NR1D1::MAML1 epithelioid and spindle cell sarcoma mimicking pseudomyogenic hemangioendothelioma in core biopsy: A case report and review of the literature

Epithelioid and spindle cell sarcomas with NR1D1::MAML1/2 gene fusions are rare and emerging entities. Only six cases of NR1D1‐rearranged mesenchymal tumors have previously been reported in the literature; they are often characterized by an epithelioid morphology, at least focal pseudogland formation, prominent cytoplasmic vacuoles, and focal to diffuse immunohistochemical expression of keratin. We herein report the first case of an NR1D1::MAML1 epithelioid and spindle cell sarcoma with dual immunohistochemical expression of ERG and FOSB, mimicking a pseudomyogenic hemangioendothelioma (PHE) on core biopsy. The sarcoma arose in the left forearm of a 64‐year‐old man. Initial biopsy showed a mesenchymal neoplasm composed of epithelioid and spindle cells dispersed in myxoid stroma with scattered stromal neutrophils. The morphologic features, combined with the dual immunohistochemical expression of ERG and FOSB, initially mimicked PHE, representing an important potential diagnostic pitfall. The patient subsequently underwent a radical resection, which showed a much more diffuse epithelioid appearance with nested architecture and pseudogland formation. Next‐generation sequencing was performed on the resection specimen, which revealed an NR1D1::MAML1 gene fusion, confirming the final diagnosis. Given the fully malignant potential of this tumor, knowledge and recognition of this rare entity are essential to ensure proper management, prevent misdiagnosis, and further characterize the clinical course of this emerging entity. Comprehensive molecular testing can help to identify these rare tumors and exclude the possibility of epithelioid mimics, including PHE.


| INTRODUCTION
The first case of a soft tissue tumor with novel NR1D1::MAML1 gene fusion was reported in 2020 by Komatsu et al. 1 The multinodular tumor, occurring in the lower leg of a 10-year-old boy, was composed of sheets of epithelioid cells with focal pseudopapillary growth patterns and immunohistochemical expression of keratin.The following year, Nakamura et al. reported the second case of a malignant epithelioid soft tissue tumor with NR1D1::MAML1 fusion, 2 which involved the subcutaneous tissue of the right waist of a 28-year-old man.The patient, unfortunately, died 3 years following resection, confirming the malignant potential of the entity.The tumor was further negative for claudin-4, arguing against true epithelial differentiation, 3 and favoring a classification of sarcoma over undifferentiated carcinoma despite keratin expression.Shortly thereafter, Lacambra et al. published the first series of mesenchymal neoplasms with NR1D1 gene rearrangements, identifying four additional cases, all of which arose in adult women. 4Half of their cases had a prominent spindle cell component in addition to the more pronounced epithelioid component.All cases demonstrated at least focal keratin expression, leading to a potential diagnostic pitfall of misclassification as a carcinoma.Two of the four cases had NR1D1::MAML1 gene fusions, while the other two cases had NR1D1::MAML2 gene fusions.Their series helped to confirm that NR1D1-rearranged mesenchymal tumors represent a distinct sarcoma with epithelioid and sometimes spindled morphology.Potential clues to the diagnosis include the formation of pseudoglandular spaces and prominent cytoplasmic vacuoles.Herein, we present the first case of an epithelioid and spindle cell sarcoma with NR1D1::MAML1 gene fusion that demonstrated immunohistochemical expression of both ERG and FOSB, mimicking a pseudomyogenic hemangioendothelioma (PHE) on core biopsy.Our case helps to expand the immunohistochemical profile of this emerging entity and reinforces that accurate classification often relies on ancillary molecular testing.

| Next-generation sequencing (NGS)
DNA was extracted from FFPE tissue using standard techniques.NGS was performed through Caris Life Sciences.Whole exome sequencing with a high-throughput assay analyzed over 22 000 DNA genes, and a 250 000 evenly spaced genome single nucleotide polymorphism backbone was used to enhance detection of genome-level alterations.

| RESULTS
A 64-year-old man with no significant past medical history presented with a 1-year history of a painless left forearm mass that arose without trauma or inciting event.The patient reported that the mass had been relatively stable in size in the year prior to presentation.MRI of the wrist confirmed a heterogeneous, enhancing soft tissue mass (2.7 cm) along the ulnar aspect of the left distal forearm (Figure 1A).
The margins were somewhat irregular with focal extension of the mass toward the distal aspect of the wrist between the ulna and extensor carpi ulnaris tendon.No bone involvement or erosion was identified, and mild surrounding inflammation was present.Initial biopsy of the mass showed a mesenchymal neoplasm composed of spindle and epithelioid cells (Figure 1B) dispersed in myxoid stroma with scattered stromal neutrophils (Figure 1C).The tumor cells had abundant eosinophilic cytoplasm with vesicular nuclei and conspicuous nucleoli (Figure 1D).Focal necrosis was present, but mitotic figures were not readily identified.

The morphologic features, combined with the co-expression of keratin, vimentin, ERG, and FOSB, initially favored a diagnosis of PHE.
Following the initial diagnosis, the patient underwent a whole-body PET/CT scan after a multidisciplinary discussion to assess for multifocality, which demonstrated hypermetabolic activity in the primary tumor but no other masses in the left upper extremity and no evidence of metastatic disease.The patient subsequently underwent resection of the forearm mass with oncologic intent by raising the tumor from the preserved ulna in a subperiosteal fashion with periosteum present at the deep margin.The resection specimen showed a malignant epithelioid and spindle cell neoplasm (4.6 cm in greatest dimension) involving the subcutis and superficial skeletal muscle.It was multinodular (Figure 3A), demonstrating variable histologic patterns including large nests and sheets of epithelioid cells with well-defined cell membranes and granular, clear-to-pale eosinophilic cytoplasm (Figure 3B).Focal areas demonstrated pseudogland formation (Figure 3C), while some F I G U R E 1 MRI of the wrist showed a heterogeneous, enhancing soft tissue mass (2.7 cm) along the ulnar aspect of the left distal forearm (A, axial, T1-weighted, fat-saturated).Biopsy of the mass showed a mesenchymal neoplasm composed of spindle and epithelioid cells in a myxoid stroma (B).Areas with prominent stromal neutrophils were present (C).The tumor cells had abundant eosinophilic cytoplasm with vesicular nuclei and conspicuous nucleoli (D).
F I G U R E 2 Immunohistochemical stains showed that the tumor cells were positive for CK cocktail (A), vimentin (B), ERG (C), and FOSB (D), while they were essentially negative for EMA, S100 protein, SMA, desmin, CD34 (E) and CD31.INI1 demonstrated retained nuclear expression (F).cells had large intracytoplasmic vacuoles (Figure 3D).Other areas showed markedly plump spindled to rhabdoid-appearing cells in a myxoid stroma (Figure 3E).Scattered neutrophils (Figure 3F) were present, as well as focal areas of necrosis (<10% of the tumor).Mitotic figures were readily identified (11 per 10 hpf).
In the setting of the patient's complete but narrow excision with tumor extending 0.1 cm from the closest deep margin, postoperative radiation (66 Gy) was planned following wound healing.Surveillance imaging at 3 months showed a few bilateral punctate lung nodules with no evidence of either local recurrence or metastatic disease.(2/2).All tested cases were negative for CK 20 (0/5), CK 5/6 (0/1), CD34 (0/6), CD31 (0/5), desmin (0/7), SMA (0/7), and SOX10 (0/6).S100 protein demonstrated rare focal expression in one case (1/7), and the current case demonstrated patchy positivity for both ERG (1/1) and FOSB (1/1).NGS was performed on all cases revealing NR1D1::MAML1 in five cases and NR1D1::MAML2 in two cases.Of the six patients with follow-up (median: 12.5 months; range: 3-36 months), half-developed lung metastasis (3/6).At last clinical follow-up, half showed no evidence of disease (3/6), two had died of disease (2/6), and one was presumed to be alive with disease (1/6).NR1D1 (nuclear receptor subfamily 1 group D member 1), also known as REV-ERBα, encoded by the NR1D1 gene on chromosome 17q21.1, is a transcription factor involved in regulating circadian rhythm and metabolism. 5,6In this group of epithelioid and spindle cell  sarcomas, NR1D1 is fused to one of a family of mastermind-like transcriptional coactivators of NOTCH receptors (MAML1-3).The most common fusion partner is MAML1.MAML1 (mastermind-like transcriptional coactivator 1), located on chromosome 5q35.3,plays an important role in the NOTCH signaling pathway. 7The putative fusion protein of NR1D1::MAML1 contains two N-terminal C4-type zinc finger domains of NR1D1 for binding specific DNA sequences and the C-terminal parts of MAML1. 1 The protein product further lacks a large proportion of important amino acid sequences (N-terminal amino acids 1-123) of MAML1 that are related to its interaction with the NOTCH receptor.The resulting fusion suggests preservation of the NR1D1 DNA binding sequences with a disruption in mesenchymal development that is independent of the NOTCH signaling pathway.

| DISCUSSION
Although rare in sarcoma, a KMT2A::MAML1 fusion has been reported in a case of infant acute lymphoblastic leukemia. 8The second most common fusion partner is MAML2.Gene fusions involving MAML2 have been reported in other tumors, including CTRC1::MAML2 in hidradenoma 9 and CRTC3::MAML2 in mucoepidermoid carcinoma. 10stly, MAML3, which has not been reported as a fusion partner of NR1D1 to date, has been shown to be a fusion partner of PAX3 in biphenotypic sinonasal sarcoma. 11 the current case, the initial core biopsy, demonstrating a spin- Then, in 2011, the entity was ultimately renamed PHE 17  Another neoplasm in the differential, which typically lacks FOSB staining 13 but may mimic PHE, is epithelioid sarcoma.Epithelioid sarcoma typically has more necrosis and cytologic atypia than PHE.Epithelioid sarcoma may also show immunoreactivity for ERG and CD34, which can be a diagnostic pitfall 30 ; however, they do not express CD31.Loss of INI1/SMARCB1 expression is the most useful feature, which is observed in more than 90% of both conventional and proximal-type epithelioid sarcomas. 31The distinction is important as epithelioid sarcomas carry a worse prognosis than PHE with a high risk for local recurrence (75%) and metastasis (45%). 19The differential diagnosis of epithelioid soft tissue tumors of uncertain differentiation also includes myoepithelial tumors, metastatic carcinoma, alveolar soft part sarcoma, and perivascular epithelioid cell tumor among others.
Immunohistochemistry, as well as NGS, can help to exclude these other possibilities.
Molecular analysis, along with the distinct morphologic features evident on resection, helped to identify this novel epithelioid and spindle cell sarcoma with NR1D1::MAML1 fusion.Given the tumor's malignant potential and clinical course, which appears distinct from either PHE or epithelioid sarcoma, it is important to identify this emerging entity so that its prognosis and biological behavior can be better understood.While a few cases of this entity have been reported in the literature, we report the first known case to demonstrate patchy expression of both ERG and FOSB, representing an important pitfall by mimicking PHE, especially on limited sampling on core biopsy.Furthermore, the selection of inclusive molecular testing is important as most targeted sarcoma gene panels usually only cover MAML2 and frequently lack both NR1D1 and MAML1.Comprehensive molecular testing, such as whole exome sequencing, can help to identify these rare cases in the future and enhance our understanding of this emerging sarcoma with epithelioid morphology.
NR1D1::MAML1/2 gene fusions have recently been described in a novel group of previously unclassified epithelioid and spindle cell sarcomas.The first case of an epithelioid soft tissue tumor with novel NR1D1::MAML1 fusion was reported in a pediatric patient in 2020 by Komatsu et al.1 Since then, six additional cases have been reported in the literature, including the current case (Table1).Clinically, tumors frequently involve the subcutaneous tissue of the extremities and abdominal wall with two cases extending into the deep soft tissue, and one with superficial bone invasion.The tumors affected patients over a wide age range (median: 62 years; range: 10-70 years) with an almost equal gender distribution (three male: four female).The median tumor size was 4.6 cm (range: 1.1-12.0cm).Original diagnostic considerations included undifferentiated carcinoma, myoepithelial carcinoma, epithelioid sarcoma, and PHE among others.Consideration for diagnosis of undifferentiated carcinoma arose in part from the fact that all cases demonstrated immunohistochemical expression of keratin, including CK AE1/AE3 (7/7; 4 focal, 3 diffuse) and CK 7 (4/5; 2 focal, 2 diffuse).Two showed expression of EMA (2/3) and MAML1

F I G U R E 3
The resection specimen showed a multinodular soft tissue mass (A) composed of large nests and sheets of epithelioid cells (B).Focal areas demonstrated pseudogland formation (C) with some cells having large intracytoplasmic vacuoles (D).Other areas showed spindled cells in a prominent myxoid stroma (E) with scattered neutrophils.T A B L E 1 Reported cases of NR1D1::MAML1/2 epitheloid and spindle cell sarcomas.
Abbreviations: DOD, died of disesae; F, female; FISH, fluorescence in situ hybridization; M, male; N/A, not applicable; NED, no evidence of disease; NGS, next-generation sequencing; y, years.a Multifocal.