Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model

Gaspar J. Kitange, Brett L. Carlson, Ann C. Mladek, Paul A. Decker, Mark A. Schroeder, Wenting Wu, Patrick T. Grogan, Caterina Giannini, Karla V. Ballman, Jan Craig Buckner, C. David James, Jann N Sarkaria

Research output: Contribution to journalArticle

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Abstract

CpG methylation within the O 6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in ∼50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kg × 5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r = -0.75; P = 0.003) and directly correlated with qMS-PCR (r = 0.72; P = 0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r = 0.78, P = 0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.

Original languageEnglish (US)
Pages (from-to)23-31
Number of pages9
JournalJournal of Neuro-Oncology
Volume92
Issue number1
DOIs
StatePublished - 2009

Fingerprint

temozolomide
Methyltransferases
Glioblastoma
Heterografts
Methylation
Polymerase Chain Reaction
DNA
Protein Methyltransferases
DNA Methylation
Survival
O(6)-Methylguanine-DNA Methyltransferase
O-(6)-methylguanine

Keywords

  • Glioblastoma
  • Methylation
  • MGMT
  • Orthotopic xenografts

ASJC Scopus subject areas

  • Clinical Neurology
  • Cancer Research
  • Oncology
  • Neurology

Cite this

Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model. / Kitange, Gaspar J.; Carlson, Brett L.; Mladek, Ann C.; Decker, Paul A.; Schroeder, Mark A.; Wu, Wenting; Grogan, Patrick T.; Giannini, Caterina; Ballman, Karla V.; Buckner, Jan Craig; David James, C.; Sarkaria, Jann N.

In: Journal of Neuro-Oncology, Vol. 92, No. 1, 2009, p. 23-31.

Research output: Contribution to journalArticle

Kitange, Gaspar J. ; Carlson, Brett L. ; Mladek, Ann C. ; Decker, Paul A. ; Schroeder, Mark A. ; Wu, Wenting ; Grogan, Patrick T. ; Giannini, Caterina ; Ballman, Karla V. ; Buckner, Jan Craig ; David James, C. ; Sarkaria, Jann N. / Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model. In: Journal of Neuro-Oncology. 2009 ; Vol. 92, No. 1. pp. 23-31.
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abstract = "CpG methylation within the O 6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in ∼50{\%} of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kg × 5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r = -0.75; P = 0.003) and directly correlated with qMS-PCR (r = 0.72; P = 0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r = 0.78, P = 0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10{\%} had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.",
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AU - Kitange, Gaspar J.

AU - Carlson, Brett L.

AU - Mladek, Ann C.

AU - Decker, Paul A.

AU - Schroeder, Mark A.

AU - Wu, Wenting

AU - Grogan, Patrick T.

AU - Giannini, Caterina

AU - Ballman, Karla V.

AU - Buckner, Jan Craig

AU - David James, C.

AU - Sarkaria, Jann N

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AB - CpG methylation within the O 6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in ∼50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kg × 5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r = -0.75; P = 0.003) and directly correlated with qMS-PCR (r = 0.72; P = 0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r = 0.78, P = 0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.

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