For diagnostic neuropathology the main potential of MGMT IHC is in the identification of tumors with loss of MGMT expression and expected better response
to alkylating agents. For glioblastoma, several stud- ies have demonstrated significant associations of IHC MGMT expression and overall survival (Anda et al., 2003; Belanich et al., 1996; Capper et al., 2008;
Chinot et al., 2007; Jaeckle et al., 1998; Metellus et al., 2009). Cut-off values in these series to differ- entiate low expressing from high expressing glioblas- tomas was either median (35%, Chinot et al., 2007), arbitrary set at 10% (Metellus et al., 2009) or 20%
(Anda et al., 2003) or calculated to best separate prognostic groups (15%, Capper et al., 2008; 60,000 molecules/nucleus in quantitative immunofluorescence microscopy Belanich et al.,1996; Jaeckle et al.,1998).
In these studies 36–56% of investigated glioblastomas were scored as low MGMT expressing cases. Other studies with concordant metholodical approaches and cut-offs did not observe significant associations of survival and MGMT IHC (Cahill et al.,2007; Karayan- Tapon et al., 2009; Preusser et al., 2008; Rodriguez et al., 2008). In these series 44–72% of analyzed glioblastomas were scored as low expressing tumors.
In previous discussions, variations of cut-off values and the combination of various tumor grades have been discussed as possible causes for lack of asso- ciation (Capper et al., 2008). Preusser et al. (2008) and Karayan-Tapon et al. (2009) both retrospectively investigated a large cohort of homogenously treated glioblastoma patients, demonstrating that this is not the cause of the discrepant results. Further, the num- bers of negatively scored cases are also in a comparable range, making differences of antibody dilution or stain- ing procedure unlikely causes. The reasons for the discordant results remain unclear. Despite some indi- cation of an association of MGMT IHC and survival, the method can currently not be recommended for evaluation of patient outcome or possible response to alkylating agents in glioblastoma.
Only little data exists concerning MGMT IHC of anaplastic astrocytomas. In an analysis of 24 anaplastic astrocytomas treated with bis-chloroethylnitrosourea overall median survival for cases with high MGMT levels was 14 months versus 62 months when MGMT expression was low (Jaeckle et al.,1998), while Capper et al. (2008) observed no association of MGMT IHC and outcome for anaplastic astrocytomas. Brell et al. (2005) reported an association of high IHC MGMT expression and worse survival of patients with anaplastic glioma (also including anaplas- tic oligodendroglioma and oligoastrocytoma) after
treatment with alkylating agents (Brell et al., 2005).
For diffuse astrocytomas grade II, MGMT IHC also did not demonstrate satisfying associations with patient outcome. While Capper et al. (2008) observed an association of high MGMT with worse survival;
this was strongly dependent on patient age. Nakasu et al. (2007) could not detect an association of overall survival and IHC MGMT expression, while a signifi- cant association was observed with tumor progression:
Tumors scored MGMT negative showed a more rapid tumor progression (Nakasu et al.,2007).
Pediatric high grade gliomas have been investigated for MGMT IHC by two major studies (Pollack et al., 2006; Schlosser et al.,2010) yielding partly conflicting results. Polack et al. (2006) demonstrated that over- expression of MGMT compared to normal brain was associated with worse prognosis in 12 of 109 malig- nant gliomas treated with alkylating agents (Pollack et al., 2006). In a series of 24 relapsed high grade gliomas Schlosser et al. (2010) observed an association of survival with MGMT promoter methylation but not with IHC protein expression (Schlosser et al.,2010).
As with glioblastoma, while there is some indi- cation for prognostic properteis of MGMT IHC for anaplastic gliomas, diffuse astrocytomas WHO grade II and pediatric diffuse gliomas, published data is con- flicting. At the current stage MGMT IHC can not be recommended as a reliable marker of patient outcome or response to alkylating agents. Cao et al. (2009) have recently proposed combining analysis of MGMT pro- moter methylation with MGMT IHC for prognostic evaluation. Indeed, the combination of MGMT pro- moter methylation with negative MGMT IHC expres- sion could reduce the shortcomings observed for both methods (Cao et al.,2009).
References
Anda T, Shabani HK, Tsunoda K, Tokunaga Y, Kaminogo M, Shibata S, Hayashi T, Iseki M (2003) Relationship between expression of O6-methylguanine-DNA methyltrans- ferase, glutathione-S-transferase pi in glioblastoma and the survival of the patients treated with nimustine hydrochloride:
an immunohistochemical analysis. Neurol Res 25:241–248 Belanich M, Pastor M, Randall T, Guerra D, Kibitel J, Alas L,
Li B, Citron M, Wasserman P, White A, Eyre H, Jaeckle K, Schulman S, Rector D, Prados M, Coons S, Shapiro W, Yarosh D (1996) Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival
of brain tumor patients treated with carmustine. Cancer Res 56:783–788
Brell M, Tortosa A, Verger E, Gil JM, Viñolas N, Villá S, Acebes JJ, Caral L, Pujol T, Ferrer I, Ribalta T, Graus F (2005) Prognostic significance of O6-methylguanine-DNA methyl- transferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas. Clin Cancer Res 11:5167–5174
Brent TP, von Wronski M, Pegram CN, Bigner DD (1990) Immunoaffinity purification of human O6-alkylguanine- DNA alkyltransferase using newly developed monoclonal antibodies. Cancer Res 50:58–61
Cahill DP, Levine KK, Betensky RA, Codd PJ, Romany CA, Reavie LB, Batchelor TT, Futreal PA, Stratton MR, Curry WT, Iafrate AJ, Louis DN (2007) Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment. Clin Cancer Res 13:2038–2045
Cao VT, Jung TY, Jung S, Jin SG, Moon KS, Kim IY, Kang SS, Park CS, Lee KH, Chae HJ (2009) The correlation and prognostic significance of MGMT promoter methyla- tion and MGMT protein in glioblastomas. Neurosurgery 65:
866–875
Capper D, Mittelbronn M, Meyermann R, Schittenhelm J (2008) Pitfalls in the assessment of MGMT expression and in its correlation with survival in diffuse astrocytomas: proposal of a feasible immunohistochemical approach. Acta Neuropathol 115:249–259
Chinot OL, Barrié M, Fuentes S, Eudes N, Lancelot S, Metellus P, Muracciole X, Braguer D, Ouafik L, Martin PM, Dufour H, Figarella-Branger D (2007) Correlation between O6- methylguanine-DNA methyltransferase and survival in inop- erable newly diagnosed glioblastoma patients treated with neoadjuvant temozolomide. J Clin Oncol 25:1470–1475 Christmann M, Nagel G, Horn S, Krahn U, Wiewrodt D,
Sommer C, Kaina B (2010) MGMT activity, promoter methylation and immunohistochemistry of pre-treatment and recurrent malignant gliomas: a comparative study on astrocy- toma and glioblastoma. Int J Cancer 127:2106–2118 Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman
JG (1999) Inactivation of the DNA repair gene O6- methylguanine-DNA methyltransferase by promoter hyper- methylation is a common event in primary human neoplasia.
Cancer Res 59:793–797
Grasbon-Frodl EM, Kreth FW, Ruiter M, Schnell O, Bise K, Felsberg J, Reifenberger G, Tonn JC, Kretzschmar HA (2007) Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic spec- imens from anaplastic astrocytomas and glioblastomas. Int J Cancer 121:2458–2464
Jaeckle KA, Eyre HJ, Townsend JJ, Schulman S, Knudson HM, Belanich M, Yarosh DB, Bearman SI, Giroux DJ, Schold SC (1998) Correlation of tumor O6 methylguanine- DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea:
a southwest oncology group study. J Clin Oncol 16:
3310–3315
Karayan-Tapon L, Quillien V, Guilhot J, Wager M, Fromont G, Saikali S, Etcheverry A, Hamlat A, Loussouarn D, Campion L, Campone M, Vallette FM, Gratas-Rabbia-Ré C (2009) Prognostic value of O(6)-methylguanine-DNA
methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 97:311–322 Margison GP, Povey AC, Kaina B, Santibáñez Koref MF (2003)
Variability and regulation of O6-alkylguanine-DNA alkyl- transferase. Carcinogenesis 24:625–635
Maxwell JA, Johnson SP, Quinn JA, McLendon RE, Ali-Osman F, Friedman AH, Herndon JE 2nd, Bierau K, Bigley J, Bigner DD, Friedman HS (2006) Quantitative analysis of O6-alkylguanine-DNA alkyltransferase in malignant glioma.
Mol Cancer Ther 5:2531–2539
McLendon RE, Cleveland L, Pegram C, Bigner SH, Bigner DD, Friedman HS (1998) Immunohistochemical detection of the DNA repair enzyme O6-methylguanine-DNA methyltrans- ferase in formalin-fixed, paraffin-embedded astrocytomas.
Lab Invest 78:643–644
Metellus P, Coulibaly B, Nanni I, Fina F, Eudes N, Giorgi R, Barrie M, Chinot O, Fuentes S, Dufour H, Ouafik L, Figarella-Branger D (2009) Prognostic impact of O6- methylguanine-DNA methyltransferase silencing in patients with recurrent glioblastoma multiforme who undergo surgery and carmustine wafer implantation: a prospective patient cohort. Cancer 115:4783–4794
Mineura K, Yanagisawa T, Watanabe K, Kowada M, Yasui N (1996) Human brain tumor O(6)-methylguanine-DNA methyltransferase mRNA and its significance as an indica- tor of selective chloroethylnitrosourea chemotherapy. Int J Cancer 69:420–425
Nakasu S, Fukami T, Baba K, Matsuda M (2004) Immunohistochemical study for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas. J Neurooncol 70:333–340 Nakasu S, Fukami T, Jito J, Matsuda M (2007) Prognostic signif-
icance of loss of O6-methylguanine-DNA methyltransferase expression in supratentorial diffuse low-grade astrocytoma.
Surg Neurol 68:603–608
Pegg AE (1990) Mammalian O6-alkylguanine-DNA alkyltrans- ferase: regulation and importance in response to alkylat- ing carcinogenic and therapeutic agents. Cancer Res 50:
6119–6129
Pollack IF, Hamilton RL, Sobol RW, Burnham J, Yates AJ, Holmes EJ, Zhou T, Finlay JL (2006) O6-methylguanine- DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: results from the CCG-945 Cohort. J Clin Oncol 24:3431–3437
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 (2008) Anti-O6- methylguanine-methyltransferase (MGMT) immunohisto- chemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as clinical biomarker. Brain Pathol 18:520–532
Rodriguez FJ, Thibodeau SN, Jenkins RB, Schowalter KV, Caron BL, O’neill BP, James CD, Passe S, Slezak J, Giannini C (2008) MGMT immunohistochemical expres- sion and promoter methylation in human glioblastoma. Appl Immunohistochem Mol Morphol 16:59–65
Sasai K, Akagi T, Aoyanagi E, Tabu K, Kaneko S, Tanaka S (2007) O6-methylguanine-DNA methyltransferase is down- regulated in transformed astrocyte cells: implications for anti-glioma therapies. Mol Cancer 5(6):36
Sasai K, Nodagashira M, Nishihara H, Aoyanagi E, Wang L, Katoh M, Murata J, Ozaki Y, Ito T, Fujimoto S, Kaneko S, Nagashima K, Tanaka S (2008) Careful exclusion of non-neoplastic brain components is required for an appro- priate evaluation of O6-methylguanine-DNA methyltrans- ferase status in glioma: relationship between immunohisto- chemistry and methylation analysis. Am J Surg Pathol 32:
1220–1227
Schlosser S, Wagner S, Mühlisch J, Hasselblatt M, Gerss J, Wolff JE, Frühwald MC (2010) MGMT as a potential strat- ification marker in relapsed high-grade glioma of children:
the HIT-GBM experience. Pediatr Blood Cancer 54:228–237 Spiegl-Kreinecker S, Pirker C, Filipits M, Lötsch D, Buchroithner J, Pichler J, Silye R, Weis S, Micksche M, Fischer J, Berger W (2010) O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients.
Neuro Oncol 12:28–36
Yachi K, Watanabe T, Ohta T, Fukushima T, Yoshino A, Ogino A, Katayama Y, Nagase H (2008) Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas. Int J Oncol 33:469–475
Yuan Q, Matsumoto K, Nakabeppu Y, Iwaki T (2003) A com- parative immunohistochemistry of O6-methylguanine-DNA methyltransferase and p53 in diffusely infiltrating astrocy- tomas. Neuropathology 23:203–209