David E. Elder

A tumorigenic subpopulation with stem cell properties in melanomas

Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSCs have been identified in several malignancies, including those of blood, brain, and breast. Here, we test whether stem cell-like populations exist in human melanomas. In approximately 20% of the metastatic melanomas cultured in growth medium suitable for human embryonic stem cells, we found a subpopulation of cells propagating as nonadherent spheres, whereas in standard medium, adherent monolayer cultures were established. Individual cells from melanoma spheres (melanoma spheroid cells) could differentiate under appropriate conditions into multiple cell lineages, such as melanocytic, adipocytic, osteocytic, and chondrocytic lineages, which recapitulates the plasticity of neural crest stem cells. Multipotent melanoma spheroid cells persisted after serial cloning in vitro and transplantation in vivo, indicating their ability to self-renew. Furthermore, they were more tumorigenic than adherent cells when grafted to mice. We identified similar multipotent spheroid cells in melanoma cell lines and found that the stem cell population was enriched in a CD20+ fraction of melanoma cells. Based on these findings, we propose that melanomas can contain a subpopulation of stem cells that contribute to heterogeneity and tumorigenesis. Targeting this population may lead to effective treatments for melanomas.

A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma

Six evident lesional steps of tumor progression form the neoplastic system that affects the human epidermal melanocyte: 1) the common acquired melanocytic nevus; 2) a melanocytic nevus with lentiginous melanocytic hyperplasia, i.e., aberrant differentiation; 3) a melanocytic nevus with aberrant differentiation and melanocytic nuclear atypia, i.e., melanocytic dysplasia; 4) the radial growth phase of primary melanoma; 5) the vertical growth phase of primary melanoma; and 6) metastatic melanoma. The common acquired melanocytic nevus is viewed as a focal proliferation of melanocytes, destined in most instances to follow a programmed pathway of differentiation that leads to disappearance of the nevus. If the pathway of differentiation is not followed, characteristic lesions result, and such lesions are regarded as the formal histogenetic precursors of melanoma. Such a developmental flaw is termed aberrant differentiation, and the resultant precursor lesion is designated melanocytic dysplasia. The vast majority of melanocytic nevi showing melanocytic dysplasia are terminal lesions that do not progress to melanoma. If melanoma is to develop via a precursor lesion, however, the nevus with melanocytic dysplasia is that precursor. When melanomas do develop, they develop focally within the precursor. The resultant primary melanoma itself does not follow a pathway of inexorable expansion of a population of melanoma cells in space and time. Rather, primary melanomas, with the exception of nodular melanoma, also evolve in a stepwise fashion. The first step, termed the radial growth phase, is characterized by the net enlargement of the tumor at its periphery, along the radii of an imperfect circle. Tumors in this stage of development show a characteristic pattern of growth within the epidermis and a distinctive form of invasion of the papillary dermis. Such melanomas are not associated with metastasis, and it is hypothesized that such tumors do not have competence for metastasis. For a melanoma to acquire competence for metastasis it must progress to the next step of tumor progression--the vertical growth phase. This lesional step is characterized by the appearance of a new population of cells within the melanoma, not an expansion of the cells forming the pre-existing radial growth phase. The net growth of the cells of the vertical growth phase is perpendicular to the directional growth of the radial growth phase. As a rule, the cells of the vertical growth phase grow in an expansile fashion, expansile as a balloon expands: a growth form characteristic of metastases.(ABSTRACT TRUNCATED AT 400 WORDS)

High Risk of Malignant Melanoma in Melanoma-Prone Families with Dysplastic Nevi

The risk of hereditary cutaneous malignant melanoma was evaluated in 401 members of 14 families with an autosomal dominant form of melanoma. We documented 127 primary melanomas in 69 family members, including 39 new melanomas diagnosed in 22 study participants from the time of first examination through a maximum of 8 years of follow-up. The 39 newly diagnosed melanomas occurred only in family members with dysplastic nevi, a known precursor of familial melanoma. Of 77 patients with dysplastic nevus syndrome without prior melanomas, 4 developed their first melanoma during prospective follow-up, as compared with 0.03 cases expected. The prospective age-adjusted incidence for melanoma was 14.3/1000 patients with dysplastic nevus per year, with a cumulative melanoma risk (+/- SE) of 7.2% (+/- 3.6) at 8 years. The actuarial probability of melanoma developing in family members with dysplastic nevi was 56.0% (+/- 10.1) from age 20 to age 59. This study confirms that dysplastic nevi are clinical markers of high risk for, and precursors of, hereditary melanoma.

Dysplastic nevus syndrome: a phenotypic association of sporadic cutaneous melanoma.

Clinical photographs of 79 prospectively studied cases of non‐familial cutaneous malignant melanoma were reviewed; special attention was directed to the distribution pattern of coexistent melanocytic lesions. A group of 15 patients had moles on the covered buttock area. Seven of these patients had large clinically atypical nevi, and biopsies of these nevi showed severe melanocytic dysplasia. Residual elements of melanocytic dysplasia were identified in five of the primary melanomas in this group of patients. It is suggested that these patients represent a distinctive syndrome, the Dysplastic Nevus Syndrome (DNS) and that they are at increased risk for development of primary cutaneous malignant melanoma. The clinically and histologically distinctive dysplastic nevi of these patients are identical to the precursor lesion for melanoma that we have previously described in a familial context, the B‐K mole syndrome. This paper represents the first description of this form of dysplasia in non‐familial melanoma.

Genome-wide association study identifies three loci associated with melanoma risk

We report a genome-wide association study of melanoma conducted by the GenoMEL consortium based on 317K tagging SNPs for 1,650 selected cases and 4,336 controls, with replication in an additional two cohorts (1,149 selected cases and 964 controls from GenoMEL, and a population-based case-control study in Leeds of 1,163 cases and 903 controls). The genome-wide screen identified five loci with genotyped or imputed SNPs reaching P < 5 × 10−7. Three of these loci were replicated: 16q24 encompassing MC1R (combined P = 2.54 × 10−27 for rs258322), 11q14-q21 encompassing TYR (P = 2.41 × 10−14 for rs1393350) and 9p21 adjacent to MTAP and flanking CDKN2A (P = 4.03 × 10−7 for rs7023329). MC1R and TYR are associated with pigmentation, freckling and cutaneous sun sensitivity, well-recognized melanoma risk factors. Common variants within the 9p21 locus have not previously been associated with melanoma. Despite wide variation in allele frequency, these genetic variants show notable homogeneity of effect across populations of European ancestry living at different latitudes and show independent association to disease risk.

High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of familys mutations) and the Netherlands (c.225_243del19, 90% of familys mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available.

Acquired precursors of cutaneous malignant melanoma: the familial dysplastic nevus syndrome

THE incidence of cutaneous malignant melanoma is rising rapidly throughout the world.1 The most current data from the National Cancer Institutes Surveillance Epidemiology and End Results (SEER) sy...

E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.

In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation.

Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents

Background: The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer. Methods: These four features were examined in 385 families with ⩾3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents. Results: Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, ⩾2 patients with MPM, median age at melanoma diagnosis ⩽40 years and ⩾6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only ⩾1 patient with MPM and age at diagnosis ⩽40 years simultaneously predicted the mutation risk. Conclusions: The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer–CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.

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.