Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference

Abstract

Background: Epigenetic silencing of the MGMT gene by promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.

Methods: An analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The SNURF promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.

Results: Concordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). MGMT promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT methylation (p = 0.003). This difference was retained using other cut off values for MGMT methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of MGMT methylated allele was used as cut-off.

Conclusions: We report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated MGMT alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of MGMT promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.

Figure 1

CpG island of SNURF promoter. The upper lane identifies the exact sequence before bisulfite modification. The bottom lane represents the bisulfite treated methylated forward sequence where all C's are changed to T's, except for those followed by a G. Primer sequences are underlined by >. The internal probe covers two consecutive CpGs (marked as +).

Figure 2

Direct Sequencing of SNURF promoter. Direct sequencing after bisulfite modification of two CpGs of SNURF for case BF215. The arrows indicates the simultaneous presence of C/T (C: black peak; T: blue peak) due to a balanced amount of methylated and unmethylated cytosine in the CpGs amplified with our primer set.

Figure 3

Standard curves for MS-qLNAPCR with LNA primers. MS-qLNAPCR standard curves with serial dilution mixtures of SssI-treated DNA:untreated DNA (100%, 50%, 10%, 1%, 0.1%, 0.01%, 0.001%; each containing 1.5 μg of template DNA) for LNA primers specific for mMGMT. LNA modified primers for mMGMT show higher PCR efficiency (slope: -3.271; efficiency: 102%) than conventional primers (slope: -4.339; efficiency: 69%, see Fig.4). The analytical detection limit of 0.01% is reached only with LNA primers.

Figure 4

Standard curves for MS-qPCR with conventional primers. MS-qPCR standard curves with serial dilution mixtures of SssI-treated DNA:untreated DNA (100%, 50%, 10%, 1%, 0.1%, 0.01%, 0.001%; each containing 1.5 μg of template DNA) for conventional primers[35] specific for mMGMT. Conventional primers for mMGMT show lower PCR efficiency (slope: -4.339; efficiency: 69%) than LNA modified primers (see Fig.3). The analytical detection limit with the use of conventional primers was 0.1%.

Figure 5

A representative MS-qLNAPCR plot. A representative MS-qLNAPCR plot (GBM case BF240) showing an MGMT methylated ratio value of 0.46.

Figure 6

Bimodal distribution of methylated and unmethylated alleles. Normalized ratio value groups between methylated and unmethylated alleles of the 70 MGMT methylated GBMs identified with our MS-qLNAPCR protocol. There is a bimodal distribution of cases with two prevalent groups showing ratio values between 0.001-0.33 and 0.67-1, respectively.

Figure 7

Kaplan-Meier survival curves. Survival curves of GBM patients with methylated MGMT promoter (thick line) and unmethylated MGMT promoter (thin line) determined by MS-qLNAPCR. Patients with tumors with unmethylated MGMT promoter have lower overall survival (Log rank test p = 0.003).