The Journal of Dermatology Vol. 19: 696-701, 1992
WS
n-s Dysplastic Nevus Syndrome: Melanoma-prone Disease Hidekazu Tsukamoto, Kazuhito Hayashibe and Masamitsu Ichihashi Abstract
Regardless of subsequent clinical courses of patients with dysplastic nevi (DN), substantial evidence supporting DN as one of the melanoma-prone diseases is not yet available, especially in sporadic DN, due to the lack of genetic information other than retrospective studies in clinical observation. This study aimed at the immunohistological characterization of sporadic DN distinct from common nevi (CN) and at the evaluation of the potentiality of sporadic DN for malignant transformation. We considered our results together with previous immunological and epidemiological reports. We noted the following three immunohistological characteristics. I) Proliferating cell nuclear antigen (PCNA), one of the markers for active cell division, could be detected on DN cells in junctional nests of only one among ten DN examined but not on CN cells at all. 2) The altered expression of a-smooth muscle actin (o-Sma), often observed in melanoma cells, could not be detected in DN cells. However, anti-o-Sma monoclonal antibody (MoAb) clearly demonstrated distinctive hypervascularity in the stroma surrounding DN when compared with CN. 3) ME491 antigen, which is known to be expressed mainly in the radial growth phase of melanoma, was detected with similar intensity on both DN and CN. These data indicate that DN has a somewhat higher potentiality than CN for cell division and secretion of some cytokines which can induce hypervascularity in the surrounding stroma, but that DN has not yet undergone the significant phenotypic changes observed in melanoma cells. Further advancements in understanding molecular events in DN cells will be of great benefit in setting DN in the multiple oncogenic spc:;ctrum from pigment cells to melanoma.
Key words: dysplasia; melanoma; a-actin; PCNA; ME491
Introduction
The concept of dysplastic nevus (DN) was first introduced by Clark et al. (I) through clinical observation of melanoma-prone families. Greene et al. (2) confirmed the extremely high risk of hereditary cutaneous melanoma in 401 members of 14 families with an autosomal dominant form. Their report proposed the actuarial probability ofmelanoma developing in family members with DN as 56.0% (±IO.I) from age 20 to age 59 (2). The sporadic form of DN has been estimated by other groups to occur in around 5% of white Americans with no familial history of melanoma (3-5). The prevalence of DN, which was surveyed retrospectively in the cases of sporadic (non-familial) melanoma, was 39% (41/105 cases examined), according to the recent report by Department of Dermatology, KobeUniversity Schoolof Medicine, Kobe,Japan. Reprint requests to: Masamitsu Ichihashi, M.D., Department of Dermatology, Kobe University School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe650,Japan.
Halpern et al. (6). The above results support strongly the significance of sporadic DN as markers of increased risk for non-familial melanoma. A possible dose-response relationship between increasing numbers of DN and melanoma risk is also inferred statistically (7). In addition, histologically contiguous DN features were seen at the lateral sites of primary melanoma (3, 8, 9). These reports indicate that both DN and familial history may act as risk markers for melanoma development (10). The characteristics of pathological features in DN are 1) melanocytic dysplasia, 2) elongated, bridging junctional nests, and 3) stromal responses, such as patchy lymphocytic infiltrate, new blood vessel formation, and perirete fibroplasia (II), which indicate active mobility of junctional nests in DN containing surrounding tissues under the possible stimuli of some cytokine factors. The purpose of this study is to evaluate the clinical significance of sporadic DN by reviewing the reports describing pathological and biological characteristics of DN and also by presenting a few unique biological
Dysplastic Nevus Syndrome
Fig. 1. Histological appearance of dysplastic nevus. Junctional nests are irregularly shaped and extend parallel to the epidermal surface. Stromal responses such as perirete lamellar fibroplasia and patchy lymphoid cell infiltration are also observed (H&E, x200).
697
Fig. 2. ME491 antigen expression in sporadic DN. Not only dysplastic melanocytes within junctional nests but individual atypical melanocytes (arrow) of DN were strongly positive. Intradermal nests of DN also reacted strongly (x200). Macrophages in dennis served as an internal positive control
events that may not be observed in junction and/or compound common nevi, but which may be observed in sporadic DN cells and surrounding stroma, in order to further characterize sporadic DN as a risk factor for cutaneous malignant melanoma. Materials and Methods Tissues: Ten dysplastic nevi and ten common nevi (junction nevus-5, compound nevus-5), surgically removed and histologically confirmed at our department, were used for the present' study: Dysplastic nevus was diagnosed by the following histopathological features: I) lentiginous or epithelioid melanocytic hyperplasia with nuclear atypia and abundant cytoplasm, 2) elongated and interconnected junctional nests, 3) stromal responses consisting of patchy dermal lymphocytic infiltrate, vascular proliferation, and lamellar or amorphous zones of fibroplasia surrounding rete ridges (Fig. 1). All dysplastic nevi examined in this study were sporadic without familial history of melanoma. Primary antibodies: 1) Murine anti-melonoma-associated glycoprotein monoclonal antibody (MoAb), designated ME491, was kindly provide by Dr. Hotta at the Department of Microbiology, Kobe University School of Medicine. ME491 antigen -is an integral membrane glycoprotein detected by the broad 30-60 kD band immunoprecipitated from melanoma cells (12). Tissue macrophages are consistently strongly reactive with ME491. 2) Murine anti-proliferating cell nuclear antigen (PCNA) MoAb was
Fig. 3. PCNA expression in sporadic DN. A few atypical melanocytes (arrow) within junctional nests were positive in one of ten sporadic DN (x200).
obtained commercially from DAKO (Denmark). PCNA is an intranuclear polypeptide which is present particularly in the S-phase of the cell cycle (13). 3) Murine anti-a-smooth muscle actin (o-Sma) MoAb, which is specific for smooth muscle cells, pericytes, and myoepithelial cells (14), was purchased from Sigma Chemical Co. (St. Louis, MO). Immunohistochemical staining: An avidin-biotinperoxidase complex (ABC) technique described by Hsu et aI. (15) was used in this study. Formalin-fixed, paraffin-embedded sections were deparaffinized in
698
Tsukamoto et al Table 1. Characteristic profile of ME491 antigen, PCNA, and hypervascularity in tumor and stromal cells of DN and CN DN
±
+
CN
±
+
ME491 antigen 0 1110 9/10 0 1110 9/10* PCNA 9/10 0 1110 10/10 0 0 Hypervascularity** 0 0 10/10 1110 6/10 3/10 *Junctional nests and A-type nevus cellsstained intensely, while B- and C-type nevus cells stained moderately or weakly in the lower dermis. **Increase (+) or decrease (-) was estimated as compared to adjacent normal sites.
After washing with PBS, the sections were incubated with biotinylated horse antimouse IgG and ABC for 30 min each. The reactions were visualized with 3amino-9-ethyIcarbazole (AEC). The specimens were finally counterstained with hematoxylin for subsequent observations. Results
Fig. 4. o-Sma expression in surrounding stroma of sporadic DN. Anti-o-Sma MoAb-positive round or dilated microvessels (arrowheads) increased at papillary and upper dermis, with a tendency to lie parallel to epidermal surface (A, x200), as compared to lateral normal site in the same specimen (B, x200). xylene, and endogenous peroxidase activity was blocked with 0.3% H~O~ in methanol for 20 min. After washing in phosphate-buffered saline (PBS) and treating with 3% fetal calf serum in PBS for 20 min, the sections were incubated with ME491 at 1:400, anti-PCNA MoAb at 1:20, or anti-a-Sma MoAb at 1:1000 dilution for 60 min at room temperature.
ME491 antigen expression in DN and CN: Not only dysplastic melanocytes within junctional nests but individual atypical melanocytes of DN showed strong positive reactivity with ME491 (Fig. 2). Similar moderate or strong positive reactivity was also observed in junctional nests and dermal A-type nevus cells, whereas the antigen expression decreased in B- and C-type nevus cells along with neural differentiation in the lower dermis, agreeing with the previous report (16). We could not demonstrate a significant difference in positive intensity between DN and CN. The antigen, however, was not expressed by normal melanocytes in lateral normal sites. PCNA expression in DN and CN: Expression of PCNA was detected in a few atypical melanocytes within junctional nests of one of ten DN (Fig. 3), suggesting the proliferative capability ofDN cells, as seen more frequently in the plaque-phase of superficial spreading melanoma (SSM) and acral lentiginous melanoma (ALM). None of CN, however, reacted positively with anti-PCNA MoAb. Anti-PCNA MoAb-positive basal cells were observed as an internal positive control. a-Sma expression in tumor and surrounding stroma of DN and CN: The altered expression of o-Sma, as previously reported in melanoma cells, could not be detected in DN cells themselves. However, we could find a distinctively increased number of anti-n-Sma MoAb-positive round or dilated microvessels in the papillary and upper dermis in all cases of DN
Dysplastic Nevus Syndrome
Table 2. MoAbs
Immunogen or defined antigen
699
Profiles of MoAbs to pigment cell-associated antigens Molecular weight
HMSA-2 NK1C3 2-139-1 6-26-3 PAL-M, PAL-M. 2G5 ME492
Melanosome 53kD Melanoma cell membrane ? Metastatic melanoma ? Metastatic melanoma ? Metastatic melanoma 95-1ookD Metastatic melanoma ? EGF-R ? Melanoma-associated 30-60kD glycoprotein (MAG) RASK-3,4 Ki-, H-, Ha-ras21 12kD A-1-43 GP130, Integrin VlA2 130kD TNKH-1,2 Human melanoma (treated ? with n-butyric acid)
Location of binding CP CP CP CP CP (diffuse) CP CM CP CM CM? CP&CM
Reference
Isotype
IgGl (k) Maeda K et al, 1987 Mackie RM et al, 1984 IgGl Imam A et al, 1986 IgGl IgG2 " IgGl Ruiter D] et al, 1985 IgGl " ? Moretti S et al, 1989 IgGl Atkinson B et al, 1984 ? IgGl IgG2
Shiku H, 1987 Briiggen] & Sorg C, 1983 Nakanishi T & Hashimoto K, 1987
CP: cytoplasm, CM: cell membrane Table 3. MoAbs HMSA-2 NK1C3 2-139-1 6-26-3 PAL-M, PAL-M. 2G5 ME492 RASK-3,4 A-I-43 TNKH-1,2
Normal MC
+
Immunohistochemical reactivity of DN cells with MoAbs CN D ]
- -»+ + -
±
-
±
±
+
?
--+ (6%)
**
DN (sporadic)
+-1t (89%) + ? ?
--+ (25%) --+ (15%) ? ? --± ? ?
DN (familial) ND*
+ --1t --+ --+ (27%) +
* ?
(33%)
--+ (31%) +
SSM (& other MM)
* ** +
(100%)
*-* *-* ** (-±)**
*
+ (53-87%) ±--
MC: melanocyte, CN: common nevus, D: dermal,]:junction, DN: dysplastic nevus, SSM: superficial spreading melanoma, MM: malignant melanoma, *ND: not done, **Focally strong or weak in metastatic melanoma
studied (Fig. 4A), as compared to adjacent lateral normal sites (Fig. 4B). Alternatively, most CN possessed an equivalent or decreased number of anti-aSma MoAb-positive vessels. Discussion
Clinical evaluation of the sporadic form of DN as a melanoma risk marker is controversial. There are two kinds of reports, one supporting sporadic DN as a risk factor for melanoma development (6, 8) and the other criticizing this theory (17). It is expected that different new antigens may be involved in different steps of tumor progression, even in the precancerous cells. A large number of monoclonal .antibodies (MoAbs) against various pigment cellaSSociated antigens have been established (Table 2).
Their binding characteristics to normal epidermal melanocytes, CN, DN and malignant melanoma at different progressive stages have been studied extensively to clarify the clinical importance of sporadic and familial DN as a high risk marker for malignant melanoma (Table 3) and also to distinguish immunohistopathologically sporadic DN from CN and malignant melanoma. These MoAbs are categorized into 2 classes as follows: 1) The first group reacts positively with malignant melanoma, but negatively or less positively with benign CN and epidermal normal melanocytes. MoAb HMSA-2, raised against the melanosomal component of human melanoma, has been shown to be a useful tool in distinguishing immunohistochemically sporadic DN from CN in-
700
Tsukamoto et al
eluding junctional melanocytic nevi (18, 19). In addition, MoAb HMSA-2 may discriminate DN from normal melanocytes and CN by the demonstration of the altered melanosomes. MoAbs 2-139-1, 6-26-3, 2G5, ME492, RASK-3,4 and A-I-43 have spectra of reactivity with DN similar to that of MoAb HMSA-2 (20-24). 2) The second group, represented by TNKH-I (25), reacts positively with CN, but negatively with malignant melanoma cells. In the present paper, we selected three MoAbs directed at different cellular biological activities. I) Human melanoma-associated ME491 antigen is speculated to be a cell surface receptor acting as a tumor suppressor factor (26). 2) Proliferating cell nuclear antigen is a marker of cell proliferation. 3) a-smooth muscle actin is related to tumor and stromal morphological changes and might detect another biological characteristic of sporadic DN different from CN. Sporadic DN and CN had similar positive reactivity with ME 491, suggesting that ME491 antigen might be retained as a tumor suppressor in both sporadic DN and CN, since the antigen gradually diminished, or sometimes disappeared, in metastatic lesions along with malignant transformation. In understanding proliferative features of DN, genetical instability was reported in DN as precursor lesions, although mitotic figures are usually rare (8). Recently, an abnormal gene for susceptibility to melanoma has been identified in chromosome 1 (lp36) of familial DN (27,28). In the sporadic form of DN, partial deletion of chromosome 11 has been demonstrated by Mcdonagh et al. (29). The presence of ras mutations in benign atypical nevi may suggest the occurrence of oncogene activation in a rather early stage of melanoma development, together with the frequent presence of DNA aneuploidy in DN (30, 31). We confirmed the prominent capillary proliferation and dilatation in the papillary and upper dermis of DN in comparison with CN by staining with anti-o-Sma MoAb, which is capable of reacting with myofibroblasts and/or proliferative pericytes as the stromal response to neoplasia (32, 33). Further, application of anti-o-Sma MoAb clearly showed that most of the papillary and upper dermal vessels of DN ran parallel to the epidermal surface. Recently, the increases of bFGF were shown to be correlated with enhanced angiogenesis and maintenance ofincreased a-Sma-positive microvessels by analyzing the proliferative characteristics of angiofibroma of tuberous sclerosis (34). Increased bFGF may stimulate fibroblasts to synthesize collagen to
form perirete fibroplasia, one of the typical characteristics ofDN architecture, although an increase of bFGF in DN has not yet been shown. Further, some factor like bFGF, which stimulates proliferation of normal melanocytes, may be responsible for the positive reactivity of DN cells with anti-PCNA MoAb, if DN cells express a higher population of bFGF receptors than CN. References 1) Clark WHJr, Reimer RR, Greene M, Ainsworth AM, Mastrangelo MJ: Origin of familial malignant melanomas from heritable melanocytic lesions: 'the B-KMole syndrome', ArchDermatol, 114: 732-738, 1978. 2) Greene M, Clark WH Jr, Tucker MA, Kraemer KH, Elder DE, Fraser MC: High risk of malignant melanoma in melanoma-prone families with dysplastic nevi, Ann Intern Med, 102: 458-465,1985. 3) Rhodes AR, Harrist 1), Day CL, Mihm MC Jr, Fitzpatrick TB, Sober AJ: Dysplastic melanocytic nevi in histologic association with 234 primary cutaneous melanomas,] Am &ad Dermatol, 9: 563-574, 1983. 4) Crutcher WA, Sagebiel RW: Prevalence of dysplastic nevi in a community practice, Lancet, 1: 729, 1984. 5) SchneiderJS, Sagebiel RW, Moore DH, et a1: Melanoma surveillance and earlier diagnosis, Lancet, 1: 1435, 1987. 6) Halpern AC, Guerry D IV, Elder DE, et a1: Dysplastic nevi as risk markers of sporadic (Nonfamilial) melanoma: A case-control study, Arch Dermatol, 127: 995-999,1991. 7) Holly EA, KellyJW, Shpall SN, Chiu S-H: Number of melanocytic nevi as a major risk factor for malignant melanoma,] Am Acad Dermatol, 17: 459-468, 1987. 8) Elder DE, Goldman LI, Goldman SC, Greene MH, Clark WH Jr: Dysplastic nevus syndrome: a phenotypic association of sporadic cutaneous melanoma, Cancer, 46: 1787-1794,1980. 9) Duray PH, ErnstoffMS: Dysplastic nevus in histologic contiguity with acquired nonfamilial melanoma; clinicopathologic experience in a 100-bed hospital, ArchDermatol, 123: 80-84, 1987. 10) Sagebiel RW: The dysplastic melanocytic nevus,] Am Acad Dermatol, 20: 496-501, 1989. 11) Rhodes AR, MelskiJW, Sober AJ, Harrist TJ, Mihm MC, Fitzpatrick TB: Increased intraepidermal melanocyte frequency and size in dysplastic melanoeyrie nevi and cutaneous melanoma. A comparative quantitative study of dysplastic melanocytic nevi, superficial spreading melanoma, nevocellular nevi, and solar lentigines,] Invest Dermatol, 80: 452-459, 1983. 12) Atkinson B, Ernst CS, Ghrist BFD, et al: Identification of melanoma-associated antigens using fixed tissue screening of antibodies, Cancer Res, 44: 2577-2581,
Dysplastic Nevus Syndrome 1984. 13) Hall PA, Levison DA, Woods AL, et al: Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms,] PalJwl, 162: 285-294, 1990. 14) Skalli 0, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G: A monoclonal antibody against a-smooth muscle actin: A new probe for smooth muscle differentiation,] Cell Biol, 103: 27872796,1986. 15) Hsu SM, Raine 1., Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures,] Histochem Cywchem, 29: 577-580, 1981. 16) Atkinson B, Ernst CS, Ghrist BID, et al: Monoclonal antibody to a highly glycosylated protein reacts in fixed tissue with melanoma and other tumors, Hybridoma, 4: 243-255, 1985. 17) Grob lJ, Andrae 1., Romano MH, et al: Dysplastic naevus in non-familial melanoma: A clinicopathological study oflOl cases, Br] Dermatol, 118:745-752, 1988. 18) Maeda K, Maeda K, Jimbow K: Positive reactivity of dysplastic melanocytes with a monoclonal antibody against melanoma melanosomes, MoAb HMSA-2,] Invest Dermatol, 91: 247-250, 1988. 19) Jimbow K, Horikoshi T, Takahashi H, Akutsu Y, Maeda K: Fine structural and immunohistochemical properties of dysplastic melanocytic nevi: Comparison with malignant melanoma,] InvestDermatol, 92: 3048-309S,1989. 20) Imam A, Mitchell MS, Modlin RL, Taylar CR, Kempf RA, Kan-MitchellJ: Human monoclonal antibodies that distinguish cutaneous malignant melanomas from benign nevi in fixed tissue sections, J Invest Dermatol, 86: 145-148,1986. 21) Moretti S, Lammers PI, Kwaspen FH1., et al: Epidermal growth factor receptor expression is associated with tumor progression in human melanocytic lesions,] Invest lJemuJtol, 92: 485, 1989. 22) Atkinson B, Ernst CS, Ghrist BFB, et al: Monoclonal antibody to a highly glycosylated protein reacts in fixed tissue with melanoma and other tumors, Hybridoma, 4: 243-255, 1985. 23) Yamamura K, Mishima Y: Antigen dynamics in melanocytic and nevocytic melanoma oncogenesis: anti-ganglioside and anti-ras p21 antibodies as
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markers of tumor progression,] Invest Dermatol, 94: 174-182,1990. 24) Holzmann B, Brocker EB, Lehmann JM, et al: Tumor progression in human malignant melanoma: five stages defined by their antigenic phenotypes, Int ] Cancer, 39: 466-471, 1987. 25) Nakanishi T, Hashimoto K: The differential reactivity of benign and malignant nevomelanocytic lesions with mouse monoclonal antibody TNKHl, Cancer, 59: 1340-1344, 1987. 26) Hotta M, Hara I, Miyamoto H, Homma M: Overexpression of the human melanoma-associated antigen ME491 partially suppresses in vivo malignant phenotypes of H-ras-transfonned NIH3T3 cells in athymic nude mice, Melanoma Res, 1: 125-132, 1991. 27) Bale SJ, Dracopoli NC, Tucker MA, et al: Mapping the gene for hereditary cutaneous malignant melanoma-dysplastic nevus to chromosome Ip, N Engl] Med, 320: 1367-1372, 1989. 28) Cannon-Albright lA, Godgar DE, Wright EC, et al: Evidence against the reported linkage of the cutaneous melanoma-dysplastic nevus syndrome locus to chromosome Ip36, Am] Hum Genet, 46: 912-918, 1990. 29) Mcdonagh J\JG, Wright AL, Messenger AG: Dysplastic naevi in association with partial deletion of chromosome 11, Clin Exp lJemuJtol, 15: 44-45, 1990. 30) Shukla VI{, Hughes DC, Hughes LE, McConnick F, Padua RA: ras Mutations in human melanotic lesions: K-ras activation is a frequent and early event in melanoma development, Oncogene Res, 5: 121-127, 1989. 31) Newton JA, Camplejohn RS, McGibbon DH: The flow cytometry of melanocytic skin lesions, Br ] Cancer, 58: 606-609, 1988. 32) Skalli 0, Schiirch W, Seemayer T, et al: Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins, Lab Invest, 60: 275285,1989. 33) Tsukamoto H, Mishima Y, Hayashibe K, Sasase A: o-Smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors,] Invest Dermatol, 98: 116-120, 1992. 34) Takanashi M, NakayamaJ, Inoue M, Urabe A, Hori Y: Increase in a-actin and basic fibroblast growth factor in angiofibromas of patients with tuberous sclerosis, ]pn] Dermatol, 101: 601-608, 1991. (in Japanese)
WS
n-s Dysplastic Nevus Syndrome: Melanoma-prone Disease Hidekazu Tsukamoto, Kazuhito Hayashibe and Masamitsu Ichihashi Abstract
Regardless of subsequent clinical courses of patients with dysplastic nevi (DN), substantial evidence supporting DN as one of the melanoma-prone diseases is not yet available, especially in sporadic DN, due to the lack of genetic information other than retrospective studies in clinical observation. This study aimed at the immunohistological characterization of sporadic DN distinct from common nevi (CN) and at the evaluation of the potentiality of sporadic DN for malignant transformation. We considered our results together with previous immunological and epidemiological reports. We noted the following three immunohistological characteristics. I) Proliferating cell nuclear antigen (PCNA), one of the markers for active cell division, could be detected on DN cells in junctional nests of only one among ten DN examined but not on CN cells at all. 2) The altered expression of a-smooth muscle actin (o-Sma), often observed in melanoma cells, could not be detected in DN cells. However, anti-o-Sma monoclonal antibody (MoAb) clearly demonstrated distinctive hypervascularity in the stroma surrounding DN when compared with CN. 3) ME491 antigen, which is known to be expressed mainly in the radial growth phase of melanoma, was detected with similar intensity on both DN and CN. These data indicate that DN has a somewhat higher potentiality than CN for cell division and secretion of some cytokines which can induce hypervascularity in the surrounding stroma, but that DN has not yet undergone the significant phenotypic changes observed in melanoma cells. Further advancements in understanding molecular events in DN cells will be of great benefit in setting DN in the multiple oncogenic spc:;ctrum from pigment cells to melanoma.
Key words: dysplasia; melanoma; a-actin; PCNA; ME491
Introduction
The concept of dysplastic nevus (DN) was first introduced by Clark et al. (I) through clinical observation of melanoma-prone families. Greene et al. (2) confirmed the extremely high risk of hereditary cutaneous melanoma in 401 members of 14 families with an autosomal dominant form. Their report proposed the actuarial probability ofmelanoma developing in family members with DN as 56.0% (±IO.I) from age 20 to age 59 (2). The sporadic form of DN has been estimated by other groups to occur in around 5% of white Americans with no familial history of melanoma (3-5). The prevalence of DN, which was surveyed retrospectively in the cases of sporadic (non-familial) melanoma, was 39% (41/105 cases examined), according to the recent report by Department of Dermatology, KobeUniversity Schoolof Medicine, Kobe,Japan. Reprint requests to: Masamitsu Ichihashi, M.D., Department of Dermatology, Kobe University School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe650,Japan.
Halpern et al. (6). The above results support strongly the significance of sporadic DN as markers of increased risk for non-familial melanoma. A possible dose-response relationship between increasing numbers of DN and melanoma risk is also inferred statistically (7). In addition, histologically contiguous DN features were seen at the lateral sites of primary melanoma (3, 8, 9). These reports indicate that both DN and familial history may act as risk markers for melanoma development (10). The characteristics of pathological features in DN are 1) melanocytic dysplasia, 2) elongated, bridging junctional nests, and 3) stromal responses, such as patchy lymphocytic infiltrate, new blood vessel formation, and perirete fibroplasia (II), which indicate active mobility of junctional nests in DN containing surrounding tissues under the possible stimuli of some cytokine factors. The purpose of this study is to evaluate the clinical significance of sporadic DN by reviewing the reports describing pathological and biological characteristics of DN and also by presenting a few unique biological
Dysplastic Nevus Syndrome
Fig. 1. Histological appearance of dysplastic nevus. Junctional nests are irregularly shaped and extend parallel to the epidermal surface. Stromal responses such as perirete lamellar fibroplasia and patchy lymphoid cell infiltration are also observed (H&E, x200).
697
Fig. 2. ME491 antigen expression in sporadic DN. Not only dysplastic melanocytes within junctional nests but individual atypical melanocytes (arrow) of DN were strongly positive. Intradermal nests of DN also reacted strongly (x200). Macrophages in dennis served as an internal positive control
events that may not be observed in junction and/or compound common nevi, but which may be observed in sporadic DN cells and surrounding stroma, in order to further characterize sporadic DN as a risk factor for cutaneous malignant melanoma. Materials and Methods Tissues: Ten dysplastic nevi and ten common nevi (junction nevus-5, compound nevus-5), surgically removed and histologically confirmed at our department, were used for the present' study: Dysplastic nevus was diagnosed by the following histopathological features: I) lentiginous or epithelioid melanocytic hyperplasia with nuclear atypia and abundant cytoplasm, 2) elongated and interconnected junctional nests, 3) stromal responses consisting of patchy dermal lymphocytic infiltrate, vascular proliferation, and lamellar or amorphous zones of fibroplasia surrounding rete ridges (Fig. 1). All dysplastic nevi examined in this study were sporadic without familial history of melanoma. Primary antibodies: 1) Murine anti-melonoma-associated glycoprotein monoclonal antibody (MoAb), designated ME491, was kindly provide by Dr. Hotta at the Department of Microbiology, Kobe University School of Medicine. ME491 antigen -is an integral membrane glycoprotein detected by the broad 30-60 kD band immunoprecipitated from melanoma cells (12). Tissue macrophages are consistently strongly reactive with ME491. 2) Murine anti-proliferating cell nuclear antigen (PCNA) MoAb was
Fig. 3. PCNA expression in sporadic DN. A few atypical melanocytes (arrow) within junctional nests were positive in one of ten sporadic DN (x200).
obtained commercially from DAKO (Denmark). PCNA is an intranuclear polypeptide which is present particularly in the S-phase of the cell cycle (13). 3) Murine anti-a-smooth muscle actin (o-Sma) MoAb, which is specific for smooth muscle cells, pericytes, and myoepithelial cells (14), was purchased from Sigma Chemical Co. (St. Louis, MO). Immunohistochemical staining: An avidin-biotinperoxidase complex (ABC) technique described by Hsu et aI. (15) was used in this study. Formalin-fixed, paraffin-embedded sections were deparaffinized in
698
Tsukamoto et al Table 1. Characteristic profile of ME491 antigen, PCNA, and hypervascularity in tumor and stromal cells of DN and CN DN
±
+
CN
±
+
ME491 antigen 0 1110 9/10 0 1110 9/10* PCNA 9/10 0 1110 10/10 0 0 Hypervascularity** 0 0 10/10 1110 6/10 3/10 *Junctional nests and A-type nevus cellsstained intensely, while B- and C-type nevus cells stained moderately or weakly in the lower dermis. **Increase (+) or decrease (-) was estimated as compared to adjacent normal sites.
After washing with PBS, the sections were incubated with biotinylated horse antimouse IgG and ABC for 30 min each. The reactions were visualized with 3amino-9-ethyIcarbazole (AEC). The specimens were finally counterstained with hematoxylin for subsequent observations. Results
Fig. 4. o-Sma expression in surrounding stroma of sporadic DN. Anti-o-Sma MoAb-positive round or dilated microvessels (arrowheads) increased at papillary and upper dermis, with a tendency to lie parallel to epidermal surface (A, x200), as compared to lateral normal site in the same specimen (B, x200). xylene, and endogenous peroxidase activity was blocked with 0.3% H~O~ in methanol for 20 min. After washing in phosphate-buffered saline (PBS) and treating with 3% fetal calf serum in PBS for 20 min, the sections were incubated with ME491 at 1:400, anti-PCNA MoAb at 1:20, or anti-a-Sma MoAb at 1:1000 dilution for 60 min at room temperature.
ME491 antigen expression in DN and CN: Not only dysplastic melanocytes within junctional nests but individual atypical melanocytes of DN showed strong positive reactivity with ME491 (Fig. 2). Similar moderate or strong positive reactivity was also observed in junctional nests and dermal A-type nevus cells, whereas the antigen expression decreased in B- and C-type nevus cells along with neural differentiation in the lower dermis, agreeing with the previous report (16). We could not demonstrate a significant difference in positive intensity between DN and CN. The antigen, however, was not expressed by normal melanocytes in lateral normal sites. PCNA expression in DN and CN: Expression of PCNA was detected in a few atypical melanocytes within junctional nests of one of ten DN (Fig. 3), suggesting the proliferative capability ofDN cells, as seen more frequently in the plaque-phase of superficial spreading melanoma (SSM) and acral lentiginous melanoma (ALM). None of CN, however, reacted positively with anti-PCNA MoAb. Anti-PCNA MoAb-positive basal cells were observed as an internal positive control. a-Sma expression in tumor and surrounding stroma of DN and CN: The altered expression of o-Sma, as previously reported in melanoma cells, could not be detected in DN cells themselves. However, we could find a distinctively increased number of anti-n-Sma MoAb-positive round or dilated microvessels in the papillary and upper dermis in all cases of DN
Dysplastic Nevus Syndrome
Table 2. MoAbs
Immunogen or defined antigen
699
Profiles of MoAbs to pigment cell-associated antigens Molecular weight
HMSA-2 NK1C3 2-139-1 6-26-3 PAL-M, PAL-M. 2G5 ME492
Melanosome 53kD Melanoma cell membrane ? Metastatic melanoma ? Metastatic melanoma ? Metastatic melanoma 95-1ookD Metastatic melanoma ? EGF-R ? Melanoma-associated 30-60kD glycoprotein (MAG) RASK-3,4 Ki-, H-, Ha-ras21 12kD A-1-43 GP130, Integrin VlA2 130kD TNKH-1,2 Human melanoma (treated ? with n-butyric acid)
Location of binding CP CP CP CP CP (diffuse) CP CM CP CM CM? CP&CM
Reference
Isotype
IgGl (k) Maeda K et al, 1987 Mackie RM et al, 1984 IgGl Imam A et al, 1986 IgGl IgG2 " IgGl Ruiter D] et al, 1985 IgGl " ? Moretti S et al, 1989 IgGl Atkinson B et al, 1984 ? IgGl IgG2
Shiku H, 1987 Briiggen] & Sorg C, 1983 Nakanishi T & Hashimoto K, 1987
CP: cytoplasm, CM: cell membrane Table 3. MoAbs HMSA-2 NK1C3 2-139-1 6-26-3 PAL-M, PAL-M. 2G5 ME492 RASK-3,4 A-I-43 TNKH-1,2
Normal MC
+
Immunohistochemical reactivity of DN cells with MoAbs CN D ]
- -»+ + -
±
-
±
±
+
?
--+ (6%)
**
DN (sporadic)
+-1t (89%) + ? ?
--+ (25%) --+ (15%) ? ? --± ? ?
DN (familial) ND*
+ --1t --+ --+ (27%) +
* ?
(33%)
--+ (31%) +
SSM (& other MM)
* ** +
(100%)
*-* *-* ** (-±)**
*
+ (53-87%) ±--
MC: melanocyte, CN: common nevus, D: dermal,]:junction, DN: dysplastic nevus, SSM: superficial spreading melanoma, MM: malignant melanoma, *ND: not done, **Focally strong or weak in metastatic melanoma
studied (Fig. 4A), as compared to adjacent lateral normal sites (Fig. 4B). Alternatively, most CN possessed an equivalent or decreased number of anti-aSma MoAb-positive vessels. Discussion
Clinical evaluation of the sporadic form of DN as a melanoma risk marker is controversial. There are two kinds of reports, one supporting sporadic DN as a risk factor for melanoma development (6, 8) and the other criticizing this theory (17). It is expected that different new antigens may be involved in different steps of tumor progression, even in the precancerous cells. A large number of monoclonal .antibodies (MoAbs) against various pigment cellaSSociated antigens have been established (Table 2).
Their binding characteristics to normal epidermal melanocytes, CN, DN and malignant melanoma at different progressive stages have been studied extensively to clarify the clinical importance of sporadic and familial DN as a high risk marker for malignant melanoma (Table 3) and also to distinguish immunohistopathologically sporadic DN from CN and malignant melanoma. These MoAbs are categorized into 2 classes as follows: 1) The first group reacts positively with malignant melanoma, but negatively or less positively with benign CN and epidermal normal melanocytes. MoAb HMSA-2, raised against the melanosomal component of human melanoma, has been shown to be a useful tool in distinguishing immunohistochemically sporadic DN from CN in-
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eluding junctional melanocytic nevi (18, 19). In addition, MoAb HMSA-2 may discriminate DN from normal melanocytes and CN by the demonstration of the altered melanosomes. MoAbs 2-139-1, 6-26-3, 2G5, ME492, RASK-3,4 and A-I-43 have spectra of reactivity with DN similar to that of MoAb HMSA-2 (20-24). 2) The second group, represented by TNKH-I (25), reacts positively with CN, but negatively with malignant melanoma cells. In the present paper, we selected three MoAbs directed at different cellular biological activities. I) Human melanoma-associated ME491 antigen is speculated to be a cell surface receptor acting as a tumor suppressor factor (26). 2) Proliferating cell nuclear antigen is a marker of cell proliferation. 3) a-smooth muscle actin is related to tumor and stromal morphological changes and might detect another biological characteristic of sporadic DN different from CN. Sporadic DN and CN had similar positive reactivity with ME 491, suggesting that ME491 antigen might be retained as a tumor suppressor in both sporadic DN and CN, since the antigen gradually diminished, or sometimes disappeared, in metastatic lesions along with malignant transformation. In understanding proliferative features of DN, genetical instability was reported in DN as precursor lesions, although mitotic figures are usually rare (8). Recently, an abnormal gene for susceptibility to melanoma has been identified in chromosome 1 (lp36) of familial DN (27,28). In the sporadic form of DN, partial deletion of chromosome 11 has been demonstrated by Mcdonagh et al. (29). The presence of ras mutations in benign atypical nevi may suggest the occurrence of oncogene activation in a rather early stage of melanoma development, together with the frequent presence of DNA aneuploidy in DN (30, 31). We confirmed the prominent capillary proliferation and dilatation in the papillary and upper dermis of DN in comparison with CN by staining with anti-o-Sma MoAb, which is capable of reacting with myofibroblasts and/or proliferative pericytes as the stromal response to neoplasia (32, 33). Further, application of anti-o-Sma MoAb clearly showed that most of the papillary and upper dermal vessels of DN ran parallel to the epidermal surface. Recently, the increases of bFGF were shown to be correlated with enhanced angiogenesis and maintenance ofincreased a-Sma-positive microvessels by analyzing the proliferative characteristics of angiofibroma of tuberous sclerosis (34). Increased bFGF may stimulate fibroblasts to synthesize collagen to
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