Abstract
Purpose
New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma.
Experimental design
The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease ≤14 months) and 17 slowly progressing (OS ≥60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. χ2 exact trend test and logistic regression analysis were used for statistical analysis.
Results
Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness.
Conclusion
Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.
Keywords: iNOS, COX-2, Metastatic lymph node, Stage III melanoma, Survival, Prognostic factor
Introduction
The 2002 staging system for melanoma developed by the American Joint Committee on Cancer represents a new staging system that was developed and validated in separate cohorts of patients with cutaneous melanoma [2]. In order to accurately stage a melanoma, patients must have a complete examination with respect to the histopathology of the primary tumor and the regional lymph nodes, using the sentinel node biopsy technique along with an examination to detect possible distant metastatic disease. Routine imaging studies such as computed tomography scanning, and magnetic resonance imaging are performed in patients with evidence of stage III metastatic disease. The number of involved regional lymph nodes has historically been the most consistent prognostic factor in stage III melanoma [3, 34]. Although improvements have been made in clinical staging modalities and prognostic models for stage III melanoma patients, molecular markers are needed, since the outcome for patients within the same clinical group or subgroup varies, indicating the presence of different biologic subtypes of disease.
In response to various stimuli, arachidonic acid can be mobilized from phospholipid pools and converted to bioactive eicosanoids through the cyclooxygenase (COX), lipoxygenase (LOX), or P-450 epoxygenase pathway. Five major prostanoids (PGD2, PGE2, PGF2, PHI2, and TXA2) are synthesized by the COX pathway. Today, three different COX enzymes have been described; COX-1, COX-2, and COX-3. COX-1 is constitutively expressed in most mammalian cells and tissues [15]. In contrast, COX-2 is absent in most normal tissues, but can be readily induced by numerous stimuli like TNFα and phorbol ester. The expression of COX-2 can be detected in several tumors, but it is also found in multiple types of non-neoplastic cells like epithelial, endothelial, and stromal cells. In addition, COX-2 has been found to be abundant in activated macrophages and other cells at the sites of inflammation [31]. COX-2 and PGE2 synthase have been well documented in the regulation of various aspects of tumor progression and metastasis. Aberrant or increased expression of COX-2 has been implicated in malignant tumors, especially in colon cancer [16, 17]. Elevated levels of COX-2 have been demonstrated in many other cancer types as well [14, 29, 41]. In melanoma, COX-2 is suggested to be associated with tumor progression [4, 8, 13]. Several reports have demonstrated that the COX-2 product PGE2 has a wide range of effects including induction of cellular proliferation, promotion of angiogenesis, inhibition of apoptosis, stimulation of tumor invasion, and suppression of immune responses [42].
Nitric oxide (NO) is a pleiotropic and important bioregulatory mediator involved in a variety of biological processes including vasorelaxation, neurotransmission, and cytotoxicity. NO production from l-arginine was first identified in endothelial cells and macrophages [23, 24], and later it was demonstrated that inflammatory stimuli induced the expression of a specific isoform of NO synthase (iNOS) in myeloid cells and other cell types [22]. One such cell type, termed myeloid derived suppressor cells (MDSC), controls T cell functions through down-regulation of TCR expression and suppression of antigen-specific T cell responses [6, 33]. There are multiple roles for iNOS in different disease processes [5]. Although a number of reports have suggested that very high levels of NO are cytotoxic for cancer cells, the constitutive production of low levels of intracellular NO has been shown to promote tumor progression and survival as well as inducing anti-apoptotic effects in many tumor types including melanoma [35, 38–40]. Moreover, iNOS is expressed constitutively in most cultured melanoma cells and is present in over 60% of human melanoma samples [19, 27]. Recently, it was reported that tumor iNOS expression is a strong predictor of disease-specific and overall survival (OS) for stage III melanoma patients [18].
Activating mutations in BRAF (mostly at codon 600) and NRAS (mostly at codon 61) proto-oncogenes are often detected in melanomas [11, 12, 21] and they are almost always mutually exclusive. However, rare human melanomas harboring both NRAS and BRAF mutations have also been described [1, 26, 37]. Activated NRAS contributes to neoplastic transformation of human melanomas and to development of invasive melanomas [7]. Similarly, activated BRAF is a transforming oncogene in immortalized melanocytes and the BRAFV600E mutation is detected in over 60% of melanomas [12, 43]. Interestingly, it has been shown that premalignant cells from other tissues can activate a senescence program in response to oncogenic RAS, and that activated BRAF triggers senescence in melanocytes from nevi [28].
In this historical cohort we wanted to compare the prognostic value of iNOS to that of COX-2 and therefore we examined their levels of expression as well as the presence of activating BRAF/NRAS mutations in metastatic lymph nodes from stage III cutaneous melanoma patients. Here, we report that iNOS expression correlates significantly with the presence of BRAF mutations, and independently and significantly predicts a shortened survival in these patients with a higher odds ratio (OR) than that of COX-2. Furthermore, iNOS maintained or increased its OR and was always higher than that of COX-2 in the multivariate logistic regression analysis together with stage IIIB/C, ulceration, metastatic lymph nodes, and Breslow tumor thickness. This suggests that iNOS is an independent and stronger adverse prognostic factor for OS than COX-2 in stage III cutaneous melanoma patients.
Materials and methods
Patients
This study was approved by the Regional Ethics Board of Karolinska Institutet. Thirty-eight stage III cutaneous melanoma patients fulfilled the inclusion criteria and could be included in this retrospective cohort, for whom paraffin-embedded tumor material was available in the archival melanoma tissue bank at the pathology department, and for whom information on survival and other prognostic data were recorded. Eligibility for inclusion in the study was the diagnosis of stage III melanoma and the availability of paraffin-embedded metastatic lymph node biopsies from two groups of patients differing significantly in terms of OS. These patient groups had survival from time of diagnosis of stage III melanoma of either ≤14 months (short survival group) or ≥5 years (long survival group). The following information was collected from the medical records: gender, age at diagnosis, histopathologic type, thickness of primary tumor according to Breslow, presence of ulceration, number of metastatically involved lymph nodes, stage III subgroup (A, B or C), and the date of death or date of last follow-up.
Immunohistochemistry
Paraffin-embedded sections of lymph nodes were examined for iNOS and COX-2 expression by immunohistochemistry (IHC) using an anti-iNOS rabbit monoclonal antibody (1:50) (Labvision, CA, USA) and anti-COX-2 mouse monoclonal antibody (1:50) (Transduction Laboratories, Lexington, KY). Tissue sections were deparaffinized and rehydrated, then placed in a 0.01 M citrate buffer, pH 6, and microwaved intermittently for a total of 20 min. After cooling, the slides were placed in 3% H2O2 in H2O for 30 min. An avidin–biotin–peroxidase complex (ABC) kit (Vectastain, Vector Laboratories) was then used for antigen detection. After 30 min of blocking in 1% BSA, the primary antibody was applied overnight at +8°C, followed by 30 min incubation with secondary biotinylated antibody, and the ABC reagent. The immunolabeling was developed with the chromogen 3-amino-9-ethylcarbazole for 6 min. Hematoxylin was applied as a counter stain. Tonsil tissue was used as positive control sample for COX-2 and iNOS staining and isotype controls for each primary antibody as well as application of secondary antibody only were used as negative controls. Immunolabeling was scored separately for two variables; first, for number of iNOS and COX-2 positive cells; second, for the overall intensity of immunoreactivity of the positive cells. Briefly, scoring for number of positive cells was defined as follows: “0”, <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”, weak staining; “2”, moderate staining; and “3”, intense staining. The slides were independently evaluated by two different readers who were blinded for the clinical outcome of patients. To prepare the data for multivariate analysis, combinations (“0” vs. “1 or 2 or 3”) of the score categories were done to simplify the scoring format, while maintaining biological/clinical relevance.
NRAS and BRAF mutation analysis
Lymph node metastases were analyzed for NRAS exon 2 and BRAF exon 15 mutations using direct DNA sequencing. DNA extracts from frozen tumor sections were used for screening of mutations in exon 2 of NRAS gene and exon 15 of BRAF gene. The DNA extractions were performed using QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) followed by removal of melanin from the DNA extracts when PCR amplification directly was unsuccessful as previously described [36]. The PCR for NRAS was performed using primers NRASx2FP 5′-gggcttgaatagttagatgct-3′ (intronic forward primer) and N2B 5′-atacacagaggaagccttcg-3′ (internal exon 2 reverse) resulting in a 248-bp amplicon and primers 61A1 5′-gattcttacagaaaacaagtg-3′ (internal exon 2 forward) and 61B1 5′-atgacttgctattattgatgg-3′ resulting in a 157-bp amplicon. Both amplicons include the commonly mutated codon 61 in melanoma. The PCR for BRAF was performed using primers published previously [29] resulting in a 224-bp PCR amplicon. The PCR protocol for both amplifications was 95°C 3 min (initial denaturation) followed by 94°C 20 s, 55°C/58°C (NRAS/BRAF) 20 s and 72°C 30 s for 38 cycles then 72°C for 5 min followed by final soak at 10°C. All PCR reagents were from Invitrogen (Invitrogen, Carlsbad, CA, USA). The specificity and quantity of the PCR fragments were confirmed by agarose gel electrophoresis (1.8%, 4 V/cm), cleaned prior to sequencing using Qiagen Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). The PCR fragments were analyzed by direct bi-directional sequencing using same primers as for PCR and BigDye v.1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) according to the kit manual. Electropherograms were analyzed in the Mutation Explorer software (SoftGenetics LLC, State College, PA, USA) and by manual inspection. All mutated DNA extracts were submitted for another PCR and sequencing for confirmation. Mutation was regarded to be present when confirmed in two analyses derived from independent PCRs. For positive and negative control samples DNA extracts from melanoma cell lines 224 and A375 were utilized.
Statistical analysis
The objective of the statistical analysis was to assess the prognostic effects of melanoma tumor COX-2 and iNOS expression, presence of BRAF/NRAS mutations, gender, age at diagnosis, number of metastatic lymph nodes, and tumor thickness according to Breslow on OS. OS from the date of diagnosis of stage III malignant melanoma was the end point for the study. For OS, censored patients included those remaining alive at last follow-up. Survival time was calculated using the date of diagnosis of stage III malignant melanoma and date of last follow-up or date of death. The χ 2 exact trend test was utilized to compare patient characteristics for discrete categorical variables or factors between groups. Univariate logistic regression analysis was performed to evaluate the prognostic effects for each of the earlier noted factors alone. OR and 95% confidence intervals (CI) were calculated. Furthermore, multivariate logistic regression analysis was carried out to simultaneously evaluate the predictive effects of all factors. P values <0.05 were considered statistically significant. All analyses were performed with the program SPSS.
Results
Patient characteristics
A summary of the patient characteristics is given in Table 1. This study was conducted using tumor material from two distinct groups of patients; one group of patients with survival time of 14 months or less and the other group with survival time of 60 months or more. The χ 2 exact trend test (Table 2) was utilized to compare patient and tumor characteristics between groups regarding age, gender, stage IIIB/C (there were no patients in stage IIIA), tumor ulceration, Breslow tumor thickness, and number of metastatic lymph nodes. There was a significant difference between groups regarding stage IIIC [P = 0.008; OR = 7.6 (95% CI: 1.7–32.2)] and ulceration [P = 0.015; OR = 8.3 (95% CI: 1.7–40)]. We observed no significant difference (P = 0.709) in tumor thickness according to Breslow depth using 1 mm as the cut-off with an OR of 0.7 (95% CI: 0.1–3.4) between the patient groups. However, there was almost a significant (P = 0.058) difference in Breslow depth between groups using a cut-off at 2 mm with an OR of 3.9 (95% CI: 1.0–15). Breslow depth with a cut-off at 2 mm was used in the logistic regression analysis. The number of metastatic nodes (>1 node) showed a tendency (P = 0.052) towards increased numbers in the short survival patient group with an OR of 4.3 (95% CI: 1.1–17.2). Next, univariate logistic regression analysis showed that stage IIIC [P = 0.009; OR = 7.5 (95% CI: 1.6–34)], ulceration [P = 0.015; OR = 8.2 (95% CI: 1.49–45.4)] and the presence of multiple metastatic lymph nodes (1 vs. 2 or more nodes) were [P = 0.042; OR = 4.3 (95% CI: 1.1–16.7)] significantly more frequent in the short survival patient group and predicted OS, whereas Breslow depth (cut-off at 2 mm) almost reached statistical significance (P = 0.051) (Table 3a).
Table 1.
Patient characteristics
| ≤14 months survival (N = 21) | ≥5 years survival (N = 17) | |
|---|---|---|
| Gender | ||
| Male | 14 (67%) | 9 (53%) |
| Female | 7 (33%) | 8 (47%) |
| Age at diagnosis (years) | 53 | 59 |
| (range) | 24–77 | 28–86 |
| Histopathologic type | ||
| SSM | 11 (52%) | 11 (65%) |
| NM | 4 (19%) | 2 (12%) |
| LMM | – | 1 (6%) |
| ALM | 1 (5%) | – |
| Unclass | 5 (24%) | 3 (18%) |
| Ulceration of primary tumor | ||
| Yes | 11 (52%) | 2 (12%) |
| No | 10 (48%) | 15 (88%) |
| Stage III subgrouping | ||
| Stage IIIA | – | – |
| Stage IIIB | 8 (38%) | 14 (82%) |
| Stage IIIC | 13 (62%) | 3 (18%) |
| Breslow (mm) | ||
| Mean ± SD | 3.68 ± 4.34 | 1.88 ± 1.55 |
| Median | 2.6 | 1.3 |
| Number of positive nodes | ||
| 1 node | 9 (43%) | 13 (76%) |
| 2 or 3 nodes | 8 (38%) | 3 (18%) |
| ≥4 nodes | 4 (19%) | 1 (6%) |
| Non-surgical treatment modalities | ||
| R | 3 (14%) | – |
| C | 2 (9.5%) | 1 (6%) |
| I | – | 2 (12%) |
| C + I | 2 (9.5%) | – |
| R + C | 6 (29%) | – |
| R + C + I | 3 (14%) | – |
| Untreated | 5 (24%) | 14 (82%) |
SSM superficial spreading melanoma, NM nodular melanoma, LMM lentigo maligna melanoma, ALM acral lentiginous melanoma, R radiotherapy, C chemotherapy, I immunotherapy
Table 2.
χ2 exact trend test for differences in prognostic factors between short survivors (Group 1) and long survivors (Group 2)
| All N = 38 | Group 1 | Group 2 | χ2 | OR | 95% CI | P value | ||
|---|---|---|---|---|---|---|---|---|
| N | Frequency | N | Frequency | |||||
| Age >55 | 10 | 0.48 | 9 | 0.53 | 0.11 | 0.8 | 0.1–2.8 | 1 |
| Gender M/F | 14 | 0.67 | 9 | 0.53 | 0.74 | 1.8 | 0.5–6.6 | 0.509 |
| Stage IIIC | 13 | 0.62 | 3 | 0.18 | 7.54 | 7.6 | 1.7–32.2 | 0.008 |
| Ulceration | 11 | 0.52 | 2 | 0.88 | 6.88 | 8.3 | 1.7–40 | 0.015 |
| Breslow >1 mm | 16 | 0.76 | 14 | 0.82 | 0.21 | 0.7 | 0.1–3.4 | 0.709 |
| Breslow >2 mm | 13 | 0.61 | 5 | 0.29 | 3.9 | 3.9 | 1.0–15 | 0.058 |
| Lymph nodes >1 | 12 | 0.57 | 4 | 0.24 | 4.35 | 4.3 | 1.1–17.2 | 0.052 |
| NRAS | 3 | 0.14 | 6 | 0.35 | 2.20 | 0.3 | 0.1–1.4 | 0.251 |
| BRAF | 13 | 0.61 | 7 | 0.41 | 10.09 | 2.3 | 1.3–3.9 | 0.194 |
| COX-2 | 9 | 0.43 | 2 | 0.12 | 4.42 | 5.6 | 1.1–28.1 | 0.070 |
| iNOS | 16 | 0.76 | 4 | 0.24 | 10.45 | 10.4 | 2.5–43 | 0.003 |
OR, odds ratio; 95% CI, 95% confidence interval on OR
Table 3.
Univariate and multivariate logistic regression analysis for overall survival from date of diagnosis of stage III malignant cutaneous melanoma regarding COX-2 and iNOS and other known prognostic factors
| a. Logistic regression: univariate | |||||
|---|---|---|---|---|---|
| Strata | Factors | OR | 95% CI | P value | |
| Dependent variable | Stage IIIC versus IIIB | 7.5 | 1.6–34 | 0.009 | |
| Breslow > 1 versus otherwise | 1.8 | 0.4–6.7 | 0.41 | ||
| Breslow > 2 versus otherwise | 3.9 | 1.0–15.2 | 0.051 | ||
| Ulceration versus otherwise | 8.2 | 1.5–45.4 | 0.015 | ||
| Lymph nodes > 1 versus otherwise | 4.3 | 1.1–17.8 | 0.042 | ||
| COX-2 positive versus negative | 5.6 | 1.0–31.3 | 0.048 | ||
| COX-2 intensity high versus low | 3.0 | 0.5–17.3 | 0.21 | ||
| iNOS positive versus negative | 10.4 | 2.3–47.6 | 0.002 | ||
| iNOS intensity high versus low | 3.2 | 0.8–12.4 | 0.09 | ||
| b. Logistic regression: multivariate | |||||
|---|---|---|---|---|---|
| Strata for iNOS dependent variable | Stage IIIC versus IIIB | Ulceration | Lymph nodes >1 | Breslow >2 | iNOS: exposed variable |
| OR | – | – | – | – | 10.4 |
| CI | – | – | – | – | 2.3–47.6 |
| OR | 9.9 | – | – | – | 13 |
| CI | 1.5–65 | – | – | – | 2.1–79 |
| OR | – | 12.4 | – | – | 14.5 |
| CI | – | 1.6–100 | – | – | 2.3-89 |
| OR | – | – | 6.8 | – | 14 |
| CI | – | – | 1.1–42 | – | 2.4–85 |
| OR | – | – | – | 4 | 10 |
| CI | – | – | – | 0.8–20 | 2.1–52 |
| OR | 0.8 | 13.6 | 6.5 | – | 19.5 |
| CI | 0.02–28 | 0.3–669 | 0.7–58 | – | 2.4–153 |
| c. Logistic regression: multivariate | |||||
|---|---|---|---|---|---|
| Strata for COX-2 dependent variable | Stage IIIC versus IIIB | Ulceration | Lymph nodes >1 | Breslow >2 | COX-2: exposed variable |
| OR | – | – | – | – | 5.6 |
| CI | – | – | – | – | 1.0–31 |
| OR | 9.5 | – | – | – | 7.6 |
| CI | 1.8–50 | – | – | – | 1.1–51 |
| OR | – | 8.1 | – | – | 5.5 |
| CI | – | 1.4–48 | – | – | 0.9–34 |
| OR | – | – | 5.3 | – | 8.5 |
| CI | – | – | 1.3–31 | – | 1.3–56 |
| OR | – | – | – | 3.3 | 4.8 |
| CI | – | – | – | 0.8–14 | 0.8–28 |
| OR | 2.1 | 5.2 | 6.4 | – | 11.9 |
| CI | 0.11–37 | 0.2-136 | 0.9–47 | – | 1.3–106 |
OR, odds ratio; 95% CI, 95% confidence interval on OR
Expression of COX-2 in metastatic lymph nodes from stage III melanoma patients
We examined the expression levels of COX-2 in paraffin-embedded sections of metastatic lymph nodes from the short- and long-surviving stage III melanoma patients by IHC (Fig. 1). Positive COX-2 expression was established as more than 5% of positive cells (with an intensity score of 1 or more). COX-2 expression was observed in 11 (29%) of the 38 samples and 82% (9/11) of these COX-2 positive patients were found within the group of patients with survival time of 14 months or less. The data in Table 2 represent the frequency of COX-2 expression (fewer than 5% vs. more than 5% COX-2 positive cells staining) in the different patient groups. Similar data were obtained using intensity of COX-2 immunoreactivity (data not shown).
Fig. 1.
COX-2 protein expression in metastatic lymph nodes from stage III melanoma patients. Representative IHC stainings of lymph node tissues displaying the following controls or immunostaining: a anti-COX-2, tumor 1 with moderate staining and >75% positive cells; b isotype control, tumor 1; c anti-COX-2, tumor 2 with intense staining and 25–75% positive cells; d negative control, tumor 2; e anti-COX-2, tumor 3 with weak staining and 5–25% positive cells; f negative control, tumor 3. Scale bar 50 μm
Correlation of COX-2 expression with survival
Initially, the χ 2 exact trend test was used to examine whether there was a significant difference in COX-2 expression between the two patient groups. As can be seen in Table 2, the increased COX-2 expression in the short surviving group nearly reached statistical significance (P = 0.070) with an OR of 5.6 (95% CI: 1.1–28.1). Next, a univariate logistic regression analysis was used to determine whether there was a correlation between COX-2 expression and OS. Results of the univariate analysis for OS from the diagnosis of stage III disease are shown in Table 3a. A significant (P = 0.048) correlation between COX-2 expression and survival was observed with an OR of 5.6 (95% CI: 1.0–31.3). Next, we examined whether the grade of intensity of COX-2 immunoreactivity (“0” or “1” vs. “2” or “3”) correlated with OS. As shown in Table 3a, intensity of COX-2 staining did not significantly correlate with OS as reflected with a lowered OR (3.0).
Expression of iNOS in metastatic lymph nodes from stage III melanoma patients
We went on to explore the expression of iNOS in metastatic lymph nodes from the same stage III melanoma patients that were screened for COX-2 expression in order to compare the prognostic value of iNOS to that of COX-2. Paraffin-embedded sections of metastatic lymph nodes were stained for iNOS using a rabbit anti-iNOS antibody by IHC with antigen retrieval (Fig. 2). Positive iNOS expression (as for COX-2, established as more than 5% of positive cells with an intensity score of 1 or more), was observed in 20 (53%) of 38 samples. Sixteen of these 20 (80%) iNOS-positive patients were found among the short survivors. Interestingly, high COX-2 frequencies paralleled high iNOS frequencies in all patients. As for COX-2, the data in Table 2 represent the frequency of iNOS expression (fewer than 5% iNOS positive cells staining vs. more than 5%) in the different patient groups. Similar data were obtained using intensity of iNOS immunoreactivity (data not shown).
Fig. 2.
iNOS protein expression in metastatic lymph nodes from stage III melanoma patients. Representative IHC stainings of lymph node tissues displaying the following controls or immunostaining: a anti-iNOS, tumor 1 with intense staining and >75% positive cells; b isotype control, tumor 1; c anti-iNOS, tumor 3 with weak staining and 25–75% positive cells; d negative control, tumor 3; e anti-iNOS, tumor 4 with intense staining and >75% positive cells; f negative control, tumor 4. Scale bar 50 μm
Correlation of iNOS expression with survival
A χ 2 exact trend test was used to examine whether there was any significant difference in iNOS expression between the two patient groups. As can be seen in Table 2, the increased iNOS expression in the short survival group reached statistical significance [P = 0.003; OR 10.4 (95% CI: 2.5–43)]. A univariate logistic regression analysis was performed to examine whether there was a correlation between iNOS expression and OS. Results of the univariate analysis for OS from the diagnosis of stage III disease are shown in Table 3a. The presence of iNOS in a patient’s tumor, graded on the basis of % of positive cells, correlated with a significant (P = 0.002) increase in the OR (OR = 10.4; 95% CI: 2.3–47.6) of death from melanoma. Next, we examined whether the grade of intensity of iNOS immunoreactivity (“0” or “1” vs. “2” or “3”) correlated with OS. The intensity of iNOS staining showed a tendency (P = 0.09) towards a correlation with OS (Table 3a).
BRAF and NRAS mutational analysis
Next, we examined for the presence of activating BRAF and NRAS mutations in metastatic lymph nodes from the short and long survival groups of stage III melanoma patients using direct DNA sequencing. The χ 2 exact trend test was used to examine whether there was a significant difference in frequency of the observed BRAF/NRAS mutations between the two patient groups. As shown in Table 2, no significantly increased frequencies of BRAF (P = 0.194) or NRAS (P = 0.251) mutations were observed in the short surviving group as compared to the long surviving group. A univariate logistic regression analysis was not performed on the BRAF/NRAS mutations, since there was no significant difference between groups as assessed by the χ 2 exact trend test. Interestingly, logistic regression analysis showed a significant (P = 0.013) correlation between BRAF mutations and expression of iNOS, whereas COX-2 expression did not show this pattern of correlation with BRAF.
Multivariate logistic regression analysis of COX-2 and iNOS and other predictors of survival
Information regarding other known prognostic factors for stage III melanoma was gathered for the study population. This included ulceration, stage IIIA/B/C, tumor thickness according to Breslow and number of metastatically involved lymph nodes (Table 1). Since the population used in this study consists of two distinct groups of patients (patients with short survival times vs. patients with long survival times) the power of the multivariate statistical analysis is limited. As mentioned in the section for patient characteristics and shown in Table 2, Breslow depth was not significantly (P = 0.709) different between the two patient groups when a cut-off at 1 mm was used, whereas a Breslow depth cut-off at 2 mm showed a tendency (P = 0.058) towards a difference between groups. Therefore, Breslow depth with a cut-off at 2 mm was included in the multivariate analysis. The number of metastatic lymph nodes showed a tendency towards a difference between patient groups as observed by the χ 2 exact trend test (P = 0.052) that became statistically significant in the logistic regression analysis: (P = 0.042). Stage IIIB/C, ulceration, Breslow depth, the number of metastatic lymph nodes, COX-2, and iNOS expression were included in the multivariate logistic regression analysis. Multivariate analysis was utilized to determine whether COX-2 and iNOS expression predicted poor survival independently of other established prognostic factors. As mentioned earlier combined scoring categories for COX-2 and iNOS were used for this analysis. In the multivariate analysis, all the factors were analyzed separately with COX-2 and iNOS as well as all factors together. The results from the multivariate analysis showed that iNOS (Table 3b) and COX-2 (Table 3c) were independently prognostic for OS (stable ORs) when co-analyzed together with the other factors as well as when all factors were co-analyzed simultaneously. In addition, iNOS showed a higher OR as compared to COX-2 as well as a higher statistical significance and both COX-2 and iNOS displayed a stable or an increased OR in the multivariate analysis.
Discussion
Advanced melanoma is a devastating disease with a very poor OS prognosis. To date, there are only two agents that are approved by the United States Federal Drug Agency (FDA) for use in patients with metastatic melanoma: dacarbazine and IL-2. Both agents have an overall response rate well below 20%, with only rare long-term responders noted. Metastatic melanoma is known to be one of the most resistant malignancies to a variety of treatment modalities. Therefore, intense efforts should be made in order to generate alternative strategies such as immunotherapy to improve the survival rates for this group of patients. Also, the ability to more accurately determine an individual patient’s likelihood of dying from his or her disease may improve patient selection for novel adjuvant therapies or other aggressive interventions aimed at disease control.
The outcome for patients within the same clinical group or subgroup varies, indicating the presence of different biologic variants of melanoma. Therefore there is a great need for new prognostic markers and a greater understanding of the disease at the molecular level. Inducible NOS and COX-2, among other factors, are mediators of immune suppression and have been associated with cancer progression in melanoma as well as other cancers. Previous reports demonstrated that tumor iNOS expression is associated with a poor prognosis in post-treatment (investigational neo-adjuvant therapy) biopsy samples from stage III melanoma patients as well as in newly diagnosed untreated stage III melanoma patients [18, 19]. Regarding COX-2, there are reports suggesting that an increased expression of COX-2 is associated with progression of malignant melanoma [4, 8, 13]. In this retrospective cohort we determined the expression of both COX-2 and iNOS in metastatic lymph nodes from stage III melanoma patients and explored their prognostic utility. Expression of COX-2 and iNOS in metastatic lymph nodes from stage III melanoma patients showed a significant correlation with OS in the univariate logistic regression analysis, as indicated by both COX-2 and iNOS having increased ORs. Furthermore, iNOS may be a stronger and better prognostic factor than COX-2, since iNOS had a higher OR (OR = 10.4) as compared to COX-2 (OR = 5.6) as well as a higher statistical significance. The ORs for both COX-2 and iNOS expression computed in the univariate analysis were stable in the multivariate analysis together with stage IIIB/C, ulceration, Breslow depth and number of positive lymph nodes. This indicates that the addition of a known risk factor, i.e., number of metastatic lymph nodes, does not affect the significance of COX-2 and iNOS as individual predictors of OS. Interestingly, MDSCs, an immune-suppressive cell population that recently has gained much attention in cancer immunology and cancer progression, expresses iNOS [22]. One may then ask whether there was a correlation between iNOS expression and frequency of MDSCs. However, future studies will have to address this question, since an IHC analysis of MDSC would require a combination of several cell surface markers (CD14, MHC class II, CD11b, IL-4Rα and other markers), and would therefore be better carried out using FACS analysis of fresh samples. The “cross-talk” between COX-2 and iNOS is not completely clear as reflected by many conflicting reports with respect to whether NO activates or inhibits PG production. Thus, it has been reported that NO inhibits COX-2-derived PG production, but it has also been demonstrated that NO is an activator of COX-2 activity [9, 10].
Various functions of iNOS and COX-2 that promote tumor progression are well-documented [25, 30, 32, 42]. Hence, both COX-2 and iNOS that are expressed in the metastatic lymph node can separately and negatively influence the survival of melanoma patients in many ways and when working in concert COX-2 and iNOS may have a more pronounced effect favoring tumor progression.
Here, we show that iNOS and COX-2 expression in metastatic lymph nodes correlate with poor survival in stage III cutaneous melanoma patients. The prognostic value (higher OR) of iNOS was more pronounced as compared to that of COX-2, suggesting that iNOS is a better predictor for OS for these patients than is COX-2. We observed a significant correlation between BRAF mutations and iNOS. It has been reported that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, possibly contributing to the poor survival of these patients [20]. In conclusion, the data presented here show that tumor iNOS is a better predictor of poor OS in stage III cutaneous melanoma patients as compared to COX-2, and that activating BRAF mutations are implicated in driving the expression of iNOS in human melanoma. Tumor iNOS may thus be a good target for development of new therapeutic modalities for malignant cutaneous melanoma.
Acknowledgments
This work was supported by grants to R.K. and J.H. from the Swedish Cancer Society, the Cancer Society of Stockholm, the European Union (Grants “ENACT” and “DC-THERA”), the Karolinska Institutet, and “ALF-Project” grants from the Stockholm City Council.
Conflict of interest statement
The authors have no financial conflict of interest.
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