Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma

Hamid Nikbakht, Eshini Panditharatna, Leonie G Mikael, Rui Li, Tenzin Gayden, Matthew Osmond, Cheng-Ying Ho, Madhuri Kambhampati, Eugene I Hwang, Damien Faury, Alan Siu, Simon Papillon-Cavanagh, Denise Bechet, Keith L Ligon, Benjamin Ellezam, Wendy J Ingram, Caedyn Stinson, Andrew S Moore, Katherine E Warren, Jason Karamchandani, Roger J Packer, Nada Jabado, Jacek Majewski, Javad Nazarian, Hamid Nikbakht, Eshini Panditharatna, Leonie G Mikael, Rui Li, Tenzin Gayden, Matthew Osmond, Cheng-Ying Ho, Madhuri Kambhampati, Eugene I Hwang, Damien Faury, Alan Siu, Simon Papillon-Cavanagh, Denise Bechet, Keith L Ligon, Benjamin Ellezam, Wendy J Ingram, Caedyn Stinson, Andrew S Moore, Katherine E Warren, Jason Karamchandani, Roger J Packer, Nada Jabado, Jacek Majewski, Javad Nazarian

Abstract

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M--including H3.2K27M--mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.

Figures

Figure 1. Oncogenic alterations in 41 sub-regions…
Figure 1. Oncogenic alterations in 41 sub-regions from nine DIPG patients from whole exome sequencing data.
Samples representing different anatomical locations within each patient are represented in columns. The mutations (in rows) were selected based on published datasets in paediatric glioblastoma and specifically DIPG. Mutations were divided into two subgroups; driver mutations which are essential for tumour initiation/maintenance and accessory driver mutations, which can further promote and accelerate tumour growth, but are not absolutely essential for tumour initiation or maintenance.
Figure 2. Selected examples of clonal evolution…
Figure 2. Selected examples of clonal evolution within DIPG tumours.
Left: histograms show the raw allele frequencies (whole exome sequencing data) for each somatic mutation in different autopsy regions within each tumour. Red: ubiquitous mutations across regions; yellow: mutations shared in at least two regions; blue: mutations seen in only one region. Right: phylogenetic trees constructed from the mutation allele frequencies of deep amplicon sequencing data showing the order of evolution along with support probabilities (upper portions of graphs) and clonal mixing proportions within samples (lower portions). For clarity, only mutations selected to be likely oncogenic are shown. (a) DIPG5: a rare case harboring both TP53 and PPM1D mutations, which are generally found to be mutually exclusive. PPM1D and TP53 mutations occur in distinct clones and are both secondary to H3K27M. ATRX is also secondary and subclonal. (b) DIPG6: while it is impossible to resolve the order of H3/TP53/ATRX mutations' appearance, PIK3CA is clearly sub-clonal and appears in the later stages of evolution within this tumour. (c) DIPG2: the H3.1 K27M and ACVR1 main driver mutations are ubiquitous, occur at similar frequencies across all samples, and their mutation order cannot be resolved. Conversely, other accessory driver mutations are clearly secondary in order of appearance, and are present only in distinct subclones.
Figure 3. Tumour spread in DIPG.
Figure 3. Tumour spread in DIPG.
(a) Tumour spread in DIPG2 in the thalamus, cerebellum and brainstem. Tumour extension in the thalamus harbors secondary mutation PIK3CA, MAX and PTEN, which indicates late spread from both Pons 1 and Pons 2. Extension towards the cerebellum is relatively early in the tumour evolution as it lacks secondary mutations found in the primary tumour and other brainstem spread. (b) Evolution of tumour in patient DIPG3. Autopsy revealed two morphologically and histologically distinct regions of the tumour, indicated as DIPG3 Pons 1 (low-grade) and DIPG3 Pons 2 (high-grade). Exome sequencing identified 11 SNVs and several large scale CNAs common to both regions. Shared alterations included H3.2 K27M and ACVR1 G328V mutations that are likely the main driver mutations in this patient. The analysis also indicated a clear clonal substructure of the two regions, with 18 SNVs and 1 CNA found only in DIPG3 Pons 1, and 11 SNVs and 3 CNAs unique to DIPG3 Pons 2. Intriguingly, DIPG3 Pons 2 carries the activating PIK3CA H1047R mutation, which occurs early in the evolution of this sub-clone judging by its high allelic frequency. PIK3CA H1047R is associated with multi-potency and PI3K activation with angiogenesis and growth and this mutation likely contributes to tumour aggressiveness and high-grade features of Pons 2 compared with Pons 1 in DIPG3. Scale bar=500 μm.

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