Sclerosing Epithelioid Fibrosarcoma presenting as intra-abdominal “sarcomatosis” with a novel EWSR1-CREB3L1 gene fusion David L. Stockman MD, Siraj M. Ali MD,PhD, Jie He PhD, Jeffrey S. Ross MD, Jeanne M. Meis MD PII: DOI: Reference:
S0046-8177(14)00224-X doi: 10.1016/j.humpath.2014.05.006 YHUPA 3323
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
19 December 2013 19 April 2014 5 May 2014
Please cite this article as: Stockman David L., Ali Siraj M., He Jie, Ross Jeffrey S., Meis Jeanne M., Sclerosing Epithelioid Fibrosarcoma presenting as intra-abdominal “sarcomatosis” with a novel EWSR1-CREB3L1 gene fusion, Human Pathology (2014), doi: 10.1016/j.humpath.2014.05.006
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ACCEPTED MANUSCRIPT Sclerosing Epithelioid Fibrosarcoma presenting as intra-abdominal “sarcomatosis” with a novel EWSR1CREB3L1 gene fusion 1
David L. Stockman, M.D., 2Siraj M. Ali, M.D., Ph.D., 2Jie He, Ph.D., 2,3Jeffrey S. Ross, M.D., 1Jeanne M. Meis, M.D.
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1 – Departments of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd Unit 085, Houston, Texas 77030, United States 2 – Foundation Medicine, Inc., 150 Second Street, Cambridge, MA 02141 3 – Department of Pathology and Laboratory Medicine, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208
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Correspondence: David L. Stockman, M.D. Department of Pathology University of Texas M.D. Anderson Cancer Center 1515 Holcombe Ave, Unit 085 Houston, TX 77030 Email: [email protected] Phone: (713) 893-8282 Fax: (713) 745-8228
RUNNING TITLE: Sarcomatosis in SEF with EWSR1-CREB3L1
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Abstract We report a case of intra-abdominal sclerosing epithelioid fibrosarcoma(SEF) with a t(11;22)(p11.2;q12.2)EWSR1CREB3L1 translocation.
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A 43-year old man presented with massive ascites and shortness of breath. Imaging studies revealed a large mesenteric based mass with extensive omental/peritoneal disease. After resection and cytoreductive surgery, the tumor recurred with metastasis to the lungs; the patient is still alive with disease.
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Histologically, there was a uniform population of epithelioid cells arranged in cords and nests, embedded in
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a dense collagenous matrix; no areas of low-grade fibromyxoid sarcoma (LGFMS) were identified. All immunohistochemical markers were non-reactive. Fluorescence in-situ hybridization(FISH) studies showed
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rearrangement of EWSR1. Genomic profiling by clinical-grade next generation sequencing(NGS) revealed a fusion gene between intron11 of EWSR1(22q12.2) and intron5 of CREB3L1(11p11.2). This is the first report of “pure” or true SEF presenting as intra-abdominal sarcomatosis with confirmation
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of the recently described unique EWSR1-CREB3L1 gene fusion in SEF without areas of LGFMS.
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Introduction
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Sclerosing epithelioid fibrosarcoma (SEF) is a rare, distinctive variant of fibrosarcoma, typically occurring in middle-aged adults. Despite its bland appearance, it is a fully malignant neoplasm usually characterized by local
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recurrences and late metastases. Since its formal description by Meis et al in 1995(1), SEF has been the subject of considerable interest with regard to its relationship to low-grade fibromyxoid sarcoma (LGFMS).(2-4) The classical histological picture of SEF is that of a sclerosing or densely hyalinized tumor with nests and cords of angulated to round epithelioid cells with scant cytoplasm that ultrastructurally have been shown to have features of fibroblasts.(1) However, the morphologic diagnosis of SEF continues to be challenging, particularly in biopsy specimens, owing to its typically low cellularity, abundant collagen, and banal cytologic features.
A possible genetic relationship between SEF and a subset of LGFMS possessing SEF-like histology has been postulated based on the identification of a t(7;16)(q32-34; p11) FUS-CREB3L2 translocation.(2, 3, 5-8) However, rearrangements of FUS were later shown to be rare in cases showing a predominantly SEF morphology.(4) SEF-like
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al areas can be seen in other sarcomas besides LGFMS. To date, no cases of true or “pure” SEF (i.e., cases showing primarily SEF histology) have shown the specific translocations seen in LGFMS.(2-4, 9)
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Most SEFs have been reported to occur in the deep musculature, associated with fascia and periosteum. Review of the literature has revealed rare cases occurring within the abdominal cavity.(1, 4, 10-14) The differential diagnosis of intra-abdominal cases, particularly those associated with disseminated peritoneal disease, includes gastrointestinal
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stromal tumor (GIST), mesothelioma, and disseminated carcinoma.
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Herein, we report a case of “pure” or true SEF presenting as sarcomatosis harboring a novel gene fusion involving
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EWSR1 and CREB3L1.
Materials and methods
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The histologic slides of this previously unpublished case were received in consultation at the University of Texas M.D. Anderson Cancer Center and the patient was subsequently referred for further treatment. All subsequent
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biopsies and resection specimens in addition to clinical data were reviewed.
Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections using the
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streptavidin-biotinylated horseradish peroxidase complex method in a Dako AutoStainer (Carpinteria, CA, USA). The primary antibodies used were AE1/AE3 (DAKO, Carpinteria, CA, USA, AE1/AE3, 1:4), Calretinin (Zymed, South San Francisco, CA, USA, 1:20), Cam 5.2 (BD Biosciences, San Jose, CA, USA, CAM 5.2, 1:50), CD34 (BD Biosciences, San Jose, CA, USA. my10, 1:40), CDX2 (BioGenex, San Ramon, CA, USA, CDX2-88, 1:50), CD117(c-kit) (DAKO, Carpinteria, CA, USA, A4502 1:200), CEA (Lab Vision, Freemont, CA, 1:200), (D2-40, Signet Laboratories, Dedham, MA, USA, D2-40, 1:50), CK5/6 (DAKO, Carpinteria, CA, USA, D5/16B4, 1:50), Desmin (DAKO, Carpinteria, CA, USA, D33, 1:75), DOG1 (Novacastra, Newcastle, UK, K9, 1:200), EMA (DAKO, Carpinteria, CA, USA, 1:100), MDM2 (Calbiochem, San Diego, CA, USA, 1F2, 1:50), MOC31 (DAKO, Carpinteria, CA, USA, 1:50), Pancytokeratin (DAKO, Carpinteria, CA, USA, 1:100), S-100 protein (DAKO, Carpinteria, CA, USA, 1:500), Smooth muscle actin (Ventana-Roche Diagnostics, 1:250), WT1 (DAKO, Carpinteria, CA, USA, 6F-H2, 1:40), Vimentin (DAKO, Carpinteria, CA, USA, V9 OR VIM 3B4, 1:400). .
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al
Rearrangements of FUS on chromosome 16p11 were assessed using the LSI FUS dual-color, break-apart probe (Abbott Molecular, Des Plaines, IL) and rearrangements of EWSR1 on chromosome 22q12 was assessed using the
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LSI EWSR1 dual-color, break-apart probes (Abbott Molecular, Des Plaines, IL) according to the manufacturer’s recommendations. Tissue sections and procedure was carried out as previously described.(4)
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Next generation sequencing (NGS) was performed using 200 ng of genomic DNA extracted from formalin-fixed
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paraffin embedded tissue sections cut at 10µm-thick for this specific case. DNA sequencing was performed for 3769 exons of 236 cancer-related genes and 47 introns of 19 genes frequently rearranged in cancer on an indexed, adaptor
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ligated, hybridization captured library and fully sequenced using 49 base paired reads (Illumina HiSeq 2000) to an average depth of 877X with high, uniform coverage. The specimen was evaluated for genomic alterations including base substitutions, insertions, deletions, copy number alterations (amplifications and homozygous deletions), and
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select gene fusions/rearrangements as previously described.(15) Sequence reads were mapped to the reference human genome (hg19) analyzed for all classes of genomic alterations. The bioinformatics process included Bayesian
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algorithms to detect base substitutions, local assembly algorithms to detect short insertions and deletions, a
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comparison with process-matched normal control samples to detect gene copy number alterations, and an analysis of
Results
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chimeric read pairs to identify gene fusions.
Clinical Features
A 43-year-old male presented with a two month history of increasing abdominal girth and pain associated with shortness of breath. Imaging studies detected multiple masses within the abdominal cavity with diffuse ascites. CT guided fine needle aspiration of the intra-abdominal mass was diagnosed at the outside institution as a low-grade mesenchymal neoplasm. The patient sought treatment at the University of Texas M.D. Anderson Cancer Center (MDACC) after four weeks where reimaging studies (Figs. 1A, 1B) showed progression of disease with extensive involvement of the small bowel, omentum, mesentery, and peritoneum, as well as increased ascites. The primary tumor was centered in the mesentery with multiple lesions and implants throughout the abdomen. Core biopsies at MDACC revealed a sclerosing sarcoma after carcinoma and mesothelioma were excluded. The patient subsequently
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al underwent complete omentectomy, resection of mesenteric nodules, pelvic peritonectomy, and en bloc resection of a pelvic mass and segment of small bowel, right colon and portion of psoas muscle involved with tumor as well as intraperitoneal cisplatin chemotherapy. Eight months after resection, there was local recurrences in the diaphragm,
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mesentery, and peritoneum with clinical metastases to the left upper lobe of the lung and pericardium.
Gross Features
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Tumors diffusely involved the omentum, mesentery, peritoneum and serosal surfaces of bowel with greater than 100
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intra-abdominal tumor deposits identified, ranging in size from 0.2 to 25.6 cm. Lobulated white firm tumor masses
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coalesced into large tumefactions encircling the small bowel and studding peritoneal surfaces (Figs. 1C, D).
Histologic Findings
The tumors histologically appeared as circumscribed and lobulated lesions with tumor cells arranged in strands,
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nests, or pseudo-alveoli embedded in a densely hyalinized collagenous matrix (Figs. 1E, F). The majority of tumor cells displayed an epithelioid appearance and contained round to oval to angulated nuclei with stippled chromatin
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and small basophilic nucleoli (Figs. 1G, H).
Immunohistochemical Findings
not performed.
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Immunohistochemical study results were negative for all antibodies assessed. Immunohistochemistry for MUC4 was
Molecular Genetic Findings Fused in sarcoma (FUS) showed no rearrangement by FISH, but subsequent testing with EWSR1 showed rearrangment. Genomic profiling revealed a fusion gene between EWSR1 (22q12.2) and CREB3L1 (11p11.2). The in-frame fusion occurred between intron 11 on EWSR1 and intron 5 on CREB3L1 (Fig. 2A). In addition to the gene fusion, the tumor also featured a T1246M mutation in MLL2.
Discussion
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Sclerosing epithelioid fibrosarcomas are rare tumors typically involving deep soft tissue, particularly dense fibrous connective tissue or periosteum. Intra-abdominal occurrences are extremely uncommon (Table 1) with cases reported in the pelvis,(13) cecum,(11) omentum(12) and retroperitoneum.(1, 4, 10) No cases to date have presented
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as sarcomatosis. Additionally, no cases of “pure” SEF have demonstrated FUS gene rearrangements(4) and only recently have rare cases been shown to possess translocations involving EWSR1.(16) Herein, we present a case of “pure” SEF exhibiting an EWSR1-CREB3L1 translocation. This finding strongly suggests that sclerosing epithelioid
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fibrosarcoma is distinct, not only histologically from LGFMS, but at the molecular level.
Gene rearrangements involving FUS have been shown to be present in 90% of LGFMS by FISH, (17-19) and FUS-
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CREB3L1 and FUS-CREB3L2 fusion transcripts have been reported in 96% of LGFMS by RT-PCR.(3, 19-22) Recently, two cases of FUS negative LGFMS without SEF-like areas by Lau et al,(9) were shown to possess the same fusion transcript seen in our tumor. Interestingly, a case of small cell osteosarcoma with features eerily
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reminiscent of SEF possessing an EWSR1-CREB3L1 fusion transcript was reported in 2011 by Debelenko and colleagues.(23) Additionally, the genomic breakpoints described by Debelenko et al, intron 11 of EWSR1 and intron
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5 of CREB3L1 are exactly those seen in our current case. Collectively, these findings suggest that true SEF rarely if
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ever harbors FUS gene rearrangements,(4) and may indeed be separable from LGFMS by detection of EWSR1.
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The Ewing sarcoma breakpoint region 1 (EWSR1) gene product is a member of the TET family of transcription factors and rearrangements involving this gene have been implicated in numerous other tumors.(24, 25) The partner gene, CREB3L1 (cAMP responsive element binding protein 3 like 1) is a member of the CREB transcription factor family (which also includes ATF1 and CREB1)(26) whose activity is normally regulated by the phosphorylation of a kinase inducible domain.(26, 27) It contains a bZiP DNA-interacting domain, that when deleted or mutated has been shown to abolish the inhibition of cell division.(28) When CREB3L1 is juxtaposed next to EWSR1 (Figs. 2B, C), the formation of a chimeric transcription factor with a strong EWSR1-derived activation domain replaces the regulatory kinase-inducible domain of the CREB family and comes under the control of a strong EWSR1 promoter.(27, 29-31) Additionally, the N-terminal of EWSR1 also contributes transactivating and oncogenic properties as in the case of other EWSR1-containing fusion oncoproteins.(32) In SEF, the EWSR1-CREB3L1 chimeric transcription factor may target a cAMP responsive element promoter different from that seen in LGFMS. This difference in gene/promoter
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al targeting could be explained in part by promoter/enhancer context specificity constrained by regulatory proteins already present in the cell of origin where the chromosomal translocation occurs.(27) CREB3L1 has already been shown to directly bind to the COL1A1 promoter region regulating collagen formation, including direct transcription
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of the pro-alpha1 chains of type I collagen found in most connective tissues such as bone, cornea, dermis and tendon. Recently, Kondo et al showed that mouse embryonic fibroblasts with CREB3L1-stabilized (ubiquination inhibited) cell lines induced increased collagen fiber production.(33) Additionally, enhanced expression of collagen
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fibers was significantly decreased in mouse embryonic fibroblasts by knocking-down CREB3L1 levels using siRNA.(33) Electrophoretic analysis of type I collagen extracted from mouse embryonic fibroblasts with increased
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levels of CREB3L1 had significantly higher levels of type 1 collagen (Col1α1 and Col1α2) compared with wild-type
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mouse embryonic fibroblasts.(33) Thus, the EWSR1-CREB3L1 rearrangement potentially produces hyperactive transcription factor that turns on the expression of various collagen producing target genes. Constitutive expression of the chimeric CREB3L1 transcription factor might be implicated in the overproduction of collagen that is an
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integral part of SEF.
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The morphologic features of our case of SEF are similar to those seen in the original description of this tumor.(1)
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However, the diagnosis may be extremely difficult, particularly in core biopsy specimens given the blandness of tumor cell nuclei and abundant hyalinization. Difficulties in diagnosis are compounded by the clinical presentation
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within the abdomen when there is disseminated disease and ascites. Clinically, the differential diagnosis includes GIST, carcinomatosis and mesothelioma. Histologically, SEF should be distinguished not only from these tumors but also from dedifferentiated liposarcoma, sclerosing mesenteritis, and desmoid fibromatosis.
Mesothelioma and poorly differentiated carcinomas can usually be distinguished based on immunohistochemical studies for keratin. Keratin positivity should prompt further clinical workup and clinical assessment for possible metastases. Additionally, electron microscopy for the presence of microvilli should be performed in cases of suspected mesothelioma, in addition to the usual immunohistochemical panel of WT1, cytokeratin, calretinin, and D2-40.(34) Sclerosing mesenteritis could display sclerotic zones similar to SEF, however, inflammatory elements should aid in the distinction. Fibromatosis may display paucicellular, keloid-like zones, and the presence of elongated, thin-walled ectatic vascular channels with a scar-like background are sometimes helpful. Moreover,
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al alterations of the CTNNB1 gene encoding beta-catenin can often be detected. Gastrointestinal stromal tumors may pose a pitfall due to mixed spindled to epithelioid morphologies. These tumors can also be diagnosed by immunohistochemical studies for CD117, DOG1, and CD34; all three markers are absent in SEF. Dedifferentiated
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liposarcomas may involve the mesentery or retroperitoneum. To date, SEF-like areas have not been reported in the dedifferentiated component of liposarcoma. However, limited sampling of the high grade sarcomatous component
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could lead to misclassification. Additionally, FISH studies to assess for amplification of MDM2 are often helpful.
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While SEF has an appearance histologically that is distinct, its sometimes broad areas of sclerosis surround small epithelioid cells may suggest the diagnosis of desmoplastic small round cell tumor (DSRCT). Given that both
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tumors appear in younger individuals and the propensity for DSRCT to involve the abdomen diffusely, this may serve as a diagnostic pitfall given both tumors show rearrangement of EWSR1 if studied by FISH. Further studies in
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scenarios where the alternative diagnosis is suspected warrant illustration of the fusion partner by RTPCR.
The diagnosis of SEF should be entertained in cellular intra-abdominal tumors displaying small epithelioid to oval
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cell populations and lacking immunohistochemical positivity for any of the aforementioned markers. A delicate
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deposition of deeply acidophilic collagen reminiscent of osteoid or broad zones of hyalinization or fibrosis with calcifications should prompt a molecular study to confirm involvement of EWSR1 chromosomal rearrangement as
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well as identifying the fusion partner gene. A combined approach utilizing immunohistochemistry and molecular studies is recommended to separate SEF from its mimic as well as LGFMS.
In summary, we have reported the clinical, histopathologic, and molecular features of a case of SEF presenting intraabdominally as sarcomatosis and harboring a previously unreported translocation EWSR1-CREB3L1 unique to these tumors.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Figure 1. Imaging studies, gross photographs, and microscopic images of intra-abdominal SEF. A, Axial view of mesenteric and omental tumor nodules; the largest tumor in the mesentery contains coalescing course calcification (white arrow head). B, Additional coronal view of tumor masses showing additional bowel involvement. C, Gross
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photograph of largest intact lobulated tumor mass. D, Sclerotic / fibrous cut surface of coalescing tumor masses. E, Sclerosing epithelioid fibrosarcoma shows cords of relatively small tumor cells separated by collagen. F, Areas of dense sclerosis juxtaposed to cellular areas of tumor. G, Files and small nests of epithelioid-to-clear cells separated
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by a collagenous matrix (appearance may be indistinguishable from mimickers without immunohistochemistry). H,
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Nests and cords of epithelioid to polygonal cells with clear cytoplasm embedded in a fibrous stroma.
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Figure 2. A, Chimeric gene product with illustrated breakpoint area occurring between introns 11 and 5 of EWSR1 and CREB3L1, respectively. The fused in-frame product occurs between exon 6 of CREB3L1 and exon 11 of EWSR1 B, Illustrated schematic of EWSR1 and CREB3L1 breakpoints; Ewing sarcoma breakpoint region 1
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(EWSR1) and cAMP responsive element binding protein 3 like 1 (CREB3L1) genes; bZiP – Basic Leucine Zipper DNA interacting domain; RXXL – recognition sequence for protease S1P; PXXP – recognition sequence for
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protease S2P; TAD – transactivation domain; RGG – domains rich in arginine and glycine; RMM – domain rich in
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arginine and methionine responsible for binding single filaments of RNA and DNA; Zn – Zinc ring finger binding
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domain. C, Diagram of putative EWSR1-CREB3L1 fusion protein.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al References 1. Meis-Kindblom JM, Kindblom LG, Enzinger FM. Sclerosing epithelioid fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol 19, 979-993, 1995. Doyle LA, Wang WL, Dal Cin P, Lopez-Terrada D, Mertens F, Lazar AJ, Fletcher CD, Hornick JL. MUC4 is a sensitive and extremely useful marker for sclerosing epithelioid fibrosarcoma: association with FUS gene rearrangement. Am J Surg Pathol 36, 1444-1451, 2012.
3.
Guillou L, Benhattar J, Gengler C, Gallagher G, Ranchere-Vince D, Collin F, Terrier P, Terrier-Lacombe MJ, Leroux A, Marques B, Aubain Somerhausen Nde S, Keslair F, Pedeutour F, Coindre JM. Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group. Am J Surg Pathol 31, 1387-1402, 2007.
4.
Wang WL, Evans HL, Meis JM, Liegl-Atzwanger B, Bovee JV, Goldblum JR, Billings SD, Rubin BP, Lopez-Terrada D, Lazar AJ. FUS rearrangements are rare in 'pure' sclerosing epithelioid fibrosarcoma. Mod Pathol 25, 846-853, 2012.
5.
Rekhi B, Folpe AL, Deshmukh M, Jambhekar NA. Sclerosing epithelioid fibrosarcoma - a report of two cases with cytogenetic analysis of FUS gene rearrangement by FISH technique. Pathol Oncol Res 17, 145148, 2011.
6.
Antonescu CR, Rosenblum MK, Pereira P, Nascimento AG, Woodruff JM. Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of a clinicopathologically distinct tumor. Am J Surg Pathol 25, 699-709, 2001.
7.
Eyden BP, Manson C, Banerjee SS, Roberts IS, Harris M. Sclerosing epithelioid fibrosarcoma: a study of five cases emphasizing diagnostic criteria. Histopathology 33, 354-360, 1998.
8.
Yoon N, Kwon JW, Seo SW, Ahn G, Choi YL. Sclerosing epithelioid fibrosarcoma: cytogenetic analysis of FUS rearrangement. Pathol Int 62, 65-68, 2012.
9.
Lau PP, Lui PC, Lau GT, Yau DT, Cheung ET, Chan JK. EWSR1-CREB3L1 gene fusion: a novel alternative molecular aberration of low-grade fibromyxoid sarcoma. Am J Surg Pathol 37, 734-738, 2013.
CE
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MA
NU
SC
RI P
T
2.
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10. Jiao YF, Nakamura S, Sugai T, Uesugi N, Habano W, Ogata M, Fujioka T. Overexpression of MDM2 in a sclerosing epithelioid fibrosarcoma: genetic, immunohistochemical and ultrastructural study of a case. Pathol Int 52, 135-140, 2002. 11. Frattini JC, Sosa JA, Carmack S, Robert ME. Sclerosing epithelioid fibrosarcoma of the cecum: a radiationassociated tumor in a previously unreported site. Arch Pathol Lab Med 131, 1825-1828, 2007. 12. Smith PJ, Almeida B, Krajacevic J, Taylor B. Sclerosing epithelioid fibrosarcoma as a rare cause of ascites in a young man: a case report. J Med Case Rep 2, 248, 2008. 13. Arya M, Garcia-Montes F, Patel HR, Emberton M, Mundy AR. A rare tumour in the pelvis presenting with lower urinary symptoms: 'sclerosing epithelioid fibrosarcoma'. Eur J Surg Oncol 27, 121-122, 2001. 14. Bai S, Jhala N, Adsay NV, Wei S. Sclerosing epithelioid fibrosarcoma of the pancreas. Ann Diagn Pathol 17, 214-216, 2013. 15. Ross JS, Ali SM, Wang K, Palmer G, Yelensky R, Lipson D, Miller VA, Zajchowski D, Shawver LK, Stephens PJ. Comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencingbased diagnostic assay reveals new routes to targeted therapies. Gynecologic oncology 130, 554-559, 2013.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al 16. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, Tayebwa J, Nord KH, Kindblom LG, Mertens F. Recurrent EWSR1-CREB3L1 Gene Fusions in Sclerosing Epithelioid Fibrosarcoma. Am J Surg Pathol, 2014.
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17. Downs-Kelly E, Goldblum JR, Patel RM, Weiss SW, Folpe AL, Mertens F, Hartke M, Tubbs RR, Skacel M. The utility of fluorescence in situ hybridization (FISH) in the diagnosis of myxoid soft tissue neoplasms. Am J Surg Pathol 32, 8-13, 2008. 18. Patel RM, Downs-Kelly E, Dandekar MN, Fanburg-Smith JC, Billings SD, Tubbs RR, Goldblum JR. FUS (16p11) gene rearrangement as detected by fluorescence in-situ hybridization in cutaneous low-grade fibromyxoid sarcoma: a potential diagnostic tool. Am J Dermatopathol 33, 140-143, 2011.
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19. Doyle LA, Moller E, Dal Cin P, Fletcher CD, Mertens F, Hornick JL. MUC4 is a highly sensitive and specific marker for low-grade fibromyxoid sarcoma. Am J Surg Pathol 35, 733-741, 2011.
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20. Matsuyama A, Hisaoka M, Shimajiri S, Hashimoto H. DNA-based polymerase chain reaction for detecting FUS-CREB3L2 in low-grade fibromyxoid sarcoma using formalin-fixed, paraffin-embedded tissue specimens. Diagn Mol Pathol 17, 237-240, 2008.
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21. Matsuyama A, Hisaoka M, Shimajiri S, Hayashi T, Imamura T, Ishida T, Fukunaga M, Fukuhara T, Minato H, Nakajima T, Yonezawa S, Kuroda M, Yamasaki F, Toyoshima S, Hashimoto H. Molecular detection of FUS-CREB3L2 fusion transcripts in low-grade fibromyxoid sarcoma using formalin-fixed, paraffinembedded tissue specimens. Am J Surg Pathol 30, 1077-1084, 2006.
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22. Mertens F, Fletcher CD, Antonescu CR, Coindre JM, Colecchia M, Domanski HA, Downs-Kelly E, Fisher C, Goldblum JR, Guillou L, Reid R, Rosai J, Sciot R, Mandahl N, Panagopoulos I. Clinicopathologic and molecular genetic characterization of low-grade fibromyxoid sarcoma, and cloning of a novel FUS/CREB3L1 fusion gene. Lab Invest 85, 408-415, 2005.
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23. Debelenko LV, McGregor LM, Shivakumar BR, Dorfman HD, Raimondi SC. A novel EWSR1-CREB3L1 fusion transcript in a case of small cell osteosarcoma. Genes Chromosomes Cancer 50, 1054-1062, 2011. 24. Boland JM, Folpe AL. Cutaneous neoplasms showing EWSR1 rearrangement. Adv Anat Pathol 20, 75-85, 2013.
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25. Thway K, Fisher C. Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status. Am J Surg Pathol 36, e1-e11, 2012. 26. Omori Y, Imai J, Suzuki Y, Watanabe S, Tanigami A, Sugano S. OASIS is a transcriptional activator of CREB/ATF family with a transmembrane domain. Biochem Biophys Res Commun 293, 470-477, 2002. 27. Davis IJ, Kim JJ, Ozsolak F, Widlund HR, Rozenblatt-Rosen O, Granter SR, Du J, Fletcher JA, Denny CT, Lessnick SL, Linehan WM, Kung AL, Fisher DE. Oncogenic MITF dysregulation in clear cell sarcoma: defining the MiT family of human cancers. Cancer Cell 9, 473-484, 2006. 28. Denard B, Seemann J, Chen Q, Gay A, Huang H, Chen Y, Ye J. The membrane-bound transcription factor CREB3L1 is activated in response to virus infection to inhibit proliferation of virus-infected cells. Cell Host Microbe 10, 65-74, 2011. 29. Brown AD, Lopez-Terrada D, Denny C, Lee KA. Promoters containing ATF-binding sites are de-regulated in cells that express the EWS/ATF1 oncogene. Oncogene 10, 1749-1756, 1995. 30. Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 10, 423-431, 1995.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al 31. Fujimura Y, Ohno T, Siddique H, Lee L, Rao VN, Reddy ES. The EWS-ATF-1 gene involved in malignant melanoma of soft parts with t(12;22) chromosome translocation, encodes a constitutive transcriptional activator. Oncogene 12, 159-167, 1996.
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32. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, Beroukhim R, Milner DA, Granter SR, Du J, Lee C, Wagner SN, Li C, Golub TR, Rimm DL, Meyerson ML, Fisher DE, Sellers WR. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117-122, 2005.
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33. Kondo S, Hino SI, Saito A, Kanemoto S, Kawasaki N, Asada R, Izumi S, Iwamoto H, Oki M, Miyagi H, Kaneko M, Nomura Y, Urano F, Imaizumi K. Activation of OASIS family, ER stress transducers, is dependent on its stabilization. Cell Death Differ 19, 1939-1949, 2012.
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34. Stoebner P, Miech G, Sengel A, Witz JP. [Concepts on pleural ultrastructure. II. Mesothelioma]. Presse Med 78, 1403-1408, 1970.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al TABLE 1. Summary of clinicopathologic features of intrabdominal SEF A/G
Location
Size
Ascites
Met.
Follow up
2000.Arya et al
55 M
Pelvis
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No
No
NED @ 48mo.
2007.Frattini et al
62 M
Colon
7.5
No
Yes
NED
2013.Bai et al
67 M
Pancreas
1
No
No
NED
1995.Meis et al
41 M
Retroperitoneum
2002.Jiao et al
75 F
Retroperitoneum
2012.Wang et al.2
48 M
Retroperitoneum
2012.Wang et al.6
78 M
Retroperitoneum
2012.Wang et al.18
38 M
Retroperitoneum
Present case
43 M
Omentum and mesentery
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Case
No
Yes
DOD @ 56mo.
8.5
No
No
NED @ 20mo.
NR
NR
NR
NFU
NR
NR
NR
NFU
NR
NR
NR
NFU
25.6
Yes
Yes
AWD @12 mo.
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A/G – age / gender; AWD – alive with disease; DOD – dead of disease; NED – no evidence of disease; NR - not reported; NFU - no follow up
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S0046-8177(14)00224-X doi: 10.1016/j.humpath.2014.05.006 YHUPA 3323
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
19 December 2013 19 April 2014 5 May 2014
Please cite this article as: Stockman David L., Ali Siraj M., He Jie, Ross Jeffrey S., Meis Jeanne M., Sclerosing Epithelioid Fibrosarcoma presenting as intra-abdominal “sarcomatosis” with a novel EWSR1-CREB3L1 gene fusion, Human Pathology (2014), doi: 10.1016/j.humpath.2014.05.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Sclerosing Epithelioid Fibrosarcoma presenting as intra-abdominal “sarcomatosis” with a novel EWSR1CREB3L1 gene fusion 1
David L. Stockman, M.D., 2Siraj M. Ali, M.D., Ph.D., 2Jie He, Ph.D., 2,3Jeffrey S. Ross, M.D., 1Jeanne M. Meis, M.D.
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1 – Departments of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd Unit 085, Houston, Texas 77030, United States 2 – Foundation Medicine, Inc., 150 Second Street, Cambridge, MA 02141 3 – Department of Pathology and Laboratory Medicine, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208
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Correspondence: David L. Stockman, M.D. Department of Pathology University of Texas M.D. Anderson Cancer Center 1515 Holcombe Ave, Unit 085 Houston, TX 77030 Email: [email protected] Phone: (713) 893-8282 Fax: (713) 745-8228
RUNNING TITLE: Sarcomatosis in SEF with EWSR1-CREB3L1
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Abstract We report a case of intra-abdominal sclerosing epithelioid fibrosarcoma(SEF) with a t(11;22)(p11.2;q12.2)EWSR1CREB3L1 translocation.
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A 43-year old man presented with massive ascites and shortness of breath. Imaging studies revealed a large mesenteric based mass with extensive omental/peritoneal disease. After resection and cytoreductive surgery, the tumor recurred with metastasis to the lungs; the patient is still alive with disease.
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Histologically, there was a uniform population of epithelioid cells arranged in cords and nests, embedded in
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a dense collagenous matrix; no areas of low-grade fibromyxoid sarcoma (LGFMS) were identified. All immunohistochemical markers were non-reactive. Fluorescence in-situ hybridization(FISH) studies showed
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rearrangement of EWSR1. Genomic profiling by clinical-grade next generation sequencing(NGS) revealed a fusion gene between intron11 of EWSR1(22q12.2) and intron5 of CREB3L1(11p11.2). This is the first report of “pure” or true SEF presenting as intra-abdominal sarcomatosis with confirmation
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of the recently described unique EWSR1-CREB3L1 gene fusion in SEF without areas of LGFMS.
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Introduction
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Sclerosing epithelioid fibrosarcoma (SEF) is a rare, distinctive variant of fibrosarcoma, typically occurring in middle-aged adults. Despite its bland appearance, it is a fully malignant neoplasm usually characterized by local
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recurrences and late metastases. Since its formal description by Meis et al in 1995(1), SEF has been the subject of considerable interest with regard to its relationship to low-grade fibromyxoid sarcoma (LGFMS).(2-4) The classical histological picture of SEF is that of a sclerosing or densely hyalinized tumor with nests and cords of angulated to round epithelioid cells with scant cytoplasm that ultrastructurally have been shown to have features of fibroblasts.(1) However, the morphologic diagnosis of SEF continues to be challenging, particularly in biopsy specimens, owing to its typically low cellularity, abundant collagen, and banal cytologic features.
A possible genetic relationship between SEF and a subset of LGFMS possessing SEF-like histology has been postulated based on the identification of a t(7;16)(q32-34; p11) FUS-CREB3L2 translocation.(2, 3, 5-8) However, rearrangements of FUS were later shown to be rare in cases showing a predominantly SEF morphology.(4) SEF-like
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al areas can be seen in other sarcomas besides LGFMS. To date, no cases of true or “pure” SEF (i.e., cases showing primarily SEF histology) have shown the specific translocations seen in LGFMS.(2-4, 9)
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Most SEFs have been reported to occur in the deep musculature, associated with fascia and periosteum. Review of the literature has revealed rare cases occurring within the abdominal cavity.(1, 4, 10-14) The differential diagnosis of intra-abdominal cases, particularly those associated with disseminated peritoneal disease, includes gastrointestinal
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stromal tumor (GIST), mesothelioma, and disseminated carcinoma.
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Herein, we report a case of “pure” or true SEF presenting as sarcomatosis harboring a novel gene fusion involving
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EWSR1 and CREB3L1.
Materials and methods
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The histologic slides of this previously unpublished case were received in consultation at the University of Texas M.D. Anderson Cancer Center and the patient was subsequently referred for further treatment. All subsequent
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biopsies and resection specimens in addition to clinical data were reviewed.
Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections using the
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streptavidin-biotinylated horseradish peroxidase complex method in a Dako AutoStainer (Carpinteria, CA, USA). The primary antibodies used were AE1/AE3 (DAKO, Carpinteria, CA, USA, AE1/AE3, 1:4), Calretinin (Zymed, South San Francisco, CA, USA, 1:20), Cam 5.2 (BD Biosciences, San Jose, CA, USA, CAM 5.2, 1:50), CD34 (BD Biosciences, San Jose, CA, USA. my10, 1:40), CDX2 (BioGenex, San Ramon, CA, USA, CDX2-88, 1:50), CD117(c-kit) (DAKO, Carpinteria, CA, USA, A4502 1:200), CEA (Lab Vision, Freemont, CA, 1:200), (D2-40, Signet Laboratories, Dedham, MA, USA, D2-40, 1:50), CK5/6 (DAKO, Carpinteria, CA, USA, D5/16B4, 1:50), Desmin (DAKO, Carpinteria, CA, USA, D33, 1:75), DOG1 (Novacastra, Newcastle, UK, K9, 1:200), EMA (DAKO, Carpinteria, CA, USA, 1:100), MDM2 (Calbiochem, San Diego, CA, USA, 1F2, 1:50), MOC31 (DAKO, Carpinteria, CA, USA, 1:50), Pancytokeratin (DAKO, Carpinteria, CA, USA, 1:100), S-100 protein (DAKO, Carpinteria, CA, USA, 1:500), Smooth muscle actin (Ventana-Roche Diagnostics, 1:250), WT1 (DAKO, Carpinteria, CA, USA, 6F-H2, 1:40), Vimentin (DAKO, Carpinteria, CA, USA, V9 OR VIM 3B4, 1:400). .
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Rearrangements of FUS on chromosome 16p11 were assessed using the LSI FUS dual-color, break-apart probe (Abbott Molecular, Des Plaines, IL) and rearrangements of EWSR1 on chromosome 22q12 was assessed using the
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LSI EWSR1 dual-color, break-apart probes (Abbott Molecular, Des Plaines, IL) according to the manufacturer’s recommendations. Tissue sections and procedure was carried out as previously described.(4)
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Next generation sequencing (NGS) was performed using 200 ng of genomic DNA extracted from formalin-fixed
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paraffin embedded tissue sections cut at 10µm-thick for this specific case. DNA sequencing was performed for 3769 exons of 236 cancer-related genes and 47 introns of 19 genes frequently rearranged in cancer on an indexed, adaptor
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ligated, hybridization captured library and fully sequenced using 49 base paired reads (Illumina HiSeq 2000) to an average depth of 877X with high, uniform coverage. The specimen was evaluated for genomic alterations including base substitutions, insertions, deletions, copy number alterations (amplifications and homozygous deletions), and
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select gene fusions/rearrangements as previously described.(15) Sequence reads were mapped to the reference human genome (hg19) analyzed for all classes of genomic alterations. The bioinformatics process included Bayesian
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algorithms to detect base substitutions, local assembly algorithms to detect short insertions and deletions, a
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comparison with process-matched normal control samples to detect gene copy number alterations, and an analysis of
Results
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chimeric read pairs to identify gene fusions.
Clinical Features
A 43-year-old male presented with a two month history of increasing abdominal girth and pain associated with shortness of breath. Imaging studies detected multiple masses within the abdominal cavity with diffuse ascites. CT guided fine needle aspiration of the intra-abdominal mass was diagnosed at the outside institution as a low-grade mesenchymal neoplasm. The patient sought treatment at the University of Texas M.D. Anderson Cancer Center (MDACC) after four weeks where reimaging studies (Figs. 1A, 1B) showed progression of disease with extensive involvement of the small bowel, omentum, mesentery, and peritoneum, as well as increased ascites. The primary tumor was centered in the mesentery with multiple lesions and implants throughout the abdomen. Core biopsies at MDACC revealed a sclerosing sarcoma after carcinoma and mesothelioma were excluded. The patient subsequently
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mesentery, and peritoneum with clinical metastases to the left upper lobe of the lung and pericardium.
Gross Features
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Tumors diffusely involved the omentum, mesentery, peritoneum and serosal surfaces of bowel with greater than 100
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intra-abdominal tumor deposits identified, ranging in size from 0.2 to 25.6 cm. Lobulated white firm tumor masses
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coalesced into large tumefactions encircling the small bowel and studding peritoneal surfaces (Figs. 1C, D).
Histologic Findings
The tumors histologically appeared as circumscribed and lobulated lesions with tumor cells arranged in strands,
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nests, or pseudo-alveoli embedded in a densely hyalinized collagenous matrix (Figs. 1E, F). The majority of tumor cells displayed an epithelioid appearance and contained round to oval to angulated nuclei with stippled chromatin
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and small basophilic nucleoli (Figs. 1G, H).
Immunohistochemical Findings
not performed.
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Immunohistochemical study results were negative for all antibodies assessed. Immunohistochemistry for MUC4 was
Molecular Genetic Findings Fused in sarcoma (FUS) showed no rearrangement by FISH, but subsequent testing with EWSR1 showed rearrangment. Genomic profiling revealed a fusion gene between EWSR1 (22q12.2) and CREB3L1 (11p11.2). The in-frame fusion occurred between intron 11 on EWSR1 and intron 5 on CREB3L1 (Fig. 2A). In addition to the gene fusion, the tumor also featured a T1246M mutation in MLL2.
Discussion
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Sclerosing epithelioid fibrosarcomas are rare tumors typically involving deep soft tissue, particularly dense fibrous connective tissue or periosteum. Intra-abdominal occurrences are extremely uncommon (Table 1) with cases reported in the pelvis,(13) cecum,(11) omentum(12) and retroperitoneum.(1, 4, 10) No cases to date have presented
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as sarcomatosis. Additionally, no cases of “pure” SEF have demonstrated FUS gene rearrangements(4) and only recently have rare cases been shown to possess translocations involving EWSR1.(16) Herein, we present a case of “pure” SEF exhibiting an EWSR1-CREB3L1 translocation. This finding strongly suggests that sclerosing epithelioid
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fibrosarcoma is distinct, not only histologically from LGFMS, but at the molecular level.
Gene rearrangements involving FUS have been shown to be present in 90% of LGFMS by FISH, (17-19) and FUS-
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CREB3L1 and FUS-CREB3L2 fusion transcripts have been reported in 96% of LGFMS by RT-PCR.(3, 19-22) Recently, two cases of FUS negative LGFMS without SEF-like areas by Lau et al,(9) were shown to possess the same fusion transcript seen in our tumor. Interestingly, a case of small cell osteosarcoma with features eerily
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reminiscent of SEF possessing an EWSR1-CREB3L1 fusion transcript was reported in 2011 by Debelenko and colleagues.(23) Additionally, the genomic breakpoints described by Debelenko et al, intron 11 of EWSR1 and intron
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5 of CREB3L1 are exactly those seen in our current case. Collectively, these findings suggest that true SEF rarely if
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ever harbors FUS gene rearrangements,(4) and may indeed be separable from LGFMS by detection of EWSR1.
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The Ewing sarcoma breakpoint region 1 (EWSR1) gene product is a member of the TET family of transcription factors and rearrangements involving this gene have been implicated in numerous other tumors.(24, 25) The partner gene, CREB3L1 (cAMP responsive element binding protein 3 like 1) is a member of the CREB transcription factor family (which also includes ATF1 and CREB1)(26) whose activity is normally regulated by the phosphorylation of a kinase inducible domain.(26, 27) It contains a bZiP DNA-interacting domain, that when deleted or mutated has been shown to abolish the inhibition of cell division.(28) When CREB3L1 is juxtaposed next to EWSR1 (Figs. 2B, C), the formation of a chimeric transcription factor with a strong EWSR1-derived activation domain replaces the regulatory kinase-inducible domain of the CREB family and comes under the control of a strong EWSR1 promoter.(27, 29-31) Additionally, the N-terminal of EWSR1 also contributes transactivating and oncogenic properties as in the case of other EWSR1-containing fusion oncoproteins.(32) In SEF, the EWSR1-CREB3L1 chimeric transcription factor may target a cAMP responsive element promoter different from that seen in LGFMS. This difference in gene/promoter
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al targeting could be explained in part by promoter/enhancer context specificity constrained by regulatory proteins already present in the cell of origin where the chromosomal translocation occurs.(27) CREB3L1 has already been shown to directly bind to the COL1A1 promoter region regulating collagen formation, including direct transcription
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of the pro-alpha1 chains of type I collagen found in most connective tissues such as bone, cornea, dermis and tendon. Recently, Kondo et al showed that mouse embryonic fibroblasts with CREB3L1-stabilized (ubiquination inhibited) cell lines induced increased collagen fiber production.(33) Additionally, enhanced expression of collagen
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fibers was significantly decreased in mouse embryonic fibroblasts by knocking-down CREB3L1 levels using siRNA.(33) Electrophoretic analysis of type I collagen extracted from mouse embryonic fibroblasts with increased
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levels of CREB3L1 had significantly higher levels of type 1 collagen (Col1α1 and Col1α2) compared with wild-type
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mouse embryonic fibroblasts.(33) Thus, the EWSR1-CREB3L1 rearrangement potentially produces hyperactive transcription factor that turns on the expression of various collagen producing target genes. Constitutive expression of the chimeric CREB3L1 transcription factor might be implicated in the overproduction of collagen that is an
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integral part of SEF.
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The morphologic features of our case of SEF are similar to those seen in the original description of this tumor.(1)
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However, the diagnosis may be extremely difficult, particularly in core biopsy specimens given the blandness of tumor cell nuclei and abundant hyalinization. Difficulties in diagnosis are compounded by the clinical presentation
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within the abdomen when there is disseminated disease and ascites. Clinically, the differential diagnosis includes GIST, carcinomatosis and mesothelioma. Histologically, SEF should be distinguished not only from these tumors but also from dedifferentiated liposarcoma, sclerosing mesenteritis, and desmoid fibromatosis.
Mesothelioma and poorly differentiated carcinomas can usually be distinguished based on immunohistochemical studies for keratin. Keratin positivity should prompt further clinical workup and clinical assessment for possible metastases. Additionally, electron microscopy for the presence of microvilli should be performed in cases of suspected mesothelioma, in addition to the usual immunohistochemical panel of WT1, cytokeratin, calretinin, and D2-40.(34) Sclerosing mesenteritis could display sclerotic zones similar to SEF, however, inflammatory elements should aid in the distinction. Fibromatosis may display paucicellular, keloid-like zones, and the presence of elongated, thin-walled ectatic vascular channels with a scar-like background are sometimes helpful. Moreover,
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al alterations of the CTNNB1 gene encoding beta-catenin can often be detected. Gastrointestinal stromal tumors may pose a pitfall due to mixed spindled to epithelioid morphologies. These tumors can also be diagnosed by immunohistochemical studies for CD117, DOG1, and CD34; all three markers are absent in SEF. Dedifferentiated
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liposarcomas may involve the mesentery or retroperitoneum. To date, SEF-like areas have not been reported in the dedifferentiated component of liposarcoma. However, limited sampling of the high grade sarcomatous component
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could lead to misclassification. Additionally, FISH studies to assess for amplification of MDM2 are often helpful.
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While SEF has an appearance histologically that is distinct, its sometimes broad areas of sclerosis surround small epithelioid cells may suggest the diagnosis of desmoplastic small round cell tumor (DSRCT). Given that both
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tumors appear in younger individuals and the propensity for DSRCT to involve the abdomen diffusely, this may serve as a diagnostic pitfall given both tumors show rearrangement of EWSR1 if studied by FISH. Further studies in
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scenarios where the alternative diagnosis is suspected warrant illustration of the fusion partner by RTPCR.
The diagnosis of SEF should be entertained in cellular intra-abdominal tumors displaying small epithelioid to oval
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cell populations and lacking immunohistochemical positivity for any of the aforementioned markers. A delicate
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deposition of deeply acidophilic collagen reminiscent of osteoid or broad zones of hyalinization or fibrosis with calcifications should prompt a molecular study to confirm involvement of EWSR1 chromosomal rearrangement as
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well as identifying the fusion partner gene. A combined approach utilizing immunohistochemistry and molecular studies is recommended to separate SEF from its mimic as well as LGFMS.
In summary, we have reported the clinical, histopathologic, and molecular features of a case of SEF presenting intraabdominally as sarcomatosis and harboring a previously unreported translocation EWSR1-CREB3L1 unique to these tumors.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al Figure 1. Imaging studies, gross photographs, and microscopic images of intra-abdominal SEF. A, Axial view of mesenteric and omental tumor nodules; the largest tumor in the mesentery contains coalescing course calcification (white arrow head). B, Additional coronal view of tumor masses showing additional bowel involvement. C, Gross
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photograph of largest intact lobulated tumor mass. D, Sclerotic / fibrous cut surface of coalescing tumor masses. E, Sclerosing epithelioid fibrosarcoma shows cords of relatively small tumor cells separated by collagen. F, Areas of dense sclerosis juxtaposed to cellular areas of tumor. G, Files and small nests of epithelioid-to-clear cells separated
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by a collagenous matrix (appearance may be indistinguishable from mimickers without immunohistochemistry). H,
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Nests and cords of epithelioid to polygonal cells with clear cytoplasm embedded in a fibrous stroma.
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Figure 2. A, Chimeric gene product with illustrated breakpoint area occurring between introns 11 and 5 of EWSR1 and CREB3L1, respectively. The fused in-frame product occurs between exon 6 of CREB3L1 and exon 11 of EWSR1 B, Illustrated schematic of EWSR1 and CREB3L1 breakpoints; Ewing sarcoma breakpoint region 1
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(EWSR1) and cAMP responsive element binding protein 3 like 1 (CREB3L1) genes; bZiP – Basic Leucine Zipper DNA interacting domain; RXXL – recognition sequence for protease S1P; PXXP – recognition sequence for
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protease S2P; TAD – transactivation domain; RGG – domains rich in arginine and glycine; RMM – domain rich in
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arginine and methionine responsible for binding single filaments of RNA and DNA; Zn – Zinc ring finger binding
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domain. C, Diagram of putative EWSR1-CREB3L1 fusion protein.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al References 1. Meis-Kindblom JM, Kindblom LG, Enzinger FM. Sclerosing epithelioid fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol 19, 979-993, 1995. Doyle LA, Wang WL, Dal Cin P, Lopez-Terrada D, Mertens F, Lazar AJ, Fletcher CD, Hornick JL. MUC4 is a sensitive and extremely useful marker for sclerosing epithelioid fibrosarcoma: association with FUS gene rearrangement. Am J Surg Pathol 36, 1444-1451, 2012.
3.
Guillou L, Benhattar J, Gengler C, Gallagher G, Ranchere-Vince D, Collin F, Terrier P, Terrier-Lacombe MJ, Leroux A, Marques B, Aubain Somerhausen Nde S, Keslair F, Pedeutour F, Coindre JM. Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group. Am J Surg Pathol 31, 1387-1402, 2007.
4.
Wang WL, Evans HL, Meis JM, Liegl-Atzwanger B, Bovee JV, Goldblum JR, Billings SD, Rubin BP, Lopez-Terrada D, Lazar AJ. FUS rearrangements are rare in 'pure' sclerosing epithelioid fibrosarcoma. Mod Pathol 25, 846-853, 2012.
5.
Rekhi B, Folpe AL, Deshmukh M, Jambhekar NA. Sclerosing epithelioid fibrosarcoma - a report of two cases with cytogenetic analysis of FUS gene rearrangement by FISH technique. Pathol Oncol Res 17, 145148, 2011.
6.
Antonescu CR, Rosenblum MK, Pereira P, Nascimento AG, Woodruff JM. Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of a clinicopathologically distinct tumor. Am J Surg Pathol 25, 699-709, 2001.
7.
Eyden BP, Manson C, Banerjee SS, Roberts IS, Harris M. Sclerosing epithelioid fibrosarcoma: a study of five cases emphasizing diagnostic criteria. Histopathology 33, 354-360, 1998.
8.
Yoon N, Kwon JW, Seo SW, Ahn G, Choi YL. Sclerosing epithelioid fibrosarcoma: cytogenetic analysis of FUS rearrangement. Pathol Int 62, 65-68, 2012.
9.
Lau PP, Lui PC, Lau GT, Yau DT, Cheung ET, Chan JK. EWSR1-CREB3L1 gene fusion: a novel alternative molecular aberration of low-grade fibromyxoid sarcoma. Am J Surg Pathol 37, 734-738, 2013.
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SC
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T
2.
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10. Jiao YF, Nakamura S, Sugai T, Uesugi N, Habano W, Ogata M, Fujioka T. Overexpression of MDM2 in a sclerosing epithelioid fibrosarcoma: genetic, immunohistochemical and ultrastructural study of a case. Pathol Int 52, 135-140, 2002. 11. Frattini JC, Sosa JA, Carmack S, Robert ME. Sclerosing epithelioid fibrosarcoma of the cecum: a radiationassociated tumor in a previously unreported site. Arch Pathol Lab Med 131, 1825-1828, 2007. 12. Smith PJ, Almeida B, Krajacevic J, Taylor B. Sclerosing epithelioid fibrosarcoma as a rare cause of ascites in a young man: a case report. J Med Case Rep 2, 248, 2008. 13. Arya M, Garcia-Montes F, Patel HR, Emberton M, Mundy AR. A rare tumour in the pelvis presenting with lower urinary symptoms: 'sclerosing epithelioid fibrosarcoma'. Eur J Surg Oncol 27, 121-122, 2001. 14. Bai S, Jhala N, Adsay NV, Wei S. Sclerosing epithelioid fibrosarcoma of the pancreas. Ann Diagn Pathol 17, 214-216, 2013. 15. Ross JS, Ali SM, Wang K, Palmer G, Yelensky R, Lipson D, Miller VA, Zajchowski D, Shawver LK, Stephens PJ. Comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencingbased diagnostic assay reveals new routes to targeted therapies. Gynecologic oncology 130, 554-559, 2013.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al 16. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, Tayebwa J, Nord KH, Kindblom LG, Mertens F. Recurrent EWSR1-CREB3L1 Gene Fusions in Sclerosing Epithelioid Fibrosarcoma. Am J Surg Pathol, 2014.
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17. Downs-Kelly E, Goldblum JR, Patel RM, Weiss SW, Folpe AL, Mertens F, Hartke M, Tubbs RR, Skacel M. The utility of fluorescence in situ hybridization (FISH) in the diagnosis of myxoid soft tissue neoplasms. Am J Surg Pathol 32, 8-13, 2008. 18. Patel RM, Downs-Kelly E, Dandekar MN, Fanburg-Smith JC, Billings SD, Tubbs RR, Goldblum JR. FUS (16p11) gene rearrangement as detected by fluorescence in-situ hybridization in cutaneous low-grade fibromyxoid sarcoma: a potential diagnostic tool. Am J Dermatopathol 33, 140-143, 2011.
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19. Doyle LA, Moller E, Dal Cin P, Fletcher CD, Mertens F, Hornick JL. MUC4 is a highly sensitive and specific marker for low-grade fibromyxoid sarcoma. Am J Surg Pathol 35, 733-741, 2011.
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20. Matsuyama A, Hisaoka M, Shimajiri S, Hashimoto H. DNA-based polymerase chain reaction for detecting FUS-CREB3L2 in low-grade fibromyxoid sarcoma using formalin-fixed, paraffin-embedded tissue specimens. Diagn Mol Pathol 17, 237-240, 2008.
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21. Matsuyama A, Hisaoka M, Shimajiri S, Hayashi T, Imamura T, Ishida T, Fukunaga M, Fukuhara T, Minato H, Nakajima T, Yonezawa S, Kuroda M, Yamasaki F, Toyoshima S, Hashimoto H. Molecular detection of FUS-CREB3L2 fusion transcripts in low-grade fibromyxoid sarcoma using formalin-fixed, paraffinembedded tissue specimens. Am J Surg Pathol 30, 1077-1084, 2006.
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22. Mertens F, Fletcher CD, Antonescu CR, Coindre JM, Colecchia M, Domanski HA, Downs-Kelly E, Fisher C, Goldblum JR, Guillou L, Reid R, Rosai J, Sciot R, Mandahl N, Panagopoulos I. Clinicopathologic and molecular genetic characterization of low-grade fibromyxoid sarcoma, and cloning of a novel FUS/CREB3L1 fusion gene. Lab Invest 85, 408-415, 2005.
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23. Debelenko LV, McGregor LM, Shivakumar BR, Dorfman HD, Raimondi SC. A novel EWSR1-CREB3L1 fusion transcript in a case of small cell osteosarcoma. Genes Chromosomes Cancer 50, 1054-1062, 2011. 24. Boland JM, Folpe AL. Cutaneous neoplasms showing EWSR1 rearrangement. Adv Anat Pathol 20, 75-85, 2013.
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25. Thway K, Fisher C. Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status. Am J Surg Pathol 36, e1-e11, 2012. 26. Omori Y, Imai J, Suzuki Y, Watanabe S, Tanigami A, Sugano S. OASIS is a transcriptional activator of CREB/ATF family with a transmembrane domain. Biochem Biophys Res Commun 293, 470-477, 2002. 27. Davis IJ, Kim JJ, Ozsolak F, Widlund HR, Rozenblatt-Rosen O, Granter SR, Du J, Fletcher JA, Denny CT, Lessnick SL, Linehan WM, Kung AL, Fisher DE. Oncogenic MITF dysregulation in clear cell sarcoma: defining the MiT family of human cancers. Cancer Cell 9, 473-484, 2006. 28. Denard B, Seemann J, Chen Q, Gay A, Huang H, Chen Y, Ye J. The membrane-bound transcription factor CREB3L1 is activated in response to virus infection to inhibit proliferation of virus-infected cells. Cell Host Microbe 10, 65-74, 2011. 29. Brown AD, Lopez-Terrada D, Denny C, Lee KA. Promoters containing ATF-binding sites are de-regulated in cells that express the EWS/ATF1 oncogene. Oncogene 10, 1749-1756, 1995. 30. Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 10, 423-431, 1995.
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al 31. Fujimura Y, Ohno T, Siddique H, Lee L, Rao VN, Reddy ES. The EWS-ATF-1 gene involved in malignant melanoma of soft parts with t(12;22) chromosome translocation, encodes a constitutive transcriptional activator. Oncogene 12, 159-167, 1996.
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32. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, Beroukhim R, Milner DA, Granter SR, Du J, Lee C, Wagner SN, Li C, Golub TR, Rimm DL, Meyerson ML, Fisher DE, Sellers WR. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117-122, 2005.
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33. Kondo S, Hino SI, Saito A, Kanemoto S, Kawasaki N, Asada R, Izumi S, Iwamoto H, Oki M, Miyagi H, Kaneko M, Nomura Y, Urano F, Imaizumi K. Activation of OASIS family, ER stress transducers, is dependent on its stabilization. Cell Death Differ 19, 1939-1949, 2012.
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34. Stoebner P, Miech G, Sengel A, Witz JP. [Concepts on pleural ultrastructure. II. Mesothelioma]. Presse Med 78, 1403-1408, 1970.
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Figure 1A
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Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al
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Figure 1B
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Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al
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Figure 1C
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Figure 1D
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Figure 2A
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Figure 2BC
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ACCEPTED MANUSCRIPT Sarcomatosis in SEF with EWSR1-CREB3L1 • Stockman et al TABLE 1. Summary of clinicopathologic features of intrabdominal SEF A/G
Location
Size
Ascites
Met.
Follow up
2000.Arya et al
55 M
Pelvis
13
No
No
NED @ 48mo.
2007.Frattini et al
62 M
Colon
7.5
No
Yes
NED
2013.Bai et al
67 M
Pancreas
1
No
No
NED
1995.Meis et al
41 M
Retroperitoneum
2002.Jiao et al
75 F
Retroperitoneum
2012.Wang et al.2
48 M
Retroperitoneum
2012.Wang et al.6
78 M
Retroperitoneum
2012.Wang et al.18
38 M
Retroperitoneum
Present case
43 M
Omentum and mesentery
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T
Case
No
Yes
DOD @ 56mo.
8.5
No
No
NED @ 20mo.
NR
NR
NR
NFU
NR
NR
NR
NFU
NR
NR
NR
NFU
25.6
Yes
Yes
AWD @12 mo.
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13
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A/G – age / gender; AWD – alive with disease; DOD – dead of disease; NED – no evidence of disease; NR - not reported; NFU - no follow up
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