Expression of cyclins A and E in melanocytic skin lesions and its correlation with some clinicopathologic features

Cyclins play a fundamental role in the cell cycle. Recent studies have focused on their role in the development of various malignancies. The objective of this study was to evaluate and compare the expression of cyclins A and E in common nevi, dysplastic nevi and malignant melanomas, and to investigate the relationship between cyclin expression and some pathological parameters such as tumor thickness, ulceration, regression, and mitotic rate, as well as several clinical and phenotypic parameters such as skin phototype, hair and eye color, number of nevi, personal or family melanoma history, and personal history of nonmelanoma skin cancer (NMSC). A total of 102 melanocytic skin lesions, including 30 common nevi, 38 dysplastic nevi and 34 melanomas, were examined. Expression of cyclins was detected by immunohistochemistry and quantified as a percentage of immunostained cell nuclei in each sample. Significant differences in expression of both cyclins were found between all lesion types: the median percentage of cyclin A-positive nuclei was 8.2% in melanomas, 3.4% in dysplastic nevi, and 0.95% in common nevi (p < 0.001). The corresponding percentages for cyclin E were 9.5%, 4.25% and 1.44% (p < 0.001). Expression of both cyclins was significantly higher among patients with a personal history of NMSC. Cyclin A was also significantly overexpressed in patients with a high total nevus count (TNC) compared to moderate and low TNC. Expression of cyclins did not significantly correlate with the other clinicopathologic features investigated. These findings indicate the possible involvement of cyclins A and E in the pathogenesis of malignant melanoma. Our results also show a potential diagnostic significance of these cyclins as markers allowing discrimination between dysplastic nevi and melanoma.     

Expression of cell cycle and apoptosis regulatory proteins and telomerase in melanocitic lesions

To gain insight into the role and association of cell cycle and apoptosis regulatory proteins and telomerase activity in the course of progression of melanocitic lesions we have examined immunohistochemicaly, expression and the distribution of p53, bcl-2, Ki-67 and telomerase in 25 samples of common and dysplastic nevi, and 45 samples of primary invasive melanomas. Protein p53 expression was significantly increased in dysplastic as compared with common nevi and melanomas (p < 0.001). Bcl-2 protein expression was significantly increased in melanomas as compared with common aquired and dysplastic nevi (p = 0.001). Nevi and melanomas exhibited clear-cut differences in terms of Ki-67 expression. Telomerase expression was significantly increased in melanomas as compared with common acquired (p = 0.014) and dysplastic nevi (p < 0.001). Enhanced telomerase activity in association with increased bcl-2 expression in the course of melanoma progression could contribute to development and progression of melanoma.     

DNA ploidy and nuclear morphometry for the classification of dysplastic nevi

OBJECTIVE: To examine the diagnostic value of DNA ploidy and nuclear morphometric features in sporadic dysplastic nevi as compared to those in compound nevi and melanoma. STUDY DESIGN: DNA ploidy profiles plus seven direct and three derived nuclear features were obtained in a series of 120 melanocytic skin neoplasms (30 dysplastic nevi [DN], 30 melanomas [MM], 60 compound nevi [CN]) and the results compared. RESULTS: DNA ploidy separated melanomas from benign melanocytic skin neoplasms with 96.5% accuracy in classifying the grouped cases. The derived nuclear shape factor Form PE and nuclear axis ratio were the most successful discriminants separating DN from MM but allowed only 73.3% correct classification of cases. Separation of DN from CN was best achieved using Form PE and mean nuclear area (74.4% correctly classified). Results from compound nevi in subjects < 25 years of age fell between those for DN and MM. CONCLUSION: Quantitative nuclear cytologic characteristics in sporadic dysplastic nevi span a range seen in common nevi through to those in thin melanomas. Cytologic changes in sporadic dysplastic nevi overlap those seen in other melanocytic skin neoplasms. Therefore, other reproducible morphometric features need to be assessed in order to further refine the histopathologic diagnosis of this entity.     

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.     

C-Terminal Tensin-Like Protein Is a Novel Prognostic Marker for Primary Melanoma Patients

Background

C-terminal tensin-like protein (Cten) is a focal adhesion protein originally identified as a tumor suppressor in prostate cancer. It has since been found to be overexpressed and function as an oncogene in numerous other cancers, but the expression status of Cten in melanoma is still unknown.

Methods

Using tissue microarrays containing 562 melanocytic lesions, we evaluated Cten protein expression by immunohistochemistry. The association between Cten expression and patient survival was examined using Kaplan-Meier survival analysis, and univariate and multivariate Cox regression analyses were used to estimate the crude and adjusted hazard ratios.

Results

Strong Cten expression was detected in 7%, 24%, 41%, and 46% of normal nevi, dysplastic nevi, primary melanoma, and metastatic melanoma samples, respectively, and Cten expression was found to be significantly higher in dysplastic nevi compared to normal nevi (P = 0.046), and in primary melanoma compared to dysplastic nevi (P = 0.003), but no difference was observed between metastatic and primary melanoma. Cten staining also correlated with AJCC stages (P = 0.015) and primary tumor thickness (P = 0.002), with Cten expression being induced in the transition from thin (<1mm) to thick (≥1mm) melanomas. Strong Cten expression was significantly associated with a worse 5-year overall (P = 0.008) and disease-specific survival (P = 0.004) for primary melanoma patients, and multivariate Cox regression analysis revealed that Cten expression was an independent prognostic marker for these patients (P = 0.038 for overall survival; P = 0.021 for disease-specific survival).

Conclusion

Our findings indicate that induction of Cten protein expression is a relatively early event in melanoma progression, and that Cten has the potential to serve as a prognostic marker for primary melanoma patients.     

Novel Multiple Markers to Distinguish Melanoma from Dysplastic Nevi

Background

Distinguishing melanoma from dysplastic nevi can be challenging.

Objective

To assess which putative molecular biomarkers can be optimally combined to aid in the clinical diagnosis of melanoma from dysplastic nevi.

Methods

Immunohistochemical expressions of 12 promising biomarkers (pAkt, Bim, BRG1, BRMS1, CTHRC1, Cul1, ING4, MCL1, NQO1, SKP2, SNF5 and SOX4) were studied in 122 melanomas and 33 dysplastic nevi on tissue microarrays. The expression difference between melanoma and dysplastic nevi was performed by univariate and multiple logistic regression analysis, diagnostic accuracy of single marker and optimal combinations were performed by receiver operating characteristic (ROC) curve and artificial neural network (ANN) analysis. Classification and regression tree (CART) was used to examine markers simultaneous optimizing the accuracy of melanoma. Ten-fold cross-validation was analyzed for estimating generalization error for classification.

Results

Four (Bim, BRG1, Cul1 and ING4) of 12 markers were significantly differentially expressed in melanoma compared with dysplastic nevi by both univariate and multiple logistic regression analysis (p < 0.01). These four combined markers achieved 94.3% sensitivity, 81.8% specificity and attained 84.3% area under the ROC curve (AUC) and the ANN classified accuracy with training of 83.2% and testing of 81.2% for distinguishing melanoma from dysplastic nevi. The classification trees identified ING4, Cul1 and BRG1 were the most important classification parameters in ranking top-performing biomarkers with cross-validation error of 0.03.

Conclusions

The multiple biomarkers ING4, Cul1, BRG1 and Bim described here can aid in the discrimination of melanoma from dysplastic nevi and provide a new insight to help clinicians recognize melanoma.     

Dermatoscopy and High Frequency Sonography: Two Useful Non-Invasive Methods to Increase Preoperative Diagnostic Accuracy in Pigmented Skin Lesions

Dermatoscopy and high frequency sonography have recently been combined to increase diagnostic preoperative accuracy in the treatment of pigmented skin lesions. In this monocentric study 80 patients with pigmented skin lesions were evaluated clinically, by dermatoscopy, and 20 MHz-sonography followed by dermatohistopathological evaluation; 39 malignant melanomas, 37 common nevi, 3 dysplastic nevi, and 1 nevus Spitz were diagnosed histologically. In 72 of the 80 cases (91.3%) dermatoscopical diagnoses were confirmed by histopathology, compared to only 79% correct clinical diagnoses. For the mere clinical diagnosis of melanoma sensitivity was 79%, specificity was 78% and diagnostic accuracy was 65%. All diagnostic values increased by dermatoscopy: sensitivity reached 90%, specificity was 93%, and diagnostic accuracy was 83%. In order to determine tumor thickness preoperatively tumor thickness was measured by 20 MHz sonography. The correlation of tumor thickness between histometric and sonographic results was determined for nevi (r = 0.93) and melanoma (r = 0.95); 74.3% of melanomas were diagnosed correctly within an 0.2 mm range. Regarding the clinical important limit of 1 mm tumor thickness, 87.2% were diagnosed in accordance with histometric evaluation. An increase of 18% in diagnostic accuracy by dermatoscopy and 87.2% of correctly diagnosed cases of tumor thickness of malignant melanoma by high frequency sonography clearly demonstrate that these methods should be considered standard procedures in the diagnosis of pigmented skin lesions and will facilitate the decision on necessary surgical treatment.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Cytoplasmic Skp2 expression is increased in human melanoma and correlated with patient survival

Background

S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation. Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival. In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial.

Methods

To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas. The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival. The univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up.

Results

Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas. Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II–IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005). Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05).

Conclusion

This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival. Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease.     

Evaluation of tubulin β‐3 as a novel senescence‐associated gene in melanocytic malignant transformation

Malignant melanoma might develop from melanocytic nevi in which the growth‐arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth‐arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β‐3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β‐3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β‐3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β‐3 as a candidate marker.     

Collagen XVII is expressed in malignant but not in benign melanocytic tumors and it can mediate antibody induced melanoma apoptosis

The 180?kDa transmembrane collagen XVII is known to anchor undifferentiated keratinocytes to the basement membrane in hemidesmosomes while constitutively shedding a 120?kDa ectodomain. Inherited mutations or auto-antibodies targeting collagen XVII cause blistering skin disease. Collagen XVII is down-regulated in mature keratinocytes but re-expressed in skin cancer. By recently detecting collagen XVII in melanocyte hyperplasia, here we tested its expression in benign and malignant melanocytic tumors using endodomain and ectodomain selective antibodies. We found the full-length collagen XVII protein in proliferating tissue melanocytes, basal keratinocytes and squamous cell carcinoma whereas resting melanocytes were negative. Furthermore, the cell-residual 60?kDa endodomain was exclusively detected in 62/79 primary and 15/18 metastatic melanomas, 8/9 melanoma cell lines, HT199 metastatic melanoma xenografts and atypical nests in 8/63 dysplastic nevi. The rest of 19 nevi including common, blue and Spitz subtypes were also negative. In line with the defective ectodomain, sequencing of COL17A1 gene revealed aberrations in the ectodomain coding region including point mutations. Collagen XVII immunoreaction-stained spindle cell melanomas, showed partly overlapping profiles with those of S100B, Melan A and HMB45. It was concentrated at vertical melanoma fronts and statistically associated with invasive phenotype. Antibody targeting the extracellular aa507-529 terminus of collagen XVII endodomain promoted apoptosis and cell adhesion, while inhibiting proliferation in HT199 cells. These results suggest that the accumulation of collagen XVII endodomain in melanocytic tumors is associated with malignant transformation to be a potential marker of malignancy and a target for antibody-induced melanoma apoptosis.     

Apaf-1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf-1, an important mediator of mitochondrial-dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf-1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf-1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf-1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf-1 mRNA by fluorescence in situ hybridization and of Apaf-1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf-1. A significant decrease in Apaf-1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf-1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf-1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf-1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf-1 may be an important contributory factor in the development of the disease.     

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.     

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

The dysplastic nevus: recognition and management

The recognition of atypical or dysplastic nevomelanocytic nevi potentially provides clinicians with another means of identifying individuals at increased risk for cutaneous malignant melanoma. However, a great deal of controversy still surrounds these lesions, their significance, and the clinical and histologic criteria needed for their diagnosis at present. In general, dysplastic nevi tend to be asymmetrical and larger (greater than 5 mm) than ordinary acquired nevi, have a macular component, irregular and ill-defined borders, and haphazard (variegate) coloration. A clinical diagnosis of dysplastic nevi must be confirmed by histopathology, since not all clinically atypical nevi are dysplastic. While precise histopathologic criteria for dysplastic nevi are lacking, most authorities agree that an abnormal nevomelanocytic proliferative pattern as manifested by increased numbers of basilar melanocytes and/or abnormal junctional nevomelanocytic nesting in the setting of lentiginous epidermal hyperplasia, variable degrees of nevomelanocytic nuclear atypia, and a lymphocytic host response are consistent with a histologic diagnosis of dysplastic nevi. Current data for individuals with dysplastic nevi and a family history of cutaneous malignant melanoma (at least two family members with cutaneous malignant melanoma) indicate a relative risk for cutaneous malignant melanoma about 148 times that of the general population. In comparison, cutaneous malignant melanoma risk seems lower for individuals with familial dysplastic nevi (but without familial cutaneous malignant melanoma) and "sporadic" dysplastic nevi. With respect to progression to melanoma, probably the vast majority of dysplastic nevi remain stable or possibly regress. Management of individuals with histologically confirmed dysplastic nevi involves periodic skin examinations. Regional overview and life-size photography are helpful in following these patients. Patients should also be instructed in the examination of their own skin. While a definite relationship between sun exposure and dysplastic nevi remains unproved, the use of sunscreens and avoidance of unnecessary sun exposure are advised. Examination of family members for atypical melanocytic lesions is also recommended.     

DNA-methylation profiling distinguishes malignant melanomas from benign nevi

DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma. We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi. Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray. Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥ 0.2. Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of > 0.95. Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons. This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.     

Characteristics of the DNA content in the cells of human pigmented nevi and of beginning and developed malignant melanomas of the skin

The comparative microspectrophotometric study of the DNA content in histological samples of 5 pigmented nevi, 3 dysplastic, 9 pigmented nevi with traits of malignization, and 10 malignant melanomas with the I-V invasion level by Clark has permitted determining a direct relationship between the expression degree and quality of the proliferation processes in neoplasia, on the one hand, and DNA quantity in nuclei, on the other hand.     

Appearance of prolactin-releasing peptide-producing cells in the area postrema of postnatal rats

Growth hormone–releasing hormone (GHRH) is secreted by the hypothalamus and upon binding to specific GHRH receptors in the pituitary stimulates growth hormone production and release. In addition to its neuroendocrine action GHRH plays a role in tumorigenesis. Consistently with this latter role, the splice variant 1 (SV1) of GHRH receptor, which is widely expressed in non-pituitary normal tissues and cancers, can mediate the proliferative effects of GHRH and even in the absence of GHRH is capable of eliciting mitogenic signals in the tissues in which it is expressed. The aim of the present study was to investigate the expression of GHRH and its tumoral receptor SV1 in primary human melanomas and dysplastic nevi by immunohistochemistry. None of the specimens tested expressed GHRH. Only 1 of 12 (8%) dysplastic nevi expressed SV1 but 14 of 23 (61%) melanomas showed moderate or strong staining for SV1 (association p < 0.005). This is the first report demonstrating the involvement of SV1 in the pathogenesis of melanomas. Our work implies that the progression from a state of dysplasia into malignancy is accompanied by expression of SV1 receptor. Our findings also suggest that treatment with GHRH antagonists should be further explored for the management of malignant melanomas.     

The metabolic microenvironment of melanomas: Prognostic value of MCT1 and MCT4

BRAF mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. BRAF mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. NRAS mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.