Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas

Abstract

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Figure 1

Plot of the number of non-synonymous mutations per megabase in craniopharyngiomas in comparison to a broad range of pediatric and adult tumors. Data for all other tumor types, as well as the figure design were taken from Lawrence et al. Each dot in this plot corresponds to a matched tumor–normal pair. The vertical position indicates the frequency of somatic mutations in that exome. Tumor types are ordered based on their median non-synonymous frequency and within each tumor type tumor-normal pairs are ordered from lowest to highest frequency. The relative proportions of six different possible base-pair substitutions are indicated in the bottom panel. Craniopharyngioma data are derived from whole exome sequencing of 15 tumor-normal pairs including 12 adamantinomatous and 3 papillary craniopharyngioma and marked in red. AML – Acute myelogenous leukemia, Cranio – Craniopharyngioma, DLBCL – Diffuse large B-cell lymphoma, LUAD – Lung adenocarcinoma; LUSC – Lung squamous cell carcinoma.

Figure 2

Mutations in adamantinomatous and papillary craniopharyngiomas. Panel a depicts the number of mutations per megabase in each of 15 craniopharyngioma whole exome sequencing data sets (12 adamantinomatous and 3 papillary samples). Specific genes are listed bearing non-synonymous somatic mutations in genes listed in Cancer Gene Census. At the right we show the false discovery rate q-values providing the significance of mutations in each listed gene. Cancer relevant genes that are not listed in the Cancer Gene Census are not shown. The q value is an evaluation of whether a gene is significantly mutated above the expected basal rate of mutation. The q values for BRAF and CTNNB1 are <0.00001. The q values of other genes in the plot are equal to 1. A full list of mutated genes is provided in Supplementary Table 2. Colors indicate the type of genetic change identified. Only one mutation is indicated even if multiple mutations were found in a particular gene. The cancer cell fraction (CCF, 0–1.0) for each indicated mutation is shown in white type in the corresponding box. The allelic fraction for mutations in each sample is shown in box plots with the median indicated by a red line (the allelic fraction for each mutation is listed in Supplementary Table 2. For each sample, the relative frequencies of six different possible base-pair substitutions are presented in the bottom panel. In Panel b, schematics for CTNNB1 and BRAF indicating the location of identified mutations (11 in CTNNB1 and 3 in BRAF) are shown.

Figure 3

Beta-catenin localization is different in adamantinomatous and papillary craniopharyngiomas. Immunohistochemistry for beta-catenin was performed. In Panel a, beta-catenin is localized to the cytoplasm and the nucleus in an adamantinomatous craniopharyngioma. In Panel b, beta catenin is localized to the cell membrane in a papillary craniopharyngioma. Scale bars, 50 µm. Beta-catenin localization in many samples from the discovery and validation cohort is reported in Supplementary Table 1.

Figure 4

BRAF and CTNNB1 mutations are clonal in craniopharyngiomas. In Panel a (left), a violin plot shows the cancer cell fraction (CCF) for BRAF (orange) and CTNNB1 (pink) mutations in each tumor analyzed with whole exome sequencing. The median CCF of all non-synonymous somatic mutations for each sample is represented by a black dot. The bar graph (Panel a, right) shows the computed purity for each sample (see Methods sections); error bars represent standard error of the mean. In Panel b, we show hematoxylin and eosin (H&E) staining of adamantinomatous and papillary craniopharyngiomas. Immunohistochemistry (IHC) shows that adamantinomatous craniopharyngiomas are negative for BRAF V600E but that there is a diffuse distribution of BRAF V600E mutant protein in the neoplastic epithelium of papillary craniopharyngiomas. Stromal elements in the fibrovascular cores of the papillary tumors are negative for the BRAF V600E mutant protein. Scale bars, 100 µm.