A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites

Abstract

The classification of epithelioid vascular tumors remains challenging, as there is considerable morphological overlap between tumor subtypes, across the spectrum from benign to malignant categories. A t(1;3)(p36.3;q25) translocation was reported in two cases of epithelioid hemangioendothelioma (EHE), however, no follow-up studies have been performed to identify the gene fusion or to assess its prevalence in a larger cohort of patients. We undertook a systematic molecular analysis of 17 EHE, characterized by classic morphological and immunophenotypic features, from various anatomical locations and with different malignant potential. For comparison, we analyzed 13 epithelioid hemangiomas, five epithelioid angiosarcomas, and four epithelioid sarcoma-like EHE. A fluorescence in situ hybridization (FISH) positional cloning strategy, spanning the cytogenetically defined regions on chromosomes 1p36.3 and 3q25, confirmed rearrangements in two candidate genes from these loci in all EHE cases tested. None of the other benign or malignant epithelioid vascular tumors examined demonstrated these abnormalities. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) confirmed in three EHE the WWTR1-CAMTA1 fusion product. CAMTA1 and WWTR1 have been previously shown to play important roles in oncogenesis. Our results demonstrate the presence of a WWTR1-CAMTA1 fusion in all EHE tested from bone, soft tissue, and visceral location (liver, lung) in keeping with a unique and specific pathological entity. Thus, FISH or RT-PCR analysis for the presence of WWTR1-CAMTA1 fusion may serve as a useful molecular diagnostic tool in challenging diagnoses.

Figure 1

Morphologic appearance of epithelioid vascular tumors analyzed in this study. A. Malignant EHE of the arm, with cords and single cells of epithelioid cells with moderate nuclear atypia, embedded in a hyalinized stroma (EHE#1, x200 magnification); B. Classic EHE of the liver with bland epithelioid cells with readily visible intra-cytoplasmic vacuoles (EHE#16, x 200); C. EH of the penis in a 48 year-old man, who presented as multiple cutaneous and subcutaneous nodules, and showed large epithelioid cells with abundant eosinophilic cytoplasm surrounding vascular lumina (x200); D. Radiation-induced angiosarcoma of breast, composed of predominantly epithelioid morphology and showing high grade cytologic atypia, with prominent nucleoli, as well as vascular channel formation (x200).

Figure 2

FISH positional cloning strategy using BAC probe sets on 1p36.33~1p36.11. Three sets of experiments identified the breakpoint in 1p36.23. Underlined genes have been previously reported in other chromosomal translocations.

Figure 3

Distribution of BAC probe sets tested spanning the 3q24~27 region. Three rounds of FISH experiments illustrated in this diagram were able to narrow down the break-apart region between 3q24~25.1. Underlined genes have been previously reported in other chromosomal translocations.

Figure 4

Identification of candidate genes on 1p36.23 and 3q25 by FISH. A. Grey area showed 1p36.23 breakpoint location within CAMTA1 gene. Three-color FISH showed a break-apart between green-RP11-1120I14 and Orange-RP11-338N10 (inset). B. Two-color FISH (orange-RP11-2G17 and Red-580-RP11-255N4) identified a split red signal associated with the orange signal (inset). This pattern narrowed the breakpoint at chr.3: 149270000-149360000 (hg.19), which localized in WWTR1 exon 4 to exon 8 (ENST00000465804) (Scale was downloaded from UCSC genome browser for accuracy consideration).

Figure 5

RT-PCR detection of WWTR1-CAMTA1 fusion transcript variants and FISH demonstration of fused CAMTA1 and WWTR1 signals. A. Gel electrophoresis showing amplified products in lanes 1–3, of two distinct sizes (M, size marker, lane 1, EHE#4, lane 2, EHE#6, lane 3, EHE#1, lane 4, negative control no-RNA). B. Sequencing of the 3 amplicons identified two molecular variants, with exon 4 of WWTR1 being fused in-frame to either exon 8 (variant 1, upper panel) or exon 9 (variant 2, lower panel) of CAMTA1. C. FISH demonstration of fused signals, using probes centromeric to CAMTA1 and telomeric to WWTR1.