Real-world experience with circulating tumor DNA in cerebrospinal fluid from patients with central nervous system tumors.

The characterization of genetic alterations in tumor samples has become standard practice for many human cancers to achieve more precise disease classification and guide the selection of targeted therapies. Cerebrospinal fluid (CSF) can serve as a source of tumor DNA in patients with central nervous system (CNS) cancer. We performed comprehensive profiling of CSF circulating tumor DNA (ctDNA) in 711 patients using an FDA-authorized platform (MSK-IMPACT™) in a hospital laboratory. We identified genetic alterations in 489/922 (53.0%) CSF samples with clinically documented CNS tumors. None of 85 CSF samples from patients without CNS tumors had detectable ctDNA. The distribution of clinically actionable somatic alterations was consistent with tumor-type specific alterations across the AACR GENIE cohort. Repeated CSF ctDNA examinations from the same patients identified clonal evolution and emergence of resistance mechanisms. ctDNA detection was associated with shortened overall survival following CSF collection. Next-generation sequencing of CSF, collected through a minimally invasive lumbar puncture in a routine hospital setting, provides clinically actionable cancer genotype information in a large fraction of patients with CNS tumors.

Establishing the utility of multi-platform liquid biopsy by integrating the CSF methylome and proteome in CNS tumours.

BACKGROUND: Liquid biopsy represents a major development in cancer research, with significant translational potential. Similarly, it is increasingly recognized that multi-omic molecular approaches are a powerful avenue through which to understand complex and heterogeneous disease biology. We hypothesize that merging these two promising frontiers of cancer research will improve the discriminatory capacity of current models and allow for improved clinical utility. METHODS: We have compiled a cohort of patients with glioblastoma, brain metastasis, and primary central nervous system lymphoma. Cell-free methylated DNA immunoprecipitation (cfMeDIP) and shotgun proteomic profiling was obtained from the cerebrospinal fluid (CSF) of each patient and used to build tumour-specific classifiers. RESULTS: We show that the DNA methylation and protein profiles of cerebrospinal fluid can be integrated to fully discriminate lymphoma from its diagnostic counterparts with perfect AUC of 1 (95% confidence interval 1-1) and 100% specificity, significantly outperforming single-platform classifiers. CONCLUSIONS: We present the most specific and accurate CNS lymphoma classifier to date and demonstrates the synergistic capability of multi-platform liquid biopsies. This has far-reaching translational utility for patients with newly diagnosed intra-axial brain tumours.

[«Masks¼ of CNS demyelinating diseases. Primary lymphoma]. / «Maski¼ demieliniziruyushchikh zabolevanii TsNS. Pervichnaya limfoma.

Primary central nervous system lymphoma (PCNSL) is a rare neoplasm that can affect the brain, eyes, and, rarely, the spinal cord. Clinical presentation and MRI findings can mimic a variety of diseases, including high-grade gliomas, infectious and granulomatous diseases, and demyelinating diseases. We describe three cases where the diagnosis of PCNSL was difficult due to an ambiguous clinical, radiological and laboratory results. The role of stereotactic biopsy remains leading in differential diagnosis; however, the invasiveness and frequent limitations of this method determine the search for additional biological markers of the disease. New evidence suggests a potential role for cerebrospinal fluid (CSF) cytokine profiles and proteomic analysis in differential diagnosis, disease progression, and treatment response.

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.

Primary diffuse large B-cell lymphoma of the central nervous system identified with CSF biomarkers.

BACKGROUND: Diagnosis of primary diffuse large B-cell lymphoma of the central nervous system (PCNSL) is challenging and often delayed. MRI imaging, CSF cytology and flow cytometry have a low sensitivity and even brain biopsies can be misleading. We report three cases of PCNSL with various clinical presentation and radiological findings where the diagnosis was suggested by novel CSF biomarkers and subsequently confirmed by brain biopsy or autopsy. CASE PRESENTATIONS: The first case is a 79-year-old man with severe neurocognitive dysfunction and static ataxia evolving over 5 months. Brain MRI revealed a nodular ventriculitis. An open brain biopsy was inconclusive. The second case is a 60-year-old woman with progressive sensory symptoms in all four limbs, evolving over 1 year. Brain and spinal MRI revealed asymmetric T2 hyperintensities of the corpus callosum, corona radiata and corticospinal tracts. The third case is a 72-year-old man recently diagnosed with primary vitreoretinal lymphoma of the right eye. A follow-up brain MRI performed 4 months after symptom onset revealed a T2 hyperintense fronto-sagittal lesion, with gadolinium uptake and perilesional edema. In all three cases, CSF flow cytometry and cytology were negative. Mutation analysis on the CSF (either by digital PCR or by next generation sequencing) identified the MYD88 L265P hotspot mutation in all three cases. A B-cell clonality study, performed in case 1 and 2, identified a monoclonal rearrangement of the immunoglobulin light chain lambda (IGL) and kappa (IGK) gene. CSF CXCL-13 and IL-10 levels were high in all three cases, and IL-10/IL-6 ratio was high in two. Diagnosis of PCNSL was later confirmed by autopsy in case 1, and by brain biopsy in case 2 and 3. CONCLUSIONS: Taken together, 5 CSF biomarkers (IL-10, IL-10/IL-6 ratio, CXCL13, MYD88 mutation and monoclonal IG gene rearrangements) were strongly indicative of a PCNSL. Using innovative CSF biomarkers can be sensitive and complementary to traditional CSF analysis and brain biopsy in the diagnosis of PCNSL, potentially allowing for earlier diagnosis and treatment.

Logistic regression modeling of cytokines for cerebrospinal fluid evaluation in primary central nervous system lymphoma.

BACKGROUND: The diagnostic utility of cerebrospinal fluid (CSF) cytology encounters impediments stemming from variability in cell collection techniques and pathologists' morphological acumen, resulting in wide-ranging CSF positivity rates for primary central nervous system lymphomas (PCNSL). Such disparity impacts patient evaluation, treatment stratagem, and prognostication. Thus, this study endeavors to explore liquid biomarkers complementary to CSF cytology or immunophenotype analysis in the diagnosis of CSF involvement. METHODS: 398 newly diagnosed PCNSL patients were categorized into CSF involvement and non-involvement groups based on CSF cytology and immunophenotype analysis. Binary logistic regression analysis was performed on 338 patients to investigate factors predicting CSF involvement and to develop a joint prediction model. An additional cohort of 60 PCNSL patients was recruited for model validation. Statistical analyses included the Mann-Whitney U test for comparing various CSF parameters between two groups. ROC curve analyses were performed for each biomarker to identify PCNSL CSF involvement. RESULTS: The cytokine IL-10 level in CSF has emerged as the most promising biomarker for CSF evaluation, boasting an ROC AUC of 0.922. C-TNFα and soluble C-IL2R demonstrate efficacy in quantifying tumor burden within the CSF. Logistic regression identified C-IL10lg (OR = 30.103, P < 0.001), C-TNC (OR = 1.126, P < 0.001), C-IL2Rlg (OR = 3.743, P = 0.029) as independent predictors for CSF involvement, contributing to a joint predictive model with an AUC of 0.935, sensitivity of 74.1 %, and specificity of 93.0 %. Validation of the model in an independent cohort confirmed its effectiveness, achieving an AUC of 0.9713. CONCLUSIONS: The identification of these feasible biomarkers and the development of an accurate prediction model may facilitate the precise evaluation of CSF status in PCNSL, offering significant advancements in patient management.

Preoperative Corticosteroids Reduce Diagnostic Accuracy of Stereotactic Biopsies in Primary Central Nervous System Lymphoma: A Systematic Review and Meta-Analysis.

BACKGROUND AND OBJECTIVES: Despite general acceptance that corticosteroid therapy (CST) should be withheld before biopsy for suspected primary central nervous system lymphoma (PCNSL), there remains conflicting evidence surrounding the precise impact of preoperative CST on the histopathological diagnosis. The objective of this systematic review and meta-analysis was to describe and quantify the effects of preoperative CST on the diagnostic accuracy of biopsies for PCNSL. METHODS: Primary articles were screened from Ovid MEDLINE, Embase, Web of Science, and Scopus databases. Meta-analysis was performed for immunocompetent patients with histologically confirmed PCNSL. Subgroup and regression analyses were performed to assess the effects of biopsy type, CST duration, dose, and preoperative taper on the diagnostic accuracy. In addition, the sensitivity of cerebrospinal fluid (CSF) analyses for PCNSL was assessed. RESULTS: Nineteen studies, comprising 1226 patients (45% female; mean age: 60.3 years), were included. Preoperative CST increased the risk of nondiagnostic biopsy with a relative risk (RR) of 2.1 (95% CI: 1.1-4.1). In the stereotactic biopsy subgroup, the RR for nondiagnostic biopsy was 3.0 (95% CI: 1.2-7.5). CST taper, duration, and dose did not significantly influence diagnostic biopsy rates. The sensitivity of CSF cytology, including flow cytometry, for PCNSL was 8.0% (95% CI: 6.0%-10.7%). CONCLUSION: Our results suggest that preoperative CST reduces the diagnostic yield of stereotactic biopsies for PCNSL. We found no evidence that tapering CST before biopsy improves diagnostic rates. CSF analysis currently has a poor sensitivity for the diagnosis of PCNSL.

Prognostic Value of CSF IL-10 at Early Assessment of Induction Chemotherapy in Primary CNS Lymphomas: A LOC Network Study.

OBJECTIVES: Despite a high response rate at the first evaluation during induction chemotherapy, the risk of early relapse remains high and unpredictable in primary CNS lymphomas (PCSNLs). We aimed to assess the prognostic value of early IL-10 levels in CSF (e-IL-10) after 2 months of induction chemotherapy. METHODS: We retrospectively selected from the LOC (Lymphomes Oculo-Cérébraux) network database patients with PCSNLs who had complete or partial response at the 2-month evaluation of a high-dose methotrexate-based first-line chemotherapy for whom e-IL-10 was available. RESULTS: Thirty patients (median age: 62 years, brain involvement in 30/30, CSF involvement in 10/30, median baseline CSF IL-10: 27.5 pg/mL) met the selection criteria. e-IL-10 was undetectable in 22 patients and detectable in 8 patients. At the end of induction treatment, 7 of 8 and 4 of 22 of the patients with detectable and undetectable e-IL-10 had experienced progressive disease, respectively (p = 0.001, OR: 26.8, 95% CI 2-1,478). The median progression-free survival times were 5.8 months (95% CI 2.8-8.8) and 28.7 months (95% CI 13.4-43.9) in the groups with detectable and undetectable e-IL-10, respectively (p < 0.001). DISCUSSION: Our results suggest that despite an objective response, the persistence of detectable e-IL-10 is associated with a high risk of early relapse in PCNSL. A closer follow-up of such patients is warranted.

Rapid tumor DNA analysis of cerebrospinal fluid accelerates treatment of central nervous system lymphoma.

ABSTRACT: Delays and risks associated with neurosurgical biopsies preclude timely diagnosis and treatment of central nervous system (CNS) lymphoma and other CNS neoplasms. We prospectively integrated targeted rapid genotyping of cerebrospinal fluid (CSF) into the evaluation of 70 patients with CNS lesions of unknown cause. Participants underwent genotyping of CSF-derived DNA using a quantitative polymerase chain reaction-based approach for parallel detection of single-nucleotide variants in the MYD88, TERT promoter, IDH1, IDH2, BRAF, and H3F3A genes within 80 minutes of sample acquisition. Canonical mutations were detected in 42% of patients with neoplasms, including cases of primary and secondary CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma, and H3K27M-mutant diffuse midline glioma. Genotyping results eliminated the need for surgical biopsies in 7 of 33 cases (21.2%) of newly diagnosed neoplasms, resulting in significantly accelerated initiation of disease-directed treatment (median, 3 vs 12 days; P = .027). This assay was then implemented in a Clinical Laboratory Improvement Amendments environment, with 2-day median turnaround for diagnosis of CNS lymphoma from 66 patients across 4 clinical sites. Our study prospectively demonstrates that targeted rapid CSF genotyping influences oncologic management for suspected CNS tumors.

Systematic Review of Cerebrospinal Fluid Biomarker Discovery in Neuro-Oncology: A Roadmap to Standardization and Clinical Application.

Effective diagnosis, prognostication, and management of CNS malignancies traditionally involves invasive brain biopsies that pose significant risk to the patient. Sampling and molecular profiling of cerebrospinal fluid (CSF) is a safer, rapid, and noninvasive alternative that offers a snapshot of the intracranial milieu while overcoming the challenge of sampling error that plagues conventional brain biopsy. Although numerous biomarkers have been identified, translational challenges remain, and standardization of protocols is necessary. Here, we systematically reviewed 141 studies (Medline, SCOPUS, and Biosis databases; between January 2000 and September 29, 2022) that molecularly profiled CSF from adults with brain malignancies including glioma, brain metastasis, and primary and secondary CNS lymphomas. We provide an overview of promising CSF biomarkers, propose CSF reporting guidelines, and discuss the various considerations that go into biomarker discovery, including the influence of blood-brain barrier disruption, cell of origin, and site of CSF acquisition (eg, lumbar and ventricular). We also performed a meta-analysis of proteomic data sets, identifying biomarkers in CNS malignancies and establishing a resource for the research community.

Liquid biopsy for improving diagnosis and monitoring of CNS lymphomas: A RANO review.

BACKGROUND: The utility of liquid biopsies is well documented in several extracranial and intracranial (brain/leptomeningeal metastases, gliomas) tumors. METHODS: The RANO (Response Assessment in Neuro-Oncology) group has set up a multidisciplinary Task Force to critically review the role of blood and cerebrospinal fluid (CSF)-liquid biopsy in CNS lymphomas, with a main focus on primary central nervous system lymphomas (PCNSL). RESULTS: Several clinical applications are suggested: diagnosis of PCNSL in critical settings (elderly or frail patients, deep locations, and steroid responsiveness), definition of minimal residual disease, early indication of tumor response or relapse following treatments, and prediction of outcome. CONCLUSIONS: Thus far, no clinically validated circulating biomarkers for managing both primary and secondary CNS lymphomas exist. There is need of standardization of biofluid collection, choice of analytes, and type of technique to perform the molecular analysis. The various assays should be evaluated through well-organized central testing within clinical trials.

Mass spectrometry-based proteomics of cerebrospinal fluid in pediatric central nervous system malignancies: a systematic review with meta-analysis of individual patient data.

BACKGROUND: The cerebrospinal fluid (CSF) proteome could offer important insights into central nervous system (CNS) malignancies. To advance proteomic research in pediatric CNS cancer, the current study aims to (1) evaluate past mass spectrometry-based workflows and (2) synthesize previous CSF proteomic data, focusing on both qualitative summaries and quantitative re-analysis. MAIN: In our analysis of 11 studies investigating the CSF proteome in pediatric patients with acute lymphoblastic leukemia (ALL) or primary brain tumors, we observed significant methodological variability. This variability negatively affects comparative analysis of the included studies, as per GRADE criteria for quality of evidence. The qualitative summaries covered 161 patients and 134 non-tumor controls, while the application of validation cohort varied among the studies. The quantitative re-analysis comprised 15 B-ALL vs 6 "healthy" controls and 15 medulloblastoma patients vs 22 non-tumor controls. Certain CSF proteins were identified as potential indicators of specific malignancies or stages of neurotoxicity during chemotherapy, yet definitive conclusions were impeded by inconsistent data. There were no proteins with statistically significant differences when comparing cases versus controls that were corroborated across studies where quantitative reanalysis was feasible. From a gene ontology enrichment, we observed that age disparities between unmatched case and controls may mislead to protein correlations more indicative of age-related CNS developmental stages rather than neuro-oncological disease. Despite efforts to batch correct (HarmonizR) and impute missing values, merging of dataset proved unfeasible and thereby limited meaningful data integration across different studies. CONCLUSION: Infrequent publications on rare pediatric cancer entities, which often involve small sample sizes, are inherently prone to result in heterogeneous studies-particularly when conducted within a rapidly evolving field like proteomics. As a result, obtaining clear evidence, such as CSF proteome biomarkers for CNS dissemination or early-stage neurotoxicity, is currently impractical. Our general recommendations comprise the need for standardized methodologies, collaborative efforts, and improved data sharing in pediatric CNS malignancy research. We specifically emphasize the possible importance of considering natural age-related variations in CSF due to different CNS development stages when matching cases and controls in future studies.

Intrathecal IgM synthesis as a diagnostic marker in patients with suspected CNS lymphoma.

For CNS lymphomas (CNSL), there is a high need for minimally invasive and easily obtainable diagnostic markers. Intrathecal IgM synthesis can easily be determined in routine CSF diagnostics. The aim of this study was to systematically investigate the diagnostic potential of intrathecal IgM synthesis in primary and secondary CNSL (PCNSL and SCNSL). In this retrospective study, patients with a biopsy-proven diagnosis of PCNSL or SCNSL were compared with patients with other neurological diseases in whom CNSL was initially the primary radiological differential diagnosis based on MRI. Sensitivity and specificity of intrathecal IgM synthesis were calculated using receiver operating characteristic curves. Seventy patients with CNSL were included (49 PCNSL and 21 SCNSL) and compared to 70 control patients. The sensitivity and specificity for the diagnosis of CNSL were 49% and 87%, respectively, for the entire patient population and 66% and 91% after selection for cases with tumor access to the CSF system and isolated intrathecal IgM synthesis. In cases with MRI-based radiological suspicion of CNSL, intrathecal IgM synthesis has good specificity but limited sensitivity. Because of its low-threshold availability, analysis of intrathecal IgM synthesis has the potential to lead to higher diagnostic accuracy, especially in resource-limited settings, and deserves further study.

Patterns and prognostic impact of CNS infiltration in adults with newly diagnosed acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is highly associated with central nervous system (CNS) infiltration and can be associated with higher risk of relapse. Conventional cytology (CC) is the traditional method for diagnosing CNS infiltration, although the use of immunophenotyping by flow cytometry (FC) has gained prominence in recent years due to its higher sensitivity. Also, some authors have proposed that CSF contamination by a traumatic lumbar puncture (TLP) could have a clinical impact. This retrospective study accessed the impact of CNS infiltration by CC or FC on overall survival, event-free survival, and relapse rate. In a cohort of 105 newly diagnosed ALL patients, CNS1, CNS2, and CNS3 status were found in 84%, 14%, and 2%, respectively. We found that extramedullary disease at the diagnosis, higher leukocyte counts, and higher blast percentage were associated with a positive CC. Sensitivity and specificity of CC were 53% and 98%, respectively. Three-year overall survival was 42.5%. Conversely, TLP was not associated with a positive CC nor had an impact on relapse rates. In multivariate analysis, a positive CC was associated with an increased relapse rate (HR 2.074, p = 0.037), while its detection by FC did not associate with this endpoint. Survival rates seem to be increasing over the last years by the adoption of a stratified CNS prophylaxis risk strategy. CSF contamination does not represent a major concern according to our report, as it did not increase CNS involvement or relapse rates.

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.

Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid.

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.

Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases.

BACKGROUND: Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. METHODS: In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. RESULTS: Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). CONCLUSIONS: These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Phosphoproteome analysis of cerebrospinal fluid extracellular vesicles in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare but highly aggressive extra-nodal non-Hodgkin's lymphoma, mostly of the diffuse large B-cell lymphoma (DLBCL) type. The present invasive diagnosis and poor prognosis of PCNSL propose an urgent need to develop molecular markers for early detection, real-time monitoring and treatment evaluation. Cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) are promising biomarker carriers for liquid biopsy of CNS diseases and brain tumors; however, research remains challenging due to the low concentration of EVs in the limited available volume of CSF from each individual patient and the low efficiency of existing methods for EV enrichment. Here, we introduce functionalized magnetic beads called EVTRAP (extracellular vesicles total recovery and purification) for rapid and efficient EV isolation from CSF. By coupling with high-performance mass spectrometry, over 19 000 peptides representing 1841 proteins were identified from just 30 µL of CSF. Furthermore, up to 3000 phosphopeptides representing over 1000 phosphoproteins were identified from about 2 mL of CSF. Finally, we analyzed the EV phosphoproteomics of CSF samples from PCNSL patients and non-PCNSL controls. Among them, multiple phosphoproteins related to PCNSL, including SPP1, MARCKS, NPM1 and VIM, were shown to be up-regulated in the PCNSL group. These results demonstrated the feasibility of the EVTRAP-based analytical strategy in CSF EV phosphoproteomic analysis of PCNSL molecular markers.

A review on genetic alterations in CNS metastases related to breast cancer treatment. Is there a role for liquid biopsies in CSF?

Acquired mutations or altered gene expression patterns in brain metastases (BM) and/or leptomeningeal metastases (LM) of breast cancer may play a role in therapy-resistance and offer new molecular targets and treatment options. Despite expanding knowledge of genetic alterations in breast cancer and their metastases, clinical applications for patients with central nervous system (CNS) metastases are currently limited. An emerging tool are DNA-techniques that may detect genetic alterations of the CNS metastases in the cerebrospinal fluid (CSF). In this review we discuss genetic studies in breast cancer and CNS metastases and the role of liquid biopsies in CSF.

Ex vivo NMR metabolomics approach using cerebrospinal fluid for the diagnosis of primary CNS lymphoma: Correlation with MR imaging characteristics.

PURPOSE: Primary central nervous system lymphoma (PCNSL) is an uncommon extranodal non-Hodgkin's lymphoma. Here, the feasibility of nuclear magnetic resonance (NMR) metabolomics for the diagnosis and prognosis prediction of PCNSL, as well as its correlation with magnetic resonance imaging (MRI) characteristics, was assessed. PATIENTS AND METHODS: Cerebrospinal fluid (CSF) samples from PCNSL and normal groups (n = 41 for each) were obtained along with MRI data including pre- and postcontrast as well as T1-, T2-, and diffusion-weighted imaging for the treatment-naïve PCNSL patients (n = 24). The CSF samples were analyzed using nuclear magnetic resonance (NMR). RESULTS: The CSF NMR metabolomic exhibited clear differences with a diagnostic sensitivity of 100% and a specificity of 97.6%. The citrate level of the leptomeningeal enhancement (LE) (+) group was significantly lower than that of the LE (-) group (p = 0.018). In addition, the MRI apparent diffusion coefficient (ADC) value of the tumor was positively correlated with the glucose level (p = 0.025). However, none of the marker metabolites were significant prognosis predictors in univariate analysis. CONCLUSIONS: In conclusion, the NMR metabolomics could be helpful to diagnose PCNSL, but not for the prognosis, and MRI features (LE or ADC) can reflect the metabolic profiles of PCNSL.