Gene expression profiling of Group 3 medulloblastomas defines a clinically tractable stratification based on KIRREL2 expression

Abstract

Medulloblastomas (MB) molecularly designated as Group 3 (Grp 3) MB represent a more clinically aggressive tumor variant which, as a group, displays heterogeneous molecular characteristics and disease outcomes. Reliable risk stratification of Grp 3 MB would allow for appropriate assignment of patients to aggressive treatment protocols and, vice versa, for sparing adverse effects of high-dose radio-chemotherapy in patients with standard or low-risk tumors. Here we performed RNA-based analysis on an international cohort of 179 molecularly designated Grp 3 MB treated with HIT protocols. We analyzed the clinical significance of differentially expressed genes, thereby developing optimal prognostic subdivision of this MB molecular group. We compared the transcriptome profiles of two Grp 3 MB subsets with various outcomes (76 died within the first 60 months vs. 103 survived this period) and identified 224 differentially expressed genes (DEG) between these two clinical groups (Limma R algorithm, adjusted p-value < 0.05). We selected the top six DEG overexpressed in the unfavorable cohort for further survival analysis and found that expression of all six genes strongly correlated with poor outcomes. However, only high expression of KIRREL2 was identified as an independent molecular prognostic indicator of poor patients' survival. Based on clinical and molecular patterns, four risk categories were outlined for Grp 3 MB patients: i. low-risk: M0-1/MYC non-amplified/KIRREL2 low (n = 48; 5-year OS-95%); ii. standard-risk: M0-1/MYC non-amplified/KIRREL2 high or M2-3/MYC non-amplified/KIRREL2 low (n = 65; 5-year OS-70%); iii. high-risk: M2-3/MYC non-amplified/KIRREL2 high (n = 36; 5-year OS-30%); iv. very high risk-all MYC amplified tumors (n = 30; 5-year OS-0%). Cross-validated survival models incorporating KIRREL2 expression with clinical features allowed for the reclassification of up to 50% of Grp 3 MB patients into a more appropriate risk category. Finally, KIRREL2 immunopositivity was also identified as a predictive indicator of Grp 3 MB poor survival, thus suggesting its application as a possible prognostic marker in routine clinical settings. Our results indicate that integration of KIRREL2 expression in risk stratification models may improve Grp 3 MB outcome prediction. Therefore, simple gene and/or protein expression analyses for this molecular marker could be easily adopted for Grp 3 MB prognostication and may help in assigning patients to optimal therapeutic approaches in prospective clinical trials.

Keywords: Expression; Group 3; KIRREL2; Medulloblastoma; Prognosis.

Fig. 1

a Heat-map generated for a set of the top most-confident genes differentially expressed between survivors (n = 103) and non-survivors (n = 76) in Grp 3 MB. b–g Box plots for 6 top genes differentially expressed between survivors (Alive; blue boxplots) and non-survivors (Death; red boxplots); Among them are MYC (b); KIRREL2 (c); ITPRIPL1 (d); DCAF4 (e); NPW (f); CDT1 (g); (all Limma algorithm; p < 0.01)

Fig. 2

Progression-free (a) and overall (b) survival analysis revealed that high levels of KIRREL2 expression (a cut-off log2 RPKM > 2.5; green line) are significantly associated with worse outcomes in Grp 3 MB (log-rank test; p < 0.01). c–h Scatter graphs show an absence of correlation between KIRREL2 and MYC expression (c; correlation coefficient: − 0.074; p = 0.374), whereas expression of ITPRIPL1 (d), DCAF4 (e), NPW (f), CDT1 (g), MAB21L2 (h) was correlated strongly with MYC expression (all p < 0.01). i. KIRREL2 expression was higher in Grp 3 MB (red boxplot; n = 179) as compared to WNT-MB (violet boxplot; n = 20), SHH-MB (blue boxplot; n = 188) and Grp 4 MB (green boxplot; n = 260) in the current/screening RNA_seq MB set (t-test; p < 0.01). j KIRREL2 expression was also higher in Grp 3 MB (red boxplot; n = 46) as compared to SHH-MB (blue boxplot; n = 51) and Grp 4 MB (green boxplot; n = 188) in independent/validation set generated with Affymetrix platform (t-test; p < 0.01). k. KIRREL2 expression is quite similar in these main second generation II (light-green boxplot), III (dark-green boxplot) and IV (gray boxplot) subgroups which composed 85% of Grp 3 MB (t-test; p = 0.323)

Fig. 3

Area under the time-dependent receiver operating characteristic (AUC and ROC) curves for Grp 3 MB disease outcome (a, b) and progression (c, d) applying clinical variables alone (CSI RT and M stage blue line for PFS and OS), and the molecular marker KIRREL2 (gold line for OS and red line for PFS). Thus, inclusion of KIRREL2 expression in current stratification model significantly improves outcome prediction and reduced prediction error. For Grp 3 MB, four risk categories were outlined in terms of PFS (e) and OS (f): i. low-risk (line 0): M0-1/MYC non-amplified/KIRREL2 low (n = 48; OS—95%); ii. standard-risk (line 1): M0-1/MYC non-amplified/KIRREL2 high or M2-3/MYC non-amplified/KIRREL2 low (n = 65; OS—70%); iii. high-risk (line 2): M2-3/MYC non-amplified/ KIRREL2 high (n = 36; OS—30%); iv. very high risk (line 3)—all MYC amplified tumors (n = 30; OS—0%)

Fig. 4

Progression-free (a) and overall (b) survival for Grp 3 MB combining various HIT regimens (RT/CHT vs. CHT alone) and KIRREL2 expression levels. High KIRREL2 was associated with adverse outcomes for patients treated either with CHT alone or combined RT/CHT as compared to those with low levels of KIRREL2

Fig. 5

a Diffuse KIRREL2 membranous-cytoplasmic immuno-expression in Grp 3 MB accompanied with high gene expression at mRNA level. b Completely KIRREL2-negative Grp3 MB sample with low gene expression at mRNA level. Progression-free (left) and overall (right) survival analysis revealed that KIRREL2 immunopositivity (green line) is significantly associated with worse outcomes in both screening/whole sections (c, d) and validation/TMA sections (e, f) sets of Grp 3 MB (log-rank test; p < 0.01)