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Solitary Fibrous Tumors/Hemangiopericytomas with Different Variants of the NAB2-STAT6 Gene Fusion Are Characterized by Specific Histomorphology and Distinct Clinicopathological Features

2014; Elsevier BV; Volume: 184; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2013.12.016

ISSN

1525-2191

Autores

Sarah Barthelmeß, Helene Geddert, Carsten Boltze, Evgeny A. Moskalev, Matthias Bieg, Horia Sı̂rbu, Benedikt Brors, Stefan Wiemann, Arndt Hartmann, Abbas Agaimy, Florian Haller,

Tópico(s)

Oral and Maxillofacial Pathology

Resumo

Recurrent somatic fusions of the two genes, NGFI-A–binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors (SFT). Herein, we evaluated a cohort of 52 SFTs/hemangiopericytomas (HPCs) by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary SFTs with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical HPCs from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary SFT and deep-seated HPC are separate entities that share common features but correlate to different clinical outcome. Recurrent somatic fusions of the two genes, NGFI-A–binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors (SFT). Herein, we evaluated a cohort of 52 SFTs/hemangiopericytomas (HPCs) by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary SFTs with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical HPCs from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary SFT and deep-seated HPC are separate entities that share common features but correlate to different clinical outcome. In 1942, Stout and Murray1Stout A.P. Murray M.R. Hemangiopericytoma: a vascular tumor featuring Zimmermann's pericytes.Ann Surg. 1942; 116: 26-33Crossref PubMed Google Scholar described a series of nine tumors sharing features of endothelial-lined tubes and sprouts and a pattern of well-developed branching staghorn thick-walled vessels with perivascular fibrosis, which they named hemangiopericytoma (HPC). In a follow-up study, Stout2Stout A.P. Hemangiopericytoma: a study of 25 cases.Cancer. 1949; 2: 1027-1054Crossref PubMed Scopus (204) Google Scholar reported on 25 cases with similar features. The concept of HPC as a specific tumor entity was later consolidated in a series of 106 cases by Enzinger and Smith,3Enzinger F.M. Smith B.H. Hemangiopericytoma: an analysis of 106 cases.Hum Pathol. 1976; 7: 61-82Abstract Full Text PDF PubMed Scopus (754) Google Scholar who refined the diagnostic criteria and established parameters for assessment of malignancy. Also in 1942, Stout and Murray4Stout A.P. Murray M.R. Localized pleural mesothelioma.Arch Pathol. 1942; 34: 951-964Google Scholar published a case of a solitary mesenchymal tumor of the lung and pleura, which they named solitary (localized) mesothelioma of the pleura, citing a first report on a series of similar tumors by Klemperer and Rabin.5Klemperer P. Rabin C.B. Primary neoplasms of the pleura: a report of five cases.Arch Pathol. 1931; 11: 385-412Google Scholar These reports were followed up later in another study establishing the characteristic features of spindle-shaped tumor cells with prominent diffuse connective tissue fibers within these mostly well-circumscribed tumors, later renamed as solitary fibrous tumor (SFT).6Stout A.P. Himadi G.M. Solitary (localized) mesothelioma of the pleura.Ann Surg. 1951; 133: 50-64Crossref PubMed Scopus (48) Google Scholar Although, in the initial descriptions, only tumors from the pleural cavity and lungs were included,7England D.M. Hochholzer L. McCarthy M.J. Localized benign and malignant fibrous tumors of the pleura: a clinicopathologic review of 223 cases.Am J Surg Pathol. 1989; 13: 640-658Crossref PubMed Scopus (1171) Google Scholar, 8Moran C.A. Suster S. Koss M.N. The spectrum of histologic growth patterns in benign and malignant fibrous tumors of the pleura.Semin Diagn Pathol. 1992; 9: 169-180PubMed Google Scholar it was not until the 1990s that comparable tumors were reported for other anatomical locations,9Goodlad J.R. Fletcher C.D. Solitary fibrous tumour arising at unusual sites: analysis of a series.Histopathology. 1991; 19: 515-522Crossref PubMed Scopus (310) Google Scholar, 10Suster S. Nascimento A.G. Miettinen M. Sickel J.Z. Moran C.A. Solitary fibrous tumors of soft tissue: a clinicopathologic and immunohistochemical study of 12 cases.Am J Surg Pathol. 1995; 19: 1257-1266Crossref PubMed Scopus (339) Google Scholar and the concept of extrapleural SFT was established.11Chilosi M. Facchettti F. Dei Tos A.P. Lestani M. Morassi M.L. Martignoni G. Sorio C. Benedetti A. Morelli L. Doglioni C. Barberis M. Menestrina F. Viale G. bcl-2 Expression in pleural and extrapleural solitary fibrous tumours.J Pathol. 1997; 181: 362-367Crossref PubMed Scopus (170) Google Scholar, 12Vallat-Decouvelaere A.V. Dry S.M. Fletcher C.D. Atypical and malignant solitary fibrous tumors in extrathoracic locations: evidence of their comparability to intra-thoracic tumors.Am J Surg Pathol. 1998; 22: 1501-1511Crossref PubMed Scopus (575) Google Scholar Enzinger and Smith3Enzinger F.M. Smith B.H. Hemangiopericytoma: an analysis of 106 cases.Hum Pathol. 1976; 7: 61-82Abstract Full Text PDF PubMed Scopus (754) Google Scholar had already emphasized that the most prominent feature of HPC, the ramifying vessels, can also be found in other mesenchymal neoplasms. An HPC-like vascular pattern has been reported for many well-defined other soft tissue sarcomas.13Tsuneyoshi M. Daimaru Y. Enjoji M. Malignant hemangiopericytoma and other sarcomas with hemangiopericytoma-like pattern.Pathol Res Pract. 1984; 178: 446-453Crossref PubMed Scopus (49) Google Scholar, 14Nappi O. Ritter J.H. Pettinato G. Wick M.R. Hemangiopericytoma: histopathological pattern or clinicopathologic entity?.Semin Diagn Pathol. 1995; 12: 221-232PubMed Google Scholar Since then, it has been debated for a long time whether HPC represents a distinct clinicopathological entity or only a non-specific vascular pattern.14Nappi O. Ritter J.H. Pettinato G. Wick M.R. Hemangiopericytoma: histopathological pattern or clinicopathologic entity?.Semin Diagn Pathol. 1995; 12: 221-232PubMed Google Scholar, 15Fletcher 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 (105) Google Scholar, 16Gengler C. Guillou L. Solitary fibrous tumour and haemangiopericytoma: evolution of a concept.Histopathology. 2006; 48: 63-74Crossref PubMed Scopus (490) Google Scholar On the basis of histomorphological and clinical similarities and the lack of clear separating diagnostic criteria, it has been proposed that HPC and SFT represent different points on a broad morphological spectrum of the same entity.16Gengler C. Guillou L. Solitary fibrous tumour and haemangiopericytoma: evolution of a concept.Histopathology. 2006; 48: 63-74Crossref PubMed Scopus (490) Google Scholar, 17Park M.S. Araujo D.M. New insights into the hemangiopericytoma/solitary fibrous tumor spectrum of tumors.Curr Opin Oncol. 2009; 21: 327-331Crossref PubMed Scopus (137) Google Scholar Although some authors still separate fibrous and cellular variants of SFT, resembling classic SFT and HPC, respectively,16Gengler C. Guillou L. Solitary fibrous tumour and haemangiopericytoma: evolution of a concept.Histopathology. 2006; 48: 63-74Crossref PubMed Scopus (490) Google Scholar these tumors were merged together in the fourth edition of the World Health Organization classification of tumors of soft tissue.18Fletcher C.D.M. Bridge J.A. Lee J.C. Extrapleural solitary fibrous tumour.in: Fletcher C.D.M. Bridge J.A. Hogendoorn P.C.W. Mertens F. World Health Organisation Classification of Tumours of Soft Tissue and Bone. ed 4. IARC Press, Lyon2013: 80-82Google Scholar Furthermore, pleural and extrapleural SFTs are distinguished according to their anatomical localization. Features suggestive of malignancy are increased mitotic counts, nuclear atypia, tumor necrosis, and infiltrative margins.3Enzinger F.M. Smith B.H. Hemangiopericytoma: an analysis of 106 cases.Hum Pathol. 1976; 7: 61-82Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 7England D.M. Hochholzer L. McCarthy M.J. Localized benign and malignant fibrous tumors of the pleura: a clinicopathologic review of 223 cases.Am J Surg Pathol. 1989; 13: 640-658Crossref PubMed Scopus (1171) Google Scholar, 8Moran C.A. Suster S. Koss M.N. The spectrum of histologic growth patterns in benign and malignant fibrous tumors of the pleura.Semin Diagn Pathol. 1992; 9: 169-180PubMed Google Scholar, 19Schirosi L. Lantuejoul S. Cavazza A. Murer B. Yves Brichon P. Migaldi M. Sartori G. Sgambato A. Rossi G. Pleuro-pulmonary solitary fibrous tumors: a clinicopathologic, immunohistochemical, and molecular study of 88 cases confirming the prognostic value of de Perrot staging system and p53 expression, and evaluating the role of c-kit, BRAF, PDGFRs (alpha/beta), c-met, and EGFR.Am J Surg Pathol. 2008; 32: 1627-1642Crossref PubMed Scopus (100) Google Scholar, 20Cranshaw I.M. Gikas P.D. Fisher C. Thway K. Thomas J.M. Hayes A.J. Clinical outcomes of extra-thoracic solitary fibrous tumours.Eur J Surg Oncol. 2009; 35: 994-998Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Rao N. Colby T.V. Falconieri G. Cohen H. Moran C.A. Suster S. Intrapulmonary solitary fibrous tumors: clinicopathologic and immunohistochemical study of 24 cases.Am J Surg Pathol. 2013; 37: 155-166Crossref PubMed Scopus (76) Google Scholar Only recently, recurrent fusion events of the two genes, NGFI-A–binding protein 2 (NAB2) and STAT6, both located at chromosomal region 12q13, have been identified by next-generation sequencing and RT-PCR in most SFTs and likely represent an initial tumorigenic event.22Robinson 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 (582) Google Scholar, 23Chmielecki 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 (432) Google Scholar, 24Mohajeri A. Tayebwa J. Collin A. Nilsson J. Magnusson L. von Steyern F.V. Brosjö O. Domanski H.A. Larsson O. Sciot R. Debiec-Rychter M. Hornick J.L. Mandahl N. Nord K.H. Mertens F. Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.Genes Chromosomes Cancer. 2013; 52: 873-886Crossref PubMed Scopus (206) Google Scholar Different fusion variants occurring at varying frequencies have been found in the three studies published so far, but, to our knowledge, no characteristic association with clinicopathological features has been reported.22Robinson 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 (582) Google Scholar, 23Chmielecki 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 (432) Google Scholar, 24Mohajeri A. Tayebwa J. Collin A. Nilsson J. Magnusson L. von Steyern F.V. Brosjö O. Domanski H.A. Larsson O. Sciot R. Debiec-Rychter M. Hornick J.L. Mandahl N. Nord K.H. Mertens F. Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.Genes Chromosomes Cancer. 2013; 52: 873-886Crossref PubMed Scopus (206) Google Scholar In contrast, the overall frequent finding of NAB2-STAT6 gene fusions and specific nuclear expression of STAT6 in benign and malignant SFTs of pleuropulmonary and different extrapleural sites is considered as further confirmation of the unifying concept of SFT as a single entity.22Robinson 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 (582) Google Scholar, 23Chmielecki 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 (432) Google Scholar, 24Mohajeri A. Tayebwa J. Collin A. Nilsson J. Magnusson L. von Steyern F.V. Brosjö O. Domanski H.A. Larsson O. Sciot R. Debiec-Rychter M. Hornick J.L. Mandahl N. Nord K.H. Mertens F. Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.Genes Chromosomes Cancer. 2013; 52: 873-886Crossref PubMed Scopus (206) Google Scholar, 25Doyle LA Vivero M Fletcher CD Mertens F Hornick JL Nuclear expression of STAT6 distinguishes solitary fibrous tumor from histologic mimics.Mod Pathol. 2013; 27: 390-395Crossref PubMed Scopus (520) Google Scholar For the current study, we evaluated a cohort of 52 SFTs/HPCs for the presence of NAB2-STAT6 gene fusions. First, we performed whole-exome sequencing of a recurrent pleural SFT and identified an identical NAB2-STAT6 gene fusion in two independent metachronous local recurrences. Second, we established a multiplex RT-PCR screening assay that interrogates all possible fusion combinations of NAB2 (seven exons) and STAT6 (23 exons) on the cDNA level, potentially amplifying a total of 161 different fusion variants. Characterizing the whole cohort of 52 tumors, we were able to identify 12 different fusion variants in 48 cases, confirming the high frequency of this recurrent genetic event. The different fusion variants were grouped according to their potential functional effects among the predicted chimeric fusion proteins. Strikingly, the tumors within the two most frequent categories of fusion variants, NAB2ex4-STAT6ex2/3 and NAB2ex6-STAT6ex16/17, displayed specific recurrent histomorphological characteristics and a significant association with relevant clinicopathological features, reminiscent of the original descriptions of SFT and HPC, respectively.1Stout A.P. Murray M.R. Hemangiopericytoma: a vascular tumor featuring Zimmermann's pericytes.Ann Surg. 1942; 116: 26-33Crossref PubMed Google Scholar, 2Stout A.P. Hemangiopericytoma: a study of 25 cases.Cancer. 1949; 2: 1027-1054Crossref PubMed Scopus (204) Google Scholar, 3Enzinger F.M. Smith B.H. Hemangiopericytoma: an analysis of 106 cases.Hum Pathol. 1976; 7: 61-82Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 4Stout A.P. Murray M.R. Localized pleural mesothelioma.Arch Pathol. 1942; 34: 951-964Google Scholar, 5Klemperer P. Rabin C.B. Primary neoplasms of the pleura: a report of five cases.Arch Pathol. 1931; 11: 385-412Google Scholar, 6Stout A.P. Himadi G.M. Solitary (localized) mesothelioma of the pleura.Ann Surg. 1951; 133: 50-64Crossref PubMed Scopus (48) Google Scholar, 7England D.M. Hochholzer L. McCarthy M.J. Localized benign and malignant fibrous tumors of the pleura: a clinicopathologic review of 223 cases.Am J Surg Pathol. 1989; 13: 640-658Crossref PubMed Scopus (1171) Google Scholar, 8Moran C.A. Suster S. Koss M.N. The spectrum of histologic growth patterns in benign and malignant fibrous tumors of the pleura.Semin Diagn Pathol. 1992; 9: 169-180PubMed Google Scholar Tumors with histological and immunohistochemical (IHC) features consistent with a diagnosis of SFT or HPC were retrieved from the surgical pathology archives of three German institutions (Institute of Pathology, University Hospital Erlangen, Erlangen; Institute of Pathology, St Vincent's Hospital, Karlsruhe; and Institute of Pathology, SRH-Klinikum, Gera). Primary meningeal and sinonasal tumors were excluded from the current series, as well as tumors displaying classic features of myofibroma, myopericytoma, or giant-cell angiofibroma. All tumors were reclassified by an experienced soft tissue pathologist (A.A.) after review of the available slides, without knowledge of the molecular findings. The IHC for STAT6 was performed as described recently by Doyle et al,25Doyle LA Vivero M Fletcher CD Mertens F Hornick JL Nuclear expression of STAT6 distinguishes solitary fibrous tumor from histologic mimics.Mod Pathol. 2013; 27: 390-395Crossref PubMed Scopus (520) Google Scholar using a polyclonal antibody directed against STAT6 (1:1000 dilution, sc-621; Santa Cruz Biotechnology, Santa Cruz, CA). Only unequivocal strong and diffuse nuclear staining for STAT6 was considered as positive. Paucicellular cases with spindled tumor cells and uniform fusiform or spindled nuclei with scanty cytoplasm, arranged in a wavy and patternless pattern within a prominent diffuse fibrous stroma displaying typical cracking artifacts, were classified as classic fibrous SFTs (Figure 1, A–C). Conventional (paucicellular) pleuropulmonary SFTs were considered the prototype of this variant. Tumors with a higher cellularity and occasionally more rounded cells, with round to oval chromatin-rich nuclei but prominent perivascular fibrosis, were considered cellular SFTs (Figure 1, D–F). This cellular variant is generally of similar architecture as the paucicellular fibrous variant, with the gaping staghorn vessels being more prominent at the periphery of the lesion. One case (case 25) with mixed patterns of paucicellular and more cellular areas was included in this category. In contrast, highly cellular tumors, with a well-developed net of thin-walled, dilated, or slit-like communicating vessels without perivascular fibrosis/hyalinization, were designated as HPCs (Figure 1, G–I). These tumors generally looked more primitive, without any fibrous stroma but occasionally with myxoid changes. Their vessels lacked any prominent connective tissue layer, and the lining endothelial cells seem to merge directly with the surrounding tumor cells, forming the vascular wall. Most of the latter tumors displayed characteristic branching staghorn-like vessels that were not confined to the tumor periphery but were frequently seen throughout the whole width. Mitoses were counted in 10 high-power fields (HPFs). Tumors with more than four mitoses in 10 HPFs, marked nuclear atypia, tumor necrosis, and/or infiltrative margins, were termed histologically malignant, in contrast to histologically benign tumors lacking these features.3Enzinger F.M. Smith B.H. Hemangiopericytoma: an analysis of 106 cases.Hum Pathol. 1976; 7: 61-82Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 7England D.M. Hochholzer L. McCarthy M.J. Localized benign and malignant fibrous tumors of the pleura: a clinicopathologic review of 223 cases.Am J Surg Pathol. 1989; 13: 640-658Crossref PubMed Scopus (1171) Google Scholar, 8Moran C.A. Suster S. Koss M.N. The spectrum of histologic growth patterns in benign and malignant fibrous tumors of the pleura.Semin Diagn Pathol. 1992; 9: 169-180PubMed Google Scholar, 19Schirosi L. Lantuejoul S. Cavazza A. Murer B. Yves Brichon P. Migaldi M. Sartori G. Sgambato A. Rossi G. Pleuro-pulmonary solitary fibrous tumors: a clinicopathologic, immunohistochemical, and molecular study of 88 cases confirming the prognostic value of de Perrot staging system and p53 expression, and evaluating the role of c-kit, BRAF, PDGFRs (alpha/beta), c-met, and EGFR.Am J Surg Pathol. 2008; 32: 1627-1642Crossref PubMed Scopus (100) Google Scholar, 20Cranshaw I.M. Gikas P.D. Fisher C. Thway K. Thomas J.M. Hayes A.J. Clinical outcomes of extra-thoracic solitary fibrous tumours.Eur J Surg Oncol. 2009; 35: 994-998Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Rao N. Colby T.V. Falconieri G. Cohen H. Moran C.A. Suster S. Intrapulmonary solitary fibrous tumors: clinicopathologic and immunohistochemical study of 24 cases.Am J Surg Pathol. 2013; 37: 155-166Crossref PubMed Scopus (76) Google Scholar Clinical follow-up was available for 33 (63%) of 52 patients, including 13 with tumor recurrences. Four patients with recurrent tumors died of their disease 3, 5, 18, and 26 years after initial diagnosis, respectively. Twenty patients had no signs of tumor recurrence after a median of 5 years (±3 years; range, 1 to 15 years). One of these patients died of another cause 5 years after the diagnosis. This study has been approved by the local ethics committee of the University Hospital Erlangen, Germany (number 217_12 B, 19.09.2012). Signed informed consent was obtained from all participating patients in this study. DNA was isolated from two fresh-frozen local recurrences of a pleural SFT and from a matched blood sample of the same patient serving as control. DNA was fragmented (Covaris E220; Covaris, Woburn, MA) to 150 to 200 bp, followed by enrichment of exonic regions and index tagging (Agilent Sure Select, version 4; Agilent Technologies, Santa Clara, CA). Paired-end sequencing (2 × 100 bp) was performed using a HiSeq2000 (Illumina, Inc., San Diego, CA) to a mean coverage of >120×. [Mapping and target coverage calculation were done using Burrows-Wheeler Alignment (BWA) tool and the OnTarget tool (http://www.dkfz.de/gpcf/ontarget/ontarget2.tar.gz)].26Hotz-Wagenblatt A. Kats I. Scharfenberger-Schmeer M. Haldeman B. Glatting K.H. OnTarget: a tool for analysing enrichment data derived from next generation sequencing.Presented at the 11th International Conference on Intelligent Systems Design and Applications (ISDA). 2011; : 873-876Google Scholar, 27Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinf. 2009; 25: 1754-1760Crossref PubMed Scopus (29919) Google Scholar Breakpoints of translocations were identified using the clipping reveals structure (CREST) algorithm, which uses soft-clipped reads to discover genomic locations with many partially mapped reads.28Wang J. Mullighan C.G. Easton J. Roberts S. Heatley S.L. Ma J. Rusch M.C. Chen K. Harris C.C. Ding L. Holmfeldt L. Payne-Turner D. Fan X. Wei L. Zhao D. Obenauer J.C. Naeve C. Mardis E.R. Wilson R.K. Downing J.R. Zhang J. CREST maps somatic structural variation in cancer genomes with base-pair resolution.Nat Methods. 2011; 8: 652-654Crossref PubMed Scopus (389) Google Scholar The soft-clipped portions of the reads were assembled into a consensus sequence that was afterward remapped against the reference. The genomic location of the remapped consensus sequence was thus identified as the translocation position. Total RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples using the RNeasy FFPE Kit and Deparaffinization Solution (Qiagen, Hilden, Germany). cDNA was synthesized from 700 ng of total RNA per sample with the Quantitect Reverse Transcription Kit (Qiagen) containing a combination of oligo-dT and random primers. A multiplex RT-PCR protocol was established using forward primers located in NAB2 exons 1 to 7 and reverse primers positioned in STAT6 exons 1 to 22 (primer sequences available on request) and performed by HotStarTaq DNA Polymerase reagents (Qiagen) with a temperature profile consisting of an initial step of 95°C for 15 minutes, 50 cycles of 95°C for 30 seconds, 57.6°C for 45 seconds, and 72°C for 1 minute, and a terminal step of 72°C for 5 minutes. RT-PCR of hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as control for RNA integrity. Samples with PCR products in the multiplex assay were subsequently analyzed by omitting different forward and reverse primers until a specific NAB2-STAT6 fusion variant was amplified. All detected NAB2-STAT6 fusion variants were confirmed by Sanger sequencing on a cDNA level. Analysis of the whole-exome sequencing data, derived from two independent metachronous local recurrences of a malignant pleural SFT (patient 24) (Table 1), revealed an NAB2-STAT6 gene fusion in both samples, which was not present in the blood control (Figure 2). The genomic breakpoints were identified using the CREST software and were located on chr12:57486804 (NAB2 intron 4) and chr12:57508845 (upstream of STAT6). The predicted fusion protein consisted of the N-terminal portion of NAB2 encoded by exons 1 to 4, which was fused to the whole STAT6 protein.Table 1Patient and Tumor CharacteristicsCaseAge/sexHistological featuresLocationFollow-upSize (cm)/mitotic counts (10 HPFs)Estimated dignity∗The dignity was estimated according to published criteria mitotic counts (>4/10 HPFs), marked nuclear atypia, tumor necrosis, and/or infiltrative margins.3,7,8,19–21NAB2-STAT6 fusion variantSTAT6 IHCTumors with NAB2ex4-STAT6ex2/3 fusion variants 174/MFibrous SFTPleuraDOOC (5 years)2.8/0Benign4–2+ 261/MFibrous SFTPleuraNA3.5/0Benign4–2+ 341/FFibrous SFTPleuraAWOD (2 years)4/0Benign4–2+ 448/FFibrous SFTPleuraNA4/0Benign4–2+ 569/MFibrous SFTPleuraAWOD (4 years)4/0Benign4–2+ 669/MFibrous SFTLungNA6/0Benign4–2+ 743/FFibrous SFTPleuraAWOD (1 year)7/0Benign4–2+ 879/FFibrous SFTPleuraAWOD (4 years)8/0Benign4–2+ 967/FFibrous SFTLungNA8.5/0Benign4–2+ 1072/MFibrous SFTCervicalAWOD (6 years)8.5/0Benign4–2+ 1171/FFibrous SFTPleuraNA9.5/0Benign4–2+ 1283/MFibrous SFTPleuraAWD (6 years)10/0Benign4–2+ 1360/MFibrous SFTPleuraAWOD (6 years)11/0Benign4–2+ 1472/FFibrous SFTPleuraNA13/0Benign4–2+ 1560/MFibrous SFTPleuraNA15.5/0Benign4–2+ 1673/FFibrous SFTLungAWOD (2 years)15.5/1Benign4–2+ 1761/MFibrous SFTPleuraAWOD (4 years)18/1Benign4–2+ 1883/FFibrous SFTPleuraAWOD (1 year)21/0Benign4–2+ 1961/MFibrous SFTPleuraNA25/1Benign4–2+ 2062/FFibrous SFTPleuraAWOD (4 years)26.5/0Benign4–2+ 2173/MCellular SFTPleuraNA13/0Benign4–2+ 2284/FCellular SFTPleuraAWD (11 years)7/7Malignant4–2+ 2379/FCellular SFTLungNA5/8Malignant4–2+ 2465/FCellular SFTPleuraAWD (7 years)12/14Malignant4–2+ 2578/FCellular SFTPleuraDOT (3 years)11.5/45Malignant4–2+ 2669/FFibrous SFTLungAWOD (1 year)1.6/0Benign4–3+ 2758/MCellular SFTInguinalNA3.5/0Benign4–3+Tumors with NAB2ex6-STAT6ex16/17 fusion variants 2833/FCellular SFTParaurethralAWOD (8 years)8/3Benign6–16+ 2946/MHPCPelvisAWOD (6 years)2.2/3Benign6–16+ 3049/FHPCParavertebralDOT (26 years)5/23Malignant6–16+ 31†Both patients experienced recurrent disease for several years, but their age at the first tumor manifestation could not be ascertained./FHPCParavertebralAWD3/8Malignant6–16+ 3231/FHPCParotid glandDOT (18 years)2.7/11Malignant6–16+ 3347/MHPCParavertebralAWD (15 years)4.5/9Malignant6–16+ 3452/MHPCParavertebralAWD6/22Malignant6–16+ 3561/FCellular SFTLower legNA1.9/0Benign6–17+ 3635/MHPCNuchalAWOD (4 years)1/1Benign6–17+ 37†Both patients experienced recurrent disease for several years, but their age at the first tumor manifestation could not be ascertained./FCellular SFTTrunk wallAWD8.5/8Malignant6–17+ 3882/MHPCInguinalAWD (1 year)9/53Malignant6–17+Tumors with other NAB2-STAT6 fusion variants 3933/MFibrous SFTOrbitaNA2.5/0Benign3–17+ 4045/FFibrous SFTCheekAWOD (5 years)0.6/1Benign3–18+ 4165/MFibrous SFTTrunk wallAWD (8 years)5.5/2Benign3–19+ 4254/MCellular SFTOrbitaAWOD (15 years)1/0Benign3–19+ 4339/MCellular SFTParotid glandNA3.5/1Benign3–19+ 4454/FCellular SFTLungAWOD (4 years)3/3Benign4–18+ 4567/FHPCSublingualNA2.9/0Benign5–18+ 4649/FCellular SFTTrunk wallAWOD (7 years)0.9/2Benign7–2+ 4767/FCellular SFTRetroperitonealNA12/0Benign7–18+ 4872/FCellular SFTRetroperitonealNA4.2/0Benign7–20+Tumors without detectable NAB2-STAT6 fusions 4968/FFibrous SFTLower legDOT (5 years)7/1BenignNone+ 5048/MFibrous SFTTongueNA2/0BenignNone+ 5157/FFibrous SFTLungNA7/0BenignNone+ 5247/FHPCParapharyngealAWOD (2 years)4/0BenignNone+F, female; M, male; AWD, alive with disease; AWOD, alive without disease; DOOC, died of other cause; DOT, died of tumor; NA, not available; +, positive nuclear staining.∗ The dignity was e

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