Int J Clin Exp Med 2016;9(9):17356-17366www.ijcem.com /ISSN:1940-5901/IJCEM0032220

Original ArticleHeterogeneity of MGMT gene promoter methylation and protein expression in serial specimens in newly diagnosed glioblastoma

Qiang Pan1*, Xue-Jun Yang3*, Lin Zhu2*, Chun-Yu Song1, Xing-Tao Diao1, Yong Gao1, Jun Zhu1, Xiao Yue4, Hu-Guang Li5, Wei-Ling Cheng6, Yi-Ping Ning6

Departments of 1Neurosurgery, 2ICU, Laiwu City People’s Hospital (Laiwu Hospital Affilliated to Taishan Medical College), Laiwu 271199, Shandong Province, P. R. China; 3Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, P. R. China; 4Tianjin Huanhu Hospital, Tianjin Neurosurgery Institute, Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases, Tianjin 300052, P. R. China; Departments of 5Neurosurgery, 6ICU, Lishui People’s Hospital, Lishui 323000, Zhejiang Province, P. R. China. *Equal contributors.

Received May 15, 2016; Accepted August 2, 2016; Epub September 15, 2016; Published September 30, 2016

Abstract: O6-methylguanine-DNA methyltransferase (MGMT) is known as a predictive marker, which has been asso-ciated with longer survival in glioblastoma patients who were treated with alkylating agents. However, little is known about heterogeneity of MGMT gene promoter methylation and its protein expression across different regions within the same tumor. This study was aimed to compare the status of MGMT gene promoter methylation and protein ex-pression in different sites’ specimens of newly diagnosed glioblastoma. Two to four specimens were obtained from different regions in the same tumor during surgical procedure in 20 newly diagnosed glioblastoma patients guided by neuronavigation system(overall number of specimens: 60, all of which were prepared for the following analysis except one specimen showed necrotic obiviously). MGMT promoter profile was analyzed by Methylation-specific polymerase-Chain-reaction analysis (MSP). The MGMT protein expression was assessed by immunohistochemistry (IHC) and western blotting (WB). MGMT promoter Methylation was detected in 45.76% (27/59) specimens. IHC and WB analysis exhibited high concordant findings (r=0.93, P<0.001). There was no significant correlation be-tween MGMT promoter methylation and protein expression (IHC and MSP, x2=3.18 P=0.074; WB and MSP, Z=-1.712 P=0.087), and this study also demonstrated the regional heterogeneity of MGMT status of methylation in 10.00% (2/20) patients and MGMT protein expression in 40.00% (8/20) patients. MGMT promoter methylation is prob-ably not the only modulating factor in MGMT protein expression. Our research questions the guiding significance of MGMT promoter methylation or its protein expression to the therapeutic scheme, which detected only in a small piece of specimen in clinical practice.

Keywords: O6-methylguanine-DNA methyltransferase, heterogeneity, gene promoter methylation, protein expres-sion, glioblastoma multiforme

Introduction

Glioblastoma (GBM WHO IV) is the most com-mon and malignant intracranial tumor with dis-mal prognosis. Its median survival time ranges from 12 to 15 months despite of aggressive surgery combined with radiation, chemothera-py [1] and biological therapy [2]. Less than 3% of glioblastoma patients survive 5 years after initial diagnosis [3]. The highly lethal nature of this tumor partly originates from its chemore-sistance of GBM tumor cells, moreover, the association between MGMT and chemoresis-

tance is most concerned. MGMT is a ubiquitous DNA-repair enzyme in normal human tissues and many cancers. However, the expression level of MGMT varies widely [4]. In the past years, through molecular and genetic profiling efforts, the promoter hypermethylation of the MGMT gene has been shown to be a common epigenetic event in several cancers, including gliomas [5-11]. In the current study, an associa-tion of MGMT promoter methylation status, clinical outcome [12-19] and the response of tumors to alkylating agents (e.g. carmustine, lomustine, temozolomide) [16, 20] of glioblas-

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toma patients have been revealed in some independent investigations. Based on these findings, MGMT promoter methylation is con-sidered an ideal predictive factor for chemo-therapy response and longer survival in glio-blastoma. Underlying mechanisms had focused on the possible association with epigenetic silencing of the MGMT by promoter hypermeth-ylation. However, some groups’ studies failed to

show any relation between MGMT promoter methylation and overall survival or response to alkylating agents [21, 22], but found prognostic significance of MGMT protein expression in glioblastoma by immunohistochemistry (IHC) [23, 24]. For example, inactivation of MGMT protein by the substrate analog O6-benzyl- guanine (O6-BG), sensitized tumor cells to alkyl-ating agents [25]. The marked heterogeneity of

Table 1. Clinical parameters and specimen protocols of 20 patients with newly diagnosed glioblastoma Age Region A Region B Region C Region D Glioblastoma

ID Sex (year) WB IHC MSP WB IHC MSP WB IHC MSP WB IHC MSP Location1 F 69 0.03±0.01 1 M 0.07±0.02 1 M 0.65±0.18 2 M N.a. N.a. N.a. Right temporal

2 M 42 0.14±0.03 1 M N.c. N.c. N.c. 0.05±0.02 1 M N.c. N.c. N.c. Left parietal

3 F 64 0.50±0.08 2 U N.c. N.c. N.c. 0.56±0.05 2 U 0.52±0.07 2 U Right frontal

4 M 63 0.62±0.16 2 M 1.00±0.11 3 M 0.10±0.01 1 M 0.47± 0.17 2 M Right temporal

5 M 63 0.15±0.01 1 M N.c. N.c. N.c. 0.07±0.04 1 M 0.15± 0.05 1 M Left frontal

6 F 55 0.97±0.07 3 U 0.41±0.13 2 U N.c. N.c. N.c. 0.57±0.08 2 U Left frontal

7 M 80 0.85±0.13 3 M 1.12±0.09 3 M 1.14±0.15 3 M 0.56±0.20 2 M Left temporal

8 F 62 0.11±0.07 1 M N.c. N.c. N.c. 0.17±0.10 1 M N.c. N.c. N.c. Left parietal

9 M 71 0.08±0.01 1 M N.c. N.c. N.c. 0.17±0.08 1 M 0.16±0.04 1 M Right temporal

10 F 56 0.93±0.17 3 U 0.92±0.11 3 U 0.86±0.20 3 U 0.19±0.10 1 M Left frontal and parietal

11 F 69 0.32±0.02 2 M N.c. N.c. N.c. 0.42±0.08 2 M N.c. N.c. N.c. Left frontal and parietal

12 M 69 N.c. N.c. N.c. 0.47±0.09 2 U 0.42±0.12 2 U 0.22±0.06 2 U Right temporal

13 M 72 N.c. N.c. N.c. N.c. N.c. N.c. 0.09±0.01 1 U 0.12±0.03 1 U Left parietal

14 M 57 0.75±0.15 2 U 0.68±0.18 2 U 1.12±0.17 3 U N.c. N.c. N.c. Right frontal

15 F 59 0.28±0.13 2 M N.c. N.c. N.c. N.c. N.c. N.c. 0.37±0.11 2 M Right frontal and parietal

16 M 46 1.66±0.12 3 U 0.62±0.16 2 U 1.29±0.16 3 U 0.53±0.02 2 U Right frontal

17 F 59 0.12±0.01 1 U 0.07±0.02 1 U 0.10±0.01 1 U N.c. N.c. N.c. Right temporal

18 M 58 0.20±0.05 1 M 0.12±0.08 1 U 0.89±0.04 3 U N.c. N.c. N.c. Left temporal

19 F 70 0.13±0.02 1 U 0.07±0.01 1 U 0.10±0.01 1 U N.c. N.c. N.c. Right parietal

20 M 67 0.32±0.07 2 U 0.36±0.05 2 U N.c. N.c. N.c. 0.37±0.11 2 U Left frontalMGMT promoter methylation (M = methylated, U = unmethy-lated) was assessed by Methylation-specific polymerase-Chain-reaction analysis (MSP). MGMT protein expres-sion was evaluated by western bloting (WB) and immunohistochemistry (IHC), the level of MGMT expression assessed by WB was quantitied by Densitometry and data were expressed as (mean ± SD) , all tests were repeated three times (n=3); MGMT protein expression was assessed and scored by IHC in each section according to the following scale: (1 = negative or <10% positive tumor cells, 2=≥10%≤50%, 3≥50%). N.c.: the regional sample not collected; N.a.: sample not analyzed.

Figure 1. T1 weighted Gd-DTPA enhanced MRI image of a newly diagnosed glioblastoma from which two to four specimens were taken for analysis during operation under assistance of neuronavigation system. Letters represent the regions from which samples were taken (patient ID-10). Which were histologically confirmed to be glioblastoma.

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every patient for obtaining best specimens, which could highly control collecting solid tumor specimens from different sites of the tumor, and guarantee the specimens not or only mini-mally contaminated with tumor necrosis or non-neoplastic cells. MGMT promoter profile was analyzed by Methylation-specific poly-merase-Chain-reaction analysis (MSP). The MGMT protein expression was assessed by IHC and WB. The purposes of the present prospec-tive study are, (1) To investigate the distribution of both MGMT promoter methylation and MGMT protein expression of different sites within the tumor; (2) To explore whether MGMT protein expression and MGMT promoter methylation have intrinsic correlations; (3) To evaluate the predictive significance of MGMT promoter methylation and MGMT protein expression.

Material and methods

Patients and specimens collection

Between June 2009 and August 2010, sixty tumor specimens were obtained from twenty

Figure 2. Four specimens taken from different regions (A-D) of one tumor (patient ID-10) were subsequently histo-pathologically confirmed to glioblastoma (WHO IV) without obvious necrotic and normal brain tissues (HE dyeing, original magnification: 200×). (A-D): represent samples from sites.

MGMT gene promoter methylation and its pro-tein expression and the relationship of both remain controversial.

It is known that there is existence of histologi-cal and genetic heterogeneity in glioblastoma. Furthermore, there is no study to examine whether and to which extent MGMT protein expresses, and whether MGMT promoter meth-ylation varies throughout the same newly diagonsed glioblastoma. If findings of heteroge-neity exist, it would possibly influence diagnos-tic methods, clinical treatment strategies and prognostic assessments. In this study, 2-4 solid tumor samples per tumor were collected from different sites within the tumor of 20 patients with the assistance of neuronavigation system during the surgical treatment on the basis of the coregistration of magnetic resonance con-trast-enhanced imaging (MRI) (3.0T EXCITE® HD, GE, American). At surgery, once the patient’s head was fixed to the Mayfield head holder, the process of registration was carried out. Multiplanar reconstructions were used in

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patients with newly diagnosed glioblastoma undergoing surgery at the department of Ne- urosurgery, Tianjin Medical University General Hospital. The clinical information relating to these patients is summarized in Table 1. Inclusion criteria were as follows, (1) Newly diagnosed glioblastoma locates in cerebral hemisphere; (2) Histopathological diagnosis of glioblastoma (WHO IV) follows the WHO criteria; (3) No chemotherapy or radiotherapy was per-formed on the patients before resection; (4) The study was approved by the Tianjin Medical University General Hospital Research Ethics Committee; (5) All enrolled patients have writ-ten informed the consent according to the com-mittee’s regulations. About 2-4 tumor solid specimens were obtained from different regions within the same glioblastoma during operation under assistance of neuronavigation system (Vector Vision, BrainLAB, Germany) on the basis of the coregistration of contrast-enhanced MRI. An example is shown in Figure 1. Every specimen was divided into two pieces: one was formalin-fixed and paraffin-embeded for histological studies and IHC analysis, and the specimen was subsequently confirmed to be glioblastoma on histological evaluation. The other part immediately snap-frozen and stored in liquid nitrogen for MSP and WB analysis.

DNA extraction and bisulfite modification of DNA

DNA was isolated from each frozen or fresh glioblastoma tissue (Wizard® Genomic DNA Purification Kit A1120, Promega). The quantity and purity of DNA were assessed using the NanoDrop® ND-1000 Spectrophotometer (Na- noDrop, Wilmington, NC). Extracted tumor DNA (1 μg) was denatured with sodium hydroxide (0.3 M), and subjected to bisulfite treatment for 18 hours at 50°C and then purified (CpGenomeTM Fast DNA Modification Kit S7824, Chemicon). In this reaction, unmethylated cyto-sine resides, but not methylated counterpart, is modified into uracil by bisulfite treatment.

Methylation-specific polymerase-Chain-reac-tion analysis, MSP assay

DNA methylation patterns in the CpG island of MGMT (Gene Bank accession no. X-61657) was determined by MSP. Primers (5 μM) were designed for either methylated or unmethyl-ated versions for MGMT gene according to pre-viously described [26, 27], MGMT sense

5’-TTTCGACGTTCGTAGGTTTTCGC-3’ and MGMT antisense 5’-GCACTCTTCCGAAAACGAAACG-3’ for methylated sequences; MGMT sense 5’- TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3’ and MGMT antisense 5’-AACTCCACACTCTTCCAAA- AACAAAACA-3’ for unmethylated sequences. The reaction mixture (25 μL) contained tem-plate DNA (100 ng), U or M primer (5 μM), dNTP (2.5 mM), “hot start” enzyme (1 U), with the 10× Universal PCR buffer (2.5 μL), and PCR performed under the following conditions: an initial melting step of 2 min at 95°C; followed by 36 cycles of 45 s at 95°C, 45 s at 60°C and 60 s at 72°C; and a final extension at 72°C for 8 min (CpG WIZ® MGMT Amplification Kit S7803, Chemicon). DNA from normal lymphocytes was used as negative control for methylated alleles of MGMT, and placental DNA treated in vitro with SssI methyltransferase (CpG Methyl- transferase, M.SssI, M0226S, NEB) was used as positive control for methylated alleles of MGMT. The PCR products were separated on 4% agarose gels with ethidium bromide. The investigators who selected and analyzed the glioblastoma specimens were blinded all other information including clinical data and the results of IHC and WB.

Western blotting assay

The glioblastoma specimens were triturated in liquid nitrogen and extracted by 1% Nonidet P-40 lysis buffer. Homogenates were clarified by centrifugation at 20 000×g for 15 min at 4°C and protein concentrations were deter-mined using a bicinchoninic acid protein assay kit (Pierce, USA). The quantity and purity of pro-tein were also assessed using the NanoDrop® ND-1000 Spectrophotometer. Samples were adjusted to equal protein concentration and volume, and subjected to SDS-PAGE. Separate proteins were transferred to PVDF membranes (Millipore, USA) followed by blocking. The mem-branes were incubated with anti-MGMT anti-bodies (1:100 dilution, MT3.1, Chemicon) against MGMT protein, followed by incubation with HRP-conjugated secondary antibody (1:1000 dilution, Zymed). The specific protein was detected using a Super Signal protein detection kit (Pierce, USA). After being washed with stripping buffer, the membrane was rep-robed with antibody against β-actin (1:100 dilu-tion, Santa Cruz Biotechnology) using the same procedures described above. Each test was repeated in triplicates. The level of WB test of MGMT was expressed as mean integrated gray

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± standard deviation. The band density of spe-cific proteins was quantified after normaliza-tion with the density of β-actin.

MGMT immunohistochemistry assay

Sections of formalin-fixed, paraffin-embeded tissues (6 μm thick) were dewaxed with xylenes, dehydrated by using graded ethanol and endog-enous peroxidase activity was blocked by 0.3% H2O2 in methanol for 10 min. To unmask anti-gens, sections were treated with 10 mM citrate buffer (pH 6.0) at 100°C in a pressure vessel. After cooling at a room temperature, sections were washed three times with water and brought to PBS for 5 min followed by incubation with 5% BSA for 10 min. The sections incubat-ed with appropriate primary antibody (1:200 dilution, MT3.1, Chemicon) overnight at 4°C. After being washed with PBS, biotinylated sec-ondary antibody (1:200 dilution, Zymed) was added at a room temperature for 1 h, followed by the incubation with ABC-peroxidase for addi-tional 1 h. After washing with Tris-buffer, the sections were incubated with DAB for 5 min, then rinsed in water and counterstained with Hematoxylin. As a control, isotyping to the pri-mary antibody and omission of the primary antibody were done, and both gave no staining. Lymphocytes and endothelial cells served as internal positive control. MGMT was consid-

ered positive when uniform MGMT staining was detected in cell nuclei [28, 29]. Cytoplasmic only immunoreactivity and granular nuclear reactivity were considered negative [29, 30]. MGMT expression was assessed and scored in tumor cells by three pathologists who blinded to molecular and clinical data examined slides, using 3-tiered scale (1= negative or approxi-mately limited to <10% positive tumor cells, 2=≥10%≤50% staining, 3≥50% staining) in the same tumor area from which the MSP and WB samples were tested.

Statistical analysis

All data are expressed as mean values ± stan-dard deviation(

_x±s); the Chi-square statistics

and Fisher’s extact test were used to compare MGMT expression analyzed by IHC with meth-ylation status evaluated by MSP; the relation-ship between tumor MGMT protein expression analyzed by WB and MSP results was assessed by Mann-Whitney Rank Sum Test; the correla-tion between MGMT protein expression ana-lyzed by IHC and by WB was assessed by Spearman Test; the MGMT protein expression of different sites within same tumor tested by WB was assessed by One-Way ANOVA. All anal-ysis with any values blow 0.05 considered sta-tistically significant. The statistical package SPSS 13.0 (SPSS, Inc) was used for this analysis.

Results

MGMT promoter hypermethylation

DNA was successfully extracted from tumor specimens and modified by bisulfite. All the cytosines in the unmethylated product were converted to thymines after bisulfite treatment and amplification, which suggests that the MGMT gene is unmethylated. However, the

cytosines in the CpG dinucleotides of methyl-ated product remained unchanged, as methyl-ated cytosines cannot be modified by bisulfite,

which indicated that the CpG islands of the gene are methylated. MSP products were ana-lyzed on an agarose gel for the MGMT gene. MSP yielded interpretable results in 59 (of 60) specimens (98.33%), and one test failed due to the specimen was contaminated by necrotic tissue. MGMT promoter methylation was detected in 45.76% (27/59) specimens (Table 1).

Figure 3. MGMT protein expression was assessed by Western blot. Top figure: representative Western blot. Bottom figure: densitometry; the data are presented as (mean ± SD). Sites A-C displayed high expression, whereas D was low or negative expression. And there are no differences among sites A-C (P>0.05), but site D is significantly lower than sites A-C (*P<0.05) (ANOVA). The test was repeated three times.

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MGMT protein expression

WB and IHC assays were used to determine MGMT protein expression of all specimens (except one sample was contaminated by necrotic tissue). The level of WB test of MGMT expresses as mean integrated gray (

_x±s), range-

ing from 0.03±0.01 to 1.66±0.12. MGMT pro-tein was detected primarily in the nuclei by IHC test. MGMT expression was present in tumor cells with a score of 1, 2, 3, respectively in 1 (42.37%), 2 (37.29%), 3 (20.34%) specimens. MGMT protein expression level assessed by WB was well correlated with IHC (r=0.931, P<0.001). The results were shown in Table 1.

Association between MGMT promoter methyla-tion and its protein expression

There was no significant correlation between MGMT promoter methylation and its protein expression: the relationship between tumor MGMT protein expression by WB and MSP

results was assessed by Mann-Whitney Rank Sum Test, WB and MSP (Z=-1.712 P=0.087); The x2 test and the Fisher’s extact test were used to compare MGMT expression analyzed by IHC with methylation status evaluated by MSP (x2=3.18 P=0.074) (Table 1).

Analysis of the series of intra-tumoral speci-mens

The results of MGMT promoter methylation (evaluated by MSP) and MGMT protein expres-sion (assessed by WB and IHC) analysis of mul-tiple sites within the same tumor are summa-rized in Table 1. There are regional heterogene-ity within the same tumor in 10.00% (2/20) patients’ status of methylation and 40.00% (8/20) patients’ protein expression. For exam-ple, histopathological diagnosis of four speci-mens from different regions (A, B, C, D) of one tumor (patient ID-10) (Figure 1) were glioblas-toma (WHO IV) without obviously necrotic and normal brain tissues (Figure 2). The MGMT pro-

Figure 4. MGMT protein level evaluated by IHC performed on sections from four sites within the same tumor (patient ID-10) displayed three of four sites (A-C) strong nuclear immunoexpression (3), but weaker staining (1) was seen in the site (D) (DAB dyeing, original magnification: 200×). This corresponded with WB analysis.

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tein level was assessed by WB showed sites A, B, C high expression, whereas D was low or no expression. There were no differences among sites A, B, C (P>0.05), but site D is significantly lower than sites A, B, C (P<0.05); Similarly, which was assessed by IHC displayed A, B, C strong immunoexpression (3), but with D no immunostaining (1). MGMT promoter methyla-tion evaluated by MSP demonstrated regions A, B, C unmethylation, whereas D was methyla-tion, this region corresponded with MGMT pro-tein expression assessed by WB and IHC (Table 1; Figures 3-5).

Discussion

O6-methylguanine-DNA methyltransferase (M- GMT) is a key enzyme in the DNA repair net-work, which catalyzes the transfer of the methyl

cantly influence the treatment strategies and prognostic judgement, and interpret the extant controversry.

In contrast to the previous studies, our study is the first investigation aimed at heterogeneity of MGMT in a series of specimens accurately col-lected from different regions within the tumor with the assistance of neuronavigation system in newly diagnosed glioblastomas (WHO IV), in which both tumor MGMT promoter methylation status and MGMT protein expression were simultaneously evaluated. Thus, the combina-tion of the assistance of neuronavigation sys-tem and molecular genetic techniques might be an ideal approach for analyzing the intratu-moral profile of MGMT status in glioblastoma. Our results show that the proportion of tumors exhibiting either MGMT promoter methylation

Figure 5. MGMT promoter methylation was evaluated by MSP. Unmethylation was observed in regions A-C, whereas methylation was observed in region D (patient ID-10). This corresponded with MGMT protein expression assessed by WB and IHC. UC: DNA from normal peripheral blood lymphocytes (PBL) was used as a control for the unmethylated MGMT promoter (U); MC: In vitro, meth-ylated DNA as positive control for methylation (M) and water control for PCR. A DNA maker ladder (DNA Marker A, DMA-50, SBS) was loaded to estimate molecular size, as shown in the left scale.

Figure 6. The repair mechanism of MGMT.

group from O6-methylgua- nine to a cysteine residue of its active site. In this sin-gle step reaction, DNA-lesions caused by alkylat-ing substances are repaired (Figure 6). MGMT subse-quently is irreversibly inac-tivated and degraded [5- 8], thus MGMT is believed to function as the utmost importance for maintaining cell integrity. MGMT gene is located on chromosome 10q26 and encodes the DNA-repair protein MGMT [31]. Nowadays, more and more studies focused on the relationship between MGMT status and chemore-sistance. However, accord-ing to conflicting data hav-ing been reported [12-25], the choice of the ideal pre-dictive marker and the

appropriate detection me- thod is still a matter of debate. In this context, it is vital to elucidate whether the MGMT promoter meth-ylation and MGMT protein expression are homoge-neously distributed or het-erogeneity exists within the same tumor. Finding of het-erogeneity might signifi-

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or MGMT protein expression did not significant-ly differ from previously reported studies [7, 12, 16, 27, 32-34], and MGMT protein expression level assessed by WB was well correlated with which by IHC (r=0.931, P<0.001). However, we found no significant correlation between MGMT protein expression and promoter methylation (all P>0.05). Moreover, this inconsistency was also observed in other neoplasms [35] and not only limited to the MGMT gene [36], so the reg-ulation of MGMT protein expression is a more complex procedure.

Our study has also demonstrated the extent of heterogeneity of MGMT promoter methylation in 10.00% (2/20) patients and MGMT protein expression in 40.00% (8/20) patients ranging from no immunostaining to strong immunoex-pression among different regions of the same tumor. Glioblastoma is known to be heteroge-nous not only at the macroscopic and micro-scopic levels, but also at the molecular level [37, 38], which implies that this heterogeneity is a sampling error that might occur at the time of histological diagnosis, protein level evalutat-ed by IHC and WB or promoter methylation sta-tus analyzed by MSP. In melanoma, a similar variability in MGMT promoter methylation has been reported [39].

There are a number of potential explanations for the lack of correlation between MGMT pro-tein expression and MGMT promoter methyla-tion, and heterogeneity of both among different regions of the same tumor, in which abnormal methylation of the promoter is not the only determining factor [40]. For example, in vitro experiments wild-type p53 seems to act as an inhibitor of MGMT expression, so that tumors with normal p53 would be more likely to have low or absent MGMT expression, independently of promoter methylation [41, 42]. On the other hand, some groups suggest that mutant p53 may be associated with decreased MGMT expression and /or methylation [37, 43], other-wise, some studies displayed loss of heterozy-gosity (LOH) on chromosomes 1p and 19q and MGMT inactivation were relevant [44, 45]. Lavon etal found a significant correlation between the extent of NF-κB activation and MGMT expression in the glioma cell lines and the human glial tumors and showed that it was independent of MGMT promoter methylation. The findings of this study strongly suggest that NF-κB plays a major role in MGMT regulation

[46]. In addition, all these processes of replica-tion and cell growth, angiogenesis, apoptosis, invasion, immunomodulation change, monoal-lelic promoter methylation and methylation of a small proportion of malignant cells are possible influence factors.

Besides above factors, it is very practical diffi-culty to avoid the non-neoplastic components, such as normal glia, microglia, oligodendrog-lias, endothelial cells, ependymal cells, tumor infiltrating lymphocytes and CD64-positive cells et al [47] and unobvious necrosis tissue contaminated. Moreover, the limited number of patients’ specimens, variation in age and observer of test results, the differences in clini-cal treatment (for instance, dexamethasone treatment can upregulate both MGMT mRNA and protein levels of glioma cell lines in vitro study [48]) and so on, all above factors might also interfere with evaluation of MGMT promot-er status and protein expression.

In summary, we found MGMT promoter methyl-ation is probably not the only modulating ele-ment in MGMT protein expression. The regional heterogeneity of both MGMT promoter methyla-tion and its protein expression in the same tumor questions the significance of the guiding role of examination as a result of one specimen collected from tumors for the choice of clinic therapy scheme. Based on our study, the results of molecular genetic and protein analy-sis can not yet be formally considerd for making clinical decision, so it is important to keep in mind of the heterogeneity when deciding on a chemotherapeutic strategy. Whether we can analyze the MGMT status of serum [49, 50] or multi-specimens were collected from different regions with the assistance of neuronavigation system, combing with patients’ age, clinical data et al to guide alkylating agents treatment in newly diagnosed glioblastoma before we come to understand the disease better and identify biologic markers that enable us to pre-dict prognosis and guide clinical chemothera-pies, which need further evaluation in future prospective clinical trials.

Acknowledgements

This work was supported by National Natural Science foundation of china (No. 30571904, 30772228 and 81272782) and College Foundation of Laiwu Hospital Affiliated to

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Taishan Medical College (No. YYLX2013019, YYLX2013016 and YYLX2014011) to which the authors wish to express their thanks. We thank Dr Lei Han and other members of Lab of Neuro-oncology, Tianjin Neurological Institute for their technical assistance.

Disclosure of conflict of interest

None.

Address correspondence to: Yi-Ping Ning, Depart- ment of ICU, Lishui People’s Hospital, No. 15 Dazhong Road, Liandu Area, Lishui 323000, Zhe- jiang Province, P. R. China. Tel: +86-0578-2780104; E-mail: yiping_ning@yeah.net

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