Targeted multiplex validation of CSF proteomic biomarkers: implications for differentiation of PCNSL from tumor-free controls and other brain tumors.

Introduction: Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin's lymphoma that affects brain parenchyma, eyes, cerebrospinal fluid, and spinal cord. Diagnosing PCNSL can be challenging because imaging studies often show similar patterns as other brain tumors, and stereotactic brain lesion biopsy conformation is invasive and not always possible. This study aimed to validate a previous proteomic profiling (PMID: 32610669) of cerebrospinal fluid (CSF) and develop a CSF-based proteomic panel for accurate PCNSL diagnosis and differentiation. Methods: CSF samples were collected from patients of 30 PCNSL, 30 other brain tumors, and 31 tumor-free/benign controls. Liquid chromatography tandem-mass spectrometry targeted proteomics analysis was used to establish CSF-based proteomic panels. Results: Final proteomic panels were selected and optimized to diagnose PCNSL from tumor-free controls or other brain tumor lesions with an area under the curve (AUC) of 0.873 (95%CI: 0.723-0.948) and 0.937 (95%CI: 0.807- 0.985), respectively. Pathways analysis showed diagnosis panel features were significantly enriched in pathways related to extracellular matrices-receptor interaction, focal adhesion, and PI3K-Akt signaling, while prion disease, mineral absorption and HIF-1 signaling were significantly enriched with differentiation panel features. Discussion: This study suggests an accurate clinical test panel for PCNSL diagnosis and differentiation with CSF-based proteomic signatures, which may help overcome the challenges of current diagnostic methods and improve patient outcomes.

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.

Biomarkers in Cerebrospinal Fluid for the Diagnosis and Monitoring of Gliomas.

Gliomas are the most common type of malignant brain tumor and are characterized by a plethora of heterogeneous molecular alterations. Current treatments require the emergence of reliable biomarkers that will aid personalized treatment decisions and increase life expectancy. Glioma tissues are not as easily accessible as other solid tumors; therefore, detecting prominent biomarkers in biological fluids is necessary. Cerebrospinal fluid (CSF) circulates adjacent to the cerebral parenchyma and holds promise for discovering useful prognostic, diagnostic, and predictive biomarkers. In this review, we summarize extensive research regarding the role of circulating DNA, tumor cells, proteins, microRNAs, metabolites, and extracellular vesicles as potential CSF biomarkers for glioma diagnosis, prognosis, and monitoring. Future studies should address discrepancies and issues of specificity regarding CSF biomarkers, as well as the validation of candidate biomarkers.

Deciphering the causal relationship between plasma and cerebrospinal fluid metabolites and glioblastoma multiforme: a Mendelian Randomization study.

BACKGROUND: Glioblastoma Multiforme (GBM) is one of the most aggressive and fatal brain cancers. The study of metabolites could be crucial for understanding GBM's biology and reveal new treatment strategies. METHODS: The GWAS data for GBM were sourced from the FinnGen database. A total of 1400 plasma metabolites were collected from the GWAS Catalog dataset. The cerebrospinal fluid (CSF) metabolites data were collected from subsets of participants in the WADRC and WRAP studies. We utilized the inverse variance weighting (IVW) method as the primary tool to explore the causal relationship between metabolites in plasma and CSF and glioblastoma, ensuring the exclusion of instances with horizontal pleiotropy. Additionally, four supplementary analytical methods were applied to reinforce our findings. Aberrant results were identified and omitted based on the outcomes of the leave-one-out sensitivity analysis. Conclusively, a reverse Mendelian Randomization analysis was also conducted to further substantiate our results. RESULTS: The study identified 69 plasma metabolites associated with GBM. Of these, 40 metabolites demonstrated a significant positive causal relationship with GBM, while 29 exhibited a significant negative causal association. Notably, Trimethylamine N-oxide (TMAO) levels in plasma, not CSF, were found to be a significant exposure factor for GBM (OR = 3.1627, 95% CI = (1.6347, 6.1189), P = 0.0006). The study did not find a reverse causal relationship between GBM and plasma TMAO levels. CONCLUSIONS: This research has identified 69 plasma metabolites potentially associated with the incidence of GBM, among which TMAO stands out as a promising candidate for an early detectable biomarker for GBM.

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.

Development and validation of a nomogram to predict leptomeningeal metastases in lung adenocarcinoma: Cervical lymph node metastasis is an important association factor.

BACKGROUND: The goal of this study was to create a nomogram using routine parameters to predict leptomeningeal metastases (LMs) in advanced lung adenocarcinoma (LAC) patients to prevent needless exams or lumbar punctures and to assist in accurately diagnosing LMs. METHODS: Two hundred and seventy-three patients with LMs and brain metastases were retrospectively reviewed and divided into derivation (n = 191) and validation (n = 82) cohorts using a 3:7 random allocation. All LAC patients with LMs had positive cerebrospinal fluid cytology results and brain metastases confirmed by magnetic resonance imaging. Binary logistic regression with backward stepwise selection was used to identify significant characteristics. A predictive nomogram based on the logistic model was assessed through receiver operating characteristic curves. The validation cohort and Hosmer-Lemeshow test were used for internal validation of the nomogram. RESULTS: Five clinicopathological parameters, namely, gene mutations, surgery at the primary lung cancer site, clinical symptoms of the head, N stage, and therapeutic strategy, were used as predictors of LMs. The area under the curve was 0.946 (95% CI 0.912-0.979) for the training cohort and 0.861 (95% CI 0.761-0.961) for the internal validation cohort. There was no significant difference in performance between the two cohorts (p = 0.116). In the internal validation, calibration plots revealed that the nomogram predictions were well suited to the actual outcomes. CONCLUSIONS: We created a user-friendly nomogram to predict LMs in advanced lung cancer patients, which could help guide treatment decisions and reduce unnecessary lumbar punctures.

Distribution of Bevacizumab into the Cerebrospinal Fluid of Children and Adolescents with Recurrent Brain Tumors.

BACKGROUND: To date, evidence has been lacking regarding bevacizumab pharmacokinetics in the cerebrospinal fluid (CSF). OBJECTIVE: This study assessed the penetration of bevacizumab, as part of a metronomic antiangiogenic treatment regimen, into the CSF of children, adolescents, and young adults with recurrent brain tumors. PATIENTS AND METHODS: Serum and CSF concentrations, malignant cells, and vascular endothelial growth factor A (VEGF-A) were analyzed in 12 patients (5-27 years) following 10 mg/kg bevacizumab intravenous biweekly administration (EudraCT number 2009-013024-23). A population pharmacokinetic model including body weight, albumin, and tumor type as influential factors was extended to quantify the CSF penetration of bevacizumab. RESULTS: Apart from in serum (minimum concentration/maximum concentration [Cmin/Cmax] 77.0-305/267-612 mg/L, median 144/417 mg/L), bevacizumab could be quantified in the CSF (0.01-2.26 mg/L, median 0.35 mg/L). The CSF/serum ratio was 0.16 and highly variable between patients. Malignant cells could be detected in CSF before initiation of treatment in five of 12 patients; after treatment, the CSF was cleared in all patients. VEGF-A was detected in three patients before treatment (mean ± SD: 20 ± 11 pg/mL), and was still measurable in one of these patients despite treatment (16 pg/mL). CONCLUSIONS: This pharmacokinetic pilot study indicated penetration of bevacizumab into the CSF in a population of children, adolescents, and young adults with recurrent brain 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.

Single-cell atlas reveals the immunosuppressive microenvironment and Treg cells landscapes in recurrent Glioblastoma.

Patients diagnosed with glioblastoma (GBM) have the most aggressive tumor progression and lethal recurrence. Research on the immune microenvironment landscape of tumor and cerebrospinal fluid (CSF) is limited. At the single-cell level, we aim to reveal the recurrent immune microenvironment of GBM and the potential CSF biomarkers and compare tumor locations. We collected four clinical samples from two patients: malignant samples from one recurrent GBM patient and non-malignant samples from a patient with brain tumor. We performed single-cell RNA sequencing (scRNA-seq) to reveal the immune landscape of recurrent GBM and CSF. T cells were enriched in the malignant tumors, while Treg cells were predominately found in malignant CSF, which indicated an inhibitory microenvironment in recurrent GBM. Moreover, macrophages and neutrophils were significantly enriched in malignant CSF. This indicates that they an important role in GBM progression. S100A9, extensively expressed in malignant CSF, is a promising biomarker for GBM diagnosis and recurrence. Our study reveals GBM's recurrent immune microenvironment after chemoradiotherapy and compares malignant and non-malignant CSF samples. We provide novel targets and confirm the promise of liquid CSF biopsy for patients with GBM.

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.

Angiogenic potential of the cerebrospinal fluid (CSF) of patients with high-grade gliomas measured with the chick embryo chorioallantoic membrane assay (CAM)

High-grade gliomas are highly vascularized tumors. Neo-angiogenesis plays a key role in tumor growth and resistance to therapy. A cerebrospinal fluid (CSF) sample could be a useful way to obtain pro-angiogenic predictive or prognostic markers at different stages of the disease. As a first step we looked for pro-angiogenic activity in the CSF of patients with high-grade gliomas. We performed the chicken embryo chorio-allantoic membrane (CAM) assay to study the angiogenic potential of the cerebrospinal fluid (CSF), obtained either by lumbar puncture (LP) or craniotomy from six patients with high-grade brain tumors (three glioblastoma (WHO grade IV), one anaplastic oligodendroglioma (WHO grade III), two anaplastic ganglioglioma (WHO grade III)), and four healthy controls. Significantly increased neo-angiogenesis was observed on the surface of the growing CAM in the 6 patients with high-grade gliomas compared to controls (3.69 ± 1.23 versus 2.16 ± 0.97 capillaries per area (mean ± SD), p<0.005). There was no statistical difference related to the hystological grade of the tumor (WHO grade III or IV), previous treatment (radio-chemotherapy plus temozolomide, temozolomide alone or no treatment), or the site of CSF sample (surgery or lumbar puncture). Our results suggest a pro-angiogenic potential in the CSF of patients with high-grade gliomas.

Actividad angiogénica comparada de líquido cefalorraquídeo de pacientes con tumores primarios del sistema nervioso central / Compared angiogenic activity of cerebrospinal fluid of patients with primary central nervous system tumors

Introducción: Los tumores cerebrales continúan siendo una patología de mal pronóstico, lo que se refleja en la baja expectativa de vida de quienes los padecen. La angiogénesis es un proceso cardinal en el crecimiento tumoral y en la producción de metástasis. No obstante el conocimiento de los mecanismos subyacentes a la oncogénesis, las terapias continúan obteniendo resultados modestos. Está demostrado que el parénquima tumoral secreta factores que estimulan la cascada angiogénica. Sin embargo, no existen trabajos que evalúen el efecto angiogénico del líquido cefalorraquídeo (LCR). La hipótesis propuesta en este trabajo es que el LCR de los pacientes con tumores primarios del sistema nervioso central (SNC), posee en sí mismo propiedades angiogénicas. Material y Método: Estudio experimental preclínico. Se utilizó, en ciento ochenta huevos de gallina White Leghorn, el modelo de membrana alantocoriónica (MAC) de pollo. Estos huevos fueron incubados y sobre la membrana se implantaron filtros de metilcelulosa con líquidos cefalorraquídeos de pacientes con diferentes tumores cerebrales. Al grupo control se le adicionó suero fisiológico. Tras diez días de incubación se realizaron cortes histológicos de las muestras y se procedió al conteo de vasos sanguíneos en un microscopio con rejilla graduada. Resultados: Se demostró con significancia estadística (p<0,05) que existe efecto angiogénico en el LCR de pacientes con tumores primarios del SNC. Discusión: A partir de los resultados obtenidos, podemos proyectar futuros trabajos hacia nuevas terapias enfocadas en la angiogénesis diferencial. Los factores angiogénicos presentes en el LCR podrían constituir un nuevo blanco terapéutico contra los tumores cerebrales.

Introduction: Nowadays, brain tumors remains being a poor-prognosis pathology, which is reflected in the low life expectancy of the patients. The angiogenesis, is a fundamental process in the tumoral growing and the metastatic feasibility. In spite of knowing of the underlying mechanism of the oncogenesis, therapies still get poor results. It is demonstrated that tumoralparenchyma secretes factors that enhance the angiogenic cascade. However, any work has ever evaluated the angiogenic effect in the cerebrospinal fluid (CSF) itself. We hypothesize that CSF belonging of patients with primary central nervous system (CNS) brain tumors, presents angiogenic properties on their own. Material and Method: Preclinical experimental trial. In 180 eggs of White Legorn chicken it was used the chorioallantoic membrane (CAMA) assay. This eggs were incubated for tendays and over the membrane were implanted methylcellulose filters containing cerebrospinal fluid coming from patients affected by different brain tumors. The control group was instilledwith saline solution. It was performed different histological sections of the samples and then proceeded to the counting of the vessels in a microscope with a squared grille inside it. Results:We demonstrated with statistical significance (p<0.05) that CSF from primary brain tumor’s affected patients presents angiogenic effect. Discussion: Owing the presented results, we can plan future investigations targeting new therapies focused on the differential angiogenesis. The angiogenic factors in the CSF could represent a new therapeutic target against brain tumors.

Líquido cefalorraquiano no diagnóstico de metástase cérebro-meníngea de melanoma maligno derivado de nevo melanocítico gigante congênito: relato de caso / Cerebrospinal fluid in the diagnosis of the cerebro-meningeal metastasis from malignant melanoma arising from giant congenital melanocytic nevus: case report

Relatamos o caso de um paciente, de 28 anos, portador de nevo melanocítico gigante congênito(NMGC), com transformaçao em melanoma e metátase no sistema nervoso central (SNC). Descreveremos resumidamente as características patológicas de ambas as lesoes (NMGC e melanoma), a frequência de malignizaçao no NMGC, os órgaos mais frequentemente acometidos pelas metástases do melanoma - dando ênfase ao acometimento do SNC - além dos fatores que podem levar à malignizaçao do NMGC, os exames usados para o diagnóstico das metástases no SNC - ressaltando a importância do líquido cefalorraquiano - e algumas modalidades terapêutica para o melanoma com acometimento do SNC.

Cerebrospinal fluid cytomorphologic findings in 41 intracranial tumors

O objectivo fundamental desta revisäo retrospectiva dos dados clínicos e do estudo do líquido céfalo-raquidiano (LCR) de 41 doentes com tumores intracranianos diagnosticados entre 1975 e 1989 é análisar a importância do achado de células neoplásicas no LCR, principalmente quando o recurso à tomografia computadorizada e ressônancia magnética cerebrais era raro. Outro objectivo diz respeito ao estudo das alteraçöes proteicas do LCR nos tumores cerebrais. A análise do LCR compreendeu a contagem celular, a observaçäo dos quadros citomorfológicos obtidos após sedimentaçäo, o doseamento das proteínas totais e a determinaçäo dos perfis electroforéticos. Encontraram-se células tumorais em 12 doentes (29 por cento): meduloblastomas - 6, meningites carcinomatosas - 3, glioblatoma multiforme - 1, ependimoma - 1, metástases cerebrais -1; em 2 casos este achado foi inesperado. Na maior parte dos casos a localizaçäo do tumor perto do sistema ventricular favoreceu a esfoliaçäo celular. Embora a pleocitose fosse rara e näo se correlacionasse com a presença de células neoplásicas, verificamos que na maioria dos casos, incluindo todos aqueles com etiologia "positiva", os quadro citomorfológicos eram patológicos. Os nossos resultados mostram que a pesquisa de células tumorais no LCR continua a ser util e o seu achado particularmente relevante quando inesperado

Linfoma primario cerebral post trasplante renal: comunicación de un caso y revisión de la literatura / Primary brain lymphoma post renal transplantation: report of a case and literature review

We report a 30 years old male, presenting eight years after a kidney transplant with intracraneal hypertension and two hyperdense masses detected in a brain CAT scan, whose histopathological study revealed a giant cell immnunoblastic lymphoma. The patient was successfully treated with chemo and radiotherapy and after 18 months of follow up there is no evidence of tumoral relapse. Immunocompromised patients specially transplant recipients, had a several fold higher incidence of malignant tumors, specially primary lymphomas of the central nervous system. These are generally of B type, are associated to Epstein Barr virus and have a high mortality. Cancer must be considered in the differential diagnosis of masses of uncertain origin in transplant recipients

Presentación primaria de reticulohistiocitosis maligna meníngea: caso clínico / Primary presentation of malignant meningeal reticulohistiocytosis: clinical case

Se presenta en detalle el caso clínico de un paciente con una meningitis reticulohistiocitaria maligna primaria, entidad no descrita previamente y tratada exitosamente con metotrexato y citosina arabinósido, administrados intratecalmente. Se destacan las múltiples posibilidades de diagnóstico diferencial y se pone énfasis en la importancia diagnóstica del estudio citomorfológico e inmunocitoquímico del líquido cefalorraquídeo

Eosinofilorraquia em tumor maligno: apresentaçäo em caso / CSF eosinophils in malignant tumor: a case report

A presença de grnaulócitos cosinófilos associados a tumores malignos que acometem o sistema nervoso tem sido raramente comunicada. O propósito deste registro é apresentar o caso de um paciente com 47 anos de idade com tumor cerebral maligno (astrocitoma grau III), cujo estudo citológico do LCR revelou quadro inflamatório com presença de elevada percentagem de granulócitos cosinófilos