Diagnostic biomarkers from proteomic characterization of cerebrospinal fluid in patients with brain malignancies.

Recent technological advances in molecular diagnostics through liquid biopsies hold the promise to repetitively monitor tumor evolution and treatment response of brain malignancies without the need of invasive surgical tissue accrual. Here, we implemented a mass spectrometry-based protein analysis pipeline which identified hundreds of proteins in 251 cerebrospinal fluid (CSF) samples from patients with four types of brain malignancies (glioblastoma, lymphoma, brain metastasis, and leptomeningeal disease [LMD]) and from healthy individuals with a focus on glioblastoma in a retrospective and confirmatory prospective observational study. CSF proteome deregulation via disruption of the blood brain barrier appeared to be largely conserved across brain tumor entities. CSF analysis of glioblastoma patients identified two proteomic clusters that correlated with tumor size and patient survival. By integrating CSF data with proteomic analyses of matching glioblastoma tumor tissue and primary glioblastoma cells, we identified potential CSF biomarkers for glioblastoma, in particular chitinase-3-like protein 1 (CHI3L1) and glial fibrillary acidic protein (GFAP). Key findings were validated in a prospective cohort consisting of 35 glioma patients. Finally, in LMD patients who frequently undergo repeated CSF work-up, we explored our proteomic pipeline as a mean to profile consecutive CSF samples. Therefore, proteomic analysis of CSF in brain malignancies has the potential to reveal biomarkers for diagnosis and therapy monitoring.

A rapid genotyping panel for detection of primary central nervous system lymphoma.

Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N = 1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0 to 617 days. Permanent histopathology confirmed PCNSL in 142 of 152 biopsies (93.4%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6% sensitivity and 100% specificity (95% confidence interval [CI]: 44.1% to 70.4% and 87.2% to 100%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8% sensitivity and 100% specificity (95% CI: 56.2%-74.5% and 83.9%-100%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL.

MYD88 L265P mutation and interleukin-10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study.

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut-MYD88) and interleukin-10 (IL-10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra-CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut-MYD88 was assessed by TaqMan-based polymerase chain reaction. IL-6 and IL-10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme-linked immunosorbent assay. Mut-MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue-CSF samples. IL-10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL-6 transcripts was negligible. In CSF samples, mut-MYD88 and high IL-10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL-6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL-10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut-MYD88 and IL-10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Extracellular Release of ILEI/FAM3C and Amyloid-ß Is Associated with the Activation of Distinct Synapse Subpopulations.

BACKGROUND: Brain amyloid-ß (Aß) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aß deposition in Alzheimer's disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aß generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aß suppressor. OBJECTIVE: This study aimed to access the mechanism underlying ILEI secretion and its effect on Aß production in the brain. METHODS: ILEI and Aß levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aß precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA. RESULTS: Extracellular release of ILEI and Aß was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aß revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aß levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aß levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aß and were reduced in AD and mild cognitive impairment. CONCLUSION: ILEI and Aß are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aß production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aß.

Prospective detection of mutations in cerebrospinal fluid, pleural effusion, and ascites of advanced cancer patients to guide treatment decisions.

Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non-small-cell lung cancer and melanoma patients. Cell-free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA-Q-PCR or next-generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty-three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine-kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies.

How we manage Bing-Neel syndrome.

Bing-Neel syndrome (BNS) is an uncommon presentation of Waldenström macroglobulinaemia (WM), seen during the course of the disease in about 1% of patients. BNS occurs when WM cells gain access to the central nervous system (CNS) causing neurological deficits. The diagnosis of BNS is suggested by the presence of radiological abnormalities, such as leptomeningeal enhancement on magnetic resonance imaging and confirmed by the presence of clonal lymphoplasmacytic cells and MYD88 L265P in the cerebrospinal fluid. The treatment of BNS requires agents with good penetration into the CNS, such as fludarabine, methotrexate and cytarabine. The novel Bruton Tyrosine Kinase inhibitor ibrutinib has shown CNS-penetrating properties, and recent data suggest a therapeutic role in BNS. In this review, we will discuss the clinical and pathological features, diagnostic criteria, treatment options and outcomes of patients with BNS.

Assessment of ctDNA in CSF may be a more rapid means of assessing surgical outcomes than plasma ctDNA in glioblastoma.

We aimed to develop a high-throughput deep DNA sequencing assay of cerebrospinal fluid (CSF) to identify clinically relevant oncogenic mutations that contribute to the development of glioblastoma (GBM) and serve as biomarkers to predict patients' responses to surgery. For this purpose, we recruited five patients diagnosed with highly suspicious GBM according to preoperative magnet resonance imaging. Subsequently, patients were histologically diagnosed with GBM. CSF was obtained through routine lumbar puncture, and plasma from peripheral blood was collected before surgery and 7 days after. Fresh tumor samples were collected using routine surgical procedures. Targeted deep sequencing was used to characterize the genomic landscape and identify mutational profile that differed between pre-surgical and post-surgical samples. Sequence analysis was designed to detect protein-coding exons, exon-intron boundaries, and the untranslated regions of 50 genes associated with cancers of the central nervous system. Circulating tumor DNAs (ctDNAs) were prepared from the CSF and plasma from peripheral blood. For comparison, DNA was isolated from fresh tumor tissues. Non-silent coding variants were detected in CSF and plasma ctDNAs, and the overall minor allele frequency (MAF) of the former corresponded to an earlier disease stage compared with that of plasma when the tumor burden was released (surgical removal). Gene mutation loads of GBMs significantly correlated with overall survival (OS, days) (Pearson correlation = -0.95, P = 0.01). We conclude that CSF ctDNAs better reflected the sequential mutational changes of driver genes compared with those of plasma ctDNAs. Deep sequencing of the CSF of patients with GBM may therefore serve as an alternative clinical assay to improve patients' outcomes.

Biomarkers of Cerebral Damage in Fatal Hypothermia: Preliminary Results.

The identification of hypothermia as the cause of death remains challenging in forensic pathology because of unspecific radiological, morphological, and biochemical results. Hyperemia, edema, and petechial hemorrhages within the cerebral parenchyma were described in cases of death by hypothermia. On the other hand, the effect of low temperatures in the brain has been speculated to cause local injuries on a cellular level with potential occurrences of necrosis and inflammation. In the study herein described, endocan, alkaline phosphatase, neuron-specific enolase, S100 protein subunit B, glial fibrillary acidic protein, and C-reactive protein were measured in postmortem serum from femoral blood and cerebrospinal fluid in a series of hypothermia fatalities and control cases. The combination of data collected failed to identify a specific biochemical profile for death by hypothermia in postmortem serum and/or the cerebrospinal fluid, thus suggesting that an alternative panel of brain damage biomarkers indicative of diffuse hypoxic brain injury needs to be defined in hypothermia fatalities.

Proteomic Biomarker Identification in Cerebrospinal Fluid for Leptomeningeal Metastases with Neurological Complications.

Leptomeningeal metastases (LM) from solid tumours, lymphoma and leukaemia are characterized by multifocal neurological deficits with a high mortality rate. Early diagnosis and initiation of treatment are essential to kerb neurological deterioration. However, this is not always possible as 25% of cerebrospinal fluid samples produce false-negative results at first cytological examination. The identification of biomarkers that allow stratification of individuals according to risk for developing LM would be a major benefit. Proteomic-based approaches are now in increasing use for this purpose, and these are reviewed in this chapter with a focus on cerebrospinal fluid (CSF) analyses. The construction of a CSF proteome disease database would also facilitate analysis of other neurological disorders.

Circulating glioma biomarkers.

Validated biomarkers for patients suffering from gliomas are urgently needed for standardizing measurements of the effects of treatment in daily clinical practice and trials. Circulating body fluids offer easily accessible sources for such markers. This review highlights various categories of tumor-associated circulating biomarkers identified in blood and cerebrospinal fluid of glioma patients, including circulating tumor cells, exosomes, nucleic acids, proteins, and oncometabolites. The validation and potential clinical utility of these biomarkers is briefly discussed. Although many candidate circulating protein biomarkers were reported, none of these have reached the required validation to be introduced for clinical practice. Recent developments in tracing circulating tumor cells and their derivatives as exosomes and circulating nuclear acids may become more successful in providing useful biomarkers. It is to be expected that current technical developments will contribute to the finding and validation of circulating biomarkers.

The relationship between cerebrospinal fluid osteopontin level and central nervous system involvement in childhood acute leukemia.

The aim of this study was to evaluate the relationship between cerebrospinal fluid (CSF) osteopontin (OPN) levels and central nervous system (CNS) involvement in children with a diagnosis of acute leukemia. The study sample consisted of 62 patients who had been diagnosed with acute leukemia. The control group consisted of 16 patients that had presented and had no malignant disease, CNS infection or chronic disease. CSF OPN levels were studied with enzyme-linked immunosorbent assay (ELISA) method. The mean CSF OPN level was 32.76±49.22 ng/ml in the patients at the time of diagnosis and 14.93±6.84 ng/ml in the control group (p>0.05). The mean CSF OPN level was 27.68±32.73 ng/ml at the time of diagnosis in the group without CNS involvement and 53.48±89.21 ng/ml in the group with CNS involvement (p>0.05). However, the CSF OPN level at the time of CNS relapse in patients who developed CNS involvement during follow-up (127.4±52 ng/ml) was significantly higher than in the group without CNS involvement at diagnosis and follow-up (mean CSF OPN level: 27.68±32.73 ng/ml) (p<0.001). The analysis of CSF OPN levels at the time of diagnosis-before relapse and at the periods of relapse and remission in patients who had CNS involvement at diagnosis and/or follow-up revealed statistically significant differences between the time points (p<0.001). High CSF OPN levels in childhood acute leukemia patients may be used as evidence for CNS involvement, and any increases found in CSF OPN levels may be a preliminary predictor for CNS involvement.

Secretome analysis of Glioblastoma cell line--HNGC-2.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary malignant tumor of the central nervous system. We have carried out a deep analysis of the secretome of a rapidly proliferating and tumorigenic cell line HNGC-2, representing GBM, in an effort to identify proteins, which may be targeted in the plasma of GBM patients as markers for diagnosis and disease surveillance. Prefractionation of the proteins from the conditioned medium of HNGC-2 cells in SDS gels followed by LC-MS/MS analysis using an ESI-IT mass spectrometer (LTQ) led to a total of 996 protein identifications with ≥2 peptides each. Of them, 664 proteins were observed in the transcriptome of HNGC-2 cells. The dataset of 996 proteins was mapped to important functional groups, such as cellular assembly and organisation, DNA recombination and repair, and other classes. Actin cytoskeleton signalling, phosphatidyl inositol 3 kinase (PI3K/AKT) and integrin linked kinase (ILK) signalling pathways were seen as enriched pathways. Comparisons with the published secretome of cell lines from 12 different cancers, including GBM, revealed that 348 proteins shared a commonality with a secretome of at least one other cell line, 321 of which were found to contain signal sequences or transmembrane domains and 335 could be linked to a plasma membrane or extracellular localization. Through intergration of this data we arrived at a non-redundant list of 597 protein identifications with the potential for secretion either by classical secretory pathways or by non-secretory processes; 233 of them have been detected in cerebrospinal fluid or plasma as per the published literature, and 172 have been implicated in GBM or other cancers. The HNGC-2 secretome dataset could serve as a useful resource for designing a targeted investigation of GBM biomarkers in plasma.

Proteome analysis of human cerebrospinal fluid as a diagnostic biomarker in patients with meningioma.

BACKGROUND: To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a meningioma and 4 patients with a non-brain tumorous lesion were analyzed. MATERIAL/METHODS: Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spectrometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with human meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21 kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (ß2M). RESULTS: The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and ß2M were decreased. CONCLUSIONS: The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.

Diagnosis of acute leukemia in cerebrospinal fluid (CSF-acute leukemia).

Cerebrospinal fluid-acute leukemia (CSF-acute leukemia) is a frequent and serious complication in patients with acute leukemia. One of the major problems of this complication is the diagnosis process itself. CSF cytology is currently considered the gold standard for establishing the diagnosis, a technique which presents various processing limitations, seriously impacting the predictive values. In the last 11 years, studies of CSF flow cytometry analysis done in patients with acute leukemia have demonstrated superiority in comparison with CSF cytology. Although comparative studies between these two techniques have been reported since 2001, no new consensus or formal changes to the gold standard have been established for the CSF acute leukemia diagnosis. The evidence suggests that positive flow cytometry cases, considered as indeterminate cases, will behave like disease in the central nervous system (CNS). Nevertheless, we think there are some variables and considerations that must be first evaluated under research protocols before CNS relapse can be established with only one positive flow cytometry analysis in the setting of indeterminate CSF samples. This paper proposes a diagnostic algorithm and complementary strategies.

Case series: CSF LDH, proteins and electrolyte levels in patients of acute lymphocytic leukemia.

BACKGROUND: Central nervous system (CNS) involvement is common in hemoncologic diseases especially in patients with acute lymphocytic leukemia (ALL). Currently available modalities have limitations in diagnosing CNS involvement in early stages of disease and have a limited prognostic value. Raised cerebrospinal fluid (CSF) total lactate dehydrogenase (LDH) levels can predict CNS involvement in patients with various neurological disorders including CNS leukemia. METHODS: This study was conducted in 23 consecutive freshly diagnosed patients of ALL without any previous CNS disease. Analysis of CSF was done for total LDH, proteins and electrolytes in all the patients before the start of chemotherapy and when the patients were in remission or 6 weeks after chemotherapy whichever was earlier. Twenty-three age and sex matched controls were also studied to set the normal reference range. The results were analyzed statistically by Student's t test and coefficient of co-relation between CSF LDH and protein in patients with raised CSF LDH at the time of presentation was also calculated. RESULTS: CSF LDH was increased in 4 out of 6 patients with signs and symptoms of CNS involvement (Group A) and 3 of these patients also had increased CSF protein levels. 2 out of 17 patients without signs and symptoms of CNS involvement (Group B) had both elevated CSF LDH and protein levels. The increased levels came down to normal reference values after chemotherapy except in one Group A patient in whom CSF LDH remained high. However, no significant change in CSF electrolytes was noted in these patients. CONCLUSION: Raised CSF LDH and CSF protein levels may indicate CNS involvement in patients with ALL.

Intracranial germ cell tumours. A review with special reference to endocrine manifestations.

EPIDEMIOLOGY: Intracranial germ cell tumours (icGCTs) represent 3-15% of primary paediatric intracranial neoplasms with a considerable geographical variation in incidence. Ninety percent of patients diagnosed with icGCTs are under 20 years of age. PATHOLOGY: Histologic characteristics and investigation of the tumour markers ß-human chorionic gonadotropin (ß-hCG) and alpha-fetoprotein (AFP) help define the different categories of icGCTs. The tumours are divided into two major groups called germinomas and non-germinomatous GCTs (NGGCTs). CLINICAL PRESENTATION: The clinical symptoms depend on the size and location of tumour in the brain, which is most commonly in the pineal or suprasellar region. Pineal GCTs often present with neurological symptoms because of their tendency to cause increased intracranial pressure. Suprasellar GCTs are often accompanied by endocrine abnormalities such as diabetes insipidus (DI), growth retardation and precocious or delayed puberty. DIAGNOSIS: A combination of clinical findings, endocrine and tumour marker evaluation, spinal fluid cytology, magnetic resonance imaging (MRI) and biopsy helps verifying the diagnosis of an icGCT. A summary of published data (n = 97) revealed that >90% of patients at diagnosis had at least one endocrine abnormality, DI being the most common (>80%). TREATMENT: Classification of tumour is important for choice of treatment and for prognosis. A combination of chemotherapy and radiotherapy is often used, since most icGCTs have a great sensitivity to these treatment modalities. CONCLUSION: Endocrine symptoms are very frequently appearing in patients with icGCTs and they can present long before neuroimaging verification of tumour is possible. It is of the outmost importance to have the diagnosis of icGCTs in mind when children, adolescents and young adults are presenting with endocrine irregularities, because most icGCTs are very sensitive to radiotherapy and chemotherapy, and early onset of treatment is important in order to minimize morbidity and mortality.

Proteomic analysis of cerebrospinal fluid from patients with idiopathic temporal lobe epilepsy.

Proteomic analysis of cerebrospinal fluid (CSF) from patients with temporal lobe epilepsy (TLE) and controls was carried out using two-dimensional gel electrophoresis followed by liquid chromatography electrospray ionization tandem mass spectrometry. Five protein spots showed significant differential expression (p<0.05): vitamin D-binding protein (DBP) was elevated in the CSF of TLE patients whereas cathepsin D, apolipoprotein J, Fam3c, and superoxide dismutase 1 (SOD1) were decreased in the CSF of TLE patients. Additional six protein spots presented only in the CSF of epilepsy patients were identified as tetranectin (TN), talin-2, apolipoprotein E, immunoglobulin lambda light chain (IGL@), immunoglobulin kappa variable light chain 1-5 (IGKV1-5), and procollagen C-endopeptidase enhancer 1 (PCOLCE). Expression of DBP, SOD1 and talin-2 was validated by western blot. Our results may provide better understanding of the pathophysiologic mechanisms underlying epileptogenesis and possible epilepsy biomarkers.

Molecular detection of human mammaglobin in cerebrospinal fluid from breast cancer patient with leptomeningeal carcinomatosis.

Leptomeningeal (LM) carcinomatosis is an increasing clinical complication in patients with advanced breast cancer (BC). The LM carcinomatosis diagnostic procedures rely mainly on cerebrospinal fluid (CSF) cytology, although both the amount of CSF and the number of malignant cells remain limiting factors. Therefore, efforts should be made to design new highly sensitive diagnostic tools to detect malignant cells in CSF of BC patients with LM carcinomatosis. In this study, the human Mammaglobin (hMAM) mRNA amplification by RT-PCR was employed to detect metastatic cells in CSF and thus, to diagnose LM carcinomatosis in a BC patient. Our data demonstrate that hMAM transcripts are expressed in the CSF of a BC patient with LM carcinomatosis, hence making RT-PCR for hMAM a potentially suitable test to identify occult BC cells in the brain.

Protein biomarker identification in the CSF of patients with CNS lymphoma.

PURPOSE: Elucidation of the CSF proteome may yield insights into the pathogenesis of CNS disease. We tested the hypothesis that individual CSF proteins distinguish CNS lymphoma from benign focal brain lesions. METHODS: We used a liquid chromatography/mass spectrometry-based method to differentially quantify and identify several hundred CSF proteins in CNS lymphoma and control patients. We used enzyme-linked immunosorbent assay (ELISA) to confirm one of these markers in an additional validation set of 101 cases. RESULTS: Approximately 80 CSF proteins were identified and found to be present at significantly different concentrations, both higher and lower, in training and test studies, which were highly concordant. To further validate these observations, we defined in detail the expression of one of these candidate biomarkers, antithrombin III (ATIII). ATIII RNA transcripts were identified within CNS lymphomas, and ATIII protein was localized selectively to tumor neovasculature. Determination of ATIII concentration by ELISA was significantly more accurate (> 75% sensitivity; > 98% specificity) than cytology in the identification of cancer. Measurement of CSF ATIII levels was found to potentially enhance the ability to diagnose and predict outcome. CONCLUSION: Our findings demonstrate for the first time that proteomic analysis of CSF yields individual biomarkers with greater sensitivity in the identification of cancer than does CSF cytology. We propose that the discovery of CSF protein biomarkers will facilitate early and noninvasive diagnosis in patients with lesions not amenable to brain biopsy, as well as provide improved surrogates of prognosis and treatment response in CNS lymphoma and brain metastasis.