Improvements in Quality Control and Library Preparation for Targeted Sequencing Allowed Detection of Potentially Pathogenic Alterations in Circulating Cell-Free DNA Derived from Plasma of Brain Tumor Patients.

Malignant gliomas are the most frequent primary brain tumors in adults. They are genetically heterogenous and invariably recur due to incomplete surgery and therapy resistance. Circulating tumor DNA (ctDNA) is a component of circulating cell-free DNA (ccfDNA) and represents genetic material that originates from the primary tumor or metastasis. Brain tumors are frequently located in the eloquent brain regions, which makes biopsy difficult or impossible due to severe postoperative complications. The analysis of ccfDNA from a patient's blood presents a plausible and noninvasive alternative. In this study, freshly frozen tumors and corresponding blood samples were collected from 84 brain tumor patients and analyzed by targeted next-generation sequencing (NGS). The cohort included 80 glioma patients, 2 metastatic cancer patients, and 2 primary CNS lymphoma (PCNSL) patients. We compared the pattern of genetic alterations in the tumor DNA (tDNA) with that of ccfDNA. The implemented technical improvements in quality control and library preparation allowed for the detection of ctDNA in 8 out of 84 patients, including 5 out of 80 glioma patients. In 32 out of 84 patients, we found potentially pathogenic genetic alterations in ccfDNA that were not detectable in tDNA. While sequencing ccfDNA from plasma has a low efficacy as a diagnostic tool for glioma patients, we concluded that further improvements in sample processing and library preparation can make liquid biopsy a valuable diagnostic tool for glioma patients.