Prospective collection of DIPG samples at autopsy allowed us to perform genome-wide studies on a sizable cohort of tumors for which samples are extremely limited. This study shows the advantages and disadvantages of using postmortem specimens. CNAs in autopsy samples collected after adjuvant therapy likely present a combination of the initial mutations driving DIPG tumorigenesis as well as secondary changes induced by irradiation and/or chemotherapy. Our findings support the relevance of molecular analysis of tissue obtained at autopsy. PDGFRA
amplifications have been identified both in post-treatment autopsy samples in the current study as well as in a previous report12
and in small numbers of pretreatment samples.6,7,12
The overall number of large-scale and focal gains and losses was not significantly different between samples obtained at diagnosis and autopsy and was consistent with the spectrum of CNAs observed in 71 pretreatment pediatric nonbrainstem HGGs,6
indicating that post-treatment samples did not show increased widespread genomic instability. Amplifications of a number of the genes that were identified in DIPG have been previously identified in pediatric or adult HGG.5–8,14,22,23
Thus, it seems likely that analyses of autopsy samples identify CNAs relevant to oncogenesis.
An advantage of the current study was the availability of large tissue samples obtained at autopsy that enabled the identification of small focal regions of tumor containing therapeutically relevant CNAs, which may have been missed had analysis been performed in rather small biopsies. Finally, autopsy samples allow analysis of end-stage disease, which is almost universally fatal. It is essential to identify potential therapeutic targets in this final stage of disease, even if they arise as late events in the evolution of DIPG.
There are shared molecular features between DIPG and nonbrainstem pediatric glioblastoma, including similar frequencies of some large-scale gains and losses. The proneural, proliferative, and mesenchymal gene expression subgroups originally identified in adult glioblastoma can also be readily identified in pediatric glioblastoma, regardless of tumor site.6,22
However, there were significant differences in the frequency of some large-scale genomic imbalances as well as focal deletion of CDKN2A
between DIPG and nonbrainstem pediatric glioblastoma. This suggests that the selective pressure driving DIPG development and growth may be different in the brainstem compared with the tumor microenvironment outside the brainstem or that DIPG may be most similar to only a subset of nonbrainstem pediatric glioblastomas. PCA showed that gene expression signatures from DIPG seemed to comprise a distinct subgroup compared with nonbrainstem pediatric HGG. Interestingly, DIPG showed significantly higher expression of specific genes regulating developmental processes and transcription factors, with coordinate upregulation of multiple HOX
family genes including HOXA1
, which play important roles in hindbrain development24,25
(Data Supplement). The multiple specific HOX
family members that are upregulated in DIPG are distinct from those previously shown to be differentially expressed in therapy-resistant adult glioblastoma or associated with decreased survival in pediatric HGGs.26,27
Previous studies showed that LGGs arising in different regions of the brain did not cluster independently in an unsupervised analysis, but location-dependent signatures could be identified in supervised comparisons.28
Consistent with this result, brainstem and nonbrainstem LGGs did not cluster separately in a PCA comparison (Appendix Fig A1, online only); however, we could identify differential expression of signature genes previously shown to associate with LGGs arising in the posterior fossa28
within the brainstem and cerebellar LGGs (data not shown). The brainstem and nonbrainstem LGGs were also similar on the genomic level, showing minimal CNAs and recurrent gains of 7q34, including BRAF
The identification of focal amplifications of RTKs and/or cell-cycle regulatory genes in approximately half of all DIPGs has potential therapeutic relevance. Several recent clinical trials have employed small-molecule inhibitors in the treatment of this tumor; however, the choice of targeted agents was based primarily on experience in the treatment of adult glioblastoma, not on the unique genetic characteristics of DIPG.29–31
On the basis of our genomic studies, it may be useful to integrate broad, as well as selective, inhibitors of RTKs32–35
in addition to agents that block cell-cycle progression through the G1 phase, such as selective inhibitors of CDK4 and CDK6, which inhibit intracranial tumor growth of glioblastoma xenografts.36
Although biopsy of DIPG at diagnosis has been routinely performed in some European countries,3
this is not standard practice in North America. A recent study showed that gene-specific assays can be performed in biopsy samples of DIPG tissue,37
and our results may provide a rationale for biopsy at diagnosis to determine if specific targets are amplified and to stratify treatment.
However, our findings also indicate that tumor heterogeneity could create substantial challenges in using molecular diagnosis to guide personalized therapy of DIPG. We found tumor subclones containing different genomic amplifications in a subset of samples obtained at autopsy. It is not clear if this intratumoral variation occurrs at similar frequencies before therapy because of the limited numbers of pretreatment samples analyzed. Tumors constantly evolve, with subclones acquiring different mutations and competing for the greatest selective advantage. Treating a heterogeneous tumor with selective inhibitors may effectively ablate only one subpopulation within the tumor. Depending on the composition of the tumor, small-molecule inhibitors of multiple RTKs and/or cell-cycle regulatory components may have a dramatic effect on overall tumor survival or allow a rapid outgrowth of tumor cells lacking the amplification. It is noteworthy that some tumors lacking amplification of PDGFRA or IGF1R still show strong overexpression of these genes. Thus, analysis of copy-number imbalances may provide insight into critical pathways underlying DIPG growth, which may be altered by varying mechanisms in different tumors.