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Histone 3.3 Hotspot Mutations in Conventional Osteosarcomas

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Histone 3.3 Hotspot Mutations in Conventional Osteosarcomas Koelsche et al. Clin Sarcoma Res (2017) 7:9 DOI 10.1186/s13569-017-0075-5 Clinical Sarcoma Research RESEARCH Open Access Histone 3.3 hotspot mutations in conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases Christian Koelsche1,2*† , Daniel Schrimpf1,2†, Lars Tharun3, Eva Roth4, Dominik Sturm4,5, David T. W. Jones4,5, Eva‑Kristin Renker6, Martin Sill7, Annika Baude8, Felix Sahm1,2, David Capper1,2, Melanie Bewerunge‑Hudler9, Wolfgang Hartmann10, Andreas E. Kulozik4, Iver Petersen11, Uta Flucke12, Hendrik W. B. Schreuder13, Reinhard Büttner3, Marc‑André Weber14, Peter Schirmacher15, Christoph Plass8, Stefan M. Pfster4,5, Andreas von Deimling1,2*‡ and Gunhild Mechtersheimer15*‡ Abstract Background: Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, his‑ topathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown. Methods: In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteo‑ sarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs. Results: Six osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberra‑ tions that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profles of H3F3A G34W/R mutant osteosarcomas were clearly diferent from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most diferentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/ PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005). *Correspondence: [email protected]‑heidelberg.de; [email protected]‑heidelberg.de; [email protected]‑heidelberg.de †Christian Koelsche and Daniel Schrimpf shared frst authorship ‡Andreas von Deimling and Gunhild Mechtersheimer shared last authorship 1 Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany 15 Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koelsche et al. Clin Sarcoma Res (2017) 7:9 Page 2 of 11 Conclusions: H3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R muta‑ tions are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB. Keywords: Mutation, H3.3, H3F3A, HIST1H2BB, KLLN, PTEN, Osteosarcoma, Giant cell tumor of bone, DNA methylation, Copy number alteration Background (α-thalassaemia/mental retardation syndrome X-linked) Osteosarcoma is a malignant bone forming neoplasm mutations, which result in a loss of its nuclear expression, with a broad spectrum of morphologies [1]. Conventional were found in osteosarcomas at a frequency of 20–30% high-grade osteosarcoma is the most common subtype [8, 16]. Loss of DAXX (death-domain associated protein) and typically arises in the long bones of the extremities expression has not yet been observed in any sarcoma with a predilection to the metaphysis, although any bone [17]. However, a high frequency of mutations in the two of the skeleton may be afected. Te incidence of osteo- genes encoding histone 3 variant 3 (H3.3) was revealed in sarcomas has a bimodal age distribution with a frst peak certain bone tumors. Giant cell tumors of bone (GCTBs) in the second decade of life and a second smaller peak in almost always carry mutations in H3F3A at codon 34, older adults [1, 2]. whereas chondroblastomas are characterized by a high Te etiology of osteosarcomas is still largely unknown. frequency of H3F3B mutations at codon 36 [18–20]. Lit- However, several genetic syndromes (e.g. Li-Fraumeni tle is known about H3.3 mutations in osteosarcomas. and hereditary retinoblastoma) greatly increase the Previous studies described single high-grade osteosarco- risk of developing osteosarcomas, especially in younger mas carrying H3.3 mutations, whereas other series did patients [3, 4]. Osteosarcomas in older patients often not. So the question arises whether osteosarcomas carry- develop from a precursor bone lesion that is prone for ing H3.3 mutations really exist or might rather be malig- malignant transformation (i.e. Paget disease) or may nant variants of GCTBs [8, 18–21]. occur after radiation treatment for other diseases, typi- Te present study addresses these issues by determin- cally after a latency period of several years [1, 5, 6]. ing the frequency of H3.3 mutations across the entire Te genomic landscape of osteosarcomas is com- age spectrum in de novo high-grade osteosarcomas of plex [7]. A remarkable attribute of primary and second- the conventional type. Genome-wide DNA methylation ary conventional osteosarcomas is their chromosomal and DNA copy number profling was applied to gain fur- instability leading to high aneuploidy [8]. Chromosomal ther molecular insight into osteosarcomas carrying H3.3 regions frequently afected by somatic structural vari- mutations. ations and copy number alterations often contain cell- cycle regulating genes such as TP53, RB1, or c-MYC [9, Methods 10]. Sequencing studies revealed recurrent mutations Tissues specimens afecting these genes, supporting their major role in Tissue samples of 106 de novo high-grade osteosarcomas osteosarcoma development [8]. However, the mutational of the conventional type (86 primary tumors, 3 recur- make-up of osteosarcomas is versatile, as some osteosar- rences, 17 metastases) and 14 GCTBs, one of them with comas also present with molecular signatures reminis- signs of malignancy in the local relapse two years after cent of BRCA1/2 defcient tumors [10]. Chromothripsis, surgical treatment, serving as H3.3 G34 mutant bone a single cellular catastrophic event resulting in extensive tumor control group were collected from the Institutes DNA rearrangements and DNA copy number changes, of Pathology of the University Hospital Heidelberg, has been observed in roughly one-third of osteosarcomas Cologne, Jena (Germany), and Nijemegen (the Neth- [11]. erlands). Patients’ characteristics are outlined in Addi- Alternative lengthening of telomeres (ALT), a tel- tional fle 1: Table S1. Tissue samples were banked in omere maintaining mechanism compensating progres- the archives of the Department of Pathology and admin- sive telomere attrition by homologous recombination istered by the tissue bank of the National Center for of telomere sequences, is associated with chromosomal Tumor Diseases (NCT), Heidelberg. Initial diagnoses instability in osteosarcomas [12]. Almost all osteosarco- were established by interdisciplinary oncology boards mas utilize ALT, highlighting its particular importance and were based on radiological fndings, standard histo- in these tumors [8, 13]. Somatic mutations in the H3.3- pathological criteria in conjunction with immunohisto- ATRX-DAXX chromatin-remodeling pathway have been chemical and molecular analyses according to the current linked to ALT in diferent tumor entities [14, 15]. ATRX WHO classifcation [1]. H3.3 mutant osteosarcomas were Koelsche et al. Clin Sarcoma Res (2017) 7:9 Page 3 of 11 correlated with clinical information and were histologi- Copy number profles were generated from the 450 k cally and radiologically re-evaluated where available. Te raw data using the ‘conumee’ R package and assessed local ethics committee approved these analyses. manually. Te dmpFinder method of the R package minf was used for the identifcation of diferentially methyl- DNA extraction and Sanger sequencing of H3F3A/B ated regions [22]. Both packages are available from the mutational hotspots Bioconductor repository (http://www.bioconductor.org). Source material for DNA extraction was formalin-fxed and parafn-embedded (FFPE) tumor tissue. Representa- Statistical analysis tive tumor tissue with highest available tumor content A two-sided t test was used to test for diferences was chosen for genomic DNA isolation, as microscopi- between the mean values for continuous variables. P val- cally documented on H&E stained parafn sections. ues of less than 0.05 were considered signifcant.
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