Cerebrospinal Fluid Supernatant Research Articles

Subarachnoid haemorrhage or traumatic lumbar puncture. Differentiation by cerebrospinal fluid parameters in a multivariable approach

Lumbar puncture (LP) is recommended in patients with thunderclap headache and negative computed tomography to rule out spontaneous subarachnoid haemorrhage (SAH). Blood contamination of cerebrospinal fluid (CSF) due to traumatic LP poses a diagnostic dilemma. Therefore, routine CSF parameters were investigated to distinguish between SAH and a traumatic LP. CSF red blood cell (RBC), white blood cell (WBC) count, total protein, CSF colour and supernatant were used for group comparisons of patients with SAH and ‘symptomatic controls’. Due to variable time intervals between bleeding onset and LP in SAH patients in contrast to patients with traumatic LP, where blood contamination of CSF occurs at the time of LP, CSF variables were adjusted for decay in time to allow comparability. Logistic regression analysis identified bloody CSF [odds ratio (OR) 32.6], xanthochromic supernatant [OR 15.5] and WBCadjusted [OR 4.5 (per increase of 100/µl)] as predictors of SAH, while age, sex and CSF total proteinadjusted were no predictors. Optimal cut-point of RBCadjusted (determined at day 1 after bleeding) was > 3667/µl to identify SAH patients with a 97% sensitivity and 94% specificity. Combination of low RBC and clear CSF supernatant was found in none of SAH patients. Combined CSF RBC count and CSF supernatant reliably distinguished traumatic LP from SAH.

Comparison of mutation detection rate between cell pellet DNA and cell-free DNA from cerebrospinal fluid in patients with advanced lung cancer.

e21014 Background: Liquid biopsy of cerebrospinal fluid (CSF) has been approved to be a promising strategy for molecular pathology testing in patients with lung cancer. Numerous studies showed that CSF-derived DNA can recapitulate the molecular characteristics of metastatic lung cancer. However, differences in mutation detection rate and the overall mutational profile between cell pellet DNA and supernatant cell-free DNA (cfDNA) from CSF in patients with lung cancer have rarely been reported. Methods: Matched CSF cell pellet and supernatant sample sets were selected from 51 lung cancer patients in our sequencing center. CSF was centrifuged (1000g, 10 min) to separate the cell pellet and supernatant components. Cellular DNA and cfDNA were extracted and underwent library preparation procedures for next-generation sequencing respectively. Cut adapter, mapping, fusion calling and cnv calling were performed for sequencing data analysis. Results: At the sample level, gene mutation could be detected in both CSF cell pellet and cfDNA, and the positive detection rate of gene mutation was consistent. The positive percent agreement (PPA) of CSF cfDNA sequencing compared with CSF cell pellet DNA was 93%. At the genetic variation level, 223 gene mutation sites were detected, and the positive rate of gene mutation in the CSF cfDNA (32.51%) was significantly higher than that in the CSF cell pellet (10.74%, p＜0.01). Additionally，the variation rate of less than 1% was defined as the low-frequency mutation range. Results showed that variant allele frequency (VAF) in CSF cfDNA were remarkably higher than those in the CSF cell pellet in both low-and high-frequency mutation ranges ( p＜0.01, p＜0.01). Conclusions: Both cell pellet DNA and cfDNA isolated from CSF can be used for molecular subtyping of lung cancer. Given that CSF is inevitably contaminated with blood cells in clinical procedures, the dynamic range of tumor cells in CSF could be quite large. CSF cfDNA better recapitulate the mutational status and is recommended to consider first for liquid biopsy in advanced lung cancer with brain metastases or suspicious brain metastases. Key words: Next-generation sequencing; CSF; cell pellet DNA; cfDNA; advanced lung cancer.

Cerebrospinal Fluid Biomarkers Associated with Intrathecal Cell Therapy in Patients with Progressive Multiple Sclerosis (P1-3.001)

<h3>Objective:</h3> To identify cerebrospinal fluid (CSF) biomarkers of clinical response to intrathecal (IT) mesenchymal stem cell-neural progenitor (MSC-NP) treatment in patients with progressive multiple sclerosis (MS). <h3>Background:</h3> MSC-NPs are a bone marrow-derived population of cells with trophic and immunomodulatory properties with therapeutic potential in MS. A phase II randomized, double-blind, placebo-controlled clinical trial was performed to investigate whether multiple intrathecal (IT) injections of autologous MSC-NPs was associated with clinical efficacy. Study subjects received either 6 IT injections of autologous MSC-NPs or 6 placebo injections spaced 2 months apart. In the crossover study design, subjects treated with placebo in the first year were treated with MSC-NPs in the second year, and vice versa. Safety and clinical efficacy will be reported elsewhere. <h3>Design/Methods:</h3> For biomarker discovery, CSF was collected from 36 study subjects at the time of their first and sixth IT-MSC-NP treatment reflecting pre-treatment and post-treatment timepoints, respectively. CSF supernatants were analyzed using SOMAScan multiplex proteomic assay. Differentially expressed proteins were identified using a multivariate linear mixed effect model. Biomarkers were validated using ELISA/Luminex assays. Treatment responders were defined by improvement in any of the following outcomes: EDSS, timed 25-foot walk, 9-hole peg test, 6-minute walk test, or muscle strength. <h3>Results:</h3> Proteomic analysis of CSF identified matrix metalloproteinase-9 (MMP-9), complement C3a and the chemokine CCL2 as potential biomarkers of IT-MSC-NP treatment. MMP-9 was significantly increased post-treatment and was observed independent of clinical response. Relative concentration of C3a, C3 and C3b were decreased post-treatment and subgroup analysis demonstrated significance only in the responder group. CCL2 was significantly decreased post-treatment with higher significance associated with clinical response. Decreased CCL2 was validated in an independent cohort of patients treated with IT-MSC-NPs. <h3>Conclusions:</h3> These results identify multiple CSF biomarkers of IT-MSC-NP treatment that may correlate with therapeutic response to this novel cell therapy in MS. <b>Disclosure:</b> Miss Kizilbash has nothing to disclose. Ms. Carlson has nothing to disclose. Violaine K. Harris, PhD has nothing to disclose. Dr. Sadiq has nothing to disclose.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Exploring genetic alterations in circulating tumor DNA from cerebrospinal fluid of pediatric medulloblastoma

Medulloblastoma (MB) is the most common type of brain malignancy in children. Molecular profiling has become an important component to select patients for therapeutic approaches, allowing for personalized therapy. In this study, we successfully identified detectable levels of tumor-derived cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) samples of patients with MB. Furthermore, cfDNA from CSF can interrogate for tumor-associated molecular clues. MB-associated alterations from CSF, tumor, and post-chemotherapy plasma were compared by deep sequencing on next-generation sequencing platform. Shared alterations exist between CSF and matched tumor tissues. More alternations were detected in circulating tumor DNA from CSF than those in genomic DNA from primary tumor. It was feasible to detect MB-associated mutations in plasma of patients treated with chemotherapy. Collectively, CSF supernatant can be used to monitor genomic alterations, as a superior technique as long as tumor-derived cfDNA can be isolated from CSF successfully.

Utility of Cerebrospinal Fluid Cell-Free DNA in Patients with EGFR-Mutant Non-Small-Cell Lung Cancer with Leptomeningeal Metastasis.

Leptomeningeal metastasis (LM) is a fatal complication of advanced non-small-cell lung cancer (NSCLC). The aim of this study was to evaluate the utility of cerebrospinal fluid (CSF) as a medium for epidermal growth factor receptor (EGFR) mutation testing in clinical practice. We prospectively enrolled patients with EGFR-mutant NSCLC who underwent CSF sampling for suspected LM. The supernatant of CSF after routine cytology examination was collected. The diagnosis of LM was established according to EANO-ESMO criteria. CSF and plasma cell-free DNA (cfDNA) were retrieved for EGFR mutation testing. Fifty-one patients with a median age of 62.7 years were enrolled. The median duration from initial diagnosis to CSF sampling was 23.0 months and most patients (94.1%) had received at least one EGFR-tyrosine kinase inhibitor. Adenocarcinoma cells were found in 37 CSF samples (72.5%), and 48 (94.1%) patients had confirmed or probable LM. Thirty-five of these 48 patients (72.9%) had valid EGFR mutation-testing results using CSF cfDNA and tended to have higher white blood cell counts and positive cytology in their CSF compared to those with invalid mutation testing results. The overall detection rate of EGFR mutation in CSF cfDNA was 68.8%, and the T790M detection rate was 14.6%. In 37 patients with paired CSF and plasma samples, the concordance rate of the EGFR mutation results was 29.7%. For patients with EGFR-mutant NSCLC with LM, CSF supernatant is a valuable source for EGFR mutation testing and may provide important information.

Immunomodulatory lipid mediator profiling of cerebrospinal fluid following surgery in older adults

Arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) derived lipids play key roles in initiating and resolving inflammation. Neuro-inflammation is thought to play a causal role in perioperative neurocognitive disorders, yet the role of these lipids in the human central nervous system in such disorders is unclear. Here we used liquid chromatography–mass spectrometry to quantify AA, DHA, and EPA derived lipid levels in non-centrifuged cerebrospinal fluid (CSF), centrifuged CSF pellets, and centrifuged CSF supernatants of older adults obtained before, 24 h and 6 weeks after surgery. GAGE analysis was used to determine AA, DHA and EPA metabolite pathway changes over time. Lipid mediators derived from AA, DHA and EPA were detected in all sample types. Postoperative lipid mediator changes were not significant in non-centrifuged CSF (p > 0.05 for all three pathways). The AA metabolite pathway showed significant changes in centrifuged CSF pellets and supernatants from before to 24 h after surgery (p = 0.0000247, p = 0.0155 respectively), from before to 6 weeks after surgery (p = 0.0000497, p = 0.0155, respectively), and from 24 h to 6 weeks after surgery (p = 0.0000499, p = 0.00363, respectively). These findings indicate that AA, DHA, and EPA derived lipids are detectable in human CSF, and the AA metabolite pathway shows postoperative changes in centrifuged CSF pellets and supernatants.

Accumulation of Cerebrospinal Fluid Glycerophospholipids and Sphingolipids in Cognitively Healthy Participants With Alzheimer's Biomarkers Precedes Lipolysis in the Dementia Stage.

Insight into lipids’ roles in Alzheimer’s disease (AD) pathophysiology is limited because brain membrane lipids have not been characterized in cognitively healthy (CH) individuals. Since age is a significant risk factor of AD, we hypothesize that aging renders the amyloid precursor protein (APP) more susceptible to abnormal processing because of deteriorating membrane lipids. To reflect brain membranes, we studied their lipid components in cerebrospinal fluid (CSF) and brain-derived CSF nanoparticle membranes. Based on CSF Aβ42/Tau levels established biomarkers of AD, we define a subset of CH participants with normal Aβ42/Tau (CH-NAT) and another group with abnormal or pathological Aβ42/Tau (CH-PAT). We report that glycerophospholipids are differentially metabolized in the CSF supernatant fluid and nanoparticle membrane fractions from CH-NAT, CH-PAT, and AD participants. Phosphatidylcholine molecular species from the supernatant fraction of CH-PAT were higher than in the CH-NAT and AD participants. Sphingomyelin levels in the supernatant fraction were lower in the CH-PAT and AD than in the CH-NAT group. The decrease in sphingomyelin corresponded with an increase in ceramide and dihydroceramide and an increase in the ceramide to sphingomyelin ratio in AD. In contrast to the supernatant fraction, sphingomyelin is higher in the nanoparticle fraction from the CH-PAT group, accompanied by lower ceramide and dihydroceramide and a decrease in the ratio of ceramide to sphingomyelin in CH-PAT compared with CH-NAT. On investigating the mechanism for the lipid changes in AD, we observed that phospholipase A2 (PLA2) activity was higher in the AD group than the CH groups. Paradoxically, acid and neutral sphingomyelinase (SMase) activities were lower in AD compared to the CH groups. Considering external influences on lipids, the clinical groups did not differ in their fasting blood lipids or dietary lipids, consistent with the CSF lipid changes originating from brain pathophysiology. The lipid accumulation in a prodromal AD biomarker positive stage identifies perturbation of lipid metabolism and disturbances in APP/Amyloid beta (Aβ) as early events in AD pathophysiology. Our results identify increased lipid turnover in CH participants with AD biomarkers, switching to a predominantly lipolytic state in dementia. This knowledge may be useful for targeting and testing new AD treatments.

BIOM-08. TARGETSELECTORTM CEREBROSPINAL FLUID (CSF) CIRCULATING TUMOR CELLS AND BIOMARKER ANALYSIS: IMPROVING SENSITIVITY AND TARGETED TREATMENT OPTIONS IN BREAST AND NSCLC CANCER PATIENTS WITH CNS INVOLVEMENT

Abstract BACKGROUND Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal disease (LMD). Here we report the analytical and clinical validation of Target Selector™ CSF assays. METHODS CSF was collected prospectively from pts with a prior solid tumor diagnosis and confirmed or clinical/ radiological suspicion of BM or LMD. CTCs were captured in microfluidic channel and classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) NSCLC and breast multi-gene testing. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software. RESULTS The Target Selector™ CTC platform assays performed on clinical samples (n = 89) resulted in clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0% at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%. CONCLUSIONS Target Selector™ is a viable and sensitive platform for CTC detection and molecular analysis of CSF samples from patients with NSCLC or breast cancer with CNS metastases compared to the current standard of care (CSF cytology) and when imaging findings are equivocal. Identifying CTCs and actionable biomarkers can help to confirm CNS involvement when clinically suspected, guide targeted therapy selection and potentially monitor treatment response.

Interleukin-6–Mediated Inflammation May Cause Methotrexate-Induced Leukoencephalopathy

Children with leukemia treated with methotrexate (MTX) may develop MTX-induced leukoencephalopathy, which can present as seizures or focal neurological deficits. However, the precise pathophysiology has not been fully elucidated. Differences in cytokine/chemokine profiles in cerebrospinal fluid (CSF) between children with MTX-induced leukoencephalopathy and those with posterior reversible encephalopathy syndrome (PRES), an acute neurological condition associated with hypertension, were investigated. Interleukin (IL)-1β, 2, 4, 5, 6, 7, 8, 10, 12, 13, and 17, tumor necrosis factor-alpha, interferon-gamma, granulocyte monocyte colony-stimulating factor, granulocyte colony-stimulating factor, macrophage inflammatory protein-1β, and monocyte chemoattractant protein-1 concentrations were measured in CSF supernatants from 3 children with acute leukemia with MTX-induced leukoencephalopathy, 3 children with acute leukemia with PRES, 6 children with acute leukemia without neurological complications, and 8 children with acute encephalopathy. CSF IL-6 concentrations were higher in children with MTX-induced leukoencephalopathy than in children with acute leukemia with PRES, with acute leukemia without neurological complications, and with acute encephalopathy. We concluded that IL-6 may be involved in the pathogenesis of MTX-induced leukoencephalopathy.

The next generation of cerebrospinal fluid (CSF)-based molecular diagnostics: Improving sensitivity and actionability in breast and lung cancer patients with CNS involvement.

e14502 Background: Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with surgical biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal metastases (LM). Here we report the analytical and clinical validation of Target Selector CSF assays. Validation testing included pre-analytical and analytical steps. Methods: CSF was collected prospectively from pts with a prior solid tumor diagnosis and suspected BM or LM. CTCs were captured utilizing a primary ten antibody cocktail followed by biotinylated secondary antibodies that bind selectively to CTCs followed by staining with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs were captured in a microfluidic channel,classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) lung and breast multi-gene testing to assess for molecular alterations. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software. Results: The Target Selector CTC platform assays performed on clinical samples (n = 89) resulted in clinical accuracy = 85.4%, clinical precision (intra-assay, inter-assay, inter-operator, and inter-instrument) = 100% for each measure, clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0%, and analytical specificity = 96.0% (acceptance criteria was 95%) at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical accuracy = 87.4%, clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%. Conclusions: Target Selector is a viable, sensitive, reproducible platform for CTC detection and molecular analysis of CSF samples from patients with breast or lung cancer with CNS metastases especially as the sensitivity of CSF cytology is low and MRI findings can be equivocal. Identifying CTCs and molecular alterations can help characterize both tumor genomic evolution as well as guide treatment following cancer metastasis to the CNS.

Study of epidermal growth factor receptor gene mutation in liquid biopsy of cerebrospinal fluid supernatant from patients with lung adenocarcinoma

Objective To study the detection protocol of epidermal growth factor receptor(EGFR)gene mutations in cerebrospinal fluid(CSF)specimens of lung adenocarcinoma by establishing a liquid biopsy technique, and to analyze clinical value of the detection findings for clinical therapy. Methods In the retrospective study, a total of 26 CSF specimens from lung adenocarcinoma patients who harbored EGFR gene mutations(L858R and EGFR exon 19 deletion)were collected.The included patients were treated with first-generation EGFR TKIs, and had brain metastasis detected by imaging examinations.Five CSF specimens from non-tumor patients confirmed by clinical findings and cytological and pathological diagnosis were collected during the same period.EGFR gene mutations in cell-free CSF supernatant fluid were detected by using the protocol recommended in this study. Results EGFR mutations were detected in 21/26(80.8%)of cell-free supernatants from CSF specimens of lung adenocarcinoma patients, including 13 cases(61.9%)with EGFR L858R mutations and 8 cases(38.1%)with EGFR exon 19 deletion.No T790M mutation was detected in cell-free supernatants from all CSF specimens.No EGFR mutation was detected in cell-free supernatants from all CSF specimens from non-tumor patients. Conclusions Our study-established protocol could be used to detect EGFR mutations in cell-free supernatants from CSF specimens with high sensitivity and specificity.Perhaps due to the characteristics of first-generation EGFR TKIs, the detection rate of T790M mutation is low.The detection of T790M mutation in cell-free supernatants from CSF specimen may have a limited clinical value for choice of 3rd generation EGFR TKIs. Key words: Lung neoplasms; Neoplasm metastasis; Receptor, epidermal growth factor; DNA mutational analysis

2036. Plasma (1→3)-β-D-Glucan Levels Correlate with Neurocognitive Functioning in HIV-Infected Adults

BackgroundAlthough antiretroviral therapy (ART) has improved survival and morbidity, HIV-infected adults still have higher rates of non-AIDS disorders, such as neurocognitive impairment, than HIV-uninfected adults. (1–3)-b-D-Glucan (BDG) is a fungal cell wall component which serves as a plasma biomarker for fungal infection and—in the absence of fungal infections—for gut barrier integrity failure and microbial translocation. The objective of this study was to determine whether higher plasma and cerebrospinal fluid (CSF) levels of BDG are associated with neurocognitive impairment [evaluated by global deficit score (GDS)] in HIV-infected adults.MethodsWe measured levels of BDG in paired plasma and CSF samples, and compared levels with GDS, soluble urokinase plasminogen activator receptor (suPAR; a marker of monocyte activation and chronic inflammation that has previously been associated with non-AIDS disorders) and plasma CD4/CD8 ratio in a cohort of 61 HIV+ adults on suppressive ART. Study samples were collected as part of the prospective CHARTER study between 2005 and 2015 at the University of California San Diego and were stored at −80°C on the day of collection. BDG testing of blood plasma and CSF supernatant was performed at the Associates of Cape Cod, Inc., research laboratories using the Fungitell assay.ResultsMedian plasma BDG level was 18 pg/mL (range: 2–60 pg/mL), median CSF BDG level was 20 pg/mL (range: 0–830 pg/mL). Higher levels of plasma BDG were associated with more severe cognitive impairment as measured by the GDS (Spearman r = 0.35; P = 0.006, Figure). Individuals with neurocognitive impairment (i.e., GDS > 0.5, n = 33) had higher plasma BDG levels compared with unimpaired individuals (P = 0.027). Plasma levels of BDG and suPAR correlated significantly (r = 0.31, P = 0.016), while all other correlations were nonsignificant (e.g., CSF BDG and GDS [r = 0.23], plasma suPAR and GDS [r = 0.19], CSF suPAR and GDS [r = −0.022], CD4/CD8 ratio and GDS [r = −0.028]).ConclusionElevated plasma levels of BDG may be an indicator of gut barrier integrity failure and an independent biomarker associated with neurocognitive functioning in HIV+ adults on suppressive ART. Disclosures M. Finkelman, Associated of Cape Cod: Employee, Salary.

Viral antigens detectable in CSF exosomes from patients with retrovirus associated neurologic disease: functional role of exosomes

BackgroundHTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences.ResultsExosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis.ConclusionCumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.

Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) (“early ART”), and 7 participants started ART >14 months after EDI (“late ART”). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2–3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

Increased cell-free mitochondrial DNA is a marker of ongoing inflammation and better neurocognitive function in virologically suppressed HIV-infected individuals.

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (<50 copies/mL) by droplet digital PCR. We measured soluble CD14, soluble CD163, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), neopterin, and neurofilament light chain (NFL) by immunoassays in CSF supernatant or blood plasma. Higher levels of mtDNA in CSF were associated with higher levels of MCP-1 (r=0.56, p<0.01) in CSF and TNF-α (r=0.43, p<0.01) and IL-8 (r=0.44, p<0.01) in blood plasma. Subjects with a previous diagnosis of AIDS showed significantly higher levels of mtDNA (p<0.01) than subjects without AIDS. The associations between mtDNA and MCP-1 in CSF and TNF-α in blood remained significant after adjusting for previous diagnosis of AIDS (p<0.01). Additionally, higher levels of mtDNA were associated with a lower CD4 nadir (r=-0.41, p<0.01) and lower current CD4% (r=-0.34, p=0.03). Paradoxically, higher levels of mtDNA in CSF were significantly associated with better neurocognitive performance (r=0.43, p=0.02) and with less neuronal damage (i.e. lower NFL). Higher cell-free mtDNA is associated with inflammation during treated HIV infection, but the impact on neurocognitive functioning and neuronal damage remains unclear and may differ in the setting of suppressive ART.

Compartmentalized HIV rebound in the central nervous system after interruption of antiretroviral therapy.

To design effective eradication strategies, it may be necessary to target HIV reservoirs in anatomic compartments other than blood. This study examined HIV RNA rebound following interruption of antiretroviral therapy (ART) in blood and cerebrospinal fluid (CSF) to determine whether the central nervous system (CNS) might serve as an independent source of resurgent viral replication.Paired blood and CSF samples were collected longitudinally from 14 chronically HIV-infected individuals undergoing ART interruption. HIV env (C2-V3), gag (p24) and pol (reverse transcriptase) were sequenced from cell-free HIV RNA and cell-associated HIV DNA in blood and CSF using the Roche 454 FLX Titanium platform. Comprehensive sequence and phylogenetic analyses were performed to search for evidence of unique or differentially represented viral subpopulations emerging in CSF supernatant as compared with blood plasma.Using a conservative definition of compartmentalization based on four distinct statistical tests, nine participants presented a compartmentalized HIV RNA rebound within the CSF after interruption of ART, even when sampled within 2 weeks from viral rebound. The degree and duration of viral compartmentalization varied considerably between subjects and between time-points within a subject. In 10 cases, we identified viral populations within the CSF supernatant at the first sampled time-point after ART interruption, which were phylogenetically distinct from those present in the paired blood plasma and mostly persisted over time (when longitudinal time-points were available). Our data suggest that an independent source of HIV RNA contributes to viral rebound within the CSF after treatment interruption. The most likely source of compartmentalized HIV RNA is a CNS reservoir that would need to be targeted to achieve complete HIV eradication.

Assessment of Central Nervous System Involvement in Pediatric Acute Lymphoblastic Leukemia Patients Using Next-Generation Sequencing Method

Background: Successful outcome in childhood acute lymphoblastic leukemia (ALL) relies upon appropriate central nervous system (CNS)-directed therapy for treatment of subclinical or overt CNS leukemia. Patients with leukemia blasts detected in the cerebrospinal fluid (CSF) at diagnosis have poorer survival compared with patients without blasts in the CSF and require intensified intrathecal therapy to avert a higher rate of relapse. Traditionally, CSF assessment is performed by morphological analysis of CSF smears and the blasts confirmed by immunohistochemistry or flow cytometry. The confirmation tests are done at few institutions and rely on subjective operator judgment for identification of leukemic blasts. A precise definition of CNS involvement is necessary to avoid over- or under- treatment. Next-generation sequencing (NGS) is currently being used for minimal residual disease (MRD) assessment in lymphoid malignancies (Faham et al., Blood 2012). In this study, we assessed whether the NGS method could be used to detect leukemic clonotypes in CSF samples from 89 newly diagnosed pediatric ALL patients.Methods: Diagnostic bone marrow samples and paired CSF samples were obtained from 89 patients. CSF samples were collected in one tube and fractionated for Wright stained cytospins (1ml), TdT immunohistochemistry (1ml), and the remainder (3-5ml) for NGS-based MRD studies (Adaptive Biotechnologies, South San Francisco, CA). Briefly, using universal primer sets, we amplified variable, diversity, and joining gene segments from immunoglobulin (Ig) heavy chain (IGH), Ig kappa chain (IGK), and T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) loci from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then quantitated in CSF (cell pellet and supernatant) samples obtained at diagnosis. A quantitative and standardized measure of leukemic clonotype level per million leukocytes in each follow-up sample was determined.Results: Diagnostic bone marrow samples from 89 pediatric ALL patients were used to identify the tumor-specific clonotypes. At least one tumor-specific clonotype was identified in 86/89 (97%) of the patients. The IGH-VDJ assay was the most frequent gene rearrangement: at least one IGH-VDJ clonal rearrangement was detected in 66 of the diagnostic ALL samples. TRD was the second most informative receptor with a clonal rearrangement found in 43 patients, followed by TRG in 38 patients, IGK in 31 patients, TCB in 16 patients and IGH-DJ in 13 patients. The level of tumor burden associated with each leukemic clonotype was then assessed in CSF samples drawn at diagnosis from each of the 86 patients. All 40 CNS1 patients had no detectable or minimal (<10) leukemic cell equivalents. Of the 24 patients with CNS2 but TDT negative status, only 2 had >10 leukemic cell equivalents in the CSF. By contrast, 8 of 20 (40%) patients with CNS2 and TDT positive status and both patients with CNS3 had CSF with >10 leukemic cell equivalents (Figure 1).Conclusions: This study demonstrates that the NGS-based method can be used to assess CNS involvement in pediatric patients with ALL. Based on these results, NGS-based CNS assessment will be used for patient stratification in future clinical trials for pediatric ALL. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Carlton:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Validation of cerebrospinal fluid findings in aneurysmal subarachnoid hemorrhage

BackgroundRecently proposed cutoff criteria for cerebrospinal fluid (CSF) analyses might safely exclude a diagnosis of aneurysmal subarachnoid hemorrhage (aSAH). ObjectiveThe objective of this study was to examine the sensitivity of a CSF red blood cell (RBC) count greater than 2000 × 106/L (ie, 2000 RBCs per microliter) or the presence of visible CSF xanthochromia in identifying patients with aSAH. MethodsWe identified a retrospective case series of patients diagnosed with aSAH after lumbar puncture (LP) in an integrated health delivery system between January 2000 and June 2013 by chart review. All identified patients had at least 1 cerebral aneurysm that was treated with a neurosurgical or endovascular intervention during the index hospitalization. The lowest CSF RBC count was used for validation analysis. Cerebrospinal fluid color was determined by visual inspection. Xanthochromia was defined as pink, orange, or yellow pigmentation of CSF supernatant. ResultsSixty-four patients met study inclusion criteria. Of these, 17 (33%) of 52 underwent LP within 12 hours of headache onset, and 49 (84%) of 58 exhibited CSF xanthochromia. The median CSF RBC count was 63250 × 106/L. The sensitivity of a CSF RBC count of greater than 2000 × 106/L in identifying aSAH was 96.9% (95% confidence interval, 89.3%-99.1%). Additional consideration of CSF xanthochromia resulted in a sensitivity of 100% (95% confidence interval, 94.3%-100%). ConclusionsAll patients in this case series of patients with aSAH had either a CSF RBC count greater than 2000 × 106/L or visible CSF xanthochromia, increasing the likelihood that this proposed cutoff strategy may safely identify patients who warrant further investigation for an aneurysmal cause of subarachnoid hemorrhage.

Assessment of Central Nervous System Involvement in Pediatric Acute Lymphoblastic Leukemia Patients Using Next-Generation Sequencing Method

Background: Successful outcome in childhood acute lymphoblastic leukemia (ALL) relies upon appropriate central nervous system (CNS)-directed therapy for treatment of subclinical or overt CNS leukemia. Patients with leukemia blasts detected in the cerebrospinal fluid (CSF) at diagnosis require intensified intrathecal therapy to avert a higher rate of relapse and poorer survival compared with patients without blasts in the CSF. Traditionally, CSF assessment is performed by morphological analysis of CSF smears with or without TdT assay or via flow cytometry; however, both of these methods are limited in their sensitivity. Moreover, these modalities rely on subjective operator judgment for identification of leukemic blasts, which can result in limited assay reproducibility. A precise definition of CNS involvement is necessary to avoid over- or under- treatment. The LymphoSIGHT™ platform is a next-generation sequencing-based method that is being used for minimal residual disease assessment in lymphoid malignancies (Faham et al., Blood 2012). The LymphoSIGHT™ platform has a sensitivity to detect one leukemic cell per million leukocytes in peripheral blood and/or bone marrow and has been shown to have prognostic value in adult ALL patients (Logan et al., Biol Blood Marrow Transplant 2014). In this pilot study, we assessed whether this sequencing method could be used to detect leukemic clonotypes in CSF samples from newly diagnosed pediatric ALL patients.Methods: Diagnostic bone marrow samples, and paired CSF and peripheral blood samples were obtained from two patients. Using universal primer sets, we amplified variable, diversity, and joining gene segments from immunoglobulin (Ig) heavy chain (IGH), Ig kappa chain (IGK), and T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) loci from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then quantitated in CSF (cell pellet and supernatant) and peripheral blood samples obtained at diagnosis and interim treatment time points. A quantitative and standardized measure of leukemic clonotype level per million leukocytes in each follow-up sample was determined.Results: Diagnostic bone marrow samples from the two pediatric ALL patients were used to identify the tumor-specific clonotypes. Three IGH tumor clonotyes, one IGK, and one TRD tumor clonotype were observed in the first patient. Three IGH tumor clonotypes were observed in the second patient. The level of tumor burden associated with each leukemic clonotype was then assessed in serial CSF samples from each of the two patients. In the first patient, both the CSF pellet and supernatant were positive by sequencing at diagnosis. Consistent with the sequencing results, this patient was determined to have CNS involvement at the time of diagnosis based on standard testing methods. The patient cleared at Day 22, and the CSF pellet and supernatant from this time point were also negative by sequencing. Serial samples from the second patient, who was shown to lack CNS involvement by standard testing methods, were also tested. Both the CSF cell pellet and supernatant were negative at the Day 0 and Day 22 time points for this patient, showing concordance with available clinical data (Table 1).Conclusions: This study demonstrates proof of principle that the LymphoSIGHT sequencing method can be used to assess CNS involvement in pediatric patients with ALL. Further studies are warranted to assess the diagnostic and prognostic value of the sequencing method for assessment of CNS disease. The study cohort has now been expanded to include an additional 35 pediatric patients with ALL, and data will be presented.Abstract 5343. Table 1Comparison of results in CSF samples using sequencing and traditional methods.PatientTime PointStandard analysis of CSF samplesSequencing analysis of CSF samplesWBC count/µLRBC count/µLCell numberPercent blastsLeukemia molecules/1M diploid genomes (cell pellet)Leukemia molecules/1M diploid genomes (supernatant)1Day 000857431,100366,8001Day 2200330002Day 000250002Day 220032000 DisclosuresWeng:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.