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Integrating transcriptomic sequencing with accepted cytogenetics, we articular WWTR1 (WW domain–containing archetype regulator 1) (3q25) and CAMTA1 (calmodulin-binding archetype activator 1) (1p36) as the two genes circuitous in the t(1;3)(p36;q25) chromosomal about-face that is appropriate of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. This WWTR1/CAMTA1 gene admixture is beneath the transcriptional ascendancy of the WWTR1 apostle and encodes a accepted chimeric archetype agency that joins the amino aals of WWTR1, a protein that is awful bidding in endothelial cells, in-frame to the carboxyl aals of CAMTA1, a protein that is frequently bidding alone in brain. Thus, CAMTA1 announcement is activated afield through a promoter-switch mechanism. The gene admixture is present in around all EHEs activated but is absent from all added vascular neoplasms, demonstrating it to be a disease-defining abiogenetic alteration. A acute and specific break-apart fluorescence in situ admixture appraisal was additionally developed to ascertain the about-face and will abetment in the appraisal of this diagnostically arduous neoplasm. The chimeric WWTR1/CAMTA1 archetype agency may represent a ameliorative ambition for EHE and offers the befalling to afford ablaze on the functions of two ailing characterized proteins.

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Little is accepted about the appraisal of epithelioid hemangioendothelioma (EHE), an cryptic vascular (endothelial cell) bump that was aboriginal declared about afresh (1). EHE occurs over a advanced age range, affects both es equally, and can arise in bendable tissue, bone, and belly organs, in accurate alarmist and lungs. The mainstay of appraisal for localized ache is surgical resection. However, EHE that occurs in alarmist and lungs artlessly presents with multifocal ache and is a cogent annual of anguish and mortality, because transplantation is generally the alone appraisal advantage available. Furthermore, no appraisal options abide for patients with metastatic EHE, accent the charge to bigger accept the pathogenesis of this neoplasm.

In general, sarcomas are classified according to their bandage of adverse (that is, their affinity to normal, nonneoplastic tissues). EHE is classified as a vascular bump because the neoplastic beef accept characteristics in accepted with normal, nonneoplastic endothelial cells. Vascular adverse in EHE is difficult to analyze histologically, because the neoplastic beef do not adapt themselves into admirable claret vessels. Instead, EHE is characterized by a admeasurement of annular (epithelioid) beef that about anatomy cord-like structures anchored aural an edematous, proteoglycan-rich extracellular cast (Fig. 1A). Endothelial corpuscle adverse is accurate alone by the announcement of CD31 [also alleged platelet endothelial corpuscle adherence atom (PECAM-1)] and CD34 (a corpuscle apparent glycoprotein circuitous in cell-cell adhesion), which are additionally bidding by accustomed endothelial beef (Fig. 1B), as able-bodied as by ultrastructural appearance admiring of endothelial adverse (2). Consequently, EHE is arduous to analyze and was accustomed as a audible article alone aural the aftermost 30 years, a artefact of accurate histological appraisal and advancements fabricated in immunohistochemistry. The seminal cardboard aboriginal anecdotic EHE as a audible blight emphasized its adeptness to actor blight histologically, which is a abounding analytic pitfall (1).

Morphological, immunohistochemical, and cytogenetic appearance of EHE: Characterization of RNA/cDNA breakpoints and anatomy of chimeric protein. (A) Hematoxylin and eosin (H&E) area of EHE shows a admeasurement of the annular EHE beef abiding in appropriate cords of cells. The beef are amidst by a appropriate edematous proteoglycan-rich matrix. (B) CD31 announcement in EHE, as apparent by immunohistochemistry performed with the avidin-biotin circuitous (ABC) method, highlights alone neoplastic EHE cells. (C) A fractional karyotype shows the t(1;3)(p36;q25) about-face in EHE1. The pairs of chromosomes are abounding as “1” and “3.” The acquired chromosomes are amid on the appropriate ancillary of anniversary pair, with an arrow pointing to the translocated chromosomal fragment. (D) Schematic representations of blazon 1 and 2 admixture transcripts. The numbers announce exons. (E) Diagram that shows the assorted RNA/cDNA breakpoints in EHE1 to EHE5 aural exon 9 of CAMTA1. (F) RNA/cDNA breakpoints of EHE1 to EHE5, assuming CAMTA1 alloyed in-frame to WWTR1 (for EHE4, WW, WWTR1; CAM, CAMTA1). (G) Schematic representation of RNA in situ hybridization. A red arresting indicates the attendance of the wild-type WWTR1 transcript. A blooming arresting indicates the attendance of the wild-type CAMTA1 transcript. The attendance of a admixture WWTR1/CAMTA1 archetype is adumbrated by a chicken fluorescence signal. (H) Abrogating ascendancy tissue (epithelioid hemangioma) that shows announcement of wild-type WWTR1 alone (red). Note that no wild-type CAMTA1 archetype is present. (I and J) Announcement of the WWTR1/CAMTA1 archetype in EHE5, apparent as a chicken admixture arresting at low (I) and aerial (J) power. (K) Diagram of the accepted admixture protein. WW, WW domain; CC, coiled-coil domain; PDZ, PDZ bounden motif; CG-1, CG-1 DNA bounden domain; TIG, archetype agency immunoglobulin domain; ANK, ankyrin repeats; IQ, IQ calmodulin-binding motifs.

The appearance of immunohistochemistry did not absolutely dness the adversity in diagnosing EHE. Added vascular neoplasms that are histological mimics of EHE, including epithelioid hemangioma (a amiable vascular neoplasm) and epithelioid angiosarcoma (which has a added advancing analytic beforehand than EHE), additionally accurate CD31 and CD34 antigens. To date, no specific biomarker exists for EHE, and appropriate this blight from added epithelioid vascular neoplasms (epithelioid hemangioma and epithelioid angiosarcoma) is based alone on the identification of appropriate histological features. Recently, the attendance of a alternate t(1;3)(p36;q25) translocation—which after-effects in the abbreviate arm of chromosome 1, bandage 3, sub-band 6 (1p36) actuality alloyed to the continued arm of chromosome 3, bandage 2, sub-band 5 (3q25)—was activate in two of three EHEs with arise karyotypes (3, 4); however, the genes circuitous in this about-face accept not been elucidated. Traditional approaches to characterize the genes circuitous in translocations are arduous and time-consuming. Attempts to analyze the genes at the abiogenetic breakpoint in EHE accept been complicated by the cancer’s low cellularity (Fig. 1A), surrounding nonneoplastic tissues, and the absence of accessible EHE corpuscle lines, all of which accomplish a of nucleic acids from a accurate citizenry of neoplastic beef difficult. By base several contempo advances in technology and application a new methodology, we accept articular the genes circuitous in the EHE translocation.

First, we complete a commutual DNA (cDNA) library from absolute RNA abandoned from a ahead karyotyped arctic bump (EHE1) with a accepted t(1;3)(p36;q25) about-face (Fig. 1C and fig. S1A). The cDNA library was deep-sequenced via paired-end whole-transcriptome sequencing (RNA-seq) and analyzed application FusionSeq, a afresh declared algorithm advised to admit gene admixture transcripts (5). A applicant admixture mRNA archetype was articular that absolute exon 2 of WWTR1 (WW domain–containing archetype regulator 1) amid on 3q25 and exon 9 of CAMTA1 (calmodulin-binding archetype activator 1) amid on 1p36, the absolute chromosomal locations articular by classical cytogenetics (see table S1 for a annual of the top 30 rank-ordered admixture archetype candidates generated by FusionSeq).

To validate the abysmal sequencing results, we performed about-face transcription–polymerase alternation acknowledgment (RT-PCR) appraisal on the EHE1 bump application PCR primers in exon 2 of WWTR1 and exon 9 of CAMTA1; the after-effects accepted an in-frame admixture of the end of exon 2 of WWTR1 to a breakpoint aural exon 9 of CAMTA1. We again analyzed by RT-PCR four added arctic bump samples from audible patients (EHE2 to EHE5), two of which (EHE2 and EHE5) had additionally been karyotyped and apparent to accept a t(1;3)(p36;q25) about-face (fig. S1, B and C). All four cases showed one of two accessible abnormal mRNA transcripts generated from admixture of the end of either exon 2 (type 1 admixture transcript) or exon 3 (type 2 admixture transcript) of WWTR1 in-frame to exon 9 of CAMTA1 (Fig. 1, D to F). All bristles fusions resulted in the in-frame announcement of the C aals of CAMTA1 and appropriately an complete admixture protein. Announcement of the WWTR1/CAMTA1 chimeric archetype accurately aural bump beef was accepted by RNA in situ admixture (Fig. 1, G to J). Notably, the admixture archetype was not bidding in adjoining accustomed tissue, which indicates that the gene admixture was a actual event.

WWTR1 is composed of seven exons and encodes a transcriptional coactivator protein that contains a bounden armpit for 14-3-3 signaling proteins (exon 2), a WW (tryptophan-tryptophan) area (which binds to proline-rich peptides) (exons 2 and 3), a coiled-coil protein dimerization area (exons 4 and 5), and a PDZ-binding motif, which is believed to collaborate with the PDZ domains of added proteins (exon 7) (6). CAMTA1 contains 23 exons that encode a transcriptional authoritative protein with a CG-1 area anticipation to bind CG-rich DNA sequences (exons 3 to 7), a archetype agency immunoglobulin (TIG) accepted DNA bounden area (exons 9 to 11), a alternation of ankyrin repeats (which arbitrate protein-protein interactions) (exons 13 to 15), and IQ calmodulin-binding motifs [IQ refers to the aboriginal two amino acids of the motif, isoleucine (I) and glutamine (Q)] (exons 19 and 20) (7). The chimeric protein acquired from the admixture 1 archetype would appropriately accommodate the 14-3-3 bounden armpit and a allocation of the WW area (protein bounden domain) from WWTR1 alloyed to the TIG area (putative DNA bounden domain), ankyrin repeats, and calmodulin-binding domains/IQ domains of CAMTA1 (Fig. 1M). The chimeric protein acquired from the admixture 2 archetype is about the same, but would accommodate the absolute WW area from WWTR1 (Fig. 1K).

Using RT-PCR performed with primers specific for the alternate acclimatization of the admixture transcript, we bootless to ascertain announcement of the alternate CAMTA1/WWTR1 archetype in EHE1, EHE2, and EHE5 (the three EHEs that were activated for this orientation). Added affirmation for announcement of the WWTR1/CAMTA1 archetype was provided by appraisal of the arresting clue of CAMTA1 (the cardinal of paired-end reads that maps to a accustomed exon aural CAMTA1) acquired from cDNA sequencing, which demonstrates that announcement of CAMTA1 began anon 3′ to the breakpoint; these allegation beforehand that CAMTA1 is bidding alone as the WWTR1/CAMTA1 transcript, and not as the alternate CAMTA1/WWTR1 archetype (Fig. 2).

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Differential announcement of CAMTA1 5′ and 3′ to the breakpoint: Affirmation in abutment of the WWTR1/CAMTA1 acclimatization of the admixture transcript. The top console letters the arresting clue (that is, the cardinal of reads that awning anniversary nucleotide position, normalized per actor mapped nucleotides) in exons 7 to 11 of CAMTA1 as able-bodied as the intronic regions. The abject bandage illustratively separates the bidding arresting from the accomplishments noise. The basal console zooms into exon 9 of CAMTA1 and shows the area of the breakpoint aural the exon as adumbrated by the vertical red line. In both panels, the regions that are allotment of the WWTR1/CAMTA1 gene admixture are adumbrated aerial by a accumbent red bar. Exons that are allotment of the WWTR1/CAMTA1 admixture transcript, starting from exon 9 (top panel), accept a college announcement arresting than those that are not allotment of the admixture archetype (for example, exon 8). Close appraisal of exon 9 of CAMTA1 (bottom panel) shows that the announcement arresting is abounding college alpha anon afterwards the breakpoint. These abstracts announce that CAMTA1 is bidding alone as the WWTR1/CAMTA1 archetype and argues adjoin announcement of the alternate CAMTA1/WWTR1 transcript.

Because the arctic EHE bump samples absolute ample amounts of accustomed tissue that had been infiltrated by neoplastic EHE cells, we adopted to use a PCR-based action rather than Southern (DNA) blots to ascertain the abiogenetic breakpoints at the genomic level. Application a about-face PCR album amid aural exon 9 of CAMTA1 and assorted advanced primers present aural introns 2 and 3 of WWTR1 (see Supplementary Material, Annual of primers), we articular all bristles genomic DNA breakpoints (fig. S2) in EHE1 to EHE5 (Fig. 3A). Atypical braid acceptor sites were apparent by these translocations, which resulted in the RNA/cDNA breakpoints declared in Fig. 1F. The genomic admixture armpit was accepted application fluorescence in situ admixture (FISH) imaging with bring-together probes that alloyed to sequences that belted the WWTR1 and CAMTA1 genomic loci (see Materials and Methods). In a accustomed cell, alone abstracted orange and blooming signals are visualized on complete chromosomes. In a corpuscle absolute a translocation, the orange and blooming signals are alloyed (because the two genes are alloyed together), bearing a chicken signal. Formalin-fixed, paraffin-embedded (FFPE) sections of EHE1 to EHE5 were probed and arise admixture of WWTR1 to CAMTA1 in interphase chromosomes aural neoplastic beef (Fig. 3, B and D).

Genomic breakpoints approved by PCR and FISH: Accident of the WWTR1/CAMTA1 gene admixture in vascular neoplasms. (A) Schematic representation of genomic DNA breakpoints in EHE. Ex, exon. (B and C) DNA FISH. Diagrammatic representations of the admixture FISH appraisal (B) and break-apart FISH appraisal (C). (D) DNA FISH admixture appraisal after-effects in neoplastic EHE beef (performed on one EHE sample). The two blooming signals represent WWTR1, which is not circuitous in the translocation. Two blooming signals are present as a aftereffect of aneusomy of chromosome 3 (an added chromosome 3), which is altered to the translocation. The orange arresting represents CAMTA1, which is present on the accustomed chromosome. The chicken arresting (composed of alloyed orange and blooming signals) represents the WWTR1/CAMTA1 admixture at the genomic level. (E and F) Break-apart probes assuming rearrangements of WWTR1 (E) and CAMTA1 (F). Arrows announce the break-apart signals (split orange and blooming signals) in neoplastic EHE cells. A accustomed corpuscle absolute two complete signals (yellow signals) is apparent for allegory in the lower larboard bend of (F). The break-apart appraisal for anniversary gene was performed at atomic already on anniversary case (duplicate cores were evaluated on the tissue microarray). (G) Summary of after-effects for break-apart FISH assays in EHE and added vascular neoplasms. NOS, not contrarily specified.

To actuate the accident of the WWTR1/CAMTA1 about-face in FFPE tissue sections from a accomplice of 47 EHE patients as able-bodied as in a adumbrative alternation of around all added vascular neoplasms/proliferations, we generated dual-color break-apart FISH probes that belted the WWTR1 or CAMTA1 gene regions (Fig. 3, C, E, and F; Materials and Methods). These probes are acclimated in pairs: Back a chromosome is intact, a chicken arresting is observed; back a about-face is present, the two probes are breach apart, and the alone orange and blooming beaming probes can be visualized. The appraisal showed that 39 of 45 EHE tumors (87%) approved barter of the CAMTA1 arena (Fig. 3G and table S2), and 42 of the aforementioned 45 added 2 added EHE bump samples (total = 47) (89%) (Fig. 3G and table S2) harbored a barter of the WWTR1 gene region, advertence a aerial accident (87 to 89%) of the t(1;3)(q36;q25) about-face in EHE. Alone one bump (lung) displayed a barter in WWTR1 gene but not in the CAMTA1 gene. This could represent a noncanonical about-face involving a gene added than CAMTA1, a cryptic about-face involving CAMTA1, or conceivably a acuteness affair with the delving (the CAMTA1 delving was technically added difficult to assignment with than the WWTR1 probe). None of the 118 added vascular neoplasms/proliferations showed barter of WWTR1 or CAMTA1, including epithelioid angiosarcoma and epithelioid hemangioma, the neoplasms that best frequently actor EHE histologically. Back evaluating vascular neoplasms, the absolute predictive amount (PPV) for a WWTR1 gene barter is 100%, the abrogating predictive amount (NPV) is 96%, the acuteness is 89%, and the specificity is 100%. A CAMTA1 barter has a PPV of 100%, a NPV of 95%, acuteness of 87%, and specificity of 100% (table S3).

To activate to analyze the action of the chimeric oncogene, we advised the abstract on the cogwheel announcement of WWTR1 and CAMTA1 in assorted primary endothelial cells. WWTR1 is acerb bidding in kidney, lung, liver, and heart, which are all endothelial cell–rich organs, as able-bodied as in animal umbilical attitude endothelial beef (HUVECs), admitting CAMTA1 is bidding about alone aural academician [Genomics Novartis Foundation 1 (GNF1) Album data, Fig. 4, A and B; GNF2 Album data, fig. S3, A and B] (8–10). We again performed quantitative RT-PCR on several altered types of primary endothelial beef (Materials and Methods) and showed that WWTR1 announcement was 103 to 106 times college than that of CAMTA1, depending on the corpuscle blazon (Fig. 4C). These allegation beforehand that CAMTA1 is aberrantly bidding in EHE through a alleged promoter-switch mechanism, whereby CAMTA1 announcement is apprenticed by the WWTR1 promoter, which is acerb alive in endothelial cells.

Expression of WWTR1 and CAMTA1 in altered tissues, corpuscle lines, and primary endothelial cells. (A) Calefaction maps generated from DNA microarray abstracts appearance WWTR1 mRNA announcement in assorted animal tissues and corpuscle ambit (GNF Announcement Album 1 Animal Abstracts from Affy 95 chips) (8, 9). Note aerial announcement of WWTR1 in HUVECs. (B) Calefaction maps generated from DNA announcement microarray abstracts appearance CAMTA1 mRNA announcement in assorted animal tissues and corpuscle ambit (GNF Announcement Album 1 Animal Abstracts from Affy 95 chips) (8, 9). Note low announcement of CAMTA1 in HUVECs. Announcement of CAMTA1 is about bound to the academician and analgesic cord. (C) Cogwheel announcement of WWTR1 and CAMTA1 mRNAs in several primary endothelial corpuscle ambit (HUVECs, HAECs, PAECs, and DMVECs) as abstinent by qPCR. At atomic two qRT-PCRs were performed evaluating WWTR1 and CAMTA1 announcement in anniversary of the primary endothelial corpuscle types. The RNA/cDNA from anniversary primary endothelial corpuscle blazon was evaluated in leash during anniversary quantitative RT-PCR. The absurdity confined represent 1 SD acquired from the ethics for the leash samples. P ethics were bent with an unpaired two-tailed t test.

Here, we abode the attendance of a atypical WWTR1/CAMTA1 gene admixture that corresponds with t(1;3)(p36;q25) detected in EHE. Our allegation appearance that the break-apart FISH appraisal for WWTR1 and CAMTA1 is a awful acute and specific appraisal for EHE that can be readily activated in the analytic ambience to FFPE tissue, bushing a analytic void. Vascular tumors are the best acceptable substrate for the break-apart appraisal acclimated in analytic practice. However, we apprehend that the break-apart FISH appraisal will additionally be advantageous in attenuate situations in which a nonvascular bump may be in the cogwheel diagnosis, because the t(1;3)(p36;q25) about-face has not been articular in any added bump (11).

Our allegation additionally abode important biological questions accompanying to EHE. Some accept appropriate that a biological continuum exists amid epithelioid angiosarcoma and EHE. However, our allegation altercate otherwise, assuming that EHE and epithelioid angiosarcoma are genetically audible entities, which we ahead will appearance added appraisal of both cancers.

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By anecdotic the WWTR1/CAMTA1 gene admixture in EHE, we can activate to abode the atomic underpinnings of this sarcoma. On the base of what is currently accepted about the two genes, we accept that the WWTR1/CAMTA1 admixture protein functions as a chimeric archetype factor. WWTR1 encodes a transcriptional coactivator that contains several protein bounden domains but no DNA bounden domains. The protein is called afterwards its WW domain, which refers to two conserved tryptophan residues and is accepted to admit added archetype factors with a PPXY burden (for example, RUNX1 and RUNX2). Wwtr1 acts as a adorning about-face in murine mesenchymal axis cells, authoritative whether they affectation an adipocytic or osteogenic phenotype (12), and studies in mice beforehand that the protein plays an important role in the pathogenesis of cystic branch ache (13, 14). WWTR1 is accepted to collaborate with DNA bounden archetype factors, including those of the Runx ancestors in mice and thyroid archetype agency 1 (TTF-1) in humans, which are important in the development of cartilage (Runx2) and lung (TTF-1), appropriately (6, 12). WWTR1 is overexpressed in animal blight (15) and papillary thyroid blight (16).

CAMTA1 encodes a archetype agency that is activate in all multicellular bacilli activated and is evolutionarily conserved from Arabidopsis to humans. Affirmation of CAMTA1’s role as a transcriptional authoritative protein stems from studies performed in Drosophila and Arabidopsis (7). Little is accepted about the protein’s action in beastly cells, but in humans, the gene is bidding about alone aural the academician and has been alive in anamnesis because aerial amounts of CAMTA1 mRNA accept been articular in memory-related regions (17). CAMTA1 has been alive in cancer, primarily because of its area aural chromosome 1p36, one of the best frequently deleted chromosomal regions in animal cancers, including neuroblastoma (18) and oligodendroglioma (19), in which CAMTA1 has been proposed as a applicant bump suppressor. However, there is additionally acute affirmation that CHD5, which encodes a DNA helicase, may be the bump suppressor gene amid at 1p36 (20).

Although studies (18, 19) accept appropriate that CAMTA1 encodes a bump suppressor, several ambit of affirmation announce that the gene is action as an oncogene aural the WWTR1/CAMTA1 fusion. Bristles of the six EHEs that accept been karyotyped [three from the abstract (3, 4) and three from the accepted abstraction (fig. S1, A to C)] anchorage the t(1;3) translocation, usually with few added cytogenetic alterations. These observations beforehand that the about-face is an aboriginal adroit accident in EHE tumorigenesis. Furthermore, sequencing of the RNA breakpoint showed that the C aals of CAMTA1 is alloyed in-frame to WWTR1, arguing adjoin the achievability that the about-face after-effects in a accident of action of CAMTA1 (an accepted award if CAMTA1 is a bump suppressor in the ambience of EHE). Together, these allegation acerb beforehand that aural this context, the C aals of CAMTA1 is behaving as an oncogene.

We accept that at atomic allotment of the action of the WWTR1/CAMTA1 oncogene manifests via a promoter-switch mechanism. Depending on which primary endothelial beef are evaluated, WWTR1 is bidding at abounding (103 to 106 times) college levels than CAMTA1. The admixture accident acceptable after-effects in overexpression of the C aals of the CAMTA1 gene because it is placed beneath the ascendancy of the WWTR1 promoter, which is awful alive in endothelial cells. Furthermore, announcement of any allotment of CAMTA1 is ectopic in endothelial cells, because the gene is usually alone bidding in brain. Together, these allegation beforehand that the admixture gene encodes a protein circuitous in activating a atypical transcriptional program. In accretion to a promoter-switch mechanism, accretion accessible access of oncogenesis includes accident of accepted authoritative domains encoded by the N aals of the CAMTA1 protein (for example, the CG-1 domain) or the C aals of the WWTR1 protein (for example, PDZ bounden motif). We ahead that compassionate the apparatus of WWTR1/CAMTA1 oncogenesis will be active adjoin developing targeted appraisal for EHE, for which none currently exists.

In accretion to address ablaze on the tumorigenesis of EHE, the WWTR1/CAMTA1 gene admixture represents the aboriginal accepted abiogenetic alterations involving either WWTR1 or CAMTA1, anon implicating them in the alpha of a ache process. These two genes arise to be important from a adorning standpoint (6, 7, 12), and our allegation as able-bodied as those of others (15, 16, 18, 19) beforehand that they ability comedy an important role in blight as well. An compassionate of whether and how the admixture gene artefact alters the announcement of added specific genes may crop added insights into the accustomed functions of these two proteins.

Finally, our access of amalgam abysmal sequencing of the transcriptome with accepted cytogenetics represents an important translational beforehand because it can be acclimated to analyze genes circuitous in translocations in added attenuate tumors for which a karyotype is known, but few arctic bump samples or no corpuscle ambit exist. Our appraisal represents a aboriginal footfall adjoin compassionate cancers that are contrarily difficult to study.

Standard cytogenetic techniques were acclimated for chromosome appraisal (21), and karyotypes are declared in accordance with the International Arrangement for Animal Cytogenetic Nomenclature (22).

Annotation for WWTR1 and CAMTA1 is based on the Animal Feb. 2009 (GRCh37/hg19) Assembly accessible on the University of California, Santa Cruz, genome browser (8). The gene accretion cardinal acclimated for WWTR1 is RefSeq NM_015472, which has the afterward genomic coordinates: chr3:149,235,022–149,375,888. The gene accretion cardinal acclimated for CAMTA1 is RefSeq NM_015215, which has the afterward genomic coordinates: chr1:6,845,384–7,829,766.

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RNA sequencing was performed according to the accepted Illumina mRNA paired-end library agreement (Illumina). mRNA was extracted with oligo(dT) alluring chaplet from absolute bump RNA (1 μg) abandoned from EHE1. The mRNA was burst by evolution at 94°C for 5 min in breach absorber (Illumina) and adapted to single-stranded cDNA with SuperScript II about-face transcriptase, which was followed by second-strand cDNA amalgam with Escherichia coli DNA polymerase I. Double-stranded cDNA was end-repaired with T4 DNA polymerase and T4 polynucleotide kinase, monoadenylated with Klenow DNA polymerase I (3′-5′ exonuclease activity), and ligated to adapter sequences with T4 DNA ligase. The adaptor-ligated cDNA library was again fractioned on a 2% agarose gel, and a apply agnate to the 200-nucleotide admeasurement ambit was excised, purified, and PCR-amplified with high-fidelity Phusion DNA polymerase (Finnzymes, Thermo Scientific). The library was quantified with the Agilent 1000 kit on an Agilent 2100 Bioanalyzer according to the manufacturer’s instructions. The cDNA library (a 1- to 4-pM sample) was again loaded assimilate an alone breeze corpuscle lane of an Illumina GAIIx platform, arrangement bearing (clonal accession of distinct cDNA species) was performed, and the sample was sequenced, breeding 36-base reads. A absolute of about 69.1 actor paired-end reads were generated.

All reads were apart accumbent with the ELAND (efficient bounded alignment of nucleotide data) software provided by Illumina. As allotment of the FusionSeq algorithm, a alternation of three modules were afterwards acclimated in allegory the paired-end reads. Module 1: Pairs that mapped to altered genes [according to the University of California, Santa Cruz Accepted Genes comment set (23)] were called as advertence abeyant admixture candidates. Module 2: A alternation of filters were again acclimated to advice annihilate affected admixture archetype candidates, such as those represented by awful agnate paralogous genes, cryptic mapping consistent from repetitive regions, accidental bond of archetype bits generated during sample preparation, genes that resembled awful abounding ribosomal RNA, or PCR artifacts. Module 3: Afterwards anecdotic regions amid two commutual genes wherein the breakpoint acceptable occurred, we complete a fusion-junction library by accoutrement the arena with a capricious cardinal of “tiles” spaced 1–base brace apart; we called the best acceptable alliance arrangement afterwards adjustment the reads adjoin this library. Already we generated a assured annual of admixture candidates (after the Module 2 step), they were ranked according to several statistics: A SPER (supportive paired-end reads) annual was affected on the base of the arrangement amid the cardinal of intertranscript reads of a accurate applicant and the absolute cardinal of mapped paired-end reads. This annual was again compared to two expectations, computed in altered ways: A DASPER (difference amid the empiric and analytically affected accepted SPER) annual was affected based on the likelihood that the two genes in the admixture could accomplish a admixture archetype by accidental chance, accustomed the announcement levels of the corresponding genes. Genes with aerial announcement levels are decumbent to accomplish bogus chimeric transcripts that aftereffect from accidental bond during sample alertness [see (5) for added details]. A college DASPER annual indicates a greater likelihood that the admixture applicant is accurate and did not action randomly. A RESPER (ratio of empirically computed SPERs) annual compared the empiric SPER of a applicant with those of added candidates. This annual takes into annual the altitude of a sequencing-specific run (for example, fragment size). In addition, the applicant fusions were classified in agreement of whether they represented two genes on altered chromosomes (interchromosomal) or two genes on the aforementioned chromosome (intrachromosomal). See (5) for added details.

RT-PCR appraisal of arctic bump samples (RNA/cDNA breakpoints). Arctic primary bump specimens for atomic studies were stored at −80°C afore analysis. Absolute RNA was abandoned from bump samples with TRIzol Reagent (Invitrogen). cDNA was actinic with SuperScript II (Invitrogen) with 1 μg of absolute RNA and 250 ng of accidental primers (Promega). PCR accession was performed with GoTaq DNA polymerase (Promega) and the afterward PCR conditions: 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1.5 min, and a final addendum of 72°C for 10 min. PCR articles were visualized by agarose gel analysis, gel-purified, and submitted for sequencing anon or cloned into a topoisomerase-containing agent with TA overhangs by agency of the TA cloning arrangement (Invitrogen). The PCR accession primers acclimated for anniversary EHE are declared in the Annual of primers (Supplementary Material).

RNA in situ hybridization. Tissue sections from FFPE primary bump specimens were decrepit with the RNAscope FFPE appraisal kit (Advanced Corpuscle Diagnostics) with modifications for the fluorescence apprehension of admixture transcripts. Briefly, 5-μm-thick sections were advised with calefaction and a protease, followed by admixture with a admixture that absolute ambition probes that were commutual to regions aural exon 1 of WWTR1 or exon 9 of CAMTA1. WWTR1- and CAMTA1-specific admixture signals were detected with Alexa 546 or Alexa 488 beaming dyes, respectively. Decrepit tissue sections were visualized with a Zeiss AxioImager M1 microscope and a QuantaMAX QMAX FITC/TRITC clarify set (Omega Optical). Images were captured with the Nuance FX multispectral imaging arrangement (CRi) and candy with the supplied software to abstract absolute fluorescence signals from tissue autofluorescence. WWTR1/CAMTA1 admixture transcripts were articular by the attendance of chicken brindled dots, and wild-type WWTR1 and CAMTA1 transcripts were authentic by red or blooming brindled dots, respectively.

Genomic DNA was abandoned from arctic bump samples with the DNeasy Claret and Tissue Kit (Qiagen). PCR accession was performed with Phire Hot Start DNA polymerase (Finnzymes) and the afterward PCR conditions: 98°C for 30 s followed by 35 cycles of 98 for 5 s, 60 for 5 s, 72 for 1.7 min, and a final addendum of 72°C for 1 min. PCR articles were visualized by agarose gel electrophoresis, gel-purified, and submitted for sequencing. The PCR accession primers for anniversary EHE are declared in the Annual of primers (Supplementary Material).

Bacterial bogus chromosome (BAC) clones that belted WWTR1 (3q24) and CAMTA1 (1p36) were acquired from Children’s Hospital Oakland Research Institute (Oakland, CA). WWTR1 3′ BACs included RP11-10901O19 and RP11-787H9 (267 kb), and WWTR1 5′ BACs included RP11-1036D23, RP11-1044P19, RP11-636H10, and RP11-597O6 (595 kb). CAMTA1 3′ BACs included RP11-748N23, RP11-11I19, RP11-260L14, and RP11-44F9 (955 kb), and CAMTA1 5′ BACs included RP11-71M19, RP11-780G9, and RP11-262K2 (630 kb). A bring-together FISH delving was additionally complete by labeling the 3′ end of CAMTA1 in spectrum orange and the 5′ end of WWTR1 in spectrum green. DNA isolation, nick translation, and admixture were performed as ahead declared (24). For the break-apart FISH assay, tissues were denticulate by two absolute board and advised absolute if added than 10% of 200 beef showed break-apart signals. FISH images were captured with a Leica DM 6000 ambit (Leica) with Cytovision software (Genetix).

To assemble the tissue microarray, we arrayed 182 vascular tumors in alike in two arrangement blocks consisting of 0.6-mm cores taken from FFPE tissue and accumulated them application a tissue arrayer from Beecher Instruments as ahead declared (25). The vascular tumors acclimated in the arrangement were advised by two pathologists (B.P.R. and R.B.W.) and classified according to World Health Organization belief (26). We were able to accomplish WWTR1 break-apart FISH on 165 of the vascular tumors in the arrangement and CAMTA1 break-apart FISH on 151 of the vascular tumors in the array. Alone tumors with accordant absolute after-effects from both cores were advised positive.

Total RNA was abandoned from HUVECs, HAECs (human aortic endothelial cells), PAECs (pulmonary avenue endothelial cells), and DMVECs (dermal microvascular endothelial cells) with TRIzol Reagent (Invitrogen) and subjected to electrophoresis on an agarose gel to affirm RNA integrity. cDNA was actinic from these primary endothelial beef ambit in alongside with SuperScript III (Invitrogen) starting with 1 μg of absolute RNA and 250 ng of accidental primers (Promega). PCR accession was performed in leash on the LightCycler 480 SW 1.5 qPCR (quantitative PCR) apparatus (Roche Activated Science) with monocolor hydrolysis probes from the Universal Delving Library Arrangement (Roche) and a LightCycler 480 qPCR admixture (Roche). Appraisal of the bridge beginning point (CT) for the accession curves for anniversary case was bent by the additional acquired best adjustment (27). Absolute quantitation was performed with an in-run accepted ambit (27) for every qPCR and bidding as the cardinal of copies per 1 μg of absolute RNA. The primers and probes acclimated in the qPCRs for WWTR1 and CAMTA1 were as follows: WWTR1 advanced primer, 5′-TATCCCAGCCAAATCTCGTG-3′; WWTR1 about-face primer, 5′-TTCTGCTGGCTCAGGGTACT-3′; WWTR1 probe, 5′-GCAGCAGA-3′ (probe #84); CAMTA1 advanced primer, 5′-TTAATAACATTTGAGAAACACGAAGAA-3′; CAMTA1 about-face primer, 5′-CATTGAGCCATTCTGTGGTC-3′; CAMTA1 probe, 5′-CCACCTCC-3′ (probe #10).

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qPCR cycling altitude for both WWTR1 and CAMTA1 were as follows: 95°C for 10 min, followed by 50 cycles of 95°C for 10 s, 60°C for 30 s, 72°C for 1 s, followed by 40°C for 10 s. PCR articles were subjected to electrophoresis on an agarose gel to affirm the absence of all-embracing PCR products.

Quantitative RT-PCR abstracts were performed in leash for anniversary sample (primary endothelial corpuscle type) a minimum of two times with a altered cDNA alertness anniversary time. An SD was affected for anniversary leash sample. The statistical acceptation (P value) of the cogwheel announcement of WWTR1 and CAMTA1 was bent by the unpaired two-tailed t test.

www.sciencetranslationalmedicine.org/cgi/content/full/3/98/98ra82/DC1

Materials and Methods

Fig. S1A. Karyotype of EHE1.

Fig. S1B. Karyotype of EHE2.

Fig. S1C. Karyotype of EHE5.

Fig. S2. Arrangement of DNA breakpoints for EHE1 to EHE5.

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Fig. S3A. Announcement of WWTR1 in altered tissues/cell ambit (GNF2 atlas).

Fig. S3B. Announcement of CAMTA1 in altered tissues/cell ambit (GNF2 atlas).

Table S1. Admixture archetype candidates detected by FusionSeq algorithm.

Table S2. Analytic characteristics of EHE and break-apart FISH results.

Table S3. Statistical appraisal of the after-effects of the WWTR1and CAMTA1 break-apart FISH assays.

S. W. Weiss, J. R. Goldblum, Enzinger and Weiss’s Bendable Tissue Tumors (Mosby Elsevier, Philadelphia, ed. 5, 2008), pp. 681–687.

R. S. Verma, A. Babu, Animal Chromosomes: Manual of Basic Techniques (Pergamon Press, New York, 1989).

L. G. Shaffer, L. J. Campbell, M. L. Slovak, Eds., ISCN 2009: An International Arrangement for Animal Cytogenetic Nomenclature (S. Karger AG, Basel, Switzerland, 2009).

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S. W. Weiss, J. A. Bridge, Epithelioid hemangioendothelioma, in World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Bendable Tissue and Bone, C. D. Fletcher, K. K. Unni, F. Mertens, Eds. (IARC Press, Lyon, France, 2002), pp. 173–175.

R. Rasmussen, Quantification on the LightCycler instrument, in Rapid Cycle Real-Time PCR: Methods and Applications, S. Meuer, C. Wittwer, K. Nakagawara, Eds. (Springer, Heidelberg, Germany, 2001), pp. 21–34.

Citation: M. R. Tanas, A. S, A. M. Oliveira, M. R. Erickson-Johnson, J. Hespelt, P. J. Hanwright, J. Flanagan, Y. Luo, K. Fenwick, R. Natrajan, C. Mitsopoulos, M. Zvelebil, B. L. Hoch, S. W. Weiss, M. Debiec-Rychter, R. Sciot, R. B. West, A. J. Lazar, A. Ashworth, J. S. Reis-Filho, C. J. Lord, M. B. Gerstein, M. A. Rubin, B. P. Rubin, Identification of a Disease-Defining Gene Admixture in Epithelioid Hemangioendothelioma. Sci. Transl. Med. 3, 98ra82 (2011).

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