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Lipomatous Solitary Fibrous Tumors Harbor Rare NAB2-STAT6 Fusion Variants and Show Up-Regulation of the Gene PPARG, Encoding for a Regulator of Adipocyte Differentiation

2021; Elsevier BV; Volume: 191; Issue: 7 Linguagem: Inglês

10.1016/j.ajpath.2021.03.012

ISSN

1525-2191

Autores

Florian Haller, Lea D. Schlieben, Fulvia Ferrazzi, Michael Michal, Robert Stöhr, Evgeny A. Moskalev, Matthias Bieg, Judith V.M.G. Bovée, Philip Ströbel, Naveed Ishaque, Robert Grützmann, Norbert Meidenbauer, Roland Eils, Stefan Wiemann, Arndt Hartmann, Michal Michal, Abbas Agaimy,

Tópico(s)

Sarcoma Diagnosis and Treatment

Resumo

Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture–based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4–STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs. Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture–based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4–STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs. Solitary fibrous tumors (SFTs) encompass a morphologic spectrum of tumors ranging from collagen-rich spindled tumors (classic fibrous SFTs) to cellular spindled or round-cell tumors with prominent vessels (formerly known as hemangiopericytomas).1WHO Classification of Tumours: Soft Tissue and Bone Tumours. ed 5. IARC, Lyon, France2020Google Scholar This unifying concept is endorsed by the nearly ubiquitous finding of NAB2-STAT6 gene fusions in SFTs.2Robinson D.R. Wu Y.M. Kalyana-Sundaram S. Cao X. Lonigro R.J. Sung Y.S. Chen C.L. Zhang L. Wang R. Su F. Iyer M.K. Roychowdhury S. Siddiqui J. Pienta K.J. Kunju L.P. Talpaz M. Mosquera J.M. Singer S. Schuetze S.M. Antonescu C.R. Chinnaiyan A.M. Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing.Nat Genet. 2013; 45: 180-185Crossref PubMed Scopus (483) Google Scholar,3Chmielecki J. Crago A.M. Rosenberg M. O'Connor R. Walker S.R. Ambrogio L. Auclair D. McKenna A. Heinrich M.C. Frank D.A. Meyerson M. Whole-exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors.Nat Genet. 2013; 45: 131-132Crossref PubMed Scopus (361) Google Scholar We first proposed a correlation between morphologic variants, anatomic localization, and distinct variants of the NAB2-STAT6 gene fusion,4Barthelmeß S. Geddert H. Boltze C. Moskalev E.A. Bieg M. Sirbu H. Brors B. Wiemann S. Hartmann A. Agaimy A. Haller F. Solitary fibrous tumors/hemangiopericytomas with different variants of the NAB2-STAT6 gene fusion are characterized by specific histomorphology and distinct clinicopathological features.Am J Pathol. 2014; 184: 1209-1218Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar which has since been confirmed by others.5Akaike K. Kurisaki-Arakawa A. Hara K. Suehara Y. Takagi T. Mitani K. Kaneko K. Yao T. Saito T. Distinct clinicopathological features of NAB2-STAT6 fusion gene variants in solitary fibrous tumor with emphasis on the acquisition of highly malignant potential.Hum Pathol. 2015; 46: 347-356Crossref PubMed Scopus (85) Google Scholar,6Tai H.C. Chuang I.C. Chen T.C. Li C.F. Huang S.C. Kao Y.C. Lin P.C. Tsai J.W. Lan J. Yu S.C. Yen S.L. Jung S.M. Liao K.C. Fang F.M. Huang H.Y. NAB2-STAT6 fusion types account for clinicopathological variations in solitary fibrous tumors.Mod Pathol. 2015; 28: 1324-1335Crossref PubMed Scopus (89) Google Scholar,7Nakada S. Minato H. Nojima T. Clinicopathological differences between variants of the NAB2-STAT6 fusion gene in solitary fibrous tumors of the meninges and extra-central nervous system.Brain Tumor Pathol. 2016; 33: 169-174Crossref PubMed Scopus (31) Google Scholar,8Huang S.C. Li C.F. Kao Y.C. Chuang I.C. Tai H.C. Tsai J.W. Yu S.C. Huang H.Y. Lan J. Yen S.L. Lin P.C. Chen T.C. The clinicopathological significance of NAB2-STAT6 gene fusions in 52 cases of intrathoracic solitary fibrous tumors.Cancer Med. 2016; 5: 159-168Crossref PubMed Scopus (51) Google Scholar,9Chuang I.C. Liao K.C. Huang H.Y. Kao Y.C. Li C.F. Huang S.C. Tsai J.W. Chen K.C. Lan J. Lin P.C. NAB2-STAT6 gene fusion and STAT6 immunoexpression in extrathoracic solitary fibrous tumors: the association between fusion variants and locations.Pathol Int. 2016; 66: 288-296Crossref PubMed Scopus (32) Google Scholar Apart from the most frequent collagen-rich and cellular variants, further distinct but uncommon morphologic variants of SFTs have been described, including SFTs with prominent myxoid changes, SFTs with giant cells (previously referred to as giant cell angiofibroma), as well as a fat-forming variant of SFT (eg, lipomatous SFT). The first description of a lipomatous SFT dates back to 1990, when Theunissen et al10Theunissen P.H. Ariëns A.T. Pannebakker M.A. Blaauw G. [Late recurrence of a hemangiopericytoma with lipomatous components]. German.Pathologe. 1990; 11: 346-349PubMed Google Scholar described a case of a paravertebral hemangiopericytoma that contained fat. This was followed by a 1993 report of a fat-forming SFT in the thyroid,11Taccagni G. Sambade C. Nesland J. Terreni M.R. Sobrinho-Simões M. Solitary fibrous tumour of the thyroid: clinicopathological, immunohistochemical and ultrastructural study of three cases.Virchows Arch A Pathol Anat Histopathol. 1993; 422: 491-497Crossref PubMed Scopus (91) Google Scholar as well as another case mentioned by Fletcher.12Fletcher C.D.M. Haemangiopericytoma- a dying breed? -reappraisal of an "entity" and its variants: a hypothesis.Curr Diagn Pathol. 1994; 1: 19-23Abstract Full Text PDF Scopus (103) Google Scholar In 1995, Nielsen et al13Nielsen G.P. Dickersin G.R. Provenzal J.M. Rosenberg A.E. Lipomatous hemangiopericytoma: a histologic, ultrastructural and immunohistochemical study of a unique variant of hemangiopericytoma.Am J Surg Pathol. 1995; 19: 748-756Crossref PubMed Scopus (92) Google Scholar described three cases and were the first to propose the name lipomatous hemangiopericytoma. The first series of 16 lipomatous SFTs was reported by Folpe et al14Folpe A.L. Devaney K. Weiss S.W. Lipomatous hemangiopericytoma: a rare variant of hemangiopericytoma that may be confused with liposarcoma.Am J Surg Pathol. 1999; 23: 1201-1207Crossref PubMed Scopus (76) Google Scholar in 1999, followed by another 13 cases by Guillou et al15Guillou L. Gebhard S. Coindre J.M. Lipomatous hemangiopericytoma: a fat-containing variant of solitary fibrous tumor? clinicopathologic, immunohistochemical, and ultrastructural analysis of a series in favor of a unifying concept.Hum Pathol. 2000; 31: 1108-1115Crossref PubMed Scopus (119) Google Scholar in 2000. In summary, the reported lipomatous SFTs are large, located mostly in the deep soft tissue of the trunk, retroperitoneum, and the extremities, and contain a variable amount of fat. Overall, they follow a rather favorable clinical course. Apart from their fatty component, lipomatous SFTs are morphologically similar to the cellular variant of SFT, also indicated by the initial name lipomatous hemangiopericytoma. In contrast, only few single case reports of pleuropulmonary SFTs with a fatty component have been described.16Yamazaki K. Eyden B.P. Pulmonary lipomatous hemangiopericytoma: report of a rare tumor and comparison with solitary fibrous tumor.Ultrastruct Pathol. 2007; 31: 51-61Crossref PubMed Scopus (8) Google Scholar,17Park C.Y. Rho J.Y. Yoo S.M. Jung H.K. Fat-forming variant of solitary fibrous tumour of the pleura: CT findings.Br J Radiol. 2011; 84: e203-e205Crossref PubMed Scopus (5) Google Scholar,18Hui C.L.Y. Asmat A. Ali Akbar F. Rajapaksha K. Chau C.Y.P. Ahmed A.D.B. Pleural fat-forming variant of solitary fibrous tumor.Asian Cardiovasc Thorac Ann. 2015; 23: 1103-1105Crossref PubMed Scopus (1) Google Scholar As reported by Lee and Fletcher,19Lee J.C. Fletcher C.D. Malignant fat-forming solitary fibrous tumor (so-called "lipomatous hemangiopericytoma"): clinicopathologic analysis of 14 cases.Am J Surg Pathol. 2011; 35: 1177-1185Crossref PubMed Scopus (51) Google Scholar of the 81 unpublished SFTs, 14 were fat-forming and had malignant features. They proposed mitoses in ≥4 of 10 high-power fields, hypercellularity, at least moderate atypia, and necrosis as indicators of malignancy. Nuclear STAT6 immunostaining has emerged as a valuable surrogate marker for the presence of NAB2-STAT6 gene fusion in SFTs,20Doyle L.A. Vivero M. Fletcher C.D. Mertens F. Hornick J.L. Nuclear expression of STAT6 distinguishes solitary fibrous tumor from histologic mimics.Mod Pathol. 2014; 27: 390-395Crossref PubMed Scopus (393) Google Scholar and nuclear STAT6 expression occurs in lipomatous SFTs.21Creytens D. Libbrecht L. Ferdinande L. Nuclear expression of STAT6 in dedifferentiated liposarcomas with a solitary fibrous tumor-like morphology: a diagnostic pitfall.Appl Immunohistochem Mol Morphol. 2015; 23: 462-463Crossref PubMed Scopus (23) Google Scholar,22Yoshida A. Tsuta K. Ohno M. Yoshida M. Narita Y. Kawai A. Asamura H. Kushima R. STAT6 immunohistochemistry is helpful in the diagnosis of solitary fibrous tumors.Am J Surg Pathol. 2014; 38: 552-559Crossref PubMed Scopus (195) Google Scholar Notably, nuclear and/or cytoplasmic STAT6 expression has been observed in up to 14% of dedifferentiated liposarcomas,20Doyle L.A. Vivero M. Fletcher C.D. Mertens F. Hornick J.L. Nuclear expression of STAT6 distinguishes solitary fibrous tumor from histologic mimics.Mod Pathol. 2014; 27: 390-395Crossref PubMed Scopus (393) Google Scholar,21Creytens D. Libbrecht L. Ferdinande L. Nuclear expression of STAT6 in dedifferentiated liposarcomas with a solitary fibrous tumor-like morphology: a diagnostic pitfall.Appl Immunohistochem Mol Morphol. 2015; 23: 462-463Crossref PubMed Scopus (23) Google Scholar,22Yoshida A. Tsuta K. Ohno M. Yoshida M. Narita Y. Kawai A. Asamura H. Kushima R. STAT6 immunohistochemistry is helpful in the diagnosis of solitary fibrous tumors.Am J Surg Pathol. 2014; 38: 552-559Crossref PubMed Scopus (195) Google Scholar,23Demicco E.G. Harms P.W. Patel R.M. Smith S.C. Ingram D. Torres K. Carskadon S.L. Camelo-Piragua S. McHugh J.B. Siddiqui J. Palanisamy N. Lucas D.R. Lazar A.J. Wang W.L. Extensive survey of STAT6 expression in a large series of mesenchymal tumors.Am J Clin Pathol. 2015; 143: 672-682Crossref PubMed Scopus (116) Google Scholar,24Ouladan S. Trautmann M. Orouji E. Hartmann W. Huss S. Büttner R. Wardelmann E. Differential diagnosis of solitary fibrous tumors: a study of 454 soft tissue tumors indicating the diagnostic value of nuclear STAT6 relocation and ALDH1 expression combined with in situ proximity ligation assay.Int J Oncol. 2015; 46: 2595-2605Crossref PubMed Scopus (32) Google Scholar most likely due to co-amplification of the STAT6 gene locus as part of the MDM2/CDK4 12q13-15 amplicon.25Doyle L.A. Tao D. Mariño-Enríquez A. STAT6 is amplified in a subset of dedifferentiated liposarcoma.Mod Pathol. 2014; 27: 1231-1237Crossref PubMed Scopus (85) Google Scholar Therefore, the analysis of the NAB2-STAT6 gene fusion may be preferable in lipomatous SFTs, and few individual cases of lipomatous SFTs with NAB2-STAT6 gene fusions have been reported as part of larger SFT series.6Tai H.C. Chuang I.C. Chen T.C. Li C.F. Huang S.C. Kao Y.C. Lin P.C. Tsai J.W. Lan J. Yu S.C. Yen S.L. Jung S.M. Liao K.C. Fang F.M. Huang H.Y. NAB2-STAT6 fusion types account for clinicopathological variations in solitary fibrous tumors.Mod Pathol. 2015; 28: 1324-1335Crossref PubMed Scopus (89) Google Scholar,8Huang S.C. Li C.F. Kao Y.C. Chuang I.C. Tai H.C. Tsai J.W. Yu S.C. Huang H.Y. Lan J. Yen S.L. Lin P.C. Chen T.C. The clinicopathological significance of NAB2-STAT6 gene fusions in 52 cases of intrathoracic solitary fibrous tumors.Cancer Med. 2016; 5: 159-168Crossref PubMed Scopus (51) Google Scholar,9Chuang I.C. Liao K.C. Huang H.Y. Kao Y.C. Li C.F. Huang S.C. Tsai J.W. Chen K.C. Lan J. Lin P.C. NAB2-STAT6 gene fusion and STAT6 immunoexpression in extrathoracic solitary fibrous tumors: the association between fusion variants and locations.Pathol Int. 2016; 66: 288-296Crossref PubMed Scopus (32) Google Scholar,26Kao Y.C. Lin P.C. Yen S.L. Huang S.C. Tsai J.W. Li C.F. Tai H.C. Lan J. Chuang I.C. Yu S.C. Huang H.Y. Clinicopathological and genetic heterogeneity of the head and neck solitary fibrous tumours: a comparative histological, immunohistochemical and molecular study of 36 cases.Histopathology. 2016; 68: 492-501Crossref PubMed Scopus (33) Google Scholar,27Vogels R.J. Vlenterie M. Versleijen-Jonkers Y.M. Ruijter E. Bekers E.M. Verdijk M.A. Link M.M. Bonenkamp J.J. van der Graaf W.T. Slootweg P.J. Suurmeijer A.J. Groenen P.J. Flucke U. Solitary fibrous tumor - clinicopathologic, immunohistochemical and molecular analysis of 28 cases.Diagn Pathol. 2014; 9: 224Crossref PubMed Scopus (77) Google Scholar However, no systematic analysis of NAB2-STAT6 gene fusion variants or specific gene expression in lipomatous SFTs has been published to date. Therefore, a multi-institutional series of 11 lipomatous SFTs was collected, and a comprehensive molecular analysis, including NAB2-STAT6 fusion variant detection and gene expression profiling, was performed. The archives of the Institutes of Pathology in Erlangen, Germany, Göttingen, Germany, Leiden, the Netherlands, and Plzen, Czech Republic, were screened for SFTs with a lipomatous component. After initial review of potential cases by a specialized soft tissue pathologist (A.A.), 11 tumors with morphologic features consistent with SFT, displaying a strong and specific homogeneous nuclear STAT6 immunostaining and at least 5% fat cells, were accepted as lipomatous SFTs for the current study. The amount and pattern of fat cells was semiquantitatively evaluated on hematoxylin and eosin–stained slides, as well as the presence and amount of inflammatory cells and myxoid changes. Ten SFTs without a lipomatous component from a recent study were used as a control for the comparison of gene expression.28Bieg M. Moskalev E.A. Will R. Hebele S. Schwarzbach M. Schmeck S. Hohenberger P. Jakob J. Kasper B. Gaiser T. Ströbel P. Wardelmann E. Kontny U. Braunschweig T. Sirbu H. Grützmann R. Meidenbauer N. Ishaque N. Eils R. Wiemann S. Hartmann A. Agaimy A. Fritchie K. Giannini C. Haller F. Gene expression in solitary fibrous tumors (SFTs) correlates with anatomical localization and NAB2-STAT6 gene fusion variants.Am J Pathol. 2021; 191: 602-617Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Samples were used in accordance with ethical guidelines for the use of retrospective tissue samples provided by the local ethics committee of the Friedrich-Alexander University Erlangen-Nuremberg (ethics committee statements 24.01.2005 and 18.01.2012). Immunohistochemistry was performed on freshly cut sections (3 μm thick) from paraffin-embedded tissue blocks using a fully automated system (Benchmark XT System; Ventana Medical Systems Inc., Tucson, AZ) and the following antibodies: anti-STAT6 [rabbit polyclonal, sc-621 (S-20), 1:1000 dilution, heat pretreatment for epitope retrieval in citrate buffer (CC1), incubation at 37°C for 32 minutes; Santa Cruz Biotechnology, Inc., Dallas, Texas); anti–peroxisome proliferator activated receptor-γ [PPARγ; mouse monoclonal, sc-7273 (E8), 1:100 dilution, heat pretreatment for epitope retrieval in citrate buffer, incubation at room temperature overnight; Santa Cruz Biotechnology, Inc.]; anti-MDM2 [mouse monoclonal, IF2, 1:50 dilution, heat pretreatment for epitope retrieval in citrate buffer (CC1), incubation at 37°C for 32 minutes; Merck KGaA, Darmstadt, Germany]; and anti-S100 [mouse monoclonal, 4C4.9, 1:3000 dilution, heat pretreatment for epitope retrieval in citrate buffer (CC1), incubation at 37°C for 32 minutes; Zytomed Systems GmbH, Berlin, Deutschland]. STAT6 immunostaining was considered positive if a moderate to strong nuclear staining was observed, whereas any cytoplasmic staining was regarded as unspecific. For PPARγ immunostaining, nonneoplastic retroperitoneal fat was used as external positive control, and only unequivocal nuclear staining was considered positive. All lipomatous SFTs were analyzed by fluorescence in situ hybridization (FISH) on freshly cut slides from conventional formalin-fixed, paraffin-embedded blocks for the presence of MDM2 amplification using an MDM2/CEN 12 dual-color probe (ZytoLight SPEC MDM2/CEN 12 Dual Color Probe, Z-2013-50; ZytoVision GmbH, Bremerhaven, Germany), according to the manufacturer's standard protocol (ZytoLight FISH-Tissue Implementation Kit, Z-2028-5; ZytoVision GmbH). Reverse transcription reactions were performed with 1 μg of total RNA using QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Quantitative RT-PCR was performed in triplicate with the C1000 Thermal Cycler and the CFX96 Real-Time System (BioRad, Hercules, CA) using the QuantiFast SYBR Green PCR kit (Qiagen). Primer sequences were as follows: PPARG (5′-GGAGTTCATGCTTGTGAAGGA-3′ and 5′-TTTTTGTGGATCCGACAGTT-3′); and LMNB1 (5′-CTGGAAATGTTTGCATCGAAGA-3′ and 5′-GCCTCCCATTGGTTGATCC-3′). The thermal cycler conditions were as follows: 95°C for 3 minutes, followed by a two-step PCR of 40 cycles at 95°C for 10 seconds and 55°C for 30 seconds. Universal human reference total RNA was used as a calibration control (Stratagene, La Jolla, CA). LMNB1 expression served as an internal control for normalization employing the ΔΔCt method. Tumor DNA and normal control DNA from Case 1 were isolated of fresh-frozen tumor tissue and blood with the DNeasy Blood and Tissue Kit (Qiagen), whereas RNA was isolated from the tumor tissue using the RNeasy Plus MiniKit (Qiagen). Quality of the DNA was analyzed using an Agilent 2200 Tapestation (Agilent, Waldbronn, Germany), and quality of the RNA was assessed with an Agilent Bioanalyzer 2100 (Agilent). Libraries for sequencing were prepared employing the TruSeq Nano DNA Library Prep Kit (Illumina, Inc., San Diego, CA) for DNA, and the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina) for RNA. For whole-genome sequencing, paired-end sequencing (2 × 150 bp) was performed using one lane of a HiSeqX (Illumina) for every sample, whereas RNA sequencing was done with paired-end sequencing (2 × 100 bp) employing a HiSeq2000 (Illumina). Bioinformatic analysis of whole-genome sequencing and RNA-sequencing data was done as described previously in detail.29Haller F. Bieg M. Moskalev E.A. Barthelmeß S. Geddert H. Boltze C. Diessl N. Braumandl K. Brors B. Iro H. Hartmann A. Wiemann S. Agaimy A. Recurrent mutations within the amino-terminal region of β-catenin are probable key molecular driver events in sinonasal hemangiopericytoma.Am J Pathol. 2015; 185: 563-571Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar NAB2-STAT6 fusion detection and gene expression profiling were performed for 10 of the 11 lipomatous SFTs (Cases 1 through 10) by using a targeted hybrid-capture RNA-sequencing panel (TruSight RNA Fusion Panel; Illumina) covering 507 genes frequently involved in gene fusions. In addition, another next-generation sequencing panel focused on 26 genes recurrently involved in gene fusions in sarcomas using the Anchored Multiplex PCR-based enrichment method (Archer FusionPlex Sarcoma Panel; ArcherDX, Boulder, CO) was employed to search for NAB2-STAT6 gene fusions in five of the cases (Cases 2 through 5 and 11). For each panel, a total of 500 ng RNA was used for the library preparation, according to the manufacturer's protocol. Up to eight libraries were pooled and sequenced on a MiSeq instrument (Illumina) by using the MiSeq reagent kit v3, 150 cycle (Illumina) and 2 × 75 bp paired-end sequencing, generating a minimum of one million reads per sample. For data analysis, the resulting FASTQ files were uploaded to Illumina's Basespace (Illumina) or the Archer Analysis 6.2 software (ArcherDX). The RNA-Seq alignment app (Illumina) was employed to call fusions using the TopHat-Fusion algorithm, and to generate raw read counts for each of the targeted 507 genes. The Integrative Genomics Viewer, version 2.2.13, was used for data visualization of fusions.30Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative Genomics Viewer.Nat Biotechnol. 2011; 29: 24-26Crossref PubMed Scopus (6812) Google Scholar Differential expression analysis was performed within the R/Bioconductor environment v.4.0.3 (https://www.R-project.org, last accessed January 31, 2021)31Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. Hornik K. Hothorn T. Huber W. Iacus S. Irizarry R. Leisch F. Li C. Maechler M. Rossini A.R. Sawitzki G. Smith C. Smyth G. Tierney L. Yang J.Y.H. Zhang J. Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar,32R Core Team. RA Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria2020Google Scholar relying on the DESeq2 package v.1.30.0.33Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (25756) Google Scholar Genes with an adjusted P value (Benjamini-Hochberg correction) 10 cm/int.Exon 4–exon 4†Analyzed with TruSight RNA fusion panel.‡Analyzed with Archer FusionPlex Sarcoma Panel.Focal cluster (40%)FewPresent3M/67Pelvis15 cm/int.Exon 7–intron 1†Analyzed with TruSight RNA fusion panel.‡Analyzed with Archer FusionPlex Sarcoma Panel.Single fat cells (15%)ProminentAbsent4M/51Paravertebral4.5 cm/lowExon 2–intron 1†Analyzed with TruSight RNA fusion panel.‡Analyzed with Archer FusionPlex Sarcoma Panel.Focal cluster (40%)ProminentAbsent5F/51Paravertebral5 cm/lowExon 7–intron 1†Analyzed with TruSight RNA fusion panel.‡Analyzed with Archer FusionPlex Sarcoma Panel.Diffuse (75%)ProminentPresent6M/47Lower leg13 cm/lowExon 5–exon 16†Analyzed with TruSight RNA fusion panel.Focal cluster (10%)FewAbsent7M/34Lower leg8 cm/lowExon 2–exon 5†Analyzed with TruSight RNA fusion panel.Single fat cells (5%)FewPresent8M/46Infraclavicular5 cm/lowExon 4–exon 4†Analyzed with TruSight RNA fusion panel.Focal cluster (20%)FewPresent9F/53Pelvis12 cm/lowExon 4–exon 4†Analyzed with TruSight RNA fusion panel.Diffuse (85%)ProminentPresent10M/57Upper leg5 cm/lowExon 2–intron 1†Analyzed with TruSight RNA fusion panel.Focal cluster (30%)AbsentProminent11M/79Pelvis6 cm/lowExon 4–exon 4‡Analyzed with Archer FusionPlex Sarcoma Panel.Focal cluster (25%)AbsentPresentF, female; M, male; int., intermediate; SFT, solitary fibrous tumor.∗ Analyzed by whole-genome sequencing.† Analyzed with TruSight RNA fusion panel.‡ Analyzed with Archer FusionPlex Sarcoma Panel. Open table in a new tab Figure 2Morphologic spectrum of lipomatous solitary fibrous tumors. A: Representative image of an abrupt transition between fatty and nonfatty components in SFTs [hematoxylin and eosin (H&E)]. B: Representative image of few solitary fat cells scattered between the classic spindled cells (H&E). C: Representative image showing a strong predominance of the fatty component (H&E). D: Representative image showing prominent myxoid changes (H&E). E: S100 immunostaining indicating the adipocytic differentiation of the fat cells (H&E). F: Presence of nuclear STAT6 immunostaining in some fat cells (asterisks), and absence in other fat cells (arrowheads) (H&E). Original magnifications, ×100 (A and B); ×200 (C–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) F, female; M, male; int., intermediate; SFT, solitary fibrous tumor. Whole-genome sequencing of Case 1 revealed no copy number changes or structural genomic changes at the gene loci for MDM2, CDK4, and HMGA2 (Figure 3), but indicated a 14.5-kb intrachromosomal inversion with breakpoints at chr12:57486789 in STAT6 intron 4, and at chr12:57501307 in NAB2 intron 4 (Figure 4). RNA sequencing of the tumor tissue revealed a NAB2 exon 4–STAT6 exon 4 fusion at the RNA level (Figure 4). None of the lipomatous SFTs displayed an MDM2 amplification in the FISH analysis. RNA level panel-based next-generation sequencing analysis of all 11 lipomatous SFTs confirmed the NAB2-STAT6 gene fusion in the index case, and revealed NAB2-STAT6 gene fusions in the 10 other cases. There was full concordance of the respective fusion variants in the five samples analyzed with two different methods (Cases 1 through 5). In total, five tumors harbored a NAB2 exon 4–STAT6 exon 4 fusion variant (45%), two tumors had a NAB2 exon 7–STAT6 intron 1 fusion variant (18%), and two tumors had NAB2 exon 2–STAT6 intron 1 fusion variant (18%) (Table 1). One tumor each had a NAB2 exon 5–STAT6 exon 16 or a NAB2 exon 2–STAT6 exon 5 fusion var

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