Pathologic and genomic characteristics of myoepithelioma-like tumor of the vulvar region: three case reports

Abstract

Background

Myoepithelioma-like tumor of the vulvar region (MELTVR) is a rare type of soft tissue mesenchymal tumor. While MELTVR exhibits histological characteristics similar to soft tissue myoepithelial tumors, its immunohistochemical and genetic features differ significantly. To date, no comprehensive genomic analysis of this tumor has been conducted.

Case presentation

We present the clinicopathological features, imaging characteristics, and immunophenotypes of three patients with MELTVR, along with their genomic characterization through high-throughput sequencing. Immunohistochemical analysis revealed that these tumors were negative for SMARCB1, S-100, CD34, CD31, SMA, Desmin, and Keratin. The Ki-67 proliferation index for tumor cells ranged from 10 to 35%. Genomic analyses showed copy number deletions in the SMARCB1 gene in all three patients. The tumor mutational burden was relatively low, ranging from 1.35 to 4.33. Additionally, two tumors exhibited fusion mutations involving PPP6R3::FHDC1 and MYH9::MYH6, while no fusions involving EWSR1, NR4A3, or FUS were detected.

Conclusions

This study reports the first comprehensive genomic analysis of three patients with MELTVR, potentially identifying therapeutic targets for this rare tumor.

Background

Myoepithelioma-like tumor of the vulvar region (MELTVR) is a rare soft tissue tumor derived from mesenchymal cells in the vulvar region of women [1, 2]. In 2015, Yoshida et al. reported nine patients of myoepithelioma-like tumors occurring in the vulva accompanied by a loss of SMARCB1 expression and named these tumors MELTVR [2]. To date, approximately 20 patients of MELTVR have been reported in the English literature [3]. The age at diagnosis ranges from 24 to 70 years, with a mean age of 44 years [3]. Generally, the tumor exhibits well-defined borders and a lobulated, solid-cut surface with colors varying from yellow-white to brown. The cut surface appears gelatinous and glossy in some patients. At low magnification, the tumor displays clear boundaries, with multiple nodular formations separated by areas of transparent degenerative fibrous tissue. In some patients, fibrous pseudocapsules are observed, accompanied by evidence of tumor infiltration beyond the capsule [2, 4]. The tumor comprises myxoid and non-myxoid areas with varying cellularity [2, 3]. Myxoid areas contain spindle or epithelioid cells in loose reticular patterns peripherally and solid sheets centrally. Non-myxoid areas display diffuse sheets of epithelioid cells or interlacing fascicles of spindle cells. Notably, these histological features closely resemble those found in soft tissue myoepitheliomas.

Due to the clinical rarity of this condition and the lack of distinctive histological features, the misdiagnosis rate is relatively high. Its diagnosis requires a combination of immunophenotypic and molecular characteristics. MELTVR typically shows positivity for SMA, MUC1, and ER. In contrast, Keratin, Calponin, Desmin, Myogenin, S-100, glial fibrillary acidic protein (GFAP), and CD34 proteins consistently test negative. The most distinguishing features of MELTVR include the absence of SMARCB1 protein expression and EWSR1 gene rearrangement [2, 5, 6]. Conversely, in soft tissue myoepitheliomas, approximately 50% of patients exhibit EWSR1 gene rearrangement, while only about 10% show SMARCB1 deficiency [7, 8].

While several case reports have documented characteristic genetic alterations in MELTVR, a comprehensive genomic profile of this entity has yet to be reported. In this study, we conducted whole-exome sequencing on three patients of MELTVR, analyzing their clinicopathological features, immunophenotype, and genomic characteristics.

Case presentation

Three patients with MELTVR were collected from the Department of Pathology, Chongqing University Cancer Hospital. All tumors were located in the vulva and showed an absence of SMARCB1 expression. The tumor cells exhibited a short spindle or epithelial-like morphology, accompanied by varying degrees of a mucinous background. The tumor cells were positive for MUC1, ER, and focally positive for calponin and SMA, but negative for Keratin, CD34, and S-100. Two senior pathologists independently reviewed these patient cases.

Case 1

A 45-year-old woman accidentally discovered a lump in the mons pubis and sought medical attention at our hospital in 2016. Ultrasonography revealed a 7 mm × 17 mm hypoechoic nodule in the subcutaneous tissue of the mons pubis, characterized by clear boundaries, regular morphology, encapsulation, and posterior acoustic enhancement. An abnormal echo in the subcutaneous soft tissue suggested enlarged lymph nodes or cysts with an inflammatory reaction (Fig. 1A). Grossly, the tumor measured approximately 15 mm × 10 mm × 10 mm and appeared gray-white with a soft, solid texture. Microscopically, the tumor boundary was clearly defined at low magnification and was encapsulated by a pseudofibrous capsule (Fig. 1B). At high magnification, the tumor displayed two distinct morphologies: one type consisted of either epithelioid or short spindle-shaped tumor cells with eosinophilic cytoplasm, uniform-sized nuclei, and eccentrically located nucleoli, situated within a stroma characterized by mucoid degeneration or loose edema (Fig. 1C, D). The second pattern involved short spindle-shaped tumor cells exhibiting bundled growth within a non-mucinous background (Fig. 1E, F). Notably, the mucoid areas comprised approximately 80% of the entire tumor volume. A mitotic figure was observed at a rate of one per 10 high-power fields (HPFs).

Fig. 1

Ultrasound imaging reveals a solid, hypoechoic mass with clear boundaries, exhibiting an unevenly thickened pseudocapsule at the margins (A). Tumor is focally encased by fibrous pseudocapsules (B). In myxoid areas, tumor cells exhibit either an epithelioid or spindle-shaped morphology, proliferating in a loosely cohesive reticular or clustered arrangement (C, D). In non-myxoid areas, spindle-shaped tumor cells exhibit bundled growth pattern, forming sheets (E, F). The tumor cells demonstrate focal positivity for MUC1 (G), as well as diffuse positivity for ER (H) and Calponin (I). In contrast, the tumor cells are negative for Keratin (J) and S-100 (K). SMARCB1 expression is completely absent in all tumor cells (L, vascular reactivity used as an internal positive control)

Immunohistochemical staining showed that the tumor cells were positive for MUC1 (Fig. 1G), vimentin, estrogen receptor (ER, Fig. 1H), progesterone receptor (PR), Calponin (Fig. 1I), and focally positive for SMA, but negative for Keratin (Fig. 1J), S-100(Fig. 1K), Desmin and CD34. In addition, the tumor cells demonstrated a loss of SMARCB1 expression (Fig. 1L). The Ki-67 proliferation index was approximately 10%. Given the histological morphology of soft tissue myoepithelioma, which shows striated muscle-like cells with cytoplasm displacement within a mucinous matrix, and co-expresses Keratin and S100, soft tissue myoepithelioma was ruled out. Our case demonstrated an absence of expression of S-100, CD34, Desmin, and Keratin, with varying degrees of MUC1, calponin, and SMA expression. Meanwhile, the case exhibited diffuse expression of ER and a lack of SMARCB1 protein expression, leading to a diagnosis of MELTVR. Following tumor resection, the patient was followed for 89 months, with no recurrence or metastasis observed.

Case 2

A 35-year-old woman discovered a labium majus mass four months prior and sought treatment at our hospital in 2021. Ultrasound demonstrated a 28 mm × 41 mm hypoechoic nodule in the right labium majus, presenting as a micro-lobulated solid mass with an irregular pseudocapsule and clear boundaries. Internal cord-like slightly hyperechoic areas and microcystic anechoic regions were observed, with posterior acoustic enhancement. Color Doppler flow imaging showed abundant short rod-like and strip-like blood flow signals, predominantly peripheral.

Magnetic resonance imaging (MRI) showed an abnormal mass in the right vulvar region. The mass displayed heterogeneous signal intensity, being isointense on T1-weighted imaging (T1WI) and slightly hyperintense on T2-weighted imaging (T2WI) (Fig. 2A). It contained multiple focal areas of higher signal intensity, restricted diffusion on diffusion-weighted imaging (DWI), and measured approximately 40 mm × 38 mm at its largest dimension. Additionally, the lesion exhibited persistent heterogeneous enhancement, suggesting a hypervascular neoplastic lesion in the right vulvar region.

Fig. 2

T2-weighted imaging (T2W1) displays slightly hyperintense signals within the mass, with multiple punctate and patchy areas of increased signal intensity observed (red arrow, A). Tumor is focally encased by fibrous pseudocapsules (B). In non-myxoid areas, tumor cells exhibit either an epithelial-like or spindle-shaped morphology, with biphasic cytoplasm, moderate cellular pleomorphism, prominent nucleoli, and a diffuse, sheet-like arrangement (C, D). In myxoid areas, epithelioid or spindle-shaped tumor cells proliferate in a loosely cohesive reticular or clustered manner (E, F). The tumor cells show diffuse positivity for MUC1 (G) and ER (H) but are negative for Keratin (I) and CD34 (J). SMARCB1 expression remains absent in all tumor cells (K, vascular reactivity used as an internal positive control). The Ki-67 proliferation index is approximately 15% (L)

Macroscopically, the tumor measured approximately 55 mm × 50 mm × 40 mm and had a gray-white appearance with a soft solid texture. Microscopically, histological images revealed the tumor was surrounded by a fibrous pseudocapsule with clear boundaries at low magnification (Fig. 2B). At high magnification, the tumor cells exhibited some degree of atypia. The histological morphology displayed two distinct cellular patterns. One pattern was characterized by tumor cells exhibiting either an epithelial-like or spindle-shaped morphology, with biphasic cytoplasm, moderate cellular pleomorphism, prominent nucleoli, and a diffuse, sheet-like arrangement (Fig. 2C, D). Another pattern showed epithelial-like tumor cells distributed sporadically in the mucous matrix (Fig. 2E, F). Notably, the mucoid areas comprised approximately 10% of the entire tumor volume, with a mitotic figure observed at a rate of 7 per 10 HPFs.

Immunohistochemical staining showed that the tumor cells were positive for MUC1 (Fig. 2G), ER (Fig. 2H), PR, and vimentin, with focal positivity for SMA. However, the tumor cells tested negative for Keratin (Fig. 2I), S-100, Desmin, and CD34 (Fig. 2J). In addition, the tumor cells demonstrated a loss of SMARCB1 expression (Fig. 2K). The Ki-67 proliferation index was approximately 15% (Fig. 2L). The tumor had clear boundaries and was not accompanied by necrosis. Although the tumor cells exhibited an epithelial-like morphology, they did not express Keratin, S-100, CD34, or SMARCB1. This morphology, along with the immunohistochemical profile, effectively ruled out the possibility of epithelial sarcoma, leading to a diagnosis of MELTVR. Following tumor resection, the patient underwent a vulvar wide excision and was followed for 35 months, with no recurrence or metastasis observed.

Case 3

A 45-year-old woman accidentally discovered a lump in the mons pubis. An ultrasound examination revealed a hypoechoic nodule measuring 21 mm × 6 mm within the adipose layer of the right mons pubis. The patient underwent tumor resection at an outside hospital and later sought a pathology consultation at our facility.

Macroscopically, the surgical specimen consisted of multiple fragments, totaling approximately 80 mm × 80 mm × 25 mm. The specimen appeared pale brown with a soft, solid texture. Microscopically, the tumor tissue was fragmented, making it challenging to assess its boundaries at low magnification (Fig. 3A). At high magnification, the tumor cells exhibited varying degrees of atypia. The histological morphology resembled that of Case 2 and was characterized by two distinct cellular patterns. One pattern featured tumor cells with epithelial-like or spindle-shaped morphology, biphasic cytoplasm, moderate cellular pleomorphism, prominent nucleoli, and a diffuse arrangement of tumor cells in a sheet-like pattern (Fig. 3B-D). Another pattern showed scattered arrangements of short spindle-shaped or epithelial-like tumor cells in a mucinous background (Fig. 3E, F). The mucoid areas comprised approximately 10% of the entire slide, with a mitotic figure observed at a rate of 12 per 10 HPFs.

Fig. 3

The tumor tissue is fragmented (A). In non-myxoid areas, tumor cells exhibit either an epithelial-like or spindle-shaped morphology, with biphasic cytoplasm, moderate cellular pleomorphism, prominent nucleoli, and a diffuse, sheet-like arrangement. (B-D). In myxoid areas, epithelioid or spindle-shaped tumor cells proliferate singly or in a loosely cohesive reticular or clustered manner (E, F). The tumor cells show focal positivity for MUC1 (G), as well as diffuse positivity for ER (H). In contrast, the tumor cells are negative for Keratin (I) and CD34 (J). SMARCB1 expression is completely absent in all tumor cells (K, vascular reactivity used as an internal positive control). The Ki-67 proliferation index is approximately 20% (L)

Immunohistochemical staining revealed that the tumor cells were focally positive for MUC1 (Fig. 3G) and diffusely positive for vimentin and ER (Fig. 3H). The cells were negative for Keratin (Fig. 3I), CD34 (Fig. 3J), P63, and SMA. In addition, the tumor cells demonstrated a loss of SMARCB1 expression (Fig. 3K). The Ki-67 proliferation index was approximately 20% (Fig. 3L). The tumor cells displayed an epithelial-like morphology and did not express Keratin, S-100, or SMARCB1, effectively ruling out the possibility of soft tissue myoepithelioma and epithelial sarcoma. Consequently, the diagnosis was confirmed as MELTVR. Following tumor resection, the patient was followed for 8 months, with no recurrence or metastasis observed.

Genomic characteristics

Whole-exome sequencing (WES) was performed on DNA isolated from tumor tissue for all three patients. Additionally, peripheral blood was collected from Patient 2 (C122201) to identify germline mutations. Due to severe RNA degradation in Patient 1 (C122202), gene fusion analysis based on RNA sequencing was only performed for Patients 2 (C122201) and 3 (C122203).

WES revealed that missense mutations were the most prevalent type of mutation observed, with the tumor mutational burden (TMB) in the three patients being relatively low (ranging from 1.35 to 4.33). Copy number variation (CNV) analysis indicated that gene deletions were the predominant type, while gene amplifications were rare (Fig. 4A). All three patients exhibited deletions in the SMARCB1 gene (Fig. 4B-D). Additionally, RNA sequencing facilitated the identification of two novel fusions: PPP6R3::FHDC1 and MYH9::MYH6 (Fig. 4E); however, no fusions involving genes such as EWSR1, NR4A3, or FUS were detected.

Fig. 4

Genomic characteristics of MELTVRs. Distribution of the top 20 genes with CNVs (A). Copy number distribution of the SMARCB1 gene in patients (B-D). The red line represents the threshold, the pink line indicates the logarithmic values of copy numbers, and the points represent the bins. Schematic diagram of gene fusion (E)

Discussion and Conclusions

MELTVR, as a rare SMARCB1-deficient mesenchymal neoplasm of the vulvar region, exhibits distinctive immunophenotypic and genomic characteristics. The clinical and pathological features of the reported 21 patients in the English literature and our cases are summarized in Table 1. The tumor consists of mucinous and non-mucinous areas, with a distribution of cellular-rich regions [2, 4, 9,10,11]. The histological morphology observed in the three patients presented in this study is consistent with the descriptions reported in the literature.

Table 1 Case reports of MELTVR in english literature

Immunohistochemically, the tumor cells are positive for vimentin, MUC1, ER, and PR, with ER and PR exhibiting either partial or diffuse positivity. All tumor cells also show a deficiency in SMARCB1 expression. In some patients, the tumor cells are also positive for SMA, ERG, STAT6, CD34, Calponin, and Keratin [1,2,3,4, 9, 12], but completely negative for Desmin, Myogenin, SOX10, p63, S-100, and GFAP. All three patients presented in this study exhibited MUC1 and ER expression, with loss of SMARCB1 expression. Patients 1 and 2 also demonstrated calponin expression, which is consistent with existing literature [3, 9].

Although MELTVR can be differentiated from other SMARCB1-deficient tumors through immunophenotypic and genomic analysis, our understanding of the comprehensive genomic features of MELTVR remains limited. In this report, we present, for the first time, an analysis of the genomic characteristics of three patients of MELTVR utilizing high-throughput sequencing technology. The TMB of MELTVR was relatively low. In all three patients, there was a deletion of the SMARCB1 gene copy number. Additionally, fusion mutations involving the PPP6R3::FHDC1 and MYH9::MYH6 genes were identified in two of these tumors, respectively. FHDC1 is an actin-regulating factor with scaffolding functions, associated with organ development and tumorigenesis. The MYH9 has been shown to inhibit the migration and invasion of tumor cells in various cancers, including ovarian cancer, osteosarcoma, hepatocellular carcinoma, and malignant melanoma, exerting an anti-tumor effect [13,14,15,16]. However, the fusion mutations involving PPP6R3::FHDC1 and MYH9::MYH6 are exceedingly rare and have not been previously reported to date.

MELTVR should be differentiated from a spectrum of neoplasms, including soft tissue myoepithelioma, extraskeletal myxoid chondrosarcoma (EMC), epithelioid sarcoma (ES), extrarenal malignant rhabdoid tumor (E-MRT), and benign mesenchymal neoplasms with a predilection for the women genital tract (such as cellular angiofibroma, aggressive angiomyxoma, and angiomyofibroblastoma).

MELTVR has a similar histological pattern to soft tissue myoepithelioma; however, their immunophenotypic and genetic profiles differ substantially. Firstly, the majority of MELTVR patients demonstrate negative Keratin expression, and all tumors lack reactivity for S-100 protein and GFAP. In contrast, 90% of myoepithelioma express Keratin, and 85% express S-100 protein [17]. Secondly, while some myoepitheliomas may show loss of SMARCB1 protein expression, all MELTVR consistently lack SMARCB1 protein immunoreactivity [7]. Furthermore, approximately 50% of soft tissue myoepitheliomas harbor EWSR1 gene rearrangements, whereas EWSR1 gene rearrangements have not been identified in any MELTVR [8]. The IHC results for our three tumors demonstrated an absence of expression of S-100, CD34, CD31, SMA, Desmin, and Keratin, with varying degrees of MUC1 expression. All three tumors exhibited a lack of SMARCB1 protein expression, and the Ki-67 proliferation index ranged from 10 to 35%.

EMC usually occurs in the deep soft tissues of the limbs in middle-aged and elderly patients, with rare involvement in the superficial perineal area. Microscopically, it displays a lobulated pattern composed of tumor cells distributed in a cord-like or mesh-like fashion within a myxoid matrix, with sparse interstitial vessels. The cytoplasm of tumor cells is deeply eosinophilic, showing fine granular or vacuolated features, and mitotic figures are rare. Expression of MUC1 and ER in EMC is uncommon, with 90% showing NR4A3 gene rearrangement [18, 19].

ES, particularly the proximal type, can involve the vulvar region and exhibits infiltrative growth with areas of necrosis. The tumor is composed of epithelioid tumor cells with vesicular nuclei, evident atypia, prominent nucleoli, frequent mitotic figures, and pathological mitoses. ES almost always expresses Keratin, 50–70% expresses CD34, 90% exhibit SMARCB1 deficiency, and concurrent SMARCB1 gene deletion or mutation is present [1, 7].

E-MRT predominantly affects the midline deep soft tissues of infants and children, comprising sheets of round or polygonal rhabdoid cells. The tumor cells have large nuclei with chromatin in a vacuolated pattern, prominent nucleoli, and frequent mitotic figures, and are often associated with hemorrhage and necrosis. Tumor cells express Keratin, Vimentin, MUC1, and SALL4, with loss of SMARCB1 protein expression, and concurrent occurrence of homozygous SMARCB1 gene deletion or mutation [20].

The imaging manifestations of tumors are closely related to the histopathology; however, there are currently scarce imaging reports on this tumor. Patient 1 appeared as a well-defined hypoechoic mass on ultrasound, with thick and uneven linear hyperechoic lines at the margins, possibly representing the fibrous pseudocapsule of the tumor. The internal short linear hyperechoic lines corresponded to fibrous structures of varying widths, with some areas showing cystic hypoechoic regions related to tumor hemorrhage and necrosis [2, 4]. In patient 2, the tumor had indistinct borders, with fine spicules at the periphery indicative of infiltration into the surrounding fat tissue from outside the tumor capsule [2, 4]. Histologically, the tumor stroma was rich in blood vessels with elongated branching vessels, showing sclerosis in the walls of medium and small vessels, visualized on ultrasound as abundant short rod-shaped and linear blood flow signals, a feature observed in both Patient 1 and Patient 2. The Patient 1’s MRI-enhanced scan showed sustained uneven enhancement. The imaging features of this disease are difficult to differentiate from vascular-rich tumors in soft tissues such as hemangiomas, vascular fibromas, and sarcomas, necessitating immunohistochemistry and genetic molecular studies for a definitive diagnosis.

In conclusion, we identified for the first time the presence of PPP6R3::FHDC1 and MYH9::MYH6 gene fusions in MELTVR, potentially providing therapeutic targets for the treatment of recurrent MELTVR. Although no common mutations were found among the three patients, this study marks the first revelation of MELTVR’s genomic characteristics. We aim to collect more patient data to develop a more comprehensive genomic atlas of MELTVR.

Materials

Immunohistochemistry

Immunohistochemical analysis was performed on 4-µm FFPE tissue sections using the following antibodies and conditions: Keratin (clone: MX005, Maixin Biotech, Fuzhou, China), vimentin (clone: MX034, Maixin Biotech, Fuzhou, China), S-100 (clone: MXR034, Maixin Biotech, Fuzhou, China), MUC1 (clone: E29, Maixin Biotech, Fuzhou, China), Calponin, (clone: MX023, Maixin Biotech, Fuzhou, China), SMA (clone: MX097, Maixin Biotech, Fuzhou, China), CD34 (clone: MX123, Maixin Biotech, Fuzhou, China), estrogen receptor (ER, clone: SP1, Maixin Biotech, Fuzhou, China), progesterone receptor (PR, clone SP2, Maixin Biotech, Fuzhou, China), SMARCB1 (clone MRQ27, Maixin Biotech, Fuzhou, China), Ki-67 (clone MXR002, Maixin Biotech, Fuzhou, China), Desmin (clone MX046, Maixin Biotech, Fuzhou, China), and P63 (clone MX013, Maixin Biotech, Fuzhou, China). The EnVision Plus detection system (Dako, Glostrup, Denmark) was used for all antibodies. Standard procedures and antigen retrieval protocols were followed. Appropriate positive and negative controls were included throughout.

Next generation sequencing

Simultaneous extraction of RNA and DNA from tissue samples, and gDNA from peripheral blood, was performed using the Nucleic Acid Isolation Kit (Novogene, Tianjin, China). The concentration of DNA and RNA was tested with a Qubit 4.0 Fluorometer (Life Technologies, CA, USA). In addition, DNA and RNA integrity were tested in the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). DNA and RNA library hybridization capture was conducted using the Novogene WES Plus Panel and RNA Fusion Panel, respectively (Novogene, Tianjin, China). All libraries were sequenced on the NovaSeq X Plus platform (Illumina, CA, USA). For data analysis, BWA and STAR were used to align DNA and RNA sequence data to the reference genome (hg19), respectively. VarScan, Samtools, and GATK were employed to call DNA SNVs/Indels, while Delly was used for DNA fusion detection. STAR-Fusion was utilized for RNA fusion calling.