Molecular Analysis of Melanoma Precursor Lesions 1

Atypical (dysplastic) nevi are melanocytic lesions, which are precursors of melanoma as well as markers of increased melanoma risk. Although these lesions exhibit distinct clinical and histological features, their molecular features are largely unknown. To determine whether atypical, compared to benign nevi, from patients with a clinical history of malignant melanoma reveal molecular changes, we analyzed these lesions for the expression of two growth factors (basic fibroblast growth factor and transforming growth factor a), their receptors (fibroblast growth factor receptor-I and epidermal growth factor receptor), and two cell adhesion molecules (MUC18 and avfi3 integrin), all of which are expressed in primary and metastatic melanomas. The results demonstrated a statistically significant correlation (P = 0.02) between increasing degrees of histological atypia and expression of epidermal growth factor receptor in the epidermal keratinocytes of atypical melanocytic lesions. Furthermore, both atypical and benign nevi revealed considerably high levels of overall gene activity in their dermal melanocytic and epidermal keratinocytic compartments. In contrast, the epidermal-dermal junction wherein melanoma evolves showed little gene activity, suggesting that molecular events occurring adjacent to this junction may be important for melanocytic transformation. Introduction Human melanoma represents the end point in a series of progressive stages of melanocyte transformation. In a sigAeceived 5/28/96; revised 8/14/96; accepted 9/24/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 Supported by grants from the NIH and the Cari J. Herzog Foundation (to 0. B.). 2 To whom requests for reprints should be addressed, at Department of Pathology, University of Pittsburgh BST El 050, 21 1 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 624-1047; Fax: (412) 624-7794; E-mail: dbecker+@pitt.edu. nificant number of patients, this transformation process begins with melanocytes of cutaneous nevi designated as atypical (dysplastic) nevi, which can progress to primary melanoma in the radial growth phase to primary melanoma in the vertical growth phase to metastatic melanoma (Refs. 1-3; reviewed in Ref. 4). In the setting of familial melanoma, comprising about 1 0% of the overall incidence of melanoma, the presence of atypical nevi is associated with a nearly 100% risk of developing primary melanoma by age 70 (5, 6). However, atypical novi also occur outside the familial melanoma setting (7, 8), and it is currently estimated that 40-60% of sporadic melanomas develop from these melanocytic precursor lesions. For example, patients with a clinical history of primary melanoma and two or more atypical nevi are at an 8-fold greater risk for developing a second primary melanoma (9). The criteria for identifying and distinguishing atypical from benign nevi are based upon a combination of clinical and histopathological features (Ref. 1 0; reviewed in Ref. 1 1). The most apparent clinical features of atypical nevi are manifest in their size ( 5 mm in diameter), irregular borders, and variegated pigmentation. Their prevalent architectural features are basalar melanocytic hyperplasia and bridging and confluence of the rote ridges. Furthermore, atypical nevi exhibit cytological features of nuclear pleomorphism and hyperchromatism of nuclei and lymphocytic infiltrate of the dermis (11). In contrast, benign nevi are generally less than 4 mm in diameter, displaying regular and defined borders and uniform coloration. Moreover, benign molanocytic lesions do not exhibit architectural and cytological features of atypia. The clinical prognosis for patients with melanoma is directly related to the depth of invasion of the primary lesion at the time of diagnosis. Thus, when recognized early in the biological course of the disease, the patient is often cured by a wide and deep excision of the melanoma. However, once a primary melanoma in the vertical growth phase metastasizes to regional lymph nodes or distant sites, the prognosis is grave because conventional chemotherapy or radiation treatment do not reliably affect the course of the disease. For this reason, it is of great importance to gain an insight into molecular events that govern the early stages of molanocytic atypia, thereby making it possible to implement strategies that will interfere with progression to advanced-stage melanomas. To date, little information is available regarding genes that are activated and expressed in atypical nevi. In contrast, over the past decade, significant information has accumulated regarding molecular features of primary and metastatic melanomas. In particular, these investigations have focused upon genes required for the proliferation and cell adhesion properties of malignant melanomas (reviewed in Ref. 1 2). For Table 1 Clinical and histopathological features of atypical nevi Clinical features of atypical nevi Asymmetry Irregular borders Variegated pigmentation Size 5 mm in diameter Macubar surface component Histopathologicab features of atypical nevi Architectural features Asymmetry Number of junctional nests Melanocytic proliferation along the basal layer Contour of junctional nests Fusion of nests Telangiectasia of papillary dermis Presence of bymphocytic infiltrate Cytological features Cellular pleomorphism Abundance of cytoplasm Enlarged nucleoli Intracellular, dispersed melanin Loss of cellular cohesion within junctional nests Rare mitotic activity Occasional pagetoid spread in center of lesion 3 The abbreviations used are: bFGF, basic fibrobbast growth factor; FGFR, fibroblast growth factor receptor; TGF, transforming growth factor EGFR, epidermal growth factor receptor; EKT, epidermal keratmnocyte; JCT, epidermal-dermab junction; ONV, dermal nevocyte; 5Th, stroma; VAS, vasculature. 4 V. Wang and 0. Becker. Antisense targeting of bFGF/FGFR-i in human melanomas inhibits intratumoral angiogenesis and tumor growth, submitted for publication. 1734 Growth Factors and Adhesion Molecules in Nevi example, bFGF3 and one of its receptors, FGFA-l , are oxpressed in all primary and metastatic molanomas analyzed to date. Their expression is essential for the proliferation of malignant melanomas, both in vitro and in vivo (1 3-1 5)#{149}4 On the other hand, TGF-a and its receptor, EGFR, are expressed in some but not all melanomas. However, previous studios suggested that the expression of TGF-a and EGFR increases with progression to advanced-stage melanoma (1 6, 17). Likewise, MUC1 8, a member of the cell adhesion molecules of the immunoglobulin superfamily (1 8), and the avp3 subunit of integrin (1 9) were reported previously to represent progression markers of primary and metastatic melanomas (18, 20-22). To determine whether activation and expression of any of these six genes would correlate with the onset of melanocytic progression in atypical nevi, we obtained atypical nevi and, as a control, benign nevi from patients with a clinical history of melanoma. By analyzing the epidermal, junctional, dermal, stromal, and vascular compartments of these nevi for the expression of each of these genes, we observed a statistically significant correlation between increasing dogrees of histopathological atypia and expression of EGFR in the EKTs of atypical nevi. Furthermore, atypical as well as benign nevi revealed high levels of gene activity in their EKTs and DNVs, which are clusters of molanocytos. In contrast, overall gene activity in the SIR, VAS, and specifically, the JCT of these melanocytic lesions was low. The latter observation is particularly surprising because melanoma is thought to evolve in the junction. Results Clinical and Histopathological Assessment of Atypical and Benign Nevi. As listed in Table 1 , the established olincal features of atypical nevi are: size 5 mm in diameter, asymmetry, irregular borders, variegated pigmentation, and the presence of a macular surface component (1 1). In contrast, benign nevi are typically smaller than 5 mm in diameter, have regular contours and smooth borders, and exhibit uniform pigmentation. In concordance with those features, we obtained a total of 78 novi from 30 patients with a clinical history of melanoma who had not undergone systemic treatmont for their disease prior to the removal of nevi. In the case of each patient, we removed at least one nevus that exhibited the most pronounced features of clinical atypia and one novus that showed no clinical signs of atypia. Upon excision, each nevus was divided, with one portion sot aside for histopathological analysis, while the other was snap-frozen for subsequent molecular analyses. The degree of architectural and cytological atypia was determined for each nevus specimen by applying a grading scheme on a scale from 0-3 for the histopathological features listed in Table 1. Expression of Growth Factors/Growth Factor ReceptoN and Cell Adhesion Molecules in Atypical and Benign Nevi. To determine whether atypical compared to benign melanocytic lesions express genes demonstrated previously or suggested to play an important role in the proliferation and cell adhesion of advanced-stage melanomas, nevus specimens, representing both categories, were analyzed for the expression of two growth factors (bFGF and TGF-a), their corresponding receptors (FGFR-l and EGFR), and two cell adhesion molecules (MUC18 and avf33 integrin). Given the biological functions of those six molecules in the developmont of advanced-stage melanomas, we analyzed each of the 78 novus specimens for expression of these genes. To probe for expression of TGF-a, EGFR, MUC18, and avIJ3 intogrin, we performed immunohistochemistry with antibodios specific for each of these proteins. To detect oxprossion of bFGF and FGFR-l , we carried out in situ