TCF12 is mutated in anaplastic oligodendroglioma

Abstract

Anaplastic oligodendroglioma (AO) are rare primary brain tumours that are generally incurable, with heterogeneous prognosis and few treatment targets identified. Most oligodendrogliomas have chromosomes 1p/19q co-deletion and an IDH mutation. Here we analysed 51 AO by whole-exome sequencing, identifying previously reported frequent somatic mutations in CIC and FUBP1. We also identified recurrent mutations in TCF12 and in an additional series of 83 AO. Overall, 7.5% of AO are mutated for TCF12, which encodes an oligodendrocyte-related transcription factor. Eighty percent of TCF12 mutations identified were in either the bHLH domain, which is important for TCF12 function as a transcription factor, or were frameshift mutations leading to TCF12 truncated for this domain. We show that these mutations compromise TCF12 transcriptional activity and are associated with a more aggressive tumour type. Our analysis provides further insights into the unique and shared pathways driving AO.

Figure 1. Significantly mutated genes in anaplastic oligodendroglioma by molecular subtype.

Significantly mutated genes (Q-value<0.1) identified by exome sequencing are listed by Q-value. The percentage of AO samples with mutation detected by automated calling is detailed on the left. Samples are displayed as columns, with the mutation rate plotted at the top. Samples are arranged to emphasize mutual exclusivity. Mutation types are indicated in different colours (see legend). White colour indicates no information available. Also shown is the relative proportion of base-pair substitutions within mutation categories for each tumour.

Figure 2. FM-biased genes and gene modules in AO identified by Oncodrive-fm using data from this study and tumours profiled by TCGA.

Heatmap shows tumours in columns and genes in rows, the colour reflecting the MutationAssessor (MA) scores of somatic mutations. FM ext. qv, corrected P values of the FM bias analysis using the external null distribution.

Figure 3. TCF12 mutations altering the bHLH domain result in impaired transactivation.

(a) Schematic view of the wild-type and mutant TCF12 proteins for which the transactivation capacity has been assessed. Upper panel: wild-type human TCF12, functional domains in grey—activation domain 1 (AD1), activation domain 2 (AD2), repressor domain (Rep) and bHLH domain (bHLH). Lower panel: resulting truncated proteins. Black boxes indicate non-related amino-acid sequences resulting from frameshift mutations (fs), and truncated proteins size is in italic. (b) Schematic structure of the bHLH domain of TCF12 (blue) bound to DNA (grey). WT R602 (yellow) and mutant M602 (purple) residues are indicated. (c) E-box-luciferase reporter plasmid (Eb) was transfected alone or in combination with TCF12 wild-type or mutant expression plasmids. Both frameshift mutants that lack the bHLH DNA binding domain completely abolish TCF12 transcriptional activity. All samples were run in triplicate in four independent experiments. Data were normalized to control renilla luciferase. Values are mean±s.d. ***P=0.0002, **P=0.0018 (Student’s t-test).

Figure 4. TCF12 is highly expressed in a subset of anaplastic oligodendroglioma.

Representative TCF12 immunostainings are shown: (a) wild-type TCF12 tumours show nuclear staining in a heterogeneous cell population. (b–e) Mutant TCF12 tumours show strong nuclear and cytoplasmic staining. (f) Mutant M260fs (resulting in a truncated protein) is associated with 15q21.3 LOH and shows no staining. Scale bar, 50 μm.

Figure 5. TCF12 mutation correlates with a higher necrotic and mitotic index.

(a) Percentage of palisading necrosis in tumours with wild-type TCF12, all tumours mutated for TCF12 or only altered bHLH TCF12 mutants; *P=0.02, **P=0.004. (b) Mitotic index in TCF12 wild-type, TCF12-mutated and altered bHLH TCF12 mutants; *P=0.039, mean±s.e.m. CN, copy number; LOH, loss of heterozygosity; HPF, high-power field. The number of samples is indicated in parenthesis.