1. The Clinical Implications of Inconsistently Methylated Results from Glioblastoma MGMT Testing by Replicate Methylation-Specific PCR 
Daniel Xia, David A. Reardon, Jacqueline L. Bruce, Neal I. Lindeman 
The Journal of Molecular Diagnostics 
Volume 18, Issue 6, Pages (November 2016)
DOI: /j.jmoldx
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
An inconsistently methylated result on methylation-specific PCR (MSP). A: At the Center for Advanced Molecular Diagnostics (Brigham and Women's Hospital, Boston, MA), MSP is performed with duplicate bisulfite reactions, followed by duplicate PCRs with methylation-specific primers. An inconsistently methylated result is reported when the methylated peak is seen in some but not all replicates. B: An inconsistently methylated glioblastoma (GBM) case. The capillary electrophoresis results from an inconsistently methylated (2/4) GBM case. The top two PCR reactions are replicates from one bisulfite reaction; the bottom two PCR reactions are replicates from a second bisulfite reaction. The left peak is the methylated PCR product; the right peak is the unmethylated PCR product. Incomplete bisulfite conversion does not easily explain discrepancies between PCR replicates within a bisulfite reaction. al, allele; ht, height of peak; sz, size (bp).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
MGMT promoter methylation status is strongly correlated with overall survival (OS) in World Health Organization grade IV cases. The median survival times in the methylated (Meth), inconsistently methylated, and unmethylated (Unmeth) groups are 34.1, 20.0, and 17.0 months, respectively. There is a statistically significant difference in survival between the inconsistently methylated and methylated groups and between the unmethylated and methylated groups. The survival difference between the inconsistently methylated and unmethylated groups is not statistically significant. P = between the inconsistently methylated and methylated groups (log-rank test); P = 2.1 × 10−6 between the unmethylated and methylated groups; P = 0.59 between the inconsistently methylated and unmethylated groups.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
There is a possible dose–response relationship between survival and the extent of promoter methylation (M) in the inconsistent World Health Organization grade IV cases. Cases with two or more M replicates have a median survival of 27.4 months. Cases with one M replicate have a median survival of 15.7 months. P = (log-rank test). OS, overall survival.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
A possible model to explain partial methylation in methylation-specific PCR (MSP). A: A small sample with very few fully methylated (M) templates (yellow circles). B: A larger sample with low overall methylation and very few fully methylated templates. Note: this diagram is simplified to better illustrate the potential impact of small tumor samples and intermediately methylated tumors on MSP results. U, unmethylated.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Supplemental Figure S1
MGMT promoter methylation status is not significantly associated with sample tumor percentage in World Health Organization grade IV cases from 2010 to The numbers of methylated (M), inconsistently methylated (I), and unmethylated (U) cases are in black above the blue lines corresponding to the medians. The mean tumor percentages in M, I, and U cases are in red below the blue median lines. P = 0.21 (Kruskal-Wallis test).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Supplemental Figure S2
MGMT promoter methylation status is not significantly associated with sample tumor necrosis in World Health Organization grade IV cases from 2010 to The numbers of methylated (M), inconsistently methylated (I), and unmethylated (U) cases are in black above the blue lines corresponding to the medians. The mean necrosis (% of sample) in M, I, and U cases are in red below the blue median lines. Please note that the number of cases (n = 78) in this analysis is low. P = 0.38 (Kruskal-Wallis test).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions