GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi.

BACKGROUND: Mutations in GNAQ/11 genes are considered an early event in the development of uveal melanoma that may derive from a pre-existing nevus. The Hippo pathway, by way of YAP activation, rather than MAP kinase, has a role in the oncogenic capacity of GNAQ/11 mutations. METHODS: We investigated 16 nevi from 13 human eyes for driver GNAQ/11 mutations using droplet digital PCR and determined whether nevi are clonal by quantifying mutant nevus cell fractions. Immunohistochemistry was performed on 15 nevi to analyse YAP activation. RESULTS: For 15 out of 16 nevi, a GNAQ/11 mutation was detected in the nevus cells albeit at a low frequency with a median of 13%. Nuclear YAP, a transcriptional co-activator in the Hippo tumour-suppressor pathway, was detected in 14/15 nevi. CONCLUSIONS: Our analysis suggests that a mutation in GNAQ/11 occurs in a subset of choroidal nevus cells. We hypothesise that GNAQ/11 mutant-driven extracellular mitogenic signalling involving YAP activation leads to accumulation of wild-type nevus cells.

Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds.

The p16 gene (CDKN2) which is localized on chromosome 9p21, is deleted in a significant number of sporadic cancers. Moreover, germline mutations identified in some melanoma-prone kindreds last year suggested that CDKN2 is identical to the 9p21-linked melanoma susceptibility gene (MLM); however, failure to identify p16 mutations in all melanoma kindreds putatively linked to 9p21 left some doubts. We have analysed CDKN2 coding sequences in 15 Dutch familial atypical multiple mole-melanoma (FAMMM) syndrome pedigrees, and identified a 19 basepair (bp) germline deletion in 13 of them. All 13 families originate from an endogamous population. The deletion causes a reading frame shift, predicted to result in a severely truncated p16 protein. Interestingly, two family members are homozygous for the deletion, one of whom shows no obvious signs of disease. This surprising finding demonstrates that homozygotes for this CDKN2 mutation are viable, and suggests the presence of a genetic mechanism that can compensate for the functional loss of p16. Our results also greatly strengthen the notion that p16 is indeed MLM.

The impact of dermoscopy on the management of pigmented lesions in everyday clinical practice of general dermatologists: a prospective study.

BACKGROUND: Dermoscopy greatly improves the clinical diagnosis of pigmented lesions. Few studies have investigated, however, how dermoscopy is guiding management decisions in everyday clinical practice. In addition, most studies have been performed in the setting of dermoscopy experts working in pigmented lesion clinics. OBJECTIVES: To assess the impact of dermoscopy on clinical diagnosis and management decisions for pigmented lesions in everyday practice of general dermatologists. METHODS: We performed a prospective study in general dermatology clinics in community hospitals run by dermatologists with intermediate dermoscopy experience and expertise. Each clinician independently included suspicious lesions from consecutive patients. Pre- and postdermoscopy diagnoses and management decisions were recorded. Pathology was used as reference diagnosis. RESULTS: In total, 209 suspicious lesions were included in the study by 17 dermatologists. Fourteen lesions were histologically proven in situ or invasive malignant melanomas. Based on clinical diagnoses, dermoscopy improved sensitivity from 0.79 to 0.86 (P = 1.0). All 14 melanomas were intended to be excised based on naked eye examination alone, independent of dermoscopic evaluation. Specificity increased from 0.96 to 0.98 (P = 0.22). Dermoscopy resulted in a 9% reduction of the number of excisions. CONCLUSIONS: Dermoscopy reduced the number of excisions, but did not improve the detection of melanomas. Our results suggest that in everyday clinical practice of general dermatologists the main contribution of dermoscopy is a reduction of unnecessary excisions.

Is statin use associated with a reduced incidence, a reduced Breslow thickness or delayed metastasis of melanoma of the skin?

BACKGROUND: Statins show anticancer activity in melanoma cells. We investigated the association between statins and incidence and Breslow thickness of cutaneous melanoma (CM). METHODS: Data were used from PHARMO, a pharmacy database, and PALGA, a pathological database, in the Netherlands. Cases had a primary CM diagnosis between January 1st 1991 and December 14th 2004, were 18 years and had 3 years of follow up in PHARMO before CM diagnosis. Controls were matched for gender, date of birth and geographic region. Analyses were adjusted for age, gender, year of diagnosis, number of medical diagnoses and the use of NSAIDs and oestrogens. FINDINGS: Finally, 1318 cases and 6786 controls were selected. CM risk was not associated with statin use (> or = 0.5 years) (adjusted odds ratio (OR)=0.98, 95% confidence interval (CI)=0.78-1.2). However, statin use was associated with a reduced Breslow thickness (-19%, 95% CI=-33, -2.3, p=0.03). CONCLUSION: Our study suggests protective effects of statins on melanoma progression.

Immunohistochemical analysis of malignant melanomas and nevocellular nevi with monoclonal antibodies to distinct monomorphic determinants of HLA antigens.

Using an indirect immunoperoxidase technique, 20 nevocellular nevi, 5 dysplastic nevi, 14 primary cutaneous melanomas, and 24 metastatic melanomas were tested with a panel of monoclonal antibodies to monomorphic determinants of Class I (HLA-A,B,C) and Class II (la-like) major histocompatibility complex antigens. Class I HLA and beta 2-microglobulins were not detected on the majority of nevus cells but were expressed by 3 of 5 dysplastic nevi, by the majority of tumor cells in 12 of 14 primary cutaneous melanomas, and in 13 of 24 metastases. The different expression of Class I HLA and beta 2-microglobulins in primary and metastatic lesions suggests that loss of these antigens may be associated with progression of malignancy. Class II HLA were not detected in common nevi but were locally present in 1 of 5 dysplastic nevi, 7 of 14 cases of primary cutaneous melanoma, and all 24 cases of metastatic lesions tested. These findings suggest that increase in Class II HLA expression may be associated with progression of malignancy. The staining patterns obtained with monoclonal antibodies to distinct determinants of Class I HLA and Class II HLA were superimposable within each type of antigen. Therefore, the discrepancies in the literature about the expression of histocompatibility antigens by lesions of melanocytic origin are not likely to reflect the different specificity of the antibodies used by the various investigators.

Promoter hypermethylation: a common cause of reduced p16(INK4a) expression in uveal melanoma.

Tumors often display unrestricted cell cycling attributable to a dysfunctional G(1)-S checkpoint. One of the mechanisms leading to such a defect is the inactivation of the cyclin-dependent kinase inhibitor p16(INK4a). Although inactivation of p16(INK4a) is observed in a wide range of tumors, including cutaneous melanoma, genetic alteration of p16(INK4a) is reportedly uncommon in uveal melanoma. Here we show that the p16(INK4a) promoter is hypermethylated in 6 of 12 uveal melanoma cell lines and in 7 of 22 primary uveal melanomas analyzed. Five of seven patients with a methylated primary tumor died of metastatic disease compared with 2 of 15 patients with a nonmethylated primary tumor. We also show that all uveal melanoma cell lines with a hypermethylated p16(INK4a) promoter have lost p16(INK4a) expression but have maintained the expression of p14(ARF). Treatment of uveal melanoma cell lines with 5-aza-2'-deoxycytidine results in demethylation of p16(INK4a) and in reexpression of p16(INK4a) mRNA, which is maintained upon withdrawal of the 5-aza-2'-deoxycytidine. In conclusion, p16(INK4a) promoter methylation appears to be a common event in uveal melanoma and is accompanied by the loss of p16(INK4a) expression.

[Recognition and follow-up of the atypical naevus: benign marker or precursor of melanoma?] / Herkenning en follow-up van de atypische naevus.

Dysplastic (atypical) naevi have long been subject to discussion with regard to their nature; even their existence has been doubted. Recently, the atypical naevus has been formally identified by molecular genetic characteristics as an intermediate lesion between banal naevi and melanoma. However, the atypical naevus, as long as it is stable and asymptomatic, is regarded as a benign lesion that does not warrant excision. Atypical naevi can occur in families in which melanoma is prevalent, and those patients need to be included in regular surveillance by a dermatologist. Two male 30-year-old patients who presented with atypical naevi in different contexts are used as illustrations in this clinical lesson.

Genetics of seven Dutch familial atypical multiple mole-melanoma syndrome families: a review of linkage results including chromosomes 1 and 9.

Familial atypical multiple mole-melanoma syndrome is characterized by the familial occurrence of malignant melanoma of the skin in combination with multiple atypical precursor nevi; its pattern shows a dominant inheritance in pedigrees. During the last 5 years we have performed linkage analysis in seven Dutch familial atypical multiple mole-melanoma families to define the locus of the underlying gene defect. In 1989 it was reported that in familial melanoma families in the USA a disease-gene was located on chromosome 1p. However, in the Dutch families we could exclude this chromosome from harboring the Dutch familial atypical multiple mole-melanoma gene. Very recently a new candidate locus was found on chromosome 9p, which could be confirmed in our family material. A melanoma-associated gene was linked to several markers on chromosome 9p21. In a linkage analysis in which only melanoma patients were considered as affected, marker D9S171 showed a maximum lod score of 3.11 (theta 0.0). After introducing family members with 10 or more, or five or more, atypical nevi as affected in addition to the melanoma patients, the maximum lod score rose to 4.88 (theta 0.05) and 3.79 (theta 0.07), respectively. Interestingly, the sharing of a unique chromosome 9p21 haplotype among most melanoma patients in the families from two different villages suggests that an old common mutation is present in the Leiden region.

Analysis of major histocompatibility antigens and the mononuclear cell infiltrate in halo nevi.

A series of monoclonal antibodies was used to characterize the nevomelanocytes and the inflammatory infiltrate of 11 halo nevi in different stages of resolution, employing an immunoperoxidase technique. Three of the 11 halo nevi histologically showed signs of mild or moderate nevomelanocytic atypia. It was found that the vast majority of the nevomelanocytes in halo nevi with a dense inflammatory infiltrate markedly expressed HLA-A,B,C antigens, while expression was not demonstrable in nevocellular nests not adjacent to the mononuclear infiltrate. No difference in expression of HLA-A,B,C antigens was found between the 3 cases with mild or moderate nevomelanocytic atypia and the other cases lacking atypia. Expression of HLA-DR (Ia-like) antigens was found on few nevomelanocytes in only 2 of 11 lesions. The cellular composition of the mononuclear inflammatory infiltrate showed a predominance of T cells (80% or more) with a relatively high proportion of cytotoxic/suppressor T cells. Most of the T cells showed signs of activation as judged by staining for HLA-DR antigens. These results demonstrate that the expression of HLA-A,B,C antigens on the nevomelanocytes and the cellular composition of the mononuclear inflammatory infiltrate in halo nevi are very similar to that in malignant melanomas and dysplastic neiv. These findings also indicate the expression of HLA-A,B,C antigens on nevomelanocytes is primarily dependent on the presence of T-cell immune response and not necessarily related to the presence of nevomelanocytic atypia.

Are atypical nevi a risk factor for uveal melanoma? A case-control study.

Atypical nevi and other potential risk factors for uveal melanoma were studied in 109 uveal melanoma patients and 149 controls. Information concerning employment, medical history, drug use, family history of cancer, excess sun exposure, and blistering sunburn before and after the age of 15 was obtained. A total skin examination was performed and skin type, hair color, eye color, freckles, actinic damage, the total number of common acquired nevi, and the number of clinically atypical nevi were noted. More atypical nevi were found in uveal melanoma patients than in controls (age- and sex-adjusted odds ratio of 2.9 [95% confidence interval 1.2-6.3] for one or two atypical nevi versus none; odds ratio of 5.1 [95% CI 1.3-20.0] for three or more atypical nevi versus none). Light skin types and freckling also prevailed in uveal melanoma cases. In our study, atypical nevi are more common in uveal melanoma patients than in controls. Further studies will have to indicate whether risk factors comparable to those for cutaneous melanoma really exist for uveal melanoma.

Chronic sun exposure and age are inversely associated with nevi in adult renal transplant recipients.

In 126 adult renal transplant recipients who had survived their transplantation for at least 8 years, we determined whether numbers of nevi and the presence of clinically atypical nevi were related to chronic sun exposure. On the basis of a skin examination, three groups were defined: patients with at least one clinically a typical nevus; patients with only clinically normal nevi: and patients without any nevi. The prevalence odds ratio of having any clinically atypical nevi as compared to having only clinically normal nevi was calculated in a logistic model, in relation to gender, skin type, age, sun exposure, and number of keratotic skin lesions present. Similarly, the prevalence odds ratio of having 30 or more nevi compared to fewer than 30 nevi was calculated. We found an inverse association between chronic sun exposure and age with numbers of nevi in adult renal transplant recipients. The presence of clinically atypical nevi was also inversely associated with chronic sun exposure, but this association disappeared after adjustment for age. We did not observe an association of nevi with the number of keratotic skin lesions, nor with humoral immune responses against human papillomavirus and the presence of certain HLA antigens, which are factors associated with nonmelanoma skin cancer in renal transplant recipients. Chronic sun exposure and age appeared to be strong determinants for decreased numbers of nevi in adult renal transplant recipients. Infection with human papillomaviruses does not appear to play an important role.

Excess cancer mortality in six Dutch pedigrees with the familial atypical multiple mole-melanoma syndrome from 1830 to 1994.

An increased incidence of systemic cancers has been described in some reports of familial atypical multiple mole-melanoma kindreds. If the gene defect underlying the familial atypical multiple mole-melanoma syndrome is not only important for the development of melanoma of the skin, the impact of the defect on life expectancy may be much higher than previously thought. We investigated all-cause mortality from 1830 to the present and causes of death from 1941 to 1994 in proven, obligate, and potential CDKN2 mutation carriers to obtain an estimate of the impact of a hereditary defect of the CDKN2 gene on mortality. From 1830 to 1994 there were 65 deaths, although only 42 deaths were expected [standardized mortality ratio (SMR) 1.6, 95% confidence interval (CI) 1.2-2.0] and the SMR doubled with calendar time. Excess mortality was shown in most of the families, but was confined to ages 35-70 y (SMR 2.1, 95%CI 1.5-2.9). Excess mortality could be fully attributed to cancer mortality, especially to pancreatic carcinoma and melanoma of the skin. There appeared to be some heterogeneity among the families, especially due to the specific cancer pattern within a family. The impact of the defect of the CDKN2 gene is rising over calendar time, mainly because the mortality in the general population has been falling. Excess mortality was not only due to melanoma, but also to pancreatic carcinoma. Therefore, follow-up programs of affected family members should not be confined to a regular check of the atypical nevi.

Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color.

Individuals carrying melanocortin 1 receptor gene variants have an increased risk for the development of cutaneous melanoma. Melanocortin 1 receptor gene variants are also associated with other risk factors for melanoma such as fair skin and red hair. We evaluated the relationship of melanocortin 1 receptor gene variants, fair skin, red hair and the development of melanoma in 123 patients with cutaneous melanoma and 385 control subjects. To analyze the association between melanocortin 1 receptor gene variants and skin type or hair color we also made use of 453 patients with nonmelanoma skin cancer. We analyzed the coding sequence of the melanocortin 1 receptor gene region by single-stranded conformation polymorphism analysis, followed by DNA sequence analysis. Risk of melanoma dependent on the various melanocortin 1 receptor variant alleles was estimated by exposure odds ratios. The analyses of all different melanocortin 1 receptor gene variants combined, showed that the presence of melanocortin 1 receptor gene variants amounted to a higher melanoma risk, which, in stratified analyses, was independent of skin type and hair color. The odds ratios after adjusting for skin type were 3.6 (95% CI 1.7-7.2) for two variants and 2.7 (95% CI 1.5-5.1) for one variant, respectively. Compound heterozygotes and homozygotes for the Val60Leu, Val92Met, Arg142His, Arg151Cys, Arg160Trp, Arg163Gln, and His260Pro variants had odds ratios of about 4 to develop melanoma, whereas heterozygotes for these variants had half the risk. The presence of the melanocortin 1 receptor gene variant Asp84Glu appeared to impose the highest risk for cutaneous melanoma with odds ratios of 16.1 (95% CI 2.3-139.0) and 8.1 (95% CI 1.2-55.9) in compound heterozygotes and heterozygotes, respectively. The broad confidence intervals, when the different variants were analyzed separately, however, do not allow drawing definite conclusions about the magnitude of these risks. Of the more frequently occurring melanocortin 1 receptor variant alleles the Asp84Glu, Arg142His, Arg151Cys, Arg160Trp, His260Pro, and Asp294His variants were strongly associated with both fair skin and red hair. The Val60Leu, Val92Met, and Arg163Gln variant alleles, however, were only weakly or not associated with fair skin type and/or red hair, which further illustrates the finding that skin type, hair color, and melanoma are independent outcomes of the presence of melanocortin 1 receptor gene variants. We conclude that numerous melanocortin 1 receptor variants predispose to cutaneous melanoma and that possibly the Asp84Glu variant confers the highest risk. This predisposition is largely independent of skin type and hair color.

Monoclonal antibodies selected to discriminate between malignant melanomas and nevocellular nevi.

Two monoclonal antibodies (MoAbs), PAL-M1 and PAL-M2, are described that were selected to discriminate between melanomas and nevocellular nevi (NN) in frozen sections. MoAb PAL-M1 reacted with all 15 melanoma metastases (MM), with 14 of 19 primary cutaneous melanomas (PCM), 9 of 35 dysplastic nevi (DN), and 2 of 26 NN. The 2 NN stained were removed from patients with the dysplastic nevus syndrome. MoAb PAL-M2 reacted with 9 of 15 MM, 5 of 19 PCM, 3 of 35 DN, and did not react with 26 NN after usual staining conditions. The proportion of melanocytic cells stained was low in DN and much higher in PCM and especially in MM. Staining in DN was restricted to intraepidermal or subepidermal nests of atypical melanocytes. In PCM, staining with PAL-M2 was observed only in tumors with a Breslow thickness of 0.76 mm or higher. PAL-M1 and PAL-M2 may be immunohistochemical markers for tumor progression in melanocytic proliferations.

Transvenous occlusion of clinical and subclinical varicoceles.

Forty-four subfertile men with a palpable or clinically suspected varicocele underwent physical examination, nuclear scan, and testicular venography. The ability of these tests to detect a varicocele was compared. The sensitivity of physical examination was 0.94 on the left and 0.35 on the right. Specificity was 0.22 on the left and 0.77 on the right. The sensitivity of nuclear scan was 0.97 on the left and 0.73 on the right. Specificity was 0.56 on the left and 0.50 on the right. Patients with varicoceles demonstrated by venography were treated with testicular vein embolization. There were no complications except two asymptomatic subintimal injections, both of which occurred on the right side. A total of 19 patients had pre- and post-embolization semen analyses performed. The total motile sperm count increased from 27.0 million to 35.5 million. The overall pregnancy rate in the embolization-treated group was 43.7 percent.