Tumor iNOS predicts poor survival for stage III melanoma patients

Authors

  • Suhendan Ekmekcioglu,

    1. Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    Search for more papers by this author
  • Julie A. Ellerhorst,

    1. Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    Search for more papers by this author
  • Victor G. Prieto,

    1. Department of Pathology and Dermatology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    Search for more papers by this author
  • Marcella M. Johnson,

    1. Department of Biostatistics and Applied Mathematics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    Search for more papers by this author
  • Lyle D. Broemeling,

    1. Department of Biostatistics and Applied Mathematics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    Search for more papers by this author
  • Elizabeth A. Grimm

    Corresponding author
    1. Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
    • Department of Experimental Therapeutics, Unit 362, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
    Search for more papers by this author
    • Fax: +713-792-2070.


Abstract

Inducible nitric oxide synthase (iNOS) produces nitric oxide, which has growth promoting activity in melanoma. A preliminary study of tumors from patients with Stage III melanoma who had received neo-adjuvant therapy revealed an association of tumor iNOS expression with shortened survival. The objective of the present study was to determine whether iNOS expression in tumors of newly diagnosed, untreated Stage III patients is predictive of survival. iNOS expression was examined by immunohistochemistry in tumors from 132 patients. The staining was evaluated for percentage of positive cells (Number score) and the intensity of staining (Intensity score). The association of iNOS expression with overall and disease-specific survival was tested in univariate and multivariate Cox proportional hazards regression models that included other known prognostic factors. Results of the univariate analysis demonstrated that the presence of iNOS in a patient's tumor, whether graded on the basis of Number or Intensity score, was associated with a significant increase in the hazard ratio of death from melanoma. These findings were corroborated by median survival data estimated from Kaplan Meier analysis. In the multivariate model including iNOS number or intensity, gender, age, number of lymph nodes, macroscopic disease and in-transit disease, only iNOS expression predicted survival. We conclude that a significant association exists between tumor iNOS expression and shortened survival in untreated Stage III melanoma patients. The ability of iNOS to predict outcomes for these patients may be independent of other known prognostic factors, providing a new molecular marker with significant potential for clinical utility. © 2006 Wiley-Liss, Inc.

Nitric oxide (NO) is a pleiotropic regulatory and signaling molecule, which plays a critical role in various biological processes, such as vasodilatation, neurotransmission, and immunity. This free radical gas is synthesized from L-arginine by a group of enzymes termed NO synthases (NOS). Because of the obvious difficulty in detecting the short-lived NO, expression of the NOS enzymes is commonly used experimentally as a surrogate for NO production. The inducible NOS isoform, iNOS, is expressed by many cell types upon stimulation by inflammatory cytokines and is able to rapidly generate high amounts of NO.1 Throughout the past decade, considerable interest has been generated in the contribution of aberrant, constitutive expression of iNOS and low amounts NO to the biology of some malignancies.2 Although a number of reports have suggested that very high levels of NO are cytostatic or cytotoxic for cancer cells, in many tumor types the constitutive production of low levels of intracellular NO promotes tumor growth and survival.3, 4, 5 Such is the case for melanoma, a malignancy in which iNOS and NO have been examined both in laboratory and clinical studies. In vitro data indicate that constitutive intracellular NO induces anti-apoptotic effects, prolonging the survival of melanoma cells.6, 7, 8 Data from our laboratory and from others have confirmed that iNOS is expressed constitutively in most cultured melanoma cells and is present in over 60% of paraffin embedded human melanoma samples.9, 10, 11

In a previous neoadjuvant biochemotherapy trial for melanoma patients with Stage III disease, we found that the presence of iNOS in the melanoma tumor cells of post-treatment biopsy samples was associated with significantly shortened patient survival.11 It was unclear from those studies whether iNOS was present prior to treatment or was induced as an inflammatory consequence of the therapy. These data, however, raised the possibility that iNOS might also be detected in melanoma cells from newly diagnosed Stage III patients, prior to receiving any treatment, and utilized as a predictor of outcome.

Currently, the major predictors of survival incorporated into the 2002 American Joint Committee on Cancer (AJCC) Staging System for Stage III melanoma patients include the number of involved lymph nodes, the presence of macroscopic vs. microscopic (sentinel lymph node or otherwise clinically undetectable) nodal disease, the presence of in transit metastases, and in some cases, the ulceration status of the primary tumor.12 The number of positive lymph nodes has historically been the most consistent prognostic factor.13, 14, 15 In spite of recent improvements in clinical staging techniques and prognostic models for Stage III melanoma patients, the variability of outcomes for individuals within the same group or subgroup suggests the existence of distinct biologic subtypes, yet to be identified by molecular markers.

In light of our earlier data suggesting that tumor iNOS expression is associated with poor outcomes for treated Stage III melanoma patients, we hypothesized that the presence of tumor iNOS is predictive of survival for newly diagnosed, untreated Stage III patients. Accordingly, we have conducted a study to examine the association of iNOS expression with survival in this group of patients. Here we report that iNOS expression is a strong predictor of disease-specific and overall survival (OS) for Stage III melanoma patients, potentially providing an easily detected molecular marker to strengthen the current list of predictors of outcome.

Abbreviations:

CI, confidence interval; DSS, disease-specific survival; HR, hazard ratio; IHC, immunohistochemistry; iNOS, inducible nitric oxide synthase; LL, lower limit; NO, nitric oxide; OS, overall survival; UL, upper limit.

Patients and methods

Patient samples and data collection

The study was approved by the M. D. Anderson Cancer Center (MDACC) Institutional Review Board and was conducted in compliance with HIPAA regulations. Power calculations, based on 5-year survivals of 80% for iNOS negative patients and 20% for iNOS positive patients, determined that 150 subjects would be sufficient to test the hypothesis with an alpha of 0.05 and power in excess of 0.95. The study subjects were sequential Stage III patients from 1994 through 1997 at our institution for whom tumor material was identified as available in our Melanoma Informatics, Tissue Resource and Pathology Core, and for whom survival and other AJCC prognostic data were considered reliable. Eligibility for inclusion in the study included the diagnosis of stage III melanoma, no prior systemic therapy, and the availability of paraffin-embedded metastatic tumor tissue from which the stage III diagnosis was made. The following information was gathered from the medical records of study subjects: gender; age at melanoma diagnosis; date of stage III diagnosis, defined as the date of pathologic confirmation; administration of adjuvant interferon; features known to influence survival of Stage III melanoma patients including number of positive nodes, macroscopic vs. microscopic disease, the presence or absence of in-transit disease, and ulceration of the primary tumor; the date and cause of death, or date of last follow-up. Patient follow-up and survival were last updated in July, 2004.

Immunohistochemical studies

Paraffin-embedded sections of tumor samples were examined for melanoma tumor iNOS expression by immunohistochemistry (IHC) using an anti-iNOS monoclonal antibody (Transduction Laboratories, Lexington, KY). Pre-immune normal mouse IgG (Vector Laboratories, Burlingame, CA) and anti-vimentin antibody (BioGenex Laboratories, San Ramon, CA) were used as negative and positive controls, respectively. Tissue sections were deparaffinized and rehydrated, then placed in Antigen Unmasking Solution (Vector Laboratories) and microwaved intermittently for a total of 10 min, to maintain boiling temperature. After cooling, the slides were placed in 3% H2O2 in cold methanol for 15 min, and then 0.05% Triton X-100 (Sigma, St. Louis, MO) for 15 min. An avidin-biotin-peroxidase complex (ABC) kit (Vectastain, Vector Laboratories) was then used for antigen detection. After a 30-min incubation with blocking serum, the primary antibody was applied for 2 hr at room temperature, followed by 30 min incubations with secondary biotinylated antibody, and the ABC reagent. The immunolabeling was developed with the chromogen 3-amino-9-ethylcarbazole for 10 min. Hematoxylin was applied as a counter stain.

Immunolabeling was scored separately for 2 variables; first, for number of positive melanoma cells; second, for the overall intensity of immunoreactivity of the positive cells.16 Briefly, scoring for number of positive tumor cells was defined as follows: “0,” less than 5% positive cells; “1,” 5–25% positive cells; “2,” >25–75% positive cells; “3,” greater than 75% positive cells. Intensity scoring was defined as follows; “0,” no staining; “1,” light staining; “2,” moderate staining and “3,” intense staining. The slides were independently interpreted by 2 readers without knowledge of the clinical data. Any discrepancies in scores were subsequently reconciled.

Statistical analysis

The objective of the statistical analysis was to simultaneously assess the prognostic effects of melanoma tumor iNOS expression, gender, age at melanoma diagnosis, number of nodes, macroscopic disease and in-transit disease in regard to disease-specific survival (DSS) and overall survival (OS). Kaplan–Meier methodology was used to compute survival in months from the date of Stage III diagnosis to the date of death (for patients who died) or to the date of last follow-up (for those still alive). For DSS, censored patients included those still alive at last follow-up, those known to have died of causes other than melanoma, and those dead of unknown causes. For OS, censored patients included only those remaining alive at last follow-up. Age was dichotomized based on the median age of the entire cohort for further analyses. Univariate Cox proportional hazards regression models were fit to evaluate the prognostic effects for each of the earlier noted factors alone. Furthermore, multivariate Cox models were fit to simultaneously evaluate the predictive effects of all factors, separately for iNOS Number and iNOS Intensity. The full multivariate model was reduced iteratively 1 factor at a time such that all factors remaining in the final model were statistically significant at a 5% significance level. The log-rank test was utilized to compare median survival between groups. All analyses were performed using SAS and S-PLUS at a significance level of 5%.17, 18

Results

Patient characteristics

The initial study group included 164 patients with Stage III melanoma. Thirty-two subjects, for whom follow-up or other critical data were missing, were later excluded, leaving a final cohort of 132 subjects. The median age at melanoma diagnosis was 51 years (range 14–84 years). Fifty-five percent of the patients were male and 45% female. The median follow-up from diagnosis of Stage III disease was 49.5 months for all patients and 74 months for those remaining alive. Seventy-two (54.6%) patients were alive or had died from causes other than melanoma at the last evaluation date. The estimated median DSS from stage III diagnosis was 80 months (95% CI: 50, upper limit not attained); the estimated median OS was 62 months (95% CI: 46, 98).

Detection of iNOS in tumors samples

IHC methodology, first optimized on melanoma cell lines, was adapted with antigen retrieval for use in the staining of formalin-fixed paraffin-embedded metastatic tumor sections from all 132 patients (Fig. 1). Definitive iNOS expression, established as >5% of tumor cells positive, was found in 63 (47.7%) of the 132 tumors examined. iNOS immunoreactivity was consistently localized to the cytoplasm. Expression was scored separately for the percentage of iNOS-expressing melanoma tumor cells and for the intensity of immunoreactivity, as described in Methods.

Figure 1.

iNOS immunoreactivity in human melanoma tissues. (a) Representative IHC staining of a Stage III melanoma sample. The 3 panels represent the following controls or immunostaining: (a) Isotype-matched IgG control; (b) anti-Vimentin positive control and (c) anti-iNOS. (40×). (b) The bottom panel represents scoring scale of immunostainings from various tumor samples as follows: (a) “0” for less than 5% positive tumor cells, (b) “1” for 5–25% positive tumor cells and (c) “2” for 25–75% positive tumor cells. (20×).

Correlation of iNOS expression with survival

A univariate Cox proportional hazards model was used to examine the association of iNOS expression with DSS and OS. Results of the univariate analysis for DSS from the diagnosis of stage III disease are shown in Table I; similar results were found for OS (data not shown). The presence of iNOS in a patient's tumor, whether graded on the basis of Number or Intensity score, was associated with a significant increase in the hazard ratio (HR) of death from melanoma. For increasing Number scores of 1, 2 and 3, the corresponding HRs were 2.62, 6.03 and 6.73. Similarly, Intensity scores of 1, 2 and 3 were associated with HRs of 2.75, 6.73 and 9.69, respectively. For both scores, a high level of significance was reached.

Table I. Univariate Cox Proportional Hazards Modelfor iNOS Expression and Disease-Specific Survival From Stage III Diagnosis
 n1HR95% LL95% ULp-value
  •  HR, hazard ratio; 95% LL, lower limit of 95% confidence interval on hazard ratio; 95% UL, upper limit of 95% confidence interval on hazard ratio.

  • 1

    Total number may be less than 132 patients due to missing data.

Individual categories
iNOS Number
 0691.00   
 1212.621.215.680.015
 2296.033.2011.36<0.0001
 3136.733.0214.99<0.0001
iNOS Intensity
 0531.00   
 1402.751.335.680.0062
 2316.733.2913.75<0.0001
 389.693.7225.23<0.0001
Combined categories for multivariate analysis
iNOS Number
 0691.00   
 1 or 2 or 3634.602.638.06<0.0001
iNOS Intensity
 0531.00   
 1402.751.335.680.0062
 2 or 3397.233.6414.36<0.0001

The results of the Cox proportional hazards model were corroborated by Kaplan–Meier survival analysis (Fig. 1a and 1c), which again demonstrated a clear survival advantage for patients whose tumors lacked iNOS expression. Median survivals were not reached for individuals with Number or Intensity scores of “0.” Shortened median survivals of 59 (95% CI: 25, not attained), 27 (95% CI: 13, 46) and 29 (95% CI: 19, not attained) months were found for patients with iNOS Number scores of “1”, “2” and “3,” respectively. Intensity scores of “1,” “2” and “3” were associated with corresponding median survivals of 67 (95% CI: 46, not attained), 21 (95% CI: 15, 43) and 29 (95% CI: 12, not attained) months.

To prepare these data for multivariate analysis, different combinations of the iNOS score categories were examined in an attempt to improve the sample distribution and simplify the scoring format, while maintaining the statistical significance and biologic/clinical relevance. For Number scores, the best model appeared to be “0” vs. “1 or 2 or 3,” i.e. fewer than 5% of the cells staining vs. >5% (Table I and Fig. 2b). For patients with iNOS Number “0,” the median DSS was not attained (95% CI: not attained, not attained) whereas for patients with iNOS number “1 or 2 or 3,” the median DSS was 29 months (95% CI: 20, 45). In terms of Intensity, the optimal categorization appeared in separation of “0” vs. “1” vs. “2 or 3,” i.e., no staining vs. light staining vs. moderate to marked staining intensity (Table I and Fig. 2d). For iNOS Intensity “0,” the median DSS was not attained (95% CI: not attained, not attained). In contrast, the median DSS was 67 months (95% CI: 46, not attained) and 27 months (95% CI: 15, 43) for patients with iNOS Intensity “1” and “2 or 3,” respectively.

Figure 2.

Kaplan–Meier survival curves, illustrating the influence of iNOS expression on disease-specific survival from the diagnosis of Stage III melanoma. For iNOS Number, the effect of each category separately and of the combined categories used in the multivariate analysis are displayed in (a) and (b), respectively. Similarly, for iNOS Intensity, the individual categories are shown in (c), and the combination used in the multivariate analysis, in (d). N = 132 for each survival curve. Associated median survivals are described in the text. Number of events per number of cases are as follows: iNOS Number “0,” 18 deaths/69 patients; iNOS Number “1,” 10 deaths/21 patients; iNOS Number “2,” 22 deaths/29 patients; iNOS Number “3,” 10 deaths/13 patients; INOS Intensity “0,” 12 deaths/53 patients; INOS Intensity “1,” 19 deaths/40 patients; INOS Intensity “2,” 22 deaths/31 patients; INOS Intensity “3,” 7 deaths/8 patients.

Multivariate analysis of iNOS and other predictors of survival

Information regarding other known prognostic factors for Stage III melanoma was gathered for the study population. This included the number of involved lymph nodes, the presence of macroscopic disease, the presence of in-transit disease, ulceration of the primary tumor and administration of adjuvant interferon (Table II). Noteworthy is the fact that information regarding ulceration and adjuvant interferon could not be obtained for a portion of the study group. Upon univariate analysis, all established prognostic factors displayed the expected trends in HR (Table II), although significance was reached only for number of involved lymph nodes, likely a consequence of the relatively small number of study subjects.

Table II. Univariate Cox Proportional Hazards Model for Disease-Specific Survival From Stage III Diagnosis, Incorporating Established Prognostic Factors
FactorUnivariate model
n1HR95% LL95% ULp-value
  •  HR, hazard ratio; 95% LL, lower limit of 95% confidence interval on hazard ratio; 95% UL, upper limit of 95% confidence interval on hazard ratio.

  • 1

    Total number may be less than 132 patients due to missing data.

Age at melanoma diagnosis (y)
 ≤51671.00   
 >51651.260.762.100.37
Gender
 Female601.00   
 Male720.970.591.610.91
Number of positive nodes
 1 node (N1)491.00   
 2 or 3 nodes (N2)401.130.572.210.73
 ≥4 nodes (N3)431.921.053.520.03
Macroscopic disease
 No431.00   
 Yes891.330.772.320.31
In-transit disease
 No1041.00   
 Yes281.080.581.990.81
Ulceration of primary
 No231.00   
 Yes491.320.622.830.47
Adjuvant Interferon
 Yes361.00   
 No841.480.802.710.21

Multivariate analysis was used to determine if iNOS expression predicted poor survival in a manner independent of the other established prognostic factors. For this analysis, the revised iNOS Number and Intensity groupings shown in Table I were used. Because the Number and Intensity scores were found to be highly correlated (p < 0.0001), potentially diminishing or negating the effect of either in the multivariate analysis, it was decided to examine iNOS Number and Intensity in separate analyses. Furthermore, as stated earlier, there were missing data in the categories of “Ulceration” and “Adjuvant Interferon” for some of the patients. In the interest of maintaining adequate subject numbers, these 2 categories were excluded from the multivariate analysis.

The final multivariate model included iNOS Number or Intensity score, gender, age at melanoma diagnosis, number of nodes, macroscopic disease and in-transit disease. In the analysis including iNOS Number score, only iNOS Number was prognostic for DSS from Stage III diagnosis (Table IIIa). Using backward reduction, the final reduced model was the univariate model in which iNOS Number was prognostic for DSS (HR = 4.60; 95% CI: 2.63, 8.06; p < 0.0001). iNOS Intensity was analyzed in a similar manner in the multivariate model (Table IIIb). Again, using backward reduction, the final reduced model was the univariate model in which iNOS Intensity was prognostic for DSS (for Intensity “1,” HR = 2.75; 95% CI: 1.33, 5.68; p = 0.0062; for Intensity “2 or 3,” HR = 7.23; 95% CI: 3.64, 14.36; p < 0.0001). No other prognostic factors were significant in the multivariate analysis, most notable being the loss of significance for involved lymph node number. Similar results were obtained when the analysis was performed for OS, with the exception that age at melanoma diagnosis retained significance in the model after backward reduction (data not shown). These findings provide support for the independence of iNOS expression in predicting disease-specific survival for patients with stage III melanoma.

Table III. Multivariate Cox Proportional Hazards Model for Disease-Specific Survival From Stage III Diagnosis (n = 132): (A) iNOS Number, (B) iNOS Intensity
FactorMultivariate model
nHR95% LL95% ULp-value
  1.  HR, hazard ratio; 95% LL, lower limit of 95% confidence interval on hazard ratio; 95% UL, upper limit of 95% confidence interval on hazard ratio.

(A) iNOS Number
 0691.00   
 1 or 2 or 3634.632.608.25<0.0001
Age at melanoma diagnosis (y)
 ≤51 years671.00   
 >51 years651.440.832.480.19
Gender
 Female601.00   
 Male720.770.461.290.32
Number of positive nodes
 1 node (N1)491.00   
 2 or 3 nodes (N2)400.890.441.810.75
 ≥4 nodes (N3)431.680.773.670.19
Macroscopic disease
 No431.00   
 Yes891.000.521.920.99
In-transit disease
 No1041.00   
 Yes280.720.341.520.39
(B) iNOS Intensity
 0531.00   
 1402.911.406.050.004
 2 or 3397.693.7615.74<0.0001
Age at melanoma diagnosis (y)
 ≤51671.00   
 >51651.190.702.040.52
Gender
 Female601.00   
 Male720.660.381.120.12
Number of positive nodes
 1 node (N1)491.00   
 2 or 3 nodes (N2)400.870.431.750.69
 ≥4 nodes (N3)431.780.823.890.15
Macroscopic disease
 No431.00   
 Yes891.070.572.020.84
In-transit disease
 No1041.00   
 Yes280.810.381.750.59

Discussion

The data which we present demonstrate the utility of tumor iNOS determination as a marker of poor survival in Stage III melanoma. Univariate analysis reveals a strong effect of iNOS expression on the HR of death from melanoma. Multivariate analysis suggests that iNOS, as a predictor of survival, may be independent of the other known prognostic factors. Limiting the multivariate analysis is the fact that, although all of the established prognostic factors demonstrated the expected trends in HR, most did not reach significant p values, likely due to the relatively small number of study subjects. Consequently, these data should be considered preliminary until confirmed in a larger study. Supporting the independence of iNOS, however, are the extremely low p values (generally <0.0001) which do not differ substantially between the univariate and multivariate analyses. Similarly, the HRs for iNOS expression computed in the univariate analysis are remarkably stable in the multivariate analysis, indicating that the addition of known risk factors does not affect the significance of iNOS as a predictor of survival. These data extend our original report of iNOS in melanoma, which included only 20 patients who had received investigational neoadjuvant therapy prior to surgical excision of tumor, raising the possibility that iNOS expression in the tumor was a result of treatment.11 Our current study eliminates this confounding factor and is more relevant to the typical Stage III patient for whom tumor samples are normally obtained for diagnostic purposes prior to the administration of any therapy.

Decisions regarding adjuvant therapy for individuals with Stage III disease are often presented to the patient with predictions of outcome. As a specific example, the decision to administer high-dose interferon, currently the standard of care in this setting, is made with consideration of the anticipated risk of relapse.19 The ability to determine more accurately an individual patient's likelihood of dying from his or her disease may improve patient selection for this form of therapy and for other aggressive interventions aimed at disease control. Beyond interferon, Stage III melanoma is commonly targeted for adjuvant vaccine trials and other novel, investigative immunotherapeutic approaches for which analysis requires stratification by predictive factors. Thus, our data showing the prognostic value of iNOS expression for Stage III melanoma patients may be highly valuable in a practical clinical sense.

The fact that this study was conducted using archived tumor and retrospective patient data provided the advantage that all patients had long follow-up, thereby strengthening our ability to draw conclusions regarding the effects of the presence of iNOS on survival. However, the same study design also provided the major shortcoming of the study, that being missing data. Information regarding ulceration of the primary tumor and the administration of adjuvant therapy were particularly problematic. The introduction of primary tumor ulceration to the staging of localized and regionally advanced melanoma is a relatively recent event, and thus this information was commonly missing for patients whose diagnosis predated the routine use of this marker.12 Information regarding the administration of adjuvant therapy could not be obtained for a smaller number of patients who were lost to follow-up. By eliminating these 2 relatively weak prognostic factors from the multivariate analysis, the largest number of tumors was preserved for the comparison of iNOS to the other predictive markers considered to be of higher value. These shortcomings, however, and the known limitations of retrospectively collected data underscore the need for a confirmatory study conducted in a prospective manner. Accordingly, a prospective study is being initiated, which will incorporate recently diagnosed patients, focus on careful documentation of all known prognostic markers including primary tumor ulceration and adjuvant therapeutic interventions, and include the analysis of iNOS in the primary cutaneous tumors. The prospectively collected data will permit greater accuracy of survival information and should minimize the number of patients lost to follow-up. This study will also recruit adequate subject numbers so that the results of the multivariate analysis can be confirmed. Additionally, issues such as optimization of the scoring system and concordance between IHC interpreters will be addressed.

A number of new questions arise from these data, many of which are currently in the process of being examined. First, the analysis needs to be expanded to include patients with localized disease (Stages I and II) and those with systemic metastases (Stage IV), to determine if iNOS has predicative value for these groups, as well. Another issue is the validity of iNOS expression as a surrogate for NO production in this setting. To address this question, we are presently examining the tissue sections for nitrated proteins, by means of IHC for nitrotyrosine (NT). NT is widely accepted as the stable end product of the intracellular reaction of NO with reactive oxygen species, forming peroxynitrite, which directly nitrates tyrosines on proteins; the reactive oxygen species upon which this reaction depends are known to exist in human melanoma cells.20 Although still circumstantial, a correlation of iNOS with NT, as shown in our earlier data, would strengthen the assumption that NO is the mediator of the observed effects.11 Finally, the major challenge arising from our study is to determine the mechanism by which iNOS, and presumably NO, negatively influence the survival of melanoma patients. NO can inhibit apoptosis, contribute to tumor angiogenesis, induce DNA damage, interfere with DNA repair mechanisms and modify the activities of critical proteins.6, 8, 21, 22, 23 The regulation of tumor growth by endogenous intracellular NO represents an important new dimension in melanoma research. The data in this report support the conclusion that iNOS expression yields significant prognostic information for patients with Stage III metastatic melanoma, providing a novel biologic basis for the development of new therapeutic tools based on regulation of this enzyme.

Acknowledgements

The authors thank Dr. Jeffrey E. Gershenwald for his critical reading and review of the manuscript, Sandra A. Kinney for outstanding technical work and Sandra Ortega-Mendez and Sandra Yekell for assistance in tumor sample procurement from the MDACC Melanoma Informatics, Tissue Resource and Pathology Core.

This publication was made possible by Grant Number R01 CA90282 (E.A.G, S.E.); P50 CA093459 from the U.T. M.D. Anderson Cancer Center SPORE in Melanoma (E.A.G, S.E., J.A.E.); the Cancer Foundation for Melanoma Research (S.E.); as well as the continuing support from the Golfer's Against Cancer. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Ancillary