Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma
The utility of O -methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significa...
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| Veröffentlicht in: | Clinical epigenetics 2020-11, Vol.12 (1), p.174-174, Article 174 |
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| creator | Hanihara, Mitsuto Miyake, Kunio Watanabe, Atsushi Yamada, Yuriko Oishi, Naoki Kawataki, Tomoyuki Inukai, Takeshi Kondo, Tetsuo Kinouchi, Hiroyuki |
| description | The utility of O
-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values.
We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively).
The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection. |
| doi_str_mv | 10.1186/s13148-020-00968-5 |
| format | Article |
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-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values.
We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively).
The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.</description><identifier>ISSN: 1868-7075</identifier><identifier>ISSN: 1868-7083</identifier><identifier>EISSN: 1868-7083</identifier><identifier>EISSN: 1868-7075</identifier><identifier>DOI: 10.1186/s13148-020-00968-5</identifier><identifier>PMID: 33203454</identifier><language>eng</language><publisher>Germany: BioMed Central Ltd</publisher><subject>Bisulfite ; Brain cancer ; Chemotherapy ; Chromatography ; Clinical trials ; CpG islands ; Deoxyribonucleic acid ; DNA ; DNA methylation ; DNA methyltransferase ; Epigenetics ; Gene silencing ; Glioblastoma ; Glioblastoma multiforme ; High performance liquid chromatography ; Isocitrate dehydrogenase ; Medical prognosis ; Methylation ; Methylguanine ; O6-methylguanine-DNA methyltransferase ; Polymerase chain reaction ; Prognosis ; Sulfites ; Survival ; Temozolomide ; Transferases ; Tumors</subject><ispartof>Clinical epigenetics, 2020-11, Vol.12 (1), p.174-174, Article 174</ispartof><rights>COPYRIGHT 2020 BioMed Central Ltd.</rights><rights>2020. This work is licensed under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>The Author(s) 2020</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c563t-ccd890d2be73804de4934cc531cb78002f7f7aa120a690ef02848baccf5e04c73</citedby><cites>FETCH-LOGICAL-c563t-ccd890d2be73804de4934cc531cb78002f7f7aa120a690ef02848baccf5e04c73</cites><orcidid>0000-0001-9196-2229</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672949/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672949/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33203454$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hanihara, Mitsuto</creatorcontrib><creatorcontrib>Miyake, Kunio</creatorcontrib><creatorcontrib>Watanabe, Atsushi</creatorcontrib><creatorcontrib>Yamada, Yuriko</creatorcontrib><creatorcontrib>Oishi, Naoki</creatorcontrib><creatorcontrib>Kawataki, Tomoyuki</creatorcontrib><creatorcontrib>Inukai, Takeshi</creatorcontrib><creatorcontrib>Kondo, Tetsuo</creatorcontrib><creatorcontrib>Kinouchi, Hiroyuki</creatorcontrib><title>Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma</title><title>Clinical epigenetics</title><addtitle>Clin Epigenetics</addtitle><description>The utility of O
-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values.
We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively).
The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.</description><subject>Bisulfite</subject><subject>Brain cancer</subject><subject>Chemotherapy</subject><subject>Chromatography</subject><subject>Clinical trials</subject><subject>CpG islands</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA methylation</subject><subject>DNA methyltransferase</subject><subject>Epigenetics</subject><subject>Gene silencing</subject><subject>Glioblastoma</subject><subject>Glioblastoma multiforme</subject><subject>High performance liquid chromatography</subject><subject>Isocitrate dehydrogenase</subject><subject>Medical prognosis</subject><subject>Methylation</subject><subject>Methylguanine</subject><subject>O6-methylguanine-DNA methyltransferase</subject><subject>Polymerase chain reaction</subject><subject>Prognosis</subject><subject>Sulfites</subject><subject>Survival</subject><subject>Temozolomide</subject><subject>Transferases</subject><subject>Tumors</subject><issn>1868-7075</issn><issn>1868-7083</issn><issn>1868-7083</issn><issn>1868-7075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptkk1v1DAQhi0EotXSP8ABWeLCJWX8kcS5IK0qKEituJSz5Th24sqxt3YC2n-Ply0LRdgHW57nndGMX4ReE7gkRDTvM2GEiwooVABdI6r6GTovAVG1INjz072tz9BFzvdQFuu6jsBLdMYYBcZrfo7SNmeT82zCgqPFt9e3d3g2y7T3anEx4LyoZc14zS6MeHLjVO1MsjHNKmiDvXtY3YD1lOKsljgmtZv22AUczA-_x4NTY4jZDHj0LvZe5aVwr9ALq3w2F4_nBn379PHu6nN18_X6y9X2ptJ1w5ZK60F0MNDetEwAHwzvGNe6ZkT3rQCgtrWtUoSCajowFqjgolda29oA1y3boA_HvLu1n82gS4tJeblLblZpL6Ny8mkkuEmO8btsm5Z2pdoGvXtMkOLDavIiZ5e18V4FE9csKW_KkGkZZUHf_oPexzWF0t6BorR8lSB_qFF5I12wsdTVh6Ry29TAOwBRF-ryP1TZg5mdjsFYV96fCOhRoFPMORl76pGAPJhFHs0ii1nkL7PIg-jN39M5SX5bg_0ERX27pg</recordid><startdate>20201117</startdate><enddate>20201117</enddate><creator>Hanihara, Mitsuto</creator><creator>Miyake, Kunio</creator><creator>Watanabe, Atsushi</creator><creator>Yamada, Yuriko</creator><creator>Oishi, Naoki</creator><creator>Kawataki, Tomoyuki</creator><creator>Inukai, Takeshi</creator><creator>Kondo, Tetsuo</creator><creator>Kinouchi, Hiroyuki</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-9196-2229</orcidid></search><sort><creationdate>20201117</creationdate><title>Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma</title><author>Hanihara, Mitsuto ; Miyake, Kunio ; Watanabe, Atsushi ; Yamada, Yuriko ; Oishi, Naoki ; Kawataki, Tomoyuki ; Inukai, Takeshi ; Kondo, Tetsuo ; Kinouchi, Hiroyuki</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c563t-ccd890d2be73804de4934cc531cb78002f7f7aa120a690ef02848baccf5e04c73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Bisulfite</topic><topic>Brain cancer</topic><topic>Chemotherapy</topic><topic>Chromatography</topic><topic>Clinical trials</topic><topic>CpG islands</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA methylation</topic><topic>DNA methyltransferase</topic><topic>Epigenetics</topic><topic>Gene silencing</topic><topic>Glioblastoma</topic><topic>Glioblastoma multiforme</topic><topic>High performance liquid chromatography</topic><topic>Isocitrate dehydrogenase</topic><topic>Medical prognosis</topic><topic>Methylation</topic><topic>Methylguanine</topic><topic>O6-methylguanine-DNA methyltransferase</topic><topic>Polymerase chain reaction</topic><topic>Prognosis</topic><topic>Sulfites</topic><topic>Survival</topic><topic>Temozolomide</topic><topic>Transferases</topic><topic>Tumors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hanihara, Mitsuto</creatorcontrib><creatorcontrib>Miyake, Kunio</creatorcontrib><creatorcontrib>Watanabe, Atsushi</creatorcontrib><creatorcontrib>Yamada, Yuriko</creatorcontrib><creatorcontrib>Oishi, Naoki</creatorcontrib><creatorcontrib>Kawataki, Tomoyuki</creatorcontrib><creatorcontrib>Inukai, Takeshi</creatorcontrib><creatorcontrib>Kondo, Tetsuo</creatorcontrib><creatorcontrib>Kinouchi, Hiroyuki</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Clinical epigenetics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hanihara, Mitsuto</au><au>Miyake, Kunio</au><au>Watanabe, Atsushi</au><au>Yamada, Yuriko</au><au>Oishi, Naoki</au><au>Kawataki, Tomoyuki</au><au>Inukai, Takeshi</au><au>Kondo, Tetsuo</au><au>Kinouchi, Hiroyuki</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma</atitle><jtitle>Clinical epigenetics</jtitle><addtitle>Clin Epigenetics</addtitle><date>2020-11-17</date><risdate>2020</risdate><volume>12</volume><issue>1</issue><spage>174</spage><epage>174</epage><pages>174-174</pages><artnum>174</artnum><issn>1868-7075</issn><issn>1868-7083</issn><eissn>1868-7083</eissn><eissn>1868-7075</eissn><abstract>The utility of O
-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values.
We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively).
The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.</abstract><cop>Germany</cop><pub>BioMed Central Ltd</pub><pmid>33203454</pmid><doi>10.1186/s13148-020-00968-5</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0001-9196-2229</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Bisulfite Brain cancer Chemotherapy Chromatography Clinical trials CpG islands Deoxyribonucleic acid DNA DNA methylation DNA methyltransferase Epigenetics Gene silencing Glioblastoma Glioblastoma multiforme High performance liquid chromatography Isocitrate dehydrogenase Medical prognosis Methylation Methylguanine O6-methylguanine-DNA methyltransferase Polymerase chain reaction Prognosis Sulfites Survival Temozolomide Transferases Tumors |
| title | Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma |
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