MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy

Journal of Translational Medicine

Antonia Aránega⁶  Fernando Rodríguez-Serrano⁶  Rodrigo López¹  Jaime Antonio Oliver³  Gloria Perazzoli³  Roberto Madeddu⁵  Pablo Juan Alvarez³  Angel Concha¹  Raul Ortiz⁴  Beatriz González²  Jose Prados⁶  Consolación Melguizo⁶ 

[1] Anatomopathological Service, Virgen de las Nieves Hospital, Granada, 18014, Spain;Service of Medical Oncology, Virgen de las Nieves Hospital, Granada, 18014, Spain;Institute of Biopathology and Regenerative Medicine (IBIMER), Granada 18100, Spain;Department of Health Science, University of Jaén, Jaén, 23071, Spain;National Institute of Biostructures and Biosystems, INBB, Sassari, Italy;Department of Anatomy and Embriology, University of Granada, Granada 18012, Spain

关键词: CD133;    Methylation;    MGMT;    Temozolomide;    Radiotherapy;    Glioblastoma;   

Others  :  828310
DOI  :  10.1186/1479-5876-10-250

 received in 2012-10-21, accepted in 2012-12-10,  发布年份 2012

【 摘 要 】

Background

The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM). The O⁶-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated.

Methods

Seventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan–Meier method.

Results

The MGMT gene promoter was found to be methylated in 34 patients (44.7%) and unmethylated in 42 patients (55.3%). A significant correlation was observed between MGMT promoter methylation and patients’ survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative.

Conclusions

Our results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these patients. Future studies will be necessary to determine its clinical utility.

【 授权许可】

   
2012 Melguizo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Quick A, Patel D, Hadziahmetovic M, Chakravarti A, Mehta M: Current therapeutic paradigms in glioblastoma. Rev Recent Clin Trials 2010, 5:14-27.
• [2]Brennand J, Margison GP: Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene. Proc Natl Acad Sci USA 1986, 83:6292-6296.
• [3]Weller M, Stupp R, Reifenberger G, Brandes AA, Van den Bent MJ, Wick W, Hegi ME: MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 2010, 6:39-51.
• [4]Hegi ME, Diserens C, Gorila 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.
• [5]Bleeker FE, Molenaar RJ, Leenstra S: Recent advances in the molecular understanding of glioblastoma. J Neurooncol 2012, 108:11-27.
• [6]Okita Y, Narita Y, Miyakita Y, Ohno M, Fukushima S, Kayama T, Shibui S: Pathological findings and prognostic factors in recurrent glioblastomas. Brain Tumor Pathol 2012.
• [7]Nakasu S, Fukami T, Baba K, Matsuda M: Immunohistochemical study for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas. J Neurooncol 2004, 70:333-334.
• [8]Cao VT, Jung TY, Jung S, Jin SG, Moon KS, Kim IY, Kang SS, Park CS, Lee KH, Chae HJ: The correlation and prognostic significance of MGMT promoter methylation and MGMT protein in glioblastomas. Neurosurgery 2009, 65:866-875.
• [9]Nakagawa T, Ido K, Sakuma T, Takeuchi H, Sato K, Kubota T: Prognostic significance of the immunohistochemical expression of O6-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas. Neuropathology 2009, 29:379-388.
• [10]Sonoda Y, Yokosawa M, Saito R, Kanamori M, Yamashita Y, Kumabe T, Watanabe M, Tominaga T: O(6)-Methylguanine DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression is correlated with progression-free survival in patients with glioblastoma. Int J Clin Oncol 2010, 15:352-358.
• [11]Preusser M, Charles Janzer R, Felsberg J, Reifenberger G, Hamou MF, Diserens AC, Stupp R, Gorlia T, Marosi C, Heinzl H, Hainfellner JA, Hegi M: Anti‐O6‐methylguanine methyltransferase (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as clinical biomarker. Brain Pathol 2008, 18:520-532.
• [12]Reya T, Morrison SJ, Clarke MF, Weissman IL: Stem cells, cancer, and cancer stem cells. Nature 2001, 414:105-111.
• [13]Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB: Identification of a cancer stem cell in human brain tumors. Cancer Res 2003, 63:5821-5828.
• [14]Liu L, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, Lu L, Irvin D, Black KL, Yu JS: Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 2006, 5:67-78. BioMed Central Full Text
• [15]Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444:756-760.
• [16]Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, Baylin SB, Herman JG: Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000, 343:1350-1354.
• [17]Macdonald DR, Cascino TL, Schold SC, Cairncross JG: Response criteria for phase II studies of supratentorial malignant glioma. J Clin Oncol 1990, 8:1277-1280.
• [18]Stupp R, Mason WP, Van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352:987-996.
• [19]Cairncross G, Berkey B, Shaw E, Jenkins R, Scheithauer B, Brachman D, Buckner J, Fink K, Souhami L, Laperierre N, Mehta M, Curran W: Phase III trial of chemotherapy plus radiotherapy compared with radiotherapy alone for pure and mixed anaplastic oligodendroglioma: Intergroup Radiation Therapy Oncology Group Trial 9402. J Clin Oncol 2006, 24:2707-2714.
• [20]Stupp R, Hegi ME, Mason WP, Van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO: Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10:459-466.
• [21]Paz MF, Yaya-Tur R, Rojas-Marcos I, Reynes G, Pollan M, Aguirre-Cruz L, Garcia-Lopez JL, Piquer J, Safont MJ, Balana C, Sanchez-Cespedes M, Garcia-Villanueva M, Arribas L, Esteller M: CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas. Clin Cancer Res 2004, 10:4933-4938.
• [22]Criniere E, Kaloshi G, Laigle-Donadey F, Lejeune J, Auger N, Benouaich-Amiel A, Everhard S, Mokhtari K, Polivka M, Delattre JY, Hoang-Xuan K, Thillet J, Sanson M: MGMT prognostic impact on glioblastoma is dependent on therapeutic modalities. J Neurooncol 2007, 83:173-179.
• [23]Weller M, Felsberg J, Hartmann C, Berger H, Steinbach JP, Schramm J, Westphal M, Schackert G, Simon M, Tonn JC, Heese O, Krex D, Nikkhah G, Pietsch T, Wiestler O, Reifenberger G, Von Deimling A, Loeffler M: Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma: a prospective translational study of the German Glioma Network. J Clin Oncol 2009, 27:5743-5750.
• [24]Minniti G, Salvati M, Arcella A, Buttarelli F, D’Elia A, Lanzetta G, Esposito V, Scarpino S, Maurizi Enrici R, Giangaspero F: Correlation between O6-methylguanine-DNA methyltransferase and survival in elderly patients with glioblastoma treated with radiotherapy plus concomitant and adjuvant temozolomide. J Neurooncol 2011, 102:311-316.
• [25]Kim YS, Kim SH, Cho J, Kim JW, Chang JH, Kim DS, Lee KS, Suh CO: MGMT gene promoter methylation as a potent prognostic factor in glioblastoma treated with temozolomide-based chemoradiotherapy: a single-institution study. Int J Radiat Oncol Biol Phys 2012.
• [26]Brandes AA, Tosoni A, Franceschi E, Sotti G, Frezza G, Amista P, Morandi L, Spagnolli F, Ermani M: Recurrence pattern alter temozolomide concomitant with and adjuvant to radiotherapy in newly diagnosed patients with glioblastoma: correlation with MGMT promoter methylation status. J Clin Oncol 2009, 27:1275-1279.
• [27]Brandes AA, Franceschi E, Tosoni A, Bartolini S, Bacci A, Agati R, Ghimenton C, Turazzi S, Talacchi A, Skrap M, Marucci G, Volpin L, Morandi L, Pizzolitto S, Gardiman M, Andreoli A, Calbucci F, Ermani M: O(6)-methylguanine DNA-methyltransferase methylation status can change between first surgery for newly diagnosed glioblastoma and second surgery for recurrence: clinical implications. Neuro Oncol 2010, 12:283-288.
• [28]Rivera AL, Pelloski CE, Gilbert MR, Colman H, De La Cruz C, Sulman EP, Bekele BN, Aldape KD: MGMT promoter methylation is predictive of response to radiotherapy and prognostic in the absence of adjuvant alkylating chemotherapy for glioblastoma. Neuro Oncol 2010, 12:116-121.
• [29]Gilbert MR, Wang M, Aldape KD, Stupp R, Hegi M, Jaeckle KA, Armstrong TS, Wefel JS, Won M, Blumenthal DT, Mahajan A, Schultz CJ, Erridge SC, Brown PD, Chakravarti A, Curran WJ, Mehta MP: RTOG 0525: a randomized phase IIII trial comparing standard adjuvant temozolomide (TMZ) with a dose-dense (dd) schedule in newly diagnosed glioblastoma (GBM) [abstract]. J Clin Oncol 2011., 29suppl; abstr 2006
• [30]Blanc JL, Wager M, Guilhot J, Kusy S, Bataille B, Chantereau T, Lapierre F, Larsen CJ, Karayan-Tapon L: Correlation of clinical features and methylation status of MGMT gene promoter in glioblastomas. J Neurooncol 2004, 68:275-283.
• [31]Yachi K, Watanabe T, Ohta T, Fukushima T, Yoshino A, Ogino A, Katayama Y, Nagase H: Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas. Int J Oncol 2008, 33:469-475.
• [32]Tang K, Jin Q, Yan W, Zhang W, You G, Liu Y, Jiang T: Clinical correlation of MGMT protein expression and promoter methylation in Chinese glioblastoma patients. Med Oncol 2012, 29:1292-1296.
• [33]Christians A, Hartmann C, Benner A, Meyer J, von Deimling A, Weller M, Wick W, Weiler M: Prognostic value of three different methods of MGMT promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma. PLoS One 2012, 7:e33449.
• [34]Dunn J, Baborie A, Alam F, Joyce K, Moxham M, Sibson R, Crooks D, Husband D, Shenoy A, Brodbelt A, Wong H, Liloglou T, Haylock B, Walker C: Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy. Br J Cancer 2009, 101:124-131.
• [35]Chinot OL, Barrie M, Fuentes S, Eudes N, Lancelot S, Metellus P, Muracciole X, Braguer D, Ouafik L, Martin PM, Dufour H, Figarella-Branger D: Correlation between O6-methylguanine-DNA methyltransferase and survival in inoperable newly diagnosed glioblastoma patients treated with neoadyuvant temozolomide. J Clin Oncol 2007, 25:1470-1475.
• [36]Nakasu S, Fukami T, Jito J, Matsuda M: Prognostic significance of loss of O6-methylguanine-DNA methyltransferase expression in supratentorial diffuse low-grade astrocytoma. Surg Neurol 2007, 68:603-608.
• [37]Capper D, Mittelbronn M, Meyermann R, Schittenhelm J: Pitfalls in the assessment of MGMT expression and in its correlation with survival in diffuse astrocytomas: proposal of a feasible immunohistochemical approach. Acta Neuropathol 2008, 115:249-259.
• [38]Watanabe R, Nakasu Y, Tashiro H, Mitsuya K, Ito I, Nakasu S, Nakajima T: O6-methylguanine DNA methyltransferase expression in tumor cells predicts outcome of radiotherapy plus concomitant and adjuvant temozolomide therapy in patients with primary glioblastoma. Brain Tumor Pathol 2011, 28:127-135.
• [39]Arazi Y, Mizoguchi M, Yoshimoto K, Shono T, Amano T, Nakamizo A, Suzuki SO, Iwaki T, Sasaki T: Quantitative digital assessment of MGMT immunohistochemical expression in glioblastoma tissue. Brain Tumor Pathol 2011, 28:25-31.
• [40]Rodriguez FJ, Thibodeau SN, Jenkins RB, Schowalter KV, Caron BL, O’Neill BP, James CD, Passe S, Slezak J, Giannini C: MGMT immunohistochemical expression and promoter methylation in human glioblastoma. Appl Immunohistochem Mol Morphol 2008, 16:59-65.
• [41]Uno M, Oba-Shinjo SM, Camargo AA, Moura RP, Aguiar PH, Cabrera HN, Begnami M, Rosemberg S, Teixeira MJ, Marie SK: Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma. Clinics 2011, 66:1747-1755.
• [42]Kreth S, Thon N, Eigenbrog S, Lutz J, Ledderose C, Egensperger R, Tonn JC, Kretzschmar HA, Hinske LC, Kreth FW: O-methylguanine-DNA methyltransferase (MGMT) mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation. PloS One 2011, 18:e17156.
• [43]Shah N, Lin B, Sibenaller Z, Ryken T, Lee H, Yoon JG, Rostad S, Foltz G: Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM. PLoS One 2011, 6:e16146.
• [44]Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL: Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 2000, 97:14720-14725.
• [45]Beier D, Wischhusen J, Dietmaier W, Hau P, Proescholdt M, Brawanski A, Bogdahn U, Beier CP: CD133 expression and cancer stem cells predict prognosis in high-grade oligodendroglial tumors. Brain Pathol 2008, 18:370-377.
• [46]Zhang M, Song T, Yang L, Chen R, Wu L, Yang Z, Fang J: Nestin and CD133: valuable stem cell-specific markers for determining clinical outcome of glioma patients. J Exp Clin Cancer Res 2008, 27:85-92. BioMed Central Full Text
• [47]Zeppernick F, Ahmadi R, Campos B, Dictus C, Helmke BM, Becker N, Lichter P, Unterberg A, Radlwimmer B, Herold-Mende CC: Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 2008, 14:123-129.
• [48]Murat A, Migliavacca E, Gorlia T, Lambiv WL, Shay T, Hamou MF, de Tribolet N, Regli L, Wick W, Kouwenhoven MC, Hainfellner JA, Heppner FL, Dietrich PY, Zimmer Y, Cairncross JG, Janzer RC, Domany E, Delorenzi M, Stupp R, Hegi ME: Stem cell-related “self-renewal” signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J Clin Oncol 2008, 26:3015-3024.
• [49]Metellus P, Nanni-Metellus I, Delfino C, Colin C, Tchogandjian A, Coulibaly B, Fina F, Loundou A, Barrie M, Chinot O, Ouafik L, Figarella-Branger D: Prognostic impact of CD133 mRNA expression in 48 glioblastoma patients treated with concomitant radiochemotherapy: a prospective patient cohort at a single institution. Ann Surg Oncol 2011, 18:2937-2945.
• [50]Pallini R, Ricci-Vitiani L, Banna GL, Signore M, Lombardi D, Todaro M, Stassi G, Martini M, Maira G, Larocca LM, De Maria R: Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme. Clin Cancer Res 2008, 14:8205-8212.
• [51]Pallini R, Ricci-Vitiani L, Montano N, Mollinari C, Biffoni M, Cenci T, Pierconti F, Martini M, De Maria R, Larocca LM: Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis. Cancer 2011, 117:162-174.
• [52]Joo KM, Kim SY, Jin X, Song SY, Kong DS, Lee JI, Jeon JW, Kim MH, Kang BG, Jung Y, Jin J, Hong SC, Park WY, Lee DS, Kim H, Nam DH: Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastoma. Lab Invest 2008, 88:808-815.
• [53]Kim KJ, Lee KH, Kim HS, Moon KS, Jung TY, Jung S, Lee MC: The presence of stem cell marker-expressing cells is not prognostically significant in glioblastomas. Neuropathology 2011, 31:494-502.