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.
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 = 718.5 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 groups 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 Fishers exact test and the MantelHaenszel 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 KaplanMeier 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.
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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 1020 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.
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Manuscript received April 23, 2002; revised September 12, 2002; accepted September 25, 2002.
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