Rapid detection of mutations in CSF-cfTNA with the Genexus Integrated Sequencer.

PURPOSE: Genomic alterations are fundamental for molecular-guided therapy in patients with breast and lung cancer. However, the turn-around time of standard next-generation sequencing assays is a limiting factor in the timely delivery of genomic information for clinical decision-making. METHODS: In this study, we evaluated genomic alterations in 54 cerebrospinal fluid samples from 33 patients with metastatic lung cancer and metastatic breast cancer to the brain using the Oncomine Precision Assay on the Genexus sequencer. There were nine patients with samples collected at multiple time points. RESULTS: Cell-free total nucleic acids (cfTNA) were extracted from CSF (0.1-11.2 ng/µl). Median base coverage was 31,963× with cfDNA input ranging from 2 to 20 ng. Mutations were detected in 30/54 CSF samples. Nineteen (19/24) samples with no mutations detected had suboptimal DNA input (< 20 ng). The EGFR exon-19 deletion and PIK3CA mutations were detected in two patients with increasing mutant allele fraction over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in one patient with prior EGFR inhibitor treatment. Additionally, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was < 24 h. CONCLUSION: In summary, our results indicate that CSF-cfTNA analysis with the Genexus-OPA can provide clinically relevant information in patients with brain metastases with short TAT.

CSF-based liquid biopsy pointing to a diagnosis of diffuse glioma in a patient with supposed neurodegenerative disorder.

INTRODUCTION: The differential diagnosis of brain diseases becomes challenging in cases where imaging is not sufficiently informative, and surgical biopsy is impossible or unacceptable to the patient. METHODS: An elderly patient with progressive short-term memory loss and cognitive impairment presented with a normal brain CT scan, a brain FDG-PET that indicated symmetrical deterioration of the white matter in the frontal lobes, and inconclusive results of a molecular marker analysis of suspected dementia in cerebrospinal fluid (CSF). Brain MRI suggested the diagnosis of lower grade glioma. The patient refused surgical biopsy. In order to investigate whether somatic mutations associated with gliomas existed, we performed a "liquid biopsy" by the targeted sequencing of cell-free DNA (cfDNA) from his CSF. RESULTS: Deep sequencing of the cfDNA from CSF revealed somatic mutations characteristically found in gliomas, including mutations of the TP53 (Arg282Trp), BRAF (Val600Glu), and IDH1 (Arg132His) genes. The patient is currently treated with temozolomide, and his clinical and MRI findings suggest the stabilization of his disease. CONCLUSION: Neurological patients may benefit from liquid biopsy diagnostic work-up as it can reveal therapeutically targetable mutations.

Prognostic value of cell-free DNA in cerebrospinal fluid from lung cancer patients with brain metastases during radiotherapy.

BACKGROUND: During the last decades, radiotherapy (RT) for non-small cell lung cancer (NSCLC) with brain metastases (BM) has been developed. However, the lack of predictive biomarkers for therapeutic responses has limited the precision treatment in NSCLC-BM. PATIENTS AND METHODS: In order to find the predictive biomarkers for RT, we investigated the influence of RT on the cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) and the frequency of T cell subsets of NSCLC patients with BM. A total of 19 patients diagnosed as NSCLC with BM were enrolled. The CSF from 19 patients and matched plasma samples from 11 patients were collected before RT, during RT, and after RT. The cfDNA from CSF and plasma were extracted, and the cerebrospinal fluid tumor mutation burden (cTMB) was calculated after through next-generation sequencing. The frequency of T cell subsets in peripheral blood was using flow cytometry. RESULTS: The detection rate of cfDNA was higher in CSF compared to plasma in the matched samples. The mutation abundance of cfDNA in CSF was decreased after RT. However, no significant difference was observed in cTMB before and after RT. Although the median intracranial progression-free survival (iPFS) has not yet been reached in patients with decreased or undetectable cTMB, there was a trend that these patients possessed longer iPFS compared to those with stable or increased cTMB (HR 0.28, 95% CI 0.07-1.18, P = 0.067). The proportion of CD4+T cells in peripheral blood was decreased after RT. CONCLUSION: Our study indicates that cTMB can serve as a prognostic biomarker in NSCLC patients with BMs.

Low-coverage whole-genome sequencing of cerebrospinal-fluid-derived cell-free DNA in brain tumor patients.

This protocol summarizes the pipeline for analysis of tumor-derived cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) using low-coverage whole-genome sequencing (lcWGS). This approach enables resolution of chromosomal and focal copy-number variations (CNVs) as oncologic signatures, particularly for patients with central nervous system tumors. Our strategy tolerates sub-nanogram cfDNA input and is thus optimized for CSF samples where cfDNA yields are typically low. Overall, the detection of tumor-specific signatures in CSF-derived cfDNA is a promising biomarker for personalization of brain-tumor therapy. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).

Next-generation sequencing of cerebrospinal fluid for clinical molecular diagnostics in pediatric, adolescent and young adult brain tumor patients.

BACKGROUND: Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests. METHODS: We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. RESULTS: We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient's disease course. CONCLUSIONS: We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care.

Serial assessment of measurable residual disease in medulloblastoma liquid biopsies.

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.

Comprehensive methylation profile of CSF cfDNA revealed pathogenesis and diagnostic markers for early-onset Parkinson's disease.

Background: Early-onset Parkinson's disease (EOPD) is one uncommon Parkinson's disease subtype with characteristic clinicopathological features. The full epigenomic profile of EOPD is largely unknown. Methods: We performed the first study to investigate the EOPD full methylation profile of cerebrospinal fluid (CSF) cell-free DNA (cfDNA) from 26 EOPD patients and 10 control patients. Results: 2220 differentially methylated genes were identified in EOPD. Hypermethylation far outweighed hypomethylation in gene numbers. Clustering and enrichment analyses identified aberrant neuronal function and immune response. Weighted correlation network analysis demonstrated significant correlation between methylation signatures and clock drawing test (CDT), mini-mental state examination (MMSE), education, working status, alcohol drinking history and Hamilton anxiety scale (HAMA). Several key networking genes in EOPD aberrant methylation were also identified. Conclusions: The methylation profile and signatures of CSF cfDNA were revealed for the first time in EOPD. Aberrant methylation signatures were correlated with education, working status, alcohol drinking history, CDT, MMSE and HAMA.

Lay abstract Early-onset Parkinson's disease (EOPD), a progressive disease of the nervous system marked by tremor, muscular rigidity and slow and imprecise movement, is generally found in people under the age of 40 years. In this study, we performed methylation test on cerebrospinal fluid (CSF). The test showed that specific CSF methylation profiles could be used to help EOPD diagnosis. A subset of differentially methylated genes were also identified, which could also be used as markers for EOPD diagnosis. In future, we will validate the methylation test in blood and try to find blood-based diagnostic markers to facilitate the diagnosis of EOPD by a single blood draw.

Liquid biopsy of cerebrospinal fluid for MYD88 L265P mutation is useful for diagnosis of central nervous system lymphoma.

The current standard of diagnosing central nervous system (CNS) lymphoma is stereotactic biopsy, however the procedure has a risk of surgical complication. Liquid biopsy of the CSF is a less invasive, non-surgical method that can be used for diagnosing CNS lymphoma. In this study, we established a clinically applicable protocol for determining mutations in MYD88 in the CSF of patients with CNS lymphoma. CSF was collected prior to the start of chemotherapy from 42 patients with CNS lymphoma and matched tumor specimens. Mutations in MYD88 in 33 tumor samples were identified using pyrosequencing. Using 10 ng each of cellular DNA and cell-free DNA (cfDNA) extracted from the CSF, the MYD88 L265P mutation was detected using digital PCR. The conditions to judge mutation were rigorously determined. The median Target/Total value of cases with MYD88 mutations in the tumors was 5.1% in cellular DNA and 22.0% in cfDNA. The criteria to judge mutation were then determined, with a Target/Total value of 0.25% as the cutoff. When MYD88 mutations were determined based on these criteria, the sensitivity and specificity were 92.2% and 100%, respectively, with cellular DNA; and the sensitivity and specificity were 100% with cfDNA. Therefore, the DNA yield, mutated allele fraction, and accuracy were significantly higher in cfDNA compared with that in cellular DNA. Taken together, this study highlights the importance of detecting the MYD88 L265P mutation in cfDNA of the CSF for diagnosing CNS lymphoma using digital PCR, a highly accurate and clinically applicable method.

Tumor DNA Mutations From Intraparenchymal Brain Metastases Are Detectable in CSF.

Discordant responses between brain metastases and extracranial tumors can arise from branched tumor evolution, underscoring the importance of profiling mutations to optimize therapy. However, the morbidity of brain biopsies limits their use. We investigated whether cell-free DNA (cfDNA) in CSF could serve as an effective surrogate marker for genomic profiling of intraparenchymal (IP) brain metastases. METHODS: CSF and blood were collected simultaneously from patients with progressive brain metastases undergoing a craniotomy or lumbar puncture. Mutations in both biofluids were measured using an error-suppressed deep sequencing method previously published by our group. Forty-three regions of 24 cancer-associated genes were assayed. RESULTS: This study enrolled 14 patients with either IP brain metastases (n = 12) or cytology-positive leptomeningeal disease (LMD, n = 2) and two controls with normal pressure hydrocephalus. Primary cancer types were lung, melanoma, renal cell, and colorectal. cfDNA was measurable in all sixteen samples of CSF. Cancer-associated mutations were found in the CSF of ten patients (eight with IP [67%] and two with LMD [100%]) and plasma of five patients (five with IP [42%] and none with LMD). All patients with plasma cfDNA had extracranial tumors. Among the five patients in the cohort who also had mutation data from time-matched brain metastasis tissue, four patients (80%) had matching mutations detected in CSF and brain, whereas only one patient (20%) had matching mutations detected in plasma and brain. CONCLUSION: The detection of mutational DNA in CSF is not restricted to LMD and was found in two thirds of patients with IP brain metastases in our cohort. Analysis of CSF can be a viable alternative to biopsy for detection of somatic mutations in brain metastases.

Detection of Brain Somatic Mutations in Cerebrospinal Fluid from Refractory Epilepsy Patients.

Brain mosaic mutations are a major cause of refractory focal epilepsies with cortical malformations such as focal cortical dysplasia, hemimegalencephaly, malformation of cortical development with oligodendroglial hyperplasia in epilepsy, and ganglioglioma. Here, we collected cerebrospinal fluid (CSF) during epilepsy surgery to search for somatic variants in cell-free DNA (cfDNA) using targeted droplet digital polymerase chain reaction. In 3 of 12 epileptic patients with known somatic mutations previously identified in brain tissue, we here provide evidence that brain mosaicism can be detected in the CSF-derived cfDNA. These findings suggest future opportunities for detecting the mutant allele driving epilepsy in CSF. ANN NEUROL 2021;89:1248-1252.

Detection of Aneuploidy in Cerebrospinal Fluid from Patients with Breast Cancer Can Improve Diagnosis of Leptomeningeal Metastases.

PURPOSE: Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis. EXPERIMENTAL DESIGN: cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; n = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; n = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology. RESULTS: Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival. CONCLUSIONS: Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.

Time to focus on circulating nucleic acids for diagnosis and monitoring of gliomas: A systematic review of their role as biomarkers.

Gliomas are diffusely growing tumours arising from progenitors within the central nervous system. They encompass a range of different molecular types and subtypes, many of which have a well-defined profile of driver mutations, copy number changes and DNA methylation patterns. A majority of gliomas will require surgical intervention to relieve raised intracranial pressure and reduce tumour burden. A proportion of tumours, however, are located in neurologically sensitive areas and a biopsy poses a significant risk of a deficit. A majority of gliomas recur after surgery, and monitoring tumour burden of the recurrence is currently achieved by imaging. However, most imaging modalities have limitations in assessing tumour burden and infiltration into adjacent brain, and sometimes imaging is unable to discriminate between tumour recurrence and pseudo-progression. Liquid biopsies, obtained from body fluids such as cerebrospinal fluid or blood, contain circulating nucleic acids or extracellular vesicles containing tumour-derived components. The studies for this systematic review were selected according to PRISMA criteria, and suggest that the detection of circulating tumour-derived nucleic acids holds great promises as biomarker to aid diagnosis and prognostication by monitoring tumour progression, and thus can be considered a pathway towards personalized medicine.

Competing Endogenous RNA Networks as Biomarkers in Neurodegenerative Diseases.

Protein aggregation is classically considered the main cause of neuronal death in neurodegenerative diseases (NDDs). However, increasing evidence suggests that alteration of RNA metabolism is a key factor in the etiopathogenesis of these complex disorders. Non-coding RNAs are the major contributor to the human transcriptome and are particularly abundant in the central nervous system, where they have been proposed to be involved in the onset and development of NDDs. Interestingly, some ncRNAs (such as lncRNAs, circRNAs and pseudogenes) share a common functionality in their ability to regulate gene expression by modulating miRNAs in a phenomenon known as the competing endogenous RNA mechanism. Moreover, ncRNAs are found in body fluids where their presence and concentration could serve as potential non-invasive biomarkers of NDDs. In this review, we summarize the ceRNA networks described in Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis and spinocerebellar ataxia type 7, and discuss their potential as biomarkers of these NDDs. Although numerous studies have been carried out, further research is needed to validate these complex interactions between RNAs and the alterations in RNA editing that could provide specific ceRNET profiles for neurodegenerative disorders, paving the way to a better understanding of these diseases.

Circulating cell-free mitochondrial DNA levels in Parkinson's disease are influenced by treatment.

Several studies have linked circulating cell-free mitochondrial DNA (ccf-mtDNA) to human disease. In particular, reduced ccf-mtDNA levels in the cerebrospinal fluid (CSF) of both Alzheimer's and Parkinson's disease (PD) patients have raised the hypothesis that ccf-mtDNA could be used as a biomarker for neurodegenerative disease onset and progression. However, how a reduction of CSF ccf-mtDNA levels relates to neurodegeneration remains unclear. Many factors are likely to influence ccf-mtDNA levels, such as concomitant therapeutic treatment and comorbidities. In this study we aimed to investigate these factors, quantifying CSF ccf-mtDNA from the Parkinson's Progression Markers Initiative in 372 PD patients and 159 matched controls at two time points. We found that ccf-mtDNA levels appear significantly reduced in PD cases when compared to matched controls and are associated with cognitive impairment. However, our data indicate that this reduction in ccf-mtDNA is also associated with the commencement, type and duration of treatment. Additionally, we found that ccf-mtDNA levels are associated with comorbidities such as depression and insomnia, however this was only significant if measured in the absence of treatment. We conclude that in PD, similar to reports in HIV and sepsis, comorbidities and treatment can both influence ccf-mtDNA homeostasis, raising the possibility that ccf-mtDNA may be useful as a biomarker for treatment response or the development of secondary phenotypes. Given that, clinically, PD manifests often decades after neurodegeneration begins, predicting who will develop disease is important. Also, identifying patients who will respond to existing treatments or develop secondary phenotypes will have increased clinical importance as PD incidence rises.

Comparison of paired cerebrospinal fluid and serum cell-free mitochondrial and nuclear DNA with copy number and fragment length.

BACKGROUND: Most studies on cell-free DNA (cfDNA) were only for single body fluids; however, the differences in cfDNA distribution between two body fluids are rarely reported. Hence, in this work, we compared the differences in cfDNA distribution between cerebrospinal fluid (CSF) and serum of patients with brain-related diseases. METHODS: The fragment length of cfDNA was determined by using Agilent 2100 Bioanalyzer. The copy numbers of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were determined by using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) with three pairs of mitochondrial ND1 and nuclear GAPDH primers, respectively. RESULTS: There were short (~60 bp), medium (~167 bp), and long (>250 bp) cfDNA fragment length distributions totally obtained from CSF and serum using Agilent 2100 Bioanalyzer. The results of both qPCR and ddPCR confirmed the existence of these three cfDNA fragment ranges in CSF and serum. According to qPCR, the copy numbers of long cf-mtDNA, medium, and long cf-nDNA in CSF were significantly higher than in paired serum. In CSF, only long cf-mtDNA's copy numbers were higher than long cf-nDNA. But in serum, the copy numbers of medium and long cf-mtDNA were higher than the corresponding cf-nDNA. CONCLUSION: The cf-nDNA and cf-mtDNA with different fragment lengths differentially distributed in the CSF and serum of patients with brain disorders, which might serve as a biomarker of human brain diseases.

Rapid Diagnosis of Tuberculosis Meningitis by Detecting Mycobacterium tuberculosis Cell-Free DNA in Cerebrospinal Fluid.

OBJECTIVES: Tuberculosis meningitis (TBM) is one of the most severe forms of tuberculosis. However, TBM diagnosis is quite challenging due to nonspecific clinical presentation and the paucity of the pathogen in cerebrospinal fluid (CSF) samples. In this study, we report a new method for detecting cell-free Mycobacterium tuberculosis DNA (cf-TB) in CSF and evaluate its clinical value for TBM diagnosis. METHODS: Of 68 patients prospectively recruited, 46 were diagnosed as having TBM and 22 as non-TBM. We compared the cf-TB method with CSF smear microscopy, mycobacterial culture, and the Xpert MTB/RIF assay (Xpert) using the consensus case definition for TBM proposed in 2009 as a reference standard. RESULTS: The sensitivity of the cf-TB test was 56.5% (26/46) in patients with TBM, and it was significantly higher than other methods: microscopy (2.2%, 1/46; P < .001), mycobacterial culture (13.0%, 6/46; P < .001), and Xpert (23.9%, 11/46; P = .001). For specificity, none of the four methods reported false-positive results in the non-TBM group. CONCLUSIONS: The new method detecting cell-free M tuberculosis DNA in CSF is rapid and accurate for diagnosis of TBM and easily incorporated into regular laboratory tests.

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

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.

Cell-Free Mitochondrial DNA in the CSF: A Potential Prognostic Biomarker of Anti-NMDAR Encephalitis.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune inflammatory brain disease that can develop a variety of neuropsychiatric presentations. However, the underlying nature of its inflammatory neuronal injury remains unclear. Mitochondrial DNA (mtDNA) is recently regarded as a damage-associated molecular pattern molecule (DAMP) that can initiate an inflammatory response. In the presenting study, we aimed to evaluate the levels of cell-free mtDNA in cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis and to determine a potential role of cell-free mtDNA in the prognosis of anti-NMDAR encephalitis. A total of 33 patients with NMDAR encephalitis and 17 patients with other non-inflammatory disorders as controls were included in this study. The CSF levels of cell-free mtDNA were measured by quantitative polymerase chain reaction (qPCR). Cytokines including interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α) were measured by ELISA. The modified Rankin scale (mRS) score was evaluated for neurologic disabilities. Our data showed that the CSF levels of cell-free mtDNA and inflammation-associated cytokines were significantly higher in the patients with anti-NMDAR encephalitis compared with those in controls. Positive correlations were detected between the CSF levels of cell-free mtDNA and mRS scores of patients with anti-NMDAR encephalitis at both their admission and 6-month follow up. These findings suggest that the CSF level of cell-free mtDNA reflects the underlying neuroinflammatory process in patients with anti-NMDAR encephalitis and correlates with their clinical mRS scores. Therefore, cell-free mtDNA may be a potential prognostic biomarker for anti-NMDAR encephalitis.

Cell-free mitochondrial DNA in progressive multiple sclerosis.

Recent studies have linked cell-free mitochondrial DNA (ccf-mtDNA) to neurodegeneration in both Alzheimer's and Parkinson's disease, raising the possibility that the same phenomenon could be seen in other diseases which manifest a neurodegenerative component. Here, we assessed the role of circulating cell-free mitochondrial DNA (ccf-mtDNA) in end-stage progressive multiple sclerosis (PMS), where neurodegeneration is evident, contrasting both ventricular cerebral spinal fluid ccf-mtDNA abundance and integrity between PMS cases and controls, and correlating ccf-mtDNA levels to known protein markers of neurodegeneration and PMS. Our data indicate that reduced ccf-mtDNA is a component of PMS, concluding that it may indeed be a hallmark of broader neurodegeneration.

Screening system for epidermal growth factor receptor mutation detection in cytology cell-free DNA of cerebrospinal fluid based on assured sample quality.

BACKGROUND: The cobas® epidermal growth factor receptor (EGFR) Mutation Test v2 designed for cell-free DNA (cfDNA) is approved as a companion diagnostic for osimertinib therapy. The aim of this study was to evaluate the concordance of EGFR mutation detection between paired primary or recurrent samples, and cerebrospinal fluid (CSF) cytology samples of lung cancer patients. METHODS: In total, 26 lung cancer patients with supernatant cytology cfDNA in CSF were analysed for EGFR mutations using the cobas® EGFR Mutation Test v2.0 designed for cfDNA, and the concordance rates between CSF cfDNA and primary or recurrent samples were investigated. RESULTS: Of the 26 CSF cytology cfDNA samples, 46.1% (12/26) were valid and 53.9% (14/26) were invalid. Sensitivity, specificity and accuracy between the valid CSF cfDNA samples and primary or recurrent samples for detection of EGFR mutation, including T790M were 87.5%, 100.0% and 91.7%, respectively. Amounts of both inflammatory cells and tumour cells in CSF cytology were higher in the valid evaluation samples than in the invalid samples (P < .05), and mutant EGFR was detected in 80.0% (4/5) of the valid CSF cytology cfDNA samples with a negative cytology diagnosis. CONCLUSIONS: The cobas® EGFR Mutation Test v2.0 can accurately detect EGFR mutations, including T790M, from supernatant cfDNA of CSF cytology samples. Utilisation of supernatant cytology cfDNA in CSF will allow us to perform both EGFR mutation analysis and cytopathological diagnosis at the same time. This represents a new role of cytology in patient treatment, based on assured sample quality.