The clinicopathological features of epithelioid undifferentiated sarcoma with TFE3 amplication: one case report and literature review

Background: Strong nuclear expression of TFE3 protein resulting from gene fusion has been reported in some neoplasms. TFE3 amplication has been proven to be a novel mechanism leading to increased protein level only in several cases of perivascular epithelioid cell tumor and renal cell carcinoma. Such rare genetic alteration might be associated with poor prognosis or aggressive course. Herein, we rst reported a case of undifferentiated sarcoma with epithelioid features harboring TFE3 amplication. Case presentation: A 66-year-old woman with a history of chronic lymphocytic leukemia and chemotherapy presented with a 4 cm palpable nodule in the left lower leg. Magnetic resonance imaging revealed an oval juxtacortical lesion to the anterolateral left tibia. Microscopically, the large epithelioid cells with marked pleomorphism and the small round cells intermingled with each other in a diffuse sheet or a hemangiopericytoma-like vascular growth pattern. Myxoid stromal change was evident focally, imparting a hypocellular appearance. Atypical mitotic gures and lymph node metastasis were identied while tumor necrosis was absent. Immunohistochemically, the tumor was positive for vimentin, TFE3, CD68 and CD34. TFE3 gene amplication was identied by uorescence in situ hybridization. Surgical resection was performed. The patient was alive without recurrence 8 month after surgery. Conclusions: The present case might represent a novel entity. Our report expands the scope of tumors carrying TFE3 amplication, and raises more attention to this rare genetic alteration and its association with potential aggressive behavior of the tumor.


Background
As a number of microphthalmia/transcription factor E (MiT/TFE) family, the transcription factor E3 (TFE3) protein plays an important role in tumorigenesis 1,2 . Strong nuclear expression of TFE3 protein is characteristically identi ed in several tumors including alveolar soft part sarcoma (ASPS), perivascular epithelioid cell tumor (PEComas), Xp11.2 renal cell carcinomas, granular cell tumor, and epithelioid haemangioendothelioma [3][4][5][6][7] . The genetic or epigenetic alterations involving TFE3 expression are complicated and have not been fully elucidated. Apart from gene translocation, TFE3 gene ampli cation has been proven to be an additional, novel mechanism leading to increased TFE3 protein expression in several cases of PEComas 8-10 and renal cell carcinoma 11 . In this article, we rst presented a case of undifferentiated sarcoma with epithelioid features harboring TFE3 ampli cation, its clinicopathological features, and differential diagnosis.

Case presentation
A 66-year-old woman presented with a 4 cm palpable and painful nodule in the left lower leg that was detected approximately 1 month before consultation. The patient was diagnosed with chronic lymphocytic leukemia (CLL) 8 years ago. Clinic staging was not available. Oral chlorambucil was attempted once per week for 4 years, combining with thymalfasin injection twice a month to improve the immune condition. The patient had a good response to therapy with no sign of recurrence or blastic transformation. The familial history was uneventful. Magnetic resonance imaging revealed an oval juxtacortical lesion with a well-de ned margin to the anterolateral left tibia, measuring 4.9×2.4×1.2 cm.
Periosteal reaction of the tibia was identi ed, indicating its aggressiveness. Neither cortical erosion nor marrow in ltration was observed ( Figure.1A-E). Surgical resection was performed.
Grossly, the resected specimen consisted of multiple fragments with gray or white on cut surface, measuring 6×3×2 cm in size. Microscopically, the well-circumscribed but unencapsulated lesion displayed variable cellularity. Two morphologically distinct types of tumor cells intimately intermingled with each other and were arranged in a diffuse sheet or a hemangiopericytoma-like vascular pattern ( Figure. Figure S1), and MPO. The Ki-67 index was up to 60% in the hot spot area ( Figure.3D). Fluorescence in situ hybridization (FISH) analysis with TFE3 Probe was breakapart designed to detect gene rearrangement and could be also used for the detection of polyploidy or target gene ampli cation. In normal cells of the present case (female), the copy of TFE3 gene is 2. But in tumor cells, TFE3 Probe show more copies, and the average copy number in each cell is 7. Of which, the number of Green probe and Orange probe is equal in each ampli ed cell. One hundred consecutive nuclei were counted, 1R1G1F accounted for 1%, and the polyploid proportion was 68% ( Figure.4A). Gene fusion of TFE3 with PRCC, ASPSCR1 or NONO was not identi ed by FISH with gene fusion probe. One hundred consecutive nuclei were counted, 1R1G1F accounted for 1%, 2% and 3%, the polyploid proportion was 68%, 65% and 61%, respectively ( Figure.4B-D). Therefore, no TFE3 rearrangement but TFE3 gene ampli cation was identi ed in the present case. We made the diagnosis of undifferentiated sarcoma with epithelioid features harboring TFE3 ampli cation. The patient was alive and well at 10 month follow-up information after surgery by radiological examination.

Discussions And Conclusions
Based on the morphological and genetic features, PEComa is a strong consideration for the diagnosis. PEComas can be composed of epithelioid and spindle cells arranged in a hemangiopericytoma-like vascular pattern. TFE3 is positively expressed in a number of PEComas resulting from gene fusion 12 .
TFE3 gene ampli cation, a characteristic nding in the present case, has also been documented in three cases of PEComas; all of these PEComas were found in adults and showed predominant or pure epithelioid morphology. Among these, two cases displayed marked atypical large cells with abundant eosinophilic cytoplasm, nuclear anaplasia, and multipolar mitotic gures, sharing histological overlaps with the present case. However, tumor cells in PEComas are commonly positive for melanocytic and smooth muscle markers; all the three cases were positive for Melan-A and actin/SMA, two were positive for HMB45 [8][9][10] , whereas none of Melan-A, HMB45 and actin/SMA were expressed in the present case. Therefore, evidence for classifying the tumor as PEComa is insu cient.
Several sarcomas should also be taken into consideration owing to their morphologic resemblance, including solid variant of ASPS, epithelioid sarcoma, epithelioid angiosarcoma, and epithelioid rhabdomyosarcoma. ASPS typically is composed of large eosinophilic cells in nest or pseudoalveolar growth pattern delineated by brous septa. In solid variant of ASPS, tumor cells arrange in a diffuse sheet growth pattern without conspicuous nesting, brous septa, or dilated sinusoidal vascular channels.
Increased cytologic pleomorphism and nuclear pleomorphism are uncommon but have been described 13 .
ASPS demonstrates nuclear expression of TFE3, resulting from the oncogenic fusion or rearrangement of TFE3 5 . However, such characteristic chromosomal translocation was not detected by FISH in the present tumor. The intracytoplasmic accumulation of rod-shaped crystals, which is another speci c character of ASPS, could be found in a majority of the cases, was not identi ed. Both epithelioid sarcoma and epithelioid angiosarcoma are composed of diffusely arranged large cells with abundant eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. As for epithelioid sarcoma, especially in the proximaltype, pleomorphism is prominent 14 . It is one of the few mesenchymal neoplasms that metastasize to lymph nodes. CD 34 is expressed in 50% of cases. However, epithelioid sarcoma is positive for CK and EMA, and usually loss of nuclear INI1 expression 15 . Epithelioid angiosarcoma presents irregular vascular spaces lined by protuberant epithelioid tumor cells. Endothelial differentiation should be demonstrated by the expression of endothelial markers such as CD31, CD34, ERG and FLI-1 13 . In contrast to the obvious cytologic pleomorphism in the present case, cells and nuclei in epithelioid rhabdomyosarcoma tend be relatively uniform in size. Skeletal muscle differentiation should be proven based on immunostaining for myogenin and myoD1 16 . Therefore, the diagnoses of ASPS, epithelioid sarcoma, epithelioid angiosarcoma, and epithelioid rhabdomyosarcoma were easily excluded.
Immunochemical panels were used to explore possible differentiation of the present tumor. Apart from vimentin, TFE3, and INI-1, only CD68 and CD34 were positive. Because of the non-speci city, whether such immunophenotype would be a diagnostic feature remained to be illuminated. CD68 expression might indicate histiocytic proliferation and be found in brous histiocytomas and granular cell tumor 13 . However, the tumor cells were negative for CD163, a biomarker that is truly histocyte speci c superior to CD68, rendering the evidence for histiocytic differentiation weak. CD34 is a non-speci c biomarker that should always be used in conjunction with morphology and other markers. Diffuse expression is usually signi cant; weak or focal expression is often nonspeci c. Tumors with broblastic/ myo broblastic or peripheral nerve sheath differentiation might be positive for CD34. Most of them should simultaneously express other more speci c markers, such as SMA and/or desmin, and S-100 protein respectively. Other markers explored in the present case include MiTF, STAT6, CDK4, MDM2 and MPO, all of which were negative, demonstrating the undifferentiated nature of the tumor. TFE3 immunoreactivity has been identi ed in several neoplasms, ranging from weak to intense expression levels ( Table 1). The most common chromosomal abnormalities involving TFE3 are translocations and rearrangements, and are among the earliest reported gene fusion in tumorigenesis 17,18 . TFE3 gene fusion can occur with different partners, such as PRCC, ASPSCR1, SFPQ/PSF, and NONO, all of which provide active gene promoters, and therefore, lead to higher levels of TFE3 fusion proteins than wild-type TFE3 5,19,20 . Apart from ASPS, PEComas, and renal cell carcinoma, which were most extensively studies, epithelioid hemangioendothelioma with YAP1-TFE3 gene fusion 21 and malignant chondroid syringoma with PHF1-TFE3 gene fusion 22 were also identi ed respectively.
Although nuclear immunoreactivity for TFE3 protein by immunohistochemistry staining was initially considered as a highly sensitive and speci c diagnostic tool for neoplasms bearing TFE3 gene fusions 23 , subsequent FISH analysis demonstrated that there was no relationship between immunoreactivity and gene fusion 24,25 . For instance, although TFE3 was positive in 53%-91% of granular cell tumors and 93.5% of desmoid-type bromatosis, gene rearrangement was absent according to FISH analysis, whether the immunohistochemical staining was weak or strong 24,26,27 . Therefore, molecular and/or cytogenetic analysis should be performed to avoid false positive staining 28 . TFE3 gene ampli cation has only been identi ed in several cases. Apart from PEComas, it has also been con rmed in four cases of renal cell carcinomas with moderate to strong nuclear expression of TFE3 11 . TFE3 is located on X-chromosome, aneuploidy for X-chromosome was detected and resulted in increasing TFE3 copy numbers 10,11 . In the present case, we also inferred that it was the X polyploidy that caused TFE3 gene ampli ed. An interesting nding is that such genetic alteration might be associated with aggressive biological behavior [8][9][10][11] . As for renal cell carcinoma, patients with TFE3 ampli cation exhibited a signi cantly poorer cancer-speci c survival rate than those with translocation 11 . As for PEComas, distant metastasis and recurrence were documented [8][9][10] . In the present case, tumor aggressiveness has been suggested by radiological examination, and one lymph node metastasis was identi ed.
The latency time between the diagnosis of CLL and the present sarcoma was 8 years. There is no strong evidence of the relationship between these two tumors in this patient. Studies have shown increased risks of second malignancies after CLL. Langerhans cell sarcoma 29 , myeloid sarcoma 30 , and histiocytic/dendritic cell sarcomas 31 would transdifferentiate from CLL. Skin neoplasm was also reported preceding the diagnosis CLL, including malignant melanoma 32 , Kaposi sarcoma [33][34][35] , Merkel cell carcinoma, malignant brous histiocytoma, dermato brosarcoma protuberans, sebaceous carcinoma 36 , and leiomyosarcoma 37 . However, the morphological and immunochemical features of the present case did not support any of these neoplasms. The most common adverse effect of chlorambucil is myelosuppression. One conjunctival Kaposi's sarcoma was developed in one patient with sympathetic ophthalmia treated with high-dose, short-term chlorambucil therapy 38 . No study has documented undifferentiated sarcoma resulting from chlorambucil therapy.
In conclusion, we present a case of undifferentiated sarcoma with characteristic genetic alteration. Even comprehensive immunohistochemistry and molecular examination could not help in classifying the tumor under any of the existing soft tissue tumor classi cation. Therefore, we designated it as epithelioid undifferentiated sarcoma with TFE3 ampli cation. Our report indicated TFE3 ampli cation as an additional chromosomal abnormality might result in increased expression of protein, and suggested the possible relationship between the genetic alteration and biological behavior of tumor.

Declarations
Ethics approval and consent to participate The patients provided informed consent. The study was approved by the Ethics Committee of Clinical Research and Experimental Animal of the First A liated Hospital, Sun Yat-Sen University.

Consent for publication
Written informed consents for publication of clinical details and clinical images were obtained from the patient. A copy of the consent form is available for review by the Editor of this journal.

Availability of data and materials
Please contact author for data requests.

Competing interests
The authors declare that they have no competing interests.

Funding
None.

Authors' contributions
Yuejiao Lang and Xiaojuan Li contributed equally to this work. Yuejiao Lang wrote the manuscript.
Xiaojuan Li collected and analyzed the pathological data. Shaoyu Chen performed and analyzed FISH. Pei Xiang analyzed the radiological examination and wrote the coincident part. Anjia Han analyzed the data and revised the manuscript.