From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients

Abstract

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

Keywords: adult glioma; childhood brain cancer; circulating free DNA (cfDNA); circulating tumor DNA (ctDNA); droplet digital PCR (ddPCR); glioblastoma; histone mutations.

Figure 1

ddPCR histone mutation assay. Mutant synthetic gBlock DNA mixed with genomic DNA from healthy donor PBMCs was used as a template for ddPCR. Clear separation of the various indicated mutant and wild-type alleles is demonstrated. Green dots define droplets with wild-type template detected, blue dots define droplets with the indicated mutant detected.

Figure 2

H3.1-K27M detection in DIPG patient 1. (A) 1D graphical data of a H3.1-K27M assay is shown for ctDNA samples from DIPG patient 1, extracted from plasma and cerebral spinal fluid (CSF) alongside synthetic gBlock template H3.1-K27M DNA (positive control) concentrated at 2 × 10⁻⁴ pg (POS2) or 1000-fold diluted (POS1), genomic DNA from healthy donor PBMCs (negative control) was used independently to demonstrate wild-type (WT) allele detection. Visual separation of samples by dotted line. Plasma 1—at diagnosis; Plasma 2—post erlotinib and radiotherapy treatment; CSF1—during erlotinib and radiotherapy treatment; CSF2—during erlotinib and radiotherapy treatment, two days after CSF1; POS1—positive control 1, 1:1000 diluted; POS2—positive control 2 1:1; NEG—negative control (healthy donor PBMCs); NTC—non-template control. (B) 2D graph of H3.1-K27M detection in the CSF1 sample taken during erlotinib and radiotherapy treatment. Green dots define droplets with wild-type template detected, blue dots define droplets with the indicated mutant (H3.1-K27M) detected.

Figure 3

Histone mutation testing of adult patient tissue DNA. Depicted are histone mutation assay 2D graphs for the one DHG patient validating H3.3-G34R status and for a patient from the adult glioma cohort with no mutations detected, representative of the overall negative histone mutation status of the 89 glioma patients. Green dots define droplets with wild-type template detected. Blue dots define droplets with the indicated mutant detected. Orange dots define droplets with both mutant and wild-type template detected.