Skip to main content

MGMT methylation assessment in glioblastoma: MS-MLPA versus human methylation 450K beadchip array and immunohistochemistry

Clinical and Translational Oncology volume 18, pages 391–397 (2016)Cite this article

Abstract

Purpose

The MGMT gene encodes a DNA repair enzyme that counteracts with chemotherapy efficiency, specifically with alkylating agents such as temozolomide (TMZ). It is well established that MGMT methylation should be screened as a predictive marker for TMZ in glioblastoma, and we thus aimed to determine a reliable and practical diagnostic method of MGMT methylation detection.

Patients and methods

55 glioblastomas were investigated for MGMT methylation status using methylation-specific multiplexed ligation probe amplification (MS-MLPA), illumina human methylation 450K BeadChip array (HM450 K) analysis, and compared to MGMT protein expression by immunohistochemistry (IHC) staining. The methylation status of promoter, intron and all MGMT CpG targeted sites were separately correlated to patient’s survival.

Results

In addition to MS-MLPA and 450 K concordance, our results showed significantly higher overall survival (OS) of patients receiving TMZ and presenting MGMT methylated promoter (mean OS = 21.5 months, p = 0.046). Including all glioblastoma cases and regardless of chemotherapy, MS-MLPA showed significant survival difference between MGMT methylated and unmethylated cases (mean OS = 13, p = 0.021).

Conclusion

We concluded that in glioblastoma, MGMT promoter methylation predicts TMZ sensitivity. This current comparative analysis leads to consider that MS-MLPA is a valuable as HM450 K array for MGMT methylation status screening.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2

References

  1. Pegg AE. Repair of O(6)-alkylguanine by alkyltransferases. Mutat Res. 2000;462(2–3):83–100.

    CAS  Article  PubMed  Google Scholar 

  2. Karran P, Bignami M. DNA damage tolerance, mismatch repair and genome instability. BioEssays. 1994;16(11):833–9.

    CAS  Article  PubMed  Google Scholar 

  3. Herfarth KK, Brent TP, Danam RP, Remack JS, Kodner IJ, Wells SA Jr, et al. A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers. Mol Carcinog. 1999;24(2):90–8.

    CAS  Article  PubMed  Google Scholar 

  4. Ogino S, Meyerhardt JA, Kawasaki T, Clark JW, Ryan DP, Kulke MH, et al. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch. 2007;450(5):529–37.

    CAS  Article  PubMed  Google Scholar 

  5. Wolf P, Hu YC, Doffek K, Sidransky D, Ahrendt SA. O(6)-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancer. Cancer Res. 2001;61(22):8113–7.

    CAS  PubMed  Google Scholar 

  6. Wu X, Wilson TE, Lieber MR. A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events. Proc Natl Acad Sci USA. 1999;96(4):1303–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Oue N, Shigeishi H, Kuniyasu H, Yokozaki H, Kuraoka K, Ito R, et al. Promoter hypermethylation of MGMT is associated with protein loss in gastric carcinoma. Int J Cancer. 2001;93(6):805–9.

    CAS  Article  PubMed  Google Scholar 

  8. Goldenberg D, Harden S, Masayesva BG, Ha P, Benoit N, Westra WH, et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg. 2004;130(1):39–44.

    Article  PubMed  Google Scholar 

  9. Maruya S, Issa JP, Weber RS, Rosenthal DI, Haviland JC, Lotan R, et al. Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications. Clin Cancer Res. 2004;10(11):3825–30.

    CAS  Article  PubMed  Google Scholar 

  10. Steinmann K, Sandner A, Schagdarsurengin U, Dammann RH. Frequent promoter hypermethylation of tumor-related genes in head and neck squamous cell carcinoma. Oncol Rep. 2009;22(6):1519–26.

    CAS  PubMed  Google Scholar 

  11. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med. 2000;343(19):1350–4.

    CAS  Article  PubMed  Google Scholar 

  12. Mellai M, Caldera V, Annovazzi L, Chio A, Lanotte M, Cassoni P, et al. MGMT promoter hypermethylation in a series of 104 glioblastomas. Cancer Genom Proteom. 2009;6(4):219–27.

    CAS  Google Scholar 

  13. Shamsara J, Sharif S, Afsharnezhad S, Lotfi M, Raziee HR, Ghaffarzadegan K, et al. Association between MGMT promoter hypermethylation and p53 mutation in glioblastoma. Cancer Invest. 2009;27(8):825–9.

    CAS  Article  PubMed  Google Scholar 

  14. Lahtz C, Pfeifer GP. Epigenetic changes of DNA repair genes in cancer. J Mol Cell Biol. 2011;3(1):51–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Bady P, Sciuscio D, Diserens AC, Bloch J, van den Bent MJ, Marosi C, et al. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status. Acta Neuropathol. 2012;124(4):547–60.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Lee JY, Park CK, Park SH, Wang KC, Cho BK, Kim SK. MGMT promoter gene methylation in pediatric glioblastoma: analysis using MS-MLPA. Childs Nerv Syst. 2011;27(11):1877–83.

    Article  PubMed  Google Scholar 

  17. Park CK, Kim J, Yim SY, Lee AR, Han JH, Kim CY, et al. Usefulness of MS-MLPA for detection of MGMT promoter methylation in the evaluation of pseudoprogression in glioblastoma patients. Neuro Oncol. 2010;13(2):195–202.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Jeuken JW, Cornelissen SJ, Vriezen M, Dekkers MM, Errami A, Sijben A, et al. MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest. 2007;87(10):1055–65.

    CAS  Article  PubMed  Google Scholar 

  19. Chakravarti A, Erkkinen MG, Nestler U, Stupp R, Mehta M, Aldape K, et al. Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms. Clin Cancer Res. 2006;12(15):4738–46.

    CAS  Article  PubMed  Google Scholar 

  20. van den Bent MJ, Dubbink HJ, Sanson M, van der Lee-Haarloo CR, Hegi M, Jeuken JW, et al. MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951. J Clin Oncol. 2009;27(35):5881–6.

    Article  PubMed  PubMed Central  Google Scholar 

  21. van den Bent MJ, Erdem-Eraslan L, Idbaih A, de Rooi J, Eilers PH, Spliet WG, et al. MGMT-STP27 methylation status as predictive marker for response to PCV in anaplastic Oligodendrogliomas and Oligoastrocytomas. A report from EORTC study 26951. Clin Cancer Res. 2009;19(19):5513–22.

    Article  Google Scholar 

  22. Burke E, Grobler M, Elderfield K, Bond F, Crocker M, Taylor R, et al. Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours. Acta Neuropathol Commun. 2013;1:22.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We would like to thank the help given by the Division of Molecular Pathology and Cancer Therapeutics at the Institute of Cancer Research Sutton UK.

Author information

Affiliations

  1. Laboratory of Cytogenetics, Molecular Genetics and Reproductive Biology, Farhat Hached University Hospital, Street Ibn Eljazzar, 4000, Sousse, Tunisia

    S. Trabelsi, M. Haddaji Mastouri, M. Chourabi, A. Saad & D. H’mida Ben Brahim

  2. Department of Imagery, Sahloul University Hospital, Sousse, Tunisia

    N. Mama & K. Tlili

  3. Department of Neurosurgery, Sahloul University Hospital, Sousse, Tunisia

    M. Ladib, N. Karmeni & H. Krifa

  4. Department of Cytopathology, Farhat Hached University Hospital, Sousse, Tunisia

    M. Mokni & M. T. Yacoubi

Authors
  1. S. Trabelsi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N. Mama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Ladib
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. Karmeni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Haddaji Mastouri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Chourabi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Mokni
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. K. Tlili
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. H. Krifa
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. T. Yacoubi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. A. Saad
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. D. H’mida Ben Brahim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Trabelsi.

Ethics declarations

Ethical standards

The manuscript has been approved by the responsible authorities of our laboratory and by the ethics committee of Farhat Hached University Hospital of Sousse, Tunisia. We have no substantial direct or indirect commercial or financial incentive associated with publishing this article, and there is no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (TIFF 345 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Trabelsi, S., Mama, N., Ladib, M. et al. MGMT methylation assessment in glioblastoma: MS-MLPA versus human methylation 450K beadchip array and immunohistochemistry. Clin Transl Oncol 18, 391–397 (2016). https://doi.org/10.1007/s12094-015-1381-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12094-015-1381-0

Keywords

  • Methylation
  • MGMT
  • MS-MLPA
  • HM450 K beadchip array