Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors

Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors Jotaro On, Manabu Natsumeda, Haruhiko Takahashi, Akihide Koyama, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3589356/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2024 Read the published version in Journal of Neuro-Oncology → Version 1 posted 8 You are reading this latest preprint version Abstract Purpose Liquid biopsy of cyst fluid in brain tumors has not been extensively studied to date. The present study was performed to see whether diagnostic genetic alterations found in brain tumor tissue DNA could also be detected in cell-free DNA (cfDNA) of cyst fluid in cystic brain tumors. Methods Cyst fluid was obtained from 17 patients undergoing surgery for a cystic brain tumor with confirmed genetic alterations in tumor DNA. Pathological diagnoses based on WHO 2021 classification and diagnostic alterations in the tumor DNA, such as IDH1 R132H and TERT promoter mutation for oligodendrogliomas, were detected by Sanger sequencing. The same alterations were analyzed by both droplet digital PCR (ddPCR) and Sanger sequencing in cyst fluid cfDNA. Results Twenty genetic alterations were found in 17 tumor samples. All (100%) alterations were detected in cyst fluid cfDNA by ddPCR. Sixteen of the 20 (80%) alterations were also detected by Sanger sequencing of cyst fluid cfDNA. Variant allele frequency (VAF) in cyst fluid cfDNA was comparable to that of tumor DNA (R = 0.67, Pearson’s correlation). Conclusion Cell-free DNA obtained from cyst fluid in cystic brain tumors is a reliable alternative to tumor DNA when diagnosing brain tumors. cell-free DNA cyst fluid liquid biopsy brain tumors Figures Figure 1 Figure 2 Figure 3 Introduction Liquid biopsy is used to detect diagnostic markers in body fluids and to detect genetic alterations derived from circulating tumor DNA (ctDNA). The main sources of ctDNA include blood, urine, and cerebrospinal fluid (CSF), 1 and has recently attracted attention as a minimally invasive screening method that can lead to diagnosis and determination of disease status. In brain tumors, the usefulness of liquid biopsy has been reported in the detection of genetic abnormalities such as MYD88 L265P in primary central nervous system lymphoma (PCNSL). 2 However, in gliomas and other types of tumors, it is difficult to detect genetic alterations from body fluids except in advanced stages such as those with disseminated foci. 3 Meanwhile, genetic diagnosis is becoming increasingly important, as the World Health Organization Classification of Central Nervous System Tumours 2016 4 and 2021 5 call for the analysis of certain genetic and chromosomal alterations for the diagnosis of gliomas. To date, there have been no large-scale reports showing the usefulness of liquid biopsy of cyst fluid in brain tumors. Although having a cystic component is not uncommon in brain tumors, it is not known how reliably genetic alterations can be detected from cyst fluid. In the present study, we detected genetic alterations from cell-free DNA of cyst fluid (cyst fluid cfDNA) taken from 12 brain tumor patients and found that in all patients, genetic abnormalities found in tumor tissue DNA (tumor DNA) were also detected in cyst fluid. Materials and methods Patients and sample collection Tumor samples and cyst fluid were collected from 38 patients who underwent surgery at the Department of Neurosurgery, Niigata University, from January 2019 to January 2023 for a cystic brain tumor. In 17 out of 38 (45%) cases, diagnostic point mutations were identified in tumor DNA by routine Sanger sequence analysis. The location of the cyst was confirmed by preoperative imaging. Cysts were defined as having low signal intensity on MRI diffusion-weighted images (DWI) and low signal intensity on contrast-enhanced T1-weighted MRI and uniform signal intensity or fluid-fluid lines on T1-weighted or T2-weighted MRI and more than 1 ml of these areas. 5 – 7 One to 20 ml of cyst fluid was aspirated manually by inserting a plastic needle or biopsy needle into the cyst to avoid CSF contamination during surgery with care to avoid blood contamination (Fig. S1 A). Cyst fluid was promptly centrifuged at 1,500 G for 10 minutes, and the supernatant was stored at − 80°C (Fig. S1 B, C). Tumor DNA and cell-free DNA (cfDNA) of cyst fluid was extracted as previously reported. 2 In brief, tissue DNA was extracted from fresh frozen tissue using the QIAamp Blood & Tissue Kit (Qiagen, Valencia, CA, USA), and ctDNA was extracted using the Maxwell RSC ccfDNA Plasma Kit (RSC; Promega, Leiden, The Netherlands), according to the manufacturer’s instructions. For all samples, DNA was stored at − 20°C until further use. The concentration of extracted DNA was measured with a spectrometer (Eppendorf, Tokyo, Japan). The surgical specimens were fixed with 10% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-µm-thick sections stained with hematoxylin and eosin. Pathologic diagnoses were made by 3 experienced neuropathologists (A.K., H.S. and M.T.) according to the 2021 World Health Organization classification system. 5 Immunohistochemistry was performed as described previously using primary antibodies against IDH1 R132H (1:100, monoclonal, clone H09, Dianova, Hamburg, Germany) and H3F3A K27M (1:3200, monoclonal, clone RM192, Sigma-Aldrich, STL, USA) . 9 Based on the histological diagnosis, tissues pathologically diagnosed as gliomas were screened for eight mutation hotspots ( IDH1 R132, IDH2 R172, HIST 1H3B K27M, H3F3A K27M/G34, BRAF V600, and pTERT C228/C250) by direct sequencing or ddPCR methods. Similarly, pTERT mutations were screened for in a malignant meningioma case. Cases in which alterations were identified in tissue DNA were also searched for in cyst fluid cfDNA, using direct sequencing and ddPCR methods. This study was approved by the Ethics Committee of Niigata University School of Medicine (Approval #: 2018 − 0353) and written informed consent for liquid biopsy and use of the resected tissues for research purposes was obtained from all patients. Genetic analysis Direct sequencing of IDH1 R132H, IDH2 R172, pTERT C228T/C250T, H3F3A K27M/G34, HIST1H3B K27M and KRAS G12D was performed as reported previously. 2 , 9 – 12 A total of 2–20 ng of DNA was used as a template for a single DNA sequencing. The sequences of the primers used in the study are listed in Table S1 . The amplified products were fractionated on 2.0% agarose gel at 100 V for 45 minutes. The PCR products were then sequenced on a 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA) with a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer’s instructions. Detailed methods of ddPCR have been previously published. 2 , 10 Briefly, ddPCR reagents and primer/probe mix for IDH1 R132H, pTERT C228T, H3F3A K27M, and KRAS G12D were purchased from Bio-Rad (Hercules, CA, USA). Alternatively, primer/probe mix for pTERT C228T was purchased from Integrated DNA Technologies Inc (Table S2, Coralville, IA, USA). When detecting IDH1 R132H, H3F3A K27M, and KRAS G12D, a 20 µL PCR mix, composed of 10 µL 2× ddPCR Supermix for Probes (no deoxyuridine triphosphate; Bio-Rad), 1 µL ddPCR Mutation Assay (Bio-Rad) and 9 µL DNA, was loaded into sample wells of an eight-channel disposable droplet generator cartridge (Cat. No. 1864007, Bio-Rad). When detecting pTERT C228T, a 20 µL PCR mix, composed of 10 µL 2× ddPCR Supermix for Probes (no deoxyuridine triphosphate; Bio-Rad), 1 µL ddPCR Mutation Assay (Bio-Rad), and 6.75 µL DNA, 2 µL 5M Betaine, and 0.25 µL 80mM EDTA was loaded into sample wells, in the same manner. An additional 50 µL of droplet generation oil (Cat. No. 189005 Bio-Rad) was loaded into the oil well for each channel. After droplet generation, the droplets were transferred into a 96-well PCR plate and then thermal cycled using the thermal cycler Dice Gradient (Takara, Shiga, Japan) or MiniAmp™ Plus thermal cycler (Applied Biosystems). Thermal cycling conditions were carried out as follows: 95˚C 10 min, 94˚C 30 sec. 60˚C 30–60 sec. (for 40 cycles) 98˚C 10 min. and hold at 4˚C. After PCR, the 96-well PCR plate was subjected to the QX-200 droplet reader (Bio-Rad), and data were analyzed by QX Manager 1.2 standard edition software (Bio-Rad). Mutation-specific signals were generated in the hexachloro-fluorescein channel. We considered definite mutant cases to have a fractional abundance of 0.1% or more and to have three or more mutant droplets and/or wildtype droplets detected. Variant allele frequency (VAF) was calculated as follows: VAF% = (Nmt/(Nmt + Nwt))×100), where Nmt is number of mutant events and Nwt is number of wildtype events per reaction. Statistical analysis The Mann-Whitney U test was conducted for comparison of the medians and correlation of VAF between cyst fluid cfDNA and tumor DNA were analyzed using Pearson's correlation coefficients. One way analysis of variance (ANOVA) with Tukey’s method for multiple comparisons was used to compare the mean values of three groups. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 10 software (GraphPad Software, La Jolla, CA, USA). Results Patient characteristics and genetic analysis In 17 brain tumor cases in which cystic fluid was collected, 20 diagnostic hotspot mutations were detected in the tumor DNA by Sanger sequencing and/or ddPCR (Table 1 , Fig. S2). The median age of the 17 patients was 52 years (range 22–83 years), 16 harbored diffuse gliomas (including one diffuse midline glioma) and one had metastatic brain tumor. 12 Six patients had recurrent brain tumors at the time of cyst fluid collection. Importantly, all (100%) 20 hotspot point mutations detected from tumor DNA were also detected in cyst fluid cfDNA by ddPCR. Table 1 Summary of patient characteristics and genetic analysis Tumor DNA Cyst DNA Pt. Age Sex Pathological Mutation ddPCR DNA Sanger* ddPCR diagnosis VAF (%) concentration (ng/µL) MT droplets WT droplets VAF (%) 1 38 M Oligodendroglioma, grade 3 IDH1 R132H 43 21.0 DHP 1414 2509 36 pTERT C228T 47 DLP 206 778 21 2 61 F Oligodendroglioma, grade 3 IDH1 R132H 44 1.7 DLP 31 30 51 pTERT C228T 32 DHP 13 19 41 3 74 F Oligodendroglioma, grade 3 IDH1 R132H pTERT C228T 50 85 27.0 DHP DHP 2093 662 2275 820 48 45 4 22 M Astrocytoma, grade 4 IDH1 R132H 31 3.6 DHP 4 11 27 5 37 M Astrocytoma, grade 4 IDH1 R132H 33 7.5 DLP 29 452 6 6 37 M Astrocytoma, grade 4 IDH1 R132H 11 10.0 DLP 35 533 6 7 45 M Astrocytoma, grade 4 IDH1 R132H 39 6.7 DHP 1571 2270 41 8 46 F Astrocytoma, grade 4 IDH1 R132H 52 7.4 DLP 52 60 46 9 67 M Astrocytoma, grade 4 IDH1 R132H 43 1.8 DLP 48 245 16 10 34 F Glioblastoma, IDH-wildtype pTERT C228T 21 25.2 ND 213 1946 10 11 44 F Glioblastoma, IDH-wildtype pTERT C228T 55 34.0 DHP 704 686 51 12 52 F Glioblastoma, IDH-wildtype pTERT C228T 38 1.0 DHP 8 14 36 13 72 F Glioblastoma, IDH-wildtype pTERT C228T 12 533.3 ND 217 2335 9 14 83 F Glioblastoma, IDH-wildtype pTERT C228T 64 264.8 DHP 668 1197 36 15 68 F Gliosarcoma pTERT C228T 21 1.5 ND 6 9 40 16 55 M Diffuse midline glioma H3F3A K27M 6 1.2 ND 4 37 10 17 69 F Enterogenous carcinoma KRAS G12D 22 31.0 DLP 286 2969 9 Abbreviations: VAF, valiant allele frequency; MT, mutant; WT, wildtype; DHP, detectable high peak; DLP, detectable low peak; ND, not detected. * DHP was defined as a mutant peak amplitude half or more than the wildtype peak amplitude. DLP was defined as a mutant peak amplitude that is less than half of the wildtype amplitude signal but is clearly distinguishable from the baseline. The median VAF of cyst fluid cfDNA (cVAF) was comparable to that of tumor DNA (tVAF) (36% vs 39%, P = 0.20, Mann-Whitney U test) (Fig. 1 A). Additionally, there was a positive correlation between tVAF and cVAF ( R = 0.67, P = 0.0012) (Fig. 1 B). Sixteen of the 20 gene alterations (80%) were confirmed by the Sanger method, although the mutant peaks were small (< 50% amplitude of wildtype peak; detectable low peak (DLP)) in 7 out of 16 (44%) mutations (Table 1 ). The average VAF (%) determined by ddPCR was significantly higher in cases with high mutant peaks (50–100% amplitude of wildtype peak; detectable high peak (DHP)) compared to those with DLP (40% vs. 22%, P < 0.05, one-way ANOVA) and cases with no detectable mutant peak (not detected (ND)) (40% vs. 17%, P = 0.03, one-way ANOVA) by Sanger sequencing (Fig. 1 C and Fig. S3). Cases in which liquid biopsy of cyst fluid was especially useful are presented below. Genetic diagnosis with cyst fluid was useful for surgical planning in Case 1 (Patient 1, Fig. 2 ) and assisted in the pathological diagnosis of Case 2 (Patient 16, Fig. 3 ). Case 1 A 38-year-old man presented with increasing headaches. MR images showed a large, cystic mass lesion with mural nodules at the left frontal lobe (Fig. 2 A). He was scheduled for surgery to remove the tumor, but due to the rapidly enlarging mass lesion, the patient became drowsy. Therefore, an Ommaya reservoir was placed urgently (Fig. 2 B) and cyst fluid was evacuated and collected. IDH1 R132H and pTERT C228T were detected from cfDNA of the fluid (Fig. 2 E, F). The tumor was later removed, and the pathological diagnosis was oligodendroglioma, IDH-mutant and 1p/19q-codeleted, WHO grade 3 (Fig. 2 C), as IDH1 R132H and pTERT C228T mutations were also detected in tumor DNA. Case 2 We performed a stereotactic needle biopsy of a 55-year-old man presenting with progressive right hemiplegia with MR images showing a cystic mass lesion located at the cerebral peduncle to midbrain (Fig. 3 A, B), but pathological diagnosis was difficult due to contamination of normal tissue (Fig. 3 C). Initially, we failed to detect H3F3A K27M in DNA extracted from tumor tissue by Sanger sequencing (Fig. 3 D). However, H3F3A K27M was detected in the cfDNA of the cyst fluid at this time (Fig. 3 E). Later, we found H3F3A K27M-positive tumor cells by immunohistochemistry (Fig. 3 F) of frozen specimens used for rapid diagnosis. Discussion In this study, we showed that the same genetic abnormalities found in tumor DNA were detected from cfDNA extracted from cyst fluid. In all cases in which genetic alterations were found in tumor DNA, the same genetic abnormalities were detected in cyst fluid cfDNA by ddPCR. Also, in 12 out of 16 (75%) cases, the Sanger method was sufficient to detect the same genetic abnormalities in cyst fluid. The frequency of the cystic component in brain tumors depends on the type of tumor. 14 While some tumors, such as hemangioblastoma, 15 are commonly associated with cysts, 8–10% are reported in glioblastoma, 16 20% in ependymoma, 17 and 2–4% in meningioma 18 . The etiologies of cyst formation can be different from case to case and are generally not well understood. A study on the composition of the fluid content report that cyst fluid is a nutrient source for tumors; 19 a study analyzing cystic components in brain tumors using MR Spectroscopy report that cyst fluid is related to tumor malignancy. 20 Although the number of cases is small, the present study also analyzed the cyst component biochemically. The electrolyte component of cyst was similar to that of blood, and the sugar component was lower than that of blood, probably because it was consumed (Fig. S1 D). Genetic studies on cyst content fluid are scarce. A study of cystic lesions in the pancreas has reported that next generation sequencing (NGS) of cystic content improve the diagnostic accuracy of cytology; 21 in a case series on liquid biopsy of diffuse midline gliomas (DMG), cyst fluid was examined in one of the cases, and in that case, a high VAF of 43% was detected for H3F3A K27M mutation. 22 In the present study, we found that VAF of cyst fluid (cVAF) was comparable to that of tumor DNA (tVAF). CSF is commonly used as a source of liquid biopsy in brain tumors patients. 1 , 23 – 26 However, the detection is difficult even by sensitive methods such as ddPCR or NGS, due to low cfDNA concentration in CSF, except for tumors with rapid cell turnover, such as lymphomas, 2 or for advanced stage gliomas that show leptomeningeal dissemination. 3 , 22 , 27 Genetic alterations were found in CSF obtained by lumbar puncture in only 42 out of 85 patients (49.4%) harboring gliomas by NGS. 2 , 3 We previously reported that H3F3A K27M mutations are detected in disseminated and advanced stages of DMG. 27 The low cfDNA concentration may be due to the extremely large volume of the CSF cavity. Recent morphological analysis using MR imaging has shown that the volume of CSF in the intracranial region alone is about 300 ml in adults over 60 years of age. 28 In contrast, the cyst cavity is a very small space relative to the CSF cavity and is located close to or inside the tumor, therefore genetic alterations in cyst fluid cfDNA were detected at high VAF and were detected even by the Sanger method. In addition, ctDNA is fragmented DNA and has been shown to be shorter (90–150 bp) than cfDNA, which is approximately 167 bp in size. 29 , 30 The primers used in the Sanger method in this study all produced PCR products of 160 bp or more (Table S1 ), suggesting that cyst fluid cfDNA may be longer than ctDNA in CSF or plasma. Collection of cyst fluid is not minimally invasive because it is usually performed during the surgical removal of tumor. However, in certain situations, a cyst fluid aspiration can be done during minimally invasive surgery. There is a report of navigation-guided puncture of the cyst cavity prior to tumor resection to reduce the cyst volume and safely remove the tumor with minimal damage to the surrounding brain tissue. 31 In another report, patients with metastatic brain tumors who are unable to undergo removal surgery due to poor general condition underwent a cyst fluid aspiration to reduce tumor volume prior to radiation therapy. 32 In these situations, histological diagnosis of the tumor may be difficult, and a genetic search of the cyst fluid may be useful. In addition, as we have shown in this study, a genetic search of cyst fluid may assist the pathological diagnosis even when a small amount of sample is collected in stereotactic brain biopsy. There are some limitations of this study. First, since our study was focused on specific genetic abnormalities and was not comprehensive, it is unclear whether cyst fluid cfDNA harbors all genetic abnormalities found in tumor DNA. Additionally, it is difficult to determine malignancy (WHO grade 2 and 3) in lower grade gliomas just by analysis of the cyst fluid. However, it is easy to collect and store the liquid, and analyzing it comprehensively by NGS or other methods shows great promise in the future. We should stress that at the presently, the utility of liquid biopsy of cyst fluid in brain tumors are restricted to cases that have very large cystic components and relatively small solid portion. In this preliminary study, we show the feasibility of liquid biopsy of cyst fluid in cystic brain tumors. Conclusion In the present report, we found that certain genetic abnormalities are commonly found in both the cyst fluid and the tumor itself. Moreover, in many cases, the genetic abnormality was also detected in cyst fluid by the Sanger method. Cyst fluid cfDNA in brain tumors may be considered as an alternative source of tumor DNA in brain tumors with cystic components. Declarations Funding This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI grants to M.N. (19K09476, 21KK0156), J.W. (19K18418), Y.T. (22K16679), R.O. (22K16652), M.Ok. (20K17955), and J.A (22K09216). Acknowledgments The authors would like to thank Akiko Yoshii and Shingo Nigorikawa for technical assistance. Conflict of interest statement. The authors have no conflicts of interest to disclose. Authorship statement. Study conception and design: J.O. and M.N. Acquisition of data: J.O., H.T., A.Ko., Sh.S., J.W., Sa.S., Y.K., T.E. Analysis and interpretation of data: J.O., M.N., M.T., H.S., and A.K. Administrative and material support: M.N., Y.T., M.Ok., R.O., J.A., K.M., R.N. Writing- original draft: J.O. and M.N. Writing- review and editing: J.O., M.N., A.Ko, Y.T. Study supervision: A.Ka. and M.Oi. References Eibl RH, Schneemann M. Liquid Biopsy and Primary Brain Tumors. Cancers (Basel) . 2021;13(21):5429. Watanabe J, Natsumeda M, Okada M, et al. 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Supplementary Files CystbiopsySupplementaryFile.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2024 Read the published version in Journal of Neuro-Oncology → Version 1 posted Editorial decision: Revision requested 21 Nov, 2023 Reviews received at journal 10 Nov, 2023 Reviewers agreed at journal 10 Nov, 2023 Reviewers agreed at journal 10 Nov, 2023 Reviewers invited by journal 10 Nov, 2023 Submission checks completed at journal 10 Nov, 2023 Editor assigned by journal 10 Nov, 2023 First submitted to journal 10 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3589356","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":247909629,"identity":"7931c26a-2fcf-4062-a002-b9ee974651be","order_by":0,"name":"Jotaro On","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jotaro","middleName":"","lastName":"On","suffix":""},{"id":247909632,"identity":"fb96e44f-8a19-4e4a-8327-4119dd61003e","order_by":1,"name":"Manabu Natsumeda","email":"data:image/png;base64,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","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Manabu","middleName":"","lastName":"Natsumeda","suffix":""},{"id":247909634,"identity":"73ec4f4f-4b02-4474-805d-d946bbbab974","order_by":2,"name":"Haruhiko Takahashi","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haruhiko","middleName":"","lastName":"Takahashi","suffix":""},{"id":247909635,"identity":"8e45e269-fb70-4886-af2f-b67e577add3b","order_by":3,"name":"Akihide Koyama","email":"","orcid":"","institution":"Niigata University Graduate School of Medical and Dental Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akihide","middleName":"","lastName":"Koyama","suffix":""},{"id":247909637,"identity":"aad766ca-2d32-4c90-878e-de707411c635","order_by":4,"name":"Satoshi Shibuma","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Shibuma","suffix":""},{"id":247909640,"identity":"757a32c0-b1b7-412d-b0f1-bff2e48db5b2","order_by":5,"name":"Jun Watanabe","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Watanabe","suffix":""},{"id":247909642,"identity":"32df8a26-1136-49b6-b803-472db1fa467b","order_by":6,"name":"Shoji Saito","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shoji","middleName":"","lastName":"Saito","suffix":""},{"id":247909644,"identity":"1546bd28-3980-4d6b-a48d-540259bc2c43","order_by":7,"name":"Yu Kanemaru","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Kanemaru","suffix":""},{"id":247909645,"identity":"f681b195-f4a5-4da9-8839-9855ec6d35d0","order_by":8,"name":"Yoshihiro Tsukamoto","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Tsukamoto","suffix":""},{"id":247909646,"identity":"a1c667be-495a-4d0e-86bc-cd3c96c35a65","order_by":9,"name":"Masayasu Okada","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masayasu","middleName":"","lastName":"Okada","suffix":""},{"id":247909648,"identity":"3ab9a644-3b18-47d7-bbe0-01d5901f8410","order_by":10,"name":"Ryosuke Ogura","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Ogura","suffix":""},{"id":247909649,"identity":"5520b189-602f-4b2e-9661-9b39ccacf0c0","order_by":11,"name":"Takeyoshi Eda","email":"","orcid":"","institution":"Niigata University Graduate School of Medical and Dental Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takeyoshi","middleName":"","lastName":"Eda","suffix":""},{"id":247909650,"identity":"3120a3d3-7276-4655-8dff-53424abb9c4e","order_by":12,"name":"Mari Tada","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mari","middleName":"","lastName":"Tada","suffix":""},{"id":247909651,"identity":"4c7dcd0c-fdab-454b-a675-16e7350ae2c0","order_by":13,"name":"Hiroshi Shimizu","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Shimizu","suffix":""},{"id":247909652,"identity":"81c93560-bf79-49ae-ac43-cc43101d9511","order_by":14,"name":"Jun-ichi Adachi","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun-ichi","middleName":"","lastName":"Adachi","suffix":""},{"id":247909653,"identity":"2cab983b-f35b-40ee-94c4-a537634cfca5","order_by":15,"name":"Kazuhiko Mishima","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazuhiko","middleName":"","lastName":"Mishima","suffix":""},{"id":247909654,"identity":"6b2b80c1-2204-4199-a26e-97fc7835cca9","order_by":16,"name":"Ryo Nishikawa","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Nishikawa","suffix":""},{"id":247909655,"identity":"8355446e-986e-4d33-a91d-204c84173d54","order_by":17,"name":"Akiyoshi Kakita","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akiyoshi","middleName":"","lastName":"Kakita","suffix":""},{"id":247909656,"identity":"94f08727-c877-4513-bfa2-29319ca7e59d","order_by":18,"name":"Makoto Oishi","email":"","orcid":"","institution":"Niigata University, Brain Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Oishi","suffix":""}],"badges":[],"createdAt":"2023-11-10 07:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3589356/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3589356/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11060-023-04555-5","type":"published","date":"2024-01-16T15:01:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":46384513,"identity":"c05907a8-71c2-41f7-ab40-b5c2f41d48ae","added_by":"auto","created_at":"2023-11-14 03:25:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145628,"visible":true,"origin":"","legend":"\u003cp\u003eThere was no significant difference between VAF in tumor DNA and cyst DNA (P = 0..20)(A). cVAF was strongly related to tVAF (R = 0.67, P = 0.0012) (B). Mean VAF was higher in DHP cases compared to that of DLP (P \u0026lt; 0.05, ANOVA) and ND (P = 0.03, ANOVA) cases (C).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3589356/v1/873c616e535d77b7fdcf81bc.png"},{"id":46384514,"identity":"e48d0cb0-a02c-4245-8a27-7b3d0e74400d","added_by":"auto","created_at":"2023-11-14 03:25:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2109564,"visible":true,"origin":"","legend":"\u003cp\u003ePatient 1. A 38-year-old man with a huge cystic lesion in the left frontal lobe who presented with symptoms of elevated intracranial pressure (A). During the initial surgery, the cyst fluid was aspirated to alleviate intracranial pressure and an Ommaya reservoir was placed in the cyst cavity (B). A total removal was performed in the second surgery, and the histopathological diagnosis was anaplastic oligodendroglioma (C). \u003cem\u003epTERT \u003c/em\u003eC228T and \u003cem\u003eIDH1\u003c/em\u003e R132H mutations were detected in cyst fluid collected at the initial surgery by Sanger (D) and ddPCR (E) methods, respectively. Scale bar C: 100 µm\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3589356/v1/11b462327afa4c55cc919f09.png"},{"id":46385437,"identity":"2df50c8c-add0-47b8-9af3-8e9afdca95b1","added_by":"auto","created_at":"2023-11-14 03:33:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5759805,"visible":true,"origin":"","legend":"\u003cp\u003ePatient 16. A 55-year-old man with a small cystic lesion in the left midbrain presented with right hemiplegia (A). A stereotactic needle biopsy was performed (B), and cystic fluid and a few tissue fragments were collected. The rapid histology (C) was glioma, but paraffin-embedded tissues failed to reveal H3F3A K27M-positive tumor cells immunohistochemically. Sanger sequencing of preserved tissue failed to detect the \u003cem\u003eH3F3A\u003c/em\u003e K27M mutation (D). However, \u003cem\u003eH3F3A\u003c/em\u003e K27M mutation was detected from the cyst fluid (E). The mutation was then confirmed by immunohistochemical staining of the frozen section that had been used intraoperative diagnosis (F). Scale bar C, F: 100 µm\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3589356/v1/f680f78dd36eedf0580ae066.png"},{"id":49978625,"identity":"13355257-f555-4524-99c2-9b3784629d4b","added_by":"auto","created_at":"2024-01-22 15:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2631842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3589356/v1/40de2ce7-b5df-40d4-9660-24557d67676d.pdf"},{"id":46384515,"identity":"ce3205b1-7dd9-4acb-a040-d90d5bb84d02","added_by":"auto","created_at":"2023-11-14 03:25:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":605957,"visible":true,"origin":"","legend":"","description":"","filename":"CystbiopsySupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3589356/v1/15f8749dbe0c9e341376410c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiquid biopsy is used to detect diagnostic markers in body fluids and to detect genetic alterations derived from circulating tumor DNA (ctDNA). The main sources of ctDNA include blood, urine, and cerebrospinal fluid (CSF),\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and has recently attracted attention as a minimally invasive screening method that can lead to diagnosis and determination of disease status.\u003c/p\u003e \u003cp\u003eIn brain tumors, the usefulness of liquid biopsy has been reported in the detection of genetic abnormalities such as \u003cem\u003eMYD88\u003c/em\u003e L265P in primary central nervous system lymphoma (PCNSL).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e However, in gliomas and other types of tumors, it is difficult to detect genetic alterations from body fluids except in advanced stages such as those with disseminated foci.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Meanwhile, genetic diagnosis is becoming increasingly important, as the World Health Organization Classification of Central Nervous System Tumours 2016\u003csup\u003e4\u003c/sup\u003e and 2021\u003csup\u003e5\u003c/sup\u003e call for the analysis of certain genetic and chromosomal alterations for the diagnosis of gliomas.\u003c/p\u003e \u003cp\u003eTo date, there have been no large-scale reports showing the usefulness of liquid biopsy of cyst fluid in brain tumors. Although having a cystic component is not uncommon in brain tumors, it is not known how reliably genetic alterations can be detected from cyst fluid. In the present study, we detected genetic alterations from cell-free DNA of cyst fluid (cyst fluid cfDNA) taken from 12 brain tumor patients and found that in all patients, genetic abnormalities found in tumor tissue DNA (tumor DNA) were also detected in cyst fluid.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePatients and sample collection\u003c/p\u003e \u003cp\u003eTumor samples and cyst fluid were collected from 38 patients who underwent surgery at the Department of Neurosurgery, Niigata University, from January 2019 to January 2023 for a cystic brain tumor. In 17 out of 38 (45%) cases, diagnostic point mutations were identified in tumor DNA by routine Sanger sequence analysis. The location of the cyst was confirmed by preoperative imaging. Cysts were defined as having low signal intensity on MRI diffusion-weighted images (DWI) and low signal intensity on contrast-enhanced T1-weighted MRI and uniform signal intensity or fluid-fluid lines on T1-weighted or T2-weighted MRI and more than 1 ml of these areas.\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e One to 20 ml of cyst fluid was aspirated manually by inserting a plastic needle or biopsy needle into the cyst to avoid CSF contamination during surgery with care to avoid blood contamination (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Cyst fluid was promptly centrifuged at 1,500 G for 10 minutes, and the supernatant was stored at \u0026minus;\u0026thinsp;80\u0026deg;C (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, C). Tumor DNA and cell-free DNA (cfDNA) of cyst fluid was extracted as previously reported.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In brief, tissue DNA was extracted from fresh frozen tissue using the QIAamp Blood \u0026amp; Tissue Kit (Qiagen, Valencia, CA, USA), and ctDNA was extracted using the Maxwell RSC ccfDNA Plasma Kit (RSC; Promega, Leiden, The Netherlands), according to the manufacturer\u0026rsquo;s instructions. For all samples, DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use. The concentration of extracted DNA was measured with a spectrometer (Eppendorf, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eThe surgical specimens were fixed with 10% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-\u0026micro;m-thick sections stained with hematoxylin and eosin. Pathologic diagnoses were made by 3 experienced neuropathologists (A.K., H.S. and M.T.) according to the 2021 World Health Organization classification system.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Immunohistochemistry was performed as described previously using primary antibodies against IDH1 R132H (1:100, monoclonal, clone H09, Dianova, Hamburg, Germany) and H3F3A K27M (1:3200, monoclonal, clone RM192, Sigma-Aldrich, STL, USA) .\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Based on the histological diagnosis, tissues pathologically diagnosed as gliomas were screened for eight mutation hotspots (\u003cem\u003eIDH1\u003c/em\u003e R132, \u003cem\u003eIDH2\u003c/em\u003e R172, \u003cem\u003eHIST 1H3B\u003c/em\u003e K27M, \u003cem\u003eH3F3A\u003c/em\u003e K27M/G34, \u003cem\u003eBRAF\u003c/em\u003e V600, and \u003cem\u003epTERT\u003c/em\u003e C228/C250) by direct sequencing or ddPCR methods. Similarly, \u003cem\u003epTERT\u003c/em\u003e mutations were screened for in a malignant meningioma case. Cases in which alterations were identified in tissue DNA were also searched for in cyst fluid cfDNA, using direct sequencing and ddPCR methods.\u003c/p\u003e \u003cp\u003eThis study was approved by the Ethics Committee of Niigata University School of Medicine (Approval #: 2018\u0026thinsp;\u0026minus;\u0026thinsp;0353) and written informed consent for liquid biopsy and use of the resected tissues for research purposes was obtained from all patients.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis\u003c/h2\u003e \u003cp\u003eDirect sequencing of \u003cem\u003eIDH1\u003c/em\u003e R132H, \u003cem\u003eIDH2\u003c/em\u003e R172, \u003cem\u003epTERT\u003c/em\u003e C228T/C250T, \u003cem\u003eH3F3A\u003c/em\u003e K27M/G34, \u003cem\u003eHIST1H3B\u003c/em\u003e K27M and \u003cem\u003eKRAS\u003c/em\u003e G12D was performed as reported previously.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e A total of 2\u0026ndash;20 ng of DNA was used as a template for a single DNA sequencing. The sequences of the primers used in the study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The amplified products were fractionated on 2.0% agarose gel at 100 V for 45 minutes. The PCR products were then sequenced on a 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA) with a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eDetailed methods of ddPCR have been previously published.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Briefly, ddPCR reagents and primer/probe mix for \u003cem\u003eIDH1\u003c/em\u003e R132H, \u003cem\u003epTERT\u003c/em\u003e C228T, \u003cem\u003eH3F3A\u003c/em\u003e K27M, and \u003cem\u003eKRAS\u003c/em\u003e G12D were purchased from Bio-Rad (Hercules, CA, USA). Alternatively, primer/probe mix for \u003cem\u003epTERT\u003c/em\u003e C228T was purchased from Integrated DNA Technologies Inc (Table S2, Coralville, IA, USA). When detecting \u003cem\u003eIDH1\u003c/em\u003e R132H, \u003cem\u003eH3F3A\u003c/em\u003e K27M, and \u003cem\u003eKRAS\u003c/em\u003e G12D, a 20 \u0026micro;L PCR mix, composed of 10 \u0026micro;L 2\u0026times; ddPCR Supermix for Probes (no deoxyuridine triphosphate; Bio-Rad), 1 \u0026micro;L ddPCR Mutation Assay (Bio-Rad) and 9 \u0026micro;L DNA, was loaded into sample wells of an eight-channel disposable droplet generator cartridge (Cat. No. 1864007, Bio-Rad). When detecting \u003cem\u003epTERT\u003c/em\u003e C228T, a 20 \u0026micro;L PCR mix, composed of 10 \u0026micro;L 2\u0026times; ddPCR Supermix for Probes (no deoxyuridine triphosphate; Bio-Rad), 1 \u0026micro;L ddPCR Mutation Assay (Bio-Rad), and 6.75 \u0026micro;L DNA, 2 \u0026micro;L 5M Betaine, and 0.25 \u0026micro;L 80mM EDTA was loaded into sample wells, in the same manner. An additional 50 \u0026micro;L of droplet generation oil (Cat. No. 189005 Bio-Rad) was loaded into the oil well for each channel. After droplet generation, the droplets were transferred into a 96-well PCR plate and then thermal cycled using the thermal cycler Dice Gradient (Takara, Shiga, Japan) or MiniAmp\u0026trade; Plus thermal cycler (Applied Biosystems). Thermal cycling conditions were carried out as follows: 95˚C 10 min, 94˚C 30 sec. 60˚C 30\u0026ndash;60 sec. (for 40 cycles) 98˚C 10 min. and hold at 4˚C. After PCR, the 96-well PCR plate was subjected to the QX-200 droplet reader (Bio-Rad), and data were analyzed by QX Manager 1.2 standard edition software (Bio-Rad). Mutation-specific signals were generated in the hexachloro-fluorescein channel. We considered definite mutant cases to have a fractional abundance of 0.1% or more and to have three or more mutant droplets and/or wildtype droplets detected. Variant allele frequency (VAF) was calculated as follows: VAF% = (Nmt/(Nmt\u0026thinsp;+\u0026thinsp;Nwt))\u0026times;100), where Nmt is number of mutant events and Nwt is number of wildtype events per reaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Mann-Whitney \u003cem\u003eU\u003c/em\u003e test was conducted for comparison of the medians and correlation of VAF between cyst fluid cfDNA and tumor DNA were analyzed using Pearson's correlation coefficients. One way analysis of variance (ANOVA) with Tukey\u0026rsquo;s method for multiple comparisons was used to compare the mean values of three groups. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were performed using JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 10 software (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePatient characteristics and genetic analysis\u003c/p\u003e \u003cp\u003eIn 17 brain tumor cases in which cystic fluid was collected, 20 diagnostic hotspot mutations were detected in the tumor DNA by Sanger sequencing and/or ddPCR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. S2). The median age of the 17 patients was 52 years (range 22\u0026ndash;83 years), 16 harbored diffuse gliomas (including one diffuse midline glioma) and one had metastatic brain tumor.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Six patients had recurrent brain tumors at the time of cyst fluid collection. Importantly, all (100%) 20 hotspot point mutations detected from tumor DNA were also detected in cyst fluid cfDNA by ddPCR.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of patient characteristics and genetic analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eTumor DNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003eCyst DNA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePt.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePathological\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eddPCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSanger*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003eddPCR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ediagnosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVAF (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003econcentration (ng/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMT droplets\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eWT droplets\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eVAF (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOligodendroglioma, grade 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOligodendroglioma, grade 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOligodendroglioma, grade 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2093\u003c/p\u003e \u003cp\u003e662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2275\u003c/p\u003e \u003cp\u003e820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e48\u003c/p\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAstrocytoma, grade 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDH1\u003c/em\u003e R132H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlioblastoma, IDH-wildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlioblastoma, IDH-wildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e34.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlioblastoma, IDH-wildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlioblastoma, IDH-wildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e533.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlioblastoma, IDH-wildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e264.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGliosarcoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epTERT\u003c/em\u003e C228T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiffuse midline glioma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eH3F3A\u003c/em\u003e K27M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEnterogenous carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eKRAS\u003c/em\u003e G12D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eAbbreviations: VAF, valiant allele frequency; MT, mutant; WT, wildtype; DHP, detectable high peak; DLP, detectable low peak; ND, not detected.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e* DHP was defined as a mutant peak amplitude half or more than the wildtype peak amplitude. DLP was defined as a mutant peak amplitude that is less than half of the wildtype amplitude signal but is clearly distinguishable from the baseline.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe median VAF of cyst fluid cfDNA (cVAF) was comparable to that of tumor DNA (tVAF) (36% vs 39%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.20, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, there was a positive correlation between tVAF and cVAF (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.67, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Sixteen of the 20 gene alterations (80%) were confirmed by the Sanger method, although the mutant peaks were small (\u0026lt;\u0026thinsp;50% amplitude of wildtype peak; detectable low peak (DLP)) in 7 out of 16 (44%) mutations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average VAF (%) determined by ddPCR was significantly higher in cases with high mutant peaks (50\u0026ndash;100% amplitude of wildtype peak; detectable high peak (DHP)) compared to those with DLP (40% vs. 22%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, one-way ANOVA) and cases with no detectable mutant peak (not detected (ND)) (40% vs. 17%, P\u0026thinsp;=\u0026thinsp;0.03, one-way ANOVA) by Sanger sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCases in which liquid biopsy of cyst fluid was especially useful are presented below. Genetic diagnosis with cyst fluid was useful for surgical planning in Case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Patient 1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and assisted in the pathological diagnosis of Case \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (Patient 16, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 1\u003c/strong\u003e \u003cp\u003eA 38-year-old man presented with increasing headaches. MR images showed a large, cystic mass lesion with mural nodules at the left frontal lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). He was scheduled for surgery to remove the tumor, but due to the rapidly enlarging mass lesion, the patient became drowsy. Therefore, an Ommaya reservoir was placed urgently (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and cyst fluid was evacuated and collected. \u003cem\u003eIDH1\u003c/em\u003e R132H and \u003cem\u003epTERT\u003c/em\u003e C228T were detected from cfDNA of the fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). The tumor was later removed, and the pathological diagnosis was oligodendroglioma, IDH-mutant and 1p/19q-codeleted, WHO grade 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), as \u003cem\u003eIDH1\u003c/em\u003e R132H and \u003cem\u003epTERT\u003c/em\u003e C228T mutations were also detected in tumor DNA.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 2\u003c/strong\u003e \u003cp\u003eWe performed a stereotactic needle biopsy of a 55-year-old man presenting with progressive right hemiplegia with MR images showing a cystic mass lesion located at the cerebral peduncle to midbrain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), but pathological diagnosis was difficult due to contamination of normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Initially, we failed to detect \u003cem\u003eH3F3A\u003c/em\u003e K27M in DNA extracted from tumor tissue by Sanger sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, \u003cem\u003eH3F3A\u003c/em\u003e K27M was detected in the cfDNA of the cyst fluid at this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Later, we found \u003cem\u003eH3F3A\u003c/em\u003e K27M-positive tumor cells by immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) of frozen specimens used for rapid diagnosis.\u003c/p\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we showed that the same genetic abnormalities found in tumor DNA were detected from cfDNA extracted from cyst fluid. In all cases in which genetic alterations were found in tumor DNA, the same genetic abnormalities were detected in cyst fluid cfDNA by ddPCR. Also, in 12 out of 16 (75%) cases, the Sanger method was sufficient to detect the same genetic abnormalities in cyst fluid.\u003c/p\u003e \u003cp\u003eThe frequency of the cystic component in brain tumors depends on the type of tumor.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e While some tumors, such as hemangioblastoma,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e are commonly associated with cysts, 8\u0026ndash;10% are reported in glioblastoma,\u003csup\u003e16\u003c/sup\u003e 20% in ependymoma,\u003csup\u003e17\u003c/sup\u003e and 2\u0026ndash;4% in meningioma\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The etiologies of cyst formation can be different from case to case and are generally not well understood. A study on the composition of the fluid content report that cyst fluid is a nutrient source for tumors;\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e a study analyzing cystic components in brain tumors using MR Spectroscopy report that cyst fluid is related to tumor malignancy.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Although the number of cases is small, the present study also analyzed the cyst component biochemically. The electrolyte component of cyst was similar to that of blood, and the sugar component was lower than that of blood, probably because it was consumed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Genetic studies on cyst content fluid are scarce. A study of cystic lesions in the pancreas has reported that next generation sequencing (NGS) of cystic content improve the diagnostic accuracy of cytology;\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e in a case series on liquid biopsy of diffuse midline gliomas (DMG), cyst fluid was examined in one of the cases, and in that case, a high VAF of 43% was detected for \u003cem\u003eH3F3A\u003c/em\u003e K27M mutation.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e In the present study, we found that VAF of cyst fluid (cVAF) was comparable to that of tumor DNA (tVAF).\u003c/p\u003e \u003cp\u003eCSF is commonly used as a source of liquid biopsy in brain tumors patients.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e However, the detection is difficult even by sensitive methods such as ddPCR or NGS, due to low cfDNA concentration in CSF, except for tumors with rapid cell turnover, such as lymphomas,\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e or for advanced stage gliomas that show leptomeningeal dissemination.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Genetic alterations were found in CSF obtained by lumbar puncture in only 42 out of 85 patients (49.4%) harboring gliomas by NGS.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e We previously reported that \u003cem\u003eH3F3A\u003c/em\u003e K27M mutations are detected in disseminated and advanced stages of DMG.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The low cfDNA concentration may be due to the extremely large volume of the CSF cavity. Recent morphological analysis using MR imaging has shown that the volume of CSF in the intracranial region alone is about 300 ml in adults over 60 years of age.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In contrast, the cyst cavity is a very small space relative to the CSF cavity and is located close to or inside the tumor, therefore genetic alterations in cyst fluid cfDNA were detected at high VAF and were detected even by the Sanger method. In addition, ctDNA is fragmented DNA and has been shown to be shorter (90\u0026ndash;150 bp) than cfDNA, which is approximately 167 bp in size.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The primers used in the Sanger method in this study all produced PCR products of 160 bp or more (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), suggesting that cyst fluid cfDNA may be longer than ctDNA in CSF or plasma.\u003c/p\u003e \u003cp\u003eCollection of cyst fluid is not minimally invasive because it is usually performed during the surgical removal of tumor. However, in certain situations, a cyst fluid aspiration can be done during minimally invasive surgery. There is a report of navigation-guided puncture of the cyst cavity prior to tumor resection to reduce the cyst volume and safely remove the tumor with minimal damage to the surrounding brain tissue.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In another report, patients with metastatic brain tumors who are unable to undergo removal surgery due to poor general condition underwent a cyst fluid aspiration to reduce tumor volume prior to radiation therapy.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e In these situations, histological diagnosis of the tumor may be difficult, and a genetic search of the cyst fluid may be useful. In addition, as we have shown in this study, a genetic search of cyst fluid may assist the pathological diagnosis even when a small amount of sample is collected in stereotactic brain biopsy.\u003c/p\u003e \u003cp\u003eThere are some limitations of this study. First, since our study was focused on specific genetic abnormalities and was not comprehensive, it is unclear whether cyst fluid cfDNA harbors all genetic abnormalities found in tumor DNA. Additionally, it is difficult to determine malignancy (WHO grade 2 and 3) in lower grade gliomas just by analysis of the cyst fluid. However, it is easy to collect and store the liquid, and analyzing it comprehensively by NGS or other methods shows great promise in the future.\u003c/p\u003e \u003cp\u003eWe should stress that at the presently, the utility of liquid biopsy of cyst fluid in brain tumors are restricted to cases that have very large cystic components and relatively small solid portion. In this preliminary study, we show the feasibility of liquid biopsy of cyst fluid in cystic brain tumors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present report, we found that certain genetic abnormalities are commonly found in both the cyst fluid and the tumor itself. Moreover, in many cases, the genetic abnormality was also detected in cyst fluid by the Sanger method. Cyst fluid cfDNA in brain tumors may be considered as an alternative source of tumor DNA in brain tumors with cystic components.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI grants to M.N. (19K09476, 21KK0156), J.W. (19K18418), Y.T. (22K16679), R.O. (22K16652), M.Ok. (20K17955), and J.A (22K09216).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Akiko Yoshii and Shingo Nigorikawa for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest statement.\u003c/strong\u003e The authors have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthorship statement.\u003c/strong\u003e Study conception and design: J.O. and M.N. Acquisition of data: J.O., H.T., A.Ko., Sh.S., J.W., Sa.S., Y.K., T.E. Analysis and interpretation of data: J.O., M.N., M.T., H.S., and A.K. Administrative and material support: M.N., Y.T., M.Ok., R.O., J.A., K.M., R.N. Writing- original draft: J.O. and M.N. Writing- review and editing: J.O., M.N., A.Ko, Y.T. Study supervision: A.Ka. and M.Oi.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEibl RH, Schneemann M. 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Alternative Treatment of Stereotactic Cyst Aspiration and Radiosurgery for Cystic Brain Metastases. \u003cem\u003eStereotact Funct Neurosurg\u003c/em\u003e. 2014;92(4):234-241. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cell-free DNA, cyst fluid, liquid biopsy, brain tumors","lastPublishedDoi":"10.21203/rs.3.rs-3589356/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3589356/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLiquid biopsy of cyst fluid in brain tumors has not been extensively studied to date. The present study was performed to see whether diagnostic genetic alterations found in brain tumor tissue DNA could also be detected in cell-free DNA (cfDNA) of cyst fluid in cystic brain tumors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCyst fluid was obtained from 17 patients undergoing surgery for a cystic brain tumor with confirmed genetic alterations in tumor DNA. Pathological diagnoses based on WHO 2021 classification and diagnostic alterations in the tumor DNA, such as \u003cem\u003eIDH1\u003c/em\u003e R132H and \u003cem\u003eTERT\u003c/em\u003e promoter mutation for oligodendrogliomas, were detected by Sanger sequencing. The same alterations were analyzed by both droplet digital PCR (ddPCR) and Sanger sequencing in cyst fluid cfDNA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwenty genetic alterations were found in 17 tumor samples. All (100%) alterations were detected in cyst fluid cfDNA by ddPCR. Sixteen of the 20 (80%) alterations were also detected by Sanger sequencing of cyst fluid cfDNA. Variant allele frequency (VAF) in cyst fluid cfDNA was comparable to that of tumor DNA (R\u0026thinsp;=\u0026thinsp;0.67, Pearson\u0026rsquo;s correlation).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCell-free DNA obtained from cyst fluid in cystic brain tumors is a reliable alternative to tumor DNA when diagnosing brain tumors.\u003c/p\u003e","manuscriptTitle":"Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-14 03:25:28","doi":"10.21203/rs.3.rs-3589356/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2023-11-21T14:15:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-11-10T13:29:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0c9f7d91-ba6f-4e6b-98f9-600519ce1232","date":"2023-11-10T11:34:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0b43d968-354b-4115-89c2-64be0632d582","date":"2023-11-10T11:28:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-10T10:56:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-10T09:10:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-10T09:10:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuro-Oncology","date":"2023-11-10T07:47:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ee77eac0-5334-4e5e-ab60-2cd15486f934","owner":[],"postedDate":"November 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T15:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-3589356","link":"https://doi.org/10.1007/s11060-023-04555-5","journal":{"identity":"journal-of-neuro-oncology","isVorOnly":false,"title":"Journal of Neuro-Oncology"},"publishedOn":"2024-01-16 15:01:09","publishedOnDateReadable":"January 16th, 2024"},"versionCreatedAt":"2023-11-14 03:25:28","video":"","vorDoi":"10.1007/s11060-023-04555-5","vorDoiUrl":"https://doi.org/10.1007/s11060-023-04555-5","workflowStages":[]},"version":"v1","identity":"rs-3589356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3589356","identity":"rs-3589356","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}