Myxoid epithelioid smooth muscle tumor of the vulva: A distinct entity with MEF2D::NCOA2 gene fusion

Smooth muscle tumors are the most common mesenchymal tumors of the female genital tract, including the vulva. Since vulvar smooth muscle tumors are rare, our understanding of them compared to their uterine counterparts continues to evolve. Herein, we present two cases of morphologically distinct myxoid epithelioid smooth muscle tumors of the vulva with novel MEF2D::NCOA2 gene fusion. The tumors involved 24 and 37‐year‐old women. Both tumors presented as palpable vulvar masses that were circumscribed, measuring 2.8 and 5.1 cm in greatest dimension. Histologically, they were composed of epithelioid to spindle‐shaped cells with minimal cytologic atypia and prominent myxoid matrix. Rare mitotic figures were present (1–3 mitotic figures per 10 high‐power field (HPF)), and no areas of tumor necrosis were identified. By immunohistochemistry, the neoplastic cells strongly expressed smooth muscle actin, calponin, and desmin, confirming smooth muscle origin. Next‐generation sequencing identified identical MEF2D::NCOA2 gene fusions. These two cases demonstrate that at least a subset of myxoid epithelioid smooth muscle tumors of the vulva represent a distinct entity characterized by a novel MEF2D::NCOA2 gene fusion. Importantly, recognition of the distinct morphologic and genetic features of these tumors is key to understanding the biological potential of these rare tumors.


| INTRODUCTION
Smooth muscle tumors are the most common mesenchymal tumors of the female genital tract, 1 including within the vulva. 2 Most smooth muscle tumors of the female genital tract occur in the uterus, and the World Health Organization (WHO) has well-established uterine criteria to distinguish benign from malignant smooth muscle tumors in this location.Unlike the more common uterine tumors, vulvar smooth muscle tumors are rare, with vulvar leiomyomas comprising only 4.2% of cutaneous leiomyomas 3 and 0.07% of vulvar tumors. 4They can originate from a variety of tissues, including the smooth muscle in erectile tissue, blood vessels, round ligament, dartos muscle, and erector pili muscles, 5 and affect patients over a wide age range but most commonly occur in the fourth or fifth decade. 6Vulvar leiomyomas frequently present as a slow-growing, painless mass that has been present for several years.Clinically, they are often assumed to be a Bartholin gland cyst or abscess.Most patients undergo a simple, conservative excision with clinical follow-up.Furthermore, their clinical behavior differs from the morphologically similar uterine tumors, leading to the proposal of different site-specific criteria for malignancy. 5,7o better understand these rare tumors, we herein report two distinct myxoid epithelioid smooth muscle tumors of the vulva with novel MEF2D::NCOA2 gene fusion.Since vulvar smooth muscle tumors with myxoid and epithelioid features may have different thresholds for malignancy, as has been shown in their uterine counterparts, our awareness of this unique variant is critical to improve our understanding of the biology and clinical course of these rare tumors, leading to appropriate classification and optimal patient management.

| Case selection
This study was approved by the Institutional Review Board of Indiana University (protocol #17625, approved January 20, 2023).Both cases were identified through routine clinical practice, and the clinicopathologic features of each case were assessed by the authors.

| Next-generation sequencing
Case 1 underwent testing with the Sarcoma Targeted Gene Fusion/ Rearrangement (SARCP) panel, which was performed at Mayo Clinic Laboratories (Rochester, MN).The SARCP panel is a custom-designed, polymerase chain reaction (PCR) D-based panel developed in collaboration with Qiagen.The panel was designed to detect 138 gene fusions (>280 fusion variants) in 38 sarcoma types.Next-generation sequencing (NGS) is performed on an Illumina MiSeq instrument, and the NGS data are analyzed using an in-house developed bioinformatics pipeline called SeekFusion that uses a combination of traditional alignment and de novo assembly-based approaches.
Case 2 had NGS 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.

| Case 1
A 24-year-old nulligravid woman with no significant past medical history presented with a several-month history of a palpable vulvar mass, which was mildly tender.Physical examination showed normal external genitalia with a smooth and mobile subcutaneous mass palpated in the right periclitoral area of the upper vulva.The vaginal vault was clear; the cervix, uterus, and bilateral adnexal structures were all within normal limits.Clinically, the differential for the vulvar mass included a Bartholin gland cyst and lipoma.The patient requested resection of the mass, which showed a solid, well-circumscribed mass (2.8 cm in greatest dimension).No further intervention was recommended, and she is alive without evidence of disease or recurrence at 2 years post resection.

| Case 2
A 37-year-old woman with a history of five Cesarean sections presented with a 6-month history of pelvic pain and a palpable vulvar mass that was first noticed 1.5 years ago, before she was last pregnant.It had grown slightly in size over that time.Imaging studies revealed an encapsulated, circumscribed lesion (5.1 cm in greatest dimension) adjacent to the left posterolateral vaginal wall, abutting the anus and pelvic floor with mass effect (Figure 1).She underwent biopsy of the mass, and surgical excision was recommended.

| Morphologic findings
Patient 1 underwent excision, which showed a circumscribed, solid smooth muscle tumor (2.8 cm) surrounded by a fibrous pseudo capsule (Figure 2A).Prominent myxoid stromal change was present, resulting in the formation of microcystic spaces lined by a peripheral condensation of epithelioid tumor cells (Figure 2B,C).The more solidappearing areas were composed of epithelioid to spindle-shaped tumor cells with minimal to mild cytologic atypia (Figure 2D).The myxoid matrix, when not forming well-defined cystic spaces, intermixed with the tumor cells, creating a bubbly appearance (Figure 2E,F).A few mitotic figures (up to 3 per 10 HPF) were present (Figure 2F, inset); however, no atypical mitotic figures and no areas of necrosis were identified.
Core biopsy of case 2 demonstrated identical morphologic features to case 1.One mitotic figure was present; however, no atypical mitotic figures and no areas of necrosis were identified.Cytologic atypia was mild at most.

| Immunohistochemical findings
Immunohistochemical study of both cases showed that the tumor cells strongly expressed multiple myogenic markers including smooth muscle actin (Figure 3A), calponin (Figure 3B), and desmin (Figure 3C), whereas they were negative for caldesmon, myogenin, and MyoD1.
The strong, diffuse expression of both smooth muscle actin and calponin was consistent with smooth muscle origin.ER was strongly and diffusely positive (Figure 3D), whereas PR was negative.Both vimentin (Figure 3E) and EMA (Figure 3F) demonstrated focal expression.CK AE1/AE3, CAM5.2, S100 protein, SOX10, HMB45, Melan A, CD34, glial fibrillary acidic protein, and MUC4 were all negative.INI1 F I G U R E 1 Imaging studies.CT abdomen/pelvis showed a heterogeneous mass likely arising from the left posterior lateral vaginal wall (A: CT, axial).Followup MRI showed an encapsulated, wellcircumscribed mass (5.1 cm) adjacent to the left posterior lateral vaginal wall, abutting the anus and pelvic floor with mass effect (B: MRI, T2-weighted, axial).The mass was mildly T2 hyperintense and T1 hypointense with thin internal septations.CT, computed tomography; MRI, magnetic resonance imaging.
F I G U R E 2 Morphologic findings.Both cases demonstrated identical morphologic features.One patient underwent an excision, which showed a well-circumscribed smooth muscle tumor surrounded by a fibrous pseudo capsule (A).Prominent myxoid stromal change was present, resulting in the formation of microcystic spaces lined by a peripheral condensation of epithelioid tumor cells (B, C).The more solid-appearing areas were composed of epithelioid to spindle-shaped tumor cells with minimal cytologic atypia (D).The myxoid matrix, when not forming welldefined cystic spaces, intermixed with the tumor cells, creating a bubbly appearance (E, F).A few mitotic figures (up to 3 per 10 HPF) were present (F, inset); however, no atypical mitotic figures and no areas of necrosis were identified.
showed retained expression in the tumor cells, and all control slides were stained appropriately.

| DISCUSSION
We report the first two cases of myxoid epithelioid smooth muscle tumors of the vulva with identical morphologic features and novel MEF2D:: NCOA2 gene fusion.One of the fusion partners, MEF2D, which maps to chromosome 1q23, is a member of the myocyte-specific enhancer factor acetyltransferases and methyltransferases to specific enhancer/ promoter regions, facilitating chromatin remodeling and stimulating transcription in a hormone-dependent manner.NCOA2 is the 3 0 partner, and the NCOA2 breakpoints are clustered in a small region between the NID and AD1 domains.The C-terminal part of NCOA2, including the AD1 and AD2 domains, is consistently preserved in the fusion protein.NCOA2 rearrangements have been shown to occur in both hematologic and mesenchymal neoplasms.The first identified NCOA2 fusion was MYST3::NCOA2, which was discovered in a rare subset of acute myeloid leukemia. 11KAT6A::NCOA2 and ETV6::NCOA2 have also been reported in acute leukemia.In the gynecologic tract, NCOA2 fusions have been reported in the following: ESR1::NCOA2 in uterine tumors resembling ovarian sex cord tumors (UTROSCT) 12,13 and adenosarcoma 14 ; GREB1::NCOA2 in UTROSCT 12 and undifferentiated uterine sarcoma 15 ; and MEIS1::NCOA2 in spindle cell sarcomas [16][17][18] and adenosarcoma. 19Other NCOA2 fusions in mesenchymal neoplasms include AHRR::NCOA2 and GTF2::NCOA2 in soft tissue angiofibromas 20,21 ; SRF:: NCOA2, TEAD1::NCOA2, and VGLL2::NCOA2 in congenital/infantile spindle cell rhabdomyosarcomas 22,23 ; MEIS1::NCOA2 in intraosseous rhabdomyosarcoma 24 ; PAX3::NCOA2 in primary CNS rhabdomyosarcoma 25 and biphenotypic sinonasal sarcoma 26 ; and HEY1::NCOA2 in mesenchymal chondrosarcoma. 27A C11orf95::NCOA2 fusion has also been reported in an ependymoma-like tumor. 28 addition to a novel gene fusion, both vulvar smooth muscle tumors demonstrated prominent myxoid change.Tavassoli and Norris 7 described 10 of 32 smooth muscle tumors of the vulva with extensive myxoid change, which they initially attributed to pregnancy.
The pregnant patients further noticed that their tumors tended to grow noticeably during gestation, likely due to hormonal influence.Both of our cases, as well as the myxoid smooth muscle tumors described by Newman and Fletcher, 29 arose in patients that were either nulligravid or not pregnant at diagnosis, countering this previous association.Newman and Fletcher 29 further noted that prominent myxoid stromal change was more common in vulvar smooth muscle tumors.Myxoid smooth muscle tumors have a prominent extracellular matrix rich in proteoglycans and glycosaminoglycans 30 that can be highlighted by Alcian blue. 31The abundant myxoid stroma often results in the discohesion or separation of individual cells, creating the appearance of a corded or plexiform growth pattern.
For smooth muscle tumors of the vulva, one of the main challenges is distinguishing between benign and malignant lesions.In contrast to vulvar tumors, the criteria for distinguishing between benign and malignant smooth muscle tumors in the uterus are well established but differ according to whether the tumor is spindle cell, epithelioid, or myxoid.The threshold for malignancy in myxoid tumors is lower than for either conventional spindle cell or epithelioid tumors.Thus, spindle cell cases require two or more of the following: significant cytologic atypia, ≥10 mitotic figures per 10 HPF, and tumor cell necrosis.For myxoid smooth muscle tumors, the threshold is lowered to any significant cytologic atypia, tumor necrosis, a mitotic index of >1 mitotic figure per 10 HPF, or infiltrative margins.Whether myxoid vulvar smooth muscle tumors should be evaluated by the same criteria as for the nonmyxoid counterparts remains an open question. 32garding vulvar smooth muscle tumors, the first site-specific criteria for malignancy were published in 1979 by Tavassoli and Norris, 7 and included two of the following features for low-grade leiomyosarcoma: size ≥5 cm, ≥5 mitotic figures per 10 HPF, and infiltrative margins. 7When all three criteria were met, a diagnosis of conventional leiomyosarcoma was made.Subsequently, in 1996, the vulvar criteria were modified to require at least three of the following features: size ≥5 cm, moderate-to-severe cytologic atypia, ≥5 mitotic figures per 10 HPF, and infiltrative margins. 5When these two site-specific vulvar criteria were compared to the 2014 WHO uterine smooth muscle tumor criteria in a large series of vulvar smooth muscle tumors, based on patient outcome, the sensitivity for diagnosing leiomyosarcoma was the same; however, the WHO uterine smooth muscle tumor criteria proved to be the most specific, followed by the 1996 and 1979 criteria, respectively. 33Therefore, it has been recommended to apply the WHO uterine criteria to conventional vulvar smooth muscle tumors.
The differential diagnosis for these lesions includes myxoid leiomyoma, myxoid leiomyosarcoma, glomus tumor, myoepithelial tumor, melanoma, and adenomatoid tumor.Vulvar smooth muscle tumors with extensive myxoid change can also mimic aggressive angiomyxoma. 34Myxoid leiomyosarcomas of both the vulva 35 and Bartholin gland 36 have been reported, with leiomyosarcoma representing approximately 1% of primary vulvar malignancies. 7While myxoid change in vulvar leiomyosarcoma can simulate its benign counterpart due to a deceptively low mitotic count, 37 leiomyosarcoma frequently occurs in postmenopausal women (>50 years of age), has infiltrative margins, and follows an aggressive clinical course.In addition to leiomyosarcoma, a case of purely myxoid endometrial stromal sarcoma has been described. 38e identification in this study of an MEF2D::NCOA2 fusion in two vulvar myxoid smooth muscle tumors should allow the analysis of similar cases as a distinct entity.Open questions include if this fusion is present in some nonmyxoid vulvar smooth muscle tumors, what proportion of vulvar smooth muscle tumors carry this fusion, and what is the biologic behavior of these neoplasms.Currently, given the rarity of these tumors, we feel that these neoplasms should be considered of uncertain malignant potential due to the paucity of data.For well-circumscribed lesions, conservative excision is recommended with possible re-excision for involved margins.Since vulvar smooth muscle tumors can recur from several months to more than 10 years after excision, 6,39 long-term clinical follow-up is essential, and that is not currently available.Nevertheless, awareness of the fusion in vulvar smooth muscle tumors should allow for additional molecular determination of valid cases and allow for accrual of additional cases to address these important questions.

2 (
MEF2) family of transcription factors, which are involved in muscle and neuronal cell differentiation and are regulated by class II histone deacetylases.The MEF2A-D proteins contain N-terminal MEF and MADS-box domains that are responsible for the recognition of A/T-rich DNA motifs and the recruitment of transcription cofactors.MEF2C and MEF2D are key components of the transcriptional complex that orchestrates B-cell development.Recurrent MEF2D fusions have been identified in a small subset (<5%) of B-cell precursor acute lymphocytic leukemia (ALL) with poor clinical outcomes. 8Nine different types of MEF2D fusions have been reported in B-cell ALL including the following: MEF2D::BCL9, MEF2D::HNRNPUL1, 8 MEF2D::DAZAP1, MEF2D::CSF1R, MEF2D::FOXJ2, MEF2D::STAT6, MEF2D::SS18, 9 MEF2D::HNRNPH1, and MEF2D:: HNRNPM.To the best of our knowledge, MEF2D::NCOA2 fusions have not been previously reported in B-cell ALL or mesenchymal neoplasms.The other fusion partner, NCOA2, maps to chromosome 8q13 and is a member of the p160 steroid receptor coactivator gene family, 10 which includes three members: NCOA1 (SRC-1), NCOA2 (SRC-2), and SRC-3.The family of coactivators interacts with ligandbound nuclear receptors, through its nuclear receptor interaction (NID) and its C-terminal transcription activation domains, activation domain 1 (AD1) and AD2, to, respectively, recruit histone F I G U R E 3 Immunohistochemical findings.The tumor cells strongly expressed multiple myogenic markers including smooth muscle actin (A), calponin (B), and desmin (C), whereas they were negative for caldesmon, myogenin, and MyoD1.The strong and diffuse expression of both smooth muscle actin and calponin was consistent with smooth muscle origin.Estrogen receptor (ER) was strongly and diffusely positive (D), whereas progesterone receptor (PR) was negative.Both vimentin (E) and EMA (F) demonstrated focal expression, whereas CK AE1/AE3 and CAM5.2 were negative.F I G U R E 4 Molecular findings.In both cases, next-generation sequencing revealed a novel gene fusion involving exon 9 of MEF2D (chr1: 156444900) and exon 13 of NCOA2 (chr8:71053634).