Complete mimicry: Rhabdomyosarcoma with FUS::TFCP2 fusion masquerading as carcinoma—diagnostic challenge and report of two cases

Rhabdomyosarcomas (RMS) are malignant mesenchymal tumors with skeletal muscle differentiation which are classified into alveolar, embryonal, pleomorphic, and spindle cell/sclerosing RMS. Within the spindle cell/sclerosing RMS tumor type there is a recently recognized sub‐type categorized as intraosseous spindle cell RMS with TFCP2/NCOA2 gene fusion. This rare tumor is highly aggressive with predominant involvement of the craniofacial and pelvic bones with approximately 30 cases reported to date. Histopathologic features include spindle cell and epithelioid morphology with a characteristic co‐expression of epithelial markers, myogenic markers, and ALK1 expression. We report two cases of gnathic spindle cell/sclerosing RMS with FUS::TFCP2 gene fusion that were initially interpreted as carcinomas by referring institutions and later reclassified when encountered in our practice after additional work‐up and molecular characterization.


| INTRODUCTION
Rhabdomyosarcomas (RMS) are malignant mesenchymal tumors with skeletal muscle differentiation. 1 The current 2020 WHO classification of tumors of soft tissue and bone subdivides RMS according to their morphology into alveolar, embryonal, pleomorphic, and spindle cell/sclerosing RMS (ssRMS). 2 Recent advances in molecular genetic diagnostics have made it possible to identify new RMS subgroups within the traditional morphological entities. ssRMS has been recently classified into three subgroups based on molecular abnormalities: (1) MYOD1-mutant ssRMS, (2) congenital/infantile spindle cell RMS with VGLL2/NCOA2/CITED2 gene fusion, and (3) intraosseous spindle cell RMS (IORMS) with TFCP2/NCOA2 gene fusion. 3,4 Although these tumors belong to the same morphological group, their clinical behavior is heterogenous. 5,6 Primary IORMS is defined by EWSR1/FUS::TFCP2 or, less commonly, MEIS1::NCOA2 gene fusion. 7 This rare neoplasm accounts for 5%-13% of all RMS 8 and is characterized by an aggressive clinical course, with predominant involvement of the craniofacial and pelvic bones. 9,10 This entity is characterized by a proliferation of spindle and epithelioid cells arranged in sheets and fascicles, with expression of myogenic markers, ALK1, and cytokeratin. 6    gene fusion, which affects mainly young female patients. 3,9,10 RMS with TFCP2-fusion is a rare tumor with a mixed spindle and epithelioid morphology, and a unique immunophenotypic profile. 6 We report two cases of RMS with FUS::TFCP2 fusion (Figure 3), which were classified as carcinoma during initial examination, but later re-classified after additional IHC and molecular work-up.

| DIAGNOSTIC CHALLENGE
In Case 1, initial work-up showed positive AE1/AE3, SMA, calponin, and S100; while negative for myogenin and p63. Myoepithelial carcinomas may show a variety of morphologic growth patterns including epithelioid cells with a high nuclear to cytoplasmic ratio and spindle cell morphology, which could bring the differential diagnosis of a sarcoma. However, in this case, the tumor location along with morphology and immunophenotype led to an initial interpretation of high-grade myoepithelial carcinoma with spindle cell morphology. In Case 2, initial studies showed positive AE1/AE3, CK7 and CAM5.2; while negative for p63, p40, CK5/6, EMA, desmin, PAX8, and TTF1.
Given the significant expression of keratin markers, case was interpreted as poorly differentiated carcinoma with spindle cell features and favored to be metastatic carcinoma.
Nevertheless, after additional specific IHC work-up to assess co-expression of epithelial markers, myogenic markers, and ALK1, and molecular analysis a final diagnosis of RMS with FUS::TFCP2 fusion was made in both cases. RMS with TFCP2 fusion is characterized by a spindle and epithelioid morphology, as well as keratin expression. The epithelioid morphology in combination with keratin expression raises the possibility of a carcinoma. Differential diagnosis includes spindle cell carcinoma (sarcomatoid carcinoma). 10 However, these are usually associated with mucosal dysplasia as well as in situ or invasive squamous cell carcinoma. 23 In the absence of these characteristics, these two reported cases highlight the importance to include this entity in the differential diagnosis. The distinction can be approached by evaluation for coexpression of epithelial markers, myogenic markers, and ALK1 expression by IHC, and the use of ancillary testing including FISH or NGS as a complementary tool to detect TFCP2 or NCOA2 gene fusion. 10,18 Considering the change of tumor type diagnosis with the understanding that RMS with TFCP2-fusion tumors behave aggressively and have a poor prognosis, 19 24 Further studies are needed to determine the potential of ALK inhibitors in this patient population.
In summary, RMS with TFCP2 fusion is a recently described subtype of ssRMS, with a propensity for craniofacial bones and aggressive clinical course. It is characterized by epithelioid and spindle cell morphology, arranged in sheets and fascicles. Its epithelioid morphology along with significant keratin expression may be deceiving and lead to a diagnosis of carcinoma. Co-expression of epithelial markers, myogenic markers, and ALK1 can aid in the diagnostic process. Fusion detection by FISH or molecular testing is essential for the diagnosis of this entity, as the presence of FUS::TFCP2 or EWSR1::TFCP2 fusions should be demonstrated.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.