Prevalence of TERT Promoter Mutations in Orbital Solitary Fibrous Tumors

The orbital manifestation of a solitary fibrous tumor (SFT) is exceptionally rare and poses specific challenges in diagnosis and treatment. Its rather exceptional behavior among all SFTs comprises a high tendency towards local recurrence, but it rarely culminates in metastatic disease. This raises the question of prognostic factors in orbital SFTs (oSFTs). Telomerase reverse transcriptase (TERT)-promoter mutations have previously been linked to an unfavorable prognosis in SFTs of other locations. We analyzed the prevalence of TERT promoter mutations of SFTs in the orbital compartment. We performed a retrospective, descriptive clinico-histopathological analysis of nine cases of oSFTs between the years of 2017 and 2021. A TERT promoter mutation was present in one case, which was classified with intermediate metastatic risk. Local recurrence or progress occurred in six cases after primary resection; no distant metastases were reported. Multimodal imaging repeatedly showed particular morphologic patterns, including tubular vascular structures and ADC reduction. The prevalence of the TERT promoter mutation in oSFT was 11%, which is similar to the prevalence of extra-meningeal SFTs of the head and neck and lower than that in other extra-meningeal compartments. In the present study, the TERT promoter mutation in oSFT manifested in a case with an unfavorable prognosis, comprising aggressive local tumor growth, local recurrence, and eye loss.

According to the fifth edition of the WHO Classification of Tumors: Soft Tissue and Bone Tumors from April 2020, the diagnosis of an SFT should desirably demonstrate the NAB2::STAT6 fusion gene, particularly in inconclusive cases [4].Yet, a typical histology combined with CD34 and/or STAT6 overexpression is sufficient for diagnosis [11][12][13].Since the initial description of a 'hemangiopericytoma' in 1942 by Stout and Murray, the terminology and the diagnostic standards have been revised multiple times, which makes the interpretation of former work challenging [2,14].Only recently, in 2021, was the term 'hemangiopericytoma' removed from the fifth edition of the WHO Classification of Tumors: Central Nervous System Tumors and the term should no longer be used to describe an SFT [4].

Ethical Approval
This study was conducted in accordance with the Ethics Committee of Charité Campus-Virchow Klinikum (protocol code EA2/182/23 8 September 2023).This study was undertaken in accordance with all rules, directives, and guidelines for good scientific practice of the University Hospital Charité Berlin and according to the Declaration of Helsinki.

Study Design and Case Research
We performed a retrospective case analysis of surgically dissected oSFTs during the years 2017 to 2019 at the Department of Ophthalmology, Campus Virchow-Klinikum, at the University Hospital Charité Berlin.Cases of surgically resected oSFTs were investigated using the medical information system of the University Hospital Charité Berlin (SAP).We researched the operation procedure codes for orbitotomy (OPS 5-160.0-5-160.4)excluding the additional code of decompression (5-169), which was used for the surgical treatment of compressive endocrine orbitopathy.The remaining cases were screened on the basis of the definitive histopathological results.Of the 469 cases of orbitotomy surgery, eight cases of oSFTs were confirmed by histology.One new case was found incidentally as a patient presented with recurrent exophthalmos and then underwent tumor surgery during the research period.The aim of our study was to evaluate the prevalence of TERT promoter mutations of oSFTs in the context of clinical appearance, histology, and molecular pathology.To our knowledge, TERT promoter mutations in SFTs have never been studied in the orbital compartment exclusively [8,30,31,41].

Ethical Approval
This study was conducted in accordance with the Ethics Committee of Charité Campus-Virchow Klinikum (protocol code EA2/182/23 8 September 2023).This study was undertaken in accordance with all rules, directives, and guidelines for good scientific practice of the University Hospital Charité Berlin and according to the Declaration of Helsinki.

Study Design and Case Research
We performed a retrospective case analysis of surgically dissected oSFTs during the years 2017 to 2019 at the Department of Ophthalmology, Campus Virchow-Klinikum, at the University Hospital Charité Berlin.Cases of surgically resected oSFTs were investigated using the medical information system of the University Hospital Charité Berlin (SAP).We researched the operation procedure codes for orbitotomy (OPS 5-160.0-5-160.4)excluding the additional code of decompression (5-169), which was used for the surgical treatment of compressive endocrine orbitopathy.The remaining cases were screened on the basis of the definitive histopathological results.Of the 469 cases of orbitotomy surgery, eight cases of oSFTs were confirmed by histology.One new case was found incidentally as a patient presented with recurrent exophthalmos and then underwent tumor surgery during the research period.

Clinical and Radiographic Analyses
Cases were analyzed regarding demographic data (age at first diagnosis, sex), symptoms at initial presentation (laterality, proptosis/exophthalmos, swelling, vision disorders, pain), best corrected visual acuity, intraocular pressure, and ophthalmologic and systemic preconditions.The patients' history and course of disease were reviewed, including the department of initial presentation (neurosurgery, ophthalmology, oral and maxillofacial surgery, otorhinolaryngeal medicine), surgical treatment (craniotomy, orbitotomy, local excision, enucleation, or exenteration), tumor-free survival, time to relapse, and overall survival.Analysis of radiographic findings included CT scans of the head, cranial MRI scans, and orbital MRI scans.Imaging was undertaken by the Department of Radiology of the University Hospital Charité Berlin.If available, we also analyzed images obtained by external outpatient services.All imaging data were transferred to MERLIN Diagnostic Workcenter (Phönix-PACS GmbH, Freiburg im Breisgau, Germany) and analyzed by a radiologist with advanced experience in orbital imaging.

Analyses of Molecular Pathology
DNA isolation from formalin-fixed paraffin-embedded tumor samples was performed using the Maxwell RSC FFPE Plus DNA Kit (Promega Corporation 2800 Woods Hollow Road•Madison, WI 53711-5399 USA) according to the manufacturer's instructions.The TERT promoter mutations (genomic position chr5, 1,295,228 C>T hg19 coordinate and chr5, 1,295,250 C>T hg19) were analyzed using Sanger sequencing (forward primer: GGATTCGCGGGCACAGAC; reverse primer: CAGCGCTGCCTGAAACTC).Details regarding the PCR protocol and conditions are available upon request.Sequencing was performed at Eurofins Genomics, Ebersberg, Germany.NAB2::STAT6 mutation and -fusion variant analyses were performed using the RNA-Archer FusionPlex sarcoma panel (Archer-Now Part of Integrated DNA Technologies, Inc. 2425 55th Street, Boulder, CO 80301, USA) according to the manufacturer's instructions.

Epidemiological and Clinical Parameters
The study included nine patients, of which four were female and five were male.The mean age at first presentation to our hospital was 55 years (23 to 83 years, median: 46).The majority of the patients reported painless intra-or periorbital swelling (8/9) leading to ipsilateral proptosis/exophthalmos (7/9) without visual alteration (Figure 2a).In the case of altered best corrected visual acuity (BCVA), a relevant non-tumor-related ocular pathology was found.Detailed information regarding demographics and clinical parameters is presented in Table 1.One patient had a history of a periorbital tumor, which was formerly surgically removed by our ORL department.This tumor was classified as an SFT 5 years before admission to our ophthalmological department.Another patient presented with recurrent orbital mass after the R1 resection of an aggressive orbital SFT 8 years before, which was treated with adjuvant stereotactic radiation at the time.One patient had a history of a gynecologic tumor of unknown dignity, which was surgically removed with no radio-or chemotherapy being administered.
5 years before admission to our ophthalmological department.Another patient presented with recurrent orbital mass after the R1 resection of an aggressive orbital SFT 8 years before, which was treated with adjuvant stereotactic radiation at the time.One patient had a history of a gynecologic tumor of unknown dignity, which was surgically removed with no radio-or chemotherapy being administered.At first presentation, the BCVA was 0.9, IOP 21 mmHg, no history of ophthalmic disease.Computed tomography with contrast media was initially performed due to better availability.(b) Axial, (c) coronary, and (d) sagittal CT revealed a solid hyperdense intraconal mass with compressive growth, with no signs of osseous infiltration.After admission to our neurosurgical department, resection was performed via the transcranial pterional approach.Because of the proximity to the optic nerve, total resection could not be achieved (R1 status).After tumor reduction, the tumor did not show any relevant growth for three years and the patient was followed up regularly in our ophthalmological department.(BCVA: best corrected visual acuity.)After admission to our neurosurgical department, resection was performed via the transcranial pterional approach.Because of the proximity to the optic nerve, total resection could not be achieved (R1 status).After tumor reduction, the tumor did not show any relevant growth for three years and the patient was followed up regularly in our ophthalmological department.(BCVA: best corrected visual acuity.)

Imaging
All patients were evaluated by cranial and/or orbital MRI.Six patients underwent computed tomography (CT).Details of the imaging are shown in Table 2.The primary manifestation was mostly intraconal (6/9) (see Figure 2b), showing a tendency of secondary extraconal growth (3/9).One tumor showed intraconal expansion after extraconal manifestation.The other tumors primarily manifested in the extraconal space (3/9).On CT, the tumors appeared hyperdense with a rather homogeneous structure (6/9) (see Figure 2b-d).MRI studies showed a homogeneous (4/9) to heterogeneous (5/9) tumor with accentuated patterns of vascularization (8/9) and mostly solid tumor growth (7/9) with the displacement of local structures (3/9) (see Figure 3a).DWI imaging showed a diffusion restriction in around half of the tumors (4/9) (see Figure 3d).In MRI scans of seven tumors, we found a signal intensity void corresponding to intratumoral vessels (Figure 3b).A chemical shift artefact was found in three tumors.As only three of the nine tumors showed this, it appeared rather inconsistently (Figure 3c).The radiographic diagnosis differed from the final histopathology, with cavernous venous malformation (formerly hemangioma) being the most important preliminary diagnosis (5/9).restriction in around half of the tumors (4/9) (see Figure 3d).In MRI scans of seven tumors, we found a signal intensity void corresponding to intratumoral vessels (Figure 3b).A chemical shift artefact was found in three tumors.As only three of the nine tumors showed this, it appeared rather inconsistently (Figure 3c).The radiographic diagnosis differed from the final histopathology, with cavernous venous malformation (formerly hemangioma) being the most important preliminary diagnosis (5/9).

Surgery and Follow-Up
The number of surgeries a single patient had to undergo varied from 1 surgery to 14 surgeries depending on the number of local recurrences.No event of distant metastatic disease was reported.Surgical procedures of tumor resection included eye preserving techniques (23/31) and definitive tumor resection (8/31).The eye preserving techniques included, among others, transcutaneous and local resection (7/31), lateral orbitotomy (9/31), medial orbitotomy with swinging eyelid (1/31), and pterional access with craniotomy (2/31).In seven of nine cases, eye preservation could be achieved.However, it was recommended that one patient of the seven should undergo orbital exenteration.Definitive tumor resection demanded enucleation of the eye (1/9) and exenteration of the orbit (1/9) as well as extended osseous dissection (6/32).Details of the tumor surgery and follow-up are shown in Table 3.The case with extended osseous tumor expanse needed continuous treatment by our ORL department because of insufficient local tumor control.This included hemimaxillotomy and repeated local tumor debulking surgeries.Aggressive local tumor progress made radiation therapy of the wound area necessary.As tumor progress involved the carotid canal, chemotherapy with Pazotanib + Doxorubicin was considered.The patient's general condition did not allow chemotherapy with Anthracycline.Hence, in case of further tumor growth, the patient was planned to receive treatment with Pazotanib only.Primary tumor resection only achieved R0 resection in one case.In four cases, residual tumor mass was present (R1, 4/9) or it was unclear whether everything had been removed (Rx, 4/9).In one case, R0 resection was achieved after local recurrence (Table 4).Tissue samples for pathology were collected during every surgery.Two patients received adjuvant treatment with radiation.For another patient, radiotherapy had been recommended, but it remained unclear whether it was performed.The shortest period until relapse was 3 months due to incomplete resection.To date, the longest tumor-free interval is 198 months (Table 3).To rule out distant metastasis, tumor staging was performed in two patients.No distant metastasis was found.

Pathology
Of all of the tissue samples from tumor surgery, we chose to analyze 17 representative specimens.The samples were chosen from relevant points of time, including first manifestation, local recurrence, or clinically significant progression that made organ preservation impossible and resulted in definitive surgery (e.g., enucleation or exenteration).H&E staining depicted a picture of mostly intermediate, sometimes high cellularity with a spindled cell or partially spindled cell configuration (8/9), with an epithelioid aspect in parts (3/9) (see Figure 4a-d).Signs of malignancy such as a high cellularity (3/9), a high mitotic count (>1/mm 2 ) (2/9), or focal necrosis (1/9) were not common, but particularly appeared in samples from tumors with a clinically aggressive growth pattern (see Figure 4c,d) and in cases of relapse [1].The histologic parameters are depicted in Table 4, while detailed histology information is shown in Supplementary Table S1.In immunohistochemistry (see Figure 4e-h), the tumors exhibited quite a homogeneous pattern with strong positivity for CD34, nuclear STAT-6, CD99 (see Table 5), and Bcl-2 (Supplementary Table S2), but little or no staining for CD31, CK MNF116, or S100 (Supplementary Table S2).In all cases, an NAB2::STAT6 fusion transcript was demonstrated.The most common fusion variant was NAB2ex3::STAT6ex19 (see Table 6).According to the metastatic risk prediction model from Demicco et al., most of the tumors (7/9) were of low risk, and only two of the nine showed intermediate metastatic potential [24] (see Table 4).A TERT promoter mutation (C250T) was present in one of the nine cases (see Figure 5).The mutation was found in all examined specimens from the patient (primary and two recurrences) and did not occur during tumor progress (see Table 6).No unaffected tissue was examined.Interestingly, the related case showed the most aggressive growth pattern by far.The patient underwent local radiation and was evaluated for systemic chemotherapy (see above).Table 5. Quantity and intensity of immunohistochemistry.Some patients had multiple resections with different specimens.We analyzed the relevant specimen with immunohistochemistry and chose the most representative samples to be depicted.Representative pictures of immunohistochemistry are shown in Figure 3.  Table 5. Quantity and intensity of immunohistochemistry.Some patients had multiple resections with different specimens.We analyzed the relevant specimen with immunohistochemistry and chose the most representative samples to be depicted.Representative pictures of immunohistochemistry are shown in Figure 3.

Discussion
oSFT is an exceptionally rare occurrence with particular properties among all extrameningeal SFTs, such as a high tendency towards local recurrence, intermediate potential of malignant transformation, and a low likelihood of metastasis [3,5].In the present study, we described the prevalence of the TERT promoter mutation in SFTs in the orbit and associated pathology in nine cases.
The clinical presentation in our study cohort was very unspecific and depicted symptoms of displacement and compressive growth, as reviewed in the literature [16,18,42].Pathognomonic paraneoplastic syndromes, like refractory hypoglycemia syndrome (Doege-Potter syndrome) or Pierre-Marie-Bamberger syndrome, did not occur.This is probably due to the tumors of the orbit being of smaller size compared to tumors in other locations, such as in the pleura or retroperitoneum [21].Heterogeneous MRI signal intensities and CT densities have previously been linked to different densities of the soft tissue of SFTs [43].Diffusion restriction is a strong predictor for malignancy in orbital neoplasms [17].Meanwhile, low to intermediate ADC values could be helpful for distinguishing SFTs from benign lesions with similar morphology (e.g., cavernous venous malformation) [17].Signal intensity void is a phenomenon arising from intra-tumoral vessels and has been shown previously in oSFTs [44].As seven of the nine tumors depicted a signal intensity void, we considered this phenomenon to be a strong predictor for SFTs in solid and well circumscribed orbital lesions with diffusion restriction.Another radiographic clue is given by the chemical shift artefact.Chemical shift refers to the phenomenon of different precessional frequencies of protons in fat and water.This results in a shift in the spatial location of fat voxels in the frequency-encoded direction [45].The artefact appears as a dark or bright band at the interface between water and fat (Figure 3c).Usually, chemical shift is suggestive of cavernous venous malformation [46].Hence, in this study, cavernous venous malformation was the most frequent differential diagnosis on imaging.However, in our collective, it appeared rather inconsistently.In CNS-related locations, vascularized meningioma shows similar imaging patterns as meningeal SFTs and needs to be considered an important differential diagnosis [47,48].Interestingly, a recent study by Liu et al. demonstrated that DWI imaging (ADC1) could differentiate between SFT and atypical meningioma with high sensitivity and specificity [49].In conclusion, the imaging of oSFTs shows a heterogeneous tumor with varying T1/T2 intensity and low to intermediate ADC values.A signal intensity void was often present, while some oSFTs had a chemical shift artifact.Since the present study included some scans that were not performed at our clinic, signal intensities may vary.
In periorbital and orbital manifestation, curative treatment demands an aggressive local resection.The recurrence rates in our study were mainly dependent on resection status.R1 status was related to the preservation of anatomical structures that are crucial for visual function.In the majority of cases (7/9), an indistinct diagnosis at first resection explains why a conservative non-aggressive resection was performed.It has previously been reported that the surgical approach is a key factor for the recurrence rate [22].In a study from Yang et al., tumor resection via lateral orbitotomy reached similar recurrence rates (83%) to those in the present study (88%) [20].As the tumor dissection surgery was performed by an 'ophthalmological' surgical approach (local excision, swinging eyelid approach, lateral orbitotomy), the possibility of dissecting periosteum, bone, and dura was limited.According to Yang et al. [20], the 'neurosurgical approach', the transfronto-orbital approach, results in much lower recurrence rates (17.6%).However, the study by Yang et al. [20] evaluated the surgical resection of SFTs exclusively localized in the retrobulbar compartment with different degrees of involvement of intracranial structures and without specifying the primary site of tumor manifestation.We propose that tumor dissection should value tumor location as well as appropriate functional and anatomical status.In our study, the tumors tended to recur locally (6/9) and showed increasingly aggressive local growth.
The diagnosis of SFT demands a typical histology with CD34 and/or STAT6: spindled to ovoid cells that arrange around a branching and hyalinized system of vessels (staghorn pattern) (see Figure 4a-d) with different degrees of stromal collagen (see Figure 4b,d) [13].Since an increased degree of dedifferentiation can lead to a loss of expression of CD34 and STAT6 (see Figure 4g), we performed an extended immunohistological panel (see Supplementary Table S2) [50].While CD99 and Bcl-2 are a common feature of SFTs, we used other markers to exclude relevant differential diagnoses, like CD31 (e.g., vascularized malignancies like spindled-cell angiosarcoma), Cytokeratin MNF116 (e.g., epithelioid sarcoma), or S100 (e.g., malignant peripheral nerve sheath tumor) (see Supplementary Table S2) [51].Nevertheless, we tested all tumors for the defining NAB2::STAT6 fusion oncogene and the fusion variant.NAB2::STAT6 fusion variants like NAB2ex4-STAT6ex2/3 occur in less aggressive tumors in elderly patients and are mostly localized in the pleura, while NAB2ex6-STAT16/17 correlates with a more aggressive phenotype in younger patients [29].Georgiesh et al. showed that these differences might depend on the mutation break points in STAT6 and, therefore, looked into STAT6 integrity [27].NAB2::STAT6 fusion variants that possess the complete STAT6 protein, namely, STAT6-ex2/3/4/5/6 (STAT6-Full), showed a better prognosis than STAT6 with only the TAD, namely, STAT6ex16/17/18/19 (STAT6-TAD).In our cohort, most of the tumors (5/8) could be considered STAT6-TAD.Although both cases where eye preservation could not be achieved were STAT6-TAD, there was no clear connection to eye preservation or local recurrence (see Table 6).In our cohort of oSFTs, TERT promoter mutations were less frequent (1/9) than in previous studies.However, the prevalence of TERT promoter mutations was never investigated exclusively in the orbit.Bahrami et al. found a TERT promoter mutation in 28% (26/94) of a collective of 94 extrameningeal SFTs [31].Although the mutation occurred in 13/31 thoracic SFTs (42%) and in 13/63 (21%) of the extra-thoracic tumors, the difference was not statistically significant (p = 0.248).Conclusively, the study found that TERT promoter mutation is significantly linked to high-risk properties (e.g., older age, larger tumor size, higher mitotic rate).A review by Liu et al., which reported both extra-meningeal and meningeal SFTs, found an overall prevalence of 24% (14/58) [30].A large study conducted by Demicco et al. found a TERT promoter mutation in 29% (54/189) of tumors, including relapses and metastatic disease [52].In this study, TERT promoter mutations were more common in high-risk tumors (45% 9/20), but also occurred in tumors of moderate (42%, 11/26) or low risk (21%, 14/67).Interestingly, the study reported a TERT promoter mutation prevalence of 2/15 (13%) of primary SFTs in the head and neck, which is similar to the prevalence in our study cohort.While these studies referred to mostly extra-meningeal SFTs, a large study from 2013 on TERT promoter mutations in 1515 CNS tumors found TERT promoter mutations in 50% (8/16) of meningeal SFTs [41].However, the authors further differentiated a meningeal hemangiopericytoma and describe a prevalence of 11% (3/27).Thus, in conclusion, the meningeal SFTs in this study have a prevalence of 26% (11/43).Because TERT promoter mutations are most common in CNS tumors, some authors hypothesize that meningeal SFTs have a higher prevalence of TERT promoter mutations than extra-meningeal SFTs [32,53].In contrast to this, the large series study carried out by Demicco et al. found no TERT promoter mutation in primary meningeal SFTs (0/14) [52].The aforementioned studies also showed that C228T mutations are more frequent than C250T mutations [31,41].In our study on oSFTs, the sole TERT promoter mutation was C250T.
OSFTs exhibit unique properties among the extra-meningeal SFT entity.While local recurrence rates are higher than in other extra-pleural or pleural locations (26% vs. 10%), the risk for metastatic disease is relatively low (2.6% vs. 5-25%), most likely due to early detection and smaller tumor size [3,16].Hence, the orbital manifestation of SFTs manifests in younger patients (mean 42 years, in our collective mean 46 years) compared to SFTs of other compartments like the pleura (60 years) and the meningeal compartment (50 years) [3,5].On the contrary, meningeal SFTs have a high rate of local recurrence and an overall poorer prognosis.This is reflected in a generally more aggressive histopathologic pattern with higher cellularity and higher mitotic activity and ki-67 index [5,20].Further factors that seem to have an influence on the outcome are mitoses per HPF and the presence of tumor necrosis.These are used in a variety of risk prediction models for extra-meningeal SFTs [25,26].Thompson et al. proposed a risk score exclusively for oSFTs, which includes tumor cellularity and cellular/nuclear polymorphism [16].Moreover, this score accounts for the risk of local recurrence rather than distant metastasis, which is much more frequent in the treatment of orbital manifestation.Interestingly, in the present study, the metastasis risk, according to the score by Demicco et al., was only low to intermediate.Nevertheless, the two cases with intermediate metastatic risk were the cases where eye preservation could not be achieved and who harbored a STAT6-TAD fusion variant.One of them showed a TERT promoter mutation.This is in accordance with Bahrami et al., where the TERT promoter mutation only occurred in moderate-to high-risk tumors when classified according to the risk score by Demicco et al. [31].In the present study, TERT promoter occurred in a case that had an unfavorable outcome, including multiple local recurrences, eye loss, and infiltrative uncontrolled local growth.
The strong association between mutations of the TERT promoter and malignant SFTs has previously been shown [54].TERT promoter mutations are more frequent in cancers derived from tissue with low potential for self-renewal and mostly occur at an early stage of molecular dedifferentiation [40].Acting as driver mutations, they facilitate molecular tumor progress by immortalizing the cell [35].Mechanistically, the mutated TERT promoter sequence forms a de novo binding site with increased affinity for an ETS, such as GABP (see Figure 1) [39].GABPA, which only possesses the ETS binding domain, must form a heterodimer with GABPB1 or GABPB2, that each only harbor the transactivation domain [40].When undergoing steric DNA alterations such as chromatin looping, multiple ETS binding sites can converge, forming heterotetramers for reinforced transcription activation [40].The induced TERT then joins the telomerase complex to complete the functional enzyme for telomere elongation [40].In contrast, a study by Demicco et al. did not find a difference in telomere length when comparing SFTs with a wildtype promoter to SFTs with a mutated TERT promoter [52].The authors hypothesize that there might be factors in wild-type tumors that equally lead to telomere elongation like posttranscriptional and epigenetic modification.Epigenetic alterations of the TERT promoter have already been described [55,56].Although germ line variants in the TERT promoter have been described in other cancers, e.g., melanoma or glioblastoma, the described hotspot mutations represent somatic mutations and a germ line hot spot mutation that, to our knowledge, has never been described [35][36][37][38].
In our cohort and due to the study's retrospective design, we only analyzed tissue retrieved during tumor surgery.Thus, we can only postulate that the C250T mutation could be a somatic mutation.As in previous studies considering SFTs in the orbital compartment, a limitation is the low number of cases, which mirrors the incidence of this rare tumor entity at this specific location.In order to increase the number of samples and draw conclusions with statistical significance, a multicenter study is actually in conception.Another limitation is the imaging availability and quality, which relied partly on scans from external facilities.This was due to the retrospective nature of the study design.
The manifestation of SFTs in the orbit is rare.Nonetheless, incidence in the early years before the recognition of NAB2::STAT6 fusion gene might be underestimated [11]; in the first two decades after the characterization of CD34 in immunohistochemistry by Westra et al. in 1994, only 80 cases of reported oSFT appeared in the English literature [11,42].Yet, as uncovering the NAB2::STAT6 fusion gene has led to increased diagnostic sensitivity and specificity in primary SFT diagnosis, case reports in the literature have appeared to snowball [1,3,20].Another bias of reporting might be that the improved diagnostic standard primarily translated into the field of thoracic pathology, while oSFTs are evaluated by ophthalmo-pathologists.These factors supposedly increase the count of false negative diagnoses of oSFT and can explain the relatively low numbers of prevalence in the early years.Our work, among others, highlights the importance of the diagnosis SFT in the orbit and helps to contextualize this diagnosis with former work.To our knowledge, this study is the first to evaluate the prevalence of TERT promoter mutations exclusively in oSFTs.Our study supports the association between TERT promoter mutation and unfavorable disease outcome.Further studies are needed to evaluate whether the routine analysis of TERT promoter mutation status in addition to the current clinical-and histopathology-based practice may improve the risk assessment for patients with oSFT.

Conclusions
SFT is a semi-malignant and heterogeneous soft tissue tumor that is becoming increasingly more well understood.To complement histomorphological analysis and to better estimate the prognosis of oSFTs, we encourage the further diagnostic use of molecular pathology.The prevalence of TERT promoter mutations in oSFT was 11% and occurred in a case with locally aggressive tumor growth and multiple uncontrollable, local recurrences.As previously shown for SFTs of other locations and supported by our results, we hypothesize that TERT promoter mutations in oSFTs might be related to unfavorable outcomes [16,52,54].Hence, TERT promoter mutation analysis could complement existing and new risk stratification scores [57,58].However, the evolution of a concept renders the interpretation of former studies more complicated.In spite of a new era of technological progress, the treatment of oSFTs remains challenging and further studies need to be undertaken in order to elucidate the role of molecular tumor biology in SFTs of the orbital compartment.

Figure 1 .
Figure 1.Schematic illustration of the DNA sequence of the TERT promoter mutation at Chromosome 5p.The TERT promoter hotspot mutations C228T and C250T occur at −146 bp and −124 bp from the translation start site of the TERT gene (ATG; reference genome hg19).C228T and C250T induce changes that create new binding sites for ETS family members, e.g., GABPs.GABPA hereby forms heterotetramers with GABPB1 or GABPB2 in order to exert a master regulatory function on the mutated TERT promoter.

Figure 1 .
Figure 1.Schematic illustration of the DNA sequence of the TERT promoter mutation at Chromosome 5p.The TERT promoter hotspot mutations C228T and C250T occur at −146 bp and −124 bp from the translation start site of the TERT gene (ATG; reference genome hg19).C228T and C250T induce changes that create new binding sites for ETS family members, e.g., GABPs.GABPA hereby forms heterotetramers with GABPB1 or GABPB2 in order to exert a master regulatory function on the mutated TERT promoter.

Figure 2 .
Figure 2. (a)A 33-year-old female patient with painless proptosis of the left eye and no subjective visual impairment.At first presentation, the BCVA was 0.9, IOP 21 mmHg, no history of ophthalmic disease.Computed tomography with contrast media was initially performed due to better availability.(b) Axial, (c) coronary, and (d) sagittal CT revealed a solid hyperdense intraconal mass with compressive growth, with no signs of osseous infiltration.After admission to our neurosurgical department, resection was performed via the transcranial pterional approach.Because of the proximity to the optic nerve, total resection could not be achieved (R1 status).After tumor reduction, the tumor did not show any relevant growth for three years and the patient was followed up regularly in our ophthalmological department.(BCVA: best corrected visual acuity.)

Figure 2 .
Figure 2. (a)A 33-year-old female patient with painless proptosis of the left eye and no subjective visual impairment.At first presentation, the BCVA was 0.9, IOP 21 mmHg, no history of ophthalmic disease.Computed tomography with contrast media was initially performed due to better availability.(b) Axial, (c) coronary, and (d) sagittal CT revealed a solid hyperdense intraconal mass with compressive growth, with no signs of osseous infiltration.After admission to our neurosurgical department, resection was performed via the transcranial pterional approach.Because of the proximity to the optic nerve, total resection could not be achieved (R1 status).After tumor reduction, the tumor did not show any relevant growth for three years and the patient was followed up regularly in our ophthalmological department.(BCVA: best corrected visual acuity.)

Figure 3 .
Figure 3. Four years later, the same patient presented with an increasing tumor growth with compressive behavior (a).Like the original tumor before (for CT scans, see Figure 2b-d), the recurrent tumor mass showed a signal intensity void (b).This phenomenon was found in 6 other tumors and is considered a radiomorphological correlate of intratumoral vessels.A chemical shift artefact was

Figure 3 .
Figure 3. Four years later, the same patient presented with an increasing tumor growth with compressive behavior (a).Like the original tumor before (for CT scans, see Figure 2b-d), the recurrent tumor mass showed a signal intensity void (b).This phenomenon was found in 6 other tumors and is considered a radiomorphological correlate of intratumoral vessels.A chemical shift artefact was present in 3 of 9 tumors (c).Low ADC values (<1000 × 10 −6 mm 2 /s) as a correlate of diffusion restriction was found in around the half of the tumors (4/9) (d).(a): 37-year-old female, contrast media T1 SE coronal MRI, arrow indicates optic nerve.(b): 33-year-old female, axial T1 TSE FS axial MRI, arrows indicate signal intensity void.(c): 27-year-old male, axial T2 TSE MRI, arrows indicate chemical shift artefact.(d): 87-year-old female, MRI, ADC, ROI indicates reduced mean ADC.

Figure 4 .
Figure 4. Representative pictures of histology and immunohistochemistry.The H&E staining (a,b) showed a storiform-like growth pattern with dense cellularity and a spindled-cell appearance that belonged to a tumor with less aggressive clinical behavior.The H&E staining (c,d) showed a dense cellularity with stranded bands of hyalinized collagenous tissue between the tumor cell nests that belonged to a tumor with very aggressive clinical behavior and a TERT promoter mutation.The diagnosis of an SFT can be given if a typical histology (a,b) and the typical immunohistochemistry with strong staining for STAT6 (e) and/or CD34 (f) is present, as was presented in the first tumor with less aggressive behavior.In contrast, the tumor with aggressive clinical behavior showed intense staining for CD34 (h), but only weak STAT6 positivity (g).(a,c): scale bar 500µm.(b,d,e-h): scale bar 50 µm.

Figure 4 .
Figure 4. Representative pictures of histology and immunohistochemistry.The H&E staining (a,b) showed a storiform-like growth pattern with dense cellularity and a spindled-cell appearance that belonged to a tumor with less aggressive clinical behavior.The H&E staining (c,d) showed a dense cellularity with stranded bands of hyalinized collagenous tissue between the tumor cell nests that belonged to a tumor with very aggressive clinical behavior and a TERT promoter mutation.The diagnosis of an SFT can be given if a typical histology (a,b) and the typical immunohistochemistry with strong staining for STAT6 (e) and/or CD34 (f) is present, as was presented in the first tumor with less aggressive behavior.In contrast, the tumor with aggressive clinical behavior showed intense staining for CD34 (h), but only weak STAT6 positivity (g).(a,c): scale bar 500 µm.(b,d,e-h): scale bar 50 µm.

Figure 5 .
Figure 5. Sanger sequencing exploring TERT promoter mutation status of our collective: (a) regular Sanger sequence.(b) At position −146 of the base pairs, starting from the transcriptional start site, the patient has an exchange of the nucleic bases cytosine to thymine (C250T mutation).

Figure 5 .
Figure 5. Sanger sequencing exploring TERT promoter mutation status of our collective: (a) regular Sanger sequence.(b) At position −146 of the base pairs, starting from the transcriptional start site, the patient has an exchange of the nucleic bases cytosine to thymine (C250T mutation).

Table 1 .
Epidemiological and clinical data at first presentation in the department of ophthalmology.(BCVA: best corrected visual acuity.IOP: intraocular pressure.)

Table 1 .
Epidemiological and clinical data at first presentation in the department of ophthalmology.

Table 2 .
Parameters and appearance of oSFT in multimodal imaging including contrast media CT and diffusion-weighted MRI imaging (DWI).
′′ Qualitative visual assessment of density or signal intensity (SI) compared to surrounding soft tissue; * CSA (chemical shift artifact); ˆReduction quantified as a mean ADC value of less than 1000 × 10 −6 mm 2 /s; n.a.(not available).

Table 3 .
Tumor surgery included eye preserving techniques and definitive tumor resection.Definitive tumor resection included bulbar enucleation and orbital exenteration.The frequency of surgery in one patient ranged between 1 and 14 surgeries.In 7 of 9 patients, eye preservation was achieved.In one case, orbital exenteration was recommended due to aggressive local recurrence.The patient refused surgery.

Table 4 .
Representative histopathological changes in 9 samples.Some patients had multiple resections with different specimens.We analyzed the relevant specimens with conventional histology and chose to present the most representative samples.Representative pictures of H&E staining are shown in Figure3.

Table 6 .
Sanger sequencing showed one TERT promoter mutation (C250T) in all of the 9 tumors.All tumors were investigated for TERT promoter mutation.If samples from different surgeries of different time points were available, we checked selected specimens to rule out molecular tumor progress.Patient H showed a consistent C250T mutation that was already shown in specimens from the first tumor resection.
+ = weak positive staining in tumour cells; ++ = moderate positive staining result in tumour cells; +++ = strong positive staining result in tumour cells; -= negative staining result in tumour cells.

Table 6 .
Sanger sequencing showed one TERT promoter mutation (C250T) in all of the 9 tumors.All tumors were investigated for TERT promoter mutation.If samples from different surgeries of different time points were available, we checked selected specimens to rule out molecular tumor progress.Patient H showed a consistent C250T mutation that was already shown in specimens from the first tumor resection.