Supplementary information. Supplementary Table S1. Antibodies used in the study. Supplementary Table S2. Primer sequences for Sanger sequencing and site-directed mutagenesis. Supplementary Table S3. SNP array analysis of 8 primary cardiac angiosarcomas. Supplementary Table S4. Mutations detected by tNGS of six primary cardiac angiosarcomas. Supplementary Table S5. Replacement and nonsense mutations detected by tNGS of six primary cardiac angiosarcomas. Supplementary Table S6. The PLCγ1-R707Q mutation increases migration and invasiveness of HUVECs. Supplementary Figure S1. Transfection efficiency with an EGFP encoding control plasmid. Supplementary Figure S2. Cell cycle distribution in HUVECs transfected with PLCγ1-R707Q, PLCγ1-WT and a control.
ARTICLE ABSTRACTPrimary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca2+-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca2+-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies. Cancer Res; 74(21); 6173–83. ©2014 AACR.