Ultra-low-input cell-free DNA sequencing for tumor detection and characterization in a real-world pediatric brain tumor cohort - Research Open Access

Sep 23, 2026

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Molecular profiling of pediatric central nervous system tumors supports diagnostic and therapeutic decision-making, yet low cell-free DNA (cfDNA) yields limit translation in pediatric neuro-oncology. Using picogram-level inputs, a low-coverage whole genome sequencing (lcWGS) assay was applied to liquid biopsies from a population-based pediatric CNS tumor cohort (n=56). Whole-genome profiles were obtained for all samples (61/61 serum, 56/56 CSF). Circulating-tumor DNA was detected by copy-number variation in CSF more often (25/56, 45%) than serum (2/61, 3%), enabling CSF lcWGS biomarker discrimination of pineal lesions and minimally invasive longitudinal monitoring, including tumor evolution and heterogeneity.

LanguageEnglish

CategoryHealthcare

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Pages19

Reading Time48 min

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Abstract

Introduction

Liquid biopsy approach and clinical context

Fischer et al. Acta Neuropathologica Communications (2025) 13:134 [https://doi.org/10.1186/s40478-025-02024-w](https://doi.org/10.1186/s40478-025-02024-w) Acta Neuropathologica Communications RESEARCH Open Access Ultra-low-input cell-free DNA sequencing for tumor detection and characterization in areal-world pediatric brain tumor cohort Tom T. Fischer 1,2,3,4,5†, Kendra K. Maaß 1,2,3,4*†, Pitithat Puranachot6,16, Markus Mieskolainen7,8, Martin Sill 1,3,4, Paulina S. Schad 1,3,4, Stefanie Volz 1,2,3,4, Fabian Rosing 1,3,4, Tatjana Wedig 1,2,3,4, Nathalie Schwarz 1,3,4, Agnes M. E. Finster 1,3,4, Florian Iser9, Jochen Meyer9,10, Felix Sahm 1,4,11,12, Olli Lohi 13,14, Ahmed El Damaty 15, Benedikt Brors4,16,17, Hannu Haapasalo8, Stefan M. Pfister 1,2,3,4, Joonas Haapasalo7,8†, Kristian W. Pajtler 1,2,3,4*† and Kristiina Nordfors 13,14† Abstract Molecular profiling of pediatric central nervous system (CNS) tumors has important clinical utility for guiding diagnostic and therapeutic strategies. Cell-free DNA (cfDNA) from liquid biopsies has been used for minimally invasive tumor profiling and longitudinal disease assessment in adult oncology and pediatric hematology. However, in pediatric neuro-oncology, low cfDNA yields pose a major barrier to translating these assays from bench to bedside. Here, we implemented a low-coverage whole genome sequencing (lcWGS) assay for picogram-level cfDNA inputs and applied it to liquid biopsies from a sizeable, population-based, cross-entity pediatric CNS tumor cohort (n = 56 patients) . Applying this protocol, cfDNA whole genome profiles were successfully acquired from all liquid biopsy samples (n = 61/61 serum, n = 56/56 CSF, 100%) . Based on copy number variations (CNVs), circulating-tumor DNA (ctDNA) was detected in 2/61 serum (3%) and in 25/56 CSF (45%) samples across various brain tumor entities. The integration of cfDNA results with clinical data demonstrated the utility of CSF lcWGS as a biomarker assay at diagnosis to distinguish cancerous from non-cancerous pineal region lesions (n = 6 patients) . Additionally, serial CSF assessment in n = 9 patients (n = 29 CSF samples) enabled minimally invasive disease monitoring, with the added value of molecular profile availability in n = 4/6 (67%) patients at relapse. Proof-of-concept data show the feasibility of serial CSF lcWGS to reveal tumor evolution, tumor heterogeneity and potential therapeutic vulnerabilities in a case of medulloblastoma and germ cell tumor. Our study underscores the clinical utility of a robust lcWGS-based liquid biopsy assay optimized for low-input samples. We identify use-cases for implementing liquid biopsies in the | †Tom T. Fischer and Kendra K. Maaß contributed equally to this work. | | --- | | †Joonas Haapasalo, Kristian W. Pajtler and Kristiina Nordfors jointly supervised the study. | *Correspondence: Kendra K. Maaß k.maass@kitz-heidelberg.de Kristian W. Pajtler k.pajtler@kitz-heidelberg.de Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creativecommons.org/l](http://creativecommons.org/l)icenses/by/4.0/. Fischer et al. Acta Neuropatholog

FAQs

Why is low cell-free DNA yield a problem in pediatric neuro-oncology?

Low cfDNA yields in pediatric neuro-oncology create a major barrier to implementing tumor profiling assays outside research settings.

What sequencing method and input level were used to enable profiling from limited cfDNA?

The study implemented a low-coverage whole genome sequencing (lcWGS) assay optimized for picogram-level cfDNA inputs.

How did copy-number variation profiling contribute to detecting circulating-tumor DNA?

Using copy number variations (CNVs), the study detected circulating-tumor DNA in serum and CSF, with substantially higher detection in CSF samples.

Sep 23, 2026

Uploaded by Rizky