Recurrent CIC Gene Abnormalities in Angiosarcomas: A Molecular Study of 120 Cases With Concurrent Investigation of PLCG1, KDR, MYC, and FLT4 Gene Alterations

Abstract

Angiosarcoma (AS) is a rare sarcoma subtype showing considerable clinicopathologic and genetic heterogeneity. Most radiation-induced AS show MYC gene amplifications, with a subset of cases harboring KDR, PTPRB, and PLCG1 mutations. Despite recent advances, the genetic abnormalities of most primary AS remain undefined. Whole-transcriptome sequencing was initiated in 2 index cases of primary soft tissue AS with epithelioid morphology occurring in young adults for novel gene discovery. The candidate abnormalities were validated and then screened by targeted sequencing and fluorescence in situ hybridization in a large cohort of 120 well-characterized AS cases. Findings were subsequently correlated with the status of KDR, PLCG1, MYC, and FLT4 gene abnormalities. The clinicopathologic relevance and prognostic significance of these genetic changes were analyzed by statistical methods. Concurrent CIC mutations and CIC rearrangements were identified in both index cases, with a CIC-LEUTX fusion detected in 1 case. Upon screening, an additional visceral AS in a young adult had a complex CIC rearrangement, whereas 6 others harbored only CIC mutations. All 3 CIC-rearranged AS cases lacked vasoformation and had a solid growth of round, epithelioid to rhabdoid cells, showing immunoreactivity for CD31 and Ets-related gene and sharing a transcriptional signature with other round cell sarcomas, including CIC-rearranged tumors. Overall, CIC abnormalities occurred in 9% (9/98) of cases, affecting younger patients with primary AS, with an inferior disease-free survival. In contrast, PLCG1 and KDR mutations occurred in both primary and secondary AS cases, accounting for 9.5% and 7%, respectively, with a predilection for breast and bone/viscera location, regardless of MYC status. MYC amplification was present in most secondary AS related to breast cancer (91%) compared with other causes (25%) or primary AS (7%). FLT4-amplified AS lacked PLCG1/KDR mutations, occurring predominantly in MYC-amplified population, and showed poor prognosis.

Figure 1. CIC gene alterations in AS

(A) The schematic diagram of chromosome 19q13 locus, encompassing CIC, LEUTX, and PEPD genes (arrows, direction of transcription; color dots indicate the BAC location used for 3-color FISH). (B) RT-PCR confirmed the chimeric CIC exon 20–LEUTX exon 3 transcript. (C) 3-color FISH confirmed the CIC and LEUTX break-apart with a complex and heterogeneous pattern in AS1: 3 different cells showing a variable pattern of rearrangement, with smaller and bigger gaps between the 3 BACs used (O, orange, CIC centromeric; G, green, CIC telomeric, R, red, PEPD; arrows indicate possible breaks) (D) CIC intra-chromosomal rearrangement in AS2 by FISH, with consistent gaps between red, orange and green BAC probes. Spectrum of CIC mutations in AS including heterozygous exon 15 mutations (p.G1190R, AS1)(E); synchronous exon 19 mutations (p.S1461F and p.R1465S, AS6)(F), and homozygous exon 19 mutations (p.Y1460H, AS7)(G).

Figure 2. Pathologic features of CIC-rearranged AS

Solid sheets of monotonous epithelioid cells, with moderate to abundant eosinophilic cytoplasm, and vesicular nuclei (A,B; AS1). Small epithelioid to round cells with scant, light eosinophilic cytoplasm and vesicular nuclei with inconspicuous nucleoli (C; AS2). Distinctive rhabdoid cells arranged in vague pseudoalveolar and nested pattern (D; AS3) All 3 cases expressed diffuse and strong CD31 (E) and ERG (F) immunoreactivity.

Figure 3. The morphologic spectrum of CIC-mutated AS

A solid growth is noted in AS6 harboring double CIC mutations (A) and AS7 bearing CIC homozygous mutations (B); pleomorphic tumor cells and inconspicuous vascular formation in AS8 with single CIC mutation (C); well-formed anastomosing vascular channels in AS4 with concurrent CIC and PLCG1 mutations (D).

Figure 4

(A) High ETV1, ETV4, ETV5 overexpression in CIC-rearranged AS (CIC-AS) and SBRCTs, but not in classic AS (AS25, AS32, AS110) (RPKM, Reads Per Kilobase per Million mapped reads). (B) CIC overexpression (160 kDa) by western blotting in AS1-2 and CIC-DUX4-positive SBRCTs (SBRCT1-2), compared to CIC-negative AS (AS25, AS32) (control actin 43 kDa, lower part).