American Journal ofPathology, Vol. 136, No. 4, April 1990 Copyright © American Association ofPathologists
Malignant Melanoma Simulants Arising in Congenital Melanocytic Nevi Do Not Show Experimental Evidence for a Malignant Phenotype
Maria-Laura Mancianti,* Wallace H. Clark,t F. Ann Hayes, and Meenhard Herlyn* From the Wistar Institute ofAnatomy and Biology, Philadelphia; Pennsylvania*; the Pigmented Lesion Study Group and the Department ofDermatology of the University ofPennsylvania, Philadelphia, Pennsylvaniat; and the Department ofHematology-Oncology, St. Jude
Children's Hospital, Memphis, Tennessee*
Proliferative neoplasms that resemble malignant melanoma may develop in large congenital melanocytic nevi, prenatally or in the neonatal period, although these lesions rarely show the progressive growth or behavioral characteristics of melanoma. This report describes the genetic, biologic, and immunologic characteristics of six tissue culture cell lines derived from two neoplasms present in congenital melanocytic nevi in two newborn infants. Both neoplasms had clinical and histologic features of malignant melanoma. Despite these features, cells from all lines were phenotypically benign, as evidenced by a normal karyotype, their expression pattern ofpigment cell-associated antigens, absence of melanoma-associated ganglioside GD2, their mitogenic response to the tumorpromoting pborbol ester 12-0-tetradecanoylphorbol- 13-myristate, their inability to grow anchorage independently in soft agar, and prolonged but finite life span. The cells did not produce tumors in nude mice, but they remained viable at the injection site for over 7 months. (Am J Pathol 1990,
136:817-829) Congenital giant nevi constitute a well-known risk factor in the development of malignant melanoma both in prepubertal and postpubertal individuals, with a reported incidence between 4.6% and 38%.'I Primary congenital malignant melanoma, with or without an associated congenital nevus, is rare. One form of congenital melanocytic
neoplasm presents as a massive, rapidly enlarging, ulcerative mass at birth and has been termed nodular proliferative neurocristic hamartoma. Such lesions arise in giant congenital melanocytic nevi. Another form, composed of epithelioid melanocytes, may be histologically indistinguishable from nodular melanoma. These nodular lesions may arise in small, intermediate, and giant congenital melanocytic nevi. To our knowledge, only 16 cases have been described in which malignant melanoma of the skin was diagnosed at birth or in the neonatal period.7-19 With the exception of the few cases in which death occurred shortly after birth and metastasis could be detected, the alarming clinical or histologic features in these congenital or neonatal tumors did not result in death due to metastasis. However, the natural history of these lesions remains unknown because their clinical presentation is an imperative for their removal, and the histologic evaluation of these lesions is difficult and inconsistent. In those lesions that are not completely removed, there is evidence of differentiation rather than progressive malignancy. Diagnostic problems at the histologic level are due to the complex cellular composition of some of the nodular overgrowths occurring in congenital melanocytic lesions. Four major histologic patterns of proliferation have been observed in congenital melanocytic nevi at birth or in the neonatal period2O: 1) simulants of superficial spreading melanoma, in which epidermis and superficial dermis are occupied by increased numbers of large epithelioid melanocytes; 2) simulants of nodular melanoma, with either multiple black nodules of epithelial melanocytes or a single black nodule of extremely large epithelioid melanocytes with uniform nuclei; 3) nodular proliferative neurocristic hamartomas, characterized by tumorous growth in fetal life, and composed of diverse tissues; the deep dermis and subcutis are occupied by neuro-sustentacular tissue and ecto-mesenchymal structures; and 4) biologic Supported by grants CA-25874, CA-10815, CA-44877, CA-25298, and CA-16520 from the National Institutes of Heath. Accepted for publication November 27, 1989. Address reprnt requests to Meenhard Herlyn, DVM, The Wistar Institute, 36th Street at Spruce, Philadelphia, PA 19104.
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Figure 1. Patient 1. Simulants of nodular melanoma. A darkly pigmented nodule of 3.5 X 1.5 cm arising within the tan background ofa congenital melanocytic nevus of6X5cm.
malignant melanoma, most of which are characterized by small blastic pleomorphic melanocytes with a high mitotic rate. The true melanomas with metastatic potential usually develop after the neonatal period. In our laboratory, we have previously studied pigmented cells from different stages of tumor progression in culture, and have characterized different biologic steps in the evolution from normal melanocytes to malignant cells.21 In the present study, we analyzed cells derived from two kinds of proliferative tumors in large congenital melanocytic nevi to determine their malignant potential. The results obtained with six cell lines are not indicative of a malignant phenotype.
scalp, ears, face, arms, legs, palms, and soles of the feet. The bulk of the large lesion was black, with some dark brown areas, especially at the periphery. The smaller lesions were dark brown to black. In the subsequent weeks, the masses progressively enlarged, with the greatest change in the subcutaneous tissue of the pubic area. Ul-
Materials and Methods
Clinical Cases Case 1 was an 8-day-old white neonate born with a congenital nevus of the right thigh. The lesion consisted of a darkly pigmented nodule of 3.5 X 1.5 cm that was rising within the tan background of a congenital melanocytic nevus that was approximately 6 X 5 cm (Figure 1). The dark lesion and part of the background lesion were excised and submitted for histologic diagnosis and for tissue culture studies. Tissue fixation, embedding, and staining was done using standard procedures. The child was alive and well at 41 months of age. Case 2 was a white male who presented at 3 weeks of age with a large congenital bathing trunk nevus extending from the umbilical area to just above the knees (Figure 2). Large masses, some soft, others firm, were palpable in the pubic-inguinal area, buttocks, and thigh. Multiple congenital nevi of varying sizes also were present on the
Figure 2. Patient 2. Large congenital bathing trunk nevus extendingfrom the umbilical area to just above the knees. Large masses werepalpable in buttock, thigh, andpubic-inguinal areas.
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ceration occurred in a few areas. Biopsies were performed on three of the nodules for histologic studies at the St. Jude Children's Research Hospital, Memphis, TN. Specimens from two of the biopsies were obtained for tissue culture. At this time, a computed tomography (CT) scan of the head showed three intracerebral nodules interpreted as metastases. At 7 weeks of age, the infant was treated with cyclophosphamide, vincristine, and actinomycin following a previously described protocol.16 Therapy was discontinued after 7 days because of hematogenous infection. The masses became softer and less prominent over the ensuing months and the cerebral lesions were indolent and calcified. The child was asymptomatic and developing normally at 18 months of age.
Tissue Culture Specimens received for tissue culture were enzymatically digested using two procedures.22 First, 1- to 2-sq mm fragments of each lesion were incubated in 0.05% collagenase type IV, 0.1% hyaluronidase, and 1.25 U/ml dispase in Hank's balanced salt solution (HBSS) without Ca++ and Mg++ and supplemented with 30 mM HEPES (modified HBSS), and digested in toto overnight at 370C. Second, fragments from the same lesions were incubated overnight at 40C in 0.25% trypsin in modified HBSS. The epidermis then was mechanically separated from the dermis and the dermis was digested as in the first procedure. The epidermis was incubated for 3 hours at 37°C in 0.1% hyaluronidase, 1.25 U/ml dispase, and 0.033% versene in modified HBSS. Cell suspensions obtained by the different procedures were sedimented, resuspended in medium, and seeded into 12-well plates. The culture medium consisted of W489 medium supplemented with 5 ,g/ml insulin, 5 ng/ml epidermal growth factor, 40,g/ml bovine pituitary extract containing fibroblast growth factor, and 2% fetal calf serum (FCS).23 Each cell line was established and maintained in the presence or absence of the phorbol ester 12-0-tetradecanoylphorbol-13-myristate (TPA) at 10 ng/ml. Cultures were maintained at 370C in 5% C02/95% air and refed biweekly. For serum-free culture, W489 medium was supplemented with insulin, epidermal growth factor, basic fibroblast growth factor (40 ng/ml), TPA, and synthetic a-melanocyte-stimulating hormone (1 to 10 ng/ml). The rationale for using these growth supplements was: 1) Insulin is a mitogen for melanocytes at different stages of tumor progression,2425 acting predominantly via the receptor for insulinlike growth factor 1;4 2) epidermal growth factor enhances the recovery rate of freshly passaged cells and is essential during the first 2 weeks in tissue culture, although it is not mitogenic for established melanocytic cell lines;23 3) basic fibroblast growth factor from bovine pituitary;26 4) TPA, an activator
for protein kinase C;27 and 5) alpha melanocyte-stimulating hormone24 are mitogenic for melanocytes. One cell line from patient 2 was initially contaminated with fibroblasts and was treated in the first passage with 200 ,g/ ml G418 (GIBCO, Grand Island, NY) for 2 days.28 Melanocytes from newborn foreskins, congenital nevus cells, and primary and metastatic melanoma cells were used as control cells, isolated and grown as described.21 Melanocytes and nevus cells were maintained in TPA-containing medium, and melanoma cells in medium without TPA.
Electron Microscopy Cells from each specimen were seeded into 35-mm plastic dishes and grown for 7 to 10 days. Cultures then were fixed for 2 hours at 350C in 3% glutaraldehyde in PIPES buffer, rinsed twice with cold 0.1 mol/I cacodylate buffer, and postfixed with 1.0% osmium tetroxide for 1 hour. Cells were dehydrated in a graded series of ethanols and embedded in Epon. Sections were cut parallel to the culture surface, stained with uranyl acetate and lead citrate, and examined under a ZEISS EM10 electron microscope (Zeiss, Oberkochen, West Germany).
Growth Analyses Cells were seeded in duplicate or triplicate in 2 sq cm wells of 24-well plates at 2 to 3 X 104 cells/sq cm. On days 1 through 10, cells were detached by trypsinization and counted in a Coulter cell counter. Anchorage-independent growth was tested using a standard agarose assay previously described.22 Briefly, 35-mm wells were prepared with an underlayer of 0.5% agarose in complete medium. Cells were seeded at 1500 and 3000 cells per well in 0.25% agarose and incubated at 370C. Fresh medium was added after 3 to 4 days and colonies were counted after 10 to 14 days. In each assay, normal melanocytes, nevus cells, and malignant melanoma cells were included as controls. Growth in nude mice was studied by subcutaneous injection of 1 to 3 x 107 cells into athymic Balb/c (nu/nu) mice. Mice were observed weekly for tumor growth. After 120 and 210 days, mice were killed and examined for evidence of tumor growth at the site of injection and in livers and lungs.
Karyotypic Analysis Karyotypes of cultured cells were analyzed at different culture passages. One to 4 days after seeding, cells were incubated with colchicine for 45 minutes at 370C. After
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Figure 3. Patient 1. Nodular melanocytic byperplasia, simulating malignant melanoma, arises in a congenital melanocytic nevus. (A) Dermal component of the basic congenital melanocytic nevus. (B) Cells deep in the epidermis that were much larger than the cells forming the congenital melanocytic nevus, formed the rapidly growing nodule observed clinically.
treatment with a KCI-sodium citrate hypotonic solution and standard fixation, air-dried slides were prepared for
Giemsa banding.29
Expression of Pigment Cell-associated Antigens Expression of melanocyte- and melanoma-associated antigens was evaluated by: 1) mixed hemadsorption assay (MHA) in which binding of monoclonal antibodies (MAbs) to live adherent target cells is visualized by sheep red blood cells preincubated with mouse anti-sheep red blood cell antibodies and agglutinated by goat anti-mouse antibodies;-: 2) fluorescence-activated cell sorting (FACS) by incubating cells in suspension with MAbs, followed by incubation with fluoresceinated goat anti-mouse immunoglobulin antibodies using an Ortho Cytofluorograf 50HH connected to an Ortho 450 data handling system
(Ortho, Westwood, MA); 3) indirect radioimmunoassay, in which the binding of MAb to adherent cells fixed with 0.1% glutaraldehyde was determined by 1251-goat antimouse F(ab)2 antibody as described;31 and 4) immunoperoxidase staining by the Vectastain ABC method, using 3-amino-9-ethyl-carbazole (AEC) as substratum since it gives a dark red color in positive cells that is distinguishable from melanin.
Results Histology Case 1
Simulant of Nodular Melanoma The basic congenital melanocytic nevus had an intraepidermal component characterized by an increased
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number of melanocytes disposed in orderly nests. Occasionally there was bridging from one rete to the next and slight atypia of this intraepidermal component, which is a standard feature of most congenital melanocytic nevi in the early months and years of life and may be seen later in the adult years. The dermal component was characterized by relatively small cells that extended throughout the dermis and into the subcutaneous tissue, especially in the fibrous trabeculae of the subcutaneous tissue (Figure 3A). These cells were uniform and formed delicate strands disposed in a random array. Sections through the rapidly growing nodule were characterized by intraepidermal melanocytic hyperplasia wherein the melanocytes were disposed as individual cells and showed some upward growth in the epidermis (Figure 3B). This upward growth was not as striking as that in adult melanomas. The pathology was dominated by replacement of the dermal components of the nevus by much larger cells than those described above. The large cells were arranged in small clusters of three to five cells within a somewhat eccentric periphery of flattened cells. The nuclei of the large cells were two to three times larger than the nuclei present in the reticular dermis of the background congenital melanocytic nevus. The lesion had some features of nodular melanoma. A major difference between simulants of nodular melanoma described here and true biologic melanoma arising in congenital melanocytic nevi was the ten-
dency for the large cells comprising the simulant nodule to blend imperceptibly at the base with the smaller cells of the background congenital melanocytic nevus. In biologic nodular melanoma arising in congenital melanocytic nevi, the melanoma is sharply delineated from the surrounding congenital melanocytic nevus.
Case 2 Nodular Proliferative Neurocristic Hamartoma The first area biopsied was from a large mass present on the lateral thigh. The changes within the epidermis and the immediately subjacent dermis were characterized by the presence of a large number of melanocytes disposed in irregular nests with upward growth of individual melanocytes to the outer reaches of the epidermis. The cells of the individual nests were large, pleomorphic, and somewhat dyshesive (Figure 4A). This portion of the tumor was virtually indistinguishable histologically from the radial growth phase of malignant melanoma of the superficial spreading type. In some sections, the epidermal component and the component immediately subjacent to the epidermis had the appearance of melanoma, forming clusters of atypical cells in the dermis (Figure 4B). The cells present in the immediately subjacent dermis also were large and atypical and associated with fibroplasia.
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Figure 4. Patient 2. (A) Large multinodular lesion present at birth. The intraepidermal component is illustrated. Large nests ofpoorly cohesive melanocytes are present at the dermal-epidermal interface. (B) Clusters of atypical melanocytes similar to those of Figure 3 have extended into the dermis. (C) The micrograph shows the neuroectodermal tissue, whichformed the bulk ofthe massive nodules in the congenital melanocytic nevus. (D) The central cells in tubular array probably represent epithelioid Schwannian tissue.
The cells then blended with the pathology of the underlying cells. This pathology extended all the way to the subcutis and was responsible for the massive nodules characterized by large, irregular cells in tubular array. These cells did not have the form of the intraepidermal component per se, and did not suggest melanoma. Their array is somewhat suggestive of nerve tissue and less probably represented Schwannian tissue. Most of these tubular collections of cells were surrounded by a concentric area of hyperplastic spindled cells variably associated with an increased amount of ground substance (Figure 4C and D). Peripheral to this, the spindle cells were associated with the overt collagen. Mature collagen formed a significant part of this neoplasm. At the very depths of the biopsy, extending into the subcutaneous tissue, smaller cells were present that were rather characteristic of the background pattern of a congenital melanocytic nevus present in both cases 1 and 2. A tri-cut biopsy of the hard, suprapubic mass was composed entirely of cells in tubular arrays similar to those just described (not shown). A biopsy from the right
iliac crest showed changes at the dermal-epidermal interface similar to those described in previous biopsies and was quite suggestive of melanoma. Areas of associated ulceration were noted. The bulk of the biopsy from the ulcerated area showed relatively small cells, some of which were synthesizing ground substance, which had the appearance of a congenital melanocytic nevus, albeit with some overgrowth of mucin-synthesizing cells. In addition, this biopsy showed an area of nodular growth composed of large pigment-synthesizing cells. The pigment was coarsely granular and the nuclei relatively uniform. On casual inspection, the histology was quite suggestive of melanoma arising in deep tissues. The coarsely granular pigment and the orderliness of the nuclei suggested against the diagnosis of melanoma, but such pattern represented a more nodular, dermal hyperplasia that can occur in congenital melanocytic nevi. The biopsies of case 2 illustrated several points. First, the intraepidermal component was hyperplastic and certainly simulated melanoma. The dermal component forming the bulk of these lesions was a proliferation of neural
823 AJPApril 1990, Vol. 136, No. 4
Neonatal Melanoma
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Figure 5. Cultured melanocytic cellsfrom patient 1. WM 1184-1 (A and B) and WM 1184-2 (C and D) cell lines had a fibroblastlike morphology when cultured in the absence of TPA (A and C), but developed a typical melanocytic shape on addition of TPA to the culture medium (B and D)
crest-derived tissue. In addition, deep in the underlying connective tissue, there was nodular proliferation of cells that simulated melanoma.
Establishment of Cell Lines in Tissue Culture Three cells lines were established from lesions of each of the two patients. Two specimens representing the congenital nevus of patient 1 yielded cell lines WM 1184-2 and WM 1184-N. Cell line WM 1184-1 was from an area representing melanoma. No morphologic differences were observed between the cell lines. They were maintained in culture for more than 30 months in the absence of TPA. Under these conditions, WM 1 184 cell lines exhibited a spindle, fibroblastlike morphology (Figures 5A and 5C). All cells examined in cultures at the 5th and 15th passages contained premelanosomes and melanosomes as assessed by electron microscopy (not shown). After the addition of TPA to the medium, all cells in the cultures accentuated their bipolar morphology, assuming a typical melanocytic shape, increased their proliferation rate, and became more pigmented (Figures 5B and 5D).
These changes were reversible on withdrawal of the phorbol ester. The nodular mass from the lateral thigh of patient 2 was divided into two parts. The superficial part, comprising epidermis, basement membrane zone, and the immediately subjacent dermis, was intensely black, and cell line WM 1491-M was obtained from this area. Cell line WM 1491-NCT was derived from the deep dermal portion of the same lesion and represented the Schwannian tissue. The congenital nevus in the iliac crest, a deep nodule of melanocytes, yielded cell line WM 1491-CN. All cell lines had a similar bipolar to tripolar morphology, with WM 1491 -M having the longest dendrites (Figure 6). The cells were bigger than normal melanocytes and heavily pigmented. Abundant premelanosomes and melanosomes could be detected by electron microscopy. Cells maintained in the absence of TPA had a bigger perinuclear region and shorter dendrites than cells cultured in the presence of TPA. Cell line WM 1491 -NCT was initially constituted of a mixed population of melanocytic cells and nonmelanocytic cells represented by fibroblastlike and epithelioid cells. After treatment with G418, it displayed a uniform melanocytic morphology similar to that of the other lines. The nature of the nonmelanocytic cells was
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(NGF) receptor, p97 melanotransferrin, and gpl 20/94 (vitronectin receptor) were strongly expressed by all cell lines as determined by mixed hemadsorption and radioimmunoassays on live cells and by immunoperoxidase assays on fixed cells. Nerve growth factor receptor and p97 melanotransferrin clearly indicate the melanocytic origin of cells from cases 1 and 2, as neither antigen is expressed by dermal fibroblasts.23 Chondroitin sulfate proteoglycan and gp120/94 vitronectin receptor may be weakly expressed by rapidly proliferating fibroblasts.23 Generally not found on normal melanocytes, HLA-DR antigen was expressed by all cultures of cases 1 and 2. The melanocyte-associated antigen gpl 45, which is not expressed on cultured melanoma cells,23 was detected on four out of five cultures. Ganglioside GD2, representing an important marker for malignancy, as it is expressed on primary and metastatic melanoma cells but not on normal melanocytes and nevus cells,' was not expressed on any cell line. Gangliosides GD3 and 9-0-acetylated GD3, which are expressed on cultured melanocytes3 and nevus cells,' were detected on all cell lines. Similar results were obtained in indirect immunoperoxidase assays on fixed cells and in indirect radioimmunoassays on live cells in suspension (data not shown). The expression of antigens remained relatively stable over serial passages in culture.
Karyotypic Analysis
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Figure 6. Morphology of cultured melanocytic cells from patient 2. (A) WM 1.491-M cells from epidermis-upper dermis; (B) WM 1491-NCT cellsfrom the dermal component of a nodular (C) WM 1491-CN cells from the congenital nevus of the iliac crest after treatment with G418 Cells were cultured in medium containing TPA. mass;
Cell lines WM 1 184-1, WM 1 184-2, and WM 1 1 84-N from patient 1, when tested after 3 to 5 and 15 passages, invariably showed a normal diploid chromosome count. Cell lines WM 1491 -NCT, WM 1491 -M, and WM 1491 -CN from patient 2 were tested at passages 1, 3, and 4 and did not show structural chromosomal alterations. However, approximately 25% of the mitotic cells in all cultures of patient 2 displayed a nearly tetraploid karyotype.
Growth Characteristics not determined: in immunoperoxidase staining, they did not express any of the pigment cell-associated antigens.
Expression of Melanocyte- and Melanoma-associated Antigens The six cell lines from patients 1 and 2 were tested for the expression of melanocyte- and melanoma-associated antigens (Table 1). For comparison, representative cultures of newborn melanocytes, congenital nevus cells, and primary melanoma cells were included in Table 1. Chondroitin sulfate proteoglycan, nerve growth factor
All cell lines from cases 1 and 2 grew rapidly during the first five passages, with doubling times of approximately 30 to 40 hours, both in the absence and presence of TPA. They then slowed to doubling times of 12 to 14 days, but continued to proliferate at this rate with delayed senescence (Table 2). Cell lines WM 1184-1 and -2 grew for more than 30 months in the absence of TPA. Cell lines WM 1491 M, WM 1491 NCT, and WM 1491 CN, when cultured without TPA in medium, slowed their growth rate to 30 days doubling time. WM 1 184-1 and WM 1 184-2 cells did not form colonies in soft agar, nor did they form tumors in athymic nude
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mice after subcutaneous injection. WM 1491-NCT, WM 1491-M, and WM 1491-CN cells also did not grow in an anchorage-independent manner; however, in nude mice, these cells formed a subcutaneous nodule, 3 mm in diameter, at the injection site at the time of inoculation, but the nodule did not enlarge during the 7-month observation period. Histologic examination of these indolent nodules revealed pigmented epithelioid melanocytic cells embedded in thick mouse stromal tissue. No mitotic figures could be identified. On reculturing, cells from the nude mouse-derived nodule were highly pigmented and then proliferated slowly, with doubling times of 3 to 4 weeks. Morphology and karyotype was the same as before the passage in nude mice. Livers and lungs of the mice carrying a melanocytic nodule of WM 1491 cells were free of metastatic foci. Prolonged survival under suboptimal conditions also was observed in WM 1184 and WM 1491 cells grown in serum-free, chemically defined medium. Cell lines WM 1184-2 and WM 1491-NCT survived and proliferated slowly in medium W489 supplemented with insulin, epidermal growth factor, fibroblast growth factor, TPA, and a-melanocyte-stimulating hormone. Both cell lines were maintained in this medium for more than 4 months with passages every third week. Similar to normal melanocytes and congenital nevus cells, WM 1184-2 and WM 1491-NCT cells grew more rapidly in the presence of TPA than in its absence (Figure 7). Primary and metastatic melanoma cells used as controls, however, were growth-inhibited by TPA in medium. The addition of TPA stimulated WM 1184 cell lines to grow, despite the fact that cell lines had been maintained for more than 30 months in the absence of TPA. On removal of TPA from the medium, WM 1184 cell lines continued to proliferate, but at approximately a 50% slower rate. Culture medium depleted of TPA, growth factors, and a-melanocyte-stimulating hormone (protein-free medium) did not support prolonged growth of WM 1 184 and WM 1491 cells. The protein-free medium allowed continuous proliferation of the metastatic melanoma control cells.
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Melanocytes derived from neonatal lesions with clinical and histopathologic features of malignancy showed no evidence for a malignant phenotype in the two cases studied here. The cells, unlike primary malignant melanoma cells of the vertical growth phase and metastatic melanoma cells,39 were unable to grow anchorage independently in soft agar; they did not proliferate in nude mice at the site of injection, nor did they produce metastases in internal organs. The melanocytic cells from cases 1 and 2 had a prolonged lifespan beyond that of neonatal
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Table 2. Growth Characteristics of Cellsfrom Neonatal Melanocytic Lesions in Comparison to Neonatal Normal Melanocytes, Congenital Nevus Cells, and Primary Melanoma Cells Neonatal melanoma Neonatal Congenital Primary WM 1491-NCT WM 1184-2 nevus cells** melanoma cells** Growth condition melanocytes** 25 to 45t 55* 60* No. of doublings 100 12t Doubling times (days) 14$ 2-6§ 1-7§ 0.7-7§ Growth in soft agar (% colony-forming
Malignant Melanoma Simulants Arising in Congenital Melanocytic Nevi Do Not Show Experimental Evidence for a Malignant Phenotype
Maria-Laura Mancianti,* Wallace H. Clark,t F. Ann Hayes, and Meenhard Herlyn* From the Wistar Institute ofAnatomy and Biology, Philadelphia; Pennsylvania*; the Pigmented Lesion Study Group and the Department ofDermatology of the University ofPennsylvania, Philadelphia, Pennsylvaniat; and the Department ofHematology-Oncology, St. Jude
Children's Hospital, Memphis, Tennessee*
Proliferative neoplasms that resemble malignant melanoma may develop in large congenital melanocytic nevi, prenatally or in the neonatal period, although these lesions rarely show the progressive growth or behavioral characteristics of melanoma. This report describes the genetic, biologic, and immunologic characteristics of six tissue culture cell lines derived from two neoplasms present in congenital melanocytic nevi in two newborn infants. Both neoplasms had clinical and histologic features of malignant melanoma. Despite these features, cells from all lines were phenotypically benign, as evidenced by a normal karyotype, their expression pattern ofpigment cell-associated antigens, absence of melanoma-associated ganglioside GD2, their mitogenic response to the tumorpromoting pborbol ester 12-0-tetradecanoylphorbol- 13-myristate, their inability to grow anchorage independently in soft agar, and prolonged but finite life span. The cells did not produce tumors in nude mice, but they remained viable at the injection site for over 7 months. (Am J Pathol 1990,
136:817-829) Congenital giant nevi constitute a well-known risk factor in the development of malignant melanoma both in prepubertal and postpubertal individuals, with a reported incidence between 4.6% and 38%.'I Primary congenital malignant melanoma, with or without an associated congenital nevus, is rare. One form of congenital melanocytic
neoplasm presents as a massive, rapidly enlarging, ulcerative mass at birth and has been termed nodular proliferative neurocristic hamartoma. Such lesions arise in giant congenital melanocytic nevi. Another form, composed of epithelioid melanocytes, may be histologically indistinguishable from nodular melanoma. These nodular lesions may arise in small, intermediate, and giant congenital melanocytic nevi. To our knowledge, only 16 cases have been described in which malignant melanoma of the skin was diagnosed at birth or in the neonatal period.7-19 With the exception of the few cases in which death occurred shortly after birth and metastasis could be detected, the alarming clinical or histologic features in these congenital or neonatal tumors did not result in death due to metastasis. However, the natural history of these lesions remains unknown because their clinical presentation is an imperative for their removal, and the histologic evaluation of these lesions is difficult and inconsistent. In those lesions that are not completely removed, there is evidence of differentiation rather than progressive malignancy. Diagnostic problems at the histologic level are due to the complex cellular composition of some of the nodular overgrowths occurring in congenital melanocytic lesions. Four major histologic patterns of proliferation have been observed in congenital melanocytic nevi at birth or in the neonatal period2O: 1) simulants of superficial spreading melanoma, in which epidermis and superficial dermis are occupied by increased numbers of large epithelioid melanocytes; 2) simulants of nodular melanoma, with either multiple black nodules of epithelial melanocytes or a single black nodule of extremely large epithelioid melanocytes with uniform nuclei; 3) nodular proliferative neurocristic hamartomas, characterized by tumorous growth in fetal life, and composed of diverse tissues; the deep dermis and subcutis are occupied by neuro-sustentacular tissue and ecto-mesenchymal structures; and 4) biologic Supported by grants CA-25874, CA-10815, CA-44877, CA-25298, and CA-16520 from the National Institutes of Heath. Accepted for publication November 27, 1989. Address reprnt requests to Meenhard Herlyn, DVM, The Wistar Institute, 36th Street at Spruce, Philadelphia, PA 19104.
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Figure 1. Patient 1. Simulants of nodular melanoma. A darkly pigmented nodule of 3.5 X 1.5 cm arising within the tan background ofa congenital melanocytic nevus of6X5cm.
malignant melanoma, most of which are characterized by small blastic pleomorphic melanocytes with a high mitotic rate. The true melanomas with metastatic potential usually develop after the neonatal period. In our laboratory, we have previously studied pigmented cells from different stages of tumor progression in culture, and have characterized different biologic steps in the evolution from normal melanocytes to malignant cells.21 In the present study, we analyzed cells derived from two kinds of proliferative tumors in large congenital melanocytic nevi to determine their malignant potential. The results obtained with six cell lines are not indicative of a malignant phenotype.
scalp, ears, face, arms, legs, palms, and soles of the feet. The bulk of the large lesion was black, with some dark brown areas, especially at the periphery. The smaller lesions were dark brown to black. In the subsequent weeks, the masses progressively enlarged, with the greatest change in the subcutaneous tissue of the pubic area. Ul-
Materials and Methods
Clinical Cases Case 1 was an 8-day-old white neonate born with a congenital nevus of the right thigh. The lesion consisted of a darkly pigmented nodule of 3.5 X 1.5 cm that was rising within the tan background of a congenital melanocytic nevus that was approximately 6 X 5 cm (Figure 1). The dark lesion and part of the background lesion were excised and submitted for histologic diagnosis and for tissue culture studies. Tissue fixation, embedding, and staining was done using standard procedures. The child was alive and well at 41 months of age. Case 2 was a white male who presented at 3 weeks of age with a large congenital bathing trunk nevus extending from the umbilical area to just above the knees (Figure 2). Large masses, some soft, others firm, were palpable in the pubic-inguinal area, buttocks, and thigh. Multiple congenital nevi of varying sizes also were present on the
Figure 2. Patient 2. Large congenital bathing trunk nevus extendingfrom the umbilical area to just above the knees. Large masses werepalpable in buttock, thigh, andpubic-inguinal areas.
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ceration occurred in a few areas. Biopsies were performed on three of the nodules for histologic studies at the St. Jude Children's Research Hospital, Memphis, TN. Specimens from two of the biopsies were obtained for tissue culture. At this time, a computed tomography (CT) scan of the head showed three intracerebral nodules interpreted as metastases. At 7 weeks of age, the infant was treated with cyclophosphamide, vincristine, and actinomycin following a previously described protocol.16 Therapy was discontinued after 7 days because of hematogenous infection. The masses became softer and less prominent over the ensuing months and the cerebral lesions were indolent and calcified. The child was asymptomatic and developing normally at 18 months of age.
Tissue Culture Specimens received for tissue culture were enzymatically digested using two procedures.22 First, 1- to 2-sq mm fragments of each lesion were incubated in 0.05% collagenase type IV, 0.1% hyaluronidase, and 1.25 U/ml dispase in Hank's balanced salt solution (HBSS) without Ca++ and Mg++ and supplemented with 30 mM HEPES (modified HBSS), and digested in toto overnight at 370C. Second, fragments from the same lesions were incubated overnight at 40C in 0.25% trypsin in modified HBSS. The epidermis then was mechanically separated from the dermis and the dermis was digested as in the first procedure. The epidermis was incubated for 3 hours at 37°C in 0.1% hyaluronidase, 1.25 U/ml dispase, and 0.033% versene in modified HBSS. Cell suspensions obtained by the different procedures were sedimented, resuspended in medium, and seeded into 12-well plates. The culture medium consisted of W489 medium supplemented with 5 ,g/ml insulin, 5 ng/ml epidermal growth factor, 40,g/ml bovine pituitary extract containing fibroblast growth factor, and 2% fetal calf serum (FCS).23 Each cell line was established and maintained in the presence or absence of the phorbol ester 12-0-tetradecanoylphorbol-13-myristate (TPA) at 10 ng/ml. Cultures were maintained at 370C in 5% C02/95% air and refed biweekly. For serum-free culture, W489 medium was supplemented with insulin, epidermal growth factor, basic fibroblast growth factor (40 ng/ml), TPA, and synthetic a-melanocyte-stimulating hormone (1 to 10 ng/ml). The rationale for using these growth supplements was: 1) Insulin is a mitogen for melanocytes at different stages of tumor progression,2425 acting predominantly via the receptor for insulinlike growth factor 1;4 2) epidermal growth factor enhances the recovery rate of freshly passaged cells and is essential during the first 2 weeks in tissue culture, although it is not mitogenic for established melanocytic cell lines;23 3) basic fibroblast growth factor from bovine pituitary;26 4) TPA, an activator
for protein kinase C;27 and 5) alpha melanocyte-stimulating hormone24 are mitogenic for melanocytes. One cell line from patient 2 was initially contaminated with fibroblasts and was treated in the first passage with 200 ,g/ ml G418 (GIBCO, Grand Island, NY) for 2 days.28 Melanocytes from newborn foreskins, congenital nevus cells, and primary and metastatic melanoma cells were used as control cells, isolated and grown as described.21 Melanocytes and nevus cells were maintained in TPA-containing medium, and melanoma cells in medium without TPA.
Electron Microscopy Cells from each specimen were seeded into 35-mm plastic dishes and grown for 7 to 10 days. Cultures then were fixed for 2 hours at 350C in 3% glutaraldehyde in PIPES buffer, rinsed twice with cold 0.1 mol/I cacodylate buffer, and postfixed with 1.0% osmium tetroxide for 1 hour. Cells were dehydrated in a graded series of ethanols and embedded in Epon. Sections were cut parallel to the culture surface, stained with uranyl acetate and lead citrate, and examined under a ZEISS EM10 electron microscope (Zeiss, Oberkochen, West Germany).
Growth Analyses Cells were seeded in duplicate or triplicate in 2 sq cm wells of 24-well plates at 2 to 3 X 104 cells/sq cm. On days 1 through 10, cells were detached by trypsinization and counted in a Coulter cell counter. Anchorage-independent growth was tested using a standard agarose assay previously described.22 Briefly, 35-mm wells were prepared with an underlayer of 0.5% agarose in complete medium. Cells were seeded at 1500 and 3000 cells per well in 0.25% agarose and incubated at 370C. Fresh medium was added after 3 to 4 days and colonies were counted after 10 to 14 days. In each assay, normal melanocytes, nevus cells, and malignant melanoma cells were included as controls. Growth in nude mice was studied by subcutaneous injection of 1 to 3 x 107 cells into athymic Balb/c (nu/nu) mice. Mice were observed weekly for tumor growth. After 120 and 210 days, mice were killed and examined for evidence of tumor growth at the site of injection and in livers and lungs.
Karyotypic Analysis Karyotypes of cultured cells were analyzed at different culture passages. One to 4 days after seeding, cells were incubated with colchicine for 45 minutes at 370C. After
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dciL--
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Figure 3. Patient 1. Nodular melanocytic byperplasia, simulating malignant melanoma, arises in a congenital melanocytic nevus. (A) Dermal component of the basic congenital melanocytic nevus. (B) Cells deep in the epidermis that were much larger than the cells forming the congenital melanocytic nevus, formed the rapidly growing nodule observed clinically.
treatment with a KCI-sodium citrate hypotonic solution and standard fixation, air-dried slides were prepared for
Giemsa banding.29
Expression of Pigment Cell-associated Antigens Expression of melanocyte- and melanoma-associated antigens was evaluated by: 1) mixed hemadsorption assay (MHA) in which binding of monoclonal antibodies (MAbs) to live adherent target cells is visualized by sheep red blood cells preincubated with mouse anti-sheep red blood cell antibodies and agglutinated by goat anti-mouse antibodies;-: 2) fluorescence-activated cell sorting (FACS) by incubating cells in suspension with MAbs, followed by incubation with fluoresceinated goat anti-mouse immunoglobulin antibodies using an Ortho Cytofluorograf 50HH connected to an Ortho 450 data handling system
(Ortho, Westwood, MA); 3) indirect radioimmunoassay, in which the binding of MAb to adherent cells fixed with 0.1% glutaraldehyde was determined by 1251-goat antimouse F(ab)2 antibody as described;31 and 4) immunoperoxidase staining by the Vectastain ABC method, using 3-amino-9-ethyl-carbazole (AEC) as substratum since it gives a dark red color in positive cells that is distinguishable from melanin.
Results Histology Case 1
Simulant of Nodular Melanoma The basic congenital melanocytic nevus had an intraepidermal component characterized by an increased
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number of melanocytes disposed in orderly nests. Occasionally there was bridging from one rete to the next and slight atypia of this intraepidermal component, which is a standard feature of most congenital melanocytic nevi in the early months and years of life and may be seen later in the adult years. The dermal component was characterized by relatively small cells that extended throughout the dermis and into the subcutaneous tissue, especially in the fibrous trabeculae of the subcutaneous tissue (Figure 3A). These cells were uniform and formed delicate strands disposed in a random array. Sections through the rapidly growing nodule were characterized by intraepidermal melanocytic hyperplasia wherein the melanocytes were disposed as individual cells and showed some upward growth in the epidermis (Figure 3B). This upward growth was not as striking as that in adult melanomas. The pathology was dominated by replacement of the dermal components of the nevus by much larger cells than those described above. The large cells were arranged in small clusters of three to five cells within a somewhat eccentric periphery of flattened cells. The nuclei of the large cells were two to three times larger than the nuclei present in the reticular dermis of the background congenital melanocytic nevus. The lesion had some features of nodular melanoma. A major difference between simulants of nodular melanoma described here and true biologic melanoma arising in congenital melanocytic nevi was the ten-
dency for the large cells comprising the simulant nodule to blend imperceptibly at the base with the smaller cells of the background congenital melanocytic nevus. In biologic nodular melanoma arising in congenital melanocytic nevi, the melanoma is sharply delineated from the surrounding congenital melanocytic nevus.
Case 2 Nodular Proliferative Neurocristic Hamartoma The first area biopsied was from a large mass present on the lateral thigh. The changes within the epidermis and the immediately subjacent dermis were characterized by the presence of a large number of melanocytes disposed in irregular nests with upward growth of individual melanocytes to the outer reaches of the epidermis. The cells of the individual nests were large, pleomorphic, and somewhat dyshesive (Figure 4A). This portion of the tumor was virtually indistinguishable histologically from the radial growth phase of malignant melanoma of the superficial spreading type. In some sections, the epidermal component and the component immediately subjacent to the epidermis had the appearance of melanoma, forming clusters of atypical cells in the dermis (Figure 4B). The cells present in the immediately subjacent dermis also were large and atypical and associated with fibroplasia.
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Figure 4. Patient 2. (A) Large multinodular lesion present at birth. The intraepidermal component is illustrated. Large nests ofpoorly cohesive melanocytes are present at the dermal-epidermal interface. (B) Clusters of atypical melanocytes similar to those of Figure 3 have extended into the dermis. (C) The micrograph shows the neuroectodermal tissue, whichformed the bulk ofthe massive nodules in the congenital melanocytic nevus. (D) The central cells in tubular array probably represent epithelioid Schwannian tissue.
The cells then blended with the pathology of the underlying cells. This pathology extended all the way to the subcutis and was responsible for the massive nodules characterized by large, irregular cells in tubular array. These cells did not have the form of the intraepidermal component per se, and did not suggest melanoma. Their array is somewhat suggestive of nerve tissue and less probably represented Schwannian tissue. Most of these tubular collections of cells were surrounded by a concentric area of hyperplastic spindled cells variably associated with an increased amount of ground substance (Figure 4C and D). Peripheral to this, the spindle cells were associated with the overt collagen. Mature collagen formed a significant part of this neoplasm. At the very depths of the biopsy, extending into the subcutaneous tissue, smaller cells were present that were rather characteristic of the background pattern of a congenital melanocytic nevus present in both cases 1 and 2. A tri-cut biopsy of the hard, suprapubic mass was composed entirely of cells in tubular arrays similar to those just described (not shown). A biopsy from the right
iliac crest showed changes at the dermal-epidermal interface similar to those described in previous biopsies and was quite suggestive of melanoma. Areas of associated ulceration were noted. The bulk of the biopsy from the ulcerated area showed relatively small cells, some of which were synthesizing ground substance, which had the appearance of a congenital melanocytic nevus, albeit with some overgrowth of mucin-synthesizing cells. In addition, this biopsy showed an area of nodular growth composed of large pigment-synthesizing cells. The pigment was coarsely granular and the nuclei relatively uniform. On casual inspection, the histology was quite suggestive of melanoma arising in deep tissues. The coarsely granular pigment and the orderliness of the nuclei suggested against the diagnosis of melanoma, but such pattern represented a more nodular, dermal hyperplasia that can occur in congenital melanocytic nevi. The biopsies of case 2 illustrated several points. First, the intraepidermal component was hyperplastic and certainly simulated melanoma. The dermal component forming the bulk of these lesions was a proliferation of neural
823 AJPApril 1990, Vol. 136, No. 4
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Figure 5. Cultured melanocytic cellsfrom patient 1. WM 1184-1 (A and B) and WM 1184-2 (C and D) cell lines had a fibroblastlike morphology when cultured in the absence of TPA (A and C), but developed a typical melanocytic shape on addition of TPA to the culture medium (B and D)
crest-derived tissue. In addition, deep in the underlying connective tissue, there was nodular proliferation of cells that simulated melanoma.
Establishment of Cell Lines in Tissue Culture Three cells lines were established from lesions of each of the two patients. Two specimens representing the congenital nevus of patient 1 yielded cell lines WM 1184-2 and WM 1184-N. Cell line WM 1184-1 was from an area representing melanoma. No morphologic differences were observed between the cell lines. They were maintained in culture for more than 30 months in the absence of TPA. Under these conditions, WM 1 184 cell lines exhibited a spindle, fibroblastlike morphology (Figures 5A and 5C). All cells examined in cultures at the 5th and 15th passages contained premelanosomes and melanosomes as assessed by electron microscopy (not shown). After the addition of TPA to the medium, all cells in the cultures accentuated their bipolar morphology, assuming a typical melanocytic shape, increased their proliferation rate, and became more pigmented (Figures 5B and 5D).
These changes were reversible on withdrawal of the phorbol ester. The nodular mass from the lateral thigh of patient 2 was divided into two parts. The superficial part, comprising epidermis, basement membrane zone, and the immediately subjacent dermis, was intensely black, and cell line WM 1491-M was obtained from this area. Cell line WM 1491-NCT was derived from the deep dermal portion of the same lesion and represented the Schwannian tissue. The congenital nevus in the iliac crest, a deep nodule of melanocytes, yielded cell line WM 1491-CN. All cell lines had a similar bipolar to tripolar morphology, with WM 1491 -M having the longest dendrites (Figure 6). The cells were bigger than normal melanocytes and heavily pigmented. Abundant premelanosomes and melanosomes could be detected by electron microscopy. Cells maintained in the absence of TPA had a bigger perinuclear region and shorter dendrites than cells cultured in the presence of TPA. Cell line WM 1491 -NCT was initially constituted of a mixed population of melanocytic cells and nonmelanocytic cells represented by fibroblastlike and epithelioid cells. After treatment with G418, it displayed a uniform melanocytic morphology similar to that of the other lines. The nature of the nonmelanocytic cells was
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(NGF) receptor, p97 melanotransferrin, and gpl 20/94 (vitronectin receptor) were strongly expressed by all cell lines as determined by mixed hemadsorption and radioimmunoassays on live cells and by immunoperoxidase assays on fixed cells. Nerve growth factor receptor and p97 melanotransferrin clearly indicate the melanocytic origin of cells from cases 1 and 2, as neither antigen is expressed by dermal fibroblasts.23 Chondroitin sulfate proteoglycan and gp120/94 vitronectin receptor may be weakly expressed by rapidly proliferating fibroblasts.23 Generally not found on normal melanocytes, HLA-DR antigen was expressed by all cultures of cases 1 and 2. The melanocyte-associated antigen gpl 45, which is not expressed on cultured melanoma cells,23 was detected on four out of five cultures. Ganglioside GD2, representing an important marker for malignancy, as it is expressed on primary and metastatic melanoma cells but not on normal melanocytes and nevus cells,' was not expressed on any cell line. Gangliosides GD3 and 9-0-acetylated GD3, which are expressed on cultured melanocytes3 and nevus cells,' were detected on all cell lines. Similar results were obtained in indirect immunoperoxidase assays on fixed cells and in indirect radioimmunoassays on live cells in suspension (data not shown). The expression of antigens remained relatively stable over serial passages in culture.
Karyotypic Analysis
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Figure 6. Morphology of cultured melanocytic cells from patient 2. (A) WM 1.491-M cells from epidermis-upper dermis; (B) WM 1491-NCT cellsfrom the dermal component of a nodular (C) WM 1491-CN cells from the congenital nevus of the iliac crest after treatment with G418 Cells were cultured in medium containing TPA. mass;
Cell lines WM 1 184-1, WM 1 184-2, and WM 1 1 84-N from patient 1, when tested after 3 to 5 and 15 passages, invariably showed a normal diploid chromosome count. Cell lines WM 1491 -NCT, WM 1491 -M, and WM 1491 -CN from patient 2 were tested at passages 1, 3, and 4 and did not show structural chromosomal alterations. However, approximately 25% of the mitotic cells in all cultures of patient 2 displayed a nearly tetraploid karyotype.
Growth Characteristics not determined: in immunoperoxidase staining, they did not express any of the pigment cell-associated antigens.
Expression of Melanocyte- and Melanoma-associated Antigens The six cell lines from patients 1 and 2 were tested for the expression of melanocyte- and melanoma-associated antigens (Table 1). For comparison, representative cultures of newborn melanocytes, congenital nevus cells, and primary melanoma cells were included in Table 1. Chondroitin sulfate proteoglycan, nerve growth factor
All cell lines from cases 1 and 2 grew rapidly during the first five passages, with doubling times of approximately 30 to 40 hours, both in the absence and presence of TPA. They then slowed to doubling times of 12 to 14 days, but continued to proliferate at this rate with delayed senescence (Table 2). Cell lines WM 1184-1 and -2 grew for more than 30 months in the absence of TPA. Cell lines WM 1491 M, WM 1491 NCT, and WM 1491 CN, when cultured without TPA in medium, slowed their growth rate to 30 days doubling time. WM 1 184-1 and WM 1 184-2 cells did not form colonies in soft agar, nor did they form tumors in athymic nude
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mice after subcutaneous injection. WM 1491-NCT, WM 1491-M, and WM 1491-CN cells also did not grow in an anchorage-independent manner; however, in nude mice, these cells formed a subcutaneous nodule, 3 mm in diameter, at the injection site at the time of inoculation, but the nodule did not enlarge during the 7-month observation period. Histologic examination of these indolent nodules revealed pigmented epithelioid melanocytic cells embedded in thick mouse stromal tissue. No mitotic figures could be identified. On reculturing, cells from the nude mouse-derived nodule were highly pigmented and then proliferated slowly, with doubling times of 3 to 4 weeks. Morphology and karyotype was the same as before the passage in nude mice. Livers and lungs of the mice carrying a melanocytic nodule of WM 1491 cells were free of metastatic foci. Prolonged survival under suboptimal conditions also was observed in WM 1184 and WM 1491 cells grown in serum-free, chemically defined medium. Cell lines WM 1184-2 and WM 1491-NCT survived and proliferated slowly in medium W489 supplemented with insulin, epidermal growth factor, fibroblast growth factor, TPA, and a-melanocyte-stimulating hormone. Both cell lines were maintained in this medium for more than 4 months with passages every third week. Similar to normal melanocytes and congenital nevus cells, WM 1184-2 and WM 1491-NCT cells grew more rapidly in the presence of TPA than in its absence (Figure 7). Primary and metastatic melanoma cells used as controls, however, were growth-inhibited by TPA in medium. The addition of TPA stimulated WM 1184 cell lines to grow, despite the fact that cell lines had been maintained for more than 30 months in the absence of TPA. On removal of TPA from the medium, WM 1184 cell lines continued to proliferate, but at approximately a 50% slower rate. Culture medium depleted of TPA, growth factors, and a-melanocyte-stimulating hormone (protein-free medium) did not support prolonged growth of WM 1 184 and WM 1491 cells. The protein-free medium allowed continuous proliferation of the metastatic melanoma control cells.
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Melanocytes derived from neonatal lesions with clinical and histopathologic features of malignancy showed no evidence for a malignant phenotype in the two cases studied here. The cells, unlike primary malignant melanoma cells of the vertical growth phase and metastatic melanoma cells,39 were unable to grow anchorage independently in soft agar; they did not proliferate in nude mice at the site of injection, nor did they produce metastases in internal organs. The melanocytic cells from cases 1 and 2 had a prolonged lifespan beyond that of neonatal
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Table 2. Growth Characteristics of Cellsfrom Neonatal Melanocytic Lesions in Comparison to Neonatal Normal Melanocytes, Congenital Nevus Cells, and Primary Melanoma Cells Neonatal melanoma Neonatal Congenital Primary WM 1491-NCT WM 1184-2 nevus cells** melanoma cells** Growth condition melanocytes** 25 to 45t 55* 60* No. of doublings 100 12t Doubling times (days) 14$ 2-6§ 1-7§ 0.7-7§ Growth in soft agar (% colony-forming
