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Genomic and transcriptomic features of dermatofibrosarcoma protuberans: Unusual chromosomal origin of the COL1A1-PDGFB fusion gene and synergistic effects of amplified regions in tumor development

Open AccessPublished:December 10, 2019DOI:https://doi.org/10.1016/j.cancergen.2019.12.001

      Highlights

      • Genomic and transcriptomic data on a cohort of dermatofibrosarcoma protuberans tumors.
      • Evaluation of genetic features in relation to various clinical features.
      • Data supporting an origin of the COL1A1-PDGFB fusion in the G2-S phase of the cell cycle.
      • Identification of potential target genes in amplified regions on chromosomes 17 and 22.

      Abstract

      The dermatofibrosarcoma protuberans family of tumors (DPFT) comprises cutaneous soft tissue neoplasms associated with aberrant PDGFBR signaling, typically through a COL1A1-PDGFB fusion. The aim of the present study was to obtain a better understanding of the chromosomal origin of this fusion and to assess the spectrum of secondary mutations at the chromosome and nucleotide levels. We thus investigated 42 tumor samples from 35 patients using chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, and/or massively parallel sequencing (gene panel, whole exome and transcriptome sequencing) methods. We confirmed the age-associated differences in the origin of the COL1A1-PDGFB fusion and could show that it in most cases must arise after DNA synthesis, i.e., in the S or G2 phase of the cell cycle. Whereas there was a non-random pattern of secondary chromosomal rearrangements, single nucleotide variants seem to have little impact on tumor progression. No clear genomic differences between low-grade and high-grade DPFT were found, but the number of chromosomes and chromosomal imbalances as well as the frequency of 9p deletions all tended to be greater among the latter. Gene expression profiling of tumors with COL1A1-PDGFB fusions associated with unbalanced translocations or ring chromosomes identified several transcriptionally up-regulated genes in the amplified regions of chromosomes 17 and 22, including TBX2, PRKCA, MSI2, SOX9, SOX10, and PRAME.

      Key words

      Introduction

      Dermatofibrosarcoma protuberans (DP) is a rare (<1/100,000 individuals per year), locally aggressive mesenchymal tumor with often recurrent local disease (up to 50%), but only low metastatic risk [
      • Mentzel T
      • Pedeutour F
      • Lazar A
      • Coindre J-M
      Dermatofibrosarcoma protuberans.
      ]. However, DP has a potential for transformation into a fibrosarcoma of higher grade. Tumors with fibrosarcomatous transformation show the same tendency for local recurrence as regular DP, but are associated with metastatic disease in about 13% of the cases [
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      ]. DP occurs most commonly on the trunk and proximal extremities of young or middle-aged adults. Due to both molecular and genetic overlap, a variety of other entities, such as Bednar tumor and giant cell fibroblastoma (GCF), are nowadays included in the DP family of tumors (DPFT).
      Genetically, DPFT is, in the vast majority of cases, characterized by a COL1A1-PDGFB gene fusion; the promoter and variable portions of the collagen 1A1 (COL1A1) gene are fused with exon 2 of the platelet-derived growth factor beta (PDGFB) gene, resulting in deregulated expression of the PDGFB protein [
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      Various regions within the alpha-helical domain of the COL1A1 gene are fused to the second exon of the PDGFB gene in dermatofibrosarcomas and giant-cell fibroblastomas.
      . At the chromosome level, the gene fusion is caused by an exchange of material between chromosome bands 17q21 (COL1A1) and 22q13 (PDGFB). This exchange may appear in the form of a balanced or unbalanced t(17;22) or as one or more supernumerary ring chromosomes [
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      ,
      • Pedeutour F
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      • Hecht F.
      • Turc-Carel C.
      Ring 22 chromosomes in dermatofibrosarcoma protuberans are low-level amplifiers of chromosome 17 and 22 sequences.
      . The latter, which may contain multiple copies of the fusion gene as well as of other portions of chromosome arms 17q and 22q, occurs more frequently in older patients [
      • Terrier-Lacombe MJ
      • Guillou L
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      Dermatofibrosarcoma protuberans, giant cell fibroblastoma, and hybrid lesions in children: clinicopathologic comparative analysis of 28 cases with molecular data - A study from the french federation of cancer centers sarcoma group.
      ] whereas the former has been described particularly in children [
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      Translocation, t(17;22)(q22;q13), in dermatofibrosarcoma protuberans: a new tumor-associated chromosome rearrangement.
      ,
      • Sirvent N
      • Maire G
      • Pedeutour F
      Genetics of dermatofibrosarcoma protuberans family of tumors: from ring chromosomes to tyrosine kinase inhibitor treatment.
      . The COL1A1-PDGFB fusion is specific for DPFT. Hence, both fluorescence in situ hybridization (FISH) and RT-PCR based methods are well established diagnostic tools [
      • O’Brien KP
      • Seroussi E
      • Dal Cin P
      • Sciot R
      • Mandahl N
      • Fletcher JA
      • et al.
      Various regions within the alpha-helical domain of the COL1A1 gene are fused to the second exon of the PDGFB gene in dermatofibrosarcomas and giant-cell fibroblastomas.
      ,
      • Terrier-Lacombe MJ
      • Guillou L
      • Maire G
      • Terrier P
      • Vince DR
      • Somerhausen N.
      • de S.A.
      • Collin F.
      • de Saint Aubain Somerhausen N
      • et al.
      Dermatofibrosarcoma protuberans, giant cell fibroblastoma, and hybrid lesions in children: clinicopathologic comparative analysis of 28 cases with molecular data - A study from the french federation of cancer centers sarcoma group.
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      Genomic gains of COL1A1-PDFGB occur in the histologic evolution of giant cell fibroblastoma into dermatofibrosarcoma protuberans.
      ,
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      • Hauke S.
      • Petersen I.
      Molecular and clinicopathological analysis of dermatofibrosarcoma protuberans.
      ,
      • Italiano A.
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      • Escande F.
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      . Rare cases combining PDGFB with other 5′-partners have been described, and recently it was shown that at least a subset of DPFT of the breast in women have a COL6A3-PDGFD fusion that, similar to the more common COL1A1-PDGFB fusion, activates the PDGFB receptor [
      • Dadone-Montaudié B.
      • Alberti L.
      • Duc A.
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      • Blay J.-.Y.
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      • Kubiniek V.
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      • Cardot-Leccia N.
      • Michot A.
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      • Tirode F.
      • Pedeutour F.
      • Ranchère-Vince D.
      • Le Loarer F.
      • Pissaloux D.
      Alternative PDGFD rearrangements in dermatofibrosarcomas protuberans without PDGFB fusions.
      ,
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      • Swanson D.
      • Zhang L.
      • Sung Y.-.S.
      • Antonescu C.R.
      Dermatofibrosarcoma protuberans with a novel COL6A3-PDGFD fusion gene and apparent predilection for breast.
      .
      Whereas the pathogenetic significance of the COL1A1-PDGFB fusion has been thoroughly documented, the information on secondary genomic changes and to what extent these may vary with age or outcome is still limited. Abnormal karyotypes have been reported in 47 cases [
      ] and a few studies have used comparative genomic hybridization (CGH) or array-based approaches to evaluate the spectrum of genomic imbalances in DPFT [
      • Kiuru-Kuhlefelt S.
      • El-Rifai W.
      • Fanburg-Smith J.
      • Kere J.
      • Miettinen M.
      • Knuutila S.
      Concomitant DNA copy number amplification at 17q and 22q in dermatofibrosarcoma protuberans.
      ,
      • Nishio J.
      • Iwasaki H.
      • Ohjimi Y.
      • Ishiguro M.
      • Isayama T.
      • Naito M.
      • Iwashita A.
      • Kikuchi M.
      Overrepresentation of 17q22-qter and 22q13 in dermatofibrosarcoma protuberans but not in dermatofibroma: a comparative genomic hybridization study.
      ,
      • Linn S.C.
      • West R.B.
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      • Zhu S.
      • Hernandez-Boussard T.
      • Nielsen T.O.
      • Rubin B.P.
      • Patel R.
      • Goldblum J.R.
      • Siegmund D.
      • Botstein D.
      • Brown P.O.
      • Gilks C.B.
      • van de Rijn M.
      Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans.
      ,
      • Kaur S.
      • Vauhkonen H.
      • Böhling T.
      • Mertens F.
      • Mandahl N.
      • Knuutila S.
      Gene copy number changes in dermatofibrosarcoma protuberans – a fine-resolution study using array comparative genomic hybridization.
      ,
      • Hong JY
      • Liu X
      • Mao M
      • Li M
      • Choi DI
      • Kang SW
      • et al.
      Genetic aberrations in imatinib-resistant dermatofibrosarcoma protuberans revealed by whole genome sequencing.
      ,
      • Stacchiotti S
      • Astolfi A
      • Gronchi A
      • Fontana A
      • Pantaleo MA
      • Negri T
      • et al.
      Evolution of dermatofibrosarcoma protuberans to DFSP-derived fibrosarcoma: an event marked by epithelial-mesenchymal transition-like process and 22q loss.
      ]. Even fewer cases have been analyzed with techniques based on massively parallel sequencing (MPS;
      • Hong JY
      • Liu X
      • Mao M
      • Li M
      • Choi DI
      • Kang SW
      • et al.
      Genetic aberrations in imatinib-resistant dermatofibrosarcoma protuberans revealed by whole genome sequencing.
      ,
      • Stacchiotti S
      • Astolfi A
      • Gronchi A
      • Fontana A
      • Pantaleo MA
      • Negri T
      • et al.
      Evolution of dermatofibrosarcoma protuberans to DFSP-derived fibrosarcoma: an event marked by epithelial-mesenchymal transition-like process and 22q loss.
      ,
      • Oh E
      • Jeong HM
      • Kwon MJ
      • Ha SY
      • Park HK
      • Song J-Y
      • et al.
      Unforeseen clonal evolution of tumor cell population in recurrent and metastatic dermatofibrosarcoma protuberans.
      ).
      Here, we present a series of 35 patients with DPFT that were analyzed with regard to genomic changes by chromosome banding, FISH, and/or high-resolution single nucleotide polymorphism (SNP) array analyses. In addition, selected cases were analyzed with regard to additional mutations and gene expression profile using MPS-based gene panel, whole exome (WES), and transcriptome sequencing (RNA-seq).

      Materials and methods

      Patients and tumors

      The study encompassed 42 tumor samples from 35 patients (27 men, 8 women; age 1–76 years) collected from 1985 to 2018 (Table 1). The patients were treated at the sarcoma centers in Lund and Stockholm, Sweden. The study was based on 28 samples from primary lesions, 12 from local recurrences, and one from a metastasis; information on the origin was missing in one patient (Case 33). All diagnoses were revised, based on the morphological picture and immune profile, according to established criteria [
      • Mentzel T
      • Pedeutour F
      • Lazar A
      • Coindre J-M
      Dermatofibrosarcoma protuberans.
      ]. Two tumors were diagnosed as GCF. Six of the tumors were classified as high-grade DP with fibrosarcomatous transformation, morphologically indicated by architectural changes, more pronounced atypia, increased mitotic activity, and in exceptional cases areas of tumor necrosis.
      Table 1Clinicopathologic features of 35 cases of dermatofibrosarcoma protuberans family of tumors subjected to genetic analyses.
      Case NoSample
      P = primary tumor; LR = local recurrence; Met =metastasis; NA = not available.
      AgeSex
      M = male; F = female.
      Site
      L = lower; Th = thoracic; Tr = trunk; U = upper; NA = not available.
      Size
      Largest diameter in cm; NA = not available.
      DX
      DX = diagnosis; GCF = giant cell fibroblastoma; LGDP = low-grade dermatofibrosarcoma protuberans; HGDP = high-grade dermatofibrosarcoma protuberans.
      KaryotypeAdditional analyses
      SNP = Single nucleotide polymorphism array (Affymetrix Cytoscan HD); FISH = fluorescence in situ hybridization; WES = whole exome sequencing; RNA-seq = mRNA sequencing; GP = Ion AmpliSeq Cancer Hot Spot Panel v2.
      1P1MTh wall4GCF46,XY,der(22)t(17;22)(q21;q13)SNP
      2P1ML armNAGCF46,XY,der(22)t(17;22)(q21;q13)FISH
      3P7MBackNALGDP46,XY,t(17;22)(q21;q13)/46,idem,der(22)t(17;22)SNP, FISH, GP
      4P16ML leg8LGDP47,XY,+18,der(22)t(17;22)(q21;q13)/47,XY,+18,der(22)r(17;22) (q21q25;p13q13)SNP, GP
      5LR19MBack6LGDP46–50,XY,+2rSNP, FISH, RNA-Seq, GP
      6LR21FTr wall3.5LGDPNot doneSNP
      7P22MThigh0.4LGDP47,XY,+?ider(22)(q10)t(17;22)(q21;q13)/47–48,idem,+2
      8aP22ML leg3.5LGDP49,XY,+8,+18,+22,der(22)t(17;22)(q21;q13)x2
      8bPLGDP49,XY,+8,+18,+22,der(22)t(17;22)(q21;q13)x2SNP, RNA-Seq, GP
      9LR30MShoulder2LGDP48,XY,+8,+der(22)r(17;22)(q12-21q24-25;p11q13)/48,XY,+der(22)r(17;22),+der(?)t(?;22)
      Previously published karyotype.
      10P33MShoulder3LGDPNot doneSNP, RNA-Seq
      11aP36MBack9LGDP47,XY,+5,−21,der(22)t(?17;22)(q21;q13),+r
      Previously published karyotype.
      11bPLGDP47–50,XY,+5,−21,der(22)t(?17;22)(q21;q13),+1–4r
      Previously published karyotype.
      12aLR140FShoulder4LGDP47,XX,+der(1)r(1;?)(p36q44;?)
      Previously published karyotype.
      12bLR22.5LGDP47,XX,+der(1)r(1;?)(p36q44;?)
      Previously published karyotype.
      13P41MTh wall6LGDP48–50,XY,+5,+8,+11,+21,+r,+marSNP, FISH, RNA-Seq, GP
      14aP42MU arm6.5HGDP50,XY,+8,+18,+21,+22,der(22)t(17;22)(q21;q13)x2
      14bPHGDP50,XY,+8,+18,+21,+22,der(22)t(17;22)(q21;q13)x2FISH
      15P43FShoulder1.6LGDP47,XX,−5,der(14)t(5;14)(q11;q31),?del(22)(q11),+r,+mar/47,idem, add(7)(p11)/47,idem,t(3;9)(q25;q13),+5,-mar
      16P47MShoulder4LGDP47,XY,+5,−22,+r/46–47,XY,add(8)(p11), +12,−14, der(16)t(14;16)(q11;q23),add(22)(q11),+rSNP, RNA-Seq
      17P48MGroin3LGDP47,XY,+r/47,XY,+mar
      18P48MTr wall5LGDP47,XY,+r
      Previously published karyotype.
      19P50ML arm1HGDP47,XY,+22,der(22)t(17;22)(q21;q13)x2/48,idem,+rFISH
      20P50FShoulder3.5LGDP46–49,XX,+4,+8,−19,−20,−22,+rFISH
      21P55MBack4HGDP46–48,XY,der(3)t(3;?5)(q29;q21),−22,+1–2rFISH
      22P57MGroin2.5LGDP87,XX,-Y,-Y,−3,−6,+8,−11,−14,−22,+r/86,idem,−9
      23LR59MTr wall6HGDPNot doneSNP
      24LR59MGroinNALGDP47,XY,+r
      Previously published karyotype.
      25P60ML leg2HGDP51–53,XY,+5,+7,+11,+12,−22,+r,+1–2marSNP, RNA-Seq
      26P60FThigh3.5LGDP47,XX,+rSNP, RNA-Seq, GP
      27P60MShoulder3LGDP46–47,XY,+rSNP, RNA-Seq, GP
      28aP64MShoulder4.5HGDP100–101,XXYY,−3,del(6)(q23),+7,−10,+15,?add(16)(q?12)x2,+18,−19,+20,+5marSNP, WES
      28bMetLung5HGDP79–83,XYY,-X,−2,−3,−4,add(5)(p15),−6,del(6)(q23),−8,−9,−9,−10,add(11)(p11), −13,−14,−15,−15,+del(17)(p11),−19,−21,+2–3marSNP, WES, RNA-Seq
      29LR64MShoulder4LGDP49–51,XY,+2,+r,+mar
      30P66FShoulder1.5LGDP46,XX,−22,+r/44–45,idem,-X/46–47,idem,+4/46–48,idem,+4,+r
      31aLR166MShoulder5LGDP45,XY,add(5)(p13),del(7)(q22q32),−10,add(20)(q12),−22,−22,+r, +der(?)t(?;22)(?;q1?)SNP, FISH, RNA-Seq, GP
      31bLR2Shoulder4.5LGDP44–45,XY,add(5)(p13),del(7)(q22q32),−10,add(20)(q12),−22,−22,+mar/44–45,idem,+r
      32LR67FGroinNALGDP49,XX,+?del(4)(q21),dic(5;22)(p12-13;p11),ins(5;?)(q31;?),+del(8)(p21),+r,+mar
      33NA69FBackNALGDP47,XX,+r
      34aP69MTr wall6LGDP47,XY,+r/48,idem,add(1)(p11),+marFISH
      34bPLGDP47–50,XY,add(?4)(p11),+8,+r,+mar,inc
      35LR76MNA0.7LGDP47,XY,+r/46,X,-Y,+r/45,X,-Y
      a P = primary tumor; LR = local recurrence; Met =metastasis; NA = not available.
      b M = male; F = female.
      c L = lower; Th = thoracic; Tr = trunk; U = upper; NA = not available.
      d Largest diameter in cm; NA = not available.
      e DX = diagnosis; GCF = giant cell fibroblastoma; LGDP = low-grade dermatofibrosarcoma protuberans; HGDP = high-grade dermatofibrosarcoma protuberans.
      f SNP = Single nucleotide polymorphism array (Affymetrix Cytoscan HD); FISH = fluorescence in situ hybridization; WES = whole exome sequencing; RNA-seq = mRNA sequencing; GP = Ion AmpliSeq Cancer Hot Spot Panel v2.
      g Previously published karyotype.

      Cytogenetic and FISH analyses

      Chromosome banding analysis was performed on 39 samples from 32 patients, as described [
      • Mandahl N.
      • Heim S.
      • Arheden K.
      • Rydholm A.
      • Willén H.
      • Mitelman F.
      Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas.
      ]. The nomenclature of the karyotypes followed the guidelines of the International System for Human Cytogenomic Nomenclature [
      • McGowan-Jordan J
      • Simons A
      • Schmid M
      ISCN 2016
      ISCN 2016. An international system for human cytogenomic nomenclature.
      ]. The karyotypes from seven samples were published before (Table 1;
      • Mandahl N.
      • Heim S.
      • Arheden K.
      • Rydholm A.
      • Willén H.
      • Mitelman F.
      Rings, dicentrics, and telomeric association in histiocytomas.
      ,
      • Mandahl N.
      • Heim S.
      • Willén H.
      • Rydholm A.
      • Mitelman F.
      Supernumerary ring chromosome as the sole cytogenetic abnormality in a dermatofibrosarcoma protuberans.
      ,
      • Mandahl N.
      • Limon J.
      • Mertens F.
      • Arheden K.
      • Mitelman F.
      Ring marker containing 17q and chromosome 22 in a case of dermatofibrosarcoma protuberans.
      ,
      • Örndal C.
      • Mandahl N.
      • Rydholm A.
      • Willén H.
      • Brosjö O.
      • Heim S.
      • Mitelman F.
      Supernumerary ring chromosomes in five bone and soft tissue tumors of low or borderline malignancy.
      ,
      • Gisselsson D.
      • Höglund M.
      • O'Brien K.P.
      • Dumanski J.P.
      • Mertens F.
      • Mandahl N.
      A case of dermatofibrosarcoma protuberans with a ring chromosome 5 and a rearranged chromosome 22 containing amplified COL1A1 and PDGFB sequences.
      ,
      • Walther C
      • Domanski HA
      • Vult von Steyern F
      • Mandahl N
      • Mertens F
      Chromosome banding analysis of cells from fine-needle aspiration biopsy samples from soft tissue and bone tumors: is it clinically meaningful?.
      ). Metaphase FISH analysis for the detection of PDGFB rearrangement and the COL1A1-PDGFB fusion gene and to identify ring chromosomes was performed in eight cases; one case was analyzed with interphase FISH for PDGFB status only (Table 1). FISH analyses were performed as described [
      • Jin Y
      • Möller E
      • Nord KH
      • Mandahl N
      • Vult Von Steyern F
      • Domanski HA
      • et al.
      Fusion of the AHRR and NCOA2 genes through a recurrent translocation t(5;8)(p15;q13) in soft tissue angiofibroma results in upregulation of aryl hydrocarbon receptor target genes.
      ], using the ZytoLight SPEC COL1A1-PDGFB Dual Color Dual Fusion Probe and the ZytoLight SPEC PDGFB Dual Color Break Apart Probe (ZytoVision, Bremerhaven, Germany). Sequences of chromosomes 17 and 22 were identified using whole chromosome painting (WCP) probes (Applied Spectral Imaging, Migdal Haemek, Israel).

      SNP array analysis

      Extracted DNA from 16 frozen tumor samples from 15 patients (Table 1) was analyzed using high-resolution single nucleotide polymorphism (SNP) arrays (Cytoscan HD, Affymetrix, Santa Clara, CA, USA), as described [
      • Walther C.
      • Mayrhofer M.
      • Nilsson J.
      • Hofvander J.
      • Jonson T.
      • Mandahl N.
      • Øra I.
      • Gisselsson D.
      • Mertens F.
      Genetic heterogeneity in rhabdomyosarcoma revealed by SNP array analysis.
      ]. The position of the probes was aligned according to the USCS hg19/NCBI Build 37 sequences. Tumor Aberration Prediction Suite (TAPS) and Rawcopy were used for segmentation of copy number shifts, copy number evaluation, and visualization of the data in the 12 samples showing copy number variation within the amplified regions of 17q and/or 22q [
      • Rasmussen M
      • Sundström M
      • Göransson Kultima H
      • Botling J
      • Micke P
      • Birgisson H
      • et al.
      Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity.
      ,
      • Mayrhofer M
      • Viklund B
      • Isaksson A
      Rawcopy: improved copy number analysis with Affymetrix arrays.
      . Constitutional copy number variations were excluded through comparison with the Database of Genomic Variants (http://projects.tcag.ca/variation/).

      MPS-based analyses

      From eight of the samples analyzed by SNP array, also targeted DNA sequencing of 50 neoplasia-associated genes was performed (Table 1), using the Ion AmpliSeq Cancer Hot Spot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) as described [
      • Hofvander J
      • Jo VY
      • Ghanei I
      • Gisselsson D
      • Mårtensson E
      • Mertens F
      Comprehensive genetic analysis of a paediatric pleomorphic myxoid liposarcoma reveals near-haploidization and loss of the RB1 gene.
      ]. Ten samples were analyzed by RNA-seq (Table 1), as described [
      • Arbajian E.
      • Puls F.
      • Antonescu C.R.
      • Amary F.
      • Sciot R.
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      • Hofvander J.
      • Mertens F.
      In-depth genetic analysis of sclerosing epithelioid fibrosarcoma reveals recurrent genomic alterations and potential treatment targets.
      ]. Gene expression levels in the DPFT were compared with those in a cohort of 64 soft tissue tumors (STT). Differences between tumor types in log2-transformed expression data were calculated using a t-test, and corrections for multiple testing were based on the Benjamini–Hochberg method (Qlucore AB). The two tumor samples and peripheral blood from Case 28 were analyzed by WES (Illumina, San Diego, CA, USA), with targeted resequencing for seven of the mutations detected at WES (Table 1); these analyses were performed as described [
      • Hofvander J.
      • Arbajian E.
      • Stenkula K.G.
      • Lindkvist-Petersson K.
      • Larsson M.
      • Nilsson J.
      • Magnusson L.
      • Vult von Steyern F.
      • Rissler P.
      • Hornick J.L.
      • Mertens F.
      Frequent low-level mutations of protein kinase D2 in angiolipoma.
      ,
      • Hofvander J.
      • Viklund B.
      • Isaksson A.
      • Brosjö O.
      • Vult von Steyern F.
      • Rissler P.
      • Mandahl N.
      • Mertens F.
      Different patterns of clonal evolution among different sarcoma subtypes followed for up to 25 years.
      . WES and targeted resequencing generated an average coverage of 99X and 145X, respectively.

      Results

      Chromosome banding and FISH analyses

      All 39 samples from the 32 patients that were analyzed by chromosome banding analysis showed abnormal karyotypes (Table 1). A balanced t(17;22)(q21;q13), 1–2 copies of an unbalanced der(22)t(17;22), and 1–4 ring chromosomes were found in one, nine, and 25 patients, respectively; one tumor (Case 3) had one clone with a balanced and one with an unbalanced translocation, and in four patients both an unbalanced der(22)t(17;22) and ring chromosomes co-existed in the same or different clones. The single case with a balanced t(17;22) was a 7-year-old boy, whereas the tumors with an unbalanced der(22)t(17;22) or ring chromosomes were found in patients aged 1–50 years and 16–76 years, respectively. One patient (Case 28) did not display any of these cytogenetic features; both samples from this high-grade DP had a complex aneuploid karyotype.
      Secondary chromosomal aberrations, here defined as rearrangements other than ring chromosomes or affecting other chromosomes than 17 and 22, were found at G-banding analysis of 21 tumors (Table 1). Most tumors had a pseudo- or hyperdiploid chromosome count; hypodiploid or triploid-hypertetraploid clones were seen in two tumors each. Among the secondary aberrations, numerical changes predominated over structural rearrangements, the most common being trisomy 8 (seven cases), loss of one or more chromosomes 22 (six cases), and trisomy 5 and 18 (four cases each). Structural rearrangements, none of which was recurrent, other than ring chromosomes or t(17;22)/der(22)t(17;22) were seen in 10 cases, including three high-grade DP.
      In seven cases, more than one sample was available for chromosome banding analysis. Intralesional clonal heterogeneity, studied in the four cases with data on both a preoperative needle biopsy and the excised primary tumor, was restricted to a variable number of ring chromosomes in one (Case 11) and different structural and numerical secondary aberrations in one (Case 34). In three patients, clonal evolution with time could be studied. Two local recurrences in Cases 12 and 31 displayed no or only subtle differences, whereas the primary tumor and a metastasis in Case 28 differed more extensively.
      Metaphase FISH analysis was performed on nine tumors, showing one fusion event on each der(22) in the samples with translocations, and up to three fusions in the ring chromosomes. In the DP from Case 19, fusion signals were seen only on the der(22)t(17;22) chromosomes, but not in the ring chromosome found in a subclone (Table 1; Fig. 1a).
      Fig 1
      Fig. 1A, FISH analysis of dermatofibrosarcoma protuberans (DP; Case 19) using a break-apart probe for PDGFB (5′ in green, 3′ in red) and a whole chromosome painting probe for chromosome 22 (blue). The two der(22)t(17;22) showed a split PDBGF signal (red, arrowhead), while the normal chromosome 22 showed an intact PDGFB locus (yellow; thick arrow). The ring chromosome was negative for PDGFB and chromosome 22 material (thin arrow). B, Circos plot showing copy number changes and non-synonymous exonic variants detected with single nucleotide polymorphism (SNP) array and whole exome sequencing (WES), respectively, in the primary tumor (PT) and a metastasis (Met) from Case 28. The circles are ordered chronologically, starting from the center with the PT. The pink/green inner circles represent the location and amplitude of the allelic imbalances in relation to the ploidy level (4n); blue is gain, gray is loss and yellow is copy neutral loss of heterozygosity. Differences between the PT and the Met are indicated by red changes in the most peripheral circle, which otherwise appears green. The light blue circles represent the location of the variants reported by the whole exome sequencing. C, Possible outcomes of a t(17;22) in different phases of the cell cycle. The two parental copies of chromosomes 17 and 22 are shown in different shades of blue and gray, respectively. (1) Trisomy 17 is followed by t(17;22) in G0-G1 and loss of der(17), resulting in UPID for the disomic part of chr 17 in 1/3 of the cases. (2) t(17;22) in G0-G1 with subsequent loss of der(17) and duplication of the normal homologue, always leading to UPID of the disomic parts of chr 17. (3) Recombinations in G2 would consistently result in retained heterodisomy for the disomic parts of chromosomes 17 and 22 in DP cases with (I) der(22)t(17;22) or (II) ring chromosomes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      SNP array analysis

      SNP array analysis revealed copy number changes in all analyzed tumors (Supplementary Table 1; Fig. 2a). All four tumors with t(17;22)/der(22)t(17;22) showed 1–2 extra copies of 17q21-qter with the copy number shift in or near the COL1A1 locus and loss of 22q13-qter with the copy number shift in or near the PDGFB locus. Gain of the centromeric part of 22q (there are no informative probes until nt position 16,877,134 in subband 22q11.1) was seen in only one of these tumors (Case 8). There was no copy number variation within the gained or deleted segments and the segment from 17pter-q21 always displayed retained heterodisomy, while the segment distal to the PDGFB locus on 22q consistently was mono-allelic. Case 6, a local recurrence 20 years after surgery for a primary tumor and for which no G-band data were available, showed a pattern similar to that of other cases with der (22) t (17;22).
      Fig 2
      Fig. 2Single nucleotide polymorphism (SNP) array and RNA-sequencing data in cases of dermatofibrosarcoma protuberans (DPFT). A, SNP array with genome-wide overview on copy number changes in the subset of the DPFT showing copy number variation within the amplified regions of 17q and/or 22q. Green signals indicate gain, blue signals loss, and gray signals copy neutral loss of heterozygosity. B and C, Copy number changes are shown in detail for chromosomes 17 and 22. D and E, Heat maps of expression levels for genes on chromosomes 17 and 22 in DPFT and other soft tissue tumors; red indicates increased and green decreased gene expression. Light blue = benign fibrous histiocytoma; dark blue = DPFT; orange = inflammatory leiomyosarcoma; green = myxoinflammatory fibroblastic sarcoma; white = myxofibrosarcoma; purple = ossifying fibromyxoid tumor; red = PRDM10-positive tumors; black = sclerosing epithelioid fibrosarcoma; yellow = undifferentiated pleomorphic sarcoma. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Also all of the eight DP with ring chromosomes showed gain of 17q21-qter, usually between 1–4 extra copies, with the shift from a normal, heterodisomic state in or near COL1A1; in contrast to the tumors with der(22)t(17;22), six of the cases showed copy number variation within the gained segment. The pattern for 22q was even more complex, with varying portions of 22q11-q13 being gained at different copy number levels; a copy number shift, corresponding to 1–4 extra copies, was always seen at the PDGFB locus. In four tumors, part of 22q11-q13 was disomic. In 5/7 tumors, the segment distal to the PDGFB locus was disomic with retained heterodisomy (Supplementary Fig. 1); in the remaining two tumors this segment was either monosomic (corresponding to the monosomy 22 observed at G-banding) or showed complex copy number variation (possibly explained by the combination of monosomy 22 and marker chromosomes at G-banding), respectively. Also Case 10, for which karyotypic data were missing, showed a profile similar to most cases with known ring chromosomes.
      SNP array data were also available for the primary tumor and the metastasis from Case 28. The primary tumor was more similar to the tumors with der(22)t(17;22) than to those with ring chromosomes: uniform low-level gain of 17q21-qter and loss of 22q13-qter, respectively. However, there was also relative gain of proximal 22q. The metastasis showed further structural rearrangements of 17p (deletion of one copy) and 22q (deletion of one copy of 22q11-qter).
      Imbalances affecting other chromosomes than 17 and 22 were mostly in good agreement with aberrations detected at G-banding analysis. For a detailed view of all observed copy number shifts in the analyzed tumors see Supplementary Table 1. The distribution of gained and lost segments on chromosomes 17 and 22 in a subset of the tumors is shown in Fig. 2b and c.

      MPS-based analyses

      RNA-seq of ten cases identified in-frame COL1A1-PDGFB fusion transcripts in all cases (Supplementary Table 2). At the gene expression level, genes mapping within the commonly amplified regions on 17q (i.e., from COL1A1 to qter) and 22q (i.e., from 22q11 to PDGFB) were expressed at higher levels than genes from the non-amplified parts of these chromosomes (Fig. 2d and e). Of the 410 and 499 genes within the amplified regions on chromosomes 17 and 22, 267 (65%) and 260 (52%) genes, respectively, showed higher expression in the DP compared to other soft tissue tumors. Among the amplified genes showing at least 5-fold increased expression in DP compared to the cohort of 64 STT were PRKCA, TBX2, MSI2, and SOX9 on chromosome 17 and PDGFB, PRAME, and SOX10 on chromosome 22 (Supplementary Tables 3 and 4).
      The gene panel analysis in eight cases revealed only one potentially pathogenic mutation (scored as deleterious by the SIFT algorithm): a c.524T>A (p.Val175Glu) at an allele frequency of 14% in the PTEN gene in a local recurrence (Case 31a). WES of the primary tumor and a metastasis from Case 28 revealed 23 non-synonymous exonic structural variants (ESV) in the primary tumor and 24 in the metastasis; 19 of these were shared (Fig. 1b and Supplementary Table 5).

      Discussion

      A unique aspect of DPFT is the association between patient age and karyotype. As noted before, the COL1A1-PDGFB fusion is in an age-dependent manner associated with either a classical translocation or with one or more supernumerary ring chromosomes [
      • Sirvent N
      • Maire G
      • Pedeutour F
      Genetics of dermatofibrosarcoma protuberans family of tumors: from ring chromosomes to tyrosine kinase inhibitor treatment.
      ]. More specifically, none of the nine previously published karyotypes from children with DPFT included a ring chromosome; instead, one (from a child aged 15 months) showed only a balanced translocation t(17;22), two (from children aged 8 months and 13 yrs) had both a balanced and an unbalanced translocation in different subclones or different samples, and six (2–13 yrs) had only an unbalanced der(22)t(17;22) or variants thereof. Of the 38 DP from adults, 32 displayed one or more ring chromosomes, the youngest patient being 30 years at diagnosis. None of the adults had a balanced t(17;22), but two, aged 38 and 47 yrs, had the same der(22)t(17;22) as seen in the pediatric setting [
      ].
      In our cohort of 32 cytogenetically analyzed DPFT, the age at diagnosis ranged from 1–76 years. All four patients aged 1–16 years had at least one clone with a balanced or unbalanced t(17;22). The only tumor with a balanced t(17;22), from a 7-year-old boy, also had a subclone with an unbalanced der(22)t(17;22). The youngest patient displaying only ring chromosomes (Case 5) was 19 years at diagnosis. In agreement with literature data, also some adult patients, the oldest being 50 years at diagnosis (Case 19), displayed a der(22)t(17;22).
      In summary, a balanced t(17;22) is a rare finding in DPFT and has so far not been observed in patients older than 13 years. The unbalanced der(22)t(17;22), or variants thereof, predominates in the pediatric/adolescent setting, but is occasionally seen also in adults up to the age of 50 years. Ring chromosomes, on the other hand, are found in the vast majority of DPFT from adults, and have so far not been observed in patients below the age of 16 years.
      The age-dependent association between the COL1A1-PDGFB fusion and three different chromosomal variants suggests that the fusion arises through different mechanisms in different age groups. On the other hand, there are several examples of one cytogenetic variant coexisting with another, suggesting that there could be a stepwise evolution from a balanced t(17;22) over der(22)t(17;22) to one or more ring chromosomes containing parts of chromosomes 17 and 22 [
      • Macarenco RS
      • Zamolyi R
      • Franco MF
      • Nascimento AG
      • Abott JJ
      • Wang X
      • et al.
      Genomic gains of COL1A1-PDFGB occur in the histologic evolution of giant cell fibroblastoma into dermatofibrosarcoma protuberans.
      ,
      . For instance, in the present cohort one DPFT had one clone with a t(17;22) and one with a t(17;22) and a der(22)t(17;22), and one case had one clone with a der(22)t(17;22) and one clone with a ring chromosome (Table 1); FISH analysis verified the presence of the COL1A1-PDGFB fusion in all these clones.
      While we, in line with previously reported data [
      ], thus in a few cases could provide direct evidence for an evolutionary relationship between the t(17;22) and the der(22)t(17;22) and between the der(22)t(17;22) and ring chromosome formation, respectively, this does not imply that the der(22)t(17;22) or ring chromosomes in DPFT generally derive from a balanced t(17;22). Indeed, the SNP array results do not support such a clonal evolution. The most common karyotype in DPFT, i.e., one with two normal copies of chromosomes 17 and 22, and one or more supernumerary der(22)t(17;22) or ring chromosomes, could arise through three different mechanisms: 1) An exchange between chromosomes 17 and 22 in the G0 or G1 phase of the cell cycle that was preceded by trisomy 17 followed by loss of the derivative chromosome 17; 2) An exchange in G0-G1 between chromosomes 17 and 22 that was followed by loss of the derivative chromosome 17 and duplication of the normal chromosome 17 homologue; or 3) An exchange between chromatids after DNA replication in S-G2, with the der(17) and the der(22) ending up in different daughter cells. Mechanism 1 would result in uniparental isodisomy (UPID) for the disomic part of chromosome 17 in one-third of the cases and mechanism 2 would always give rise to UPID for the disomic part of chromosome 17. Only scenario 3 would consistently result in retained heterodisomy for the disomic parts of chromosomes 17 and 22, which is the allelic pattern that was found in all four DPFT with der(22)t(17;22) and all seven DPFT with ring chromosome(s) analyzed here (Fig. 1c and Supplementary Fig. 1).
      Whereas it thus seems clear that the COL1A1-PDGFB fusion in most DPFT with a der(22)t(17;22) or ring chromosome(s) is due to an exchange between chromosomes 17 and 22 after DNA synthesis, the origin of the balanced t(17;22) cannot be deduced from the currently available data. Although this translocation, like most other balanced neoplasia-associated translocations, most likely arises in G0-G1, an origin in S-G2 could also result in the segregation of a der(17) and a der(22) to the same daughter cell; hence, more cases than the only tumor (Case 3) with a balanced t(17;22) analyzed so far need to be studied before an S-G2 origin can be excluded.
      The question, then, is why the COL1A1-PDGFB fusion arises through S-G2 errors in the vast majority of DPFT. The timing and origin of translocations and other types of erroneous DNA double-strand repair is still incompletely understood, but based on cytogenetic data most neoplasia-associated translocations resulting in pathogenetic fusions are due to G0-G1 errors [
      • Weinstock DM
      • Richardson CA
      • Elliott B
      • Jasin M
      Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells.
      ,
      • Biehs R.
      • Steinlage M.
      • Barton O.
      • Juhász S.
      • Künzel J.
      • Spies J.
      • Shibata A.
      • Jeggo P.A.
      • Löbrich M.
      DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination.
      . Possibly, the COL1A1-PDGFB fusion is sufficient for tumorigenesis only in a pediatric setting, whereas most children and all adults require not only the gene fusion but also the extra copies of distal 17q and/or a distorted ratio between genes centromeric and telomeric to the PDGFB locus on chromosome 22. Hence, the different types of translocation may occur at equal frequencies in all age groups, but only the unbalanced ones have a selective advantage in older patients.
      The pathogenetic impact of the copy number changes affecting chromosomes 17 and 22 is, however, not obvious. As shown before [
      • Linn S.C.
      • West R.B.
      • Pollack J.R.
      • Zhu S.
      • Hernandez-Boussard T.
      • Nielsen T.O.
      • Rubin B.P.
      • Patel R.
      • Goldblum J.R.
      • Siegmund D.
      • Botstein D.
      • Brown P.O.
      • Gilks C.B.
      • van de Rijn M.
      Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans.
      ], genes included in amplified regions are, in general, expressed at higher levels than are non-amplified genes on chromosomes 17 and 22. However, the copy number increase is relatively moderate (on average 1–3 extra copies in DPFT with ring chromosomes) with usually only minor fluctuations along the amplified segment (Fig. 2b and c). Our RNA-seq analysis showed that of 499 transcripts (long non-coding and micro RNA molecules excluded) located on chromosome 22, 344 were from the amplified region and 155 mapped to the non-amplified, sometimes deleted, region telomeric of PDGFB. While 71 (21%) of the genes in the amplified region showed increased expression (here defined as a fold change ≥2), the corresponding figure for genes in the non-amplified region was only 7 (5%). For the 410 genes located in the amplified region of chromosome 17, 125 (30%) showed increased expression (Fig. 2d and e; Supplementary Tables 3 and 4).
      RNA-seq identified a few genes (26 on chromosome 17 and 18 on chromosome 22) in the amplified regions with at least 5-fold higher expression compared to the other STT. Many of the amplified genes on chromosomes 17 and 22 that were found to be highly expressed in DPFT by Linn and co-workers [
      • Linn S.C.
      • West R.B.
      • Pollack J.R.
      • Zhu S.
      • Hernandez-Boussard T.
      • Nielsen T.O.
      • Rubin B.P.
      • Patel R.
      • Goldblum J.R.
      • Siegmund D.
      • Botstein D.
      • Brown P.O.
      • Gilks C.B.
      • van de Rijn M.
      Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans.
      ] showed high expression also in the present study. For instance, PDGFB was, as expected, one of the top up-regulated genes, showing a 20-fold higher expression in DPFT (Supplementary Table 4). For genes mapping to the amplified part of chromosome 17, we could confirm that the transcription factor-encoding gene TBX2 and the kinase-encoding gene PRKCA were relatively highly expressed (6.4- and 6.8-fold increase, respectively). Two transcriptionally upregulated genes on 17q that have not previously been implicated in DPFT were MSI2 (16.1-fold higher expression) and SOX9 (10.5-fold higher expression). From a clinical point of view, a potentially interesting target for the amplified segment on 22q was PRAME (12.2-fold higher expression in DPFT). This gene codes for a cancer-testis antigen, and it was recently suggested that overexpressed PRAME could constitute a good antigen for immunotherapy in sarcomas [
      • Roszik J.
      • Wang W.-.L.
      • Livingston J.A.
      • Roland C.L.
      • Ravi V.
      • Yee C.
      • Hwu P.
      • Futreal A.
      • Lazar A.J.
      • Patel S.R.
      • Conley A.P.
      Overexpressed Prame is a potential immunotherapy target in sarcoma subtypes.
      ]. In summary, the gene expression profiling of DPFT shows that the characteristic gain of 17q and 22q material that in most cases accompanies the COL1A1-PDGFB fusion is associated with a reproducible differential expression of many genes, some of which are potential treatment targets.
      Little is known about the mechanisms behind transformation of a low-grade into a high-grade DP. None of the previously reported karyotypes was from a tumor diagnosed as high-grade DP. In the present study there were cytogenetic data on five such cases. These five tumors did not differ in any distinct way from the cases diagnosed as low-grade DP. Similarly, SNP array analysis did not find any distinct biomarker among the few high-grade DP cases.
      It has also been assessed whether the number of fusion copies or the presence of mutations in selected cancer-associated genes could correlate with sarcomatous transformation, but no strong evidence for such an association has emerged, in line with the findings in our study [
      • Kiuru-Kuhlefelt S.
      • El-Rifai W.
      • Fanburg-Smith J.
      • Kere J.
      • Miettinen M.
      • Knuutila S.
      Concomitant DNA copy number amplification at 17q and 22q in dermatofibrosarcoma protuberans.
      ,
      • Stacchiotti S
      • Astolfi A
      • Gronchi A
      • Fontana A
      • Pantaleo MA
      • Negri T
      • et al.
      Evolution of dermatofibrosarcoma protuberans to DFSP-derived fibrosarcoma: an event marked by epithelial-mesenchymal transition-like process and 22q loss.
      • Wang J.
      • Morimitsu Y.
      • Okamoto S.
      • Hisaoka M.
      • Ishida T.
      • Sheng W.
      • Hashimoto H.
      COL1A1-PDGFB fusion transcripts in fibrosarcomatous areas of six dermatofibrosarcomas protuberans.
      ,
      • Takahira T.
      • Oda Y.
      • Tamiya S.
      • Yamamoto H.
      • Kawaguchi K.
      • Kobayashi C.
      • Oda S.
      • Iwamoto Y.
      • Tsuneyoshi M.
      Microsatellite instability and p53 mutation associated with tumor progression in dermatofibrosarcoma protuberans.
      ,
      • Abbott J.J.
      • Erickson-Johnson M.
      • Wang X.
      • Nascimento A.G.
      • Oliveira A.M.
      Gains of COL1A1-PDGFB genomic copies occur in fibrosarcomatous transformation of dermatofibrosarcoma protuberans.
      . One possible exception could be the inactivation of the tumor suppressor CDKN2A, which has been reported to occur more often, but not exclusively, in high-grade DP [
      • Stacchiotti S
      • Astolfi A
      • Gronchi A
      • Fontana A
      • Pantaleo MA
      • Negri T
      • et al.
      Evolution of dermatofibrosarcoma protuberans to DFSP-derived fibrosarcoma: an event marked by epithelial-mesenchymal transition-like process and 22q loss.
      ,
      • Eilers G.
      • Czaplinski J.T.
      • Mayeda M.
      • Bahri N.
      • Tao D.
      • Zhu M.
      • Hornick J.L.
      • Lindeman N.I.
      • Sicinska E.
      • Wagner A.J.
      • Fletcher J.A.
      • Mariño-Enríquez A.
      CDKN2A /p16 loss implicates CDK4 as a therapeutic target in imatinib-resistant dermatofibrosarcoma protuberans.
      . Among the 15 cases analyzed by SNP array in the present study, two of three high-grade DP and one of 12 low-grade DP showed deletion of sequences in 9p21, where CDKN2A resides. Thus, the significance of 9p deletions in DPFT remains to be elucidated.
      We also had the opportunity to study two separate lesions, a primary tumor and its metastasis 6 years later, with WES. In line with previously published results on other soft tissue sarcomas [
      • Hofvander J.
      • Viklund B.
      • Isaksson A.
      • Brosjö O.
      • Vult von Steyern F.
      • Rissler P.
      • Mandahl N.
      • Mertens F.
      Different patterns of clonal evolution among different sarcoma subtypes followed for up to 25 years.
      ], neither the mutational load nor the extent of the amplified regions showed much variation with time or disease progression. Thus, it may well be that tumor progression in DPFT is largely driven by epigenetic changes.

      Declaration of Competing Interest

      The authors declare that they have no conflicts of interest.

      Acknowledgments

      This study was supported by grants from the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, and Governmental Funding of Clinical Research within the National Health Service.

      Appendix. Supplementary materials

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