while melanoma is more common in women than in men prior to 50 years of age , this trend reverses after age 50.[15 ] although melanoma accounts for roughly 4% of all skin cancers , it is responsible for more than 74% of skin cancer deaths . tumor thickness , as defined by the breslow index , is the most important histological prognostic factor of melanoma and is measured vertically in millimeters from the top of the granular layer ( or base of superficial ulceration ) to the deepest point of tumoral involvement . increased tumor thickness suggests a higher metastatic potential and a poorer prognosis . the clark level on the other hand , is a measure of the anatomical invasion of the tumor and appears to affect prognosis only in thinner ( < 1 mm of depth ) melanomas . murine double minute 2 ( mdm2 ) is a 491 amino acid protein that functions as a key regulator of p53 by binding to its n - terminus , inhibiting its transcriptional activity , and promoting its degradation . mdm2 overexpression or hyperactivation directly contributes to loss of p53 function during the development of nearly 50% of human cancers . amplification of the genetic locus of mdm2 is a common event in several tumors including glioma , liposarcoma , osteosarcoma , pancreatic ductal adenocarcinoma , and germ cell tumors.[1014 ] the mdm2 immunoreactivity of tumoral cells has also been shown to be potentially correlated with the outcome . however , we used immunohistochemical techniques because finding a significant correlation between mdm2 expression and tumor thickness and invasion would introduce a predictive prognostic marker for cutaneous malignant melanoma . therefore , the relationship between mdm2 expression and tumor thickness and invasion in primary cutaneous malignant melanoma was evaluated . this was a descriptive analytical study on specimens from 43 randomly selected patients with primary cutaneous malignant melanoma who had undergone surgery or biopsy at alzahra hospital ( isfahan , iran ) during 2005 - 11 . all paraffin blocks of the patients were studied and the most appropriate ones were selected . afterwards , 2 slides were made from each block to be either stained with hematoxylin and eosin , or subjected to immunohistochemical analysis using a mouse monoclonal mdm2 antibody ( ncl - mdm2 , novocastra ) . based on manufacturer instructions , immunohistochemical staining was performed using the following stages : 1 ) h2o2 ( hydrogen peroxide ) stage ; 2 ) incubation of the tissue with the primary mdm2 antibody for 30 minutes ; 3 ) incubation with polymer ( envision ; secondary antibody ) for 30 minutes ; and 4 ) incubation with diaminobenzidine ( dab ) chromogen substrate solution for 5 minutes . at every stage , after the application of the above - mentioned materials , the slides were washed with a buffer solution or distilled water . for further staining , the percentage of immunoreactive nuclei was evaluated by counting at least 500 cells using the 40 objective lens . the scores were defined as positive when there was nuclear staining in at least 10% of neoplastic cells . such slides were categorized as weak , moderate , or strong based on their intensity . associations between the intensity of mdm2 expresion and the depth of invasion ( clark level ) and tumor thickness ( according to breslow index ) were evaluated using spearman 's correlation coefficients . of 43 patients , 24 ( 55.8% ) were men and 19 ( 44.2% ) were women . a mann - whitney test revealed no significant correlation between the intensity of mdm2 expression in men and women ( p = 0.495 ) . the mean breslow index values for men and women were 1.96  0.33 and 1.67  0.34 , respectively . an independent t - test revealed no significant difference between men and women ( p = 0.538 ) . after calculating the relative frequency distribution of mdm2 expression intensity , 31 patients ( 72.1% ) were negative , while 5 patients ( 11.6% ) , 2 patients ( 4.7% ) , and 5 patients ( 11.6% ) were positive with weak , moderate , and strong intensity staining , respectively . in terms of the frequency distribution of tumor thickness , 13 patients ( 30.2% ) had tumors < 0.76 mm thick , 10 patients ( 23.3% ) had tumors 0.76 - 1.5 mm thick , and 20 patients ( 46.5% ) had tumors thicker than 1.5 mm . spearman 's correlation analysis indicated a direct relationship between tumor thickness ( assessed by the breslow index ) and mdm2 expression intensity ( r = 0.425 ; p = 0.002 ) ( table 1 ) . association between murine double minute 2 ( mdm2 ) expression and breslow index the level of invasion ( assessed by clark level ) in the participants was classified as level i in 10 patients ( 23.3% ) , level ii in 7 patients ( 16.3% ) , level iii in 8 patients ( 18.6% ) , level iv in 8 patients , and level v in 10 patients ( 23.3% ) . spearman 's correlation analysis revealed a direct relationship between mdm2 expression intensity and clark 's level of invasion ( r = 0.43 ; p = 0.01 ) ( table 2 ) . association between murine double minute 2 ( mdm2 ) expression and clark 's level of invasion has been observed in 50% of human cancers , including glioma , osteosarcoma , and liposarcoma . a few studies have investigated the mdm2 expression rate in malignant melanoma and its association with prognostic factors and clinical outcomes . in a study conducted by hernberg et al . , the mdm2 expression rate was , on average , low , and therefore the diagnostic significance of this factor was questioned . however , there was a significant correlation between tumor development and mdm2 overexpression . in 2006 , muthusam et al . observed high - level amplification of the 12q13 - 15 region , a mdm2 locus site , in 3 of 53 patients with melanoma . their analysis of mrna and protein levels ( mdm2 ) also revealed overexpression of the mdm2 target gene . in 2000 , coupland et al . studied the prognostic significance of a number of factors , including mdm2 , in 92 patients with uveal melanoma using an immunohistochemical technique . their results revealed a significant correlation between mdm2 expression and clinical outcome ( odds ratio = 2.1 ; p = 0.13 ) . in 2008 , firoz et al . used pcr - restriction fragment length polymorphism ( rflp ) to investigate a single nucleotide polymorphism ( snp309 ) of the mdm2 gene as these previous studies have shown , increased mdm2 expression has only been observed in a limited number of patients with malignant melanoma . in addition , immunohistochemical techniques have revealed low average mdm2 expression rates . in this study , mdm2 expression rate was also low ( 28% ) , suggesting that mdm2 is not an appropriate diagnostic factor in melanoma . in two previous studies conducted to determine immunohistochemical factors prognostic for cutaneous malignant melanoma and its clinical outcome , a strong association between mdm2 expression intensity and prognosis was observed . similarly , direct associations between mdm2 expression intensity and tumor thickness ( the most important histological prognostic factor ) and clark 's level of invasion were observed in the current study . in conclusion , considering the importance of immunohistochemical approaches in determining the prognosis of a large number of tumors including melanoma , this study could be clinically useful for assessing prognostic factors among patients with malignant melanoma .
background : malignant melanoma is the most invasive cutaneous tumor which is associated with an incredibly high mortality rate 
 . the most reliable histological factors associated with melanoma prognosis are tumor thickness- measured by the breslow index- and invasion depth- measured by clark level . 
 murine double minute 2 ( mdm2 ) gene inhibits p53-dependent apoptosis . 
 an increase in mdm2 expression has been found in many tumors . 
 this study aimed to investigate mdm2 expression and its correlation with tumor thickness and invasion level in malignant melanoma.materials and methods : this study evaluated paraffin blocks from 43 randomly selected patients with primary cutaneous melanoma who referred to the main university pathology center in isfahan , iran . 
 mdm2 expression rate was assessed via immunohistochemical techniques and hematoxylin and eosin staining to determine tumor thickness and invasion level . 
 correlations between mdm2 expression and tumor thickness and invasion were analyzed using spearman 's correlation coefficient in spss17.results:the mean age of patients was 61.2  15 years . men and women constituted 55.8% and 44.2% of the participants , respectively . 
 the rate of mdm2 positivity was 28.9% . 
 mdm2 expression was directly associated with tumor thickness ( r = 0.425 ; p = 0.002 ) and weakly with invasion level ( r = 0.343 ; p = 0.01).conclusions : despite the low mdm2 expression rate observed in this study , direct relationships between mdm2 positivity and tumor thickness and invasion level were identified . 
 mdm2 expression can thus be suggested as a potential new predictive prognostic factor .