Prognostic significance of MYCN gene amplification and protein expression in primary brain tumors: Astrocytoma and meningioma

Abstract

BACKGROUND:

Astrocytoma and meningioma are the most common primary brain tumors. MYCN as a member of MYC proto-oncogenes has recently appeared as an attractive therapeutic target. Functions of MYCN are critical for growth of nervous system and neural differentiation.

OBJECTIVE:

We examined MYCN amplification and protein expression in astrocytoma and meningioma cases.

METHODS:

In this study, we used real-time PCR, FISH assay and flowcytometry to analyze DNA amplification and protein expression of MYCN.

RESULTS:

Among 30 samples of brain tumor, 14 cases (46.6%) revealed MYCN amplification. High-protein expression of MYCN was also observed in 43.3% of patients. There was a significant correlation between MYCN gene amplification and protein expression ( $r=$ 0.523; $p=$ 0.003), interestingly five case showed discrepancy between the gene amplification and protein expression. Although MYCN amplification fails to show correlation with poor prognosis ( $p=$ 0.305), protein high-expression of MYCN significantly reduce disease-free survival ( $p=$ 0.019).

CONCLUSIONS:

Our results challenge the concept of the neural specificity of MYCN by demonstrating contribution of MYCN in meningioma. Moreover, this study highlights the importance of research at both level of DNA and protein, to determine the biological functions and medical impacts of MYCN.

Keywords

MYCN amplification expression astrocytoma meningioma

1. Introduction

Brain tumors (BTs) are one of the most devastating and lethal forms of cancer that occur in both infants and adults. Neurological abnormalities caused by BTs and treatment procedures are severe challenges to patients’ quality of life [1, 2]. There are several types of BTs including astrocytoma and meningioma which are the most common primary brain tumors in adults [3, 4, 5, 6]. Meningioma originated from the meningothelial (arachnoidal) cells, which accounts for about 25–30% of primary intracranial neoplasms [7, 8]. The median survival time for meningioma is reported to be 4.9 years [9]. Astrocytoma arises from neural progenitor cells in the central nervous system (CNS) and is the most common type of glial tumors (30% of all BTs) [10]. The median survival for patients with astrocytoma is 5.2 years [11] and for glioblastoma is only 9.6 months at best [12].

So far, the causes of BTs are a mysterious puzzle. A number of genetic alterations are presumed to be critical in determining the biology of BTs and have been reported earlier such as loss of neurofibromatosis (NF2) gene in meningioma [13, 14]; and EGFR gene amplification and TP53 gene mutation in glioblastoma and other astrocytic tumors [15, 16, 17, 18]. Therapeutic resistance and tumor recurrence still restrict long-term survival of patients. Over the past decades, there are great failures in developing new drugs to treat BTs. In these regards, the establishment of new genetic biomarker which contributes to malignant progression can be beneficial for prediction of patients’ prognosis and also effective treatment.

The MYCN gene, as a member of MYC family of cellular proto-oncogenes, located at 2p24.3 and encodes nuclear phosphoprotein. MYCN is a transcription factor with basic Helix-Loop-Helix leucine zipper (bHLH-ZIP) to bind a consensus sequence in upstream of target genes. These target genes encode proteins with roles in cell proliferation, cell cycle regulation, apoptosis, and differentiation [19, 20]. Functions of MYCN are critical for growth of nervous system and neural differentiation [21, 22, 23, 24]. It seems that MYCN amplification disturbs terminal differentiation of normal neuroblast [25]. Although, gene amplification of MYCN in neuroblastoma is well established [26, 27], only a limited number of MYCN amplification have been conducted in other type of BTs.

The initial studies suggested that amplification and expression of MYCN was specific for neuroblastoma. Later studies revealed that it could be also observed in some other tumors with neural origin such as medulloblastoma and retinoblastoma [28, 29, 30]. The aim of this study was to evaluate the gene amplification and protein expression of MYCN in meningioma (as a benign and non-neuroectodermal tumor with mesenchymal origin) and astrocytoma (as a malignant type of brain tumor with neuroectodermal origin) for the first time. We also assess the correlation between MYCN gene amplification and protein expression in both types of tumors that has been barely explored. In addition, we performed the survival analysis for patient with and without MYCN amplification and/or high-expression. The amplification of MYCN is of biological and clinical interest since results of this investigation may lead to provide prognostic information and therapeutic opportunity.

2. Materials and methods

2.1 Tumor specimens and histopathologic information

The study included 30 patients with the histological and clinical diagnosis of meningioma and astrocytoma according to the WHO classification of CNS tumors [31] from the Shariati Hospital of Tehran, Iran. The histology of tumors was reviewed by an expert neuropathologist. Furthermore, two samples were taken from the deceased men without any history of malignancy in their pedigrees. All patients were untreated before the operation. Exclusion criteria had no complete clinical presentation or with a history of chemotherapy and radiotherapy. Clinical data were collected from all participating centers. Specimens from each tumor were taken at the time of the initial operation and then immediately frozen in liquid nitrogen and stored at $-$ 80 ${}^{\circ}$ C. Histological analysis was made to confirm the presence of tumor cells in all specimens.

2.2 Genomic DNA extraction

DNA was purified from brain tissue samples, using QIAamp DNA Mini kit (Qiagen) according to the manufactures’ instructions. The quantity of purified DNA was examined by spectrophotometer (NanoDrop Technologies, Washington, DE).

Table 1
Primer sequences used for real-time quantitative PCR

Gene Sequence Product length (bp) Tm (ċ)

MYCN (forward) AAGCACCCCCGGTATTAAAACG 120 59

MYCN (reverse) CTGGGTTCTTGCAGATCATGC 56

HBB (forward) GCTTCTGACACAACTGTGTTCACTAGC 120 61

HBB (reverse) CACCAACTTCATCCACGTTCACC 60

RPLP0 (forward) TGTGGCCTTCTACCTTACTG 124 57

RPLP0 (reverse) TGGGTCTCTTGTCATTTCTC 54

Gene	Sequence	Product length (bp)	Tm (ċ)
MYCN (forward)	AAGCACCCCCGGTATTAAAACG	120	59
MYCN (reverse)	CTGGGTTCTTGCAGATCATGC		56
HBB (forward)	GCTTCTGACACAACTGTGTTCACTAGC	120	61
HBB (reverse)	CACCAACTTCATCCACGTTCACC		60
RPLP0 (forward)	TGTGGCCTTCTACCTTACTG	124	57
RPLP0 (reverse)	TGGGTCTCTTGTCATTTCTC		54

2.3 Relative gene copy number quantification

Extracted DNA was quantified by NanoDrop ND-1000 (NanoDrop Technologies Wilmington, DE). Then, 20 ng of DNA from each sample was used for real-time reaction. We used quantitative real-time PCR to measure relative DNA copy number of MYCN gene. Real-time PCR reaction was carried out on rotor gen 6000 Corbett detection system using Precision 2X qPCR Mastermix SYBR Green detection kits (Primer Design company) with initial activation at 90 ${}^{\circ}$ C for 10 min. Thermo cycling included a single initial heat inactivation and denaturation incubation at 95 ${}^{\circ}$ C for 10 min, followed by 40 cycles of 95 ${}^{\circ}$ C for 5 s, and a combined annealing/extension step for 30 s at 60 ${}^{\circ}$ C. Melting curve analysis was performed to ensure that all of the primers yielded single PCR product. HBB (Hemoglobin, Beta) and RPLP0 (Ribosomal Protein Lateral Stalk Subunit P0) genes were amplified as internal control, and relative-fold differences of target were calculated using the 2 ${}^{\rm-\Delta\Delta Ct}$ method. Sequences of the primers and product size are shown in Table 1. Based on control DNA samples’ distribution in all experiment sets, normalized copy number value between 1.2 to 5 is defined as MYCN low-level amplification and values $>$ 5 is defined as high-level amplification.

2.4 Fluorescence in situ hybridization (FISH)

FISH as a complementary and conformational assay, was performed according to the protocol of Kreatech FISH probes (KBI-10706 [Poseidon™ MYCN (2p24) & LAF (2q11) Control Probe, Kreatech, The Netherlands). Probes are labeled with either a green (Platinum Bright 495) and red (Platinum Bright 550) fluorophore. The positivity of MYCN amplification by FISH ( $+$ , the mean number of MYCN signals were three or more per cell; less than three signals per cell) and also increases in the number of signals greater than 5 were recorded as high-level amplifications.

2.5 MYCN protein expression analysis by flowcytometry

Extracted cells were sustained using monoclonal mouse anti-human for MYCN (Anti-n-Myc antibody [NCM II 100]-ABCAM, UK) isotype IgG1. An average of 1000–5000 cells in each sample was washed twice with 1X PBS. After adding 10 ml of antibody, the mixture of cells was incubated at 4 ${}^{\circ}$ C for 45 minutes. The cells were washed with 1X PBS twice. The antibody was detected using anti-mouse second layer (Pe-Cy5 phycoerythrinindodicarbocyanine). Mixture was incubated at 4 ${}^{\circ}$ C for 45 minutes. The conjugated cells were assayed by BD FACS Calibur flowcytometry (BD FACSCALIBUR ${}^{\rm TM}$ Flow Cytometry System, US) and analyzed using FlowJo software. We determined the basic value of expression of the negative controls (a cut off value of 2% was used to define positivity and the matched isotype in the studied cells). Also, for confirmation of expression mode in these samples, images are provided by immunofluorescence assay according to the standard protocol which was described, previously [32].

2.6 Statistical analysis

Data was statistically analyzed using SPSS 18 (SPSS Inc, IL, USA) and GraphPad Prism (GraphPad Prism 6 Software Inc, San Diego, CA) software. Shapiro-Wilk, Mann-Whitney U, Kruskal-Wallis, Fisher Exact test, and Chi-Square tests were used. Also, Pearson’s Correlation and linear regression were performed. The Kaplan-Meier method was used to estimate disease-free survival rates and comparison of the survival between groups was performed with the log-rank test (Mantel-Cox). Disease-free survival was calculated from the date of diagnosis until death occurred or last follow-up. In all of the statistical analysis, $p$ value less than 0.05 was considered as significant.

3. Results

3.1 Clinicopathological factors in patients with astrocytoma and meningioma

Thirty samples of brain tumor (17 meningioma and 13 astrocytoma), were collected. The mean age at the time of diagnosis was 47.1 $\pm$ 19.8 years. The median age of patients was 32 years old for astrocytoma and 56 years old for meningioma. Patients with astrocytoma were significantly younger than patients with meningioma ( $p=$ 0.001). The female to male ratio was 2.7. The mean size of astrocytoma and meningioma tumors were 4.7 $\pm$ 2.2 and 3.1 $\pm$ 1.3, respectively. Thirteen patients died of disease.

Figure 1.

Distribution of MYCN relative gene copy number in (A) astrocytoma and (B) meningioma tumors. Relative gene copy number ranged from 0.5- to 10.4-fold; level of threshold were defined as 1.

3.2 MYCN gene amplification status in tumors of astrocytoma and meningioma

Figure 2.

Fluorescence in situ hybridization (FISH) with MYCN probe (red) and control probe (green). Red signals represent 2p24.3, green signals, 2q11.2 (LAF4). (A) High amplification of MYCN detected by FISH (multiple red signals). (B) Tumor cells carrying MYCN low amplification. Nuclei are stained with DAPI (blue), X100.

Real-time PCR was used to quantify relative MYCN gene copy number. As shown in Fig. 1, 13 out of 30 (43.3%) tumors uncover 1.5–4.4 fold increase in relative MYCN gene copy number which defined as low-level amplification ( $\leqslant$ 5-fold). High-level amplification ( $>$ 5-fold) was only seen in one case with 10.4 fold change. In addition, results of qPCR in some cases were confirmed by FISH analysis ( $n=$ 10, Figs 2A and B). FISH assay also detects high-level amplifications in one case, consistently agreed with the results by real-time PCR. Otherwise, it was supposed to be done for 30 samples. Clinicopathological characteristics were analyzed in relation to the MYCN gene amplification status (Table 2). The comparison between MYCN gene amplification in different types and grades are shown in Fig. 3.

Figure 3.

MYCN gene amplification status in different grades of meningioma and astrocytoma tumors. Relative gene copy number of MYCN were measured by real time PCR and normalized against RPLP0 and HBB genes on the basis of a comparative Ct (2 ${}^{\rm-\Delta\Delta ct}$ ) method. Values represent mean $\pm$ SD. Error bars 95% CI.

Table 2

Clinicopathological characteristics were analyzed in relation to the MYCN gene amplification status and its protein expression

Characteristics	MYCN gene amplification			MYCN protein expression
	OR	95 % CI	$P$ value	OR	95 % CI	$P$ value
Age	0.75	1.77–3.17	0.49	1.6	0.38–7.15	0.37
$<$ 50
$>$ 50
Gender	0.27	0.04–1.69	0.15	0.11	0.01–1.13	0.04
Male
Female
Tumor type	1.8	0.41–7.81	0.33	0.81	0.19–3.50	0.53
Astrocytoma
Meningioma
Tumor size	2.75	0.5–14.86	0.22	1.76	0.32–9.51	0.4
$<$ 5
$>$ 5

3.3 MYCN protein expression in patients with astrocytoma and meningioma

Figure 4.

Protein expression of MYCN was evaluated by flowcytometry. Total cells were isolated from tumors and control and then sustained using monoclonal mouse anti-human for MYCN (Anti-n-Myc antibody) isotype IgG1. The antibody was detected using anti-mouse second antibody (Pe-Cy5). The basic value of negative controls expression was determined. The cut-off point of 2% was used to define positivity and the matched isotype in the cells. Shown is representative single-parameter histogram. Histograms of (A) and (B) show the unstained sample of MYCN protein expression and stained meningioma tumor cell with high-expression of MYCN protein, respectively.

Figure 5.

Protein expression of MYCN in tumor cells of patients affected with brain tumors (A) Protein expression of tumor cells of a patient affected with astrocytoma. (B) Protein expression of tumor cells of a patient affected with meningioma. (A1) Tumor cells with dapi, as counter stain (A2) Tumor cells with Pe-cy5, representative of MYCN with high expression in both sporadic isolated cells and in accumulated cells (B1) Tumor cells with dapi, as counter stain (B2) Tumor cells conjugated with Pe-cy5, reflective of the low protein expression of MYCN in the majority of cells and high expression in the minority of cells. Magnification: A) X200; B) X100.

Flowcytometry was applied to measure MYCN protein and the median percentage of protein expression was 11.6% (Figs 4A and B). High-expression of MYCN was observed in 13 patients (43.3%) out of all patients ( $n=$ 30) and it was statistically significant in compare to normal samples ( $p=$ 0.00). Results of protein expression were confirmed by immunofluorescence assay (Figs 5A and B). Expression status of MYCN was analyzed in relation to the clinicopathological characteristics (Table 2). The comparison between MYCN protein expression in different types and grades of brain tumor are shown in Fig. 6. Male patients with astrocytoma did not show MYCN gene amplification and/or protein high-expression. However, 66.6% and 55.5% of female patients with astrocytoma showed MYCN protein high-expression and MYCN amplification, respectively.

Figure 6.

Protein expression of MYCN in different grades of meningioma and astrocytoma tumors. Flowcytometry was used to determine the MYCN protein expression in samples. Bars represent the mean $\pm$ SD. Error bars 95% CI; * $p<$ 0.05 vs. control.

Figure 7.

MYCN gene amplification and its protein expression status in different types of brain tumors. Two non-amplified MYCN samples (one astrocytoma and one meningioma) showed high-expression of MYCN protein. Three meningioma cases with MYCN gene amplification did not show MYCN protein high-expression.

Figure 8.

Survival curves comparing survival times of patients. (A) Whose tumors exhibited low/high-level amplification of MYCN ( $n=$ 14) (dotted line) with those from patients with non-amplified MYCN gene ( $n=$ 16) (straight line). (B) Protein expression of MYCN were divided into high-expression ( $n=$ 13) and normal-expression ( $n=$ 17) groups. Probability of disease-free survival was calculated by the Kaplan-Meier method based on the protein expression and gene amplification of MYCN and were analysed by the log rank test (Mantel-Cox).

3.4 The correlation between MYCN gene amplification and protein expression

There was a direct positive and significant correlation between MYCN gene amplification and protein expression of MYCN ( $r=$ 0.523; $P=$ 0.003) and linear regression model has been developed ( $Y=$ 20.17 $+$ 2.98 $X$ ). MYCN gene amplification and its protein expression status in different types of BTs are shown in Fig. 7. The highest and the lowest MYCN protein expression were seen in two meningioma cases. Two samples with high-expression of MYCN did not show MYCN gene amplification. In other hands, three cases with MYCN amplification did not show high protein expression. In this investigation, most of astrocytoma and meningioma (92% and 76%, respectively) reveal complete concordance between MYCN gene amplification and protein expression. As mentioned earlier, only one case depicts high amplification (10.4 fold change). This case was the 47 years old woman with tumor size of 3 $\times$ 18 meningiotheliomathous tumor. Interestingly, the protein expression of MYCN was normal in this patient.

3.5 The outcome of the patients with MYCN amplification/high-expression

We analyzed disease-free survival by classifying our patients in two groups, patients with- ( $n=$ 14) and without- ( $n=$ 16) MYCN amplification. Statistical analysis revealed that the group of patients with MYCN amplification had shorter survival than the group without amplification, although it was not statistically significant ( $p=$ 0.305, log-rank test; Fig. 8A). The observation of worse prognosis is independent of histological subtype. Moreover, the study of survival by Kaplan-Meier analysis showed that patients with MYCN protein high-expression had a significant worse mean survival compared with patients in whom the MYCN protein expressed normally ( $p=$ 0.019, log-rank test; Fig. 8B).

4. Discussion

Tumors of nervous system are genetically heterogeneous and are among the most challenging forms of neurological disease. Despite the different types of BTs, there have only been a few major prognostic biomarkers and approved medications for treatment of BTs, thus more research are critical. Notably, MYCN as an important predictive marker has appeared as an attractive target for therapy and prognostic implication [33]. MYCN plays essential roles during brain development [34]. Mutations, translocation and gene amplification are common mechanisms which can activate proto-oncogenes. The first two ones are not common activation mechanism for MYCN proto-oncogene. However, there is a case report of a novel cryptic t(2, 14) (p24; q32)/IGH-MYCN targeting MYCN in 2 blastoid mantle cell lymphomas, in which MYCN located around 545 kilobases from the breakpoint that lead to high-expression of MYCN in both cases [35]. Moreover, recent studies reported rare point mutation of MYCN in Wilms’ tumor and neuroblastoma [36, 37, 38].

Table 3

MYCN gene amplification status in different studies

Tumor type	Method	Total	High-level amplification	Low-level amplification	Country	Year	Ref
Neuroblastoma	qPCR	20	4 (20%)	6 (30%)	Hungary	2003	[27]
Neuroblastoma	qPCR	49	4 (8.1%)	22 (44.8%)	Iran	2012	[44]
Neuroblastoma	qPCR	45	6 (13.3%)	–	US	1990	[47]
Neuroblastoma	FISH	47	8 (17%)	7 (14.8%)	Japan	2011	[45]
Neuroblastoma	FISH	178	24 (13.4%)	129 (72.4%)	China	2013	[67]
Neuroblastoma	FISH	58	4 (6.8%)	43 (74.1%)	China	2012	[68]
Neuroblastoma	FISH	22	5 (22.7%)	–	Japan	1999	[69]
Neuroblastoma	qPCR	49	4 (8.1%)	22 (44.8%)	Italy	1999	[70]
Neuroblastoma	qPCR	97	16 (16.4%)	–	France	2002	[71]
Neuroblastic Tumors	qPCR	36	10 (27.7%)	–	Morocco	2013	[43]
Neuroblastic Tumors	FISH	119	18 (15.1%)	79 (66.3%)	China	2015	[72]
Gangelioneuroblastoma	FISH	32	1 (3.1%)	25 (78.1%)	China	2013	[67]
Gangelioneuroblastoma	qPCR	12	1 (8.3%)	–	Iran	2014	[73]
Medulloblastoma	qPCR	22	2 (9%)	9 (40.9%)	Russia	2008	[74]
Medulloblastoma	FISH	27	1 (3.7%)	5 (18.5%)	Czech	2011	[29]
Retinobalstoma	qPCR	25	3 (12%)	9 (36%)	UK	2002	[42]
Small Cell Lung Carcinoma	CGH	23	2 (8.7%)	–	US	2001	[75]
Gangelioneuroma	FISH	10	0 (0%)	4 (40%)	China	2013	[67]
Neurocytoma	qPCR	22	0 (0%)	18 (81.8%)	Russia	2008	[74]
PNET	MLPA	25	6 (24%)	3 (12%)	Germany	2014	[30]
Astrocytoma $+$ Meningioma	qPCR	30	1 (3.3%)	13 (43.3%)	Iran	2016	Present study

Until the first report of MYCN gene amplification in neuroblastoma by Schwab et al. [39], there are only a limited number of reports of MYCN amplification in different tumors other than neuroblastoma. In this investigation, we analyzed gene amplification of MYCN in BTs including meningioma, astrocytoma and glioblastoma which have not been, previously, reported. We used real-time PCR to quantify MYCN amplification and then apply FISH assay to confirm it. As shown in Table 3, we found amplification of MYCN in a substantial number of meningioma (52.9%) and astrocytoma (30.7%). Previously, Garson et al. described the MYCN amplification as a case report in astrocytoma tumor [40]. Also, other studies revealed that MYCN amplification is not restricted to neuroblastoma and it was found in small cell lung cancer [41], retinoblastoma [42], and other tumors (Table 3). Result of our investigation challenge the concept of the neural specificity of MYCN by demonstrating amplification of MYCN in meningioma. In this study, patients with MYCN amplification showed worsen disease-free survival however it was not statistically significant. There are controversy in published articles about significant correlation between MYCN amplification and deteriorate survival [30, 43, 44, 45]. Some findings can make this puzzle more complex. Current discoveries show that MYCN could also be transcribed from opposite strand to a long non-coding RNAs which known as NCYM, cis-antisense gene of the MYCN oncogene. NCYM is 100% co-amplified and co-expressed with MYCN in neuroblastomas, and mRNA expression of NCYM is associated with poor clinical outcome [46]. Herein, we suggest evaluating simultaneously MYCN and NCYM gene amplification and/or expression to make more precise correlation with disease-free survival.

The high-expression of the protein product would be a direct consequence of gene amplification. Our results show that approximately 46% of astrocytoma and 41% of meningioma have high-expression of MYCN protein, somewhat lower compared with that described in few reports on neuroblastoma (80.7% and 66.6%) [47, 48]. Garson et al., showed high-expression of MYCN protein in six out of eleven medulloblastoma brain tumor [49]. Our results show that glioblastoma (astrocytoma grade IV) exhibited higher expression of MYCN in compare to other types of astrocytoma and meningioma ( $P=$ 0.04). It seems that the amplification/high-expression of MYCN is involved in glial cell malignant transformation [50]. Study of Nisen, et al. uncovers the high-expression of MYCN in Wilms’ tumors and they suggested MYCN high-expression can be a marker for neoplasms that arise from primitive cell precursors [51]. In addition, it was shown that up-regulation of MYCN was occurred in the BTs that have features of primitive neuroectodermal tumors [51, 52]. MYCN is expressed at particularly high levels in the neuroblastoma, Wilms’ tumor (a neoplasm which arises from embryonal kidney cells) and medulloblastoma that all of them plus glioblastoma arises from primitive and undifferentiated cells. In addition, MYCN induce the expression of some genes involved in neuronal progenitor cells [53]. Recent studies showed MYCN has a direct role to maintain pluripotency [54, 55, 56]. The enhanced differentiation in MYCN-null cells of neural progenitor may be a result of increased levels of the p27kip1, that plays a role in differentiation and is normally degraded by SKP2 as a transcriptional target of MYCN [57]. Moreover, previous research shows that downregulation of MYCN is associated with decreased proliferation and neuronal differentiation [58]. Taken together, these documents indicate that MYCN is important for maintenance of the undifferentiated phenotype and that suppression of MYCN signaling could even lead to improved anti-cancer treatments. In this regard, new approaches base on destabilizing MYCN protein or inhibitor of MYCN would be applied for treatment of aggressive BTs such as glioblastoma [59]. Moreover, pathway analyses uncover involvement of MYCN in mTOR signaling activation, suggesting mTOR inhibitors would be an effective treatment for tumors with MYCN amplification [60].

Although MYCN amplification fails to show significant reduction in disease-free survival, protein high-expression of MYCN is meaningfully correlated with worsen disease-free survival and poor prognosis. However, our understanding of the molecular link between MYCN high-expression and poor prognosis remain elusive, some studies suggesting that gene ID2 (inhibitor of DNA binding/differentiation) is a direct target of MYCN which could be contributed to poor prognosis [61]. Totally, it seems that gene amplification and protein high-expression of MYCN would have different prognostic implications.

There are few reports in which a correlation between gene amplification and protein expression of MYCN is considered. Gene amplification could suffice to indicate the MYCN high-expression, but other mechanisms of gene activation casts doubt on a strict correlation between amplification and high-expression of MYCN. This observation highlights the importance of performing both DNA and protein analyses, to assess the biological effect of MYCN gene alterations. Our results show that, there is a significant correlation between MYCN gene amplification and its protein expression (83.3%, $P=$ 0.004). Such a relationship would be required in order to understand the function of gene amplification and MYCN protein stabilization in tumor cells. Several studies failed to detect such a significant correlations [28, 62]. Interestingly, two cases in present study (one meningotheliomatous and one glioblastoma), uncover MYCN protein high-expression without gene amplification. However, the case with meningotheliomatous tumor expired due to this kind of malignancy, other 6 patients of this group did not have significant amplification and high-expression of MYCN and they are all alive. As previously reported, MYCN protein can be methylated via PRMT5 (Protein Arginine Methyltransferase 5) which lead to increased protein stabilization and MYCN high-expression without gene amplification [63]. On the other hand, in this investigation three samples of meningioma with MYCN gene amplification did not revealed MYCN high-expression. The increased gene copy number of MYCN without protein high-expression may be explained by the down-regulation of gene transcripts, the post-transcriptional alterations (protein inactivation or disrupted protein distribution), the protein deregulation by a downstream effectors (such as PI3K inhibitor) and may be due to the promoter hypermethylation [64, 65, 66]. Herein we suggest further study is required to determine the different prognostic implications of MYCN gene amplification and protein high-expression. Molecular mechanisms involved in this discrepancy between MYCN amplification and protein high-expression remain to be clarified.

Footnotes

Acknowledgments

This research was supported by Tehran University of Medical Sciences of Iran.

Conflict of interest

There is no conflict of interest.

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