Othr-03. Classification of Brain Tumors by Nanopore Sequencing of Cell-Free Dna From Cerebrospinal Fluid

Abstract

For exact molecular diagnosis of CNS tumors, tumor tissue obtained through surgery is usually needed. However, knowledge on the molecular tumor type beforehand might influence surgical strategies and reduce surgery-associated risks. Molecular analyses of liquid biopsies have therefore gained increasing interest in order to secure diagnosis before surgery or to replace surgery in rare instances. Moreover, the minimally invasive nature of liquid biopsies enables a tight and longitudinal tumor monitoring with potentially higher sensitivity than imaging approaches. In this study, we applied Nanopore sequencing to 129 cell-free DNA (cfDNA) samples from the cerebrospinal fluid (CSF) of brain tumor patients, with 70.5% coming from pediatric patients. We analyzed copy number variations (CNV) and methylation patterns with a recently published random forest classifier (NanoDx). Circulating tumor DNA (ctDNA) was detected and successfully classified the tumor in 45% of all technically successful samples, both in pre- and early post-surgery samples as well as in samples from >14 days post-surgery, often with clinically unclear residual tumor or disease relapse. In all samples containing detectable tumor DNA, CNV analysis was more frequently successful than methylation analysis with overall detection rates of 88% and 44%, respectively. CNV analysis revealed diagnostic alterations, such as C19MC amplifications in ETMR as well as Chr.1q gain and Chr.6q loss in PFA ependymoma, which are important prognostic markers. Methylation analysis also allowed the classification of tumors with balanced genomes, like craniopharyngiomas. Finally, we were able to perform longitudinal analyses and found aberrations in the CNV profiles that were private to the tumor relapse, highlighting the potential of liquid biopsies to detect potentially relevant changes of tumor biology. Our results show that Nanopore sequencing is a promising approach to establish initial tumor diagnosis and to monitor disease courses by lumbar punctures.

Liquid Biopsy for Glioma Using Cell-Free DNA in Cerebrospinal Fluid

“…Recent technological developments have led to the application of a new generation of sequencing techniques, as exemplified through Nanopore sequencing [ 82 , 182 ]. Recently, Afflerbach et al reported a successful genome-wide methylome and copy number analysis of cfDNA in the CSF of glioma patients [ 182 ].…”

“…Recent technological developments have led to the application of a new generation of sequencing techniques, as exemplified through Nanopore sequencing [ 82 , 182 ]. Recently, Afflerbach et al reported a successful genome-wide methylome and copy number analysis of cfDNA in the CSF of glioma patients [ 182 ]. This new technology, which enables methylome analysis using extremely small amounts of DNA compared to the conventionally required DNA input, is noteworthy and may open new perspectives for liquid biopsy if it can achieve the inherently relatively high failure rate and improved sensitivity of Nanopore sequencing.…”