Subtypes of medulloblastoma have distinct developmental origins

Abstract

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1(+) precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.

Figure 1. WNT and SHH-subtypes of medulloblastoma are anatomically distinct

(a) Expression distribution in (a) E11.5 and (b) E15.5 mouse hindbrain of orthologs that distinguish human WNT and SHH-subtype medulloblastoma (Supplemental Dataset 1). Cartoons in (b) denote the position of rhombomeres relative to the cerebellum and brainstem. (c) Top=pre and bottom=post-operative MRI scans of exemplary SHH and WNT-subtype medulloblastomas. Right panels show close up views of left. Brainstem (BSt), post-operative tumor cavity (cvt.). (d) Frequency and site of post-operative surgical cavities of SHH (n=6) and WNT (n=6)-subtype medulloblastomas. Axial (left) and sagittal (right) views are shown.

Figure 2. Mutant-Ctnnb1 causes aberrant accumulation of LRL cells

(a) Low (scale=180 μm) and (b) high (scale=50 μm) power views of LRL/dorsal brainstem in Ctnnb1 mutant and wild-type E16.5 embryos. (b) Includes the corresponding adult brainstem region. (c) Volume and indicated immunoreactivity differences between Ctnnb1-mutant and wild-type LRL (n≥3 mice per group, bars=mean ±S.D). Immunofluorescence of Olig3 (d), Pax6 (e), Zic1 (f) in Ctnnb1-mutant E16.5 LRL (left) and aberrant adult dorsal brainstem masses (right, scale=180 μm). Inset=high-power views of ‘*’ (scale=5 μm). (g) Postmitotic MF precursor neurons (Zic1⁺/Ki67^-) exit the proliferating E16.5 control LRL. (h) Ctnnb1-mutant LRL contains aberrant proliferating Zic1⁺ precursors (arrows, scale=50 μm). (i) GFP-electroporated wild-type LRL marks Olig3⁺ cells (j) and migrating precursors (arrows in i) that include Zic1⁺ MF neurons (k) that form the PGN (l). GFP-fluorescence of whole (m,o) and sectioned (n,p) Ctnnb1-mutant and wild-type P0 hindbrains electroporated at E12.5. (q) Mean ± SD of LRL:PGN GFP-fluorescence in whole hindbrains of three BlBp-Cre ; Ctnnb1^+/+ and five Blbp-Cre ; Ctnnb1^+/lox(Ex3) mice (graphs, *≤0.05, **≤0.005, Exact Mann-Whitney P).

Figure 3. Mutant-Ctnnb1 and SHH-subtype mouse medulloblastomas are anatomically distinct

(a) Tumor free survival of SHH-subtype medulloblastoma mouse models (Nes-Cre^+/−; Lig4^flx/flx ; Tp53^−/−, Nes-Cre^+/−; Xrcc2^flx/flx ; Tp53^−/−, Ptch1^+/−; Ink4c^−/−, Ptch1^+/−; Tp53^−/− data from Refs.14,27,28) and Ctnnb1-mutant ; Tp53^flx/flx and Ctnnb1-mutant ; Tp53^+/flx mice. ***=Log Rank P<0.0001. Immunoflourescence of (b) Zic1 and (c) Olig3 and Ctnnb1 expression in a Ctnnb1-mutant ; Tp53^flx/flx medulloblastoma. (d) Hematoxylin and eosin stained low (i, v; scale=800 μm) and high (ii, vi; scale=25 μm) power views of mouse medulloblastomas and tumor-brainstem interface (iii, vii; scale bar=50μm). Ctnnb1 immunostaining (iv, viii; scale=10 μm, arrows indicate nuclear immunoreactivity). Boxes indicate location of high power views. (e) Frequency and anatomical site of mouse medulloblastomas.

Figure 4. Mutant-Ctnnb1 mouse medulloblastomas recapitulate the molecular characteristics of human WNT-subtype disease

(a) AGDEX comparison of Ctnnb1-mutant ; Tp53^flx/flx mouse medulloblastoma, and mouse EGL, E16.5 dorsal brainstem (DBS) and human medulloblastoma subgroups. (b) Unsupervised clustering of human WNT and SHH-subtype medulloblastoma signature ortholog expression in E16.5 DBS, Ctnnb1-mutant ; Tp53^flx/flx mouse medulloblastoma (Ctnnb1 MB), P7 GNPCs and Ptch1^+/−; Tp53^−/− medulloblastoma (Ptch1 MB). (c) Top-bottom: Nine SNP-inferred homozygous deletions in three human WNT-subtype medulloblastomas. Real-Time PCR validation of deletions in the human tumors (SD below the mean diploid copy-number). Mouse chromosomal regions syntenic for human chromosome 6. ArrayCGH-inferred copy number in Ctnnb1-mutant ; Tp53^flx/flx mouse medulloblastomas identifies common syntenic deletion of TULP4.