Edited by: Hailiang Tang, Huashan Hospital Affiliated to Fudan University, China
Reviewed by: Yujie Chen, First Affiliated Hospital of Army Medical University, China; Tianwen Li, Huashan Hospital, Fudan University, China
This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology
†These authors share first authorship
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Meningioma, which has an incidence of 6–7 in 100,000 people, has become the most common primary brain tumor, accounting for 36.3% of all primary central nervous system tumors (
However, due to the controversial results obtained between different studies, whether the ART is beneficial for the treatment of atypical meningioma (AM) patients, especially for those who underwent gross total resection (GTR, Simpson I–II), remains unclear (
During the past two decades, the WHO classification system was significantly revised in 2000 and updated in 2007 and then in 2016 (
In our institution, after surgical resection, we routinely recommend patients with AM to consult a radiotherapy specialist for further treatment. Interestingly, we found that even for those AM patients who underwent GTR and ART, there were still some differences in prognosis. Therefore, in order to investigate the correlation between radiotherapy sensitivity and miRNA expression, we conducted an extensive miRNA profiling study on tissue samples from postoperative radiotherapy-sensitive and radiotherapy-resistant AM patients who underwent GTR and ART in a single institution and searched for unique miRNA expression signatures that could distinguish radiotherapy-sensitive patients from radiotherapy-resistant patients.
All patients enrolled in the study signed an informed consent form for the current study, and the clinical study was approved by the Medical Ethics Committee of the Capital Medical University.
Patients diagnosed with AM from 2008 to 2015 were initially identified through the database of our Neurosurgery department at the Beijing Tiantan Hospital. The clinical history of the patients was gathered retrospectively by chart review. Fifty-nine AM patients who underwent gross total resection were identified and selected for further analysis. The operation notes and postoperative magnetic resonance images (MRIs) were reviewed to confirm the extent of the resection. Simpson I (macroscopically complete tumor resection with removal of affected dura and underlying bone)–II (macroscopically complete tumor resection with coagulation of affected dura only) was defined as GTR (
In recent studies, recurrence-free survival in 3 years has been a critical prognostic indicator to estimate the efficiency of radiotherapy for atypical meningioma. Since a prospective study confirmed GTR + ART could make PFS in 3 years >70% (
For every patient, immediately after surgery, tumor samples were fixed with formalin and embedded in paraffin blocks. A random selection was made to obtain 10 tumor samples for further miRNA microarray test, with five samples from the radioresistant group and five from the radiosensitive group.
miRNAs were extracted from formalin-fixed and paraffin-embedded tissues using the miRNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. The concentration and purity of the RNA were measured using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). The quality of the total RNA was accessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A total of 200 ng of small RNAs were labeled using the FlashTag biotin-HSR RNA labeling kit (Genisphere). First, poly(A) tailing was carried out at 37°C for 15 min in a volume of 15 μl of reaction mixture containing the reaction buffer, MnCl2, ATP, and poly(A) polymerase. Then, the Genisphere biotin complex was ligated at room temperature for 30 min by adding the FlashTag Ligation Mix Biotin and T4 DNA Ligase into the 15-μl reaction mix. The Stop Solution was then added to stop the reaction.
Subsequently, the microRNA cocktails were hybridized and analyzed on microRNAs microarrays version 2 or 3 (Affymetrix). Labeled RNAs were hybridized on GeneChip microarrays, washed, stained, and then scanned using the miRNA-2.0 library for microRNA microarrays version 2 and the miRNA-3.0 library for microRNA microarrays version 3, according to Affymetrix's specifications.
Analyses of clinical data were performed using the SPSS software (release version 21; IBM Corp., Armonk, NY, USA). The tumor size and Ki67 index between groups were compared by independent samples
For microRNA data, the signal intensity was loaded into the Rosetta Resolver System® (Rosetta Biosoftware, USA) for data preprocessing and application of the 75th percentile centering normalization. Simultaneously, the errors of the sample were estimated using the error-weighted approach. Both the fold change and
Fifty-five cases of AM were included in this study. For all cases, the last follow-up was in December 2018, with a median follow-up time of 57 months (range, 37–127). A summary of patient characteristics is shown in
Clinical characteristics of the atypical meningioma patients with adjuvant radiotherapy after gross total resection.
Gender (male/female) | 25/18 | 4/8 | 0.192 |
Median age at surgery | 52 | 52 | 0.673 |
Tumor location | |||
Supratentorial (yes/no) | 27/16 | 7/5 | 0.779 |
Convexity (%) | 15 (34.9%) | 3 (25.0%) | 0.519 |
Falx/parasagittal (%) | 10 (23.2%) | 2 (16.7%) | 0.625 |
Cranial base (%) | 8 (18.6%) | 3 (25.0%) | 0.624 |
Lateral ventricle trigone area (%) | 2 (4.7%) | 2 (16.7%) | 0.204 |
Posterior fossa (%) | 8 (18.6%) | 2 (16.7%) | 0.878 |
Median tumor size (cm) | 5.00 | 5.95 | 0.265 |
PTBE (with/without) | 11/32 | 6/6 | 0.158 |
Mean Ki 67 index | 8.4% | 11.5% | 0.343 |
Death by the last follow-up | 2 | 1 | 0.117 |
Median RFS (months) | 58 | 28.5 | <0.001 |
Median follow-up (months) | 57 (36–127) |
In this study, tumor samples from 10 patients were selected for miRNA microarray. Five of them suffered tumor recurrence <3 years (36 months) after total resection, which was considered as radioresistant, while the others who did not exhibit tumor recurrence during the follow-up time (>36 months) were considered as radiosensitive. The clinical characteristics of these 10 patients are shown in
Clinical characteristics of patients whose tumor was detected by microRNA (miRNA) microarray.
C1 | Female | 33 | Convexity | 4.5 | Yes | 1 | No |
C2 | Female | 53 | Convexity | 5.0 | Yes | 1 | Endometrial polyp with resection |
C3 | Female | 54 | Posterior fossa | 4.8 | No | 20 | Ovarian cyst with resection |
C4 | Male | 18 | Cranial base | 3.1 | Yes | 5 | No |
C5 | Female | 47 | Lateral ventricle trigone area | 4.9 | No | 3 | Uterine fibroids |
T1 | Female | 64 | Convexity | 4.4 | Yes | 15 | Hypertension for 5 years |
T2 | Female | 34 | Lateral ventricle trigone area | 5.9 | No | 5 | No |
T3 | Male | 24 | Posterior fossa | 2.7 | No | 1 | No |
T4 | Female | 44 | Cranial base | 4.1 | Yes | 30 | Chronic superficial gastritis |
T5 | Male | 58 | Convexity | 6.0 | Yes | 1 | Inguinal hernia with repair |
1.0 | 0.695 | 1.0 | 0.828 | 1.0 | 0.526 |
Differential expression microRNAs (miRNAs) in the radiosensitive and radioresistant groups of atypical meningioma with gross total resection plus adjuvant radiotherapy (GTR + ART).
Clustering analysis of the radioresistant and radiosensitive groups using differentially expressed miRNAs. The columns represent the cases, and the lines represent the miRNAs. Red and green indicate high and low expression levels, respectively. Cluster 1: five radiosensitive and one radioresistant; cluster 2: radioresistant.
Results from the DIANA-miRPath v3.0 predictions of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways according to the differential expression microRNAs (miRNAs) between radioresistant and radiosensitive groups.
Metabolism | Fatty acid biosynthesis (hsa00061) | <1 × 10–325 |
Fatty acid metabolism (hsa01212) | 4.21 × 10–06 | |
Environmental information processing | TGF-beta signaling pathway (hsa04350) | 0.040294 |
The use of ART for the treatment of AM after GTR has remained controversial. Maybe the single institution and relatively small study scale could be a partial reason for this contradiction. However, recently, two large-scale studies have not managed to reach a consensus. Wang C. et al. showed that ART is not associated with improved overall survival (OS) in patients who underwent GTR [adjusted hazard ratio (HR) = 1.093,
As we all know, in order to make individual treatment decisions, clinicians should weigh and balance multiple factors at different levels. At the clinical level, this study failed to find a significant difference in the characteristics of patients with AM treated with GTR + ART. To explore the factors that exert more influence on these patients, especially to find those factors that contribute to radiosensitivity, we investigated differentially expressed miRNAs. Recent studies have revealed that differences in miRNA expression could influence radiosensitivity in a series of tumors, including, but not limited to, glioblastoma (GBM), breast cancer, lung cancer, melanoma, and cervical cancer (
Among the 14 deregulated miRNAs, miR-7977, miR-4286, miR-1275, and miR-30c-1-3p have been previously reported to play a role in tumor malignancy. Horiguchi H. et al. found that miR-7977 was upregulated in acute myeloid leukemia and myelodysplastic syndrome and could reduce the expression of poly(rC) binding protein 1 to interfere with normal hematopoiesis. Moreover, miR-7977 was also reported to regulate the Hippo-YAP pathway, therefore inducing the upregulation of leukemia-supporting stroma growth (
Finally, in order to investigate the molecular pathways affected by the differentially expressed miRNAs between radiosensitive and radioresistant AM, we used the DIANA-miRPath software and found three enriched pathways. The two most significant pathways were fatty acid biosynthesis (hsa00061) and metabolism (hsa01212): fatty acid biosynthesis, biosynthesis, and TGF-β signaling pathways. These pathways have been verified relate to some common chronic disorders such as chronic inflammation, hypertension, and hyperlipidemia (
The miRNA deregulation pattern discovered in this study could help to define radioresistant AM patients properly; this is important for follow-up treatment. On the one hand, AM patients who are radioresistant and vulnerable to radiation-induced injury could choose observation after GTR. On the other hand, these radioresistant patients are more worth trying radiosensitizer to improve the effect of radiotherapy. The use of miRNA as a kind of treatment method is quite far from clinical practice, but there are still a number of drugs to improve radiosensitivity. For now, several clinical trials about radiosensitizers have been done (
Our study presents some limitations: the inherent limitation of a retrospective analysis, relatively low number of cases due to the rarity of this kind of tumor, the decision to undergo postoperative ART at the discretion of surgeons rather than objective parameters, and the small number of microRNA samples. However, the present study includes a significant follow-up, and all cases are from a single institution, which avoids the “interinstitutional” diagnostic and therapeutic discrepancies.
In summary, we found 14 differently expressed miRNAs in radiotherapy-sensitive and radiotherapy-resistant AM patients. These miRNAs may be used as candidate predictive markers for the benefit of radiotherapy in AM. Should these results be confirmed in future prospective randomized trials, the miRNA signatures may be used to identify AM patients who may not respond well to adjuvant radiotherapy and may, therefore, benefit from the addition of radiosensitizers or immunotherapy to enhance the radiation response. As such, applying the potential roles of miRNAs in individualized radiotherapy may lead to novel trends in AM therapeutic options.
The datasets (GENERATED) for this study can be found in the [Gene Expression Omnibus (GEO)] (
The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Capital Medical University. The patients/participants provided their written informed consent to participate in this study.
All authors listed have made a substantial, direct, and intellectual contribution to the work. The idea came from the discussion of SL and YW. Material preparation, data collection, and analysis were performed by XZ, GZ, and HL. The first draft of the manuscript was written by HH and XZ and reviewed by GZ and YW. All authors commented on the manuscript and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank all the patients who trusted them and all the physicians and staff who helped them in this study.