Perivascular Epithelioid Cell Tumor of Gastrointestinal Tract

Perivascular epithelioid cell tumors of gastrointestinal tract (GI PEComas) are exceedingly rare, with only a limited number of published reports worldwide. Given the scarcity of GI PEComas and their relatively short follow-up periods, our current knowledge of their biologic behavior, molecular genetic alterations, diagnostic criteria, and prognostic factors continues to be very limited. We present 2 cases of GI PEComas, one of which showed an aggressive histologic behavior that underwent multiple combined chemotherapies. We also review the available English-language medical literature on GI PEComas-not otherwise specified (PEComas-NOS) and discuss their clinicopathological and molecular genetic features. Pathologic analyses including histomorphologic, immunohistochemical, and ultrastructural studies were performed to evaluate the clinicopathological features of GI PEComas, their diagnosis, and differential diagnosis. Immunohistochemistry, semiquantitative reverse transcriptase polymerase chain reaction, and DNA sequencing assays were carried out to detect the potential molecular genetic alterations in our cases Microscopically, the tumors showed distinctive histologic features of PEComas-NOS, including fascicular or nested architecture, epithelioid or spindled cell type, and clear to eosinophilic cytoplasm. The tumor cells were immunohistochemically positive for melanocytic markers. Molecular pathological assays confirmed a PSF-TFE3 gene fusion in one of our cases. Furthermore, in this case microphthalmia-associated transcription factor and its downstream genes were found to exhibit elevated transcript levels. Knowledge about the molecular genetic alterations in GI PEComas is still limited and warrants further study.

INTRODUCTION P erivascular epithelioid cell tumors (PEComas) are a family of rare mesenchymal neoplasms histologically and immunohistochemically characterized by perivascular epithelioid cell (PEC) differentiation. 1 The PECs have variable morphologic features, with an epithelioid to spindled cell type resembling smooth muscle, clear to granular lightly eosinophilic cytoplasm, and round to oval nuclei with small nucleoli. The PECs also exhibit a distinct immunophenotype with a coexpression of melanocytic and myogenic markers, such as HMB45, Melan-A, MiTF, smooth muscle actin (SMA), and calponin. 2 The PEComa family includes angiomyolipoma (AML), clear cell ''sugar'' tumor of the lung (CCST), lymphangioleiomyomatosis (LAM), clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres (CCMMT), and unusual clear cell tumors in other locations. PEComas have been reported in various anatomic sites, with a marked female predominance. Due to their relative rarity, the diagnostic criteria, optimal treatment strategies, and prognostic factors for PEComas have not yet been confirmed at this time. We report 2 cases of PEComas arising in the gastrointestinal tract, including the clinicopathological features and potential molecular genetic alterations of this rare tumor.

Case 1
A 29-year-old Chinese woman was admitted to our hospital because of gradual onset of abdominal pain, nausea, vomiting and weight loss for 6 months. 3 The patient did not have a medical history of gastrointestinal tumors, inflammatory bowel disease, or tuberous sclerosis complex. Her family history was unremarkable. Physical examination revealed a large mass at the right lower abdomen. All blood and biochemical tests were within the normal ranges, apart from a hemoglobin reading of 85 mg/dL and a C-reactive protein (CRP) reading of 15 mg/dL. An intravenous contrast-enhanced computed tomography (CT) scan showed an ill-defined multilocular soft tissue tumor measuring 13 cm Â 8 cm Â 7 cm in the pelvis and lower abdomen ( Figure 1). During the surgical operation, a large tumor was found in the terminal ileum about 12 cm from ileocecal valve adhering tightly to the mesentery of the ileum and the right pelvic wall. Surgical resection of the tumor and the affected segment of the intestine was carried out. After surgery, the patient received 5 courses of multiple combined chemotherapies including ifosfamide 2000 mg/m 2 day 1 to 4, epidoxorubicin 30 mg/m 2 day 1 to 3, dacarbazine 350 mg/m 2 day 1 to 4, and mesna 4800 mg/m 2 day 1 to 4 once every 3 weeks (Q3W). In addition, granulocyte colony-stimulating factor (G-CSF; Filgrastim) was given at a dose of 5 pg/kg/day subcutaneously from day 5 to day 12 of each cycle. Follow-up CT scans were performed every 6 months after chemotherapy. The patient was alive and well with no signs of recurrence or metastasis for 28 months of follow-up.

Case 2
A 41-year-old Chinese woman with a history of hysterectomy for benign leiomyoma presented with progressive epigastric pain and dark stools. The patient denied any family history of gastrointestinal (GI) cancer or inflammatory bowel disease. Laboratory investigations showed a white blood cell (WBC) count of 11.6 Â 10 9 cells/L with 87.3% neutrophils and a hemoglobin reading of 100 mg/dL. Serum levels of CA19-9, CEA, AFP, and CA125 were within normal limits. The abdominal CT scan and ultrasonography revealed ileocecal intussusception with a tumor in the ileum. An enteroscopy displayed a 2-cm diameter, polypoid, submucosal tumor in the terminal ileum ( Figure 2). At laparotomy, neither celiac lymphadenectasis nor distant metastatic foci were detected. Approximately, 45 cm of the terminal ileum (20 cm) and proximal colon (25 cm) were resected. Without additional therapy, the patient remained asymptomatic and free of disease at 39 months postoperatively.

Histologic and Immunohistochemical Analyses
The tumor tissues were fixed in 10% phosphate buffered saline formalin solution and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin for microscopic examination. Immunohistochemical stainings were performed with the primary antibodies listed in Table 1

Electron Microscopy
The tumor samples were collected and fixed in 4% phosphate-buffered solution of glutaraldehyde and osmium A B   tetroxide. Ultrathin sections were cut for ultrastructural evaluation using a Tecnai G 2 Spirit TWIN electron microscope (FEI Co, Eindhoven, Netherlands).

Reverse Transcription-Polymerase Chain Reaction and Sequencing
A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed to detect related gene fusions. Total RNA was isolated from the tumor tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The first strand of cDNA was obtained using RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). PCR was performed with rTaq polymerase (Takara Shuzo, Ohtsu, Japan). The PCR primers and cycling conditions were performed as described in Table 2. The resulting PCR products were analyzed in 1.5% agarose electrophoretic gels using DL1, 000 DNA Marker (Takara Shuzo) as a size reference. Gel images were obtained with Gene Genius (Syngene, Frederick, MD). The specific PCR product was sent to Shanghai Invitrogen Biotechnology Co., Ltd (Guangzhou Office) for purification and sequencing. In the analysis of MiTF and its downstream genes expression, malignant melanoma tissue served as a positive control, and normal intestinal mucosa distant from cancer served as a negative control.

Case 1
On gross examination, an ill-defined tumor measuring 13.5 Â 7.5 Â 7.0 cm was found at the terminal ileum about 12 cm from ileocecal valve ( Figure 3). The cut surface showed a pinkish-grey solid parenchyma, with several scattered and irregular cystic spaces containing clear colorless serous fluid. The tumor mainly involved the muscularis propria, and protruded into the tunica adventitia, while the mucosa and submucosa were still intact. The right pelvic side wall and mesentery of the ileum were invaded by the tumor. Regional lymph nodes and lymphovascular invasions were void of any tumor. Microscopically, the tumor showed epithelioid cell proliferation with a vaguely nested pattern ( Figure 4A). The nests were separated by thin fibrovascular septa. Some areas showed a pseudoglandular histological appearance ( Figure 4B). The tumor cells had clear to eosinophilic granular cytoplasm.

Case 2
Grossly, a 2.2-cm diameter, polypoid, submucosal tumor was located in the terminal ileum protruding into the lumen. On cut section, the tumor was well-circumscribed but not encapsulated ( Figure 5). Histologic examination revealed clear spindle-shaped cells arranged in fascicular and nesting patterns, separated by thin fibrovascular septa ( Figure 6A-B). The nuclei were mainly round and vesicular. Neither mitosis nor necrosis was identified in the tumor. Immunohistochemical analysis revealed that the tumor cells expressed HMB45, Melan-A, SMA, HHF35, Desmin, and pathogenesis-related protein ( Figure 6C-F), and were negative for all other markers tested ( Table 1).
The above pathologic findings in our cases confirmed a final diagnosis of GI PEComas-NOS.

Electron Microscopy
Melanosomes and premelanosomes were identified in the cytoplasm of both cases, using electron microscopy ( Figure 7).

Molecular Pathological Studies
An RT-PCR assay was performed to detect related gene fusions, including PSF-TFE3, NonO-TFE3, PRCC-TFE3, and EWS-ATF-1. 4 A distinct band of 186 bp in length (lane 2) was amplified in case 1, representing the PSF-TFE3 fusion fragment ( Figure 8A). Sequencing of the amplified DNA revealed that the PSF-TFE3 fusion transcript contained an in-frame junction of exon 9 of the PSF gene to exon 6 of the TFE3 gene ( Figure 8B and C). Analyses regarding the expression levels of MiTF and its downstream genes showed that MiTF, TYR, CDK2, TBX2, and C-MET were upregulated in the tumor sample of case 1 ( Figure 8D). Case 2 was also tested here,, but no fusion genes were found.

Literature Review
English-language medical reports on GI PEComas-NOS were searched for on PubMed and Embase using the search terms PEComa and gastrointestinal tract or bowel. Their clinicopathological features were reviewed and tabulated in Table 3. 1,5 -30

DISCUSSION
According to the World Health Organization Classification of tumors, PEComas are defined as a family of rare mesenchymal neoplasms histologically and immunohistochemically characterized by PEC differentiation. 31 The PEComa family includes AML, CCST, LAM, CCMMT, and unusual clear cell tumors in other locations. [32][33][34][35] The latter subgroup, which has been collectively classified as PEComas-NOS, represents a collection of unusual, histologically and immunohistochemically distinctive tumors arising at various anatomic sites such as the uterus, gastrointestinal tract, and soft tissue. 31,35 The GI tract is the second most common site of PEComas-NOS, accounting for 20% to 25% of all PEComas-NOS cases. To the best of our knowledge, only 70 cases (including the current reports) of GI PEComas-NOS have currently been reported in the English language medical literature ( Table 3). The most common location of GI PEComas-NOS was the colon (n ¼ 32), [5][6][7][8][9][10][11][12][13][14]36 followed by the small intestine (n ¼ 20), 1,15-18,36,37 rectum (n ¼ 7), [19][20][21][22][23][24] and stomach (n ¼ 4). 25,26,36 Of the patients, 43 were females and 27 were males, with a ratio of 2:1. The age at diagnosis ranged from 5.5 to 71 years. Until now, all reported cases of GI PEComas-NOS were sporadic, and only 1 patient was associated with tuberous sclerosis syndrome. Given the rarity of GI PEComas-NOS and their relatively short follow-up periods, our current knowledge of their biologic behavior, natural history, criteria for malignancy, and prognostic factors is limited.
From recent clinical data, it appears that GI PEComas-NOS exhibit a spectrum of biologic behavior from benign to malignant. The majority of reported GI PEComas-NOS were considered to be benign or to have uncertain malignant potential, whereas only 22 cases (22/70) exhibited definite malignant behavior, with local recurrence in 4 cases, metastasis in 19 cases, and tumor-related death in 7 cases. 1,9,10,12,15,17,21,26,29,36 The proposed histologic features indicative of malignancy or high risk for aggressive clinical behavior in GI PEComas-NOS by Folpe et al 38 include infiltrative growth pattern, tumor size (>5 cm), high nuclear grade, tumor necrosis, high mitotic activity (>1/50 HPF), and lymphovascular invasion. A recent case series study of 35 GI PEComas has shown that malignant behavior was statistically significantly associated with marked nuclear atypia, diffuse pleomorphism, and mitoses !2/10 HPF, but not with tumor necrosis. 36 Optimal treatment strategies for GI PEComas-NOS have not yet been well established. Currently, surgical resection with a wide margin seems to be the mainstay of treatment. The benefit of adjuvant chemotherapy, 21 radiation, and immunotherapy 8 has not yet been established. In case 1, the tumor showed features of malignancy in the form of large size (13.5 Â 7.5 Â 7.0 cm), surrounding tissue invasion, necrosis, and high mitotic activity (3$5/50 HPF). Given the high risk of tumor aggressive behavior, the patient was treated with multiple combined chemotherapies after surgery on the basis of nonrhabdomyosarcoma soft tissue sarcoma protocol (COG-ARST0332). She was alive and well with no signs of recurrence or metastasis for 28 months of follow-up. However, the benefit of adjuvant chemotherapy is still an area of controversy that requires more evidence-based studies.
Knowledge about the molecular genetic alterations in PEComas is still limited. Some cases of AML, CCST, LAM, and CCMMT have been reported to be associated with tuberous sclerosis complex (TSC), a genetic disease caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) genes, whereas only one of the reported cases of GI PEComas-NOS showed an association with TSC until now. However, Cyclin D1 overexpression has been detected by immunohistochemistry in 5 cases of PEComas-NOS that were either malignant or had an uncertain malignant potential. 12,21,39 The role of Cyclin D1 in the pathogenesis and progression of PEComas is becoming an area of interest. However, no Cyclin D1 immunoreactivity was evident in the 2 current cases. In recent years, it has been reported that a distinctive subset of PEComas harbors TFE3 gene fusion. 40 Tanaka et al 11 reported the first case of GI PEComa-NOS with a PSF-TFE3 gene fusion. In this study, we have confirmed the PSF-TFE3 gene fusion in another GI PEComa-NOS (case 1). In addition, MiTF and its downstream genes including TYR, CDK2, TBX2, and C-MET were detected in elevated transcript levels. TFE3 and MiTF belong to the MiTF/TFE transcription factor family, which is believed to be involved in pivotal developmental and cellular processes in various cell types. In different human tissues, the ratio of expression of the MiTF/TFE family members is found to be  unique. 41 Translocations of these genes are implicated in the MiT translocation subgroup of renal cell carcinomas. 42,43 Recently, TFE3 has also been found to be an efficient regulator in melanocyte differentiation and pigment production under specific pathological conditions in vitro. 44 Also, results from several recent clinical studies support the hypothesis that TFE3 can substitute for MiTF in a subset of MiTF-negative PEComas. 10,13,38 As previously proposed by others, 10,13,40,45,46 the subset of tumors harboring TFE3 gene fusions or exhibiting TFE3 immunoreactivity share distinctive clinicopathological features including relatively young age, nested/alveolar architecture, epithelioid cells with eosinophilic cytoplasm, and negative immunoreactivity for MiTF or muscular markers. Some studies also confirmed that overexpression of the TFE3 fusion protein is necessary for proliferation, migration, invasion potential, and long-term survival of UOK-145 cell lines. 47,48 These findings suggest that TFE3 may play an important role in the tumorigenesis, and warrants further study. In summary, we report 2 cases of PEComas-NOS arising in the GI tract, one of which was confirmed to harbor a PSF-TFE3 gene fusion and to exhibit upregulation of MiTF and its downstream genes. Although the contribution of TFE3 to the pathogenesis and progression of PEComas-NOS remains poorly understood, the assessment of the TFE3 gene status may be necessary for an accurate diagnosis and prognosis of PEComas-NOS.