Annals of Oncology Advance Access published August 7, 2008
original article
Annals of Oncology doi:10.1093/annonc/mdn548
A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients M. Schaich1, L. Kestel1, M. Pfirrmann2, K. Robel3, T. Illmer1, M. Kramer1, C. Dill1, G. Ehninger1, G. Schackert3 & D. Krex3* 1
Department of Medicine I, University Hospital C. G. Carus, Dresden; 2Department of Medical Informatics, Biometry and Epidemiology (IBE), Ludwig Maximilians University Munich, Munich; 3Department of Neurosurgery, University Hospital C. G. Carus, Dresden, Germany
Received 30 June 2008; accepted 4 July 2008
aberrant promoter methylation of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT). This suggests that additional factors hamper temozolomide cytotoxicity. We aimed to confirm first that temozolomide is a target for the multidrug resistance transporter MDR1/ABCB1 and second to investigate whether genetic variants of the MDR1 gene are associated with the survival of glioblastoma patients treated with temozolomide. Materials and methods: Temozolomide-mediated cytotoxicity was determined by the colorimetric methyl-thiazoltetrazolium assay in MDR-expressing and MDR-nonexpressing cell lines. Genotypes of three single nucleotide polymorphisms (SNPs) of the MDR1 gene (C1236T, G2677T, and C3435T), MDR1 mRNA expression levels, and the MGMT promoter methylation status were analyzed in 112 glioblastoma patients who had been treated either by surgery plus radiotherapy alone or by additional temozolomide chemotherapy. Results: In vitro analysis revealed that temozolomide-mediated cytotoxicity is dependent on MDR1 expression. Multivariate analysis of MDR1 genotypes showed that the C/C variant of the exon12 C1236T SNP is predictive for survival of patients treated with temozolomide. This effect was independent of the MGMT methylation status. Patients with the C/C genotype had a 2-year overall survival of 37% compared with 8% and 10% for patients with C/T and T/T genotypes, respectively (P = 0.02). No influence was seen in the group of patients with radiotherapy only. Conclusion: The genotype of the MDR1 exon12 C1236T SNP is a novel independent predictive factor for outcome of temozolomide treatment in glioblastoma patients. Key words: glioblastoma, MDR1, MGMT, polymorphism, temozolomide
introduction Glioblastoma multiform (astrocytoma World Health Organization grade IV) is the most frequent and most malignant primary brain tumor in adults, causing 13 000 deaths per year in the United States. The median survival is still in the range of 12 months in spite of multimodal aggressive treatment, comprising surgical resection, local radiotherapy, and systemic chemotherapy [1]. Recently, temozolomide has been introduced into standard care [2]. Temozolomide is a DNA-methylating agent with a broad-spectrum antitumor activity, the ability to cross the blood–brain barrier, a good safety profile with nonoverlapping toxic effects, an oral formulation, and the ability to overcome resistance to nitrosoureas [3]. It is effective in glioblastoma treatment as *Correspondence to: Dr D. Krex, Department of Neurosurgery, Carl Gustav Carus University Hospital, University of Technology, Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany. Tel: +49-351-4584163; Fax: +49-351-4584304; E-mail:
[email protected]
shown for recurrent glioblastoma first [4, 5] and, recently, in a prospective randomized trial conducted by the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC) Clinical Trial Group (CTG), for newly diagnosed glioblastoma when administered concomitantly with and as an adjuvant after radiotherapy [6]. The DNA damage caused by methylating agents like temozolomide is reversed by the ubiquitous repair enzyme O6-methylguanine methyltransferase (MGMT) [7, 8]. The MGMT gene can be silenced epigenetically by hypermethylation of its promoter [9]. Transcriptional silencing of the MGMT gene by promoter hypermethylation is seen in 50% of glioblastomas and has been linked to a better response to alkylating chemotherapy and longer survival in glioblastoma patients [10]. Therefore, MGMT methylation status represents the first molecular marker of prognostic and predictive significance for glioblastoma. However, results still need to be confirmed in a prospective randomized trial. Moreover, MGMT promoter methylation status is unlikely to be the only
ª The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email:
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original article
Background: Some patients with glioblastoma multiform do not respond to temozolomide even though they have
original article molecular marker and mechanism that affects survival outcome for patients with glioblastoma. Another mechanism mediating chemoresistance is the expression of efflux pump proteins that selectively transport substances out of cells. The multidrug resistance gene (MDR1/ABCB1, OMIM *171050) encodes for a P-glycoprotein (P-gp), which is a well-known drug efflux pump whose overexpression has been primarily observed in human cancer that is resistant to chemotherapy [11], particularly to drugs such as vincristine, etoposide, and doxorubicin [12]. Whether temozolomide is also a target for P-gp-mediated chemoresistance has not been well documented [13]. MDR1 has been found to be expressed in the majority of brain tumors, including glioblastomas [14]. The effectiveness of the P-gp efflux pump may be influenced in several ways: an altered ability of this P-gp to recognize target substrates, by other nontarget substances inhibiting the pump transporter and by genetic variants having a functional impact on MDR1 transcription [15, 16]. Among those, the three single nucleotide polymorphisms (SNPs) C1236T, G2677T, and C3435T have been well characterized for their functional effect on disease development and drug response in various populations (reviewed by Pauli-Magnus and Kroetz [17]). In a previous study, we confirmed that those SNPs of the MDR1 gene had influence on treatment outcome in acute myeloid leukemia patients [18]. Therefore, in the present study, we aimed to confirm first that temozolomide is also a target for the P-gp efflux pump MDR1 and second to investigate whether genetic variants of the MDR1 gene are associated with the survival of glioblastoma patients receiving temozolomide treatment.
materials and methods in vitro cytotoxicity In vitro cytotoxicity was measured using the colorimetric methyl-thiazoltetrazolium (MTT) assay. Cells from the chemosensitive parental MDR1-negative chronic myeloid leukemia cell line K562 and the strongly MDR1-positive derived line K562-VP16 were cultured with RPMI medium and only for K562-VP16 with 5 lM etoposide [19]. From the parental and resistant line, 1 · 106 cells were incubated with 10 lg/ml temozolomide, 1 lM cyclosporine A, 10 lg/ml temozolomide plus 1 lM cyclosporine A and without any drugs as a control. Experiments were done eight-fold for 48 h in wells of microculture plates at 37C in humified air containing 5% CO2. After 48 h, the MTT assay was carried out as previously described [19]. Mean values and standard deviations of six independent experiments were determined.
Annals of Oncology
Table 1. Patient characteristics n Sex (male/female) Age (years) Karnofsky performance score (%) on admission 100 90 80 £70 Treatment overall Radiotherapy only Radiotherapy + temozolomide Treatment details Total radiation dosage (Gy) Cycles of temozolomide (n) Daily temozolomide dosage/cycle (mg/m2)
%
Median (range)
63/49 56/44 63 (18–89)
34 32 27 17
31 29 24 16
48 64
43 57
Patients
P
n
112 112 110a
112
112 59 (30–89) 112 4 (1–13) 64 183 (75–350) 64
a
Data not available from two patients.
DNA and mRNA isolation Tumor tissues were taken intraoperatively and were snap frozen at 280C. Frozen tumor tissue was sectioned using a cryotome (Cryostat Jung CM 1800, Leica). To assure a tumor cell content of at least 80% for nucleic acid extraction, control slides stained with hematoxylin and eosin were examined by the local neuropathologist. For DNA isolation, the QIAmp DNA Mini Kit 50 (Qiagen, Hilden, Germany) and for RNA isolation, the Invisorb Spin Tissue RNA Mini Kit 250 (Invitek, Berlin, Germany) were used according to the manufacturers’ instructions. The quantity and purity of RNA were estimated spectrophotometrically and their integrity was checked by electrophoresis in 1% agarose gel. RNA was reverse transcribed using the Omniscript RT Kit (Qiagen).
PCR of polymorphic sites of the MDR1 gene The most frequent polymorphic sites of the MDR1 gene comprising the exon12 C1236T (rs1128503), exon21 G2677T (rs2032582), and exon26 C3435T (rs1045642) SNPs were amplified by PCR. SNP positions refer to the MDR1 cDNA sequence [20], with the first base of the ATG start codon set to 1. PCR was carried out on genomic DNA using the previously described primer molecules [18]. Primer sequences and PCR conditions are provided on request. PCR products were analyzed on standard 1.5% agarose gels stained with ethidium bromide (0.5 lg/ml).
SSCP analysis and sequencing study population The tumor database of the Department of Neurosurgery, University Hospital in Dresden, was used to identify adult patients who had been operated for supratentorial glioblastoma during the years 1998–2006, had adjuvant locoregional radiotherapy, and either had no adjuvant chemotherapy or had temozolomide concomitantly and/or adjuvant to radiotherapy. All patients had given informed written consent for molecular analyses of tumor tissue, and the study had been approved by the local ethics committee. Histopathology was carried out at the Department of Neuropathology, Dresden University Hospital, and only cases with an unequivocal diagnosis of glioblastoma were selected. For patient characteristics see Table 1.
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For SSCP analysis, the PhastGel system (Amersham Pharmacia Biotech, Freiburg, Germany) was used as previously described [18]. Primer pairs covering the described sequence strings additionally may amplify polymorphic loci of exon21 and 26. However, the frequencies of these polymorphisms are low. The observed frequency for cDNA2677 G/A and T/A genotypes together is 3.8% [21] and for the heterozygous genotype of the C3396T SNP is 0.5% [15]. We therefore did not consider those changes in our survey and excluded patients with SSCP ‘extra bands’. For confirmation of SSCP results, bands from representative samples of each genotype of all three SNPs were cut out, eluted, and prepared for sequencing by an ABI 3730· (Applied Biosystems) using the same primers as for the detection of the polymorphic sites of the MDR1 gene (see above).
original article
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real-time PCR To determine the amount of MDR1 mRNA expression, real-time PCR was carried out using primers and probes according to Schiedlmeier et al. [22]. GAPDH was used as positive control. Primers and probes were purchased from TibMolBiol (TibMolBiol, Berlin, Germany). PCR reactions were run in triplicate. Amplification conditions were 2 min preincubation time at 50C, 10 min at 95C enzyme activation and 50 cycles at 95C for 15 s denaturation and 60C for 1 min annealing and extension. Quantitative results were assessed by determination of the critical threshold cycle (Ct) as described previously [18, 23].
immunohistochemistry Samples from 10 tumors each exhibiting overexpressed and low amounts of mRNA in real-time PCR, respectively, were analyzed. Frozen tissue was cut into 8-lm slices and fixed by acetone. The Streptavidin-fluorescein isothiocyanate technique was used for immunohistochemistry applying the primary goat polyclonal antibody Mdr C-19:sc-1517 (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) raised against a peptide mapping at the C-terminus of Mdr-1 of human origin and the secondary anti-goat biotin-labeled antibody. For controls, K562 and K562-VP16 cell lines (see above) were used. Tissue spins from those cells were prepared by cytocentrifuge preparations of 1 ·105cells (60 g, 7 min, Cytospin Shandon, France) in PBS. Spins were air dried for 2 h and fixed in acetone for 10 min at 4C. Slides were visualized using a spectral microscope with AxionVisionsoftware (Zeiss, Go¨ttingen, Germany).
MGMT promoter methylation analysis MGMT promoter methylation was analyzed by methylation-specific PCR as reported before [24]. The primer sequences used to detect methylated MGMT promoter sequences were 5#-gtttttagaacgttttgcgtttcgac-3# and 5#caccgtcccgaaaaaaaactccg-3# (corresponding to nucleotides 46912–47033, GenBank accession no. AL355531, fragment size: 122 bp). The primer sequences used to detect unmethylated MGMT promoter sequences were 5#-tgtgtttttagaatgttttgtgttttgat-3# and 5#-ctaccaccatcccaaaaaaaaactcca-3# (corresponding to nucleotides 46909–47037, fragment size: 129 bp). As positive control sample, we used the A172 glioma cell line, which has a completely methylated MGMT promoter. Genomic DNA from a meningioma served as an unmethylated control sample.
markers, three estimators, D, D#, and r were computed. These analyses were carried out using the free statistical computing environment R (Version 2.3.1, http://www.r-project.org/) and its library genetics for genetic analyses.
results in vitro cytotoxicity of temozolomide The results of the MTT cytotoxicity assays are presented in Figure 1. In the chemosensitive MDR1-negative parental cell line K562 10 lg/ml temozolomide resulted in pronounced cell death with only 47.1% surviving 48 h compared with the control. In contrast, in the highly MDR1-expressing resistant subline K562-VP16, cell death was significantly lower after exposure to temozolomide with 73.4% surviving 48 h (P = 0.002). Addition of a nontoxic dose of the MDR1-modulator cyclosporine A (1 lM) to temozolomide resulted in a trend towards restoration of chemosensitivity in the resistant MDR1-expressing cell line. However, this difference in survival was not statistically significant. As both cell lines were methylated at the MGMT promoter site (data not shown), these results indicate that MDR1 participates in the resistance of tumor cells to temozolomide. genotype frequencies MDR1 genotypes of exon12 C1236T, exon21 G2677T, and exon26 C3435T SNPs were analyzed by SSCP (Figure 2) in all 112 glioblastoma patients. Genotype frequencies are depicted in Table 2 and were in accordance with those predicted by the Hardy–Weinberg equilibrium (P = 0.33, 1.0, and 0.85 for exon12, 21, and 26 SNPs, respectively). All three examined gene polymorphisms were in a linkage disequilibrium to each other (P < 0.0001 for each possible pair). This is reflected by the distribution of the detected
statistical analyses The Mann–Whitney U-test was used for comparison of the results. Differences in clinical or laboratory parameters were compared between the in vitro MTT assays or between the various SNPs of the MDR1 gene using the Mann–Whitney test for continuous and the chi-square test for categorical variables. Overall survival (OS) was measured from the date of diagnosis until death (failure) or last follow-up date (censoring). The Kaplan–Meier method was used to estimate OS probabilities, which were compared between different groups using the log-rank test. The Cox model was applied to identify independent prognostic variables for survival. Only patients with complete data for all variables considered were included in multiple models. A stepwise backward-selection procedure was carried out in order to determine the final model. The significance level was 0.05. To provide quantitative information on the relevance of results, 95% confidence intervals (CIs) of odds ratios and hazard ratios (HRs) were computed. All the above mentioned statistical analyses were carried out with the software package of SPSS Version 12.0.1 (SPSS Inc., Chicago, IL) and the S-PLUS software package version 6.2 (Insightful AG, Reinach, Switzerland). The deviation from the Hardy–Weinberg equilibrium was tested using the chi-square test. For pair wise linkage disequilibrium between the genetic
Figure 1. Cytotoxicity assay with methyl-thiazol-tetrazolium. The chemosensitive parental MDR1-negative cell line K562 was exposed to 10 lg/ml temozolomide (TMZ) and the derived MDR1-positive subline K562-VP16 to 10 lg/ml temozolomide, 1 lM cyclosporine A (CSA) and a combination of both drugs (TMZ + CSA). Results are shown as percentage of living cells compared with an unexposed control.
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significant difference of the MGMT methylation status was detected between the examined SNPs of the MDR1 gene (Table 2).
Figure 2. MDR1 SNP genotypes determined by PCR-SSCP of glioma patients (1–10). Two bands indicate heterozygosity, one lower band the C/ C allele in exon26 or the T/T alleles in exon12 and 21, one upper band the T/T allele in exon26 or the C/C and G/G alleles in exon12 and 21 as confirmed by sequence analysis.
Table 2. Genotypes of MDR1 gene polymorphisms, MDR1 expression, and MGMT promoter methylation status in glioblastoma patients
Exon12 C1236T C/C C/T T/T Exon21 G2677T G/G G/T T/T Exon26 C3435T C/C C/T T/T
n
%
Median MDR1/mRNA expression (copies)
Methylated MGMT promoter (%)
40 49 23
36 44 20
4427 6315 3364
44 25 36
36 56 20
32 50 18
3449 6739 3240
46 30 21
25 54 33
22 48 30
6621 3737 7106
36 32 36
combinations of the allelic variants (D# 0.85, 0.68, and 0.76 for exon21/12, 26/12, and 26/21 combination, respectively).
MDR1 mRNA and P-gp expression mRNA with sufficient quantity and integrity was available from 99 tumor samples. The median MDR1 mRNA expression was 5119 copies with a broad interindividual range of 1–165 628 copies. MDR1 mRNA expression correlated with P-gp expression as shown in 20 tumor tissue samples by immunohistochemistry (data not shown). Interestingly, the median MDR1 expression was higher in patients with the heterozygous variant of the exon12 and 21 SNPs and lower in patients with the heterozygous state of exon26 SNP compared with all other patients (Table 2). However, these differences were not statistically significant. MGMT promoter methylation Methylation of the MGMT gene promoter was found in 37 (34%) of 110 examined patients. Methylation status was hardly different between the two treatment groups (32% for patients receiving radiotherapy only and 35% for patients receiving radiotherapy plus temozolomide; P = 0.45). Furthermore, no
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effect of MDR1 gene variants and MGMT promoter methylation on treatment outcome The median OS was 11.0 (8.1–13.8) months for all 112 glioblastoma patients. Median survival was 8.5 months for patients receiving radiation therapy only (n = 48) and 15.8 months for patients (n = 64) receiving temozolomide in addition to radiation therapy (log-rank test: P < 0.001, data not shown). Potentially prognostic factors included in a multiple Cox model were gender, age, Karnofsky performance score, postoperative treatment group, MGMT methylation status and MDR1 genotypes of exon12, 21, and 26 SNPs (n = 108 patients with complete data). As MDR1 expression had no influence on survival in the univariate analysis and data were available only for 99 patients, this factor was not included. Postoperative treatment comprising radiotherapy plus temozolomide was protective, resulting in a HR of 0.33 (0.21–0.52) (P < 0.001) (Table 3). In the group of patients receiving temozolomide in addition to radiotherapy, the MGMT methylation status was predictive of treatment outcome. Patients with methylated MGMT (n = 22) survived longer than patients with an unmethylated MGMT promoter (n = 41). The 2-year OS rates were 27% versus 13%, respectively (log-rank test: P = 0.01, data not shown). In addition, patients with the C/C allele of the exon12 C1236T SNP (n = 28) had better 2-year OS compared with patients with the heterozygous variant (n = 25) (37% versus 8%; log-rank test: P = 0.02). Patients with the less frequent T/T variant (n = 11) had a 2-year OS of 10%. Due to the low number of patients with this variant no valid statistical conclusion in comparison with patients harboring the other two variants was applicable (Figure 3A). In the Cox model including above candidate variables (n = 61 patients receiving temozolomide with complete data), the MDR1 genotype of the exon12 C1236T SNP was an independent predictive factor for outcome of temozolomide therapy (P = 0.02). The HR for death of patients with the C/C allele was 0.25 (95% CI 0.10–0.65) compared with patients with the heterozygous variant (P = 0.004) (Table 3). No influence of the exon12 C1236T SNP genotypes on survival was seen in the group of patients with radiation therapy only (n = 48) (Figure 3B).
discussion In the present study, we have shown that the MDR1 encoded P-gp efflux pump is one of those factors, which are involved in resistance to temozolomide-mediated cytoxicity. In vitro survival of MDR1 expressing K562 cells was significantly higher than in the MDR1-negative parental cell line on incubating the cells with temozolomide. Both cell lines had a methylated MGMT promoter. Jelinek et al. [13] transfected K562 cells with a retroviral vector containing MDR1 and MGMT and correlated temozolomide resistance of the cells with MGMT expression. However, they did not test the influence of MDR1
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Annals of Oncology
Table 3. Multivariate analysis for survival probabilities in glioblastoma patients with a complete dataset of the examined factors Stepwise backward Cox regression analysis All patients (n = 108) Patients receiving temozolomide (n = 61) HR 95% CI P value HR 95% CI P value Age (<60 versus ‡60 years) Sex (male versus female) Karnofsky index (>80 versus £80) Postoperative therapy (radiotherapy + temozolomide versus radiotherapy only) MGMT promoter status (methylated versus unmethylated) MDR1 exon12 C/T C/C T/T MDR1 exon21 G/T G/G T/T MDR1 exon26 C/T T/T C/C
0.99 1.30 0.97 0.33
0.96–1.01 0.85–2.01 0.61–1.55 0.21–0.52
0.37 0.23 0.90 <0.001
0.99 1.70 0.95 NA
0.95–1.02 0.84–3.46 0.47–1.92 NA
0.50 0.14 0.57 NA
0.76
0.46–1.26
0.29 0.32
0.88
0.36–2.15 –
Baseline 0.68 0.83
– 0.41–1.12 0.48–1.45
–
Baseline 1.01 1.36
– 0.39–2.62 0.43–4.36
–
Baseline 1.29 1.27
– 0.64–2.58 0.69–2.34
–
0.78 0.02 – 0.004 0.80 0.26 – 0.86 0.14 0.07 – 0.07 0.19
0.13 0.51 0.86 0.98 0.60 0.60 0.48 0.45
Baseline 0.25 0.90
0.10–0.65 0.39–2.05
Baseline 0.90 2.67
– 0.28–2.91 0.73–9.72
Baseline 2.40 0.60
– 0.92–6.25 0.28–1.30
Numbers in bold are statistically significant. CR, complete remission; HR, hazard ratio to experience death; CI, confidence interval; NA, not applicable; MGMT, O6-methylguanine methyltransferase.
expression alone on temozolomide resistance. Significant MDR1 P-gp expression was found in both low- and high-grade gliomas, suggesting an intrinsic resistance to anticancer drugs [14]. Particularly, endothelial cells within gliomas stained positive for MDR1 P-gp [25, 26] supporting the idea that chemoresistance may be caused not only by P-gp expression in cancer cells but also by its expression in the endothelial cells of brain tumor capillaries. Thus, as it is known that genetic polymorphisms of the MDR1 gene may affect the expression and function of the P-gp efflux pump [15, 18, 27], we examined three polymorphisms of the MDR1 gene in exon12, 21, and 26 in the tumor specimens of 112 patients with glioblastoma. The genotype frequencies of those SNPs in our study population was comparable to the distribution found in the normal Caucasian population [21] and is in line with a recently published survey of Miller et al. [28]. They found no significant association of the exon26 C3435T polymorphism and the occurrence of glioma. In our analysis, the exon12 C1236T polymorphism of the MDR1 gene turned out to be an independent predictor of the outcome of temozolomide treatment in glioblastoma patients. Patients treated with radiotherapy plus temozolomide harboring the more frequent C/C allele had superior 2-year OS compared with patients with the heterozygous variant. This effect was independent of well-known prognostic factors like age and Karnofsky performance score and particularly the MGMT methylation status and was not seen in the group of patients having received radiotherapy only, indicating a specific role of MDR1 in the response to temozolomide in glioblastoma patients. This is further supported by a lower MDR1 expression in the tumor samples of patients harboring the C/C allele at the examined exon12 locus compared with
heterozygous patients. It also accords with our previous study of patients with acute myeloid leukemia [18], where allelic variants of the MDR1 gene including the exon12 C1236T SNP were found to influence therapy outcome. However, the way in which the exon12 polymorphism influences MDR1 gene expression and protein function is not known yet. One explanation might be functional linkage to polymorphic positions at regulatory sites of the MDR1 promoter. This hypothesis is supported by the linkage disequilibrium detected between the exon12, 21 and 26 SNPs, indicating that linkage of polymorphisms in the MDR1 gene is a common phenomenon, as shown for healthy subjects [27] and in patients with acute myeloid leukemia [18]. Kimchi-Safarty et al. [29] have shown that substrate specificity of the encoded P-gp is altered by a synonymous SNP (C3435T) which is part of the haplotype comprising the exon12, 21, and 26 SNPs, suggesting that an altered affinity of P-gp for temozolomide might be a reason for the survival differences after temozolomide therapy in our study. Nevertheless, we cannot rule out the possibility that the C/C genotype of the exon12 C1236T SNP might be an epiphenomenon accompanying other resistance mechanisms, like mismatch repair (MMR) pathway deficiency or microsatellite instability (MSI). However, in glioblastoma, MSI and underlying MMR gene mutation were found only in subgroups of patients like long-term survivors or particularly young patients, but infrequently in common cases [30]. In conclusion, the outcome of temozolomide treatment in glioblastoma patients can be predicted by evaluating the genotype of the MDR1 exon12 C1236T SNP. This finding seems to be independent of the MGMT methylation status. However, our data have to be verified in a prospective trial,
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Figure 3. Kaplan–Meier estimates of overall survival in glioblastoma patients according to genotype of MDR1 exon12 SNP. (A) Patients receiving postoperative treatment with radiotherapy + temozolomide (n = 64) (C/C genotype versus C/T genotype: log-rank test P = 0.02). (B) Patients receiving postoperative radiotherapy only (n = 48). No influence of the C/C allele on survival was found in this group.
where the MDR1 exon12 C1236T genotype is analyzed in combination with the MGMT promoter methylation status.
acknowledgements We would like to thank Kathrin Geiger, MD, for her help in questions of neuropathology and Michelle Meredyth-Stewart for her assistance in preparing the manuscript.
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