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MGMT gene promoter methylation correlates with tolerance of temozolomide treatment in melanoma but not with clinical outcome.

Hassel JC, Sucker A, Edler L, Kurzen H, Moll I, Stresemann C, Spieth K, Mauch C, Rass K, Dummer R, Schadendorf D - Br. J. Cancer (2010)

Bottom Line: Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response.Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy.Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32-5.7)).

View Article: PubMed Central - PubMed

Affiliation: Skin Cancer Unit, German Cancer Research Center, University Hospital Mannheim, Mannheim, Germany.

ABSTRACT

Background: Despite limited clinical efficacy, treatment with dacarbazine or temozolomide (TMZ) remains the standard therapy for metastatic melanoma. In glioblastoma, promoter methylation of the counteracting DNA repair enzyme O(6)-methylguanine-DNA-methyltransferase (MGMT) correlates with survival of patients exposed to TMZ in combination with radiotherapy. For melanoma, data are limited and controversial.

Methods: Biopsy samples from 122 patients with metastatic melanoma being treated with TMZ in two multicenter studies of the Dermatologic Cooperative Oncology Group were investigated for MGMT promoter methylation. We used the COBRA (combined bisulphite restriction analysis) technique to determine aberrant methylation of CpG islands in small amounts of genomic DNA isolated from paraffin-embedded tissue sections. To detect aberrant methylation, bisulphite-treated DNA was amplified by PCR, enzyme restricted, and visualised by gel electrophoresis.

Results: Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response. A methylated MGMT promoter was observed in 34.8% of responders and 23.4% of non-responders (P=0.29). In addition, no survival advantage for patients with a methylated MGMT promoter was detectable (P=0.79). Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy. Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32-5.7)). Analysis of MGMT promoter methylation comparing primaries and different metastases over the clinical course revealed no statistical difference (P=0.49).

Conclusions: In advanced melanoma MGMT promoter, methylation correlates with tolerance of therapy, but not with clinical outcome.

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Related in: MedlinePlus

(A) COBRA results from five representative samples. Tumour DNA was extracted from paraffin-embedded tissue sections, bisulphite treated, amplified by PCR, Taq1 digested, and visualised by gel electrophoresis. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA (lower band) was estimated as 0, 10, 25, 50, 75, or 100% methylated (see numbers; M=marker); (B) Examples of bisulphite sequencing results for 10 clones of unmethylated (sample 39) and methylated (sample 648) samples in COBRA analysis: For quality control, PCR products of the COBRA analysis were extracted from the gel and cloned; each circle represents a CpG island of the MGMT promoter. The ‘unmethylated' sample by COBRA analysis revealed, on average, 3.4 methylated CpG islands, the ‘methylated' sample 11.8, indicative of reliable COBRA results.
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fig1: (A) COBRA results from five representative samples. Tumour DNA was extracted from paraffin-embedded tissue sections, bisulphite treated, amplified by PCR, Taq1 digested, and visualised by gel electrophoresis. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA (lower band) was estimated as 0, 10, 25, 50, 75, or 100% methylated (see numbers; M=marker); (B) Examples of bisulphite sequencing results for 10 clones of unmethylated (sample 39) and methylated (sample 648) samples in COBRA analysis: For quality control, PCR products of the COBRA analysis were extracted from the gel and cloned; each circle represents a CpG island of the MGMT promoter. The ‘unmethylated' sample by COBRA analysis revealed, on average, 3.4 methylated CpG islands, the ‘methylated' sample 11.8, indicative of reliable COBRA results.

Mentions: Combined bisulphite restriction analysis was performed to determine aberrant methylation of CpG islands in small amounts of genomic DNA (Xiong and Laird, 1997). We introduced methylation-dependent sequence differences by sodium bisulphite treatment of genomic DNA using the EpiTect Bisulphite Kit (Qiagen) according to manufacturer's instructions. Primers used for amplifying a 295-bp PCR product of the MGMT promoter region were as follows: forward 5-TGGTAAATTAAGGTATAGAGTTTTAGG and reverse 5-AAAACCTAAAAAAAACAAAAAAAC. The PCR reactions were performed in a volume of 20 μl containing 1x Ready mix (Abgene, Cambridge, UK), 2 μl bisulphite-treated genomic DNA, 0.2 μ of each primer, 1 m dNTPs (Stratagene, Amsterdam, NL, USA), and 1 U Thermoprime Plus DNA polymerase (Abgene). We performed 35 cycles of amplification at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s followed by a final extension at 72°C for 3 min. PCR products were digested with restriction enzyme TaqI (New England Biolabs, Ipswich, MA, USA) and separated on agarose gels. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA of the extracted amount of genomic DNA was estimated as 0, 10, 25, 50, 75, or 100% methylated (Figure 1a).


MGMT gene promoter methylation correlates with tolerance of temozolomide treatment in melanoma but not with clinical outcome.

Hassel JC, Sucker A, Edler L, Kurzen H, Moll I, Stresemann C, Spieth K, Mauch C, Rass K, Dummer R, Schadendorf D - Br. J. Cancer (2010)

(A) COBRA results from five representative samples. Tumour DNA was extracted from paraffin-embedded tissue sections, bisulphite treated, amplified by PCR, Taq1 digested, and visualised by gel electrophoresis. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA (lower band) was estimated as 0, 10, 25, 50, 75, or 100% methylated (see numbers; M=marker); (B) Examples of bisulphite sequencing results for 10 clones of unmethylated (sample 39) and methylated (sample 648) samples in COBRA analysis: For quality control, PCR products of the COBRA analysis were extracted from the gel and cloned; each circle represents a CpG island of the MGMT promoter. The ‘unmethylated' sample by COBRA analysis revealed, on average, 3.4 methylated CpG islands, the ‘methylated' sample 11.8, indicative of reliable COBRA results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2966614&req=5

fig1: (A) COBRA results from five representative samples. Tumour DNA was extracted from paraffin-embedded tissue sections, bisulphite treated, amplified by PCR, Taq1 digested, and visualised by gel electrophoresis. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA (lower band) was estimated as 0, 10, 25, 50, 75, or 100% methylated (see numbers; M=marker); (B) Examples of bisulphite sequencing results for 10 clones of unmethylated (sample 39) and methylated (sample 648) samples in COBRA analysis: For quality control, PCR products of the COBRA analysis were extracted from the gel and cloned; each circle represents a CpG island of the MGMT promoter. The ‘unmethylated' sample by COBRA analysis revealed, on average, 3.4 methylated CpG islands, the ‘methylated' sample 11.8, indicative of reliable COBRA results.
Mentions: Combined bisulphite restriction analysis was performed to determine aberrant methylation of CpG islands in small amounts of genomic DNA (Xiong and Laird, 1997). We introduced methylation-dependent sequence differences by sodium bisulphite treatment of genomic DNA using the EpiTect Bisulphite Kit (Qiagen) according to manufacturer's instructions. Primers used for amplifying a 295-bp PCR product of the MGMT promoter region were as follows: forward 5-TGGTAAATTAAGGTATAGAGTTTTAGG and reverse 5-AAAACCTAAAAAAAACAAAAAAAC. The PCR reactions were performed in a volume of 20 μl containing 1x Ready mix (Abgene, Cambridge, UK), 2 μl bisulphite-treated genomic DNA, 0.2 μ of each primer, 1 m dNTPs (Stratagene, Amsterdam, NL, USA), and 1 U Thermoprime Plus DNA polymerase (Abgene). We performed 35 cycles of amplification at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s followed by a final extension at 72°C for 3 min. PCR products were digested with restriction enzyme TaqI (New England Biolabs, Ipswich, MA, USA) and separated on agarose gels. For semiquantitative evaluation, percentage of cleaved and, therefore, methylated DNA of the extracted amount of genomic DNA was estimated as 0, 10, 25, 50, 75, or 100% methylated (Figure 1a).

Bottom Line: Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response.Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy.Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32-5.7)).

View Article: PubMed Central - PubMed

Affiliation: Skin Cancer Unit, German Cancer Research Center, University Hospital Mannheim, Mannheim, Germany.

ABSTRACT

Background: Despite limited clinical efficacy, treatment with dacarbazine or temozolomide (TMZ) remains the standard therapy for metastatic melanoma. In glioblastoma, promoter methylation of the counteracting DNA repair enzyme O(6)-methylguanine-DNA-methyltransferase (MGMT) correlates with survival of patients exposed to TMZ in combination with radiotherapy. For melanoma, data are limited and controversial.

Methods: Biopsy samples from 122 patients with metastatic melanoma being treated with TMZ in two multicenter studies of the Dermatologic Cooperative Oncology Group were investigated for MGMT promoter methylation. We used the COBRA (combined bisulphite restriction analysis) technique to determine aberrant methylation of CpG islands in small amounts of genomic DNA isolated from paraffin-embedded tissue sections. To detect aberrant methylation, bisulphite-treated DNA was amplified by PCR, enzyme restricted, and visualised by gel electrophoresis.

Results: Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response. A methylated MGMT promoter was observed in 34.8% of responders and 23.4% of non-responders (P=0.29). In addition, no survival advantage for patients with a methylated MGMT promoter was detectable (P=0.79). Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy. Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32-5.7)). Analysis of MGMT promoter methylation comparing primaries and different metastases over the clinical course revealed no statistical difference (P=0.49).

Conclusions: In advanced melanoma MGMT promoter, methylation correlates with tolerance of therapy, but not with clinical outcome.

Show MeSH
Related in: MedlinePlus