Hello, my name is Addison Barnett, and I'll be presenting on the sex-associated analysis of MGMT promoter site methylation in newly diagnosed glioblastoma. I would like to thank the AANS committee for the opportunity to present this research on behalf of my co-authors and the Cleveland Clinic Rosella Burkhardt Brain Tumor and Neuro-Oncology Center. We have no relevant disclosures. To briefly set the stage, glioblastoma is the most common primary malignant central nervous system tumor among adults, with a median survival of 15 to 18 months and a five-year survival rate of about 6%. Despite tremendous efforts in recent years, little improvement has been made in terms of improving survival outcomes in these patients. The introduction of the STEP protocol, followed by the identification of the MGMT repair enzyme and control in a patient's outcomes, really served to highlight this approach to targeting patients and better tailoring treatments for those who would receive the greatest benefit while exploring other treatments for those who would not. Those patients who were MGMT methylated had significantly improved survival outcomes than those who were unmethylated. The determination of MGMT methylation is made by the actual degree or percent of promoter methylation. And that promoter region is rich in cysteine phosphate guanine dinucleotides and is called a CpG island, which is comprised of 98 individual CpG sites. Much research has been done on these sites to identify which ones have the greatest prognostic complications. The most commonly studied regions where the best prognostic evidence exists encompasses CpGs 74 through 78, where specifically the mean value of these five sites is compared to a threshold value above or below which the determination for methylation is made. It has been shown that glioblastoma exhibits a sexual dimorphism. Perhaps the best-known aspects of this is that primary glioblastoma is more common among males than in females. And females experience a better prognosis and outcomes than males when adjusted for clinical variables. Several studies, including our own institutional cohort study, suggest that among glioblastoma patients, MGMT methylation is seen more often in females than in males, and that MGMT methylation has a greater prognostic benefit in females than in males. And I will touch on these points further on in the presentation. So our objective was to more comprehensively explore the sexually dimorphic nature of MGMT methylation in glioblastoma, the outcomes of which might influence clinical practice. More specifically, we sought to compare and analyze survival outcomes of newly diagnosed male and female glioblastoma patients by their degree of MGMT promoter methylation, both individually at each CpG site, as well as the mean value of methylation. Overall, we reviewed three and four adult patients who underwent surgery for newly diagnosed glioblastoma at Cleveland Clinic between 2015 and 2018 and had available CpG data. CpG methylation was determined using a clinically validated bisulfate conversion test followed by PCR and pyrosequencing. CpG sites 74 through 78, which I referred to before, will now be referred to as CpGs 1 through 5. On the threshold value for determining methylation, it was greater than or equal to 12. Statistically, CpG methylation values were compared using a generalized linear model and a Lecoxin signed rank test. And overall survival was compared using a Kaplan-Meier method and Cox proportional hazard model before and after propensity score matching. And we matched on age, surgery type, and exact CpG methylation value. The mean age of our cohort was 64 years. 104 patients, or 34.2%, were female, and 120 patients, or 39.5%, were MGMT methylated. On the left, where we look at the survival curves of males versus females, we see that females do better than males as expected. On the right, where we compare MGMT methylated patients versus MGMT unmethylated patients, methylated being the red line, we again see that MGMT methylated patients do much better than unmethylated, as we expect. Now when we stratify by sex and methylation status, the first thing I'd like to point out is that females are more often methylated than males, as we touched on before. Next, when we look at females by methylation status, we see that methylated females have the best survival outcomes, and that the difference between the methylated and unmethylated curve for females is very large, or the area between those two curves is very large, compared to males, where there is a survival benefit by methylation, although the difference in methylation status is not as significant as in females. Next, when we look at CpG methylation values between males and females, we again see that the difference in prevalence of methylation is much higher in females, and is statistically significantly different. When we look at the CpG values, females are more often methylated across the board, and significant at CpGs 1, 2, 4, as well as the mean, where the mean is much larger in females, 11 compared to 3. Graphically, we see the distribution of mean methylation value between males and females, where it falls more to the left in males, and more to the right in females. Here are sub-cohorts following a parentheses score matching for age, surgery type, and mean methylation value. Our N is now 152, 76 between males and females. When we look at survival, we see that the outcomes are very similar between these two groups. If anything, it slightly favors males. And when we look at those variables being controlled for, we see that the two sub-cohorts are nearly identical. Ultimately, after matching, females maintained a significant survival benefit associated with MGMT methylation that was not seen in males. When we look specifically at females, methylated females had a median survival of about 18.7 months, and a one-year survival of 78.4%, compared to unmethylated females with a median survival of 10 months and a one-year survival of 37.4%. That's almost doubled for methylated females. Whereas in males, we see almost no appreciable difference in median or one-year survival. If anything, methylation outcomes are slightly decreased compared to unmethylated males. And the difference by sex and methylation status between these groups is statistically significant. So in conclusion, our research suggests that females are more often MGMT methylated than males. In this cohort, females have a greater degree of individual and mean CpG site methylation. These were statistically significant as CpG sites 1, 2, 4, and at the mean. And again, CpG sites 1, 2, and 4 were translated to 74, 75, and 77. Finally, females confirmed a significant overall survival benefit associated with MGMT methylation that was not seen in males, even with equal degrees of mean CpG methylation and controlling for other clinical variables. And I'd really like to emphasize this last point, that with all things being equal, these critical clinical variables that we controlled for, that methylated females did far better than methylated males. And the difference between males and females by methylation status was much more significant in females. So in summary, the data suggests that the sexual dimorphism associated with MGMT exists among newly diagnosed glioblastoma patients, which requires further exploration. And this may have serious implications on the clinical management of these patients. Moving forward, I think it'd be important to control for more clinical variables in a patient's treatment course, such as radiation, the tumor location, surgical section, whether there are multiples, the patient's pharmacological course, and other key biological markers. But to do so would require a large increase in the cohort size, which may mean a cooperation between institutions. So with that, I'd like to thank you for your time. I'd like to thank my co-authors, Dr. Justin Lathia, Ms. Hong Li, Dr. Gabrielle Yeeney, Dr. David Bostler, Mr. Asad Ali, Dr. Anas Syed-Bamushmous, and my mentor, Dr. Manmeet Alawalia. I'd also like to thank the Cleveland Clinic Rosalyn Burkhardt Brain Tumor and Neuro-Oncology Center. I'd like to thank the Journal of Neuro-Oncology for recognizing our research, and for the honor of bestowing us the Journal of Neuro-Oncology Award. We're extraordinarily grateful. And I'd like to, again, thank the AANS for their tremendous efforts in organizing this conference, this virtual option, and for recognizing our research. Thank you all very much.

Addison Barnett

MGMT promoter site methylation

glioblastoma

sex differences

survival outcomes