Spatial genomic heterogeneity in diffuse intrinsic pontine and midline high-grade glioma: implications for diagnostic biopsy and targeted therapeutics

Abstract

Introduction: Diffuse intrinsic pontine glioma (DIPG) and midline high-grade glioma (mHGG) are lethal childhood brain tumors. Spatial genomic heterogeneity has been well-described in adult HGG but has not been comprehensively characterized in pediatric HGG. We performed whole exome sequencing on 38-matched primary, contiguous, and metastatic tumor sites from eight children with DIPG (n = 7) or mHGG (n = 1) collected using a unique MRI-guided autopsy protocol. Validation was performed using Sanger sequencing, Droplet Digital polymerase-chain reaction, immunohistochemistry, and fluorescent in-situ hybridization.

Results: Median age at diagnosis was 6.1 years (range: 2.9-23.3 years). Median overall survival was 13.2 months (range: 11.2-32.2 months). Contiguous tumor infiltration and distant metastases were observed in seven and six patients, respectively, including leptomeningeal dissemination in three DIPGs. Histopathological heterogeneity was evident in seven patients, including intra-pontine heterogeneity in two DIPGs, ranging from World Health Organization grade II to IV astrocytoma. We found conservation of heterozygous K27M mutations in H3F3A (n = 4) or HIST1H3B (n = 3) across all primary, contiguous, and metastatic tumor sites in all DIPGs. ACVR1 (n = 2), PIK3CA (n = 2), FGFR1 (n = 2), and MET (n = 1) were also intra-tumorally conserved. ACVR1 was co-mutated with HIST1H3B (n = 2). In contrast, PDGFRA amplification and mutation were spatially heterogeneous, as were mutations in BCOR (n = 1), ATRX (n = 2), and MYC (n = 1). TP53 aberrations (n = 3 patients) varied by type and location between primary and metastatic tumors sites but were intra-tumorally conserved.

Conclusion: Spatial conservation of prognostically-relevant and therapeutically-targetable somatic mutations in DIPG and mHGG contrasts the significant heterogeneity of driver mutations seen in adult HGG and supports uniform implementation of diagnostic biopsy in DIPG and mHGG to classify molecular risk groups and guide therapeutic strategy.

Fig. 1

Spatial histopathological and genomic landscape of DIPG and mHGG in children. Histology and WHO grade were frequently heterogeneous within the pons or between the pons and contiguous or metastatic sites. Somatic mutations (indicated by a black box or stripped overlay of a colored box if co-existing with a copy number change) in H3F3A (H3.3), HIST1H3B (H3.1), ACVR1, PIK3CA, FGFR1, and MET were conserved across all disease sites. Somatic mutations in ATRX, BCOR, MYC, and PDGFRA and PDGFRA amplification were spatially heterogeneous. WHO = World Health Organization, FFPE = formalin-fixed paraffin-embedded

Fig. 2

Intra-tumoral heterogeneity in a 4-year old male with DIPG (Patient 3). Each row represents a distinct disease location, including four different areas in the primary tumor, one contiguous lesion in the right basal ganglia, and one metastatic leptomeningeal lesion. a H&Es of each tumor location demonstrate WHO grade IV histology in all except the right basal ganglia lesion (WHO grade III), b P53 IHC demonstrates variable positivity, c PDGFRA FISH demonstrates gain or amplification in 3 of 4 sites within the pontine tumor, gain in the contiguous right basal ganglia lesion, and amplification in the metastatic leptomeningeal lesion, d PDGFRA ddPCR demonstrating PDGFRA mutation observed in the right posterior pons, e H3F3A was mutated in all samples as demonstrated by Sanger sequencing, f sample location taken from fresh (“Primary Pons”, Row 1) or fixed tissue (remaining rows) at autopsy corresponding to tumor location identified on post-mortem MRI imaging (g). Because post-mortem imaging was only performed on fixed tissue, MRI imaging for the fresh tissue sample (“Primary Pons”, Row 1) was a pre-mortem MRI performed approximately 6 weeks prior to death