Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.     

Identification of higher risk thin melanomas should be based on Breslow depth not Clark level IV

BACKGROUND: There is good prognostic correlation for the two microstaging systems, Breslow depth and Clark level, commonly used to stage melanomas. Many investigators have reported that Breslow depth is the superior microstaging method. Although Clark level has been dropped from most of the proposed American Joint Committee on Cancer (AJCC) melanoma staging system, the AJCC system still includes Clark Level IV as a criterion for upstaging thin melanomas. The authors sought to determine whether this is appropriate, based on melanoma patient data in the Duke Comprehensive Cancer Center database. METHODS: Of the 8833 patients registered between January 1, 1970 and December 31, 1995, complete data on Breslow depth and Clark level was available for 4560 patients who were without nodal or metastatic disease at presentation. Ten-year survival was measured from the date of excision of the primary tumor until death from melanoma and analyzed using Kaplan-Meier and Cox proportional hazard methodologies. RESULTS: When analyzed separately, both increased Breslow thickness and Clark level correlated with shorter survival times. During subgroup analysis, Breslow thickness remained a significant prognostic indicator of survival at Clark Levels III and IV. Conversely, at narrow levels of Breslow thickness (i.e., 0-0.75 mm, > 0.75 -1.0 mm, > 1.0-1.5 mm) survival times were indistinguishable between Clark Levels III and IV. For the broader Breslow thickness interval of 0-1.0 mm, a barely significant difference between Clark Levels III and IV could be obtained. However, for this thickness range, even greater differences in survival could be obtained by merely comparing Breslow subgroups (i.e., < or = 0.8 mm vs. > 0.8-1.0 mm, < or = 0.9 mm vs. > 0.9-1.0 mm). CONCLUSION: The authors' data suggested that, after controlling for Breslow depth, Clark level was not a good prognostic indicator for survival. If the AJCC's objective is to design a classification system that will reliably predict the higher risk melanomas, then the system should be based on tumor thickness, which is clearly a better prognostic indicator, and should not be modified because of Clark level.     

Co-Expression of Putative Cancer Stem Cell Markers,CD133 and Nestin,in Skin Tumors

Background: Cancer stem cells (CSC) are populations of cells responsible for tumor initiation, progression andtherapeutic resistance in many cancers. In the present study, we aimed to investigate the expression pattern andclinical significance of two CSC markers, CD133 and Nestin, in a series of skin tumors. Materials and Methods:One hundred and thirteen paraffin blocks from skin cancers including 16 (14%) cases of melanoma, 37 (33%)of squamous cell cancer (SCC) and 60 (53%) of basal cell cancer (BCC) were collected and assembled in a tissuemicroarray (TMA). The samples were immunohistochemically examined for the expression of CD133 and Nestin.Expression of these markers was also correlated with clinicopathological parameters. Results: A significantdifference was observed in the expression of CD133 and Nestin in melanomas, SCC and BCC (p value=0.001).Furthermore, the level of expression was significantly higher in the melanomas compared to the SCC and BCCtumors. Expression of CD133 in the melanoma was significantly associated with increased tumor invasiveness (pvalue=0.05), a higher rate of metastasis (p value=0.04) and the presence of ulceration (p value=0.02). Increasedexpression of Nestin was observed in metastatic melanoma (p value=0.04), while no statistically significantcorrelation was found with other clinicopathological parameters including Breslow thickness, Clark level andulceration. Conclusions: Elevated expression levels of CD133 and Nestin in the melanomas are associated withadvanced disease, with more aggressive and metastatic skin tumors. Therefore, these markers could be potentialtherapeutic targets for malignant tumors of the skin.     

Estrogen receptor-alpha methylation predicts melanoma progression

The role of estrogen receptor alpha (ER-alpha) in melanoma is unknown. ER-alpha expression may be regulated in melanoma via hypermethylation of promoter CpG islands. We assessed ER-alpha hypermethylation in primary and metastatic melanomas and sera as a potential tumor progression marker. ER-alpha methylation status in tumor (n = 107) and sera (n = 109) from American Joint Committee on Cancer (AJCC) stage I to IV melanoma patients was examined by methylation-specific PCR. The clinical significance of serum methylated ER-alpha was assessed among AJCC stage IV melanoma patients receiving biochemotherapy with tamoxifen. Rates of ER-alpha methylation in AJCC stage I, II, and III primary melanomas were 36% (4 of 11), 26% (5 of 19), and 35% (8 of 23), respectively. Methylated ER-alpha was detected in 42% (8 of 19) of stage III and 86% (30 of 35) of stage IV metastatic melanomas. ER-alpha was methylated more frequently in metastatic than primary melanomas (P = 0.0003). Of 109 melanoma patients' sera in AJCC stage I, II, III, and IV, methylated ER-alpha was detected in 10% (2 of 20), 15% (3 of 20), 26% (5 of 19), and 32% (16 of 50), respectively. Serum methylated ER-alpha was detected more frequently in advanced than localized melanomas (P = 0.03) and was the only factor predicting progression-free [risk ratio (RR), 2.64; 95% confidence interval (95% CI), 1.36-5.13; P = 0.004] and overall survival (RR, 2.31; 95% CI, 1.41-5.58; P = 0.003) in biochemotherapy patients. Hypermethylated ER-alpha is a significant factor in melanoma progression. Serum methylated ER-alpha is an unfavorable prognostic factor.     

Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction-based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastases = 54.5, lymph node metastases = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.     

Identification of a novel gelsolin truncate in the vertical and metastatic phase malignant melanomas

Examination of 38 human melanoma samples by Western blotting analysis with anti-gelsolin antibodies showed that a new 85 kDa truncated gelsolin (GSNp85), co-expressed with wild-type gelsolin, was frequently expressed in vertical growth phase melanomas (Clark level II-IV) and metastatic growth phase melanomas. The GSNp85 truncate was not expressed in radial growth phase melanomas (Clark level I), acquired naevi, other skin cancers or normal skin tissues. Peptide-sequencing analysis revealed that GSNp85 lacks the C-terminal domain of wild-type gelsolin at the region containing the caspase-8 recognition site IETD. Caspase-8 processing was detected in GSNp85-positive but not GSNp85-negative melanomas. These data suggest that GSNp85 is a cleavage product of caspase-8 and may be useful as a new marker for the vertical or metastatic growth phase of malignant melanoma.     

Expression of p16, p27, p53, p73 and Nup88 proteins in matched primary and metastatic melanoma cells

Cutaneous melanoma is a tumor with high metastatic potential, but the mechanisms leading to progression are still not fully understood. In this study, we examined whether p16, p27, p53, p73 and Nup88 proteins were involved in the progression from primary to metastatic melanomas by immunocytochemistry and Western blotting in eleven melanoma cell lines from five matched primary and metastatic melanomas. We demonstrated that the primary and metastatic melanomas expressed differently p16, p27, p73 and Nup88 proteins. When expressed in the primary melanoma cells p16 and p27 were lost or reduced in almost all the metastatic melanoma cell lines. In contrast, p73 and Nup88 were expressed in most of the tested melanoma cell lines and were increased in the metastatic melanomas. p53 was expressed at the same level in both the primary and metastatic melanoma cells. These data suggest that a reduced expression of p16 and p27 and an enhanced expression of p73 and Nup88 might play an important role in the progression of melanoma from primary tumor to metastasis.     

Expression of collagenases-1 and -3 and their inhibitors TIMP-1 and -3 correlates with the level of invasion in malignant melanomas.

Since proteolysis of the dermal collagenous matrix and basement membranes is required for local invasive growth and early metastasis formation of cutaneous melanomas, we have analysed the activities/expression levels of certain metalloproteinases in melanomas and cultured melanoma cells by in situ hybridization and Northern analysis. In addition to collagenases-1 and -3 that have been implicated in invasive growth behaviour of various malignant tumours, we analysed the levels of 72-kDa gelatinase and its activators MT1-MMP and TIMP-2 in cultured melanoma cells. The lesions examined included three cases of lentigo maligna and 28 cases of Clark grade I-V melanomas. The premalignant as well as the grade I tumours were consistently negative for collagenase-1 and -3 and TIMP-1 and -3. The collagenases were predominantly expressed in the cancer cells of Clark grade III and IV tumours. TIMP-1 and -3 were abundantly expressed in the cancer and/or stromal cells of grade III and IV melanomas, while TIMP-2 protein was detected also in melanomas representing lower invasive potential. Northern analysis of seven melanoma cell lines showed that the expression of collagenase-1 and TIMPs-1 and -3 was associated with 72-kDa gelatinase positivity. All melanoma cell lines were positive for MT1-MMP and TIMP-2 mRNAs. Our results suggest that overexpression of collagenases-1 and -3 and TIMPs-1 and -3 is induced during melanoma progression. Expression of TIMPs may reflect host response to tumour invasion in an effort to control MMP activity and preserve extracellular matrix integrity.     

C6 ceramide potentiates curcumin-induced cell death and apoptosis in melanoma cell lines in vitro

Purpose

The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents, and long-term survival for patients with melanoma who have metastatic disease is dismal. Consequently, the search for novel anti-melanoma agents is urgent. Here, we evaluate the potential effects of C6 ceramide to sensitize melanoma cell lines (B16 and WM-115 cells) to curcumin-induced cell death.

Methods

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to test melanoma cell viability in vitro. Hoechst 33342 fluorescence and Histone DNA ELISA was used to evaluate melanoma cell apoptosis. Apoptosis-associated proteins in melanoma cells after treatments were measured by Western blot.

Results

C6 ceramide promotes curcumin-induced cell death and apoptosis in B16 and WM-115 melanoma cell lines. Curcumin itself promotes pro-apoptosis protein Caspase 3 and Caspase 9 cleavage and anti-apoptosis protein Bcl-XL and X-IAP degradation, and combination of C6 ceramide with curcumin dramatically enhances it. Caspase inhibitors largely inhibit C6-ceramide plus curcumin induced cell death and apoptosis.

Conclusion

We suggest that C6 ceramide sensitizes melanoma cell to curcumin induced cell death and apoptosis in vitro, which is due to, at least in part, the augment of mitochondria apoptosis pathway. Combining C6 ceramide with traditional chemotherapy drugs such as curcumin may have potential to be used as a new therapeutic intervention against melanoma.     

Linear discriminant analysis of dermoscopic parameters for the differentiation of early melanomas from Clark naevi

As a first step to develop a screening system for pigmented skin lesions, we performed digital discriminant analyses between early melanomas and Clark naevi. A total of 59 cases of melanoma, including 23 melanoma in situ and 36 thin invasive melanomas (Breslow thickness < or =0.75 mm), and 188 clinically equivocal, histopathologically diagnosed Clark naevi were used in our study. After calculating 62 mathematical variables related to the colour, texture, asymmetry and circularity based on the dermoscopic findings of the pigmented skin lesions, we performed multivariate stepwise discriminant analysis using these variables to differentiate melanomas from naevi. The sensitivities and specificities of our model were 94.4 and 98.4%, respectively, for discriminating between melanomas (Breslow thickness < or =0.75 mm) and Clark naevi, and 73.9 and 85.6%, respectively, for discriminating between melanoma in situ and Clark naevi. Our algorithm accurately discriminated invasive melanomas from Clark naevi, but not melanomas in situ from Clark naevi.     

人参皂甙Rg3和核糖核酸酶抑制因子转基因对小鼠黑色素瘤的抑制作用研究

目的观察人参皂甙Rg3和核糖核酸酶抑制因子(RI)转基因对小鼠B16黑色素瘤肺转移的抑制作用和影响,探讨人参皂甙Rg3和RI抗肿瘤生长和转移作用的分子机制。方法制备转RI基因的B16黑色素瘤肺转移小鼠模型,对野生型对照组(W组)、空质粒转染组(B组)和RI转基因组(RI组)以及给予Rg3的野生型对照组(Rg3/W组)、空质粒转染组(Rg3/B组)和RI转基因组(Rg3/RI组)中,荷瘤小鼠肺重量、肿瘤转移灶数目、生存期和肿瘤组织微血管密度进行检测和分析。结果Rg3和RI转基因使荷瘤小鼠肺重量降低,肿瘤转移灶数目减少,其肺重量降低和肿瘤转移灶数目减少的程度以Rg3/RI组最明显,Rg3/B组、Rg3/W组和RI组次之,与W组和B组差异有显著性(P<0.01),Rg3和RI有一定的协同性。Rg3和RI可延长荷瘤小鼠的生存期,Rg3/RI组小鼠在观察期(1.5个月)内均存活,W组和B组小鼠全部死亡(至26d),且出现死亡的时间较早。经HE染色和第Ⅷ因子相关抗原的免疫组化分析显示,Rg3和RI使肺内瘤组织的微血管密度降低,降低的程度为Rg3/RI组>Rg3/B组>Rg3/W组>RI组>B组>W组。结论人参皂甙Rg3可增强RI转基因对小鼠黑色素瘤肺转移的抑制作用,人参皂甙Rg3和RI基因在抗肿瘤生长、转移及血管生成方面有协同作用。     

Recurrence potential of thin primary melanomas

Between April 1971 and January 1985, the University of California at Los Angeles (UCLA) Division of Surgical Oncology has seen 649 patients diagnosed with thin primary melanomas, as defined by a Clark level less than IV and Breslow depth less than 0.76 mm. Thirty-six patients, whose primary diagnosis was confirmed by UCLA pathologists, presented with or subsequently developed melanoma metastases. Twenty-four (67%) had Clark level II lesions, whereas the remaining 12 (33%) had Clark level III lesions. Metastases were noted more frequently in men than in women, with a man-to-woman ratio of 3 to 2. The site of the primary lesion in this group was most frequently on the trunk (16 of 36 cases, 44%), followed by the head and neck (9 of 36 cases, 25%) and arm and shoulder (7 of 36 cases, 19%). Metastasizing thin lesions were least likely to occur on the leg and hip (4 of 36 cases, 11%). Twenty of these lesions were recently reexamined, and 17 of 20 cases (85%) demonstrated evidence of regression. Twenty-eight patients (78%) had their first metastases to regional lymph nodes. Fifteen remained alive with no evidence of disease at a median follow-up of 99 months. Twenty patients died from systemic metastases, and one patient was alive with brain metastasis at the time of this article. Although infrequent, metastases from thin melanomas do occur, suggesting the need for careful long-term follow-up and immediate investigation of adenopathy even in low-risk patients.     

The familial dysplastic nevus syndrome: Natural history and the impact of screening on prognosis a study of nine families in the Netherlands

Since 1982, nine families with the dysplastic nevus syndrome have been identified in the Leiden area (The Netherlands). A total of 50 primary melanomas were diagnosed in 38 persons. Nineteen of these melanomas had been diagnosed before the start of the screening programme (category I), 11 were detected at the initial examination of the families (category II), and 20 were found during the course of follow-up (category III).To assess the effect of screening, we compared these categories with respect to the developmental stage of the melanomas. One of the 19 melanomas in category I, two of the 11 in category II and seven of the 20 in category III were melanoma in situ. The average thickness of the invasive melanomas in categories I, II and III was 1.75, 0.80 and 0.54 mm respectively. Sixteen of the 19 melanomas in category I (84%) were Clark III or IV, whereas 15 of the 20 melanomas in category III (75%) were Clark I or II.From these findings it may be concluded that screening can lead to the detection of melanomas at an earlier stage, which in turn can permit curative treatment and improvement of both prognosis and life expectancy.The need for supervision based on central registration of affected families to guarantee the continuity of screening is discussed.     

Breslow thickness and clark level in melanoma: support for including level in pathology reports and in American Joint Committee on Cancer Staging

BACKGROUND: Thickness is known to be an important survival prognosticator for cutaneous melanoma, but controversy exists as to whether Clark level of invasion retains prognostic significance once thickness has been accounted for. A recent proposal to eliminate Clark level from the staging system for melanoma of the American Joint Committee on Cancer (AJCC) prompted the authors to investigate whether level adds useful prognostic information to Breslow thickness. They used the data base of the New York University Melanoma Cooperative Group (NYU-MCG) Registry. METHODS: The analysis was based on 919 patients with AJCC Stage I or II melanomas diagnosed between 1972 and 1982 and followed for an average of 10.9 years. Melanoma thicknesses were divided into 4 categories (< or = 0.75, 0.76-1.50, 1.51-4.00, and >4.00 mm). Patients were cross-classified according to tumor thickness and Clark level (II-V). For each combination of thickness and level, the Kaplan-Meier survival curve and 10-year survival proportion were computed, using death from melanoma as the outcome. The impact of Clark level on survival was evaluated for each of the thickness categories. The Cox proportional hazards model was used to assess the simultaneous effect of thickness and level on survival while controlling for other important prognostic factors, i.e., age, tumor location, and presence or absence of ulceration. RESULTS: Level of invasion was a significant predictor of death from melanoma in each of the four thickness categories. Likewise, in the Cox analyses, level was a significant prognostic variable, even after thickness was included in the model and regardless of whether thickness was treated as a categoric or a continuous variable. CONCLUSIONS: These results confirm that both tumor thickness and level of invasion are important independent prognostic factors in AJCC Stage I and II melanomas. The authors recommend that Clark levels be kept as criteria in the AJCC staging system and be included in pathology reports. [See editorial on pages 491-6, this issue.] Copyright 2000 American Cancer Society.     

Monoclonal antibody 4C5 immunostains human melanomas and inhibits melanoma cell invasion and metastasis.

PURPOSE: Tumor cell metastasis constitutes a major problem in the treatment of cancer. Because the cure rate of metastatic tumors is very low, new therapeutic approaches are needed. Heat shock protein 90 (HSP90) is a molecular chaperone that is recognized as a new target for the treatment of cancer. Here, we examine the value of a monoclonal antibody (mAb) against HSP90, mAb 4C5, as a potential marker in malignant melanomas. Moreover, we investigate the possibility to use mAb 4C5 as an inhibitor of melanoma cell invasion and metastasis. EXPERIMENTAL DESIGN: Paraffin blocks of formalin-fixed human melanoma tumor tissues were used to prepare tissue microarrays. The B16 F10 melanoma cell line was used in all the in vitro experiments. To assess melanoma cell invasion, the wound-healing assay and the Matrigel invasion assay were applied. To evaluate the effect of mAb 4C5 on tumor metastasis, we used an experimental model of metastatic melanoma. RESULTS: Immunohistochemical studies done on a panel of malignant melanomas showed positive immunostaining with mAb 4C5 in all cases. mAb 4C5 inhibits B16 F10 cell invasion by binding to surface HSP90 because it is not internalized. mAb 4C5 significantly inhibits melanoma metastasis in C57BL/6 mice inoculated with B16 F10 cells. CONCLUSIONS: mAb 4C5 could be potentially used as a novel specific marker for malignant melanomas. mAb 4C5 inhibits melanoma cell invasion in vitro by binding to cell surface HSP90 expressed on B16 F10 melanoma cells. Finally, this antibody significantly inhibits melanoma metastasis, thus rendering it a potential therapeutic agent for the treatment of cancer metastasis.     

A preliminary report of a pilot trial in adjuvant chemotherapy of primary melanoma.

It is well known that level of skin invasion and tumor thickness are significant prognostic factors in the evolution of primary melanoma. The prognosis of primary melanoma Clark III to V skin invasion level and more than 1.5 mm thick confirms this statement. Even the prophylactic dissection of regional lymph nodes has not improved results. In an attempt to obtain better results in the treatment of primary melanomas, a pilot trial was carried out combining surgery and adjuvant chemotherapy. A group of 21 patients with Clark III, IV and V level primary melanoma who underwent adjuvant polychemotherapy (velba + dactinomycin + procarbazine) for 1 year after surgery showed a very low incidence of recurrences (5%) after 24 months of observation. The historical control group, with the same level of tumor skin invasion, treated only surgically had in the same follow-up period a recurrence rate of 65%. This difference was statistically significant (p less than 0.01). All patients who received adjuvant chemotherapy survived 2 years whereas survival was 77% (p less than 0.05) in the surgical historical control group. Favorable results with the same protocol of adjuvant chemotherapy were not obtained in the group of 16 patients with stage II melanoma when compared with primary tumors. However, 4 recurrences were observed after 12 months of observation; toxic side effects of adjuvant chemotherapy were mild and tolerable. Considering the insufficient number of clinical trials with adjuvant chemotherapy, as well as sometimes controversial results, further randomized clinical studies are needed to establish the actual value of this conbined method in the treatment of primary melanoma with a high risk of dissemination.     

Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 and type 2 in stage I malignant melanoma

The urokinase-type plasminogen activator (uPA) and its inhibitors type 1 (PAI-1) and type 2 (PAI-2) are considered to have a key role in the process of invasion and metastasis. We investigated the differences in uPA, PAI-1 and PAI-2 concentrations in primary cutaneous melanoma and normal skin and correlations with well-established melanoma prognostic factors. The study was performed on 43 patients (19 men, 24 women; mean age 57 years) with histologically confirmed primary melanomas <1.5 mm thick. The uPA concentrations were determined in 36 pairs of triton extracts, and the PAI-1 and PAI-2 concentrations in 43 pairs of cytosols prepared from the tumour and adjacent normal tissue samples (matched pairs). The uPA, PAI-1 and PAI-2 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Significantly higher concentrations of both uPA and PAI-1 were measured in melanomas than in normal surrounding skin (uPA: 1.08 vs 0.48 ng/mg total protein (mgp), p<0.001; PAI-1: 14.07 vs 2.07 ng/mgp, p<0.001). The melanoma uPA, PAI-1 and PAI-2 concentrations correlated significantly (p<0.05) with normal skin (r=0.73, 0.54, 0.38 respectively). The uPA concentrations positively correlated with those of PAI-1 measured in melanomas (r=0.45, p<0.01). PAI-1 values were significantly lower (p<0.001) in the melanomas of Breslow thickness < or =0.75 mm, Clark invasion 0.75 mm, Clark invasion of > or =II and < or =III, with microscopic ulceration and vascular invasion (22.25, 17.67, 27.67, 37.77, respectively). Determination of uPA and PAI-1 can provide significant additional prognostic information for melanoma patients.     

HDM2 protein overexpression, but not gene amplification, is related to tumorigenesis of cutaneous melanoma

We investigated the role of alterations of HDM2, the human homologue of murine mdm2, in the tumorigenesis and progression of cutaneous melanoma. A well-characterized cohort of 172 cases representing different points in the spectrum of melanocyte transformation (16 dysplastic nevi, 11 melanomas in situ, 107 invasive primaries, and 38 metastatic lesions), as well as 11 human melanoma cell lines were examined by immunohistochemistry and Western blotting for HDM2 protein expression, and by either Southern blotting (SB) or fluorescence in situ hybridization for HDM2 gene amplification. HDM2 overexpression, defined as >20% tumor cells showing nuclear immunoreactivity, was observed in 1 of 16 (6%) dysplastic nevi, 3 of 11 (27%) melanomas in situ, and 81 of 145 (56%) invasive primary and metastatic melanomas. Comparable frequencies of HDM2 overexpression were observed among invasive primary cases with differing tumor thicknesses as well as among the metastatic cases: 21 of 40 (53%) at < or =1.5 mm; 31 of 50 (62%) at 1.6-3.9 mm; 10 of 17 (58%) at >4 mm; and 19 of 38 (50%) metastases. HDM2 amplification was observed in 1 of 88 (1%) primary cases using fluorescence in situ hybridization, and in 0 of 12 (0%) metastatic cases that overexpressed HDM2 using SB. Melanoma cell lines expressed HDM2 protein, but there was no evidence of amplification by SB. Our data suggest that HDM2 protein overexpression is common in invasive and metastatic melanoma. Observing HDM2 overexpression in noninvasive melanoma suggests that expression of this oncogene may play an early role in melanocyte transformation. HDM2 amplification occurs infrequently, and other mechanisms that up-regulate HDM2 expression are under investigation.