—  SHORT COURSE #31  —

Precursors To Melanoma And The Problematic Nevomelanocytic Proliferation

Section 12 - Prognostication Including Microstaging of Malignant Melanoma

Neil Crowson, MD
Cynthia M. Magro, MD
Martin C. Mihm, Jr., MD


The prognosis of patients with invasive malignant melanoma in vertical growth phase can be predicted by specific light microscopic parameters including the measured depth, mitotic count, host response, sex, anatomic site, and presence or absence of regression, angioinvasion, or ulceration. All of this information should appear in the pathology report.

Level and Depth of Invasion
A critical factor is the extent of invasion into the dermis. Tumor invasion may be qualitatively expressed according to the anatomic compartment (i.e., papillary or reticular dermis or subcutis) of invasion, referred to as the Clark level, or quantitatively assessed as the so called Breslow measurement, the thickness of the melanoma from the most superficial aspect of the granular cell layer to the deepest point of invasion in the dermis. Adventitial dermal invasion is not measured unless it is the only site of dermal invasion, and one then measures from the inner luminal surface of the eccrine gland or duct or the inner aspect of the outer root sheath epithelium of the hair follicle (Breslow, 1970).

Clark 's levels are defined as follows (Clark et al, 1969):

Level I: Malignant melanocytes are confined to the epidermis

Level II: Partial infiltration of the papillary dermis by single cells or small nests of cells that do not exceed the size of any intraepidermal nest. The presence of a large nest, even if the entire depth of the papillary dermis is not occupied, may represent incipient vertical growth phase.

Level III: Tumor cells fill and expand the papillary dermis with extension of tumor to the papillary-reticular dermal interface, the latter identifiable through the routine use of a polarizer and compensator to take advantage of the birefringence patterns of the dermal collagen; the papillary dermal collagen fibers are oriented vertically whereas the reticular dermal collagen bundles have a more horizontal orientation. In level III invasion, a few cells may infiltrate the superficial reticular dermis, but the growth is not permeative. Level III penetration usually signifies vertical growth phase melanoma (Mihm and Googe, 1990).

Level IV: Melanoma cells infiltrate the reticular dermis in a significant fashion.

Level V: Melanoma cells infiltrate the subcutaneous fat.

The Breslow measurement is taken from the epidermal surface or, in the event that the surface is ulcerated, from the base of the ulcer, and is made with a calibrated ocular micrometer. It is the most important means of prognosticating mucosal melanomas that lack the anatomic compartmentalization of the corium seen in the skin. In most prognostic studies, the measured depth emerges as the most powerful independent factor for prediction of lymph nodes metastasis and survival (Vollmer and Seigler, 2000).

Other prognostic variables include:

Mitotic Count Per Square Millimeter
This index is reported as the number of mitoses in a square millimeter. From 3 to 10 high-power fields is equivalent to one square millimeter, depending upon the nature of optical equipment used. There is wide variance between different microscope makes and models and thus we recommend formal calibration using a stage micrometer (Mihm and Googe, 1990; Crowson et al, 2001).

Inflammatory Host Response
A lymphocytic response to the vertical growth phase component influences prognosis in some studies. If there is no host response the designation is "absent", while infiltration focally either along the base or within the tumor is considered to be "present, non-brisk". Infiltration either of the entire base of the tumor or diffuse permeation of the vertical growth phase is designated "present, brisk". To be significant lymphocytes must infiltrate and disrupt tumor nests and show apposition to tumor cells; lymphocytes in a perivenular array within the tumor nodule but not permeative of it are designated as an absent host response, as are lymphocytes in a cuff about the base of the tumor but not infiltrating it (Clark et al, 1989; Mihm and Googe, 1990; Crowson et al, 2001).

Regression
Complete regression is characterized by an area of absent melanocytic growth in the epidermis and dermis, often bordered on one or both sides by melanoma. This signifies a worse prognosis. The epidermis is often attenuated with loss of the retiform pattern. The subjacent dermis shows a nonlaminated fibroplasia containing a few inflammatory cells and melanophages with variable edema and telangiectasia, the vessels typically assuming a perpendicular orientation to the long axis of the epidermis. Regression of over 75% of a given tumor may be the critical volume that portends metastasis (Ronan et al, 1987; Byers and Bahwan, 1988).

Microscopic Satellites
Microscopic satellites, characterized by reticular dermal and/or subcuticular nodules of tumor greater than 0.05 mm in width separated from the main vertical growth phase component, are associated with lymph node metastasis, decreased disease-free and overall survival (Day et al, 1981; Harrist et al, 1984; Leon et al, 1991; Balch et al, 2000).

Ulceration
By convention we measure size of any ulcer (Balch et al, 1980), even though recent studies suggest that any evidence of ulceration that cannot be attributed to trauma portends a grave prognosis (Balch et al, 2000). To qualify as ulceration there must be evidence of host response, ie fibrin and cellular debris, to distinguish the process from an in vitro artifact induced by trauma of processing artifact or surgery.

Blood Vessel and Lymphatic Invasion
The presence or absence of blood vessel and lymphatic invasion should be reported, although it is unclear that angiolymphatic invasion will prove ultimately to be an independent prognostic variable. Some studies have correlated the frequency of angiolymphatic invasion with increasing depth and level of tumor invasion (Schmoeckel, et al, 1983), while others have shown vascular invasion to be a significant predictor of metastasis (Mraz-Gernhard, et al, 1998) or of reduced survival (Barnhill et al, 1996). It is perhaps the low frequency of this finding which precluded its emergence as an independent prognostic variable in many series (Clark et al, 1989).

Anatomic Site
Melanomas in the head and neck area, upper back, axial skeleton, subungual region and/or on the palms or soles have a worse prognosis than do extremity based lesions (Clark et al, 1969; Balch et al 2000).

Sex
Women have a better prognosis than men in some but not all studies (Clark et al., 1989; Balch et al, 2000). The statistical significance of gender is confounded by anatomic site.

Age
Some studies indicate that patients over age 60 years have a worse prognosis (Clark et al, 1989; Balch et al, 2000).

The Molecular Diagnosis of Melanoma through Microarray Techniques
Immunohistochemical analyses are routinely used to detect the protein products of gene expression and thereby to predict the malignant phenotype or, more commonly, to identify the melanocytic histogenesis of a given neoplasm. The use of reverse transcriptase polymerase chain reaction (RT-PCR) methodologies to detect mRNA in tissue provides similar and complimentary information. A novel scientific approach employs microarray technology to assess biomolecules in high-throughput analytical systems. These microarray technologies are broadly classed as complimentary DNA (cDNA) microarrays, oligonucleotide arrays, protein microarrays and tissue microarrays (Rimm, 2001). The information derived from such studies, compiled as large databases, permits meta-analysis of quantities of information that can be correlated mathematically for the extraction of the maximum amount of information (Khna te al, 1999).

The cDNA microarray is a miniature display of large numbers of DNA sequences on a solid support system, either a microchip or a slide. The methodology employed to create a cDNA microarray involves the generation of cDNA copies from mRNA derived from tumor samples synthesized in vitro with fluorescent-labeled nucleotides which are then hybridized into the array. Thousands of separate and distinct DNA probes applied per square centimeter of slide or microchip array are then analyzed using complex biochemical-optical systems which employ analytical computer technologies including neural nets and hierarchical data analysis and processing to decode the data obtained (Kim et al, 2000). These technologies enable the identification of amplification of genes and of chromosomes at a level of only five times above background (Heiskanen et al, 2000). Using current technologies, a single mRNA species can be detected from among 500,000 different mRNAs. For analysis using a cDNA microarray, specific sets of tissue-appropriate cDNA probes must first be generated from the mRNA derived from relevant clones. The expressed sequenced tag database (dbEST) for neural crest-derived melanocytic cDNA sets has been created to facilitate this. (Loftus et al, 1999). The Stanford University microarray database lists specific sets of tissue-appropriate cDNA for use in various human cancers and is accessible to researchers through their web site (http://genome-www.stanford.edu/microarray) (Sherlock et al, 2001). Using such technology, one group analyzed melanoma samples with an array of 7,000 discrete genes and thus identified a distinct melanoma subset capable of producing primitive tubular networks in vivo that correlated to aggressive biological behaviour (Bittner et al, 2000). This technology can also be used to identify novel aberrant tumor suppressor genes and other genes predisposing to the metastatic phenotype. Su and co-workers analyzed the expression of 3317 genes in 3 different melanoma cell lines, analyzed in pairs by cDNA microarray technology and thus identified specific tumor suppressor genes including the Cx43 suppressor gene, monocyte chemotactic protein-1 and the cystein proteinase P32- a gene responsible for apoptosis (Su et al, 2000). Transfection of the Cx43 gene suppressed anchorage-independent growth of a melanoma cell line. The ability to alter cellular phenotype in concert with the global gene expression profile enabled identification of previously unrecognized tumor suppressor genes. The cDNA microarray can also be used to establish how a neoplasm metabolizes and responds to a given cheomotherapeutic agent (Scherf et al, 2000), and to identify signalling and metabolic pathways as potential therapeutic targets (Roses, 2000).

The oligonucleotide microarray uses silicone wafers similar to those employed in the computer industry to construct an oligonucleotide array. This chip technique is best used to search for specific genetic mutations.

Complimentary to genomic initiatives is the science of proteomics, or the study of expressed proteins in tissue and cell types (Bans et al, 2000) enhanced by the use of laser capture microdissection to enable isolation of neoplastic cells under study. Proteins are the functional products of genes modified by post-translational events such as phosphorylation or glycosylation as well as by environmental or epigenetic factors that impact the aging host. Proteomic research analyzes the functional state of the protein products of genes identified by cDNA microarray technology.

Tissue micro-array involves the performance of a core biopsy of a paraffin embedded tissue block with relocation of the tissue core to an array block which is then sectioned to reveal 0.5-0.6 mm cross sections. Hundreds of unique sections thus consitute a single slide-based array that is then probed using conventional immunohistochemical reagents to detect in tissue the protein products of gene expression (Koonen et al, 1999. This technology can be applied to cytologic preparations as well.

Proteomic and genomic investigation provides a novel molecular diagnostic strategy for melanoma that may reveal new therapeutic insights. Clinical and histologic data will remain necessary adjuncts to guide proteomic/genomic analyses as they evolve.

Table 1 : Antibodies commonly used to determine melanocytic histogenesis

Antibody [Clone] Manufacturer* Antigenic Target
HMB-45 Novacastra Gp100 antigen of premelanosome complex
Mel 5 [TRP-1 and TRP-2] Signet Laboratories Gp75 antigen (75kDa tyrosinase-related glycoprotein)
Tyrosinase [T311] Lab Vision 70-80 kDa protein moities
Microphthalmia transcription factor Novacastra
Anti-S-100 PROTEIN (polyclonal) Ventana Ca++-channel regulatory protein
Anti-Melan-A [SKMEL-29] Novacastra A103; tyrosinase pathway antigen
Anti-MART-1 [M2-7C10] Oncogene Research Products tyrosinase pathway antigen
* of reagents commonly used at Regional Medical Laboatories, Tulsa, OK. This does not constitute a specific endoresment of the product or manufacturer.

Table 2: The Synoptic Report for Primary Cutaneous Malignant Melanoma, Regional Medical Laboratory, Tulsa, OK

Name: ______________________ Case #: ___________
Location: _________________________________________
Histological type:
lentigo maligna...........__
superficial spreading...__
nodular.....................__
acral lentiginous..........__
not otherwise specified..__
Clark's level: I...... __
II..... __
III.... __
IV..... __
V...... __
Thickness (Breslow): ________ mm.
Mitotic index (mitoses /mm2): ________
Radial Growth Phase Y__ N__
Vertical Growth Phase Y__ N__
Microscopic satellites Y__ N__
Regression Y__ N__
diameter ____________
Host immune response Y__ N__
Brisk
Non-brisk
Absent
Neurotropism Y__ N__
Vascular invasion Y__ N__
Microscopic Satellites Y__ N__
Precursor lesion Y__ N__
type _____________________
Margin positive Y__ N__
location if positive ________________
minimum distance from tumor if negative _____________
Lymph nodes involved............ Y__ N__
number _____________
sentinel lymph node positive Y__ N__ not assessed__
Distant metastases Y__ N__
site _____________________
This synoptic report is appended to a narrative description of all malignant melanomas except for in situ disease, where the "microstaging" of prognostic risk factors, i.e., ulceration, regression, Breslow depth, etc., is not given.

References
  1. Abernathy JL, Soyer HP, Kerl H, Jorizzo JL, White WL. Epidermotropic metastatic malignant melanoma simulating melanoma in situ. A report of 10 examples from two patients. Am J Surg Pathol 1994;18:1140.

  2. Aloi F, Pich A, Pippione M. Malignant cellular blue nevus: a clinicopathological study of 6 cases. Dermatology 1996;192:36–40.

  3. Auger M, Caraway NP, Wojcik EM, Ordonez NG. Fine needle aspiration biopsy of metastatic myxoid melanoma. Cytopathology 1994;5:118–122.

  4. Baer SC , Schultz D, Synnestvedt M, Elder DE. Desmoplasia and neurotropism. Prognostic variables in patients with stage I melanoma. Cancer 1995;76: 2242-2247.

  5. Balch CM, Buzaid AC, Atkins MB et al. A new American Joint Committee on Cancer staging system for cutaneous melanoma. Cancer 2000;88:1484-91.

  6. Balch CM, Soong SJ, Ross MI et al. Long-term results of a multi-institutional randomized trial comparing prognostic factors and surgical results for intermediate thickness melanomas (1.0 to 4.0 mm). Intergroup Surgical Trial. Ann Surg Oncol 200;7:97-97.

  7. Balch CM, Wilkerson JA, Murad TM, Soong SJ, Ingalls AL, Maddox WA. The prognostic significance of ulceration of cutaneous melanoma. Cancer 1980;45: 3012–3017.

  8. Banerjee SS, Harris M. Morphological and immunophenotypic variations in malignant melanoma. Histopathology 2000;36:387-402.

  9. Barnhill RL. Pathology of Melanocytic Nevi and Malignant Melanoma. Boston: Butterworth-Heinemann, 1995.

  10. Barnhill RL, Fine J, Roush GC, Berwick M. Predicting five-year outcome for patients with cutaneous melanoma in a population-based study. Cancer 1996;78:427-432.

  11. Barnhill RL, Argenyi ZB, From L et al. Atypical Spitz tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome. Hum Pathol 1999;30:1523-1526.

  12. Barnhill RL. Childhood melanoma. Semin Diagn Pathol 1998;15:189-194.

  13. Beardmore GL, Davis NC. Multiple primary cutaneous melanomas. Arch Dermatol 1975;111:603–609.

  14. Bengoechea-Beeby MP, Velasco-Oses A, Mourino Fernandez F, Reguilon-Rivero MC, Remon-Garijo L, Casado-Perez C. Epidermotropic metastatic melanoma. Are the current criteria adequate to differentiate primary from metastatic melanoma? Cancer 1993;72:1909–1913.

  15. Berg P, Lindelof B. Differences in malignant melanoma between children and adolescents. Arch Dermatol 1997;133:295–297.

  16. Bittner M, Meltzer P, Chen Y et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 2000;406:536-540.

  17. Blessing K, Grant JJ, Sanders DS, Kennedy MM, Husain A, Coburn P. Small cell malignant melanoma: a variant of naevoid melanoma. Clinicopathological features and histological differential diagnosis. J Clin Pathol 2000;53:591-591.

  18. Breslow A. Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg 1970;172: 902–908.

  19. Bruijn JA, Mihm MC Jr, Barnhill RL. Desmoplastic melanoma. Histopathology 1992;20:197–205.

  20. Busam KJ, Iverson K, Coplan KC, Jungbluth AA. Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic melanoma. Am J Surg Pathol 2001;25:197-204.

  21. Byers HR, Bahwan J. Pathologic parameters in the diagnosis and prognosis of primary cutaneous melanoma. Hematol Oncol Clin North Am 1988;12:717–725.

  22. Carney JA, Ferreiro JA. The epithelioid blue nevus. A multicentric familial tumor with important associations, including cardiac myxoma and psammomatous melanotic schwannoma. Am J Surg Pathol 1996;20:259–272.

  23. Carlson JA, Mihm MC. Vulvar nevi, lichen sclerosis et sclerosis, and vitiligo (letter). Arch Dermatol 1997;133: 1314–1316.

  24. Carlson JA, Mu XC, Slominski A, Weismann K, Crowson AN, Malfetano J, Mihm MC Jr. Melanocytic proliferations associated with lichen sclerosus. Arch Dermatol 2002;138:77-87.

  25. Carlson JA, Dickersin GR, Sober AJ, Barnhill RL. Desmoplastic neurotropic melanoma. A clinicopathologic analysis of 28 cases. Cancer 1995;75:478–494.

  26. Ceballos PI, Ruiz-Maldonado R, Mihm MC. Melanoma in children. N Engl J Med 1995;332: 656–662.

  27. Chang AE, Karnell LH, Menck HR. The National Cancer Data Base report on cutaneous and noncutaneous melanoma: a summary of 84,836 cases from the past decade. The American College of Surgeons Commission on Cancer and the American Cancer Society. Cancer 1998;83:1664–1678.

  28. Chang KL, Folpe AL. Diagnostic utility of microphthalmia transcription factor in malignant melanoma and other tumors. Adv Anat Pathol 2001;8:273-5.

  29. Chen JC, Petrovich Z, O'Day S et al. Stereotactic radiosurgery in the treatment of metastatic disease to the brain. Neurosurgery 2000;47:268-279.

  30. Chetty R, Slavin JL, Pitson GA, Dowling JP. Melanoma botryoides: a distinctive myxoid pattern of sino-nasal melanoma. Histopathology 1994;24:377–379.

  31. Chung AF, Woodruff JM, Lewis JL, Jr. Malignant melanoma of the vulva: A report of 44 cases. Obstet Gynecol 1975;45: 638–646.

  32. Chung EB, Enzinger FM. Malignant melanoma of soft parts. A reassessment of clear cell sarcoma. Am J Surg Pathol 1983;7:405-413.

  33. Crawford RI, Tron VA, Ma R, Rivers JK. Sinonasal malignant melanoma – a clinicopathologic analysis of 18 cases. Melanoma Res 1995;5:261-265.

  34. Clark WH Jr. A classification of malignant melanoma in man correlated with histogenesis and biological behaviour. In: Montagna W, Hu F, eds. Advances in the Biology of the Skin, vol VIII. New York: Pergamon, 1967, p. 621–647

  35. Clark WH Jr, From L, Bernardino EA, Mihm MC. Histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Res 1969;29: 705–726.

  36. Clark EH Jr, Elder DE, Guerry D IV, Epstein MN, Greene MH, van Horn M. A study of tumor progression: The precursor lesions of superficial spreading and nodular malignant melanoma. Hum Pathol 1984;15: 1147–1165.

  37. Clark WH, Elder DE, Guerry D et al. model predicting survival in stage 1 melanoma based on tumor progression. J Natl Cancer Inst 1989;81:1893-1903.

  38. Clark WH, Reimer RR, Greene M, Ainsworth AM, Mastrangelo MJ. Origin of familial malignant melanomas from heritable melanocytic lesions. "The B-K mole syndrome." Arch Dermatol 1978;114: 732–738.

  39. Clark WH, Mihm MC.Lentigo maligna and lentigo maligna melanoma. Am J Pathol 1969;55: 39–54.

  40. Collina G, Losi L, Taccagni GL, Maiorana A. Myxoid metastases of melanoma: report of three cases and review of the literature. Am J Dermatopathol 1997;19:52–57.

  41. Conley J, Lattes R, Orr W. Desmoplastic malignant melanoma (a rare variant of spindle cell melanoma). Cancer 1971;28: 914–936.

  42. Connelly J, Smith JL. Malignant blue nevus. Cancer 1991;67:2653–2657.

  43. Cox NH , Aitchison TC, MacKie RM. Extrafacial lentigo maligna melanoma: analysis of 71 cases and comparison with lentigo maligna melanoma of the head and neck. Br J Dermatol 1998;139: 439–443.

  44. Crotty KA, McCarthy SW, Palmer AA et al. Malignant melanoma in childhood: a clinicopathologic study of 13 cases and comparison with Spitz nevi. World J Surg 1992;16:179–85.

  45. Crowson AN, Magro CM, Clark WH. Pilar neurocristic hamartoma (letter). J Am Acad Dermatol 1996;34:715.

  46. Crowson AN, Magro CM, Mihm MC Jr. Malignant melanoma with prominent pigment synthesis: "animal type" melanoma. Hum Pathol 1999;30: 543–550.

  47. Darier J. Le melanome malin mesenchymateaux ou melano-sarcome. Bull Assoc Fr Cancer 1925;14: 221–249.

  48. Day CL, Harrist TJ, Gorstein F et al. Malignant melanoma. Prognostic significance of "microscopic satellites" in the reticular dermis and subcutaneous fat. Ann Surg 1981;194: 108–112.

  49. Day CL, Lew RA, Mihm MC Jr et al. A multivariate analysis of prognostic factors for melanoma patients with lesions greater than or equal to 3.65 mm in thickness. The importance of revealing alternate Cox models. Ann Surg 1982;195: 44–49.

  50. DeMatos P, Tyler D, Seigler HF. Mucosal melanoma of the female genitalia: a clinicopathologic study of forty-three cases at Duke University Medical Center. Surgery 1998;124:38–48.

  51. Egan CA, Bradley RR, Logsdon VK, Summers BK, Hunter GR, Vandrhooft SL. Vulvar melanoma in childhood. Arch Dermatol 1997;133:345–348.

  52. Egbert B, Kempson R, Sagebiel R. Desmoplastic malignant melanoma. A clinicopathologic study of 25 cases. Cancer 1988;62:2033–2041.

  53. Elder DE. Metastatic melanoma. In: Elder DE, ed: Pathobiology of Malignant Melanoma. Basel:Karger, 1987:182–204.

  54. Elder DE, Guerry D IV, Epstein MN et al. Invasive melanomas lacking competence for metastasis. Am J Dermatopathol 1984;6(Suppl 1):55–61.

  55. Enzinger FM. Clear cell sarcoma of tendons and aponeuroses: an analysis of 21 cases. Cancer 1968;18:1163-1174.

  56. Fernandez-Figueras MT, Ariza A, Calatrava A, Puig L, Fernandez-Vasalo A, Ferrandiz C. CD44 and melanocytic tumors: a possible role for standard CD44 in the epidermotropic spread of melanoma. J Cutan Pathol 1996;23:133–139.

  57. Fletcher CDM. Diagnostic Histopathology of Tumors, 2nd Ed'n. New York: Churchill Livingston 2000:1705-8.

  58. Gillham SL, Morrison RG, Hurt MA. Epidermotropic neuroendocrine carcinoma. Immunohistochemical differentiation from simulators, including malignant melanoma. J Cutan Pathol 1991;18: 120–127.

  59. Gorsky M, Epsein JB. Melanomas arising from the mucosal surfaces of the head and neck. Oral Surg Med Oral Pathol Oral Radiol Endod 1998;86: 715–719.

  60. Granter SR, McKee PH, Calonje E, Mihm MC Jr, Busam K. Melanoma associated with blue nevus and melanoma mimicking cellular blue nevus: a clinicopathologic study of 10 cases on the spectrum of so-called 'malignant blue nevus'. Am J Surg Pathol 2001;25:316-323.

  61. Guerry D 4th, Synnestvedt M, Elder DE, Schultz D. Lessons from tumor progression: the invasive radial growth phase of melanoma is common, incapable of metastasis, and indolent. J Invest Dermatol 1993;100: 342S–345S.

  62. Guitart J, Lowe L, Piepkorn M et al. Histologic characteristics of thin metastasizing melanomas: A case-control study of 43 cases. Arch Dermatol 2002 (in press)

  63. Harrist TJ, Rigel DS, Day CL Jr et al. "Microscopic satellites" are more highly associated with regional lymph nodes metastases than is primary melanoma thickness. Cancer 1984;53: 2183–2187.

  64. Heenan PJ, Clay CD. Epidermotropic metastatic melanoma simulating multiple primary melanomas. Am J Dermatopathol 1991;13:396–402.

  65. Heiskanen MA, Bittner ML, Chen Y et al. Detection of gene amplification by genomic hybridization to cDNA microarrays. Cancer Res 2000;60:799-802.

  66. Hendrickson MR, Ross JC. Neoplasms arising in congenital giant nevi: morphological study of seven cases and a review of the literature. Am J Surg Pathol 1981;5:109–135.

  67. Hernandez FJ. Malignant blue nevus: a light and electron microscopic study. Arch Dermatol 1973;107: 741.

  68. Kao GF, Helwig EB, Graham JH. Balloon cell malignant melanoma of the skin. A clinicopathologic study of 34 cases with histochemical, immunohistochemical, and ultrastructural observations. Cancer 1992;69:2942.

  69. Hitchcock MG, White WL. Malicious masquerade: myxoid melanoma. Semin Diagn Pathol 1998;15:195.

  70. Holman CD, Armstrong BK. Cutaneous malignant melanoma and indicators of total accumulated exposure to the sun: an analysis separating histogenetic types. J Natl Cancer Inst 1984;73: 75–82.

  71. Hoorweg JJ, Loftus BM, Hilgers FJ. Osteoid and bone formation in a nasal mucosal melanoma and its metastasis. Histopathology 1987;31:465–468,

  72. Iwamoto S, Odland PB, Piepkorn M, Bothwell M. Evidence that the p75 neurotrophin receptor mediates perineural spread of desmoplastic melanoma. J Am Acad Dermatol 1996;35(5 pt 1):725–731.

  73. Jimbow K, Takahashi H, Miura S, Ikeda S, Kukita A. Biological behaviour and natural course of acral malignant melanoma. Am J Dermatopathol 1984;6(Suppl 1):43–53.

  74. Kato T, Suetake T, Sugiyama Y, Tabata N, Tagami H. Epidemiology and prognosis of subungual melanoma in 34 Japanese patients. Br J Dermatol 1996;134: 383–387.

  75. Kato T, Kumasaka N, Suetake T, Tabata N, Tagami H. Clinicopathological study of acral melanoma in situ in 44 Japanese patients. Dermatology 1996;193: 192–197.

  76. Kato T, Suetake T, Tabata N, Takahashi K, Tagami H. Epidemiology and prognosis of plantar melanoma in 62 Japanese patients over a 28-year period. Int J Dermatol 1999;38: 515–519.

  77. Khan J, Bittner ML, Chen Y, Meltzer PS, Trent JM. DNA microarray technology: the anticipated impact on the study of human disease. Biochem Biophys Acta 1999;1423:M17-M28.

  78. Kim S, Dougherty ER, Chen Y et al. Multivariate measurement of gene expression relationships. Genomics 2000;67:201-209.

  79. Kornberg R, Harris M, Ackerman AB. Epidermotropically metastatic malignant melanoma. Differentiating malignant melanoma metastatic to the epidermis from malignant melanoma primary in the epidermis. Arch Dermatol 1978;114:67–69.

  80. Kurihara K, Sanada E, Yasuda S, Yamasaki H. Desmoplastic melanoma of the gingiva. Oral Surg Med Oral Pathol 1992;74: 201–205.

  81. Leon P, Daly JM, Synnestvedt M, Schultz DJ, Elder DE, Clark WH Jr. The prognostic implications of microscopic satellites in patients with clinical stage I melanoma. Arch Surg 1991;126:1461–1468.

  82. Levene A. On the natural history and comparative pathology of the blue naevus. Ann R Coll Surg Engl 1980;62:327–334.

  83. Levene A. Disseminated dermal melanocytosis terminating in melanoma : A human condition resembling equine melanotic disease. Br J Dermatol 1979;101:197–205.

  84. Levit EK, Kagen MH, Scher RK, Grossman M, Altman E. The ABC rule for clinical detection of subungual melanoma. J Am Acad Dermatol 2000;42(2Pt 1):269-274.

  85. Lodding P, Kindblom LG, Angervall L. Metastases of malignant melanoma simulating soft tissue sarcoma. A clinico-pathological, light- and electron microscopic and immunohistochemical study of 21 cases. Virchows Arch A Pathol Anat Histopathol 1990;417:377–388.

  86. Loftus SK, Chen Y, Gooden G et al. Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis. Proc Natl Acad Sci USA 1999;96:9277-9280.

  87. Massi D, Franchi A, Borgognoni L, Reali UM, Santucci M. Thin cutaneous malignant melanomas (< or = 1.5 mm): identification of risk factors indicative of progression. Cancer 1999;85:1067–1076.

  88. McCarthy SW, Crotty KA, Palmer AA, Mg AB, McCarthy WH, Shaw HM. Cutaneous malignant melanoma in teenagers. Histopathology 1994;24:453–461.

  89. Merkow LP, Burt RC, Hayeslip DW, Newton FT, Slifkin M, Pardo M. A cellular and malignant blue nevus: a light and electron microscopic study. Cancer 1969;24: 888–896.

  90. Mihm MC Jr, Googe PB. Problematic Pigmented Lesions. A Case Method Approach. Philadelphia : Lea and Febiger, 1990, p. 279–370.

  91. Mihm MC Jr, Clemente CG, Cascinelli N. Tumor infiltrating lymphocytes in lymph node melanoma metastases : a histopathologic prognostic indicator and an expression of local immune response. Lab Invest 1996;74 :43–47.

  92. Mishima Y. Cellular blue nevus : Melanogenic activity and malignant transformation. Arch Dermatol 1970;101:104–110.

  93. Montes LF, Vaughan JT, Ramer G. Equine melanoma. J Cutan Pathol 1979;6:234–235.

  94. Moreno A, Lomarca J, Martinez R, Guix M. Osteoid and bone formation in desmoplastic malignant melanoma. J Cutan Pathol 1986;13:128–134.

  95. Mraz-Gernhard S, Sagebiel RW, Kashani-Sabet M, Miller JR 3rd, Leong SP. Prediction of sentinel lymph node micrometastasis by histological features in primary cutaneous malignant melanoma. Arch Dermatol 1998;134:983–987.

  96. Nakleh RE, Wick MR, Rocamora A, Swanson PE, Dehner LP. Morphological diversity in malignant melanomas. Am J Clin Pathol 1990;93:731–740.

  97. Neimann TH, Thomas PA. Melanoma with signet-ring cells in a peritoneal effusion. Diagn Cytopathol 1995;12:241–244.

  98. Nowak M, Fatteh S, Campbell T. Glycogen-rich malignant melanomas and glycogen-rich balloon cell malignant melanomas: frequency and pattern of PAS positivity in primary and metastatic melanomas. Arch Pathol Lab Med 1998;122:353–360.

  99. Oliva E, Quinn TR, Amin MB et al. Primary malignant melanoma of the urethra. A clinicopathologic analysis of 15 cases. Am J Surg Pathol 2000;24: 785-796.

  100. Ozgur F, Akyurek M, Kayikcioglu A, Barista I, Gokoz A. Metastatic malignant blue nevus: a case report. Ann Plast Surg 1997;39:411–415.

  101. Pandey M, Mathew A, Abraham EK, Ahamed IM, Nair. Primary malignant melanoma of the mucous membranes. Eur J Surg Oncol 1998;24:303–307.

  102. Perniciaro C. Dermatopathologic variants of malignant melanoma. Mayo Clin Proc 1997;72:273–279.

  103. Piura B, Rabinovich A, Dgani R. Malignant melanoma of the vulva: report of six cases and review of the literature. Eur J Gynaecol Oncol 1999;20:182–186.

  104. Quinn MJ, Crotty KA, Thompson JF, Coates AS, O'Brien CJ, McCarthy WH. Desmoplastic and desmoplastic neurotropic melanoma: experience with 280 patients. Cancer 1998;83:1128–1135.

  105. Raber G, Mempel V, Jackish C et al. Malignant melanoma of the vulva. Report of 89 patients. Cancer 1996;78: 2353–2358.

  106. Ragnarsson-Olding BK, Kanter-Lewensohn LR, Lagerlof B, Nilsson BR, Ringborg UK. Malignant melanoma of the vulva in a nationwide, 25-year study of 219 Swedish females: clinical observations and pathological features. Cancer 1999;86:1273–1284.

  107. Ragnarsson-Olding B, Johansson H, Rutqvist LE, Ringborg U. Malignant melanoma of the vulva and vagina. Trends in incidence, age distribution, and long-term survival among 245 consecutive cases in Sweden 1960–1984. Cancer 1993;71:1893–1897.

  108. Rapini RP. Spitz nevus or melanoma? Semin Cutan Med Surg 1999;18:56–63.

  109. Reed RJ. Acral lentiginous melanoma. In: New Concepts In Surgical Pathology of the Skin. New York: Wiley, 1976, p. 89–90.

  110. Reed R, Ichinose H, Clark WC, Mihm M. Common and uncommon melanocytic nevi and borderline melanomas. Semin Oncol 1975;2: 119–147, 1975.

  111. Reed RJ. Minimal deviation melanoma. Hum Pathol 1990;21: 1206–1211.

  112. Reed R, Leonard D. Neurotropic melanoma. A variant of desmoplastic melanoma. Am J Surg Pathol 1979;3:301–311.

  113. Riccioni L, Di Tommaso L, Collina G. Actin-rich desmoplastic malignant melanoma. Report of three cases. Am J Dermatopathol 1999;21: 537-541.

  114. Rimm DL. Impact of microarray technologies on cytopathology. Acta Cytologica 2001;452:111.

  115. Ronan SG, Eng AM, Briele HA, Shioura NN, Das Gupta TK. Thin melanomas with regression and metastases. Arch Dermatol 1987;123:1326-1330.

  116. Roses AD. Pharmacogenetics and future drug development and delivery. Lancet 2000;355:1358.

  117. Saida T. Malignant melanoma on the sole: how to detect the early lesions efficiently. Pigment Cell Res 2000;3 (Suppl 8): 135-139.

  118. Scalzo DA, Hida CA, Toth G, Sober AJ, Mihm MC. Childhood melanoma : a clinicopathological study of 22 cases. Melanoma Res 1997;7:63–68.

  119. Scherf U, Ross DT, Waltham M et al. A gene expression database for the molecular pharmacology of cancer. Nat Genet 2000;24:236-244.

  120. Schmoeckel C, Castro CE, Braun-Falco O. Nevoid malignant melanoma. Arch Derm Res 1985;277: 362–369.

  121. Schmoeckel C, Bockelbrink A, Bockelbrink H et al. Low-high malignant melanoma-1. Evaluation of clinical and histological prognosticators in 585 cases. Eur J Cancer Clin Oncol 1983;19:227-235.

  122. Sherlock G, Hernandez-Boussard T, Kasarskis A et al. The Stanford microarray database. Nucleic Acids Res 2001;29:152-155.

  123. Simmons TJ, Martin SE. Fine-needle aspiration biopsy of malignant melanoma: a cytologic and immunocytochemical analysis. Diagn Cytopathol 1991;7:380–386.

  124. Skelton HG, Maciera J, Smith KJ, McCarthy WF, Lupton GP, Graham JH. HMB45 negative spindle cell melanoma. Am J Dermatopathol 1997;19:580–584.

  125. Skelton HG, Smith KJ, Laskin WB et al. Desmoplastic malignant melanoma. J Am Acad Dermatol 1995;32:717–725.

  126. Smithers BM, McLeod GR, Little JH. Desmoplastic melanoma: patterns of recurrence. World J Surg 1992;16:186–190.

  127. Spatz A, Calonje E, Handfield-Jones S, Barnhill RL. Spitz tumors in children: a grading system for risk stratification. Arch Dermatol 1999;135: 282–285.

  128. Spitz S. Melanomas in childhood. Am J Pathol 1948;24:591–609.

  129. Su YA, Bittner ML, Chen Y et al. Identification of tumor-suppressor genes using human melanoma cell lines UACC903, UACC903 (+6), and SRS3 by comparison of expression profiles. Mol Carcinogen 2000;28:119-127.

  130. Taran JM, Heenan PJ. Clinical and histological features of level 2 cutaneous malignant melanoma associated with metastasis. Cancer 2001;91:1822-1825.

  131. Toda S, Heasley DD, Mihm MC. Osteogenic melanoma: stromal metaplasia in association with subungual melanoma. Histopathology 1997;31:293–295.

  132. Tsang WY , Chan JK, Chow LT. Signet-ring melanoma mimicking adenocarcinoma. A case report. Acta Cytol 1983;37:559–562.

  133. Tuthill RJ, Clark WH, Levene A. Pilar neurocristic hamartoma. Its relationship to blue nevus and equine melanotic disease. Arch Dermatol 1982;118:592–596.

  134. White WL, Hitchcock MG. Dying dogma: the pathological diagnosis of epidermotropic metastatic malignant melanoma. Semin Diagn Pathol 1998;15:176–188.

  135. Vollmer RT, Seigler HF. A model for pretest probability of lymph node metastasis from cutaneous melanoma. Am J Clin Pathol 2000;114: 875-879.

  136. Wong TY, Duncan LM, Mihm MC Jr. Melanoma mimicking dermal and Spitz's nevus ("nevoid melanoma"). Semin Surg Oncol 1993;9:188–193.

  137. Wong TY, Suster S, Duncan LM, Mihm MC Jr. Nevoid melanoma: A clinicopathological study of seven cases of malignant melanoma mimicking spindle and epithelioid cell nevus and verrucous dermal nevus. Hum Pathol 1995;26:171–179.

  138. Van Krieken JH, Boom BW, Scheffer E. Malignant transformation in a naevus of Ito: a case report. Histopathology 1988;12:100–102.

  139. Yusuoka N, Ueda M, Ohgami Y, Hayashi K, Ichihashi M. Amelanotic acral lentiginous malignant melanoma. Br J Dermatol 1999;141: 370–372.

  140. Zelger BG, Steiner H, Wambacher B, Zelger B. Malignant melanomas simulating various types of soft tissue tumors. Dermatol Surg 1997;23:1047–1054.

  141. Zembowicz A, McCusker M, Chiarelli C et al. Morphological analysis of nevoid melanoma: a study of 20 cases. Am J Dermatopathol 2001;23:167-175.