Sex-specific clinicopathological significance of novel (Frizzled-7) and established (MGMT, IDH1) biomarkers in glioblastoma

Abstract

Background: The Wnt receptor Frizzled-7 (FZD7) promotes tumor progression and can be currently targeted by monoclonal antibody therapy. Here, we determined the prognostic value of FZD7 for the overall survival of glioblastoma (GBM) patients, both as individual marker and taken in combination with the previously-described markers MGMT and IDH1. Additionally, we tested whether these markers (alone or in combination) exhibited sex-specific differences.

Results: High levels of FZD7 (FZD7high) associated with shorter survival in GBM patients; however, FZD7high was a significant predictor of poor survival only in male patients. Mutation of IDH1 significantly associated with longer survival in male but not female patients. Methylated MGMT promoter significantly associated with longer survival only in female patients. Combination of FZD7 with MGMT enhanced the prognostic accuracy and abrogated the sex differences observed upon single marker analysis. Combination of FZD7 with IDH1 was a significant predictor of survival in male GBM patients only.

Materials and methods: Three independent cohorts of patients with primary GBM (n=120, n=108 and n=105, respectively) were included in this study. FZD7 and IDH1 were assessed by immunohistochemistry in tissue microarrays. MGMT promoter methylation was determined by methylation-specific polymerase chain reaction. Survival analysis was performed by Kaplan-Meier estimate, log-rank test and Cox proportional hazard regression.

Conclusions: Our study identifies novel individual and combination markers with prognostic and, possibly, therapeutic relevance in GBM. Furthermore, our findings substantiate the importance of sexual dimorphism in this type of cancer.

Keywords: Frizzled-7; IDH1; MGMT; glioblastoma; sex-specific biomarkers.

Figure 1. FZD7 in primary GBM: expression and scoring system

A-B. Representative micrographs showing (A) weak, medium or strong expression of FZD7 and (B) heterogenous expression of FZD7 in GBM tissues. Scale bars are indicated in the lower-left corner of each panel. C. FZD7 expression in GBM tissues (n=222) versus non-cancerous brain tissues (brain parenchyma from patients with arteriovenous malformations-AVM; n=52). The medians are shown as black lines and the percentiles (25th and 75th) as vertical boxes with error bars. Statistical analysis was performed with the chi-square test and the p-value is indicated in the upper right corner of the plot. D. Representative micrograph showing FZD7 expression in AVM tissues. The scale bar is indicated in the lower-left corner of the panel. BV= blood vessels; P=brain parenchyma.

Figure 2. FZD7 in primary GBM: impact on patients’ overall survival and sex differences

A. Kaplan–Meier 5-year survival curves were plotted for the Hannover Cohort 1 patients with low versus high levels of FZD7. Analysis was performed for the entire cohort of patients (all patients), male and female patients, respectively. The log-rank test was used for statistical testing and the p-values are indicated in the upper-right corner of each plot. The numbers of patients per group are indicated next to each curve. B. Multivariate Cox regression analysis models for the Hannover Cohort 1 patients with FZD7^low versus FZD7^high. The models were adjusted for age, Karnofsky scale (KS), therapy and extent of surgical resection, respectively. HR = hazard ratio; 95% CI = 95% confidence interval. C. Kaplan–Meier 2-year survival curves were plotted for the Hannover Cohort 2 patients with low versus high levels of FZD7. The statistical testing was performed as in (A). D. Multivariate Cox regression analysis models for the Hannover Cohort 2 patients with FZD7^low versus FZD7^high. The statistical testing was performed as in (B).

Figure 3. MGMT methylation in primary GBM: impact on patients’ overall survival and sex differences

A-B. Kaplan–Meier 5-year survival curves were plotted for the Hannover Cohort 1 patients with methylated versus unmethylated MGMT promoter. Analysis was performed for (A) all therapy groups and (B) only the group which received radiochemotherapy (RCTX) in addition to surgery. C. Kaplan–Meier 5-year survival curves were plotted for the Bologna patients with methylated versus unmethylated MGMT promoter. All patients in this cohort had received RCTX in addition to surgery. The p-values of the log-rank test are indicated in the upper-right corner of each plot. The numbers of patients per group are indicated next to each curve.

Figure 4. IDH1 mutation in primary GBM: impact on patients’ overall survival and sex differences

A. Representative micrographs showing expression of wild-type IDH1 (negative staining; left panels) and mutated IDH1 (positive staining; right panels). Scale bars are indicated in the lower-left corner of each panel. B. Kaplan–Meier 5-year survival curves were plotted for the Hannover Cohort 1 patients with mutated versus wild-type status of IDH1 and statistical testing was performed with the log-rank test.

Figure 5. Combination of FZD7 with MGMT or IDH1: impact on patients’ overall survival and sex differences

A. Kaplan–Meier 5-year survival curves and B. multivariate Cox regression analysis models for the Hannover Cohort 1 patients with FZD7^lowMGMT^methylated versus FZD7^highMGMT^unmethylated phenotypes. C. Kaplan–Meier 5-year survival curves and D. multivariate Cox regression analysis models for the Hannover Cohort 1 patients with FZD7^lowIDH1^mutated versus FZD7^highIDH1^wild-type phenotypes. (A, C) Statistical testing was performed with the log-rank test and the p-values are indicated in the upper-right corner of each plot. The numbers of patients per group are indicated next to each curve. (B, D) The multivariate models were adjusted for age, Karnofsky scale (KS), therapy and extent of surgical resection. HR = hazard ratio; 95% CI = 95% confidence interval.