Germline ATM variants predispose to melanoma: a joint analysis across the GenoMEL and MelaNostrum consortia.

PURPOSE: Ataxia-Telangiectasia Mutated (ATM) has been implicated in the risk of several cancers, but establishing a causal relationship is often challenging. Although ATM single-nucleotide polymorphisms have been linked to melanoma, few functional alleles have been identified. Therefore, ATM impact on melanoma predisposition is unclear. METHODS: From 22 American, Australian, and European sites, we collected 2,104 familial, multiple primary (MPM), and sporadic melanoma cases who underwent ATM genotyping via panel, exome, or genome sequencing, and compared the allele frequency (AF) of selected ATM variants classified as loss-of-function (LOF) and variants of uncertain significance (VUS) between this cohort and the gnomAD non-Finnish European (NFE) data set. RESULTS: LOF variants were more represented in our study cohort than in gnomAD NFE, both in all (AF = 0.005 and 0.002, OR = 2.6, 95% CI = 1.56-4.11, p < 0.01), and familial + MPM cases (AF = 0.0054 and 0.002, OR = 2.97, p < 0.01). Similarly, VUS were enriched in all (AF = 0.046 and 0.033, OR = 1.41, 95% CI = 1.6-5.09, p < 0.01) and familial + MPM cases (AF = 0.053 and 0.033, OR = 1.63, p < 0.01). In a case-control comparison of two centers that provided 1,446 controls, LOF and VUS were enriched in familial + MPM cases (p = 0.027, p = 0.018). CONCLUSION: This study, describing the largest multicenter melanoma cohort investigated for ATM germline variants, supports the role of ATM as a melanoma predisposition gene, with LOF variants suggesting a moderate-risk.

Associations of pigmentary and naevus phenotype with melanoma risk in two populations with comparable ancestry but contrasting levels of ambient sun exposure.

BACKGROUND: People at high risk of developing melanoma are usually identified by pigmentary and naevus phenotypes. OBJECTIVE: We examined whether associations of these phenotypes with melanoma risk differed by ambient sun exposure or participant characteristics in two population-based, case-control studies with comparable ancestry but different ambient sun exposure. METHODS: Data were analysed from 616 cases and 496 controls from the Australian Melanoma Family Study and 2012 cases and 504 controls from the Leeds (UK) case-control study. Questionnaire, interview and dermatological skin examination data were collected using the same measurement protocols. Relative risks were estimated as odds ratios using unconditional logistic regression, adjusted for potential confounders. RESULTS: Hair and skin colour were the strongest pigmentary phenotype risk factors. All associations of pigmentary phenotype with melanoma risk were similar across countries. The median number of clinically assessed naevi was approximately three times higher in Australia than Leeds, but the relative risks for melanoma associated with each additional common or dysplastic naevus were higher for Leeds than Australia, especially for naevi on the upper and lower limbs. Higher naevus counts on the head and neck were associated with a stronger relative risk for melanoma for women than men. The two countries had similar relative risks for melanoma based on self-reported naevus density categories, but personal perceptions of naevus number differed by country. There was no consistent evidence of interactions between phenotypes on risk. CONCLUSIONS: Classifying people at high risk of melanoma based on their number of naevi should ideally take into account their country of residence, type of counts (clinical or self-reported), body site on which the naevus counts are measured and sex. The presence of naevi may be a stronger indicator of a genetic predisposition in the UK than in Australia based on less opportunity for sun exposure to influence naevus development.

Model predicting survival in stage I melanoma based on tumor progression.

We used the lesional steps in tumor progression and multivariable logistic regression to develop a prognostic model for primary, clinical stage I cutaneous melanoma. This model is 89% accurate in predicting survival. Using histologic criteria, we assigned melanomas to tumor progression steps by ascertaining their particular growth phase. These phases were the in situ and invasive radial growth phase and the vertical growth phase (the focal formation of a dermal tumor nodule or dermal tumor plaque within the radial growth phase or such dermal growth without an evident radial growth phase). After a minimum follow-up of 100.6 months and a median follow-up of 150.2 months, 122 invasive radial-growth-phase tumors were found to be without metastases. Eight-year survival among the 264 patients whose tumors had entered the vertical growth phase was 71.2%. Survival prediction in these patients was enhanced by the use of a multivariable logistic regression model. Twenty-three attributes were tested for entry into this model. Six had independently predictive prognostic information: (a) mitotic rate per square millimeter, (b) tumor-infiltrating lymphocytes, (c) tumor thickness, (d) anatomic site of primary melanoma, (e) sex of the patient, and (f) histologic regression. When mitotic rate per square millimeter, tumor-infiltrating lymphocytes, primary site, sex, and histologic regression are added to a logistic regression model containing tumor thickness alone, they are independent predictors of 8-year survival (P less than .0005).

Primary melanoma cells of the vertical growth phase: similarities to metastatic cells.

Three primary and 16 metastatic melanoma cell lines were established from primary and metastatic lesions of 4 patients with malignant melanoma. Comparison of metastatic melanoma cells with cells of the vertical growth phase (VGP) or late primary melanoma from the same individual revealed, generally, a shorter population-doubling time, growth to a higher cell density, higher tyrosinase activity, and more pigmentation in metastatic cells. Conversely, primary and metastatic melanoma cells had similar morphology, plating efficiency, and tumorigenicity in nude mice. Karyotypic analysis revealed clonality and nonrandom abnormalities in chromosomes 1, 6, and 7 in cells of the primary and metastatic lesions of the 3 patients studied. Few differences were found in the expression of melanoma-associated antigens on short-term and long-term cultured cells by tests with monoclonal antibodies in mixed hemadsorption assays, flow cytometry, and radioimmunoassays. Our results indicate that cells cultured from the VGP but not from the radial growth phase of primary melanoma are similar to metastatic melanoma cells.

Natural killer cells in the host response to melanoma.

Using an immunoperoxidase technique, we have investigated natural killer (NK) cells in the host response to malignant melanomas and melanocytic nevi in frozen sections. Eight primary melanomas, 12 metastatic melanomas, and 31 dysplastic nevi were studied. NK cells were identified phenotypically using an antibody, B73.1, against an Fc receptor present only on NK cells and neutrophils. Rare NK cells were identified in three of 31 dysplastic nevi and in one of eight melanomas. In contrast, significant numbers of NK cells were identified in ten of 12 metastases.

Immunoperoxidase localization of lymphocyte subsets in the host response to melanoma and nevi.

Using an avidin:biotin immunoperoxidase system with monoclonal antibodies to lymphocyte subsets, we have investigated the host response to malignant melanoma and melanocytic nevi in frozen sections. Eight primary melanomas, eight metastases, three dysplastic nevi, and two dermal nevi were studied with antibodies T11, T4, T8, and B1. Sections were read in a semiquantitative manner by two observers. Virtually all lymphocytes in these lesions were T-cells (T11 positive). In all primary melanomas, in the majority of metastases, and in all dysplastic nevi, both T4- and T8-positive cells were present. In two of eight metastases, tumor cells stained with T4, and in one case, melanoma cells stained with B1 antibody. The host response to melanoma involves primarily T-cells and includes both the helper:inducer (T4) and suppressor:cytotoxic (T8) subsets.

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas.

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.

Isolation and functional characterization of the A32 melanoma-associated antigen.

Cell surface melanoma-associated antigens can mediate cell-cell or cell-substrate adhesion, signal transduction, proteolysis, or immune recognition and play a key role in determining invasive and metastatic competence of the tumor cells. The melanoma-associated antigen, A32, was defined by a murine monoclonal antibody and was immunoprecipitated as a single 113 kDa integral membrane glycoprotein containing sialic acid and HNK-1 carbohydrate moieties. Immunohistochemistry revealed the presence of A32 antigen on most melanomas and nevi but not on normal epidermal melanocytes. Of the normal tissues tested, only endothelium, smooth muscle, cerebellum, and hair follicles expressed the A32 antigen. Tryptic peptides of the A32 antigen obtained after immunoaffinity chromatography showed sequence identity to MUC18 antigen, a member of the immunoglobulin supergene family. Melanoma cells adhered to affinity-purified A32 antigen immobilized to a solid phase, and the adhesion was blocked by either soluble A32 antigen or monoclonal antibody against the HNK-1 carbohydrate moiety. These findings, together with the observation that A32 antigen is concentrated in cell-cell contact borders, suggest that this antigen is an adhesion molecule with a possible role in tumor invasion and metastasis.

Human cutaneous nevi transplanted onto nude mice: a model for the study of the lesional steps in tumor progression.

Normal human cutaneous nevi were transplanted to the skin of nude mice and some of the grafts were treated topically with 7,12-dimethylbenz[a]anthracene (DMBA, 1.6 mumol weekly). Histologically proven human skin was present in 22 grafts. In the 9 untreated control grafts, the tendency of nevic cells to form nests and the number of nevomelanocytes decreased with time; the melanocytic cells showed no signs of hypertrophy or atypia. In most of the 14 specimens treated with DMBA, the nevomelanocytes showed distinct signs of hypertrophy. The cells were enlarged and often dendritic and were filled with melanin granules for which the transfer to keratinocytes appeared to be blocked. The nevomelanocytes of 4 of the 9 specimens treated with DMBA for greater than or equal to 82 days (9-16 DMBA applications), showed atypical enlarged nuclei with mitotic figures. Since atypia is one criterion for identifying precursors of transformed cells, the model of human nevi grafted onto nude mouse skin may be useful for studying the various steps involved in the progression of benign melanocytic nevi to malignant melanoma.

Monoclonal antibody-defined correlations in melanoma between levels of GD2 and GD3 antigens and antibody-mediated cytotoxicity.

A monoclonal antibody is described that specifically detects the ganglioside antigens GD2 and GD3, binding preferentially to GD2, in melanoma. Antibody specificity was demonstrated with solid-phase radioimmunoassay and enzyme-linked immunosorbent assay as well as by immunostaining on thin-layer chromatography plates using structurally characterized gangliosides. Binding of both the IgG3 antibody and its IgG2a switch variant were assayed on live cells by cytofluorography and by immunoperoxidase staining on frozen tissue sections. The binding patterns correlated with antitumor activity in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays with human effector cells and complement in an 111In-release assay using cell lines derived from the same individual. The significant level of killing in all tumor cells tested that express GD2, GD3, or both, suggests the importance of multiple specificity towards tumor antigens, i.e., binding of a monoclonal antibody to two or more tumor-associated antigens.

Phenotypic characteristics of cells derived from precursors of human melanoma.

Of 66 specimens from benign melanocytic nevi, including common acquired and congenital nevi, Spitz tumors (epithelioid cell nevi), and melanocytic nevi with dysplasia, 57 could be grown in tissue culture. The cultured cells were identified as melanocytes by the presence of premelanosomes and melanosomes. Cells from 28 of 32 nevus cultures grew in an anchorage-independent way in soft agar with a colony-forming efficiency between 0.001 and 1%. Clones derived from single cells and soft agar-selected colonies showed marked phenotypic heterogeneity, but all had a limited life span and did not undergo transformation in culture. These cells were nontumorigenic in nude mice. Cultured nevus cells expressed antigens present on melanoma but absent on normal fibroblasts and/or melanocytes as tested with monoclonal anti-melanoma antibodies. The anti-melanoma antibodies bound equally well to dysplastic, congenital, and common acquired nevi. Antigens are released by nevus cells similar to melanoma cells.

Specific immunoreactivity of hybridoma-secreted monoclonal anti-melanoma antibodies to cultured cells and freshly derived human cells.

The specificities of monoclonal antibodies against melanoma cells were analyzed using radioimmunoassay, mixed-hemadsorption assay, and quantitative absorption on a variety of malignant and nonmalignant cells. Three of the six hybridoma-secreted antibodies bound to the majority of melanoma cell lines, melanoma tumors, and astrocytoma cell lines as well as to all normal and Epstein-Barr virus-transformed lymphocytes tested. The binding pattern coincides with the presence or absence of the DR antigen on human cells. Conversely, two other antibodies, 19-19 and Nu4B, detected two different antigens common to melanoma and astrocytoma cells only. Cloning of melanoma cells resulted in establishment of DR-positive and DR-negative clones, with the binding of Nu4B antibody retained in all.

Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression.

Since tumor progression is dependent on the ability of malignant cells to interact with the extracellular matrix, molecules on the cell surface which mediate cell-substratum interactions are likely to be important regulators of tumor invasion and metastasis. The purpose of this study was to examine the distribution of one such group of cell adhesion receptors, the integrins, in benign and malignant lesions of human melanocytes. The distribution of integrin adhesion receptors was defined on cells in culture derived from normal and malignant melanocytes and in tissue sections from benign to increasingly malignant melanocytic lesions using a panel of monoclonal antibodies against specific integrin subunits. Cells in culture expressed a large variety of integrins, including all of the previously characterized members of the beta 1 subfamily plus the alpha v/beta 3 vitronectin receptor. The expression of integrins was similar in cells cultured from either benign or malignant lesions. In contrast, consistent differences were noted in integrin expression by cells within tissues containing metastatic and vertical growth phase melanomas when compared to radial growth phase melanoma cells and cells within nevi. Most notably, the expression of the beta 3 subunit was restricted exclusively to cells within vertical growth phase and metastatic melanomas. The presence of this integrin may be important in the development of tumor invasiveness and could be useful as a marker of melanoma cells entering the more aggressive phase of the malignant process.

Characteristics of cultured human melanocytes isolated from different stages of tumor progression.

Normal melanocytes and melanocytes of normal nevi, primary melanoma in the radial (RGP) and vertical (VGP) growth phases, and metastatic melanoma exhibited and maintained phenotypic differences when grown in tissue culture or in experimental animals. Only metastatic and VGP primary melanoma cells were tumorigenic in athymic nude mice and had nonrandom chromosomal abnormalities involving chromosomes 1, 6, and 7. The colony-forming efficiency in soft agar was also highest in these two cell types. A cell line of RGP primary melanoma had characteristics of both benign and malignant cells: nevus-like morphology; nontumorigenicity in nude mice; but karyotypic abnormality of chromosome 6. It also had a ganglioside pattern similar to that of normal melanocytes but not melanomas, i.e., a high GM3 ganglioside content compared to the amounts of GM2, GD2, and GD3 gangliosides. Binding of monoclonal antibodies secreted by hybridomas generated by immunization of mice with VGP primary and metastatic melanoma was highest with cells and supernatants of cultures from advanced melanoma and least with nevus cells. There was no binding to normal melanocytes except with the monoclonal antibodies specific for nerve growth factor receptor or 9-O-acetyl-GD3 ganglioside. On the other hand, monoclonal anti-nevus antibodies bound to melanocytes, nevus cells, and RGP primary melanoma cells but not to VGP primary or metastatic melanoma cells. Cultured human melanocytic cells appear to be a unique model for the study of tumor progression.

Chromosome 9 deletion in sporadic and familial melanomas in vivo.

Twenty microsatellite loci on chromosome 9 were analysed for allelic losses in DNAs from 30 uncultured melanomas from 25 patients, relative to DNA from autologous peripheral blood lymphocytes. All patients were constitutionally heterozygous at several loci, and loss of heterozygosity (LOH) affecting 9p was observed in melanoma DNAs from 18 individuals (72%). Observations of losses of identical alleles in different metastatic lesions from the same patients, and of LOH in a vertical growth phase primary melanoma, were consistent with previous reports of chromosome 9 deletion early in melanoma development. LOH data suggested the loss of entire copies of chromosome 9 in 11 cases, and the terminal deletion of all or a portion of 9p in six cases. A somatic interstitial deletion of 9p between D9S162 and D9S169 was seen in a familial melanoma. This 21 cM deleted region corresponded with the previously reported positions of homozygous deletions in melanoma cell lines, and of the familial melanoma susceptibility locus (MLM). As 16 of the 18 cases of 9p LOH in the present study were observed in individuals with no family history of melanoma, it is likely that the MLM locus plays a role in the development of most sporadic melanomas.

Effects of topical tretinoin on dysplastic nevi.

PURPOSE: As potential precursors of melanoma and markers of increased melanoma risk, dysplastic nevi are suitable targets of strategies for melanoma chemoprevention. We report the results of a pilot study of topical retinoic acid in patients with dysplastic nevi. PATIENTS AND METHODS: Five male patients with dysplastic nevi applied tretinoin to half of the back for 6 months. Baseline photographs of dysplastic nevi were compared with posttreatment photographs and assessed for morphologic change. At study completion, each subject had four nevi excised from the treated side and four from the untreated side of the back. Biopsies were histologically evaluated for the presence of dysplasia. RESULTS: All patients developed signs of irritation as a result of treatment. One patient was not compliant with treatment due to skin irritation. The four compliant patients showed significant decreases in the clinical atypia of treated lesions, with concomitant fading and even disappearance of many treated nevi. Histologically, only four of 16 treated nevi met histologic criteria for dysplasia, in comparison to 13 of 16 untreated nevi. CONCLUSION: These results suggest that there is concomitant clinical and histologic improvement in a significant percentage of dysplastic nevi treated with topical tretinoin. However, the utility of topical tretinoin for chemoprevention of melanoma is limited by difficulty of application and associated inflammation. While new strategies in chemoprevention of melanoma are explored, sun protection and assiduous avoidance of sunburn must remain the mainstay of melanoma prevention.

Autopsy after death due to extreme prematurity.

Autopsy reports for 29 very preterm infants dying at <28 days of age were reviewed. New findings were discovered in 79% and resulted in a significant change in diagnoses in 28%. Iatrogenic lesions were identified in 41% of cases and were the main cause of death in 14%.

Human melanocytic neoplasms and their etiologic relationship with sunlight.

Two hypotheses have been presented. The first states that melanomas commonly evolve from normal melanocytes by a tumor progression pathway from a banal nevus to a nevus with dysplasia, to a micro-invasive, and then to a fully evolved, tumorigenic, primary melanoma which has competence for metastasis. It is important to note that not all melanomas follow this complete pathway. As Foulds noted long ago, tumors may bypass any of the stages of tumor progression. Thus, many melanomas do not, apparently, arise in nevi, and melanomas may evolve "fully formed" as pure tumorigenic nodules. However, from the biological point of view, study of the benign potential precursors (nevi and, especially, dysplastic nevi as well as microinvasive melanomas) may well reveal mechanisms of progression that are applicable to all melanomas, and perhaps to other solid tumors as well. From a clinical viewpoint, follow-up and education of patients at increased risk for melanoma, and early diagnosis of melanomas in their curable, microinvasive stages may result in a reduction of mortality from the disease, even without influencing its overall incidence. The melanomas that occur on plantar and palmar (acral) skin appear to progress through a microinvasive stage similar to that of other cutaneous melanomas. However, the significance of precursor and marker lesions (if any exist) in acral melanoma remains to be elucidated by clinicopathologic and epidemiologic studies. The possibility of etiologic agents other than UV light, such as chemical carcinogens and/or viruses, should be investigated in these cases. The second hypothesis presented here, that UV light is etiologic for the common cutaneous melanoma of white populations, has support from clinical, epidemiologic, and biologic observations. From a biologic viewpoint, ultraviolet light has all of the properties that might enable it to act as a complete carcinogen, and to enhance tumor progression in melanocytic "potential-precursor" lesions. Clinically, it seems appropriate to encourage patients (and members of the general population, as well) to adopt sensible attitudes to sun exposure. By such means, it is possible that some melanomas might be prevented, or that the rate and incidence of progression to more-advanced stages might be inhibited.

Lessons from tumor progression: the invasive radial growth phase of melanoma is common, incapable of metastasis, and indolent.

Primary melanoma generally evolves through three clinically and morphologically discernable tumor progression steps. Transformed melanocytes first proliferate above the epidermal basement membrane, then invade the papillary dermis (the in situ and invasive radial growth phases of melanoma), and subsequently develop the capacity to grow as a tumor (the vertical growth phase). Here, we address three aspects of the invasive radial growth phase that provide the rationale for viewing it as the critical lesion for melanoma detection and therapy. We determined the fraction of melanomas having this growth phase, tested its hypothesized incapacity to metastasize, and estimated its longevity. The high prevalence of this step in tumor progression was demonstrated in a data base of 624 patients, where at least 87% of melanomas exhibited a radial growth phase. The benignity of this lesion was evinced by the perfect metastasis-free survival of 161 patients treated for pure radial growth-phase melanomas and followed for a median of 13.7 years. Its indolence was evident in an analysis of the ages of 234 patients with superficial spreading melanomas without or with vertical growth phase: The cases with lesions having only radial growth phase were 4.3 years younger than those additionally having vertical growth phase (p < 0.05). These features of the invasive radial growth phase of primary melanoma, first described by Wallace H. Clark, make it a pivotal lesion in the evolutionary biology of melanocytic neoplasia and confirm its central place in public health programs to control melanoma mortality.