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

This study profiled the somatic genes mutations and the copy number variations (CNVs) in cerebrospinal fluid (CSF)-circulating tumor DNA (ctDNA) from patients with neoplastic meningitis (NM). A total of 62 CSF ctDNA samples were collected from 58 NM patients for the next generation sequencing. The data were bioinformatically analyzed by (Database for Annotation, Visualization and Integrated Discovery) DAVID software. The most common mutated gene was TP53 (54/62; 87.10%), followed by EGFR (44/62; 70.97%), PTEN (39/62; 62.90%), CDKN2A (32/62; 51.61%), APC (27/62: 43.55%), TET2 (27/62; 43.55%), GNAQ (18/62; 29.03%), NOTCH1 (17/62; 27.42%), VHL (17/62; 27.42%), FLT3 (16/62; 25.81%), PTCH1 (15/62; 24.19%), BRCA2 (13/62; 20.97%), KDR (10/62; 16.13%), KIT (9/62; 14.52%), MLH1 (9/62; 14.52%), ATM (8/62; 12.90%), CBL (8/62; 12.90%), and DNMT3A (7/62; 11.29%). The mutated genes were enriched in the PI3K-Akt signaling pathway by the KEGG pathway analysis. Furthermore, the CNVs of these genes were also identified in these 62 samples. The mutated genes in CSF samples receiving intrathecal chemotherapy and systemic therapy were enriched in the ERK1/2 signaling pathway. This study identified genes mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.


Background
Neoplastic meningitis (NM) refers to the dissemination of malignant cells to the leptomeninges, and is associated with very poor survival of patients [1]. The primary cancers are mostly lung and breast cancers or brain tumors, such as medulloblastoma. In clinic, early NM detection and timely treatment could significantly impact the outcome of patients. However, the present diagnosis is primarily based on the clinical signs and symptoms plus cerebrospinal fluid (CSF) cytology and neuroimaging [2]. Furthermore, although the detection of tumor cells in the CSF is the key to make NM diagnosis, the CSF cytology may not be reliable due to insufficient sensitivity and specificity. Thus, the detection of cell-free circulating tumor DNA (ctDNA) could be another diagnostic strategy [3][4][5]. This can detect cancer-associated genes and gene alterations in the plasma or CSF to monitor the tumor progression and/or treatment responses [3][4][5]. In patients with brain tumor, the plasma ctDNA analysis has revealed either its absence, or extremely low levels [5,6]. Fortunately, CSF ctDNA has been well demonstrated to be present and even abundant in brain tumor patients [6,7]. In order to better understand characterization of ctDNA in CSF of NM patients, the detection of mutated genes in the CSF could help medical oncologists identify the primary tumor and make effective treatment options for patients. Therefore, the present study aimed to investigate the gene mutations in the CSF ctDNA samples obtained from NM patients using the cutting-edge technique of next generation sequencing (NGS). This approach can help to characterize NM genetic profiles and profile of gene mutations, which can thereby be potentially applied for molecularly targeted therapy. Towards this end, a recent study [8] genetically profiled the CSF ctDNA obtained from NM patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). Therefore, characterization of gene mutations in CSF ctDNA samples can provide valuable clinical guidance for precision medicine.

Study population and samples
In the present study, a total of 58 patients with NM, who received lumbar puncture for the CSF cytology examination between October 2014 and June 2018 in the Department of Neurology, The Second Hospital of Hebei Medical University (Hebei, China), were enrolled. The diagnosis of these 58 NM patients was entirely according to the clinical signs and symptoms, positive CSF cytology, and/or neuroimaging findings, such as contrast-enhanced brain magnetic resonance imaging (MRI) or computed tomography (CT), and the results from the CSF ctDNA. The NM clinical signs and symptoms included headache, nausea, vomiting, convulsion, lower back pain, cranial nerve paralysis, paresthesia, gait disturbances, vertigo and defects. The positive CSF cytology was defined by the distinctive pattern of the neoplastic cell morphology. That is, cells that had an irregular size and shape, and contained large and polymorphic nuclei with a lobulated state and malformed buds. The chromatin size increased with the basophilic coarse particles, and the mitotic activity was enhanced with aberrant mitosis. The nuclear membrane was usually thickened with a saw-tooth-shaped and wear edge. In addition, the positive neuroimaging revealed the presence of leptomeningeal enhancement. The clinical data are presented in Table 1.
The present study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (Hebei, China), and a written informed consent was obtained from each patient or their legal surrogates.

CSF cytology examination
About 0.3 ml CSF was centrifuged at 750 r/min for 4 min (Therm-4, Shandon Cytospin, US). After naturally drying on the slide, the deposit was dyed with May-Grünwald-Giemsa liquid (Yucai, Beijing, China) and Alcian blue staining (Yucai, Beijing, China), according to the manufacturer's protocol, and observed under a light microscope (OLYMPUS-BX41, Japan). The determination of the positive result was as follows: cells that had an irregular size and shape, and contained large and polymorphic nuclei with a lobulated state and malformed buds. The chromatin size increased with the basophilic coarse particles, and the mitotic activity was enhanced with aberrant mitosis. The nuclear membrane was usually thickened with a saw-tooth-shaped and wear edge. Analysis was done using a cell medical image analysis system (MCDS-20, Chongqing, China).
Next-generation sequencing CSF samples and processing The CSF samples were collected from each patient and placed into EDTA tubes, according to our hospital routine protocols. Then, these were centrifuged for five minutes at 1000 g. The pellet was stored at − 20°C, while the supernatant was centrifuged at 10, 000 g for an additional 30 min, according to a previous study [9]. The supernatant was transferred into pre-labeled cryotubes and stored at − 80°C. Next, the ctDNA was extracted from at least 5 mL of the CSF supernatant using a QIAamp Circulating Nucleic Acid kit (QIAGEN), according kit instructions, and the ctDNA was quantified using a Qubit2.1 Fluorometer and Qubit dsDNA HS Assay kit (Life Technologies, Carlsbad, CA, USA).
Preparation of the ctDNA library and the next generation DNA sequencing The ctDNA samples were subjected to preparation of the Ion Proton library and DNA sequencing, according to the methodologies from previous studies [10][11][12]. Briefly, for each sample, an adapter-ligated library was generated using the Ion AmpliSeq Library Kit 2.0 (Life Technologies). That is, the pooled amplicons made from 10 to 20 ng of ctDNA samples were endrepaired and ligated to Ion Adapters X and P1, and purified using AMPure beads (Beckman Coulter, Brea, USA) to obtain the adapter-ligated products, followed by nick-translation and PCR-amplification for a total of five cycles. Then, the products were subjected to analysis using the Agilent 2100 Bioanalyzer and Agilent Bioanalyzer DNA High-Sensitivity LabChip (Agilent Technologies) to determine the concentration and size of the library, and sample emulsion PCR and emulsion breaking using the Ion OneTouch™  (Life Technologies) was used for the sequencing reactions. Then, the SV-OCP143-ctDNA panel (San Valley Biotech Inc., Beijing, China) was used to detect the somatic mutations of 143 cancer-related genes. Since the CSF ctDNA samples contained short DNA fragments, the amplicons in the panel were specially designed for the efficient amplification of ctDNA, and the total read counts were more than 25 million to ensure an average base coverage depth over 10,000 folds. In addition, strict quality control criteria were used to ensure that the average uniformity of the base coverage is no less than 95.5% for the reliability of the DNA sequencing and mutation detection.

Processing and analysis of DNA sequencing data
The raw DNA sequencing data were processed and analyzed using the Ion Proton platform-specific pipeline (Torrent Suite v5.0) with a specific plug-in (Variant Caller v5.0), which included the readouts of the raw DNA sequences, the trimming of the adapter sequences, and the filtering and removal of poor signal sequences according to previous studies [10][11][12]. These three filtering steps were applied to eliminate the erroneous base calling, and the final variant calling was generated. That is, the first step evaluated the DNA sequences using the following criteria: the average total coverage depth is > 10,000, each variant coverage is > 10, the variant frequency for each sample is > 0.1%, and the P-value is < 0.01. The second step was to eliminate ant potential DNA strand-specific errors after visual examination of the gene mutations using the Integrative Genomics Viewer (IGV; http//www.broadinstitute.org/igv) or Samtools (http://samtools.sourceforge.net) software. For the third step, the total amplicon read counts from the Coverage Analysis Plugin were utilized to identify the copy number variants (CNVs). Then, the read counts per amplicon of each sample was normalized to the total number of reads for a given sample, and divided by normalized counts from a composite normal male genomic DNA sample, which yielded a copy number ratio after correcting for GC content. The gene-level copy number was estimated through the determination of the coverage-weighted mean of the GC-corrected per-probe ratio, which was corrected with the expected error, according to the probe-to-probe variance [13]. Afterwards, genes with a copy number of < 1 or > 4 were regarded as loss or gain, respectively.

EGFR mutations in lung cancer tissues
Information of EGFR mutations in lung cancer tissues were obtained from patient's medical record. Lung cancer tissues of most patients were sequenced before this study by various methods.

Functional annotation and pathway enrichment analysis
Next, the mutated genes were bioinformatically analyzed using the gene ontology (GO) terms [14] and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis with the Database for Annotation, Visualization, and Integrated Discovery (DAVID; v.6.8, https://david.ncifcrf.gov/tools.jsp). Then, the data were clasisified as the functional annotation and the KEGG pathway enrichment. A P-value of < 0.05 was set to be statistically significant.

Statistical analysis
The statistical significance in gene mutation frequency between the two groups was analyzed using Fisher exact test. Pearson correlation analysis was used for correlation analysis. Nonparametric test was used for two independent samples that did not meet the normal distribution. Statistical analyses were performed using SPSS version 19, and a two tailed P-value of < 0.05 was considered statistically significant.

Patient characteristics
Clinical characteristics are shown in Table 1. For NM patients with lung cancer, the majority of these patients had lung adenocarcinoma (27/42, 64.3%), while of 55 patients had known primary malignancies and 10 patients (18.2%) had NM as the first clinical manifestation. These patients were followed up for two month or longer.
A total of 62 CSF samples were collected from these 58 NM patients, in which three CSF samples were collected from a single patient, while two CSF samples were collected from other two patients at distinct time points. Furthermore, among the 62 CSF samples, 30 CSF samples were collected from 28 patients who received intrathecal chemotherapy and systemic radiotherapy, chemotherapy, and/or molecule-targeted therapy, 11 CSF samples were obtained from 11 patients who received intrathecal chemotherapy, and 12 CSF samples were obtained from 12 patients who received systemic therapy. The remaining nine CSF samples were collected from nine patients who did not receive any anticancer therapy.
Cancer-associated genes mutations in the 62 CSF specimens, regardless of the origin of the primary cancer and the mutated genes functional enrichment analysis The 62 CSF samples were all positive for ctDNA and mutations of cancer-associated genes. Specifically, 68 (47.6%) of the 143 cancer-associated genes analyzed in the present study had mutations in at least one NM CSF sample, and 62 (100%) of the NM CSF samples carried at least one mutated gene. The most commonly mutated gene was TP53 (54/62, 87.10%), followed by EGFR (44/  (Fig. 1). Furthermore, GO (Gene Ontology) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were used to explore the potential functions of these high frequency mutated genes, and it was found that these high frequency mutated genes were rich in the PI3K-Akt signaling pathway (Fig. 2).
Furthermore, the variant allele frequency was divided into five groups: ≥50, 30-50%, 10-30%, 1-10% and 0.2-1%, respectively. Then, the variant allele frequency was associated with the detectable tumor cells in the CSF samples, and it was found that the frequency of variant allele frequency (≥1%) was higher in the group with detectable tumor cells than that in the group without detectable tumor cells (P < 0.001, Fig. 3).

Copy number variations (CNVs)
Data on the CNVs in these CSF ctDNA samples were also obtained, and it was found that high CNVs occurred in 22 of 58 NM patients. Among these 22 patients, the primary tumors were 15 lung cancers (13 lung adenocarcinoma, one squamous cell carcinoma and one unspecified), four gastric cancers, and one each of breast cancer, parotid carcinoma, and unknown primary cancer. The deletion of the CDKN2A copy number was the most frequent CNV that occurred in seven CSF ctDNA samples from six non-small cell lung cancers (6/22, 27.3%). In the increase in CDK4 copy number that occurred in five lung adenocarcinomas, four of these exhibited an increase in MDM2 copy number. In addition, an increase in MDM2 copy number was also detected from another lung adenocarcinoma patient. Two CSF ctDNA samples had a gain of ERBB2 (HER2) copy number from a parotid carcinoma patient, while an increase in CD44 copy number was identified in three patients, in which each patient has breast cancer, gastric cancer and unknown cancer, respectively. In addition, an increased EGFR copy number occurred in three lung adenocarcinoma patients. Other CNVs of tumor-associated genes were detected in five patients (six positive CSF ctDNA samples) with decreased AR copy numbers, five patients had decreased CD274 copy numbers, three patients each has a decreased PDCD1LG2 copy number, two patients each has an increase in FGFR2, CCNE1, or NKX2-1 copy numbers, respectively, and one patient had increased TIAF1, GAS6, or IL6 copy numbers, or reduced CSNK2A1, JAK2, MED12, or SMAD4 copy numbers.

Association of gene mutations with intrathecal chemotherapy and systemic therapy
The data on these unique mutated genes were summarized for each group of patients, followed by a geneannotation enrichment analysis. It was found that the ERK1/2 pathway was mostly enriched by the GO analysis in patients who received both intrathecal chemotherapy and systemic therapy ( Fig. 4 and Table 2).
The association of CSF ctDNA concentration with Karnofsky performance status (KPS) score, gene mutation and CSF tumor cells The CSF ctDNA concentration was not statistically associated with the KPS scores (r = − 0.038, P = 0.768;  Fig. 5b). Furthermore, the number of gene mutations was not associated with the KPS score (r = 0.192, P = 0.135; Fig. 5c). However, it was found that the CSF ctDNA concentration was associated with tumor cells in the CSF, when compared to that without circulating tumor cells (Z = -2.883, P = 0.004; Fig. 5d).
Cancer-associated genes mutations in the 45 CSF samples obtained from 42 NM patients with lung cancer A total of 45 CSF samples were collected from 42 NM patients with lung cancer, in which three CSF samples were collected from a single patient at distinct time   Table 3).
A number of gene mutations were statistically more common in the present cohort of NM, when compared to the lung cancer noted in the COSMIC database, and these genes were also further analyzed by GO annotation and KEGG pathway analyses (Fig. 7).

The association of EGFR mutations between lung cancer tissues and NM CSF samples
Next, EGFR mutations were associated between lung cancer tissues and the NM CSF samples available in 10 patients (Table 4). Specifically, CSF samples were collected from N033, N063, N077, N1088, N156, N331, N355 and N1286 during the TKI therapy, while N079 and N090 before the TKI. It was found that there were roughly the same EGFR mutations between lung adenocarcinoma tissues and CSF of nine patients, except for N1088, in which the EGFR mutation was undetectable in the CSF sample.

A representative case
In the present cohort, there was a lung adenocarcinoma patient who underwent surgical lung cancer resection, and tumor tissues had an EGFR 19Del mutation detected by NGS. Thus, the patient orally received 125 mg of icotinib three times a day for six months and thereafter. However, the patient had a headache during the icotinib therapy for the primary tumor. The head contrast enhanced MRI showed the linear and strip abnormal enhancement of the cerebellar sulcus (Fig. 8a) after the patient's cancer spread into the leptomeninges, and the CSF cytology examination showed tumor cells in the CSF (Fig. 8b). Furthermore, the CSF sample revealed EGFR 19Del and T790M mutations in the CSF ctDNA by NGS technology. Given such a situation, the patient was given 80 mg of AZD9291 once a day to replace the icotinib for 18 months, and the patients overall health condition improved. Furthermore, a complete response was confirmed by the contrast-enhanced brain MRI (Fig. 8c), CSF cytology (Fig. 8d), and undetectable EGFR mutations in the CSF samples. The patient has been alive for nearly 3 years since the diagnosis of NM.

Discussion
In the present study, NM patients were enrolled for the detection of CSF ctDNA, gene mutations and copy number variations. Furthermore, the present cohort of NM patients revealed that the large majority of primary cancers was lung adenocarcinoma, and 10 patients had NM as the first clinical manifestation, although seven of these 10 patients were clarified for their primary tumor. Afterwards, 62 CSF samples were acquired from 58 NM patients, and all samples contained detectable ctDNA, indicating that detection of CSF ctDNA is a sensitive biomarker for NM patients, since the ctDNA may not originate from benign tumors and non-neoplastic conditions, according to previous studies [6,7]. Indeed, a previous study of ctDNA in 640 patients with different cancers [15] revealed that plasma ctDNA can be detected in at least 75% of patients vs. less than 50% patients with brain tumors, such as glioma, suggesting that CSF ctDNA can be an alternative source of samples for brain tumor diagnosis, since the present data detected positive CSF ctDNA in all 62 CSF specimens. However, it may also be observed that all patients in the present study had NM, and tumor cells in NM can disseminate over the leptomeningeal surface, followed by neoplastic cell shedding into the CSF. Thus, it needs to be further determined whether the CSF could be used to detect early stage brain tumors. However, it is true that the CSF can be a best source to detect ctDNA in NM patients. Previous studies have also reported that all 26 patients [8] and three patients [6] had positive ctDNA in the CSF samples, and the present study further supports these previous studies. In addition, the present study further demonstrated that CSF ctDNA is a useful resource to analyze gene mutations, which can help medical oncologists identify primary tumors that can cause NM. It was found that the mutations of cancer-associated genes occurred in all 62 CSF ctDNA samples, with the highest frequency on TP53 (54/62, 87.  samples; (4) the PI3K-Akt and ERK1/2 signaling pathways are the most altered signaling pathways for these mutated genes; (5) novel gene mutations are induced by intrathecal chemotherapy and systemic therapy in NM patients; (6) lung cancer (especially lung adenocarcinoma) is the major primary tumor in the present cohort of NM patients. Future studies would investigate the usefulness of the CSF and ctDNA for the early detection of NM patients, and target these mutated genes for the therapy of NM patients or even patients with these primary tumors. Indeed, the PI3K-Akt signaling pathway, including but is not limited to TP53, EGFR, PTEN, KIT and KDR, could be crucial or at least partially crucial in mediating primary cancer for meningeal metastasis. In particular, numerous isoforms and/or spliced variants of PI3Ks participate in the regulation of various cell processes, such as cell cycle progression, cell polarization, migration, survival and metabolism, as well as tumor angiogenesis [16]. Furthermore, Akt is amenable to the vast majority of PI3K-mediated responses [17], and the alterations of Akt upstream regulators, elevated Akt expression, and/or Akt activation all result in the promotion of tumor metastasis [18]. For example, activated PI3K-Akt signaling could stimulate the translocation of α-actinin-4 from the nucleus to the cytoplasm and plasma membrane, which in turn induce changes in cell morphology and motility [19]. In human carcinogenesis, the PI3K-Akt signaling pathway inhibited the expression of tumor suppressor gene E-cadherin, which led to tumor cell epithelial mesenchymal transition and metastasis [20][21][22]. Previous studies have revealed that the PI3K-Akt signaling pathway plays a crucial role in the progression and metastasis of lung cancer [23], ovarian cancer [18], nasopharyngeal carcinoma [24], prostate cancer [25], colorectal cancer [26], and gastric cancer [27]. The present study further supports and confirms the important role of the PI3K-Akt signaling pathway in NM patients, which is novel, and to date, there has been no report in the literature. Hence, further studies are needed to verify the importance of this signaling pathway in NM. Furthermore, in the present study, ERK1/2 signaling was found to be enriched in  NM patients after intrathecal chemotherapy and systemic therapy, indicating that the change in the ERK1/2 signaling could be associated with treatment resistance. Indeed, the inhibition of the ERK1/2 signaling pathway was caused by the Dioscorea bulbifera-induced apoptosis in human colorectal carcinoma cells [28]. Conversely, the GABAergic signaling facilitated breast cancer metastasis through promotion of the ERK1/2-dependent phosphorylation [29]. This speculation needs to be further studied to determine whether this is associated with treatment resistance to both intrathecal chemotherapy and systemic therapy.
In addition, the present study also identified the CNVs of different genes in the CSF ctDNA samples, and the most affected ones were CDKN2A, CDK4 and MDM2. As it is known, the deletion of tumor suppressor CDKN2A was associated with melanoma and pancreatic neuroendocrine tumors metastasis, and the reduced survival rate of patients [30,31]. Indeed, during the DNA replication in cells, gene amplification could generally create a risk for gene overexpression, which could be involved in cancer initiation and progression [32]. Furthermore, a previous study revealed that CDK4 and MDM2 mutations occurred in melanomas and liposarcoma, while ERBB2 mutations occurred in breast cancer, EGFR mutations occurred in astrocytoma, and MYCN mutations occurred in neuroblastoma [32]. Altered CD44 expression was associated with the aggressive clinicopathological characteristics of various human cancers [33]. In addition, the present study revealed the EGFR and TP53 mutations in the CSF samples of NM patients with lung cancer, in which the COSMIC database also confirmed that these were the most frequently mutated genes in the CSF of NM patients, when compared to primary lung cancer. Furthermore, a higher mutational rate was found from EGFR , TP53, PTEN,  TET2, APC, CDKN2A, GNAQ, NOTCH1, FLT3, VHL,  BRCA2, PTCH1, CBL, MLH1, BRAF, NRAS, TSC2,  CSF1R, KIT, MAP2K1, MSH2, TSC1, HRAS, IFITM1 and BCL9 genes in NM with lung cancer, when compared to lung cancer patients without NM. A previous study revealed that EGFR mutations predisposed to the leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer [34], and the present study further supports this notion. In spite of the relatively small sample size, the rate of the above mutated genes was much higher than what was reported in patients with lung adenocarcinoma from the COSMIC database, implicating that these genes mutations and the alteration of the signaling pathways are involved in and have become risk factors for NM.
After associating these EGFR activating mutations between primary lung cancer and NM, the present data shows that the EGFR activating mutations in the CSF samples were roughly consistent with those of primary lung cancer. For example, the T790M in two CSF samples were sequentially collected during the TKI therapy.  Lastly, the representative patient in the present study provided a unique showcase. The genetic profiling of the CSF ctDNA clearly reflected the dynamic changes in these identified driver genes and treatment responses. That is, this patient was detected to have the EGFR 19Del mutation in the CSF sample, and thereby received icotinib. Thereafter, the CSF sample revealed the EGFR 19Del and T790M mutations. Hence, the treatment was switched to AZD9291, which is a third generation EGFR TKI agent. A high response rate was exhibited by patients with tumors harboring the EGFR T790M mutation, as well as a high capacity to penetrate into the CSF by crossing the blood-brain barrier [36,37]. The NGS allowed the physician to modify treatment option, in order to help the patient archive complete remission. As it is known, the EGFR T790M mutation mediates the acquired resistance to EGFR TKI [38,39], and a previous study reported that AZD9291 was designated as a powerful agent capable of overcoming the acquired EGFR T790M resistance mutation [40]. Fig. 7 The data on the GO analysis (a) and KEGG pathway analysis (b) of significant mutated genes in the CSF obtained from NM patients with lung cancer compared to mutated genes of lung cancer in COSMIC database However, the present study does have some limitations. For example, the cohort has a relatively small number of patients, and the different treatment options could not be separated to analyze the association of gene mutation, and the changes in gene copy number with treatment responses in these patients. Thus, future studies with a larger sample size from multiple institutions could help to solve these issues. Though we have found that mutated genes detected in CSF are enriched in the PI3K-Akt and ERK1/2 signaling pathways, it is hard for us to explain the detected mutations are gain of function mutations or loss of function mutations in such pathways. Thus, we will perform the functional analysis in the following studies. Due to the target amplicon sequencing, we could not address the whole genome alterations and yield the comprehensive landscape of NM. So there are any other area we will focus on to make more depth analyses in the future studies.

Conclusions
This study identified gene mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.