Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients
Abstract Background Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide t...
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| Language: | English |
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BMC
2025-01-01
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| Series: | Clinical Epigenetics |
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| Online Access: | https://doi.org/10.1186/s13148-025-01822-2 |
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| author | Halka Lhotska Karolina Janeckova Hana Cechova Jaromir Macoun Tatiana Aghova Libuse Lizcova Karla Svobodova Lucie Hodanova Dora Konecna Jiri Soukup Filip Kramar David Netuka Zuzana Zemanova |
| author_facet | Halka Lhotska Karolina Janeckova Hana Cechova Jaromir Macoun Tatiana Aghova Libuse Lizcova Karla Svobodova Lucie Hodanova Dora Konecna Jiri Soukup Filip Kramar David Netuka Zuzana Zemanova |
| author_sort | Halka Lhotska |
| collection | DOAJ |
| description | Abstract Background Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. Results A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). Conclusions Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma. |
| format | Article |
| id | doaj-art-1ff2d198ee1540669de0949fb6f16909 |
| institution | Kabale University |
| issn | 1868-7083 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Clinical Epigenetics |
| spelling | doaj-art-1ff2d198ee1540669de0949fb6f169092025-02-02T12:30:00ZengBMCClinical Epigenetics1868-70832025-01-0117111210.1186/s13148-025-01822-2Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patientsHalka Lhotska0Karolina Janeckova1Hana Cechova2Jaromir Macoun3Tatiana Aghova4Libuse Lizcova5Karla Svobodova6Lucie Hodanova7Dora Konecna8Jiri Soukup9Filip Kramar10David Netuka11Zuzana Zemanova12Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueDepartment of HLA, Institute of Hematology and Blood TransfusionThe Clinical Trials and Research Department, General University Hospital and 1st Faculty of Medicine of Charles University in PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueDepartment of Neurosurgery and Neurooncology, 1st Faculty of Medicine of Charles University and Military University Hospital PragueDepartment of Pathology, 1st Faculty of Medicine of Charles University and Military University Hospital PragueDepartment of Neurosurgery and Neurooncology, 1st Faculty of Medicine of Charles University and Military University Hospital PragueDepartment of Neurosurgery and Neurooncology, 1st Faculty of Medicine of Charles University and Military University Hospital PragueCenter of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in PragueAbstract Background Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. Results A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). Conclusions Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.https://doi.org/10.1186/s13148-025-01822-2GlioblastomaMGMTMethylationMS-MLPASanger sequencingStupp protocol |
| spellingShingle | Halka Lhotska Karolina Janeckova Hana Cechova Jaromir Macoun Tatiana Aghova Libuse Lizcova Karla Svobodova Lucie Hodanova Dora Konecna Jiri Soukup Filip Kramar David Netuka Zuzana Zemanova Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients Clinical Epigenetics Glioblastoma MGMT Methylation MS-MLPA Sanger sequencing Stupp protocol |
| title | Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients |
| title_full | Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients |
| title_fullStr | Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients |
| title_full_unstemmed | Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients |
| title_short | Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients |
| title_sort | validating a clinically based ms mlpa threshold through comparison with sanger sequencing in glioblastoma patients |
| topic | Glioblastoma MGMT Methylation MS-MLPA Sanger sequencing Stupp protocol |
| url | https://doi.org/10.1186/s13148-025-01822-2 |
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