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Evaluation of immunohistochemical expression of MDM2 protein in comparison with MDM2 gene amplification in diagnosing lipomatous tumors

ElMoneim, Hanan Mohammed Abda; El Sherbiny, Yaser Makramb

doi: 10.1097/01.XEJ.0000406597.88861.1e
ORIGINAL ARTICLES
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Objective Our goal was to compare the immunohistochemical detection of murine double minute 2 (MDM2) protein and MDM2 gene amplification in diagnosing adipose tissue tumors.

Materials and methods MDM2 protein expression was examined by immunohistochemistry in formalin-fixed paraffin-embedded tissue samples from 67 benign and malignant adipose tumors. The immunostaining findings were correlated with the MDM2 amplification status by differential PCR.

Results MDM2 immunopositivity was detected in 3.8% lipoma, 70.6% well-differentiated liposarcomas (LPS), and 75% of dedifferentiated LPS with significant difference. MDM2 amplification occurred in 21 out of the 27 (56%) positive cases for MDM2 protein expression. MDM2 amplification was found more frequently in well-differentiated LPS (58.8%) and dedifferentiated LPS (62.5%) in comparison with the pleomorphic or myxoid variants. Overall, the MDM2 positivity was not associated with MDM2 amplification in six out of 27 (22.2%) cases, whereas 21 (77.8%) of the MDM2 amplified cases displayed nuclear immunoreactivity with anti-MDM2 antibodies. A highly significant correlation was obtained between MDM2 protein expression and gene amplification.

Conclusion Evaluation of MDM2 amplification using differential PCR may be used to supplement the immunohistochemical analysis of MDM2 protein when the diagnosis of adipose tissue tumors is not possible based on the clinical and histologic information alone.

aDepartments of Pathology

bClinical Pathology, Faculty of Medicine, Minia University, Egypt

Correspondence to Hanan M. Abd ElMoneim, PhD, Department of Pathology, Faculty of Medicine, El Minia University, Mailbox 61111, Egypt Tel: +0020106860230; fax: + 0862366149; e-mail: annglasgow@hotmail.com

Received July 15, 2011

Accepted August 1, 2011

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Introduction

Liposarcomas (LPS) are aggressive malignant neoplasms. It is one of the most frequent sarcomas in late adult life, representing 25% of soft tissue sarcomas in a large European database (Kooby et al., 2004; Sandberg, 2004a, 2004b; Weiss and Goldblum, 2008). It can be subtyped into three categories according to its clinicopathological and genetic characteristics: atypical lipomatous tumor or well-differentiated liposarcomas (ALT-WDLPS)/dedifferentiated liposarcomas (DDLPS), myxoid/ round cell liposarcomas(MLPS), and pleomorphic liposarcomas (PLPS) (Fletcher et al., 2002). The discrimination of lipoma from WDLPS can be problematic, especially those with a subtle cytologic atypia or a so-called lipoma-like histology (Jacob et al., 2006; Erickson-Johnson et al., 2009; Weaver et al., 2009).

Murine double minute 2 (MDM2) gene is an oncogene, which encodes a 90-kDa oncoprotein that is overexpressed in several human cancers such as in leukemias, breast, colon, and prostate cancers, osteosarcomas, gliomas, and soft tissue sarcomas. WDLPS and DDLPS cells contain a supernumerary ring or giant marker chromosomes characterized by 12q13–15 region amplification. In contrast, this molecular event has not been reported in benign lipomas. Within the 12q13–15 chromosomal region, the MDM2, SAS, HMGA2, and cyclin-dependent kinase 4 (CDK4) genes are the most frequent targets of amplification (Fletcher et al., 2002; Arrigoni and Doglioni, 2004; Hostein, 2004; Jacob et al., 2006; Shimada et al., 2006; Sirvent et al., 2007; Weaver et al., 2008; Erickson-Johnson et al., 2009; Weaver et al., 2009). MDM2 plays a central role in cancer development and progression. It may also serve as a diagnostic marker for cancer stage and to differentiate between similar cancers (Rayburn et al., 2005).

The MDM2 protein has been identified as an important modulator along the p53 pathway, leading to control cell proliferation (Cordon Cardo, 1995). The nuclear antigen Ki-67 is related to cell proliferation and is expressed in the S, G2, and M phases of the cell cycle but absent in the G0 phase (Gerdes et al., 1983). Ki-67 was shown to be an independent prognostic marker concerning primary high-grade mixed soft tissue sarcomas (Heslin et al., 1998). Soft tissue sarcomas are often present as large and histopathologically heterogeneous tumors. Proliferation has repeatedly been identified as a prognostic factor, and immunostaining for Ki-67 represents the most commonly used proliferation marker (Fernebro et al., 2007).

This study aimed to compare MDM2 protein expression by immunohistochemistry and MDM2 gene amplification by differential PCR in adipose tissue tumors, and to evaluate its relation to some anatomical and pathological aspects including the expression of the prognostic cell proliferation marker, Ki-67.

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Materials and methods

All 67 cases available were collected from the files of the Pathology Department, Minia University, dating from 2001 to 2009. Formalin-fixed, paraffin-embedded tissue blocks of these cases were used for immunohistochemical and molecular analyses. Tumors were categorized according to the WHO standard of soft tissue tumors (Dei Tos and Pedeutour, 2002; Nielson and Mandahl, 2002). The tumors’ final diagnoses were as follows: 26 cases of lipoma and 17 cases of WDLPS (WDLPS/atypical lipoma group). Furthermore, 14 MLPS include the MLPS and the cellular MLPS. Finally, eight cases of DDLPSs and two cases of PLPSs.

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Immunohistochemical staining

Immunohistochemical staining for MDM2 and Ki-67 was performed on representative paraffin blocks. Sections (4 µm thick) were cut and mounted on previously silanized glass slides. Sections were deparaffinized in xylene and rehydrated with ethanol. They were then treated with citrate buffer (0.01 mol/l citric acid, pH 6.0), and heat-based antigen retrieval was carried out in a microwave oven at 600 W for 20 min. Endogenous peroxidase was blocked using 95 ml of methanol and 5 ml of 3% hydrogen peroxide solution. The preparations were washed in distilled water and phosphate-buffered saline solution. Unspecific protein binding was blocked with 1% BSA for 30 min. Primary antibodies MDM2 (clone 1B10, dilution 1 : 50; Novocastra) and anti-Ki-67 antibody (clone MIB-1, dilution 1/100; DAKO M7240, UK) were incubated overnight at 4°C in a humid chamber. Next, the sections were stained by the streptavidin biotin peroxidase complex method using the LSAB kit (Dako). To reveal the reaction, a diaminobenzidine solution was used and counterstaining was performed with hematoxylin. A positive control for both antibodies in each staining run was osteosarcoma. Negative controls were obtained by omitting the primary antibodies (Aleixo et al., 2009).

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Assessment of immunohistochemical staining

Scoring was based on the percentage of tumor cell nuclei staining positive in the region of tumor with the greatest density of staining. The percentage of cells expressing nuclear staining was analyzed as a categoric variable. For MDM2 overexpression, nuclear accumulation of staining for at least 20% of the cells was scored positive (Adachi et al., 2001; Aleixo et al., 2009). For Ki-67, the proliferative index was defined as the percentage of cells expressing nuclear staining and was considered categorically positive if at least 20% of the nuclei were stained (Adachi et al., 2001).

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Formalin-fixed paraffin-embedded tissue DNA extraction

DNA was extracted from a paraffin-embedded tissue section. Five sections, each 7 µm, were cut on separate clean microtome blades. Paraffin was removed with xylene, and then the sample was washed twice with 100% ethanol and subsequently dried. The tissue was suspended in digestion 10 X TBE buffer pH 8.0 (Trizma base, 108 g Boric acid, 55 g EDTA, 9.3 g to 1 l by distilled water, Sigma) containing 10 μg proteinase K, before being incubated at 37°C overnight to release the DNA, and then centrifuged; the enzyme was inactivated at 95°C for 10 min in PCR heating blocks. The DNA-containing digest was stored at −20°C before amplification (Jablkowski et al., 2005).

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Differential PCR

An aliquot of the extracted DNA (3 µl) was amplified in the total volume of reaction mixture (26.3 µl) containing Reddy-Load PCR Mix (22.5 µl) with Taq polymerase (1.25 units), HCl (75 mmol/l, pH 8.8 at 25°C), (NH4)2SO4 (20 mmol/l), MgCl2 (1.5 mmol/l), Tween 20 (0.01% (v/v), dATP, dCTP, dGTP, and dTTP (0.2 mmol/l of each), and 4 pmol of the primer: the primer that amplifies MDM2 (forward primer 5′-CCG GAT GAT CGC AGG TG-3′, reverse primer 5′-AAA AGC TGA GTC AAC CTG CCC-3′, 98-bp PCR fragment), and the Dopamine receptor (DR) as a reference gene (forward primer 5′-CCACTGAATCTGTCCTGGTATG-3′, reverse primer 5′-GCGTGGCATAGTAGTTGTAGTGG-3′, 112-bp PCR fragment) were synthesized by MWG Oligo Synthesis Report Biotech UK Ltd, UK. Differential PCR, performed on a Perkin–Elmer Gene Amplification PCR System 9600 (Cetus, Connecticut, USA), consisted of 5 min of denaturation at 94°C, followed by 30 cycles of the following: denaturation for 1 min at 94°C, annealing for 1 min at 54°C, and elongation for 1 min at 72°C. The amplification ended with a final extension for 5 min at 72°C. Each sample was analyzed at least twice in separate PCRs. The samples were loaded (5 µl) on 3% agarose gel stained with ethidium bromide.

The intensities of the MDM2 fragment and the DR reference sequence were measured using an image Gel Doc 1000 system (Bio-Rad, France). An MDM2/DR ratio above 0.7 was regarded as positive for MDM2 amplification (mean ratio + 2 SD values, calculated for several controls without amplification specimens). To determine the cut-off point for MDM2 gene amplification, the copy number ratio for MDM2 related to the dopamine gene was calculated from a panel of normal DNA (surrounded normal adipose tissue). These results were expressed as the following ratio: intensity of the MDM2 band/intensity of the DR band. The ratios determined for the tumor samples were converted into a measure of gene amplification using the ratios determined for the normal controls (Jablkowski et al., 2005).

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Statistical analysis

All statistical analyses were performed using SPSS 16 software (Chicago, Illinois, USA). The results of the immunohistochemical analysis of MDM2 and Ki-67 and MDM2 amplification were compared with the clinicopathologic features using the χ2-test. The Spearman correlation coefficients were used to study the correlation between MDM2 protein expression and MDM2 gene amplification or Ki-67. A P-value of less than or equal to 0.05 was considered to indicate statistical significance.

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Results

Patients were between 28 and 67 years of age (mean 47.40 years, median 47 and SD 11.112); there were 40 (59.7%) men and 27 (40.3%) women. The anatomical locations of the tumors were the upper limb 26 (38.8%), the lower limb 22 (32.8%), retroperitoneum 8 (11.9%), and others 11 (16.4%). Tumor sizes ranged between 2.5 cm and 17.5 cm (median, 11.0 cm) as measured on the specimens after surgical resection. The tumors were divided into three groups: less than 5 cm, 16 (23.9%); between 5 and 10 cm, 27 (40.3%); and more than 10 cm, 24 (35.8%).

On immunohistochemical examination, MDM2 nuclear staining was detected in one out of 26 (3.8%) lipomas, 12 out of 17 (70.6%) WDLPS, six out of eight (75%) DDLPS, seven out of 14 (50%) MLPS, and one out of two (50%) PLPS. The MDM2 expression of DDLPS seemed to be higher than that of WDLPS (Fig. 1). However, both show a strong and diffuse positive staining pattern with a significant difference (P<0.001). MDM2 immunoreactivity was not correlated with the patients’ age, sex, and tumor size or location (Table 1).

Fig. 1

Fig. 1

Table 1

Table 1

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.

Fig. 2

Fig. 2

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).

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Discussion

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).

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Conclusion

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.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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