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

SIMPLE SUMMARY: Malignant brain tumors present an enormous challenge due to their genetic heterogeneity, and the difficulties in accessing them impede a precise diagnosis. Numerous genetic alterations have been described, and some of them can assist in personalized therapy for glioma patients. Brain biopsy is an invasive procedure with potentially deleterious complications. Liquid biopsy from a patient’s plasma may provide a less invasive method for diagnosis and personalized therapy selection. We performed targeted next-generation sequencing of tumors and circulating cell-free DNA (ccfDNA) from 84 brain tumor patients. We detected tumor-specific genetic alterations in ccfDNA in 5 out of 80 glioma patients and potentially pathogenic alterations in ccfDNA from the plasma of 29 out of 80 glioma patients. Despite a low efficacy, with further improvements, the detection of genetic alterations in ccfDNA holds promise for noninvasive diagnosis, which may revolutionize personalized therapy for these deadly tumors. ABSTRACT: 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.