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

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 [3‐5]. This can detect cancer-associated genes and gene alterations in the plasma or CSF to monitor the tumor progression and/or treatment responses [3‐5]. 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.

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, 87.10%), EGFR (44/62, 70.97%), PTEN (39/62, 62.90%), CDKN2A (32/62, 51.61%), APC (27/62, 43.55%), and TET2 (27/62, 43.55%). These mutated genes enriched by the KEGG pathway analysis was the PI3K-Akt signaling pathway. The ERK1/2 signaling pathway was significantly activated in NM patients who received intrathecal chemotherapy and systemic therapy, indicating that intrathecal chemotherapy and systemic therapy might induce novel gene mutations in NM patients. The present study also identified the variation of gene copy numbers in these 62 samples. In conclusion, the data obtained from the present study demonstrates the following: (1) ctDNA is detectable in all CSF samples; (2) gene mutations are detectable in all CSF samples; (3) the gene copy number varies in all CSF 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 [20‐22]. 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. Furthermore, according to the gene set enrichment analysis, it was found that the ErbB, VEGF, MAPK, and m-TOR signaling pathways were significantly enriched in the 45 CSF samples obtained from NM patients with lung cancer, suggesting that the alterations of these signaling pathways might promote NM in lung cancer patients. In particular, the ErbB signaling pathway can regulate cell proliferation, migration, differentiation and apoptosis through the crosstalk with the PI3K-Akt, MAPK, or other signaling pathways [35].

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].

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.

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.