Diagnosis of pediatric central nervous system tumors using methylation profiling of cfDNA from cerebrospinal fluid.

Pediatric central nervous system tumors remain challenging to diagnose. Imaging approaches do not provide sufficient detail to discriminate between different tumor types, while the histopathological examination of tumor tissue shows high inter-observer variability. Recent studies have demonstrated the accurate classification of central nervous system tumors based on the DNA methylation profile of a tumor biopsy. However, a brain biopsy holds significant risk of bleeding and damaging the surrounding tissues. Liquid biopsy approaches analyzing circulating tumor DNA show high potential as an alternative and less invasive tool to study the DNA methylation pattern of tumors. Here, we explore the potential of classifying pediatric brain tumors based on methylation profiling of the circulating cell-free DNA (cfDNA) in cerebrospinal fluid (CSF). For this proof-of-concept study, we collected cerebrospinal fluid samples from 19 pediatric brain cancer patients via a ventricular drain placed for reasons of increased intracranial pressure. Analyses on the cfDNA showed high variability of cfDNA quantities across patients ranging from levels below the limit of quantification to 40 ng cfDNA per milliliter of CSF. Classification based on methylation profiling of cfDNA from CSF was correct for 7 out of 20 samples in our cohort. Accurate results were mostly observed in samples of high quality, more specifically those with limited high molecular weight DNA contamination. Interestingly, we show that centrifugation of the CSF prior to processing increases the fraction of fragmented cfDNA to high molecular weight DNA. In addition, classification was mostly correct for samples with high tumoral cfDNA fraction as estimated by computational deconvolution (> 40%). In summary, analysis of cfDNA in the CSF shows potential as a tool for diagnosing pediatric nervous system tumors especially in patients with high levels of tumoral cfDNA in the CSF. Further optimization of the collection procedure, experimental workflow and bioinformatic approach is required to also allow classification for patients with low tumoral fractions in the CSF.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

PURPOSE: Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. METHODS: CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. RESULTS: Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200 µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. CONCLUSIONS: Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Cerebrospinal Fluid cfDNA Sequencing for Classification of Central Nervous System Glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single-nucleotide variants (SNV), and small insertions/deletions (indel) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), nonmalignant (n = 17), and nondiagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/or cancer-specific alterations in 75.0% (n = 24) of glioblastoma and 52.6% (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26%-48%) and SNVs at predefined gene loci (44%), followed by SNVs/indels identified via uninformed variant calling (8%-14%). A molecular-guided tumor classification was possible for 23.5% (n = 4) of nondiagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma. See related commentary by Abdullah, p. 2860.

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.

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.

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.

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.

Unique genomic profiles obtained from cerebrospinal fluid cell-free DNA of non-small cell lung cancer patients with leptomeningeal metastases.

BACKGROUND: Leptomeningeal metastases (LM), associated with poor prognosis, are frequent complications of advanced non-small cell lung cancer (NSCLC) patients, especially in patients with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the mutational landscape of LM has not been comprehensively investigated in large cohorts and the underlining biology of LM remains elusive. Some studies have explored the potential of cerebrospinal fluid (CSF) in reflecting the molecular profile of LM but with limited number of patients enrolled. METHODS: In this study, we performed capture-based targeted sequencing using a panel consisting of 168 lung cancer-related genes on matched CSF and plasma samples from 72 advanced NSCLC patients with confirmed LM to interrogate the potential of CSF as a source of liquid biopsy. RESULTS: We revealed a rate of detection of 81.5% and 62.5% for CSF and plasma, respectively (p = 0.008). The maximum allelic fraction (MaxAF) was also significantly higher in CSF (43.6% vs. 4.6%) (p < 0.001). CSF, harboring a unique genomic profile by having a significant number of CSF-specific mutations, primarily copy number variations, is superior to plasma in reflecting the mutational profile of LM. Further pathway enrichment analysis revealed that most of CSF-specific mutations participated in pathways relevant to the tumorigenesis and the development of metastases. Moreover, our data also revealed that TP53 loss of heterozygosity (LOH) predominantly existed in CSF (p < 0.001). CONCLUSIONS: Collectively, we demonstrated that CSF provides a more comprehensive profile of LM than plasma in a large cohort, thus can be used as an alternative source of liquid biopsy for LM patients.

Temporal patterns of circulating cell-free DNA (cfDNA) in a newborn piglet model of perinatal asphyxia.

Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.