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Lentigo maligna and superficial spreading melanoma are different in their in situ phase: An immunohistochemical study
Lentigo Maligna and Superficial Spreading Melanoma Are Different in Their In Situ Phase: An Immunohistochemical Study SILVIU AUSLENDER, MD, AVIV BARZILAI, MD, MSC, IRIS GOLDBERG, PHD, JURI KOPOLOVIC, MD, AND HENRI TRAU, MD Clinical and pathologic observations have prompted the categorization of malignant melanoma into 4 subtypes. Although some authorities challenge the value of this classification, nevertheless it is generally accepted that lentigo maligna (LM), or melanoma on sundamaged skin, has a different biological behavior than so-called superficial spreading melanoma (SSM), at least in the early stage of its evolution. To characterize some aspects of this different behavior, the in situ phase of SSM and LM was studied using immunohistochemical methods. Seventeen cases of SSM in situ and 13 cases of LM were chosen for the study. All cases qualified with strict histologic criteria. Sections from these lesions were stained with antibodies against HMB-45 antigen, basic fibroblast growth factor (bFGF), proliferating cell nuclear antigen (PCNA), and factor VIII. Semiquantitative analysis was performed. Cases classified as either LM or SSM corresponded well to the epidemiologic and clinical characteristics as described in the literature; that is, LM appeared in older patients and occurred mostly on the face, whereas SSM occurred mostly on the trunk and lower limbs. Although no difference in HMB-45 stain was observed, melanoctyes of SSM showed greater proliferative activity,
as reflected by PCNA stain (P < 0.02) and higher levels of bFGF (P < 0.001), than melanocytes of LM. More blood vessels were counted under SSM than under LM (P < 0.05). These results are in accordance with the biological behavior of SSM and LM, that is, the longer in situ phase of the latter. bFGF is both a growth factor for melanocytes and an angiogentic factor. The differences in PCNA, a proliferation marker, and blood vessel count may be related to the bFGF effect. Thus this study reveals some of the biological differences between LM and SSM. Location and sun exposure habits may contribute to these differences, which already exist in the in situ phase. HUM PATHOL 33:1001-1005. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: malignant melanoma, lentigo maligna, superficial spreading melanoma. Abbrevations: LM, lentigo maligna; SSM, superficial spreading melanoma; MIS, melanoma in situ; MM, malignant melanoma; HMB45, human melanoma black 45; PCNA, proliferating cell nuclear antigen; bFBF, basic fibroblast growth factor; EMA, epithelial membrane antigen.
Traditionally, 4 subtypes of malignant melanoma (MM) are recognized according to their clinical characteristics and histologic features.1,2 Among these, superficial spreading melanoma (SSM) is the most common subtype and lentigo maligna (LM) is the rarest.2 Epidemiologic, clinical, histologic, ultrastructural, and probably also etiologic differences exist between the in situ phases of these 2 subtypes.1,2 LM usually appears in older patients (usually in the seventh decade) and on sun exposed areas, mainly the face. When diagnosed, it has a larger diameter and a radial growth phase lasting about 3 to 15 years. In contrast, SSM appears in younger patients (usually in the fourth or fifth decades), occurs in any area, has a smaller diameter at the time of diagnosis, and has a shorter radial growth phase (1 to 7 years). Although distinguishing between LM and SSM in situ is not always possible, and some authors claim that criteria for this are not reputable,3,4 most textbooks on dermatology and dermatopathology1,2 quote such criteria. Accordingly, LM is characterized by
proliferation of pleomorphic atypical melanocytes along the basal layer and the adnexa, whereas SSM is marked by proliferation of atypical melanocytes along the basal layer with pagetoid spread of atypical melanocytes in the epidermis. An atrophic epidermis, probably a refection of sun damage, is usually seen in LM. Further evidence to support the distinction came from an epidemiologic study showing that the relative risk of SSM is almost exclusively determined by nevi count, whereas that of LM is determined mostly by skin type (type I) and to a lesser extent by history of sun exposure.5 Numerous studies have shown that no difference exists between the subtypes in the invasive phase and that prognosis is mainly determined by the depth of invasion.6 Even so, many clinicians and dermatopathologists agree that determining the melanoma subtype is of some importance. Nevertheless, Ackerman et al3,4 suggested that only 1 type of melanoma exists and that the differences found in the intraepidermal phase are related solely to the location, sun exposure, and heterogeneity of tumors. Many studies have examined various biological factors during different stages of melanoma evolution. These factors included proliferation,7,8 angiogenesis around tumors,9-13 expression of specific markers of melanocytes,14-16 adhesion molecules,17 and oncogenes,18 as well as growth factors.19-21 However, most of these studies grouped together the various in situ melanomas. Therefore, the aim of our study was to compare some characteristics of LM and SSM in situ using
From the Department of Dermatology and Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel and the Sackler Faculty of Medicine, Tel-Aviv University, Israel. Accepted for publication January 18, 2002. The first 2 authors are equal contributors. Address correspondence and reprint requests to Henri Trau, MD, Chairman, Department of Dermatology, Sheba Medical Center, 52621 Tel-Hashomer, Israel. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3310-0008$35.00/0 doi:10.1053/hupa.2002.124014
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TABLE 1. Criteria for Classification of LM and SSM
Epidermis Dermis Melanocyte proliferation Melanocyte distribution
TABLE 3. Clinical and Histologic Data of the Study Cases
LM
SSM
Atrophic Elastotic changes Basal pleomorphic atypical
Normal Normal Monomorphic atypical, singly or in nests Pagetoid spread
Infiltrating adnexa
immunohistochemical stains and to correlate the findings with their different clinical behaviour.
Number of cases Age (years) median (range) average* Location† n(%) head trunk upper limbs lower limbs
All of the cases diagnosed as MM in situ between 1989 and 1995 were retrieved from the archive of the Institute of Pathology at the Sheba Medical Center. The original hematoxylin & eosin–stained sections were reviewed, and cases were classified as LM or SSM in situ according to the stringent criteria, summarized in Table 1. In brief, these criteria included epidermal changes (atrophic vs. normal), absence or presence of solar elastosis, and the proliferation pattern of the melanocytes and their cytologic features. Only those cases that met all criteria of either LM or SSM, as agreed on by the 3 dermatopathologists/pathologists (A.B., J.K., and H.T.) were included in the study. Cases that showed equivocal criteria, or those for which there was disagreement among the authors, were excluded. Finally, 30 cases of melanoma in situ (MIS) (out of the 56 reviewed) were chosen for the study. Thirteen cases were classified as LM and 17 as SSM. For each case, the age of the patient and location of the lesion were retrieved from the files. Four m-thick sections from the paraffin-embedded, formalin-fixed blocks, which represented the center of the lesion, were used for the immunohistochemical studies. Sections were placed on poly-L lysine– coated glass slides. After deparaffinization, bleaching was performed using 0.25% KMnO4 and 5% oxalic acid solutions. Then, labeled avidinbiotin indirect immunohistochemical staining for single and double antibodies was performed as previously described,22,23 using commercial kits (Zymed Laboratories, San Francisco, CA). All cases were stained with antibodies against Von Willebrand factor (factor VIII), HMB-45, basic fibroblast growth factor (bFGF), and proliferating cell nuclear antigen (PCNA) (Table 2). To determine whether bFGF is present in tumoral melanocytes, some cases of LM and SSM were also double stained with antibodies against bFGF and either keratin or epithelial membrane antigen (EMA), and bFGF and HMB-45. Staining evaluation was performed in the following manner:
Polyclonal anti-Von Willebrand factor (1:200; Dako, Glostrup Denmark) Monoclonal anti-human melanoma (HMB-45) (1:50; Dako) Polyclonal anti bFGF (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) Monoclonal anti-PCNA (1:50; Zymed Laboratories, San Francisco, CA) Monoclonal anti-EMA (E29, 1:50, Dako) Polyclonal anti-keratin wide spectrum (1:150, Dako)
SSM
13
17
67 (44–82) 68 ⫾ 10
43 (20–74) 45 ⫾ 15
10 (76%) 1 (8%) 1 (8%) 1 (8%)
4 (24%) 6 (35%) 1 (6%) 6 (35%)
*P value ⬍0.0001 †P value ⬍0.05
MATERIALS AND METHODS
TABLE 2. Antibodies Used for Staining
LM
1. bFGF. The staining intensity of the tumoral melanocytes was assessed and classified into 4 grades: 0, negative; 1, low; 2, intermediate; 3, strong. 2. PCNA. All positively stained tumoral melanocytes (morphologically identified) in 5 consecutive highpower fields (x400) in the central area of the tumor were counted. The percentage of positive melanocytes from the total count of the melanocytes in the same field was calculated. 3. HMB-45. The percentage of positively stained melanocytes from all tumoral melanocytes was semiquantitatively assessed into 4 grades from 0 to 3 (up to 25%, 25% to 50%, 50% to 75%, ⬎75%). 4. Blood vessels. All blood vessels stained with factor VIII were counted in 5 consecutive high-power fields (x400) using an ocular grid. To avoid influence of factors such as location, degree of solar elastosis and so on, the count of vessels under the center of the tumor was compared to the count of 5 high-power fields beyond the edge of the tumor. For the statistical analysis, consecutive variables were compared using the 2-tailed, unpaired Student t test. Singular variables were compared using the 2 test.
RESULTS The clinical data of the study population is shown in Table 3. Patients with LM were significantly older (median age, 67; average age, 68 ⫾ 10) than those with SSM (median age, 43; average age, 45 ⫾ 15), (P ⬍ 0.001 for the average). Most LM were located on the head (face and scalp, 77%) whereas most SSM were on the trunk and legs (71%) (P ⬍ 0.05). The results of the immunohistochemical stains are shown in Table 4 and are summarized as follows: 1. bFGF. The cytoplasm of both basal keratinocytes and tumoral melanocytes was stained. Within the same melanoma, all melanocytes were stained with the same degree of intensity. Double stains with bFGF and HMB-45 and with bFGF and either EMA or keratin clearly showed the presence of bFGF in tumoral melanocytes (Fig 1). Staining intensity was significantly stronger in SSM (P ⬍ 0.001). 2. PCNA. All nuclei of basal layer keratinocytes were stained. Some of the tumoral melanocytes
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TABLE 4. Immunohistochemical Staining Results
bFGF* Average staining intensity PCNA* Average % positive melanocytes HMB45 Average staining degree Factor VIII* vessel count/5 high-power fields: Center Edge P value between center and edge
LM
SSM
P value
1.2 (0.6)
2.4 (0.7)
⬍0.001
2.8 (1.7)
4.8 (2.6)
⬍0.05
1.4 (1.1)
1.8 (0.9)
0.15 ⬍ P ⬍ 0.2
25.5 (18.4) 14.2 (9.6) 0.06 ⬍ P ⬍ 0.07
37.9 (10.7) 21.6 (5.9) ⬍0.001
⬍0.05 ⬍0.05
*Significant differences
were also similarly stained. Their percentage was significantly higher in SSM than in LM (P ⬍ 0.05). 3. HMB-45. No difference was found in the percentage of melanocytes that showed reactivity for the antigen in LM and SSM (0.15 ⬍ P ⬍ 0.2). 4. Factor VIII. The average blood vessel count under the center of both MIS was higher than that beyond the margins, with significant values only for SSM. SSM showed significantly more vessels than LM, both in the center (Fig 2) and beyond margins (Table 4). DISCUSSION Division of MM into 4 subtypes is controversial, and thus consensus about the histological criteria for diagnosis is lacking.1-4 However, clinical and epidemiologic data support, at least partially, the distinction between the various subtypes. The so-called “lentigo maligna” has a longer in situ phase, appears larger, is usually located on the face and occurs in older people. In contrast, the so-called “superficial spreading” subtype has a shorter in situ phase, tends to be smaller
FIGURE 1. Double staining of SSM for bFGF (red) and HMB45 (blue). The melanocytes that express both molecules were stained deep purple (concentrated in the rete ridge on the left). (Original magnification ⫻200.)
when diagnosed, is located usually on the trunk or legs, and occurs in younger people.2 Whether one accepts or denies the validity of dividing MM into subtypes, it is generally agreed that LM (or, as termed by others, MM developing on severely sun-damaged skin),3,4 has a longer in situ phase, which may explain their different clinical characteristics. We have decided to study whether immunophenotypical differences between LM and SSM, which may explain the different behavior, exist already in their in situ phase. Because we could not find histologic consensus criteria to define LM and SSM, we used a unifying concept, adopting histopathologic characteristics of most authorities in the field to define the study sample. Thus it might be that cases typical for 1 subtype in the eyes of other experts were excluded from our study. This may also explain our small sample size. Furthermore, some may claim that our cases represent principally the differences between the in situ phase of MM developed on sun-exposed areas and those that develop on usually shaded areas. But despite all of these limitations, and although categorization of tumors was based solely on histopathologic findings, the study population represents both LM and SSM. This is shown by the clinical data of the patients, which corresponds well to differences between LM and SSM described in the literature.1,2 Therefore, the results also represent differences of the in situ phase of LM and SSM. Few studies examined the differences in proliferation between invasive MM, MM in situ, and nevi using PCNA.7,8 In general, it was found that the proliferation of melanocytes in invasive MM was higher than that of MM in situ and that in both types of MM, the proliferation was higher than in nevi. However, the values varied greatly, probably due to the differences in the cases studied and to the method of evaluation. The percentages of PCNA-positive melanocytes found in the current study (4.8% and 2.6%) correspond well to the values reported in the literature.7 The proliferative activity of the SSM melanoctyes was higher (almost double) than LM melanocytes. This explains the shorter in situ phase of the former. One factor that may explain the difference in proliferation between LM and SSM is bFGF. It is well established that bFGF is expressed by melanoma cells24 and is an autocrine growth factor and mitogen for
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FIGURE 2. Factor VIII stain of SSM (A) and LM (B) demonstrates more blood vessels under the former. (Original magnification ⫻100.)
them.25 We found higher levels of bFGF in SSM than in LM, which may contribute to the aforementioned difference in their proliferative activity. These results are in accordance with those of Ueda et al19 and Yamamura et al,20 who found that melanocytes and tumors from melanocytic origin like LM do not express bFGF, whereas those that originated from SSM melanocytes do. Many studies have shown that the solid tumors growth, invasiveness, and ability to metastasize depends on the capability of the tumor cells to induce blood vessel formation (angiogensis).26 In a variety of human tumors, including melanoma, immunohistochemistryaccentuated blood vessel counts have prognostic value.27 Trotter and Tron9 found a slightly increased number of blood vessels under LM compared to normal skin and a significant rise in their count when dermal invasion occurred (LM MM). Barnhill et al10 showed an increase in blood vessel count under melanomas in situ compared to dysplastic nevi. Graham et al11 compared thin melanomas (⬍0.76 mm) with and without metastases, and found correlation between the angiogenesis of the tumor and metastases, as well as patient death. In the current study, more blood vessels were found under SSM than under LM. This difference in angiogenesis may explain the longer in situ phase of the latter. Furthermore, bFGF may also contribute to this observation. The induction of tumoral vasculature depends on the excretion of angiogenic factors by the tumor cell.27 Besides being a growth factor for melanocytes, bFGF is known to be an angiogenic factor.28 Therefore, its higher levels in SSM may also promote blood vessel proliferation under this tumor. In this context, it is noteworthy that 2 studies performed on thin melanomas12,13 did not find any correlation between angiogenesis and metastatic potential. The issue of angiogenesis as a predictor of clinical outcome in cancer patients has recently been discussed.29 The author lists the potential causes for the discrepancy discussed in the literature. Among those that also apply to melanoma studies, including ours, are inadequate sample size, different definitions of the study group, and different assays for tumor microvessel density. Never-
theless, it may also be that angiogenesis plays a major role in early stages of melanoma development, as was shown for breast cancer.30 Interestingly, we found that more vessels were also present under the edge of SSM than under LM. Whether this represents a remote effect of the tumor, which has some implication on its aggressiveness, or is just a reflection of the tumor location or chronic sun exposure warrants further investigation. Many studies have found differences in melanin synthesis between the 2 melanomas. Electron microscopy showed that melanocytes of SSM have significant abnormalities in their melanosomes compared to LM melanocytes.31,32 An immunohistochemical study with antibodies against melanosomal proteins found similar differences between LM and SSM in regard to the disturbance in melanosome synthesis and epidermal melanin unit biology,33 including increased pheomelanin levels. Tyrosinase activity was found to be higher in LM than in SSM.34 We have found no significant differences in HMB-45, a melanosomal antigen, between LM and SSM; however, this may be due to our small sample size and the limitations of semiquantitive assays. One can not ignore that our study has some methodologic limitations, such as the small sample size, the way in which LM and SSM were defined, and the use of semiquantitive assays. However, these do not significantly interfere with the validity of our results, but studies on larger populations, the use of morphometry, and the recently developed DNA microarray assays may further support our preliminary results. These results, not previously reported, show that there are biological differences between LM (or melanoma on severely sundamaged skin) and SSM already in their in situ phase. These differences may explain their biological behavior. Thus the higher melanocyte proliferation rate and angiogenesis in SSM correspond well to its shorter in situ phase, and both may be, at least partially, result of higher levels of bFGF. Whether the cause of these differences is the different origin of the melanocytes, as implicated by the theory of Mishima and Matsunaka,31 or environmental factors, such as sun exposure and location, awaits further investigations.
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REFERENCES 1. Elder D, Elenitsas R: Benign pigmented lesions and malignant melanoma, in Elder D, Elenitsas R, Jaworsky C, et al (eds): Lever’s Histopathology of the Skin (eds). Philadelphia, PA, Lippincott-Raven 1997, pp 654-663 2. Barnhill RL, Mihm MC Jr, Fitzpatrick TB, et al: Neoplasms: Cutaneous melanoma, in Freedberg IM, Eisen AZ, Wolff K, et al (eds): Dermatology in General Medicine (ed 5). New York, NY, McGraw-Hill, 1999, pp 1085-1087. 3. Ackerman AB: Malignant melanoma: A unifying concept. HUM PATHOL 11:591-595, 1980 4. Ackerman AB, David KM: A unifying concept of malignant melanoma: Biologic aspects. HUM PATHOL 17:438-440, 1986 5. Weiss J, Bertz J, Jung EG: Malignant melanoma in southern Germany: Different predictive value of risk factors for melanoma subtypes. Dermatologica 183(2):109-113, 1991 6. Cox NH, Aitchinson TC, Sirel JM, et al: Comparison between lentigo maligna melanoma and other histogenetic types of malignant melanoma of the head and neck. Br J Cancer 73:940-944, 1996 7. Kuwata T, Kitagawa M, Kasuga T: Proliferative activity of primary cutaneous melanocytic tumors. Virchows Arch Pathol Anat Histopathol 423:359-364, 1993 8. Tokuda Y, Mukai K, Matsuno Y, et al: Proliferative activity of cutaneous melanocytic neoplasms defined by a proliferating cell nuclear antigen labelling index. Arch Dermatol Res 284:319-323, 1992 9. Trotter MJ, Tron VA: Dermal vascularity in lentigo maligna. J Pathol 173:341-345, 1994 10. Barnhill R, Ball R, Karaoli T: Melanoma in situ and tumor vascularity. American Society of Dermatopathology 34th annual meeting, March 1997, poster 65 11. Graham CH, Rivers J, Kerbel RS, et al: Extent of vascularization as a prognostic indicator in thin malignant melanomas. Am J Pathol 145:510-514, 1994 12. Guffey JM, Chaney JV, Stevens GL, et al: Immunohistochemical assessment of tumor vascularity in recurrent Clark II melanomas using antibody to type IV collagen. J Cutan Pathol 22(2):122-127, 1995 13. Busam KJ, Berwick M, Fandrey K, et al: Tumor vascularity is not a prognostic factor for malignant melanoma of the skin. Am J Pathol 147:1049-1056, 1995 14. Smoller BR, Hsu A, Krueger J: HMB-45 monoclonal antibody recognizes an inducible and reversible melanocyte cytoplasmic protein. J Cutan Pathol 18:315-322, 1991 15. Skelton HG, Smith KJ, Barrett TL, et al: HMB-45 staining in benign and malignant melanocytic lesions. A reflection of cellular activation. Am J Dermatopathol 13:543-550, 1991 16. Smoller BR, McNutt NS, Hsu A: HMB45 recognizes stimulated melanocytes. J Cutan Pathol 16(2):49-53, 1989 17. Collins KA, White WI: Intercellular adhesion molecule 1
(ICAM-1) and bcl-2 are differentially expressed in early evolving malignant melanoma. Am J Dermatopathol 1995, 17:429-438, 1995 18. Saenz-Santamaria MC, Reed JA, McNutt NS, et al: Immunohistochemical expression of BCL-2 in melanomas and intradermal nevi. J Cutan Pathol 21:393-397, 1994 19. Ueda M, Funasaka Y, Ichihashi M, et al: Stable and strong expression of basic Fibroblast growth factor in naevus cell naevus contrast with aberrant expression in melanoma. Br J Dermatol 130: 320-324, 1994 20. Yamamura K, Mishima Y: Antigen dynamics in melanocytic and nevocytic melanoma oncogenesis. J Invest Dermatol 94:174-182, 1990 21. Reed JA, McNutt SN, Albino AP: Differential expression of basic fibroblast growth factor in melanocytic lesions demonstrated by in situ hybridization. Am J Pathol 14:329-336, 1994 22. Giorno R: A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diagn Immunol 2:161, 1994 23. Guesdon JL, Ternynck T, Aurameas S: The use of avidin biotin interaction in Immunoenzymatic techniques. J Histochem 27: 1131-1139, 1979 24. Scott G, Stoler M, Sarkar S, et al: Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization. J Invest Dermatol 96:318-322, 1991 25. Halaban R, Kwan BS, Ghosh S: bFGF as an autocrine growth factor for human melanomas. Oncogene Res 3:177-186, 1988 26. Folkman J: What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82:4-6, 1990 27. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nature Med 1:27-31, 1995 28. Rifkin DB, Mascatelli D: Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 109:1-6, 1989 29. Weidner N: Angiogeneis as a predictor of clinical outcome in cancer patients (editorial). HUM PATHOL 31;403-404, 2000 30. Gasparini G, Harris A: Tumor vascularity in prognosis, in Teicher B (ed): Angiogenetic Agents in Cancer Chemotherapy. Totowa, NJ, Hunama Press, 1999, pp 317-431 31. Mishima Y, Matsunaka M: Pagetoid premalignant melanosis and melanoma: Differentiation from Hutchinson’s melanotic freckle. J Invsest Dermatol 65:434-440, 1975 32. Clark WH Jr, Ainsworth AM, Bernardino EA, et al: The developmental biology of primary human malignant melanomas. Semin Oncol 2:83-103, 1975 33. Jimbow K, Salopek TG, Dixon WT, et al: The epidermal melanin unit in the pathophysiology of malignant melanoma. Am J Dermatopathol 13:179-188, 1991 34. Haensch R: Tyrosinase activity in three types of the malignant melanoma: Superficial spreading melanoma, lentigo maligna melanoma and nodular melanoma. Arch Dermatol Forsch 252:193201, 1975
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as reflected by PCNA stain (P < 0.02) and higher levels of bFGF (P < 0.001), than melanocytes of LM. More blood vessels were counted under SSM than under LM (P < 0.05). These results are in accordance with the biological behavior of SSM and LM, that is, the longer in situ phase of the latter. bFGF is both a growth factor for melanocytes and an angiogentic factor. The differences in PCNA, a proliferation marker, and blood vessel count may be related to the bFGF effect. Thus this study reveals some of the biological differences between LM and SSM. Location and sun exposure habits may contribute to these differences, which already exist in the in situ phase. HUM PATHOL 33:1001-1005. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: malignant melanoma, lentigo maligna, superficial spreading melanoma. Abbrevations: LM, lentigo maligna; SSM, superficial spreading melanoma; MIS, melanoma in situ; MM, malignant melanoma; HMB45, human melanoma black 45; PCNA, proliferating cell nuclear antigen; bFBF, basic fibroblast growth factor; EMA, epithelial membrane antigen.
Traditionally, 4 subtypes of malignant melanoma (MM) are recognized according to their clinical characteristics and histologic features.1,2 Among these, superficial spreading melanoma (SSM) is the most common subtype and lentigo maligna (LM) is the rarest.2 Epidemiologic, clinical, histologic, ultrastructural, and probably also etiologic differences exist between the in situ phases of these 2 subtypes.1,2 LM usually appears in older patients (usually in the seventh decade) and on sun exposed areas, mainly the face. When diagnosed, it has a larger diameter and a radial growth phase lasting about 3 to 15 years. In contrast, SSM appears in younger patients (usually in the fourth or fifth decades), occurs in any area, has a smaller diameter at the time of diagnosis, and has a shorter radial growth phase (1 to 7 years). Although distinguishing between LM and SSM in situ is not always possible, and some authors claim that criteria for this are not reputable,3,4 most textbooks on dermatology and dermatopathology1,2 quote such criteria. Accordingly, LM is characterized by
proliferation of pleomorphic atypical melanocytes along the basal layer and the adnexa, whereas SSM is marked by proliferation of atypical melanocytes along the basal layer with pagetoid spread of atypical melanocytes in the epidermis. An atrophic epidermis, probably a refection of sun damage, is usually seen in LM. Further evidence to support the distinction came from an epidemiologic study showing that the relative risk of SSM is almost exclusively determined by nevi count, whereas that of LM is determined mostly by skin type (type I) and to a lesser extent by history of sun exposure.5 Numerous studies have shown that no difference exists between the subtypes in the invasive phase and that prognosis is mainly determined by the depth of invasion.6 Even so, many clinicians and dermatopathologists agree that determining the melanoma subtype is of some importance. Nevertheless, Ackerman et al3,4 suggested that only 1 type of melanoma exists and that the differences found in the intraepidermal phase are related solely to the location, sun exposure, and heterogeneity of tumors. Many studies have examined various biological factors during different stages of melanoma evolution. These factors included proliferation,7,8 angiogenesis around tumors,9-13 expression of specific markers of melanocytes,14-16 adhesion molecules,17 and oncogenes,18 as well as growth factors.19-21 However, most of these studies grouped together the various in situ melanomas. Therefore, the aim of our study was to compare some characteristics of LM and SSM in situ using
From the Department of Dermatology and Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel and the Sackler Faculty of Medicine, Tel-Aviv University, Israel. Accepted for publication January 18, 2002. The first 2 authors are equal contributors. Address correspondence and reprint requests to Henri Trau, MD, Chairman, Department of Dermatology, Sheba Medical Center, 52621 Tel-Hashomer, Israel. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3310-0008$35.00/0 doi:10.1053/hupa.2002.124014
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HUMAN PATHOLOGY
Volume 33, No. 10 (October 2002)
TABLE 1. Criteria for Classification of LM and SSM
Epidermis Dermis Melanocyte proliferation Melanocyte distribution
TABLE 3. Clinical and Histologic Data of the Study Cases
LM
SSM
Atrophic Elastotic changes Basal pleomorphic atypical
Normal Normal Monomorphic atypical, singly or in nests Pagetoid spread
Infiltrating adnexa
immunohistochemical stains and to correlate the findings with their different clinical behaviour.
Number of cases Age (years) median (range) average* Location† n(%) head trunk upper limbs lower limbs
All of the cases diagnosed as MM in situ between 1989 and 1995 were retrieved from the archive of the Institute of Pathology at the Sheba Medical Center. The original hematoxylin & eosin–stained sections were reviewed, and cases were classified as LM or SSM in situ according to the stringent criteria, summarized in Table 1. In brief, these criteria included epidermal changes (atrophic vs. normal), absence or presence of solar elastosis, and the proliferation pattern of the melanocytes and their cytologic features. Only those cases that met all criteria of either LM or SSM, as agreed on by the 3 dermatopathologists/pathologists (A.B., J.K., and H.T.) were included in the study. Cases that showed equivocal criteria, or those for which there was disagreement among the authors, were excluded. Finally, 30 cases of melanoma in situ (MIS) (out of the 56 reviewed) were chosen for the study. Thirteen cases were classified as LM and 17 as SSM. For each case, the age of the patient and location of the lesion were retrieved from the files. Four m-thick sections from the paraffin-embedded, formalin-fixed blocks, which represented the center of the lesion, were used for the immunohistochemical studies. Sections were placed on poly-L lysine– coated glass slides. After deparaffinization, bleaching was performed using 0.25% KMnO4 and 5% oxalic acid solutions. Then, labeled avidinbiotin indirect immunohistochemical staining for single and double antibodies was performed as previously described,22,23 using commercial kits (Zymed Laboratories, San Francisco, CA). All cases were stained with antibodies against Von Willebrand factor (factor VIII), HMB-45, basic fibroblast growth factor (bFGF), and proliferating cell nuclear antigen (PCNA) (Table 2). To determine whether bFGF is present in tumoral melanocytes, some cases of LM and SSM were also double stained with antibodies against bFGF and either keratin or epithelial membrane antigen (EMA), and bFGF and HMB-45. Staining evaluation was performed in the following manner:
Polyclonal anti-Von Willebrand factor (1:200; Dako, Glostrup Denmark) Monoclonal anti-human melanoma (HMB-45) (1:50; Dako) Polyclonal anti bFGF (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) Monoclonal anti-PCNA (1:50; Zymed Laboratories, San Francisco, CA) Monoclonal anti-EMA (E29, 1:50, Dako) Polyclonal anti-keratin wide spectrum (1:150, Dako)
SSM
13
17
67 (44–82) 68 ⫾ 10
43 (20–74) 45 ⫾ 15
10 (76%) 1 (8%) 1 (8%) 1 (8%)
4 (24%) 6 (35%) 1 (6%) 6 (35%)
*P value ⬍0.0001 †P value ⬍0.05
MATERIALS AND METHODS
TABLE 2. Antibodies Used for Staining
LM
1. bFGF. The staining intensity of the tumoral melanocytes was assessed and classified into 4 grades: 0, negative; 1, low; 2, intermediate; 3, strong. 2. PCNA. All positively stained tumoral melanocytes (morphologically identified) in 5 consecutive highpower fields (x400) in the central area of the tumor were counted. The percentage of positive melanocytes from the total count of the melanocytes in the same field was calculated. 3. HMB-45. The percentage of positively stained melanocytes from all tumoral melanocytes was semiquantitatively assessed into 4 grades from 0 to 3 (up to 25%, 25% to 50%, 50% to 75%, ⬎75%). 4. Blood vessels. All blood vessels stained with factor VIII were counted in 5 consecutive high-power fields (x400) using an ocular grid. To avoid influence of factors such as location, degree of solar elastosis and so on, the count of vessels under the center of the tumor was compared to the count of 5 high-power fields beyond the edge of the tumor. For the statistical analysis, consecutive variables were compared using the 2-tailed, unpaired Student t test. Singular variables were compared using the 2 test.
RESULTS The clinical data of the study population is shown in Table 3. Patients with LM were significantly older (median age, 67; average age, 68 ⫾ 10) than those with SSM (median age, 43; average age, 45 ⫾ 15), (P ⬍ 0.001 for the average). Most LM were located on the head (face and scalp, 77%) whereas most SSM were on the trunk and legs (71%) (P ⬍ 0.05). The results of the immunohistochemical stains are shown in Table 4 and are summarized as follows: 1. bFGF. The cytoplasm of both basal keratinocytes and tumoral melanocytes was stained. Within the same melanoma, all melanocytes were stained with the same degree of intensity. Double stains with bFGF and HMB-45 and with bFGF and either EMA or keratin clearly showed the presence of bFGF in tumoral melanocytes (Fig 1). Staining intensity was significantly stronger in SSM (P ⬍ 0.001). 2. PCNA. All nuclei of basal layer keratinocytes were stained. Some of the tumoral melanocytes
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TABLE 4. Immunohistochemical Staining Results
bFGF* Average staining intensity PCNA* Average % positive melanocytes HMB45 Average staining degree Factor VIII* vessel count/5 high-power fields: Center Edge P value between center and edge
LM
SSM
P value
1.2 (0.6)
2.4 (0.7)
⬍0.001
2.8 (1.7)
4.8 (2.6)
⬍0.05
1.4 (1.1)
1.8 (0.9)
0.15 ⬍ P ⬍ 0.2
25.5 (18.4) 14.2 (9.6) 0.06 ⬍ P ⬍ 0.07
37.9 (10.7) 21.6 (5.9) ⬍0.001
⬍0.05 ⬍0.05
*Significant differences
were also similarly stained. Their percentage was significantly higher in SSM than in LM (P ⬍ 0.05). 3. HMB-45. No difference was found in the percentage of melanocytes that showed reactivity for the antigen in LM and SSM (0.15 ⬍ P ⬍ 0.2). 4. Factor VIII. The average blood vessel count under the center of both MIS was higher than that beyond the margins, with significant values only for SSM. SSM showed significantly more vessels than LM, both in the center (Fig 2) and beyond margins (Table 4). DISCUSSION Division of MM into 4 subtypes is controversial, and thus consensus about the histological criteria for diagnosis is lacking.1-4 However, clinical and epidemiologic data support, at least partially, the distinction between the various subtypes. The so-called “lentigo maligna” has a longer in situ phase, appears larger, is usually located on the face and occurs in older people. In contrast, the so-called “superficial spreading” subtype has a shorter in situ phase, tends to be smaller
FIGURE 1. Double staining of SSM for bFGF (red) and HMB45 (blue). The melanocytes that express both molecules were stained deep purple (concentrated in the rete ridge on the left). (Original magnification ⫻200.)
when diagnosed, is located usually on the trunk or legs, and occurs in younger people.2 Whether one accepts or denies the validity of dividing MM into subtypes, it is generally agreed that LM (or, as termed by others, MM developing on severely sun-damaged skin),3,4 has a longer in situ phase, which may explain their different clinical characteristics. We have decided to study whether immunophenotypical differences between LM and SSM, which may explain the different behavior, exist already in their in situ phase. Because we could not find histologic consensus criteria to define LM and SSM, we used a unifying concept, adopting histopathologic characteristics of most authorities in the field to define the study sample. Thus it might be that cases typical for 1 subtype in the eyes of other experts were excluded from our study. This may also explain our small sample size. Furthermore, some may claim that our cases represent principally the differences between the in situ phase of MM developed on sun-exposed areas and those that develop on usually shaded areas. But despite all of these limitations, and although categorization of tumors was based solely on histopathologic findings, the study population represents both LM and SSM. This is shown by the clinical data of the patients, which corresponds well to differences between LM and SSM described in the literature.1,2 Therefore, the results also represent differences of the in situ phase of LM and SSM. Few studies examined the differences in proliferation between invasive MM, MM in situ, and nevi using PCNA.7,8 In general, it was found that the proliferation of melanocytes in invasive MM was higher than that of MM in situ and that in both types of MM, the proliferation was higher than in nevi. However, the values varied greatly, probably due to the differences in the cases studied and to the method of evaluation. The percentages of PCNA-positive melanocytes found in the current study (4.8% and 2.6%) correspond well to the values reported in the literature.7 The proliferative activity of the SSM melanoctyes was higher (almost double) than LM melanocytes. This explains the shorter in situ phase of the former. One factor that may explain the difference in proliferation between LM and SSM is bFGF. It is well established that bFGF is expressed by melanoma cells24 and is an autocrine growth factor and mitogen for
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FIGURE 2. Factor VIII stain of SSM (A) and LM (B) demonstrates more blood vessels under the former. (Original magnification ⫻100.)
them.25 We found higher levels of bFGF in SSM than in LM, which may contribute to the aforementioned difference in their proliferative activity. These results are in accordance with those of Ueda et al19 and Yamamura et al,20 who found that melanocytes and tumors from melanocytic origin like LM do not express bFGF, whereas those that originated from SSM melanocytes do. Many studies have shown that the solid tumors growth, invasiveness, and ability to metastasize depends on the capability of the tumor cells to induce blood vessel formation (angiogensis).26 In a variety of human tumors, including melanoma, immunohistochemistryaccentuated blood vessel counts have prognostic value.27 Trotter and Tron9 found a slightly increased number of blood vessels under LM compared to normal skin and a significant rise in their count when dermal invasion occurred (LM MM). Barnhill et al10 showed an increase in blood vessel count under melanomas in situ compared to dysplastic nevi. Graham et al11 compared thin melanomas (⬍0.76 mm) with and without metastases, and found correlation between the angiogenesis of the tumor and metastases, as well as patient death. In the current study, more blood vessels were found under SSM than under LM. This difference in angiogenesis may explain the longer in situ phase of the latter. Furthermore, bFGF may also contribute to this observation. The induction of tumoral vasculature depends on the excretion of angiogenic factors by the tumor cell.27 Besides being a growth factor for melanocytes, bFGF is known to be an angiogenic factor.28 Therefore, its higher levels in SSM may also promote blood vessel proliferation under this tumor. In this context, it is noteworthy that 2 studies performed on thin melanomas12,13 did not find any correlation between angiogenesis and metastatic potential. The issue of angiogenesis as a predictor of clinical outcome in cancer patients has recently been discussed.29 The author lists the potential causes for the discrepancy discussed in the literature. Among those that also apply to melanoma studies, including ours, are inadequate sample size, different definitions of the study group, and different assays for tumor microvessel density. Never-
theless, it may also be that angiogenesis plays a major role in early stages of melanoma development, as was shown for breast cancer.30 Interestingly, we found that more vessels were also present under the edge of SSM than under LM. Whether this represents a remote effect of the tumor, which has some implication on its aggressiveness, or is just a reflection of the tumor location or chronic sun exposure warrants further investigation. Many studies have found differences in melanin synthesis between the 2 melanomas. Electron microscopy showed that melanocytes of SSM have significant abnormalities in their melanosomes compared to LM melanocytes.31,32 An immunohistochemical study with antibodies against melanosomal proteins found similar differences between LM and SSM in regard to the disturbance in melanosome synthesis and epidermal melanin unit biology,33 including increased pheomelanin levels. Tyrosinase activity was found to be higher in LM than in SSM.34 We have found no significant differences in HMB-45, a melanosomal antigen, between LM and SSM; however, this may be due to our small sample size and the limitations of semiquantitive assays. One can not ignore that our study has some methodologic limitations, such as the small sample size, the way in which LM and SSM were defined, and the use of semiquantitive assays. However, these do not significantly interfere with the validity of our results, but studies on larger populations, the use of morphometry, and the recently developed DNA microarray assays may further support our preliminary results. These results, not previously reported, show that there are biological differences between LM (or melanoma on severely sundamaged skin) and SSM already in their in situ phase. These differences may explain their biological behavior. Thus the higher melanocyte proliferation rate and angiogenesis in SSM correspond well to its shorter in situ phase, and both may be, at least partially, result of higher levels of bFGF. Whether the cause of these differences is the different origin of the melanocytes, as implicated by the theory of Mishima and Matsunaka,31 or environmental factors, such as sun exposure and location, awaits further investigations.
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