cancers Review ctDNA-Based Liquid Biopsy of Cerebrospinal Fluid in Brain Cancer Laura Escudero 1 , Francisco Martínez-Ricarte 2,3 and Joan Seoane 1,3,4,5, * 1 Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, 08035 Barcelona, Spain; lescudero@vhio.net 2 Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron University Hospital, 08035 Barcelona, Spain; frmartinez@vhebron.net 3 Universitat Autotònoma de Barcelona (UAB), 08193 Bellaterra, Spain 4 CIBERONC (Centro de Investigación Biomédica en Red de Cáncer), Instituto de Salud Carlos III, 28029 Madrid, Spain 5 ICREA (Institució Catalana de Recerca i Estudis Avançats), 08010 Barcelona, Spain * Correspondence: jseoane@vhio.net Simple Summary: The optimal treatment and management of patients with brain cancer depend on the molecular characteristics of their tumour. Since the tumour changes with time, it is, therefore, essential to characterise the tumour of each patient at the exact time of selecting the most suitable therapeutic strategy. However, obtaining a tumour biopsy for its characterisation is a risky and invasive procedure and, sometimes, not even feasible, leading to a lack of information about the tumour. These challenges can be overcome by using a liquid biopsy of cerebrospinal fluid. Brain cancer cells release DNA into the cerebrospinal fluid, and the analysis of the cell-free circulating tumour DNA can reveal the genetic profile of brain cancer in a relatively noninvasive manner. In  this review, we revise the recent results in this field that show how circulating tumour DNA in  cerebrospinal fluid can provide diagnostic and prognostic information, identify potential therapeutic Citation: Escudero, L.; targets, monitor the tumour response or resistance to treatment, and help to identify tumour relapse. Martínez-Ricarte, F.; Seoane, J. ctDNA-Based Liquid Biopsy of Abstract: The correct characterisation of central nervous system (CNS) malignancies is crucial for Cerebrospinal Fluid in Brain Cancer. accurate diagnosis and prognosis and also the identification of actionable genomic alterations that Cancers 2021, 13, 1989. https:// can guide the therapeutic strategy. Surgical biopsies are performed to characterise the tumour; doi.org/10.3390/cancers13091989 however, these procedures are invasive and are not always feasible for all patients. Moreover, they Academic Editor: Marica Eoli only provide a static snapshot and can miss tumour heterogeneity. Currently, monitoring of CNS cancer is performed by conventional imaging techniques and, in some cases, cytology analysis of Received: 22 March 2021 the cerebrospinal fluid (CSF); however, these techniques have limited sensitivity. To overcome these Accepted: 19 April 2021 limitations, a liquid biopsy of the CSF can be used to obtain information about the tumour in a Published: 21 April 2021 less invasive manner. The CSF is a source of cell-free circulating tumour DNA (ctDNA), and the analysis of this biomarker can characterise and monitor brain cancer. Recent studies have shown that Publisher’s Note: MDPI stays neutral ctDNA is more abundant in the CSF than plasma for CNS malignancies and that it can be sequenced with regard to jurisdictional claims in to reveal tumour heterogeneity and provide diagnostic and prognostic information. Furthermore, published maps and institutional affil- analysis of longitudinal samples can aid patient monitoring by detecting residual disease or even iations. tracking tumour evolution at relapse and, therefore, tailoring the therapeutic strategy. In this review, we provide an overview of the potential clinical applications of the analysis of CSF ctDNA and the challenges that need to be overcome in order to translate research findings into a tool for clinical practice. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: central nervous system malignancies; brain cancer; circulating tumour DNA; cere- This article is an open access article brospinal fluid; liquid biopsies distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 1989. https://doi.org/10.3390/cancers13091989 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 1989 2 of 15 1. Introduction Central nervous system (CNS) malignancies affect both children and adults worldwide and are responsible for substantial morbidity and mortality. An epidemiological study of CNS cancer between 1990 and 2016 revealed that the age-standardised incidence rate has increased by 17.3% globally, with 330,000 incident cases and 227,000 deaths globally in 2016 [1]. CNS cancer consists of primary tumours and intracranial metastases. Most CNS tumours (>90%) occur in the brain, with the remaining located in the meninges, spinal cord and nerves. Depending on the anatomical region and the tumour type, the neurological signs and symptoms will vary and may include headaches, seizures, loss of vision, paralysis, speech disturbance, and motor deficits [2]. CNS tumours are diagnosed using neuroimaging techniques such as magnetic reso- nance imaging (MRI) or computed tomography; however, to obtain pathological informa- tion and molecular diagnosis, tumour biopsies are required. The treatment strategy for primary CNS tumours consists of either obtaining a biopsy or performing a surgical resec- tion, combined, when appropriate, with postoperative radiotherapy and chemotherapy [3]. CNS tumour prognosis is diverse since there are distinct entities with different histopathological characteristics and molecular profiles. Therefore, characterising the tumour specimen is essential for accurate diagnosis and prognosis, as well as to identify potential therapeutic targets. To improve disease control, primary brain tumours or single nodule brain metastases are resected. However, obtaining tumour biopsies is not always possible due to their location, particularly when CNS tumours occur in vital regions such as the basal ganglia or the brain stem. In addition, patients with disseminated disease may not be eligible for such procedures [4–6]. In cases where tumour resection or obtaining a biopsy is possible, the sample obtained may not be representative of tumour heterogeneity [7–9], and, therefore, multiple sampling may be required to confirm the pathological diagnosis in some cases. Moreover, the analysis of the sample obtained only provides a static snapshot from the time of resection. It is important to monitor the patient’s response to treatment during the course of the disease, particularly to distinguish true disease progression from a pseudoprogression induced by treatment. Sometimes a new or enlarging area of contrast enhancement is observed, but it is not easy to assess whether it is the result of tumour growth or an inflammatory response [10]. This can be challenging when using conventional imaging techniques. Tumours evolve over time, particularly under the selective pressure of therapy, which can result in the expansion of pre-existing resistant clones or the acquisition of de novo resistant alterations [7]. Thus, the genomic characteristics at relapse may differ from the genomic landscape at first occurrence. In some cases, treatment decisions at relapse are just based on the characteristics of the primary tissue obtained [11,12]. The known evolution of tumours and the absence of longitudinal tumour sampling may, therefore, lead to imprecise diagnosis and clinical management. For these reasons, there is an urgent need to develop less invasive methods to identify and validate tumour biomarkers that provide real-time information to aid in diagnosing and monitoring CNS malignancies. Overall, this will help to adjust the therapeutic strategy and guide treatment decisions based on the current tumour profile and its burden. An alternative to a tumour biopsy is a liquid biopsy (Figure 1). Liquid biopsies are emerging as noninvasive tools that can provide longitudinal information about the tumour genomic landscape and facilitate patient monitoring. It consists of the analysis of biomarkers, including circulating tumour cells, exosomes and circulating tumour nucleic acids that are present in bodily fluids such as blood, cerebrospinal fluid (CSF), urine and saliva [13,14]. Cancers 2021, 13, 1989 3 of 15 Cancers 2021, 13, x 3 of 15 Figure 1. Solid vs. liquid biopsies. Schematic representation of the tumour biopsy and CSF Figure 1. Solid vs. liquid biopsies. Schematic representation of the tumour biopsy and CSF samples samples obtained from a patient with a CNS malignancy. Advantages and limitations for each obtained from a patient with a CNS malignancy. Advantages and limitations for each methodology methodology are indicated. Definition of acronyms: standard of care (SOC), cerebrospinal fluid are indicated. (CSF), Definition circulating tumour of DNA acronyms: standard (cfDNA) of carenervous and central (SOC), cerebrospinal system (CNS).fluid (CSF), circulating tumour DNA (cfDNA) and central nervous system (CNS). In this review, we will discuss the potential applications of circulating tumour DNA In this review, we will discuss the potential applications of circulating tumour DNA (ctDNA) in CSF as a biomarker for CNS malignancies, the challenges that we need to (ctDNA) in CSF as a biomarker for CNS malignancies, the challenges that we need to overcome, and future perspectives for its implementation in the clinical setting. overcome, and future perspectives for its implementation in the clinical setting. 2.2.Circulating CirculatingCell-Free Cell-FreeDNA DNAand andCirculating CirculatingTumour TumourDNA DNA Cellsrelease Cells releaseDNA DNAthatthatthen thencirculate circulateininbodily bodilyfluids. fluids.The Thefraction fractionof ofcell-free cell-freeDNA DNA (cfDNA) that is shed by cancer cells, presumably undergoing apoptosis or necrosis, isis (cfDNA) that is shed by cancer cells, presumably undergoing apoptosis or necrosis, knownasasctDNA known ctDNAand andcarries carries genomic genomic alterations alterations that that cancanbe be detected detected using using PCR-based PCR-based or or next-generation next-generation sequencing sequencing (NGS)-based (NGS)-based methods methods [14–17]. [14–17]. Increasedconcentrations Increased concentrationsofof cfDNA cfDNA havehave beenbeen detected detected in pathological in pathological conditions conditions like trauma, infection and cancer, or even other physiological conditions like exercise [18]. [18]. like trauma, infection and cancer, or even other physiological conditions like exercise Forpatients For patientswith withananextracranial extracranialdisease, disease,plasma plasmactDNA ctDNAhas hasbeen beendetected detectedacross across differentcancer different cancertypes. types.However, However,blood bloodmay maynot notbe beaasuitable suitablesource sourcefrom frompatients patientswith with CNSmalignancies CNS malignancies since since ctDNA ctDNA levelslevels werewere infrequently infrequently detecteddetected in [19,20]. in plasma plasma ctDNA [19,20]. ctDNA was was detectable detectable in theofplasma in the plasma >75% of of patients >75% of with patients with advanced advanced cancers, cancers, such such as as bladder, bladder, colorectal, colorectal, gastroesophageal, gastroesophageal, ovarian, pancreatic, ovarian, pancreatic, breast, melanoma, breast, melanoma, hepatocel- hepatocellular, and lular,and head andneck headcancers, and neck cancers, with in contrast in contrast <10% with <10%patients of glioma of glioma patients (2/27) [19].(2/27) [19]. Theproportion The proportion of ctDNA ctDNAin inthe theblood blood is is small andand small varies depending varies on tumour depending char- on tumour characteristics, including acteristics, including type, type, grade grade andburden and burden[16,19]. [16,19].InIncontrast, contrast, the total total amount amountofof ctDNA ctDNAin inCSF CSFisisincreased, increased,making makingititan anideal idealbiofluid biofluidtotocharacterise characteriseand andmonitor monitorCNSCNS cancer [20,21]. Interestingly, the levels of ctDNA in CSF may be influenced by tumour Cancers 2021, 13, 1989 4 of 15 cancer [20,21]. Interestingly, the levels of ctDNA in CSF may be influenced by tumour burden, tumour progression and anatomical location of the tumour, with regard to the proximity to CSF reservoirs [21–23]. 3. Cerebrospinal Fluid as a Source of ctDNA CSF is a clear bodily fluid secreted by the choroid plexus that is present in the sub- arachnoid space of the brain, the spinal cord and the central canal [24]. CSF is in direct contact with the brain parenchyma, and several studies have shown that CSF is a reliable source of cell-free ctDNA, providing advantages over plasma or serum for the analysis of CNS tumours [20,21]. Several studies have reported the ability to detect ctDNA in CSF of patients with CNS malignancies. Gene mutations and molecular alterations have been detected in the CSF DNA of CNS cancer patients [25–30], followed by genomic landscape characteri- sation of CSF ctDNA with the development of high throughput sequencing technolo- gies [20–23,31–36]. CSF samples can be accessed through a lumbar puncture or obtained from the ventri- cles under certain circumstances. Patients with posterior fossa tumours tend to present with hydrocephalus, a condition in which the CSF accumulates within the cerebral ventri- cles and/or subarachnoid spaces [37–39]. A lumbar puncture is contraindicated in these patients, given the risk of brain herniation; therefore, CSF is obtained from the ventricles during procedures that are performed to alleviate intracranial pressure and drain the excess of CSF [38,40–42]. As part of the CNS staging criteria for certain brain tumours, a diagnostic lumbar puncture is routinely performed as standard of care for CSF cytology evaluation of CNS lymphoma, CNS metastasis, and medulloblastoma [43–46]. In these cases, the CSF samples collected as standard of care can be further used to characterise the ctDNA and provide information about the tumour [20,34,47]. 4. Clinical Applications of the CSF ctDNA for CNS Malignancies CNS tumours are a heterogeneous group of malignancies [48]. In most cases, tumour resection is required to reduce tumour burden and mass effect [49]. However, liquid biopsies can be used to complement histopathological diagnosis and are essential for those patients with inoperable tumours. The monitoring of CNS malignancies is currently performed by imaging techniques; however, these are not sensitive for microscopic disease [50]. A complementary liquid biopsy of CSF could, therefore, be performed to aid clinical assessment by determining the response to treatment, differentiating pseudoprogression from true progression and tracking levels of residual disease. In addition, the genomic characterisation of CSF ctDNA can facilitate the identification of actionable genomic alterations that confer sensitivity or resistance to clinically available drugs and the detection of mechanisms of resistance at relapse. The distinct clinical applications of the analysis of CSF ctDNA are discussed below for patients with distinct types of CNS cancer. 4.1. Diffuse Gliomas 4.1.1. A Diagnostic and Prognostic Tool Among diffuse gliomas, glioblastoma (GBM) is the most common malignant brain tumour in adults, with a 2-year survival of 18% and 5-year overall survival (OS) of 4% [51]. Providing an accurate molecular profile for diagnosis and prognosis is essential and can be achieved with a CSF liquid biopsy. The analysis of the mutational status of IDH1, IDH2, ATRX, TP53, TERT, H3F3A and HIST1H3B in CSF ctDNA facilitates the molecular diag- nosis of diffuse gliomas and provides prognostic information in a relatively noninvasive manner [22]. In addition, TERT promoter mutations have been detected in the CSF ctDNA of GBM patients, and shorter OS of patients with high variant allele frequency (VAF) has Cancers 2021, 13, 1989 5 of 15 been observed. The results from this pilot study suggested that VAF levels of the TERT promoter mutation could be a predictor of poor survival [52]. In more recent studies, CSF was obtained from lumbar punctures in glioma patients, and ctDNA was detected and was associated with disease burden, tumour progression, and adverse outcomes [20,23]. Moreover, most patients with detectable ctDNA had a negative cytopathologic analysis, and ctDNA was not detected in plasma [20,23]. Diffuse midline glioma (DMG) is a tumour entity characterised by a K27M mutation in either H3F3A or HIST1H3B/C; it is usually located in the brain stem, thalamus and spinal cord [48]. Within H3 K27M-mutant DMG, diffuse intrinsic pontine glioma (DIPG) is a rapidly growing tumour in the brain stem that typically arises in young children and is associated with poor survival [53]. The anatomical location of these tumours, the brainstem, makes them difficult and dangerous to biopsy. Importantly, H3 K27M mutations can be detected in the CSF ctDNA, facilitating diagnosis and opening the possibility of avoiding diagnostic surgical biopsies [22,54,55]. The molecular characterisation and understanding of DIPG biology have been im- proved from specimens obtained from rare diagnostic biopsies and postmortem tissue donations [56–59]. Indeed, the lack of surgical specimens can be overcome by the analysis of CSF ctDNA to aid in the management of patients with DIPG and contribute to the molecular study of this disease to accelerate research. An NGS panel of 68 genes commonly mutated in brainstem tumours was used to study a cohort of 57 patients with brainstem tumours, including 23 patients with DIPG. Mutations were detected in the CSF ctDNA of 82.5% of patients, and the presence of H3F3A/HIST1H3B mutations was correlated with poor OS while the IDH1 mutation predicted better OS [54]. Moreover, longitudinal analysis of CSF samples offers the possibility of monitoring and allows the tumour evolution of this dismal disease to be studied. 4.1.2. Monitoring and Therapeutic Strategies The number of actionable genomic alterations for patients with primary brain tumours is limited. The most relevant biomarker for glioma is MGMT promoter methylation status. MGMT promoter methylation causes the loss of MGMT expression, and since it is involved in DNA repair by reversing DNA alkylation, MGMT promoter methylation renders cells more susceptible to temozolomide and is associated with longer survival [60–63]. MGMT promoter methylation was detected using methylation-specific PCR from genomic DNA extracted from the CSF of glioma patients, with higher sensitivity than from serum [64]. A potential biomarker for GBM is epidermal growth factor receptor (EGFR). EGFR amplification and EGFRvIII mutation have been detected in RNA within extracellular vesicles circulating in CSF [65]. This could be of high interest as a biomarker to predict response to future EGFRvIII-targeted therapies in GBMs. Longitudinal analysis of CSF ctDNA from glioma patients showed the evolution of the cancer genome through the mutational changes detected [23]. 4.2. Brain and Leptomeningeal Metastases 4.2.1. CSF ctDNA Facilitates Diagnosis and Allows Tumour Genomic Characterisation About 20–40% of patients with advanced-stage cancers of the lung, breast and melanoma develop brain metastasis, and approximately 5–8% of these patients are diagnosed with leptomeningeal metastasis. These are devastating diseases that carry a poor prognosis and are often resistant to treatment [66–68]. In addition to brain metastasis present in the brain parenchyma, malignant cells can seed the leptomeninges, causing leptomeningeal metastasis [67,69,70]. The diagnosis of leptomeningeal metastasis is based on clinical symptoms, MRI scans, and cytology analysis of CSF [71]. However, up to 20% of patients with positive clinical and radiographic signs presented false-negative CSF cytology [72,73]. Several studies have shown that ctDNA can be detected in the CSF of patients with negative cytology analysis [20,27,32,74]. Cancers 2021, 13, 1989 6 of 15 Cytology has limited sensitivity, and the analysis of ctDNA can complement the diagnosis of leptomeningeal metastases. Brain metastasis can present different genomic alterations compared to their pri- mary extracranial tumour [12]. Several studies have shown that the analysis of CSF ctDNA enables the characterisation of the genomic complexity of CNS metastases, in- cluding intratumour heterogeneity, revealed with the identification of trunk and private genomic alterations. Moreover, the genomic landscape of CNS metastasis, including the brain lesion’s private alterations, was better represented from the ctDNA in the CSF than plasma [20,32,75]. Analysis of CSF ctDNA from a cohort of 26 patients with leptomeningeal metastases from non-small cell lung cancer (NSCLC) revealed their unique genetic profiles, including mutations in several driver genes, copy number variations (CNVs) in MET, ERBB2, KRAS, ALK, and MYC, and loss of heterozygosity in TP53 [76]. 4.2.2. Patient Monitoring and Identification of Therapeutic Targets There are several targeted therapies for brain metastases [77–86]. For EGFR-mutated brain metastases from NSCLC, first-, second- and third- generation EGFR tyrosine kinase inhibitors (TKIs) are available [87–90]. In addition, for NSCLC with ALK gene rearrange- ment, CNS penetration and therapeutic potential were exhibited by second-generation ALK inhibitors [84–86]. There are also targeted therapies for patients diagnosed with HER2+ breast cancer and melanoma patients, including BRAF and MEK inhibitors [82,83,91,92]. For the treatment of leptomeningeal metastases, targeted therapies for the aforementioned actionable genomic alterations may also be effective [93]. The availability of targeted therapies highlights the importance of the identifica- tion of actionable genomic alterations or resistance mutations in genes, including EGFR, ALK, BRAF and HER2, which have been detected from CSF ctDNA in several stud- ies [20,21,31,32,94–96]. For example, EGFR–TKI resistance mutation EGFR T790M has been detected in the CSF ctDNA of lung cancer patients [94,97]. A study of 21 patients with brain metastasis from NSCLC compared the NGS results obtained from different samples to reveal the mutation pattern of driver genes for each patient. Mutations were detected in 95.2%, 66.7% and 39% of patients from CSF ctDNA, plasma ctDNA, and plasma circulating tumour cells, respectively. The most mutated gene was EGFR, followed by KIT, PIK3CA, TP53, SMAD4, ATM, SMARCB1, PTEN and FLT3 (all >15%). For EGFR mutations, the detection rate was 57.1% (12/21) from CSF ctDNA, which, interestingly, was higher for patients with leptomeningeal (81.8%; 9/11) compared with brain parenchymal (30%; 3/10) metastases [98]. The analysis of CSF ctDNA can also contribute to monitoring response to treatment. Metastasis in the CNS developed in a patient with HER2+ breast cancer. Analysis of baseline CSF ctDNA revealed mutations in TP53 and PIK3CA and amplification in ERBB2 and cMYC. Following treatment with T-DM1, extracranial disease control was achieved, and marker levels in plasma decreased. However, the levels increased in CSF ctDNA, consistent with poor treatment benefit to the CNS [99]. Altogether, these results indicate that CSF is a more suitable fluid than plasma to reveal the mutational profile of CNS metastases and can aid in diagnosis, tailored treatment selection and monitoring. 4.3. CNS Lymphoma Malignant B-cells can infiltrate the CNS and are associated with poor prognosis, particularly at relapse [100]. Primary CNS lymphoma is defined by the absence of systemic disease in contrast to secondary CNS lymphoma that presents infiltration into the CNS with previous or concomitant systemic lymphoma [101,102]. ctDNA has only been detected in the plasma of a minority of patients with restricted CNS lymphoma [103,104]. In contrast, several studies of patients with CNS lymphomas detected ctDNA in CSF [47,105–109]. Cancers 2021, 13, 1989 7 of 15 Diagnosis and monitoring of CNS lymphoma are challenging, given the difficulties of tumour biopsies and the lack of sensitivity of CSF standard analysis (cytology and flow cytometry) and neuroimaging. The detection of the MYD88 L265P mutation strongly suggests the diagnosis of primary CNS lymphoma, and this mutation has been detected in the CSF ctDNA of patients with CNS lymphoma [47,106,108,109], showing that the analysis of CSF ctDNA could complement the diagnosis. An NGS-based analysis of the CSF cfDNA of 8 patients with CNS lymphoma, at recurrence, detected tumour-derived genetic alterations and showed that the clearance of ctDNA from CSF was associated with sustained tumour responses [105]. The comparison of CSF ctDNA with plasma ctDNA and CSF standard analysis (cy- tology and flow cytometry) revealed that the analysis of CSF ctDNA better detected CNS disease in patients with B-cell lymphoma [47]. Moreover, longitudinal analysis of CSF ctDNA levels allowed the monitoring of response to treatment, the detection of residual disease and predicted relapse. The dynamic changes observed in CSF ctDNA recapitulated the evolution of the disease for patients with CNS lymphoma [47]. 4.4. Medulloblastoma CNS tumours are the leading cause of cancer-related mortality in children and ado- lescents due to the aggressiveness of certain subtypes, including medulloblastoma and high-grade gliomas such as DMG [110,111]. Medulloblastoma (MB), an embryonal tumour of the CNS, is the most aggressive brain tumour in childhood that can also occur in adults, although this is less common [112]. MB is a complex and evolving heterogeneous disease that can be divided into four molecular consensus subgroups (WNT, SHH, Group 3 and Group 4), with further subtypes identi- fied [113–117]. The lack of sufficient sample, intratumour heterogeneity or the presence of disseminated disease make diagnosis and monitoring difficult [8,118–121]. However, hydrocephalus is common amongst these patients, and CSF samples can be obtained prior to tumour surgical resection or biopsy [40,42]. In addition, CSF samples are routinely col- lected through a lumbar puncture for cytology analysis to assess metastatic dissemination according to Chang’s M-staging system, in combination with brain and spinal MRIs [45]. The study of paediatric patients with MB showed that ctDNA was more abundant in CSF (76.9%, 10/13 patients) than plasma (1/13 patients) for patients with negative CSF cytology results. Moreover, exome sequencing of CSF ctDNA recapitulated the tumour mutational burden and the genomic alterations, including MB common mutations (PTCH1, TP53), CNVs (MYCN and GLI2 amplification) and arm-level chromosomal aberrations (chromosome 17p loss), providing diagnostic and prognostic information [34]. Longitudinal CSF samples were also collected, and ctDNA analysis detected residual disease, identified intratumour and interlesion heterogeneity, and revealed a genomic transformation of the tumour at relapse [34]. More recently, another study reported the detection of ctDNA in CSF; however, shared genetic mutations between CSF and the tumour specimen were only identified in 22% (2/9) of patients. The authors suggested that this could be explained by the time-interval differences between tumour and CSF collection [122]. MB also presents abnormal DNA methylation changes, with distinct epigenetic signa- tures identified across MB subtypes that can be altered during tumour progression and treatment [114,123]. The epigenetic analysis of CSF ctDNA from 4 MB patients (3 with matching tumour samples) was attained. A positive correlation of tumour and CSF sam- ples was identified, suggesting that CSF ctDNA could be used to monitor changes in MB tumour DNA methylomes and hydroxymethylomes. In addition, DNA methylation markers of diagnostic and prognostic value could be detected in the CSF ctDNA [124]. In summary, CSF ctDNA analysis could facilitate the clinical management of paediatric patients with MB. Cancers 2021, 13, 1989 8 of 15 5. Challenges and Limitations Obtaining CSF samples is less invasive than surgery; however, in some cases, a lumbar puncture is not feasible. Contraindications to performing a lumbar puncture include risk for cerebral herniation, abnormal intracranial pressure and coagulation abnormalities [41]. The analysis of ctDNA could be used as a biomarker of residual disease. However, it will be important to establish the dates of sample collection, particularly postsurgery, since the abundance of trauma-induced cfDNA, up to 4 weeks from the surgical procedure, could dilute the fraction of ctDNA and influence the results [125]. Another limitation is that ctDNA is not detected in all patients with CNS malignan- cies. Detection of ctDNA may be influenced by tumour burden, tumour progression and anatomical location [21–23]. Therefore, further research to determine the biological factors involved and improve technological sensitivity will be required. For patient monitoring through the analysis of specific mutations by sensitive tech- niques such as droplet digital PCR (ddPCR), prior knowledge of the tumour genetic profile is required, and new mutations (not present in the primary sample) can appear in the relapse setting. In contrast, whole-exome sequencing (WES), shallow whole-genome sequencing (WGS) or specific gene panels might provide more information to aid with tumour characterisation and monitor residual disease. However, it is important to consider sensitivity, turnaround time and cost-effectiveness. In addition, imaging techniques may sometimes reflect either inflammatory processes from treatment or neoplastic progression [10]. Further research is needed to investigate whether the analysis of CSF ctDNA can help distinguish between true progression from pseudoprogression. To determine the impact of the results and translate them into a tool for clinical practise, standardisation of protocols and larger studies with more patients will be required. The implementation of well-designed and controlled clinical trials will be essential to validate the use of CSF ctDNA as a liquid biopsy for the clinical management of patients. 6. Conclusions and Future Insights CNS cancer is a dismal disease. It has elevated mortality and disabling effects on patients and is a massive burden on global health care systems. However, early detection and treatment can result in improved outcomes [1]. Several studies of the CSF ctDNA of patients with different types of primary and metastatic CNS tumours have been performed and show promising results, highlighting the potential of CSF ctDNA as a biomarker. The challenges will be to translate these findings into clinically validated assays to im- prove patient healthcare. Standardisation of protocols and further studies and clinical trials will be necessary to translate the current results into a feasible tool for its implementation in the clinical setting. A liquid biopsy of CSF can characterise the tumour for diagnosis and provide prog- nostic information, also complementing the information obtained from the tumour sample if a biopsy or resection is feasible (Figure 1). During patient follow-up, it could be used to monitor the response to treatment through the levels of ctDNA and the detection of mini- mal residual disease. Importantly, it can facilitate early detection of relapse and identify therapeutic targets or mechanisms of resistance in order to adjust the therapeutic strategy at relapse (Figure 2). Altogether, the analysis of CSF ctDNA remains a promising strategy to improve the clinical management of patients with CNS malignancies, and further studies are required to make liquid biopsies a standard clinical tool. Cancers 2021, 13, 1989 9 of 15 Cancers 2021, 13, x 9 of 15 Figure 2. Liquid biopsies in the clinical setting. Schematic representation of the standard of care for patients with CNS Figure 2. Liquid biopsies in the clinical setting. Schematic representation of the standard of care for patients with CNS malignancies and the complementation with a longitudinal analysis of CSF ctDNA. malignancies and the complementation with a longitudinal analysis of CSF ctDNA. Altogether, the analysis of CSF ctDNA remains a promising strategy to improve the Author management Writing—original clinicalContributions: of patients with draft CNSpreparation, L.E.;and malignancies, writing—review and are further studies editing, F.M.- required R. and J.S. All authors have read and agreed to the published version of the manuscript. to make liquid biopsies a standard clinical tool. Funding: This work was supported by Fundación Asociación Española contra el Cáncer (AECC), Authorthe FERO, Contributions: Ramón Areces Writing—original Foundation, thedraft preparation, Cellex L.E.; Foundation, writing—review BBVA (CAIMI), theand editing, ISCIII, FIS F.M.-R. and J.S. (PI19/00318) andAll theauthors Juan dehave read and la Cierva agreed (L.E.). fellowship to the published version of the manuscript. Funding: This Institutional work was Review Boardsupported by Not Statement: Fundación Asociación Española contra el Cáncer (AECC), applicable. FERO, the Ramón Areces Foundation, the Cellex Foundation, BBVA (CAIMI), the ISCIII, FIS Informed Consent (PI19/00318) and the Juan de laNot Statement: applicable. Cierva fellowship (L.E.). Data Availability Institutional Statement: Review No new data Board Statement: Notwere created or analyzed in this study. Data sharing is applicable. not applicable to this article. Informed Consent Statement: Not applicable. Acknowledgments: The authors would like to thank the patients and their families who took part in Data the Availability research Statement: studies. Nofigures Parts of the new data were were created created orBioRender.com with analyzed in this(accessed study. Data sharing on 14 Marchis not applicable 2021). to this article. Acknowledgements: Conflicts Theisauthors of Interest: J.S. wouldof a cofounder like to thank Mosaic the patients Biomedicals andand hastheir families interests ownership who tookfrom part in the research Mosaic studies. Biomedicals andParts of the Biologics. Northern figures were J.S.created with BioRender.com has received grant/research(accessed on 14Mosaic support from March 2021). Biomedicals, Northern Biologics, Roche/Glycart and Hoffmann la Roche. The remaining authors declare no of Conflicts competing Interest: interests. J.S. is a cofounder of Mosaic Biomedicals and has ownership interests from Mosaic Biomedicals and Northern Biologics. J.S. has received grant/research support from Mosaic Biomedicals, Northern Biologics, Roche/Glycart and Hoffmann la Roche. The remaining authors declare no competing interests. Cancers 2021, 13, 1989 10 of 15 References 1. Patel, A.P.; Fisher, J.L.; Nichols, E.; Abd-Allah, F.; Abdela, J.; Abdelalim, A.; Fitzmaurice, C. Global, regional, and national burden of brain and other CNS cancer, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019, 18, 376–393. [CrossRef] 2. Alentorn, A.; Hoang-Xuan, K.; Mikkelsen, T. Presenting signs and symptoms in brain tumors. Handb. Clin. Neurol. 2016, 134, 19–26. [CrossRef] 3. Preusser, M.; Marosi, C. Neuro-oncology in 2016: Advances in brain tumour classification and therapy. Nat. Rev. Neurol. 2017, 13, 71–72. [CrossRef] 4. Owen, S.; Souhami, L. The management of brain metastases in non-small cell lung cancer. Front. Oncol. 2014, 4, 248. [CrossRef] 5. Ferguson, S.D.; Wagner, K.M.; Prabhu, S.S.; McAleer, M.F.; McCutcheon, I.E.; Sawaya, R. Neurosurgical management of brain metastases. Clin. Exp. Metastasis 2017, 34, 377–389. [CrossRef] [PubMed] 6. Wijdicks, E.F.M. Historical awareness of the brainstem: From a subsidiary structure to a vital center. Neurology 2020, 95, 484–488. [CrossRef] [PubMed] 7. Dagogo-Jack, I.; Shaw, A.T. Tumour heterogeneity and resistance to cancer therapies. Nat. Rev. Clin. Oncol. 2018, 15, 81–94. [CrossRef] 8. Morrissy, A.S.; Cavalli, F.M.G.; Remke, M.; Ramaswamy, V.; Shih, D.J.H.; Holgado, B.L.; Farooq, H.; Donovan, L.K.; Garzia, L.; Agnihotri, S.; et al. Spatial heterogeneity in medulloblastoma. Nat. Genet. 2017, 49, 780–788. [CrossRef] [PubMed] 9. Sottoriva, A.; Spiteri, I.; Piccirillo, S.G.; Touloumis, A.; Collins, V.P.; Marioni, J.C.; Curtis, C.; Watts, C.; Tavaré, S. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc. Natl. Acad. Sci. USA 2013, 110, 4009–4014. [CrossRef] 10. Thust, S.C.; van den Bent, M.J.; Smits, M. Pseudoprogression of brain tumors. J. Magn. Reason. Imaging 2018, 48, 571–589. [CrossRef] [PubMed] 11. Johnson, B.E.; Mazor, T.; Hong, C.; Barnes, M.; Aihara, K.; McLean, C.Y.; Fouse, S.D.; Yamamoto, S.; Ueda, H.; Tatsuno, K.; et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 2014, 343, 189–193. [CrossRef] [PubMed] 12. Brastianos, P.K.; Carter, S.L.; Santagata, S.; Cahill, D.P.; Taylor-Weiner, A.; Jones, R.T.; Van Allen, E.M.; Lawrence, M.S.; Horowitz, P.M.; Cibulskis, K.; et al. Genomic Characterization of Brain Metastases Reveals Branched Evolution and Potential Therapeutic Targets. Cancer Discov. 2015, 5, 1164–1177. [CrossRef] 13. Diaz, L.A., Jr.; Bardelli, A. Liquid biopsies: Genotyping circulating tumor DNA. J. Clin. Oncol. 2014, 32, 579–586. [CrossRef] 14. Siravegna, G.; Mussolin, B.; Venesio, T.; Marsoni, S.; Seoane, J.; Dive, C.; Papadopoulos, N.; Kopetz, S.; Corcoran, R.B.; Siu, L.L.; et al. How liquid biopsies can change clinical practice in oncology. Ann. Oncol. 2019, 30, 1580–1590. [CrossRef] 15. Schwarzenbach, H.; Hoon, D.S.; Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 2011, 11, 426–437. [CrossRef] [PubMed] 16. Wan, J.C.M.; Massie, C.; Garcia-Corbacho, J.; Mouliere, F.; Brenton, J.D.; Caldas, C.; Pacey, S.; Baird, R.; Rosenfeld, N. Liquid biopsies come of age: Towards implementation of circulating tumour DNA. Nat. Rev. Cancer 2017, 17, 223–238. [CrossRef] [PubMed] 17. Heitzer, E.; Haque, I.S.; Roberts, C.E.S.; Speicher, M.R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet. 2019, 20, 71–88. [CrossRef] 18. Crigna, A.T.; Samec, M.; Koklesova, L.; Liskova, A.; Giordano, F.A.; Kubatka, P.; Golubnitschaja, O. Cell-free nucleic acid patterns in disease prediction and monitoring-hype or hope? EPMA J. 2020, 11, 1–25. [CrossRef] 19. Bettegowda, C.; Sausen, M.; Leary, R.J.; Kinde, I.; Wang, Y.; Agrawal, N.; Bartlett, B.R.; Wang, H.; Luber, B.; Alani, R.M.; et al. Detection of circulating tumor DNA in early-and late-stage human malignancies. Sci. Transl. Med. 2014, 6, 224ra224. [CrossRef] 20. De Mattos-Arruda, L.; Mayor, R.; Ng, C.K.Y.; Weigelt, B.; Martínez-Ricarte, F.; Torrejon, D.; Oliveira, M.; Arias, A.; Raventos, C.; Tang, J.; et al. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat. Commun. 2015, 6, 8839. [CrossRef] [PubMed] 21. Wang, Y.; Springer, S.; Zhang, M.; McMahon, K.W.; Kinde, I.; Dobbyn, L.; Ptak, J.; Brem, H.; Chaichana, K.; Gallia, G.L.; et al. Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc. Natl. Acad. Sci. USA 2015, 112, 9704–9709. [CrossRef] 22. Martínez-Ricarte, F.; Mayor, R.; Martínez-Sáez, E.; Rubio-Pérez, C.; Pineda, E.; Cordero, E.; Cicuéndez, M.; Poca, M.A.; López- Bigas, N.; Ramon, Y.C.S.; et al. Molecular Diagnosis of Diffuse Gliomas through Sequencing of Cell-Free Circulating Tumor DNA from Cerebrospinal Fluid. Clin. Cancer Res. 2018, 24, 2812–2819. [CrossRef] 23. Miller, A.M.; Shah, R.H.; Pentsova, E.I.; Pourmaleki, M.; Briggs, S.; Distefano, N.; Zheng, Y.; Skakodub, A.; Mehta, S.A.; Campos, C.; et al. Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid. Nature 2019, 565, 654–658. [CrossRef] [PubMed] 24. Ghersi-Egea, J.F.; Strazielle, N.; Catala, M.; Silva-Vargas, V.; Doetsch, F.; Engelhardt, B. Molecular anatomy and functions of the choroidal blood-cerebrospinal fluid barrier in health and disease. Acta Neuropathol. 2018, 135, 337–361. [CrossRef] 25. Schmitt-Gräff, A.; Hummel, M.; Anagnostopoulos, I.; Stoltenburg, G.; Stein, H. Primary brain lymphoma in acquired immunodefi- ciency syndrome. Immunophenotype and molecular pathologic characterization in stereotactic biopsy, autopsy and cerebrospinal fluid cytology. Pathologe 1995, 16, 75–80. [CrossRef] [PubMed] Cancers 2021, 13, 1989 11 of 15 26. Rhodes, C.H.; Honsinger, C.; Sorenson, G.D. PCR-detection of tumor-derived p53 DNA in cerebrospinal fluid. Am. J. Clin. Pathol. 1995, 103, 404–408. [CrossRef] 27. Swinkels, D.W.; de Kok, J.B.; Hanselaar, A.; Lamers, K.; Boerman, R.H. Early detection of leptomeningeal metastasis by PCR examination of tumor-derived K-ras DNA in cerebrospinal fluid. Clin. Chem. 2000, 46, 132–133. [CrossRef] [PubMed] 28. Shi, W.; Lv, C.; Qi, J.; Zhao, W.; Wu, X.; Jing, R.; Wu, X.; Ju, S.; Chen, J. Prognostic value of free DNA quantification in serum and cerebrospinal fluid in glioma patients. J. Mol. Neurosci. 2012, 46, 470–475. [CrossRef] [PubMed] 29. Yang, H.; Cai, L.; Zhang, Y.; Tan, H.; Deng, Q.; Zhao, M.; Xu, X. Sensitive detection of EGFR mutations in cerebrospinal fluid from lung adenocarcinoma patients with brain metastases. J. Mol. Diagn. 2014, 16, 558–563. [CrossRef] 30. Touat, M.; Duran-Peña, A.; Alentorn, A.; Lacroix, L.; Massard, C.; Idbaih, A. Emerging circulating biomarkers in glioblastoma: Promises and challenges. Expert Rev. Mol. Diagn. 2015, 15, 1311–1323. [CrossRef] 31. Pan, W.; Gu, W.; Nagpal, S.; Gephart, M.H.; Quake, S.R. Brain tumor mutations detected in cerebral spinal fluid. Clin. Chem. 2015, 61, 514–522. [CrossRef] [PubMed] 32. Pentsova, E.I.; Shah, R.H.; Tang, J.; Boire, A.; You, D.; Briggs, S.; Omuro, A.; Lin, X.; Fleisher, M.; Grommes, C.; et al. Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid. J. Clin. Oncol. 2016, 34, 2404–2415. [CrossRef] [PubMed] 33. Mouliere, F.; Mair, R.; Chandrananda, D.; Marass, F.; Smith, C.G.; Su, J.; Morris, J.; Watts, C.; Brindle, K.M.; Rosenfeld, N. Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients. EMBO Mol. Med. 2018, 10. [CrossRef] [PubMed] 34. Escudero, L.; Llort, A.; Arias, A.; Diaz-Navarro, A.; Martínez-Ricarte, F.; Rubio-Perez, C.; Mayor, R.; Caratù, G.; Martínez-Sáez, E.; Vázquez-Méndez, É.; et al. Circulating tumour DNA from the cerebrospinal fluid allows the characterisation and monitoring of medulloblastoma. Nat. Commun. 2020, 11, 5376. [CrossRef] 35. Seoane, J.; De Mattos-Arruda, L.; Le Rhun, E.; Bardelli, A.; Weller, M. Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases. Ann. Oncol. 2019, 30, 211–218. [CrossRef] [PubMed] 36. Le Rhun, E.; Seoane, J.; Salzet, M.; Soffietti, R.; Weller, M. Liquid biopsies for diagnosing and monitoring primary tumors of the central nervous system. Cancer Lett. 2020, 480, 24–28. [CrossRef] 37. Nguyen, T.T.; Smith, M.V.; Rodziewicz, G.S.; Lemke, S.M. Hydrocephalus caused by metastatic brain lesions: Treatment by third ventriculostomy. J. Neurol. Neurosurg. Psychiatry 1999, 67, 552–553. [CrossRef] 38. Marx, S.; Reinfelder, M.; Matthes, M.; Schroeder, H.W.S.; Baldauf, J. Frequency and treatment of hydrocephalus prior to and after posterior fossa tumor surgery in adult patients. Acta Neurochir. 2018, 160, 1063–1071. [CrossRef] 39. Won, S.Y.; Dubinski, D.; Behmanesh, B.; Bernstock, J.D.; Seifert, V.; Konczalla, J.; Tritt, S.; Senft, C.; Gessler, F. Management of hy- drocephalus after resection of posterior fossa lesions in pediatric and adult patients-predictors for development of hydrocephalus. Neurosurg. Rev. 2020, 43, 1143–1150. [CrossRef] 40. Sainte-Rose, C.; Cinalli, G.; Roux, F.E.; Maixner, R.; Chumas, P.D.; Mansour, M.; Carpentier, A.; Bourgeois, M.; Zerah, M.; Pierre-Kahn, A.; et al. Management of hydrocephalus in pediatric patients with posterior fossa tumors: The role of endoscopic third ventriculostomy. J. Neurosurg. 2001, 95, 791–797. [CrossRef] 41. Engelborghs, S.; Niemantsverdriet, E.; Struyfs, H.; Blennow, K.; Brouns, R.; Comabella, M.; Dujmovic, I.; van der Flier, W.; Frölich, L.; Galimberti, D.; et al. Consensus guidelines for lumbar puncture in patients with neurological diseases. Alzheimer Dement. 2017, 8, 111–126. [CrossRef] 42. Lee, M.; Wisoff, J.H.; Abbott, R.; Freed, D.; Epstein, F.J. Management of hydrocephalus in children with medulloblastoma: Prognostic factors for shunting. Pediatr. Neurosurg. 1994, 20, 240–247. [CrossRef] 43. Balhuizen, J.C.; Bots, G.T.; Schaberg, A.; Bosman, F.T. Value of cerebrospinal fluid cytology for the diagnosis of malignancies in the central nervous system. J. Neurosurg. 1978, 48, 747–753. [CrossRef] [PubMed] 44. Cohen, N.R.; Phipps, K.; Harding, B.; Jacques, T.S. Is CSF cytology a useful diagnostic procedure in staging paediatric CNS tumours? Cytopathology 2009, 20, 256–260. [CrossRef] 45. Chang, C.H.; Housepian, E.M.; Herbert, C., Jr. An operative staging system and a megavoltage radiotherapeutic technic for cerebellar medulloblastomas. Radiology 1969, 93, 1351–1359. [CrossRef] 46. Rahimi, J.; Woehrer, A. Overview of cerebrospinal fluid cytology. Handb. Clin. Neurol. 2017, 145, 563–571. [CrossRef] [PubMed] 47. Bobillo, S.; Crespo, M.; Escudero, L.; Mayor, R.; Raheja, P.; Carpio, C.; Rubio-Perez, C.; Tazón-Vega, B.; Palacio, C.; Carabia, J.; et al. Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas. Haematologica 2021, 106, 513–521. [CrossRef] [PubMed] 48. Louis, D.N.; Perry, A.; Reifenberger, G.; von Deimling, A.; Figarella-Branger, D.; Cavenee, W.K.; Ohgaki, H.; Wiestler, O.D.; Kleihues, P.; Ellison, D.W. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: A summary. Acta Neuropathol. 2016, 131, 803–820. [CrossRef] 49. Rees, J.H. Diagnosis and treatment in neuro-oncology: An oncological perspective. Br. J. Radiol. 2011, 84, S82–S89. [CrossRef] [PubMed] 50. Aquino, D.; Gioppo, A.; Finocchiaro, G.; Bruzzone, M.G.; Cuccarini, V. MRI in Glioma Immunotherapy: Evidence, Pitfalls, and Perspectives. J. Immunol. Res. 2017, 2017, 5813951. [CrossRef] [PubMed] 51. Poon, M.T.C.; Sudlow, C.L.M.; Figueroa, J.D.; Brennan, P.M. Longer-term (≥ 2 years) survival in patients with glioblastoma in population-based studies pre- and post-2005: A systematic review and meta-analysis. Sci. Rep. 2020, 10, 11622. [CrossRef] Cancers 2021, 13, 1989 12 of 15 52. Juratli, T.A.; Stasik, S.; Zolal, A.; Schuster, C.; Richter, S.; Daubner, D.; Juratli, M.A.; Thowe, R.; Hennig, S.; Makina, M.; et al. TERT Promoter Mutation Detection in Cell-Free Tumor-Derived DNA in Patients with IDH Wild-Type Glioblastomas: A Pilot Prospective Study. Clin. Cancer Res. 2018, 24, 5282–5291. [CrossRef] 53. Hoffman, L.M.; Veldhuijzen van Zanten, S.E.M.; Colditz, N.; Baugh, J.; Chaney, B.; Hoffmann, M.; Lane, A.; Fuller, C.; Miles, L.; Hawkins, C.; et al. Clinical, Radiologic, Pathologic, and Molecular Characteristics of Long-Term Survivors of Diffuse Intrinsic Pontine Glioma (DIPG): A Collaborative Report from the International and European Society for Pediatric Oncology DIPG Registries. J. Clin. Oncol. 2018, 36, 1963–1972. [CrossRef] 54. Pan, C.; Diplas, B.H.; Chen, X.; Wu, Y.; Xiao, X.; Jiang, L.; Geng, Y.; Xu, C.; Sun, Y.; Zhang, P.; et al. Molecular profiling of tumors of the brainstem by sequencing of CSF-derived circulating tumor DNA. Acta Neuropathol. 2019, 137, 297–306. [CrossRef] 55. Azad, T.D.; Jin, M.C.; Bernhardt, L.J.; Bettegowda, C. Liquid biopsy for pediatric diffuse midline glioma: A review of circulating tumor DNA and cerebrospinal fluid tumor DNA. Neurosurg. Focus 2020, 48, E9. [CrossRef] 56. Kambhampati, M.; Perez, J.P.; Yadavilli, S.; Saratsis, A.M.; Hill, A.D.; Ho, C.Y.; Panditharatna, E.; Markel, M.; Packer, R.J.; Nazarian, J. A standardized autopsy procurement allows for the comprehensive study of DIPG biology. Oncotarget 2015, 6, 12740–12747. [CrossRef] 57. Kambhampati, M.; Panditharatna, E.; Yadavilli, S.; Saoud, K.; Lee, S.; Eze, A.; Almira-Suarez, M.I.; Hancock, L.; Bonner, E.R.; Gittens, J.; et al. Harmonization of postmortem donations for pediatric brain tumors and molecular characterization of diffuse midline gliomas. Sci. Rep. 2020, 10, 10954. [CrossRef] [PubMed] 58. Baugh, J.; Bartels, U.; Leach, J.; Jones, B.; Chaney, B.; Warren, K.E.; Kirkendall, J.; Doughman, R.; Hawkins, C.; Miles, L.; et al. The international diffuse intrinsic pontine glioma registry: An infrastructure to accelerate collaborative research for an orphan disease. J. Neurooncol. 2017, 132, 323–331. [CrossRef] 59. Veldhuijzen van Zanten, S.E.M.; Lane, A.; Heymans, M.W.; Baugh, J.; Chaney, B.; Hoffman, L.M.; Doughman, R.; Jansen, M.H.A.; Sanchez, E.; Vandertop, W.P.; et al. External validation of the diffuse intrinsic pontine glioma survival prediction model: A collaborative report from the International DIPG Registry and the SIOPE DIPG Registry. J. Neurooncol. 2017, 134, 231–240. [CrossRef] [PubMed] 60. Hegi, M.E.; Diserens, A.C.; Gorlia, T.; Hamou, M.F.; de Tribolet, N.; Weller, M.; Kros, J.M.; Hainfellner, J.A.; Mason, W.; Mariani, L.; et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N. Engl. J. Med. 2005, 352, 997–1003. [CrossRef] [PubMed] 61. Hegi, M.E.; Liu, L.; Herman, J.G.; Stupp, R.; Wick, W.; Weller, M.; Mehta, M.P.; Gilbert, M.R. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J. Clin. Oncol. 2008, 26, 4189–4199. [CrossRef] 62. Szopa, W.; Burley, T.A.; Kramer-Marek, G.; Kaspera, W. Diagnostic and Therapeutic Biomarkers in Glioblastoma: Current Status and Future Perspectives. Biomed. Res. Int. 2017, 2017, 8013575. [CrossRef] 63. Weller, M.; van den Bent, M.; Tonn, J.C.; Stupp, R.; Preusser, M.; Cohen-Jonathan-Moyal, E.; Henriksson, R.; Le Rhun, E.; Balana, C.; Chinot, O.; et al. European Association for Neuro-Oncology (EANO) guideline on the diagnosis and treatment of adult astrocytic and oligodendroglial gliomas. Lancet Oncol. 2017, 18, e315–e329. [CrossRef] 64. Wang, Z.; Jiang, W.; Wang, Y.; Guo, Y.; Cong, Z.; Du, F.; Song, B. MGMT promoter methylation in serum and cerebrospinal fluid as a tumor-specific biomarker of glioma. Biomed. Rep. 2015, 3, 543–548. [CrossRef] 65. Figueroa, J.M.; Skog, J.; Akers, J.; Li, H.; Komotar, R.; Jensen, R.; Ringel, F.; Yang, I.; Kalkanis, S.; Thompson, R.; et al. Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients. Neuro Oncol. 2017, 19, 1494–1502. [CrossRef] 66. Tabouret, E.; Chinot, O.; Metellus, P.; Tallet, A.; Viens, P.; Gonçalves, A. Recent trends in epidemiology of brain metastases: An overview. Anticancer Res. 2012, 32, 4655–4662. [PubMed] 67. Le Rhun, E.; Weller, M.; Brandsma, D.; Van den Bent, M.; de Azambuja, E.; Henriksson, R.; Boulanger, T.; Peters, S.; Watts, C.; Wick, W.; et al. EANO-ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up of patients with leptomeningeal metastasis from solid tumours. Ann. Oncol. 2017, 28, iv84–iv99. [CrossRef] [PubMed] 68. Beauchesne, P. Intrathecal chemotherapy for treatment of leptomeningeal dissemination of metastatic tumours. Lancet Oncol. 2010, 11, 871–879. [CrossRef] 69. Remon, J.; Le Rhun, E.; Besse, B. Leptomeningeal carcinomatosis in non-small cell lung cancer patients: A continuing challenge in the personalized treatment era. Cancer Treat. Rev. 2017, 53, 128–137. [CrossRef] 70. Dudani, S.; Mazzarello, S.; Hilton, J.; Hutton, B.; Vandermeer, L.; Fernandes, R.; Ibrahim, M.F.; Smith, S.; Majeed, H.; Al-Baimani, K.; et al. Optimal Management of Leptomeningeal Carcinomatosis in Breast Cancer Patients-A Systematic Review. Clin. Breast Cancer 2016, 16, 456–470. [CrossRef] 71. Pan, Z.; Yang, G.; He, H.; Yuan, T.; Wang, Y.; Li, Y.; Shi, W.; Gao, P.; Dong, L.; Zhao, G. Leptomeningeal metastasis from solid tumors: Clinical features and its diagnostic implication. Sci. Rep. 2018, 8, 10445. [CrossRef] 72. Glantz, M.J.; Cole, B.F.; Glantz, L.K.; Cobb, J.; Mills, P.; Lekos, A.; Walters, B.C.; Recht, L.D. Cerebrospinal fluid cytology in patients with cancer: Minimizing false-negative results. Cancer 1998, 82, 733–739. [CrossRef] 73. Clarke, J.L.; Perez, H.R.; Jacks, L.M.; Panageas, K.S.; Deangelis, L.M. Leptomeningeal metastases in the MRI era. Neurology 2010, 74, 1449–1454. [CrossRef] Cancers 2021, 13, 1989 13 of 15 74. Zhao, Y.; He, J.Y.; Zou, Y.L.; Guo, X.S.; Cui, J.Z.; Guo, L.; Bu, H. Evaluating the cerebrospinal fluid ctDNA detection by next-generation sequencing in the diagnosis of meningeal Carcinomatosis. BMC Neurol. 2019, 19, 331. [CrossRef] [PubMed] 75. Ge, M.; Zhan, Q.; Zhang, Z.; Ji, X.; Zhou, X.; Huang, R.; Liang, X. Different next-generation sequencing pipelines based detection of tumor DNA in cerebrospinal fluid of lung adenocarcinoma cancer patients with leptomeningeal metastases. BMC Cancer 2019, 19, 143. [CrossRef] [PubMed] 76. Li, Y.S.; Jiang, B.Y.; Yang, J.J.; Zhang, X.C.; Zhang, Z.; Ye, J.Y.; Zhong, W.Z.; Tu, H.Y.; Chen, H.J.; Wang, Z.; et al. Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: A new medium of liquid biopsy. Ann. Oncol. 2018, 29, 945–952. [CrossRef] [PubMed] 77. Rochet, N.M.; Kottschade, L.A.; Markovic, S.N. Vemurafenib for melanoma metastases to the brain. N. Engl. J. Med. 2011, 365, 2439–2441. [CrossRef] 78. Welsh, J.W.; Komaki, R.; Amini, A.; Munsell, M.F.; Unger, W.; Allen, P.K.; Chang, J.Y.; Wefel, J.S.; McGovern, S.L.; Garland, L.L.; et al. Phase II trial of erlotinib plus concurrent whole-brain radiation therapy for patients with brain metastases from non-small-cell lung cancer. J. Clin. Oncol. 2013, 31, 895–902. [CrossRef] 79. Seoane, J.; De Mattos-Arruda, L. Brain metastasis: New opportunities to tackle therapeutic resistance. Mol. Oncol. 2014, 8, 1120–1131. [CrossRef] 80. Long, G.V.; Trefzer, U.; Davies, M.A.; Kefford, R.F.; Ascierto, P.A.; Chapman, P.B.; Puzanov, I.; Hauschild, A.; Robert, C.; Algazi, A.; et al. Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): A multicentre, open-label, phase 2 trial. Lancet Oncol. 2012, 13, 1087–1095. [CrossRef] 81. Margolin, K.; Ernstoff, M.S.; Hamid, O.; Lawrence, D.; McDermott, D.; Puzanov, I.; Wolchok, J.D.; Clark, J.I.; Sznol, M.; Logan, T.F.; et al. Ipilimumab in patients with melanoma and brain metastases: An open-label, phase 2 trial. Lancet Oncol. 2012, 13, 459–465. [CrossRef] 82. Lin, N.U.; Diéras, V.; Paul, D.; Lossignol, D.; Christodoulou, C.; Stemmler, H.J.; Roché, H.; Liu, M.C.; Greil, R.; Ciruelos, E.; et al. Multicenter phase II study of lapatinib in patients with brain metastases from HER2-positive breast cancer. Clin. Cancer Res. 2009, 15, 1452–1459. [CrossRef] [PubMed] 83. Bachelot, T.; Romieu, G.; Campone, M.; Diéras, V.; Cropet, C.; Dalenc, F.; Jimenez, M.; Le Rhun, E.; Pierga, J.Y.; Gonçalves, A.; et al. Lapatinib plus capecitabine in patients with previously untreated brain metastases from HER2-positive metastatic breast cancer (LANDSCAPE): A single-group phase 2 study. Lancet Oncol. 2013, 14, 64–71. [CrossRef] 84. Gadgeel, S.M.; Gandhi, L.; Riely, G.J.; Chiappori, A.A.; West, H.L.; Azada, M.C.; Morcos, P.N.; Lee, R.M.; Garcia, L.; Yu, L.; et al. Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): Results from the dose-finding portion of a phase 1/2 study. Lancet Oncol. 2014, 15, 1119–1128. [CrossRef] 85. Costa, D.B.; Shaw, A.T.; Ou, S.H.; Solomon, B.J.; Riely, G.J.; Ahn, M.J.; Zhou, C.; Shreeve, S.M.; Selaru, P.; Polli, A.; et al. Clinical Experience With Crizotinib in Patients with Advanced ALK-Rearranged Non-Small-Cell Lung Cancer and Brain Metastases. J. Clin. Oncol. 2015, 33, 1881–1888. [CrossRef] 86. Crinò, L.; Ahn, M.J.; De Marinis, F.; Groen, H.J.; Wakelee, H.; Hida, T.; Mok, T.; Spigel, D.; Felip, E.; Nishio, M.; et al. Multicenter Phase II Study of Whole-Body and Intracranial Activity with Ceritinib in Patients With ALK-Rearranged Non-Small-Cell Lung Cancer Previously Treated With Chemotherapy and Crizotinib: Results From ASCEND-2. J. Clin. Oncol. 2016, 34, 2866–2873. [CrossRef] 87. Iuchi, T.; Shingyoji, M.; Sakaida, T.; Hatano, K.; Nagano, O.; Itakura, M.; Kageyama, H.; Yokoi, S.; Hasegawa, Y.; Kawasaki, K.; et al. Phase II trial of gefitinib alone without radiation therapy for Japanese patients with brain metastases from EGFR-mutant lung adenocarcinoma. Lung Cancer 2013, 82, 282–287. [CrossRef] 88. Porta, R.; Sánchez-Torres, J.M.; Paz-Ares, L.; Massutí, B.; Reguart, N.; Mayo, C.; Lianes, P.; Queralt, C.; Guillem, V.; Salinas, P.; et al. Brain metastases from lung cancer responding to erlotinib: The importance of EGFR mutation. Eur. Respir. J. 2011, 37, 624–631. [CrossRef] 89. Schuler, M.; Wu, Y.L.; Hirsh, V.; O’Byrne, K.; Yamamoto, N.; Mok, T.; Popat, S.; Sequist, L.V.; Massey, D.; Zazulina, V.; et al. First-Line Afatinib versus Chemotherapy in Patients with Non-Small Cell Lung Cancer and Common Epidermal Growth Factor Receptor Gene Mutations and Brain Metastases. J. Thorac. Oncol. 2016, 11, 380–390. [CrossRef] 90. Remon, J.; Steuer, C.E.; Ramalingam, S.S.; Felip, E. Osimertinib and other third-generation EGFR TKI in EGFR-mutant NSCLC patients. Ann. Oncol. 2018, 29, i20–i27. [CrossRef] 91. Swain, S.M.; Baselga, J.; Miles, D.; Im, Y.H.; Quah, C.; Lee, L.F.; Cortés, J. Incidence of central nervous system metastases in patients with HER2-positive metastatic breast cancer treated with pertuzumab, trastuzumab, and docetaxel: Results from the randomized phase III study CLEOPATRA. Ann. Oncol. 2014, 25, 1116–1121. [CrossRef] [PubMed] 92. Bartsch, R.; Berghoff, A.S.; Preusser, M. Breast cancer brain metastases responding to primary systemic therapy with T-DM1. J. Neurooncol. 2014, 116, 205–206. [CrossRef] [PubMed] 93. Taillibert, S.; Chamberlain, M.C. Leptomeningeal metastasis. Handb. Clin. Neurol. 2018, 149, 169–204. [CrossRef] 94. Jiang, B.-Y.; LI, Y.; Chuai, S.; Zhang, Z.; Yang, J.-J.; Zhong, W.; Zhou, Q.; Wu, Y.-L. NGS to reveal heterogeneity between cerebrospinal fluid and plasma ctDNA among non-small cell lung cancer patients with leptomeningeal carcinomatosis. J. Clin. Oncol. 2017, 35, 9022. [CrossRef] Cancers 2021, 13, 1989 14 of 15 95. Zhao, Y.; He, J.Y.; Cui, J.Z.; Meng, Z.Q.; Zou, Y.L.; Guo, X.S.; Chen, X.; Wang, X.; Yan, L.T.; Han, W.X.; et al. Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next generation sequencing. BMC Cancer 2020, 20, 690. [CrossRef] [PubMed] 96. Ma, C.; Zhang, J.; Tang, D.; Ye, X.; Li, J.; Mu, N.; Li, Z.; Liu, R.; Xiang, L.; Huang, C.; et al. Tyrosine Kinase Inhibitors Could Be Effective Against Non-small Cell Lung Cancer Brain Metastases Harboring Uncommon EGFR Mutations. Front. Oncol. 2020, 10, 224. [CrossRef] 97. Kawahara, A.; Abe, H.; Murata, K.; Ishii, H.; Azuma, K.; Takase, Y.; Hattori, S.; Naito, Y.; Akiba, J. Screening system for epidermal growth factor receptor mutation detection in cytology cell-free DNA of cerebrospinal fluid based on assured sample quality. Cytopathology 2019, 30, 144–149. [CrossRef] [PubMed] 98. Ma, C.; Yang, X.; Xing, W.; Yu, H.; Si, T.; Guo, Z. Detection of circulating tumor DNA from non-small cell lung cancer brain metastasis in cerebrospinal fluid samples. Thorac. Cancer 2020, 11, 588–593. [CrossRef] 99. Siravegna, G.; Geuna, E.; Mussolin, B.; Crisafulli, G.; Bartolini, A.; Galizia, D.; Casorzo, L.; Sarotto, I.; Scaltriti, M.; Sapino, A.; et al. Genotyping tumour DNA in cerebrospinal fluid and plasma of a HER2-positive breast cancer patient with brain metastases. ESMO Open 2017, 2, e000253. [CrossRef] [PubMed] 100. Yamamoto, W.; Tomita, N.; Watanabe, R.; Hattori, Y.; Nakajima, Y.; Hyo, R.; Hashimoto, C.; Motomura, S.; Ishigatsubo, Y. Central nervous system involvement in diffuse large B-cell lymphoma. Eur. J. Haematol. 2010, 85, 6–10. [CrossRef] 101. Swerdlow, S.H.; Campo, E.; Harris, N.L.; Jaffe, E.S.; Pileri, S.A.; Stein, H.; Thiele, J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed.; IARC: Lyon, France, 2017. 102. El-Galaly, T.C.; Cheah, C.Y.; Bendtsen, M.D.; Nowakowski, G.S.; Kansara, R.; Savage, K.J.; Connors, J.M.; Sehn, L.H.; Goldschmidt, N.; Shaulov, A.; et al. Treatment strategies, outcomes and prognostic factors in 291 patients with secondary CNS involvement by diffuse large B-cell lymphoma. Eur. J. Cancer 2018, 93, 57–68. [CrossRef] 103. Fontanilles, M.; Marguet, F.; Bohers, É.; Viailly, P.J.; Dubois, S.; Bertrand, P.; Camus, V.; Mareschal, S.; Ruminy, P.; Maingonnat, C.; et al. Non-invasive detection of somatic mutations using next-generation sequencing in primary central nervous system lymphoma. Oncotarget 2017, 8, 48157–48168. [CrossRef] 104. Hattori, K.; Sakata-Yanagimoto, M.; Suehara, Y.; Yokoyama, Y.; Kato, T.; Kurita, N.; Nishikii, H.; Obara, N.; Takano, S.; Ishikawa, E.; et al. Clinical significance of disease-specific MYD88 mutations in circulating DNA in primary central nervous system lymphoma. Cancer Sci. 2018, 109, 225–230. [CrossRef] 105. Grommes, C.; Tang, S.S.; Wolfe, J.; Kaley, T.J.; Daras, M.; Pentsova, E.I.; Piotrowski, A.F.; Stone, J.; Lin, A.; Nolan, C.P.; et al. Phase 1b trial of an ibrutinib-based combination therapy in recurrent/refractory CNS lymphoma. Blood 2019, 133, 436–445. [CrossRef] [PubMed] 106. Hiemcke-Jiwa, L.S.; Minnema, M.C.; Radersma-van Loon, J.H.; Jiwa, N.M.; de Boer, M.; Leguit, R.J.; de Weger, R.A.; Huibers, M.M.H. The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid. Hematol. Oncol. 2018, 36, 429–435. [CrossRef] 107. Hiemcke-Jiwa, L.S.; Leguit, R.J.; Snijders, T.J.; Bromberg, J.E.C.; Nierkens, S.; Jiwa, N.M.; Minnema, M.C.; Huibers, M.M.H. MYD88 p.(L265P) detection on cell-free DNA in liquid biopsies of patients with primary central nervous system lymphoma. Br. J. Haematol. 2019, 185, 974–977. [CrossRef] 108. Hickmann, A.K.; Frick, M.; Hadaschik, D.; Battke, F.; Bittl, M.; Ganslandt, O.; Biskup, S.; Döcker, D. Molecular tumor analysis and liquid biopsy: A feasibility investigation analyzing circulating tumor DNA in patients with central nervous system lymphomas. BMC Cancer 2019, 19, 192. [CrossRef] [PubMed] 109. Rimelen, V.; Ahle, G.; Pencreach, E.; Zinniger, N.; Debliquis, A.; Zalmaï, L.; Harzallah, I.; Hurstel, R.; Alamome, I.; Lamy, F.; et al. Tumor cell-free DNA detection in CSF for primary CNS lymphoma diagnosis. Acta Neuropathol. Commun. 2019, 7, 43. [CrossRef] [PubMed] 110. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2016. CA Cancer J. Clin. 2016, 66, 7–30. [CrossRef] 111. Udaka, Y.T.; Packer, R.J. Pediatric Brain Tumors. Neurol. Clin. 2018, 36, 533–556. [CrossRef] [PubMed] 112. Smoll, N.R.; Drummond, K.J. The incidence of medulloblastomas and primitive neurectodermal tumours in adults and children. J. Clin. Neurosci. 2012, 19, 1541–1544. [CrossRef] 113. Taylor, M.D.; Northcott, P.A.; Korshunov, A.; Remke, M.; Cho, Y.J.; Clifford, S.C.; Eberhart, C.G.; Parsons, D.W.; Rutkowski, S.; Gajjar, A.; et al. Molecular subgroups of medulloblastoma: The current consensus. Acta Neuropathol. 2012, 123, 465–472. [CrossRef] [PubMed] 114. Northcott, P.A.; Buchhalter, I.; Morrissy, A.S.; Hovestadt, V.; Weischenfeldt, J.; Ehrenberger, T.; Gröbner, S.; Segura-Wang, M.; Zichner, T.; Rudneva, V.A.; et al. The whole-genome landscape of medulloblastoma subtypes. Nature 2017, 547, 311–317. [CrossRef] 115. Cavalli, F.M.G.; Remke, M.; Rampasek, L.; Peacock, J.; Shih, D.J.H.; Luu, B.; Garzia, L.; Torchia, J.; Nor, C.; Morrissy, A.S.; et al. Intertumoral Heterogeneity within Medulloblastoma Subgroups. Cancer Cell 2017, 31, 737–754.e736. [CrossRef] 116. Schwalbe, E.C.; Lindsey, J.C.; Nakjang, S.; Crosier, S.; Smith, A.J.; Hicks, D.; Rafiee, G.; Hill, R.M.; Iliasova, A.; Stone, T.; et al. Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: A cohort study. Lancet Oncol. 2017, 18, 958–971. [CrossRef] 117. Hovestadt, V.; Ayrault, O.; Swartling, F.J.; Robinson, G.W.; Pfister, S.M.; Northcott, P.A. Medulloblastomics revisited: Biological and clinical insights from thousands of patients. Nat. Rev. Cancer 2020, 20, 42–56. [CrossRef] Cancers 2021, 13, 1989 15 of 15 118. Koschmann, C.; Bloom, K.; Upadhyaya, S.; Geyer, J.R.; Leary, S.E. Survival After Relapse of Medulloblastoma. J. Pediatr. Hematol. Oncol. 2016, 38, 269–273. [CrossRef] [PubMed] 119. Zapotocky, M.; Mata-Mbemba, D.; Sumerauer, D.; Liby, P.; Lassaletta, A.; Zamecnik, J.; Krskova, L.; Kyncl, M.; Stary, J.; Laughlin, S.; et al. Differential patterns of metastatic dissemination across medulloblastoma subgroups. J. Neurosurg. Pediatr. 2018, 21, 145–152. [CrossRef] [PubMed] 120. Ramaswamy, V.; Remke, M.; Bouffet, E.; Faria, C.C.; Perreault, S.; Cho, Y.J.; Shih, D.J.; Luu, B.; Dubuc, A.M.; Northcott, P.A.; et al. Recurrence patterns across medulloblastoma subgroups: An integrated clinical and molecular analysis. Lancet Oncol. 2013, 14, 1200–1207. [CrossRef] 121. Hill, R.M.; Kuijper, S.; Lindsey, J.C.; Petrie, K.; Schwalbe, E.C.; Barker, K.; Boult, J.K.; Williamson, D.; Ahmad, Z.; Hallsworth, A.; et al. Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease. Cancer Cell 2015, 27, 72–84. [CrossRef] [PubMed] 122. Sun, Y.; Li, M.; Ren, S.; Liu, Y.; Zhang, J.; Li, S.; Gao, W.; Gong, X.; Liu, J.; Wang, Y.; et al. Exploring genetic alterations in circulating tumor DNA from cerebrospinal fluid of pediatric medulloblastoma. Sci. Rep. 2021, 11, 5638. [CrossRef] [PubMed] 123. Hovestadt, V.; Jones, D.T.; Picelli, S.; Wang, W.; Kool, M.; Northcott, P.A.; Sultan, M.; Stachurski, K.; Ryzhova, M.; Warnatz, H.J.; et al. Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing. Nature 2014, 510, 537–541. [CrossRef] [PubMed] 124. Li, J.; Zhao, S.; Lee, M.; Yin, Y.; Li, J.; Zhou, Y.; Ballester, L.Y.; Esquenazi, Y.; Dashwood, R.H.; Davies, P.J.A.; et al. Reliable tumor detection by whole-genome methylation sequencing of cell-free DNA in cerebrospinal fluid of pediatric medulloblastoma. Sci. Adv. 2020, 6. [CrossRef] [PubMed] 125. Henriksen, T.V.; Reinert, T.; Christensen, E.; Sethi, H.; Birkenkamp-Demtröder, K.; Gögenur, M.; Gögenur, I.; Zimmermann, B.G.; Dyrskjøt, L.; Andersen, C.L. The effect of surgical trauma on circulating free DNA levels in cancer patients-implications for studies of circulating tumor DNA. Mol. Oncol. 2020, 14, 1670–1679. [CrossRef] [PubMed]