Brain tumor mutations detected in cerebral spinal fluid.

BACKGROUND: Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood-brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative "liquid biopsy" of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined. METHODS: We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease. RESULTS: We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from <5 to nearly 3000 copies/mL CSF. We identified 7 somatic mutations from the CSF of a patient with leptomeningeal disease by use of cancer panel sequencing, and the result was concordant with genetic testing on the primary tumor biopsy. CONCLUSIONS: Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.

Molecular diagnosis of central nervous system opportunistic infections in HIV-infected Zambian adults.

BACKGROUND: Knowledge of central nervous system (CNS) opportunistic infections (OIs) among people living with human immunodeficiency virus (HIV) in sub-Saharan Africa is limited. METHODS: We analyzed 1 cerebrospinal fluid (CSF) sample from each of 331 HIV-infected adults with symptoms suggestive of CNS OI at a tertiary care center in Zambia. We used pathogen-specific primers to detect DNA from JC virus (JCV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) types 1 and 2, Mycobacterium tuberculosis, and Toxoplasma gondii via real-time polymerase chain reaction (PCR). RESULTS: The patients' median CD4(+) T-cell count was 89 cells/µL (interquartile range, 38-191 cells/µL). Of 331 CSF samples, 189 (57.1%) had at least 1 pathogen. PCR detected DNA from EBV in 91 (27.5%) patients, M. tuberculosis in 48 (14.5%), JCV in 20 (6.0%), CMV in 20 (6.0%), VZV in 13 (3.9%), HSV-1 in 5 (1.5%), and HSV-2 and T. gondii in none. Fungal and bacteriological studies showed Cryptococcus in 64 (19.5%) patients, pneumococcus in 8 (2.4%), and meningococcus in 2 (0.6%). Multiple pathogens were found in 68 of 189 (36.0%) samples. One hundred seventeen of 331 (35.3%) inpatients died during their hospitalization. Men were older than women (median, 37 vs 34 years; P = .01), more recently diagnosed with HIV (median, 30 vs 63 days; P = .03), and tended to have a higher mortality rate (40.2% vs 30.2%; P = .07). CONCLUSIONS: CNS OIs are frequent, potentially treatable complications of AIDS in Zambia. Multiple pathogens often coexist in CSF. EBV is the most prevalent CNS organism in isolation and in coinfection. Whether it is associated with CNS disease or a marker of inflammation requires further investigation. More comprehensive testing for CNS pathogens could improve treatment and patient outcomes in Zambia.

Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease.

The cerebrospinal fluid of patients with Parkinson's disease was shown to contain extracellular DNA. Extracellular DNA concentration in the cerebrospinal fluid was 3.3-fold lower than in blood plasma from these patients. HPLC-mass spectrometry analysis showed that the pool of extracellular DNA from the liquor is characterized by a lower content of deoxythymidine, but greater amounts of deoxycytidine and deoxyguanosine than the pool of extracellular DNA from the plasma. The level of deoxyguanosine was 2 times lower than that of deoxycytidine (as differentiated from plasma extracellular DNA with similar content of these substances). Our findings indicate that extracellular DNA from the cerebrospinal fluid contains considerable amounts of modified deoxyguanosine. These data attest to significant differences in the quantitative and qualitative characteristics of extracellular DNA from the blood and cerebrospinal fluid of patients. Specific features of extracellular DNA from the cerebrospinal fluid of patients suggest its involvement in the pathogenesis of Parkinson's disease.

Hypertrophic pachymeningitis in an immunocompetent adult with positive Aspergillus DNA in the cerebrospinal fluid.

A 42-year-old immunocompetent man presented with subacute onset unilateral headache and associated lower cranial nerve palsies. Cranial magnetic resonance imaging showed enhancing thickened tentorium cerebelli and subtentorial dura mater. Cerebrospinal fluid examination revealed lymphocytic pleocytosis and positive polymerase chain reaction assay of Aspergillus DNA. While on voriconazole treatment a progressive increase was noted in subtentorial pachymeningeal hypertrophy, which was excised because of critical compression of the medulla. The excision material showed extensive fibrosis, cellular infiltrates and no organisms. With combination therapy with anti-fungal agents and corticosteroids, pachymeningitis showed regression. We hypothesised that intact immune status and less burden of Aspergillus infection in this patient may have resulted in a chronic progressive hypertrophic pachymeningitis.

[Assay of extracellular low-molecular DNA in blood as a diagnostic technique for clinical and experimental studies].

It was shown experimentally that ionizing radiation and low-frequency noise increase the level of extracelular low-molecular DNA in rat's blood plasma. Growth of this parameter has been observed in acute stroke patients at the onset of the disease. Low-molecular DNA assay is proposed for evaluation of the unfavorable effects of physical factors on organism.

Relationships of Cerebrospinal Fluid Alzheimer's Disease Biomarkers and COMT, DBH, and MAOB Single Nucleotide Polymorphisms.

The noradrenergic and dopaminergic systems are affected in Alzheimer's disease (AD). Polymorphisms in genes encoding enzymes and proteins that are components of these systems can affect products of transcription and translation and lead to altered enzymatic activity and alterations in overall dopamine and noradrenaline levels. Catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAOB) are the enzymes that regulate degradation of dopamine, while dopamine ß-hydroxylase (DBH) is involved in synthesis of noradrenaline. COMT Val158Met (rs4680), DBH rs1611115 (also called -1021C/T or -970C/T), and MAOB rs1799836 (also called A644G) polymorphisms have been previously associated with AD. We assessed whether these polymorphisms are associated with cerebrospinal fluid (CSF) AD biomarkers including total tau (t-tau), phosphorylated tau proteins (p-tau181, p-tau199, and p-tau231), amyloid-ß42 (Aß42), and visinin-like protein 1 (VILIP-1) to test possible relationships of specific genotypes and pathological levels of CSF AD biomarkers. The study included 233 subjects: 115 AD, 53 mild cognitive impairment, 54 subjects with other primary causes of dementia, and 11 healthy controls. Significant decrease in Aß42 levels was found in patients with GG compared to AG COMT Val158Met genotype, while t-tau and p-tau181 levels were increased in patients with AA compared to AG COMT Val158Met genotype. Aß42 levels were also decreased in carriers of A allele in MAO-B rs1799836 polymorphism, while p-tau181 levels were increased in carriers of T allele in DBH rs1611115 polymorphism. These results indicate that COMT Val158Met, DBH rs1611115, and MAOB rs1799836 polymorphisms deserve further investigation as genetic markers of AD.

Mutation detection in DNA isolated from cerebrospinal fluid and urine: Clinical utility and pitfalls of multiple displacement amplification.

This study describes the use of cerebral spinal fluid (CSF) and/or urine as source of DNA for mutation analysis combined with multiple displacement amplification. The findings illustrate the opportunities and pitfalls of these methods in the search for identification of the pathogenic mutations in the case that only scarce material is available such as CSF.

Chlamydophila pneumoniae DNA and mRNA transcript levels in peripheral blood mononuclear cells and cerebrospinal fluid of patients with multiple sclerosis.

Chlamydophila pneumoniae DNA and mRNA transcripts were investigated by PCR and RT-PCR in fresh CSF and PBMC specimens co-cultured in Hep-2 cell lines and collected from 14 patients with definite RR MS and 19 patients with other inflammatory (OIND) and non-inflammatory (NIND) neurological controls. A positivity for C. pneumoniae DNA and mRNA was detected in CSF and PBMCs of 9 RR MS patients (64.2%) with evidence of disease activity, whereas only 3 controls were positive for Chlamydial DNA. These preliminary findings suggest that C. pneumoniae may occur in a persistent and metabolically active state at both peripheral and intrathecal levels in MS, but not in OIND and NIND.

DNA versus RNA oxidation in Parkinson's disease: Which is more important?

BACKROUND: 8-hydroxy-2 deoxyguanosine (8-OHdG) and the 8-hydroxyguanosine (8-OHG) are the most widely used biomarkers of nucleoside oxidation affecting DNA and RNA and are considered reliable markers of oxidative stress. Increased levels of these markers are found in the various biological fluids of patients with neurodegenerative disorders. OBJECTIVE: The primary aim of our study was to assess the differences of investigated markers between patient groups and subsequently study the influence of clinical factors that might modify the levels of investigated markers during the disease progression. METHODS: In this study, we analysed the 8-OHdG and 8-OHG levels in the cerebrospinal fluid (CSF) and serum from 44 patients with Parkinson's disease (PD) and 32 controls using an ELISA. RESULTS: There were significantly higher CSF levels of both investigated markers in Parkinson's disease patients as compared to controls (p=0.02 and p=0.04). Significantly higher CSF values of 8-OHdG were found in PD patients without dementia (p=0.05), whereas patients with dementia recorded lower 8-OHG CSF levels compared to controls (p=0.04). The disease duration and age influenced the levels of both markers within investigated groups. CONCLUSION: Oxidative DNA damage plays an important role in the early stages of PD, whereas during the progression of the disease the process is more complex, and other mechanisms are in the foreground. The measurement of 8-OHdG might be used as an "early-stage marker", whereas the decrease of 8-OHG in CSF might reflect the degree of neurodegeneration during the disease progression, suggesting its utility as a prognostic marker of advanced PD stages.

Cerebrospinal fluid biomarkers of malignancies located in the central nervous system.

CNS malignancies include primary tumors that originate within the CNS as well as secondary tumors that develop as a result of metastatic cancer. The delicate nature of the nervous systems makes tumors located in the CNS notoriously difficult to reach, which poses several problems during diagnosis and treatment. CSF can be acquired relatively easy through lumbar puncture and offers an important compartment for analysis of cells and molecules that carry information about the malignant process. Such techniques have opened up a new field of research focused on the identification of specific biomarkers for several types of CNS malignancies, which may help in diagnosis and monitoring of tumor progression or treatment response. Biomarkers are sought in DNA, (micro)RNA, proteins, exosomes and circulating tumor cells in the CSF. Techniques are rapidly progressing to assess these markers with increasing sensitivity and specificity, and correlations with clinical parameters are being investigated. It is expected that these efforts will, in the near future, yield clinically relevant markers that aid in diagnosis, monitoring and (tailored) treatment of patients bearing CNS tumors. This chapter provides a summary of the current state of affairs of the field of biomarkers of different types of CNS tumors.

Good outcome of brain stem progressive multifocal leukoencephalopathy in an immunosuppressed renal transplant patient: Importance of early detection and rapid immune reconstitution.

Progressive multifocal leukoencephalopathy (PML) is a rare, opportunistic and often fatal disease of the CNS which may occur under immunosuppression in transplant patients. Brain stem PML is associated with a particularly bad prognosis. Here, we present a case of a renal transplant patient treated with mycophenolate mofetil (MMF) and tacrolimus who developed brain stem PML with limb ataxia, dysarthria and dysphagia. Diagnosis was established by typical MRI features and detection of JCV-DNA in the CSF. Immune reconstitution after stopping MMF and tacrolimus led to a complete and sustained remission of symptoms with improvement of the brain stem lesion over a follow-up over 20months. In summary, early detection of PML and consequent treatment may improve neurological outcomes even in brain stem disease with a notorious bad prognosis.

Psychiatric symptoms and cognitive dysfunction caused by Epstein-Barr virus-induced encephalitis.

Epstein-Barr virus (EBV) encephalitis is rare and shows a wide range of clinical manifestations. We report an immunocompromised patient with EBV encephalitis diagnosed by EBV-specific PCR and antibody testing in the cerebrospinal fluid who presented with psychiatric symptoms and cognitive dysfunction in the absence of any neurological impairments or infectious signs. Clinical recovery and clearance of cerebrospinal fluid EBV DNA appeared following ganciclovir treatment within 6 weeks.

Drug-associated progressive multifocal leukoencephalopathy: a clinical, radiological, and cerebrospinal fluid analysis of 326 cases.

The implementation of a variety of immunosuppressive therapies has made drug-associated progressive multifocal leukoencephalopathy (PML) an increasingly prevalent clinical entity. The purpose of this study was to investigate its diagnostic characteristics and to determine whether differences herein exist between the multiple sclerosis (MS), neoplasm, post-transplantation, and autoimmune disease subgroups. Reports of possible, probable, and definite PML according to the current diagnostic criteria were obtained by a systematic search of PubMed and the Dutch pharmacovigilance database. Demographic, epidemiologic, clinical, radiological, cerebrospinal fluid (CSF), and histopathological features were extracted from each report and differences were compared between the disease categories. In the 326 identified reports, PML onset occurred on average 29.5 months after drug introduction, varying from 14.2 to 37.8 months in the neoplasm and MS subgroups, respectively. The most common overall symptoms were motor weakness (48.6 %), cognitive deficits (43.2 %), dysarthria (26.3 %), and ataxia (24.1 %). The former two also constituted the most prevalent manifestations in each subgroup. Lesions were more often localized supratentorially (87.7 %) than infratentorially (27.4 %), especially in the frontal (64.1 %) and parietal lobes (46.6 %), and revealed enhancement in 27.6 % of cases, particularly in the MS (42.9 %) subgroup. Positive JC virus results in the first CSF sample were obtained in 63.5 %, while conversion after one or more negative outcomes occurred in 13.7 % of cases. 52.2 % of patients died, ranging from 12.0 to 83.3 % in the MS and neoplasm subgroups, respectively. In conclusion, despite the heterogeneous nature of the underlying diseases, motor weakness and cognitive changes were the two most common manifestations of drug-associated PML in all subgroups. The frontal and parietal lobes invariably constituted the predilection sites of drug-related PML lesions.

Molecular diagnosis of leukemic cerebrospinal fluid cells in children with newly diagnosed acute lymphoblastic leukemia.

Cytomorphology and IgH/T-cell receptor g clonal gene rearrangements detected by polymerase chain reaction (PCR) homo/heteroduplex analysis and direct sequencing were evaluated in cerebrospinal fluid (CSF) free of red-blood cells at diagnosis of 37 children with acute lymphoblastic leukemia. Molecular CSF involvement was greater as detected by molecular analysis than observed by morphologic criteria (45.9% vs 5.4%). The 4-year event-free survival was lower in the group with molecularly detected CSF involvement (p = 0.01).

Fetal cell-free nucleic acids in the maternal circulation: new clinical applications.

Six years after the demonstration of the presence of cell-free fetal nucleic acids in maternal plasma, perinatal clinical applications continue to expand. The focus of this article is on advances that have occurred since the CNAPS II conference held in Hong Kong in 2001. Circulating fetal DNA levels (fDNA) are elevated in pregnancies complicated by fetal trisomies 13 and 21 but not 18. Measurement of fDNA levels improves the performance of the current standard maternal serum screen, by increasing the detection of Down syndrome cases by 5% with no increase in the false-positive rate. fDNA levels are elevated in women who have developed clinical symptoms of preeclampsia, but they are also elevated by the early second trimester in women who will eventually develop preeclampsia. fDNA and mRNA gamma globin measurement may have clinical utility as markers for fetomaternal hemorrhage in the late first trimester. Cell-free fetal DNA levels are quite high in the amniotic fluid, permitting fetal genomic isolation and analysis using comparative genomic hybridization techniques. Fetal DNA crosses the blood-brain barrier and is detectable in maternal cerebrospinal fluid in a subset of pregnant women. The biological implications of this are currently unknown. Review of the literature suggests that the placenta is the predominant source of the circulating fetal nucleic acids. However, detection of gamma globin mRNA sequences in the plasma of pregnant women suggests that fetal blood cells also contribute to the pool of nucleic acids. Widespread incorporation of fetal nucleic acid measurement into routine prenatal care depends on the identification of a readily accessible gender-independent fetal marker.

3H-thymidine autoradiography of the CSF cells in cases of non-neoplastic disease.

Human CSF cells in cases of non-neoplastic disease of the central nervous system (CNS) were examined in vitro by 3H-thymidine autoradiography. Immediately after withdrawl by lumbar or ventricular puncture, the CSF was incubated in a sedimentation chamber at 37 degrees C for 1 hr with an admixture of 3H-thymidine at a concentration of 1-2 muCi/ml CSF. In a few cases the CSF withdrawn was incubated in a glass tube in the same condition as in a sedimentation chamber, and the CSF cells were collected by centrifugation. The CSF cells collected were fixed in methanol and the microautoradiographic procedure was performed. Labeled CSF cells were found in 21 cases out of 22. The average labeling index of the total nucleated cells was 0.22% with the highest labeling of 0.74%. Almost all the labeled cells were thought to be medium to large sized lymphocytes and monocytoids. Peripheral blood was examined by a similar method and the results were compared with those of the CSF. It may be noteworthy that thre exist DNA synthesizing cells in the CSF even in a non-neoplastic state of the CNS, although the number is not large.

Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography.

Detecting and quantifying generalized mitochondrial heteroplasmy is essential if the field of mitochondrial genetics is to advance in the arena of complex genetic disorders. The majority of techniques used to detect and quantify mitochondrial heteroplasmy focus on a known mutation or polymorphism. The necessity of knowing the mitochondrial DNA (mtDNA) change beforehand means that non-specific heteroplasmy in general cannot be assessed. In this study, we assessed the extent that denaturing high-performance liquid chromatography (dHPLC) could detect and quantify mitochondrial heteroplasmy from cerebrospinal fluid (CSF). Although we used a known polymorphism to assess reliability and sensitivity of this technique, a distinct advantage to using dHPLC for heteroplasmy detection is that the entire fragment is screened for variability and any unique fragments will be detected regardless of the placement or type of change. Our results demonstrate that dHPLC can consistently and reliably detect mitochondrial heteroplasmy in a CSF sample down to 0.01%. In addition, the level of heteroplasmy was consistent with peak height for each homoduplex, giving a reliable method to quantify level of heteroplasmy.

Applications of flow cytometric DNA analysis to diagnostic cytology.

Flow cytometry is a rapid method for measuring DNA content. Its high sensitivity and specificity in detecting transitional cell carcinomas in bladder washings should make it a routinely applied adjunct to cytology once universal laboratory standards are firmly established. Diagnostic and prognostic information on solid tumors can be obtained at least as reliably on fine-needle aspirations as on surgically resected specimens. At the present time, the sensitivity of FCM in the evaluation of effusions, peritoneal washings, and CSF is relatively low, and although in some cases malignant cells in these specimens that are missed by conventional cytology are detected by FCM, the number of such cases is small. Investigation continues into the application of FCM to the screening of cervical specimens.

Evaluation of in-house optimized semi-nested PCR and EIA for direct detection of mycobacterial DNA in CSF.

A rapid and correct diagnosis of mycobacterial infections is important for effective patient treatment. Semi-nested-PCR with Fl-16 SOL, 16SOR and 16SNSR primers based on the 16S rRNA gene, under optimized conditions, can detect 499 bp amplified products from all tested mycobacteria. The assay could detect as little as 100 fg of mycobacterial DNA except for rapid growing mycobacteria, whose detection limits ranged from 1 ng to 10 pg. The specificities of the capture probes were assessed with 96 mycobacterial strains (22 species) and 33 nonmycobacterial strains (30 species). The specificities of pAll1, pTbc1 and pMar1 were 94%, 93% and 82%, respectively, and that of pAvi1, pInt1, pChe1 and pFor1 were 100%. The pTbc1 and pAvi1 were tested with DNA from 108 CSF samples, and the sensitivity and specificity of the detection method were 56% and 84% compared to culture and patient histories. The assay should be used for rapid detection and concurrent identification of slow growing mycobacteria without parallel conventional culture verification.

[Applications of the polymerase chain reaction (PCR) to the diagnosis of central nervous system infections]. / Aplicaciones de la reacción en cadena de polimerasa (PCR) al diagnóstico de infecciones del sistema nervioso central.

The utility of polymerase chain reaction (PCR) is described for the diagnosis in three patients suffering from central nervous system infections, tuberculous meningitis, herpetic encephalitis and cerebral toxoplasmosis. PCR was performed in the cerebrospinal fluid after processing the specimen by two methods, proteinase K digestion and phenol extraction of DNA. Amplification was realized using primers previously described that amplify specific DNA fragments of each microorganisms (insertion sequence IS6110 of Mycobacterium tuberculosis, B1 gene of Toxoplasma gondii, and DNA polymerase gene of Herpes simplex virus). In all three cases, PCR was positive after amplification of the specimen extracted with proteinase K, as well as when a complete DNA extraction with phenol was realized. In all cases a band of amplified products was observed in agarose gels. In conclusion, in all three patients described, PCR would had allowed the diagnosis in seven hours, and PCR should be consider a rapid sensitive and relatively simple method.