Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions

Abstract

Angiosarcoma (AS) is a distinct group of sarcomas characterized by upregulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, TEK, and FLT1. In keeping with the clinical heterogeneity, gene-expression profiling distinguishes two AS genomic clusters, which correlate with anatomical location and prior exposure to radiation. Furthermore, a high percentage of secondary AS, but not primary AS, shows distinct 8q24 chromosomal gains, due to MYC amplification. In this study, we mined the transcriptional output of 10 secondary and 11 primary AS to better define the dichotomy in the pathogenesis of these two clinical subsets. The oncogenic role of MYC was investigated further in secondary AS as well as in radiation-induced atypical vascular lesions (AVL) and other radiation-associated sarcomas. High-level MYC amplification was found in 100% of secondary AS, but in none of the AVL or other radiation-associated sarcomas. Coamplification of FLT4 (encoding VEGFR3) was identified in 25% of secondary AS, but not in other types. Our findings reinforce the distinct pathogenesis of AS subtypes, with MYC amplification being an early, but necessary event in secondary AS. Secondary genetic hits, such as FLT4 gene coamplification or KDR mutations, may play a role in tumor progression as well as potential therapeutic targeting.

Fig. 1

MYC and TPD52 mRNA expression levels were upregulated in secondary compared to primary AS tumors. MYC downstream genes, ODC1 and DKC1 were also upregulated in secondary AS.

Fig. 2

Atypical vascular lesion (AVL) microscopic appearance with dilated channels lined by hobnailed endothelium infiltrating dermis (A) and showing immunoreactivity for D2-40 (B). D2-40 expression is also seen in a subset of AS, occasionally with a strong pattern staining, as seen in this scalp AS, associated with brisk lymphoid infiltrate (C) or in a spleen AS (D). FISH analysis showed no abnormalities of MYC or FLT4 by FISH in the AVL (E), however the adjacent RT-induced AS diagnosed synchronously showed evidence of MYC amplification (F).

Fig 3

FISH analysis showing evidence of high level MYC amplification in an RT-associated AS carrying a KDR exon 15 mutation (A); co-amplification of MYC and FLT4 in a RT-associated AS; in contrast no evidence of copy number changes of MYC or FLT4 were noted in primary AS (C) or in RT-induced sarcoma (without an angiosarcoma morphology)(D).

Fig. 4

MYC isoforms in AS tumors. Western Blotting with anti-MYC antibody demonstrated that MYC-II (62KDa) expression was abundant in secondary AS tumors (RT-BR, post-radiation breast; PL, post-lymphedema), SKBR3 and MYC-transfected NIH3T3 cells, while not detected in primary AS (BR, primary breast AS). MYC-I (67KDa) was expressed in AS tumors, regardless to RT-exposure, but not in the tested cell lines (SKBR3 and MYC-transfected NIH3T3).

Fig. 5

MYC/MAX heterodimers were detected in secondary AS tumors (RT-BR, post-radiation breast; PL, post-lymphedema), MYC amplified SKBR3 and MYC-transfected NIH3T3 cells, but not in primary AS (BR, primary breast AS) and GIST cell line. AS tumor lysates were co-IP with anti-MAX antibody and the immunoprecipitates were detected with anti-MYC and anti-MAX antibodies separately. MAX was detected in all tested samples.