Abstract
O6-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O6-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to November 2013, 22 specimens came from Peking Union Medical College Hospital during September 2004 to April 2010. MGMT protein was detected with EnVision immunohistochemical staining method. Clinicopathological factors were also collected and analyzed. We observed that the overall expression rate of MGMT was 83.9%. Over expression of MGMT protein was not associated with sex, age, functional status, primary tumor location, grading, classification, TNM stage and metastasis (P > 0.05). Kaplan-Meier analysis revealed that there was no significant difference in survival between MGMT-positive and MGMT-negative tumors of GEP-NEN patients (χ2 = 0.887, P = 0.346). In multivariate analyses carried out by Cox proportional hazards regression model, MGMT expression was also not an independent predictors of survival. These results demonstrated that MGMT protein was highly expressed in GEP-NEN. MGMT deficiency rate was similar in pancreatic NEN and in gastrointestinal NEN. MGMT expression was not correlated with prognosis of GEP-NEN.
Keywords: Gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN), O5-methylguanine-DNA methyltransferase (MGMT), immunohistochemisty, survival
Introduction
O6-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme which is critically important in maintaining genomic stability. The suicide enzyme MGMT repairs O6-methylguanine (O6-MG) related mispairing DNA lesion caused by O6-alkylating agents, thus preventing genetic changes leading to malignant transformation of cells. The special anti-carcinogenic mechanisms made MGMT a double-edged sword. On one hand, it protects normal tissue from chemotherapy-induced cell injury or carcinogenesis. On the other hand, MGMT weakens the anti-tumor effect and may cause tumor resistance to guanine O6-alkylating agents [1].
Human MGMT gene stably exists in all normal tissues. Its expression was relatively higher in liver, lymphoid, gastrointestinal-tract and other active site of gene transcription [1,2]. Several studies have found that silencingof MGMT and decreasing of the protein expression could be associated with tumorigenesis [1]. In most tumors, MGMT deficiency rate was about 10%-20%, only a small part, for example gliomas, could reach and exceeded 20% [2,3]. MGMTdeficiency was a strong predictor of favorable treatment and survival in glioblastomas patients treated with temozolomide [4], but no evidence supported survival benefit in those receiving surgery alone [5]. The prognosis predictive value of MGMT varies in different tumors. For instance, low expression of MGMT was regarded as poor prognosis predictor in gastric cancer, hepatocellular carcinoma (HCC) and lung cancer [6-8]. Though, MGMT deficiency predicted poor outcome in breast duct adenocarcinoma [6], the basal-like breast cancer (BLBC) went to the opposite side that positive expression of MGMT was associated with poor survival [9]. Besides, there was no evidence to support MGMT expression correlated with the prognosis in colorectal cancer [10].
Gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) is a group of rare heterogeneous malignancy originating from neuroendocrine cells of the digestive system, mainly in gastrointestinal (GI) tract and pancreas. There wereonly a few small-sample studies reported the expression of MGMT in GEP-NEN at present. A study from North America suggested that MGMT immunohistochemical deficiency was more common in pancreatic neuroendocrine neoplasm (p-NEN) than in gastrointestinal neuroendocrine neoplasm (GI-NEN) (19/37, 51% vs. 0/20, 0%) [11]. A study from Sweden showed that low MGMT expression rate was 43% (10/23) in 23 cases of NEN [12]. Another study from Sweden and Norway reported a relatively low MGMT deficiency rate of 6% (1/17) in neuroendocrine carcinoma (NEC) [13]. As for the prognostic value of MGMT in GEP-NEN, Matthew H. Kulke et al observed a trend toward longer progression-free-survival (PFS) and overall survival (OS) in MGMT-deficient patients compared with MGMT-positive patients treated with temozolomide (median PFS: 19.2 m vs. 9.3 m, P = 0.11; median OS: not reached vs.19.1 m). However, the possibility of independent effect of MGMT on survival cannot be ruled out [11]. So far, there has been no study about the independent prognostic value of MGMT in GEP-NEN. Therefore, we analyzed 174 cases of GEP-NEN patients retrospectively, detecting the expression of MGMT in the tumor tissues and investigating its correlation with sex, age, functional status, primary tumor location, grading, classification, TNM stage, metastasis and prognosis.
Materials and methods
Patients and sample collection
174 paraffin slices of histopathological confirmed GEP-NEN were collected, of which 152 specimens came from the First Affiliated Hospital, Sun Yat-sen University during October 1995 to November 2013, 22 specimens came from Peking Union Medical College Hospital during September 2004 to April 2010. MGMT protein were detected with Envision immunohistochemical staining method, clinicopathological factors were also collected and analyzed. World Health Organization (WHO) 2010 classification (the 4th edition) was adopted to name and classify the GEP-NEN specimens [14]. 2012 European Neuroendocrine Tumor Society (ENETS) TNM staging criteria [15,16] was used to evaluate tumor stage, while 2012 American Joint Committee on Cancer (AJCC) Cancer Staging Atlas [17] served as supplementary when the original site of tumor was not included in ENETS criteria. Patients’ clinicopathological factors were collected, including age at diagnosis, sex, tumor functional status, primary site, tumor grade (G1, G2, G3), tumor type (neuroendocrine tumor, NET; neuroendocrine carcinoma, NEC; mixed adenoneuroendocrine carcinomas, MANEC), TNM stage and metastasis at diagnosis, treatment (radical treatment, palliative treatment, supportive treatment), etc.
Immunohistochemical staining for MGMT and scoring method for its expression
To detect the expression of MGMT in GEP-NEN tumor tissues, immunohistochemical studies were performed on paraffin sections using an EnVision method. Appropriate antibody dilute concentration and antigen repairing method were decided according to preliminary experiment result. MGMT strongly positive stained sections of GEP-NEN patient in preliminary experiment was served as positive control. Phosphate buffer saline (PBS) instead of the first antibody was applied as negative control. Paraffin tissue sections (4 μm) were incubated for 60 min at 60°C, deparaffinized, and rehydrated in graded ethanol solutions to water. After retrieval in the EDTA buffer (1 mmol/L, pH 8.0, ZSGB-BIO, Beijing, China) for 2.5 min, the sections were incubated with a 1:50 dilution of primary mouse anti-MGMT monoclonal antibody (Fuzhou Maxim Biotech, Ltd, Fujian, China) overnight at 4°C. On next morning, the slides were brought to room temperature and exposed to 3% H2O2 for 10-20 min so as to block endogenous peroxidase activity. Antibody reactivity was detected using the DAKO DAB (3.3’-diaminobenzidine) detection system (Dako K5007 HRP/DAB+, Rabbit/Mouse). The sections were incubated with secondary antibody for 30 min at 37°C. DAB staining time was up to 10 min. Then the sections were counterstained with hematoxylin, differentiated by 1% hydrochloric alcohol, washed to blue-back in saturated lithium carbonate, and finally sealed with neutral balsam. Slides were observed at 100 ×, 200 ×, and 400 × using Olympus BX-50 microscope and images were captured under an Olympus DP72 camera (Olympus, Tokyo, Japan) using Olympus DP2-BSW microscope digital-imaging software.
The MGMT positive staining refers to either cytoplasm or nucleus staining to yellow or dark brown. Nonneoplastic cells (lymphocytes, stromal cells, endothelial cells and liver cells) served as an internal positive control in all tissuesections. The immunoreactivity of MGMT was evaluated microscopically under 5 different high-powered fields (× 400) in a blinded fashion by 2 observers. We counted one hundred tumor cells in each area and calculated the mean percentage of positive tumor cells as well as the intensity of staining. The percentage of positive tumor cells was ranked as follows: 0% - 5%, 0; 6% - 25%, 1; 26% - 50%, 2; 51% - 75%, 3; 76% - 100%, 4. The intensity of immunostaining was scored as follows: weak or only cytoplasm stained, 1; moderate, 2; intense, 3. The immunoreactive score (IRS) was the product of the two scores. IRS ≤ 2 were defined as negative and otherwise defined as positive. Among them, 2-5 point represented weak positive (+), 5-9 points represented moderate positive (++), and above 9 points represented strong positive (+++) [18]. If the result was difficult to judge, the paraffin would be cut and stained again.
Statistical analysis
Statistical analyses were performed using SPSS software (version 16.0). Measurement data with normal distribution were reported as the mean ± standard error (X̅ ± S). For enumeration data, the constituent ratio was reported. Patient’s survival was described with median survival time and survival rate. The clinicopathologic characteristics were compared with MGMT-positive and MGMT-negative groups, and the significance of associations was determined with chi-square test. Survival was defined as the time from diagnosis of GEP-NEN until death. NEN-related deaths were considered as events for survival analysis. Patients who died of other reasons or alive at the last follow-up were censored. Overall survival analyses were performed using the Kaplan-Meier method and Cox regression analyses. The log-rank test was used for univariate analyses of potentially prognostic factors. P value < 0.05 was considered statistically significant; all tests were 2-sided.
Ethics
The study was approved by the ethics committee of the First Affiliated Hospital Sun Yat-sen University and complied with the Declaration of Helsinki. Informed consent was obtained from each patient before the study.
Results
Clinicopathological features
In total, 174 patients with histologically confirmed sporadic GEP-NEN were analyzed retrospectively. 100 (57.5%) were men and 74 (42.5%) were women. The mean age at initial diagnosis was 49.6 ± 13.8 years (range 18-85 years). 127 tumors (73.0%) were nonfunctional, while 47 patients (27.0%) had functional tumors, including 36 insulinoma, 1 glucagonoma, 2 vasoactive intestinal peptide tumor, 2 gastrinoma, 2 p-NEN with diarrhea and 4 small intestine NEN with flushing or diarrhea. Primary tumors were pancreas (74/174, 42.5%), gastrointestinal tract (83/174, 47.7%) and other (17/174, 9.8%). In detail, GI-tract NEN consisted of esophagus (4/178, 2.3%), stomach (20/174, 11.5%), duodenum (14/174, 8.0%), jejunum-ileum (7/174, 4.0%), appendix (3/174, 1.7%), colon and rectum (35/174, 20.1%). Other sites included gallbladder (3/174, 1.7%), common bile duct (1/174, 0.6%), Vater’s ampulla (2/174, 1.1%), liver (2/174, 1.1%) and unknown primary (9/174, 5.2%). The numbers of G1, G2, G3 tumors were 86 (49.4%), 41 (23.6%), 47 (27.0%), respectively. As for tumor type, neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC) and mixed adenoendocrine carcinoma (MANEC) accounted for 70.3% (127/174), 25.3% (44/174) and 1.7% (3/174), respectively. Acording to ENET and AJCC TNM staging criteria, 49 patients (28.2%) were on stage I, 38 (21.8%) stage II, 22 (12.6%) stage III and 65 (42.1%) stage IV. 65 (42.1%) patients had distant meta- stases when diagnosed, most of them (58/65, 89.2%) were liver metastases. 137 out of 174 patients received long-term follow up from the time of diagnosis to February 28, 2014. The median follow-up time was 2.65 years (range: 14+ days - 11.4 years).
MGMT expression profiles
The overall expression rate of MGMT was 83.9% in the tumor tissue of 174 GEP-NEN patients. The yellow brown staining granules majorly distributed in tumoral nuclei, few in cytoplasm. 28 cases (16.1%) showed negative MGMT immunoexpression, others included 27 cases (15.1%) of weak positive (+), 49 cases (28.2%) of moderate positive (++) and 70 cases (40.2%) of strong positive (+++). Totally 55 tumors (31.6%) showed low-level MGMT immunoexpression (Figure 1).
Figure 1.
Immunohistochemical staining of MGMT in GEP-NEN (EnVision). A. p-NET, G1, MGMT negative (-); B. p-NET, G2, MGMT weak positive (+); C. Rectal NET, G1, MGMT moderate positive (++); D. Ileum NET, G1, MGMT strong positive (+++) (× 200).
Relationship between expression of MGMT and clinicopathological factors
None of the characteristics analyzed, including sex, age, tumor functional status, primary site, tumor grade, tumor type, tumor stage and metastasis status, was significantly correlated with MGMT expression (P > 0.05, Table 1). Particularly, 60 of 74 cases (81.8%) in p-NEN exhibited positive MGMT expression, were not significantly less compared with 71 of 83 cases (85.5%) in GI-NEN (χ2 = 0.030, P = 0.863 > 0.05). In addition, MGMT expression positive percentage of grade 1, grade 2 and grade 3 GEP-NEN tissues were 84.9%, 78.0%, 87.2%, respectively, there being no significant difference (P > 0.05).
Table 1.
Correlation between clinicopathological factors and expression of MGMT in GEP-NEN (N = 174)
| Characteristics | n | MGMT | Positive rate (%) | χ2 | P value | |||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| - | + | ++ | +++ | |||||
| Sex | 1.602 | 0.206 | ||||||
| Male | 100 | 18 | 13 | 30 | 39 | 82.0 | ||
| Female | 74 | 10 | 14 | 19 | 31 | 86.6 | ||
| Age(years) | 0.287 | 0.592 | ||||||
| < 50 | 85 | 15 | 12 | 25 | 33 | 82.4 | ||
| ≥ 50 | 89 | 13 | 15 | 24 | 37 | 85.4 | ||
| Functional status | 0.525 | 0.469 | ||||||
| Nonfunctional | 127 | 22 | 22 | 37 | 46 | 82.7 | ||
| Functional | 47 | 6 | 5 | 12 | 24 | 87.2 | ||
| Site | 0.031 | 0.985 | ||||||
| GI tract | 83 | 12 | 11 | 25 | 35 | 85.5 | 0.030 | 0.863* |
| Pancreas | 74 | 14 | 11 | 21 | 28 | 81.1 | ||
| Other | 17 | 2 | 5 | 3 | 7 | 88.2 | ||
| Tumor grade | 1.418 | 0.492 | ||||||
| G1 | 86 | 13 | 11 | 26 | 36 | 84.9 | ||
| G2 | 41 | 9 | 7 | 10 | 15 | 78.0 | ||
| G3 | 47 | 6 | 9 | 13 | 19 | 87.2 | ||
| Tumor type | 0.525 | 0.469 | ||||||
| NET | 127 | 22 | 18 | 36 | 51 | 82.7 | ||
| NEC | 44 | 6 | 9 | 12 | 17 | 86.4 | ||
| MANEC | 3 | 0 | 0 | 1 | 2 | 100.0 | ||
| Tumor stage | 0.153 | 0.696 | ||||||
| I + II | 87 | 13 | 11 | 25 | 38 | 85.1 | ||
| III + IV | 87 | 13 | 14 | 23 | 32 | 82.8 | ||
| Metastasis | 0.098 | 0.755 | ||||||
| No | 109 | 17 | 13 | 32 | 47 | 84.4 | ||
| Yes | 65 | 11 | 14 | 17 | 23 | 83.1 | ||
Abbreviations: GI tract, gastrointestinal tract; NET, neuroendocrine tumor; NEC, neuroendocrine carcinoma; MANEC, mixed adenoneuroendocrine carcinomas.
The χ2 and P value were computed by the contrast between gastrointestinal tract and pancreas.
The primary and metastasis tumor tissues were both studied in 6 patients. At metastasis lesions, MGMT immunoexpression were stronger in 3 specimens, deficient in 2 and no change in 1 when compared with the primary foci (Table 2).
Table 2.
MGMT expression in primary and metastasis site (N = 6)
| NO. | Primary | Metastasis | MGMT | ||||
|---|---|---|---|---|---|---|---|
|
|
|
|
|||||
| Site | Grade | Site | Grade | Primary | Metastasis | Change | |
| 1 | Pancreas | 2 | Liver | 2 | - | + | Stronger |
| 2 | Pancreas | 1 | Scalp | 2 | ++ | +++ | Stronger |
| 3 | Stomach | 3 | Liver | 3 | ++ | +++ | Stronger |
| 4 | Ileum | 1 | Liver | 1 | +++ | +++ | Unchanged |
| 5 | Jejunum | 3 | Liver | 3 | ++ | - | Deficient |
| 6 | Pancreas | 2 | Liver | 2 | +++ | - | Deficient |
Survival and prognostic factors
During the observation period, 32 patients had died at the last follow-up (23.4%, 32/137). Two of them died of postoperative complications, other 30 patients’ death were associated with GEP-NEN. Median survival was not reached during the observation period. As univariate analysis showed, the overall 1-, 2- and 5-year survival rates between MGMT negative and positive group were 96% and 88%, 91% and 80%, 84% and 71%, respectively, and there was no statistical significance between the two groups (χ2 = 0.887, P = 0.346). MGMT expression status of GEP-NEN and clinicopathologic variables were introduced in Cox’s model to identify independent prognostic factors of survival. Multivariate analysis for overall survival drew the same conclusion as the Kaplan-Meier method done that the expression status of MGMT was not a predictor of survival in patients with GEP-NEN. However, tumor type, TNM stage and treatment were the major prognostic factors. Survival curves were displayed in Figure 2. Multivariate Cox proportional hazard model were provided in Table 3.
Figure 2.
A. Kaplan-Meier survival curves (N =137); B. Cox Multivariate analysis survival curve (N = 137).
Table 3.
Multivariate Cox proportional hazard model (N =137)
| Characteristic | B | SE | P | OR | OR 95% CIa |
|---|---|---|---|---|---|
| Tumor type (NEC+MANEC vs. NET) | 1.115 | 0.393 | 0.005 | 3.049 | 1.411-6.589 |
| Stage (III+IV vs. I+II) | 3.121 | 1.083 | 0.004 | 22.668 | 2.712-189.478 |
| Treatment (vs. radical treatment) | 0.000 | ||||
| palliative treatment | -0.074 | 0.434 | 0.865 | 0.929 | 0.397-2.174 |
| supportive treatment | 3.554 | 0.805 | 0.000 | 34.947 | 7.212-169.335 |
Abbreviations: NET, neuroendocrine tumor; NEC, neuroendocrine carcinoma; MANEC, mixed adenoneuroendocrine carcinomas.
CI, confidence interval.
Discussion
As the critical enzyme to repair O6-mG related DNA lesion, MGMT has double-acting effect when defending against mutation caused by O6-alkylating agents. MGMT gene silence and MGMT protein deficiency was confirmed to be associated with tumorigenesis, and was regarded as a predictor of treatment response to a given therapy in glioma and melanoma. Until now, we know only little about the expression of MGMT in GEP-NEN according to several small sample studies. Our study systematically examined the expression of MGMT in 174 cases of GEP-NEN tissues and analyzed its relationship with the clinical features and prognosis.
Previous studies from Europe and America reported that MGMT negative or low expression rate in GEP-NEN ranged from 6% to 43% [11-13]. Moreover, MGMT deficiency rate was significantly higher in p-NEN (51%) than in GI-NEN (0%) [11]. In our study, overall MGMT deficiency rate was 16.1% (28/174) in GEP-NEN. Low expression rate (include negative and weak positive) was 31.6% (55/174), which was similar to the low expression rate in esophageal cancer, gastric cancer, and colorectal cancer. MGMT deficiency rate was slightly higher in p-NEN (18.9%, 14/74) compared with GI-NEN (14.5%, 12/83), but the difference was not statistically significant. Our study was the first study detected the expression of MGMT in Chinese GEP-NEN patients with a relatively large sample (N = 174). We found no significant difference of MGMT expression between p-NEN and GI-NEN. The characteristic of MGMT immunoexpression in Chinese GEP-NEN patients was different from previous studies. The different expression pattern of MGMT in GEP-NEN between Chinese patients and patients in western countries may be due to race differences, but it still need to be confirmed by further studies.
MGMT expression deficiency was associated with an improvement in survival among patients with melanoma and glioblastoma treated with temozolomide [5,19]. Several studies from Europe and America also found that MGMT deficient GEP-NEN patients would have better response to temozolomide-based chemotherapy, and would have higher objective response rate (ORR) [11,13]. Especially for p-NEN, ORR can reach 33%-70% [11,12,20-23], much higher than the ORR of GI-NEN (0%-7%) [11,20,23]. Thus temozolomide was recommended as one of the second-line treatment for p-NEN in European and American NEN guidelines. The better treatment effect of temozolomide in p-NEN may due to the high deficiency rate of MGMT in p-NEN patients. However, in our study, MGMT deficiency rate was similar in p-NEN and GI-NEN, thus we suppose that in Chinese patients, temozolomide may have comparable effect on p-NEN and GI-NEN.
The expression of MGMT was highly heterogeneous. MGMT protein was highly expressed in some solid tumors of brain, breast, colon, bladder, gastric and lung, but was expressed lower in other tumors, for example, testis and livercancer. The expression level of MGMT also varies in different stage of disease and different stage of treatment. In basal-like breast cancer and ovarian cancer, MGMT activity increased with the disease progression, and the level of MGMT declined during the treatment of alkylating drugs in glioma. Moreover, previous studies even showed that in melanoma, the expression of MGMT was different in primary and metastases tumors [2]. In our study, we investigatedthe MGMT expression in 6 patients with both primary and metastasis tumor specimens. 5 of 6 had different MGMT expression between primary and metastasis tissues. Among them, 3 patients had stronger MGMT expression in metastasis, 2 had MGMT deficiency in metastasis while primary foci was positive (Table 2). It is noteworthy that NO.5 patient, jejunum NEC with liver metastasis, whose primary tumor expressed MGMT but liver metastasis had MGMT deficiency, acquired partial remission from second-line chemotherapy with capecitabine and temozolomide. It suggested that when we choose temozolomide to treat NEN patients, it is necessary to detect the expression of MGMT in both primary and metastasis tumors.
The independent prognostic value of MGMT varies in different tumors. For high-grade gliomas, MGMT was not related to prognosis for the patients receiving surgery alone [5]. In gastric cancers, MGMT-negative tumors invaded deeper into the stomach wall, easier to have lymph node and distant metastasis, had a higher ratio of diffuse type, and had a poor outcome [6]. In HCC, the overall 5-year survival rate of MGMT-negative patients was lower than thatof MGMT-positive patients (46.9% vs. 89.1%, P = 0.005), and multivariate analysis of survival confirmed MGMT deficiency was an independent predictor of poor prognosis [6]. In breast infiltrative ductal carcinoma, MGMT-negative tumors had a higher recurrence rate (6/21 vs. 0/25, P = 0.004), and shorter 10-year relapse-free survival rates (35.7% vs. 100%, P < 0.001) than those of MGMT-positive tumors [6]. However in another type of breast cancer, the basal-like breast cancer, MGMT-positive was a poor prognosis predictor. The recurrence rate in MGMT-positive tumors had increased 2.81 time (95% HR CI 1.18-6.72, P = 0.02) than MGMT-negative tumors [9]. In colorectal cancer, MGMT status was not correlated with prognosis [6]. As far as we known, our study was the first study explored the prognosis value of MGMT in GEP-NEN patients. We analyzed 137 cases of GEP-NEN patients who acquired long-time follow-up, using both Kaplan-Meier method and Cox Multivariate analysis of survival, the expression of MGMT was not related to patients’ overall survival. Thus MGMT may not be an independent prognostic factor in patients with GEP-NEN.
In conclusion, our study for the first time demonstrated that MGMT protein was highly expressed in both p-NEN and GI-NEN in Chinese patients. The expression of MGMT was not a prognostic factor in GEP-NEN patients.
Acknowledgements
The authors are grateful to Drs. Zeng Jing and Lu Jiabin (Department of Pathology, Sun Yat-Sen University Cancer Center) for skillful technical assistance.
Disclosure of conflict of interest
None.
References
- 1.Sharma S, Salehi F, Scheithauer BW, Rotondo F, Syro LV, Kovacs K. Role of MGMT in tumor development, progression, diagnosis, treatment and prognosis. Anticancer Res. 2009;29:3759–3768. [PubMed] [Google Scholar]
- 2.Christmann M, Verbeek B, Roos WP, Kaina B. O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: enzyme activity, promoter methylation and immunohistochemistry. Biochim Biophys Acta. 2011;1816:179–190. doi: 10.1016/j.bbcan.2011.06.002. [DOI] [PubMed] [Google Scholar]
- 3.Brell M, Ibanez J, Tortosa A. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction. BMC Cancer. 2011;11:35. doi: 10.1186/1471-2407-11-35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JE, Hau P, Mirimanoff RO, Cairncross JG, Janzer RC, Stupp R. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352:997–1003. doi: 10.1056/NEJMoa043331. [DOI] [PubMed] [Google Scholar]
- 5.Olson RA, Brastianos PK, Palma DA. Prognostic and predictive value of epigenetic silencing of MGMT in patients with high grade gliomas: a systematic review and meta-analysis. J Neurooncol. 2011;105:325–335. doi: 10.1007/s11060-011-0594-5. [DOI] [PubMed] [Google Scholar]
- 6.Matsukura S, Miyazaki K, Yakushiji H, Ogawa A, Harimaya K, Nakabeppu Y, Sekiguchi M. Expression and prognostic significance of O6-methylguanine-DNA methyltransferase in hepatocellular, gastric, and breast cancers. Ann Surg Oncol. 2001;8:807–816. doi: 10.1007/s10434-001-0807-9. [DOI] [PubMed] [Google Scholar]
- 7.Wu PF, Kuo KT, Kuo LT, Lin YT, Lee WC, Lu YS, Yang CH, Wu RM, Tu YK, Tasi JC, Tseng HM, Tseng SH, Cheng AL, Lin CH. O(6)-Methylguanine-DNA methyltransferase expression and prognostic value in brain metastases of lung cancers. Lung Cancer. 2010;68:484–490. doi: 10.1016/j.lungcan.2009.08.010. [DOI] [PubMed] [Google Scholar]
- 8.Liu Y, Wang J, Zhang H, Fu XM, Wang HQ. The expression of O6-methylguanine DNA methyltransferase and p53 in non-small cell lung cancer and the association with the prognosis. Zhonghua Jie He He Hu Xi Za Zhi. 2010;33:427–431. [PubMed] [Google Scholar]
- 9.Alkam Y, Mitomi H, Nakai K, Himuro T, Saito T, Takahashi M, Arakawa A, Yao T, Saito M. Protein expression and methylation of DNA repair genes hMLH1, hMSH2, MGMT and BRCA1 and their correlation with clinicopathological parameters and prognosis in basal-like breast cancer. Histopathology. 2013;63:713–725. doi: 10.1111/his.12220. [DOI] [PubMed] [Google Scholar]
- 10.Shima K, Morikawa T, Baba Y, Nosho K, Suzuki M, Yamauchi M, Hayashi M, Giovannucci E, Fuchs CS, Ogino S. MGMT promoter methylation, loss of expression and prognosis in 855 colorectal cancers. Cancer Causes Control. 2011;22:301–309. doi: 10.1007/s10552-010-9698-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kulke MH, Hornick JL, Frauenhoffer C, Hooshmand S, Ryan DP, Enzinger PC, Meyerhardt JA, Clark JW, Stuart K, Fuchs CS, Redston MS. O6-methylguanine DNA methyltransferase deficiency and response to temozolomide-based therapy in patients with neuroendocrine tumors. Clin Cancer Res. 2009;15:338–345. doi: 10.1158/1078-0432.CCR-08-1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ekeblad S, Sundin A, Janson ET, Welin S, Granberg D, Kindmark H, Dunder K, Kozlovacki G, Orlefors H, Sigurd M, Oberg K, Eriksson B, Skogseid B. Temozolomide as monotherapy is effective in treatment of advanced malignant neuroendocrine tumors. Clin Cancer Res. 2007;13:2986–2991. doi: 10.1158/1078-0432.CCR-06-2053. [DOI] [PubMed] [Google Scholar]
- 13.Welin S, Sorbye H, Sebjornsen S, Knappskog S, Busch C, Oberg K. Clinical effect of temozolomide-based chemotherapy in poorly differentiated endocrine carcinoma after progression on first-line chemotherapy. Cancer. 2011;117:4617–4622. doi: 10.1002/cncr.26124. [DOI] [PubMed] [Google Scholar]
- 14.Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO classification of tumours of the digestive system. 4th edition. Lyon: nternational Agency for Research on Cancer Press; 2010. p. 417. [Google Scholar]
- 15.Rindi G, Kloppel G, Alhman H, Caplin M, Couvelard A, de Herder WW, Erikssson B, Falchetti A, Falconi M, Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O, Perren A, Scarpa A, Scoazec JY, Wiedenmann B. TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2006;449:395–401. doi: 10.1007/s00428-006-0250-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Rindi G, Kloppel G, Couvelard A, Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O, Perren A, Scarpa A, Scoazec JY, Wiedenmann B. TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2007;451:757–762. doi: 10.1007/s00428-007-0452-1. [DOI] [PubMed] [Google Scholar]
- 17.Compton CC, Byrd DR, Garcia-Aguilar J, Kurtzman SH, Olawaiye A, Washington MK. AJCC Cancer Staging Atlas. Springer; 2012. [Google Scholar]
- 18.Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000;6:127–134. [PubMed] [Google Scholar]
- 19.Busch C, Geisler J, Lillehaug JR, Lonning PE. MGMT expression levels predict disease stabilisation, progression-free and overall survival in patients with advanced melanomas treated with DTIC. Eur J Cancer. 2010;46:2127–2133. doi: 10.1016/j.ejca.2010.04.023. [DOI] [PubMed] [Google Scholar]
- 20.Kulke MH, Stuart K, Enzinger PC, Ryan DP, Clark JW, Muzikansky A, Vincitore M, Michelini A, Fuchs CS. Phase II study of temozolomide and thalidomide in patients with metastatic neuroendocrine tumors. J Clin Oncol. 2006;24:401–406. doi: 10.1200/JCO.2005.03.6046. [DOI] [PubMed] [Google Scholar]
- 21.Strosberg JR, Fine RL, Choi J, Nasir A, Coppola D, Chen DT, Helm J, Kvols L. First-line chemotherapy with capecitabine and temozolomide in patients with metastatic pancreatic endocrine carcinomas. Cancer. 2011;117:268–275. doi: 10.1002/cncr.25425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Fine RL, Gulati AP, Krantz BA, Moss RA, Schreibman S, Tsushima DA, Mowatt KB, Dinnen RD, Mao Y, Stevens PD, Schrope B, Allendorf J, Lee JA, Sherman WH, Chabot JA. Capecitabine and temozolomide (CAPTEM) for metastatic, well-differentiated neuroendocrine cancers: The Pancreas Center at Columbia University experience. Cancer Chemother Pharmacol. 2013;71:663–670. doi: 10.1007/s00280-012-2055-z. [DOI] [PubMed] [Google Scholar]
- 23.Chan JA, Stuart K, Earle CC, Clark JW, Bhargava P, Miksad R, Blaszkowsky L, Enzinger PC, Meyerhardt JA, Zheng H, Fuchs CS, Kulke MH. Prospective study of bevacizumab plus temozolomide in patients with advanced neuroendocrine tumors. J Clin Oncol. 2012;30:2963–2968. doi: 10.1200/JCO.2011.40.3147. [DOI] [PMC free article] [PubMed] [Google Scholar]