Cancer and Metastasis Reviews 9: 101-112, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Human melanoma: Development and progression Meenhard Herlyn

The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, USA Key words: melanoma, progression, growth factors, monoclonal antibodies Abstract Clinical and histopathological evidence suggests that melanoma develops in a sequence of steps, progressing from benign proliferative lesions, to primary melanomas that do not show evidence for metastasis, to invasive primary lesions, and to metastases. This review focuses on the experimental studies examining the phenotypic characteristics of cultured primary melanoma cells as they relate to cells from non-malignant nevi and metastases. Genetic, biologic, and immunologic criteria have been established to distinguish melanocytes from different steps of tumor development. These include non-random chromosomal abnormalities, expression of melanocyte- and melanoma-specific antigens, requirements for exogenous growth factors, production of endogenous growth factors, and expression of receptors for growth factors. The transformation of melanocytes and nevus cells with viral oncogenes has facilitated studies on the malignant phenotype. Variants have been developed through successive selections from primary melanoma cell populations that have one or several characteristics of metastatic cells. The study of melanocytes isolated from various stages of tumor development and the generation of cell variants with specific properties should enable a long-term search for the molecular mechanisms of melanoma development and progression.

Introduction The etiology, progression, diagnosis and therapy of human malignant cutaneous melanomas have been intensely investigated over recent years. Major advances have been made in the definition of different steps of tumor development using clinical, histopathological, and experimental approaches. Better diagnosis of melanoma has led to treatments in earlier stages of malignancy, resulting in higher survival rates of patients with the early forms of the disease. However, the poor prognosis of advanced melanoma, due to its general resistance to the various forms of therapy, and the increase of melanoma by 5% to 7% per year have not resulted in an overall reduction of this malignancy. After a brief discussion of etiological and clinical studies, this review focuses on experimental studies with primary and metastatic melanoma cells that were iso-

lated and cultured from the same or different patients, and on factors related to progression and the metastatic phenotype.

Etiology of melanoma Melanocytes are neural crest-derived cells. Precursor cells of melanocytes have been identified in human skin [1], but these cells have been minimally characterized. It has been suggested that at least two precursor stages of melanocytes, early and intermediate, exist in the skin [2, 3]. Functional maturation of melanocytes, i.e., the process by which cells express all the specific properties characteristic of this cell type, may occur through these preliminary defined stages (Table 1). Cells of each maturation form may transform to either a nevus or a malignant melanoma. Nevi may further progress

102 to melanoma, or they can regress by differentiating to cell type(s) that have been histopathologically defined as (non-malignant) Schwann cells [4]. The molecular mechanism leading to transformation and progression or to differentiation is poorly understood.

patients who were monitored for 10 years after removal of the primary tumor [7]. The vertical growth phase (VGP) primary melanoma arises as a focal subpopulation showing a different growth pattern within the RGP lesions. VGP lesions are associated with risk for metastasis. Six tumor- and host-related factors associated with survival of patients with clinical stage I VGP primary melanomas have recently been defined ([8]; Table 3). Tumor thickness is the most significant criterion for evaluating risk of death due to metastatic disease. Patients with thin melanomas (< 0.76 mm) have an 8-year survival rate of 93%. If the tumor thickness increases to more than 3.6 mm, survival rate drops to 33%. A similar decrease in survival may occur in lesions with a high mitotic rate. Other attributes included in the multivariable regression model of Clark et al. [8] were the anatomical site of the lesion, the presence of tumor-infiltrating lymphocytes, the sex of the patient, and histologic regression. The survival prediction with the prognostic model was 89% accurate. Metastasis is the final step of tumor progression. In most cases, metastases develop after the epidermal lesions have undergone several or all of the steps of tumor progression. Only 8% of melanoma cases that are metastatic at first presentation apparently fail to traverse any of these tumor progression steps and appear to originate de n o v o [4,

Clinical and histopathological characteristics of melanoma development and progression Extensive histopathological and clinical studies of sporadic and familial melanocytic lesions [4, 5] have led to the distinction of sequential stages of neoplastic development in the melanocytic system (Table 2). The common acquired nevus usually appears in the first two decades of life, and may grow from 0.5mm to 0.5cm over the course of many years before it differentiates along Schwannian lines and gradually disappears. In contrast to the common acquired nevus, the dysplastic nevus shows architectural and cytological atypia [4, 6]. Dysplastic lesions appear histogenetically as precursors of melanomas. The radial growth phase (RGP) primary melanoma, representing the first malignant step, develops competence for autonomous growth and local invasion. However, RGP lesions do not show competence for metastatic spread, as demonstrated by a 100% survival rate of Table 1.

Melanocytematurationand transformation

Maturation

Pathogenesis

Transformation

Metastasis Melanoblast

}

V

}

~ Nevus

}

~

Nevus

Earlypremelanocyte late

Intermediate or premelanocyles

Melanocyte

Melanoma

!

t

Progression

Melanoma{

{

Differentiation Normal

cell

103 Table 2. Tumor progression in the human melanocytic system

Step

Melanocytic lesion Common acquired and congenital nevus (no cytologic atypia) Dysplastic nevus (persistent architectural and cytologic atypia) Radial growth phase of primary melanoma (no competence for metastasis) Vertical growth phase of primary melanoma (competence for metastasis) Metastatic melanoma

5]. Lymph node metastases of melanomas are the most common source of establishing cell lines, with brain- and liver-derived metastatic cell lines being the exceptions.

Culture of melanocytic cells

The procedure for isolating melanocytic cells from normal skin, benign melanocytic lesions, and primary melanomas consists of separating the epider-

mal portion of the skin from the dermal portion using trypsin, followed by further disaggregation of each layer of cells with the enzymes, dispase, collagenase, and hyaluronidase [9]. Improved selective media to counter contamination by fibroblasts and keratinocytes can yield pure melanocytic cultures. However, in complex melanocytic lesions, such as dysplastic nevi and RGP primary melanomas, the cells representing the various stages of tumor progression are difficult to separate from each other. There have been reviews [10-12] of the success rates for establishing cell lines, the morphologies of cells from each stage, the cytogenetic evidence for chromosomal abnormalities, the growth characteristics, and the expression of antigens associated with normal melanocytes, non-malignant nevus cells and primary and metastatic melanoma cells. Here we will summarize recent studies on primary and metastatic melanoma cells that point to similarities and differences in the phenotype of both cell types and to those factors that could be involved in the metastatic progression of primary lesions. Table 4 lists primary melanoma cell lines that have been established in our laboratory. Meta-

Table 3. Tumor and host factor associated with survival of melanoma patients with clinical stage I vertical growth phase (VGP) primary melanoma

Attribute

Category

Survival Decrease in % after 8 yrs.

Thickness (ram)

Mitotic Rate (per mm2)

Site

Tumor infiltrating lymphocytes (TIL)

Sex Regression

Modified from Clark et al. [8].

3.60 0.0 0.1-6.0 >6.0

60

I

Extremities Head, Neck, and Trunk Volar or Subungual Brisk Nonbrisk Absent Female Male

~1 27

Absent or incomplete Present

~1 17

104 static cells were also obtained and cultured from two groups of patients (intermediate and advanced). These metastatic cell lines (and approximately 150 additional ones) allowed a systematic evaluation of tumor-related factors that are associated with either the primary malignancy or with metastasis. Earlier studies [13] demonstrated that after tissue culture for multiple passages melanoma cells retain the same chromosomal abnormalities and have a stable antigenic phenotype. Early primary melanomas were from patients without evidence of recurrence. Only WM793, WM902B, and WM35 from this group have been characterized in more detail, because the patients remained disease-free for more than 80 months. Intermediate primary

melanoma cells were from patients who developed metastases after 9 to 84 months. The advanced group (five cases) represents primary melanomas that were obtained simultaneously with metastases. No differences could be observed between cell lines from primary melanomas of the early and intermediate groups, but primary melanomas of the advanced groups were indistinguishable from metastatic cells [14].

Chromosomal abnormalities Cytogenetic studies revealed not only non-random chromosomal abnormalities involving chromosomes 1, 6, and 7, but commonly identical dele-

Table 4. Primary and metastatic melanoma cell lines isolated from lesions of patients Primary melanoma

Metastatic melanoma

Group

Cell line a

Recurrence (months)

Early

WM 793 WM 902B WM 35 WM 1341D WM 39c WM 747

None None None None None None

Intermediate

WM 75 WM 115

33 9 16 18

WM 278 WM 98-1 WM 853-2 WM 74OV ~

84 60 36 9 11

Advanced

WM 983A WM 1361A WM 1650Vf WM 1791A WM811P ~

0 0 0 0 0 6

Cell line

(2/82) b (5/83) (2/79) (7/86) (12/78) (10/82) WM 373 WM 165-1, WM 239A, WM 266-1, WM 266-3, WM 1617 None a None d WM 858 WM873-1, WM 873-3

WM 165-2 WM 239B WM 266-2 WM 266-4

WM 873-2,

WM 983B, WM 983C WM 1361, WM 1361C None a WM 1791C WM811M WM 862

a Sources were VGP or complex (RGP and VGP) primary melanoma lesions except WM 35, which is from RGP. b Month and year of isolation. c Patient died from non-melanoma related cause. d Establishment of permanent cell line unsuccessful or material unavailable. Poor growth. f Cell line has characteristics of RGP primary melanoma.

105 tions, translocations, or additions when comparing primary and metastatic cells from the same patients (Table 5). Metastatic melanomas often have additional random abnormalities [15, 16]. The similarities in chromosomal abnormalities of primary and metastatic cells represent strong evidence for a clonal evolution of metastases in melanoma.

Antigens on melanoma cells

Approximately 60 different antigenic systems have been identified on melanoma cells with monoclonal antibodies (MAbs) (reviewed in [17, 18]). Immunological, biological, and molecular studies have revealed at least eight groups of antigens on malignant melanocytes in vivo and in situ (Table 6). In contrast to differences in the expression of antigens on melanocytes and nevus cells in vitro versus in situ, cultured melanoma cells express tumorassociated antigens at similar levels as in situ. HLA

class II antigens are highly expressed on melanoma cells. HLA-DR is involved in the immunological recognition of tumor cells by the host immune system. Of the growth factor receptors, the nerve growth factor (NGF) receptor and the epidermal growth factor (EGF) receptor have been most extensively studied. A biological role in melanoma development for the NGF receptor is not known. The EGF receptor may be utilized for autocrine and paracrine stimulation (see below). Other growth factor receptors on melanoma cells are the insulin-like growth factor (IGF)-I receptor, the basic fibroblast growth factor (bFGF) receptor, and the receptor for a-melanocyte stimulating hormone (¢t-MSH). Cation binding and transport proteins on melanomas include p97 melanotransferrin (iron), S-100 (calcium), and ceruloplasmin (copper). Approximately 60% of anti-melanoma MAbs produced by different laboratories detect antigens involved in cell-cell and cell-substrate interactions.

Table 5. Similarities of chromosomal abnormalities in primary and metastatic melanoma lesions Patient

1

2

3

4

5

Melanoma

Primary WM 115 Metastasis WM 165-1 5 others Primary WM 75 Metastasis WM 373 Primary WM 740 Metastasis WM 858 3 others Primary WM983A Metastasis WM 983B WM 983C Primary WM 1361A Metastasis WM 1361C

Abnormality of chromosome #1

#6

#7

t(lp; 9q)

6p-

+7

same same

same same

same

0

0

7q-

lq-

0

same

0

6q-

0

lpsame

same same

+7 same

lp ÷, l q -

6q +, 6q-

+ 7q-

same same

same same

same same

t(1; 14)

6q-

+7

same

same

same

same

106 Table 6. Antigens on melanoma cells

FILA class II for antigen recognition

\

Growth factor receptors for autocrine and paracrine stimulation.

Binding and transport of cations: .calcium *iron

Pigmentation antigens and related enzymes. Nudeus

Intermediate filaments and other cytoskeletal

• copper

structures.

Extracellular matrix proteins for. • attachment • motility

~ • adhesion? • suppression?

Gangliosides for:.

Miscellaneous and undefined

~

• growth regulation?

Adhesion receptors for. • matrix • tumor cells •lymphocytes • endothelium

Melanoma cells express at least six, or possibly seven, integrins (VLA-1 through VLA-6), the vitronectin receptor, and the ligand for lymphocyte attachment, intercellular-adhesion molecule-1 (ICAM-1). The list of adhesion receptors on melanoma cells, especially receptors that do not bind the RGD peptide sequence, may possibly increase. Another prominent antigen group on melanoma cells is the gangliosides GD2, GD3, 9-0-acetyl GD3, GM2 and acetylated G M 2. Antibodies to GD3 and GD2 are currently in several clinical trials in the Table 7. Significantly higher expression of antigens on advanced melanomas (VGP and metastases) versus early (non-invasive) melanomas (RGP) and dysplastic nevi

Higher expression on VGP melanomas and metastases Type of Antigen

Antigen

Adhesion receptor Adhesion ligand Extracellular matrix protein Ganglioside Growth factor receptor Fc Receptor

Vitronectin receptor ICAM-1 Tenascin GD2 EGF receptor B73.1 determinant on NK cells

* Antigens not identified: A-10-33, ME27.2; and PALMI.

therapy of melanoma. The role of gangliosides in melanoma is not entirely dear: they may be involved in adhesion, host immune suppression, and/ or growth regulation. Melanoma cells secrete basement membrane proteins and other extracellular matrix proteins. Fibronectin and tenascin are most strongly secreted, followed by collagens, proteoglycans, and laminin. MAbs have been used to 'stage' non-malignant and malignant melanocytes. Holzmann et aL [19], Elder et al. [20], and others have attempted to define each step of melanoma progression with MAbs, Normal melanocytes express very few, if any, antigens in situ. Mature nevus cells from common acquired nevi weakly express the gangliosides GD3 and 9-0-acetyl GD3, NGF receptor and chondroitin sulfate proteoglycan. Dysplastic nevus cells and RGP primary melanoma cells express the same antigens and cannot be distinguished with MAbs. On the other hand, major antigenic differences exist between non-invasive (RGP) and invasive (VGP) primary melanoma cells. Table 7 summarizes those antigens that are more prominently expressed on invasive cells from advanced primary and metastatic lesions. Interestingly, four out of six highly significantly discriminating antigens (vitro-

107 Table 8. R e q u i r e m e n t s for growth factors and other mitogens by melanocytes Melanocyte

IGF-I/Insulin

bFGF

ct-MSH

TPA

Calcium

Normal Nevus Primary m e l a n o m a Early and Intermediate Advanced Metastatic m e l a n o m a

++++" ++++

++++ ++

++++ ++++

++++ ++

++++ ++++

++++ + +

0 0 0

0 0 0

0 ~, 0 J, 0 J,

+ + +

+ + + + , essential; + + , not required in all cultures but generally mitogenic; + , mitogenic but not required; 0, not mitogenic; a n d ~,, inhibitory.

nectin receptor, ICAM-1, tenascin, and ganglioside GD2) are related to adhesion, reflecting the increased mobility of invasive melanoma cells. Antigenic differences between advanced primary and metastatic melanoma cells are minimal and statistically insignificant [20]. Little is known about suppressed antigens on melanoma cells. The only two examples are the loss of expression on melanoma ceils of adenosine deaminase binding protein [21] and gp145 [22]. Both are expressed on melanocytes and nevus ceils, but not on melanoma cells. This group is potentially far larger, and may include additional antigens related to terminal differentiation of nevus and melanoma cells.

Growth factors in melanoma Melanocytes isolated from normal skin, non-malignant nevi, and primary and metastatic melanomas show marked differences in their requirements for exogenous growth factors (see [11] for review). As summarized in Table 8, normal melanocytes require at least four mitogens for growth: IGF-I or insulin, bFGF, a-MSH, and the phorbol ester TPA [23]. These factors are growth stimulatory only in the presence of at least 0.8 mM calcium (optimum at 2.0mM). EGF is needed for melanocytic cells only during the first 2 to 3 weeks in culture. After this time, melanocytes of all progression stages are independent of EGF. Nevus cells are less dependent on bFGF and TPA than melanocytes (M. Man-

Table 9. Growth factors and growth factor receptors on m e l a n o m a cells Growth factor

bFGF PDGFA PDGFB NGF TGF-ct TGFI~ IL-1 MGSA/MIF-2 EGF IGF-I/II

Production 0 to + + + a

+++ +++ + 0 +++ ++ ++ +++ 0 +

a + + + , > 1 0 ng/ml; + + , 0.1-1"ng/ml. b N . D . = not determined. c Variable with culture conditions.

% Positive cell fines (approx.)

>90 70 20 60 60 40 100 -

Receptor Expression 0 to + + + +

% Positive cell lines

++++ N.D.b 0 + ++ +~ N.D. + N.D. see TGF-ce +

100 _ 90 30 ~ N.D. N.D. 30~ 100

108 Table 10. Regulatory pathways for growth and invasion in melanoma

Growth factors ~

Proteolytic enzymes

PDGFA PDGFB TGF a TGF~ IL-1 IL-8

Adhesion receptors

uPA Collagenase IV

heparanase and Enzyme inhibitors PAI-I PAI-2 TIMP

~

v

Extracellular matrix proteins

VLA.1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6 VNR ICAM-1

Fibronectin Tenascin Collagens Laminin Proteoglycan

cianti et al., manuscript in preparation). Primary melanoma cells of the early and intermediate groups (see Table 4) require at least IGF-I or insulin [24]. Both factors, IGF-I and insulin, bind to the IGF-I receptor. Primary melanoma cells from the advanced group, as well as metastatic melanoma cells, grow for several months to years in a medium devoid of any exogenous growth factors or other proteins. TPA is inhibitory for melanoma cells. For better attachment, cells are seeded on gelatin as substrate. Gelatin binds fibronectin with high affinity. Melanoma cells express the fibronectin receptor and also secrete fibronectin, allowing a cheap and effective procedure for better attachment in serum- and growth factor-free (proteinfree) media. Metastatic cells grown in protein-free

medium remain responsive to exogenous growth factors including IGF-I/insulin, EGF, and transferrin.

Melanoma cells produce a variety of growth factors, but normal melanocytes do not (Table 9). With the exception of bFGF, all factors are secreted in vitro into the spent medium, bFGF is found in the extracellular matrix. It is produced by 9 out of 10 cell lines. We have also found bFGF in extracts of cultured nevus cells, explaining the decreased need of nevus cells for this growth factor. In melanoma, bFGF is produced for autocrine stimulation because anti-bFGF antibodies, incorporated into cells, inhibit growth [25]. A similar growth inhibitory effect was also achieved with anti-sense deoxynucleotides of bFGF [26].

Table 11. Characteristics of experimentally and spontaneously transformed melanocytes Transforming agent

Soft agar growth

Growth factor independence

Stimulation by phorbol ester

Indefinite life span

Tumorigenicity in nude mice

Adl2/SV40 hybrid virus SV40 T antigen gene MSV (Ha-ras) SV40 T + n-ras Spontaneous (nevus) Spontaneous (VGP melanoma) Spontaneous (metastatic melanoma)

+ +a ++ 0 N.D. ++ ++ ++

++ ++ 0 ++ 0 0 ++

0 0 ++ 0 ++ 0 0

++ ++ 0 ++ 0 ++ ++

0 0 N.D. ++ 0 ++ ++

a + + , yes; 0, no; N.D., not determined.

109 Table 12. Progression of primary VGP melanoma cells by natural selection

Table 13. In vitro characteristics of a melanoma cell line before and after selection for metastasis formation in nude mice

Type

Cell line

Stepwiseselection for

Characteristic

In vivo

WM793

Metastasis in nude mice (s.c. to lungs and lumph nodes)

In vitro

WM 75 WM 793 WM 278 WM 902B WM 115

Invasion of basement membranes

WM 75 WM 793 WM 278 WM 902B

Growth factor independence

WM 75 WM 793

Independence from growth factors and high invasiveness

In vitro

In vitro

Another autocrine growth factor in melanoma has been identified by A. Richmond and colleagues [27]. Melanocyte-growth stimulatory activity (MGSA) is, apparently, identical to macrophageinflammatory factor 2 (MIF-2). MGSA/MIF-2 is produced by all melanoma cells and most nevus cells tested. In normal skin, this factor is also produced by keratinocytes. The role of PDGF in melanoma remains to be clarified. PDGF-A is secreted by most melanoma cells as a homodimer, while PDGF-B is less frequently secreted, if at all. Transforming growth factors (TGF)-ct and TGF-[3 are secreted by approximately 50% to 60% of melanomas, and IL-1 is secreted by approximately 40% of melanoma cells. The roles of PDGFs, TGFs, and IL-1 remain to be clarified. The complex interactions of growth factors, proteolytic enzymes, enzyme inhibitors, extracellular matrix proteins, and adhesion receptors are indicated in Table 10. It is currently impossible to account for all the variables between the four compartments, and only a fraction of the relationships have been investigated, bFGF, for example, stimulates production of plasminogen activators (PAs), whereas TGF-~ stimulates production of PA inhibitors, fibronectin and tenascin, and modulates the expression of integrins.

Growth in serum (Population doublings in h) Growth in protein-free medium (Population doublings in h) Colony formation in soft agar (%) In vitro invasiveness (cells/field) Collagenase type IV (ng/ml) Tissue plasminogen activator (mPu/mg)

Parental cell line WM 164

48

Metastatic variant 451Lu

72

221 2.0 9.2 0.05

94 17 25.0 0.6

8.5

25.1

Experimental progression Normal melanocytes and common acquired nevus cells can be transformed with SV40 T antigen, using either a hybrid virus between SV40 and adenovirus 12 (Ad 12/SV40) or the SV40 T antigen gene [28, 29]. As shown in Table 11, SV40-transformed melanocytes and nevus cells have several characteristics of spontaneously transformed melanocytes, including growth in soft agar, independence from exogenous growth factors, inhibition by TPA, and an infinite lifespan (more than 100 doublings). However, the cells are not tumorigenic in nude mice, in contrast to spontaneously transformed VGP and metastatic cells. If SV40-transformed cells are supertransfected with n-ras, they form tumors after subcutaneous injection (L. Diamond and K. Melber, unpublished). The infection of melanocytes with an amphotrophic murine sarcoma virus increases the expression of HLA-DR and GD3 but does not lead to growth factor independence nor an infinite lifespan. VGP primary melanoma cell variants can be selected from uncloned mass cultures through stepwise procedures that have characteristics of metastatic cells. Table 12 outlines four approaches in vivo and three in vitro that have been used by our laboratory to obtain cell variants with invasive and metastatic properties through natural selection. The WM793 primary melanoma cell line of the early group has been selected through successive

110 intravenous and subcutaneous injections in nude mice to metastasize to the lungs after subcutaneous injection (D. Herlyn et al., unpublished). The same cell line has also been selected to highly invade the basement membrane reconstruct, 'Matrigel' (J. Jambrosic, unpublished), and to grow independently from all exogenous growth factors (R. Kath et al., unpublished). The two procedures, selection of growth factor independence and invasiveness, can also be combined. We have previously shown [30, 31] that melanoma cells from a metastatic lesion can be selected in vitro for invasiveness and can be metastatic in nude mice. In vivo selected melanoma cells that metastasize in nude mice show a number of differences from the parental cells that help to elucidate the various factors involved in metastasis [30, 31]. Metastatic cells isolated and cultured from the lungs of nude mice grow more slowly than parental cells in the presence of serum but more rapidly in protein-free medium, and they also grow with higher colonyforming efficiency in soft agar (Table 13). Metastatic cells have higher activities of tissue plasminogen activator and collagenase. Their invasion of Matrigel can be inhibited with antibodies against tissue plasminogen activator.

Conclusion Melanocytes, nevus cells, and primary and metastatic melanoma cells have distinct characteristics in vitro that allow the delineation of each stage of tumor development and progression. Unfortunately, dysplastic nevus cells and RGP (early/primary melanoma cells) have not been studied in detail because of difficulties in their in vitro growth. Melanocytes from different stages have in vitro properties that resemble their phenotype in situ. Characteristic differences between cultured normal melanocytes and highly malignant metastatic melanoma cells are: 1) limited lifespan for normal melanocytes and non-malignant cells; 2) inability to grow anchorage-independently versus high colonyforming efficiency in soft agar; 3) non-tumorigenicity versus tumorigenicity in athymic nude mice; 4) dependence on exogenous growth factors and

other mitogens versus autonomous growth in protein-flee medium; 5) production of endogenous growth factors for autocrine growth stimulation; 6) expression of melanocyte-associated antigens versus expression of melanoma-associated antigens; and 7) diploid karyotype versus non-random chromosomal abnormalities. The only major distinction found between primary and metastatic melanomas was that only metastatic melanoma cells proliferated continuously in the absence of growth factors or other proteins. However, primary melanoma cells could be clearly distinguished from nevus cells by their growth behavior and growth factor requirements. The availability of cells from sequential steps of tumor progression in the human melanocytic system offers a unique experimental model for the study of malignant transformation. Cultured normal melanocytes can be experimentally transformed by transforming viruses (SV40). Primary melanoma of the vertical growth phase cells can undergo phenotypic changes to cell type(s) resembling metastasis. However, the naturally selected variants are genetically unstable and need constant selective pressure. Several questions remain regarding the molecular mechanisms of melanoma development. Ontogenes or suppressor genes that might possibly be involved in the etiology of melanoma have not been identified, nor have we investigated the regulatory genes that control progression. A multitude of approaches must be taken to define the phenotypic properties of melanocytes from different stages of tumor progression, in order to develop new tools for diagnosis and therapy and to better understand the mechanisms of differentiation of melanoma cells to a cell with a 'normal' phenotype. As nevus and melanoma cells spontaneously differentiate in situ, they may be susceptible to experimental induction of differentiation - even after progression to metastasis.

Acknowledgements These studies were only made possible through the enthusiastic collaboration of my colleagues, D. Herlyn, W.H. Clark, H. Koprowski, D. Guerry,

111

D.E. Elder, P. Nowell, U. Rodeck, J. Jambrosic, M.L. Mancianti, R. Kath, H. Menssen, D. Iliopoulos, A. Linnenbach, and I. Valyi-Nagy. These studies were supported, in part, through grants CA-25874, CA-44877, and CA-47159 from the National Institutes of Health.

13.

14.

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16.

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26.

27.

human melanocytic system. Anticancer Res 9: 865-872, 1989 Herlyn M, Balaban G, Bennicelli J, Guerry D IV, Halaban R, Herlyn D, Elder DE, Maul GG, Steplewski Z, Nowell PC, Clark Jr WH, Koprowski H: Primary melanoma cells of the vertical growth phase: Similaritiesto metastatic cells. J Natl Cancer Inst 74: 283-289, 1985 Kath R, Rodeck U, Parmiter A, Jambrosic J, I-Ierlyn M: Increased growth factor independence of primary melanoma cells from advanced but not early or intermediate lesions• Cancer Therapy and Control, 1990, in press Balaban GB, Herlyn M, Clark Jr WH, Nowell PC: Karyotypic evolution in human malignant melanoma• Cancer Genet Cytogenet 19: 113-122, 1986 Balaban G, Herlyn M, Guerry D IV, Bartolo R, Koprowski H, Clark Jr WH, Nowell PC: Cytogenetics of human malignant melanoma and premalignant lesions• Cancer Genet Cytogenet 11: 429-439, 1984 Herlyn M, Koprowski H: Melanoma antigens: Immunological and biological characterization and clinical significance. Ann Rev Immunol 6: 283-308, 1988 Kath R, Herlyn M: Molecular biology of tumor antigens. Curt Opinion Immunol 1: 863-866, 1989 Holzmann B, Broecker EB, Lehmann JM, Ruites DJ, Sorg C, Riethmueller C, Johnson JP: Tumor progression in human malignant melanoma: Five stages defined by their antigenic phenotypes. Int J Cancer 39: 466-471, 1987 Elder DE, Rodeck U, Thurin J, Cardillo F, Clark Jr WH, Stewart R, Herlyn M: Pigmented lesion-associatedantigens distinguish between benign and malignant melanocytic lesions• Cancer Res 49: 5091-5096, 1989 Houghton AN, Albino AP, Cordon-Cardo C, Davis LJ, Eisinger M: Cell surface antigens of human melanocytes and melanoma: Expression of adenosine deaminase binding protein is extinguished with melanocyte transformation. J Exp Med 167: 197-212, 1988 Herlyn M, Rodeck U, Mancianti ML, Cardillo FM, Lang A, Ross AH, Jambrosic J, Koprowski H: Expression of melanoma-associated antigens in rapidly dividing human melanocytes in culture. Cancer Res 47: 3057-3061, 1987 Hedyn M, Mancianti ML, Jambrosic J, Bolen JB, Koprowski H: Regulatory factors that determine growth and phenotype of normal human melanocytes. Exp Cell Res 179: 322-331, 1988 Rodeck U, Herlyn M, Menssen HD, Furlanetto RW, Koprowski H: Metastatic but not primary melanoma cell lines grow in vitro independently of exogenous growth factors. Int J Cancer 40: 687-690, 1987 Halaban R, Kwon BS, Ghosh S, Delli Bovi P, Baird A: bFGF as an autocrine growth factor for human melanomas. Oncogene Res 3: 177-186, 1988 Becker D, Meier CB, Herlyn M: Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor. EMBO J 8: 3685-3691, 1989 Richmond A, Balentien E, Thomas HG, Flaggs G, Barton

112 DE, Speiss J, Bordoni R, Francke U, Derynck R: Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin. EMBO J 7: 20252031, 1988 28. Jambrosic J, Mancianti ML, Ricciardi RP, Sela BA, Koprowski H, Herlyn M: Transformation of normal human melanocytes and non-malignantnevus cells by adenovirus 12-SV40 hybrid virus. Int J Cancer 44: 1117-1123, 1989 29. Melber K, Zhu G, Diamond L: SV40-transfected human melanocyte sensitivity to growth inhibitionby the phorbolester 12-O-tetradecanoyl-phorbol-13-acetate. Cancer Res 49: 3650-3655, 1989 30. Iliopoulos D, Ernst C, Steplewski Z, Jambrosic JA, Ro-

deck U, Herlyn M, Clark Jr WH, Koprowski H, Herlyn D: Inhibitionof metastasis of a human melanoma xenograft by monocional antibody to the GD2/GD3 gangliosides. J Natl Cancer Inst 81: 440--444, 1989 31. Herlyn D, Iliopoulos D, Jensen PJ, Parmiter A, Baird J, Hotta H, Ross AH, Jambrosic J, Koprowski H, Herlyn M: In vitro properties of human melanoma cells metastatic in nude mice. Cancer Res 50: 2296--2302, 1990

Address for offprints: M. Herlyn, The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, USA