Med Oncol (2012) 29:1272–1279 DOI 10.1007/s12032-011-9835-x
ORIGINAL PAPER
Prognostic significance of kappaB-Ras1 expression in gliomas Hong Lin • YanGang Wang • Xiang Zhang BoLin Liu • Wei Zhang • JinXiang Cheng
•
Received: 17 September 2010 / Accepted: 17 January 2011 / Published online: 8 February 2011 Ó Springer Science+Business Media, LLC 2011
Abstract Nuclear factor (NF)-kappa-B is a pleiotropic transcriptional regulator that plays important roles in cell differentiation, growth, tumorigenesis, and apoptosis. Constitutive NF-kappa-B is overexpressed and activated in various tumors, including gliomas. Here, we investigated the expression of NF-kappa-B inhibitor interacting ras-like protein 1 (jB-Ras1), which is one of the most important negative modulators of NF-kappa-B, and a well-known proliferation biomarker survivin protein. We performed immunohistochemistry and western blot analysis on 154 glioma specimens and 3 non-neoplastic brain parenchyma specimens. Immunohistochemistry showed a strong-toweak range of jB-Ras1 staining with increasing pathologic grade of glioma (P = 0.000). Immunoreactivity scores of jB-Ras1 were 8.15 ± 0.72 in non-neoplastic brain parenchyma, 5.00 ± 0.29 in low-grade gliomas, 3.89 ± 0.30 in anaplasia astrocytomas, and 2.78 ± 0.25 in glioblastomas. In contrast, the immunoreactivity of survivin increased with pathological grade in gliomas. The immunohistochemical data were in line with the results from western blot analysis. Moreover, a non-parametric analysis revealed that the attenuated jB-Ras1 expression was correlated with elevated survivin expression, large tumor diameter, frequent intra-tumor necrosis, and worse overall survival. These results indicated that jB-Ras1 was downregulated in gliomas compared to non-neoplastic brain The authors Hong Lin and YanGang Wang contribute equally to this work. H. Lin Y. Wang X. Zhang (&) B. Liu W. Zhang J. Cheng Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, No.17 Changle Western Road, 710032 Xi’an, China e-mail:
[email protected]
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parenchyma, and the expression was even lower in glioblastomas. In addition, multivariate analysis showed that jB-Ras1 expression and intra-tumor necrosis were two important prognostic factors identified by the Cox proportional hazards model. Taken together, our study suggests that glioma patients with lower jB-Ras1 expression have a worse prognosis, which is partly due to NF-kappa-B pathway-mediated aberrant proliferation of tumor cells. Keywords NF-kappa-B jB-Ras1 Survivin Glioma Tumorigenesis Abbreviations jB-Ras1 Nuclear factor kappa-B inhibitor interacting Ras-like protein 1 NF-kappa-B Nuclear factor kappa-B IRS Immunoreactivity score SI Staining intensity PP Percentage of positive cell
Introduction Gliomas account for about 50–60% of intracranial neoplasms [1, 2], with glioblastomas (WHO IV) being the most malignant entity [3]. Although therapeutic strategies have been greatly improved, the prognosis of glioma patients remains depressingly poor [4–6]. Despite receiving selective tumor resection and ordinary chemotherapy, more than 70% low-grade glioma patients suffer from tumor recurrence and malignant progression that require additional resection [7]. Early diagnosis and clinical intervention are of the utmost importance in the treatment of glioma. Patients with young
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age, low malignancy grade, and wise management decision achieve long-term benefits such as longer life expectancies [8]. Therefore, there is a need to further study the molecular mechanisms of glioma and identify the specific proteins that cause tumor progression and predict prognosis. Indeed, molecular therapies targeting at these tumor-supportive proteins have been widely investigated. The eukaryotic transcription factor nuclear factor kappaB (NF-kappa-B) exhibits its transcriptional effect on the intracellular signaling proteins by binding to the targeting DNA through specific DNA sequences [9]. NF-kappa-B complexes regulate multiple cellular processes such as inhibition of apoptosis, promotion of cell growth tumorigenesis and neoplastic angiogenesis in pancreatic cancer [10], oral squamous cell carcinoma [11], colorectal cancer [12], and breast cancer [13]. NF-kappa-B is sequestered through its interactions with the kappa-B inhibitor proteins (IjB) [9]. Seven IjBs have been identified as follows: IjB-alpha, IjB-beta, IjB-gamma, IjB-epsilon, Bcl-3, p100, and p105. IjB-beta (IjBb) is a dominant inhibitor of NF-kappa-B that works by complexing with and trapping NF-kappa-B in the cytoplasm. The unphosphorylated status of IjBb is critical for its steady state and function. However, the signals stimulating NF-kappa-B activation could precedingly cause its phosphorylation, disassociation with NF-kappa-B, and subsequent degradation [14]. NF-kappaB inhibitor interacting Ras-like protein 1 (jB-Ras1) is a potent regulator that prevents the stimulus-dependent phosphorylation and degradation of IjBb, thus maintaining the inactive and cytoplasm-localized NF-kappa-B [15–18]. We hypothesize that reduced expression of jB-Ras1 may lead to the development of gliomas. To test this hypothesis, we investigated the endogenous expression of the jB-Ras1 protein in glioma samples of different pathological grades. Furthermore, we analyzed the correlation between jB-Ras1 expression and several clinical parameters including overall survival. Our study aimed at determining whether there were correlations between jB-Ras1 expression and the outcome of glioma patients, and to improve the understanding of bioactivity and prognostic indication of the jB-Ras1 in gliomas.
Materials and methods
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(n = 3), which were used as controls in this study, were obtained from chronic epilepsy patients who underwent surgery. All tumor tissues were obtained from the initial surgery for glioma. Samples were collected from 154 patients (88 men and 66 women; median age, 43 years; age range, 14–69 years) and included 44 low-grade gliomas [23 pilocytic astrocytomas (WHO I) and 21 diffuse astrocytomas (WHO II)], 48 anaplasia astrocytomas (WHO III), and 62 primary glioblastomas (WHO IV). The treatment strategies of all the patients were indicated in Table 1. Four years follow-up was performed, and all patients had complete follow-up until death. Immunohistochemistry Each tissue sample was consecutively cut into six slices (4-lm thick), so that both jB-Ras1 and survivin expression were detected on three consecutive sections. Then, sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and blocked with blocking solution. The mouse anti-human jB-Ras1 monoclonal antibody and the goat anti-human survivin polyclonal antibody c-19 (both purchased from Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in 0.1 M phosphatebuffered solution (PBS, 1:200) and applied onto the sections. The sections were then incubated with biotinylated anti-mouse and anti-goat IgG antibodies (Sigma, St. Louis, MO) that were diluted in 0.1 M PBS (1:500) and incubated with an ABC kit (Sigma, St. Louis, MO). Evaluation of jB-Ras1 and survivin immunoreactivity Two pathologists who were blind to the original clinical and histological diagnoses interpreted the slides. For each slide, 10 high-power (4009) fields were randomly picked for quantification. Immunoreactivity scores (IRS) of jB-Ras1 and survivin proteins (IRS: negative 0; weak 1–3; moderate to strong 4–12) were determined by multiplication of the values for their staining intensity (SI) (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentages of positive tumor cells (PP) (0, \1%; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, [75%). Because of the heterogeneous staining intensity of tumor cells, SI was determined according to the staining intensity of most cells.
Patients Western blot analysis All tissue specimens were obtained according to institutional review board-approved procedures for consent. All of the archival paraffin blocks and frozen tissue specimens, as well as their clinical data, were provided by the specimen bank of Xijing Institute of Clinical Neuroscience between 2000 and 2005. Non-neoplastic brain parenchyma
Eighty-nine frozen specimens (3 non-neoplastic brain parenchymas, 7 pilocytic astrocytomas, 15 diffuse astrocytomas, 16 anaplasia astrocytomas, and 48 glioblastomas) were obtained and then were homogenized and lysed with RIPA lysis buffer. Protein concentration was determined with the micro-BCA
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Table 1 Patient demographics for the human glioma samples WHO I
WHO II
WHO III
WHO IV
All tumors
Cases
23
21
48
62
154
Mean age (year)
36.4
46.2
42.5
44.8
43
Age range (year)
25-56
32-46
28-69
14-54
14-69
Male
15
8
29
36
88
Female
8
13
19
26
66
Gender
KPS score [70
18
15
12
17
62
\70
5
6
36
45
92
Gross total resection Partial resection
23 0
21 0
34 12
40 14
124 20
Biopsy
0
0
2
8
10
RT alone
0
1
28
15
44
CT alone
0
2
0
6
8
RT and CT combination
0
0
6
25
31
Pilocytic astrocytoma
Diffuse astrocytoma
Anaplasia astrocytoma
Glioblastoma
–
Surgery
Adjuvant treatment
Pathological classification
RT radiotherapy, CT chemotherapy
protein assay (Pierce, Rockford, IL). Tissue protein lysates (20 lg) were resolved by electrophoresis on a 12% SDS– PAGE gel. The proteins were then transferred onto a nitrocellulose membrane, and non-specific binding was blocked by incubating with 5% non-fat milk in Tris Buffer Saline Tween20 buffer (TBST) at room temperature for one hour. The anti-jB-Ras1 antibody (1:600), anti-survivin antibody (1:600), and anti-b-actin antibody (1:400, mouse monoclonal C-2, Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in TBST with 5% non-fat dry milk and incubated with the membranes for 1.5 h at room temperature or 4°C overnight. The antibodies were probed with HRP-anti-goat IgG (1:5,000, Santa Cruz, CA) and HRP-anti-mouse IgG (1:5,000, Santa Cruz, CA) secondary antibodies. Proteins were finally developed by an ECL system (Cell Signaling Technology, Beverly, MA, USA). The grayscale values of each band on the blots were measured using BandScan4.3. Statistical analysis To clarify the prognostic significance of the intracellular expression of jB-Ras1 protein for the management of gliomas, its correlations with the following clinical indices were assessed with SPSS 13.0 software: age (1, B43 years old; 2, [43 years old) and gender (1, male; 2, female) of patients, cell proliferation (evidenced by the largest diameter of the tumor measured when first operation: 1,\6 cm; 2, C6 cm), neoplastic vascular apoptosis and involution (evidenced by the intra-tumor necrosis: 0, no necrosis; 1, necrosis), and overall survival (OS, calculated from the date of the
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diagnosis to death from glioma). Because it was difficult for us to confirm a normal distribution for the clinical indices and because most of indices were translated into ranked data, we used non-parametric Spearman’s rank correlation for the statistical analysis. In addition, statistical differences between the levels of jB-Ras1 expression in pathological grades were evaluated by the non-parametric Kruskal– Wallis test, and those differences in the bi-nominal clinical categories were evaluated by the non-parametric Mann– Whitney test. A life table was calculated according to the Kaplan–Meier method. Hazard ratios for the time-to-eventendpoint were estimated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of multiple independent prognostic factors on survival outcome [age, gender, Karnofsky performance status, largest tumor diameter, extent of resection, type of adjuvant treatment, intra-tumor necrosis, and expression of jB-Ras1 (1, IRS C 4; 2, IRS B 3) and survivin (1, IRS C 3; 2, IRS B 2)]. Data are expressed as the mean ± the standard error of mean (SEM) of separate experiments.
Results jB-Ras1 protein expression is reduced in glioma specimens We investigated the endogenous jB-Ras1 protein expression in normal brain tissue and a series of glioma specimens. Immunoreactivity of intracellular jB-Ras1 was
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evaluated in 154 gliomas, of which 59.7% (92/154) had weak staining and 40.3% (62/154) had moderate to strong staining. Immunoreactivity was also measured in 3 nonneoplastic brain parenchyma samples, all of which had strong staining. Reduced expression of jB-Ras1 protein with cytoplasmic localization was observed in gliomas of different pathological grades when compared to normal brain tissue (Fig. 1). The IRS of jB-Ras1 was 8.15 ± 0.72 in non-neoplastic brain parenchyma, 6.24 ± 0.16 in Pilocytic astrocytomas (WHO I), 4.65 ± 0.24 in diffuse astrocytomas (WHO II), 3.89 ± 0.30 in anaplasia astrocytomas (WHO III), and 2.78 ± 0.25 in glioblastomas (WHO IV). The median IRS value of jB-Ras1 in all the
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glioma samples was 3.57 ± 0.48. Based on these results, we determined that gliomas with an IRS \ 4 were more malignant and divided the glioma specimens into two categories (high score, IRS C 4; low score, IRS B 3) for the survival analysis. Consistent with the IRS results, we found that the jB-Ras1 expression level was gradually decreased with tumor grades as assessed by western blot analysis (Fig. 2). The relative level of jB-Ras1 was calculated as the ratio of the densitometric value of the jB-Ras1 band to the b-actin band. The jB-Ras1/b-actin ratio was 105.4 ± 2.1% in nonneoplastic brain parenchyma specimens and decreased in gliomas with increasing pathological grade. The jB-Ras1/ b-actin ratio was 64.4 ± 2.8%, 27.8 ± 3.1, 26.5 ± 2.6%, and 11.3 ± 1.5% in pilocytic astrocytomas, diffuse astrocytomas, anaplasia astrocytomas, and glioblastomas, respectively. Increased expression of survivin protein in glioma specimens Endogenous survivin protein expression was also detected in the same specimens. In contrast to the decreasing jB-Ras1, immunohistochemistry and western blot analysis indicated that survivin expression increased with pathological grade. The IRS of survivin was 0.15 ± 0.03, 0.54 ± 0.11, 0.66 ± 0.08, 2.56 ± 0.17, and 4.78 ± 0.26 in non-neoplastic brain parenchyma, pilocytic astrocytomas, diffuse astrocytomas, anaplasia astrocytomas, and glioblastomas, respectively. The median IRS value of survivin in all the glioma samples was 3.02 ± 0.48. The survivin/b-actin ratio was 42.3 ± 3.1%, 42.5 ± 4.6%, 85.2 ± 6.1%, 94.2 ± 3.2%, and 98.8% ± 6.3% in nonneoplastic brain parenchyma, pilocytic astrocytomas, diffuse astrocytomas, anaplasia astrocytomas, and glioblastomas, respectively. The expression of jB-Ras1 and survivin proteins as determined by immunohistochemistry and western blot analysis is shown in Fig. 1 and 2, respectively. Correlation of jB-Ras1 expression with clinical parameters
Fig. 1 jB-Ras1 and survivin protein expression as investigated by immunohistochemistry (9400). Brown staining identified positive jB-Ras1 or survivin staining and blue staining by hematoxylin counterstain represented cell nuclei. jB-Ras1 expression was observed in glioma specimens and non-neoplastic brain parenchyma and decreased with their pathological grade, as shown in the upper row. Survivin expression increased with their pathological grade, as shown in the lower row. Histograms for IRS values of jB-Ras1 and survivin protein were provided closely to their Immunohistochemistry images, respectively
jB-Ras1 expression was significantly lower in gliomas than in normal brain parenchyma and was much more lower in more malignant gliomas than in less malignant gliomas (P \ 0.01). jB-Ras1 expression was inversely correlated with survivin expression (r = -0.611, P \ 0.01), large tumor diameter (r = -0.436, P \ 0.01), and frequent intra-tumorous necrosis (r = 0.494, P \ 0.01) in gliomas. Spearman’s rank correlation analysis did not show a statistically significant correlation between jB-Ras1 and patient gender (P = 0.666). In addition, no statistically
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Discussion
Fig. 2 jB-Ras1 and survivin protein expression as investigated by western blot analysis. a Western blot analysis, b-actin was used as loading control. b Statistical analysis
significant correlation was found between jB-Ras1, and age at diagnosis when 43 (median age of selected patients) was used to distinguish young and old patients (P = 0.369). Correlation of jB-Ras1 expression with overall survival Spearman’s rank correlation analysis showed that jB-Ras1 expression was significantly correlated with the overall survival of glioma patients (r = 0.756, P \ 0.01). In addition, the Kruskal–Wallis test indicated that patients with IRS C4 for jB-Ras1 had longer overall survival than those patients with IRS B3 of jB-Ras1 (19.4 ± 1.2 months vs. 11.3 ± 0.3 months, respectively, P \ 0.01). Moreover, the one-year survival was 48.5% in the low IRS score category compared with 78.9% in the high IRS score category. Survival curves for the two categories according to their pathological grades are shown in Fig. 3. Multivariate analysis Cox stepwise proportional hazards model involving the IRS score of jB-Ras1 and survivin expression and seven clinical parameters identified two prognostic variables including intra-tumor necrosis (P = 0.044) and jB-Ras1 expression (P = 0.032). Statistical values of the expression of jB-Ras1 and survivin and seven clinical parameters derived from Cox stepwise proportional hazards model were indicated in Table 2.
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Several studies have presented evidence that overstimulated survival signaling is one of the key factors in the development and progression of gliomas [19–21]. Hyperproliferation that is induced by aberrant survival signaling and apoptosis regulation has also been investigated in gliomas [22, 23]. The transcription factor NF-jB regulates many genes that are involved in the immune response, cell adhesion, differentiation, proliferation, angiogenesis, and apoptosis [9, 24] Overexpressed NF-jB, which correlated with pathological grade in gliomas [23, 25], is important in the oncogenic and cytodifferentiation processes. Many molecules, such as histone deacetylase-1 and -2 and ING4 (inhibitor of growth family, member 4), inhibit the expression and activation of NF-jB [26, 27]; however, the biological activity of NF-jB may be mainly regulated by the interaction among jB-Ras1, IjBb, and NF-jB. Under physiological conditions, these three molecules form a ternary complex, the stability of which depends on the cellular concentrations of these proteins. Despite emerging studies indicating that the imbalance of IjBb and NF-jB expression is associated with tumorigenesis [28], little is known about the relationship between jB-Ras1 expression and tumorigenesis. Our data provided evidence that the jB-Ras1 protein expression is significantly reduced in glioma specimens than in their non-neoplastic counterpart, non-neoplastic brain parenchyma. In addition, reduced jB-Ras1 expression was correlated with increasing pathological grade of gliomas, underlining a connection between down-regulation of jB-Ras1 and development of gliomas. In addition to more rodust NF-jB expression in gliomas, we suspected that abnormal expression of jB-Ras1 contributed to the pro-survival signaling. Because of their opposite expression, the jB-Ras1/IjBb/NF-jB ternary complex is replaced by the binary IjBb/NF-jB complex in gliomas. Because the stability of the binary complex was significant lower than that of the ternary complex in the tumors [29], where the pro-survival signal predominated, we hypothesize that the reduced jB-Ras1 expression leads to more frequent phosphorylation and degradation of IjBb and hence NF-jB activation in gliomas compared to their nonneoplastic counterpart. This finding was consistent with a previous study that showed that removal of cellular jB-Ras enhanced, whereas excess jB-Ras reduced, IjBb degradation. In our study, increased pro-survival signaling caused by reduced jB-Ras1 expression was demonstrated by investigating survivin protein expression in the same series of glioma specimens. As a chromosomal passenger protein, survivin regulates cell mitosis and promotes cell proliferation; nuclear survivin has also been suggested to be a validated prognostic indicator of gliomas [30].
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Fig. 3 Survival rates of glioma patients according to their jB-Ras1 expression a All data. B WHO grade 1. c WHO grade 2. d WHO grade 3. e WHO grade 4 Table 2 Statistical values of clinical parameters and expression of jB-Ras1 and survivin derived from Cox stepwise proportional hazards model Variables
b
SE of b
Sig. (P)
Exp. (b)
95% CI for Exp. (b)
Age
0.627
0.186
0.104
1.254
1.048-1.462
Gender Karnofsky performance status
0.584 0.794
0.141 0.222
0.482 0.215
1.465 1.849
1.228-1.712 1.457-2.189
Largest tumor diameter
0.454
0.028
0.072
1.374
1.125-1.652
Extent of resection
0.536
0.110
0.165
1.226
1.104-1.339
Type of adjuvant treatment
0.483
0.039
0.053
1.424
1.132-1.775
Intra-tumor necrosis
0.562
0.281
0.044
1.528
1.264-1.803
jB-Ras1 expression
0.723
0.157
0.032
1.472
1.210-1.728
Survivin expression
0.652
0.241
0.089
1.274
1.091-1.536
Reduced jB-Ras1 correlated with survivin expression and increasing pathological grades of gliomas. Down-regulation of jB-Ras1 expression was not a transient alteration that occurred in the primary stage of low-grade glioma
growth (WHO I and II) but in fact persisted even after malignancy (WHO III and IV), suggesting that jB-Ras1 may play an important role not only in the development but also in the progression of gliomas. However, further studies
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are needed to test whether the expression pattern of jB-Ras1 is different between primary glioblastoma and secondary glioblastoma and whether recombinant jB-Ras1 can inhibit the growth and progression of gliomas. Intra-tumorous necrosis commonly occurs in malignant tumors due to the imbalance between neoplastic vascularization and growth and invasion of gliomas [8]. As the tumor diameter increases, angiogenesis in the core of the tumors becomes so poor that the glioma cells suffer from an ischemic and anoxemic microenvironment, causing intra-tumorous necrosis [31]. Large tumor diameter and frequent intra-tumorous necrosis are commonly presumed as characteristic and prognostic indicators of malignant gliomas. Our results also establish a weak but statistically significant inverse correlation between lower jB-Ras1 expression and large tumor diameter (r = -0.436, P \ 0.01) as well as frequent intra-tumorous necrosis (r = -0.494, P \ 0.01). These relationships may be explained due to lower jB-Ras1 expression, leading to enhanced activation of NF-jB signaling, which modulates corresponding genes targeting at tumor proliferation and angiogenesis. Multivariate analysis showed that jB-Ras1 expression and intra-tumor necrosis were two important prognostic factors identified by the Cox proportional hazards model. Age, gender, Karnofsky performance status, largest tumor diameter, extent of resection, type of adjuvant treatment, and survivin expression did not reach statistical significance associated with survival (P [ 0.05). As such, our study highlights the prognostic significance of lower jB-Ras1 expression in gliomas than that in non-neoplastic brain parenchyma. Expression of jB-Ras1, which was semi-quantitated using the IRSs calculated in immunohistochemistry, decreased with the increasing pathological grades. The difference in expression was statistically significant between low-grade and high-grade glioma groups. Patients with low jB-Ras1 expression (IRS B 3) were more likely to suffer from malignant gliomas than those with high jB-Ras1 expression (IRS C 4). In addition, an inverse correlation between low jB-Ras1 expression and overall survival was demonstrated by Spearman’s rank correlation analysis (r = 0.756, P \ 0.01). Patients with abundant jB-Ras1 expression (IRS C 4) had better overall survival. Furthermore, jB-Ras1 expression did not correlated with age (cutoff value = 43) or gender, suggesting that the prognostic significance of low jB-Ras1 on overall survival was independent of these factors. In summary, we investigated the intracellular expression of jB-Ras1 in both non-neoplastic brain parenchyma and glioma specimens. Significant down-regulation of jB-Ras1 expression in gliomas was observed by immunohistochemistry and western blot analysis. The down-regulation of jB-Ras1 was statistically associated with increasing
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pathological grades of gliomas and expression of survivin protein. Glioma patients with lower jB-Ras1 expression had significant intra-tumorous necrosis, and worse overall survival, indicating the prognostic significance of jB-Ras1 for gliomas. Taken together, our data not only provided a novel understanding of the genetic character of gliomas, but also identified a potential target for molecular therapies. Acknowledgments The authors would like to thank the patients who participated in the study. Conflict of interest Supported by Chinese National Natural Science Foundation (Grant number: 20502035).
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