Gene Expression in Solitary Fibrous Tumors (SFTs) Correlates with Anatomic Localization and NAB2-STAT6 Gene Fusion Variants

Solitary fibrous tumors (SFTs) harbor recurrent NAB2-STAT6 gene fusions, promoting constitutional up-regulation of oncogenic early growth response 1 (EGR1)-dependent expression. SFTs with the most common canonical NAB2 exon 4–STAT6 2 fusion variant are often located in thorax (pleuropulmonary) and less cellular abundant collagen. In contrast, 6–STAT6 16/17 variants typically display a round to ovoid cell morphology deep soft tissue retroperitoneum intra-abdominal pelvic region or meninges. Here, we employed next-generation sequencing–based expression profiling identify significant differences associated anatomic localization variants. showed transcriptional signature enriched for genes involved DNA binding, transcription, nuclear localization, whereas were tyrosine kinase signaling, proliferation, cytoplasmic localization. Specific transcription factor binding motifs among differentially expressed different variants, implicating co–transcription factors modification chimeric NGFI-A protein (NAB2)-STAT6–dependent deregulation EGR1-dependent summary, this study establishes potential molecular biologic basis clinicopathologic distinct rare mesenchymal fibroblastic characterized by haphazardly arranged spindled cells within variably collagenous stroma characteristic branching thin-walled vasculature.1WHO Classification Tumours: Soft Tissue Bone Tumours. ed 5. International Agency Research on Cancer (IARC), Lyon, France:2020Google Scholar initially described occur pleuropulmonary region, but more research confirms that arise at all sites, including A wide morphologic spectrum exists encompasses low cellularity embedded collagen bundles as well highly slit-like vascular spaces. 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Signed informed consent obtained participating study, other anonymized before further biological analysis. Clinicopathologic data follow-up pathology files archives contributing Open reading frames 4-2 6-16 synthesized cloned pUC57-Kan plasmid Genewiz (South Plainfield, NJ). NAB exons 4 ending 5′-end 4, followed 22 beginning 3′-end 2. spanned 6, 16 22. Minimal Kozak sequence ACC introduced start codon. shuttled lentiviral vector rwpLX305 (German Center, Heidelberg, Germany) under control constitutive cytomegalovirus immediate using Gateway cloning (ThermoFisher, Braunschweig, Germany). After propagation, endotoxin-free constructs (EndoFree Plasmid Maxi Kit; Qiagen, Hilden, generation pseudoviral particles HEK293T (Dulbecco's Eagle's medium supplemented 10% fetal bovine serum 5% sodium pyruvate), manufacturer's protocol (System Biosciences, Palo Alto, CA). total 200 μL crude unconcentrated viral supernatant μL/mL polybrene mixed × 105 CCD-1137Sk cells. Cells then seeded 6-well plates grown minimum essential Iscove's Dulbecco's (Life Technologies, Carlsbad, CA), respectively, (Pan Biotech, Aidenbach, μg/mL blasticidin (Thermo Fisher Scientific, Waltham, MA). detection cancer-relevant performed targeted hybrid-capture RNA-sequencing panels. TruSight Pan-Cancer Panel (Illumina, San Diego, 1385 genes, detect determine (cohort 1). Fusion (Illumina), subset 507 frequently used same purposes tissue, experiments. each panel, 500 ng library preparation, protocol. Up eight libraries pooled sequenced MiSeq instrument reagent kit version 3, 150 cycle (Illumina) paired-end 75 bp direction, generate least million reads per sample. analysis, FASTQ uploaded Illumina's Basespace (Illumina). alignment application call TopHat-Fusion algorithm, raw counts genes. Total isolated miRNeasy Mini (Qiagen, Germany), suggested manufacturer. concentration quantified ND1000 spectrophotometer (ThermoFisher). Reverse reactions μg QuantiTect Transcription (Qiagen). Quantitative RT-PCR triplicate C1000 Thermal Cycler CFX96 Real-Time System (BioRad, Hercules, CA) QuantiFast SYBR Green PCR Primer sequences follows: COL1A1, 5′-CTCCTCTTAGCGGCCACC-3′ 5′-TACCTGAGGCCGTTCTGTAC-3′; ERG, 5′-GCAGGATTGGCTGTCTCAAC-3′ 5′-TTGGCCACACTGCATTCATC-3′; IGF1, 5′-ATCAGCAGTCTTCCAACCCA-3′ 5′-AGCGCCAGGTAGAAGAGATG-3′; IGF2, 5′-GACACCCTCCAGTTCGTCT-3′ 5′-GGAAACAGCACTCCTCAACG-3′; PAPPA, 5′-CAGGGCCAGGAAATGACAC-3′ 5′- CTTGATTGGGCGTGAAGGAG-3′; LMNB1, 5′-CTGGAAATGTTTGCATCGAAGA-3′ 5′-GCCTCCCATTGGTTGATCC-3′. Universal human reference calibration (Stratagene, La Jolla, LMNB1 served internal control. thermal cycler conditions 95°C minutes, two-step 40 cycles 10 seconds 55°C 30 seconds. analyzed ΔΔ threshold method.29Livak Schmittgen T.D. relative real-time quantitative 2(-Delta Delta C(T)) method.Methods. 25: 402-408Crossref (111501) two-sided U-test test versus 6-16/17 Differential read R package deSeq2.30Love M.I. 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Chapman B.A. Cox Dalke Friedberg Hamelryck Kauff Wilczynski de Hoon Biopython: freely available Python tools computational biology bioinformatics.Bioinformatics. 1422-1423Crossref (1991) exact overrepresented underrepresented sets. contingency tables serving input tests containing one TFBS TFBS, TFBS. comprised patients. 36 SFTs, 9 13 14 (Table pelvis retroperitoneal (nine cases), lower extremity head neck each.