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Updated: Aug 30, 2025
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Improvements in Quality Control and Library Preparation for Targeted Sequencing Allowed Detection of Potentially
Paulina Szadkowska1,2, Adria-Jaume Roura1, Bartosz Wojtas1
1Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
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.
Insights
Liquid biopsy using circulating cell-free DNA (ccfDNA) shows promise for detecting genetic alterations in brain tumors. While current ctDNA detection rates are low in glioma patients, further improvements could establish liquid biopsy as a valuable diagnostic tool.
Area of Science:
- Neuro-oncology
- Molecular Diagnostics
- Genomics
Background:
- Malignant gliomas are common primary adult brain tumors, characterized by genetic heterogeneity and recurrence due to incomplete resection and treatment resistance.
- Biopsies of brain tumors, often in critical areas, pose significant risks; thus, noninvasive methods like circulating cell-free DNA (ccfDNA) analysis are desirable.
- Circulating tumor DNA (ctDNA) is a fraction of ccfDNA derived from tumor cells, offering a potential window into tumor genetics without direct tissue sampling.
Purpose of the Study:
- To assess the utility of analyzing ccfDNA for detecting genetic alterations in brain tumor patients, particularly gliomas.
- To compare genetic profiles between tumor DNA (tDNA) and ccfDNA using targeted next-generation sequencing (NGS).
- To evaluate the potential of liquid biopsy as a noninvasive diagnostic approach for brain tumors.
Main Methods:
- Collected freshly frozen tumor and matched blood samples from 84 brain tumor patients (80 glioma, 2 metastatic, 2 PCNSL).
- Performed targeted next-generation sequencing (NGS) on tumor DNA (tDNA) and circulating cell-free DNA (ccfDNA).
- Implemented quality control and library preparation enhancements for improved ctDNA detection.
Main Results:
- Detected ctDNA in 8 out of 84 patients (5 out of 80 glioma patients).
- Identified potentially pathogenic genetic alterations in ccfDNA in 32 out of 84 patients, which were not found in their corresponding tDNA.
- Sequencing ccfDNA from plasma demonstrated low efficacy as a current diagnostic tool for glioma patients.
Conclusions:
- Liquid biopsy via ccfDNA sequencing currently has limited diagnostic efficacy for glioma patients due to low ctDNA detection rates.
- The presence of genetic alterations in ccfDNA not found in tDNA suggests complex tumor biology or limitations in tissue sampling.
- Further advancements in sample processing and library preparation techniques are crucial to enhance liquid biopsy's diagnostic value for brain tumors.
