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Small blue round cell tumor of the interosseous membrane bearing a t(2;22)(q34;q12)/EWS-CREB1 translocation:… Pacheco, Marina; Horsman, Douglas E; Hayes, Malcolm M; Clarkson, Paul W; Huwait, Hassan; Nielsen, Torsten O Jul 2, 2010

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CASE REPORT Open AccessSmall blue round cell tumor of the interosseousmembrane bearing a t(2;22)(q34;q12)/EWS-CREB1translocation: a case reportMarina Pacheco1, Douglas E Horsman2, Malcolm M Hayes1,2, Paul W Clarkson3, Hassan Huwait1,Torsten O Nielsen1*AbstractBackground: The group of small blue round cell tumors encompasses a heterogeneous group of neoplasmscharacterized by primitive appearing round cells with few distinguishing histologic features.Results: We report the case of a small blue round cell tumor with an EWS gene rearrangement detected byfluorescent in situ hybridization (FISH) analysis that mimicked Ewing sarcoma, but with unusual histology andimmunohistochemical features. Multi-color karyotyping identified the presence of a t(2;22)(q34;q12) that wasinitially expected to represent a variant EWSR1-FEV translocation. After an extensive workup, the lesion isconsidered to represent a clear cell sarcoma harboring an EWSR1-CREB1 fusion transcript.Conclusions: This case appears to represent a rare variant of clear cell sarcoma arising in peripheral soft tissueswith unusual histology and unique immunophenotype. In this circumstance, FISH for all EWSR1 translocationpartners or RT- PCR for a spectrum of possible transcript variants is critically important for diagnosis, sincecytogenetic analysis or clinical FISH assay using only commercial EWSR1 probes will be misleading.BackgroundThe accurate diagnosis of small blue round cell neo-plasms can be difficult. The differential diagnosisincludes sarcomas (such as the Ewing family of tumors(EFT), alveolar rhabdomyosarcoma, poorly differentiatedsynovial sarcoma, myxoid/round cell liposarcoma, des-moplastic small round cell tumor, and cellular variantsof extraskeletal myxoid chondrosarcoma), small cell andlymphoblastic lymphomas, neuroblastoma, melanomaand small cell carcinoma among others.Although certain histologic features may be useful indifferentiating these entities, their general morphology isgeneric by light microscopy and a large battery of ancil-lary studies is required. Immunohistochemistry is thefirst line supplemental methodology and is sufficient fordiagnosis in many cases of small round cell tumors. Forexample, myogenin and myoD1 are specific and sensi-tive for the diagnosis of rhabdomyosarcoma [1] andlymphoid markers such as CD20, CD3, CD30 and CD45are very useful in the diagnosis of lymphoma. However,many other markers, although helpful, are not so speci-fic and require interpretation in the context of animmunohistochemical panel. For example, epithelialmarkers are essential for the diagnosis of carcinoma, butthey can also be positive in poorly differentiated synovialsarcoma, Merkel cell carcinoma and in rare Ewingfamily tumors [2]. S-100 is positive in melanoma butalso in clear cell sarcoma, myxoid liposarcoma, extra-skeletal myxoid chondrosarcoma and some Ewing familytumors; desmin is strongly positive in rhadomyosarcomabut is also positive in desmoplastic small round celltumor; and CD99 immunoreactivity is seen in EFT, butalso in mesenchymal chondrosarcoma and lymphoblasticlymphoma [3].FISH analyses for chromosomal translocations can beextremely helpful in this setting, but even these findingsmay not be specific. EWSR1 gene rearrangement is char-acteristic of EFT, but is also present in extraskeletalmyxoid chondrosarcoma, desmoplastic small round celltumor, and a subset of myxoid/round cell liposarcomas* Correspondence: torsten@interchange.ubc.ca1Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, CanadaPacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12© 2010 Pacheco et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.[4]. FUS rearrangements are seen in a majority of myx-oid/round cell liposarcomas but also in EFT [5,6].Karyotype analysis is a global genome scan that hasthe ability to detect gross chromosomal alterations suchas translocations, but the precise chromosomal bandsand breakpoints involved in the identified translocationsmay be inaccurate due to the very low resolution of thistechnique.Here we report on a small blue round cell tumor withan unusual combination of histological and immuno-histochemical findings. Results from standard first linemolecular cytogenetic studies turned out to be mislead-ing for both diagnosis and therapy. A complete workupincluding karyotype analysis, multicolor FISH and con-struction of new FISH probes was required for the defini-tive diagnosis of what we consider to represent a variantof clear cell sarcoma bearing an EWSR1-CREB1 fusiontranscript and expressing an aberrant immunophenotype.Case presentationClinical details54-year-old female presented with pain and swelling ofone year duration in her left leg. An MRI scan revealeda 7 cm enhancing mass lying in the posterior calf, at thelevel of popliteus muscle and extending through theinterosseous membrane. The tibial nerve and poplitealvessels were encased in the tumor. Systemic imagingrevealed no metastases.A core needle biopsy was taken, but proved insuffi-cient for a definitive diagnosis. An open biopsy was per-formed and a working diagnosis of soft tissue Ewingsarcoma rendered. The patient received pre-operativesystemic chemotherapy as for Ewing sarcoma, but failedto respond with any tumor shrinkage.For local treatment of her tumor she was advised toundergo above knee amputation due to the anticipatedpoor functional results of limb salvage in this situation.Despite extensive counseling and corroborating secondopinions she refused amputation. As limb salvage wastechnically feasible, she underwent pre-operative radiationtherapy and a complex wide resection of the tumor wasperformed, with tibial nerve resection, vascular reconstruc-tion with saphenous vein grafts, allograft reconstructionand internal fixation of the tibial defect, as well as recon-struction of the soft tissue defect with a free tissue transferfrom the scapular region. Wide margins were achieved.The patient developed a wound infection with methicillin-resistant S. aureus 14 days postoperatively. At day 22, shesuffered an anastomotic leak of the vessel reconstruction.Although vascularity was restored and her limb at thisstage was still viable, she was very disappointed with thefunctional results of her procedure and requested an aboveknee amputation. She was clear of disease at last follow up.ResultsCore needle biopsySections showed a small blue round cell tumor growingin poorly cohesive sheets. The tumor cells had uniformplasmacytoid cytology (Fig. 1). A morphologically sus-pected diagnosis of plasma cell myeloma was ruled outbased on the lack of immunoreactivity for kappa orlambda light chains. Instead, the tumor cells showedstriking CD99, synapthophysin and desmin immunor-eactivity. In contrast, other immunohistochemical mar-kers were all negative, including melanocytic markerssuch as S-100, HMB-45 and Melan-A. The results ofimmunohistochemistry are shown in Table 1 with somerelevant immunohistochemical staining patterns illu-strated in Figure 1.The main differential diagnoses were considered to beEwing family tumor and alveolar rhabdomyosarcoma.Alveolar rhabdomyosarcoma was ruled out by negativemyogenin and myoD1 and by absence of PAX3/PAX7-FKHR translocations by FISH. EWSR1 FISH, in contrast,showed a break-apart signal pattern in most of theinterphase nuclei. A provisional diagnosis of Ewing sar-coma was rendered. However, the histology, the absenceof Fli1 staining and the strong desmin immunoreactivitywere all considered unusual by several local and consul-tant pathologists who reviewed the case.Additional FISH assays for WT1, CHN and DDIT3 (3′partners of EWSR1 in desmoplastic round cell tumor,extraskeletal myxoid chondrosarcoma and round cellliposarcoma, respectively) were negative. All core needlebiopsy tissue had been consumed after these assays.In view of the major therapeutic implications engen-dered by a diagnosis of Ewing sarcoma, a decision wasmade to perform an open biopsy to obtain more tissueincluding fresh tissue for cytogenetics.Open biopsyAn open biopsy was performed 6 weeks after the origi-nal core needle procedure. Histological examination ofthis specimen revealed plasmacytoid cells with small,eccentric, uniform, moderately vesicular nuclei, mostwith a single central nucleolus. The tumor cells werearranged in poorly cohesive sheets with no associatednecrosis and occasional mitotic figures were evident.Results of immunohistochemical studies were similar tothose found on the core biopsy. Other immunohisto-chemical markers were ordered and found to be nega-tive, including EMA, CD68, PNL2 and WT1.The cultured tumor specimen did not yield metaphasesof sufficient quality to be analyzed by G-banding. Multi-color karyotyping identified a few metaphases that con-tained a reciprocal translocation between 2 q and 22 q inthe context of additional numerical and structuralPacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 2 of 8changes. The M-FISH stem line karyotype was estab-lished as 46, XX, t(2;22)(q?36;q?12), +5, t(14;21)(q?;q?),-21 [5] (Fig. 2). On this basis the diagnosis of Ewing sar-coma was considered confirmed as these findings wereconsistent with the variant t(2;22) EWS-FEV fusion. Atthis point neoadjuvant chemotherapy was initiated forEwing sarcoma.Characterization of the translocation t(2;22)To confirm the suspected EWS-FEV translocation, adual-color break-apart probe for FEV on chromosomeband 2q36 [6] was applied to the cultured specimen. Thescoring with this probe revealed two intact signals in theinterphase nuclei, with one metaphase showing an intactcopy of FEV abnormally located on der(22)t(2;22). FISHfor EWSR1 was performed on the same slide and showedrearrangement in most of the interphase nuclei with the3′ probe signal located on der(2)t(2;22) (Fig. 2b). Thesefindings indicated that FEV was not rearranged asexpected, and that the breakpoint on the der(2)t(2;22)was in fact centromeric to the FEV locus.A candidate gene approach was initiated to establishthe variant EWSR1 fusion partner. CREB1 is the onlyother gene in this region previously identified in sar-coma translocations, and a break-apart CREB1 probewas created and applied to the cultured specimen. Withthis FISH experiment, break-apart signals in ~80% ofthe interphase nuclei were observed (Fig. 2c). The reci-procal t(2;22)(q34;q12) was confirmed using a EWSR1-CREB1 dual-fusion probe that revealed the expectedfusion signals.Agarose gel electrophoresis of the RT-PCR productshowed a ~120 bp band (Fig. 3a). The size of the pro-duct corresponded to the predicted product size of thefragment spanning the fusion transcript breakpointbased on the primer design from published cDNAsequences for EWSR1 (NM_013986.2) and CREB1(NM_004379.3). Cloning and sequencing of this productshowed an in-frame fusion between EWSR1 exon 7 andCREB1 exon 7 (Fig. 3b).Following this workup and considering also the lack ofresponse to neoadjuvant therapy, a diagnosis of clearFigure 1 Microscopic appearance and immunohistochemical features of the tumor. A) Representative area of the core needle biopsyspecimen showing a homogeneous plasmacytoid appearance of the tumor cells (H&E; ×100 magnification). B) Desmin and C) CD99 strongimmunoreactivity (×200 magnification). D) S100 and E) HMB-45 immunohistochemical staining (×200 magnification). F) Representative area ofthe resection specimen showing nests of tumor cells with clear cytoplasm divided by thin fibrous septa (×100 magnification).Pacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 3 of 8cell sarcoma with unusual histology and variant immu-nophenotype was considered.Surgical specimenThe resection specimen revealed a high-grade sarcomawith areas similar to those found in the biopsies, butalso other areas with neoplastic cells with clear cyto-plasm arranged in a nested pattern (Fig. 1f). Extensiveinfiltration of malignant cells through soft tissues andthe dense fascia of the periosteum and interosseousmembrane was evident as was extensive lymphovascularinvasion. Immunohistochemistry was repeated and whileHMB45 remained negative and CD99 strongly positive,S-100 and Melan-A now revealed patchy positive stain-ing. These new findings led us to favor a final diagnosisof clear cell sarcoma.DiscussionClear cell sarcoma (CCS) is an aggressive neoplasm ofuncertain histogenesis, accounting for 1% of soft tissuesarcomas. The deep soft tissues of the distal extremitiesare most frequently involved, often in association withtendons and aponeuroses [7]. Unlike most sarcomas,CCS has a high propensity for lymph nodes metastasis.The tumor cells show immunoreactivity for melanocy-tic markers [8,9] as they contain melanosomes in differ-ent stages of development [10], and display melanocyticgene expression signatures [11]. Despite its similaritywith melanoma, CCS is a distinct entity genetically char-acterized by the presence of a chromosomal transloca-tion involving EWSR1 most frequently partnered withATF1 [10,12-14]. More recently, the alternative chimerictranscript EWSR1-CREB1 has been described in threecases of clear cell sarcoma of the gastrointestinal tractthat, interestingly, did not show overt melanocytic dif-ferentiation [15]. The same chimeric transcript, resultingfrom a presumed t(2;22)(q34;q12) has been described inthree cases of CCS of soft tissue to date [13,14]. Hisaokaet al [13] found this chimeric transcript in 2 of 33 casesof CCS (6%). One presented as a 1.5 cm mass in the fin-ger of a 67-year-old male, and the other as a 15 cmmass in the pelvis of a 31-year-old female. Wang et alTable 1 Methods and results of immunohistochemistryAntibody Source Dilution/antigen retrieval Detection method ReactivityCK AE1/AE3 Dako 1:200/protease digestion SA/Bi -CK7 Dako 1:200/protease digestion SA/Bi -CK20 Dako 1:500/protease digestion SA/Bi -TTF1 Dako 1:100/CC1/95°C/30 min SA/Bi -EMA Dako 1:200/CC1/95°C/30 min SA/Bi -Vimentin Biogenex 1:10000/CC1/95°C/30 min SA/Bi -CD20 Dako 1:250/CC1/95°C/30 min SA/Bi -CD30 Dako 1:50/CC1/95°C/30 min SA/Bi -CD34 Cell Marque 1:50/CC1/95°C/30 min SA/Bi -Kappa light chain Dako 1:5000/protease digestion SA/Bi -Lambda light chain Dako 1:10000/protease digestion SA/Bi -CD68 Dako 1:800/CC1/95°C/8 min SA/Bi -S-100 Univ. of Toronto 1:1000/CC1/95°C/30 min SA/Bi - *HMB-45 Dako 1:100/untreated SA/Bi -Melan-A Cell Marque 1:50/CC1/95°C/30 min SA/Bi - *PNL2 Private source RTU/untreated Polymer based -SMA Dako 1:200/untreated SA/Bi -Desmin Dako 1:200/CC1/95°C/30 min SA/Bi +++H-Caldesmon Dako 1:200/CC1/95°C/30 min SA/Bi -Myogenin Dako 1:50/CC1/95°C/60 min Polymer based -Myo-D1 Dako 1:60/citrate buffer (pH6.0) Polymer based -CD99 Signet 1:20/CC1/95°C/30 min SA/Bi +++Synaptophysin Cell Marque 1:250/CC1/95°C/30 min SA/Bi +++Fli1 BD Pharmingen 1:20/CC1/95°C/60 min Polymer based -WT1 Dako 1:50/CC1/95°C/60 min Polymer based -Ki 67 Lab Vision 1:200/CC1/95°C/30 min SA/Bi >10%Dako, Carpinteria, CA, USA; Biogenex, San Ramon, CA, USA; Cell Marque, Rocklin, CA, USA; Signet, Dedham, MA, USA; BD Pharmingen, Franklin Lakes, NJ, USA.SA/Bi: Streptavidin/biotin method; RTU: ready to use; CC1: Cell conditioning 1 (pH8.0) (Ventana medical systems, Tucson, AZ, USA).*Negative on the core and open biopsies, but patchy positive staining was identified in the resection specimen.Pacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 4 of 8Figure 2 Characterization of the t(2;22) by molecular cytogenetic techniques. A) Partial multicolor karyotype showing normal chromosome2, normal chromosome 22 and both derivative chromosomes resulting from t(2;22)(q?36;q?12). B) Metaphase FISH with EWSR1 break-apart probe.An intact dual-color EWSR1 signal can be seen on normal chromosome 22, a green signal on the der(2) and a red signal on the der(22). C)Interphase FISH with the CREB1 break-apart probe showing break-apart signals (1 fused, 1 green and 1 red signals).Figure 3 Agarose gel of EWSR1-CREB1 RT-PCR product and sequence of the junction point. A) RT-PCR product of ~120 bp correspondingto the predicted size of the fragment spanning the break point based on the pair of primers used. M, 100 bp molecular marker; NC, negativecontrol (molecularly confirmed clear cell sarcoma with an EWSR1-ATF1 translocation); NTC, no template control. B) Sequence electropherogramshowing an EWSR1 exon7/CREB1 exon 7 in-frame fusion.Pacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 5 of 8[14] found the EWSR1-CREB1 chimeric transcript in 1of 15 cases (~7%), a superficial tumor in the palm of a66-year-old female.EWSR1-ATF1 and EWSR1-CREB1 are not exclusivelyfound in CCS. They also are the most common genefusions in angiomatoid fibrous histiocytoma, a mesench-ymal neoplasm of borderline malignancy of children andyoung adults [16,17] that typically presents as a smallsuperficial mass with a distinctive histology (nodules ofhistiocytoid cells, pseudoangiomatoid blood-filled spaces,fibrous pseudocapsule with a pericapsular lymphoplas-macytic infiltrate). None of these typical clinical featuresnor morphologic hallmarks were present in the currenttumor.Typically, CCS is composed of nests of plump spindlecells with clear to pale eosinophilic cytoplasm, separatedby delicate fibrous septa. The histology of the presenttumor was misleading as it presented as a monotonoussmall round cell neoplasm, with most cells exhibitingplasmacytoid characteristics. Deviation from the usualhistology has been seen in some cases of CCS[10,13,18]. Areas with rhabdoid tumor cells weredetected in 24% and 16% of the molecularly confirmedcases reported by Antonescu et al [10] and Hisaokaet al [13]. However, to the best of our knowledge, des-min expression by CCS cells has not been describedpreviously [13,19] and CD99 immunoreactivity has beenfound only in one molecularly confirmed case of CCS ofthe stomach [20]. Nevertheless, these are markers thatare not consistently tested in the clinical workup forclear cell sarcoma, as small blue round cell tumors suchas Ewing sarcoma and rhabdomyosarcoma are not partof the usual histological differential diagnosis of CCS.CCS of soft tissue is typically characterized by theexpression of S-100 and melanosome-associated markers[10,13,14,18,21]. In this regard too, the present tumorhad a unique immunoprofile, as S-100, HMB45 andMelan-A were all negative in both the core needle andincisional biopsies. It was only in the resection specimenthat some patchy staining for S-100 and Melan-A wasseen. Lack of vimentin expression was also an unex-pected finding.In a setting of supportive histomorphology andimmunohistochemistry, detection of EWSR1 rearrange-ments by FISH is a very useful diagnostic tool that cansupport the diagnosis for the known set of EWSR1-translocation bearing tumors (Ewing family tumors,clear cell sarcoma, extraskeletal myxoid chondrosar-coma, desmoplastic round cell tumor, and variant myx-oid/round cell liposarcomas) [4]. Nevertheless, in lightof the lack of specificity of EWSR1 break apart probes inthe differential diagnosis of small round cell sarcomas,the search for a 3′ partner is very important in thefollow-up analysis when the histology is not entirelyclassic for one of these diagnoses. The combined gen-ome wide-screen provided by multicolor FISH, and theadvantage of metaphase FISH to detect rearrangementswith reference to specific chromosomal bands allowedus to interrogate the involvement of CREB1 on chromo-some band 2q34 as the EWSR1 3′ partner in the reci-procal t(2;22).The rate of detection of diagnostic chimeric tran-scripts in CCS by RT-PCR ranges between 91%-100%among published studies [10,12-14,18]. This minor dif-ference could be explained by the fact that not all thestudies were intended to cover all the transcript variants,and the negative cases may well represent tumors withoverlooked alternative chimeric transcripts. Interestingly,the rate of detection of EWSR1 rearrangement in CCSby FISH has been variously reported to be 70%, 88% or100% [18,22,23], raising the possibility of EWSR1 beingsubstituted by other genes as a 5′partner in a small sub-set of CCS.Our case would represent the fourth soft tissue CCSreported harboring this fusion transcript variant, andthe first in which the cytogenetic features of this reci-procal translocation t(2;22)(q34;q12) are detailed. Thepredicted structure of EWSR1-CREB1 in this case issimilar to that described previously [14-16] in whichthe oncogenic chimeric transcript retains its CREB1carboxyl-terminal basic leucine zipper DNA bindingand dimerization domain, fused to the amino-terminaltranscriptional activation domain of EWS whichconfers oncogenic properties by transcriptionaldysregulation.ConclusionsThis case supports the fact that EWSR1-CREB1 is not atranslocation variant exclusive to clear cell sarcomasarising in the gastrointestinal tract.The described variation in histology and immunohis-tochemical features displayed by CCS must be takeninto account when considering the differential diagnosisfor an unusual small blue round cell tumor. In this set-ting, immunohistochemistry and even karyotype can bemisleading, and FISH for both of the translocation part-ners or PCR primers accounting for all of the fusiontranscript variants is important for accurate diagnosis.MethodsTissue handlingThe core needle biopsy was fixed in neutral-bufferedformalin and processed for routine histology. Unstained6 μm paraffin sections were submitted for interphaseFISH. Representative tissue from the incisional biopsyand from the excision specimen were submittedfresh for cytogenetic analysis and a portion was snapfrozen for molecular studies. The remaining tissuePacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 6 of 8was fixed and submitted for routine histology andimmunohistochemistry.ImmunohistochemistryStandard immunohistochemical studies were done usinga Ventana Benchmark XT Instrument (Ventana medicalsystems, Tucson, AZ, USA). The source and dilution ofthe antibodies, antigen retrieval and the detection meth-ods are presented in Table 1.Cytogenetic studies, FISH and multicolor FISHChromosome analysis was performed by standard meth-ods after 6 days culture in RPMI 1640 medium supple-mented with 20% fetal calf serum and L-glutamine.Metaphase chromosomes were banded by the GTGmethod and the karyotypes were described according tothe International System for Human CytogeneticNomenclature 2005 [24].FISH was performed on sections from the core needlebiopsy and on cell preparations from the cultured inci-sional biopsy specimen. Commercial probes for EWSR1,FUS and DDIT3 (Abbott Molecular, Des Plaines, IL,USA) and “in-house” dual-color break-apart (CHN,WT1, FEV) and dual-color dual-fusion (PAX3-FKHRand PAX7-FKHR) bacterial artificial chromosome (BAC)probes were used. An “in house” dual-color break-apartprobe was prepared for the detection of CREB1 rearran-gements using BACs RP11-354H1 and RP11-135B21.An “in-house” dual-color, dual-fusion EWSR1-CREB1probe was prepared to confirm the reciprocal t(2;22)(q34;q12) using BACs RP11-135B21/RP11-354H1 (chro-mosome 2) and RP11-945M21/RP11-1126O13 (chromo-some 22).The BAC probes were directly labeled by nick transla-tion using either Spectrum Green or Spectrum Orange(Abbott Laboratories, Abbott Park, IL, USA). The chro-mosomal locations of the BACs were initially confirmedby hybridization to normal metaphases from a periph-eral blood culture. Each probe was scored by counting200 interphase nuclei under fluorescent microscopy. Forconfirmation of true breakapart, >10% of cells showing aclear pattern of one fused, one red and one green signalwas required. For confirmation of dual fusions, >5% ofnuclei with a clear two fused, one red, one green patternwas required.Multicolor FISH was performed using the 24 XCytecolor kit (Metasystems, Altlussheim, Germany) followingthe manufacturer’s protocol.Reverse transcription-PCR of the fusion transcriptTotal RNA from the frozen open biopsy tumor tissuewas isolated using the RNeasy mini kit (Qiagen, Mary-land, USA). Two micrograms of total RNA were reversetranscribed using the qScript cDNA SuperMix system(Quanta Biosciences, Maryland, USA) and used as tem-plate for PCR amplification of the EWS-CREB1 fusionbreakpoints using the following primers [14] EWSex7-F1 primer (5′-TCCTACAGCCAAGCTCCAAGTC -3′)and CREB1ex7-REVC primer specific for CREB1 (5′-GTACCCCATCGGTACCATTGT -3′). The PCR ampli-fication started with 5 minutes at 95°C; followed by 35cycles of 30 seconds at 95°C, 30 seconds at 58°C, and 45seconds at 72°C; and a final extension of 10 minutes at72°C. PCR product was detected by 1.5% agarose gelelectrophoresis.cDNA cloning and sequencing analysis of the fusiontranscriptThe cDNA of the break-point-crossing fragment of theEWS-CREB1 chimeric transcript was first amplified withHindIII-EWSex7-FW (5′TATCaagcttTCCTACAGC-CAAGCTCCAAGTC) and XhoI-CREB1ex7-RV (5′-TTTTctcgagGTACCCCATCGGTACCATTGT), thensubcloned into pcDNA3.1 (+) vector. Purified plasmidDNA was verified using the same restriction enzymes.The clones with an insert of the appropriate size werethen submitted for cDNA sequencing using a T7-FWprimer.ConsentWritten informed consent was obtained from the patientfor publication of this case report. A copy of the writtenconsent is available for review by the Editor-in-Chief ofthis journal.AcknowledgementsWe thank Ms. S. Clemens and Ms. C. Salski for FISH and MFISH analyses, Ms.J. Senz for sequencing, and Drs. H. Cheng and S. Liu for assistance withcloning and RT-PCR.Author details1Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, Canada. 2Department of Pathology, British ColumbiaCancer Agency, Vancouver, Canada. 3Division of Surgical Oncology, BritishColumbia Cancer Agency, Vancouver, Canada.Authors’ contributionsMP carried out the candidate gene approach and the preparation of the “in-house” FISH probes, led the molecular studies, performed the cloning of thefusion transcript and drafted the manuscript. DEH led the cytogenetic andmolecular cytogenetic studies, their analysis and interpretation and reviewedthe manuscript. MMH was involved in the histopathologic diagnosis of thepatient’s sample and the review of the manuscript. PWC provided theclinical data of the patient and helped drafting the manuscript. HHorganized the original histopathologic diagnosis of the patient. TON initiatedand led the pathologic investigations and co-wrote the manuscript. Allauthors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 20 April 2010 Accepted: 2 July 2010 Published: 2 July 2010Pacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 7 of 8References1. Kumar S, Perlman E, Harris CA, Raffeld M, Tsokos M: Myogenin is a specificmarker for rhabdomyosarcoma: an immunohistochemical study inparaffin embedded tissues. Mod Pathol 2000, 13:988-993.2. Gu M, Antonescu CR, Guiter G, Huvos AG, Ladanyi M, Zakowski MF:Cytokeratin immunoreactivity in Ewing’s sarcoma: prevalence in 50cases confirmed by molecular diagnostic studies. Am J Surg Pathol 2000,24:410-416.3. Coindre JM: Immunohistochemistry in the diagnosis of soft tissuetumours. Histopathology 2003, 43:1-16.4. Romeo S, Dei Tos AP: Soft tissue tumors associated with EWSR1translocation. Virchows Arch 2010, 456:219-234.5. 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Granter SR, Weilbaecher KN, Quigley C, Fletcher CD, Fisher DE: Clear cellsarcoma shows immunoreactivity for microphthalmia transcriptionfactor: further evidence for melanocytic differentiation. Mod Pathol 2001,14:6-9.22. Patel RM, Downs-Kelly E, Weiss SW, Folpe AL, Tubbs RR, Tuthill RJ,Goldblum JR, Skacel M: Dual-color, break-apart fluorescence in situhybridization for EWS gene rearrangement distinguishes clear cellsarcoma of soft tissue from malignant melanoma. Mod Pathol 2005,18:1585-1590.23. Tanas MR, Goldblum JR: Fluorescence in situ hybridization in thediagnosis of soft tissue neoplasms: a review. Adv Anat Pathol 2009,16:383-391.24. Shaffer LG, Tommerup N: ISCN. An International System for HumanCytogenetic Nomenclature. Basel: S. Karger 2005.doi:10.1186/1755-8166-3-12Cite this article as: Pacheco et al.: Small blue round cell tumor of theinterosseous membrane bearing a t(2;22)(q34;q12)/EWS-CREB1translocation: a case report. Molecular Cytogenetics 2010 3:12.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitPacheco et al. Molecular Cytogenetics 2010, 3:12http://www.molecularcytogenetics.org/content/3/1/12Page 8 of 8


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