Giant congenital melanocytic nevus with vascular malformation and epidermal cysts associated with a somatic activating mutation in BRAF.

Giant congenital melanocytic nevi may be symptomatically isolated or syndromic. Associations with capillary malformations are exceptional, and development of epidermal cysts has not been described. A 71-year-old patient with a giant congenital melanocytic nevus (CMN) of the lower back, buttocks, and thighs was asymptomatic except for unexpected hemorrhage during partial surgical excision years before. Blunt trauma at age 64 initiated recurrent, severe pain under the nevus; multiple large epidermal cysts then developed within it. Imaging and biopsy showed a large, non-pulsatile venous malformation intermingled with the deep nevus. A low-abundance, heterozygous BRAF c.1799T>A (p.V600E) mutation was present in both gluteal and occipital congenital nevi; additional mutations in NRAS, GNAQ, GNA11, HRAS, or PIK3CA were undetectable. This is the first demonstration of a recurrent BRAF mutation in multiple large congenital nevi from the same individual, confirming that this malformation can have multiple genetic origins. Early constitutive activation of BRAF can therefore cause unusual associations of giant nevi with vascular malformations, indicating that both pigment and endothelial cell physiology may be affected by mosaic RASopathies.

The BRAF and NRAS mutation prevalence in dermoscopic subtypes of acquired naevi reveals constitutive mitogen-activated protein kinase pathway activation.

BACKGROUND: Acquired naevi can have unique dermoscopic patterns that correspond to distinct microanatomical growth patterns. Previous studies on acquired naevi stratified according to dermoscopic pattern focused on the frequency of somatic BRAF mutations, whereas NRAS mutations remained to be elucidated. OBJECTIVES: To investigate the BRAF and NRAS mutation prevalence and activation of the mitogen-activated protein kinase (MAPK) pathway in distinct dermoscopic subtypes of acquired naevi. METHODS: Common mutations present in BRAF and NRAS were assessed in 40 globular, reticular and peripheral rim of globules (PG) subtypes of acquired naevi from 27 participants (19 male, 8 female; mean age 46·7 years) selected from 1261 eligible volunteers. Mutations were determined using the highly sensitive and quantitative QX200 droplet digital™ polymerase chain reaction (ddPCR) system. RESULTS: The BRAF V600E (c.1799T>A or c.1799_1800delTGinsA) and BRAF V600K mutations were detected in 85% (n = 34/40) of naevi. All BRAF wild-type naevi (15%; n = 6/40) harboured an NRAS codon 12/13 or 61 mutation. BRAF mutations were present in 92% (n = 12/13) of globular and 100% (n = 12/12) of PG naevi, whereas reticular naevi were 67% (n = 10/15) BRAF- and 33% (n = 5/15) NRAS-mutant (P = 0·037). CONCLUSIONS: We discovered that 100% of the assessed acquired naevi had either a BRAF or NRAS mutation. Using sensitive techniques capable of single-cell mutation detection, it is likely that all acquired naevi will be mutated for BRAF or NRAS. Because both of these mutations are prevalent in distinct dermoscopic naevus subsets, our study supports the role of the MAPK pathway in the development of benign melanocytic proliferations, indicating that additional genomic events besides somatic mutations in BRAF or NRAS are required for melanoma development.

Dysregulation of redox-state-regulating enzymes in melanocytic skin tumours and the surrounding microenvironment.

AIMS: To investigate redox-regulating enzymes that may have a special role in melanoma pathogenesis due to continuous exposure to microenvironment-produced and ultraviolet radiation-induced oxidative stress. METHODS AND RESULTS: We assessed immunohistochemically the expression of antioxidant enzymes peroxiredoxins (Prxs) I-IV, sulfiredoxin (Srx) and redox-regulated proto-oncogene DJ-1 in material consisting of 30 benign naevi, 14 lentigo malignas and 67 malignant melanomas. Evaluation of immunostaining was performed with special attention paid to protein expression in different tumour compartments. In particular, the expression patterns of nuclear Prx I and Prx II and cytoplasmic DJ-1 were decreased significantly in melanomas compared with dysplastic and benign naevi. In multivariate analysis, several prognostic factors were identified: Prx III expression in the cytoplasm of stromal fibroblasts was associated with shortened melanoma-specific survival [hazard ratio (HR) 6.730; 95% confidence interval (CI) 1.579-28.689], while cytoplasmic Prx IV expression in endothelial cells (HR 6.563; 95% CI 1.750-24.620) and Srx expression in the cytoplasm of keratinocytes (HR 6.988; 95% CI 1.559-31.324) were associated with better prognosis independently of ulceration, thickness of melanoma or its diagnostic type. CONCLUSIONS: Redox-regulating enzymes have the potential to serve as novel prognostic factors and targeting them may offer new therapeutic options in malignant melanoma.

Chronically sun-damaged melanomas express low levels of nuclear glutathione-S-transferase-π: an epidemiological and clinicopathological study in Italy.

The detoxifying enzyme glutathione-s-transferase pi (GST-π) is present in keratinocytes and melanocytes and exerts a protective role against tumour progression. Melanomas close to melanocytic naevus remnants occur less frequently on sun-exposed areas, whereas solar dermal elastosis, hallmark of chronic sun-damage, characterise melanomas on sun-exposed skin. We evaluated the expression of GST-π in 113 melanomas associated to melanocytic naevus remnants or to solar dermal elastosis, classified according to clinical characteristics, history of sun exposure, histological subtypes and AJCC staging. Chronically sun-damaged melanomas, identified by moderate-severe solar dermal elastosis, showed a lower nuclear GST-π expression and a higher thickness than those related to melanocytic naevus remnants (p < 0.03). Multivariate logistic regression analysis demonstrated that male gender and chronic sun-exposure are independent risk factors significantly associated to melanomas localised on the trunk (OR = 3.36, 95% CI: 1.31-8.65; OR = 5.97, 95% CI: 1.71-20.87). If confirmed on a larger series, lower expression of nuclear GST-π in melanoma cells could represent a possible marker of chronically sun-damaged melanoma pathogenesis.

Value of dopachrome tautomerase detection in the assessment of melanocytic tumors.

Dopachrome tautomerase (DCT) and tyrosinase (Tyr) are melanogenic enzymes and structurally related melanosomal proteins. The present study investigates DCT expression comparatively with Tyr, the most tested melanoma biomarker, aiming to evaluate DCT potential in the assessment of melanocytic tumors and gain insights into the molecular and pathological characterization of DCT-phenotype in tumor progression. DCT and Tyr are simultaneously analyzed in melanoma cell lines by semiquantitative RT-PCR, western blot, and N-glycan analysis, and in cell populations of melanocytic tumors by immunohistofluorescence using a novel anti-hDCT antibody against an extended sequence within DCT luminal domain. DCT, unlike Tyr, is fully processed along the secretory pathway in both pigmented and amelanotic melanoma cells. In 53 nevi and 116 primary malignant melanomas, 81% and 52%, respectively, are DCT+/Tyr+, showing that DCT is a stable antigen, retained by most tumors and partially expressed in Tyr-negative cell populations. The DCT/Tyr disjunction is a process correlated with melanocyte neoplastic transformation and malignant progression. A tumor architecture--DCT-phenotype-containing DCT+/Tyr- cell populations selected into the innermost dermis from double-positive cells is detected in 35% of DCT+/Tyr+ specimens. The DCT-phenotype is associated with enhanced neurotization in benign nevi and with ulceration in thin malignant melanomas. The intradermal DCT+/Tyr- clones in superficial melanomas acquire the expression and specific subcellular distribution of unfavorable prognostic markers. DCT assessment shows specific antigen patterns with potential significance in the outcome of melanocytic lesions, connecting DCT, a mediator of a melanoma stress-resistant pathway, and an antiapoptotic molecule to DCT- phenotypes that are possibly more stable and stress resistant.

Genomewide RNAi screen identifies protein kinase Cb and new members of mitogen-activated protein kinase pathway as regulators of melanoma cell growth and metastasis.

A large-scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole-genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen-activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C ß (PKCß) as candidate genes. Knockdown of PKCß most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCß showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCß-specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCß-shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCß seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.

[Cellular and molecular mechanisms of carcinogenic side effects and resistance to BRAF inhibitors in metastatic melanoma with BRAFV600 mutation: state of the knowledge]. / Effets secondaires carcinogènes et résistances aux thérapies ciblées anti-BRAF.

Cutaneous melanoma is a malignant tumor with a high metastatic potential. If an early treatment is associated with a favorable outcome, the prognosis of metastatic melanoma remains poor. Advances in molecular characterization of cancers, notably the discovery of BRAF gene mutations in metastatic melanoma, allowed to the recent development of targeted therapies against mutated BRAF protein. Despite high tumor response rates observed in clinical trials, these new drugs are associated with frequent secondary tumor resistance occurrence and paradoxical carcinogenic side effects. The cellular and molecular mechanisms of these carcinogenic side effects and secondary resistance are not yet fully elucidated and are actually intensely studied. This review of the literature focus on the mechanisms of these carcinogenic side effects and on the tumor resistance associated with anti-BRAF targeted therapies.

Predictors of BRAF mutation in melanocytic nevi: analysis across regions with different UV radiation exposure.

BACKGROUND: BRAF mutations have been implicated in initiating promutagenic cellular melanocytic proliferation mostly based on homogeneous Western-based cohorts. Data addressing the possible interaction between exposure to different solar ultraviolet radiation (UVR) magnitudes and BRAF mutation rate (BMR) in melanocytic nevi are limited. DESIGN: Extended BRAF testing for 9 mutations in 225 melanocytic nevus (MN) cases derived from 211 patients from 4 different UVR regions: Lebanon (n = 95; 110 kJ · m(-2) · yr), Syria (n = 23; 93.5 kJ · m(-2) · yr), Kingdom of Saudi Arabia (n = 70; 139 kJ · m(-2) · yr), and Pakistan (n = 37; 118 kJ · m(-2) · yr) was performed. Data collected included age, gender, anatomic location, and lesion size. Histological parameters recorded were MN type (junctional, compound, intradermal, classical blue, cellular blue, compound and intradermal spitz, and congenital) solar elastosis grade, and nevus pigmentation degree. Cumulative 21-year erythemally effective UV averages were derived from The National Center for Atmospheric Research. RESULTS: BRAF mutation status was obtained in 210 cases (6.7% failed polymerase chain reaction). Overall, BMR was 62.4% (131/210) with V600E mutation accounting for 98.5% of cases. Discordant mutation status was found in 2 of 10 patients with multiple nevi. BMR differed significantly, yet nonsystematically, among UVR regions; the highest was detected in nevi coming from Syria (18/23 cases, 78%), followed by Pakistan (21/30 cases, 70%), Kingdom of Saudi Arabia (47/70 cases, 67%), and Lebanon (45/87 cases, 52%). Mutation rates varied significantly across MN type (P < 0.001); the highest rate was recorded in the intradermal nevus type (33/39 cases, 84.6%), followed by the compound (26/32 cases, 81.2%) and congenital (60/74 cases 81.0%) nevi. Stratified by anatomic location, nevi occurring on the face (61/82, 74%) and trunk (58/78, 74%) had more frequent BMRs compared with those occurring on the upper (7/26, 27%) and lower extremities (5/24, 21%, P < 0.001). Severe pigmentation was less frequent in BRAF mutation-positive nevi [5/131 (4%) vs. 34/79 (43%); P < 0.001]. Multivariate independent predictors of BRAF mutation in MN were age [odds ratio (95% confidence interval ) = 1.43 (1.13-1.74) per 10 years; P = 0.004], anatomic location [P = 0.043 overall], and nevus type [P < 0.001 overall]. UVR region was not an independent predictor of BRAF mutation. CONCLUSIONS: Increased BRAF mutation with age along with the lack of a UVR magnitude-BRAF mutation association suggests that duration of exposure rather than UVR exposure dose is the more likely link to acquiring the mutation.

C-kit expression of melanocytic neoplasm and association with clinicopathological parameters and anatomic locations in Chinese people.

Distinct genetic aberrations between melanomas in different anatomical locations have been confirmed in recent years. However, the associations between immunohistochemical expression, tumor sites, and clinical parameters are not clear. We examined the correlation of protein expression and gene mutation of c-kit with clinicopathological parameters and lesion locations in patients with malignant melanoma (MM). We collected 170 melanocytic lesions, including 106 cutaneous MM from acral melanoma (AM) and nonacral melanoma (NAM) sites, 24 dysplastic nevi, and 40 common melanocytic nevi. Tissue microarray was constructed, and immunohistochemical expression for c-kit was assessed with correlation with clinical parameters. Mutation in exons 11, 13, 17, and 18 of KIT gene in genomic DNA by polymerase chain reaction sequencing was also analyzed. Immunostaining scores for c-kit were found to be statistically higher in Dysplastic Nevi than in common melanocytic nevi and MM. In addition, cytoplasmic c-kit staining was significantly correlated with poor survival in patients with AM but not in those with NAM. Twenty-nine cases of MM (including 9 NAM and 20 AM) are analyzed for mutation in exons 11, 13, 17, and 18 of KIT gene in genomic DNA by polymerase chain reaction sequencing, and no genetic mutation is found. Our findings confirm that KIT mutations, in contrast to previous white cohorts, are not common in both AM and NAM of the Chinese and do not necessarily correlate with c-kit expression. The significantly different association between the expression of c-kit immunoreactivities and the mortality risks of melanomas on acral versus nonacral sites might change site-specific targeted therapeutic concepts in melanoma in the future.

Mast cells in melanocytic skin lesions. An immunohistochemical and quantitative study.

The mast cells participate in inflammation and possibly in carcinogenesis. The aim of the study was to study mast cells in melanocytic lesions. The material consisted of 24 pigmented nevi, 18 dysplastic nevi and 19 melanomas. The sections were stained immunohistochemically for tryptase and chymase. Positive cells were counted inside the lesions and at the interface between the lesion and dermis. The mean intralesional tryptase+ count was 15.75 for nevi, 21.78 for dysplastic nevi, and 8.07 for melanomas. The chymase+ intralesional count was 14.89 for nevi, 21.88 for dysplastic nevi, and 11.34 for melanomas. The tryptase+ perilesional count was 16.89 for nevi, 15.93 for dysplastic nevi, and 15.71 for melanomas. The chymase+ perilesional count was 16.52 for nevi, 16.16 for dysplastic nevi, and 14.77 for melanomas. The tryptase/chymase intralesional ratio was 0.93 for nevi, 1.05 for dysplastic nevi, and 1.67 for melanomas. The tryptase/chymase perilesional ratio was 1.02 for nevi, 1.09 for dysplastic nevi, and 1.00 for melanomas. The differences between intralesional mast cells, both tryptase+ and chymase+, were statistically significant. The intralesional tryptase+ count showed an inverse correlation to age (R = -0.42); this correlation was the strongest in melanomas. The results obtained in our study suggest a possible correlation between mast cells and the pathogenesis of cutaneous melanoma.

Expression of fatty acid synthase (FASN) in oral nevi and melanoma.

OBJECTIVE: The aim of this study was to determine the expression of fatty acid synthase (FASN) in oral nevi and melanomas, comparing the results with correspondent cutaneous lesions. MATERIALS AND METHODS: Expression of FASN was evaluated by immunohistochemistry in 51 oral melanocytic lesions, including 38 intramucosal nevi and 13 primary oral melanomas, in 10 cutaneous nevi and in 14 melanomas. RESULTS: Fatty acid synthase was strongly expressed only in melanomas, either of the oral mucosa or cutaneous. On the other hand, most oral and cutaneous nevi were negative, with a few oral cases showing focal and weak expression. CONCLUSION: Fatty acid synthase is expressed in malignant melanocytes, and it can be a helpful marker to distinguish oral melanomas from oral melanocytic nevi.

Immunohistochemical expression patterns of the microRNA-processing enzyme Dicer in cutaneous malignant melanomas, benign melanocytic nevi and dysplastic melanocytic nevi.

Dicer is an essential cytosolic enzyme necessary for processing pre-microRNAs into mature microRNAs (miRNAs). Although a variety of malignancies have been attributed to perturbations in the miRNA machinery, there has been little research conducted on the role of miRNAs in cutaneous malignant melanoma and its premalignant lesions. In this small pilot study, we therefore investigated the distribution of Dicer by immunohistochemistry in cutaneous malignant melanomas, as well as in benign and dysplastic melanocytic nevi. Dicer was assessed in ten cutaneous malignant melanomas (CMM), benign melanocytic nevi (BMN), and dysplastic melanocytic nevi (DMN), by standard immunohistochemical staining. Semiquantitative analyses determined expression indices (EIs), which associate the conventional area fraction of labeled cells with immunostaining intensity scores, based on visual qualitative examination by two independent observers. Mean EI scores were significantly higher in the CMM group compared to those in the BMN group (p < 0.05). However, EI differences between BMN and DMN as well as between CMM and DMN were not significant (p > 0.05). For CMM we observed a significant correlation of Breslow tumor thickness and Dicer EI (r â€Š=  0.84, p â€Š=  0.022). For all three groups investigated, Dicer-positive staining was primarily located in the epidermis, specifically in melanocytes. By immunohistochemistry, Dicer staining was significantly higher in melanoma cells than in benign melanocytes. This preliminary study indicates that alterations in the miRNA machinery could exist and should be subject of further investigation.

Cyclooxygenase-2 immunohistochemistry in human melanoma: differences between results obtained with different antibodies.

Several groups have reported that cyclooxygenase-2 (COX-2) expression is significantly enhanced in human melanomas, and that the expression of this protein may be useful as diagnostic and prognostic marker for the disease. At the same time, collective analysis of immunohistochemical data on the COX-2 expression in melanomas, presented by different researchers, shows a clear lack of consistency of reported results commonly assigned to differences in protocols used for the staining. This paper describes a study involving the parallel use of three different primary anti-COX-2 antibodies targeting different COX-2 epitopes. A surprising outcome is that although the three antibodies gave very consistent results for the COX-2 expression in keratinocytes, they showed significant differences in immunoreactivity for both melanocytic naevi and melanomas. This phenomenon has not been described before, and has implications for the selection of antibodies for studies on the diagnostic potential of COX-2 for melanoma.

Phosphatidylinositol-3-kinase as a therapeutic target in melanoma.

PURPOSE: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002. EXPERIMENTAL DESIGN: Using Automated Quantitative Analysis, we quantified expression of p85 and p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. RESULTS: p85 and p110alpha tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively). CONCLUSIONS: Expression of p85 and p110alpha subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.

Inducible nitric oxide synthase expression in melanoma: implications in lymphangiogenesis.

Cutaneous melanoma preferentially metastasizes via the lymphatic route. However, the mechanisms of lymphatic invasion and metastasis to regional lymph nodes are poorly understood. Nitric oxide is a free radical molecule synthesized from L-arginine by nitric oxide synthases that plays a critical role in various physiological and pathological processes, including tumor growth and angiogenesis. We have tested whether inducible nitric oxide synthase expression correlates with lymphatic vessel density identified with D2-40 antibody and/or blood microvessel density identified with CD105/endoglin in a series of melanocytic nevi (n=28) and cutaneous melanomas (n=38), representative of various pT. Inducible nitric oxide synthase expression was significantly lower in melanocytic nevi in comparison with primary and metastatic melanomas (P<0.001). Mean microvessel density was significantly higher in primary and metastatic melanomas in comparison with melanocytic nevi (P<0.001 for intratumoral and P=0.001 for peritumoral vessels). Vertical growth phase melanomas showed a higher intratumoral microvessel density in comparison with radial growth phase melanomas (P=0.02). The number of peritumoral lymphatics was significantly lower in nevi as compared with primary and metastatic melanomas (P=0.01). No correlation between microvessel or lymphatic vessel and clinical outcome was found in melanomas. A significant direct correlation was observed between inducible nitric oxide synthase immunostaining in melanocytic tumor cells and the density of lymphatic vessels (peritumoral: P=0.001; intratumoral: P=0.08), and the density of peritumoral blood microvessel (P=0.02). Our findings support the hypothesis that inducible nitric oxide synthase is implicated not only in blood, but also in lymphatic vascular neoformation in melanoma. Mechanistic studies are needed to address the possibility that inducible nitric oxide synthase controls lymphangiogenesis, dissemination and lymphatic borne metastases.

Immunohistochemical expression of matrix metalloproteinase-2 and -9 in melanocytic nevi is altered by ultraviolet B.

BACKGROUND: Ultraviolet B (UVB) radiation can cause morphological and biological alterations similar to those of melanoma in situ in irradiated melanocytic nevi. matrix metalloproteinase (MMP-2) and -9 appear to be involved with tumour invasion, the formation of metastases and neoangiogenesis in melanomas. The effects of UVB on the immunohistochemical expression of gelatinases in different cell types in melanocytic nevi have not been completely studied. PURPOSE: Evaluate the effects of UVB on the immunohistochemical expression of MMP-2 and -9 in different cell types in melanocytic nevi. METHODS: Forty-two melanocytic nevi had one half irradiated with 2 MEDs of UVB and were excised 1 week later. Three different observers compared the intensity of the immunohistochemical expression of gelatinases in keratinocytes, melanocytes, endothelial cells and fibroblasts on the irradiated (IS) and non-irradiated sides (NIS). RESULTS: All the cell types showed an increase in the expression of MMP-2 on the IS, especially the epidermal melanocytes. No significant increase in the expression of MMP-9 in keratinocytes was detected on the IS; significant increase was observed in all the remaining cells. CONCLUSIONS: One single irradiation of UVB increases the immunohistochemical expression of gelatinases in almost all evaluated cell lines, with the exception of MMP-9 in keratinocytes.