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. S1A). Cyst fluid was promptly centrifuged at 1,500 G for 10 minutes, and the supernatant was stored at − 80°C (Fig. S1B, 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).