Children Brain Tumour Project

Adult cancer

Biomarker evaluation in paediatric high-grade and diffuse intrinsic pontine glioma: a SIOPE HGG/DIPG working group study

Background

High-grade astrocytomas (gliomas) in children are different from those that arise in adults. They occur in distinct locations – in the cerebral hemispheres (cortex), midline structures such as the thalamus, and in the brainstem, where they are known as diffuse intrinsic pontine glioma (DIPG). In all locations, the clinical outcome is dismal, with a median survival of 9-18 months. They also have unique biological features, with specific mutations in histone H3 genes defining robust subgroups which appear to have a different age of incidence, anatomical location and response to therapy. Despite this, molecular subgroups are not yet integrated into clinical practice, and there remains around 50% of tumours with no histone mutation for whom the biological drivers are not known. We have recently carried out a molecular meta- analysis of ~1000 pHGG and DIPG in order to robustly define disease subgroups and to identify prognostic and predictive biomarkers. Some of these may also represent novel therapeutic targets for children with these incurable tumours.

  • – Aims

We wish to validate our histone-based subgrouping and novel prognostic/predictive biomarkers in a large retrospective validation cohort collected across Europe.

  • – Research Plan

As Chair of the International Society of Paediatric Oncology (SIOP) Europe HGG/DIPG Biology Subgroup, I aim to collect on behalf of the collaboration a large series of these rare tumours from all participating countries. We will carry out specific assays aimed at evaluating the most promising biomarkers associated with, subgroup definition, clinical outcome and response to therapy. We will do this through high-throughput targeted resequencing using the IonTorrent AmpliSeq platform for a limited number of gene mutations, in parallel with Illumina 450K methylation profiling to determine DNA copy number events as well as CpG island methylation (DKFZ Heidelberg). All data will be carried out blinded to clinical annotation and analysed by an experienced bioinformatician in the laboratory.

  • – Expected outputs

We will have systematically evaluated a series of the most promising biomarkers in pHGG and DIPG, and integrated this data with clinical variables, in order to develop a novel risk stratification in these diseases. We will be able to identify which children may benefit most from traditional therapies, and give prioritisation of novel targets for therapeutic development in these poor outcome tumours.

  • – Further technical details

Cases

The SIOPE HGG/DIPG biology group comprises clinicians and biologists from more than 25 countries, comprising a total number of paediatric patients with these tumours of more than 400-450 per year. As Chair of the group, I will collect, through the designated National Coordinators, approximately 500 cases of clinically annotated HGG and DIPG from the pathology archives of paediatric oncology centres throughout Europe. Whilst frozen tumour material and matching blood for constitutional DNA will be prioritised, the reality that the majority of cases will be provided in the form of formalin-fixed paraffin-embedded blocks poses no problems for our analyses. As surgical resection of DIPG is not undertaken, we will specifically target centres and research groups with open biopsy and/or autopsy protocols in order to collect these specimens. I have already carried out a SIOPE-wide census of HGG/DIPG material as part of the collaborative group to ascertain the feasibility this approach. Collecting, database management, processing and, where necessary, returning samples will be the responsibility of the post-holder associated with this funding request. They will also either carry out individual assays or ship samples to a central laboratory for analysis.

Biomarkers

As a standardised assay for molecular profiling and putative subgroup assignment, we will carry out Illumina 450k methylation profiling on all the samples in collaboration with the DKFZ in Heidelberg, Germany (Stefan Pfister and David Jones). The specific subgroups to be assessed are:

  • ※ 3 G34R/V
  • ※ 3 K27M
  • ※ 1 K27M
  • ※  IDH1/2
  • ※  PXA/BRAF
  • ※  HGG_NOS 
This assay has the advantage of additionally providing robust DNA copy number assessment in order to detect large-scale chromosomal gains and losses as well as focal amplifications and deletions. The specific copy number changes to be investigated will be defined by published markers as well as our ongoing meta- analysis of more than 1000 paediatric HGG and DIPG. These will include:
  • ※  Receptor tyrosine kinases e.g. PDGFRA, EGFR, MET
  • ※  Cell cycle genes e.g. CCND1, CCND2, CDK4, CDK6
  • ※  Transcription factors e.g. MYCN, MYC
  • ※  Subgroup-specific chromosomal alterations e.g. 1q, 2 gain; 3q, 4q, 13q, 14q loss
  • ※  Novel amplifications/deletions e.g. SIAH2, CDKN2AIP, CCNG1, PARP10, 
HRAS,FOXP2, TOP3A 
We will additionally design a custom AmpliSeq panel for mutation assessment on the IonTorrent instrument available at the Tumour Profiling Unit as the ICR. The final list will be similarly based on published and novel putative markers, and will include:
  • ※  Histones, e.g. H3F3A, HIST1H3B, HIST1H3C, HIST2H3C
  • ※  DNA repair pathways, e.g. TP53, ATRX, PPM1D, ATM, ATR
  • ※  RTK/PI3-kinase pathways, e.g. PDGFRA, PIK3CA, PIK3R1, PTEN, NF1

 Subgroup-specific markers, e.g. IDH1, IDH2, BRAF
 Novel somatic mutations, e.g. ACVR1, CRIPAK, PI4KA, BCOR, BCLAF1,

NOTCH2

All data will be carried out blinded to clinical annotation and analysed by an experienced bioinformatician in the laboratory.

Research dissemination

We will keep the HGG/DIPG biology group fully informed of progress through presentation at regular SIOPE brain tumour working group meetings (annual meeting plus 1-2 tumour-specific meetings annually). Raw data will be circulated to all participants and made open-access for the entire research community.

A final report will be prepared for our funders and for circulation to the SIOPE working group. We anticipate that at least one high quality manuscript will be prepared based on the data of this unique collaborative analysis, which will be drafted by the laboratory and finalised on a collaborative basis by all participants.

References

1-14

  • 1  Bax, D. A. et al. A distinct spectrum of copy number aberrations in paediatric high-grade gliomas. Clinical cancer research: an official journal of the American Association for Cancer Research 16, 3368-3377, doi:10.1158/1078-0432.CCR-10-0438 (2010). 

  • 2  Bender, S. et al. Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant paediatric high-grade gliomas. Cancer cell 24, 660-672, doi:10.1016/j.ccr.2013.10.006 (2013). 

  • 3  Bjerke, L. et al. Histone H3.3 Mutations Drive Paediatric Glioblastoma through Upregulation of MYCN. Cancer discovery, doi:10.1158/2159-8290.CD-12-0426 (2013). 

  • 4  Fontebasso, A. M. et al. Recurrent somatic mutations in ACVR1 in paediatric midline high-grade astrocytoma. Nature genetics 46, 462-466, doi:10.1038/ng.2950 (2014). 

  • 5  Jones, C. & Baker, S. J. Unique genetic and epigenetic mechanisms driving paediatric diffuse high-grade glioma. Nature reviews. Cancer 14, doi:10.1038/nrc3811 (2014). 

  • 6  Korshunov, A. et al. Integrated analysis of paediatric glioblastoma reveals a subset of biologically favourable tumours with associated molecular prognostic markers. Acta neuropathological 129, 669-678, doi:10.1007/s00401-015-1405-4 (2015). 

  • 7  Paugh, B. S. et al. Genome-wide analyses identify recurrent amplifications of receptor tyrosine kinases and cell-cycle regulatory genes in diffuse intrinsic pontine glioma. Journal of clinical oncology: Official Journal of the American Society of Clinical Oncology 29, 3999-4006, doi:10.1200/JCO.2011.35.5677 (2011). 

  • 8  Paugh, B. S. et al. Integrated molecular genetic profiling of paediatric high-grade gliomas reveals key differences with the adult disease. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 28, 3061-3068, doi:10.1200/JCO.2009.26.7252 (2010). 

  • 9  Schwartzentruber, J. et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482, 226-231, doi:10.1038/nature10833 (2012). 

  • 10  Sturm, D. et al. Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge. Nature reviews. Cancer 14, 92-107, doi:10.1038/nrc3655 (2014). 

  • 11  Sturm, D. et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer cell 22, 425-437, doi:10.1016/j.ccr.2012.08.024 (2012). 

  • 12  Taylor, K. R. et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nature genetics 46, 457-461, doi:10.1038/ng.2925 (2014). 

  • 13  Wu, G. et al. Somatic histone H3 alterations in paediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nature genetics 44, 251-253, doi:10.1038/ng.1102 (2012). 

  • 14  Wu, G. et al. The genomic landscape of diffuse intrinsic pontine glioma and paediatric non-brainstem high-grade glioma. Nature genetics 46, 444-450, doi:10.1038/ng.2938 (2014). 


 

Prof. Chris Jones, Institute of Cancer Research