Apps, JR

Chalker, J

Forshew, T

Gonzalez-Meljem, JM

Gutteridge, A

Hölsken, A

Jacques, TS

Martinez-Barbera, JP

Stache, C

English

UCL Discovery

Scientific CorrespondenceCTNNB1 mutations are clonal inadamantinomatous craniopharyngiomaAdamantinomatous craniopharyngiomas (ACPs) aretumours of the sellar region and although histologicallybenign, they frequently invade the hypothalamus,visual tracts and local vascular structures. This aggres-sive behaviour can result in profound chronic morbid-ity, poor quality of life and increased mortality in long-term follow-up [1].ACPs are histologically complex tumours with vari-able cystic, calcified and solid components often sur-rounded by a florid glial and inflammatory reactivetissue. Within tumour epithelia, a peripheral layer ofpalisading epithelium (PE) encloses more loosely packedstellate cells (i.e. stellate reticulum (SR)) and epithelialwhorls [2]. Since the first identification of activatingCTNNB1 mutations in ACP [3], several cohorts haverecapitulated this finding and mutations have beenidentified anywhere between 39 and 100% of thetumours. Nuclear accumulation of b-catenin andimmunohistochemical evidence of activation of theWNT pathway (e.g. Axin2 expression) is limited to onlya small proportion of cells, which in many cases corre-late with epithelial whorls (from now on referred to asclusters) [1-2,4].Several studies have investigated the distribution ofCTNNB1 mutations within the different tumour cellcompartments. Using Sanger sequencing mutationswere identified in both epithelial and ‘mesenchymal’components [3]. However, Kato et al. did not identifymutations outside of the tumour epithelia [5]. Morerecently, using laser capture microdissection (LCM) andsingle-strand conformational polymorphism analysis,CTNNB1 mutations were identified in all the eightcases [6]. Surprisingly, two cases harboured more thanone CTNNB1 mutations and in four cases, differentCTNNB1 mutations were found in b-catenin-accumu-lating and non-accumulating tumour cells [6]. Subse-quent studies have failed to identify ACP tumoursharbouring more than one CTNNB1 mutation and onlyone report has described ACP with coexistence ofCTNNB1 and BRAF-V600E mutations [7]. Genetictracing experiments in genetically engineered mousemodels of ACP have revealed a non-cell autonomousmechanism of pathogenesis, whereby not all tumourcells contain CTNNB1 mutations [1]. Together, thesefindings have brought into question whether CTNNB1mutations are present in all ACP tumour cells or onlyin those accumulating nuclear b-catenin.To further investigate the occurrence and cell distri-bution of CTNNB1 and BRAF mutations in ACPs, weassessed the mutational status of the CTNNB1 exon 3and the BRAF V600E locus, as well as other commonlymutated brain tumour genes including H3F3A,HIST1H3B, IDH1 and IDH2 in 22 tumours [8]. In addi-tion to Sanger sequencing of exon 3 of CTNNB1, weused a highly sensitive next generation targeted ampli-con sequencing panel (TAm-Seq) [8]. Compared withconventional targeted sequencing TAm-Seq replicateamplification of the regions of interest followed by bar-coding of separate replicates and deep sequencing,enabling the identification of mutations at variant allelefrequencies well below those reliably detected by San-ger sequencing (limited to ~20%) [9]. In three cases, inwhich cryopreserved tumour tissue was available, weperformed LCM using the Zeiss PALM MicroBeam sys-tem. We analysed by TAm-Seq specific tumour cellcompartments including the clusters (C), PE and SR aswell as local reactive glial tissue as previously described[10](Appendix S1). To confirm the sequencing results,we used immunofluorescence staining using antibodiesagainst specific CTNNB1 mutations (Appendix S1).Sanger sequencing analysis was successful in 19 of22 cases of ACP, but confirmed the presence of aCTNNB1 mutation only in 12 of 19 tumours (Table 1).In contrast, TAm-Seq identified CTNNB1 mutations inall cases (22 of 22), with mutation allele frequenciesvarying from 3 to 47% (Table 1). The higher detectionrate reflects a greater sensitivity of the TAm-Seqmethod relative to Sanger sequencing in the identifica-tion of mutations with lower allele frequency.All CTNNB1 mutations have previously beendescribed in ACP, that is D32, S33, I35, S37 and T41substitutions, which are expected to prevent phospho-rylation and therefore disrupt the degradation of© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltdon behalf of British Neuropathological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited.510Neuropathology and Applied Neurobiology (2020), 46, 510–514 doi: 10.1111/nan.12613b-catenin [1,4]. No tumours were found to carry morethan one CTNNB1 pathogenic mutation and no muta-tions in the hotpots of BRAF, H3F3A, HIST1H3B,IDH1 or IDH2 were identified in ACP.The CTNNB1 mutation allele frequency in ACP cor-related with the percentage of tumour epithelia asassessed by nuclear content (r = 0.70, P = 0.0004;Appendix S1, Table S1; Figure S1). Indeed, theCTNNB1 mutation allele frequencies observed acrossthe tumour samples were consistent with the presenceof heterozygous CTNNB1 mutations throughout alltumour epithelial cells. For instance, in case 9, thevariant allele frequency of the S33C mutation was39%, consistent with 78% of the cells harbouring aheterozygous mutation and closely correlating with thehistological assessment of tumour epithelia of around80% (Table 1).The previous results were confirmed by TAm-Seqanalysis of specific tumour cell compartments isolatedby LCM (Appendix S1; Figure S2). CTNNB1 mutationfrequencies were 47–55%, which are consistent withheterozygosity within all tumour compartments (clus-ters, PE, SR) but nearly zero within the surroundingglial tissue (max 0.65%) and germline DNA (availablein two of the three cases) (Table S1).Finally, we used immunofluorescence staining withspecific antibodies against the S33F, S37F and T41Isubstitutions in b-catenin on cases of ACP (cases:S33F, n = 2; S37F, n = 4; T41I, n = 6; Figure 1).These studies confirmed the expression mutant b-cate-nin protein across the tumour epithelium with nuclearaccumulation limited to the cell clusters (Figure 1).Negative control immunofluorescence staining on ACPtumours separately known to harbour other CTNNB1mutations confirmed the specificity of the antibodiesused (Figure 1) [10].In conclusion, we confirm a high prevalence ofCTNNB1 mutations in ACP. The high sensitivity ofTAm-Seq has helped identify CTNNB1 mutations in allACP samples analysed, including those with very lowallele frequencies. This suggests that failure to identifyCTNNB1 mutations in a low proportion of ACPtumours in previous studies using Sanger sequencing,single-strand conformation polymorphism analysis,exome sequencing and targeted next generationsequencing may reflect methodological limitations.Table 1. CTNNB1 exon 3 sequencing analysis of DNA from 22 cases of ACPCase No:Age at diagnosis(years)*Tumour content(% nuclear area)DNA conc(copies/ul)CTNNB1 mutationby Tam-SeqCTNNB1 mutationby Sanger SeqAverage mutationallele frequency1 8 20 1100 T41I T41I 17%2 14 70 2315 T41I T41I 28%3 6 70 390 S37F No mutation 15%4 14 60 1155 S45F Failed** 35%5 8 10 525 S33F No mutation 3%6 12 50 680 S37A No mutation 16%7 46 70 770 T41I No mutation 21%8 71 80 8854 T41I T41I 35%9 adult 80 395 S33C S33C 39%10 adult 80 2273 T41I T41I 29%11 60 70 143 T41I T41I 15%12 83 45 2087 S33C S33C 19%13 22 80 439 T41I T41I 26%14 87 90 1573 D32N D32N 31%15 61 50 195 S33C S33C 30%16 65 40 237 T41A T41A 25%17 53 40 520 S37A S37A 17%18 adult 90 470 I35S Failed** 43%19 adult 60 260 S33F No mutation 25%20 adult 25 1256 S37F No mutation 3%21 adult 20 1460 S33F No mutation 18%22 adult 70 4505 S37C Failed** 47%*Age not available in all patients.**Sanger sequencing reaction failed to give readable trace.© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltdon behalf of British Neuropathological SocietyNAN 2020; 46: 510–514Scientific Correspondence 511We reveal that all cellular components of the tumourepithelium harbour the same CTNNB1 mutation andfound no evidence of second CTNNB1 mutations, evenat low allele frequencies. Additionally, we show thepresence of CTNNB1 mutations throughout the tumourepithelia, which is consistent with the early stages oftumourigenesis in the embryonic mouse model of ACP.In this model, oncogenic Ctnnb1 mutation is presentthroughout the entire tumoural pituitary, but nuclearaccumulation of b-catenin is limited to only a smallproportion of cells mostly forming clusters, which aremolecularly analogous to the human clusters [11,12].The mechanism behind why b-catenin is accumulatedonly in single cells or clusters despite the presence ofCTNNB1 mutations in other epithelial tumour cells (i.e.palisading epithelium and stellate reticulum) is notknown.AcknowledgementsTissue samples were obtained from Plymouth HospitalsNHS trust as part of the UK Brain Archive InformationNetwork (BRAIN UK), which is funded by the MedicalResearch Council and brains trust. We thank the CCLGTissue Bank for access to samples, and contributingCCLG Centres, including members of the ECMC Paedi-atric network. The CCLG Tissue Bank is funded by Can-cer Research UK and CCLG. Tissue samples wereprovided by the Imperial College Healthcare NHS TrustTissue Bank. Other investigators may have receivedS37F tumourS33F tumourT41I tumourG34E tumourG34E tumourD32Y tumourDAPIDAPIDAPIβ-cateninβ-cateninβ-cateninS37F β-catenin S33F β-catenin T41I β-catenin MergeMergeMergeFigure 1. Mutant b-catenin is expressed throughout the tumour epithelia. Double immunofluorescence stainings on FFPE histologicalsection of ACP tumours harbouring different mutations (shown on the left) using specific antibodies against either S37F, S33F or T41Imutant b-catenin (red) as well as mutation non-specific b-catenin antibody (shown within the panels). Boxes highlight nuclearaccumulation within clusters. Scale bar = 100 µm.© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltdon behalf of British Neuropathological SocietyNAN 2020; 46: 510–514512 Scientific Correspondencesamples from these same tissues. The research sup-ported by the National Institute for Health Research(NIHR) Biomedical Research Centre based at ImperialCollege Healthcare NHS Trust and Imperial CollegeLondon. The views expressed are those of the author(s)and not necessarily those of the NHS, the NIHR or theDepartment of Health. This work is supported by theNIHR GOSH BRC. The views expressed are those of theauthor(s) and not necessarily those of the NHS, theNIHR or the Department of HealthEthicsExperiments were performed under NHS ResearchEthics Committee approval (14/LO/2265) or approvalfrom individual biobanks. Where required, informedconsent was obtained from all individual participantsincluded in the study.FundingFunding for this research was provided by CancerResearch UK, the Children’s Cancer and LeukaemiaGroup, Children with Cancer UK (15/190), MRC (MR/M125/1), the Brain Tumour Charity (SIGNAL andEVEREST), Great Ormond Street Hospital Children’sCharity and the National Institute of Health ResearchBiomedical Research Centre at Great Ormond StreetHospital for Children NHS Foundation Trust and Univer-sity College London. J.R.A. is supported by a CancerResearch UK Clinical Trials Fellow. JMGM is part of theTranslational OMICS Strategic Focus Group at Tecno-logico de Monterrey. J.P.M.-B. is a Great Ormond StreetHospital for Children’s Charity Principal Investigator.Conflict of InterestThe Editors of Neuropathology and Applied Neurobiology arecommitted to peer-review integrity and upholding the high-est standards of review. As such, this article was peer-re-viewed by independent, anonymous expert referees, andthe authors (including TSJ and JPMB) had no role in eitherthe editorial decision or the handling of the paper.J. R. Apps*†‡,1C. Stache*,1J. M. Gonzalez-Meljem§A. Gutteridge¶J. Chalker**T. S. Jacques***T. Forshew¶A. H€olsken††J. P. Martinez-Barbera**Developmental Biology and Cancer Research and TeachingDepartment, Birth Defects Research Centre, UCL GreatOrmond Street Institute of Child Health, University CollegeLondon, London, UK, †Cancer Research UK Clinical TrialsUnit, University of Birmingham, Birmingham, UK, ‡Birming-ham Women’s and Children’s Hospital NHS Foundation Trust,Birmingham, UK, §Bioengineering Department, School ofEngineering and Science, Tecnologico de Monterrey, Monter-rey, Mexico, ¶UCL Cancer Institute,University College London,London, UK, **Great Ormond Street Hospital for ChildrenNHS Foundation Trust, London, UK and ††Department ofPathology, REGIOMED-Klinikum Coburg, Coburg, Germany1These authors contributed equally.References1 Muller HL, Merchant TE, Puget S, Martinez-Barbera JP.New outlook on the diagnosis, treatment and follow-upof childhood-onset craniopharyngioma. Nat Rev Endo-crinol 2017; 13: 299–3122 Martinez-Barbera JP, Buslei R. Adamantinomatous cran-iopharyngioma: pathology, molecular genetics andmouse models. J Pediatr Endocrinol Metab 2015; 28: 7–173 Sekine S, Sato S, Takata T, Fukuda Y, Ishida T,Kishino M, et al Beta-catenin mutations are frequentin calcifying odontogenic cysts, but rare in ameloblas-tomas. Am J Pathol 2003; 163: 1707–124 Larkin SJ, Ansorge O. Pathology and pathogenesis ofcraniopharyngiomas. Pituitary 2013; 16: 9–175 Kato K, Nakatani Y, Kanno H, Inayama Y, Ijiri R,Nagahara N, et al Possible linkage between specific his-tological structures and aberrant reactivation of theWnt pathway in adamantinomatous craniopharyn-gioma. J Pathol 2004; 203: 814–216 Holsken A, Kreutzer J, Hofmann BM, Hans V, Oppel F,Buchfelder M, et al Target gene activation of the Wntsignaling pathway in nuclear beta-catenin accumulat-ing cells of adamantinomatous craniopharyngiomas.Brain Pathol 2009; 19: 357–647 Larkin SJ, Preda V, Karavitaki N, Grossman A,Ansorge O. BRAF V600E mutations are characteristicfor papillary craniopharyngioma and may coexist withCTNNB1-mutated adamantinomatous craniopharyn-gioma. Acta Neuropathol 2014; 127: 927–98 Stone TJ, Keeley A, Virasami A, Harkness W, TisdallM, Izquierdo Delgado E, et al Comprehensive molecularcharacterisation of epilepsy-associated glioneuronaltumours. Acta Neuropathol 2018; 135: 115–29© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltdon behalf of British Neuropathological SocietyNAN 2020; 46: 510–514Scientific Correspondence 5139 Forshew T, Murtaza M, Parkinson C, Gale D, Tsui DW,Kaper F, et al Noninvasive identification and monitor-ing of cancer mutations by targeted deep sequencing ofplasma DNA. Sci Transl Med 2012; 4: 136ra6810 Apps JR, Carreno G, Gonzalez-Meljem JM, Haston S,Guiho R, Cooper JE et al Tumour compartment tran-scriptomics demonstrates the activation of inflamma-tory and odontogenic programmes in humanadamantinomatous craniopharyngioma and identifiesthe MAPK/ERK pathway as a novel therapeutic target.Acta Neuropathol 2018; 135: 757–7711 Gonzalez-Meljem JM, Haston S, Carreno G, Apps JR,Pozzi S, Stache C, et al Stem cell senescence drivesage-attenuated induction of pituitary tumours inmouse models of paediatric craniopharyngioma. NatCommun 2017; 8: 181912 Gaston-Massuet C, Andoniadou CL, Signore M, Jayak-ody SA, Charolidi N, Kyeyune R, et al Increased Wing-less (Wnt) signaling in pituitary progenitor/stem cellsgives rise to pituitary tumors in mice and humans.Proc Natl Acad Sci USA 2011; 108: 11482–7Supporting informationAdditional Supporting Information may be found in theonline version of this article at the publisher’s web-site:Figure S1. A. Correlation between mutation allele fre-quency and histologically assessed tumour content (%nuclei). Arrows indicate cases 9 and 17. B. Examples ofACP FFPE histological sections stained with haema-toxylin and eosin.Figure S2. Representative images of laser capturemicrodissection (LCM): A. Areas selected for LCM arehighlighted by colours: Clusters (green), stellate reticu-lum (black), palisading epithelium (blue), reactive glialtissue (yellow). B. Example showing the excision of acluster. C. Example of multiple clusters pooled beforeDNA extraction. Scale bar =100lm.Table S1. Laser capture microdissection identifiesCTNNB1 mutations in all ACP tumour compartments.Appendix S1. Supplementary MethodsReceived 18 October 2019Accepted after revision 16 January 2020Published online Article Accepted on 3 March 2020© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltdon behalf of British Neuropathological SocietyNAN 2020; 46: 510–514514 Scientific Correspondence

CTNNB1 mutations are clonal in adamantinomatous craniopharyngioma

https://discovery.ucl.ac.uk/10093507/1/Apps_nan.12613.pdf

CTNNB1 mutations are clonal in adamantinomatous craniopharyngioma

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