Minimally invasive diagnostic approaches in brain tumours using circulating cell-free nucleic acids

Introduction. Tumours of the central nervous system (CNS) are a group of tumours that develop in the brain and spinal cord, including the meninges, pituitary gland, pineal gland and optic nerve. The majority of primary CNS tumours are aggressive neoplasms, which despite radical treatment (usually a combination of surgery, radiotherapy and chemotherapy) frequently recur, often in a more malignant form. Advances in the molecular characterization of primary brain tumours have led to an extensive revision of the WHO classification of CNS tumours and to an update of the neuropathological assessment of brain tumours, which integrates histopathological features and molecular genetic tests for classification, prediction of biological behaviour and treatment decisions. One of the main problems in the management of patients with primary brain tumours is the lack of effective strategies that could detect the disease at an early stage. In addition, these tumours are clinically and molecularly extremely heterogeneous. Often several genes are altered, both at the genomic and transcriptomic level. Due to high tumour heterogeneity, the correct diagnosis and therapy selection can therefore be difficult and inadequate, and may lead to poor prognosis. Currently, CNS tumour diagnostics require surgical removal of the tumour, a highly invasive procedure, which sometimes needs to be performed repeatedly (for primary and recurrent tumours), and, on the other hand, is not always possible due to the tumour location or patient’s general condition. Objectives. The main goal of the proposed research project is the implementation of minimally invasive diagnostics of brain tumours in adults and children by analysing cell-free tumour DNA and RNA in plasma and cerebrospinal fluid (liquid biopsy). We will also compare detailed genetic data from patients' tumour tissue and fluids to study the heterogeneity and evolution of tumours. By using a next-generation sequencing panel, we will try to identify and monitor acquired treatment resistance and the efficiency of treatment. Moreover, with the use of ultrasensitive digital droplet PCR, we will monitor minimal residual disease and recurrence. We aim that the results will provide us with standardised tests and techniques needed for an early diagnosis/recurrence of brain tumours and for determining the response to treatment and follow-up. Patients and methods. For the study, we will prospectively recruit 150-200 patients with primary brain tumour and follow them from the initial diagnostic phase, through the treatment phase and during the monitoring phase. In each phase, we will collect plasma and cerebrospinal fluid (when available) samples, together from 4-6 samples (before surgery, before radiation and/or chemotherapy and then at 3-6 months’ intervals). Surgically removed brain tumour samples will be used for standard histopathological diagnostic procedures and analysed by next-generation sequencing panels. We will develop new methods, such as digital droplet PCR-based custom assays for the ultrasensitive detection of cell-free tumour DNA and RNA in the plasma and cerebrospinal fluid of the patient. For a broader biomarker detection in plasma and cerebrospinal fluid, we will use a method of next-generation sequencing pan-cancer panel. In addition, we will perform non-coding RNA and methylation profiling on tissue samples using RNA sequencing and methylation array profiling.