstream in biomarker detection assays. On-going work includes expanding the tested cohort to CNS tumor samples using a developed ddPCR assay to detect the H3F3A p.K27M mutation. We anticipate the establishment of a reliable CSF cfDNA detection method to enable the study of CNS tumor for genomic evolution, minimal residual disease monitoring and the characterization of tumors at recurrence.
Citation Format: Dennis J. O'Rourke, Danyi Wang, Zheng Feng. Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5110.