Molecular tumor analysis and liquid biopsy: a feasibility investigation analyzing circulating tumor DNA in patients with central nervous system lymphomas

Abstract

Background Central nervous system lymphomas (CNSL) is a devastating disease. Currently, a confirmatory biopsy is required prior to treatment. Objective Our investigation aims to prove the feasibility of a minimally-invasive diagnostic approach for the molecular characterization of CNSL. Methods Tissue biopsies from 6 patients with suspected CNSL were analyzed using a 649gene next-generation sequencing (NGS) tumor panel (tumor vs. reference tissue (EDTA-blood)). The individual somatic mutation pattern was used as a basis for the digital PCR analyzing circulating tumor DNA (ctDNA) from plasma and cerebrospinal fluid (CSF) samples, identifying one selected tumor mutation during this first step of the feasibility investigation. Results NGS-analysis of biopsy tissue revealed a specific somatic mutation pattern in all confirmed lymphoma samples ( n = 5, NGS-sensitivity 100%) and none in the sample identified as normal brain tissue (NGS-specificity 100%). cfDNA-extraction was dependent on the extraction-kit used and feasible in 3 samples, in all of which somatic mutations were detectable (100%). Analysis of CSF-derived cfDNA was superior to plasma-derived cfDNA and routine microscopic analysis (lymphoma cells: n = 2, 40%). One patient showed a divergent molecular pattern, typical of Burkitt-Lymphoma (HIV+, serologic evidence of EBV-infection). Lumbar puncture was tolerated without complications, whereas biopsy caused 3 hemorrhages. Conclusions Our investigation provides evidence that analysis of cfDNA in central nervous system tumors is feasible using the described protocol. Molecular characterization of CNSL could be achieved by analysis of CSF-derived cfDNA. Knowledge of a tumor’s specific mutation pattern may allow initiation of targeted therapies, treatment surveillance and could lead to minimally-invasive diagnostics in the future. Electronic supplementary material The online version of this article (10.1186/s12885-019-5394-x) contains supplementary material, which is available to authorized users.

Molecular profiling for precision cancer therapies

“…An emerging field that may ameliorate some tumor sample issues is the testing of circulating tumor-specific markers. These include circulating tumor cells (CTC) or circulating tumor DNA (ctDNA), as well as RNAs, proteins, or metabolites, that are present in body fluids such as the blood, urine, and peritoneal or cerebrospinal fluid [50][51][52][53][54]. Liquid biopsies are easily accessible through minimally invasive procedures that can be repeated to provide a dynamic and longitudinal assessment of tumor-specific diagnostic, prognostic, or predictive biomarkers.…”

Beyond the Blood: CSF-Derived cfDNA for Diagnosis and Characterization of CNS Tumors

“…Special cell-stabilizing blood collection tubes have been used to minimize genomic DNA contamination, which allows processing to be delayed for multiple days (Norton et al, 2013;Diaz et al, 2016;Merker et al, 2018). Although some labs are using these for CSF (Hickmann et al, 2019), the practice is not standard, and the value of cell-stabilizing tubes has not been systematically investigated. As with plasma, CSF specimens can be stored frozen after processing, but multiple freeze-thaw cycles should be avoided (Merker et al, 2018).…”

“…Multiple methods for purifying cell free DNA exist, but none of these have been systematically studied in CSF. For example, Hickmann et al (2019) reported that in their general experience, the QIAamp Circulating Nucleic Acid Kit provided better results with CSF. Likewise, Huang et al (2017) reported that centrifugation for 10 min at 1000 × g is optimal for selection of DNA fragments of ∼150 bp, but others have not confirmed this.…”

Pre-analytical issues in liquid biopsy – where do we stand?

“…plasma/serum and lymphatic fluid) but also in all examined body fluids [7]. Among them is urine [8][9][10], cerebrospinal fluid (CSF) [11,12], pleural fluid including ascites [13,14], saliva [15,16], bronchial lavage fluid [17] and the cell-free supernatant from fine needle aspiration of thyroid nodules [18]. The ubiquitous occurrence of cell-free nucleic acids leads one to assume that these molecules might have a biological meaning.…”