Study subjects.
Written informed consent has been obtained from all patients or their legal surrogates. The study protocol has been approved by the Ethics Committee of the Second Hospital of Hebei Medical University, Hebei, China. The patients with MC were enrolled from Department of Neurology in the Second Hospital of Hebei Medical University. In total, 35 patients with MC who underwent lumbar puncture for CSF cytology examination, CSF ctDNA extraction and cancer-associated gene mutations detection by next-generation sequencing (NGS) at the same time and underwent contrast-enhanced brain MRI and/or CT were enrolled in this study between October 2014 and September 2017. The diagnosis of MC had been established by clinical signs and symptoms in addition to positive CSF cytology and/or neuroimaging findings (contrast-enhanced brain MRI or CT) consistent with MC. The clinical signs and symptoms included headache, nausea and vomiting, convulsion, lower back pain, cranial nerve paralysis, paresthesia, gait disturbances, vertigo and defects in mental functioning. The positive CSF cytology result was defined by the morphology of neoplastic cells such as different size with irregular-shape, big nucleus with malignant signs such as lobulated state and malformed buds, increasing chromatin with basophilic coarse particles, mitotic activity with aberrant mitosis, obvious and polymorphic nucleolus accounting for the majority of chromatin and thickening nuclear membrane with saw-tooth-shaped and wear edge. The positive neuroimaging finding was defined as the presence of leptomeningeal enhancement.
Next-Generation Sequencing
Sample processing
Cerebrospinal fluid samples were collected from each patient. CSF samples in EDTA tubes were centrifuged for 5 minutes at 1,000g. The pellet was stored at -20°C, while the supernatant was centrifuged at 10,000g for another 30 minutes [16]. The supernatant was aseptically transferred to pre-labeled Cryotubes stored at -80°C. The ctDNA was extracted from at least 5ml CSF supernatant using the QIAamp Circulating Nucleic Acid kit (QIAGEN) as per the manufacturer’s instructions. Finally, ctDNA was quantified with the Qubit2.1 Fluorometer and Qubit dsDNA HS Assay kit (Life Technologies, Carlsbad, CA) following the recommended protocol.
Ion Proton Library Preparation and Sequencing
Preparation of the Ion Proton library and DNA sequencing were performed as described in our previous publications [17-19]. For each sample type, an adapter-ligated library was generated with the Ion AmpliSeq Library Kit 2.0 (Life Technologies) according to the manufacturer’s protocol. Briefly, pooled amplicons made from 10~20ng ctDNA were end-repaired and ligated to Ion Adapters X and P1. AMPure beads (Beckman Coulter, Brea, CA) were used to purify adapter-ligated products, followed by nick-translation and PCR-amplification for a total of 5 cycles. AMPure beads were used to purify the resulting library. And the Agilent 2100 Bioanalyzer and Agilent Bioanalyzer DNA High-Sensitivity LabChip (Agilent Technologies) were used to determine the concentration and size of the library. Sample emulsion PCR and emulsion breaking were performed using the Ion OneTouchTM2 system (Life Technologies) with the Ion PI Template OT2 200 Kit v3 (Life Technologies) as per the manufacturer’s instructions. Ion Sphere Particles (ISPs) were recovered, and template-positive ISPs were enriched with Dynabeads MyOne Streptavidin C1 beads (Life Technologies) on the Ion One Touch ES (enrichment system) (Life Technologies). ISPs enrichment was confirmed using the Qubit 2.0 Fluorometer (Life Technologies). The Ion Proton System using Ion PI v2 Chips (Life Technologies) were used to sequence barcoded samples for 100 cycles and the Ion PI Sequencing 200 Kit v3 (Life Technologies) was used for sequencing reactions following the recommended protocol.
We used the SV-OCP143-ctDNA panel (San Valley Biotech Inc., Beijing, China), which is capable of detecting somatic mutations from plasma or tissue samples on 143 cancer-related genes. Since the ctDNA in CSF is comprised of short DNA fragments, amplicons in the panel are specially designed for efficient amplification of ctDNA. For CSF samples, the total read count was more than 25 million to ensure the average base coverage depth over 10,000 x. Additionally, the average uniformity of base coverage was 95.5%. These strict quality control criteria ensured the reliability of sequencing.
Variant Calling.
Initial data from the sequencing runs were processed with the Ion Proton platform-specific pipeline software Torrent Suite v5.0 including generating sequencing reads, trim adapter sequences, filtering and removing poor signal-profile reads as described in our previous publications [17-19]. Initial variant calling from the sequencing data was generated with the TorrentSuite Software with a plug-in ‘variant caller v5.0’. Three filtering steps were used to eliminate erroneous base calling and generate final variant calling. For the first-step, the following filtering criteria were defined for CSF ctDNA: the average total coverage depth>10000; each variant coverage>10; a variant frequency of each sample>0.1%; and p value<0.01. The second-step utilized the Integrative Genomics Viewer (IGV) software (http//www.broadinstitute.org/igv) or Samtools software (http://samtools.sourceforge.net) to eliminate possible DNA strand-specific errors after visual examination of called mutations.