Quantification of tumor-derived cell free DNA(cfDNA) by digital PCR (DigPCR) in cerebrospinal fluid of patients with BRAFV600 mutated malignancies.

Tumor-derived cell free DNA (cfDNA) can be detected in plasma. We hypothesized that mutated BRAF V600 cfDNA could be quantified in the cerebrospinal fluid (CSF) of patients with central nervous system (CNS) metastases. We collected CSF from patients with BRAF V600E or K-mutated melanoma (N=8) or BRAF V600E mutated Erdheim-Chester Disease (ECD) (N=3) with suspected central nervous system (CNS) involvement on the basis of neurological symptoms (10/11), MRI imaging (8/11), or both. Tumor-derived cfDNA was quantified by digital PCR in the CSF of 6/11 patients (range from 0.15-10.56 copies/µL). Conventional cytology was negative in all patients except in the two patients with markedly elevated levels of tumor-derived cfDNA. In 2 patients with serial measurements, CSF tumor-derived cfDNA levels reflected response to treatment or progressive disease. CSF tumor-derived cfDNA has the potential to serve as a diagnostic tool that complements MRI and may be more sensitive than conventional cytology.

Utility of CSF Cytokine/Chemokines as Markers of Active Intrathecal Inflammation: Comparison of Demyelinating, Anti-NMDAR and Enteroviral Encephalitis.

BACKGROUND: Despite the discovery of CSF and serum diagnostic autoantibodies in autoimmune encephalitis, there are still very limited CSF biomarkers for diagnostic and monitoring purposes in children with inflammatory or autoimmune brain disease. The cause of encephalitis is unknown in up to a third of encephalitis cohorts, and it is important to differentiate infective from autoimmune encephalitis given the therapeutic implications. AIM: To study CSF cytokines and chemokines as diagnostic biomarkers of active neuroinflammation, and assess their role in differentiating demyelinating, autoimmune, and viral encephalitis. METHODS: We measured and compared 32 cytokine/chemokines using multiplex immunoassay and APRIL and BAFF using ELISA in CSF collected prior to commencing treatment from paediatric patients with confirmed acute disseminated encephalomyelitis (ADEM, n = 16), anti-NMDAR encephalitis (anti-NMDAR E, n = 11), and enteroviral encephalitis (EVE, n = 16). We generated normative data using CSF from 20 non-inflammatory neurological controls. The sensitivity of CSF cytokine/chemokines to diagnose encephalitis cases was calculated using 95th centile of control values as cut off. We correlated CSF cytokine/chemokines with disease severity and follow up outcome based on modified Rankin scale. One-way hierarchical correlational cluster analysis of molecules was performed in different encephalitis and outcome groups. RESULTS: In descending order, CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 had the best sensitivity (>79.1%) when all encephalitis patients were included. The combination of IL-6 and IFN-α was most predictive of inflammation on multiple logistic regression with area under the ROC curve 0.99 (CI 0.97-1.00). There were no differences in CSF cytokine concentrations between EVE and anti-NMDAR E, whereas ADEM showed more pronounced elevation of Th17 related (IL-17, IL-21) and Th2 (IL-4, CCL17) related cytokine/chemokines. Unlike EVE, heat map analysis showed similar clustering of cytokine/chemokine molecules in immune mediated encephalitis (ADEM and anti-NMDAR E). Th1 and B cell (CXCL13 and CXCL10) molecules clustered together in patients with severe encephalopathy at admission and worse disability at follow up in all encephalitis. There was no correlation between CSF neopterin and IFN-γ or IFN-α. CONCLUSION: A combination panel of cytokine/chemokines consisting of CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 measured using multiplex immunoassay may be used to diagnose and monitor intrathecal inflammation in the brain. Given their association with worse outcome, certain key chemokines (CXCL13, CXCL10) could represent potential therapeutic targets in encephalitis.