We found an amplification of the MDM2 gene in 21 out of 27 (56%) positive cases for MDM2 protein. For all normal DNA controls, the intensity of the higher-molecular-weight DR band was greater than the intensity of the lower-molecular-weight MDM2 band. In comparison with the pleomorphic or myxoid variants, MDM2 amplification (Fig. 2) occurred more frequently in (58.8%) WDLPS and (62.5%) DDLPS, with a statistically significant difference (P=0.001). MDM2 amplification was not associated with the patients’ age, sex, and tumor size or location.
The average Ki-67 nuclear staining was 17.6% in WDLPS, 25% in DDLPS, 21.4% in MLPS, and 50% in PLPS, whereas it was totally negative in all lipoma cases; P-value=0.08. There was no statistical significance in its expression among different age groups, tumor size, or location of the tumors, except for the sex (P=0.05) (Table 1).
In the same consideration, 21 (77.8%) MDM2 amplified cases displayed nuclear immunoreactivities with anti-MDM2 antibodies. Six out of 27 (22.2%) cases with elevated levels of MDM2 proteins showed no detectable amplified MDM2 gene. A highly significant correlation between MDM2 protein and gene expression was found (r=0.822, P<0.001). The nuclear accumulation of MDM2 proteins and Ki-67 (MDM2 positive, K-i67 positive immunoreactivity) was present in nine out of 27 (33.3%) cases. However, MDM2 immunoreactivity dissociated from Ki-67 expression (MDM2 positive, Ki-67 negative immunophenotype) was present in 18 out of 27 (66.7%) cases. A significant correlation was shown between the expression of both antibodies (r=0.479, P<0.001).
Immunohistochemistry identifies a tumor accurately as being of mesenchymal or nonmesenchymal origin. Once a mesenchymal origin has been established, histologic subtyping according to the specific cell lineage may be achieved using lineage-specific markers (Heim-Hall, Yohe, 2008).
In this study, immunohistochemical detection of MDM2 in a series of lipomas and LPS revealed that only one case of lipoma (3.8%) was positive, whereas 70.6% of WDLPS, 75% of DDLPS, and 50% of MLPS cases were positive. Benign lipomatous lesions were generally negative for MDM2. When WDLPS dedifferentiates, the high-grade component usually retains MDM2 and CDK4 positivity (Binh et al., 2005, 2006). In the primary extremity MLPS group, 90% of the patients were negative for MDM2 and 10% showed weak staining (De Vreeze et al., 2009). A high frequency of MDM2 protein detected in different histological subtypes of LPS suggests a preliminarily high sensitivity of this marker (Júnior et al., 2008). MDM2 immunohistochemistry can be used to differentiate WDLPS and DDLPS from their stimulators. MDM2 alone has a very high sensitivity in the identification of WDLPS among lipomas, but has a low specificity in diagnosing DDLPS among several pleomorphic sarcomas and other mesenchymal tumors (Aleixo et al., 2009). Another method to separate DDLPS from other sarcomas is to combine MDM2/CDK4 testing and consider a case positive, when strong and diffuse immunoreactivity is seen in more than 30% of the neoplastic cells. Several tumors expressed these two proteins despite the absence of gene amplification, suggesting a mechanism of protein overexpression other than gene amplification (Sirvent et al., 2007).
The detection of MDM2 protein overexpression by immunohistochemistry and MDM2 gene amplification can be used for clinical purposes to diagnose WDLPS/DDLPS. The use of MDM2 immunostaining alone to differentiate pleomorphic sarcomas from DDLPS has a low specificity, even in cases with a strong and diffuse immunoreactivity (Sirvent et al., 2007). This confirms that MDM2 immunohistochemical analysis is an exportable technique and can be used as a routine test. However, it is important to calibrate the immunohistochemical technique accurately by using the genetic data obtained from fluorescent in-situ hybridization, quantitative PCR, or comparative genomic hybridization (Vincent Salomon et al., 2003; Binh et al., 2006).
In our study, an amplification of the MDM2 gene was detected in 56% of the positive cases for MDM2 protein. Alterations of the MDM2 gene are known to be a common mechanism in the tumorigenesis of LPS. Similar results claimed that MDM2 gene amplification was found in 53.8% of LPS (Ladanyi et al., 1993); however, a lower frequency of gene amplification (15.8%) was found in other studies (Florenes et al., 1994; Schneider Stock et al., 1999). WDLPS was associated with an amplification of MDM2 gene (Dei Tos and Pedeutour, 2002; Pedeutour et al., 2004; Sandberg, 2004a, 2004b). ALT-WDLPS and DDLPS share the same basic genetic abnormality characterized by a simple genomic profile with a 12q14–15 amplification involving MDM2 gene (Coindre et al., 2010). MDM2 (12q15) and HMGA2 (12q14.3) were consistently amplified in WDLPS/DDLPS (Italiano et al., 2008; Italiano et al., 2009). MDM2 gene amplification appeared to be a valid target to distinguish between lipoma and liposarcoma groups. The specificity of MDM2 amplification in LPS was 98.2% (Pilotti et al., 2000; Hostein, 2004).
In the current study, MDM2 amplification occurred more frequently in WDLPS (58.8%) and DDLPS (62.5%) than in pleomorphic or myxoid variants. Increased MDM2 expression or MDM2 amplification can be used to differentiate these entities with a very high sensitivity and specificity (Weaver et al., 2009). Other sarcomas can show positivity for the MDM2 immunostaining and/or amplification of the MDM2 gene, including malignant peripheral nerve sheath tumors, myxofibrosarcomas (Binh et al., 2005), and rhabdomyosarcomas (Takahashi et al., 2004). In addition, other tumors besides sarcomas can show MDM2 amplification, including melanoma (Muthusamy et al., 2006). Thus, MDM2 may be more helpful in deciding whether a lesion is malignant, and may not be as helpful in specifically subtyping a neoplasm as an LPS. MDM2 amplification is a key feature of WDLPS/DDLPS and is amplified and overexpressed in a number of other cancers, highlighting its importance in tumorigenesis (Toledo and Wahl, 2006).
However, when looking at mesenchymal malignancies, it is uncertain whether the MDM2 amplification truly occurs in a subset of pleomorphic sarcomas other than LPS, or whether this group simply represents DDLPS (Coindre et al., 2004; Chung et al., 2009). A series of malignant fibrous histiocytomas were diagnosed in the retroperitoneum, and most of these were reclassified as DDLPS after careful analysis, including examination for MDM2 and CDK4 amplification (Coindre et al., 2003; Al-Maghraby et al., 2010). The main differential diagnosis for DDLPS is pleomorphic sarcoma, not otherwise specified, and both entities display MDM2 amplification. MDM2 have been reported to be amplified in cases of pleomorphic sarcoma. In fact, recent studies suggest that most pleomorphic sarcomas are actually DDLPS on the basis of histological review, immunoprofile, and genomic profile (Coindre et al., 2003, 2004).
In our series, 77.8% of MDM2 amplified cases displayed positive expression of MDM2 protein, and 22.2% of the cases with elevated levels of MDM2 proteins showed no amplified MDM2 gene. Although immunohistochemistry can be used to demonstrate MDM2 overexpression, direct correlation between the gene amplification and protein overexpression is not the rule (Hostein, 2004). Amplification of the MDM2 gene results in an increased mRNA level, but it is not necessarily associated with an increased accumulation of MDM2 protein (Bartel et al., 2001). MDM2 protein, when overexpressed, acts as a stimulator of the cell cycle through the G1-S phase. Overexpression of this protein could lead to a loss of cell-cycle control with consequent development of neoplastic growth (Momand et al., 1998; Albertson, 2006).
MDM2 amplification is a predictor of the sensitivity to current MDM2 antagonists (Muller et al., 2007). Given that MDM2 is consistently amplified in WDLPS/DDLPS, and sensitivity to MDM2 antagonists (such as Nutlin-3a) is predicted by MDM2 amplification, it is an appealing therapeutic target (Vassilev et al., 2004; Muller et al., 2007). A major challenge with the use of molecularly targeted therapeutics is to translate disease control into disease eradication (Conyers et al., 2011).
Ki-67 was negative in lipoma cases but nuclear staining was 17.6% in WDLPS, 25% in DDLPS, and 21.4% in MLPS. There is no significant difference in the immunoreactivity of Ki-67 between benign and malignant adipocytic tumors. The same results were obtained by another study conducted by Adachi et al. (2001). Comparing the expression profile in malignant and benign adipocytic tumors, ki-67 was positive in 57.1% of the WDLPS cases and all lipomas were negative for MDM2, p53, and ki-67 (Hatano et al., 2004). Nuclear accumulation of Ki-67 in more than 20% of the tumor cells in primary, high-grade extremity soft tissue sarcomas was demonstrated to be an independent negative prognostic factor for the development of distant metastasis and tumor mortality (Heslin et al., 1996; Pisters et al., 1996). Ki-67 overexpression is associated with an increased risk of distant metastasis and tumor mortality (Heslin et al., 1998). Ki-67 was the highest in malignant fibrous histiocytomas and lowest in LPS (Huuhtanen et al., 1999). In LPS, the Mib-1 proliferation index were associated with a poor prognosis. Moreover, their prognostic value was higher in myxoid than in pleomorphic LPS. Ki-67 overexpression was associated with a more aggressive clinical behavior in MLPS (Drobnjak et al., 1994; Schneider Stock et al., 1999).
We concluded that the immunohistochemical study of MDM2 could be used to diagnose problematic adipocytic tumors and suspicious cases of ALT/WDLPS or DDLPS. It may not be necessary to perform any complementary molecular genetic tests in cases without major diagnostic difficulties. In case of a query, unexpected immunohistochemical staining results, or confusing histopathological features, a complementary simple molecular analysis, preferably differential PCR, is useful to obtain reproducible results.
There are no conflicts of interest.
Adachi T, Oda Y, Sakamoto A, Saito T, Tamiya S, Masuda K, et al. Immunoreactivity of p53, mdm2 and p21WAF1 in dedifferentiated liposarcoma: special emphasis on the distinct immunophenotype of the well-differentiated component. Int J Surg Pathol. 2001;9:99–109
Albertson DG. Gene amplification in cancer. Trends Genet. 2006;22:447–455
Aleixo PB, Hartmann AA, Menezes IC, Meurer RT, Oliveira AM. Can MDM2 and CDK4 make the diagnosis of well differentiated/ dedifferentiated liposarcoma? An immunohistochemical study on 129 soft tissue tumours. J Clin Pathol. 2009;62:1127–1135
Al-Maghraby H, Khalbuss W, Rao U, Cieply K, Dacic S, Monaco S. Fine needle aspiration biopsy diagnosis of dedifferentiated liposarcoma: Cytomorphology and MDM2 amplification by FISH. Cyto Journal. 2010;7:5–18
Arrigoni G, Doglioni C. Atypical lipomatous tumor: molecular characterization. Curr Opin Oncol. 2004;16:355–358
Bartel F, Meye A, Wurl P, Kappler M, Bache M, Lautenschlager C, et al. Amplification of the MDM2 gene, but not expression of splice variants of MDM2 MRNA, is associated with prognosis in soft tissue sarcoma. Int J Cancer. 2001;95:168–175
Binh MB, Sastre Garau X, Guillou L, de Pinieux G, Terrier P, Lagace R, et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 2005;29:1340–1347
Binh MB, Garau XS, Guillou L, Aurias A, Coindre JM. Reproducibility of MDM2 and CDK4 staining in soft tissue tumors. Am J Clin Pathol. 2006;125:693–697
Chung L, Lau SK, Jiang Z, Loera S, Bedel V, Ji J, et al. Overlapping features between dedifferentiated liposarcoma and undifferentiated high-grade pleomorphic Sarcoma. Am J Surg Pathol. 2009;33:1594–1600
Coindre JM, Mariani O, Chibon F, Mairal A, De Saint Aubain Somerhausen N, Favre Guillevin E, et al. Most malignant fibrous histiocytomas developed in the retroperitoneum are dedifferentiated liposarcomas: a review of 25 cases initially diagnosed as malignant fibrous histiocytoma. Mod Pathol. 2003;16:256–262
Coindre JM, Hostein I, Maire G, Derre J, Guillou L, Leroux A, et al. Inflammatory malignant fibrous histiocytomas and dedifferentiated liposarcomas: histological review, genomic profile and MDM2 and CDK4 status favour a single entity. J Pathol. 2004;203:822–830
Conyers R, Young S, Thomas DM. Liposarcoma: Molecular genetics and therapeutics. Sarcoma. 2011;27:1–13
Coindre JM, Pédeutour F, Aurias A. Well-differentiated and dedifferentiated liposarcomas. Virchows Arch. 2010;456:167–179
Cordon Cardo C. Mutations of cell cycle regulators. Biological and clinical implications for human neoplasia Am J Pathol. 1995;147:545–560
Dei Tos AP, Pedeutour F, Ruijter HJ, Nederlof PM, Haas RLUnni K, Mertens F. Atypical lipomatous tumor/well-differentiated liposarcoma. Pathology and genetics of tumours of soft tissue and bone WHO classification of tumours, CDM Fletcher. 2002 Lyon IARC Press:35–46
De Vreeze RSA, De Jong D, Tielen IHG, Ruijter HJ, Nederlof PM, Haas RL, et al. Primary retroperitoneal myxoid/round cell liposarcoma is a nonexisting disease: an immunohistochemical and molecular biological analysis. Mod Pathol. 2009;22:223–231
Drobnjak M, Latres E, Pollack D, Karpeh M, Dudas M, Woodruff JM, et al. Prognostic implications of p53 nuclear overexpression and high proliferation index of Ki-67 in adult soft-tissue sarcomas. J Natl Cancer Inst. 1994;86:549–554
Erickson-Johnson MR, Seys AR, Roth CW, King AA, Hulshizer RL, Wang X, et al. Carboxypeptidase M: a biomarker for the discrimination of well-differentiated liposarcoma from lipoma. Mod Pathol. 2009;22:1541–1547
Fernebro J, Engellau J, Persson A, Rydholm A, Nilbert M. Standardizing evaluation of sarcoma proliferation higher Ki-67 expression in the tumor periphery than the center. Acta Pathol Microbiol Immunol Scandi. 2007;115:707–712
Fletcher CDM, Unni K, Mertens F. Pathology and genetics of tumours of soft tissue and bone. WHO classification of tumours. 2002 Lyon IARC Press
Florenes VA, Maelandsmo GM, Forus A, Andreassen A, Myklebost O, Fodstad O. MDM2 gene amplification and transcript levels in human sarcomas: Relationship to TP53 gene status. J Natl Cancer Inst. 1994;86:1297–1302
Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983;31:13–20
Hatano H, Morita T, Ogose A, Hotta T, Kobayashi H, Honma K. Well-differentiated liposarcoma associated with benign lipoma. Anticancer Res. 2004;24(2C):1039–1044
Heim-Hall J, Yohe SL. Application of immunohistochemistry to soft tissue neoplasms. Arch Pathol Lab Med. 2008;132:476–489
Heslin MJ, Woodruff JM, Brennan MF. Prognostic significance of a positive microscopic margin in high-risk extremity soft tissue sarcoma: Implications for management. J Clin Oncol. 1996;14:473–478
Heslin MJ, Cordon Cardo C, Lewis JJ, Woodruff JM, Brennan MF. Ki-67 detected by MIB-1 predicts distant metastasis and tumor mortality in primary, high grade extremity soft tissue sarcoma. Cancer. 1998;83:490–497
Hostein I, Pelmus M, Aurias A, Pedeutour F, Mathoulin Pelissier S, Coindre JM. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas. The J Pathol. 2004;202:95–102
Huuhtanen RL, Blomqvist CP, Wiklund TA, Bohling TO, Virolainen MJ, Tukiainen EJ, et al. Comparison of the Ki-67 score and S-phase fraction as prognostic variables in soft-tissue sarcoma. Br J Cancer. 1999;79:945–951
Italiano A, Bianchini L, Keslair F, Bonnafous S, Cardot-Leccia N, Coindre JM, et al. HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon. Int J Cancer. 2008;122:2233–2241
Italiano A, Bianchini L, Gjernes E, Keslair F, Ranchere-Vince D, Dumollard JM, et al. Clinical and biological significance of CDK4 amplification in well-differentiated and dedifferentiated liposarcomas. Clin Cancer Res. 2009;15:5696–5703
Jablkowski M, Bocian A, Bialkowska J, Bartkowiak J. A comparative study of P53/MDM2 genes alterations and P53/MDM2 proteins immunoreactivity in liver cirrhosis and hepatocellular carcinoma. J Exp Clin Cancer Res. 2005;24:117–125
Jacob EK, Erickson-Johnson MR, Wang X. Assessment of MDM2 amplification using fluorescence in situ hybridization of paraffin embedded tissues discriminates atypical lipomatous tumors from lipomas. Mod Pathol. 2006;19(13A):45–57
Júnior RZ, De Camargo OP, De Oliveira GCM, Zon Filippi R, Baptista AM, Caiero MT. Prognostic factors and expression of MDM2 in patients with primary extremity liposarcoma. Clinics. 2008;63:157–164
Kooby DA, Antonescu CR, Brennan MF, Singer S. Atypical lipomatous tumor/well-differentiated liposarcoma of the extremity and trunk wall: Importance of histological subtype with treatment recommendations. Ann Surg Oncol. 2004;11:78–84
Ladanyi M, Cha C, Lewis R, Jhanwar SC, Huvos AG, Healey JH. MDM2 gene amplification in metastatic osteosarcoma. Cancer Res. 1993;53:16–18
Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification database. Nucleic Acids Res. 1998;26:3453–3459
Muller CR, Paulsen EB, Noordhuis P, Pedeutour F, Saeter G, Myklebost O. Potential for treatment of liposarcomas with the MDM2 antagonist Nutlin-3A. Int J Cancer. 2007;121:199–205
Muthusamy V, Hobbs C, Nogueira C, Cordon Cardo C, McKee PH, Chin L, et al. Amplification of CDK4 and MDM2 in malignant melanoma. Genes Chromosomes Cancer. 2006;45:447–454
Nielson GP, Mandahl NFletcher CDM, Unni K, Mertens. F. Lipoma. Pathology and genetics of tumours of soft tissue and bone, WHO classification of tumours. 2002 Lyon IARC Press:20–22
Pedeutour F, Maire G, Sirvent N. From cytogenetics to cytogenomics of adipose tissue tumors: 2. Malignant adipose tissue tumors. Bull Cancer. 2004;91:317–323
Pilotti S, Della Torre G, Mezzelani A, Tamborini E, Azzarelli A, Sozzi G, et al. The expression of MDM2/CDK4 gene product in the differential diagnosis of well differentiated liposarcoma and large deep-seated lipoma. Br J Cancer. 2000;82:1271–1275
Pisters PW, Leung DH, Woodruff J, Shi W, Brennan MF. Analysis of prognostic factors in 1,041 patients with localized soft tissue sarcomas of the extremities. J Clin Oncol. 1996;14:1679–1689
Rayburn E, Zhang R, He J, Wang H. MDM2 and human malignancies: expression, clinical pathology, prognostic markers and implications for chemotherapy. Current cancer drug targets 5. 2005;1:27–41
Sandberg AA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: lipoma. Cancer Genet Cytogenet. 2004a;150:93–115
Sandberg AA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: liposarcoma. Cancer Genet Cytogenet. 2004b;155:1–24
Schneider Stock R, Ziegeler A, Haeckel C, Franke DS, Rys J, Roessner A. Prognostic relevance of p53 alterations and Mib-1 proliferation index in subgroups of primary liposarcomas. Clin Cancer Res. 1999;5:2830–2835
Shimada S, Ishizawa T, Ishizawa K, Matsumura T, Hasegawa T, Hirose T. The value of MDM2 and CDK4 amplification levels using real-time polymerase chain reaction for the differential diagnosis of liposarcomas and their histologic mimickers. Hum Pathol. 2006;37:1123–1129
Sirvent N, Coindre JM, Maire G, Hostein I, Keslair F, Guillou L, et al. Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: Utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR. Am J Surg Pathol. 2007;31:1476–1489
Takahashi Y, Oda Y, Kawaguchi K, Tamiya S, Yamamoto H, Suita S, et al. Altered expression and molecular abnormalities of cell-cycle-regulatory proteins in rhabdomyosarcoma. Mod Pathol. 2004;17:660–669
Toledo F, Wahl GM. Regulating the p53 pathway: In vitro hypotheses, in vivo veritas. Nat Rev Cancer. 2006;6:909–923
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 2004;303:844–848
Vincent Salomon A, MacGrogan G, Couturier J, Arnould L, Denoux Y, Fiche M, et al. Calibration of immunohistochemistry for assessment of HER2 in breast cancer: results of the French multicentre GEFPICS study. Histopathology. 2003;42:337–347
Weaver J, Downs-Kelly E, Goldblum JR, Turner S, Kulkarni S, Tubbs RR, et al. Fluorescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatous neoplasms. Mod Pathol. 2008;21:943–949
Weaver J, Goldblum JR, Turner S, Tubbs RR, Wang WL, Lazar AJF, et al. Detection of MDM2 gene amplification or protein expression distinguishes sclerosing mesenteritis and retroperitoneal fibrosis from inflammatory well-differentiated liposarcoma. Mod Pathol. 2009;22:66–70
Weiss SW, Goldblum JRWeiss SW, Goldblum JR. Atypical lipomatous neoplasm/ALT/WDL. Dedifferentiated liposarcoma. Enzinger and Weiss’s soft tissue tumors. 2008 London Mosby:482–498