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-potency²⁴ 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.