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.

Keywords: CNS-lymphoma; Cerebrospinal fluid; Circulating DNA; Liquid biopsy; Personalized medicine; Targeted therapies.

Fig. 1

Study design

Fig. 2

Cranial imaging of # 1, 2: a, b/e, f) MRI T2 flair showing multifocal hyperintense lesions with contact to the CSF space in both patients; c, d/g, h) MRI T1 with contrast showing only sparse enhancement in both patients

Fig. 3

Cranial imaging of #3–6: a) CT with contrast (#3), b) MRI T1 with contrast, c) CT with contrast (#5); In contrast to the images in Fig. 2, homogenous, strong contrast enhancement is visible. d) MRI T1 with contrast (#6) showing homogenously thickened meninges and hypervascularity over the left hemisphere

Fig. 4

Venn diagram: Overlap of mutation profiles of tumor samples #1, 3, 4, 5. Note that this diagram shows the mutated genes. Therefore, the total number differs from Table 2, which shows all somatic mutations (2 independent mutations may occur within the same gene). Large copy number aberrations (CNAs) were not analyzed genome-wide, as the design of the NGS panel is optimized for the detection of single nucleotide variants (SNVs) and small copy number aberrations. All genes are listed in Table 3, their details are listed in the supplement (Additional file 1). Patients 2 and 6 were excluded from this diagram: The lymphoma sample #2 shows a divergent somatic mutation pattern (only 1 somatic mutation in MDM2) than the 4 other lymphomas and patient 6 had no somatic mutations at all, which is in accordance with the histopathologic diagnosis of absent malignancy. The mean coverage was >1000x in all tumor samples (ultra-deep sequencing)

Fig. 5

cfDNA concentration in plasma vs. CSF. Fluorometric measurement of cfDNA concentration in CSF and plasma. For plasma and csf samples #1 and #2 no cfDNA could be detected, most probably due to technical reasons (explanation see text)

Fig. 6

Mutant Allele Fraction in plasma and CSF of patients with feasible extraction. For plasma and csf samples #1 and #2 no cfDNA could be detected, most probably due to technical reasons (explanation see text)