Abstract
Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema1. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma1. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.
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Acknowledgements
We thank M. Taylor and R. Leek for sample preparation. We are grateful to the patients for participating in the research and to the clinicians and support staff involved in their care from the Oxford University Hospitals NHS Trust, the University of Texas MD Anderson Cancer Center Sarcoma Program and the London Sarcoma Service. This work was supported by funding from the Wellcome Trust (grant reference 077012/Z/05/Z). The material was obtained from the Royal National Orthopaedic Hospital Musculoskeletal Research Program and Biobank and from the Oxford Radcliffe Biobank. Support was provided to A.M.F. by the National Institute for Health Research, the University College London Hospital Biomedical Research Centre and the Cancer Research UK University College London Experimental Cancer Medicine Centre. Support was provided to A.H. by Cancer Research UK, the Oxford Biomedical Research Centre and the Breast Cancer Research Foundation. P.J.C. is personally funded through a Wellcome Trust Senior Clinical Research Fellowship (grant reference WT088340MA). P.V.L. is a postdoctoral researcher of the Research Foundation–Flanders (FWO). H.K.M.V. is supported by the Norwegian Radium Hospital's Foundation. S.B. is funded through the Wellcome Trust PhD Programme for Clinicians. P.A.F. is supported by the Cancer Prevention Research Institute of Texas and the Welch Foundation.
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Contributions
S.B. and P.S.T. performed analyses of sequencing data. H.S. performed in vitro experiments. P.V.L. performed copy number analysis. D.C.W. and I.M. performed statistical analyses. S.L.C. performed rearrangement analysis. G.G., N.P., M.R., H.K.M.V. and E.P. contributed to data analysis. H.K., T.D.B. and M.K. contributed structural analyses. C.H., O.R.J., L.M., H.M., A.P., J.T., L.L., Y.C. and C.G. coordinated sample processing and technical investigations. S.M. coordinated sample acquisition. A.B., J.W.T., S.S., X.S. and J.Z. coordinated informatics analyses. B.K., D.H., D.B., M.P., G.S., I.R., R.T., M.F.A., A.M.F., C.B., V.R., K.E.T., D.I., A.J.L., G.A.A.S. and A.H. provided samples and clinical data. P.J.C., M.R.S., A.H., P.A.F., U.M. and A.M.F. directed the research. M.R.S., P.J.C., S.B. and P.S.T. wrote the manuscript, with contributions from A.H., A.M.F. and P.A.F.
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Integrated supplementary information
Supplementary Figure 1 Genome-wide structural rearrangements.
CIRCOS plots representing rearrangement in the three genomes studied at the whole-genome level. Colors represent rearrangement class: orange, inversion type (tail to tail); blue, inversion type (head to head); green, deletion type; black, tandem duplication type; purple, interchromosomal.
Supplementary Figure 2 Mutation frequency in tumor suppressor genes.
Publicly available catalogs of somatic mutations in cancers (n = 4,073) were analyzed for the frequency of 2 mutations (point mutations or LOH) in established tumor suppressor genes. For every gene, we selected all those samples in which the gene had a truncating mutation (nonsense, essential splice site or out-of-frame indel) and quantified the frequency of a second mutation, truncating, LOH, missense or in-frame indel, in the gene. Error bars represent the 95% confidence intervals of the total fraction of two-hit samples (using a Chi-square approximation, as implemented in the function prop.test in R (version 3.0.1).
Supplementary Figure 3 Assessment of PTPRB silencing.
(A) PTPRB levels after silencing demonstrating ~80% reduction in PTPRP transcript levels. (B) Following PTPRB silencing, VE-cadherin expression is greatly reduced. (C) PTPRB silencing increases the phosphorylation levels of VEGFR2.
Supplementary Figure 4 Activation of PLCγ enzymes by loss of autoinhibition.
(A) Diagram showing the domains in PLCγ. The regulatory region (comprising the spPH, nSH2, cSH2 and SH3 domains; amino acids 465–952 in PLCγ1) is inserted in a loop that connects two halves (X and Y boxes) of the catalytic domain (purple). The cSH2 domain (red), involved in autoinhibition, is in direct contact with the catalytic domain. Disruption of the cSH2 domain by deletion or point mutation can cause constitutive PLCγ activation. The R707Q alteration in the cSH2 domain is indicated in blue. Residue numbering is per human PLCγ1. (B) Structure of the PLCγ1 cSH2 domain (PDB 4EY0). The interface involved in autoinhibition and residue R707 are shown. (C) Possible impact of R707Q alterations on the PLCγ1 cSH2 domain. R707 is involved in interactions with several residues (left, doted lines) that are likely to be important for domain stability and conformational changes. These interactions are disrupted when R707 is replaced by glutamine (right).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 and Supplementary Table 6 (PDF 3604 kb)
Supplementary Table 1
Clinical features of cases included in this study with likely driver mutations. (XLSX 72 kb)
Supplementary Table 2
Coding mutations in 11 angiosarcomas subjected to whole-genome or whole-exome sequencing. (XLSX 88 kb)
Supplementary Table 3
Substitutions and indels identified in three angiosarcoma genomes. (XLSX 1137 kb)
Supplementary Table 4
Rearrangements identified in three angiosarcoma genomes. (XLSX 105 kb)
Supplementary Table 5
Amplification and homozygous deletions identified in three angiosarcoma genomes. (XLSX 17122 kb)
Supplementary Table 7
Cancer genes screened by targeted sequencing. (XLSX 69 kb)
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Behjati, S., Tarpey, P., Sheldon, H. et al. Recurrent PTPRB and PLCG1 mutations in angiosarcoma. Nat Genet 46, 376–379 (2014). https://doi.org/10.1038/ng.2921
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DOI: https://doi.org/10.1038/ng.2921
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