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Teaching cases
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Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm夽
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Alexandra von Baer a , Alexander Ehrhardt b , Daniel Baumhoer c , Regine Mayer-Steinacker d , Markus Schultheiss a , Thair Abdul-Nou e , Thomas Mentzel f , Falko Fend g , Peter Möller h , Gernot Jundt c , Thomas F.E. Barth h,∗ a
Department of Trauma and Orthopaedic Surgery, University of Ulm, Germany Department of Radiology, University of Ulm, Germany Bone Tumor Reference Center at the Institute of Pathology, University Hospital Basel, Switzerland d Department of Internal Medicine III, Germany e Institute of Pathology, Hospital of Memmingen, Germany f Institute of Dermatopathology, Friedrichshafen, Germany g Institute of Pathology, University of Tübingen, Germany h Institute of Pathology, University of Ulm, Germany b c
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Article history: Received 27 February 2014 Received in revised form 5 May 2014 Accepted 15 May 2014
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Keywords: Extraskeletal osteosarcoma Differential diagnosis Histology Molecular pathology
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Introduction
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Extraskeletal osteosarcoma is a rare neoplasia within the broad differential diagnostic spectrum of calcifying intramuscular lesions. We present a case of a slowly increasing mass within the left vastus lateralis muscle. At first presentation the patient showed a partially calcified well defined mass with a diameter of 5 cm and with no direct contact to the femur. A biopsy from the periphery revealed an ossifying lesion compatible with myositis ossificans. The patient returned 18 months later with the lesion having increased to a diameter of 25 cm. The resection specimen revealed a well delimitated tumor with a central core of partially necrotic neoplastic bone. Besides, histology showed high mitotic areas with pleomorphic spindle cells and regions with cartilaginous differentiation. Immunohistochemistry demonstrated: vimentin+, CD34−, desmin−, actin−, EMA− and pancytokeratin− with focal S100 protein positivity and a Ki-67 index of 20%. Comparative genomic hybridization (CGH) revealed a gain of chromosomal material on 12q; FISH analyses for the CDK4 and MDM2 region showed high level amplifications. Consequently, a high-grade dedifferentiated extraskeletal osteosarcoma was diagnosed. In conclusion, analysis of the MDM2 and CDK4 status is a powerful discriminating diagnostic tool to distinguish dedifferentiated extraskeletal osteosarcoma from other benign/malignant ossifying lesions in the skeletal muscle. © 2014 Published by Elsevier GmbH.
Differential diagnosis of intramuscular fibrous and ossifying lesions is challenging for the clinician as well as for the pathologist. These mesenchymal lesions comprise a variety of benign
夽 This case was presented at the 81st meeting of the Arbeitsgemeinschaft Knochentumoren e.V./Working Group on Bone Tumors in Munich, October 19, 2013 receiving the AGKT-award as best presentation. ∗ Corresponding author at: Institute of Pathology, University of Ulm, AlbertEinstein Allee 23, D-89070 Ulm, Germany. Tel.: +49 0731 50056340. E-mail address: [email protected] (T.F.E. Barth).
conditions such as metaplastic ossifications as well as myositis ossificans. Examples for malignant lesions include extraskeletal osteosarcoma (low-/high-grade) as well as other soft tissue sarcomas with metaplastic/neoplastic ossification (e.g. synovial sarcoma) [5,12]. The histological differential diagnosis can even be more difficult on small biopsies since these lesions may have heterogeneous patterns of differentiation. In the following we present the clinical history, the imaging results, the histological and immunohistological profile, and the molecular pathology of a slowly growing mass with a wide range of histological patterns that ultimately was diagnosed as a high-grade dedifferentiated extraskeletal osteosarcoma.
http://dx.doi.org/10.1016/j.prp.2014.05.010 0344-0338/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Clinical history A 46 year old male presented with a swelling of the left lateral thigh causing him modest pain comparable to muscle soreness after physical exercise. No trauma was reported. At physical examination there was neither inducible palpable pain nor signs of a disturbed peripheral blood flow or symptoms of an altered sensor-motoric function. Blood work was normal. Imaging by Rx and magnetic resonance imaging (MRI) revealed an intramuscular mass within the left vastus lateralis muscle with a diameter of 5 cm; there was no contact to the femoral bone. A biopsy was obtained from the periphery of the tumor. After having consulted a bone tumor reference center the lesion was classified as most probably compatible with myositis ossificans and a “wait and see” strategy was adopted by the local Oncological advisory Board for sarcomas. 18 months later the patient showed up complaining about the continuously increasing mass in the left vastus lateralis muscle. Imaging by Rx and MRI revealed a partially ossified intramuscular lesion without contact to the femur or the skin, now measuring 25 cm × 13 cm × 8 cm. The case was presented again at the Oncological advisory Board for Sarcomas and another biopsy was taken. Since the histological report of the biopsy was suspicious for malignancy a complete resection of the tumor was advised by this interdisciplinary board. Materials and methods Biopsies and surgical specimen were fixed in 4% paraformaldehyd in phosphate-buffered saline and embedded in paraffin. Calcified tissue was treated with EDTA (Ethylendiamintetraacetate; Triplex Merk, Mannheim, Germany) before embedding. Sections of 2–3 m were stained with heamtoxylin-eosin and with antibodies for vimentin (VIM3B4, Dako, Glostrup, Denmark; dilution 1:300), CD34 (QBEnd10, dilution 1:100, Dako), desmin (DE-R-11, Menarini, Milan, Italy, dilution 1:50), actin (HHF35, Dako, dilution 1:500), EMA (E29, Dako, dilution 1:500), pancytokeratin (AE1 + AE3; Dako, dilution 1:100), S100 protein (Z0311, Dako, dilution 1:1000) and Ki-67 (Mib-1, Dako, dilution 1:200). CGH and FISH was performed using standard protocols [6]. For FISH analyses commercially available probes were used: a dual color break-apart probe for 18q11.2 (SYT 18, Abbott, IL, USA) and dual color probe sets for CDK4 (12q13; Kreatech, Amsterdam, The Netherlands) and MDM2 (12q15; Kreatech). Results At first presentation a biopsy of 1.8 cm × 1.7 cm × 0.4 cm was obtained. Histology revealed a matrix rich lesion consisting of a spindle cell proliferation with intermingled ossified trabeculae. There was neither clear osteoblastic rimming nor higher polymorphism and no atypical mitoses or necrosis (Fig. 2C1). A clear zonal phenomenon in terms of immature cellular areas in the center and more mature, ossifying areas with osteoblastic rimming in the periphery was not present in the biopsy. Morphology was interpreted to be compatible with myositis ossificans. At second presentation imaging revealed a partially calcified intramuscular mass still without contact to the femoral bone (Fig. 1A, A1–A3). Two core biopsies measuring 3 cm in length showed a very similar morphology to the former biopsy with a spindle cell proliferation and intermingled bony trabeculae. However, polymorphy of nuclei was focally pronounced. No necrosis was detected. By immunohistochemistry the lesion was positive for vimentin and focally for S100 protein. CD34, desmin, actin, EMA, and pancytokeratin were
negative. To definitely rule out a biphasic, partially ossifying synovial sarcoma, FISH analysis was performed with a commercial break-apart probe for the SYT region on chromosomal band 18q11.2; no rearrangement was detected, practically excluding synovial sarcoma. Therefore the diagnosis was adjusted to an intramuscular lesion “highly suspicious for extraskeletal osteosarcoma”. Since there were no signs of metastasis the patient was staged as limited disease and surgery was advised by the Oncological advisory Board for Sarcomas. The resection specimen weighed 4.2 kg and measured 30 cm × 20 cm × 15 cm. On cross section a 25 cm × 13 cm × 8 cm solid yellowish tumor emerged that was well delimitated from the surrounding muscle and fat tissue (Fig. 1B, B12). The central part was ossified, measured 9 cm in diameter and was partly necrotic. 30 representative tissue blocks were performed for histologic analysis. Histology revealed three patterns of differentiation: (1) A zone of irregular trabeculae of bone embedded in a spindle cell stroma with only minimal atypia reminiscent of a low-grade parosteal osteosarcoma adjacent to high-grade pleomorphic cells with a lace-like bone formation (Fig. 2C1/2). (2) An area of cartilaginous differentiation with polymorphic tumor cells (Fig. 2C3); (3) A highly proliferating spindle cell region with accompanying extracellular matrix deposition, few pleomorphic cells and with up to 14 (partially atypical) mitoses/high power field (Fig. 2C4). The central part of the lesion was ossified, while the periphery revealed predominantly atypical spindle cell proliferations, thus resembling a reverse zoning phenomena with central deposition of neoplastic bone and atypical spindle cell proliferation in the periphery characteristic for extraskeletal osteosarcoma. In none of the sections we detected a lipogenic differentiation in terms of lipoblast or fat tissue with atypical stromal cells. DNA was extracted from the spindle cell and osteoblastic areas and comparative genomic hybridization (CGH) was performed [6]. In both areas the same gain of chromosomal material on 12q was detected including a high-level gain on chromosomal band 12q13-12q15 (Fig. 2D1–D3). In a separate step we analysed the low-grade compartment by CGH. CGH analysis of the low- and high-grade compartment revealed as the sole overlapping aberration a gain of chromosomal material on chromosome 12 while the high-grade sarcoma showed a high level amplification on 12q (data not shown). In a candidate gene approach we performed double color fluorescence in situ hybridization (FISH) using commercial probe sets for the CDK4 and MDM2 region both mapping to chromosomal bands 12q13 and 12q15; as a reference a probe for the centromere of chromosome 12 was used. The spindle cell compartment as well as the osteogenic area were analysed by FISH. In both respective areas we detected a high-level amplification of CDK4 and MDM2 with ratio values of 6.4 and up to 7.1 (in analogy to the Her2 FISH signal estimation) with up to twenty signals for CDK4 and MDM2 (Fig. 2E1 and E2). Immunohistochemistry for CDK4 and MDM2 revealed a strong nuclear accumulation of CDK4 and a less strong nuclear staining for MDM2 in all three compartments of the lesion (Fig. 2F1, F2). The Ki-67 index was up to 20% (Fig. 2F3). We then performed FISH and immunohistochemistry on the initial biopsy taken 18 month ago and detected close to identical FISH based amplification ratios for CDK4 and MDM2. Immunohistochemistry was positive for CDK4, however, MDM2 was negative (data not shown). Ultimately, the tumor was diagnosed as a dedifferentiated osteosarcoma grade 3 with a sum score of 6 (3-3-0) according to the FNCLCC system [7]. The final TNM classification was: pT2b, V0, L0, Nx, Mx, R0 with a minimal resection margin of 5 mm. Accordingly, the patient was enrolled in an adjuvant chemotherapy regimen following the guidelines of the EURO-B.O.S.S. therapy protocol [3]. He finished chemotherapy in December 2013 with no evidence of disease during followup.
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Fig. 1. Comparison of imaging and macroscopic aspects of the resection specimen. (A1) Rx shows a calcified extraskeletal mass of the lateral left thigh. Magnetic resonance imaging (A2 and A3) reveals a heterogeneous intramuscular lesion without contact to the bone and a central calcified core. Figure B, B1 and B2 show the corresponding macroscopic aspects of the resection specimen well delineated from the adjacent subcutaneous fat tissue (B1) and muscle (B2). B1 and B2 correspond to the spindle cell compartment while the central part (B3) presents the ossified central core of the lesion.
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Discussion Intramuscular calcifying masses comprise a broad spectrum of differential diagnoses, including various benign and malignant low and high-grade lesions [4,9]. The definitive diagnosis is crucial for prognostic prediction and an accurate choice of the highly varying therapeutic options ranging from “watch and wait” to simple excision to wide resection and high-dose neoadjuvant chemotherapy [3]. Low-grade extraskeletal osteosarcoma is rare with only few case listed in the literature (see Table 1). The patient presented here had a progressively growing intramuscular mass well delimitated from the surrounding tissue. Only the resection specimen allowed sampling of the whole lesion and finally revealed the full spectrum of its complex differentiation. This pattern was in large
aspects similar to a low-grade paraosteal osteosarcoma of bone. Since the lesion was located in the soft tissues outside and without any direct contact to the bone, an extraskeletal osteosarcoma was diagnosed. Conventional extraskeletal osteosarcoma is a rare highgrade entity with an aggressive course of disease [7]. However, within the spectrum of those tumors, a low-grade variant with features of classical paraosteal osteosarcoma has been described [8,17; Table 1]. This variant is characterized by a high-level amplification on 12q and is associated with a favorable prognosis [11]. The case shown here showed at least partly the histomorphological features of a low-grade parosteal osteosarcoma with a high histological heterogeneity and presented with a 12q amplification identical to those found in paraosteal osteosarcoma [5,17,18]. However, the presented lesion also disclosed patterns of a high-grade sarcoma
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Fig. 2. Histology, immunohistochemistry, and comparative genomic hybridization (CGH), and FISH analysis. Histology of the initial biopsy reveals a spindle cell proliferation with intermingled ossified trabeculae and structures like woven bone (C1). The central part of the resection specimen shows areas with osteoblastic differentiation with lace-like neoplastic bone (C2), a cartilaginous differentiation (C3), and a high-grade spindle cell lesion with atypical mitoses in the periphery (C4, insert); (C1, bar = 100 m Q3 C1, C2, C4, insert; bar = 40 m; C3 = 50 m). CGH analysis shows examples of DNA hybridization of control (D1) and tumor (high-grade spindle cell compartment) (D2) DNA on chromosome 12 detected via red and green fluorescence with an enhanced green signal (tumor DNA) on the long arm of chromosome 12 as compared to the red signal (control DNA). D3 shows the corresponding ratio profile resulting in a shift of the profile to the left of the central line indicating a DNA amplification. Figure E1 and E2 shows corresponding FISH images with multiple signals for the genomic regions of CDK4 (E1) and MDM2 (E2) detected in red and single green signals for the centromere region of chromosome 12, respectively (red arrows mark signals of CDK4 and MDM2, respectively; green arrows mark centromere 12). Immunohistochemistry for CDK4 (F1) and MDM2 (F2) shows strong nuclear signals for CDK4 and some positive nuclei for MDM2 in the sarcoma. The proliferation is enhanced to approximately 20% in a Ki-67 staining (F3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Table 1 Summary of reported cases with well-differentiated extraskeletal osteosarcomas. Case
Year
First author
Age
Sex
Site
Treatment
Follow-up and outcome
1
1953a
Umiker [16]
44
M
Rt. thigh
5
Resection
Lt. knee
24
Resection
F
Lt. axilla
15
Resection
63
F
Lt. tigh
7
Resection
Okada [13]
35
F
Lt. leg
11
Resection
2005
Abramovici [1]
40
F
Lt. upper back
9×6×5
Resection
7
2005a
Abramovici [1]
32
M
8
2010
Arai [2]
62
F
Bil. thighs, buttocks and lt. paravertebral area (max. lesion) Retroperitoneum
Died with widespread metastasis after 5 years Alive 5 years after surgery Alive 2 years after surgery Alive 12 months after surgery Alive 4 years after surgery Alive 2 months after surgery lost in follow-up Alive with widespread metastasis 4 years after initial surgery
2
1989
Present [14]
57
F
3
1991
Yi [17]
74
4
2003
Fukunaga [8]
5
2003
6
9
2010
Sabatier [15]
30
M
Chest wall
10
2014
Japuria [10]
38
M
Retroperitoneum
Present case
2013
von Baer
46
M
Lt. lateral tigh
a
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231
Size (cm)
16 × 7 × 8
14 × 11 × 8
30
25 × 13 × 8
Resection and chemotherapy
Resection
Chemotherapy Resection and chemotherapy Resection and chemotherapy
Died with widespread metastasis after 33 months of dedifferentiated spindle cell sarcoma Died 5 years after first symptoms Disease free after 3 years of follow-up Alive one year after surgery/chemotherapy
Recurred as a high-grade malignancy.
with necrosis and pleomorphic nuclei that finally led to the diagnosis of a dedifferentiated extraskeletal high-grade osteosarcoma most probably arising from an extraskeletal low-grade osteosarcoma with features close to identical of a parosteal osteosarcoma. The assumption of tumor progression to a high-grade sarcoma is strengthened by the observation that the initial lesion and histology of the resection specimen already revealed an amplification of MDM2 and CDK4 on the genomic level; a full gene dosage effect, however, with nuclear protein accumulation of MDM2 and CDK4 protein was detected only in the dedifferentiated sarcoma, while the initial biopsy was convincingly positive for CDK4 protein only. These findings may be suggestive of a progressive and probable metachronous upregulation of MDM2 and CDK4 protein expression during the process of dedifferentiation. This assumption is further strengthened by the fact, that CGH analysis of the low- and high-grade compartment revealed as the sole aberration a gain on chromosome 12 while the high-grade sarcoma showed a high level amplification on 12q. Since no further clonal genetic aberrations were detected by CGH these findings may support the concept of a clonal evolution. Nevertheless, the immunohistological detection of CDK4 in the initial biopsy together with amplification of MDM2 and CDK4 as detected by FISH already pointed at a malignant lesion. A further possible differential diagnosis of the presented case is the recently described well-differentiated liposarcoma with lowgrade osteosarcomatous component. This tumor is characterized by a biphasic appearance with lipogenic and osteogenic areas and is CDK4 and MDM2 positive by immunohistochemistry [19]. In the presented case, however, lipogenic differentiation was excluded by extensive histological analysis, thus ruling out this rare tumor. Summing up, integration of FISH analysis of MDM2 and CDK4 combined with immunohistochemistry for these two proteins is a highly discriminating diagnostic tool in the diagnostic algorithm of
extraskeletal ossifying lesions and clearly allows the differentiation from benign intramuscular mimics.
Acknowledgements We thank Michaela Buck and Iwona Nerbas for excellent technical help.
References [1] L.C. Abramovici, P. Hytiroglou, R.M. Klein, et al., Well differentiated extraskeletal osteosarcoma: report of 2 cases, 1 with dedifferentiation, Hum. Pathol. 36 (2005) 439–443. [2] H. Arai, Y. Rino, T. Nishii, N. Yukawa, N. Wada, H. Oshiro, T. Ishida, N. Nakaigawa, M. Masuda, Well-differentiated extraskeletal osteosarcoma arising from the retroperitoneum that recurred as anaplastic spindle cell sarcoma, Case Rep. Med. (2010) 1–5. [3] D. Andreou, S.S. Bielack, D. Carrle, M. Kevric, R. Kotz, W. Winkelmann, G. Jundt, et al., The influence of tumor- and treatment-related factors on the development of local recurrence in osteosarcoma after adequate surgery. An analysis of 1355 patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols, Ann. Oncol. 22 (May (5)) (2011) 1228–1235. [4] J.M. Coindre, D. Ranchère, F. Collin, How to classify a soft tissue sarcoma in 2006, Ann. Pathol. November (SpecNo 1) (2006), 1S98–1S109. [5] F. Dujardin, M.B. Binh, C. Bouvier, A. Gomez-Brouchet, F. Larousserie, A. Muret, et al., MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary fibro-osseous lesions of the bone, Mod. Pathol. 24 (5) (2011) 624–637. [6] J. Ellegast, T.F. Barth, M. Schulte, S.S. Bielack, M. Schmid, R. Mayer-Steinacker, Metastasis of osteosarcoma after 16 years, J. Clin. Oncol. 29 (January (3)) (2011) e62–e66, http://dx.doi.org/10.1200/JCO.2010. [7] C.D. Fletcher, D.M. Bridge, C.W. Pancas, P.C. Hogendoorn, F. Mertens, WHO Classification of Tumours of Soft Tissue and Bone, 4th ed., 2013, pp. 161–162. [8] M. Fukunaga, Extraskeletal osteosarcoma histologically mimicking parosteal osteosarcoma, Pathol. Int. 52 (July (7)) (2002) 492–496. [9] R.J. Grimer, S.R. Carter, D. Spooner, R.S. Sneath, Diagnosing musculoskeletal tumours, Sarcoma 5 (2) (2001) 89–94.
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[10] J. Jaipuria, A. Kumar, A.S. Rao, S.P. Kataria, Large retroperitoneal low-grade extraskeletal osteosarcoma, BMJ Case Rep. (2014), pii:bcr-2014-203745. 267 [11] A.I. Kyriazoglou, J. Vieira, E. Dimitriadis, N. Arnogiannaki, M.R. Teixeira, N. Pan268 dis, 12q amplification defines a subtype of extraskeletal osteosarcoma with 269 good prognosis that is the soft tissue homologue of parosteal osteosarcoma, 270 Cancer Genet. 205 (June (6)) (2012) 332–336. 271 [12] H.J. Mankin, T.A. Lange, S.S. Spanier, The hazards of biopsy in patients with 272 malignant primary bone and soft-tissue tumors, J. Bone Joint Surg. Am. 64 273 (October (8)) (1982) 1121–1127. 274 [13] K. Okada, H. Ito, N. Miyakoshi, M. Sageshima, J. Nishida, E. Itoi, A low-grade 275 exdtraskelatal osteosarcoma, Skel. Radiol. 32 (2003) 165–169. 276 [14] D. Present, M. Bertoni, M. Laus, P. Bacchini, M. Bocanera, Case report 565, Skel. 277 Radiol. 18 (1989) 471–474. 278 [15] R. Sabatier, C. Bouvier, G. de Pinieux, A. Sarran, I. Brenot-Rossi, F. PedeuQ2 tour, B. Chetaille, P. Viens, P.J. Weiller, F. Bertucci, Low-grade extraskeletal 265 266
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osteosarcoma of the chest wall: case report and review of the literature, BMC Cancer 10 (2010) 645–650. W. Umiker, H.L. Jaffe, Ossifying fibrosarcoma (extraskletal osteogenic sarcoma) of the muscle, Ann. Surg. 138 (1953) 789–795. E.S. Yi, B.M. Shmookler, M.M. Malawer, D.E. Sweet, Well-differentiated extraskeletal osteosarcoma. A soft-tissue homologue of parosteal osteosarcoma, Arch. Pathol. Lab. Med. 115 (September (9)) (1991) 906–909. A. Yoshida, T. Ushiku, T. Motoi, T. Shibata, Y. Beppu, M. Fukayama, H. Tsuda, Immunohistochemical analysis of MDM2 and CDK4 distinguishes low-grade osteosarcoma from benign mimics, Mod. Pathol. 23 (September (9)) (2010) 1279–1288. A. Yoshida, T. Ushiku, T. Motoi, T. Shibata, M. Fukiyama, H. Tsuda, Well differentiated liposarcoma with low-grade osteosarcomatous component: an underrecognized variant, Am. J. Surg. Pathol. 34 (2010) 1361–1366.
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Teaching cases
1
Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm夽
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Alexandra von Baer a , Alexander Ehrhardt b , Daniel Baumhoer c , Regine Mayer-Steinacker d , Markus Schultheiss a , Thair Abdul-Nou e , Thomas Mentzel f , Falko Fend g , Peter Möller h , Gernot Jundt c , Thomas F.E. Barth h,∗ a
Department of Trauma and Orthopaedic Surgery, University of Ulm, Germany Department of Radiology, University of Ulm, Germany Bone Tumor Reference Center at the Institute of Pathology, University Hospital Basel, Switzerland d Department of Internal Medicine III, Germany e Institute of Pathology, Hospital of Memmingen, Germany f Institute of Dermatopathology, Friedrichshafen, Germany g Institute of Pathology, University of Tübingen, Germany h Institute of Pathology, University of Ulm, Germany b c
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Article history: Received 27 February 2014 Received in revised form 5 May 2014 Accepted 15 May 2014
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Keywords: Extraskeletal osteosarcoma Differential diagnosis Histology Molecular pathology
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Introduction
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Extraskeletal osteosarcoma is a rare neoplasia within the broad differential diagnostic spectrum of calcifying intramuscular lesions. We present a case of a slowly increasing mass within the left vastus lateralis muscle. At first presentation the patient showed a partially calcified well defined mass with a diameter of 5 cm and with no direct contact to the femur. A biopsy from the periphery revealed an ossifying lesion compatible with myositis ossificans. The patient returned 18 months later with the lesion having increased to a diameter of 25 cm. The resection specimen revealed a well delimitated tumor with a central core of partially necrotic neoplastic bone. Besides, histology showed high mitotic areas with pleomorphic spindle cells and regions with cartilaginous differentiation. Immunohistochemistry demonstrated: vimentin+, CD34−, desmin−, actin−, EMA− and pancytokeratin− with focal S100 protein positivity and a Ki-67 index of 20%. Comparative genomic hybridization (CGH) revealed a gain of chromosomal material on 12q; FISH analyses for the CDK4 and MDM2 region showed high level amplifications. Consequently, a high-grade dedifferentiated extraskeletal osteosarcoma was diagnosed. In conclusion, analysis of the MDM2 and CDK4 status is a powerful discriminating diagnostic tool to distinguish dedifferentiated extraskeletal osteosarcoma from other benign/malignant ossifying lesions in the skeletal muscle. © 2014 Published by Elsevier GmbH.
Differential diagnosis of intramuscular fibrous and ossifying lesions is challenging for the clinician as well as for the pathologist. These mesenchymal lesions comprise a variety of benign
夽 This case was presented at the 81st meeting of the Arbeitsgemeinschaft Knochentumoren e.V./Working Group on Bone Tumors in Munich, October 19, 2013 receiving the AGKT-award as best presentation. ∗ Corresponding author at: Institute of Pathology, University of Ulm, AlbertEinstein Allee 23, D-89070 Ulm, Germany. Tel.: +49 0731 50056340. E-mail address: [email protected] (T.F.E. Barth).
conditions such as metaplastic ossifications as well as myositis ossificans. Examples for malignant lesions include extraskeletal osteosarcoma (low-/high-grade) as well as other soft tissue sarcomas with metaplastic/neoplastic ossification (e.g. synovial sarcoma) [5,12]. The histological differential diagnosis can even be more difficult on small biopsies since these lesions may have heterogeneous patterns of differentiation. In the following we present the clinical history, the imaging results, the histological and immunohistological profile, and the molecular pathology of a slowly growing mass with a wide range of histological patterns that ultimately was diagnosed as a high-grade dedifferentiated extraskeletal osteosarcoma.
http://dx.doi.org/10.1016/j.prp.2014.05.010 0344-0338/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Clinical history A 46 year old male presented with a swelling of the left lateral thigh causing him modest pain comparable to muscle soreness after physical exercise. No trauma was reported. At physical examination there was neither inducible palpable pain nor signs of a disturbed peripheral blood flow or symptoms of an altered sensor-motoric function. Blood work was normal. Imaging by Rx and magnetic resonance imaging (MRI) revealed an intramuscular mass within the left vastus lateralis muscle with a diameter of 5 cm; there was no contact to the femoral bone. A biopsy was obtained from the periphery of the tumor. After having consulted a bone tumor reference center the lesion was classified as most probably compatible with myositis ossificans and a “wait and see” strategy was adopted by the local Oncological advisory Board for sarcomas. 18 months later the patient showed up complaining about the continuously increasing mass in the left vastus lateralis muscle. Imaging by Rx and MRI revealed a partially ossified intramuscular lesion without contact to the femur or the skin, now measuring 25 cm × 13 cm × 8 cm. The case was presented again at the Oncological advisory Board for Sarcomas and another biopsy was taken. Since the histological report of the biopsy was suspicious for malignancy a complete resection of the tumor was advised by this interdisciplinary board. Materials and methods Biopsies and surgical specimen were fixed in 4% paraformaldehyd in phosphate-buffered saline and embedded in paraffin. Calcified tissue was treated with EDTA (Ethylendiamintetraacetate; Triplex Merk, Mannheim, Germany) before embedding. Sections of 2–3 m were stained with heamtoxylin-eosin and with antibodies for vimentin (VIM3B4, Dako, Glostrup, Denmark; dilution 1:300), CD34 (QBEnd10, dilution 1:100, Dako), desmin (DE-R-11, Menarini, Milan, Italy, dilution 1:50), actin (HHF35, Dako, dilution 1:500), EMA (E29, Dako, dilution 1:500), pancytokeratin (AE1 + AE3; Dako, dilution 1:100), S100 protein (Z0311, Dako, dilution 1:1000) and Ki-67 (Mib-1, Dako, dilution 1:200). CGH and FISH was performed using standard protocols [6]. For FISH analyses commercially available probes were used: a dual color break-apart probe for 18q11.2 (SYT 18, Abbott, IL, USA) and dual color probe sets for CDK4 (12q13; Kreatech, Amsterdam, The Netherlands) and MDM2 (12q15; Kreatech). Results At first presentation a biopsy of 1.8 cm × 1.7 cm × 0.4 cm was obtained. Histology revealed a matrix rich lesion consisting of a spindle cell proliferation with intermingled ossified trabeculae. There was neither clear osteoblastic rimming nor higher polymorphism and no atypical mitoses or necrosis (Fig. 2C1). A clear zonal phenomenon in terms of immature cellular areas in the center and more mature, ossifying areas with osteoblastic rimming in the periphery was not present in the biopsy. Morphology was interpreted to be compatible with myositis ossificans. At second presentation imaging revealed a partially calcified intramuscular mass still without contact to the femoral bone (Fig. 1A, A1–A3). Two core biopsies measuring 3 cm in length showed a very similar morphology to the former biopsy with a spindle cell proliferation and intermingled bony trabeculae. However, polymorphy of nuclei was focally pronounced. No necrosis was detected. By immunohistochemistry the lesion was positive for vimentin and focally for S100 protein. CD34, desmin, actin, EMA, and pancytokeratin were
negative. To definitely rule out a biphasic, partially ossifying synovial sarcoma, FISH analysis was performed with a commercial break-apart probe for the SYT region on chromosomal band 18q11.2; no rearrangement was detected, practically excluding synovial sarcoma. Therefore the diagnosis was adjusted to an intramuscular lesion “highly suspicious for extraskeletal osteosarcoma”. Since there were no signs of metastasis the patient was staged as limited disease and surgery was advised by the Oncological advisory Board for Sarcomas. The resection specimen weighed 4.2 kg and measured 30 cm × 20 cm × 15 cm. On cross section a 25 cm × 13 cm × 8 cm solid yellowish tumor emerged that was well delimitated from the surrounding muscle and fat tissue (Fig. 1B, B12). The central part was ossified, measured 9 cm in diameter and was partly necrotic. 30 representative tissue blocks were performed for histologic analysis. Histology revealed three patterns of differentiation: (1) A zone of irregular trabeculae of bone embedded in a spindle cell stroma with only minimal atypia reminiscent of a low-grade parosteal osteosarcoma adjacent to high-grade pleomorphic cells with a lace-like bone formation (Fig. 2C1/2). (2) An area of cartilaginous differentiation with polymorphic tumor cells (Fig. 2C3); (3) A highly proliferating spindle cell region with accompanying extracellular matrix deposition, few pleomorphic cells and with up to 14 (partially atypical) mitoses/high power field (Fig. 2C4). The central part of the lesion was ossified, while the periphery revealed predominantly atypical spindle cell proliferations, thus resembling a reverse zoning phenomena with central deposition of neoplastic bone and atypical spindle cell proliferation in the periphery characteristic for extraskeletal osteosarcoma. In none of the sections we detected a lipogenic differentiation in terms of lipoblast or fat tissue with atypical stromal cells. DNA was extracted from the spindle cell and osteoblastic areas and comparative genomic hybridization (CGH) was performed [6]. In both areas the same gain of chromosomal material on 12q was detected including a high-level gain on chromosomal band 12q13-12q15 (Fig. 2D1–D3). In a separate step we analysed the low-grade compartment by CGH. CGH analysis of the low- and high-grade compartment revealed as the sole overlapping aberration a gain of chromosomal material on chromosome 12 while the high-grade sarcoma showed a high level amplification on 12q (data not shown). In a candidate gene approach we performed double color fluorescence in situ hybridization (FISH) using commercial probe sets for the CDK4 and MDM2 region both mapping to chromosomal bands 12q13 and 12q15; as a reference a probe for the centromere of chromosome 12 was used. The spindle cell compartment as well as the osteogenic area were analysed by FISH. In both respective areas we detected a high-level amplification of CDK4 and MDM2 with ratio values of 6.4 and up to 7.1 (in analogy to the Her2 FISH signal estimation) with up to twenty signals for CDK4 and MDM2 (Fig. 2E1 and E2). Immunohistochemistry for CDK4 and MDM2 revealed a strong nuclear accumulation of CDK4 and a less strong nuclear staining for MDM2 in all three compartments of the lesion (Fig. 2F1, F2). The Ki-67 index was up to 20% (Fig. 2F3). We then performed FISH and immunohistochemistry on the initial biopsy taken 18 month ago and detected close to identical FISH based amplification ratios for CDK4 and MDM2. Immunohistochemistry was positive for CDK4, however, MDM2 was negative (data not shown). Ultimately, the tumor was diagnosed as a dedifferentiated osteosarcoma grade 3 with a sum score of 6 (3-3-0) according to the FNCLCC system [7]. The final TNM classification was: pT2b, V0, L0, Nx, Mx, R0 with a minimal resection margin of 5 mm. Accordingly, the patient was enrolled in an adjuvant chemotherapy regimen following the guidelines of the EURO-B.O.S.S. therapy protocol [3]. He finished chemotherapy in December 2013 with no evidence of disease during followup.
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Fig. 1. Comparison of imaging and macroscopic aspects of the resection specimen. (A1) Rx shows a calcified extraskeletal mass of the lateral left thigh. Magnetic resonance imaging (A2 and A3) reveals a heterogeneous intramuscular lesion without contact to the bone and a central calcified core. Figure B, B1 and B2 show the corresponding macroscopic aspects of the resection specimen well delineated from the adjacent subcutaneous fat tissue (B1) and muscle (B2). B1 and B2 correspond to the spindle cell compartment while the central part (B3) presents the ossified central core of the lesion.
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Discussion Intramuscular calcifying masses comprise a broad spectrum of differential diagnoses, including various benign and malignant low and high-grade lesions [4,9]. The definitive diagnosis is crucial for prognostic prediction and an accurate choice of the highly varying therapeutic options ranging from “watch and wait” to simple excision to wide resection and high-dose neoadjuvant chemotherapy [3]. Low-grade extraskeletal osteosarcoma is rare with only few case listed in the literature (see Table 1). The patient presented here had a progressively growing intramuscular mass well delimitated from the surrounding tissue. Only the resection specimen allowed sampling of the whole lesion and finally revealed the full spectrum of its complex differentiation. This pattern was in large
aspects similar to a low-grade paraosteal osteosarcoma of bone. Since the lesion was located in the soft tissues outside and without any direct contact to the bone, an extraskeletal osteosarcoma was diagnosed. Conventional extraskeletal osteosarcoma is a rare highgrade entity with an aggressive course of disease [7]. However, within the spectrum of those tumors, a low-grade variant with features of classical paraosteal osteosarcoma has been described [8,17; Table 1]. This variant is characterized by a high-level amplification on 12q and is associated with a favorable prognosis [11]. The case shown here showed at least partly the histomorphological features of a low-grade parosteal osteosarcoma with a high histological heterogeneity and presented with a 12q amplification identical to those found in paraosteal osteosarcoma [5,17,18]. However, the presented lesion also disclosed patterns of a high-grade sarcoma
Please cite this article in press as: A. von Baer, et al., Immunohistochemical and FISH analysis of MDM2 and CDK4 in a dedifferentiated extraskeletal osteosarcoma arising in the vastus lateralis muscle: Differential diagnosis and diagnostic algorithm, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.05.010
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Fig. 2. Histology, immunohistochemistry, and comparative genomic hybridization (CGH), and FISH analysis. Histology of the initial biopsy reveals a spindle cell proliferation with intermingled ossified trabeculae and structures like woven bone (C1). The central part of the resection specimen shows areas with osteoblastic differentiation with lace-like neoplastic bone (C2), a cartilaginous differentiation (C3), and a high-grade spindle cell lesion with atypical mitoses in the periphery (C4, insert); (C1, bar = 100 m Q3 C1, C2, C4, insert; bar = 40 m; C3 = 50 m). CGH analysis shows examples of DNA hybridization of control (D1) and tumor (high-grade spindle cell compartment) (D2) DNA on chromosome 12 detected via red and green fluorescence with an enhanced green signal (tumor DNA) on the long arm of chromosome 12 as compared to the red signal (control DNA). D3 shows the corresponding ratio profile resulting in a shift of the profile to the left of the central line indicating a DNA amplification. Figure E1 and E2 shows corresponding FISH images with multiple signals for the genomic regions of CDK4 (E1) and MDM2 (E2) detected in red and single green signals for the centromere region of chromosome 12, respectively (red arrows mark signals of CDK4 and MDM2, respectively; green arrows mark centromere 12). Immunohistochemistry for CDK4 (F1) and MDM2 (F2) shows strong nuclear signals for CDK4 and some positive nuclei for MDM2 in the sarcoma. The proliferation is enhanced to approximately 20% in a Ki-67 staining (F3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Table 1 Summary of reported cases with well-differentiated extraskeletal osteosarcomas. Case
Year
First author
Age
Sex
Site
Treatment
Follow-up and outcome
1
1953a
Umiker [16]
44
M
Rt. thigh
5
Resection
Lt. knee
24
Resection
F
Lt. axilla
15
Resection
63
F
Lt. tigh
7
Resection
Okada [13]
35
F
Lt. leg
11
Resection
2005
Abramovici [1]
40
F
Lt. upper back
9×6×5
Resection
7
2005a
Abramovici [1]
32
M
8
2010
Arai [2]
62
F
Bil. thighs, buttocks and lt. paravertebral area (max. lesion) Retroperitoneum
Died with widespread metastasis after 5 years Alive 5 years after surgery Alive 2 years after surgery Alive 12 months after surgery Alive 4 years after surgery Alive 2 months after surgery lost in follow-up Alive with widespread metastasis 4 years after initial surgery
2
1989
Present [14]
57
F
3
1991
Yi [17]
74
4
2003
Fukunaga [8]
5
2003
6
9
2010
Sabatier [15]
30
M
Chest wall
10
2014
Japuria [10]
38
M
Retroperitoneum
Present case
2013
von Baer
46
M
Lt. lateral tigh
a
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231
Size (cm)
16 × 7 × 8
14 × 11 × 8
30
25 × 13 × 8
Resection and chemotherapy
Resection
Chemotherapy Resection and chemotherapy Resection and chemotherapy
Died with widespread metastasis after 33 months of dedifferentiated spindle cell sarcoma Died 5 years after first symptoms Disease free after 3 years of follow-up Alive one year after surgery/chemotherapy
Recurred as a high-grade malignancy.
with necrosis and pleomorphic nuclei that finally led to the diagnosis of a dedifferentiated extraskeletal high-grade osteosarcoma most probably arising from an extraskeletal low-grade osteosarcoma with features close to identical of a parosteal osteosarcoma. The assumption of tumor progression to a high-grade sarcoma is strengthened by the observation that the initial lesion and histology of the resection specimen already revealed an amplification of MDM2 and CDK4 on the genomic level; a full gene dosage effect, however, with nuclear protein accumulation of MDM2 and CDK4 protein was detected only in the dedifferentiated sarcoma, while the initial biopsy was convincingly positive for CDK4 protein only. These findings may be suggestive of a progressive and probable metachronous upregulation of MDM2 and CDK4 protein expression during the process of dedifferentiation. This assumption is further strengthened by the fact, that CGH analysis of the low- and high-grade compartment revealed as the sole aberration a gain on chromosome 12 while the high-grade sarcoma showed a high level amplification on 12q. Since no further clonal genetic aberrations were detected by CGH these findings may support the concept of a clonal evolution. Nevertheless, the immunohistological detection of CDK4 in the initial biopsy together with amplification of MDM2 and CDK4 as detected by FISH already pointed at a malignant lesion. A further possible differential diagnosis of the presented case is the recently described well-differentiated liposarcoma with lowgrade osteosarcomatous component. This tumor is characterized by a biphasic appearance with lipogenic and osteogenic areas and is CDK4 and MDM2 positive by immunohistochemistry [19]. In the presented case, however, lipogenic differentiation was excluded by extensive histological analysis, thus ruling out this rare tumor. Summing up, integration of FISH analysis of MDM2 and CDK4 combined with immunohistochemistry for these two proteins is a highly discriminating diagnostic tool in the diagnostic algorithm of
extraskeletal ossifying lesions and clearly allows the differentiation from benign intramuscular mimics.
Acknowledgements We thank Michaela Buck and Iwona Nerbas for excellent technical help.
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