Ossifying fibromyxoid tumor of soft parts

Cancer Genetics and Cytogenetics 127 (2001) 1–6

Ossifying fibromyxoid tumor of soft parts: report of a case with novel cytogenetic findings V. Sovania,1, G.V.N. Velagaletia,b,*, E. Filipowicza, Z. Gatalicaa, A.S. Kniselya,2 a

Department of Pathology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0359, USA Department of Pediatrics, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0359, USA Received 18 September 2000; accepted 18 October 2000

b

Abstract

A slowly growing tumor of the left thenar region in a 40-year-old man had the classic features of an ossifying fibromyxoid tumor of soft parts, including an incomplete shell of lamellar bone; a center composed of nodular aggregates of small spindled, oval, and stellate cells in abundant myxoid stroma; and strong expression of vimentin, S-100, and neuron-specific enolase by the tumor cells. Clonal chromosomal abnormalities included loss of a chromosome 6, extra material of unknown origin attached to the long arm of chromosome 12, and an unbalanced translocation involving the short arm of a chromosome 6 and the long arm of a chromosome 14. The karyotype was interpreted as 45,XY, der(6;14)(p10;q10),add(12)(q24.3). The chromosomal abnormalities suggest osteochondroblastic rather than neuronal or schwannian lineage. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction

2. Case report

Ossifying fibromyxoid tumor of soft parts (OFMT), first described in 1989 [1], is characterized by nodules or lobules of round to fusiform cells in a myxoid matrix, encased in a discontinuous, generally hypocellular shell of metaplastic bone. It is uncommon; one recent review identified fewer than 110 reported cases [2]. The histologic origin of the tumor is not clearly known. Various features suggest schwannian, neuronal, or chondroid origin. Recurring, non-random chromosome abnormalities are often hallmarks of tumor cells. Such specific clonal abnormalities can become important diagnostic as well as prognostic factors. Correlation of specific chromosome abnormalities with specific tumors may also help in delineating the histogenesis of those tumors. To date, chromosome abnormalities have not been reported in OFMT. We present a case of typical OFMT with hitherto undescribed clonal cytogenetic abnormalities.

A 40-year-old Caucasian male sought medical attention for an anterior right shoulder soft tissue mass and a left palm soft tissue mass. The right shoulder mass, 9  7 in greatest dimensions, was clinically assessed as a lipoma; the products of liposuction were not submitted for pathologic study. The left palm mass had been present for five years, with slower growth in the three most recent years. No history of an inciting event was elicited. The mass was 3 cm in greatest dimension and protruded from the center of the palm. Sonography did not identify cystic changes or a vascular component; the mass was not seen to involve structures deep to the subcutis. The mass was excised. In the eight months since surgery, the mass has not recurred locally, and secondary lesions have not been identified.

* Corresponding author. Tel.: 409-772-3466; fax: 409-772-9595. E-mail address: [email protected] (G.V.N. Velagaleti). 1 Present address: Department of Pathology, Louisiana State University Medical Center, Shreveport, LA 71130. 2 Present address: Institute for Liver Studies, King’s College Hospital, London SE5 9RS, UK.

3. Materials and methods The excised tumor measures 3  2.5  2.5 cm and appeared encapsulated and lobulated (Fig. 1a). Incision encountered resistance at the periphery and a rim of bone was identified. The cut surface was otherwise fairly homogenous, tan-white, and glistening. Tissue was submitted for ultrastructural and cytogenetic studies as well as for routine processing, including decalcification. Routinely processed formalin-fixed, paraffin-embedded tissue samples were used for light-microscopic and immu-

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Fig. 1. (a) An exterior view of the encapsulated-appearing, bosselated mass. (b) Mature lamellar bone lies immediately within the collagenous, fibrous capsule. (c) The tumor cells are disposed in lobules defined by fibrovascular septa. (d) Small, rounded or ovoid nuclei of tumor cells are seen against a palely amphophilic myxoid background.

nohistochemical studies. Immunohistochemical studies were performed using automated staining procedures (DAKO, Carpinteria, CA) with diaminobenzidine as a chromogen [3]. Table I lists the sources and dilutions of primary

antibodies used in the study. Fresh tissue submitted for ultrastructural study was fixed in 2% glutaraldehyde/2% paraformaldehyde, postfixed in 1% osmium tetroxide, and block-stained with uranyl acetate. Thin sections of resin-

Fig. 2. Immunohistochemical studies (diaminobenzidine chromogen/hematoxylin counterstain [DAB/H]). (a) Tumor cell cytoplasm marks strongly for vimentin. (b) Tumor nuclei mark strongly on immunostaining for S-100 protein. (c) Tumor cell cytoplasm marks strongly for neuron-specific enolase. (d) Tumor cell cytoplasm marks focally for matrix metalloproteinase 13.

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Table 1 Immunohistochemical findings Antigen

Antibody (clone)

Source/Dilution

Reactivity in OFMT

Vimentin NSE Synaptophysin S-100 MMP-13 Desmin Cytokeratin AE1/3 Cytokeratin CAM5.2 CD31 CD34 Smooth muscle actin Myosin CD57 GFAP HMB-45 Chromogranin EMA Myoglobin MIB-1

Vim3B4 BBS/NC/VI-H14 Polyclonal Polyclonal 181-15A12 D33 AE1/3 Monoclonal JC/70A QBEND/10 1A4 SMM S-1 HNK-1 Polyclonal HMB-45 DAK-A3 E 29 Polyclonal MIB-1

Dako/1:400 Dako/1:100 Novocastraa/1:80 Dako/1:6000 Chemiconb/1:500 Dako/1:200 Dako/1:100 Becton-Dickinsonc/1:8 Dako/1:80 Signetd/1:40 Dako/1:400 Dako/1:400 Becton-Dickinson/1:20 Dako/1:1000 Dako/1:100 Dako/1:100 Dako/1:1600 Dako/1:1600 Immunoteche/1:100

Strong, uniform Strong, near-uniform Moderate, focal Strong, near-uniform Moderate, focal Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent 5%–10% of cells

a

Novocastra, c/o Vector Laboratories, Burlingame, CA. Chemicon, Temecula, CA. c Becton-Dickinson, Franklin Lakes, NJ. d Signet, Dedham, MA. e Immunotech, c/o Beckman Coulter, Fullerton, CA. b

embedded material were stained with lead citrate and examined by transmission electron microscopy. Cytogenetic studies were carried out using standard procedures [4]. Tissue samples were rinsed several times in balanced salt solution and were disaggregated with 1 m/mL collagenase solution for 15 minutes at 37C. At the end of incubation, 10 mL of fresh Amniogrow medium (Rainbow Scientific Inc., Windsor, CT) was added to the sample, which was then triturated to yield a single-cell suspension. After centrifugation at 70  g for 10 minutes, the cell pellet was resuspended in 1 mL of fresh Amniogrow medium. The cell suspension was thoroughly mixed and two primary cultures were inoculated in T-25 culture flasks. The cultures were fed after the fifth day. Harvesting was done on day 8 using routine procedures including hypotonic treatment with 0.075M KCl solution and fixation in 3:1 methanol : glacial acetic acid. The harvested cell suspension was dropped onto pre-cleaned, chilled slides. The slides were artificially aged by baking at 80C for 30 minutes. The chromosomes were G-banded using pancreatin [5] (0.05%) for 3 minutes followed by Giemsa staining for 1.5 minutes. 4. Results 4.1. Light-microscopic and immunohistochemical studies Light microscopy revealed benign lamellar bone and hyaline cartilage at the periphery of the mass (Fig. 1b), partially cuffing moderately cellular, lobulated portions of the tumor (Fig. 1c), which were composed of small ovoid or spindled cells, arranged in cords, trabeculae, and fascicles within a myxoid background (Fig. 1d). Cell chromatin was

finely stippled, without nucleoli. Mitotic figures, significant variation in nuclear size or staining intensity, and necrosis were not found. Occasional interspersed mast cells were noted. The tumor cells strongly expressed vimentin (Fig. 2a), S-100 antigen (Fig. 2b), and neuron-specific enolase (NSE) (Fig. 2c). They marked faintly for synaptophysin and matrix metalloproteinase-13 (MMP-13, also known as collagenase 3, and considered a marker of cartilaginous differentiation [6]) (Fig. 2d). Cytokeratins AE1/3 and CAM 5.2, epithelial membrane antigen (EMA), chromogranin, glial fibrillary acidic protein (GFAP), HMB-45, actin, desmin, myoglobin, and CD antigens 31, 34, and 57 were not found. Five to 10% of cells exhibited proliferative activity as assessed on MIB-1 (Ki67) immunostaining. Results are summarized in Table 1. 4.2. Ultrastructural studies Collagen bundles interspersed with amorphous, relatively electron-lucent material separated cells with moderate quantities of cytoplasm and smooth-contoured nuclei of uniform cross-sectional area (Fig. 3a). Most nuclei contained a single small chromocenter. Cell margins were irregular, with frequent pseudopodia (Fig. 3b), some of which abutted other cells; tight junctions, components of a desmosomal apparatus, and basal lamina were not identified. In the cytoplasm, unremarkable mitochondria, peroxisomes, and primary lysosomes accompanied dilated cisterns of rough endoplasmic reticulum whose contents resembled the amorphous material present between cells. Loosely aggregated intermediate filaments cuffed the nuclei (Fig. 3c). Dense-core granules were not seen.

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Fig. 3. Ultra structural studies (osmium tetroxide/uranyl acetate/lead citrate). (a) Palely staining intercellular matrix containing occasional collagen fibrils surrounds cells that exhibit few markers of differentiation and have rounded, smooth-contoured nuclei (2,950  original magnification). (b) Cytoplasmic cisterns contain material similar to intercellular matrix; cell borders are irregular, bearing pseudopod-like processes (15,500  original magnification). (c) Intermediate filaments and a lipid droplet are present; rough endoplasmic reticulum can be seen (28,500  original magnification).

4.3. Cytogenetic studies The mitotic index was extremely poor. Eleven Giemsabanded metaphases in all were analyzed. Almost all the metaphases (10/11) showed a model count of 45 chromosomes, with consistent abnormalities that included loss of chromosome 6, extra material of unknown origin attached to the long arm of a chromosome 12, and an unbalanced translocation involving the short arm of a chromosome 6 and the long arm of a chromosome 14 (Fig. 4). The remaining cell showed a normal 46,XY karyotype. The karyotype was interpreted as 45,XY,der(14)t(6;14)(p10;q10),add(12)(q24.3)[10]/ 46,XY[1][7]. 5. Discussion The clinical, light-microscopic, immunohistochemical, and electron-microscopic findings in this case are entirely typical of usual OFMT, which behave in benign fashion [2]. A few cases of OFMT have been interpreted as atypical or malignant owing to increased cellularity, frequent mitotic activity (2 mitotic figures/10 400 microscopic fields), and randomly interspersed osteoid not in association with

the bony shell [8]. Review of reported cases has identified one patient with OFMT who developed a tumor of the contralateral lower extremity that might have been either a metastatic deposit or a second primary OFMT [2] and one recurrent tumor, with metastasis, that owing to the metastasis was re-classified as well-differentiated osteosarcoma [8]. Overtly sarcomatous regions [9], invasion from neck into mediastinum [6], and repeated recurrence with cellular atypism, interpreted as malignancy [10], have been described. Our review of reported cases suggests that 3% to 5% of OFMT may be malignant or behave aggressively. Derivation and differentiation in OFMT are not understood. The tumor cells have been considered morphologically similar to atypical osteoblasts, and have been shown to express alkaline phosphatase activity strongly [9]. Given the results of immunohistochemical studies [2], however, it can be speculated that OFMT represents a variant of peripheral nerve sheath tumor, with focal cartilaginous differentiation. The possibility of multilineage differentiation of an immature mesenchymal stem cell remains open [2]. A novel finding, that of clonal cytogenetic abnormalities with a karyotype of 45,XY,der(6;14)(p10;q10),add(12) (q24.3), was detected in this patient’s tumor. This is the first report, to our knowledge, of clonal abnormalities in OFMT. These abnormalities overlap in part with those described in malignant peripheral nerve sheath tumor (MPNST), in which abnormalities of chromosomes 6 and 12, especially deletions, have been found [11,12]. The origin of extra material attached to 12q24.3 could not be identified in our patient’s tumor, but the abnormality may represent an unbalanced translocation with loss of the terminal long arm of chromosome 12. In most MPNSTs, the deletion on chromosome 12 has involved a longer region with deletions extending through bands q12 to q24. In light of the results of immunohistochemical studies, and given the involvement of chromosomes 6 and 12, the possibility of neural origin can be considered. However, the most common karyotypic abnormalities in MPNST include anomalies of chromosome 1p and gain of chromosome 7, with recurrent involvement of chromosomes 17 and 22 [11,12]. None of these typical MPNST-associated abnormalities was present in the tumor that we describe. Most reported schwanommas exhibit chromosome 22 deletions, sex-chromosome loss, and gain of a chromosome 7 [12]. The abnormalities in our patient’s tumor were quite different. We infer that, in our patient, OFMT was not a phenotypic variant of either MPNST or schwannoma. The possibility of osteoblastic origin seems more likely in the present tumor, based on the karyotypic findings. In primary malignant neoplasms of the bone such as osteosarcoma and chondrosarcoma, similar chromosome abnormalities have been reported [13,14], with deletion of the long arm of chromosome 6 seen in chondrosarcoma and rearrangements involving the distal long arm of chromosome 12 and anomalies of chromosome 14 seen in osteosarcoma. These observations are consonant with the karyotypic ab-

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Fig. 4. Karyotype from the cultured tumor biopsy. Arrows point to the clonal abnormalities.

normalities seen in our patient’s tumor, and permit the inference of possible osteochondroblastic origin. Our hypothesis is further strengthened by the fact that clonal abnormalities such as rearrangements of chromosome 6 and 12 are also seen in patients with chondromyxoid fibroma [15–18], a rare benign cartilaginous tumor with varying degrees of myxoid and fibrous tissue elements. Monosomy or loss of the long arm of chromosome 6 has been described in chondrosarcoma, while the abnormalities of chromosomes 12 and 14 are reported in other malignant osteogenic tumors. However, the specific type of abnormality or the breakpoint on chromosome 6 varied considerably in these tumors. The heterogeneity of these recurring anomalies may be due in part to the underlying malignant processes within these histologically different tumors. Because of the limited number of cases with such karyotypic abnormalities, it is difficult to understand the significance of the chromosome 6 abnormalities in these tumors; however, the presence of these recurring chromosome 6 abnormalities may serve as a cytogenetic marker to indicate the primitive embryological origin of these tumors. As this is the first report of clonal chromosomal abnormalities in OFMT, data from other cases will be required to assess whether consistent karyotypic lesions characterize the tumor. The prognostic implications of this karyotype are unclear as well; while most tumors with multiple chromosomal abnormalities are malignant, with adverse prognoses,

the tumor in our patient was not atypical for OFMT, nor has it to date evinced aggressive behavior.

Acknowledgments The case was presented for discussion in the Kansas Histopathology Club in March 2000, and the consensus of participants (panel of pathologists from 10 medical schools) was that the lesion indeed represents an OFMT.

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