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Randomized Controlled Trial
. 2019 Jul 31;9(1):11125.
doi: 10.1038/s41598-019-47642-2.

Pyrosequencing versus methylation-specific PCR for assessment of MGMT methylation in tumor and blood samples of glioblastoma patients

Anna Estival  1 , Carolina Sanz  2 , Jose-Luis Ramirez  1 , Jose Maria Velarde  3 , Marta Domenech  1 , Cristina Carrato  2 , Ramón de Las Peñas  4 , Miguel Gil-Gil  5 , Juan Sepúlveda  6 , Roser Armengol  7 , Isaac Cardiel  7 , Alfonso Berrocal  8 , Raquel Luque  9 , Ana Herrero  10 , Carmen Balana  11
Affiliations
Randomized Controlled Trial

Pyrosequencing versus methylation-specific PCR for assessment of MGMT methylation in tumor and blood samples of glioblastoma patients

Anna Estival et al. Sci Rep. .

Abstract

Circulating biomarkers in blood may provide an interesting alternative to risky tissue biopsies in the diagnosis and follow-up of glioblastoma patients. We have assessed MGMT methylation status in blood and tissue samples from unresected glioblastoma patients who had been included in the randomized GENOM-009 trial. Paired blood and tissue samples were assessed by methylation-specific PCR (MSP) and pyrosequencing (PYR). After establishing the minimum PYR cut-off that could yield a significant difference in overall survival, we assessed the sensitivity, specificity, positive predictive value and negative predictive value (NPV) of the analyses. Methylation could be detected in cfDNA by both MSP and PYR but with low concordance with results in tissue. Sensitivity was low for both methods (31% and 38%, respectively), while specificity was higher for MSP in blood than for PYR in plasma (96% vs 76%) and NPV was similar (56 vs 57%). Concordance of results in tissue by MSP and PYR was 84.3% (P < 0.001) and correlated with outcome. We conclude that detection of cfDNA in the blood of glioblastoma patients can be an alternative when tumor tissue is not available but methods for the detection of cfDNA in blood must improve before it can replace analysis in tumor tissue.

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Conflict of interest statement

Dr. C.B. has been funded by a grant of the GEINO group for this project. Dr. C.B. has received support from Pfizer, AbbVie, and Servier for attending scientific conferences and had an advisory role for Abbvie and Celgene. The rest of the authors declare that they have no competing interests None authors have received compensation of any profit company or non-profit company for carrying out this project.

Figures

Figure 1
Figure 1
CONSORT diagram showing patients and analyses in the study. (A) Numbers in shaded boxes indicate the patients included in the comparisons between the results of different analyses of MGMT methylation. (B) Numbers indicate patients evaluable for outcome in the GENOM 009 trial and with informative results for the MGMT methylation analyses.
Figure 2
Figure 2
Progression-free survival (PFS) according to the results of the MGMT methylation analyses by (A) MSP-tumor, (B) PYR-tumor, (C) MSP-blood, and (D) PYR-plasma. Solid lines indicate methylated MGMT (MET); broken lines indicate unmethylated MGMT (UNMET).
Figure 3
Figure 3
Overall survival (OS) according to the results of the MGMT methylation analysis by (A) MSP-tumor, (B) PYR-tumor, (C) MSP-blood, and (D) PYR-plasma. Solid lines indicate methylated MGMT (MET); broken lines indicate unmethylated MGMT (UNMET).
See this image and copyright information in PMC

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