Supernumerary ring chromosomes in dermatofibrosarcoma protuberans may contain sequences from 8q11.2∼qter and 17q21∼qter

Supernumerary ring chromosomes in dermatofibrosarcoma protuberans may contain sequences from 8q11.2∼qter and 17q21∼qter

Cancer Genetics and Cytogenetics 129 (2001) 102–106 Supernumerary ring chromosomes in dermatofibrosarcoma protuberans may contain sequences from 8q11...

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Cancer Genetics and Cytogenetics 129 (2001) 102–106

Supernumerary ring chromosomes in dermatofibrosarcoma protuberans may contain sequences from 8q11.2qter and 17q21qter: a combined cytogenetic and comparative genomic hybridization study Jun Nishioa,*, Hiroshi Iwasakia, Yuko Ohjimia, Masako Ishiguroa, Teruto Isayamab, Masatoshi Naitob, Yasuhiko Kanekoc, Masahiro Kikuchia b

Abstract

a Department of Pathology, School of Medicine, Nanakuma, Jonan-Ku, Fukuoka University, Fukuoka, Japan Department of Orthopedics, School of Medicine, Nanakuma, Jonan-Ku, Fukuoka University, Fukuoka, Japan c Department of Laboratory Medicine, Saitama Cancer Center, Saitama, Japan Received 5 January 2001; received in revised form 8 February 2001; accepted 13 February 2001

Dermatofibrosarcoma protuberans (DFSP) presents with characteristic cytogenetic features such as reciprocal t(17;22)(q22;q13) or, more commonly, supernumerary ring chromosomes containing sequences from chromosomes 17 and 22. Here, we report the identification of a novel abnormality in a 43-year-old woman with DFSP. Cytogenetic analysis of tumor cells showed the presence of a supernumerary ring chromosome as the sole anomaly. Amplification of 8q11.2qter and 17q21qter sequences was confirmed by comparative genomic hybridization (CGH); the present case apparently lacked amplification of chromosome 22. To our knowledge, this is the first case indicating that the ring chromosome in DFSP is possibly associated with amplified material from chromosomes 8 and 17. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction Dermatofibrosarcoma protuberans (DFSP) is a lowgrade malignant tumor arising in the dermis, which produces an elevated nodular mass with frequent ulcerations on the surface of the epidermis [1]. Although it tends to recur, it rarely gives rise to distant metastases. Histologically, DFSP is composed of uniform but atypical spindle cells forming a characteristic storiform pattern. The pathogenesis of this tumor is uncertain. Based on light and electron microscopic examinations, some investigators have proposed a fibroblastic or histiocytic origin [2,3]. Others have proposed a neurogenic or perineural origin, a view supported also by immunohistochemical analysis [4,5]. To our knowledge, 29 cases of DFSP with chromosomal abnormalities have been reported in the literature [6–25], including 21 cases with ring chromosomes [6–10,12,14– 17,20–23]. Using fluorescence in situ hybridization (FISH) analysis with whole chromosome painting (WCP) and/or comparative genomic hybridization (CGH), recent studies have shown that the ring chromosomes contain segments from chromosomes 17 and 22 [10,12,14–17,20]. Here, we report the first case of a DFSP in which supernumerary ring * Corresponding author. Tel. 81-92-801-1011; fax: 81-92-861-7300.

chromosome was composed of amplified material from chromosomes 8 and 17. 2. Materials and methods 2.1. Case report A 43-year-old woman presented with a 7-year history of a painless solitary nodule on the back. Physical examination showed an otherwise healthy woman with an elevated nodular lesion in the dermis, which extended to the subcutaneous tissue. The mass was surgically removed and at the last follow-up examination 3 years after removal of the tumor, the patient was doing well and there was no evidence of recurrence or metastasis. Gross examination of the excised tumor showed a solid nodule measuring 21.51 cm, whitishgray in color, well-demarcated but nonencapsulated. 2.2. Morphology and immunohistochemistry For light microscopy, the surgically removed tumor was fixed in 10% formalin and embedded in paraffin. Dewaxed paraffin sections were stained with hematoxylin and eosin. Immunohistochemical staining was performed by using a labeled streptavidin biotin system and an alkaline phosphatase technique described earlier [26]. The primary anti-

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bodies used included broad spectrum vimentin (monoclonal, 1:50, DAKO JAPAN, Kyoto), cytokeratins (AE1/ AE3, monoclonal, 1:50; DAKO and CAM 5.2, monoclonal, kit; Becton Dickinson, San Jose, CA, USA), epithelial membrane antigen (EMA, monoclonal, 1:50; DAKO), desmin (monoclonal, 1:50; DAKO), alpha-smooth muscle actin (monoclonal, 1:50, DAKO), muscle-specific actin (HHF35, monoclonal, 1:50; Enzo Biochem, New York, NY, USA), S-100 protein (polyclonal, 1:1000; DAKO), KP-1 (monoclonal, 1:200; DAKO) and CD34 (HPCA-1, monoclonal, 1:40; Becton Dickinson). 2.3. Cytogenetic analysis For cell cultures, the fresh tumor tissue was minced with fine scissors and disaggregated for 1 h in 400 U/ml type II collagenase (Worthington Biochemical Corporation, Freehold, NJ, USA). Disaggregated cell clusters were cultured in plastic flasks (Falcon 3013, Becton Dickinson Japan, Tokyo), and filled with RPMI 1640 medium or 1:1 mixtures of DMEM and Ham’s F-12 (DF medium, Kyokuto Co., Tokyo) containing 20% fetal bovine serum (Cell Culture Laboratories, Cleveland, OH, USA) in an incubator (BL-120, Astec Co., Fukuoka, Japan) under 5% CO2 and 5% O2 at 36.5C. After 24 h and/or 8 or 10 days, adherent cells were exposed to 0.02 g/ml colcemid (Life Technologies, Gaithersburg, MD, USA) for 3 h, lifted from the flasks with trypsin-ethylene diamine tetraacetic acid (EDTA), treated in 0.075 mol/L KCl hypotonic solution for 30 min and then fixed with three changes of 3:1 methanol-acetic acid solution. Slides were made by conventional techniques using steam to assist in chromosome spreading. After 7 days in a desiccator at 45C, the chromosomes were banded by the standard trypsin-Giemsa banding technique [27,28]. Karyotypes were described on the basis of the short system of the International System for Human Cytogenetic Nomenclature [29]. 2.4. Comparative genomic hybridization (CGH) CGH was performed as described previously [30] with minor modifications. Briefly, normal and tumor-cell DNAs were labeled with Spectrum Red-dUTP (Vysis, Downers Grove, IL, USA) and fluorescein-12-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation, with the use of a commercial kit (Vysis). Subsequently, equal amounts of normal and tumor-labeled probes (200 ng) and 20 g of Cot-1 DNA (GIBCO Brl, Gaithersburg, MD, USA) were coprecipitated with the use of ethanol. The precipitated DNA was dissolved in 10 L of hybridization buffer and denatured at 75C for 8 min. Normal metaphase spreads (Vysis) were denatured for 5 min at 75C and hybridized with the DNA mixture in a moist chamber for 3 days. Slides were washed according to the protocol supplied by the manufacturer. Chromosomes were counterstained with 4,6-diamino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) and embedded in antifade solution (Vectashield, Vector Laboratories, Burlingame, CA, USA). Location of

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aberrant CGH signals was analyzed using an image analysis system (Isis, Carl Zeiss Vision, Oberkochen, Germany). The average ratios and their standard deviation were determined in control CGH studies, using differentially labeled control DNA. A change in copy number in a sample was indicated when the average fluorescence ratio from at least 10 metaphase spreads lay outside the standard deviation from the control, here found to be 1.17 for gain and 0.85 for loss. The heterochromatic regions in chromosomes 1, 9, and 16, the p-arms of the acrocentric chromosomes, and Y chromosome were excluded from the analysis because of suppression of hybridization with Cot-1 DNA in these regions. 2.5. Fluorescence in situ hybridization (FISH) The same slides presented 8 years at room temperature were analyzed for FISH (chromosome painting), by using a digoxigenin-labeled total chromosome 17 probe (COATASOME 17 total chromosome probe p5217-DG.5, Oncor, Gaithersburg, MD, USA) and a biotin-labeled total chromosome 8 probe (Chromosome Painting System, Cambio, Cambridge, UK), as described previously [21,31]. 3. Results Histologically, the tumor was composed of uniform but atypical spindle cells arranged in a characteristic storiform pattern (Fig. 1A). The tumor cells invaded the whole thickness of the dermis and extended to the subcutaneous fatty tissue. The majority of spindle cells exhibited strong positive staining for vimentin and CD34 (Fig. 1B), but were uniformly negative for other antibodies. Based on these features, the tumor was diagnosed as dermatofibrosarcoma protuberans. For cytogenetic analysis, a total of 11 G-banded metaphases were studied. Six metaphases had an apparently normal female karyotype and five cells showed karyotype 47,XX,r (Fig. 2). CGH analysis of tumor DNA showed amplification of chromosomes 8q11.2qter and 17q21 qter sequences (Figs. 3 and 4). In FISH analysis, we could not find any analyzable metaphases with ring chromosomes on these slides. The negative FISH results were probably due to the deterioration of the chromosome preparation after the long time stage at room temperature. 4. Discussion Cytogenetic studies of DFSP have usually demonstrated ring chromosomes to be either the only anomaly [7,9,15,17,21] or associated with other types of abnormalities [6,8–10,12,14,16,17,20,22,23]. A number of recent studies have also demonstrated that the ring chromosomes of DFSP contain the chromosome 22 centromere along with interstitial sequences from chromosomes 17 and 22, specifically from regions 17q23q24 and 22q11q12 [15–18]. In addition, more recent studies have demonstrated that DFSP

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Fig. 2. Representative G-banded karyotype of a DFSP showing a ring chromosome as the sole anomaly. A loss of chromosome 21 was seen in this metaphase only.

the primary cytogenetic abnormality in DFSP. Naeem et al. [17] also suggested that trisomy 8 is a non-random aberration that might be acquired before formation of the ring chromosome in some DFSPs. In the present case, while the role of chromosome 8 remains to be determined, its involvement in the tumorigenesis of DFSP cannot be ruled out. Recent molecular analyses have shown a specific gene fusion between the collagen type I1 gene (COL1A1) on

Fig. 1. (A) DFSP is composed of uniform but atypical spindle cells arranged in a characteristic storiform pattern. Original magnification  120. (B) Tumor cells exhibit strong immunopositivity for CD34 (streptavidin-biotin procedure,  180).

is characterized by t(17;22)(q22;q13) [18,19,25]. These findings, therefore, suggest that either 17q or 22q, or both, are involved in the pathogenesis of DFSP. However, our CGH analysis demonstrated that amplification of 8q11.2 qter and 17q21qter is present in DFSP with supernumerary ring chromosome. However, there is the possibility that the ring chromosome might be composed of chromosomes 17 and 22 sequences, but with the chromosome 22 sequences being too short for detection by CGH, and that the chromosome arm 8q gains might represent isochromosome 8q present in only a subpopulation of the cells and therefore undetected by the banding studies. Although this is the first description of the involvement of chromosome 8 in a DFSP ring, trisomy 8 has been described as either the only anomaly [6,14] or accompanied by ring chromosomes or various numerical and structural abnormalities [6,10,12,14,17,18,20,25]. Bridge et al. [6] and Stenman et al. [14] suggested that trisomy 8 appears to be

Fig. 3. Representative three-color metaphase demonstrating DNA sequence gains (green fluorescence) of 8q11.2qter (large arrows) and 17q21qter (small arrows), detected by CGH in DFSP. The corresponding profile is presented in Fig. 4. Three colors: DAPI for chromosome counterstain, fluorescein for tumor DNA, and SpectrumRed for reference DNA.

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Fig. 4. CGH profile of DFSP showing the gain of chromosome 8q11.2qter (large arrow) and chromosome 17q21qter (small arrow). The line in the middle (gray) is the baseline ratio (1.0); the left (red) and the right (green) lines indicate ratio values of 0.85 and 1.17. Bars on the left (red) and on the right (green) of each frame indicate losses and gains, respectively. 1/11 means that on chromosome 1, 11 aberrations were detected. The same applies to other chromosomes.

chromosome 17 and the platelet-derived growth factor -chain gene (PDGFB) on chromosome 22 in DFSP with a ring chromosome or translocation [32–34]. These findings suggest that a chimeric COL1A1-PDGFB gene may play an important role in the tumorigenesis of DFSP. However, Navarro and coworkers [23] suggested that assessments for cryptic molecular events should be performed in other variant DFSP rearrangements. Therefore, further studies are necessary to determine whether molecular rearrangements of yet unknown genes on the chromosome arm 8q other than COL1A1 or PDGFB related to tumorigenesis are present in DFSP. In conclusion, chromosomes other than chromosomes 17 and 22, such as chromosome 8, might participate in ring chromosomes in some cases of DFSP. However, further studies are needed to confirm this finding. References [1] Enzinger FM, Weiss SW. Fibrohistiocytic tumors in intermediate malignancy. In: Soft tissue tumors, 3rd Ed. FM Enzinger, SW Weiss, eds. Mosby-Year Book, Inc., St. Louis, 1995. pp. 325–49. [2] Ozzello L, Hamels J. The histiocytic nature of dermatofibrosarcoma protuberans: tissue culture and electron microscopic study. Am J Clin Pathol 1976;65:136–48. [3] Gutierrez G, Ospina JE, De Baez NE. Dermatofibrosarcoma protuberans. Int J Dermatol 1984;23:396–401. [4] Hashimoto K, Brownstein MH, Jakobiec FA. Dermatofibrosarcoma protuberans. A tumor with perineural and endoneural cell features. Arch Dermatol 1974;110:874–85.

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