Recurrent PTPRB and PLCG1 mutations in angiosarcoma

Journal name:
Nature Genetics
Volume:
46,
Pages:
376–379
Year published:
DOI:
doi:10.1038/ng.2921
Received
Accepted
Published online

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.

At a glance

Figures

  1. Distribution of alterations in PTPRB.
    Figure 1: Distribution of alterations in PTPRB.

    Each symbol represents a mutation. Red, truncating mutations; blue, missense mutations.

  2. Sensitivity of PTPRB-driven angiogenesis to VEGF inhibition.
    Figure 2: Sensitivity of PTPRB-driven angiogenesis to VEGF inhibition.

    (a) HUVEC spheroids embedded in a fibrin gel were photographed after 24 h of treatment. Scale bar, 0.2 mm (10× magnification). (b) Quantification of spheroid sprouting area. Error bars, 1 s.d.; 5,000 cells were counted for each treatment condition. *P < 0.0001.

  3. Driver variants in angiosarcoma.
    Figure 3: Driver variants in angiosarcoma.

    Likely driver variants are indicated by colored squares. Truncating variants include nonsense variants, essential splice-site variants and frameshift indels. Secondary angiosarcomas are either clinically classified as secondary or are unclassified with MYC amplification.

  4. Genome-wide structural rearrangements.
    Supplementary Fig. 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.

  5. Mutation frequency in tumor suppressor genes.
    Supplementary Fig. 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).

  6. Assessment of PTPRB silencing.
    Supplementary Fig. 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.

  7. Activation of PLC[gamma] enzymes by loss of autoinhibition.
    Supplementary Fig. 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).

References

  1. Young, R.J., Brown, N.J., Reed, M.W., Hughes, D. & Woll, P.J. Angiosarcoma. Lancet Oncol. 11, 983991 (2010).
  2. Fachinger, G., Deutsch, U. & Risau, W. Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2. Oncogene 18, 59485953 (1999).
  3. Winderlich, M. et al. VE-PTP controls blood vessel development by balancing Tie-2 activity. J. Cell Biol. 185, 657671 (2009).
  4. Bamford, S. et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br. J. Cancer 91, 355358 (2004).
  5. Guo, T. et al. Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions. Genes Chromosom. Cancer 50, 2533 (2011).
  6. Dominguez, M.G. et al. Vascular endothelial tyrosine phosphatase (VE-PTP)-null mice undergo vasculogenesis but die embryonically because of defects in angiogenesis. Proc. Natl. Acad. Sci. USA 104, 32433248 (2007).
  7. Bäumer, S. et al. Vascular endothelial cell–specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development. Blood 107, 47544762 (2006).
  8. Broermann, A. et al. Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo. J. Exp. Med. 208, 23932401 (2011).
  9. Carra, S. et al. Ve-ptp modulates vascular integrity by promoting adherens junction maturation. PLoS ONE 7, e51245 (2012).
  10. Hayashi, M. et al. VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation. Nat. Commun. 4, 1672 (2013).
  11. Li, Z. et al. Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis. Proc. Natl. Acad. Sci. USA 106, 2239922404 (2009).
  12. Mellberg, S. et al. Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis. FASEB J. 23, 14901502 (2009).
  13. Nawroth, R. et al. VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts. EMBO J. 21, 48854895 (2002).
  14. Nottebaum, A.F. et al. VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF. J. Exp. Med. 205, 29292945 (2008).
  15. Saharinen, P., Eklund, L., Pulkki, K., Bono, P. & Alitalo, K. VEGF and angiopoietin signaling in tumor angiogenesis and metastasis. Trends Mol. Med. 17, 347362 (2011).
  16. Zhou, Q. et al. A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am. J. Hum. Genet. 91, 713720 (2012).
  17. Everett, K.L. et al. Characterization of phospholipase Cγ enzymes with gain-of-function mutations. J. Biol. Chem. 284, 2308323093 (2009).
  18. Ombrello, M.J. et al. Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions. N. Engl. J. Med. 366, 330338 (2012).
  19. Bunney, T.D. et al. Structural and functional integration of the PLCγ interaction domains critical for regulatory mechanisms and signaling deregulation. Structure 20, 20622075 (2012).
  20. Covassin, L.D. et al. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development. Dev. Biol. 329, 212226 (2009).
  21. Lawson, N.D., Mugford, J.W., Diamond, B.A. & Weinstein, B.M. Phospholipase Cγ-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev. 17, 13461351 (2003).
  22. Liao, H.J. et al. Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice. J. Biol. Chem. 277, 93359341 (2002).
  23. Antonescu, C.R. et al. KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors. Cancer Res. 69, 71757179 (2009).
  24. Behjati, S. et al. Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone. Nat. Genet. 45, 14791482 (2013).
  25. Tarpey, P.S. et al. Frequent mutation of the major cartilage collagen gene COL2A1 in chondrosarcoma. Nat. Genet. 45, 923926 (2013).
  26. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589595 (2010).
  27. Ye, K., Schulz, M.H., Long, Q., Apweiler, R. & Ning, Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25, 28652871 (2009).
  28. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 11051111 (2009).
  29. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511515 (2010).
  30. Van Loo, P. et al. Allele-specific copy number analysis of tumors. Proc. Natl. Acad. Sci. USA 107, 1691016915 (2010).
  31. Greenman, C., Wooster, R., Futreal, P.A., Stratton, M.R. & Easton, D.F. Statistical analysis of pathogenicity of somatic mutations in cancer. Genetics 173, 21872198 (2006).

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Author information

  1. These authors contributed equally to this work.

    • Sam Behjati,
    • Patrick S Tarpey &
    • Helen Sheldon

Affiliations

  1. Cancer Genome Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK.

    • Sam Behjati,
    • Patrick S Tarpey,
    • Inigo Martincorena,
    • Peter Van Loo,
    • Gunes Gundem,
    • David C Wedge,
    • Manasa Ramakrishna,
    • Susanna L Cooke,
    • Hans Kristian M Vollan,
    • Elli Papaemmanuil,
    • Claire Hardy,
    • Olivia R Joseph,
    • Sancha Martin,
    • Laura Mudie,
    • Adam Butler,
    • Jon W Teague,
    • Ultan McDermott,
    • Michael R Stratton,
    • P Andrew Futreal &
    • Peter J Campbell
  2. Department of Paediatrics, University of Cambridge, Cambridge, UK.

    • Sam Behjati
  3. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

    • Helen Sheldon &
    • Adrian Harris
  4. Human Genome Laboratory, Department of Human Genetics, VIB and KU Leuven, Leuven, Belgium.

    • Peter Van Loo
  5. Histopathology, Royal National Orthopaedic Hospital National Health Service (NHS) Trust, Stanmore, UK.

    • Nischalan Pillay,
    • Bhavisha Khatri,
    • Dina Halai,
    • Roberto Tirabosco,
    • M Fernanda Amary &
    • Adrienne M Flanagan
  6. University College London Cancer Institute, London, UK.

    • Nischalan Pillay,
    • Chris Boshoff &
    • Adrienne M Flanagan
  7. Department of Oncology, Oslo University Hospital, Oslo, Norway.

    • Hans Kristian M Vollan
  8. KG Jebsen Center for Breast Cancer Research, University of Oslo, Oslo, Norway.

    • Hans Kristian M Vollan
  9. Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK.

    • Hans Koss,
    • Tom D Bunney &
    • Matilda Katan
  10. Division of Molecular Structure, Medical Research Council (MRC) National Institute for Medical Research, London, UK.

    • Hans Koss
  11. Department of Pathology, John Radcliffe Hospital, Oxford, UK.

    • Meena Patil,
    • Graham Steers,
    • Ioannis Roxanis &
    • Adrian Harris
  12. Department of Genomic Medicine, MD Anderson Cancer Center, University of Texas, Houston, Texas, USA.

    • Yu Cao,
    • Curtis Gumbs,
    • Davis Ingram,
    • Alexander J Lazar,
    • Latasha Little,
    • Harshad Mahadeshwar,
    • Alexei Protopopov,
    • Ghadah A Al Sannaa,
    • Sahil Seth,
    • Xingzhi Song,
    • Jiabin Tang,
    • Jianhua Zhang,
    • Vinod Ravi,
    • Keila E Torres &
    • P Andrew Futreal
  13. Bone Tumour Reference Centre, Institute of Pathology, University Hospital Basel, Institute for Applied Cancer Science, Basel, Switzerland.

    • Daniel Baumhoer
  14. Pfizer Oncology, La Jolla, California, USA.

    • Chris Boshoff
  15. Department of Haematology, Addenbrooke's Hospital, Cambridge, UK.

    • Peter J Campbell
  16. Department of Haematology, University of Cambridge, Cambridge, UK.

    • Peter J Campbell

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.

Competing financial interests

C.B. is an employee of Pfizer.

Corresponding author

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Genome-wide structural rearrangements. (54 KB)

    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.

  2. Supplementary Figure 2: Mutation frequency in tumor suppressor genes. (142 KB)

    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).

  3. Supplementary Figure 3: Assessment of PTPRB silencing. (97 KB)

    (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.

  4. Supplementary Figure 4: Activation of PLCγ enzymes by loss of autoinhibition. (137 KB)

    (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).

PDF files

  1. Supplementary Text and Figures (3,690 KB)

    Supplementary Figures 1–4 and Supplementary Table 6

Excel files

  1. Supplementary Table 1 (74 KB)

    Clinical features of cases included in this study with likely driver mutations.

  2. Supplementary Table 2 (90 KB)

    Coding mutations in 11 angiosarcomas subjected to whole-genome or whole-exome sequencing.

  3. Supplementary Table 3 (1,164 KB)

    Substitutions and indels identified in three angiosarcoma genomes.

  4. Supplementary Table 4 (107 KB)

    Rearrangements identified in three angiosarcoma genomes.

  5. Supplementary Table 5 (17 KB)

    Amplification and homozygous deletions identified in three angiosarcoma genomes.

  6. Supplementary Table 7 (71 KB)

    Cancer genes screened by targeted sequencing.

Additional data