KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors

Abstract

Angiosarcomas (AS) represent a heterogeneous group of malignant vascular tumors occurring not only in different anatomic locations but also in distinct clinical settings, such as radiation or associated chronic lymphedema. Although representing only 1% to 2% of soft tissue sarcomas, vascular sarcomas provide unique insight into the general process of tumor angiogenesis. However, no molecular candidates have been identified to guide a specific therapeutic intervention. By expression profiling, AS show distinct up-regulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, SNRK, TEK, and FLT1. Full sequencing of these five candidate genes identified 10% of patients harboring KDR mutations. A KDR-positive genotype was associated with strong KDR protein expression and was restricted to the breast anatomic site with or without prior exposure to radiation. Transient transfection of KDR mutants into COS-7 cells showed ligand-independent activation of the kinase, which was inhibited by specific KDR inhibitors. These data provide a basis for the activity of vascular endothelial growth factor receptor-directed therapy in the treatment of primary and radiation-induced AS.

Fig. 1

Fig 1A: Heat map of unsupervised clustering of U133A genechip transcripts reveals that the AS tumors formed a tight genomic cluster (yellow branches) distinct from all other soft tissue sarcoma types (red branches). Fig 1B: Further unsupervised clustering of AS samples showed two distinct genomic groups, based on anatomic location and prior exposure to radiation therapy. Cluster 1 (right side) includes all primary breast angiosarcoma (BREAST), visceral (VISC), head and neck (H&N) and 5 of the 6 soft tissue & bone (ST&B) AS. Cluster 2 (left side) includes all radiation-induced breast (BREAST RX) and post-lymphedema AS.

Fig 2

Morphologic appearance and KDR expression of AS carrying KDR mutations: A. Post-radiation AS of breast with a KDR kinase domain A1065T mutation, showing a conventional vasoformative growth and high grade cytology (H&E, 200x); B. High grade post-radiation breast AS, spindle cell type, harboring a T717V KDR mutation (H&E, 200x); all above tumors showing strong and diffuse KDR immunoreactivity (C), while the KDR immunofluorescence highlights the membranous staining pattern (D).

Fig 3

KDR^D717V and KDR^A1056T showed constitutive auto-phosphorylation on tyrosine in the absence of serum and rhVEGF for 6 hours, whereas wild type KDR was only phosphorylated after rhVEGF stimulation.

Fig. 4

Sensitivity of KDR^D717V, KDR^A1056T and wild-type KDR transfected COS-7 cells with sunitinib and sorafenib exposure for 90 minutes in the absence of serum and growth factors (0, 0.5 and 1 μM). Both mutants showed decreased phosphorylation at 0.5 μM, while kinase activity was abrogated at 1μM of either drug.