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Multicenter Study
. 2019 Mar;25(3):314-322.
doi: 10.1111/cns.13040. Epub 2018 Aug 16.

Combinations of four or more CpGs methylation present equivalent predictive value for MGMT expression and temozolomide therapeutic prognosis in gliomas

Rui-Chao Chai  1   2 , Ke-Nan Zhang  1   2   3 , Yu-Qing Liu  1   2 , Fan Wu  1   2 , Zheng Zhao  1   2 , Kuan-Yu Wang  1   2   3 , Tao Jiang  1   2   3 , Yong-Zhi Wang  1   2   3
Affiliations
Multicenter Study

Combinations of four or more CpGs methylation present equivalent predictive value for MGMT expression and temozolomide therapeutic prognosis in gliomas

Rui-Chao Chai et al. CNS Neurosci Ther. 2019 Mar.

Abstract

Aims: The pyrosequencing (PSQ) has been regarded as the gold standard for MGMT promoter methylation testing in gliomas. However, various CpG combinations are currently used in clinical practice. We aimed to clarify how and how many CpGs combined is robust enough to predict MGMT mRNA expression and therapeutic prognosis of patients.

Methods: Total 223 patients with WHO III/IV gliomas were enrolled from Chinese Glioma Genome Atlas, including two independent cohorts, the eight-site cohort (with CpGs 75-82 tested) and the seven-site cohort (with CpGs 72-78 tested). Spearman's correlation and ROC curves were employed to investigate the value of different CpG combinations on predicting MGMT mRNA expression. The ROC curves and Kaplan-Meier steps were performed to compare the TMZ therapeutic prognostic values of different CpG combinations.

Results: The methylation level of all individual CpG and CpG combinations for the eleven CpGs (CpGs 72-82), significantly correlated to MGMT mRNA expression (Spearman, all P < 0.0001), could effectively predict the mRNA expression (AUC, 0.86-0.91 in the eight-site cohort, 0.83-0.90 in the seven-site cohort). Moreover, the correlation coefficients and the predictive values presented equivalent when four or more CpGs combinedly used (AUC, 0.88-0.90 in the eight-site cohort, 0.87-0.88 in the seven-site cohort). Finally, similar results were also observed when using selected CpG combinations to predict therapeutic prognosis of patients.

Conclusions: Four-CpG combinations of pyrosequencing are sufficient for evaluating the methylation status of MGMT and predicting therapeutic prognosis in gliomas.

Keywords: M GMT; CpGs; glioma; pyrosequencing; temozolomide.

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Conflict of interest statement

There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1
Figure 1
Schematic diagram of CpG studied in previous and current studies. The distribution of CpG in the MGMT 5′ CpG island is shown in the upper panel. CpGs that had been used in pyrosequencing (PSQ) testing of the published literature are shown in the middle panel. The CpGs tested in the current study are shown in the lower panel
Figure 2
Figure 2
Correlation of MGMT mRNA expression to the methylation level of MGMT promoter individual CpG or CpG combinations. A and B, The methylation level of individual CpG or CpG combinations is applied to heatmap in the eight‐site cohort (A) and the seven‐site cohort (B). The methylation level of a CpG combination is the average methylation level of all CpGs included in this combination. “r” is the Spearman r value, and the 95% confidence interval of the “r” is shown in the following bracket, respectively. ****P < 0.0001
Figure 3
Figure 3
Predictive value of different individual CpG or selected CpG combinations methylation levels for MGMT mRNA expression. A and B, The area under the ROC (AUC) and corresponding Standard error (SDE) of different individual CpG or selected CpG combinations in the eight‐site cohort (A), and the seven‐site cohort (B) is arranged along with the CpG numbers contained in each combination. The CpG combinations with convergent AUCs are highlighted with the gray background
Figure 4
Figure 4
Comparison of the therapeutic prognostic value of different CpG combinations for glioblastoma patient survival. A‐C, Therapeutic prognostic effects of the MGMT methylation statuses determined by CpGs 75‐82 (A), CpGs 76‐79 (B), and CpGs 75‐78(C) for glioblastoma patient OS were evaluated by ROC in the eight‐site cohort. D‐F,Therapeutic prognostic effects of the MGMT methylation statuses determined by CpGs 72‐78 (D), CpGs 74‐79 (E), and CpGs 75‐78(F) for glioblastoma patient OS were evaluated by ROC in the seven‐site cohort
Figure 5
Figure 5
Kaplan‐Meier steps for the OS of glioblastoma patients stratified by the methylation level of different CpG combinations. A‐C, Kaplan‐Meier step for the OS of glioblastoma patients in the eight‐site cohort. The MGMT methylation statuses determined by CpGs 75‐82 (A), CpGs 76‐79 (B), and CpGs 75‐78(C) were used as the stratification reference, respectively. D‐F, Kaplan‐Meier curves for the OS of glioblastoma patients in the eight‐site cohort. The MGMT methylation statuses determined by CpGs 72‐78 (D), CpGs 74‐79 (E), and CpGs 75‐78(F) were used as the stratification reference, respectively
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