Solitary fibrous tumor – clinicopathologic, immunohistochemical and molecular analysis of 28 cases

Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic type that can affect virtually any region of the body [1],[2]. The neoplastic cells are arranged in a patternless architecture with alternating hypo- and hypercellular areas and a prominent branching vasculature. These lesions occur predominantly in middle-aged adults with equal gender distribution [1]. Most tumors present as well defined, slow growing masses, which can be cured by surgery. A small percentage of SFTs, between 10-20%, behave in a more aggressive way, with local recurrence and/or distant metastasis for which systemic therapy (chemotherapy or targeted treatment with e.g. sunitinib) can be given [1],[3]-[5]. Prediction of behavior is difficult, with tumor size above 15 cm, positive surgical margins, tumor site and high mitotic count (>4/10 high power fields, HPF) being the most useful indicators for malignancy [3],[6]-[8].

Recently, a recurrent gene fusion NAB2-STAT6 has been identified as molecular hallmark of SFT, encoding a chimeric protein that combines the EGR-binding domain of NAB2, a repressor of early growth response (EGR) transcription factors that regulate differentiation and proliferation, with the transactivation domain of STAT6, a transcription factor that mediates cytokine signaling [2],[9]. Molecular detection of the fusion gene and immunohistochemical expression of nuclear STAT6 can be helpful in diagnosing SFT, especially in cases not unequivocally classifiable [2],[10]-[13].

In this study, molecular analysis and immunohistochemical staining of STAT6 protein was performed in 28 cases of SFT. In addition, as the NAB2-STAT6 fusion leads to EGR1 (early growth response protein 1) activation and transcriptional deregulation of EGR1-dependent target genes, we immunohistochemically evaluated the expression of EGR1 in our tumor samples in order to semi-quantify EGR1 protein levels in SFT [2],[14].

In 1942 and 1949, Stout and Murray defined hemangiopericytoma as a neoplasm with prominent branching vessels surrounded by pericytic tumor cells [16],[17]. The lesions included in their studies comprised true pericytic tumors, nowadays termed myofibroma/myopericytoma, and conventional hemangiopericytoma, which was afterwards accepted as cellular variant of SFT [18]. The unified entity SFT now also contains the lipomatous variant, giant cell angiofibroma, and at the malignant end, dedifferentiated SFT showing abrupt transition from classical areas into high-grade sarcoma [1].

With the advent of whole-genome sequencing, the fusion gene NAB2-STAT6 has been detected as the driver mutation of SFT [2],[9],[10],[14]. NAB2 and STAT6, adjacent genes on 12q13, fuse following genomic inversion of STAT6 with consecutive transcription in a common direction [2],[9]. Expression of the chimeric protein NAB2-STAT6 leads to activation of EGR-mediated signaling via distorted NAB2 activity. Modulation of STAT6 dependent gene expression seems to be an alternative mechanism but was shown to play a minor role [2],[9].

NAB2-STAT6 fusion is a distinct molecular feature of SFT since it has not been detected in other tumors (so far) and its frequency ranges from 55% to 100%, irrespective of dignity [2],[9],[13],[14]. There are several fusion variants, with conjunction of NAB2 exon 4 - STAT6 exon 3 and NAB2 exon 6- STAT6 exon 17 being the most common [2],[13],[14]. In our study, 68% of SFTs carried a NAB2-STAT6 fusion transcript, mostly with a fusion between NAB2 exon 4 and STAT6 exon 3 in accordance with the results by Mohajeri et al. and Barthelmess et al. [13],[14]. Negativity for this fusion in 25% of our cases could possibly be explained by alternative or complex genetic rearrangements involving other exons or inversions and deletions, not detectable with our primer combination. In 2 cases (7%), adequate interpretation of RT-PCR results was not possible due to the presence of complex breakpoints. Furthermore, in single cases other fusion genes have been reported, e.g. STAT6-TRAPPC5[14] and probably additional fusion genes will be detected in the future.

Doyle et al. demonstrated nuclear expression of STAT6 in 98% of a large series of SFTs indicating the presence of the NAB2-STAT6 fusion protein in the nucleus [11]. STAT6 is therefore a highly sensitive and specific immunohistochemical marker for SFT [10]-[13],[19],[20]. This is in concert with our results, where 100% of the cases showed strong and diffuse nuclear positivity for STAT6 in comparison to the control group being 100% negative. Thereby, STAT6 was diffusely expressed in 7 tumors without a detected NAB2-STAT6 fusion, suspecting limitations in our RT-PCR approach in which NAB2-STAT6 fusions could be missed as mentioned above. Potential diagnostic pitfalls could be STAT6 expression in, for example, the morphologic mimics deep benign fibrous histiocytoma and dedifferentiated liposarcoma, especially in retroperitoneal and abdominal localization [11]. As known, MDM2 and CDK4 immunohistochemistry or MDM2 FISH are useful in identifying dedifferentiated liposarcoma. Expression of STAT6 in ca. 10% of dedifferentiated liposarcomas is based on amplification of the corresponding gene located in proximity to MDM2 and CDK4[11],[12],[21],[22]. Deep benign fibrous histiocytoma may also show a hemangiopericytoma-like vasculature and may express CD34 [1]. In such cases, molecular analysis of NAB2-STAT6 may be helpful. Other differential diagnoses and their histopathological characteristics are listed in Table 5.

Previously, CD34, CD99 and bcl-2 were the most useful positive immunohistochemical markers for SFT. However, they are less specific and absence of CD34 does not rule out SFT [1],[28]-[31].

Cell line experiments supported that in contrast to the known transcriptional repressor activity of NAB2, the NAB2-STAT6 fusion protein leads to activation of EGR1 and expression of its target genes. In addition, the increased proliferation of the NAB-STAT6-expressing cell lines could be inhibited by small interfering RNA (siRNA) knockdown of EGR1 expression [2]. This prompted us to investigate EGR1 protein expression in our tumor samples which showed low-level variable nuclear reactivity in all stained samples, including 47 control samples of possible mimickers of SFT. This result is in line with EGR1 protein activation without high-level expression mainly due to altered NAB2 function resulting in deregulation of target genes as mentioned above [2],[14].

Correlation of clinicopathologic parameters and outcome is difficult. This could be due to, at least in part, relatively small cohorts of SFT studied. Therefore, no statistical correlation could be made between pathological findings and clinical data. To date, no definitive markers have been identified that classify malignant SFT. Recently, efforts have been made to define a risk assessment model based on patient age, tumor size and mitotic index with promising results [7],[8]. From the genetic point of view, Mohajeri et al. [14] did not find any clinical associations including genetic changes beyond the fusion-genes. In contrast, Barthelmess et al. showed that the most common fusion variant NAB2 exon 4-STAT6 exon 3 corresponded to classic pleuropulmonary SFT as we found in our study: all 9 pleuropulmonary SFTs had a fusion between NAB2 exon 4 and STAT6 exon 3.

NAB2 exon 6-STAT6 exon16/17 fusions were detected in cellular soft tissue SFTs with more aggressive behavior in younger patients [13].

Three of the 18 patients (17%) of whom we had adequate clinical follow-up data (cases 1, 3, 4) behaved in a malignant fashion with metastases. Only one of them showed a high mitotic index (case 1). Locations were abdomen, pelvis and retroperitoneum in accordance with known parameters for malignant potential but sizes were below 10 cm [6]-[8].

Therapeutic options for more advanced or aggressive tumors are scarce and no standard modality for metastasized SFT has currently been accepted. Few chemotherapeutical drugs have been studied with different outcome. Anti-angiogenic targeted treatment shows interesting results [4],[5]. The potential role of EGR1 and its downstream targets in treatment of (metastasized SFT) is not clearly defined so far [2],[9].

In summary, STAT6 immunohistochemistry is a powerful tool in diagnosing SFTs. Also, the identification of the NAB2-STAT6 fusion gene can provide important diagnostic information, even in formalin-fixed and paraffin-embedded tissue or when biopsy material is limited. In accordance with Barthelmess et al., the most common fusion variant NAB2 exon 4-STAT6 exon 3 corresponded mostly to pleuropulmonary SFT.

EGR1 immunohistochemistry indicates low-level protein expression in accordance with EGR1 activation due to distorted NAB2 activity resulting in deregulation of EGR1 target genes.