Page 0004

private, subclonal mutations associated with the progression and relapse ("branch") 1416

.

In adult GBM, studies of intratumoral heterogeneity demonstrated a marked subclonal

diversity across time and space17

. These data provided evidence for differential

mechanisms of treatment-induced and spontaneous acquisition of the malignant

phenotype in the transformation from low- to high grade glioma18

. In children, the

initiating genetic lesions and cells of origin are intrinsically linked to

neurodevelopmental processes5,19

, and markedly different evolutionary dynamics are

likely in tumours that arise some decades prior to their adult counterparts. We have

provided some limited evidence for a non-random distribution of genetic heterogeneity

within tumours, with subclonal populations associated with differential tissue

distribution and cellular morphologies11,20

. Although these and other data hint at key

functionally distinct roles, the precise nature of the subclonal genotype-phenotype

relationships that may exist have not been elucidated.

We now wish to extend our ongoing work in this area to understand the complex

evolutionary patterns of DIPG and assigning specific functional roles to the subclonal

cell populations will allow us to design novel therapeutic strategies aimed at

overcoming the deficiencies evident in current treatment approaches.

iv. Research design

How does the subclonal architecture of DIPG differ across time and space?

We intend to apply a combination of next generation sequencing (NGS) approaches to

a series of DIPG samples and derived cells, in order to quantitate the subclonal

diversity and infer the evolutionary history of the tumours. This will involve the

following:

Advanced sampling of primary DIPG

We have well-established collaborations locally (Royal Marsden Hospital, St Georges

and Kings College Hospitals), nationally (INSTINCT network - Newcastle Royal

Infirmary and Great Ormond Street Hospital), and internationally (SIOPE Europe, as

well as individual centres in Brazil, France, Spain, Australia and North America) for the

collection of DIPG samples. We are extending these initiatives to include longitudinal

studies of matched biopsy/autopsy pairs as well as multiple sampling, i.e. more than

one topographical region within a single tumour post-mortem. We will collect both

fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) material as availability

dictates, with recent experience in our lab producing high quality reads from as little as

50ng FFPE-derived DNA. In all instances, we obtain fresh material for sequencing as

well as establishment of primary cell cultures. In total we aim to fully characterise

samples from approximately 10 patients, as described below, and compare this data

to an ongoing program in the Jones laboratory looking at paediatric and adult GBM.

High-depth sequencing of tumour DNA

Tumour material and matched normal DNA will undergo targeted resequencing using

a custom Agilent SureSelect capture set of 435 genes representing all recurrent gene

mutations found within ~200 pGBM/DIPG genomes and exomes from our recent

studies7-9. All samples from a single patient will be run on a single lane of an Illumina

HiSeq2500, giving an estimated >2000-fold coverage of somatic calls. This will be

Index

  1. Page 0004
  2. Page 0004
  3. Page 0004
  4. Page 0004
  5. Page 0004
  6. Page 0004
  7. Page 0004
  8. Page 0004
  9. Page 0004
  10. Page 0004
  11. Page 0004
  12. Page 0004

Related Issues

powered by PageTiger