key: cord-1009601-sz0jrhqd
authors: Tsamis, Konstantinos I.; Sakkas, Hercules; Giannakis, Alexandros; Ryu, Han Suk; Gartzonika, Constantina; Nikas, Ilias P.
title: Evaluating Infectious, Neoplastic, Immunological, and Degenerative Diseases of the Central Nervous System with Cerebrospinal Fluid-Based Next-Generation Sequencing
date: 2021-03-01
journal: Mol Diagn Ther
DOI: 10.1007/s40291-021-00513-x
sha: 826f2720c8ebe8a79c1f486e44919feefbb89a14
doc_id: 1009601
cord_uid: sz0jrhqd

Cerebrospinal fluid (CSF) is a clear and paucicellular fluid that circulates within the ventricular system and the subarachnoid space of the central nervous system (CNS), and diverse CNS disorders can impact its composition, volume, and flow. As conventional CSF testing suffers from suboptimal sensitivity, this review aimed to evaluate the role of next-generation sequencing (NGS) in the work-up of infectious, neoplastic, neuroimmunological, and neurodegenerative CNS diseases. Metagenomic NGS showed improved sensitivity—compared to traditional methods—to detect bacterial, viral, parasitic, and fungal infections, while the overall performance was maximized in some studies when all diagnostic modalities were used. In patients with primary CNS cancer, NGS findings in the CSF were largely concordant with the molecular signatures derived from tissue-based molecular analysis; of interest, additional mutations were identified in the CSF in some glioma studies, reflecting intratumoral heterogeneity. In patients with metastasis to the CNS, NGS facilitated diagnosis, prognosis, therapeutic management, and monitoring, exhibiting higher sensitivity than neuroimaging, cytology, and plasma-based molecular analysis. Although evidence is still rudimentary, NGS could enhance the diagnosis and pathogenetic understanding of multiple sclerosis in addition to Alzheimer and Parkinson disease. To conclude, NGS has shown potential to aid the research, facilitate the diagnostic approach, and improve the management outcomes of all the aforementioned CNS diseases. However, to establish its role in clinical practice, the clinical validity and utility of each NGS protocol should be determined. Lastly, as most evidence has been derived from small and retrospective studies, results from randomized control trials could be of significant value.

Cerebrospinal fluid (CSF) is a clear and paucicellular fluid of low protein concentration, derived from the blood plasma and produced by the ependymal cells of the choroid plexus. In normal conditions, ependymal cells can produce around 500 mL/day of CSF, of which 150 mL circulates within the ventricular system and the subarachnoid space of the central nervous system (CNS) [1, 2] . Being a crucial component of the CNS structure, any change in the cellular/biochemical composition, volume, and flow of the CSF could affect CNS function, and CNS disorders may alter CSF characteristics [2] . These changes are caused by various diseases that impact the CNS either primarily or secondarily, while CSF examination often helps to identify the cause [1, 2] .

Physicians sample CSF by performing a procedure called a lumbar puncture, where they insert a needle within the L3-L4 or L4-L5 intervertebral space of the patient, and subsequently send the samples to the laboratory for analysis [3] . Currently, the main diagnostic indications of CSF testing include confirming or excluding CNS infections that cause meningitis or meningoencephalitis, CNS primary or metastatic neoplasms, subarachnoid hemorrhage, and neuroinflammatory disorders such as multiple sclerosis (MS) [2, 3] . For instance, identifying numerous neutrophils in the CSF supports a bacterial infection, whereas malignant cells confirm the presence of a cancer disseminating within the subarachnoid space [1, 2] .

Although vital for everyday clinical practice, conventional CSF testing has significant drawbacks, the most important of them being suboptimal sensitivity. Morphologic evaluation of infectious agents (e.g., using Gram stain), cultures, or serologic testing often leave patients undiagnosed, while polymerase chain reaction (PCR) testing needs a formal hypothesis for the potential infectious agents implicated to select the most suitable primers. As a result, antimicrobial treatment could cause delay or even be inappropriate, with significant impact on morbidity and mortality [4, 5] . Likewise, CSF cytology shows sensitivity of < 50% to detect cancer dissemination into the subarachnoid space, even if it is highly specific [6, 7] . While the presence of oligoclonal bands-which represent increased concentrations of IgG immunoglobulins-in the CSF characterizes most MS cases, these bands could also be detected in other inflammatory or even neoplastic CNS conditions [8] . Of interest, CSF examination could provide answers where other diagnostic modalities fail or may harm the patients; for instance, when a brain biopsy fails to diagnose and assess the molecular profile of a CNS tumor or it is contraindicated due to the tumor location or the condition of the patient's health, CSF testing is a viable option [9] .

In contrast to Sanger sequencing, next-generation sequencing (NGS) can process multiple nucleic acid fragments in parallel and within a single run, at both genomic (DNA) and transcriptomic levels (coding or noncoding RNA) [10] . Selection from various existing NGS-based methods depends on the clinical scenario. Targeted NGS using predefined panels is commonly applied in oncology [11] , while metagenomic NGS (mNGS) is an emerging application in microbiology laboratories; mNGS is able to identify all pathogens present in a sample and in an unbiased way, without depending on a hypothesis, such as with PCR [12] . MicroRNA (miRNA) NGS can identify short, singlestranded, non-coding RNAs that regulate gene expression at the post-transcriptional level, and have acted as diagnostic and prognostic biomarkers in various disorders [13] [14] [15] . This review aims to highlight the value of applying NGS in the CSF of patients with infectious, neoplastic, neuroimmune, and neurodegenerative CNS diseases.

Infections of the CNS-associated with bacteria, viruses, parasites, fungi, and prions-are a major cause of morbidity and mortality, especially in immunocompromised individuals [16, 17] . Based on the predominant anatomic site of infection, CNS diseases are usually classified as meningitis, encephalitis (or meningoencephalitis), or myelitis (or encephalomyelitis). Acute meningitis may be caused by bacteria or viruses, while fungi, mycobacteria, or atypical bacteria are usually involved in chronic meningitis [17] . The rapid identification of the pathogen is essential for the process of antimicrobial therapy and its impact on clinical outcomes [18] . More than 100 pathogens, commonly viruses, have been reported as causative agents of encephalitis; however, most of the cases remain undiagnosed or even underestimated and the selection of an appropriate diagnostic method must be the ultimate objective [19, 20] .

It has been reported that in approximately 75-80% of pediatric CNS infections, microbiological diagnosis is usually obtained with molecular laboratory methods [21] . Recently, mNGS technology of the CSF has been suggested as a fast, sensitive, affordable but also costly and laborintensive diagnostic tool, detecting unidentified pathogens in a single run, since culture, serologic procedures, and PCR have occasionally been proven ineffective [5, 16, [21] [22] [23] [24] [25] [26] . This technology allows rapid sequencing of full pathogen genomes, even in point-of-care diagnostics, overcoming the limitations of available targeted PCR methods. It is useful in the examination of a large number of cases or small outbreaks, through accurate genotyping and molecular epidemiology, and also by characterization of genes mediating drug resistance [22, [27] [28] [29] . Interestingly, in a recent study, NGS of ancient dental calculus samples from a prehistoric site revealed high levels of Neisseria meningitidis and low levels of Haemophilus influenzae, which was interpreted as an ancient case of meningococcal disease associated with incipient endocranial lesions and pronounced meningeal grooves [30] . Nevertheless, clinical application is still in an early phase and there are several limitations [31] as most of the published reports consist of single case reports or small retrospective case series [32] . In addition, direct comparison between conventional testing and mNGS should rely on the definition of the gold standard method, further adding to the difficulty in validating such a new technology [31] . It has been shown that several background and contaminating bacteria (e.g., Propionibacterium, Burkholderia, Acinetobacter species) [33] and DNA contamination from CSF pleocytosis, a common feature in meningitis patients, considerably reduce the sensitivity of mNGS in the detection of pathogens [34] . Furthermore, several challenges including the improvement of bioinformatics software tailored for clinical diagnostic use and concerns over the quality and comprehensiveness of available reference databases still exist in making mNGS more applicable into laboratory practice [16] . Laboratory diagnosis of CNS infections with mNGS and the characteristics of the studies are summarized in Table 1 and Fig. 1. 

Streptococcus pneumoniae followed by Streptococcus agalactiae, N. meningitidis, H. influenzae, and Listeria monocytogenes have been reported as the most common causes of acute bacterial meningitis [35] . Rapid diagnosis and treatment reduce mortality and neurological consequences, but this can be delayed by atypical clinical presentation, evaluation of the lumbar puncture safety, and poor sensitivity of conventional diagnostic methods [36] . Common pathogens including N. meningitidis, Streptococcus spp., L. monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae [26, 28, 37] , but also rare opportunistic pathogens such as Psychrobacter spp. [38] have been successfully detected in CSF specimens of pediatric and adult patients. Zhang et al. analyzed the diagnostic value of mNGS for identifying S. pneumoniae in pediatric bacterial meningitis. Results obtained by mNGS were compared to those from CSF culture, which is recognized as the "gold standard" method [39] , although culture-based methods are less sensitive than molecular techniques, because most patients usually receive antibiotics prior to admission [40] . The sensitivity and specificity of mNGS for detecting S. pneumoniae were 73.1% and 88.1%, respectively, whereas the positive predictive value (PPV) and negative predictive value (NPV) were 81.4% and 89.3%, respectively. In addition, the difference in number of unique reads of bacteria from CSF samples collected up to 14 days from disease onset was significantly higher than the pathogen load from the sample collected over 14 days from the clinical onset [39] . In another study, NGS was applied for the investigation of two N. meningitidis serogroup C isolates from a meningococcal outbreak, classified within the same N. meningitidis sequence type ST-11 by conventional molecular techniques. Results demonstrated subtle genetic differences among the above mentioned isolates [28] , providing useful information about the epidemic potential and dynamics of geographic distribution of distinct meningococcal serotypes [41] .

L. monocytogenes is also an important bacterial cause of meningoencephalitis in immunocompromised individuals, with a high mortality rate. The positivity rates of Gram stain and CSF culture have been reported to be 14% and 41%, respectively, and NGS recently identified and sequenced L. monocytogenes from three cases of clinically suspected meningoencephalitis [26] . In another study, four patients with a clinically suspected CNS infection and non-specific clinical manifestations were diagnosed with neurobrucellosis using NGS of the CSF, while only one patient (25%) had a positive CSF culture. Furthermore, it is estimated that approximately 28% and 15% of patients with CNS brucellosis have positive blood cultures and CSF cultures, respectively, and although serological procedures are both more sensitive and faster than conventional culture, seropositivity is high in endemic areas and may not easily distinguish active from past infections [33, 42] .

Infections with Mycobacterium tuberculosis (MTB) may occur with CNS comorbidities in approximately 1% of cases and are associated with significant mortality and adverse neurologic outcomes [17] . In a retrospective analysis, 51 inpatients with suspected tuberculous meningitis were examined with mNGS and four other assays in CSF. The sensitivity, specificity, PPV, and NPV of mNGS were 84.4%, 100%, 100%, and 46.1%, respectively, while sensitivity was significantly higher than that of acid-fast staining (0%), CSF culture (22.2%), MTB RT-PCR (24.4%), and Xpert MTB/ RIF (40%) [43] . In another study, the sensitivity of mNGS, acid-fast staining, CSF culture, and MTB RT-PCR in the first CSF samples from patients with definite tuberculous meningitis were 66.7%, 33.3%, 8.3%, and 25%, respectively, whereas the specificity of all procedures was 100% [44] . Lately, mNGS has also been evaluated for Ureaplasma parvum detection in the CSF of a neonate with meningitis [45] .

In more than half of cases, the etiological cause of CNS viral diseases remains unknown. Non-specific culture assays, serological tests (e.g., ELISA) and molecular techniques with high sensitivity and specificity (e.g., quantitative PCR) have been used as diagnostic tools, but they are compromised by several limitations since they are targeted against individual viruses or subsets of similar viruses [24] . NGS seems to be a promising method for the diagnosis of both DNA and RNA viral infections as it enables the detection and identification of known as well as novel pathogens [46, 47] . Although viruses in terms of genome size may be small, and their viral load may be undetectable due to the high concentration of background nucleic acids, viral metagenomics [57] overcomes this by the enrichment of the viral genomic content in the sample [24] . Herpes viruses, measles virus, Coxsackieviruses, and Polyomaviruses have been identified by mNGS from CSF, pooled nucleic acids from CNS specimens and brain biopsies as known causes of encephalitis. Arenaviruses, Astroviruses, Bornaviruses, Coronaviruses, and Parvoviruses are considered novel causes of encephalitis. Cycloviruses, Densoviruses, and Gemycircularviruses have been isolated as unexpected pathogens from clinical specimens, demonstrating an unclear association with the reported infections [22] . Especially in low-and middle-income countries, unbiased mNGS can complement conventional diagnostic methods in order to identify potentially pathogenic viruses that cause epidemics, such as Chikungunya virus and as well as other RNA viruses, but also may be useful for the conduction of surveillance studies, and prediction of outbreaks [48] . Although techniques to reduce host DNA contamination from CSF samples are still lacking, several effective approaches have been used in order to improve the detection sensitivity of pathogens [34] . Using negative controls with predetermined DNA composition has been shown to be a necessary option to define the expected microbial nucleic acid background [29] . Nevertheless, mNGS should be performed in combination with conventional methods in order to diagnose a viral infection in patients presenting with mildto-moderate lymphocytic pleocytosis [32] .

In contrast to bacterial and viral infections, laboratory diagnosis of some parasitic infections cannot be primarily accomplished by conventional methods. Parasites are more frequently found in immunocompromised than immunocompetent patients with meningoencephalitis, while the most common CNS protozoan infection in the former is Toxoplasma gondii [17] . In a recent study, toxoplasmic encephalitis with atypical brain imaging features and negative results of Toxoplasma IgM antibodies test, was rapidly diagnosed by an mNGS method, in a critically ill HIV-infected individual [19] . In addition, NGS technology was successfully used in the accurate diagnosis of primary amoebic meningoencephalitis, an acute and life-threatening disease caused by Naegleria fowleri [49] .

Regarding helminths, neurocysticercosis caused by Taenia solium is the most common infection of the CNS. The clinical manifestations and MRI findings may vary due to several factors, such as the number, stage, size, and different locations of the encysted larvae, thus the diagnosis usually becomes quite challenging. Also, considering the variable PPV of the available serological experiments, detection of T. solium DNA in CSF with NGS could facilitate diagnosis [50, 51] . In such a case, a patient who presented for several months with atypical clinical symptoms including headache and paroxysmal left face and arm numbness, was diagnosed with neurocysticercosis using NGS in the CSF [52] . Furthermore, when compared with CNS bacterial infections, the NGS results for neurocysticercosis are readily interpreted, since they are not influenced by the existing background or contaminating bacteria [50] . Angiostrongylus cantonensis is the most common parasitic nematode cause of eosinophilic meningitis. Since the diagnosis of angiostrongyliasis also lacks standardized and confirmatory assays including serological tests and CSF cytology, the development of a precise diagnostic method is a challenge. A recent report has described a case of angiostrongyliasis confirmed by NGS in the CSF of a patient with ELISA-negative results [53] . Similarly, NGS was considered to have higher sensitivity and specificity when compared to microscopy, serological tests, and MRI in the detection of A. cantonensis eosinophilic meningoencephalitis in two infants [54] .

Fungal CNS infections with either yeasts or molds can cause severe neurological manifestations of either meningitis or focal abscess and cause life-threatening complications in the immunocompromised host. Most of these pathogens are acquired through the respiratory tract and secondarily disseminate though the hematogenous route to the CNS [17] . Among fungal infections, the opportunistic infection with Cryptococcus neoformans/Cryptococcus gattii species complexes has become the leading cause of meningitis among HIV-infected patients and is associated with high morbidity and mortality rates [55, 56] . mNGS has been successfully applied as an important diagnostic tool for rapid and specific diagnosis of cryptococcal infection in CSF samples, especially for accurate species identification, in conjunction with the traditional diagnostic methods including India ink staining, CSF culture, and cryptococcal antigen detection [57] . Recently, mNGS has been reported as a more sensitive procedure than conventional CSF culture and cryptococcal antigen test for the detection of Cryptococcus spp. at even low abundance in CSF of HIV-infected individuals. In this study, three HIV-seropositive, cryptococcal antigenemic patients with clinical features of meningitis and negative CSF microbiological procedures were finally diagnosed with cryptococcal meningitis by mNGS [58] . Diverse fungal pathogens including members of the genera Aspergillus, Histoplasma, and Candida have been also successfully identified by NGS technology [59] .

As most primary and metastatic tumors involving the CNS show poor prognosis, their prompt diagnosis along with effective personalized therapy and close follow-up are imperative [60] [61] [62] . To this end, tumor molecular profiling is increasingly used in the clinic as an integral part of precision oncology [63, 64] . Molecular tumor data are often collected with tissue-based testing, either small biopsy or resection specimens [62] . However, invasive CNS procedures could carry significant risk, especially when sampled in patients with medical comorbidities or from critical areas such as the brainstem, prohibiting serial testing to monitor tumor evolution or treatment response. Furthermore, brain tumors might be inaccessible to biopsy, while, if reachable, the tissue specimens could be inadequate, show hypocellularity, abundant necrosis, or poor preservation that hamper subsequent molecular analysis [62, 65] . Of interest, both primary and metastatic CNS tumors display significant intra-tumor heterogeneity. This means that processing a small biopsy might not represent the whole tumor. Likewise, molecular testing of a primary extracranial cancer could be insufficient to guide treatment decisions for its CNS metastasis [9, 63, 66, 67] .

Liquid molecular analysis could facilitate tumor diagnosis, monitoring, and therapy selection, and overcome inherent problems related to a tissue biopsy. Similar to tissues, cancer mutational testing can be performed in body fluids such as the blood, urine, sputum, pleural fluid, and CSF [9, [68] [69] [70] . Even if it often fails to reach the diagnostic capacity of its tissue counterpart and lacks sufficient evidence of clinical validity and utility, the liquid biopsy is minimally invasive, cheaper than tissue biopsy, easier to obtain, and free from preservatives and fixatives. In addition, it depicts intra-tumor heterogeneity effectively and allows tumor follow-up with serial testing [69, 71, 72] . Any liquid counterpart including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), non-coding RNAs (e.g., miRNAs), or exosomes can be analyzed with diverse molecular techniques [69, 73] . Of the latter, NGS is especially appealing, as it offers the capacity to examine comprehensively multiple cancer-related molecular alterations (point mutations, [112] insertions, deletions, copy number variations, and gene rearrangements) in a single run [74] . This section describes the recent evidence regarding CSFbased NGS testing in patients with primary or metastatic CNS tumors. We summarize this evidence in Table 2 and illustrate its importance in Fig. 2. 

Regarding primary CNS cancers, GLOBOCAN estimated 296,851 (1.6% of all sites) new cases and 241,037 deaths (2.5% of all sites) globally in 2018 [75] . Gliomas represent the most common primary CNS malignancy, while glioblastomas account for most gliomas in the adults, exhibiting low resectability and high recurrence rates [64] . In children, CNS tumors follow leukemias in incidence yet they lead all pediatric cancers in morbidity and mortality; of them, the embryonal tumor medulloblastoma is the most common malignant lesion [76] . Prognosis and management of primary CNS tumors largely depend on their grade. Grading measures diverse tumor characteristics including cellular atypia, mitoses, necrosis, and endothelial proliferation. The higher the grade, the worse the prognosis generally is; for instance, glioblastomas and medulloblastomas are both grade IV tumors of dismal prognosis [60] . Molecular profiling of CNS tumors is essential for their accurate diagnosis and should be performed together with morphologic evaluation, according to the latest WHO classification [60] . Gliomas are classified as IDH-mutant and IDH-wild type, as their prognosis differs significantly, being worse in the wild type. In addition, 1p/19q co-deletion is a molecular alteration characterizing oligodendrogliomas, while a diffuse glioma of lower grade (II or III) and an IDHwild type status is upgraded to "molecular glioblastoma" (integrated grade IV glioma) when alterations such as EGFR amplification or TERT promoter alterations are found [60, 64, 77] . TERT promoter mutation is associated with a dismal prognosis, while MGMT promoter methylation is associated with a favorable response to chemotherapy and prolonged survival [64, 78, 79] . Likewise, hypermutation after chemotherapy is a major factor of tumor progression and therapy resistance [64, 80] .

CSF-based molecular analysis in primary CNS tumor patients could provide valuable information concerning accurate diagnosis and classification, alterations linked with prognosis and therapy selection, tumor evolution, and response to treatment ( Table 2) . Besides testing 20 tissue biopsies and CSFs from glioma patients, Martinez-Ricarte et al. also analyzed 648 diffuse gliomas from the TCGA Atlas and developed an NGS platform that can genotype seven genes (including IDH1, IDH2, TP53, TERT, and H3F3A); this platform could facilitate the classification of gliomas with CSF ctDNA, avoiding the risk of invasive sampling [81] . Zhao et al. applied targeted NGS and identified driver mutations from the CSF ctDNA in both newly diagnosed and relapsed glioma patients; of interest, the concordance rate with their matched tumor tissue samples in this study was high [82] . As CSF sampling is minimally invasive, CSF-derived ctDNA analysis is not only promising for defining gliomas' initial molecular signature but also for subsequent serial testing to monitor potential tumor progression [83, 84] .

Concerning the value of NGS as an element of CSF molecular analysis, several teams have compared the molecular signatures of the CSF-derived ctDNA to the tissuederived DNA. In brief, CSF-based NGS analysis was largely concordant with the tissue-based analysis. Furthermore, it also spotted additional mutations, reflecting intratumoral heterogeneity, while it overcame the under-sampling bias inherent to small biopsies [9, 82, [84] [85] [86] . NGS diagnostic capacity was directly correlated to the tumor grade and location. High-grade tumors (e.g., glioblastomas) with a location next to the CSF space were more likely to excrete ctDNA into the CSF circulation [81, 82, 87, 88] . Likewise, NGS in CSF exhibited superior performance to plasma molecular analysis, revealing higher mutation detection rates [83, 84, 86 ].

Metastatic spread is a frequent complication and substantial cause of mortality in patients with a cancer that first develops outside the CNS, most commonly in the lung, breast, or skin (melanoma). Prognosis in such patients is generally poor, hence there is an emerging need to integrate molecular data into personalized treatment strategies [27] . Recent guidelines recommend that patients with advanced non-small-cell lung cancer (NSCLC) should undergo predictive biomarker testing for potential EGFR, ALK, ROS1, BRAF molecular alterations, as targeted therapies exist for them. In addition, mutations such as the EGFR T790M are associated with resistance to therapy [63, 89, 90] . Likewise, patients with breast cancer and melanoma should be tested for the presence of HER2 amplification or BRAF V600E mutation to assess their prognosis and evaluate their eligibility for trastuzumab or the BRAF inhibitor vemurafenib, respectively [63, 91] . While delivery of traditional chemotherapy into the CNS lesion is significantly limited by the blood-brain barrier, targeted therapies seem to outweigh chemotherapy in terms of efficacy and safety [62, 63] . As the metastatic cancer molecular profile might differ from its primary counterpart, testing of only the primary site could be insufficient as it ignores heterogeneity and tumor evolution. In fact, the European Association of Neuro-Oncology recommends that, even if a cancer is previously assessed in its primary extracranial location, predictive biomarker testing should also be performed in the CNS metastatic site [62, 63, 91, 92] .

Similar to primary CNS tumors, the application of CSFbased NGS testing in metastatic tumors could also facilitate diagnosis and prognosis, identify targetable mutations, assess intra-tumor heterogeneity, monitor tumor evolution and therapy response, and identify mechanisms of drug resistance ( Table 2 ). Diagnosis and follow-up of metastatic CNS lesions are typically performed with neuroimaging and CSF cytology; however, the latter suffer from low sensitivity [93, 94] . NGS was found to outweigh the diagnostic performance of imaging and cytology in terms of sensitivity for the detection of brain metastases [62, [95] [96] [97] [98] . It was also able to detect targetable mutations in metastatic lung (e.g., EGFR, ALK, or ROS1) [62, 96, 97, [99] [100] [101] [102] , melanoma [95] [96] [97] , and breast cancer [96] , also identify mechanisms of acquired resistance to therapy, such as the EGFR T790M mutation [96, [103] [104] [105] [106] . Von Baumgarten et al. tested the CSF of 27 patients with metastatic and primary CNS tumors and reported that, by applying NGS to analyze the CSFderived ctDNA, they could identify targetable mutations and change the treatment strategy in some of their patients. They also identified six cases with several molecular alterations suggesting high tumor mutational burden (TMB), which is known to be predictive of response to immune checkpoint inhibitors [62] . Many groups have also suggested the potential value of CSF-based NGS testing to follow-up patients with brain metastases and evaluate their response to treatment or identify disease progression, which could be spotted earlier compared to clinical presentation, imaging, or CSF cytology [42, [107] [108] [109] [110] . While most studies have used ctDNA for their NGS experiments, Kopkova et al. demonstrated differences in the miRNA levels of the CNS tumors compared to controls by examining the CSF samples of 70 patients with both metastatic and primary lesions [111] . Similar to primary CNS tumors, CSF-based NGS testing was also largely concordant with tissue molecular analysis in metastatic CNS cancer patients, while it further detected additional mutations that were potentially missed due to tissue under-sampling. [9, 42, 100, [102] [103] [104] . Likewise, NGS in CSF was more sensitive than plasma molecular analysis in metastatic cancer patients [86, 97, 100, 102, 104, 107, 109, 110, 112, 113] .

Genetics of neuro-psychiatric disorders involve diseases in the CNS controlled by either monogenic inheritance or complex polygenic inheritance and associated with an interplay of genetic risk and environmental influence [114, 115] . The rapid advancement of molecular testing during the last decades with NGS and genome-wide association studies (GWAS) has revolutionized the area, facilitating the research of disease mechanisms and enhancing the accuracy of diagnosis. Genetic studies in neurologic and psychiatric disorders are focused on the investigation of the genome, the transcriptome, or the epigenome. While for disorders with monogenic type of inheritance, testing for genomic variations in the peripheral blood is adequate, for diseases with complex polygenic inheritance transcriptome and epigenome analysis is equally important [116] . Brain tissue from autopsy or biopsy and peripheral blood are the most commonly used sources for this kind of testing, but during recent years the CSF has also been investigated [117] . To date, the CSF has been used as a source of genetic material for NGS studies, mainly for transcriptomics in autoimmune neurological disorders and neurodegenerative disorders. The characteristics of these studies are summarized in Table 3 and Fig. 3 . NGS has also been used in CSF of patients with acute ischemic stroke and several brain-enriched miRNAs have been detected 3 days after stroke, mostly associated with large ischemic strokes [118] . Finally, in recent years, the genetic basis of psychiatric disorders has begun to be identified [116, [119] [120] [121] [122] , but the only NGS study in the CSF contacted so far is related to the detection of known and novel viruses in patients with schizophrenia [123] .

Multiple sclerosis (MS) is the most common autoimmune disease of the CNS with still unknown etiology. The interaction between genetic risk factors and environmental influence is believed to be the key feature of the disease pathogenesis [124] . Initial genetic studies revealed strong associations in MS for genes within the major histocompatibility complex (MHC) including the human leukocyte antigen (HLA) class I and II [125] [126] [127] . More recently, during the last two decades, the advent of GWASs revealed more than 200 genetic risk variants for MS located in introns or exons, most of which reside in non-MHC regions [128] [129] [130] [131] . The functional consequences of these variants are not yet fully understood, apart from a small number that are proven to be implicated in the regulation of the immune system [132] [133] [134] [135] . On the way to better characterize the pathogenetic cascade that is related to the genetic risk variants, transcription and epigenetic mechanisms have been investigated as well, adding meaningful information to pathway analysis [135] [136] [137] [138] [139] . To this extent, the genetic material isolated from CSF, tested with NGS, provides valuable information both for research and for diagnostic purposes. B and T cells have a central role in the pathogenesis of MS, as they interact and infiltrate the CNS causing local pathology and demyelination [140] . Lymphocytes of MS patients seem to undergo extensive clonal expansion inside the CNS [141, 142] , but there is still a long debate about the mechanisms and the location of B-and T-cell activation, the invasion of CNS, their maturation and their reactivation. Findings of NGS in B and T cells of the peripheral blood, the CSF and the brain lesions have largely contributed to the understanding of the disease pathogenesis. Initial NGS studies on the repertoire of IgG heavy chain variable region genes showed that B cells may experience ongoing stimulation and maturation in the CNS, but they exchange across the blood-brain barrier and they can be identified in the peripheral blood [141, 143, 144] . Another NGS study, however, claimed that most of their maturation takes place outside the CNS in the draining cervical lymph nodes [145] . Further NGS analysis of CSF-derived B cells from MS patients has shown rearranged IgG variable heavy chain family 4 genes, suggestive of pronounced affinity maturation in the CNS, a feature that can be used as a biomarker for the diagnosis of MS [146] [147] [148] [149] . In line with these findings, other studies revealed persistence over time of intrathecal oligoclonal B cells and IgG [150] and intrathecal somatic hypermutation of IgM chains that are not observed in the peripheral blood [151] . Similarly, for T cells, initial NGS studies have shown that CD8+ and CD4+ cells isolated from brain autopsy lesions can be also found in the CSF [152] , and that clonally expanded CD8+ T cells are located in brain lesions, the CSF, and the peripheral blood [153] . However, further high-throughput sequencing of the T-cell receptor repertoires of the CSF revealed that they are largely distinct from the blood in MS patients [154, 155] , and that there is intrathecal enrichment of EBV-reactive CD8+ T cells and CD8+CD161 T cells [156, 157] . Interestingly, NGS of CD8+ and CD4+ T-cell receptor gene repertoires from CSF was used recently to support the diagnosis of MS [158] and to test the efficacy of treatment with autologous hematopoietic stem cell transplantation [159] . Furthermore, recently single-cell transcriptomics revealed increased transcriptional diversity in blood, and increased cell-type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells clonally expanded B cell in MS [160, 161] .

Neuromyelitis optica (NMO) is a rare autoimmune disease of the CNS characterized by inflammatory response against aquaporin-4 (AQP4). A recent study comparing NGS analysis of B cells in the CSF and the peripheral blood revealed that CSF AQP4-specific B cells are closely related to an expanded population of double negative B cells of the peripheral blood that may undergo maturation within the CNS [162] .

During the past few decades, tremendous progress on the understanding of neurodegenerative diseases has been achieved. However, neurodegenerative diseases constitute a heterogenic cluster with various degrees of overlapping in terms of clinical features, imaging findings, serum and CSF parameters, pathological characteristics, and associated genes [163] [164] [165] [166] [167] [168] . While pathological brain changes [120] may begin up to 30 years before the symptomatic phase in some of them [169, 170] , only symptomatic treatments exist, all trying to counterbalance the neurotransmitter disorganization and none to hamper disease progression [171] . Furthermore, symptomatic treatments are effective only in the early disease stages [172] . Thus, early detection is crucial, but accurate diagnosis is a real challenge for the clinical neurologist, and it is not uncommon for the diagnosis to be established only post-mortem [173] . Alzheimer disease (AD) is the most common neurodegenerative disease. While AD is characterized by an age-related pathology, there are a variety of genes related to early-onset AD [174] . Among them, the most common is Allele 4 of apolipoprotein E (ApoE4), which is also the most important risk factor for sporadic AD [175] . The pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles in brain tissue, reflected by a decrease in amyloid-β peptide 1-42 (Aβ42), and increase in total Tau and phosphorylated Tau protein (p-tau) in CSF [176] . Αβ42 is considered to be responsible for triggering a downstream that leads to Tau accumulation within neurons and, eventually, apoptosis [177] . Nevertheless, several other factors are implicated in AD pathogenesis, including mitochondrial, metabolic, inflammatory, and oxidative stress mechanisms [178] [179] [180] [181] . Recently, genetic studies in the CSF significantly contributed to the clarification of these pathways. MicroR-NAs (miRNAs) seem to play an important role in abnormal gene expression for genes associated with AD pathology [174] . During the past few years, several studies have identified with RT-PCR numerous miRNAs implicated in AD pathogenesis, some of them detected in the CSF [182] [183] [184] [185] . Additionally, transcriptome analysis of the CSF with NGS in AD managed to identify a variety of miRNAs and other small noncoding RNAs considered to be candidate biomarkers for AD, while changes in miRNA expression were shown to correlate well with aspects of disease severity [186, 187] .

Parkinson disease (PD) is the second most common neurodegenerative disease. It is characterized by motor symptoms, including bradykinesia, rigidity, and tremor, along with non-motor symptoms [169] . Its pathological hallmark is the progressive neuronal loss in the substantia nigra pars compacta due to accumulation of protein α-synuclein into cytoplasmic inclusions [188] . From there, the pathology spreads to the basal ganglia and the brain cortex. This could explain the variety of motor and non-motor symptoms associated with PD, including REM-sleep behavior disorder, dysautonomia, depression, and, finally, dementia. Thus, similar to AD patients, PD patients also seem to express Fig. 3 The role of CSF-based next-generation sequencing (NGS) in the diagnosis on neurodegenerative and neuroinflammatory diseases. CSF cerebrospinal fluid, CNS central nervous system, CNS central nervous system, CNS central nervous system cognitive impairment, especially in the late stages of the disease, when advanced neurodegeneration has taken place [165, 166] . This common disease progression, at least in the late stages of the PD, implies that AD and PD might share some common pathological features [167, 168] . So, the initiative of transcriptomics in AD has also motivated similar studies in PD. Burgos et al. compared miRNAs from the periphery and the CSF from patients with PD or AD and normal controls [187] . They found miRNA dysregulation in both AD and PD patients compared to normal controls. Interestingly, some of the miRNAs were differentially expressed in both PD and AD, while others were expressed only in AD or PD patients. After that, a few other miRNAs in the CSF have been identified as potential PD biomarkers. Some of them were related to genes that are associated with PD, such PARK7, LRRK2, and SNCA [189] .

Amyotrophic lateral sclerosis (ALS) is another neurodegenerative disease, whose main characteristic is the progressive loss of upper and lower motor neurons, commonly in association with cognitive and behavioral symptoms [190] . Indeed, an association between ALS and certain types of frontotemporal dementia has been well established [164] . Otake et al., using NGS transcriptome technology, analyzed exosomal mRNAs in human CSF [191] . In comparison with CSF of normal controls, 543 genes were found to have mutations and most of them were involved in the ubiquitin-proteasome, unfolded protein, and oxidative stress pathways.

Several studies suggest that CSF-based NGS has a role in the evaluation of infectious and neoplastic CNS diseases, while evidence concerning immunological and neurodegenerative diseases is at a more rudimentary level. NGS is a highthroughput, fast, and sensitive, albeit labor-intensive and costly, technique able to process multiple nucleic acid fragments in parallel and within a single run, at both genomic (DNA) or transcriptomic levels, without requiring prior knowledge of the exact nucleic acid sequence(s). This contrasts with PCR applications, which entail a hypothesis of potential targets in order to select the most suitable primers [10, 192] . Of interest, most CSF-based next-generation transcriptomic analyses applying RNA-Seq have been reported in MS, AD, or PD studies (Table 3) , whereas only a minority of oncology studies have utilized this methodology instead of DNA-Seq (Table 2) . Targeted NGS is often used in oncological applications [11] , and mNGS in microbiological applications [12] . The latter could especially be of value when conventional, less sensitive microbiologic methodssuch as morphologic evaluation (e.g., with Gram stain), cultures, or serologic testing-fail to diagnose the cause of a CNS infection, delaying treatment and impacting clinical outcome [4, 5] . In addition, because of its high-throughput nature, mNGS could help characterize genes responsible for antimicrobial drug resistance [29] or identify pathogens causing outbreaks [28] . However, despite its reported high diagnostic accuracy, mNGS clinical application is still at an early phase and there are several limitations [31] , while most evidence has been derived from small retrospective studies [32] .

The application of NGS in the CSF can facilitate diagnosis and classification, prognosis, selection of therapy, and follow-up of primary and metastatic CNS tumors (Fig. 1) . Of interest, CSF liquid biopsy could supplement or even replace tissue-based molecular analysis in the right clinical setting (e.g., patients with severe comorbidities, CNS tumors in critical or inaccessible locations, indication for sequential sampling to monitor evolution or therapy response), as it minimizes operational risk, is cheaper, and provides molecular signatures concordant to tissue analysis [9, 62, 73] . In addition, CSF liquid biopsy does not suffer from the inherent problems of tissue biopsy that commonly hamper subsequent molecular analysis, such as inadequacy and formalin fixation, and could depict intra-tumor heterogeneity more comprehensively, especially when compared to small biopsies [62, 63, 66, 193] . Likewise, it exhibits higher sensitivity to detect leptomeningeal metastases than imaging or CSF cytology [62, [95] [96] [97] [98] . Evidence suggests that CSF liquid biopsy should be favored over its plasma counterpart when evaluating a CNS tumor, either primary or metastatic, as it shows a higher concentration of ctDNA and a higher mutation detection rate, and it depicts the CNS lesion molecular profile more accurately, especially when dealing with brain metastases. Plasma liquid biopsy exhibits reduced diagnostic capacity since the blood-brain-barrier hampers the shedding of CNS tumor-derived genetic material into the blood. In addition, as blood contains more normal circulating DNA derived from its higher population of non-malignant cells than the paucicellular CSF, ctDNA comprises only a small fraction of the total circulating DNA, resulting in lower sensitivity of the NGS assay [9, 62, 73, 84, 96, 97] .

In the era of personalized oncology, laboratories are often asked to test minimal samples (such as the CSF) for multiple cancer biomarkers to assess patients' eligibility for targeted therapies. To this end, NGS is an attractive option, as it allows high-throughput testing in a single run saving at the same time precious diagnostic material [74, 89, [194] [195] [196] . Notably, NGS is an excellent tool to identify potential mechanisms of therapy resistance, such as the EGFR-T790M or C797S in metastatic NSCLC, in addition to concurrent alterations associated with shorter survival [197, 198] , or high TMB, which is known to be predictive of response to immune checkpoint inhibitors [62] . Although existing evidence seems promising, this is mostly derived from small and retrospective studies of low statistical power (similar to infectious diseases). Concerning future directions, relevant NGS oncology assays should ideally be designed specifically for CSF and validated at both pre-analytical and analytical levels [9] . In addition, as most published studies describing CSF-based molecular analysis have tested ctDNA (Table 2) , it would be interesting to see more studies researching DNA methylation, miRNAs [111] , or exosomes. Research directed on the molecular subtypes of diverse primary or metastatic cancers such as glioblastoma [199] or breast cancer [200] could potentially improve the clinical management and patient outcomes.

As far as neuroimmunological and neurodegenerative diseases are concerned, NGS in the CSF is still in an early stage. CSF-based, single-cell NGS at genomic and transcriptomic levels has added valuable mechanistic insight for the understanding and the diagnosis of MS [201] . Compartmentalization between blood and CSF and maturation of B and T cells have extensively been studied in MS, while NGS in the CSF has offered significant help for our understanding about the development and the progression of this disease [145, 153, 154, 157] . During the past two decades there was a boom in the use of genomics and transcriptomics for the study of neurodegenerative diseases [202] . However, a limited number of studies has been performed so far with NGS in the CSF of patients with AD, PD, and ALS, evaluating the potential role of miRNAs as biomarkers for disease progression. Despite their promising results, use of miRNAs as a routine examination in the CSF is still impractical, not only due to the cost but also their still undetermined significance [186, 187, 189, 191] . Moreover, studies that include other common neurodegenerative diseases, such as Frontotemporal and Lewy body dementias, are still lacking. Hence, further investigation is needed towards a wider spectrum of neurodegenerative diseases sorting out their most significant and common findings.

In conclusion, clinical application of NGS in the CSF of patients with CNS diseases can help physicians make accurate diagnoses, thus planning treatments effectively and reducing adverse effects and mortality rates. Metagenomic NGS has shown improved sensitivity-compared to traditional microbiological methods-to detect bacterial, viral, parasitic, and fungal infections, while overall diagnostic performance could be maximized when all modalities were applied. In patients with primary CNS cancers, NGS findings in the CSF were largely concordant with the molecular signatures derived from tissue-based molecular analysis, showing the potential to supplement and even replace tissuebased molecular profiling in the right clinical context, while additional mutations were detected in the CSF in some studies, reflecting intratumoral heterogeneity. In patients with metastatic CNS cancers, NGS could facilitate diagnosis and classification, prognosis, therapy selection, and follow-up, exhibiting higher sensitivity than neuroimaging, cytology, or plasma molecular analysis. Although most evidence has derived from studies dealing with infectious or neoplastic CNS disorders, NGS has shown potential to help diagnosing or understanding the pathogenesis of MS and neurodegenerative diseases, such as AD, PD, and ALS. However, as all aforementioned applications are still in an early phase, validation of NGS for each clinical scenario at both preanalytical and analytical levels is vital. Lastly, as most evidence stems from small and retrospective series, studies in the form of randomized controlled trials would be of paramount importance to establish clinical validity and utility.

Funding No funding was received to assist with the preparation of this article.

Ethics approval Not applicable.

Author contributions KIT, HS, AG, HSR, CG and IPN analyzed the bibliography, prepared all tables and wrote the manuscript. IPN designed the manuscript, prepared all figures and supervised the preparation of the manuscript.

Anatomy and physiology of the leptomeninges and CSF space

Cerebrospinal fluid and lumbar puncture: a practical review

Diagnostic lumbar puncture

State-of-the-art microbiologic testing for community-acquired meningitis and encephalitis

Next-generation sequencing in neuropathologic diagnosis of infections of the nervous system

Diagnostic tools for neoplastic meningitis: detecting disease, identifying patient risk, and determining benefit of treatment

Detection of cancer cells in the cerebrospinal fluid: current methods and future directions

Cerebrospinal fluid humoral immunity in the differential diagnosis of multiple sclerosis

Beyond the blood: CSF-derived cfDNA for diagnosis and characterization of CNS tumors

Next-generation sequencing-an overview of the history, tools, and "Omic" applications. Next generation sequencing advances, applications and challenges

Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls

Clinical metagenomic next-generation sequencing for pathogen detection

MicroRNAs in Alzheimer's disease: diagnostic markers or therapeutic agents?

Next generation sequencing for profiling expression of miRNAs: technical progress and applications in drug development

The role of MicroRNAs in human cancer

Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid

Central nervous system infections. Microbiol Spectrosc

Cerebrospinal fluid MinION sequencing of 16S rRNA gene for rapid and accurate diagnosis of bacterial meningitis

Metagenomic next-generation sequencing as a diagnostic tool for toxoplasmic encephalitis

Next-generation sequencing (NGS) in the identification of encephalitis-causing viruses: unexpected detection of human herpesvirus 1 while searching for RNA pathogens

Differential diagnosis of acute central nervous system infections in children using modern microbiological methods

Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases

Clinical metagenomics

Viral metagenomics on cerebrospinal fluid. Genes (Basel)

Identification of viruses in cases of pediatric acute encephalitis and encephalopathy using next-generation sequencing

Detection of Listeria monocytogenes in CSF from three patients with meningoencephalitis by next-generation sequencing

Brain metastases

Genomic comparative analysis and gene function prediction in infectious diseases: application to the investigation of a meningitis outbreak

From the pipeline to the bedside: advances and challenges in clinical metagenomics

A probable prehistoric case of meningococcal disease from San Francisco Bay: next generation sequencing of Neisseria meningitidis from dental calculus and osteological evidence

Two years of viral metagenomics in a tertiary diagnostics unit: evaluation of the first 105 cases. Genes (Basel)

Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis

Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis

Reduction of human DNA contamination in clinical cerebrospinal fluid specimens improves the sensitivity of metagenomic next-generation sequencing

Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion

Dilemmas in the diagnosis of acute community-acquired bacterial meningitis

Rapid sequencing-based diagnosis of infectious bacterial species from meningitis patients in Zambia

Fatal Psychrobacter sp. infection in a pediatric patient with meningitis identified by metagenomic next-generation sequencing in cerebrospinal fluid

The diagnostic value of metagenomic next-generation sequencing for identifying Streptococcus pneumoniae in paediatric bacterial meningitis

Aetiologies of central nervous system infection in Viet Nam: a prospective provincial hospital-based descriptive surveillance study

Toward a global genomic epidemiology of meningococcal disease

Cell-cycle and DNA-damage response pathway is involved in leptomeningeal metastasis of non-small cell lung cancer

Metagenomic next-generation sequencing (mNGS) in cerebrospinal fluid for rapid diagnosis of Tuberculosis meningitis in HIV-negative population

The Feasibility of metagenomic next-generation sequencing to identify pathogens causing Tuberculous meningitis in cerebrospinal fluid

Neonatal Ureaplasma parvum meningitis: a case report and literature review

Sensitivity of next-generation sequencing metagenomic analysis for detection of RNA and DNA viruses in cerebrospinal fluid: the confounding effect of background contamination

Current trends in diagnostics of viral infections of unknown etiology

Unbiased metagenomic sequencing for pediatric meningitis in Bangladesh reveals neuroinvasive chikungunya virus outbreak and other unrealized pathogens. mBio

A case of Naegleria fowleri related primary amoebic meningoencephalitis in China diagnosed by next-generation sequencing

Next-generation sequencing of cerebrospinal fluid for the diagnosis of neurocysticercosis

Next-generation sequencing of cerebrospinal fluid for the diagnosis of neurocysticercosis

Next generation sequencing based pathogen analysis in a patient with neurocysticercosis: a case report

Angiostrongyliasis detected by next-generation sequencing in a ELISA-negative eosinophilic meningitis: a case report

Next-generation sequencing specifies Angiostrongylus eosinophilic meningoencephalitis in infants: two case reports

Diagnosis and management of central nervous system cryptococcal infections in HIVinfected adults. J Fungi (Basel)

New approaches for cryptococcosis treatment. Microorganisms

Apparent performance of metagenomic next-generation sequencing in the diagnosis of cryptococcal meningitis: a descriptive study

Detection of cryptococcus DNA by metagenomic next-generation sequencing in symptomatic cryptococcal antigenemia

Chronic meningitis investigated via metagenomic next-generation sequencing

The 2016 World Health Organization classification of tumors of the central nervous system: a summary

Recent advances in the biology and treatment of brain metastases of non-small cell lung cancer: summary of a multidisciplinary roundtable discussion

Therapeutic management of neuro-oncologic patients-potential relevance of CSF liquid biopsy

Genetic characterization of brain metastases in the era of targeted therapy

Sequencing the next generation of glioblastomas

Success and failure rates of tumor genotyping techniques in routine pathological samples with nonsmall-cell lung cancer

Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma

Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics

Non-blood sources of cellfree DNA for cancer molecular profiling in clinical pathology and oncology

Liquid biopsy: general concepts

Molecular profiling of malignant pleural effusions with next generation sequencing (NGS): evidence that supports its role in cancer management

Comparison of liquid-based to tissue-based biopsy analysis by targeted next generation sequencing in advanced non-small cell lung cancer: a comprehensive systematic review

Circulating tumor DNA analysis in patients with cancer

The value of cell-free DNA for molecular pathology

Next-generation sequencing in oncology: genetic diagnosis, risk prediction and cancer classification

Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

Pediatric brain tumors

Molecular pathology of tumors of the central nervous system

MGMT gene silencing and benefit from temozolomide in glioblastoma

TERT promoter mutation detection in cell-free tumorderived DNA in patients with IDH wild-type glioblastomas: a pilot prospective study

Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma

Molecular diagnosis of diffuse gliomas through sequencing of cell-free circulating tumor DNA from cerebrospinal fluid

Applications of cerebrospinal fluid circulating tumor DNA in the diagnosis of gliomas

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

Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid

Assessment of circulating tumor DNA in cerebrospinal fluid by whole exome sequencing to detect genomic alterations of glioblastoma

Brain tumor mutations detected in cerebral spinal fluid

Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients

Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord

Molecular testing guideline for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical Practice Guideline Update

Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology

Diagnosis and treatment of brain metastases from solid tumors: guidelines from the European Association of Neuro-Oncology (EANO)

Viewing the Eph receptors with a focus on breast cancer heterogeneity

Diagnostic accuracy of cerebrospinal fluid (CSF) cytology in metastatic tumors: an analysis of consecutive CSF samples

Brain metastases: neuroimaging

Evaluating circulating tumor DNA from the cerebrospinal fluid of patients with melanoma and leptomeningeal disease

Evaluating cancer of the central nervous system through next-generation sequencing of cerebrospinal fluid

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

Evaluating the cerebrospinal fluid ctDNA detection by next-generation sequencing in the diagnosis of meningeal Carcinomatosis

Different next-generation sequencing pipelines based detection of tumor DNA in cerebrospinal fluid of lung adenocarcinoma cancer patients with leptomeningeal metastases

Detection of circulating tumor DNA from non-small cell lung cancer brain metastasis in cerebrospinal fluid samples

Tyrosine kinase inhibitors could be effective against non-small cell lung cancer brain metastases harboring uncommon EGFR mutations

Clinical utility of cerebrospinal fluid cell-free DNA as liquid biopsy for leptomeningeal metastases in ALK-rearranged NSCLC

Detection of driver and resistance mutations in leptomeningeal metastases of NSCLC by next-generation sequencing of cerebrospinal fluid circulating tumor cells

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-smallcell lung cancer: a new medium of liquid biopsy

Afatinib helped overcome subsequent resistance to osimertinib in a patient with NSCLC having leptomeningeal metastasis baring acquired EGFR L718Q mutation: a case report

Longterm survival with targeted therapy in an advanced non-small cell lung cancer patient based on genetic profiling

Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma

Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases

Genotyping tumour DNA in cerebrospinal fluid and plasma of a HER2-positive breast cancer patient with brain metastases. ESMO Open

Osimertinib quantitative and gene variation analyses in cerebrospinal fluid and plasma of a non-small cell lung cancer patient with leptomeningeal metastases

Cerebrospinal fluid MicroRNA signatures as diagnostic biomarkers in brain tumors

Circulating tumor DNA analysis for patients with oncogene-addicted NSCLC with isolated central nervous system progression

Unique genomic profiles obtained from cerebrospinal fluid cell-free DNA of non-small cell lung cancer patients with leptomeningeal metastases

The genetics of neuropsychiatric disorders

Advances in genetic diagnosis of neurological disorders

Circulating microRNAs as potential biomarkers for psychiatric and neurodegenerative disorders

Genomics in neurological disorders

Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke

Autism spectrum disorder genomics: the progress and potential of genomic technologies

Dissecting diagnostic heterogeneity in depression by integrating neuroimaging and genetics

Heritability of specific cognitive functions and associations with schizophrenia spectrum disorders using CAN-TAB: a nation-wide twin study

Independent replications and integrative analyses confirm TRANK1 as a susceptibility gene for bipolar disorder. Neuropsychopharmacology

Virus discovery analyses on post-mortem brain tissue and cerebrospinal fluid of schizophrenia patients

The genetics of multiple sclerosis

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Gene map of the extended human MHC

B-cell alloantigen Ag 7a in multiple sclerosis

Genetics of multiple sclerosis: an overview and new directions

Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1. PLoS One

Genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci

A non-synonymous single-nucleotide polymorphism associated with multiple sclerosis risk affects the EVI5 interactome

Interleukin 7 receptor alpha chain (IL7R) shows allelic and functional association with multiple sclerosis

IL2RA genetic heterogeneity in multiple sclerosis and type 1 diabetes susceptibility and soluble interleukin-2 receptor production

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation

A gene pathway analysis highlights the role of cellular adhesion molecules in multiple sclerosis susceptibility

International Multiple Sclerosis Genetics C. Network-based multiple sclerosis pathway analysis with GWAS data from 15,000 cases and 30,000 controls

Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis

Cell-free mitochondrial DNA in progressive multiple sclerosis

B and T cells driving multiple sclerosis: identity, mechanisms and potential triggers

B cell exchange across the blood-brain barrier in multiple sclerosis

Detailed characterization of T cell receptor repertoires in multiple sclerosis brain lesions

In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses

Immunoglobulin class-switched B cells form an active immune axis between CNS and periphery in multiple sclerosis

B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes

The antibody genetics of multiple sclerosis: comparing next-generation sequencing to sanger sequencing

MSPrecise: a molecular diagnostic test for multiple sclerosis using next generation sequencing

Intrathecal BCR transcriptome in multiple sclerosis versus other neuroinflammation: Equally diverse and compartmentalized, but more mutated, biased and overlapping with the proteome

Statistical classifiers for diagnosing disease from immune repertoires: a case study using multiple sclerosis

Persistence of intrathecal oligoclonal B cells and IgG in multiple sclerosis

Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation

Central role of Th2/Tc2 lymphocytes in pattern II multiple sclerosis lesions

Expanded CD8 T-cell sharing between periphery and CNS in multiple sclerosis

Disease etiology and diagnosis by TCR repertoire analysis goes viral

Intrathecal T-cell clonal expansions in patients with multiple sclerosis

High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8+ T cells

An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8(+) T cells involved in multiple sclerosis

CTLA4 as immunological checkpoint in the development of multiple sclerosis

Extensive intrathecal T cell renewal following hematopoietic transplantation for multiple sclerosis

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis

A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis

CNS Aquaporin-4-specific B cells connect with multiple B-cell compartments in neuromyelitis optica spectrum disorder

Can cognitive assessment really discriminate early stages of Alzheimer's and behavioural variant frontotemporal dementia at initial clinical presentation?

movement disorders and genetics in frontotemporal dementia

Cognitive and pathological influences of tau pathology in Lewy body disorders

A broader view of dementia: multiple co-pathologies are the norm

Regional neuropathology distribution and verbal fluency impairments in Parkinson's disease

Neurodegenerative disease concomitant proteinopathies are prevalent, age-related and APOE4-associated

Staging of brain pathology related to sporadic Parkinson's disease

Early intervention is key to successful management of Alzheimer disease

Alzheimer's disease: diagnosis and prediction of progression

Current and future treatments in Alzheimer disease: an update

Association between clinical symptoms and post-mortem neuropathology in dementia with Lewy bodies

Alzheimer's disease in the omics era

APOE and Alzheimer disease: a major gene with semi-dominant inheritance

The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease

The amyloid hypothesis of Alzheimer's disease at 25 years

Early diagnostic accuracy and pathophysiologic relevance of an autopsy-confirmed Alzheimer's disease peripheral biomarker

Oxidative stress hypothesis in Alzheimer's disease: a reappraisal

Bidirectional interactions between diabetes and Alzheimer's disease

Impaired resolution of inflammation in Alzheimer's disease: a review

microRNA (miRNA) speciation in Alzheimer's disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Reduced expression of hsa-miR-27a-3p in CSF of patients with Alzheimer disease

Epigenetic alterations in Alzheimer's disease

MicroRNA expression levels are altered in the cerebrospinal fluid of patients with young-onset Alzheimer's disease

A combined miRNA-piRNA signature to detect Alzheimer's disease

Profiles of extracellular miRNA in cerebrospinal fluid and serum from patients with Alzheimer's and Parkinson's diseases correlate with disease status and features of pathology

Parkinson's disease

Transcriptomic profiling of extracellular RNAs present in cerebrospinal fluid identifies differentially expressed transcripts in Parkinson's disease

Amyotrophic lateral sclerosis: moving towards a new classification system

Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid

Integrated morphologic and molecular analysis of Trichomonas vaginalis, Mycoplasma hominis, and human papillomavirus using cytologic smear preparations

Cerebrospinal fluid detection of enterovirus genome in ALS: a study of 242 patients and 354 controls

Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology

Molecular pathology of lung cancer cytology specimens: a concise review

Invited review-next-generation sequencing: a modern tool in cytopathology

Biomarkers of osimertinib response in patients with refractory, EGFR-T790M-positive non-small cell lung cancer and central nervous system metastases: the APOLLO study

Genotyping of cerebrospinal fluid associated with osimertinib response and resistance for leptomeningeal metastases in EGFR-mutated NSCLC

Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1

Intrinsic subtypes and gene expression profiles in primary and metastatic breast cancer

Single-cell high-throughput technologies in cerebrospinal fluid research and diagnostics

Omics in neurodegenerative disease: hope or hype?

Molecular profiling of tumors of the brainstem by sequencing of CSF-derived circulating tumor DNA. Acta Neuropathol

Phase 1b trial of an ibrutinib-based combination therapy in recurrent/refractory CNS lymphoma

Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next generation sequencing

Association of cerebrospinal fluid tumor DNA genotyping with survival among patients with lung adenocarcinoma and central nervous system metastases

Multiple combination therapy based on intrathecal pemetrexed in nonsmall cell lung cancer patients with refractory leptomeningeal metastasis

Afatinib for advanced non-small cell lung cancer in a case with an uncommon epidermal growth factor receptor mutation (G719A) identified in the cerebrospinal fluid

The therapeutic value of cerebrospinal fluid ctDNA detection by nextgeneration sequencing for meningeal carcinomatosis: a case report