Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild-type DIPG samples. This study presents the first comprehensive multidimensional protein, mRNA, and methylation profiling of pediatric brain tumor specimens, detecting the presence of two subgroups within our DIPG cohort. This multidimensional analysis of DIPG provides increased analytical power to more fully explore molecular signatures of DIPGs, with implications for evaluating potential molecular subtypes and biomarker discovery for assessing response to therapy.

Fig. 1. Protein and mRNA profiles of DIPGs are Distinct from other Pediatric Brain Tumors and Exhibit Two Molecular Subtypes

1a Principal component analysis of high-grade astrocytoma protein expression values (DIPG n=14, Yellow; pediatric supratentorial GBM n=4, Green) demonstrates unique protein profiles by tumor location and Histone 3 K27M mutation status (H3.1 Red, H3.3 Orange, H3 wild type Gray). H3 mutant DIPGs cluster closely compared to H3 wild type or GBMs. 1b Top Left Panel Western blot analysis of DIPG and normal tissue lysate reveals tumorspecific expression of Talin-1 (TLN1), Eukaryotic Elongation Factor 2 (EF2) and Clusterin (CLU), with GAPDH was used as a loading control 1b Top Right Panel Protien expression levels were measured by densitometry: average values for each group (tumor and normal) are represented by bar graphs. * = p-value < 0.05 1b Bottom Panel Immunohistochemical staining of DIPG tumor and adjacent normal brainstem tissue (Patient ID 7) validated cytosolic upregulation of TLN1, EF2 and CLU in tumor regions as indicated by H&E staining. Scale bar=200μm; Inset scale bar=25μm 1cTop Panel Supervised hierarchical clustering of DIPG tumor protein expression profiles (n=8 pairs, pairwise tumor vs. normal FC values) based on subgroups identified through mRNA expression profile analysis revealed 65 significantly differentially expressed proteins between Subgroup 1 and 2 (ANOVA, p-value<0.05 and FC≤-2 or ≥2) 1c Middle Panel Principal component analysis after unsupervised hierarchical clustering of DIPG mRNA expression profiles depicts two distinct subgroups with differential expression of 1,918 genes (n=8 pairs, pairwise tumor vs. normal FC≤-2 or ≥2). Subgroup 1=Red, n=3, Patient IDs 3, 8 and 10; Subgroup 2=Blue, n=5, PIDs 6, 7, 11, 13 and 14 1c Bottom Panel Overall concordance between mRNA and protein expression profiles is seen across 53% of molecules detected

Fig. 1. Protein and mRNA profiles of DIPGs are Distinct from other Pediatric Brain Tumors and Exhibit Two Molecular Subtypes

1a Principal component analysis of high-grade astrocytoma protein expression values (DIPG n=14, Yellow; pediatric supratentorial GBM n=4, Green) demonstrates unique protein profiles by tumor location and Histone 3 K27M mutation status (H3.1 Red, H3.3 Orange, H3 wild type Gray). H3 mutant DIPGs cluster closely compared to H3 wild type or GBMs. 1b Top Left Panel Western blot analysis of DIPG and normal tissue lysate reveals tumorspecific expression of Talin-1 (TLN1), Eukaryotic Elongation Factor 2 (EF2) and Clusterin (CLU), with GAPDH was used as a loading control 1b Top Right Panel Protien expression levels were measured by densitometry: average values for each group (tumor and normal) are represented by bar graphs. * = p-value < 0.05 1b Bottom Panel Immunohistochemical staining of DIPG tumor and adjacent normal brainstem tissue (Patient ID 7) validated cytosolic upregulation of TLN1, EF2 and CLU in tumor regions as indicated by H&E staining. Scale bar=200μm; Inset scale bar=25μm 1cTop Panel Supervised hierarchical clustering of DIPG tumor protein expression profiles (n=8 pairs, pairwise tumor vs. normal FC values) based on subgroups identified through mRNA expression profile analysis revealed 65 significantly differentially expressed proteins between Subgroup 1 and 2 (ANOVA, p-value<0.05 and FC≤-2 or ≥2) 1c Middle Panel Principal component analysis after unsupervised hierarchical clustering of DIPG mRNA expression profiles depicts two distinct subgroups with differential expression of 1,918 genes (n=8 pairs, pairwise tumor vs. normal FC≤-2 or ≥2). Subgroup 1=Red, n=3, Patient IDs 3, 8 and 10; Subgroup 2=Blue, n=5, PIDs 6, 7, 11, 13 and 14 1c Bottom Panel Overall concordance between mRNA and protein expression profiles is seen across 53% of molecules detected

Fig. 1. Protein and mRNA profiles of DIPGs are Distinct from other Pediatric Brain Tumors and Exhibit Two Molecular Subtypes

1a Principal component analysis of high-grade astrocytoma protein expression values (DIPG n=14, Yellow; pediatric supratentorial GBM n=4, Green) demonstrates unique protein profiles by tumor location and Histone 3 K27M mutation status (H3.1 Red, H3.3 Orange, H3 wild type Gray). H3 mutant DIPGs cluster closely compared to H3 wild type or GBMs. 1b Top Left Panel Western blot analysis of DIPG and normal tissue lysate reveals tumorspecific expression of Talin-1 (TLN1), Eukaryotic Elongation Factor 2 (EF2) and Clusterin (CLU), with GAPDH was used as a loading control 1b Top Right Panel Protien expression levels were measured by densitometry: average values for each group (tumor and normal) are represented by bar graphs. * = p-value < 0.05 1b Bottom Panel Immunohistochemical staining of DIPG tumor and adjacent normal brainstem tissue (Patient ID 7) validated cytosolic upregulation of TLN1, EF2 and CLU in tumor regions as indicated by H&E staining. Scale bar=200μm; Inset scale bar=25μm 1cTop Panel Supervised hierarchical clustering of DIPG tumor protein expression profiles (n=8 pairs, pairwise tumor vs. normal FC values) based on subgroups identified through mRNA expression profile analysis revealed 65 significantly differentially expressed proteins between Subgroup 1 and 2 (ANOVA, p-value<0.05 and FC≤-2 or ≥2) 1c Middle Panel Principal component analysis after unsupervised hierarchical clustering of DIPG mRNA expression profiles depicts two distinct subgroups with differential expression of 1,918 genes (n=8 pairs, pairwise tumor vs. normal FC≤-2 or ≥2). Subgroup 1=Red, n=3, Patient IDs 3, 8 and 10; Subgroup 2=Blue, n=5, PIDs 6, 7, 11, 13 and 14 1c Bottom Panel Overall concordance between mRNA and protein expression profiles is seen across 53% of molecules detected

Fig. 2. Molecular Expression in Tumor Tissue Validates Differential Hh Signaling Between DIPG Subgroups

2a Immunohistochemical staining of DIPG tumor and normal tissue demonstrates increased PTCH staining in DIPG tumor tissue from patients with implicated Hh signaling (center and right, Patient IDs 7 and 14). Patients with implicated Myc signaling (left, Patient ID 10) demonstrate a relative decrease in PTCH staining. H3.3 mutation status does not appear to affect degree of PTCH staining in tumor tissue. Scale Bar=100μm; Inset Scale Bar=12.5μm 2b Immunohistochemical staining of DIPG tumor tissue (n=4) demonstrates nuclear localization of GLI1 in patients with implicated Hh signaling (Subgroup 2, PIDs 7, 11, 14), and low levels of p53 expression in all H3 mutants tested (denoted by *). H3 mutation does not appear to affect degree of GLI1 staining in tumor tissue. ATRX expression was not detected in DIPG specimens. Scale Bar = 3μm for P53 and ATRX and 90 μm for GLI1

Fig. 2. Molecular Expression in Tumor Tissue Validates Differential Hh Signaling Between DIPG Subgroups

2a Immunohistochemical staining of DIPG tumor and normal tissue demonstrates increased PTCH staining in DIPG tumor tissue from patients with implicated Hh signaling (center and right, Patient IDs 7 and 14). Patients with implicated Myc signaling (left, Patient ID 10) demonstrate a relative decrease in PTCH staining. H3.3 mutation status does not appear to affect degree of PTCH staining in tumor tissue. Scale Bar=100μm; Inset Scale Bar=12.5μm 2b Immunohistochemical staining of DIPG tumor tissue (n=4) demonstrates nuclear localization of GLI1 in patients with implicated Hh signaling (Subgroup 2, PIDs 7, 11, 14), and low levels of p53 expression in all H3 mutants tested (denoted by *). H3 mutation does not appear to affect degree of GLI1 staining in tumor tissue. ATRX expression was not detected in DIPG specimens. Scale Bar = 3μm for P53 and ATRX and 90 μm for GLI1

Fig. 3. Patterns of DNA Methylation in DIPG Tissue

3a Analysis of DNA methylation profiles of all DIPG specimens (fold change of average methylation in tumor compared to normal) demonstrates global DNA hypomethylation in tumor (n=9) compared normal brainstem tissue specimens (n=9). Volcano plot depicts 4,808 hypermethylated (red) and 14,428 hypomethylated (blue) CpG sites in tumor relative to normal tissue specimens (ANOVA, p-value with FDR<0.05 and FC ≤-3 or ≥3). X-axis=Tumor vs. Normal Fold Change value. Y-axis = p-value 3b Unsupervised analysis of DNA methylation profiles at 486 CpG loci related to genes belonging to Myc and Hh signaling pathways in patients in whom mRNA analysis was also performed (n=6) identified statistically significant differential methylation of 29 genes between DIPG Subgroup 1 and 2 (ANOVA, p-value <0.05 and FC≤-2 or ≥2) 3c Comparison of whole genome DNA methylation profiles supervised by molecular subgroup (n=7 pairs, pairwise tumor vs. normal fold change values) revealed an additional 786 differentially methylated sites between DIPG Subgroup 1 and 2 (ANOVA, p-value <0.05 and FC≤-3 or ≥3). Functional analysis of this data set identified dysregulated genes related to Myc, WNT, and Hh signaling pathways and network interaction with Histone 3

Fig. 3. Patterns of DNA Methylation in DIPG Tissue

3a Analysis of DNA methylation profiles of all DIPG specimens (fold change of average methylation in tumor compared to normal) demonstrates global DNA hypomethylation in tumor (n=9) compared normal brainstem tissue specimens (n=9). Volcano plot depicts 4,808 hypermethylated (red) and 14,428 hypomethylated (blue) CpG sites in tumor relative to normal tissue specimens (ANOVA, p-value with FDR<0.05 and FC ≤-3 or ≥3). X-axis=Tumor vs. Normal Fold Change value. Y-axis = p-value 3b Unsupervised analysis of DNA methylation profiles at 486 CpG loci related to genes belonging to Myc and Hh signaling pathways in patients in whom mRNA analysis was also performed (n=6) identified statistically significant differential methylation of 29 genes between DIPG Subgroup 1 and 2 (ANOVA, p-value <0.05 and FC≤-2 or ≥2) 3c Comparison of whole genome DNA methylation profiles supervised by molecular subgroup (n=7 pairs, pairwise tumor vs. normal fold change values) revealed an additional 786 differentially methylated sites between DIPG Subgroup 1 and 2 (ANOVA, p-value <0.05 and FC≤-3 or ≥3). Functional analysis of this data set identified dysregulated genes related to Myc, WNT, and Hh signaling pathways and network interaction with Histone 3