The data presented in this study, generated from systematic sequencing of tumour exomes, genomes, and transcriptomes of oligodendroglioma, demonstrate the power of massively parallel next generation sequencing in uncovering novel genetic changes in cancers. We sequenced a total of 628,096,883,783 bp of tumour and 499,887,339,249 bp of normal DNA from genome, RNA-seq and exome libraries. From this, we confirmed that IDH mutations are a universal feature of oligodendrogliomas. We also found novel potential driver mutations in CIC in a majority of the tumours.
On a broad level, the data show that oligodendrogliomas carry a relatively modest and stable somatic mutational load compared to many other cancers -- much lower than that present in glioblastoma, melanoma, lymphoma, and colorectal cancer (Broad Institute, unpublished). In addition to known positive prognostic factors such as the proneural gene signature and MGMT promoter methylation, the finding that oligodendrogliomas bear mutations relatively infrequently might also explain the relatively benign clinical course of this tumour and its initial chemosensitivity, an hypothesis that has yet to be formally tested. A particularly striking finding from our analysis, however, was the identification of recurrent mutations in CIC and the apparent relationship between mutations in CIC and IDH genes, and the 1p/19q anomaly, which appear to be distinguishing features of oligodendrogliomas.
, found on chromosome 19q13.2, encodes the mammalian homolog of the Drosophila
transcriptional repressor Capicua. In Drosophila
, Capicua is a transcriptional repressor with crucial roles in development – it represses genes downstream of the RAS/MAPK pathway that signal the differentiation of wing veins, imaginal eye discs, and head and tail polarity development [40
]. Mammalian Capicua is highly expressed in the brain, particularly in the external granular cells of the developing cerebellum [43
], and its over-expression has been identified in medulloblastomas, an aggressive primitive neuroectodermal tumour of the central nervous system [44
]. However CIC
mutations in medulloblastoma have not been reported. Association of CIC
alterations with human disease is restricted to two diseases affecting cells of the neural crest lineage. Spinocerebellar Ataxia I (SCAI), a progressive neurodegenerative disease, and Ewing’s Family Tumours, a cohort of aggressive soft tissue tumours affecting children and young adults, both involve defects of the CIC
gene which in turn lead to dysregulated interactions of the CIC protein with downstream signaling partners [45
]. With respect to the Ewing’s family of tumours, rare cases of Ewing’s sarcoma have a recurrent chromosomal translocation t(4;19)(q35;q13) that generates a novel CIC –DUX4 fusion protein. The fusion oncoprotein exhibits enhanced transcriptional activation of the ETS family genes ERM/ETV5
] whose expression levels are also elevated in “traditional” Ewing’s sarcoma with the EWS-FLI1
It is intriguing that recurrent mutation in CIC
, located on chromosome 19q, is found almost exclusively in 1p/19q codeleted oligodendrogliomas with IDH1
mutation. Yet loss of chromosome 1p is more strongly associated with the oligodendrogliomatous phenotype and clinical behaviour than 19q loss [3
]. In addition, 19q loss, which is occasionally found in astrocytomas, has traditionally been considered less specific to oligodendroglioma. Thus, it was unexpected that our sequencing did not yield more recurrent candidate coding point mutations in 1p, including mutations in FUBP1
as reported by others [24
]. Since our study focused on the detection of recurrent point mutations, other types of recurrent alterations affecting the 1p region have not been excluded. Larger scale sequencing of panels of whole tumor and normal genomes would address this possibility.
High levels of cross-species sequence conservation and identification of functional domains encoded by the human CIC gene support possible functional consequences of mutations in CIC. Two highly conserved regions in CIC include the high mobility group (HMG) DNA binding domain in exon 5 and the protein-protein interaction GRO-L domain in exon 20 at the C-terminus of the protein – both these regions contained recurrent mutations in our patient series. We have confirmed that mutant CIC mRNA and mutant CIC protein are expressed. Thus, based on the known structure/function of CIC, it is likely that the oligodendroglioma mutations play a key role in the biology of the disease.
Two- thirds of CIC
somatic mutations in oligodendroglioma occurred in exon 5 within the HMG-box domain. A majority of these are clustered around several hotspots in the following order of frequency – p.Arg215 > p.Arg201 > p.Arg202, and the affected residues display a high degree of evolution conservation in all examined vertebrate homologs. Furthermore, 33% of the unique exon 5 mutations were predicted to affect protein function using POLYPHEN2 analysis which included the recurrent p.Arg201Trp and p.Arg215Trp changes. Interestingly, 5/20 exon 5 mutations affected amino acid position 215 and 3 of these caused p.Arg215Trp changes, which corresponds to the Drosophila cicArg505Trp
mutation associated with loss of CIC
function. These mutations result in aberrant Drosophila
eye development [42
]. After exon 5, the next most frequently altered CIC
region was in the C- terminal portion of exon 20. We found 4 mutations causing amino acid changes at p.Arg1515 to His, Leu, and Cys. All were predicted to be deleterious to protein function by POLYPHEN2 analysis. It is undetermined, however, whether the hemizygous CIC
mutations in oligodendroglioma result in loss or gain of protein function. Whatever the consequence, the recurrent nature of CIC
mutations in such a high percentage of tumours supports that they are driver mutations central to the genesis and/or identity of the tumours. It is clear that carefully designed functional studies taking into account genomic context (such as 1p/19q loss and IDH mutations) are necessary to delineate the role of CIC
in the pathogenesis of oligodendroglioma.
Further investigation of this complex relationship and of the downstream molecular consequences of CIC
mutation will necessitate the development of novel biological reagents and appropriate model systems. To this end, the two oligodendroglioma TIC lines analyzed here represent a unique resource to study the relationship between 1p/19q co-deletions and the special biology and clinical behaviour of this tumour. Future studies should also concentrate on sequencing of CIC
in larger cohorts of oligodendrogliomas with clinical follow-up data to better distill potential influences on outcome. This will most likely involve international multicenter collaboration similar to a recent study [49
In the minority of oligodendrogliomas where we do not find CIC
mutations, it is reasonable to speculate that other mutations in the RAS/MAPK pathway or in CIC interacting proteins may result in a phenocopy of CIC
mutants. One potential candidate is ATXN2, which we found mutated in two oligodendrogliomas in our exome dataset. CIC is known to interact with ATXN1 in human disease [46
], and ATXN1 is modulated by ATXN2 [50
]. Similarly, we and others [24
] have found a low frequency of NOTCH
mutations in oligodendrogliomas. Recent work suggests direct involvement of ATXN1 in the NOTCH signaling pathway that has critical roles in neural development and tumourigenesis [51
]. Lastly, FUBP1
was reported to be mutated in oligodendroglioma [24
], and although we did not find non-synonymous coding mutations in our tumours we did detect infrequent somatic splice site variants. The possible relationship between FUBP1
, however, remains to be elucidated. Our working model is that mutations in CIC
(in the majority of tumours) or a number of related genes such as ATXN2
, and FUBP1
(in a minority of tumours) may be functionally equivalent in the context of 1p/19q loss, IDH1/2 mutation, and the genesis of oligodendroglioma (). In our exome discovery cohort, we noted various patterns of co-occurrence of somatic mutations affecting CIC, NOTCH
genes, and ATXN2
. However, such a model has yet to be formally tested in a larger cohort of tumours and also in the context of the two predominant patterns of mutations in CIC
affecting the two functional domains.
Proposed CIC interaction network in the context of observed mutations
Our discovery that CIC mutations are a core abnormality in the majority of oligodendrogliomas has translational and clinical implications in neuro-oncology. Improved understanding of the role of CIC in a background of IDH1/2 mutations in codeleted oligodendroglioma will significantly enhance our understanding of this disease and may pave the way for the development of more targeted therapies for this common type of glioma.