Complexity of PEComas

In March 2017, a 12.5-cm large lobulated tumor without necrosis was resected from the left soleus muscle of a 58-year-old patient. Histologically, the tumor revealed a solid growth pattern of pleomorphic cells with abundant granular eosinophilic cytoplasm (Fig. 1a). The nuclei showed irregular contours and prominent nucleoli. Occasionally, nuclear inclusions could be seen and on average 1–2 mitoses/HPF were found. Immunohistochemically, the tumor showed a patchy positivity for HMB-45 and caldesmon (Fig. 1b, c) as well as smooth muscle actin. Other melanocytic markers, such as Sox-10, Melan A, MITF, and S‑100 and myogenic markers, such as desmin, MyoD1, and myogenin, were both negative. Furthermore, the tumor cells were negative for cytokeratin (AE1/AE3) and CD68 (PGM-1). TFE-3 immunohistochemistry (clone MRQ-37, Cell Marque, Rocklin, CA, USA) revealed only a weak-to-moderate nuclear staining, which is not characteristic of a TFE-3 translocated tumor. In addition, a TFE-3 translocation was absent on fluorescence in situ hybridization (FISH) analysis (ZytoLight ® SPEC TFE3 Dual Color Break Apart Probe, ZytoVision GmbH, Bremerhaven, Germany). The diagnosis of a malignant PEComa was made. Because of the narrow resection margin (0.1 cm), local radiation was performed.

In December 2017, the patient developed a left-sided pleural dissemination with tumor nodules of up to 15 cm in size and partial infiltration of the left lower lobe of the lung; the lesions were completely resected via partial pneumo- and pleurectomy. Necrosis was again not recognizable in the tumor. The patient refused the recommended postprocedural radiation. In June 2018, two new metastases (8.9 cm and 7.1 cm in diameter) in the left mediastinum were discovered on computed tomography (CT) scans and were histologically verified by biopsy (Fig. 2a). At that time, the tumor was examined immunohistochemically and molecularly in order to find possible drug targets. In a multigene panel analysis (Human Actionable Solid Tumor Panel kit, Qiagen, Venlo, The Netherlands) only a pathogenic TP53 mutation in exon 5, but no other mutations especially no melanoma-associated mutations, could be detected. Immunohistochemically, the tumor revealed a weak-to-moderate expression of phosphorylated mTOR (protein phosphorylation site SER2884, Cell Signaling Technology, Danvers, MA, USA) and a strong expression of the two main target proteins of the mTOR complex 1, namely, phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1; Clone 53H11, Cell Signaling Technology) and phosphorylated ribosomal p‑RPS6 (protein phosphorylation site SER235/236, Cell Signaling Technology; Fig. 1d–f). On the basis of the immunohistochemical results, treatment with everolimus (10 mg daily), an mTOR inhibitor, was started in July 2018. On a restaging CT scan in September 2018, a mixed response was detected, which was judged as stable disease. Because of iron-deficiency anemia, fatigue, and mucositis, the dosage of the mTOR inhibitor had to be reduced to 5 mg. In November 2018, tumor progression was visible on CT scans. The metastasis in the left mediastinum measured 13.9 and 10 cm (Fig. 2b). In December 2018, a re-thoracotomy with left-sided pneumectomy, pericardial sac resection, and tumor resection was performed. The resected tumor showed large necrotic areas, accounting for about 50% (Fig. 2c), which were evaluated as effects of mTOR inhibitor therapy.

In January of 2016, a 39-year-old male patient presented as an emergency case because of severe epistaxis. His medical record showed an embryonal carcinoma of the testis in 2006. Clinical examination revealed a bleeding fleshy polypoid mass located at the left middle nasal concha. Computed tomography scans showed pansinusitis without a clear-cut tumor formation. No distant metastases were found on positron emission tomography (PET) scans. The polypoid tumor was subsequently resected.

Macroscopically, we saw a soft brown–red polypoid tumor measuring up to 2.5 cm. Histological examination revealed a neoplasm composed of epithelioid cells in a prominent alveolar to nested architecture (Fig. 3a, c). The cytoplasm was clear to finely granular, the vesicular nuclei were atypical and showed prominent nucleoli. Of note, areas of the tumor cells were heavily pigmented with melanin (Fig. 3a, b). No suspicious PAS-positive inclusions were present, and mitoses, necrosis, or angioinvasion were likewise not detected. Immunohistochemically, the tumor cells were diffusely HMB-45 positive and showed a strong nuclear expression of TFE3 (Fig. 3d, f). The tumor cells were negative for any other melanocytic markers such as S100 (Fig. 3e), Melan A, MITF, SOX10, all myogenic markers (SMA, MSA, desmin, caldesmon) and cytokeratins. The Ki67-index was 5%. In line with the other results, Melanoma-associated mutations (BRAF, NRAS, c‑Kit) were absent and malignant melanoma was ruled out. Pursuing the nuclear TFE3 expression, FISH analysis with a TFE3 gene break-apart probe was performed, which was initially interpreted as being negative. Only a small subset of tumor cells with minimal separation of the 5′TFE3 and 3′TFE3 probe signals were detected, not meeting established cut-off values. In order to verify the immunohistochemical results and to possibly unveil the binding partner, we performed RNA-sequencing (TrueSight RNA Fusion Panel, Illumina, San Diego, CA, USA), which disclosed an extremely rare NONO–TFE3 fusion. NONO and TFE3 genes are both located on the X chromosome leading to an inverse translocation. The proximity of the two genes is the reason for the only minimal signal separation and initial misinterpretation of the TFE3 FISH analysis. In light of this specific constellation, a second FISH analysis, in which small gaps of one to two signal diameters were now correctly interpreted, confirmed the TFE3 gene disruption (Fig. 3g). Finally, the diagnosis of a melanotic TFE3-(Xp.11)-rearranged PEComa was made. The follow-up of the patient remained unremarkable 2 years after resection.

A 58-year-old woman underwent a hysterectomy in December 2010 in an external hospital under the suspicion of having uterine leiomyomas. Histologic analysis of the morcellated uterus, however, led to the diagnosis of an epithelioid, possibly malignant, PEComa of the uterus. In the follow-up examination 3 months later, the patient presented with a multinodular large recurring intraperitoneal pelvic tumor, located in the peritoneum of the cecum, sigmoid/descending colon, greater omentum, mesorectum, bladder and retroperitoneally near the right ureter. All visible tumor nodules were resected in April 2011. However, only 2 months later, a new multinodular pelvic relapse and, in addition, pulmonary metastases (Fig. 4a, b) were noted. In August 2011, the patient was transferred to the German Clinical Competence Center for Sarcomas and Mixed Tumors of the Female Genital Tract in Greifswald. Because the pelvic tumor, measuring 15 cm in diameter, was not resectable and the disease had become systemic with four pulmonary metastases in both lungs, a (palliative) chemotherapeutic approach was considered as the appropriate treatment. At that time, case reports showed that mTOR-targeted therapy could be of value because this signaling pathway is usually strongly active in this tumor type [9, 13, 25, 27]. Of note, retrospective analyses of these case reports have shown that tumors proven to be mTOR-active by immunohistochemistry are particularly prone to regression [13, 27]. Thus, a biopsy of the pelvic tumor was performed to examine the activity of mTORC1-signaling as a biomarker for subsequent therapy. Histologically, the tumor showed the typical morphology and immune profile of an epithelioid malignant PEComa, i.e., co-expression of myogenic and melanocytic markers, such as desmin, Melan-A, and HMB45. Of note, the tumor revealed a strong immunohistochemical expression of not only mTOR but also its two main target proteins of the mTOR complex 1 by use of phosphorylation-specific antibodies against the active forms of the proteins, i.e., p‑4EBP1 and p‑RPS6. We also examined the primary tumor regarding mTOR activity retrospectively and found the same pattern of alterations (Fig. 5a). These tissue biomarkers provided the scientific basis in this individual case to start everolimus therapy, an mTOR inhibitor. The patient received everolimus in a daily dose of 10 mg for four consecutive months until December 2011. At the end of therapy, the patient complained mainly of severe fatigue, a known side effect of the therapy.

Everolimus therapy was extremely successful. Imaging studies showed a complete regression of the pulmonary metastases and an astonishing shrinkage of the pelvic tumor bulk from 15 cm to 3 cm in diameter (Fig. 4c, d). As this success proved to be much better than expected, the initial approach from a palliative systemic therapy was switched to a neoadjuvant approach that was followed by the resection of the residual tumor nodule in January 2012. The 3‑cm-large tumor was resected in total and consisted of 50% vital tumor tissue (Fig. 5b) with identical histology and immune phenotype as in the prior manifestations (Fig. 5a). In necrotic areas, a strong resorptive inflammatory reaction by foamy macrophages and histiocytic giant cells was noted, intermingling with single or small clusters of damaged but vital tumor cells. Of note, the vital tumor tissue still showed strong activation of mTORC1 signaling on immunohistochemistry and also by immunoblotting of frozen tumor tissue, which has never been investigated before (Fig. 5c). As the patient was now completely free of disease, and since no data about a beneficial continuous pharmacological therapy were available, the local tumor board decided to discontinue mTOR inhibitor therapy. However, 5 months later, the patient presented with another tumor relapse, showing a large non-resectable tumor mass in the pelvic region and the right kidney as well as new lung metastases. Since the tumor still showed high mTOR activity in the last resection, the everolimus treatment was once again started. Again, the tumor showed an impressive response to the targeted therapy, lasting for about 1 year. However, in June 2013, the pelvic recurrence and the lung metastasis showed tumor progression and everolimus treatment was discontinued and switched to conventional therapy with doxorubicin monotherapy (six cycles 60 mg/m²), which was of limited benefit. The patient died in 2014 of brain metastasis 3 years after the initial diagnosis. An autopsy was not performed.

Perivascular epithelioid cell neoplasms are rare tumors occurring in many anatomic sites and, depending on the location, leading to distinct differential diagnosis. Thus, awareness of this entity is mandatory in order to arrive at the correct diagnosis, and the investigation of the molecular background may help to predict the response to targeted therapies.