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A group of 160 patients with primary glioblastoma treated with radiotherapy and temozolomide was analyzed for the impact of O6-methly-guanly-methyl-transferase (MGMT)-promoter methylation as well as isocitrate dehydrogenase (IDH)1-mutational status. Unexpectedly, overall survival or progression-free survival were not longer in the group with methylated MGMT-promoter as compared to patients without that methylation. IDH-1 mutations were significantly associated with increased overall survival. Keywords: Glioblastoma, radiation, temozolomide, MGMT, IDH Introduction mechanism contributing to a loss of MGMT-expression Over recent years, the search for outcome factors in has been described by Esteller et al. . The epigeneti- patients with glioblastomas (GBM) has identified at least cally mediated silencing of the MGMT gene in GBM two candidates that have shown to be prognostic for has been shown to correlate with an increased survival: progression-free and overall survival or predictive for Some studies have shown significant correlation with response to a particular therapeutic modality, that is MGMT-promoter methylation and outcome to alkylat- alkylating chemotherapy, in patients with high-grade ing chemotherapeutic substances such as temozolomide gliomas. The O6-methylguanine-DNA-methyltransferase (TMZ) . Moreover, a correlation with outcome inde- (MGMT) gene encodes MGMT,aproteinwithDNA pendently of treatment choice, i.e. chemotherapy or repair activity, which removes alkyl groups from several radiotherapy, has been postulated by some authors [7,8]. residues, of which the O6-position of guanine might be However, until now, most reports on the prognostic most relevant for the action of an extensively used che- value of MGMT-promoter methylation have answered motherapeutic drug, temozolomide, by an irreversible this question in a retrospective manner. Additionally, transfer of the alkyl group to a cystein residue at it’s several methods of MGMT-promoter methylation con- active side [1,2]. The MGMT expression level and its firmationhavebeenusedwithinthe different studies, activity varies widely between different tissues, cell and comparative analyses have shown substantial het- types, and in particular, between different tumors [3,4]. erogeneity in results after MGMT-testing. In the litera- It has been shown that glial brain tumors are character- ture, some authors have reported that MGMT promoter methylation might not be correlated with outcome, ized by a low expression of MGMT, however, the activ- ity of MGMT is commonly increased in relation to either after treatment with radiotherapy, or with alkylat- surrounding normal tissue [4,5]. ing chemotherapeutic substances [9,10]. MGMT-activity is partly mediated through methyla- Only recently, mutations of the IDH1 gene encoding tion of the MGMT promoter region; this epigenetic cytosolic NADP+-dependent isocitrate dehydrogenase have been show to correlate with outcome in patients with malignant gliomas [11,12]. It has been proposed * Correspondence: firstname.lastname@example.org that IDH1 mutations can be used to distinguish primary Department of Radiation Oncology, University Hospital of Heidelberg, Heidelberg, Germany from secondary GBM, since IDH1 mutations are Full list of author information is available at the end of the article © 2011 Combs et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Combs et al. Radiation Oncology 2011, 6:115 Page 2 of 5 http://www.ro-journal.com/content/6/1/115 associated with diffuse gliomas WHO Grade II and III Table 1 Patients’ characteristics of 160 patients treated with radiation and temozolomide for primary as well as with secondary GBM, whereas primary GBM glioblastoma rarely show IDH1 mutations. Characteristic N (%) We have treated a large group of patients with pri- mary GBM with radiotherapy and chemotherapy with Age - years temozolomide. To determine the prognostic value of median 56 MGMT-promoter methylation and IDH1 mutational sta- range 20-76 tus, we analyzed both markers in a homogenous group Age - no. (%) of 160 patients with primary GBM treated with radia- < 50 38 (24) tion and TMZ and correlated results with outcome. ≥ 50 122 (76) Sex - number (%) Materials and methods female 66 (41) Patient population male 94 (59) Between 1999 and 2007, 160 consecutive patients with primary, histologically confirmed GBM were treated Karnofsky Performance Score with radiation and temozolomide as reported previously ≥ 70 115 (72) [13,14]. < 70 45 (28) After neurosurgical resection, which was complete in Extent of surgery - no. (%) 51 patients and subtotal in 66 patients, patients were biopsy 43 (27) treated with 3D-conformal radiation therapy based on complete resection 51 (32) CT- and MRI-based treatment planning. The median partial resection 66 (41) age of the patients included was 56 years at primary RPA/EORTC-Classification diagnosis (range 20-76 months). Patients’ characteristics III 33 (21) are shown in table 1. IV 90 (56) Amediandoseof60Gyin 2Gysingle fractionswas applied. All patients were treated with concomitant V 37 (23) TMZ, and adjuvant TMZ was given in 34 patients. At Time from diagnosis to RT - days this time, a phase II trial evaluation radiation and che- median 24 motherapy with TMZ at a dose of 50 mg/m without range 7-98 adjuvant TMZ was performed in our institution, there- Corticosteroid therapy - no. (%) fore 124 patients had been treated according to this yes 113 (71) regimen, and 36 patients received TMZ according to the no 47 (29) Stupp regimen [13,15,16]. A detailed report on patient Anti-seizure medication - no. (%) and treatment characteristics has been published pre- yes 75 (47) viously . no 85 (53) Molecular analyses and immunohistochemistry Tumor tissue for molecular analysis of MGMT-promo- ter methylation was available from 127 out of 160 UltraWash, counterstaining with one drop of hematoxylin patients (80%). MGMT status was determined using for 4 min and one drop of bluing reagent for 4 min. For methylation-specific polymerase chain reaction . chromogenic detection UltraViewTMUniversal DAB Details are described elsewhere . Detection Kit (Ventana) was used. Slides were removed To determine the IDH1-mutational status we used from the immunostainer and mounted. A strong cytoplas- either immunohistochemistry with an antibody specifically mic immunoreaction product was scored positive. A weak binding the R132H mutational variant of IDH1 (n = 125) diffuse staining and staining of macrophages were not or direct sequencing (n = 15). To determine the IDH1 sta- scored positive. Figure 1 depicts an example of the immu- tus by immunohistochemistry, sections were cut to 4 μm, nohistochemistry as well as sequencing results. dried at 80°C for 15 min and further processed on a Ven- tana BenchMark XT immunostainer (Ventana Medical Statistical Analysis Systems, Tucson, AZ, USA). After 60 min pretreatment All patients were seen for regular follow-up and clinical with cell conditioner 2 (pH 6) the slides were incubated data was collected in the institutions’s database. For the with 1:30 diluted H09 antibody (Dianova, Hamburg, Ger- present analysis, we correlated status of MGMT-promo- many) at 37°C for 32 min. Antibody incubation was fol- ter methylation as well as IDH-1 mutational status with lowed by Ventana standard signal amplification, patients’ characteristics and outcome. Combs et al. Radiation Oncology 2011, 6:115 Page 3 of 5 http://www.ro-journal.com/content/6/1/115 Figure 1 GBM sample with an IDH1 R132H mutation demonstrated by IHC and sequencing. Overall survival (OS) was calculated from the date of patients with a positive IDH1 mutations status showed primary diagnosis until death or last observation during significantly longer OS (p = 0.002; Figure 3A), but unal- follow up (censored data). Progression-free survival tered PFS (p = 0.25) than patients with wildtype IDH1. (PFS) was determined from the time of the beginning of radiotherapy and chemotherapy until tumor progression Discussion or to last observation or death if none occurred (cen- In the present analysis we evaluated the impact of sored data). OS and PFS were calculated using the MGMT-promother methylation as well as IDH1-muta- Kaplan-Meier-Method. Survival curves for prognostic tional status on outcome in 160 patients with GBM factors were compared using a two-sided log rank test. treated with radiation and temozolomide. IDH1 muta- All statistical analyses were performed using the Statis- tions occur in approximately 60 - 80% of diffusely infil- tica 6.1 software (Statsoft, Tulsa, OK, USA). trating gliomas of the WHO grades II and III and in secondary GBM but only in around 5% of primary GBM Results [11,18-22]. In our series we identified in 4 of 140 Molecular analyses: MGMT Promoter Methylation patients (3%) IDH-1 mutations. Currently, it remains Of the 127 patients analyzed, the MGMT-promoter was unclear if at least some of the patients with clinically methylated in 43 patients (34%) and was unmethylated defined primary GBM and IDH1 mutations may actually in 84 patients (66%). have suffered from secondary GBM that rapidly pro- MGMT-promoter methylation did not correlate with gressed from less malignant precursor lesions that overall survival (OS; p = 0.18 (Figure 2A)). Additionally escaped diagnosis . In summary, IDH1 is a sufficient marker that allows a better separation of primary GBM progression-free-survival was not influenced by MGMT- promoter methylation status (p = 0.93; Figure 2B). from other malignant astrocytomas than any other mar- Looking at subgroups, we analyzed the impact of ker and will help to define more accurately this tumor MGMT- promoter methylation on OS and PFS in entity in upcoming studies. The low number of primary patients ≤ 60 yrs. (n = 85; 53%) and > 60 yrs. (n = 75; GBM exhibiting IDH1 mutations in our series indicates 47%). In the younger age group MGMT-promoter that our sample set consists indeed predominately of methylation did not influence OS (p = 0.93) or PFS (p = these tumors. IDH1mutations in GBM were found in 0.69). However, in older patients, MGMT-promoter general in younger patients and were associated with a methylation was associated with a significant increase in better prognosis [22-24]. This has been confirmed in the OS (p = 0.02), however PFS was comparable (p = 0.11). present study, showing that IDH1 mutational status, although only positive in few patients, is associated with Molecular analyses and immunohistochemistry: IDH1 younger age and lower survival times than in the group Fourofthe140patients(3%)showedanIDH1muta- of patients with wildtype IDH1. Therefore, the pattern tion; all mutations were of the R132H variant. The 4 of IDH1 mutations confirm that the present group of Combs et al. Radiation Oncology 2011, 6:115 Page 4 of 5 http://www.ro-journal.com/content/6/1/115 AB p=0.18 p=0.93 Figure 2 Correlation of overall survival (A) and progression-free survival (B) with MGMT-promotor methylation. For both endpoints, MGMT-activity did not significantly influence outcome. 160 patients with GBM is a very homogeneous group determining the role for chemoradiation with temozo- with respect to histological clasification. lomide has shown MGMT-promoter methylation to be In contrast to most studies, MGMT-promoter strongly associated with an improved outcome [7,15]. methylation was not associated with an increase in OS In contrast, other studies in anaplastic gliomas have or PFS; both endpoints were comparable in patients shown that MGMT-methylation status dose not only with active MGMT or with MGMT silencing. The only influence outcome after alkylkating chemotherapies subgroup of patients showing a significant impact of but also radiotherapy and may therefore be prognostic MGMT-promoter methylation on survival were rather than predictive. This is reported by numerous patients older than 60 years, where MGMT-promoter other studies . However, controversial results have methylation was associated with an increase in OS. also been published in groups of GBM patients, in This is in contrary to the results published by Stupp which MGMT-status is not associated with differences and colleagues [7,15]. Therefore, the strong impact on in outcome: Costa et al. reports on 90 GBM-patients MGMT-promoter methylation might not hold true for treated with temozolomide-based chemoradiation all age groups of patients with GBM. The EORTC where MGMT promoter methylation was not asso- 26981/22981/NCIC CE.3 study by Stupp et al. ciated with increased outcome . Park et al. pub- lished 48 patients treated with alkylating chemotherapy and could not confirm a significant impact of methyla- p=0.002 tion status of MGMT gene promoter . Many argu- ments may be brought forward to explain these differing clinical data, including the various methods of measurement of MGMT-activity sometimes showing discrepant results, differences between frozen or paraf- fin embedded tissues. Additionally, when analyzing dif- ferent chemotherapeutic combinations, substances such as cisplatinum might inactivate or attenuate MGMT-status thus influencing the clinical outcome when combined with alkylating chemotherapies. An important differential explanation is the variation of the treatment in our cohort as compared to the pub- lished data [7,16,17,25,26] with a lower exposure of our patients to alkylating chemotherapy both in the Figure 3 IDH-1 mutational status did influcence overall survival in concomitant phase (50 mg in 78% and 75 mg in only 160 patients treated with radiation and temozolomide (p = 0.002). 22%) as well as in the maintenance phase (no adjuvant Combs et al. Radiation Oncology 2011, 6:115 Page 5 of 5 http://www.ro-journal.com/content/6/1/115 10. Costa BM, Caeiro C, Guimaraes I, Martinho O, Jaraquemada T, Augusto I, treatment in 79% and a mean of 6 cycles in 21% of et al: Prognostic value of MGMT promoter methylation in glioblastoma patients). However, inspite of this difference, outcome patients treated with temozolomide-based chemoradiation: a between the two dosing groups of temozolomide was Portuguese multicentre study. Oncol Rep 2010, 23:1655-62. 11. Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, et al: Type identical, therefore counteracting this argument. and frequency of IDH1 and IDH2 mutations are related to astrocytic and In conclusion, controversial results exist on the impact oligodendroglial differentiation and age: a study of 1,010 diffuse of MGMT-promoter methylation status in patients with gliomas. Acta Neuropathol 2009, 118:469-74. 12. Capper D, Weissert S, Balss J, Habel A, Meyer J, Jager D, et al: GBM, and further studies will hopefully further clarify Characterization of R132H mutation-specific IDH1 antibody binding in these differences. At this time, in spite of the strong evi- brain tumors. Brain Pathol 2010, 20:245-54. dencefor ahighimpactof MGMT-promoter methyla- 13. Combs SE, Gutwein S, Schulz-Ertner D, van Kampen M, Thilmann C, Edler L, et al: Temozolomide combined with irradiation as postoperative tion, differentiating treatment strategies based on treatment of primary glioblastoma multiforme. Phase I/II study. MGMT-promoter methylation status should therefore Strahlenther Onkol 2005, 181:372-7. be applied within the framework of clinical studies only. 14. Combs SE, Wagner J, Bischof M, Welzel T, Edler L, Rausch R, et al: Radiochemotherapy in patients with primary glioblastoma comparing two temozolomide dose regimens. Int J Radiat Oncol Biol Phys 2008, 71:999-1005. Author details 15. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Department of Radiation Oncology, University Hospital of Heidelberg, et al: Radiotherapy plus concomitant and adjuvant temozolomide for Heidelberg, Germany. Department of Neurooncology, University Hospital of glioblastoma. N Engl J Med 2005, 352:987-96. Heidelberg, Heidelberg, Germany. Clinical Cooperation Unit 16. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Neuropathology, German Cancer Research Center, Heidelberg Germany. et al: Effects of radiotherapy with concomitant and adjuvant Department of Neuropathology, University Hospital of Heidelberg, temozolomide versus radiotherapy alone on survival in glioblastoma in Heidelberg, Germany. a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10:459-66. Authors’ contributions 17. Weiler M, Hartmann C, Wiewrodt D, Herrlinger U, Gorlia T, Bahr O, et al: SC, SR, WW and JD treated the patients and collected the cllinical data. SC Chemoradiotherapy of newly diagnosed glioblastoma with intensified and JD performed the clinical analysis of the dataset. CH and AVD temozolomide. Int J Radiat Oncol Biol Phys 2010, 77:670-6. performed the histopathological and molecular analysis. SC, JD, CH and AA 18. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A: analyszed the prognostic relevance of the molecular data. SC and CH wrote Analysis of the IDH1 codon 132 mutation in brain tumors. Acta the mansucript. JD, AVD, WW, SR and AA helped with manuscript finalization Neuropathol 2008, 116:597-602. and discussion. 19. Ichimura K, Pearson DM, Kocialkowski S, Backlund LM, Chan R, Jones DT, et al: IDH1 mutations are present in the majority of common adult Conflict of interests gliomas but rare in primary glioblastomas. Neuro Oncol 2009, 11:341-7. The authors declare that they have no competing interests. 20. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al: An integrated genomic analysis of human glioblastoma multiforme. Science Received: 2 April 2011 Accepted: 13 September 2011 2008, 321:1807-12. Published: 13 September 2011 21. 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Radiation Oncology – Springer Journals
Published: Sep 13, 2011
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