Pigmented perivascular epithelioid cell tumors of the orbit with NONO::TFE3 fusion: Molecular evaluation and literature review

Abstract

Purpose

To elucidate the clinical characteristics of rare orbital pigmented perivascular epithelioid cell tumors (PEComa) with transcription factor E3 (TFE3) rearrangement through a literature review.

Observations

A 49-year-old female was referred to our hospital with a mass measuring 16 × 14 mm in the extraconal space of the right orbit. Suspecting hemangioma or distended hematoma clinically, the tumor was completely excised through the right nasal conjunctiva. Although initial histological evaluation raised the suspicion of malignant melanoma, subsequent immunohistochemistry and molecular analyses led to a diagnosis of TFE3-rearranged PEComa with the NONO::TFE3 fusion gene. The patient's visual function was preserved 14 months postoperatively, with no evidence of recurrence.

Conclusions and importance

Although rare, TFE3-rearranged PEComa should be considered in the differential diagnosis of pigmented orbital tumors.

1. Introduction

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms composed of perivascular epithelioid cells characterized by smooth muscle and melanocytic differentiation.¹ PEComas occur in various anatomical locations. They are more frequently observed in females than males (M:F ratio: 1:3∼5.5), with a wide age range and a peak incidence in young to middle-aged adults.1, 2, 3 Inactivating mutations in tuberous sclerosis complex 2 (TSC2), which occur sporadically or as germline mutations in tuberous sclerosis, have been observed in most PEComa cases (89–92 %).^4,5 However, a small subset of PEComas exhibit transcription factor E3 (TFE3) rearrangements.^6,7 These two subsets are mutually exclusive, and TFE3-rearranged PEComas have distinctive pathological features from those of typical PEComas, such as an almost exclusively epithelioid morphology, minimal expression of smooth muscle proteins, and occasional melanin pigmentation.^3,6,7 Orbital TFE3-rearranged PEComa is an exceedingly rare disease, with only six patients reported to date (including the present case).8, 9, 10, 11, 12 Differentiating orbital TFE3-rearranged PEComa from other tumors is sometimes challenging. This case report describes the case of a 49-year-old female with orbital pigmented PEComa with the NONO::TFE3 fusion gene, along with a review of the literature.

2. Case report

A 49-year-old female was referred to our hospital with hyperemia and swelling of the conjunctiva of the right eye for one month. The patient had been diagnosed with hormone receptor-positive/HER2-positive invasive ductal carcinoma (pT2N2aM0, stage IIIA) of the left breast at the age of 42 years and had undergone partial mastectomy and subsequent radiotherapy, hormone therapy, anti-HER2 treatment, and chemotherapy. No relevant family history was reported. Examination revealed a subconjunctival mass that appeared bluish-black in color, and could be subcutaneously palpated from the right upper eyelid to the inner eye angle. Dilated and tortuous blood vessels were present across the surface of the mass (Fig. 1). A hemangioma or distended hematoma was initially suspected. Preoperative magnetic resonance imaging revealed a well-defined mass measuring 16× 14 mm in the right extraconal space of the right orbit. The lesion exhibited high signal intensity with no fat suppression effect on T1-weighted images (Fig. 2A), and slightly heterogeneous low signal intensity on T2-weighted images (Fig. 2B). Owing to the relatively long waiting period at our hospital, the patient underwent surgery at an affiliated hospital. Intraoperatively, the tumor was found to be confined within the capsule and was completely excised in its entirely through the right nasal conjunctiva. The surgical specimen was submitted to a private laboratory for pathology evaluation with the provisional diagnosis of malignant melanoma. The patient was referred back to our hospital for confirmation of the pathological diagnosis and additional treatment.

Fig. 1.

Photograph of the orbital lesion. The conjunctiva over the orbital mass appeared bluish-black throughout. Dilated and tortuous blood vessels were present across the surface.

Fig. 2.

Pre-operative magnetic resonance imaging. (A) A well-defined mass measuring 16 × 14 mm was observed in the right extraconal space of the right orbit. It appeared hyperintense on T1-weighted fat-saturated images (arrow). (B) A slightly heterogeneous low-intensity signal was observed on T2-weighted imaging (arrow).

Formalin-fixed paraffin-embedded tissue from the surgical specimen was subjected to hematoxylin and eosin staining and immunohistochemistry (IHC). Histological examination revealed a capsulated solid nodular lesion (Fig. 3A). The lesion consisted of relatively uniform, plump atypical epithelioid cells arranged in nests and sheets that were separated by thin fibrous septa and narrow vascular channels. The cells had an abundant, clear to partially vacuolated eosinophilic cytoplasm with melanin pigments, and round nuclei with small nucleoli (Fig. 3B). The mitotic count was one per 50 high-power fields (field diameter: 0.55 mm). Extensive infarct-type necrosis was observed. Lymphovascular invasion was not observed, and the resection margins were negative. The lack of conspicuous nuclear atypia and infrequent mitotic figures led us to consider tumors other than melanoma. The morphological features of nested growth of epithelioid cells with rich, delicate vascular connective tissue stroma suggested a differential diagnosis of alveolar soft part sarcoma (ASPS) and paraganglioma, in addition to PEComa. Immunohistochemical analysis revealed strong positivity for HMB45 (HMB45) and TFE3 (polyclonal) (Fig. 3C and D); weak positivity for cathepsin K (E-7); and negativity for Melan A (A103), S-100 (polyclonal), SOX10 (SP267), INSM-1 (A-8), SMA (1A4), and PAX8 (SP348). The Ki-67 (MIB-1) labeling index at the hotspot was 4 %. Melanoma was excluded based on the negativity for S-100, Melan A, and SOX10. ASPS was also excluded, since the strong HMB45 positivity was inconsistent with the diagnosis. INSM1 was negative, and S-100/SOX10-positive sustentacular cells were absent, ruling out paraganglioma. The tumor demonstrated diffuse and strong reactivity to HMB45 and TFE3, suggesting a diagnosis of TFE3-rearranged PEComa.

Fig. 3.

Microscopic findings. (A) At low power, a well-circumscribed solid nodular lesion with a thin fibrous capsule was visible. (B) At high power, the tumor cells were seen to proliferate in a nested pattern, separated by thin fibrous septa accompanied by rich narrow vascular channels (arrows). The tumor cells had abundant cytoplasm containing melanin pigment and round nuclei with small nucleoli (arrow heads). (C) The tumor cells showed strong and diffuse cytoplasmic reactivity to HMB45. (D) The tumor cells also showed strong and diffuse nuclear reactions for TFE3. On immunohistochemistry evaluation, cytoplasmic melanin pigment in the tumor cells appeared bluish-green with Giemsa counterstaining (arrow heads). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Molecular confirmation of TFE3 rearrangement was performed using fluorescence in situ hybridization (FISH). The TFE3 break-apart probe (Agilent Technologies) revealed separation of the 3′ (red) and 5′ (green) signals, indicating the rearrangement of TFE3 (Fig. 4). Next, RNA sequencing (RNA-seq) was performed to identify the fusion partner. SureSelect XT Human All Exome V6 (Agilent Technologies) was used to generate RNA-seq libraries. NovaSeq6000 (Illumina) was used to sequence RNA libraries at 151 bp pair-end. Arriba v2.4.0 was used to map the sequencing data to the hg38 reference genome, and the fusion transcripts were analyzed. In-frame NONO::TFE3 fusion joining the 3′ end of exon 12 of the NONO gene with the 5′ end of exon 5 of the TFE3 gene was observed (Fig. 5A). To verify the fusion point, reverse transcription polymerase chain reaction (RT-PCR) was performed, followed by Sanger sequencing. The same fusion site identified by RNA-seq was confirmed (Fig. 5B).

Fig. 4.

TFE3 break-apart fluorescence in situ hybridization. The break-apart probe separates 3′ (red) and 5′ (green) signals. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.

Details of the NONO::TFE3 fusion. (A) In-frame NONO::TFE3 fusion joining the 3′ end of exon 12 of the NONO gene with the 5′ end of exon 5 of the TFE3 gene was observed by RNA sequencing. (B) Confirmation of fusion transcripts by reverse transcription polymerase chain reaction testing.

Postoperative positron emission tomography/computed tomography revealed no renal tumors. Finally, metastasis of Xp11 translocation renal cell carcinoma was excluded, and the patient was diagnosed with pigmented TFE3-rearranged PEComa.

Thereafter, although we recommended annual imaging surveillance, the patient declined due to the medical expenses involved. The patient's visual function was preserved at 14 months postoperatively, with no clinical evidence of recurrence.

3. Discussion

3.1. Literature review of orbital PEComa

To date, six cases of orbital TFE3-rearranged PEComa have been reported, which, along with eight cases with unknown TFE3 status, lead to a total of 14 documented cases of orbital PEComa.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Table 1 presents the clinicopathological characteristics of these 14 patients. The median age of the patients was 20 years (range: 4–54 years). Among them, 10 were male and four were female. The clinical presentations included proptosis, swelling of the eyes, diplopia and reduced vision, with these conditions persisting or worsening over a period of several months in most cases. The median tumor size was 1.6 cm (range: 1.0–5.2 cm). Clinically, two cases (including the present case) were suspected to be hemangioma,¹⁰ and one case was suspected to be rhabdomyosarcoma.¹⁸ Histologically, two cases were initially misdiagnosed as ASPS,^16,20 and one case (present case) was suspected as malignant melanoma. Excluding one case with unknown clinical follow-up information, the follow-up period ranged from 3 to 120 months (median, 12 months). No recurrence was observed in 11 patients who underwent complete resection,9, 10, 11^,13, 14, 15, 16, 17^,19 of whom one patient also underwent neoadjuvant and adjuvant chemotherapy.¹⁶ Among the remaining three patients, one patient underwent postoperative local radiotherapy due to adhesion of the tumor to neighboring tissues²⁰; one patient experienced local recurrence or residual tumor¹²; and chemotherapy was administered, but surgical excision could not be performed in the third patient.¹⁸ Increased mitotic activity, nuclear atypia and infiltrative growth were observed in the cases with local recurrence or requiring additional treatments.^12,16,20

Table 1.

Clinicopathological characteristics of orbital perivascular epithelioid cell tumors in the literature (n = 14).

	Age (years) /sex	Clinical presentation [Presumptive diagnosis]	Size (cm)	Pigments	Worrisome histological features	MF [Ki-67 LI]	TFE3 IHC/Molecular analyses	Treatment	Outcome
Iyengar et al. (2005)13	9/F	Painless orbital mass (for 2 m)	1.2	+	–	Infrequent MF [<10 %]	NA/NA	Complete excision	NED, 7 m
Guthoff et al. (2008)14	54/M	Painless swelling of the right eyelid (slowly progressive)	1.5	+	–	NA [<1 %]	NA/NA	Complete excision	NED, 17 m
Lubo et al. (2016)15	47/M	Progressive diplopia (for 4 m)	2.0	+	MF > 1/50 HPF	2/50 HPF	-/NA	Complete excision	NED, 40 m
Argani et al. (2016)8	20/M	NA	NA	+	NA	NA	+/NONO::TFE3 (FISH)	NA	NA
Alam et al. (2017)16	5/M	Painless proptosis (gradually increased) [ASPS]	>5.0	–	Nuclear atypia; MF > 1/50 HPF	>1/HPF	NA/NA	Adjuvant and neoadjuvant chemotherapy + Complete excision	NED, 2 y
Paliogiannis et al. (2017)17	46/M	Painful round lesion of the right orbit	1.5	–	MF > 1/50 HPF	2/10 HPF	NA/NA	Complete excision	NED, 50 m
Varan et al. (2017)18	7/M	Progressive swelling of the right eye (for 3 m) [Rhabdomyosarcoma]	3.4	–	Necrosis	0 MF	+/NA	Incisional biopsy + sirolimus therapy attempted, followed by chemotherapy	AWD, 6 m (residual)
Nair et al. (2018)19	9/F	Painless progressive swelling of the right eye, proptosis, reduced vision (for 1 year)	5.2	–	–	NA [5–7 %]	NA/NA	Complete excision	NED, 6 m
Gao et al. (2021)9	20/M	Foreign body sensation in the right eye	1.0	+	–	NA	+/NONO::TFE3 (FISH, RNA-seq)	Complete excision	NED, 10 y
Feu-Basilio et al. (2021)10	28/M	Painless orbital mass (slow-growing) [Cavernous hemangioma]	1.6	–	–	<1 MF/10HPF	+/NONO::TFE3 (NGS)	Complete excision	NED, 3 m
Lin et al. (2023)20	4/M	Painless swelling of the right eyelid, progressive diplopia, reduced vision (for 1 m) [ASPS]	2.5	–	Necrosis; hyperchromatic chromatin; unclear boundaries; increased MF	Increased MF [10 %]	+/NA	Surgical excision (adherent to neighboring tissues) + radiotherapy	NED, 4 m
Bouzid et al. (2024)11	9/F	Painless progressive ptosis, superior palpebral mass	1.7	+	Necrosis	Low mitotic activity	+/NONO::TFE3 (FISH, RNA-seq)	Complete excision	NED, 12 m
Takahashi et al. (2025)12	35/M	Eye pain, diplopia	1.0	–	Necrosis; partial pleomorphism; MF > 1/50 HPF	6/50 HPF [11 %]	+/ZC3H4::TFE3 (FISH, RNA-seq)	Tumor enucleation + under consideration of additional treatment	AWD, 3 m (residual or local recurrence)
Present case	49/F	Hyperemia, swelling of the conjunctiva (for 1 m) [Hemangioma; malignant melanoma]	1.6	+	Necrosis	1/50 HPF [4 %]	+/NONO::TFE3 (FISH, RNA-seq, RT-PCR)	Complete excision	NED, 14 m

Abbreviations: ASPS, alveolar soft part sarcoma; AWD, alive with disease; F, female; FISH, fluorescence in situ hybridization; HPF, high power field; IHC, immunohistochemistry; LI, labeling index; m, month; M, male; MF, mitotic figure; NA, not available; NED, no evidence of disease; NGS, next-generation sequencing; RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction; y, year.

Of the six cases of TFE3-rearranged PEComa confirmed by molecular analyses,8, 9, 10, 11, 12 the fusion partners were NONO in five (including the present case) and ZC3H4 in one case. All the cases of orbital TFE3-rearranged PEComa showed pure epithelioid morphology and expression of TFE3 and HMB45 on IHC. Muscle markers were negative in four, focally positive in one, and unknown in one case. Intracytoplasmic melanin pigments were observed in more than half of the cases.

3.2. Mechanism of tumorigenesis induced by the TFE3 fusion gene

The TFE3 gene, located on chromosome Xp 11.2, is involved in chromosomal translocation in several tumors, such as Xp-translocated renal cell carcinoma and ASPS, producing fusion proteins. TFE3, the encoded protein, is a member of the MiT family of transcription factors, along with MITF, TFEB, and TFEC, which are involved in melanocyte development.^28,29 These transcription factors share a helix-loop-helix leucine zipper dimerization domain motif, a transactivation domain, and a basic region involved in DNA binding. All MiT family transcription factors bind to identical DNA recognition sequences (CA[T/C]GTG), known as E-boxes owing to their sequence homology, promoting the expression of genes downstream of transforming growth factor-beta signaling.³⁰ Most breakpoints in the TFE3 fusion gene are located between exon 4 and exon 6, which retains the helix-loop-helix and transcriptional activation domains of TFE3.^3,8,9,23,27 The NONO gene, which is located on chromosome Xq 13.1, is the most common fusion gene in orbital TFE3-rearranged PEComas.8, 9, 10, 11, 12 It encodes proteins belonging to the Drosophila behavior/human splicing (DBHS) family (such as NOPS and RRP), which are multifunctional nuclear proteins involved in mRNA splicing and various aspects of RNA metabolism.^31,32 The SFPQ gene, which is the most common fusion partner in TFE3-rearranged PEComas across various organs,^3,6,8 also encodes DBHS family proteins. In the NONO::TFE3 and SFPQ::TFE3 fusions, the rearrangement results in the fusion of almost the entire splicing factor protein with the TFE3 helix-loop-helix DNA-binding domain 33. Splicing and DNA transcriptional control interference might be involved in the tumorigenesis of TFE3-rearranged PEComa, although the details remain unknown.

3.3. Differential diagnosis of orbital TFE3-rearranged PEComa

TFE3-rearranged PEComa has the potential to arise in any organ; however, its rare occurrence in the orbit makes diagnosis difficult. The present case showed tortuous and dilated blood vessels running across the surface of the bluish-black tumor, which was clinically suspected to be a hemangioma. Feu-Basilio et al. also reported a similar case in which hemangioma was clinically suspected.¹⁰ Cavernous hemangioma is the most common primary orbital tumor producing proptosis,²¹ and TFE3-rearranged PEComa in the orbit can be clinically misinterpreted as a hemangioma because it presents with slowly progressive proptosis as a clinical manifestation. A 7-year-old boy reported by Varan et al. was clinically suspected of rhabdomyosarcoma.¹⁸ Rhabdomyosarcoma is the most common malignant mesenchymal orbital neoplasm, and it is the most common primary malignant orbital tumor in children, especially those under 10 years of age.²¹ However, compared to the rapid onset of rhabdomyosarcoma, orbital PEComas tend to progress slowly and show different clinical courses. In our literature review, two cases were histologically misdiagnosed as ASPS,^16,20 which is another rare tumor in the orbit that predominantly affects children.²² Morphological and phenotypic similarities observed between ASPS and TFE3-rearranged PEComa, such as a nested growth pattern with a rich capillary network, strong and diffuse nuclear TFE3 positivity, and negativity for muscle markers on IHC, are thought to contribute to the diagnostic misinterpretation. In ASPS, the presence of pseudoalveolar-like structures resulting from central discohesion, along with the absence of melanocytic marker expression, are distinguishing features from TFE3-rearranged PEComa. Cases of misdiagnosis of TFE3-rearranged PEComa as melanoma have also been previously reported,^23,24 as was also seen in the present case, which was initially suspected as malignant melanoma at the referring institution. The presence of melanin pigment within the tumor might have led to consideration of melanoma in the differential diagnosis. Melanoma is the most common tumor arising from the uvea, but its occurrence in the orbit is exceedingly rare.²⁵ TFE3-rearranged PEComa is reported to frequently contain melanin pigment,^3,6,7 and more than half of the orbital cases exhibit melanin pigments. Therefore, when encountering orbital tumors with melanin pigments, TFE3-rearranged PEComa should be included in the differential diagnosis.

According to the 5th edition of the WHO Classification of Soft Tissue and Bone Tumors, molecular confirmation methods, such as FISH or next-generation sequencing, are not considered essential criteria for the diagnosis of TFE3-rearranged PEComa.¹ The diagnosis might be suggested based on its characteristic morphology of epithelioid cells with granular eosinophilic to clear cytoplasm, nested architecture, expression of melanocytic markers, and strong and diffuse expression of TFE3. However, it is important to note that TFE3 immunohistochemical staining does not always correlate well with the status of translocation.²⁶ Molecular confirmation should be considered when the clinical presentations or histological features are atypical.

3.4. Prognosis of orbital TFE3-rearranged PEComa

The generally used classification of PEComa proposed by Folpe et al. considered tumors with two or more worrisome features, including a size of >5 cm, infiltrative growth, high nuclear grade and cellularity, mitotic rate of >1/50 high-power fields, necrosis, and lymphovascular invasion, as malignant.² Although not well understood due to its rarity, the prognosis of TFE3-rearranged PEComa is thought to be poorer than that of conventional PEComa.²⁷ In our literature review, all orbital TFE3-rearranged PEComas were found to be less than 5 cm in size, and no recurrence after complete resection was reported.9, 10, 11 Its conspicuous location in the eyes, early onset of subjective symptoms, and detection at a relatively smaller size might be associated with a favorable prognosis. However, despite the tumor being small, measuring 1 cm, one case showing high mitotic activity, necrosis and focal pleomorphism, developed either local recurrence or residual tumor following enucleation.¹² This suggests that even small tumors warrant intensive follow-up when multiple worrisome histological features are present. On the other hand, in another case of orbital PEComa with unknown TFE3 translocation status and multiple worrisome histological features, complete resection following combination chemotherapy led to a disease-free survival of two years.¹⁶ Thus, combination chemotherapy might be beneficial in the treatment of malignant PEComas, although the number of reported cases remains limited, and further studies are needed.

4. Conclusions

Herein, we reported a case of orbital TFE3-rearranged pigmented PEComa. Although rare, TFE3-rearranged PEComas should be considered in the differential diagnosis of orbital pigmented tumors. Orbital PEComas are typically detected at a relatively small size, and generally have a favorable prognosis following complete surgical excision. However, since the follow-up period was short in most of the patients found in the literature review, including the present case, further investigation is needed. Further, even if the tumor is small, the presence of malignant histological features might indicate a locally aggressive behavior.

CRediT authorship contribution statement

Naoko Takeda-Miyata: Writing – original draft, Formal analysis, Data curation, Conceptualization. Ken-ichi Yoshida: Writing – review & editing, Formal analysis. Miho Shirono: Writing – review & editing, Data curation. Akihide Watanabe: Writing – review & editing, Resources, Formal analysis, Data curation. Yoji Kukita: Writing – review & editing, Formal analysis. Chie Sotozono: Writing – review & editing, Supervision, Formal analysis. Eiichi Konishi: Writing – review & editing, Supervision, Formal analysis, Data curation, Conceptualization.

Patient consent

Written informed consent was obtained from the patient for publication of this case report and the accompanying images.

Funding

This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.