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

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.

Plasma ctDNA enables early detection of temozolomide resistance mutations in glioma

“…Similar results were seen in a recent publication where the majority of potentially pathogenic mutations in plasma ctDNA were not found in tumor DNA. 35 The marked intra-tumor heterogeneity and subclonal architecture in glioblastoma, 36 and the necessity for piecemeal surgical biopsies, are all factors that can lead to tissue sampling being under-representative in glioma. 37 In this context, plasma ctDNA analysis could provide valuable additional genomic information for surgical biopsy.…”

Exploring the Role of Circulating Cell-Free DNA in Disease Diagnosis and Therapy

“…Apart from NGS, PCR-based methods, such as quantitative PCR (qPCR) and digital PCR (dPCR), are employed for more targeted analysis. These methods are particularly useful when specific genetic markers or mutations are being investigated and are known for their high sensitivity and specificity [23]. Additionally, advancements in sequencing technologies continue to improve the efficiency and reduce the costs of ccfDNA analysis, making it more accessible for routine clinical use [24].…”

Next-generation sequencing impact on cancer care: applications, challenges, and future directions

“…Following DNA/RNA extraction and quality assessment, the next critical step in the NGS workflow is library preparation ( Fujii et al, 2020 ; Szadkowska et al, 2022 ; Michalska-Falkowska et al, 2023 ). This process involves fragmenting the nucleic acids, repairing the ends, adding adapters, and sometimes incorporating specific indexes for multiplexing samples ( Fujii et al, 2020 ).…”

“…The quality and integrity of the input material directly influence the efficiency of these steps and the overall complexity and quality of the final library. Libraries from high-integrity samples will more accurately reflect the genome or transcriptome of interest and are more likely to yield robust, comprehensive sequencing data ( Szadkowska et al, 2022 ).…”