Value In Melanoma Research Articles

VSIG-3/IGSF11 silencing in A2058 melanoma cells simultaneously suppresses melanoma progression and induces anti-tumoral cytokine profile in human T cells: In silico and in vitro study.

VISTA is a newly discovered immune checkpoint whose functional mechanisms have become increasingly important to study due to its brilliant results in cancer immunotherapy. Despite VSIG-3/IGSF11 being identified as an inhibitory ligand for VISTA with potential as a target for cancer immunotherapy, very little is known of its functions. This study aimed to conduct a detailed analysis of VSIG-3/IGSF11 in melanoma, as well as to study the effects of its silencing on melanoma cell line progression and human T cell functions. Online databases were used to investigate VSIG-3/IGSF11 expression, its relationships, and prognostic value in melanoma. Then, the effects of VSIG-3/IGSF11 silencing on proliferation, migration, cell cycle arrest, and apoptosis in A2058 melanoma cells were assessed using MTT, colony formation, wound healing, cell cycle, and Annexin-VFITC/PI assays, respectively. Finally, A2058 cells transfected with VSIG-3/IGSF11 siRNA were co-cultured with human T cells, and the expression levels of T cell cytokines were evaluated using qRT-PCR. VSIG-3/IGSF11 expression was significantly increased in melanoma patients and cell lines; however, no correlation was found between VSIG-3/IGSF11 expression levels and clinicopathological characteristics, survival, or immune cell infiltration. Following VSIG-3/IGSF11 silencing in A2058 cells, viability, proliferation, and migration rates were decreased, while apoptosis was increased. T cells co-cultured with VSIG-3/IGSF11 siRNA-transfected A2058 cells exhibited increased expression levels of IFN-γ and IL-12 and decreased expression levels of IL-10, TGF-β, and TNF-α. The inhibitory effect of VSIG-3/IGSF11 silencing on A2058 melanoma cell progression, along with the alteration of T cell cytokines towards a pro-inflammatory phenotype, suggests that VSIG-3/IGSF11 is primarily involved in melanoma progression and modulating immune responses. Therefore, it may be a valuable target for immunotherapy in melanoma patients.

The Controversy and Value of Mohs Micrographic Surgery for Melanoma and Melanoma in Situ on the Trunk and Extremities.

The use of Mohs surgery for melanoma on the trunk and extremities is not supported in the guidelines of dermatology, but is widely used in the real world. The purpose of this article is to expose the value of Mohs surgery for melanoma on the trunk and extremities for consideration of updating the guidelines. This was a retrospective review of a prospectively maintained database 7 to identify patients whose melanomas would likely have recurred using standard surgical margins. A prediction model was used to evaluate the value of Mohs surgery. The model predicted that 2,847 (2%) patients with melanoma on the trunk and extremities would likely recur each year with standard surgical margins even after re-excision when positive margins were identified, compared with 0.1% after Mohs surgery. This likely would result in the upstaging of 27% of melanoma in situ patients and 13% of patients with invasive melanoma. The upstaging would also result in a decrease in melanoma-specific survival and the death of 1% of patients with true local recurrences of melanoma. Mohs surgery has value for melanoma on the trunk and extremities by minimizing local recurrence and death from disease progression.

A Tumor and Immune-Related Micro-RNA Signature Predicts Relapse-Free Survival of Melanoma Patients Treated with Ipilimumab.

Despite the unprecedented advances in the treatment of melanoma with immunotherapy, there continues to be a major need for biomarkers of clinical benefits and immune resistance associated with immune checkpoint inhibitors; microRNA could play a vital role in these efforts. This study planned to identify differentially expressed miRNA molecules that may have prognostic value for clinical benefits. Patients with surgically operable regionally advanced melanoma were treated with neoadjuvant ipilimumab (10 mg/kg intravenously every 3 weeks × two doses) bracketing surgery. Tumor biospecimens were obtained at baseline and surgery, and microRNA (miRNA) expression profiling was performed on the tumor biopsies. We found that an expression profile consisting of a 4-miRNA signature was significantly associated with improved relapse-free survival (RFS). The signature consisted of biologically relevant molecules previously reported to have prognostic value in melanoma and other malignancies, including miR-34c, miR-711, miR-641, and miR-22. Functional annotation analysis of target genes for the 4-miRNA signature was significantly enriched for various cancer-related pathways, including cell proliferation regulation, apoptosis, the MAPK signaling pathway, and the positive regulation of T cell activation. Our results presented miRNAs as potential biomarkers that can guide the treatment of melanoma with immune checkpoint inhibitors. These findings warrant further investigation in relation to CTLA4 blockade and other immune checkpoint inhibitors. ClinicalTrials.gov NCT00972933.

SWI/SNF Complex Genomic Alterations as a Predictive Biomarker for Response to Immune Checkpoint Inhibitors in Multiple Cancers.

Whether there is an association between SWI/SNF genomic alterations in tumors and response to immune checkpoint inhibitors (ICI) remains unclear because prior studies have focused on either an individual gene or a predefined set of genes. Herein, using mutational and clinical data from 832 ICI-treated patients who underwent whole-exome sequencing, including sequencing of all 31 genes of the SWI/SNF complex, we found that SWI/SNF complex alterations were associated with significantly improved overall survival (OS) in melanoma, clear-cell renal cell carcinoma, and gastrointestinal cancer, as well as improved progression-free survival (PFS) in non-small cell lung cancer. Including tumor mutational burden as a variable, the multivariate Cox regression analysis showed SWI/SNF genomic alterations had prognostic value in melanoma [HR, 0.63 (95% confidence interval, CI, 0.47-0.85), P = 0.003], clear-cell renal cell carcinoma [HR, 0.62 (95% CI, 0.46-0.85), P = 0.003], and gastrointestinal cancer [HR, 0.42 (95% CI, 0.18-1.01), P = 0.053]. Furthermore, we used the random forest method for variable screening, identifying 14 genes as a SWI/SNF signature for potential clinical application. Significant correlations were observed between SWI/SNF signature alterations and improved OS and PFS in all cohorts. This suggests that SWI/SNF gene alterations are associated with better clinical outcomes in ICI-treated patients and may serve as a predictive marker for ICI therapy in multiple cancers.

Transcriptome-based network analysis related to M2-like tumor-associated macrophage infiltration identified VARS1 as a potential target for improving melanoma immunotherapy efficacy

RationaleThe M2-like tumor-associated macrophages (TAMs) are independent prognostic factors in melanoma.MethodsWe performed weighted gene co-expression network analysis (WGCNA) to identify the module most correlated with M2-like TAMs. The Cancer Genome Atlas (TCGA) patients were classified into two clusters that differed based on prognosis and biological function, with consensus clustering. A prognostic model was established based on the differentially expressed genes (DEGs) of the two clusters. We investigated the difference in immune cell infiltration and immune response-related gene expression between the high and low risk score groups.ResultsThe risk score was defined as an independent prognostic value in melanoma. VARS1 was a hub gene in the M2-like macrophage-associated WGCNA module that the DepMap portal demonstrated was necessary for melanoma growth. Overexpressing VARS1 in vitro increased melanoma cell migration and invasion, while downregulating VARS1 had the opposite result. VARS1 overexpression promoted M2 macrophage polarization and increased TGF-β1 concentrations in tumor cell supernatant in vitro. VARS1 expression was inversely correlated with immune-related signaling pathways and the expression of several immune checkpoint genes. In addition, the VARS1 expression level helped predict the response to anti-PD-1 immunotherapy. Pan-cancer analysis demonstrated that VARS1 expression negatively correlated with CD8 T cell infiltration and the immune response-related pathways in most cancers.ConclusionWe established an M2-like TAM-related prognostic model for melanoma and explored the role of VARS1 in melanoma progression, M2 macrophage polarization, and the development of immunotherapy resistance.

Effects of RNA methylation N6-methyladenosine regulators on malignant progression and prognosis of melanoma

BackgroundMelanoma is an extremely aggressive type of skin cancer and experiencing a expeditiously rising mortality in a current year. Exploring new potential prognostic biomarkers and therapeutic targets of melanoma are urgently needed. The ambition of this research was to identify genetic markers and assess prognostic performance of N6-methyladenosine (m6A) regulators in melanoma.MethodsGene expression data and corresponding clinical informations of melanoma patients as well as sequence data of normal controls are collected from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. Quantitative real-time PCR (qRT-PCR) analysis was carried out to detect the RNA expression of IGF2BP3 in A375 cell line, melanoma tissues, and normal tissues. Western blot, cell proliferation, and migration assays were performed to assess the ability of IGF2BP3 in A375 cell line.ResultsDifferently expressed m6A regulators between tumor samples and normal samples were analyzed. A three-gene prognostic signature including IGF2BP3, RBM15B, and METTL16 was constructed, and the risk score of this signature was identified to be an independent prognostic indicator for melanoma. In addition, IGF2BP3 was verified to promote melanoma cell proliferation and migration in vitro and associate with lymph node metastasis in clinical samples. Moreover, risk score and the expression of IGF2BP3 were positively associated with the infiltrating immune cells and these hub genes made excellent potential drug targets in melanoma.ConclusionWe identified the genetic changes in m6A regulatory genes and constructed a three-gene risk signature with distinct prognostic value in melanoma. This research provided new insights into the epigenetic understanding of m6A regulators and novel therapeutic strategies in melanoma.

Disease Progression in Cutaneous Squamous Cell Carcinoma Patients With Satellitosis and In-transit Metastasis.

Satellitosis/in-transit metastasis (S-ITM) has prognostic value in melanoma and Merkel cell carcinoma, but is not incorporated into cutaneous squamous cell carcinoma (cSCC) staging. From our IRB-approved registry, patients with high-risk cSCC, including patients with S-ITM, were identified. Univariate (UVA) and multivariate (MVA) analyses were performed to compare disease progression (DP) and overall survival (OS). Cumulative incidence of DP and OS analyses were performed using Fine-Gray and Kaplan-Meier methods, respectively. A total of 18 S-ITM subjects were compared to 247 high risk subjects including T3N0 (n=143), N1-N3 without extranodal extension (ENE) (n=56), N1-N3 with ENE (n=26) and M1 disease (n=22). Median follow up was 16.5 months. Three-year rates of DP were 22% for T3N0, 42% for S-ITM, 48% for T4 bone invasion, 50% for N1-N3 without extranodal extension (ENE), 53% for N1-N3 with ENE, and 66% for M1. Patients with S-ITM did not experience significantly worse DP compared to those with T3N0 (HR=1.96, 95%CI=0.8-4.9; p=0.14). Cutaneous SCC patients with S-ITM experienced outcomes similar to locally advanced non-metastatic cSCC patients. Larger studies are needed to guide incorporation into staging systems.

Optimization of an automated tumor-infiltrating lymphocyte algorithm for improved prognostication in primary melanoma.

Tumor-infiltrating lymphocytes (TIL) have potential prognostic value in melanoma and have been considered for inclusion in the American Joint Committee on Cancer (AJCC) staging criteria. However, inter-observer discordance continues to prevent the adoption of TIL into clinical practice. Computational image analysis offers a solution to this obstacle, representing a methodological approach for reproducibly counting TIL. We sought to evaluate the ability of a TIL-quantifying machine learning algorithm to predict survival in primary melanoma. Digitized hematoxylin and eosin (H&E) slides from prospectively-enrolled patients in the NYU melanoma database were scored for % TIL using machine learning and manually graded by pathologists using Clark’s model. We evaluated the association of % TIL with recurrence-free survival (RFS) and overall survival (OS) using Cox proportional hazards modeling and concordance indices. Discordance between algorithmic and manual TIL quantification was assessed with McNemar’s test and visually by an attending dermatopathologist. 453 primary melanoma patients were scored using machine learning. Automated % TIL scoring significantly differentiated survival using an estimated cutoff of 16.6% TIL (Log Rank P<0.001 for RFS; P=0.002 for OS). % TIL was associated with significantly longer RFS (adjusted HR = 0.92 [0.84–1.00] per 10% increase in % TIL) and OS (adjusted HR = 0.90 [0.83–0.99] per 10% increase in % TIL). In comparison, a subset of the cohort (n=240) was graded for TIL by melanoma pathologists. However, TIL did not associate with RFS between groups (P>0.05) when categorized as brisk, non-brisk, or absent. A standardized and automated % TIL scoring algorithm can improve the prognostic impact of TIL. Incorporation of quantitative TIL scoring into the AJCC staging criteria should be considered.

Development and characterization of CD54-targeted immunoPET imaging in solid tumors.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is an emerging therapeutic target for a variety of solid tumors including melanoma and anaplastic thyroid cancer (ATC). This study aims to develop an ICAM-1-targeted immuno-positron emission tomography (immunoPET) imaging strategy and assess its diagnostic value in melanoma and ATC models. Flow cytometry was used to screen ICAM-1-positive melanoma and ATC cell lines. Melanoma and ATC models were established using A375 cell line and THJ-16T cell line, respectively. An ICAM-1-specific monoclonal antibody (R6-5-D6) and a nonspecific human IgG were radiolabeled with 64Cu and the diagnostic efficacies were interrogated in tumor-bearing mouse models. Biodistribution and fluorescent imaging studies were performed to confirm the specificity of theICAM-1-targeted imaging probes. ICAM-1 was strongly expressed on melanoma and advanced thyroid cancer cell lines. 64Cu-NOTA-ICAM-1 immunoPET imaging efficiently delineated A375 melanomas with a peak tumor uptake of 21.28 ± 6.56 %ID/g (n = 5), significantly higher than that of 64Cu-NOTA-IgG (10.63 ± 2.58 %ID/g, n = 3). Moreover, immunoPET imaging with 64Cu-NOTA-ICAM-1 efficiently visualized subcutaneous and orthotopic ATCs with high clarity and contrast. Fluorescent imaging with IRDye 800CW-ICAM-1 also visualized orthotopic ATCs and the tumor uptake could be blocked by the ICAM-1 parental antibody R6-5-D6, indicating the high specificity of the developed probe. Finally, blocking with the human IgG prolonged the circulation of the 64Cu-NOTA-ICAM-1 in R2G2 mice without compromising thetumor uptake. ICAM-1-targeted immunoPET imaging could characterize ICAM-1 expression in melanoma and ATC, which holds promise for optimizing ICAM-1-targeted therapies in the future.

Systems analysis identifies melanoma-enriched pro-oncogenic networks controlled by the RNA binding protein CELF1

Melanomas are well-known for their altered mRNA expression profiles. Yet, the specific contribution of mRNA binding proteins (mRBPs) to melanoma development remains unclear. Here we identify a cluster of melanoma-enriched genes under the control of CUGBP Elav-like family member 1 (CELF1). CELF1 was discovered with a distinct prognostic value in melanoma after mining the genomic landscape of the 692 known mRBPs across different cancer types. Genome-wide transcriptomic, proteomic, and RNA-immunoprecipitation studies, together with loss-of-function analyses in cell lines, and histopathological evaluation in clinical biopsies, revealed an intricate repertoire of CELF1-RNA interactors with minimal overlap with other malignancies. This systems approach uncovered the oncogene DEK as an unexpected target and downstream effector of CELF1. Importantly, CELF1 and DEK were found to represent early-induced melanoma genes and adverse indicators of overall patient survival. These results underscore novel roles of CELF1 in melanoma, illustrating tumor type-restricted functions of RBPs in cancer.

Significance of serum neural precursor cell-expressed developmentally downregulated protein 9 in melanoma.

Neural precursor cell-expressed developmentally downregulated protein 9 (NEDD9) is a promoter for various cellular functions that result in tumorigenesis. The aim of the present study was to analyse the serum levels of NEDD9 in melanoma patients in order to evaluate its prognostic, predictive and diagnostic value. Data from 112 melanoma patients were retrospectively analyzed and ELISA assays were used to measure serum NEDD9 concentration. The median serum NEDD9 levels of the patients were significantly higher compared with those of the controls. Serum NEDD9 was not found to be associated with any of the clinicopathological parameters, and was also not found to be prognostic for survival in melanoma. Therefore, serum NEDD9 may be of diagnostic value in melanoma, but its usefulness in prognosis remains controversial. The important role of NEDD9 in tumor angiogenesis necessitates efforts to elucidate its interactions with the tumor microenvironment and its potential as a therapeutic target for malignancies.

Clinical Significance of Circulating Serum Cellular Heat Shock Protein 90 (HSP90) Level in Patients with Cutaneous Malignant Melanoma

Background:Cellular heat shock proteins (HSPs) play significant roles in sustaining normal cellular conditions. The stimulated expressions of HSPs result in cellular stabilization at times of stress, such as cancer. The objective of this study was to determine the clinical significance of the serum levels of HSP90 in melanoma patients.Material and Methods:A total number of 98 melanoma patients were enrolled into this study. Serum HSP90 concentrations were determined by the solid-phase sandwich ELISA method. Age and sex matched 43 healthy controls were included in the analysis.Results:The median age of patients was 51 years, ranging from 16 to 85 years. The majority of patients were male (61%), had lesions in axial localizations (54%) and had metastatic disease (61%). Moreover, most of the patients with metastatic disease had M1c diseases (73%). The baseline serum HSP90 levels of melanoma patients were significantly higher than those of the control subjects (median values 49.76 v 27.07ng/ml, respectively, p<0.001). However, clinical variables, such as age, gender, site of lesion, histology, lymph node involvement, stage, serum LDH levels and response to chemotherapy, were found not correlated with serum HSP90 concentrations (p>0.05). Moreover, serum HSP90 level was found not prognostic on survival (p=0.683).Conclusions:Serum levels of HSP90 may have a diagnostic value in melanoma. However, its predictive and prognostic values were not determined.

Association of vascular endothelial growth factor expression with patohistological parameters of cutaneous melanoma.

Melanoma is the most aggresive malignant tumor of the skin. Contradictory data was published on vascular endothelial growth factor (VGEF) in tumor samples and its role in skin melanoma progression and prognosis. The aim of this study was to investigate the significance of VEGF expression as a prognostic parameter in melanoma. The experimental group included 81 patients with primary skin melanomas treated from 2009 to 2013 at the Military Medical Academy, Belgrade. The control group included 20 patients with dysplastic and 20 with benign naevi. Stratification was done according to gender, age, clinical and patological stage, localization, histologic type, Clark's, Breslow, mitotic count, regression and ulceration, tumor infiltrating lymphocytes and metastatic spread.Immunohistochemical staining was performed on skin biopsies using DAKO anti-VEGF antibodies (Ab), LSAB+HRP, DAB and microvawe antigen (Ag) retrieval in DAKO pH 9.0 solution. For statistical data analysis was done with ANOVA, Bonferroni, Mann Whitney and Wilcox on test. The mean intensity of VEGF staining was statistically significantly higher in melanomas than in benign or dysplastic naevi. Furthermore, the highest recorded values were in Ia and IV clinical stages. The majority of melanomas with high intensity of VEGF staining were in pT1a pathological stage. Melanomas with the highest mitotic count (> 6) had a significantly higher intensity of VEGF staining than those with < 2 mitoses. The higest intensity of staining was in melanomas without significant lymphocytic infiltrate and the lowest was in those with brisk lymphocytic infiltrate, thus a statistical difference was siginifant. The mean intensity of VEGF staining was highest in melanomas with lymphovascular invasion. There was no statistically significant difference between VEGF and any other parameter. VEGF in primary skin melanomas plays an important role in tumor progression and is linked to the absence if tumor infiltrating lymphocytes and the presence of lymphovascular invasion. More detailed studies have to be done on VEGF prognostic value in melanoma on a larger number of patients.

SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma.

Background:We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3′ untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA–mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs.Methods:The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155–TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3′UTR and miR-155 were analysed by RT–qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues.Results:The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the ‘match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the ‘match' group, whereas it positively correlated with protein but not with miR-155 in the ‘mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival.Conclusion:Polymorphisms in 3′UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Correlation of prevalence of CD68+ macrophages within tumor-draining lymph node basins and overall survival in stage III melanoma patients.

e21034 Background: Although it is well-established that melanomas can elicit immune responses, they more often produce factors that adversely impact or block antitumor immunity. Suppressive immune cells such as Foxp3+ regulatory T cells (Treg) and tumor-infiltrating macrophages have been implicated as poor prognostic indicators in other cancer etiologies, but their prognostic value in melanoma has remained unclear. Methods: We performed an immunohistochemical (IHC) analysis of 62 melanoma tumors comprising a spectra of disease stages to assess tumor infiltration by T cells (CD3+, CD4+, CD8+ and Foxp3+ subsets), B cells (CD19+), and macrophages (CD68+). To assess the potential negative impact of tumors on regional immunity, we also analyzed a set of uninvolved regional lymph nodes (LN) harvested from sentinel node positive basins of 28 stage III melanoma patients. All immune markers were correlated with overall survival by univariate Cox regression analyses. Results: Overall, relatively few Foxp3+ T cells or B cells infiltrated melanoma tumors. In contrast, CD68+ macrophages were found to be abundant at all disease stages, typically comprising 5% to 20% of all cells within the tumor parenchyma. Although no significant associations were found with any immune markers in melanoma tumors, Cox regression analysis demonstrated a significant adverse association between the percentage of CD68+ macrophages in uninvolved regional LNs and overall survival (HR=1.93 per 5% increase in CD68+ cells, 95% CI (1.17, 3.18), p=0.01). Kaplan-Meier analyses revealed that stage III patients harboring >15% CD68+ cells in uninvolved regional LNs had a median survival of 2.6 yr vs. 7.7 yr for patients with <15% CD68+ cells. Conclusions: These results suggest that melanoma involvement of regional LNs can adversely impact the immune microenvironment of adjacent uninvolved regional LNs, possibly by facilitating regional immune suppression mediated by CD68+ cells. Pharmacological targeting of tumor-associated CD68+ macrophages may favorably impact survival outcomes for patients with stage III melanoma. No significant financial relationships to disclose.

Sentinel node status has high prognostic value for melanoma

Sentinel node status has high prognostic value for melanoma

Minimal residual disease in melanoma.

A number of specific genes encoding for melanosomal proteins are selectively expressed in melanocytes and melanomas. For detection of circulating melanoma cells, the expression of the tyrosinase gene is most widely used. Several cohorts of melanoma patients from single institutions have been analyzed by various research groups for the presence of circulating melanoma cells in all stages of disease. The percentage of patients with evidence of occult tumor dissemination has been correlated with stage of disease in several, but not all, reports. Two prospective analyses suggest that the PCR result is of prognostic value in melanoma. Several laboratories have found PCR evidence for circulating melanoma cells in the vast majority of untreated patients with stage IV disease, although other groups have reported much lower frequencies. Taken together, there is a wide range of results. Methodological differences likely account for this discrepancy. With the availability of true quantitative RT-PCR systems, such as the Light Cycler system, accurate quantification of tyrosinase transcripts over a range of 1-10,000 tumor cells per ml of blood is possible. Quantitative RT-PCR systems also dramatically improve quality control, since exact quantitation of housekeeping gene mRNA facilitates determination of sample quality.

Prognostic significance of nucleolar organizer regions (NORS) in malignant melanoma

Nucleolar organizer regions (NORs) are loops of ribosomal DNA (rDNA) in the nucleolus and are associated with acidic proteins. They are seen in routinely processed paraffin sections by using a one-step colloidal silver (Ag) staining method; they appear as black dots termed "AgNORs". The quantitative assay of AgNORs has been used to differentiate benign from malignant neoplasms. Melanocytic lesions differ significantly in AgNOR counts between malignant melanoma and nevi. However, conflicting results have been reported as to AgNORs' prognostic value in melanoma. A recent study showed AgNOR counts to be a more accurate prognostic indicator than Breslow's thickness. In this study, we counted the AgNORs in 26 patients with primary cutaneous melanomas (CMM) between 2.0 mm and 2.5 mm thick. Of these, 14 are alive without disease (AN) at 5 years after diagnosis (group 1) and 12 are dead of disease (DD) in less than 5 years (group 2). The AgNORs were scored in 30 nuclei per tumor, and the means were calculated. For group 1, the mean number of AgNORs per nucleus was 6.88, ranging from 3.73 to 12.70. For group 2, the mean number was 6.97, ranging from 3.63 to 11.67. Statistical analysis using analysis of variance (ANOVA) showed no significant difference between the groups (p = 0.33). In our study, AgNOR counts did not prove to be of prognostic value in malignant melanoma.