Intra-abdominal EWSR1/FUS-CREM-rearranged malignant epithelioid neoplasms: two cases of an emerging aggressive entity with emphasis on misleading immunophenotype

CREB family (CREB1, ATF1, and CREM) gene fusions are defining markers in diverse mesenchymal neoplasms (clear cell sarcoma, angiomatoid fibrous histiocytoma, and others). However, neoplasms harboring EWSR1-CREM/FUS-CREM fusions are rare and poorly characterized. We describe two cases (55-year-old male with 7.5 cm renal mass and 32-year-old female with 5.5 cm mesenteric mass) illustrating their misleading immunophenotypes. Histologically, both showed eosinophilic and focally clear epithelioid cells arranged into sheets, nests, and trabeculae. Immunohistochemistry showed ALK, EMA, and AE1/AE3 immunoreactivity suggesting ALK-rearranged renal cell carcinoma (Case 1) and coexpression of keratin, EMA, synaptophysin, and chromogranin-A, suggesting neuroendocrine neoplasm (Case 2). Targeted RNA sequencing revealed EWSR1-CREM (Case 1) and FUS-CREM (Case 2) fusions. These cases add to the spectrum of CREM fusion-positive intra-abdominal epithelioid neoplasms. Their unusual immunophenotype and unexpected sites represent major pitfalls, underline a wide differential diagnosis, and emphasize the value of molecular testing in correctly diagnosing them.


Introduction
Gene fusions involving the Ewing Sarcoma Breakpoint Region 1 gene (EWSR1 on 22q) and one of the CREB family genes (CREB1, ATF1, and CREM) have been increasingly recognized in soft tissue and visceral neoplasms with significant phenotypic and demographic diversity [1]. The major representatives of this category are clear cell sarcoma (CCS) of tendon and aponeuroses (mainly EWSR1-ATF1 fusions), malignant gastrointestinal neuroectodermal tumor (mainly EWSR1-CREB1 fusions), angiomatoid fibrous histiocytoma (mainly EWSR1-CREB1 fusions), and clear cell carcinomas of the head and neck (EWSR1-ATF1 fusions) [1]. As a group, these neoplasms differ significantly in morphology, immunophenotypes, and behavior, ranging from low-grade indolent (e. g., angiomatoid fibrous histiocytoma) to aggressive (e.g., CCS) [1]. Within the CREB fusion family, CREM-rearranged neoplasms are rare. They include subsets of CCS, angiomatoid fibrous histiocytomas, and clear cell carcinoma of the head and neck [2]. We describe two CREM-rearranged intra-abdominal malignant epithelioid neoplasms (one renal and one mesenteric) displaying confusing immunophenotypes: expression of ALK and keratin (Case 1) and keratin and neuroendocrine markers (Case 2). In both, diagnosis was only made after NGS testing uncovered the underlying fusions.

Molecular testing
RNA-based NGS was performed as described previously [3]. ALK/EWSR1 (Case 1) and FUS/TFE3 (Case 2) FISH analyses were performed using ZytoLight® SPEC Dual Color Break Apart Probes (ZytoVision, Bremerhaven, Germany) designed to detect translocations involving these genes, respectively. Case 2 was tested for SDHB mutations using the methods described previously [4].

Case 1
A 55-year-old male underwent radical nephrectomy for a renal mass. His medical history was negative for other malignancies. He is currently diagnosed with metastatic disease in the pelvis 23 months from initial diagnosis.

Case 2
A 32-year-old female without other primary malignancy presented with a mesenteric mass that was resected together with the adjacent small bowel segment. She presented 8 months later with peritoneal, pleural, and lymph node metastases. Currently (13 months from initial diagnosis), she is alive with extensive progressive disease under palliative chemotherapy.

Molecular findings
With a provisional diagnosis of ALK + renal cell carcinoma (RCC), ALK FISH failed to show rearrangement or copy number gains in Case 1. Targeted RNA sequencing revealed a EWSR1-CREM fusion (Fig. 3), which was confirmed by EWSR1 FISH. Case 2 (mesenteric tumor) showed a FUS-CREM fusion (Fig. 3) which was also confirmed by FUS FISH. NGS (Case 2) revealed no SDHB mutation (which was suggested by the immunohistochemical loss of the protein).

Discussion
With increasing use of targeted RNA NGS in routine surgical pathology practice, the numbers and variants of gene fusions detected in neoplasms have been increasing, and several new molecularly defined entities are emerging. EWSR1/FUS-CREM fusions were recognized recently in a group of unclassified epithelioid mesenchymal neoplasms showing predilection for intra-abdominal organs, not fitting any known EWSR1-CREB-rearranged entity [1,2]. We herein expand the topographic and phenotypic features of these emerging neoplasms and highlight increasing pitfalls associated with them. Including our cases, 12 EWSR1/ FUS-CREM-positive intra-abdominal epithelioid neoplasms have been reported [2,5,6]. Affected sites were omentum/ mesentery (4), stomach (2), kidney (2), pelvic organs (2) [2,7]. These neoplasms possess a definite malignant potential with propensity for peritoneal recurrences and nodal and distant metastasis [2,5,6].
The morphology of these tumors seems distinctive. They all displayed monotonous epithelioid morphology with variable cytoplasmic clearing and sparse fibrous stroma. Most cases expressed keratin and EMA [2,5,6]. ALK was positive in one (intra-abdominal [2]), synaptophysin in two (chest wall and lung [2,7]), and MUC4 in two (chest wall and gastric [2,6]) previous cases. We add the expression of ALK, MUC4, and synaptophysin in one case each and report novel chromogranin-A expression in one.
Although the MUC4-positive gastric tumor was diagnosed as sclerosing epithelioid fibrosarcoma (SEF), the depicted histology suggests similarity to the current cases with variable clear cell epithelioid pattern, lacking characteristic sclerosis of SEF [6]. Another reported EWSR1-CREMpositive SEF was not illustrated [8]. Including our cases, 3/5 tumors tested positive for MUC4. This finding is relevant given that MUC4 expression and presence of EWSR1 fusion by FISH are otherwise considered diagnostic of SEF in the context of epithelioid mesenchymal neoplasms [9]. However, distinction of the two entities seems justified to address their potentially different biology. Most SEFs are driven by EWSR1-CREB3L1 and rarely by FUS-CREB3L1 fusions [1].
Our renal case expands the spectrum of EWSR1-rearranged renal sarcomas and represents the second EWSR1-CREM fusion-positive renal case [5]. Moreover, it represents true pitfall as detection of ALK in eosinophilic RCC strongly suggests an ALK-rearranged RCC [10]. Indeed, this was our initial diagnosis. The absence of PAX8 suggests alternative diagnoses and justifies further molecular testing in such a case. ALK expression is common in several translocation sarcomas including angiomatoid fibrous histiocytoma [11] and rhabdomyosarcoma [12]. The molecular basis of ALK immunoreactivity observed in diverse fusion-associated soft tissue tumors seems heterogeneous and associates with specific types of fusions (e.g., EWSR1/FUS-TFCP2 and EWSR1 but not MEIS1-NCOA2 and other fusions [11,12]). While the ALK protein expression in alveolar rhabdomyosarcoma and neuroblastoma correlates frequently with ALK copy number gains, none of angiomatoid fibrous histiocytoma showed this feature [11]. ALK might represent a downstream target of EWSR1 and PAX-FOXO1 fusion proteins, but this remains speculative [11,12].  A, B). C Focal areas displayed rhabdoid or spindle-shaped morphology. D A pseudotrabecular pattern within desmoplastic stroma was seen focally. Immunohistochemistry revealed strong expression of synaptophysin (E) and chromogranin-A (F) Regarding Case 2, it is noteworthy that it labeled not only for synaptophysin but also for chromogranin-A, a combination that we encountered only rarely in mesenchymal neoplasms with neuroendocrine features (data not shown). Since chromogranin-A normally resides in the membrane of neurosecretory granules, we studied the tumor tissue by electron microscopy to search for neurosecretory granules but could not identify any. We conclude that chromogranin-A protein associates with other cell organelles than secretory granules in this neoplasm. Loss of SDHB in this case remained unexplained and represents a further confusing factor with neuroendocrine neoplasms. Finally, aberrant or equivocal TFE3 expression noted in Case 2 and in a previous case [7] may suggest a TFE3-rearranged neoplasm. This is particularly important in renal cases as rare Xp11-translocation RCC may harbor EWSR1-TFE3 fusions and be indistinguishable from EWSR1/FUS-CREM neoplasms by FISH alone [13]. All these observations underline the morphological and immunophenotypic heterogeneity of EWSR1/FUS-CREM fusion driven mesenchymal neoplasms.
In summary, we report two intra-abdominal malignant epithelioid mesenchymal neoplasms carrying the recently described EWSR1-CREM and FUS-CREM fusions. Besides their epithelioid morphology, these tumors displayed unusual misleading immunophenotypes. Recognition of these findings should help to avoid misinterpretation as SEF, ALK-rearranged RCC, and neuroendocrine carcinoma. The cases highlight the value of precise genotyping in these challenging lesions to ovoid misdiagnoses.
Author contribution AA, GK: conception and design of the work; acquisition, analysis, and interpretation of data; and drafting the MS and revising it critically for important intellectual content and scientific integrity.
AA, RS, MO, JHB, NP, BK, AK, AH, and GK: acquisition, analysis, and interpretation of data and reading and revising the MS critically for important intellectual content and scientific integrity. All authors have read and approved the final manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL.