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Recurrent PAX3-MAML3 fusion in biphenotypic sinonasal sarcoma

Abstract

Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Here we report a recurrent chromosomal translocation in SNS, t(2;4)(q35;q31.1), resulting in a PAX3-MAML3 fusion protein that is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, closely simulating the developmental roles of PAX3.

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Figure 1: Structure and transactivation potential of the PAX3-MAML3 fusion.
Figure 2: Unsupervised hierarchical clustering analysis showing the gene expression signature of 8 SNS and 33 other mesenchymal tumors based on the expression levels of 516 genes.

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Acknowledgements

The authors thank A. Maran, K.L. Shogren, S.L. Segovis, J.L. Herrick and X. Li for logistical support in the performance of several of these experiments. We also express gratitude to L. Jin, M.R. Erickson-Johnson, B.R. Evers, C.W. Roth, R.N. Wehrs, A.R. Seys and M.L. Lonzo for technical support in the Molecular Anatomic Pathology Laboratory and Cytogenetics Laboratory, to T.J. Flotte and L.A. Gross in the Pathology Resource Core Laboratory, to K.C. Halling and M.E. Brown for administration support, to T.L. Schmidt for secretarial assistance and to T.A. Bennett for editorial assistance. We also thank C.P. Kolbert and B.W. Eckloff at the Gene Expression Core and the Sequencing Core at the Mayo Medical Genome Facility for providing transcriptome sequencing and gene expression profiling services. This work was supported by Mayo Clinic DLMP 2012/13 Research Grants and by US National Institutes of Health grant T32 CA148073.

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Authors and Affiliations

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Contributions

X.W. and K.L.B. performed PCR, qPCR, FISH, cloning experiments, cell culture, luciferase assays, immunoblotting, DNA-binding assays and immunofluorescence experiments. J.E.L., J.T.L., R.P.G. and A.M.O. recognized the disease, reviewed pathology slides and obtained clinical information and tumor material. J.J. provided next-generation sequencing analysis. Y.W.A. performed all bioinformatics analyses. A.M.O., M.J.Y., M.M.C. and D.S.V. reviewed clinical information, performed data analysis and supervised some of the experiments. J.J.W. and A.M.O. planned and supervised the work. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to André M Oliveira.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cytogenetics and molecular genetics findings for the PAX3-MAML3 fusion.

(a) Partial karyogram showing the chromosomal translocation t(2;4)(q35;q31.1). (b,c) FISH break-apart strategy showing rearrangements of the PAX3 and MAML3 loci, respectively (see also Fig. 1b). The percentage of rearranged cells for these loci ranged from 25 to 78% (median, 60%). Endothelial cells, inflammatory cells, sinonasal epithelial cells and other non-neoplastic cells showed no rearrangement of these loci. (d) Representative RT-PCR gel showing the PAX3-MAML3 fusion in three examples of SNS (lanes 1–3). Several other tumors (lanes 4–8: alveolar rhabdomyosarcoma, dermatofibrosarcoma protuberans, melanoma, malignant peripheral nerve sheath tumor and synovial sarcoma, respectively) and normal sinonasal tissue (lane 9) were negative for the fusion product; lanes 10–11 have no template. (See also Supplementary Table 2). (e) Confirmatory chromatogram showing the PAX3-MAML3 sequence at the fusion breakpoint in case 1.

Supplementary Figure 2 The PAX3-MAML3 fusion protein localizes to the nucleus and binds DNA.

(a) Protein blots were performed with antibody to PAX3 or tubulin on lysates of HEK293T cells expressing pcDNA3 (Co), Flag-PAX3-MAML3 (P-M) or HA-PAX3. (b) Immunofluorescence images showing nuclear expression of Flag-PAX3-MAML3 in C2C12 cells. Cells were counterstained with phalloidin to detect cytoplasmic F-actin and with DAPI to detect DNA. (c) Electrophoretic mobility shift assay (EMSA) of Flag-PAX3-MAML3 binding to double-stranded DNA containing a PAX3-binding element. Lysates from COS-7 cells expressing Flag-PAX3-MAML3 or HA-PAX3 were incubated with a 32P-labeled PAX3 DNA probe in the presence or absence of 100-fold excess of the wild-type (WT) probe, a mutated (Mut) probe, control IgG, antibody to HA or antibody to Flag. The arrow points to PAX3-MAML3–DNA complexes. The arrowhead indicates PAX3-DNA complexes. The asterisk indicates free probe. (d) Transient transcription assays demonstrating the effect of PAX3-MAML3 on a Notch reporter, Hes5p-Luc, in C2C12 cells in the presence or absence of the Notch1 intracellular domain 1 (NICD1). *P < 0.01, **P < 0.0001 relative to control in unpaired t tests.

Supplementary Figure 3 Pathway enrichment analysis.

(a) Gene ontology analysis shows that the SNS expression profile is enriched in genes involved in several developmental processes, including skeletal system morphogenesis and general organ development (minimum P = 3.39 × 10–14 for skeletal system morphogenesis, false discovery rate (FDR) = 1.37 × 10–10; maximum P = 1.14 × 10–10 for organ morphogenesis, FDR = 4.64 × 10–8). (b) Enrichment process network analysis shows that SNS exhibits differential expression of several biological circuits involved in neural, cardiac, bone and other developmental processes (minimum P = 4.19 × 10–4 for neural development, FDR = 0.058; maximum P = 0.031 for olfactory transduction, FDR = 0.427). Enrichment analyses were performed using the MetaCore gene regulatory network database in order to identify networks that are significantly enriched by differentially expressed genes. Modified gene ontology terms are based on the GO database (http://www.geneontology.org/).

Supplementary Figure 4 Representative qPCR and immunohistochemistry studies confirm differential gene expression in SNS.

(a,b) High expression of NTRK3 and MYOD1 was observed in most examples of SNS. (c) MLANA and MITF (data not shown) were not expressed in SNS. (d) NTRK3 showed a strong membranous pattern of expression in virtually all SNS samples (24/25). No staining was observed in the sinonasal epithelium. (e) MYOD1 nuclear expression was focal in a few SNS tumors (4/25); the arrow indicates an example cell with focal MYOD1 expression. (f) Melan A and MITF (data not shown) expression was universally negative in all SNS tumors tested (n = 22). qPCR and/or immunohistochemistry also confirmed the overexpression of PLUNC, PCP4, GDF7, PIK3C2G, ALX1 and PAX3 and the downregulation of HOXB7 (data not shown; see also Supplementary Table 1; additional information about the SNS phenotype can be found in ref. 1). RE, relative expression; SNS, sinonasal sarcoma (n = 5); NST, normal sinonasal tissue (n = 4); ARMS, alveolar rhabdomyosarcoma (n = 3); DFSP, dermatofibrosarcoma protuberans (n = 3); MPNST, malignant peripheral nerve sheath tumor (n = 3); SS, synovial sarcoma (n = 3). *** and ** indicate Kruskal-Wallis test P < 0.01 and P < 0.05, respectively, and post-hoc Dunett's test P < 0.05; * indicates Wilcoxon-Mann-Whitney test P < 0.05; data are shown as means ± s.d.

Supplementary Figure 5 Differential expression of myogenin in SNS and ARMS.

(a) Biphenotypic SNS is a monomorphic spindle cell neoplasm that exclusively occurs in the head and neck region of middle-aged individuals, most commonly in females (hematoxylin and eosin, ×100). (b) ARMS is a pediatric small round cell tumor that shows a characteristic pseudoalveolar growth pattern (hematoxylin and eosin, ×100). (c,d) qPCR and immunohistochemical analyses show that, whereas all ARMS tumors (n = 11) express high levels of MYOG (myogenin) mRNA (c) and protein (d, inset), SNS tumors (n = 11) are consistently negative for this marker (c,d). *P < 0.0001, Wilcoxon-Mann-Whitney test; data are shown as means ± s.d.

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Wang, X., Bledsoe, K., Graham, R. et al. Recurrent PAX3-MAML3 fusion in biphenotypic sinonasal sarcoma. Nat Genet 46, 666–668 (2014). https://doi.org/10.1038/ng.2989

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