MGMT Promoter Methylation and Glioblastoma: A Comparison of Analytical Methods and of Tumor Specimens

Abstract

It is already well known that hypermethylation of the O⁶-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (88.5%, 23/26 samples), using PSQ (76.7%, 23/30 samples). Moreover, we confirmed that OS and PFS for patients carrying methylation of the MGMT promoter were longer than for patients with an unmethylated promoter. In conclusion, we considered MSP a limited technique for FFPE tissues due to the high risk of false-positive results; in contrast, our data indicated PSQ as the most powerful method to stratify methylated/unmethylated patients as it allows reaching quantitative results with high sensitivity and specificity. Furthermore, frozen tumor tissues were shown to be the best specimens for MGMT methylation analysis, due to the low DNA degradation and homogeneity in methylation throughout the tumor.

Keywords

FFPE tissue Frozen tissue Methylation-specific PCR MGMT methylation Pyrosequencing

Introduction

Glioblastoma (GBM) is the most aggressive and highly malignant among the primary brain tumors, and represents the most common subtype in adults, with an annual incidence of 3-4/100,000 (1). In 2005, Stupp and coworkers introduced concomitant and adjuvant temozolomide (TMZ) to postoperative radiotherapy in GBM patients, reporting a 2.5-month increase in median overall survival (2). After this trial, the current standard therapy for GBM patients was defined as surgical resection followed by radiotherapy (RT) plus cytotoxic chemotherapy given concomitantly with and after RT (3).

TMZ is a second-generation DNA-alkylating agent that mediates its cytotoxic effect by forming O⁶-methylguanines (O⁶-MeG) which preferentially pair with thymines rather than cytosine during DNA replication (4). These DNA adducts lead to base mispairing and double-strand breaks inducing apoptosis and cell death (5). The major complication of treating cancer patients with alkylating agents is chemotherapy resistance due to cellular intrinsic DNA repair mechanisms. Among these, the human O⁶-methylguanine DNA methyltransferase (MGMT) protein is well known to remove alkyl groups from the O⁶-position of guanine by an irreversible transfer of the alkyl group to a cysteine residue at its active site (6). Therefore a transcriptionally active MGMT gene is thought to protect tumor cells against the cytotoxic effect of TMZ (7, 8).

In human cancer, silencing of the MGMT gene is not commonly due to mutation or deletion but most frequently to epigenetic changes and specifically promoter-region methylation (9). It is already well demonstrated that in primary tumors, including GBM, MGMT methylation correlates with loss of mRNA expression, lack of MGMT protein and loss of enzymatic activity (10–12).

Many studies have already defined MGMT hypermethylation as a predictive biomarker of favorable outcome in terms of progression-free survival (PFS) and overall survival (OS) in GBM patients treated with TMZ (12, 13). Though MGMT promoter methylation is largely recognized in clinical neuro-oncology as an important prognostic marker, the choice of the analytical method to be used for its determination is still controversial. Recently, many detection methods to analyze MGMT methylation have been described: bisulfite sequencing (14), methylation-specific polymerase chain reaction (MSP) (15), combined bisulfite restriction analysis (COBRA) (16), pyrosequencing (PSQ) (17), SNuPE ion pair-reverse phase high-performance liquid chromatography (SIRPH) (18), methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) (19), real-time quantitative MSP (RT-MSP) (20), Methylation Specific PCR-Capillary Electrophoresis (MSP-CE) (21) and others. Indeed, considering the cost-effectiveness and ease of use, PSQ seems to be the most suitable method for MGMT methylation analysis (5, 6, 22), although it is not available in all laboratories for routine clinical practice. Obversely, MSP requires only a PCR machine, commonly present in all diagnostic pathology departments.

In this study we systematically compared the qualitative MSP and the quantitative PSQ methods for MGMT promoter methylation analysis performed on both snap-frozen and formalin-fixed paraffin-embedded (FFPE) samples. We discuss our results in order to define the most suitable method and sample specimen for MGMT methylation analysis in clinical settings to better tailor TMZ therapy in GBM patients.

Materials and Methods

Patients and samples

Tissue samples were collected between 2006 and 2013 from 46 patients treated at the Department of Neurosurgery at S. Croce University Hospital, Cuneo, after written informed consent by the patients and approval by the ethics committee of S. Croce University Hospital in accordance with the Helsinki Declaration. Patients included in the study were newly diagnosed for GBM and treated with standard TMZ-containing chemoradiotherapy protocols. The group of patients consisted of 35 men and 11 women, with a median age of 64.5 years (range 24-84 years). Fifteen of 46 patients were censored, while 29 of 46 are now dead. Median follow-up was 7.4 months, median OS was 10.5 months and median PFS was 7.2 months. For each patient, 2 samples of the primary tumor were obtained: 1 was collected during surgery, immersed in RNA later (Life Technologies, Carlsbad, CA, USA) and immediately snap-frozen in liquid nitrogen, and the other was assembled from biopsy in FFPE sections by use of standard procedures. FFPE tissues were selected by a single expert pathologist and microdissected to have at least a 80% tumor cell content. FFPE samples were prepared at the same time as frozen samples.

To determine the methylation cutoff value for PSQ analysis, we extracted DNA from a pool of 5 normal brain tissues derived from autopsies; the average percentage of methylation of the 5 samples was 8%; thus we considered methylated any tumor sample carrying ≥9% methylation.

DNA extraction and bisulfite treatment

Genomic DNA was extracted from frozen samples using the tissue protocol of the QIAamp DNA Blood mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. DNA from FFPE tissue sections was obtained performing standard proteinase K digestion and phenol extraction. DNA concentration and quality was assessed with a NanoDrop ND-1000 Spectrophotometer (Celbio-Euroclone, Irvine, CA, USA). Genomic DNA from frozen and FFPE tissues (300 ng and 500 ng, respectively) was subjected to bisulfite conversion using the EZ Methylation Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. A universal methylated human DNA (Zymo Research, Irvine, CA, USA) and a human placenta DNA (Sigma Aldrich, St. Louis, MO, USA) were used, respectively, as positive and negative controls for methylation in both MSP and PSQ procedures.

Methylation-Specific Polymerase Chain Reaction

To improve MSP results, we attempted a 2-step PCR approach using a first set of primers amplifying the exon 1 region of the MGMT gene including the CpG island and subsequently the specific primers for either methylated or unmethylated DNA established by Esteller et al (12). Primers used in the first PCR reaction were: 5′-GGATATGTTGGGATAGTT-3′ (forward primer, GenBank accession number AL355531, nucleotides 46891 to 46908) and 5′-CCAAAAACCCCAAACCC-3′ (reverse primer, GenBank accession number AL355531, nucleotides 47162 to 47179) amplifying a 289-bp fragment. The PCR was performed in a final volume of 10 µL containing 1 µL of ×10 buffer, 1 µL of 2 mM dNTPs, 0.6 µL of 25 mM MgCl₂, 1 µL of each primer (5 µM), 0.05 units of AmpliTaq Gold 360 DNA polymerase (Applied Biosystems, Foster City, CA, USA), 4.35 µL of high purity water and 1 µL of bisulfite-treated DNA as template. The PCR program included 10 minutes at 95°C followed by 40 cycles at 95°C for 30 seconds, 52°C for 30 seconds and 72°C for 30 seconds, and a final step at 72°C for 10 minutes. Positive (methylated DNA) and negative (placenta DNA) controls were included in the PCR and treated as well as samples. The PCR products were diluted 1:20 with high purity water and used in the second PCR reaction as templates. PCR protocol and program of the second PCR were equal to the first one except for the primers’ annealing temperature (62°C) and number of cycles (n=30). PCR products were separated on a 2% agarose gel. For each sample, the results were qualitatively interpreted as follows: a visible band in the M primer set and absence of the U primer set product indicated a positive methylation status, whereas absence of a M primer set product and presence of a band in the U primer set was evaluated as a negative methylation status.

Frozen and FFPE tissue samples were analyzed in triplicate.

Pyrosequencing

PSQ was performed using the PyroMark ID System (Biotage, Uppsala, Sweden). The PSQ primers used for amplification of bisulfite-treated DNAs were designed to cover a region including 9 CpG sites of the MGMT promoter at the beginning of the first exon, adjacent to the region covered by MSP primers (specifically, CpGs 5-6-7-8-9 in the pyrograms corresponded to CpGs included in specific M/U MSP primers). The primers were 5′-GGATATGTTGGGATAGTT-3′ (forward primer, GenBank accession number AL355531, nucleotides 46891 to 46908) and 5′-biotin- ACCCAAACACTCACCAAA-3′ (reverse primer, GenBank accession number AL355531, nucleotides 46972 to 46990), which amplified a 99-bp region. PCR was performed in a final volume of 30 µL, containing 0.5 µM of each primer, 200 µmol/L of each dNTP, 0.05 units of AmpliTaq Gold 360 DNA polymerase (Applied Biosystems, Foster City, CA, USA) in buffer containing 1.5 mmol/L MgCl₂, 3 µL of 360 GC enhancer (Applied Biosystems, Foster City, CA, USA) and 2 µL of bisulfite-treated DNA as template. The initial denaturation step (95°C, 10 minutes) was followed by 35 cycles (frozen tissues) or 40 cycles (FFPE tissues) of 30 seconds at 95°C, 30 seconds at 52°C, 40 seconds at 72°C and a final extension step at 72°C for 10 minutes. Three microliters of the PCR products was visualized by gel electrophoresis, and 25 µL was subjected to PSQ using the forward primer as sequencing primer at a final concentration of 0.5 µM. Purification and subsequent processing of the biotinylated single-strand DNA was done according to the manufacturer's instructions using the SAMPLE PREP reagents kit (Biotage, Uppsala, Sweden). Resulting data were analyzed and quantified with PyroMark CpG Software (Biotage). Positive (methylated DNA) and negative (placenta DNA) controls were included and treated as well as samples. Pyrograms of the control DNA were analyzed to confirm complete bisulfite conversion. All samples were analyzed in duplicate.

Statistical Analysis

Survival curves were estimated according to the Kaplan-Meyer method, and the differences of MGMT methylation status category were estimated using the log-rank test. We obtained patients’ life status, death date and follow-up by registry. OS was defined as the time between the date of first surgery and date of death or last follow-up. PFS was defined as the time between the date of first surgery and progression or last follow-up. A p value less than 0.05 was considered statistically significant. The statistical analysis was performed using SPSS version 13 (SPSS Inc, Chicago, IL, USA).

Results

MGMT Methylation Status Analyzed by MSP and PSQ

In the present study we investigated frozen tissue samples and FFPE tissue samples of 46 GBM patients treated with chemoradiotherapy protocols containing TMZ.

MGMT methylation data were successfully obtained in all tumor specimens when DNA was extracted from frozen samples (46/46), whereas 5 FFPE tissue samples gave no results in PCR amplification by MSP (41/46). In particular, using MSP, among frozen tissue samples, 26/46 tumors were methylation-positive (56.5%), while among FFPE tissue samples 18/41 tumors were methylated (43.9%). Using PSQ, on the basis of the methylation level based on normal brain tissues (8%), we used ≥9% as cutoff value to consider a sample methylated. Consequently, 23/46 DNAs extracted from frozen tissues were scored as methylation-positive (50%), whereas analysis of FFPE tissues resulted in 30/46 positive samples (65.2%). Representative pictures of MSP and PSQ results are shown in Figure 1.

Fig. 1

Representative methylation-specific polymerase chain reaction (MSP) (A) and pyrosequencing (PSQ) (B) analyses of MGMT promoter in glioblastoma (GBM) tissues. A) Typical results obtained by MSP: samples #80, #71 and methylated DNA present a band in the M primer set (methylation-positive), whereas samples #65, #84 and placenta DNA are unmethylated (band in the U primer set). Neg ctrl = water (no amplification). B) Typical pyrograms obtained for a methylated sample (#72, upper panel) and for an unmethylated sample (#52, lower panel); shadow squares highlight each CpG site analyzed by pyrosequencing with the corresponding percentage of methylation calculated by the software. The light gray box around the single “C” corresponds to the bisulfite control: there must be no peak in this nucleotide, meaning 0% cytosine incorporation.

Comparison of the 2 Techniques and the 2 Sample Specimens

When comparing in 2-by-2 entry diagrams for all the parameters, we did not include the 5 samples that gave no PCR amplification by MSP, making a total of 41 GBM samples.

Figure 2 shows concordant and discordant results, calculated among methylation-positive samples. In particular, comparing the 2 techniques, the highest concordance was found on frozen tissue samples (88.5%, 23/26 samples, Fig. 2A), while in the comparison between the 2 tissue specimens, the highest concordance was found using PSQ (76.7%, 23/30 samples, Fig. 2B).

Fig. 2

Comparison of the 2 techniques and of the 2 sample specimens. Diagrams summarize the numbers of methylated samples and highlight the concordance/discordance between the 2 techniques (A) and the 2 sample specimens (B). The central dotted part of the diagrams shows the concordance between groups; discordant results are highlighted in the corresponding clear part. Black boxes indicate the highest concordance for each comparison. FFPE = formalin-fixed paraffin-embedded; MSP = methylation-specific polymerase chain reaction; PSQ = pyrosequencing.

Comparison of the Results in Discordant Samples

Table I summarizes the results of MGMT methylation analysis by MSP and PSQ on frozen and FFPE tissues in discordant samples. Comparing both techniques and both tissue specimens, 28% of samples showed discordant results (13/46).

TABLE I

MGMT Methylation status in discordant samples

Patient ID	Frozen tissues		FFPE tissues
	MSP results (meth/unmeth)	PSQ results (average % meth)	MSP results (meth/unmeth)	PSQ results (average % meth)
# 14	meth	67	unmeth	37
# 16	meth	7	not available	2
# 17	meth	9	unmeth	18
# 26	meth	1	unmeth	48
# 27	meth	92	unmeth	42
# 46	meth	2	meth	42
# 48	meth	65	unmeth	43
# 50	unmeth	7	meth	10
# 56	unmeth	1	meth	43
# 59	unmeth	2	unmeth	24
# 67	unmeth	2	unmeth	11
# 85	meth	35	unmeth	71
# 90	unmeth	2	unmeth	42

Comparison of results obtained in discordant samples by MSP and PSQ. Patient ID = patient identification number; FFPE = formalin-fixed paraffin-embedded; MSP = methylation-specific PCR; PSQ = pyrosequencing; meth = methylated; unmeth = unmethylated.

Among FFPE samples, 9/13 were discordant and were methylation-positive only if analyzed by PSQ and not by MSP. Three of 13 frozen tissues were found to be methylated by MSP but not by PSQ. Five of 13 samples were shown to be methylated by all techniques except for MSP on FFPE tissues. Seven of 13 samples analyzed by PSQ showed discordant results if the 2 different specimens were examined, and in particular all of them were methylated only for FFPE tissues. Five samples (ID nos. 50, 56, 59, 67 and 90) were found to be unmethylated on frozen tissues by both techniques but methylated on FFPE tissues by PSQ.

Correlation between Methylation Status and Overall Survival

We evaluated the correlation between GBM OS and MGMT methylation status determined for 45 patients by MSP and PSQ on frozen tissues and by PSQ on FFPE tissues, and for 40 patients by MSP on FFPE tissues. Figure 3 shows Kaplan-Meier survival curves for both qualitative MSP and quantitative PSQ results. In line with results reported in the literature, our data showed that patients carrying methylation on the MGMT promoter presented a favorable prognosis compared with patients with an unmethylated MGMT promoter. Correlation between OS and methylation on frozen tissues by MSP and PSQ reached the same statistical significance (p=0.031, Fig. 3A, C), while on FFPE tissues, PSQ performed better (p=0.045, Fig. 3D) than MSP (p=0.598, not significant, Fig. 3B).

Fig. 3

Overall survival (OS) of glioblastoma patients. Kaplan-Meyer curves show the correlation between methylation status and OS, based on results from methylation-specific polymerase chain reaction (MSP) on frozen tissues (45 patients analyzed) (A); MSP on formalin-fixed paraffin-embedded (FFPE) tissues (40 patients analyzed) (B); pyrosequencing (PSQ) on frozen tissues (45 patients analyzed) (C); and PSQ on FFPE tissues (45 patients analyzed) (D). In the figures, dark gray lines: methylated; light gray lines: unmethylated; p values calculated as described in “Methods.”

Considering as optimal the analysis on frozen tissues, the median OS of patients with a methylated MGMT promoter was about 5.2 months longer than the median OS of patients with unmethylated MGMT promoter (14.1 vs. 8.9 months, respectively).

Correlation between MGMT Methylation Status and PFS

On the same cohort of patients, we determined the PFS, for 42 patients analyzed by MSP and PSQ on frozen tissues and by PSQ on FFPE, and for 39 patients by MSP on FFPE tissues (Fig. 4). Again, PSQ appeared to be the most efficient technique for MGMT methylation studies as it was shown to be not only appropriate for frozen tissues, where the 2 techniques performed the same (p=0.020, Fig. 4A, C), but also on FFPE tissues (p=0.021, Fig. 4D), where the prognostic value of methylation by MSP was lower (p=0.808, Fig. 4B).

Fig. 4

Progression-free survival data (PFS). Kaplan-Meyer curves show the correlation between methylation status and PFS, based on results from methylation-specific polymerase chain reaction (MSP) on frozen tissues (42 patients analyzed) (A); MSP on formalin-fixed paraffin-embedded (FFPE) tissues (39 patients analyzed) (B); pyrosequencing (PSQ) on frozen tissues (42 patients analyzed) (C); and PSQ on FFPE tissues (42 patients analyzed) (D). In the figures, dark gray lines: methylated; light gray lines: unmethylated; p values calculated as described in “Methods.”

The median PFS for patients with methylated MGMT promoter on frozen tissues was 4.7 months longer than the median PFS for patients with unmethylated MGMT promoter (9.9 vs. 5.2 months, respectively).

Discussion

The main focus of this study was the comparison of analytical methods for MGMT methylation analysis and sample specimens, aiming to correlate these clinically only with OS and PFS. Our results determined that PSQ on frozen tissue specimens was the most accurate method.

It is already well known that MGMT promoter methylation is of prognostic value for primary GBM (5, 23), but the optimal analytical method for its determination needs still to be defined and incorporated into clinical practice. Actually, also the choice of the ideal tissue sample is still controversial, since frozen specimens might be less often available than FFPE tissues in pathology laboratories, and furthermore the preservation method might influence MGMT methylation analysis.

In this study, we initially determined the percentage of MGMT-positive methylated samples: 56.5% and 43.9% by MSP on frozen and FFPE tissues, respectively, and 50% and 65.2% by PSQ on frozen and FFPE tissues, respectively. These data are in agreement with the literature reporting MGMT methylation in 30%-60% of GBM patients (24). We determined a threshold value of 9% for scoring samples as methylation positive by calculating the average percentage methylation of 5 normal brain tissues derived from autopsies. The resulting cutoff is similar to the one (8%) recently described by Quillien et al (25).

Afterward, we compared MSP/PSQ and frozen/FFPE tissues in terms of concordance and discordance between techniques and sample specimens. Our data suggested that the most suitable technique seems to be PSQ, as it may be used on both frozen and FFPE tissue types with 100% efficiency (46/46 results obtained). Moreover, in our analysis, the highest concordance in methylation was found on frozen tissues (88.5%, 23/26 samples) and using PSQ (76.7%, 23/30 samples). Discordant results, as expected, were found among FFPE tissues: in particular in 5 FFPE samples, we did not obtain any amplification, and 5/13 discordant samples were unmethylated when analyzed by MSP on FFPE tissue but methylated in all other cases. Considering discordant samples, 9/13 among FFPE tissues were found to be methylation-positive only by PSQ, while among frozen tissues, 3/13 were found to be methylated only by MSP; 7/13 among samples analyzed by PSQ were shown to be methylation-positive only on FFPE tissues and 5 samples were found to be unmethylated on frozen tissues by both techniques but methylated on FFPE tissues by PSQ.

It is already well known that the preservation process can influence DNA quality and integrity. Formalin fixation is the standard storage method for tissues, but induces DNA degradation, leading to a poor DNA quality; this, in addition to damage derived from bisulfite conversion could determine the false-positive results and overestimation of methylation level analysis (26). This could explain the large number of discrepant results obtained in FFPE tissues and in particular the higher number of methylated samples analyzed by PSQ: the inefficient conversion matched with the high sensitivity of PSQ could lead to false positive results. In addition, discordant results obtained by MSP on frozen or FFPE tissues could be explained by subtle bias in PCR efficiency obtained with methylated/unmethylated primers (i.e., the degradated DNA in FFPE tissues could hamper PCR amplification, and unmethylated primers, being longer than methylated ones, could be leading to false-negative results). Obversely, cryopreservation techniques protect DNA from degradation but maintenance of a frozen tissue bank is complex and costly, and such banks are lacking in many laboratories. Both techniques present advantages and pitfalls: MSP requires standard and not expensive laboratory equipment such as a PCR amplifier which is actually nowadays present in almost all diagnostic laboratories, but it is a qualitative method and attempts at quantification based on interpretation of gel band intensities may be operator-dependent. In contrast, PSQ provides quantitative levels of methylation at each individual CpG site, includes a conversion control, and is highly sensitive and specific (27), but it requires expensive equipment and specialized personnel. Recent studies have shown that not all CpG sites in the MGMT promoter are equally capable of predicting patient outcomes (5). In this contest, we considered the average methylation level in order to compare the distinct techniques (MSP and PSQ), as MSP results are expressed as a simply qualitative result (methylated/unmethylated). However, it is worth noting that samples presenting high levels (>15%) or very low levels (<5%) of methylation displayed uniform distribution of methylation percentage in all CpG sites; in contrast, samples near to the cutoff (9%) presented differences in methylation levels between single CGs. Further analysis on more patients with such intermediate profiles of methylation and correlations of single CGs with OS/PFS will give an opportunity to focus on specific CGs instead of using the average of methylation.

Moreover, it must be considered that those discrepancies may also derive from intratumoral heterogeneity of surgical sample collection within the glioblastoma. However, recent studies have demonstrated that methylation status was homogeneous in different regions of the tumor when a frozen tissue was used, even if the comparison between frozen and FFPE tissues presented discordant results (27). If MGMT protein expression is considered, some heterogeneity is present in different regions, decreasing progressively from the inner to the outer layer of tumors (28). Thus, even if frozen tissues seem to be the most suitable specimens for MGMT methylation analysis in terms of detection methods, the determination of both MGMT methylation and protein expression should actually be used by clinicians to optimize a therapy containing TMZ, for GBM patients.

We are aware that the “take-home” message of our study may be influenced by the low sample number, but we consider the following points to be strengths: (i) all tissues examined were derived from the neurosurgery and pathology departments of a single hospital; (ii) all frozen tissues were collected during surgery by a unique team of surgeons, and this actually considerably decreased the variability among samples and with regard to the quality of biopsies and radical surgery; (iii) FFPE tissues were selected by a single expert pathologist, microdissected when necessary and taken with the same biopsy used for GBM diagnosis (29); (iv) MSP and PSQ are routinely used in the laboratories of S. Croce University Hospital. Altogether, this makes our cohort of patients a valid single-center and uniformly treated GBM patient group in which to compare different techniques and tissue specimens.

Moreover, in the univariate analysis, we confirmed that OS and PFS for patients carrying methylation on the MGMT promoter were longer than for patients with an unmethylated promoter, as reported by others (13, 23, 30). Our data showed a correlation between OS/PFS and MGMT methylation that was statistically significant in all analyses except for that for MSP on FFPE tissues. We are aware that this result could derive from the low number of analyzed samples, as the prognostic role of methylation on survival has been already well established (30). In this study, the most suitable sample specimen to be used for MGMT methylation analysis seems to have been frozen tissue, as it correlated with OS and PFS with the highest statistical significance (OS: p=0.031; PFS: p=0.020). Furthermore, we suggest that the better method to use for prognosis and pseudoprogression estimation by clinicians is PSQ, since it performed well on both frozen and FFPE tissues (OS on frozen tissues: p=0.031; OS on FFPE tissues: p=0.045). Since recent studies have reported that MGMT promoter methylation status is highly concordant also between tumor tissues and plasma, and both are associated with longer survival (31), it would be of interest to extend the comparison herein proposed also to serum samples collected from patients after and during treatments.

In conclusion, our clinical and technical data obtained by MSP on FFPE tissues affirmed the limit of this technique, suggesting that, whenever possible, the opportunity should be taken to confirm the level of methylation using PSQ, when the clinician intents to use it for OS and PFS predictions and to tailor TMZ treatment in GBM patients.

Footnotes

Acknowledgements

We thank Dr. Maura Menghi (Diatech, Jesi, AN) for scientific assistance.

Financial support: No grants or funding have been received for this study.

Conflict of interest: None of the authors has any financial interest related to this study to disclose.

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