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An unusual association of malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma-like) and Ewing sarcoma
Pathology – Research and Practice 211 (2015) 688–692
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
An unusual association of malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma-like) and Ewing sarcoma Luigi Insabato a,∗ , Elia Guadagno a , Valentina Natella a , Anna Somma a , Michel Bihl b , Antonio Pizzolorusso c , Pier Paolo Mainenti a , Gaetano Apice c , Luigi Tornillo b a
Dip. Scienze Biomorfologiche e Chirurgiche, Università Federico II Napoli, Italy Department of Pathology, University Hospital of Basel, Switzerland c SSD Oncologia medica Sarcomi dei Tessuti molli e delle Ossa, Istituto Tumori, Fondazione G. Pascale, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 5 November 2014 Received in revised form 4 June 2015 Accepted 5 June 2015 Keyword: Clear cell sarcoma-like gastric tumor
a b s t r a c t Very recently a new designation of “Malignant Neuroectodermal Gastrointestinal Tumor” has been proposed for an aggressive form of neuroectodermal tumor with features similar to that of Clear Cell Sarcoma of Soft Tissue, however without a melanocytic differentiation. Also known as “clear cell sarcoma-like tumors of the gastrointestinal tract”, these tumors show some features strongly suggesting an origin from a gastrointestinal neuroectodermal precursor cell unable to differentiate along the melanocytic lineage. They occur mainly in young and middle-aged adults, and have a poor prognosis with a high rate of liver and lymphnode metastases. Histologically they are composed of epithelioid or oval-to spindle cells with a sheet-like or nested pattern of growth, strongly positive for neural markers (S-100, SOX10, and vimentin) and negative for the melanocytic ones. EWSR1 gene rearrangements including EWSR1-ATF1 or EWSR1-CREB1 GENE fusions are typically assessed in these tumors. Here we report a case of malignant neuroectodermal gastrointestinal tumor which immunophenotypically unusually expressed FLI-1, occurring in a 29-year-old man with a previous medical history of Ewing sarcoma. We finally suggest that this case might be a further evidence of a link between these two entities. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction
2. Clinical history
Malignant gastrointestinal neuroectodermal tumor (GNET) is a new disease entity, recently described [1]. It might be considered an aggressive form of a specific neuroectodermal tumor that is clinically, morphologically, and immunohistochemically different from any other primitive tumor of the gastrointestinal tract. Its morphology is similar to the rare Clear Cell Sarcoma of soft tissue (CCSST), involving the gastrointestinal tract [2], but without the evidence of melanocytic differentiation. These two entities share features of molecular genetics, harboring a distinctive chromosomal translocation involving EWSR1-ATF1 t (12;22) (q13;q12). Here is presented a rare case of GNET of the stomach, occurring in a patient with a previous diagnosis of Ewing sarcoma.
A 29 year-old man was admitted in our hospital for acute gastric pain, in January 2009. The magnetic resonance imaging showed a solid submucosal mass localized in the wall of the gastric antrum (Fig. 1a). A clinical diagnosis of gastric GIST had been done, so the patient underwent a partial gastrectomy. On the pathological examination, the diagnosis was that of a mesenchymal tumor, probably originating from a peripheral nerve. A follow-up was mandatory. In January 2012, during one of his periodic check-ups, the patient made an abdominal ultrasonography which revealed the presence of three nodules, respectively at the II, III and IV segment of the liver, the largest of which measured 24 mm; a percutaneous liver biopsy was made. In the medical record it was reported that the patient, at the age of 5 years, in 1985, after a trauma to the sternum, caused by a fall from the bike, was diagnosed a Ewing’s Sarcoma of the left XI rib; this diagnosis had been made in another hospital (Fig. 1b). Therefore, he was treated with five cycles of combined chemotherapy which led to a total remission from the disease.
∗ Corresponding author. Tel.: +39 0817463442; fax: +39 0817463475. E-mail address: [email protected] (L. Insabato). http://dx.doi.org/10.1016/j.prp.2015.06.001 0344-0338/© 2015 Elsevier GmbH. All rights reserved.
L. Insabato et al. / Pathology – Research and Practice 211 (2015) 688–692
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Switzerland. FISH analysis was performed using a commercial probe (ZytoLight® SPEC EWSR1, ZytoVision GmbH·Fischkai 1 27572 Bremerhaven·Germany). 4-mm-tick slides were pretreated for 15 with pretreatment solution (Vysis® , Abbott Laboratories, Abbott Park, IL), digested with protease I for 70 (Vysis® , Abbott Laboratories, Abbott Park, IL) and afterwards incubated overnight with the ZytoLight® SPEC EWSR1 probe, a mixture of two direct labeled probes hybridizing to the 22q12.1–q12.2 band. The orange fluorochrome direct labeled probe hybridizes the proximal end of the band and extends inward into intron 4 of the EWSR1 gene while the green fluorochrome direct labeled probe hybridizes the distal end of the band. The detection of orange and green signals separated by more than 1.5 probe diameter indicates that the locus 22q12.1–q12.2 is affected by a translocation. 100 non-overlapping nuclei were evaluated. The presence of a translocation, as defined above, in more than 15% of the nuclei was considered positive. 3.4. RT-PCR
Fig. 1. (a) MR imaging: solid submucosal mass localized at the level of the gastric antrum wall (arrow). (b) 99mTC-Methylene diphosphonate bone scintigraphy (posterior view): focal tracer uptake at the level of the left XI rib (black arrow).
3. Materials and methods 3.1. Immunohistochemical analysis Immunohistochemistry was conducted on selected formalinfixed, paraffin-embedded blocks and performed using a fully automated system. The following commercially available antibodies were used: S-100 (Medite, Nunningen, Switzerland, 1:10,000), vimentin (Boehringer Mannheim Biochemica Mannheim, Germany, 1:10), CD56 (Vector, Burlingame, CA, 1:100), NSE (Dakopatts, Glostrup, Denmark, 1:100), keratin AE1/AE3 (Dako, Carpinteria, CA, 1:300), EMA (Ventana Medical Systems, Tucson, AZ, prediluted), CD117 (Dako, 1:200), CD99 (Covance Inc., Princeton, NJ, 1:400), CD34 (Immunotech Beckam Coulter, Inc., Fullerton, CA, 1:160), CD31 (Dako, 1:40), synaptophysin (Dako, 1:25), chromogranin-A (Dako, 1:3000), HMB45 (Dakopatts, Glostrup, Denmark, 1:50), MART-1 (Dakopatts, Glostrup, Denmark, 1:50), actin (Enzo Diagnostics, New York, USA, 1:16,000), desmin (Dakopatts Glostrup, Denmark, 1:900), and FLI1 (Cell Marque, Rocklin, CA, 1:100). 3.2. Electron microscopy The formalin-fixed, paraffin-embedded tissue sample of gastric tumor was de-waxed, rehydrated, fixed in glutaraldehyde, and then processed for electron microscopy. 3.3. Florescence in situ hybridization A genetic analysis by FISH technique was carried out at the pathology laboratory of the University of Basel, in
Slides were dewaxed with xylene three times for 5 min each, hydrated through alcohol gradient ethanol (100%, 95%, 70%, 50% ethanol) and then rinsed with tap water. Cells from selected area were scraped and collected into a 2 ml Eppendorf. RNA extraction from cell pellet was performed using the Trizol/chloroform method (Invitrogen). One to two ug of the total RNA was reverse-transcribed to cDNA, using the Gene Amp® RNA PCR (Applied Biosystem) kit. The reaction was conducted at 37 ◦ C for 60 min and then the samples were heated to 95 ◦ C for 5 min to inactivate the reverse transcriptase. The cDNA was amplified using specific primers for the FIP1L1PDGFRA translocation. For the first PCR we used the following primers: FIP1L1 ex8 F1: 5 -ACCTGGTGCTGATCTTTCTGAT-3 PDGFRA ex14 R1: 5 -TGAGAGCTTGTTTTTCACTGGA-3 ; while for the nested PCR the primers were: FIP1L1 Ex8 F2: 5 -AAAGAGGTACGAATGGGACTTG-3 PDGFRA ex13 R2: 5 -GGGACCGGCTTAATCCATAG-3 . A fragment of 772 bp from the YY1 gene was also independently amplified, in order to verify the quality of the cDNA fragments. YY1 primers were: YY1-851 F: 5 -AGTGGGAGCAGAAGCAGGTGC-3 YY1-1623 R: 5 -AAACATGTCCCTTAGGTGTGTAGGATTC-3 . Forty repeated cycles of denaturation at 95 ◦ C for 20 s, the primer annealing at 55 ◦ C for 10 s (FIP1L1-PDGFRA) or at 62 ◦ C for 30 s (YY1 gene), elongation at 72 ◦ C 40 s, followed by a final elongation at 72 ◦ C 7 min, were carried out. 4. Results Microscopically, the gastric tumor was composed of epithelioid and spindle cells arranged in nests and cords, with abundant clear cytoplasm, slightly pleomorphic nuclei with occasionally prominent nucleoli, low mitotic activity, and without necrosis (Fig. 2a and b); neoplastic cells infiltrated almost the whole thickness of the gastric wall, sparing the mucosa. Immunohistochemical investigation revealed positivity for the S-100 protein and vimentin, and negativity for actin, desmin and CD117. Because of the immunoreactivity for the S-100 protein, the tumor was diagnosed as a mesenchymal tumor, possibly of peripheral nerve origin. Three years later, the microscopic examination of the percutaneous liver biopsy showed a patternless tumor, composed of round to oval cells with scant eosinophilic cytoplasm, with prominent nucleoli, and low mitotic activity (Fig. 2c and d), highly suggestive for a liver metastasis. Having got this evidence, the slides of the gastric tumor were reexamined: we came across a lesion
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L. Insabato et al. / Pathology – Research and Practice 211 (2015) 688–692
Fig. 2. Gastric and hepatic tumors. In the stomach the tumor was mainly composed of clear cells arranged in solid nests, lacking tumor necrosis (a, H-E stain, 20×) (b, H-E stain, 40×), while in the liver neoplastic cells were round-shaped with an eosinophilic cytoplasm (c, H-E stain, 10×) (d, H-E stain, 40×). Both of them were strongly immunoreactive for S100 (e, f) and for FLI-1 (g). Fish analysis: a translocation was found in more than 15% of the nuclei in both lesions (h).
that was morphologically different from the hepatic one. Infact, in the antrum, tumor cells had a clear and abundant cytoplasm. A detailed immunohistochemical study was performed on both tumors. The hepatic tumor, exactly like the gastric one, was strongly immunoreactive for S-100 protein (Fig. 2e and f), FLI-1 (Fig. 2g) and vimentin, and focally positive for 56 and NSE. Keratin AE1/AE3, EMA, CD117, CD99, CD34, CD31, synaptophysin, chromogranin, HMB45, and MART-1 were all negative. On this basis, the diagnosis of clear cell sarcoma-like tumor of the stomach with liver metastasis was suggested. On electron microscope, the gastric tumor tissue did not show the presence of melanosomes.
On FISH analysis, 100 non-overlapping nuclei were evaluated from the tissue of the gastric tumor and from the hepatic metastasis. A translocation of EWSR1 gene was found in 27% and 42% of the nuclei, respectively, indicating a clear-cut positivity (Fig. 2h), while RT-PCR for FLI-1 was negative. Finally, a diagnosis of primitive malignant gastric neuroectodermal tumor (clear cell sarcoma-like tumor) with liver metastases was made. After this diagnosis, the patient underwent a total body computerized tomography with contrast, confirming the presence of hepatic nodular lesions and showing multiple hyperdense bone lesions, thoracic lymph nodes enlargement and peritoneal thickening in correspondence of the stomach, the jejunum and the
L. Insabato et al. / Pathology – Research and Practice 211 (2015) 688–692
pancreas. In September 2012, 10 cycles of chemotherapy with etoposide–ifosfamide were started and, at the end of this therapy, the enlargement of lymph nodes and the peritoneal involvement had slightly regressed; this partial regression of disease remained stable for about 8 months, after which a treatment with 3 cycles of ifosfamide was made. On the last follow-up, in March 2015, the patient was in stable disease.
5. Discussion Among all mesenchymal tumors of the gastrointestinal tract, a small number of neoplasms with features of clear cell sarcoma (CCS) have been reported. Originally described by Enzinger in 1965, CCS is a deep soft tissue tumor associated with tendons and aponeuroses [3]. In 1993, the first visceral case of CCS in the duodenum was reported [4]; thereafter, about 36 cases have been reported [1,5–8] and their molecular features have been investigated [6–8]. Neoplastic cells usually express melanocytic markers including S-100, HMB45 and melan-A [9]; in fact, the most difficult differential diagnosis is with malignant melanoma (MM). Molecular-based tests are useful in discriminating these two tumors; CCS is consistently associated with the t(12;22) (q13;q12) translocation leading to the EWS/ATF1 fusion transcript. This has never been documented in cutaneous malignant melanoma and is considered specific for the diagnosis of CCS [7]. Many of CCS occurring in the gastrointestinal tract have lacked evidence of melanocytic differentiation, therefore, some authors have proposed the designation of clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT) as a new unrecognized entity [1,10,11]. Zambrano et al. reported 6 cases of CCSLTGT without melanocytic differentiation and with prominent osteoclast-like giant cells [12]. Very recently, Stockmann et al. described 16 cases of gastrointestinal neoplasms with morphologic, ultrastructural, immunohistochemical and molecular features similar to those described in CCSLTGT [1], characterized by sheets or nests of primitive epithelioid-to-oval or spindle tumor cells, with osteoclast-like giant cells. A primitive neural phenotype was postulated for the immunohistochemical profile (positivity for S100, SOX10, NSE, synaptophysin, CD56 and NB84 and negativity for HMB45, melan A, tyrosinase) [8]. Moreover, the ultrastructural examination revealed a neural differentiation, lacking melanocytic differentiation [1]. Genetically, they were characterized by EWSR1 gene rearrangements, including EWSR1-ATF1 or EWSR1-CREB1 fusions [6–8]. These tumors of uncertain lineage, do not have a normal cellular counterpart, however, an origin from a gastrointestinal neuroectodermal precursor cell was hypothesized, leading to the designation of “Malignant Gastrointestinal Neuroectodermal Tumor (GNET)” [1]. They occur mainly in young-aged and middle-aged adults and have a poor prognosis, usually developing lymph nodes and liver metastases. We report the case of a 29-year old man with a gastric tumor morphologically and immunohistochemically similar to those described in CCSLTGT, lacking osteoclast-like giant cells, harboring a rearrangement of EWSR1, and even with a positivity for FLI-1, which is not frequently described in this tumor. Our tumor also lacks the well-formed nests typical of CCS and any form of melanocytic differentiation. During the follow-up, hepatic metastases have been discovered. Interestingly, the patient was diagnosed with Ewing sarcoma when he was 5 year old, after a trauma to the sternum. The long-term survival of the patient is really surprising; after chemotherapic treatment, he had a long recurrence-free survival and, unexpectedly, after 24 years, he developed a GNET of the stomach. To the best of our knowledge, this is the first reported case with an association of these two tumors in the same patient. In fact,
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in the largest series of GNET is reported that “all tumors arose de novo without any history or evidence of a similar neoplasm in soft tissue or any other location” [1]. They both lack normal counterpart cell and variably display neuroectodermal differentiation. It is well known that with chemiotherapic treatment, a 5-year survival in Ewing sarcoma can reach 70% in case of localized disease [13], whereas clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT) usually displays a high local recurrence rate, nodal and visceral metastases and early death from disease [1,14]. It would have been interesting analyzing the lesion of the sternum and performing on this tissue immunohistochemical evaluation and molecular analysis, in order to confirm the diagnosis of Ewing sarcoma. Unfortunately, the paraffin blocks of the tissue related to the surgery of 1985, are no longer available (Italian law obliges laboratories to keep the blocks for a maximum of 20 years). EWSR1-CREB1 and EWSR1-ATF1 are gene fusions which have been described in five neoplasms with different morphology and behavior (angiomatoid fibrous histiocytoma, conventional CCS, CCSLTGT, hyalinizing clear cell sarcoma of the salivary gland and primary pulmonary myxoid sarcoma) [15]. EWSR1 is a prototypical “promiscuous gene,” with a propensity to fuse with a group of different genes. Its product is a member of the TET family of transcription factors. Rearrangements of this gene occur in a variety of mesenchymal tumors and other neoplasms (Ewing sarcoma, Ewing family of tumors, hyalinizing clear cell carcinoma of salivary gland, myoepithelial carcinoma, extraskeletal myxoid chondrosarcoma, myxoid liposarcoma, angiomatoid fibrous histiocytoma, and desmoplastic small round cell tumors) [1,15]. ATF1 encodes the cyclic AMP(cAMP)-dependent transcription factor ATF1, a protein member of the cAMP response element-binding protein (CREB)-ATF family of transcription factors that bind to cAMP-inducible promoters. ATF and CREB proteins are functionally related [15]. The fusion translocation EWSR1-ATF1 and EWSR1-CREB1 are reported in an increasing variety of related and unrelated neoplasms. The translocations could be an initial or early step in tumorigenesis and a secondary genetic or epigenetic event is required for the transformation to a specific tumor type [15]. All these different phenotypes of tumors with common molecular rearrangements are thus probably the result of the stage of maturation or differentiation of the affected precursor cells [1]. It appears obvious that genetic rearrangements should not be considered diagnostic or pathognomonic of specific neoplasms, in absence of other findings [1]. FLI1 is the most frequent partner of EWSR translocation in Ewing sarcoma, and it is a member of the ETS family of transcription factors, being normally expressed in hematopoietic, vascular and neural crest development [16]. The present case showed a strong immunohistochemical reactivity for FLI-1 on the primary tumor of the stomach, as well as on the liver metastases. This evidence, together with S100 immunoreactivity, could reinforce the hypothesis of a neuroectodermal origin for this tumor. The differential diagnosis of GNET includes GIST, monophasic synovial sarcoma (MSS), primary or metastatic malignant melanoma (MMA) and CCSTA. GISTs and GNETs may have a very similar morphology (epithelioid and/or spindle cells, sometimes cytoplasmic vacuoles, low mitotic rate), therefore the immunohistochemistry is reliable in distinguishing these 2 lesions, as all GIST markers (CD117, DOG1 and CD34) are negative in GNET [17]. MSS is characterized by the expression of epithelial markers, typically negative in GNET, as in our case. In cases of GNET showing an immunohistochemical profile overlapping with that of synovial sarcoma, Vilela et al. recently suggested the use of CD99 and SOX10 (positive in SS and negative in most GNET), useful for reaching a definitive diagnosis [18]. Although GNET is negative for HMB45 and MART-1, molecular studies are important to distinguish GNET from MMA (primary and metastatic) and from CCSTA.
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In conclusion, we believe that GNET should be considered in the differential diagnosis of gastrointestinal mesenchymal tumors. The coexistence of Ewing sarcoma and GNET in the same patient might be a further possible evidence that these two tumors share biological properties and have, at least partially, the same histogenesis. Therefore, further studies are essential to understand whether this tumor that is immunoreactive for S-100 protein and FLI-1, harbors EWSR1 translocations and lacks melanocytic differentiation, might represent a clinical, morphologic, immunohistochemical and genetic spectrum of the same tumor, or a new distinct entity, as recently proposed.
[8]
[9]
[10]
[11]
References [12] [1] D.L. Stockman, M. Miettinen, S. Suster, D. Spagnolo, H. Dominguez-Malagon, J.L. Hornick, V. Adsay, P.M. Chou, B. Amanuel, P. Vantuinen, E.V. Zambrano, Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reappraisal of clear cell sarcoma-like tumors of the gastrointestinal tract, Am. J. Surg. Pathol. 36 (6) (2012) 857–868. [2] K. Kosemehmetoglu, A.L. Folpe, Clear cell sarcoma of tendons and aponeuroses and osteoclast-rich-tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update, J. Clin. Pathol. 63 (5) (2010) 416–423. [3] E.B. Chung, F.M. Enzinger, Malignant melanoma of soft parts. A reassessment of clear cell sarcoma, Am. J. Surg. Pathol. 7 (1983) 405–413. [4] T.O. Ekfors, H. Kujari, M. Isomaki, Clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts) in the duodenum: the first visceral case, Histopathology 22 (1993) 255–259. [5] C.E. Comin, L. Novelli, D. Tornaboni, L. Messerini, Clear cell sarcoma of the ileum: report of a case and review of literature, Virchows Arch. 451 (2007) 839–845. [6] M. Covinsky, S. Gong, V. Rajaram, A. Perry, J. Pfeifer, EWS-ATF1 fusion transcripts in gastrointestinal tumors previously diagnosed as malignant melanoma, Hum. Pathol. 36 (2005) 74–81. [7] P.L. Lyle, C.M. Amato, J.E. Fitzpatrick, W.A. Robinson, Gastrointestinal melanoma or clear cell sarcoma? Molecular evaluation of 7 cases previously
[13]
[14]
[15] [16] [17]
[18]
diagnosed as malignant melanoma, Am. J. Surg. Pathol. 32 (2008) 858–866. C.R. Antonescu, K. Nafa, N.H. Segal, P. Dal Cin, M. Ladanyi, EWS-CREB1: a recurrent variant fusion clear cell sarcoma-association with gastrointestinal location and absence of melanocytic differentiation, Clin. Cancer Res. 12 (2006) 5356–5362. L. Taminelli, K. Zaman, C. Gengler, N. Peloponissios, H. Bouzourene, J.M. Coindre, I. Hostein, L. Guillou, Primary clear cell sarcoma of the ileum: an uncommon and misleading site, Virchows Arch. 447 (2005) 772– 777. P. Balkaransingh, S.A. Saad, S.C. Govil, P.K. Thind, C.M. Balance, A.R. Weiss, Clear cell sarcoma of the gastrointestinal tract presenting as a second malignant neoplasm following neuroblastoma in infancy, Pediatr. Blood Cancer 58 (March (3)) (2012) 481–482. P. Pauwels, M. Debiec-Rychter, R. Sciot, T. Vlasveld, B. den Butter, A. Hagemeijer, P.C. Hogendoorn, Clear cell sarcoma of the stomach, Histopathology 41 (2002) 526–530. E. Zambrano, M. Reyes-Mugica, A. Franchi, J. Rosai, An osteoclast-rich tumor of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: reports of 6 cases of a GIST simulator, Int. J. Surg. Pathol. 11 (2003) 75–81. M.A. Applebaum, J. Worch, K.K. Matthaay, R. Goldsby, J. Neuhaus, D.C. west, S.G. Dubois, Clinical features and outcomes in patients with extraskeletal, Ewing sarcoma, Cancer 117 (2011) 3027–3032. K. Kosemehmetoglu, A.L. Folpe, Clear cell sarcoma of tendons and aponeuroses, and osteoclast-rich tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update, J. Clin. Pathol. 63 (2010) 416–423. K. Thway, C. Fisher, Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status, Am. J. Surg. Pathol. 36 (7) (2012) e1–e11. S. Sankar, S.L. Lessnick, Promiscuous partnerships in Ewing’s sarcoma, Cancer Genet. 204 (July (7)) (2011) 351–365. M. Miettinem, Z.F. Wang, J. Lasota, DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 cases, Am. J. Surg. Pathol. 33 (2009) 1401–1408. D. Suarez-Vilela, F.M. Izquierdo, S. Tojo-Ramallo, J. Riera-Velasco, J. Escobar-Velasco, Letters to editor: Malignant gastrointestinal neuroectodermal tumor showing overlapped immunophenotype with synovial sarcoma. CD99 and SOX 10 antibodies are useful in differential diagnosis, Am. J. Surg. Pathol. 36 (12) (2012) 1905–1907.
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
An unusual association of malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma-like) and Ewing sarcoma Luigi Insabato a,∗ , Elia Guadagno a , Valentina Natella a , Anna Somma a , Michel Bihl b , Antonio Pizzolorusso c , Pier Paolo Mainenti a , Gaetano Apice c , Luigi Tornillo b a
Dip. Scienze Biomorfologiche e Chirurgiche, Università Federico II Napoli, Italy Department of Pathology, University Hospital of Basel, Switzerland c SSD Oncologia medica Sarcomi dei Tessuti molli e delle Ossa, Istituto Tumori, Fondazione G. Pascale, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 5 November 2014 Received in revised form 4 June 2015 Accepted 5 June 2015 Keyword: Clear cell sarcoma-like gastric tumor
a b s t r a c t Very recently a new designation of “Malignant Neuroectodermal Gastrointestinal Tumor” has been proposed for an aggressive form of neuroectodermal tumor with features similar to that of Clear Cell Sarcoma of Soft Tissue, however without a melanocytic differentiation. Also known as “clear cell sarcoma-like tumors of the gastrointestinal tract”, these tumors show some features strongly suggesting an origin from a gastrointestinal neuroectodermal precursor cell unable to differentiate along the melanocytic lineage. They occur mainly in young and middle-aged adults, and have a poor prognosis with a high rate of liver and lymphnode metastases. Histologically they are composed of epithelioid or oval-to spindle cells with a sheet-like or nested pattern of growth, strongly positive for neural markers (S-100, SOX10, and vimentin) and negative for the melanocytic ones. EWSR1 gene rearrangements including EWSR1-ATF1 or EWSR1-CREB1 GENE fusions are typically assessed in these tumors. Here we report a case of malignant neuroectodermal gastrointestinal tumor which immunophenotypically unusually expressed FLI-1, occurring in a 29-year-old man with a previous medical history of Ewing sarcoma. We finally suggest that this case might be a further evidence of a link between these two entities. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction
2. Clinical history
Malignant gastrointestinal neuroectodermal tumor (GNET) is a new disease entity, recently described [1]. It might be considered an aggressive form of a specific neuroectodermal tumor that is clinically, morphologically, and immunohistochemically different from any other primitive tumor of the gastrointestinal tract. Its morphology is similar to the rare Clear Cell Sarcoma of soft tissue (CCSST), involving the gastrointestinal tract [2], but without the evidence of melanocytic differentiation. These two entities share features of molecular genetics, harboring a distinctive chromosomal translocation involving EWSR1-ATF1 t (12;22) (q13;q12). Here is presented a rare case of GNET of the stomach, occurring in a patient with a previous diagnosis of Ewing sarcoma.
A 29 year-old man was admitted in our hospital for acute gastric pain, in January 2009. The magnetic resonance imaging showed a solid submucosal mass localized in the wall of the gastric antrum (Fig. 1a). A clinical diagnosis of gastric GIST had been done, so the patient underwent a partial gastrectomy. On the pathological examination, the diagnosis was that of a mesenchymal tumor, probably originating from a peripheral nerve. A follow-up was mandatory. In January 2012, during one of his periodic check-ups, the patient made an abdominal ultrasonography which revealed the presence of three nodules, respectively at the II, III and IV segment of the liver, the largest of which measured 24 mm; a percutaneous liver biopsy was made. In the medical record it was reported that the patient, at the age of 5 years, in 1985, after a trauma to the sternum, caused by a fall from the bike, was diagnosed a Ewing’s Sarcoma of the left XI rib; this diagnosis had been made in another hospital (Fig. 1b). Therefore, he was treated with five cycles of combined chemotherapy which led to a total remission from the disease.
∗ Corresponding author. Tel.: +39 0817463442; fax: +39 0817463475. E-mail address: [email protected] (L. Insabato). http://dx.doi.org/10.1016/j.prp.2015.06.001 0344-0338/© 2015 Elsevier GmbH. All rights reserved.
L. Insabato et al. / Pathology – Research and Practice 211 (2015) 688–692
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Switzerland. FISH analysis was performed using a commercial probe (ZytoLight® SPEC EWSR1, ZytoVision GmbH·Fischkai 1 27572 Bremerhaven·Germany). 4-mm-tick slides were pretreated for 15 with pretreatment solution (Vysis® , Abbott Laboratories, Abbott Park, IL), digested with protease I for 70 (Vysis® , Abbott Laboratories, Abbott Park, IL) and afterwards incubated overnight with the ZytoLight® SPEC EWSR1 probe, a mixture of two direct labeled probes hybridizing to the 22q12.1–q12.2 band. The orange fluorochrome direct labeled probe hybridizes the proximal end of the band and extends inward into intron 4 of the EWSR1 gene while the green fluorochrome direct labeled probe hybridizes the distal end of the band. The detection of orange and green signals separated by more than 1.5 probe diameter indicates that the locus 22q12.1–q12.2 is affected by a translocation. 100 non-overlapping nuclei were evaluated. The presence of a translocation, as defined above, in more than 15% of the nuclei was considered positive. 3.4. RT-PCR
Fig. 1. (a) MR imaging: solid submucosal mass localized at the level of the gastric antrum wall (arrow). (b) 99mTC-Methylene diphosphonate bone scintigraphy (posterior view): focal tracer uptake at the level of the left XI rib (black arrow).
3. Materials and methods 3.1. Immunohistochemical analysis Immunohistochemistry was conducted on selected formalinfixed, paraffin-embedded blocks and performed using a fully automated system. The following commercially available antibodies were used: S-100 (Medite, Nunningen, Switzerland, 1:10,000), vimentin (Boehringer Mannheim Biochemica Mannheim, Germany, 1:10), CD56 (Vector, Burlingame, CA, 1:100), NSE (Dakopatts, Glostrup, Denmark, 1:100), keratin AE1/AE3 (Dako, Carpinteria, CA, 1:300), EMA (Ventana Medical Systems, Tucson, AZ, prediluted), CD117 (Dako, 1:200), CD99 (Covance Inc., Princeton, NJ, 1:400), CD34 (Immunotech Beckam Coulter, Inc., Fullerton, CA, 1:160), CD31 (Dako, 1:40), synaptophysin (Dako, 1:25), chromogranin-A (Dako, 1:3000), HMB45 (Dakopatts, Glostrup, Denmark, 1:50), MART-1 (Dakopatts, Glostrup, Denmark, 1:50), actin (Enzo Diagnostics, New York, USA, 1:16,000), desmin (Dakopatts Glostrup, Denmark, 1:900), and FLI1 (Cell Marque, Rocklin, CA, 1:100). 3.2. Electron microscopy The formalin-fixed, paraffin-embedded tissue sample of gastric tumor was de-waxed, rehydrated, fixed in glutaraldehyde, and then processed for electron microscopy. 3.3. Florescence in situ hybridization A genetic analysis by FISH technique was carried out at the pathology laboratory of the University of Basel, in
Slides were dewaxed with xylene three times for 5 min each, hydrated through alcohol gradient ethanol (100%, 95%, 70%, 50% ethanol) and then rinsed with tap water. Cells from selected area were scraped and collected into a 2 ml Eppendorf. RNA extraction from cell pellet was performed using the Trizol/chloroform method (Invitrogen). One to two ug of the total RNA was reverse-transcribed to cDNA, using the Gene Amp® RNA PCR (Applied Biosystem) kit. The reaction was conducted at 37 ◦ C for 60 min and then the samples were heated to 95 ◦ C for 5 min to inactivate the reverse transcriptase. The cDNA was amplified using specific primers for the FIP1L1PDGFRA translocation. For the first PCR we used the following primers: FIP1L1 ex8 F1: 5 -ACCTGGTGCTGATCTTTCTGAT-3 PDGFRA ex14 R1: 5 -TGAGAGCTTGTTTTTCACTGGA-3 ; while for the nested PCR the primers were: FIP1L1 Ex8 F2: 5 -AAAGAGGTACGAATGGGACTTG-3 PDGFRA ex13 R2: 5 -GGGACCGGCTTAATCCATAG-3 . A fragment of 772 bp from the YY1 gene was also independently amplified, in order to verify the quality of the cDNA fragments. YY1 primers were: YY1-851 F: 5 -AGTGGGAGCAGAAGCAGGTGC-3 YY1-1623 R: 5 -AAACATGTCCCTTAGGTGTGTAGGATTC-3 . Forty repeated cycles of denaturation at 95 ◦ C for 20 s, the primer annealing at 55 ◦ C for 10 s (FIP1L1-PDGFRA) or at 62 ◦ C for 30 s (YY1 gene), elongation at 72 ◦ C 40 s, followed by a final elongation at 72 ◦ C 7 min, were carried out. 4. Results Microscopically, the gastric tumor was composed of epithelioid and spindle cells arranged in nests and cords, with abundant clear cytoplasm, slightly pleomorphic nuclei with occasionally prominent nucleoli, low mitotic activity, and without necrosis (Fig. 2a and b); neoplastic cells infiltrated almost the whole thickness of the gastric wall, sparing the mucosa. Immunohistochemical investigation revealed positivity for the S-100 protein and vimentin, and negativity for actin, desmin and CD117. Because of the immunoreactivity for the S-100 protein, the tumor was diagnosed as a mesenchymal tumor, possibly of peripheral nerve origin. Three years later, the microscopic examination of the percutaneous liver biopsy showed a patternless tumor, composed of round to oval cells with scant eosinophilic cytoplasm, with prominent nucleoli, and low mitotic activity (Fig. 2c and d), highly suggestive for a liver metastasis. Having got this evidence, the slides of the gastric tumor were reexamined: we came across a lesion
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Fig. 2. Gastric and hepatic tumors. In the stomach the tumor was mainly composed of clear cells arranged in solid nests, lacking tumor necrosis (a, H-E stain, 20×) (b, H-E stain, 40×), while in the liver neoplastic cells were round-shaped with an eosinophilic cytoplasm (c, H-E stain, 10×) (d, H-E stain, 40×). Both of them were strongly immunoreactive for S100 (e, f) and for FLI-1 (g). Fish analysis: a translocation was found in more than 15% of the nuclei in both lesions (h).
that was morphologically different from the hepatic one. Infact, in the antrum, tumor cells had a clear and abundant cytoplasm. A detailed immunohistochemical study was performed on both tumors. The hepatic tumor, exactly like the gastric one, was strongly immunoreactive for S-100 protein (Fig. 2e and f), FLI-1 (Fig. 2g) and vimentin, and focally positive for 56 and NSE. Keratin AE1/AE3, EMA, CD117, CD99, CD34, CD31, synaptophysin, chromogranin, HMB45, and MART-1 were all negative. On this basis, the diagnosis of clear cell sarcoma-like tumor of the stomach with liver metastasis was suggested. On electron microscope, the gastric tumor tissue did not show the presence of melanosomes.
On FISH analysis, 100 non-overlapping nuclei were evaluated from the tissue of the gastric tumor and from the hepatic metastasis. A translocation of EWSR1 gene was found in 27% and 42% of the nuclei, respectively, indicating a clear-cut positivity (Fig. 2h), while RT-PCR for FLI-1 was negative. Finally, a diagnosis of primitive malignant gastric neuroectodermal tumor (clear cell sarcoma-like tumor) with liver metastases was made. After this diagnosis, the patient underwent a total body computerized tomography with contrast, confirming the presence of hepatic nodular lesions and showing multiple hyperdense bone lesions, thoracic lymph nodes enlargement and peritoneal thickening in correspondence of the stomach, the jejunum and the
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pancreas. In September 2012, 10 cycles of chemotherapy with etoposide–ifosfamide were started and, at the end of this therapy, the enlargement of lymph nodes and the peritoneal involvement had slightly regressed; this partial regression of disease remained stable for about 8 months, after which a treatment with 3 cycles of ifosfamide was made. On the last follow-up, in March 2015, the patient was in stable disease.
5. Discussion Among all mesenchymal tumors of the gastrointestinal tract, a small number of neoplasms with features of clear cell sarcoma (CCS) have been reported. Originally described by Enzinger in 1965, CCS is a deep soft tissue tumor associated with tendons and aponeuroses [3]. In 1993, the first visceral case of CCS in the duodenum was reported [4]; thereafter, about 36 cases have been reported [1,5–8] and their molecular features have been investigated [6–8]. Neoplastic cells usually express melanocytic markers including S-100, HMB45 and melan-A [9]; in fact, the most difficult differential diagnosis is with malignant melanoma (MM). Molecular-based tests are useful in discriminating these two tumors; CCS is consistently associated with the t(12;22) (q13;q12) translocation leading to the EWS/ATF1 fusion transcript. This has never been documented in cutaneous malignant melanoma and is considered specific for the diagnosis of CCS [7]. Many of CCS occurring in the gastrointestinal tract have lacked evidence of melanocytic differentiation, therefore, some authors have proposed the designation of clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT) as a new unrecognized entity [1,10,11]. Zambrano et al. reported 6 cases of CCSLTGT without melanocytic differentiation and with prominent osteoclast-like giant cells [12]. Very recently, Stockmann et al. described 16 cases of gastrointestinal neoplasms with morphologic, ultrastructural, immunohistochemical and molecular features similar to those described in CCSLTGT [1], characterized by sheets or nests of primitive epithelioid-to-oval or spindle tumor cells, with osteoclast-like giant cells. A primitive neural phenotype was postulated for the immunohistochemical profile (positivity for S100, SOX10, NSE, synaptophysin, CD56 and NB84 and negativity for HMB45, melan A, tyrosinase) [8]. Moreover, the ultrastructural examination revealed a neural differentiation, lacking melanocytic differentiation [1]. Genetically, they were characterized by EWSR1 gene rearrangements, including EWSR1-ATF1 or EWSR1-CREB1 fusions [6–8]. These tumors of uncertain lineage, do not have a normal cellular counterpart, however, an origin from a gastrointestinal neuroectodermal precursor cell was hypothesized, leading to the designation of “Malignant Gastrointestinal Neuroectodermal Tumor (GNET)” [1]. They occur mainly in young-aged and middle-aged adults and have a poor prognosis, usually developing lymph nodes and liver metastases. We report the case of a 29-year old man with a gastric tumor morphologically and immunohistochemically similar to those described in CCSLTGT, lacking osteoclast-like giant cells, harboring a rearrangement of EWSR1, and even with a positivity for FLI-1, which is not frequently described in this tumor. Our tumor also lacks the well-formed nests typical of CCS and any form of melanocytic differentiation. During the follow-up, hepatic metastases have been discovered. Interestingly, the patient was diagnosed with Ewing sarcoma when he was 5 year old, after a trauma to the sternum. The long-term survival of the patient is really surprising; after chemotherapic treatment, he had a long recurrence-free survival and, unexpectedly, after 24 years, he developed a GNET of the stomach. To the best of our knowledge, this is the first reported case with an association of these two tumors in the same patient. In fact,
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in the largest series of GNET is reported that “all tumors arose de novo without any history or evidence of a similar neoplasm in soft tissue or any other location” [1]. They both lack normal counterpart cell and variably display neuroectodermal differentiation. It is well known that with chemiotherapic treatment, a 5-year survival in Ewing sarcoma can reach 70% in case of localized disease [13], whereas clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT) usually displays a high local recurrence rate, nodal and visceral metastases and early death from disease [1,14]. It would have been interesting analyzing the lesion of the sternum and performing on this tissue immunohistochemical evaluation and molecular analysis, in order to confirm the diagnosis of Ewing sarcoma. Unfortunately, the paraffin blocks of the tissue related to the surgery of 1985, are no longer available (Italian law obliges laboratories to keep the blocks for a maximum of 20 years). EWSR1-CREB1 and EWSR1-ATF1 are gene fusions which have been described in five neoplasms with different morphology and behavior (angiomatoid fibrous histiocytoma, conventional CCS, CCSLTGT, hyalinizing clear cell sarcoma of the salivary gland and primary pulmonary myxoid sarcoma) [15]. EWSR1 is a prototypical “promiscuous gene,” with a propensity to fuse with a group of different genes. Its product is a member of the TET family of transcription factors. Rearrangements of this gene occur in a variety of mesenchymal tumors and other neoplasms (Ewing sarcoma, Ewing family of tumors, hyalinizing clear cell carcinoma of salivary gland, myoepithelial carcinoma, extraskeletal myxoid chondrosarcoma, myxoid liposarcoma, angiomatoid fibrous histiocytoma, and desmoplastic small round cell tumors) [1,15]. ATF1 encodes the cyclic AMP(cAMP)-dependent transcription factor ATF1, a protein member of the cAMP response element-binding protein (CREB)-ATF family of transcription factors that bind to cAMP-inducible promoters. ATF and CREB proteins are functionally related [15]. The fusion translocation EWSR1-ATF1 and EWSR1-CREB1 are reported in an increasing variety of related and unrelated neoplasms. The translocations could be an initial or early step in tumorigenesis and a secondary genetic or epigenetic event is required for the transformation to a specific tumor type [15]. All these different phenotypes of tumors with common molecular rearrangements are thus probably the result of the stage of maturation or differentiation of the affected precursor cells [1]. It appears obvious that genetic rearrangements should not be considered diagnostic or pathognomonic of specific neoplasms, in absence of other findings [1]. FLI1 is the most frequent partner of EWSR translocation in Ewing sarcoma, and it is a member of the ETS family of transcription factors, being normally expressed in hematopoietic, vascular and neural crest development [16]. The present case showed a strong immunohistochemical reactivity for FLI-1 on the primary tumor of the stomach, as well as on the liver metastases. This evidence, together with S100 immunoreactivity, could reinforce the hypothesis of a neuroectodermal origin for this tumor. The differential diagnosis of GNET includes GIST, monophasic synovial sarcoma (MSS), primary or metastatic malignant melanoma (MMA) and CCSTA. GISTs and GNETs may have a very similar morphology (epithelioid and/or spindle cells, sometimes cytoplasmic vacuoles, low mitotic rate), therefore the immunohistochemistry is reliable in distinguishing these 2 lesions, as all GIST markers (CD117, DOG1 and CD34) are negative in GNET [17]. MSS is characterized by the expression of epithelial markers, typically negative in GNET, as in our case. In cases of GNET showing an immunohistochemical profile overlapping with that of synovial sarcoma, Vilela et al. recently suggested the use of CD99 and SOX10 (positive in SS and negative in most GNET), useful for reaching a definitive diagnosis [18]. Although GNET is negative for HMB45 and MART-1, molecular studies are important to distinguish GNET from MMA (primary and metastatic) and from CCSTA.
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In conclusion, we believe that GNET should be considered in the differential diagnosis of gastrointestinal mesenchymal tumors. The coexistence of Ewing sarcoma and GNET in the same patient might be a further possible evidence that these two tumors share biological properties and have, at least partially, the same histogenesis. Therefore, further studies are essential to understand whether this tumor that is immunoreactive for S-100 protein and FLI-1, harbors EWSR1 translocations and lacks melanocytic differentiation, might represent a clinical, morphologic, immunohistochemical and genetic spectrum of the same tumor, or a new distinct entity, as recently proposed.
[8]
[9]
[10]
[11]
References [12] [1] D.L. Stockman, M. Miettinen, S. Suster, D. Spagnolo, H. Dominguez-Malagon, J.L. Hornick, V. Adsay, P.M. Chou, B. Amanuel, P. Vantuinen, E.V. Zambrano, Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reappraisal of clear cell sarcoma-like tumors of the gastrointestinal tract, Am. J. Surg. Pathol. 36 (6) (2012) 857–868. [2] K. Kosemehmetoglu, A.L. Folpe, Clear cell sarcoma of tendons and aponeuroses and osteoclast-rich-tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update, J. Clin. Pathol. 63 (5) (2010) 416–423. [3] E.B. Chung, F.M. Enzinger, Malignant melanoma of soft parts. A reassessment of clear cell sarcoma, Am. J. Surg. Pathol. 7 (1983) 405–413. [4] T.O. Ekfors, H. Kujari, M. Isomaki, Clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts) in the duodenum: the first visceral case, Histopathology 22 (1993) 255–259. [5] C.E. Comin, L. Novelli, D. Tornaboni, L. Messerini, Clear cell sarcoma of the ileum: report of a case and review of literature, Virchows Arch. 451 (2007) 839–845. [6] M. Covinsky, S. Gong, V. Rajaram, A. Perry, J. Pfeifer, EWS-ATF1 fusion transcripts in gastrointestinal tumors previously diagnosed as malignant melanoma, Hum. Pathol. 36 (2005) 74–81. [7] P.L. Lyle, C.M. Amato, J.E. Fitzpatrick, W.A. Robinson, Gastrointestinal melanoma or clear cell sarcoma? Molecular evaluation of 7 cases previously
[13]
[14]
[15] [16] [17]
[18]
diagnosed as malignant melanoma, Am. J. Surg. Pathol. 32 (2008) 858–866. C.R. Antonescu, K. Nafa, N.H. Segal, P. Dal Cin, M. Ladanyi, EWS-CREB1: a recurrent variant fusion clear cell sarcoma-association with gastrointestinal location and absence of melanocytic differentiation, Clin. Cancer Res. 12 (2006) 5356–5362. L. Taminelli, K. Zaman, C. Gengler, N. Peloponissios, H. Bouzourene, J.M. Coindre, I. Hostein, L. Guillou, Primary clear cell sarcoma of the ileum: an uncommon and misleading site, Virchows Arch. 447 (2005) 772– 777. P. Balkaransingh, S.A. Saad, S.C. Govil, P.K. Thind, C.M. Balance, A.R. Weiss, Clear cell sarcoma of the gastrointestinal tract presenting as a second malignant neoplasm following neuroblastoma in infancy, Pediatr. Blood Cancer 58 (March (3)) (2012) 481–482. P. Pauwels, M. Debiec-Rychter, R. Sciot, T. Vlasveld, B. den Butter, A. Hagemeijer, P.C. Hogendoorn, Clear cell sarcoma of the stomach, Histopathology 41 (2002) 526–530. E. Zambrano, M. Reyes-Mugica, A. Franchi, J. Rosai, An osteoclast-rich tumor of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: reports of 6 cases of a GIST simulator, Int. J. Surg. Pathol. 11 (2003) 75–81. M.A. Applebaum, J. Worch, K.K. Matthaay, R. Goldsby, J. Neuhaus, D.C. west, S.G. Dubois, Clinical features and outcomes in patients with extraskeletal, Ewing sarcoma, Cancer 117 (2011) 3027–3032. K. Kosemehmetoglu, A.L. Folpe, Clear cell sarcoma of tendons and aponeuroses, and osteoclast-rich tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update, J. Clin. Pathol. 63 (2010) 416–423. K. Thway, C. Fisher, Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status, Am. J. Surg. Pathol. 36 (7) (2012) e1–e11. S. Sankar, S.L. Lessnick, Promiscuous partnerships in Ewing’s sarcoma, Cancer Genet. 204 (July (7)) (2011) 351–365. M. Miettinem, Z.F. Wang, J. Lasota, DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 cases, Am. J. Surg. Pathol. 33 (2009) 1401–1408. D. Suarez-Vilela, F.M. Izquierdo, S. Tojo-Ramallo, J. Riera-Velasco, J. Escobar-Velasco, Letters to editor: Malignant gastrointestinal neuroectodermal tumor showing overlapped immunophenotype with synovial sarcoma. CD99 and SOX 10 antibodies are useful in differential diagnosis, Am. J. Surg. Pathol. 36 (12) (2012) 1905–1907.