© 2002 by Oxford University Press
Journal of the National Cancer Institute, Vol. 94, No. 23, 1803-1806,
December 4, 2002
© 2002 Oxford University Press
BRIEF COMMUNICATION |
HDM2 Protein Overexpression and Prognosis in Primary Malignant Melanoma
Affiliations of authors: D. Polsky, K. Melzer, C. Hazan, H. Kamino, J. G. Spira, A. W. Kopf, Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York; I. Osman, Ronald O. Perelman Department of Dermatology, Department of Urology, and Kaplan Comprehensive Cancer Center, New York University School of Medicine; K. S. Panageas (Department of Epidemiology and Biostatistics), K. Busam, M. Drobnjak, C. Cordon-Cardo (Department of Pathology), A. Houghton (Department of Immunology), Memorial Sloan-Kettering Cancer Center, New York.
Correspondence to: Iman Osman, M.D., New York University Medical Center, 550 1st Ave., Department of Dermatology, H-100, New York, NY 10016 (e-mail: Iman.Osman{at}med.nyu.edu).
ABSTRACT
Overexpression of the oncogene HDM2 is observed in a substantial proportion of melanomas, including noninvasive and thin lesions, suggesting that HDM2 overexpression may be an early event in melanocyte transformation. To determine the role of HDM2 in the clinical progression of melanoma, we examined whether its expression was associated with patient survival. From November 1972 through November 1982, 134 patients with melanoma who participated in the New York University Melanoma Cooperative Group were studied, if representative tissues and follow-up were available. HDM2 protein expression was assessed immunohistochemically. Unexpectedly, we observed that HDM2 overexpression was statistically significantly associated with improved disease-free survival (relative risk [RR] = 0.47, 95% confidence interval [CI] = 0.24 to 0.89; two-sided 
2 P = .021) and overall survival (RR = 0.55, 95% CI = 0.33 to 0.94; two-sided 2 P = .027) in multivariable analysis. HDM2 overexpression appears to be an independent predictor of survival for patients with primary melanoma; however, larger prospective studies are required for validation.
HDM2, the human homologue of murine mdm2, encodes a 90-kd zinc-finger protein (HDM2) that binds to the transcriptional activation domain of p53, blocking its ability to regulate target genes and targeting p53 for proteosome-mediated enzymatic degradation by ubiquitination (1). HDM2 is a transcriptional target of p53, forming an autoregulatory feedback loop that keeps p53 levels under tight control. Transgenic mice overexpressing mdm2 are tumor-prone, developing mainly lymphomas and sarcomas (2). In humans, HDM2 gene amplification and protein overexpression have been observed in many cancers (3,4). Overexpression of the oncogene HDM2 is observed in a substantial proportion of melanomas, including noninvasive and thin lesions, suggesting that HDM2 overexpression may be an early event in melanocyte transformation (5). The association between HDM2 and clinical outcome has been studied in several human malignancies to examine the utility of HDM2 as a clinical prognostic marker (6,7). HDM2 has not been investigated previously as a prognostic marker for melanoma. Consequently, we determined whether the expression of HDM2 was associated with the survival of patients with melanoma.
We studied 134 patients with American Joint Committee on Cancer stage I, II, and III melanoma, identified by reviewing the database of the Melanoma Cooperative Group at New York University Medical Center, for the availability of representative tissues and adequate follow-up data. This prospective study enrolled patients with primary cutaneous melanoma from November 1972 through November 1982 documenting pathologic and clinical factors and clinical outcome. Of the 134 patients studied, 65 died during follow-up, and 69 were censored. Median follow-up among the survivors was 12.7 years (range = 7–18.5 years). We compared data from these 134 patients with data from those without available tissues to rule out selection bias and did not observe statistically significant differences with regard to sex, age at diagnosis, or the location, thickness, ulceration, stage, or histologic type of the primary lesion (data not shown). The Institutional Board of Research Associates at New York University Medical Center approved the tissue acquisition protocol to conduct the correlative immunohistochemical study. Samples were 5-µm sections of paraffin-embedded tissue specimens retrieved from the pathology archives of New York University. Expression of HDM2, p53, and Ki-67 was assessed by immunohistochemistry with the following antibodies and dilutions: clone 2A10 (anti-HDM2; provided by A. Levine, Rockefeller University Laboratories) used at a 1 : 500 dilution, PAB1801 (anti-p53; Calbiochem/Oncogene Science, Boston, MA) used at a 1 : 500 dilution, and MIB-1 (anti-Ki-67; Immunotech, Marseille, France) used at a 1 : 50 dilution (6,7). After analysis of the entire lesion, nuclear immunoreactivity of the tumor cells for each marker was classified on a scale from undetectable (i.e., 0%) to homogenous staining (i.e., 100%). For samples with a heterogeneous pattern of expression, the percentage of immunoreactive nuclei was obtained from regions of the tumor exhibiting the strongest immunoreactivity, as described (8). The choice of cut points was based on our group’s prior experience with these antibodies in clinical specimens of bladder, prostate, squamous cell carcinomas, and sarcomas, in which we found that the 20% cut point stratifies patients in a clinically relevant fashion (6,7,9). This study was designed to test the 20% cut point, a priori, in melanoma. We have to emphasize that the appropriate cut points for primary melanoma remain to be established in analyses of larger prospective cohorts of patients. Clinical data collection and immunohistochemical analysis were performed independently of each other, with respective investigators blinded to the clinical outcome and HDM2 result until the study was completed. To confirm the specificity of the 2A10 antibody for HDM2 in tissue specimens, 20 primary melanomas were also stained with anti-HDM2 antibody, clone 1B10 (Novocastra Laboratories, Ltd., Newcastle upon Tyne, U.K.), at a 1 : 100 dilution. This antibody recognizes an epitope that does not overlap with that recognized by 2A10. We observed similar results with both antibodies, confirming the specificity of 2A10 for HDM2 in melanoma tissue sections.
The association between HDM2 expression and disease-free survival (DFS) and overall survival (OS) was assessed with Fisher’s exact test and the Mantel–Haenszel test for trend (10). DFS was calculated from the date of initial surgery to the date of recurrence or last follow-up. OS was calculated from date of initial surgery to the date of death or last follow-up. DFS and OS were estimated with the Kaplan–Meier method and compared with the log-rank test (11). Multivariable analyses were performed with the Cox proportional hazards method to obtain estimates of relative risk (12). Assumptions for Cox proportional hazards were met for these data.
HDM2 protein overexpression was observed in 101 of 134 (75.4%) patients (Fig. 1, A and B
). This result parallels our previous observation with a different cohort of 107 patients with primary melanoma (5). We found no statistically significant association between HDM2 protein expression and baseline characteristics or p53 or Ki-67 expression. However, we did observe a trend between HDM2 overexpression and thinner lesions (P = .08) and between HDM2 overexpression and the location on the extremity (P = .07) (Table 1). Overexpression of p53 was observed in 22 of 134 (16.4%) patients. No association was observed between p53 overexpression and baseline characteristics. In addition, no correlation was observed between p53 overexpression and clinical outcome (data not shown). The detailed analysis of Ki-67 in this cohort has been recently published (13). Analysis of immunohistochemical results did not reveal any trends linking expression of the markers with the duration of specimen storage.
|
|
Univariate analyses showed that HDM2 protein overexpression was statistically significantly associated with longer DFS (two-sided log-rank, P = .021) (Fig. 1, C
) and with better OS (two-sided log-rank P = .023) (Fig. 1, D). This association remained even after controlling for other well-known prognostic factors in a multivariable analysis. The Cox regression analysis showed that HDM2 protein overexpression was associated with better DFS (relative risk [RR] = 0.47, 95% CI = 0.24 to 0.89; two-sided 2 P = .021) and OS (RR = 0.55, 95% CI = 0.33 to 0.94; two-sided 2 P = .027) (Table 2). In addition, treating HDM2 expression as a continuous variable, HDM2 expression was statistically significantly associated with OS (P = .05).
|
We speculated (5) that HDM2 overexpression was an early oncogenic event in melanocyte transformation. If HDM2 were functioning as an oncogene, we would expect its overexpression to be associated with a poorer clinical outcome. In contrast, we observed that HDM2 overexpression was statistically significantly associated with longer DFS and OS. This association was maintained after controlling for other statistically significant outcome predictors, including tumor thickness, ulceration, and anatomic location. One explanation for this result is that overexpression of HDM2 may indicate the presence of a functional p53 that exerts an inhibitory effect on tumor cell growth and activates the HDM2 promoter as part of its normal autoregulatory feedback loop. This possibility is supported by the study of Landers et al. (14) in which HDM2 transcripts in human melanoma cell lines originated from the p53-responsive promoter and transfection of two cell lines with a construct encoding a human papillomavirus E6 protein, which binds to p53 blocking its function, led to decreased levels of HDM2 mRNA and protein. Thus, these findings cast some doubt on the oncogenic role of HDM2 in melanoma.
Any analysis of prognostic factors in melanoma should include the status of regional lymph nodes when possible. Although the patients in this study were enrolled 10–20 years before the first reports describing sentinel lymph node mapping (15), 70% of patients had histopathologic examination of surgically resected lymph nodes, which was the standard of care at the time. Currently, patients with melanomas thicker than 1.0 mm are the ones most likely to undergo sentinel lymph node examination. We compared the rates of lymph nodal disease by thickness category in our cohort with rates from Gershenwald et al. (16) and found similar proportions.
In conclusion, HDM2 overexpression appears to be an independent predictor of survival for patients with primary melanoma. However, larger prospective studies are required for validation.
NOTES
Supported by grants from the American Skin Association and grant K08 AR02129 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (to D. Polsky).
We thank the Manhattan Veterans Affairs Medical Center (New York, NY) for the use of their facilities.
REFERENCES
1 Piette J, Neel H, Marechal V. Mdm2: keeping p53 under control. Oncogene 1997;15:1001–10.[CrossRef][Web of Science][Medline]
2 Jones SN, Hancock AR, Vogel H, Donehower LA, Bradley A. Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. Proc Natl Acad Sci U S A 1998;95:15608–12.
3 Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification database. Nucleic Acids Res 1998;26:3453–9.
4 Shiina H, Igawa M, Shigeno K, Yamasaki Y, Urakami S, Yoneda T, et al. Clinical significance of mdm2 and p53 expression in bladder cancer. A comparison with cell proliferation and apoptosis. Oncology 1999;56:239–47.[CrossRef][Web of Science][Medline]
5 Polsky D, Bastian BC, Hazan C, Melzer K, Pack J, Houghton A, et al. HDM2 protein overexpression, but not gene amplification, is related to tumorigenesis of cutaneous melanoma. Cancer Res 2001;61:7642–6.
6 Osman I, Drobnjak M, Fazzari M, Ferrara J, Scher HI, Cordon-Cardo C. Inactivation of the p53 pathway in prostate cancer: impact on tumor progression. Clin Cancer Res 1999;5:2082–8.
7 Osman I, Scher HI, Zhang ZF, Pellicer I, Hamza R, Eissa S, et al. Alterations affecting the p53 control pathway in bilharzial-related bladder cancer. Clin Cancer Res 1997;3:531–6.[Abstract]
8 Rieger E, Hofmann-Wellenhof R, Soyer HP, Kofler R, Cerroni L, Smolle J, et al. Comparison of proliferative activity as assessed by proliferating cell nuclear antigen (PCNA) and Ki-67 monoclonal antibodies in melanocytic skin lesions. A quantitative immunohistochemical study. J Cutan Pathol 1993;20:229–36.[CrossRef][Web of Science][Medline]
9 Osman I, Sherman E, Singh B, Scher H, Zelefsky M, Shah J, et al. Inactivation of p53-pathway in squamous cell carcinoma of the head and neck: impact on treatment outcome in patients treated with larynx preservation intent. J Clin Oncol 2002;20:2980–7.
10 Agresti A. Categorical data analysis. New York (NY): John Wiley & Sons; 1990.
11 Kaplan EL, Meier P. Nonparametric estimation for incomplete observations. J Am Stat Assoc 1958;53:457–81.[CrossRef][Web of Science]
12 Cox DR. Regression models and life-tables. J Royal Stat Soc 1972;34:187–220.
13 Hazan C, Melzer K, Panageas KS, Li E, Kamino H, Kopf AW, et al. Evaluation of the proliferative marker MIB-1 in the prognosis of cutaneous malignant melanoma. Cancer 2002;95:634–40.[CrossRef][Web of Science][Medline]
14 Landers JE, Cassel SL, George DL. Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res 1997;57:3562–8.
15 Morton DL, Wen DR, Wong JH, Economou JS, Cagle LA, Storm FK, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992;127:392–9.
16 Gershenwald JE, Thompson W, Mansfield PF, Lee JE, Colome MI, Tseng CH, et al. Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage I or II melanoma patients. J Clin Oncol 1999;17:976–83.
Manuscript received April 23, 2002; revised September 12, 2002; accepted September 25, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. E. G. Rothberg, M. B. Bracken, and D. L. Rimm Tissue Biomarkers for Prognosis in Cutaneous Melanoma: A Systematic Review and Meta-analysis J Natl Cancer Inst, April 1, 2009; 101(7): 452 - 474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. F. Firoz, M. Warycha, J. Zakrzewski, D. Pollens, G. Wang, R. Shapiro, R. Berman, A. Pavlick, P. Manga, H. Ostrer, et al. Association of MDM2 SNP309, Age of Onset, and Gender in Cutaneous Melanoma Clin. Cancer Res., April 1, 2009; 15(7): 2573 - 2580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Fecher, S. D. Cummings, M. J. Keefe, and R. M. Alani Toward a Molecular Classification of Melanoma J. Clin. Oncol., April 20, 2007; 25(12): 1606 - 1620. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Burger, Y. Gao, Y. Amemiya, H. J. Kahn, R. Kitching, Y. Yang, P. Sun, S. A. Narod, W. M. Hanna, and A. K. Seth A Novel RING-Type Ubiquitin Ligase Breast Cancer-Associated Gene 2 Correlates with Outcome in Invasive Breast Cancer Cancer Res., November 15, 2005; 65(22): 10401 - 10412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Berger, R. L. Camp, K. A. DiVito, H. M. Kluger, R. Halaban, and D. L. Rimm Automated Quantitative Analysis of HDM2 Expression in Malignant Melanoma Shows Association with Early-Stage Disease and Improved Outcome Cancer Res., December 1, 2004; 64(23): 8767 - 8772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. I. Bardos, N.-M. Chau, and M. Ashcroft Growth Factor-Mediated Induction of HDM2 Positively Regulates Hypoxia-Inducible Factor 1{alpha} Expression Mol. Cell. Biol., April 1, 2004; 24(7): 2905 - 2914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Onel and C. Cordon-Cardo MDM2 and Prognosis Mol. Cancer Res., January 1, 2004; 2(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||