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Background
Melanoma is one of the deadliest skin cancers but with increasing awareness, it can be cured in its earliest stage. It is, however, one of the fastest growing cancers in the western world . Most melanomas are discovered as a suspicious changing mole. In fact, any change in a preexisting mole should prompt a visit to a physician. Increased sun exposure is certainly an important risk factor but melanoma is often a result of a complex interaction of race, sex, and genetics.
Melanoma can be recognized by following this simple chart.
THE ABCD's OF MELANOMA
A Assymetrical B Border irregularity C Color change D Diameter enlarging The outlines below have a detailed discussion of the disease.
The pathologist's task in diagnosing melanoma is first to distinguish it from benign moles (nevi) and other melanocytic proliferations. The dysplastic nevus has varying degrees of cytologic and architectural atypia that can sometimes histologically mimic a melanoma. There are a number of atypical melanocytic proliferations have been termed minimal deviation melanomas and nevoid melanomas. These latter two entities are indeed melanomas but have subtle histologic features that mimic benign nevi. If there is any doubt about the diagnosis, a review of the original tissue slide by a dermatopathologist should be sought. However, it must be stressed that even amongst experts, differences of opinion may occur. Sometimes additional consultation with several dermatopathologists is needed.
In an effort to sharpen the diagnostic criteria to distinguish between atypical moles and melanomas, numerous studies utilizing immunoperoxidase markers and other genetic probes have been undertaken. In general, there is no convincing evidence that any special stain or marker can definitely distinguish between benign and malignant.
The next task is to determine whether the melanoma is in the radial or vertical growth phase. All melanomas except nodular melanomas have a radial growth phase (see below). Melanomas confined to the epidermis are termed melanoma in-situ. In this stage, the melanoma is completely curable if it has been completely excised. At some point, the melanoma cells may invade into the underlying papillary dermis. Even in this early stage, the melanoma has a very limited potential for metastasis and some investigators have termed this stage an invasive radial growth phase. Key features which exclude this diagnosis include the presence of mitotic figures within these dermal melanocytes, a distinctly different cytologic appearance of these dermal melanocytes as compared to the intraepidermal melanocytes, and the formation of an expansile nodule within the papillary dermis. Once a melanoma has entered into the vertical growth phase, it definitely has the capacity to metastasize.
If the melanoma is in the vertical growth phase, measurements and staging must be given (see Clark's level and Breslow thickness below). In addition, parameters such as ulceration, regression, vascular-lymphatic invasion, satellite lesions, or perineural invasion should also be commented upon. Regression may not be a familiar term. This is the host body's response to the melanoma and may be observed clinically as a scar or depigmentation in a previously pigmented mole or melanoma. Histologically, the melanoma has areas replaced by a scar with a heavy lymphoplasmacytic inflammatory cell infiltrate. Some investigators believe that the presence of regression in thin melanomas (<1.5 mm thickness) portends a poorer prognosis. One explanation may be that regression may obscure the fact the melanoma was more deeply invasive in the past, and thus more aggressive. Satellite lesions are clinically distinct pigmented lesions located apart from the main tumor. These represent an intraepithelial or in-transit metastasis of the melanoma and thus are a poor prognostic factor.
Approximately 2% of melanomas may present with no recognizable primary skin lesion. A careful search of the patient, which can be aided with an ultraviolet lamp is necessary. Occasionally a history is elicited of a mole or pigmented lesion which had disappeared. In many cases, the assumption is a primary skin melanoma has undergone regression. In addition, there are a number of rare histologic variants of melanoma usually reserved for specialized textbooks of dermatopathology and case reports in pathology journals. Some of these variants produce some of the most challenging diagnoses in all of pathology. Indeed, these variants may mimic every known malignancy. The alert pathologist can rely upon a battery of immunoperoxidase stains to differentiate between these tumors. The skin is the most common site but melanoma has been described in almost every organ, including the eye. In these rare locations, a metastasis from a primary skin tumor must always be ruled out. Even this task may not be straightforward.
OUTLINE
Epidemiology Incidence
Age
Risk factors
Race and geographyDisease Associations Basal Cell Carcinoma
Dermatomyositis
HIV
Lichen sclerosus
Lymphoma
Melanoma associated retinopathy
Pancreatic cancer
Pregnancy
TraumaPathogenesis Ultraviolet light
Angiotropism
Animal models
APC Gene
Beta catenin
BRAF gene
Cadherin
Chromosomal abnormalities
Cyclin D1
Erzin
Insulin-Like Growth Factor Binding Protein 2
Ku
Loss of heterozygosity
p27
Protease-activated Receptors
PTEN
Regression
Retinoblastoma gene
RGS1
Transforming Growth Factor-Beta
Tumor doubling time
Tyrosine kinase
Ultraviolet lightLaboratory/Radiologic/
Other Diagnostic TestingDermoscopy
CD95 circulating
Circulating melanoma cells in peripheral blood
HypercalcemiaGross Appearance and Clinical Variants Diffuse melanosis associated with metastatic disease
Multiple primary melanomas
Melanoma of unknown primary
Extracutaneous Melanomas
Small melanomas
Histopathological Features and Variants Special Stains/
Immunohistochemistry/
Electron MicroscopyDifferential Diagnosis Staging Prognosis
Treatment Commonly Used Terms Breslow thickness
Clark's level
Ocular melanoma
Pagetoid
Radial and vertical growth phaseInternet Links
DISEASE ASSOCIATIONS CHARACTERIZATION BASAL CELL CARCINOMA
- A Collision Tumor Involving Basal Cell Carcinoma and Lentigo Maligna Melanoma.
Belisle A, Gautier MS, Ghozali F, Plantier F, Wechsler J.
From the *Department of Pathology, Henri Mondor University Hospital, University Paris-Val-de-Marne, Creteil, France; dagger
Department of Dermatology,
Henri Mondor University Hospital, University Paris-Val-de-Marne, Creteil, France; and double daggerDepartment of Pathology, Tarnier-Cochin University Hospital, Paris, France.
Am J Dermatopathol. 2005 Aug;27(4):319-321. Abstract quote
Basal cell carcinomas (BCC) are known to co-exist with other cutaneous lesions, but the collision of BCC with malignant melanoma is rare.
We report on the case of an 82-year-old woman with a translucent papule set on a beige-brown plaque on the right side of the nose. Histologic examination showed lesions of lentigo maligna melanoma (LMM) in situ and invasive melanoma involving nests of BCC that invaded the dermis. Immunohistochemical studies with S100 protein, HMB-45, and Melan-A antibodies showed the melanocytic component in the epidermis and dense clusters of "atypical" melanocytes in the dermal nests of BCC.
On examination of the biopsy specimen, melanoma was still in situ because it was limited to the nests of BCC and not detectable between dermal collagen bundles. However, the re-excision of the lesion showed residual BCC and invasive LMM, level II, measuring 0.2 mm in thickness. The diagnosis, pathogenesis, and prognosis of this collision tumor are discussed.DERMATOMYOSITIS
J Am Acad Dermatol. 2006 Feb;54(2):221-6. Abstract quote
BACKGROUND: Dermatomyositis (DM) is an inflammatory connective tissue disorder well recognized as a paraneoplastic syndrome in adults.
OBJECTIVE: The objective of this study was to assess the prognosis of DM associated with malignant melanoma (MM).
PATIENTS AND METHODS: We systematically searched databases (PubMed, MEDLINE, and WEB OF SCIENCE) for articles reporting the concurrence of DM and MM. For the literature study, time of onset of DM in relation to diagnosis of MM (before, concomitant with, or after), stage of MM after restaging (according to the American Joint Committee on Cancer [AJCC] guidelines, 2001), and survival time after diagnosis of DM were recorded. Survival time studies and univariate statistical analyses were performed. Furthermore, we present our own clinical case of a patient with DM concomitantly occurring with regional lymph node metastasis of MM.
RESULTS: In 5 cases DM occurred before, in 6 cases concomitantly with, and in 6 cases after progression of MM. Univariate analysis identified the AJCC stage of MM as a significant prognostic factor. Gender, age, and the time interval between onset of DM and progression of melanoma were unrelated. The 1-year actuarial survival rate was 0% for patients with DM when occurring with MM at stage IV and 60% when occurring with MM at stage III (P < .05). The estimated mean survival time was 6.6 months for patients with MM stage IV and 57 months for stage III.
LIMITATIONS: The conclusions from this study are limited by the relatively small number of articles that reported the association of MM and DM.
CONCLUSION: DM occurring in patients with MM at stage IV is connected with an extremely poor prognosis, whereas the few reported patients with DM and MM at stage III, including our case, have a prognosis similar to stage III patients without DM.HIV-1 INFECTION
Altered Clinical Course of
Malignant Melanoma in
HIV-Positive Patients.Rodrigues LK, Klencke BJ, Vin-Christian K, Berger TG,
Crawford RI, Miller JR 3rd,
Ferreira CM, Nosrati M, Kashani-Sabet M.University of California, San Francisco, Melanoma Center,
UCSF Comprehensive Cancer
Center,
1600 Divisadero St,
San Francisco, CA 94115.
Arch Dermatol 2002 Jun;138(6):765-70 Abstract quote OBJECTIVE: To determine whether the natural history of melanoma is different in patients who test positive for human immunodeficiency virus (HIV) compared with matched control subjects.
DESIGN: Retrospective cohort analysis.
SETTING: Ambulatory care at 2 university-affiliated medical centers. PATIENTS: Each HIV-positive melanoma patient (n = 17) was randomly matched with 2 HIV-negative patients (HIV status unknown, but without risk factors for HIV) based on the melanoma subtype, tumor thickness, Clark level, tumor location, and sex and age of the patient.
MAIN OUTCOME MEASURES: Disease-free survival and overall survival of HIV-positive and HIV-negative melanoma patients were compared using a matched-pairs analysis. CD4 cell counts were recorded at the time of melanoma diagnosis and disease recurrence.
RESULTS: Melanoma patients who were HIV positive had a significantly shorter disease-free survival (P =.03) and overall survival (P =.045) compared with HIV-negative melanoma patients by matched-pairs analysis. There was an inverse relationship between CD4 cell counts and time to first melanoma recurrence.
CONCLUSIONS: The natural history of malignant melanoma in HIV-positive patients is more aggressive compared with matched HIV-negative melanoma patients. Altered immune response and comorbid disease may play a role in the poor clinical outcome of HIV-positive patients. These findings have important implications in the management of melanoma in the setting of HIV disease.
LICHEN SCLEROSUS Melanocytic Proliferations
Associated With Lichen SclerosusJ. Andrew Carlson, MD, FRCPC; Xiao C. Mu, MD, PhD; Andrzej Slominski, MD, PhD; Kaare Weismann, MD, PhD; A. Neil Crowson, MD; John Malfetano,
MD; Victor G. Prieto, MD, PhD;
Martin C. Mihm, Jr, MDArch Dermatol. 2002;138:77-87 Abstract quote
Objectives
To describe the clinicopathologic features of melanocytic proliferations associated with lichen sclerosus (LS) and to compare these findings with those in controls. Design Cohort study.Setting
Academic and private practice dermatology and dermatopathology services.Patients
Cases of melanocytic proliferations associated with LS and consecutive controls with persistent (recurrent) melanocytic nevi, persistent malignant melanomas, and compound melanocytic nevi.Main Outcome Measures
Diagnostic criteria and disease recurrence.Results
Eleven patients, all female, with a mean age of 40 years (range, 8-83 years), presented with pigmented lesions clinically suspected to be malignant melanoma or atypical melanocytic nevi affecting the vulva (7 patients), perineum (3 patients), or chest (1 patient). Lichen sclerosus was first identified in the biopsy specimen and subsequently confirmed clinically. In 10 cases, a melanocytic nevus was superimposed on LS (overlying or entrapped by sclerosis), whereas LS was found at the periphery of vulvar malignant melanoma. After complete excision, no recurrences have been reported for the melanocytic nevi in LS (mean follow-up, 29 months; range, 4-60 months). Compared with control lesions, the LS melanocytic nevi most closely resembled persistent melanocytic nevi and could be distinguished from persistent malignant melanoma histologically. Melanocytes, nevoid or malignant, proliferating contiguously with fibrotic or sclerotic collagen, contained abundant melanin, diffusely expressed HMB-45, and had a higher Ki-67 labeling index than ordinary melanocytic nevi. However, persistent malignant melanoma exhibited mitotic figures, significantly higher Ki-67 labeling index, and deep dermal HMB-45 expression compared with LS melanocytic nevi and persistent melanocytic nevi.Conclusions
Melanocytic nevi occurring in LS have features in common with persistent melanocytic nevi and can mimic malignant melanoma. An "activated" melanocytic phenotype is seen in LS melanocytic nevi, implicating a stromal-induced change.LYMPHOMA Monoclonal proliferation of
germinal center cells
(incipient follicular lymphoma)
in an axillary lymph node of a melanoma patientGiancarlo Pruneri, MD
Giovanni Mazzarol, MD
Michela Manzotti, BSc
Giuseppe Viale, MD, FRCPathHum Pathol 2002;32:1410-1413. Abstract quote
A monoclonal proliferation of germinal center cells within a lymph node follicle was incidentally discovered during the staging surgical procedures in a patient with Clark III–level cutaneous melanoma.
In one of the 19 axillary lymph nodes examined, we identified a single morphologically atypical lymphoid follicle, predominantly composed of medium-sized cells and immunoreactive for B-cell antigens and for the markers of germinal center origin CD10 and bcl-6. A monoclonal rearrangement of the immunoglobulins heavy chains (IgH) was documented by polymerase chain reaction after laser capture microdissection. The cells of the aberrant follicle expressed the bcl-2 protein at higher levels than the surrounding T lymphocytes in the absence of bcl-2 gene rearrangement.
We propose for this lesion the designation of incipient follicular lymphoma. The present findings also confirm the previously reported association between melanoma and lymphoproliferative disorders.
A single-institution case series of patients with cutaneous melanoma
and non-Hodgkin's lymphomaHensin Tsao, MD, PhD
Kimberly Kwitkiwskib
Arthur J. Sober, MD
Boston, MassachusettsJ Am Acad Dermatol 2002;46:55-61 Abstract quote
Background: Over the past two decades, cutaneous melanoma (CM) and non-Hodgkin's lymphoma (NHL) have both experienced unabated increases in incidence. Population-based studies have documented an elevated risk of subsequent NHL among patients with CM and vice versa.
Methods: To better characterize the clinical features of patients who have had both malignancies, we retrospectively identified all patients with CM at the Massachusetts General Hospital (MGH) who subsequently had NHL and all NHL patients who later developed CM.
Results: A total of 2461 CM and 2708 NHL patients were registered at MGH between 1973 and 1998. Out of these groups, 10 CM patients (0.4%) eventually developed NHL and 11 NHL patients (0.4%) subsequently had CM. The mean age at the first CM diagnosis was 56 years, wheras the mean age at the first NHL diagnosis was 67 years. The mean interval to NHL among the CM group was 119 months, whereas the mean interval to CM among the NHL patients was 36 months. All primary CMs were relatively thin (0.95 mm, first diagnosis; 1.07 mm, second diagnosis), and most NHL subtypes were indolent in nature.
Conclusion: The rates of developing a second NHL among CM patients and vice versa are low. Any interactions between CM and NHL may be due to factors such as detection bias, shared environmental ultraviolet carcinogenesis, or possibly, posttreatment effects.
MELANOMA ASSOCIATED RETINOPATHY Am J Ophthalmol 1988;106:307–11.
Invest Ophthalmol Vis Sci 2000;41:262–6.
Invest Ophthalmol Vis Sci 1993;34:91–100.Patients have autoantibodies that cross-react with the tumor cells and an unknown antigen in rod bipolar cells of the retina, leading to loss of rod function (night blindness)
Corticosteroid Treatment for Melanoma-Associated Retinopathy Effect on Visual Acuity and Electrophysiologic Findings
Caroline Jacobzone, MD ;Catherine ;
Cochard-Marianowski, MDIngrid Kupfer, MD ;Samia Bettembourg, MD ;Yves Dordain, MD ;Laurent Misery, MD ;Beatrice Cochener, MD ;Bruno Sassolas, MD
Arch Dermatol. 2004;140:1258-1261. Abstract quote Background Visual disturbance in the course of melanoma is rare. Specific localized metastases and drug toxic effects are frequently the cause. Recognition of a retinopathy raises several questions when the diagnosis of melanoma-associated retinopathy (MAR) can be confirmed. Descriptions of such patients in dermatologic literature are rare and deserve attention because therapeutic decisions are mandatory.
Observations A 70-year-old woman had a first melanoma in 1985 and a second primary melanoma in 1994. Axillary lymph node involvement occurred in November 2000, leading to surgery and chemotherapy. In December 2001, she had sudden bilateral visual loss, with shimmering blobs of color and flickering photopsias. Computed tomography and cerebral magnetic resonance imaging ruled out localized tumor on the eyes or optic nerves or evolution of disease. Ophthalmologic examination revealed a bilateral posterior uveitis, with hyalitis and progressive destruction of retinal pigment. The electrophysiologic data confirmed the diagnosis of MAR. Symptoms improved after systemic corticosteroid therapy, with no relapse after tapering doses despite worsening of melanoma.
Conclusions As a rare paraneoplastic visual syndrome possibly leading to blindness, MAR is characterized by bipolar cell involvement without photoreceptor cell impairment. Also, MAR is linked to the presence of autoantibodies directed against melanoma antigens that cross-react with the rod bipolar cells of the retina. Corticosteroid therapy is rarely beneficial. Our case of MAR is noteworthy because it involved a woman, was associated with an uveitis, and improved with corticosteroid therapy.
PANCREATIC CANCER
Pancreatic carcinoma surveillance in patients with familial melanoma.Parker JF, Florell SR, Alexander A, DiSario JA, Shami PJ, Leachman SA.
Department of Dermatology and Huntsman Cancer Institute and the Divisions of Gastroenterology and Oncology, University of Utah, Salt Lake City, Utah 84112, USA.
Arch Dermatol. 2003 Aug;139(8):1019-25. Abstract quote OBJECTIVE: To determine the optimal methods for pancreatic adenocarcinoma surveillance in high-risk patients with familial melanoma and cyclin-dependent kinase inhibitor 2A (CDKN2A) mutations.
DESIGN: Case report with pedigree analysis and literature review, with an emphasis on guideline development for high-risk kindreds with familial pancreatic adenocarcinoma.
SETTING: A university-affiliated familial melanoma research clinic.Patients The proband was referred as a participant in a research clinic protocol and was found to carry a germline CDKN2A mutation and have a history of melanoma and pancreatic adenocarcinoma. A total of 179 family members were identified through the Utah Population Database and underwent evaluation for history of melanoma and pancreatic adenocarcinoma.
Intervention/ METHODS: Comprehensive family history and pedigree analysis performed by means of personal interview, medical record review, and use of cancer registry and population database records. Mutation status was confirmed by results of DNA sequence analysis. Tumor identity was confirmed with immunohistochemical markers.
MAIN OUTCOME MEASURES: Estimated risk for pancreatic adenocarcinoma in a high-risk family with CDKN2A-positive melanoma. Guidelines for surveillance in these families were based on review of the literature.
RESULTS: Sequence analysis confirmed a CDKN2A mutation, and immunohistochemical evaluation confirmed the diagnoses of metastatic melanoma and metastatic pancreatic adenocarcinoma. Pedigree analysis showed an observed-expected ratio of 8.9 to 12.6 for pancreatic adenocarcinoma and 16.4 to 20.8 for melanoma in this family. Guidelines used for surveillance of kindreds at high risk for pancreatic adenocarcinoma were applied to families with CDKN2A melanoma.
Conclusion Patients with melanoma and a germline CDKN2A mutation should be considered for pancreatic adenocarcinoma surveillance that is based on the most recent published studies.
Phenotypic variation in eight extended CDKN2A germline mutation familial atypical multiple mole melanoma-pancreatic carcinoma-prone families: the familial atypical mole melanoma-pancreatic carcinoma syndrome.Lynch HT, Brand RE, Hogg D, Deters CA, Fusaro RM, Lynch JF, Liu L, Knezetic J, Lassam NJ, Goggins M, Kern S.
Department of Preventive Medicine, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.
Cancer 2002 Jan 1;94(1):84-96 Abstract quote BACKGROUND: Hereditary pancreatic carcinoma shows extant phenotypic and genotypic heterogeneity as evidenced by its integral association with a variety of hereditary cancer syndromes inclusive of the familial atypical multiple mole melanoma (FAMMM) syndrome in concert with CDKN2A (p16) germline mutations.
METHODS: Creighton University's familial pancreatic carcinoma resource comprises 159 families of which 19 (12%) show the FAMMM cutaneous phenotypes. The authors describe eight families with the FAMMM-pancreatic carcinoma (FAMMM-PC) association in concert with a CDKN2A germline mutation. Each family was thoroughly educated about all facets of the study, including the molecular genetics, reduced penetrance of CDKN2A mutations, and their variable expressivity. Genetic counseling was provided to each patient.
RESULTS: Diversity in cancer presentation within and among the families was noteworthy, wherein melanoma predominated in certain of the families whereas pancreatic carcinoma predominated in others. Early-onset pancreatic carcinoma (at ages 35, 45, 46, and 49 years) appeared in some of the families whereas markedly later-onset pancreatic carcinoma occurred in others. There were four incidences of melanoma and pancreatic carcinoma as double primaries in the same individuals. One patient with melanoma and pancreatic carcinoma had a third primary of breast carcinoma. Another patient had sarcoma, esophageal carcinoma, and two melanoma primaries, whereas his daughter had sarcoma and was a carrier of a CDKN2A mutation.
CONCLUSIONS: The authors suggest that these tumors may collectively, in concert with CDKN2A mutations, constitute a "new" putative hereditary carcinoma syndrome referred to as FAMMM-PC. More clinical and molecular genetic research on additional families with pancreatic carcinoma in concert with the FAMMM will be required.
PREGNANCY
Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
For many years, clinicians have been concerned about a potential adverse effect of pregnancy-associated hormones and exogenous hormones on melanocytic nevi and malignant melanoma.
Today, these issues are more significant as women have delayed childbearing into their 30's and 40's, and the likelihood of diagnosis with melanoma during pregnancy is enhanced.
More recent clinical, epidemiologic, and laboratory studies have shed some light on the relationship among hormones, nevi, and melanoma in pregnancy.TRAUMA Trauma and melanoma formation: a true association?
Kaskel P, Kind P, Sander S, Peter RU, Krahn G.
Department of Dermatology, University of Ulm, 89070 Ulm/Donau, Germany.
Br J Dermatol 2000 Oct;143(4):749-53 Abstract quote
BACKGROUND: Little is known about the role of mechanical trauma in the pathogenesis of malignant melanoma. In individual patients, traumatic events have been discussed as a causative factor for the induction of melanoma and diagnosis of melanoma following trauma may raise medico-legal questions.
OBJECTIVES: To evaluate the relationship between traumatic single or recurrent events and melanoma characteristics.
METHODS: Retrospective questionnaire in 369 melanoma patients.
RESULTS: A large number of patients (337 of 369; 91.3%) denied an association between a possible traumatic event and melanoma formation. Thirty-two of 369 patients (8.7%) considered an association of trauma and melanoma formation likely. Of these 32 patients, 22 patients (13 men, nine women) reported a single event, and 10 patients (four men, six women) a persisting irritation. An irritation of a pre-existing melanocytic naevus was reported by two patients with histologically confirmed melanoma on acquired or congenital naevus.
CONCLUSIONS: As most of the patients who mentioned a trauma in this study suffered from acral melanoma, or melanoma located on the extremities, a history of trauma should be expected more frequently at these body sites. A review of epidemiological, clinical and scientific research indicates that there seems to be no evidence for single or persistent traumatic events as a causative factor for melanoma formation.
PATHOGENESIS CHARACTERIZATION ANGIOTROPISM
Pericytic-like angiotropism of glioma and melanoma cells.Lugassy C, Haroun RI, Brem H, Tyler BM, Jones RV, Fernandez PM, Patierno SR, Kleinman HK, Barnhill RL.
Am J Dermatopathol 2002 Dec;24(6):473-8 Abstract quote We have identified in malignant melanoma an angiotumoral complex in which tumor cells occupy a pericytic location along the endothelium of microvessels without evidence of intravasation.
We have suggested that this pericytic-like angiotropism could be a marker of an extravascular migration of tumor cells along the abluminal surface of vessels. The extravascular migratory metastasis proposed for melanoma has close analogies with glioma migration.
To compare our hypothesis of extravascular migration by melanoma with the migration of glioma cells, we have used the B16 murine melanoma cell line and the GL26 murine glioma cell line in an in vivo murine brain tumor model and in vitro using endothelial cells that have formed capillary-like structures and have been cocultivated with tumor cells. In the brain tumors, a clear progression of glioma and melanoma cells was observed along the abluminal surface of vessels, where they occupied a pericytic location along the periendothelial laminin. In vitro, time-lapse videomicroscopy recorded the migration of tumor cells toward endothelial tubules. After 24 hours, both the melanoma cells and the glioma cells were localized along the external surfaces of the vascular tubules, occupying a pericytic-like location.
These similarities between glioma and melanoma support the hypothesis of an extravascular migration of melanoma cells, particularly along the abluminal surface of vessels.
ANIMAL MODELS
Malignant melanoma in the Sinclair miniature swine: an autopsy study of 60 cases.Oxenhandler RW, Adelstein EH, Haigh JP, Hook RR Jr, Clark WH Jr.
Am J Pathol 1979 Sep;96(3):707-20 Abstract quote Sinclair miniature swine spontaneously develop multiple cutaneous melanomas which have the ability to metastasize and regress.
This study, based on 60 necropsies, documents the similarity of the pathology of the cutaneous malignant melanomas and the organ distribution of metastasis to human melanoma. The invasive cutaneous melanomas have an intraepidermal component analogous to human superficial spreading melanoma. The pathology of the spontaneous regression, characterized by a series of cellular events beginning with a mononuclear inflammatory infiltrate and leading to depigmentation and fibrosis, is likewise similar to cutaneous regression in human melanoma. Just as with human melanoma,metastasis was correlated with deeply invasive cutaneous tumors.
Because of both the biologic and histologic similarity of this animal model to human melanoma, the Sinclair miniature swine should serve as an important resource in continuing the study of melanoma.
Histopathology of regression in sinclair swine model of melanoma.
Greene JF Jr, Townsend JS 4th, Amoss MS Jr.
Department of Pathology, Scott & White Clinic, Temple, Texas.
Lab Invest 1994 Jul;71(1):17-24 Abstract quote BACKGROUND: Detailed histopathologic studies of melanomas occurring in neonatal Sinclair miniature swine have demonstrated a remarkable similarity to human melanoma. A significant difference is the predictable, complete regression of primary and metastatic tumors that occurs in all animals by early adulthood (1 to 2 years). Prior histopathologic descriptions of regression in this model have been incomplete with regard to the time of onset and chronologic sequence of events. This lack of data makes it difficult to plan studies of regression mechanisms especially when requiring the harvesting of tumor tissue.
EXPERIMENTAL DESIGN: By routine histologic methods, 94 tumors from 46 piglets were evaluated for the degree of regression, presence of pigment-laden macrophages, and presence of lymphocytes. One or more punch biopsies were performed on 51 tumors before excision, for a total of 256 biopsies.
RESULTS: Regression took place in two phases. The first phase began during the 4th week after birth; was preceded by a rapid, massive infiltration of pigment-laden macrophages; and was most active during the 2nd month. Significant numbers of lymphocytes were rarely seen in tumors during this phase of regression. In the vast majority of tumors, this initial regression activity was followed by regrowth of residual tumor usually appearing as emerging clones (intralesional transformation). The second phase of regression was characterized by asymmetrically distributed lymphocytic infiltration of the residual melanoma, and progressive regression of tumor over several months. Significant numbers of lymphocytes were not present in the majority of the tumors until the beginning of the 4th month.
CONCLUSIONS: We conclude that regression of melanoma in this animal model is a complex event in which the immune system participates differentially during the natural history of the disease.
Antimelanoma antibodies in swine with spontaneously regressing melanoma.Cui J, Chen D, Misfeldt ML, Swinfard RW, Bystryn JC.
Ronald O. Perelman Department of Dermatology, New York University Medical Center, New York, USA.
Pigment Cell Res 1995 Feb;8(1):60-3 Abstract quote Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth.
To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS-PAGE analysis of 125I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68-75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68-75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P < 0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation.
These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo-like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.
ROLE OF APAF-1 GENE Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.
Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald WL, Lazebnik YA, Cordon-Cardo C, Lowe SW.
Cold Spring Harbor Laboratory, New York 11724, USA.
Nature 2001 Jan 11;409(6817):207-11 Abstract quote
Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma.
Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss.
We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.
APF GENE Analysis of adenomatous polyposis coli gene expression, APC locus-microsatellite instability and APC promoter methylation in the progression of melanocytic tumours.
Korabiowska M, Schlott T, Siems N, Muller A, Cordon-Cardo C, Fischer G, Brinck U.
1Department of Cytopathology, University of Gottingen, Gottingen, Germany.
Mod Pathol. 2004 Dec;17(12):1539-44. Abstract quote
Adenomatous polyposis coli gene (APC) defects have been demonstrated for the first time in familial adenomatous polyposis. Recent reports indicate that the APC gene is an intermediary between cell adhesion molecules and the cytoskeleton and that it may function as a gatekeeper of colonic epithelial proliferation.
The objective of this study was to analyse APC's presence in lentigos, primary melanomas and melanoma metastases. By immunohistochemistry, APC was demonstrated in all lentigos, in 75 out of 88 primary melanomas and in 16 out of 28 melanoma lymphatic metastases. The percentage of immunolabelled tumour cells (APC index) in lentigos ranged between 5 and 69%, in primary melanomas between 0 and 98% and in melanoma metastases between 0 and 52%. Statistically significant differences between lentigos and primary melanomas and between lentigos and metastases in APC expression were found.
In a multivariate analysis, APC showed an independent prognostic impact. Analysis of microsatellite instability in the APC locus was performed on 29 melanomas. Microsatellite instability was found in 5/29 melanomas and loss of heterozygosity in 1/29 melanomas. Promoter methylation of APC was found in 6/10 APC-negative primary melanomas and in 9/10 APC-negative melanoma lymphatic metastases investigated.
We conclude about important role of APC alterations for melanoma progression.BETA CATENIN
Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations.Demunter A, Libbrecht L, Degreef H, De Wolf-Peeters C, van den Oord JJ.
Department of Pathology, Laboratory of Morphology and Molecular Pathology, University Hospitals, Katholieke Universiteit Leuven, Belgium.
Mod Pathol 2002 Apr;15(4):454-61 Abstract quote beta-Catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also plays a role in the Wnt signaling pathway by activating T-cell factor- and lymphoid enhancer factor-regulated gene transcription. The level of beta-catenin in cells is tightly controlled in a multiprotein complex, and mutations in the glycogen synthase kinase 3beta (GSK-3beta) phosphorylation sites of the beta-catenin gene (CTNNB1) result in nuclear and/or cytoplasmic accumulation of beta-catenin and constitutive transactivation of T-cell factor and lymphoid enhancer factor target genes, a mechanism occurring in many cancers. Melanoma cell lines may harbor beta-catenin mutations; in vivo, however, cellular accumulation of beta-catenin is rarely caused by CTNNB1 mutations.
In our study, 43 primary cutaneous melanoma and 30 metastases were screened for CTNNB1 exon 3 mutations by using a denaturing gradient gel electrophoresis technique and sequencing. beta-Catenin mutations were found in 2 primary melanomas and 1 metastatic melanoma and were not correlated with nuclear accumulation of beta-catenin in these cases. Cellular expression of beta-catenin was evaluated by immunohistochemistry and by reverse polymerase chain reaction (RT-PCR) in 80 and 70 cases, respectively. Immunohistochemistry revealed a significant loss of membranous beta-catenin staining between the primary and metastatic melanomas as well as between radial and vertical growth phase. RT-PCR showed a significant inverse correlation between the amount of RNA and the proportion of cells with membranous expression of beta-catenin (P =.0015); no correlation existed between the amount of RNA and the number of cells with nuclear or cytoplasmic expression of beta-catenin.
In conclusion, nuclear expression of beta-catenin is seen in cutaneous melanoma but, in contrast to the case of many other cancers, does not correlate with tumor stage or mutation status. A combination of immunohistochemistry and RT-PCR showed that down-regulation of membranous beta-catenin was associated with an increased amount of beta-catenin RNA in primary or metastatic melanoma. Our results suggest that posttranslational events, rather than CTNNB1 mutations, are responsible for the altered distribution of beta-catenin in cutaneous melanoma.
BRAF GENE
- BRAF and c-kit gene copy number in mutation-positive malignant melanoma.
Willmore-Payne C, Holden JA, Hirschowitz S, Layfield LJ.
Department of Pathology, University of Utah, Salt Lake City, UT 84132, USA.
Hum Pathol. 2006 May;37(5):520-7. Abstract quote
Activating mutations in BRAF or c-kit have been reported in malignant melanoma. Because the activating mutations are dominant, it has been assumed that they are heterozygous in the affected tumors.
To test this, we have carefully examined the DNA sequencing electropherograms on 43 BRAF mutation-positive and 3 c-kit mutation-positive malignant melanomas to determine the ratio of the normal to mutant allele. Of the 43 BRAF mutation-positive tumors, we classified 26 as presumptive heterozygous. Eight cases were indeterminate. Surprisingly, 9 cases appeared to contain an excess of the mutant allele. BRAF fluorescence in situ hybridization on these 9 cases suggested the increased amount of the mutant BRAF allele was due to amplification (2 cases) or chromosome 7 polysomy (7 cases).
We have previously described the presence of the c-kit-activating mutation, L576P, in 2 of 100 malignant melanomas. In this report, we have evaluated an additional 53 cases and found 1 additional case that contained the L576P mutation. Evaluation of the DNA sequencing electropherograms from all 3 cases of L576P mutation-positive melanoma suggests a selective loss of the normal allele. Fluorescence in situ hybridization for c-kit on these 3 L576P mutation-positive tumors indicated that one showed slight amplification of the c-kit gene and the other 2 were present in a nonamplified diploid state.
These results have important implications concerning the mechanism of oncogenesis in melanoma as well as in the response of the tumor to anticancer drugs targeting BRAF or c-kit.
J Am Acad Dermatol. 2005 Jul;53(1):108-14. Abstract quote
High frequency of B-RAF gene mutations has recently been identified in benign melanocytic nevi and melanoma.
This review focuses on clinical studies that evaluate the role of B-RAF in melanocytic neoplasia.
- Human malignant melanoma: Detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis.
Willmore-Payne C, Holden JA, Tripp S, Layfield LJ.
Hum Pathol. 2005 May;36(5):486-93. Abstract quote
Summary Activating mutations in the BRAF kinase have been reported in a large number of cases of malignant melanoma. This suggests that therapy with specific RAF kinase inhibitors may find use in treating this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of malignant melanoma for the presence or absence of BRAF mutations.
High-resolution amplicon melting analysis is able to detect single base-pair changes in DNA isolated from paraffin-embedded tissue sections and obviates the need for direct DNA sequencing. Results can be available within 48 hours.
In this report, we used high-resolution amplicon melting analysis to evaluate 90 cases of malignant melanoma for BRAF mutations. Of these 90 cases, 74 were metastatic melanomas, 12 were primary cutaneous melanomas, and 4 were in situ melanomas. BRAF activation mutations were found in 43 cases (48%). Forty-one of these mutations were in exon 15. The mutations in exon 15 included V600E (34 cases), V600K (6 cases), and V600R (1 case). Two activating mutations were found in exon 11, G469V and G469R. The presence or absence of a BRAF mutation in the junctional component of an invasive melanoma was maintained in the invasive component.
We also evaluated these 90 cases, as well as an additional 10 cases (total of 100) for the expression of c-kit. The majority of invasive and metastatic malignant melanomas did not express c-kit, although all in situ lesions and the junctional components of invasive lesions were strongly c-kit positive. Surprisingly, 2 cases of metastatic malignant melanoma (2%) showed strong and diffuse c-kit expression and contained a c-kit-activating mutation, L576P, as detected by high-resolution amplicon melting analysis and confirmed by direct DNA sequencing. These 2 c-kit mutation-positive cases did not contain BRAF mutations. The presence of a c-kit-activating mutation in metastatic malignant melanoma suggests that a small number of melanomas may progress by a somatic mutation of the c-kit gene.
The presence of BRAF- and c-kit-activating mutations in malignant melanoma suggests new approaches to treating this disease involving specific tyrosine kinase inhibitors may prove worthwhile and that mutation analysis by high-resolution melting analysis might help guide therapy.
BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.
Uribe P, Wistuba II, Gonzalez S.
Department of Anatomic Pthology, Medical School, P.Universidad Catolica de Chile, Santiago, Chile.
Am J Dermatopathol. 2003 Oct;25(5):365-70. Abstract quote
BRAF mutations have recently been detected with a high frequency (66%) in cutaneous melanoma. All those mutations are activating, with a single substitution (T1796A) at codon 599 (V599E) accounting for over 90%.To investigate the stage in which those mutations occur in the currently proposed sequential malignant transformation of melanocytes, 22 benign melanocytic nevi, 23 melanocytic atypical nevi, and 25 primary cutaneous melanoma from 63 different patients were examined for BRAF mutations using DNA extracted from microdissected formalin-fixed and paraffin-embedded tissues, and a two-round PCR-RFLP-based strategy. A subset of samples was sequenced for mutation confirmation. Sixteen benign (73%) and eleven atypical (52%) melanocytic nevi, and thirteen melanoma (56%) demonstrated BRAF mutations at codon 599, and no statistically significant differences were detected among all three types of lesions. No mutations were demonstrated in microdissected epidermal keratinocytes adjacent to melanocytic lesions having BRAF mutations. No correlation was detected between BRAF mutational status and age, sun exposure, and Clark's level in malignant melanoma. However, comparing only atypical nevi and melanoma lesions the frequency of BRAF mutation is significantly greater in male (78%) than female (35%) patients (P = 0.0194). The previously described T1796A point mutation was detected in 17 of 18 mutated samples, and a novel mutation consisting of a substitution of valine for lysine (GT1795-96AA) was detected in one melanoma case.
Our findings of a high frequency of BRAF mutations at codon 599 in benign melanocytic lesions of the skin indicate that this mutation is not sufficient by itself for malignant transformation.
Mutations of the BRAF gene in human cancer.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA.
Cancer Genome Project, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK.
Nature 2002 Jun 27;417(6892):949-54 Abstract quote Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer.
The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%.
Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
CADHERIN
Cadherin expression pattern in melanocytic tumors more likely depends on the melanocyte environment than on tumor cell progression.
Krengel S, Groteluschen F, Bartsch S, Tronnier M.
Department of Dermatology, Medical University Lubeck, Lubeck, and Department of Dermatology, Municipal Hospital Hildesheim, Hildesheim, Germany.
J Cutan Pathol. 2004 Jan;31(1):1-7 Abstract quote.
BACKGROUND: Adhesion molecules have been assigned an important role in melanocytic tumor progression. By the loss of E-cadherin, melanocytes might escape the control of neighbouring keratinocytes. Although in vitro data support this hypothesis, there are yet no conclusive immunohistochemical results on cadherin expression in melanocytic tumors.
OBJECTIVE: To gain detailed insight in the expression of cadherins and their cytoplasmic binding partners, the catenins, in various types of benign and malignant melanocytic neoplasms.
METHODS: Immunohistochemical analysis of the expression of E-, P-, and N-cadherin and alpha-, beta-, and gamma-catenin in compound and dermal nevi, Spitz nevi, blue nevi, ultraviolet B (UVB)-irradiated nevi, and malignant melanomas of various tumor thickness.
RESULTS: In both nevi and melanomas, E-cadherin expression in melanocytic cells decreased, following a gradient from junctional to deeper dermal localization. The pattern of E-cadherin expression was more heterogeneous in melanomas than in nevi. In some melanomas, E-cadherin was only weakly positive in the epidermal tumor cells. P-cadherin expression was similar to that of E-cadherin. N-cadherin expression in melanocytic lesions was a rare finding, however, a small percentage of melanomas showed expression in some cell nests. Some Spitz nevi exhibited strong N-cadherin immunoreactivity. Most melanocytic cells were alpha- and beta-catenin-positive and gamma-catenin-negative. UVB irradiation did not influence the expression of cadherins and catenins in melanocytic nevi in vivo.
CONCLUSIONS: It is presumed that the gradual loss of E-cadherin expression represents a reaction of melanocytic cells to altered conditions in the dermal environment, e.g. lack of contact to keratinocytes, or new contact with dermal extracellular matrix molecules, respectively. Melanoma cells apparently are less dependent on these environmental factors and, therefore, show a more heterogeneous expression pattern. This might be of importance for the adaptation of the tumor cells to local requirements. However, in view of our results, a causative role of (loss of ) E-cadherin or (gain of ) N-cadherin for melanocytic tumor progression still remains to be proven.CDKN2A
(Tumor suppressor gene)NEJM 1998;338:879-887
Non familial cases of multiple primary melanomas have germ-line mutations (5/33-15%)Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group.
Soufir N, Avril MF, Chompret A, Demenais F, Bombled J, Spatz A, Stoppa-Lyonnet D, Benard J, Bressac-de Paillerets B.
Unite des Marqueurs Genetiques des Cancers, Institut Gustave Roussy (IGR), 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France.
Hum Mol Genet 1998 Feb;7(2):209-16 Abstract quote
Germline mutations in the p16 and CDK4 genes have been reported in a subset of melanoma pedigrees, but their prevalence is not well known. We searched for such germline mutations in 48 French melanoma-prone families selected according to two major criteria: families with at least three affected members (n = 20) or families with two affected members, one of them affected before the age of 50 (n = 28), and one additional minor criterion. Sixteen different p16 germline mutations were found in 21 families, while one germline mutation, Arg24His, was detected in the CDK4 gene.
The frequency of p16 gene mutation in our sample (44%) is among the highest rates yet reported and the CDK4 mutation is the second mutation detected in this gene worldwide. In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma-predisposing gene.
Low prevalence of germline CDKN2A and CDK4 mutations in patients with early-onset melanoma.
Tsao H, Zhang X, Kwitkiwski K, Finkelstein DM, Sober AJ, Haluska FG.
Department of Dermatology, Massachusetts General Hospital, Bartlett 622, 48 Blossom St, Boston, MA 02114.
Arch Dermatol 2000 Sep;136(9):1118-22 Abstract quote
BACKGROUND: In patients with cutaneous melanoma, early age at disease onset is characteristic in familial cases and in individuals with multiple primary melanomas. Both subsets of patients with melanoma are at risk for harboring germline CDKN2A or CDK4 mutations.
OBJECTIVE: We set out to prospectively determine the prevalence of CDKN2A and CDK4 mutations in a group of young patients with melanoma. DESIGN: We prospectively screened 913 patients over a 6-month period and identified 519 patients with invasive melanomas. We invited 172 patients with melanoma who were younger than 40 years to participate in the study, and 49 patients consented and donated peripheral blood samples. Forty-nine percent (n = 24) of our patients developed cutaneous melanoma before the age of 30 years.
SETTING: A melanoma clinic in the Boston, Mass, area.
MAIN OUTCOME MEASURE: We used a combination of single-strand conformation analysis and direct sequencing of samples of peripheral blood leukocyte DNA to search for mutations in exons 1alpha, 1beta, 2, and 3 of CDKN2A and in exon 2 of CDK4.
RESULTS: The mean and median ages at diagnosis in our group were 30 and 32 years, respectively. Among a group of 49 patients, we detected 1 (2%; 95% confidence interval, 0.07%-10.8%) Met 53 Ile CDKN2A mutation, which was found in a patient with a strong family history of melanoma. This alteration has been previously shown to impair p16 function. One patient had an Ala 148 Thr change in CDKN2A, which has also been shown to be a polymorphism. We also detected a sequence polymorphism (in the 3' untranslated region [3'UTR] of CDKN2A) in 27% of our patients. A similar incidence of this 3'UTR polymorphism was observed in a control population. We found no CDK4 mutations.
CONCLUSIONS: Germline CDKN2A and CDK4 mutations are not common in patients who develop melanoma at an early age. This finding contrasts with other cancer-predisposition syndromes, in which there is an increased incidence of germline mutations among young patients. Selection of patients with melanoma for genetic testing based solely on age at onset may not be warranted at the current time.
CHROMOSOMAL ABNORMALITIES Cancer Res 1988;58:2170-2175
Chromosome 9p21
Other candidates include 12q14 and 1p36
Increased risk of pancreatic cancerInterphase Cytogenetic Analysis of 1q 12 Satellite III DNA in Melanocytic Lesions Increased Aneuploidy With Malignant Histology
J. Dennis Lee, M.D.; Elizabeth R. Unger, Ph.D., M.D.; Cynthia Gittenger, B.S.; Daisy R. Lee, M.S.; Reneé Hebert, Ph.D.; John C. Maize, M.D.
Departments of Dermatology (J.D.L., J.C.M.), Pathology and Laboratory Medicine (C.G.), Biometry and Epidemiology (R.H.), Medical University of South Carolina, Charleston, South Carolina; Department of Health and Human Services Centers for Disease Control and Prevention, Atlanta, Georgia (E.R.U., D.R.L.).
Am J Dermatopathol 2001;23:176-180 Abstract quote
To examine the relationship of chromosome 1 copy number to melanocytic tumorigenesis, interphase cytogenetic analysis of 1q12 satellite III DNA was performed on the spectrum of melanocytic lesions comprising Clark's tumor progression model.
Results showed increased copy number in a ``step off'' pattern between melanoma in-situ and the intraepidermal component of invasive melanoma rather than a progression between each lesional group.
These findings support Clark's concept of independent clonal expansion of a cell population giving rise to the vertical growth phase and further demonstrates increased chromosome 1 copy number as a late event in melanoma tumor progression.
CD95 (FAS) Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.
Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M, French LE, Schneider P, Bornand T, Fontana A, Lienard D, Cerottini J, Tschopp J.
Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland.
Science 1996 Nov 22;274(5291):1363-6 Abstract quote Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells.
In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL.
Thus, FasL may contribute to the immune privilege of tumors.
Human melanoma cells do not express Fas (Apo-1/CD95) ligand.Chappell DB, Zaks TZ, Rosenberg SA, Restifo NP.
Howard Hughes Medical Institute-NIH Research Scholars Program, Bethesda, Maryland 20814, USA.
Cancer Res 1999 Jan 1;59(1):59-62 Abstract quote A recent report described the expression of Fas ligand (FasL) by melanoma cells as an important mechanism involved in the immune evasion by tumors [M. Hahne et al., Science (Washington DC), 274: 1363-1366, 1996].
To investigate the expression of FasL by melanomas, we screened a panel of early-passage cell lines by functional assay and reverse transcriptase-PCR. Using conditions designed to replicate those in the original report, we did not find functional FasL on any of the 19 human melanoma lines established at the National Cancer Institute. Furthermore, we additionally evaluated our melanoma lines using reverse transcriptase-PCR and found that 0 of the 26 human melanoma cell lines expressed FasL mRNA. FasL mRNA was abundantly expressed by anti-melanoma T-cell lines after activation.
These data do not support a role for FasL expression in the escape of melanoma cells from immune destruction.
Blockade of the Fas-triggered intracellular signaling pathway in human melanomas is circumvented by cytotoxic lymphocytes.Ferrarini M, Imro MA, Sciorati C, Heltai S, Protti MP, Pellicciari C, Rovere P, Manfredi AA, Rugarli C.
Laboratorio di Immunologia dei Tumori, Divisione di Medicina II, H San Raffaele Scientific Institute, Milan, Italy.
Int J Cancer 1999 May 17;81(4):573-9 Abstract quote Fas and Fas ligand (FasL) have been found both in lymphoid and in non-lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system.
Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas-induced apoptosis of human T lymphocytes clones. Conversely, cross-linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase-3 activation, pointing to an early alteration in the Fas-triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism.
This suggests that melanoma cells evade immune-mediated Fas-triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas-induced death via granzyme-mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer.
Predominant expression of Fas (CD95) ligand in metastatic melanoma revealed by longitudinal analysis.Terheyden P, Siedel C, Merkel A, Kampgen E, Brocker EB, Becker JC.
Department of Dermatology, School of Medicine, Julius-Maximilians University, Wurzburg, Germany.
J Invest Dermatol 1999 Jun;112(6):899-902 Abstract quote The expression of Fas ligand has recently been proposed as a novel tumor escape mechanism for melanoma.
To establish the characteristics of Fas ligand expression during the course of melanoma progression we performed a longitudinal study analyzing primary tumors as well as subsequently evolving metastases. In primary melanoma Fas ligand was expressed in two of 20 lesions; this expression was weak and restricted to few parts of the tumors.
The Fas ligand positive primary melanomas were rather thick, i.e., 8.5 and 3.8 mm, versus a median of 2.4 mm of the remaining tumors. In contrast, for metastatic melanoma Fas ligand expression was present in six of 11 cases investigated. The metastases of primary tumors displaying Fas ligand maintained its expression. As Fas ligand positive melanoma cells are capable of inducing apoptosis in susceptible cells, e.g., Fas positive tumor infiltrating lymphocytes, we tested for the presence of apoptotic cells in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. This analysis revealed that apoptotic cells were present within the Fas ligand positive tumors. The number of apoptotic cells, however, never exceeded 5% of the total cells.
Thus, Fas ligand mediated apoptosis does not seem to be a major immune escape mechanism for melanoma but its expression correlates with the stage of melanoma.
Alterations of Fas (Apo-1/CD95) gene in cutaneous malignant melanoma.
Shin MS, Park WS, Kim SY, Kim HS, Kang SJ, Song KY, Park JY, Dong SM, Pi JH, Oh RR, Lee JY, Yoo NJ, Lee SH.
Departments of Pathology, Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Am J Pathol 1999 Jun;154(6):1785-91 Abstract quote Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many nonlymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas resistance, may be involved in the pathogenesis of nonlymphoid malignancies as well.
In this study, we have analyzed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 44 human malignant melanomas in skin by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Overall, 3 tumors (6.8%) were found to have the Fas mutations, which were all missense variants and identified in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal.
The data presented here suggest that somatic alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human malignant melanomas.
A comparative study of Fas and Fas-ligand expression during melanoma progression.Soubrane C, Mouawad R, Antoine EC, Verola O, Gil-Delgado M, Khayat D.
Medical Oncology Department, Salpetriere Hospital, 47 boulevard de l'Hopital, 75013 Paris, France.
Br J Dermatol 2000 Aug;143(2):307-12 Abstract quote BACKGROUND: Impaired regulation of apoptosis is known to be associated with the development of various cancers. Fas receptor (APO-1/CD95) binding to its ligand, Fas-ligand (Fas-L), has been shown to trigger apoptosis in various cell types.
OBJECTIVES: In this study, we examined CD95 and Fas-L expression on primary and metastatic melanoma cells from patients to investigate a potential correlation between these measures of apoptosis and different disease stages.
PATIENTS AND METHODS: Primary melanoma cells were obtained after surgical resection from 19 patients and metastatic cells from fine-needle aspiration of lymph nodes or palpable subcutaneous lesions in 25 patients. Normal skin cells were obtained at skin biopsy of 10 healthy donors.
RESULTS: Flow cytometric analysis revealed that CD95 and Fas-L expression was detected in all the kinds of cell studied. In whole cell suspensions, CD95 expression was significantly higher (P < 0.0001) in normal skin cells than in melanoma cells, whatever the stage studied. By contrast, we observed an increase in Fas-L expression in melanoma cells compared with normal ones. Subsequently, using a double staining method, we studied these measures on HMB45+ cells, a specific marker for melanoma cells, and found that CD95 expression was significantly higher (P = 0.0005) in primary than in metastatic cells while Fas-L expression was significantly increased (P = 0. 0004) in metastatic compared with primary cells. Furthermore, a relationship was found between CD95 or Fas-L expression and Breslow thickness; as primary melanoma thickness progressively increased, the percentage of HMB45+ CD95+ cells decreased while that of HMB45+ Fas-L+ cells concurrently increased.
CONCLUSIONS: These results suggest that downregulation of CD95 and upregulation of Fas-L in melanoma might be considered as concomitant with disease progression.
Fas-mediated apoptosis of melanoma cells and infiltrating lymphocytes in human malignant melanomas.Shukuwa T, Katayama I, Koji T.
Department of Dermatology, Nagasaki University School of Medicine, Sakamoto, Japan.
Mod Pathol 2002 Apr;15(4):387-96 Abstract quote In a rodent system, melanoma cells expressing Fas ligand (FasL) could kill Fas-positive lymphocytes, suggesting that FasL expression was an essential factor for melanoma cell survival in vivo. These findings led us to investigate apoptosis, and to histochemically analyze involvement of Fas and FasL in the induction of apoptosis, in human malignant melanoma tissues.
The percentages of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL)-positive melanoma cells and of proliferating cell nuclear antigen (PCNA)-positive melanoma cells in melanoma tissues (n = 22) were greater than those in melanocytes in uninvolved skin (n = 6) and nevus cells in nevi tissues (n = 9). The infiltrating lymphocytes around melanomas were also TUNEL positive. Immunohistochemistry revealed expression of Fas and FasL in melanoma cells and lymphocytes, whereas no Fas or FasL expression was detected in normal skin melanocytes and nevus cells. There was significant correlation between Fas-positive indices and TUNEL indices in melanoma tissues. Moreover, TUNEL-, Fas-, and FasL-positive indices of melanoma cells from patients with Stage 3 melanomas were significantly lower than those with Stage 2 melanomas. The PCNA index of Stage 1 melanoma was significantly lower than that of the other stages, although the difference of PCNA index was insignificant among Stages 2 to 4. Among Stages 1 to 4, there was no difference in the PCNA, TUNEL-, and Fas-positive indices of lymphocytes, although the FasL-positive index of lymphocytes from Stage 3 melanomas was significantly lower than in that from Stage 2.
These data reveal that melanoma cells and infiltrating lymphocytes have the potential to induce their own apoptosis regulated by Fas and FasL in an autocrine and/or paracrine fashion and that the decline of Fas-mediated apoptosis of melanoma cells, rather than the apoptosis of infiltrating lymphocytes, may affect the prognosis of melanoma patients, possibly through the accumulation of more aberrant cells acquiring metastatic activity.
CLAUDIN-1
J Cutan Pathol. 2005 Sep;32(8):533-6. Abstract quote
Claudins are a family of transmembrane proteins involved in cell-to-cell adhesion and are believed to be the main component of tight junctions. Recent studies have suggested that some metastatic solid tumors lack claudin expression. It is unknown whether claudins play a role in cutaneous melanoma.
Immunohistochemical studies were performed on tissue microarrays containing 19 benign melanocytic nevi (BN), 21 dysplastic nevi (DN), 23 primary malignant melanomas (MMs), and 31 metastatic melanomas (MMMs) using a polyclonal anti-claudin-1 antibody. Immunoreactivity in tumor cells and associated vessels was graded by intensity and by percentage of reactive cells. Normal epidermis served as internal control (3+ labeling). Cases with at least 2+ labeling in more than 25% of the cells were considered positive. Claudin-1 expression was present in 37% of BN, 24% of DN, 26% of MM, and 3.2% of MMM. Tumor-associated vessels showed the following results: 11 of 19 (58%) in BN, 14 of 21 (67%) in DN, 17 of 23 (74%) in MM, and 6 of 31 (19%) in MMM. A significant loss of expression was noted between MMM and all other lesions in tumor cells and associated vessels. There was no significant difference between BN, DN, and MM.
Within primary melanomas, there was a significant correlation between expression of claudin in tumor cells and Clark level/Breslow thickness. Also significant was a decreased expression of claudin in tumor vessels of lesions with higher Breslow thickness or Clark level. These data suggest that loss of claudin-1 may play a significant role in the acquisition of metastatic phenotype in cutaneous melanoma.CYCLIN D1
- Cyclin D1 expression in melanocytic lesions of the skin.
Ramirez JA, Guitart J, Rao MS, Diaz LK.
Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Ann Diagn Pathol. 2005 Aug;9(4):185-8. Abstract quote
BACKGROUND: Progression through the cell cycle is controlled by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitory proteins. The role of cyclin D1 in the development and progression of melanomas is controversial. The goal of this study is to evaluate the role of cyclin D1 in benign and malignant melanocytic lesions of the skin.
METHODS: A total of 126 pigmented lesions of the skin including compound nevi (21), intradermal nevi (18), melanoma in situ (28), primary invasive melanomas (30), and metastatic melanoma (29) were evaluated for cyclin D1 expression. The following tiered system was used for scoring: 0% nuclear staining (score 0), 1% to 19% nuclear staining (score 1), 20% to 49% nuclear staining (score 2), and 50% or greater nuclear staining (score 3).
RESULTS: Average scores were significantly higher for primary melanomas compared with nevi and for in situ melanomas compared with primary invasive melanomas. The average score for metastatic melanomas was not significantly different compared with primary invasive melanomas. Scores for primary invasive melanomas did not correlate with depth of invasion or presence of metastases. Compound nevi exhibited a slightly higher level of cyclin D1 expression compared with intradermal nevi.
CONCLUSION: Although primary melanomas show a higher level of cyclin D1 expression compared with nevi, cyclin D1 appears to have little role in development of a metastatic phenotype. It is not clear why lesions localized near the dermal-epidermal junction express higher levels of cyclin D1. Further studies are indicated to ascertain the biologic role and practical utility of cyclin D1 in melanocytic lesions of the skin.EPIDERMAL GROWTH FACTOR
Association between functional polymorphism in EGF gene and malignant melanoma.Shahbazi M, Pravica V, Nasreen N, Fakhoury H, Fryer AA, Strange RC, Hutchinson PE, Osborne JE, Lear JT, Smith AG, Hutchinson IV.
Immunology Research Group, School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK.
Lancet 2002 Feb 2;359(9304):397-401 Abstract quote BACKGROUND: Malignant melanoma, the most serious cutaneous malignancy, has attracted substantial attention because of its rapidly increasing incidence and the poor prognosis of some tumours. Little is known of the genetic factors that mediate susceptibility to, and outcome of, sporadic malignant melanoma. Because of its role in mitogenesis, which is especially relevant to wound healing, tumorigenesis, and proliferation of epidermal tissues, epidermal growth factor (EGF) is an attractive candidate in which to look for genetic polymorphisms.
METHODS: We enrolled 135 white European patients with malignant melanoma and 99 healthy white European controls, and screened a selection of DNA samples for polymorphisms in the promoter and 5' untranslated region of the EGF gene by analysis. We then screened DNA samples from all participants for the identified polymorphism by restriction-fragment-length polymorphism (RFLP) analysis. In-vitro EGF production was measured in peripheral-blood mononuclear cells from 34 controls, and the results were compared with the individuals' EGF genotypes.
FINDINGS: We identified a single nucleotide substitution (G to A) at position 61 of the EGF gene. Allele frequencies in the controls were 56% EGF 61*A and 44% EGF 61*G. Cells from individuals homozygous for the 61*A allele produced significantly less EGF than cells from 61*G homozygotes (p=0.0004) or heterozygous A/G individuals (p=0.001). Compared with the A/A genotype, G/G was significantly associated with Breslow thickness (p=0.045) and with risk of malignant melanoma (odds ratio 4.9 [95% CI 2.3-10.2], p<0.0001).
INTERPRETATION: This study suggests that high EGF production might be important in the development of malignant melanoma.
ERZIN
- Ezrin in primary cutaneous melanoma.
Ilmonen S, Vaheri A, Asko-Seljavaara S, Carpen O.
1Department of Plastic Surgery, Helsinki University Hospital, Helsinki, Finland.
Mod Pathol. 2005 Apr;18(4):503-10. Abstract quote
Ezrin is a member of the ezrin-radixin-moesin family of proteins that link the actin-containing cytoskeleton to the plasma membrane. Ezrin is also connected to signaling molecules involved in the regulation of cell survival, proliferation and migration.
Here, we examined the expression of ezrin in 95 primary cutaneous melanomas and correlated ezrin expression with conventional prognostic factors and biomarkers. From 12 patients metastatic tissue samples were also examined. In addition to ezrin staining, Mib-1 proliferation antigen, p53 and Bcl-2 were evaluated. Ezrin immunoreactivity was seen in most tumors; only 19 (20%) melanomas were negative. A total of 48 (51%) tumors had weak immunoreactivity and 28 (29%) strong immunoreactivity. The intensity of ezrin immunoreactivity was associated with tumor thickness (Breslow, P=0.0008) and with tumor invasion level (Clark, P=0.004), thicker tumors having stronger immunoreactivity. Also, there was a correlation between higher Mib-1 index in tumors and strong ezrin expression. All metastatic samples (n=12) showed positive ezrin immunoreactivity. In univariate analysis of survival, patients (n=76) with positive ezrin immunoreactivity had worse clinical disease behavior than those (n=19) without ezrin immunoreactivity, but the difference was not significant (P=0.19). In multivariate analysis of survival, the ezrin immunoreactivity was not a significant marker.
The results indicate that ezrin is expressed in most primary melanomas of the skin and in all metastatic tumors. Ezrin expression correlates with tumor thickness and level of invasion suggesting an association between ezrin expression and tumor progression.Ets-1 PROTO-ONCOGENE
Expression of the ets-1 proto-oncogene in melanocytic lesions.Keehn CA, Smoller BR, Morgan MB.
Department of Pathology, University of South Florida College of Medicine, USA.
Mod Pathol. 2003 Aug;16(8):772-7. Abstract quote Ets-1 oncoprotein is a transcription factor known to regulate the expression of numerous genes important in extracellular matrix remodeling and angiogenesis. Up-regulation of Ets-1 has been shown to be important in a variety of human malignancies and to correlate with prognosis.
To our knowledge, this oncoprotein has not been examined in melanocytic lesions. A series of 10 cutaneous melanomas and 24 benign melanocytic lesions with patient records were independently examined for diagnosis confirmation and immunohistochemical expression by two dermatopathologists.
The immunohistochemical expression for Ets-1 (Novocastra, Newcastle upon Tyne, UK) was scored by an average of the mean labeling intensity; no nuclear staining = 0, weak nuclear staining = 1, moderate = 2, and intense = 3. Ets-1 expression was statistically assessed by the one-way analysis of variance (ANOVA) comparing the mean labeling intensity of melanoma to benign melanocytic nevi. All of the benign melanocytic lesions exhibited negative to weak nuclear staining, with an average mean labeling intensity of 0.4. Melanoma in situ exhibited moderate nuclear staining, for a mean labeling intensity of 2.0, whereas all conventional invasive melanomas exhibited moderate to strong nuclear staining, with a mean labeling intensity of 2.7. Metastatic melanoma exhibited very strong nuclear staining, with a mean labeling intensity of 3.0. Invasive desmoplastic melanoma, like melanoma in situ, showed moderate nuclear staining with a mean labeling intensity of 2.1. There was a trend toward more intense staining with melanoma progression.
A statistically significant difference in the mean labeling intensity of Ets-1 was seen between invasive melanoma and benign melanocytic nevi (P <.0001). Ets-1 oncoprotein expression, however, does not distinguish among benign melanocytic lesions. Staining intensity and pattern might be a useful adjunct with histomorphology in distinguishing invasive melanoma from benign melanocytic nevi. Furthermore, Ets-1 expression may be an important pathogenic mechanism and predictor of aggressive biologic behavior of cutaneous melanoma, with a trend toward staining intensity increasing as Clark stage increases.INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 2 Expression of insulin-like growth factor-binding protein 2 in melanocytic lesions
Huamin Wang, Steven S. Shen, Hua Wang, A. Hafeez Diwan, Wei Zhang3, Gregory N. Fuller and Victor G. Prieto
J Cutan Pathol 2003;30:599-605 Abstract quote
Background: Insulin-like growth factor-1 (IGF-1) is one of the most critical proteins required for the survival, migration, and growth of melanoma cells. IGF-binding protein 2 (IGFBP2), which binds and regulates the function of IGF-1, is upregulated in a dose-dependent manner in melanoma cells treated with IGF-1, suggesting a possible role of IGFBP2 in the pathogenesis of melanoma.Methods: Tissue microarrays were constructed using formalin-fixed, paraffin-embedded archival tissue blocks from 94 melanocytic lesions: 20 benign nevi, 20 dysplastic nevi, 23 primary melanomas, and 31 metastatic melanomas. IGFBP2 expression was evaluated immunohistochemically using a polyclonal antibody against the C-terminus of IGFBP2. The number of cells and labeling intensity were assessed semiquantitatively.
Results: Positive IGFBP2 labeling was observed in 5.0% of benign nevi, which was significantly lower than in dysplastic nevi (35.0%), primary melanomas (52.2%), or metastatic melanomas (54.8%) (p < 0.05). Among the IGFBP2-positive cases, moderate-to-strong immunostaining was observed in 64.7% of metastatic melanomas and 33.3% of primary melanomas. But none of the dysplastic nevi had moderate-to-strong immunostaining (p < 0.05).
Conclusions: Our study shows that IGFBP2 expression increases from benign and dysplastic nevi to primary and metastatic melanomas and suggests that it may play a role in melanoma progression.
KU
Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis.
Korabiowska M, Bauer H, Quentin T, Stachura J, Cordon-Cardo C, Brinck U.
Department of Cytopathology, Georg-August-University, Goettingen, Germany.
Hum Pathol. 2004 Feb;35(2):210-6 Abstract quote.
Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis.
Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01).
We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.
Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.Korabiowska M, Tscherny M, Stachura J, Berger H, Cordon-Cardo C, Brinck U.
Department of Cytopathology, University of Gottingen, Germany.
Mod Pathol 2002 Apr;15(4):426-33 Abstract quote Ku70 and Ku80 heterodimers function as regulatory subunits of the DNA-dependent protein kinase and play a very important role in the repairing of DNA double-strand breaks. Although Ku70 is proposed as a candidate for a tumor suppressor gene, not many data are available on Ku70 and Ku80 expression in human tumors.
The main aim of this study was to investigate the expression of Ku70 and Ku80 in the ultraviolet-induced lesions-nevus cell nevi, lentigos maligna, and malignant melanomas. Nineteen nevus cell nevi, 23 lentigos maligna, 76 primary melanomas, and 31 melanoma metastases were stained immunohistochemically for the presence of Ku70 and Ku80 proteins. Ku70 and Ku80 expression was preserved in about 80% of nevi, 26% of lentigo maligna, 45% of primary melanomas, and 67% of melanoma metastases. Highly significant differences in Ku70 and Ku80 expression were found between nevi, lentigo maligna, and melanomas. In Cox regression, Ku70 and Ku80 were shown to be highly significant influences on patients' prognosis. Significant correlations between Ku70 and Ku80 expressions were found in nevi, lentigo maligna, and primary melanomas. These correlations were not more present in melanoma metastases.
To summarize, earlier phases of melanoma progression seem to be connected with the loss of expression of Ku proteins. Metastatic spread is related to dysregulation of the Ku70 and Ku80 axis.
LOSS OF HETEROZYGOSITY
Comparative analysis of loss of heterozygosity and microsatellite instability in adult and pediatric melanoma.
Uribe P, Wistuba II, Solar A, Balestrini C, Perez-Cotapos ML, Gonzalez S.
From the Department of Pathology, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile.
Am J Dermatopathol. 2005 Aug;27(4):279-85. Abstract quote
Although 0.3% of melanomas occur in children, the incidence has risen in past decades. In adult melanoma, some chromosomal regions in 1p, 6q, 9p, 10q, and 11q are frequently deleted. Microsatellite instability (MSI), which reflects impaired DNA repair, has been found at low levels in adult melanoma and melanocytic nevi.
To investigate the molecular changes in pediatric melanoma, a screening for loss of heterozygosity and microsatellite instability was performed and compared with changes found in adult melanoma. Formalin-fixed, paraffin-embedded tissues from 10 adult melanomas, 9 melanocytic nevi, and 8 pediatric melanomas were microdissected and the DNA was extracted. Loss of heterozygosity and microsatellite instability were evaluated using 13 microsatellite repeat polymorphisms located in 1p36, 1q32, 2p12, 2p22-25, 2q33-37, 9p21, 10q23.3, 11q23, 13q14, 17p13, and 17q21. The overall frequency of loss of heterozygosity was 0.09 for nevi, 0.30 for adult melanoma, and 0.43 for pediatric melanoma (nevi vs. adult melanoma, P = 0.0082; nevi vs. pediatric melanoma, P = 0.0092).
Pediatric melanoma has more loss of heterozygosity (44%) in 11q23 than adult melanoma (7%, P = 0.046). The microsatellite instability overall frequency was greater in pediatric melanoma (0.24) than nevi (0.05, P = 0.0031) and adult melanoma (0.09, P = 0.0195). Our findings suggest that pediatric melanoma has a different abnormal pattern than adult melanoma. Pediatric melanoma has more microsatellite instability than adult melanoma. 11q23 could contain genes related to the early age onset of melanoma.
The high frequency of microsatellite instability is coincidental with the finding of higher levels of microsatellite instability in pediatric brain tumors and could play a role in the pathogenesis of pediatric melanoma.
Loss of heterozygosity analysis of cutaneous melanoma and benign melanocytic nevi: Laser capture microdissection demonstrates clonal genetic changes in acquired nevocellular nevi.Maitra A, Gazdar AF, Moore TO, Moore AY.
Departments of Pathology and Dermatology and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX; the Departments of Surgery and Dermatology, Baylor University Medical Center, Dallas, TX; and the Department of Pathology, Johns Hopkins Medical Center, Baltimore, MD.
Hum Pathol 2002 Feb;33(2):191-197 Abstract quote The molecular pathology of the common nevocellular nevus (NCN) and its relationship to the genetic model of malignant melanoma (MM) progression has not been fully characterized.
We used laser capture microdissection of archival formalin-fixed material to study 34 melanocytic lesions (19 MM and 15 NCN). Twelve of the 15 NCN were acquired, 3 of which were congenital; none had dysplastic features. Ten polymorphic markers on five chromosomal regions (1p36, 6q22-23.3, 8p22-24, 10q23, and 11q23) were selected for loss of heterozygosity (LOH) analysis, based on previous studies demonstrating involvement in MM pathogenesis and progression.
Loss of heterozygosity at any allelic locus was observed in 18 of 19 (95%) MM and in 9 of 15 (60%) NCN. Of the three congenital nevi analyzed, none showed LOH at any informative locus. The frequency of allelic loss was highest in the MM at 6q22-23.3 (64%), followed by 10q23 (62%), 8p22-24 (43%), 11q23 (43%), and 1p36 (13%). In the 15 NCN, the most frequent allelic losses were detected at 6q22-23.3 (29%), 1p36 (27%), and 10q23 (25%), with lower frequencies of LOH at 11q23 (10%) and 8p22-24 (7%). LOH at a single polymorphic marker, D6S1038, was detected in 70% of the MM and in 36% of the NCN, suggesting the presence of putative tumor-suppressor genes (TSGs) critical in melanocytic neoplasia at 6q22-23.3.
The presence of clonal genetic alterations in acquired NCN justifies their classification as a benign neoplasm. The pattern of LOH in NCN is not random; rather, the relative frequencies of LOH at the chromosomal regions examined are consistent with a multistep model of MM progression that begins with NCN. Molecular analysis of NCN reiterates established epidemiologic and morphologic notions that NCN are precursor lesions for MM.
MISMATCH REPAIR GENES
Loss of heterozygosity, microsatellite instability, and mismatch repair protein alterations in the radial growth phase of cutaneous malignant melanomas.Hussein MR, Sun M, Roggero E, Sudilovsky EC, Tuthill RJ, Wood GS, Sudilovsky O.
Department of Medicine (Dermatology), University of Wisconsin and William S. Middleton Memorial Veteran Hospital, Madison, Wisconsin 53715, USA.
Mol Carcinog 2002 May;34(1):35-44 Abstract quote Little is known about genomic alterations during development of the radial growth phase (RGP) of cutaneous malignant melanomas (CMMs).
In this investigation polymerase chain reaction-based microsatellite assays were applied to analyze 13 RGP-CMMs with 18 microsatellite markers at six chromosomal regions: 1p, 3p, 4q, 6q, 9p, and 10q. Loss of heterozygosity (LOH) was found in eight cases (62%), at 9p22, 1p36, and 10q11, suggesting the presence of tumor-suppressor genes at these regions. LOH was encountered frequently at the interferon-alpha (31%) and D10S249 loci (15%). Low-level microsatellite instability (MSI) (11-16% of investigated loci unstable) was noted in three cases (23%). Two MSI banding patterns were seen: band shift and the presence of additional bands.
To investigate the underlying mechanisms of the low-level MSI pattern, we analyzed the lesions for expression of mismatch repair (MMR) proteins with immunoperoxidase methods and mouse monoclonal antibodies. The average percentages of positively stained cells for human MutL homolog 1 (hMLH1), human MutS homolog 2 (hMSH2), and human MutS homolog 6 (hMSH6) in RGP-CMM (75.6 +/- 3.4%, 67.20 +/- 7.71%, and 76.6 +/- 2.1%, respectively) were reduced compared with benign nevi. No statistically significant differences in MMR protein expression were found between microsatellite-stable and low-level MSI lesions (P = 0.173, P = 0.458, and P = 0.385 for hMLH1, hMSH2, and hMSH6, respectively).
There was a direct correlation between values for percentages of positively stained cells for hMSH2 and hMSH6 (r = +0.9, P = 0.03), suggesting that common mechanisms regulate their expression. In conclusion, LOH, MSI, and reduced MMR protein expression appear to be present in at least some RGP-CMMs and may play a role in their pathogenesis. Further studies are necessary to support these finding and to determine their diagnostic and prognostic significance.
Alterations of Mismatch Repair Protein Expression in Benign Melanocytic Nevi, Melanocytic Dysplastic Nevi, and Cutaneous Malignant Melanomas
Mahmoud R. Hussein, M.D., Ph.D.; Eduardo Roggero, M.D.; Eulalia C. Sudilovsky, B.S.; Ralph J. Tuthill, M.D.; Gary S. Wood, M.D.; Oscar Sudilovsky, M.D., Ph.D.
From the Institute of Pathology, Case Western Reserve University, Cleveland, Ohio (M.R.H., E.C.S.); Instituto de Histopatología, Rosario, Prov. de Santa Fé, Argentina (E.R.); Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio (R.J.T.); Departments of Pathology and Dermatology, Case Western Reserve University, University Hospitals of Cleveland and Veterans Hospital Administration, Cleveland, Ohio (Current address: Department of Medicine (Dermatology), University of Wisconsin and Middleton VA Medical Center, Madison, Wisconsin (G.S.W.); and Institute of Pathology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio (O.S.).
Am J Dermatopathol 2001;23:308-314 Abstract quote
Immunoperoxidase-staining methods were used to examine the expression of hMLH1, hMSH2, and hMSH6 mismatch repair (MMR) proteins in 50 melanocytic lesions.
Microsatellite instability (MSI), screened previously in these lesions by polymerase chain reaction–based microsatellite assay, showed low-level microsatellite instability (MSI-L) in 11 of 22 melanocytic dysplastic nevi (MDN) and two of nine primary cutaneous malignant melanomas (CMMs) but not in the benign melanocytic nevi (BN). Mismatch repair proteins were widely expressed in the epidermis and adnexal structures.
All lesions showed positive immunoreactivity with a gradual decrease in the MMR staining values during the progression from BN to MDN to CMMs. The average percentage of positively (PP) stained cells for hMLH1, hMSH2, and hMSH6 in BN was 85.50 ± 1.95, 77.90 ± 4.50, and 87.11 ± 1.85, respectively. The PP cell values in CMMs were significantly reduced as compared with BN (75.22 ± 3.57, p= 0.01; 56.11 ± 8.73, p= 0.02; 65.22 ± 6.47, p = 0.0002 for hMLH1, hMSH2, and hMSH6, respectively). No comparable significant difference was found between microsatellite stable and MSI-L lesions (p = 0.173, p = 0.458, and p = 0.385), suggesting a lack of correlation between MMR expression and MMR function. There was a direct correlation between PP cell values of hMSH2 and hMSH6 (R = 0.39, p = 0.008), implying that their expression could be regulated by a common mechanism.
Thus, an important finding of these studies was the reduction of MMR protein levels in CMMs; whether this reflects underlying genetic or epigenetic mechanisms is still to be determined.
MXI1 GENE
Loss of heterozygosity in the MXI1 gene is a frequent occurrence in melanoma.
Ariyanayagam-Baksh SM, Baksh FK, Swalsky PA, Finkelstein SD.
Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
Mod Pathol. 2003 Oct;16(10):992-5 Abstract quote.
Melanoma development and progression is thought to be the result of a multi-step accumulation of genetic damage, with loss of heterozygosity in chromosome 9p (MTS1) frequently described. In addition, chromosome 10q allelic loss has been reported, implicating the tumor suppressor gene PTEN/MMAC1 on 10q23.3.The MXI1 gene at 10q24-25 is another candidate tumor suppressor that has only rarely been studied in melanomas, with conflicting results.
We used microdissection-based genotyping to investigate 29 melanomas from 20 patients for loss of heterozygosity in intragenic and flanking microsatellite markers for this latter gene. Concurrently, the MTS1 gene was similarly studied using two flanking microsatellites. Fifty-four percent (15 of 28) of the informative cases showed loss of heterozygosity for one or both MXI1 markers, as compared with 67% (16 of 24) of the informative cases for MTS1. MXI1 allelic loss was seen more frequently in recurrent/metastatic tumors (59%), as compared with in primary (33%) lesions. Eighty percent of the primary tumors showed loss of heterozygosity for MTS1, as well as 63% of recurrent/metastatic ones. We studied more than one tumor in eight patients, with those from three patients showing discordant genetic patterns. One patient showed a metastatic tumor with allelic loss for MXI1 that was not identified in the primary melanoma or a local recurrence. The other two patients showed clonal heterogeneity in MXI1 at synchronous and metachronous metastatic foci.
These findings support MXI1 as a putative tumor suppressor gene involved in conventional melanoma progression. Genetic heterogeneity seen in different metastases from the same primary suggests a nonlinear pattern of chromosomal damage, with the development of multiple clones within the primary tumor, each acquiring its own metastatic potential.
J Biol Chem 1999;274:23358
Cyclin dependent kinase inhibitor is significantly upregulated in cultured keratinocytes that have undergone irreversible growth arrest following onset of replicative senescence or after confluency-induced premature senescence
Senescent keratinocytes are resistant to apoptosis
May truncate or block the differentiation pathway
Conversely, inactivation is commonly present in pre-malignant and invasive SCCA of the skin, oral cavity, bladder cancers, and melanoma patients
Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients.
Ghiorzo P, Villaggio B, Sementa AR, Hansson J, Platz A, Nicolo G, Spina B, Canepa M, Palmer JM, Hayward NK, Bianchi-Scarra G.
Hum Pathol. 2004 Jan;35(1):25-33 Abstract quote.
Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations.
We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels.
Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation.
Della Torre G, Pasini B, Frigerio S, Donghi R, Rovini D, Delia D, Peters G, Huot TJ, Bianchi-Scarra G, Lantieri F, Rodolfo M, Parmiani G, Pierotti MA.
Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian, 1 20133 Milan, Italy
Br J Cancer 2001 Sep 14;85(6):836-44 Abstract quote
Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma.
We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case.
The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families.
Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.
p27
- Hypoxia regulation of the cell cycle in malignant melanoma: putative role for the cyclin-dependent kinase inhibitor p27.
Murphy M, Carlson JA, Keough MP, Claffey KP, Signoretti S, Loda M.
Division of Dermatopathology, Department of Dermatology, University of Connecticut Health Center, Farmington, CT 06030, USA.
J Cutan Pathol. 2004 Aug;31(7):477-82. Abstract quote
BACKGROUND: Intratumor hypoxia has been shown to promote more aggressive and metastatic cancer phenotypes that are associated with treatment resistance and poor prognosis. Cellular proliferation and its control are known to be important components of tumor progression. Hypoxia induces cell-cycle arrest in cultured cell lines, possibly via up-regulation of the cyclin-dependent kinase inhibitor p27. The effect of hypoxia on cell-cycle regulation in excised human tumors has not been investigated.
METHODS: We performed immunohistochemistry for p27 and Ki-67 on 10 formalin-fixed paraffin-embedded metastatic melanomas, selected on the basis of histological evidence of zonal/geographic necrosis, adjacent to areas with viable perivascular tumor cells.
RESULTS: In the majority of cases, there was a significant increase in p27 staining in cells adjacent to necrotic areas compared to perivascular zones. An inverse staining pattern between Ki-67 and p27 was identified in these tumors. Tumors with no zonal increase in p27 staining demonstrated a diffuse pattern of staining for Ki-67 within tumor nests.
CONCLUSIONS: While increased cellular proliferation is a characteristic of cancer, subsets of human melanomas may retain the ability to regulate their rate of proliferation in response to changes in the tumor microenvironment. The hypoxia-mediated cell-cycle arrest (decreased Ki-67) in these tumors may be mediated by p27 up-regulation.PCNA
Lentigo maligna and superficial spreading melanoma are different in their in situ phase: An immunohistochemical study.Auslender S, Barzilai A, Goldberg I, Kopolovic J, Trau H.
Department of Dermatology and Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel and the Sackler Faculty of Medicine, Tel-Aviv University, Israel.
Hum Pathol 2002 Oct;33(10):1001-5 Abstract quote 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 sun-damaged 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.
PROTEASE-ACTIVATED RECEPTORS 1 AND 2
- Expression of protease-activated receptors 1 and 2 in melanocytic nevi and malignant melanoma.
Massi D, Naldini A, Ardinghi C, Carraro F, Franchi A, Paglierani M, Tarantini F, Ketabchi S, Cirino G, Hollenberg MD, Geppetti P, Santucci M.
Hum Pathol. 2005 Jun;36(6):676-85. Abstract quote
Summary Protease-activated receptors (PARs) are members of the G protein-coupled receptor superfamily that are activated by the proteolytic cleavage of their amino terminal domain.
PAR-1 activation by thrombin results in several biologic effects, including platelet adhesion to other cells or extracellular matrix, fibroblast, and endothelial cell growth, whereas PAR-2, activated by trypsin, has mainly a proinflammmatory and angiogenetic role. PAR-1 and PAR-2 modulate cell proliferation in physiopathologic cell invasion processes, suggesting that they may play a role in the setting of cancer growth and metastasis.
Here, we have investigated the expression of PAR-1 and PAR-2 proteins by immunohistochemistry in a series of benign and malignant melanocytic lesions: 20 melanocytic lesions (10 common melanocytic nevi and 10 atypical or "dysplastic" melanocytic nevi) and 50 melanomas (10 in situ melanomas, 10 melanomas T1, 10 melanomas T2, 10 melanomas T3 to T4, and 10 metastatic melanomas). PAR-1 was significantly overexpressed in atypical nevi and melanomas in comparison with common melanocytic nevi. PAR-2 was strongly and diffusely expressed by immunohistochemistry in all melanocytic lesions, with no statistically significant differences between nevi and melanomas. Because we found a differential expression in PAR-1 protein, but not in PAR-2, we next investigated the expression of PAR-1 messenger RNA (mRNA) by ribonuclease protection assay in paraffin-embedded tissues using a paraffin block RNA isolation procedure. Similarly to immunohistochemical results, PAR-1 mRNA expression was significantly higher in atypical nevi and melanomas in comparison with common nevi and controls.
Overexpression of PAR-1 in atypical nevi and melanomas supports a role for PAR-1 in the initial phases of melanoma development as well as in tumor progression and metastasis. Conversely, the significance of PAR-2 up-regulation in both benign and malignant melanocytic lesions requires further research.PTEN
PTEN expression in normal skin, acquired melanocytic nevi, and cutaneous melanoma.
Tsao H, Mihm MC Jr, Sheehan C.
J Am Acad Dermatol. 2003 Nov;49(5):865-72. Abstract quote
BACKGROUND: Although various studies have shown mutations of the tumor suppressor gene, PTEN/MMAC1, in primary, metastatic, and cultured cutaneous melanoma specimens, little is known about the pattern of PTEN protein expression in early melanocytic tumor progression.
OBJECTIVE: To further investigate the role of PTEN in melanocytic tumor development.
METHODS: We assessed the level and distribution of PTEN in normal skin, 39 acquired melanocytic nevi, and 30 primary cutaneous melanomas, including lentigo malignas, by immunostaining.
RESULTS: We found high levels of PTEN expression in cutaneous muscles, nerves, and muscular arteries, and moderate-to-high amounts of PTEN in the epidermis, follicular epithelium, and sebaceous and eccrine glands. PTEN staining in cutaneous lymphatics, dermal and periadnexal adventitial fibroblasts, and chondrocytes were variably absent. Junctional melanocytes and chondrocytes frequently exhibited preferential nuclear staining. We found uniformly strong PTEN expression in the cytoplasm of almost all benign and dysplastic nevi. However, there was some evidence of nuclear PTEN loss even in the benign melanocytic proliferations. In addition, out of 30 primary cutaneous melanomas and lentigo malignas, we detected diffuse expression of PTEN in 11 (37%) tumors, widespread loss of PTEN in 11 (37%) tumors and mixed PTEN expression in 8 (27%) lesions. In the primary cutaneous melanomas, PTEN was largely localized to the cytoplasm.
CONCLUSIONS: The presence of PTEN in benign melanocytic tumors and the absence of PTEN in a significant proportion of primary cutaneous melanomas support a role for PTEN loss in the pathogenesis of melanoma.REGRESSION
Joerg Wenzel, MD, etal.
We studied 253 primary melanomas of the skin for histologic signs of regression. Detailed immunohistologic analyses, including expression of MxA (an antiviral protein specifically induced by type I interferons), the chemokine IP10/CXCL10, the chemokine receptor CXCR3, and the cytotoxic molecule granzyme B, were performed for 14 typical regressive tumors and 20 control samples (congenital nevi, halo nevi, unaffected skin).
We found high expression of MxA, indicating local type I interferon production, in inflamed regressive melanocytic lesions, along with large numbers of natural interferon-producing plasmacytoid dendritic cells, CXCR3+ lymphocytes, and granzyme B+ lymphocytes. We also detected high expression of the interferon-induced chemokine IP10/CXCL10, linking type I interferon production and recruitment of CXCR3+ lymphocytes.
Our results provide evidence that endogenous activation of type I interferons, infiltration of plasmacytoid dendritic cells, and recruitment of CXCR3+ and granzyme B+ lymphocytes are involved in spontaneous regression of melanoma and other melanocytic lesions.
We believe this cytotoxic immune response represents an evolutionarily conserved pathway against intracellular pathogens.RETINOBLASTOMA GENE
Mod Pathol. 2005 Sep;18(9):1249-57. Abstract quote
In malignant melanomas, the loss of cell cycle control is thought to be due to a lack of retinoblastoma protein (pRb)-activity. Members of the previously described family of retinoblastoma-binding proteins (RBPs) are supposed to act as pRb-modulating factors.
Based on RNA-fingerprinting of normal human melanocytes, we previously described a new family member with high sequence homology to the retinoblastoma-binding protein-2 (RBP-2), termed RBP2-Homolog 1 (RBP2-H1). Based on its UVB responsiveness, it was hypothesized that this gene may also play a role in melanocytic tumors. In the present study, we can confirm by real-time RT-PCR (six common melanocytic nevi, five advanced nodular melanomas and seven melanoma metastases) and immunohistochemistry (tissue microarrays: 52 melanocytic nevi, 60 melanomas, 60 metastases; and conventional sections: five common nevi, four advanced nodular melanomas, five melanoma metastases) that RBP2-H1 expression is progressively downregulated in advanced and metastatic melanomas in vivo with a certain intratumoral heterogeneity.
Whereas benign melanocytic nevi are RBP2-H1 positive in about 70% of the cases, a lack of RBP2-H1 expression was found in 90% of the primary malignant melanomas and 70% of the melanoma metastases, respectively. Interestingly, a similar deficiency can be found in glioblastomas, but not epithelial cancers. In accordance to the in vivo data, established melanoma cell lines exhibit low but heterogeneous levels of RBP2-H1 expression. By co-immunoprecipitation, we provide the first evidence that a subfraction of total RBP2-H1 can bind to pRb, which makes this protein a true pRb-interacting factor.
We conclude that loss of RBP2-H1 is a common finding in the progression of malignant melanomas. Since a direct interaction of RBP2-H1 and pRb seems possible, the loss of RBP2-H1 may possibly contribute to uncontrolled growth in malignant melanomas.
- Retinoblastoma-binding protein 2-homolog 1: a retinoblastoma-binding protein downregulated in malignant melanomas.
Roesch A, Becker B, Meyer S, Wild P, Hafner C, Landthaler M, Vogt T.
1Department of Dermatology, University of Regensburg, Regensburg, Germany.
Mod Pathol. 2005;18:565-572 Abstract quote
In malignant melanomas, the loss of cell cycle control is thought to be due to a lack of retinoblastoma protein (pRb)-activity. Members of the previously described family of retinoblastoma-binding proteins (RBPs) are supposed to act as pRb-modulating factors. Based on RNA-fingerprinting of normal human melanocytes, we previously described a new family member with high sequence homology to the retinoblastoma-binding protein-2 (RBP-2), termed RBP2-Homolog 1 (RBP2-H1). Based on its UVB responsiveness, it was hypothesized that this gene may also play a role in melanocytic tumors.
In the present study, we can confirm by real-time RT-PCR (six common melanocytic nevi, five advanced nodular melanomas and seven melanoma metastases) and immunohistochemistry (tissue microarrays: 52 melanocytic nevi, 60 melanomas, 60 metastases; and conventional sections: five common nevi, four advanced nodular melanomas, five melanoma metastases) that RBP2-H1 expression is progressively downregulated in advanced and metastatic melanomas in vivo with a certain intratumoral heterogeneity. Whereas benign melanocytic nevi are RBP2-H1 positive in about 70% of the cases, a lack of RBP2-H1 expression was found in 90% of the primary malignant melanomas and 70% of the melanoma metastases, respectively. Interestingly, a similar deficiency can be found in glioblastomas, but not epithelial cancers. In accordance to the in vivo data, established melanoma cell lines exhibit low but heterogeneous levels of RBP2-H1 expression. By co-immunoprecipitation, we provide the first evidence that a subfraction of total RBP2-H1 can bind to pRb, which makes this protein a true pRb-interacting factor.
We conclude that loss of RBP2-H1 is a common finding in the progression of malignant melanomas. Since a direct interaction of RBP2-H1 and pRb seems possible, the loss of RBP2-H1 may possibly contribute to uncontrolled growth in malignant melanomas.RGS1
Auerback Melanoma Research Laboratory, Cutaneous Oncology Program, UCSF Comprehensive Cancer Center, Department of Dermatology, University of California San Francisco, San Francisco, CA 94115, USA.
RGS1 (regulator of G protein signaling 1) encodes a member of the regulator of G protein family. Recently, RGS1 was found to be overexpressed in gene expression-profiling studies of melanoma. However, no analyses have been reported of its expression at the protein level in melanoma.
In this study, the potential impact of RGS1 as a molecular prognostic marker for melanoma was assessed using immunohistochemical analysis of a melanoma tissue microarray containing primary cutaneous melanomas from 301 patients. High RGS1 expression was significantly correlated with increased tumor thickness (P=0.0083), mitotic rate (P=0.04), and presence of vascular involvement (P<0.02). Kaplan-Meier analysis demonstrated a significant association between increasing RGS1 expression and reduced relapse-free survival (P=0.0032) as well as disease-specific survival (DSS) (P=0.018) survival. Logistic regression analysis showed RGS1 overexpression to be significantly correlated to sentinel lymph node metastasis (P=0.04). Multivariate Cox regression analysis showed that increasing RGS1 immunostaining had an independent impact on the relapse-free survival (P=0.0069) and DSS (P=0.0077) of this melanoma cohort. In the analysis of DSS, RGS1 expression level was the most powerful factor predicting DSS.
RGS1 immunostaining retained independent prognostic impact even when sentinel lymph node status was included in the prognostic model (P=0.0039). These results validate the role of RGS1 as a novel prognostic marker for melanoma given its impact on the survival associated with melanoma.Skp2
- Skp2 and p27 expression in melanocytic nevi and melanoma: an inverse relationship*.
Li Q, Murphy M, Ross J, Sheehan C, Carlson JA.
Department of Pathology and Laboratory Medicine, Albany Medical College, New Scotland Ave., Albany, NY, USA.
J Cutan Pathol. 2004 Nov;31(10):633-42 Abstract quote
Background: S-phase kinase associated protein-2 (Skp2) ubiquitin ligase p45(SKP2) is important in the degradation of p27(kip1) (a cyclin dependent kinase inhibitor) and progression through the G1-S cell-cycle checkpoint. Low levels of p27 and high levels of Skp2 are related to poor prognosis in some cancers.
Methods: Clinicopathologic features and immunohistochemical expression of Skp2 and p27(kip1) were investigated in 198 melanocytic proliferations: 21 melanocytic nevi, 23 melanoma in situ, 119 primary melanoma, and 35 metastatic melanoma samples. Comparative and survival analyses were performed.
Results: Progressive and significant increases and decreases in the nuclear expression of Skp2 and p27(kip1), respectively, was identified moving from melanocytic nevi (0.05 +/- 0.2/85 +/- 15) to melanoma in situ (3 +/- 2/45 +/- 20) to primary cutaneous melanoma (12 +/- 9/30 +/- 25) to metastatic melanoma (25 +/- 15/15 +/- 20) (p </= 0.006). Expression of these proteins also significantly correlated with increasing American Joint Committee on Cancer (AJCC) T (tumor) classification and AJCC stage (p </= 0.01). Moreover, the level of these two proteins exhibited a significant inverse relationship (r = -0.4, p = 0.0001). Skp2 cytoplasmic labeling index of >20% predicted worse 10-year overall survival (38% vs. 86%, p = 0.04) in primary melanoma. Neither p27 nor Skp2 nuclear expression impacted significantly on prognosis.
Conclusions: Gain of Skp2 and loss of p27(kip1) protein expression are implicated in melanoma progression where the level of p27(kip1) may be regulated by targeted proteolysis via Skp2. Cytoplasmic expression of Skp2 defines a subset of aggressive melanomas and could represent another pathway of deregulation of the cell cycle.Trk-C
Expression of neurotrophin receptor Trk-C in nevi and melanomas.Xu X, Tahan SR, Pasha TL, Zhang PJ.
Hospital of University of Pennsylvania, Philadelphia, PA, and Beth Israel Deaconess Medical Center and Harvard Medical School Boston, MA, USA.
J Cutan Pathol. 2003 May;30(5):318-22. Abstract quote BACKGROUND: Neurotrophins (NTs) are growth factors for neurons and other neural crest-derived cells. Their functions are mediated by 75-kDa low-affinity glycoprotein receptor (p75) NT receptor and a family of tyrosine kinase receptors (Trks) that includes Trk-A, -B, and -C. Signal transduction through the Trk receptors has been shown to regulate growth and apoptosis of tumors of neuronal origin. In addition, Trk oncogenes have been shown to be rearranged in some non-neuronal neoplasms. Recently, immunoexpression of NT-3 has been shown to be significantly higher in melanomas than in banal nevi on cryostat tissue.
METHODS: Since the biologic function of NT-3 is mediated primarily through Trk-C, we investigated Trk-C immunoexpression on paraffin sections of 10 compound nevi and 63 melanomas.
RESULTS: The expression of Trk-C was relatively low in compound nevi (30%). Trk-C expression was overall 62% in melanomas of various stages. Our data show that the expression of Trk-C increased as melanoma progressed from in situ (58%) to papillary dermal invasion (91%), and then declined in deeper (57%) and metastatic melanomas (31%).
CONCLUSION: These findings suggest a possible role of Trk-C in the progression of early stages of melanoma.
TRANSFORMING GROWTH FACTOR-BETA
- Transforming growth factor-beta and malignant melanoma: molecular mechanisms.
Hussein MR.
Department of Pathology, School of Medicine, Assuit University, Assuit, Egypt.
J Cutan Pathol. 2005 Jul;32(6):389-95. Abstract quote
Transforming growth factor family members (TGF-beta) are secretory polypeptides that have dual tumor-suppressor and oncogenic effects. They signal through kinase receptor complexes on the cell surface, which phosphorylate cytoplasmic mediators (SMADs). Upon phosphorylation, SMADs march to the nucleus and interact with coactivators or corepressors to mediate the transcriptional regulation of several genes resulting in diverse effects.
In tumorigenesis, malignant cells escape from the tumor-suppressive effects of TGF-beta by mutational inactivation or dysregulated expression of the molecular components in TGF-beta signaling pathway. Although melanoma cells are resistant to the tumor-suppressive effects of TGF-beta, there are no detectable defects at the receptor/SMAD level. Therefore, in these lesions, it is possible that TGF-beta effects occur independently of TGF-beta receptor/SMAD pathway.
This review seeks to examine the present knowledge about TGF-beta receptor/SMAD signaling pathway and its related genes (SMADs, SKI, Filamin, endoglin, Follistatin, and other molecules) in melanomas. Hussein MR. Transforming growth factor-beta and malignant melanoma: molecular mechanisms.TUMOR DOUBLING TIME
Tumor doubling time of cutaneous melanoma and its metastasis.Carlson JA.
Am J Dermatopathol. 2003 Aug;25(4):291-9. Abstract quote Tumor doubling time, estimated using clinical data, is one factor used to determine who will benefit from surgery for patients with pulmonary metastatic melanoma.
Reported herein are the tumor doubling times of four primary cutaneous melanomas in which the diagnosis of melanoma had been delayed, and their metastases (3 of 4). The median/mean tumor doubling times for primary melanoma and metastatic melanoma was 94/144 days (range 50-377) and 33/64 days (range 8-212), respectively.
By paired t test, metastatic tumor doubling time was significantly faster than primary tumor doubling time (P = 0.01). Extrapolating the growth curves of metastatic melanoma to the point of onset of metastasis revealed that dissemination of tumor cells occurred well before first clinical presentation and symptoms in two cases, and in one case, during the interval between clinical presentation and complete excision of the primary. At the opposite pole, extrapolated growth curves for slow tumor doubling time melanoma demonstrated that long-term follow up (>10 years) is required to determine whether patients have been cured of melanoma or not.
Overall, these estimates of melanoma tumor doubling time validate the concept of tumor progression, reinforce the tenet of early detection and curative excision, and can explain the phenomenon of late onset of metastases, respectively.TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND
Discipline of Pathology, Faculty of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia; Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, New South Wales 2050, Australia.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in melanoma by interaction with death receptors TRAIL-R1 (DR4) or TRAIL-R2 (DR5) on melanoma cells or resists apoptosis by interaction with decoy receptors TRAIL-R3 (DcR1) or TRAIL-R4 (DcR2).
Studies on cell lines suggest that there is a wide variation in TRAIL death receptor expression; however, their expression on excised human melanoma is not well documented. In view of this, we studied death receptor expression on melanomas using monoclonal antibodies specific for these receptors.
Immunohistochemical staining for DR4, DR5, and DcR1/DcR2 was performed on formalin-fixed paraffin-embedded sections of 100 cases of primary melanoma, metastatic melanoma, and benign nevi. Percentage expressions of DR4 versus DR5 in benign nevi, primary melanoma, and melanoma metastases were 40% versus 90%, 69% versus 98%, and 55% versus 66%, respectively. There were significant differences in the mean percentage of DR5-positive cells between different groups of melanocytic lesions. Percent expression was higher in thin (</=1.0 mm) compared with thick primary melanoma (88.9% versus 66.9%), and expression was less in subcutaneous metastases (49%) and lymph node metastases (30.6%) (P < .005). Expression was also higher in compound nevi (57%) than dysplastic nevi (49%). DcR1/DcR2 was found in 75% of benign nevi, 62% of primary melanomas, and 74% melanoma metastases.
The results showed a wide variation in the expression of death receptors for TRAIL between and within primary and metastatic melanoma and a decreased expression on the thick primary melanoma and metastatic melanoma.
This suggests that melanoma may not respond to treatment with TRAIL unless given with agents that increase the expression of TRAIL death receptors.TYROSINE KINASE
Analysis of protein tyrosine kinase expression in melanocytic lesions by tissue array.
Shen SS, Zhang PS, Eton O, Prieto VG.
Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Baylor College of Medicine, and Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
J Cutan Pathol. 2003 Oct;30(9):539-47. Abstract quote
BACKGROUND: It has been proposed that melanoma progression involves a multistep process from benign nevi (BN), dysplastic nevi (DN), radial and vertical growth phase melanoma (MM) to metastatic melanoma (MMM). Protein tyrosine kinases (PTKs) may participate in this progression.
METHODS: Tissue microarray blocks of 89 melanocytic lesions were evaluated by immunohistochemistry for the expression of selected PTKs: c-kit, c-abl, abl-related gene (ARG), platelet-derived growth factor receptors alpha (PDGFR-alpha) and beta (PDGFR-beta).
RESULTS: Seventeen of 31 (55%) MMM lacked expression of c-kit versus 100% expression (18/18) in DN and 96% expression (22/23) in MM; similarly, only 59% (10/17) of BN showed expression of c-kit. PDGFR-beta expression levels were similar in BN, DN, and MM, but lower in MMM. There was a trend toward lower expression of abl and ARG from BN to MMM. There was a marked decrease in staining intensity of ARG from BN to DN, MM, and MMM.
CONCLUSION: Our results support that BN is different from DN and MM and that these two are different from MMM. Metastasis appears to be associated with loss of c-kit and PDGFR-beta expression. Since malignant melanoma expresses PTK, it may be a candidate for treatment with anti-PTK, such as STI-571 (Gleevec).ULTRAVIOLET LIGHT
Morphological changes and apoptosis in radial growth phase melanoma cell lines following ultraviolet-B irradiation.
Hussein MR, Hassan M, Wood GS.
Department of Dermatology, University of Wisconsin and William S Middleton Memorial Veteran Hospital, Madison, Wisconsin 537158, USA.
Am J Dermatopathol. 2003 Dec;25(6):466-72. Abstract quote
BACKGROUND: Knowledge about the morphologic changes following ultraviolet irradiation in the earliest stages of melanomas is still lacking.
METHODS: To investigate these changes, an in vitro system consisting of radial growth phase Wistar melanoma cell lines (WM35 and WM3211) was established. Cells were irradiated with a single erythemogenic dose of UVB (10 mJ/cm2) and evaluated for morphologic changes.
RESULTS: When compared with the non-irradiated cells, inverted light microscopy revealed increased cellular branching, cytoplasmic size, and multinucleation in the irradiated cells. Transmission electron microscopy revealed the features of increased metabolic activity (hyperplasia of the mitochondria and Golgi) and those of ultrastructural atypia (pleomorphism of the nuclei and nucleoli, increased euchromatin, and nucleolar margination) in the irradiated cells. Moreover, UVB irradiation caused an increase in the apoptotic activity. These alterations were associated with up-regulation of p53, Bcl-2, and the second mismatch repair protein (hMSH2), as revealed by Western blot analysis.
CONCLUSIONS: UVB irradiation can induce apoptosis, morphologic changes, and altered expression of p53, Bcl-2, and hMSH2 in radial growth phase melanoma cell lines. Up-regulation of p53, Bcl-2, and hMSH2 suggests that these factors are involved in the altered balance between survival and apoptosis induced by UVB. Further investigation will be needed to determine if apoptosis and ultrastructural atypia reflect underlying DNA damage and genomic instability induced by UVB.The risk of melanoma in association with long-term exposure to PUVA
Robert S. Stern, etal.
J Am Acad Dermatol 2001;44:755-61 Abstract quote
Background: Oral methoxsalen (psoralen) and ultraviolet A radiation (PUVA) is a highly effective therapy for psoriasis and many other skin conditions. It is carcinogenic. Previously we reported an increased risk of melanoma that first emerged 15 years after first treatment.
Objective: Our purpose is to present additional data concerning the associations of previous exposure to PUVA, the passage of time, and the risk of malignant melanoma. Methods: We have prospectively studied a cohort of 1380 patients first treated with PUVA in 1975 and 1976. We have documented the occurrence of melanoma and in this report compare the observed and expected incidence of melanoma in this cohort, particularly melanomas developing since our earlier report (ie, after March 1996).
Results: Since 1975, 23 patients have developed 26 invasive or in situ cutaneous melanomas. In an average of 2.25 years since our last report, we detected 7 additional invasive melanomas (incidence rate ratio, 8.4; 95% confidence interval, 3.4-17.3).
Conclusion: Beginning 15 years after first exposure to PUVA, an increased risk of melanoma is observed in our cohort of PUVA-treated patients. This risk is greater in patients exposed to high doses of PUVA, appears to be increasing with the passage of time, and should be considered in determining the risks and benefits of this therapy
Ultraviolet A and melanoma: A review
Steven Q. Wang, etal.
J Am Acad Dermatol 2001;44:837-46 Abstract quote
The incidence and mortality rates of melanoma have risen for many decades in the United States. Increased exposure to ultraviolet (UV) radiation is generally considered to be responsible. Sunburns, a measure of excess sun exposure, have been identified as a risk factor for the development of melanoma. Because sunburns are primarily due to UVB (280-320 nm) radiation, UVB has been implicated as a potential contributing factor to the pathogenesis of melanoma. The adverse role of UVA (320-400 nm) in this regard is less well studied, and currently there is a great deal of controversy regarding the relationship between UVA exposure and the development of melanoma.
This article reviews evidence in the English-language literature that surrounds the controversy concerning a possible role for UVA in the origin of melanoma.
Our search found that UVA causes DNA damage via photosensitized reactions that result in the production of oxygen radical species. UVA can induce mutations in various cultured cell lines. Furthermore, in two animal models, the hybrid Xiphophorus fish and the opossum (Mondelphis domestica), melanomas and melanoma precursors can be induced with UVA. UVA radiation has been reported to produce immunosuppression in laboratory animals and in humans. Some epidemiologic studies have reported an increase in melanomas in users of sunbeds and sunscreens and in patients exposed to psoralen and UVA (PUVA) therapy. There is basic scientific evidence of the harmful effects of UVA on DNA, cells and animals.
Collectively, these data suggest a potential role for UVA in the pathogenesis of melanoma. To date evidence from epidemiologic studies and clinical observations are inconclusive but seem to be consistent with this hypothesis. Additional research on the possible role of UVA in the pathogenesis of melanoma is required.
Semin Cutan Med Surg 1996;15:336-348
There is thought to be a stepwise development of melanoma through 5 stages:1. Nevi with architectural disorder
2. Unregulated proliferation of melanocytes within the epidermis (melanoma in situ)
3. Acquisition of the competence to invade and proliferate in the dermal connective tissue (invasive melanoma)
4. Development of metastatic capacity (invasive melanoma)
5. Metastasis to distant sites (metastatic melanoma)
DNA repair, dysplastic nevi, and sunlight sensitivity in the development of cutaneous malignant melanoma.Landi MT, Baccarelli A, Tarone RE, Pesatori A, Tucker MA, Hedayati M, Grossman L.
Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892-7236, USA.
J Natl Cancer Inst 2002 Jan 16;94(2):94-101 Abstract quote BACKGROUND: Exposure to UV radiation is associated with cutaneous malignant melanoma (CMM). In mammalian cells, UV radiation induces DNA damage that can be repaired by the nucleotide excision repair system. We designed this case-control study to determine whether DNA repair capacity (DRC) is associated with the risk of CMM and to identify risk factors that may interact biologically with DRC in the development of melanoma.
METHODS: Global DRC was measured in lymphocytes with the host-cell reactivation assay. Data were analyzed by use of multiple regression models. All statistical tests were two-sided.
RESULTS: DRC could be determined for 132 case patients with incident melanoma and for 145 age- and sex-matched control subjects. No statistically significant association between melanoma risk and DRC by itself was found (odds ratio [OR] = 1.0; 95% confidence interval [CI] = 0.6 to 1.7, adjusted for age, sex, lymphocyte viability, and sample storage time). DRC, however, strongly influenced CMM risk in individuals with a low tanning ability or dysplastic nevi. Individuals with a low tanning ability and a low DRC had a higher risk for CMM (OR = 8.6; 95% CI = 2.7 to 27.5) than individuals with a higher tanning ability and a high DRC. Likewise, individuals with dysplastic nevi and a low DRC had a higher relative risk (OR = 6.7; 95% CI = 2.4 to 18.6) than those lacking dysplastic nevi and having a high DRC. Subjects with dysplastic nevi and a high DRC had an intermediate risk. A likelihood-ratio test gave statistically significant interactions between DRC and tanning response (P =.001) and between DRC and dysplastic nevus status (P =.04), which were independently associated with CMM risk.
CONCLUSIONS: DRC may modify the risk for melanoma in the presence of other strong risk factors, such as a low tanning ability and the presence of dysplastic nevi. The occurrence of melanoma in subjects without these risk factors appears to be independent of DRC.
VASCULAR ENDOTHELIAL GROWTH FACTOR
Immunologic escape and angiogenesis in human malignant melanoma.Redondo P, Sanchez-Carpintero I, Bauza A, Idoate M, Solano T, Mihm MC Jr.
Department of Dermatology, University Clinic of Navarra, School of Medicine, Pamplona, Spain
J Am Acad Dermatol. 2003 Aug;49(2):255-63. Abstract quote BACKGROUND: Melanoma escape mechanisms include immunosuppressive and angiogenic cytokine production.
OBJECTIVE: We sought to determine vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression by immunohistochemistry, and soluble circulating plasma levels of VEGF, bFGF, IL-10, and transforming growth factor-beta2 in patients with different stages of melanoma.
METHODS: Biopsy specimens from 42 patients with primary melanoma and 9 with cutaneous metastases were studied by immunohistochemistry. In another 46 patients with melanoma (8 stage I and II; 18, III; and 20, IV) and in 10 healthy control participants, bFGF, VEGF, IL-10, and transforming growth factor-beta2 circulating levels were analyzed.
RESULTS: bFGF was positive in 85% and VEGF in 47.5% of 42 primary melanomas. Of 10 patients with primary melanoma (Breslow depth 1.5-3 mm) 6 were VEGF positive and had metastases develop, whereas 4 were VEGF negative and had no metastases at 5 years of follow up. VEGF, bFGF, and IL-10 plasma levels in patients with stages III and IV melanoma were higher than the control group (P <.05 and P <.01, respectively). An inverse relationship was found between VEGF and IL-10. Specifically, in 7 patients with IL-10 levels higher than 10 pg/mL, VEGF levels were less than 49 pg/mL (P <.05); in 9 patients with VEGF levels higher than 100 pg/mL, IL-10 levels were less than 6.7 pg/mL (P <.01).
CONCLUSION: VEGF expression in 1.5- to 3.0-mm Breslow depth melanomas may be considered as an unfavorable prognostic factor. Immunosuppressive (IL-10, transforming growth factor-beta2) and proangiogenic (bFGF, VEGF) cytokines are increased in metastatic melanoma. Inverse plasma levels between IL-10 and VEGF in patients with metastatic melanoma are shown in vivo for the first time, the significance of which must be further investigated.
LABORATORY/
RADIOLOGICCHARACTERIZATION COLOR DOPPLER
SONOGRAPHY
Improved differentiation of benign and malignant lymphadenopathy in patients with cutaneous melanoma by contrast-enhanced color Doppler sonography.Schmid-Wendtner MH, Partscht K, Korting HC, Volkenandt M.
Department of Dermatology and Allergology, Ludwig-Maximilians-University, Frauenlobstr 9-11, D-80337 Munich, Germany.
Arch Dermatol 2002 Apr;138(4):491-7 Abstract quote OBJECTIVE: To evaluate whether administration of a D-galactose-based signal enhancer is useful in color Doppler sonography (CDS) for better detection of vascularity patterns, which may help to differentiate malignant from benign lymph nodes in patients with cutaneous melanomas.
DESIGN: Comparison of B-mode sonography, native CDS, and signal-enhanced CDS.
SETTING: Department of Dermatology and Allergology, Ludwig-Maximilians-University, Munich, Germany.
PATIENTS: Twenty examinations in 19 patients (median age, 60 years; 10 men) who presented with echo-poor structures suggestive of lymphadenopathy in B-mode sonography during follow-up for cutaneous melanomas.
INTERVENTIONS: Histopathologic and follow-up examinations; documentation by color prints.
MAIN OUTCOME MEASURES: Frequency of detection and description of different lymph node vascularity patterns in signal-enhanced CDS.
RESULTS: Signal-enhanced CDS revealed additional information about vascularization of lymph node metastases, reactive lymph nodes, hematomas, and seromas, which was helpful for the differential diagnosis in 15 of 20 examinations. For lymph node metastases, signal enhancement facilitated the detection of accessory peripheral vessels in most investigations. Concerning reactive lymph nodes, hilar vessels in part with branching to the lymph node periphery could be identified only after application of the contrast enhancer in most patients. Quantitative variables could not be measured in all cases and did not help to differentiate between malignant and reactive lymph nodes.
CONCLUSIONS: Administration of a D-galactose-based signal enhancer for CDS in patients with cutaneous melanomas can help to differentiate malignant from reactive lymph nodes, hematomas, or seromas. However, these promising results require confirmation in a prospective multicenter study.
COMPARATIVE GENOMIC HYBRIDIZATION J Cutan Pathol. 2009 Jun 11.
Comparative genome hybridization analysis of laser-capture microdissected in situ melanoma.
Vincek V, Xu S, Fan YS.Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Abstract quote
Background: The progression of melanoma occurs through discrete stages with known clinical and histologic features. Although many molecular events that occur during the progression of invasive and metastatic melanomas have been elucidated, there is limited knowledge of genetic changes that occur in the earliest stages of melanoma development. In this pilot study, we investigated genetic changes that happen in in situ melanoma so that we can better understand early melanoma development.
Materials and methods: DNA was extracted from five laser-capture microdissected Clark's level III melanomas, five in situ melanomas and five compound nevi all from sun exposed skin. Array-based comparative genomic hybridization was performed using Agilent 44 K platform.
Results: The group of Clark's level III melanomas was characterized with multiple large deletions and duplications. In the group of in situ melanoma, deletions and duplications were limited in size. Deletions in in situ melanomas were present only on chromosomes 13q and 16q. Compound nevi did not show any significant chromosomal aberrations.
Conclusion: In situ melanomas show characteristic chromosomal aberrations that are limited compared to melanomas that invade the dermis. Deletion of 13q found in in situ melanomas, which encompass the Rb1 tumor suppressor gene, might be one of the first events in the development of melanoma.DERMOSCOPY Significance of Dermoscopic Patterns in Detecting Malignant Melanoma on Acral Volar Skin Results of a Multicenter Study in Japan
Toshiaki Saida, MD, PhD ;Atsushi Miyazaki, MD ;Shinji Oguchi, MD, PhD ;Yasushi Ishihara, MD ;Yoriko Yamazaki, MD ;Sumio Murase, MD, PhD ;Shusuke Yoshikawa, MD ;Tetsuya Tsuchida, MD, PhD ;Yasuhiro Kawabata, MD, PhD ;Kunihiko Tamaki, MD, PhD Arch Dermatol. 2004;140:1233-1238. Abstract quote Objective To determine diagnostic variables such as sensitivity and specificity of the major dermoscopic patterns observed in melanocytic lesions on acral volar skin, with particular attention to the significance of the parallel ridge pattern and irregular diffuse pigmentation in detecting acral melanoma.
Design Multicenter, retrospective study.
Setting University hospitals in Japan.
Patients Patients with melanocytic lesions on acral volar skin. A total of 712 melanocytic lesions (103 malignant melanomas, including 36 in situ lesions, and 609 melanocytic nevi) were consecutively collected from the files of 3 hospitals. Diagnoses of all the lesions had been determined histopathologically.
Interventions Dermoscopic examination.
Main Outcome Measures The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the major dermoscopic patterns seen in benign and malignant melanocytic lesions on acral volar skin.
Results The parallel ridge pattern and irregular diffuse pigmentation showed extremely high specificity (99.0% and 96.6%, respectively) and very high negative predictive value (97.7% and 97.5%, respectively) in malignant melanoma. For melanoma in situ, the positive predictive value and diagnostic accuracy of the parallel ridge pattern were significantly higher than those of irregular diffuse pigmentation (P = .009 and P = .006, respectively). In melanocytic nevi, the specificity and positive predictive value of the parallel furrow pattern and/or the latticelike pattern were found to be very high (93.2% and 98.3%, respectively).
Conclusions Dermoscopy is immensely helpful in differentiating malignant melanomas from melanocytic nevi on acral volar skin. Moreover, the parallel ridge pattern aids in detecting acral melanomas in early, curable stages.
Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet.Argenziano G, Soyer HP, Chimenti S, Talamini R, Corona R, Sera F, Binder M, Cerroni L, De Rosa G, Ferrara G, Hofmann-Wellenhof R, Landthaler M, Menzies SW, Pehamberger H, Piccolo D, Rabinovitz HS, Schiffner R, Staibano S, Stolz W, Bartenjev I, Blum A, Braun R, Cabo H, Carli P, De Giorgi V, Fleming MG, Grichnik JM, Grin CM, Halpern AC, Johr R, Katz B, Kenet RO, Kittler H, Kreusch J, Malvehy J, Mazzocchetti G, Oliviero M, Ozdemir F, Peris K, Perotti R, Perusquia A, Pizzichetta MA, Puig S, Rao B, Rubegni P, Saida T, Scalvenzi M, Seidenari S, Stanganelli I, Tanaka M, Westerhoff K, Wolf IH, Braun-Falco O, Kerl H, Nishikawa T, Wolff K, Kopf AW.
Department of Dermatology, Second University of Naples, Italy.
J Am Acad Dermatol 2003 May;48(5):679-93 Abstract quote BACKGROUND: There is a need for better standardization of the dermoscopic terminology in assessing pigmented skin lesions.
OBJECTIVE: The virtual Consensus Net Meeting on Dermoscopy was organized to investigate reproducibility and validity of the various features and diagnostic algorithms.
METHODS: Dermoscopic images of 108 lesions were evaluated via the Internet by 40 experienced dermoscopists using a 2-step diagnostic procedure. The first-step algorithm distinguished melanocytic versus nonmelanocytic lesions. The second step in the diagnostic procedure used 4 algorithms (pattern analysis, ABCD rule, Menzies method, and 7-point checklist) to distinguish melanoma versus benign melanocytic lesions. kappa Values, log odds ratios, sensitivity, specificity, and positive likelihood ratios were estimated for all diagnostic algorithms and dermoscopic features.
RESULTS: Interobserver agreement was fair to good for all diagnostic methods, but it was poor for the majority of dermoscopic criteria. Intraobserver agreement was good to excellent for all algorithms and features considered. Pattern analysis allowed the best diagnostic performance (positive likelihood ratio: 5.1), whereas alternative algorithms revealed comparable sensitivity but less specificity. Interobserver agreement on management decisions made by dermoscopy was fairly good (mean kappa value: 0.53).
CONCLUSION: The virtual Consensus Net Meeting on Dermoscopy represents a valid tool for better standardization of the dermoscopic terminology and, moreover, opens up a new territory for diagnosing and managing pigmented skin lesions.
Modified ABC-point list of dermoscopy: A simplified and highly accurate dermoscopic algorithm for the diagnosis of cutaneous melanocytic lesions.Blum A, Rassner G, Garbe C.
Department of Dermatology, University of Tuebingen.
J Am Acad Dermatol 2003 May;48(5):672-8 Abstract quote BACKGROUND: The use of dermoscopy (epiluminescence microscopy, surface microscopy, dermatoscopy) improves clinical diagnostic sensitivity by 10% to 27%, particularly achieved by different algorithms or scores.
OBJECTIVE: We sought to develop a simplified and highly accurate dermoscopic-point list for cutaneous melanocytic lesions.
METHOD: We studied consecutive patients with suspicious melanocytic lesions, which were excised and histopathologically examined at our institution. On the basis of the ABCD rule of Stolz, Menzies score, and the modified ABCD rule of Kittler, a simplified ABC-point list was developed. Simple points were given for the following: asymmetry of outer shape (A) or differential structures inside the lesion in at least 1 axis ((A)); the abrupt cutoff of network at the border in at least one quarter of circumference (B); 3 or more colors (C); 3 or more differential structures (D); or noticed change (evolution) in the last 3 months (E). Using 20-fold magnification of computer dermoscopy, the sensitivity, specificity, and diagnostic accuracy were examined in 269 cutaneous melanocytic lesions. Of these, 84 (31.2%) were cutaneous melanomas. Also, the sensitivity, specificity, and diagnostic accuracy were investigated with a 7-point checklist and the 7 features for melanoma.
RESULTS: With the ABC-point list for the diagnosis of cutaneous melanoma, sensitivity was 90.5%, specificity was 87%, and diagnostic accuracy was 88.1%, confirmed by cross-validation. The ABCD rule resulted in 90.5%, 72.4%, and 78.1%; Menzies score in 95.2%, 77.8%, and 83.3%; 7-point checklist in 90.5%, 87%, and 88.1%; and 7 features for melanoma in 94%, 74.6%, and 80.7%, respectively.
CONCLUSIONS: The ABC-point list is simpler than the already established algorithms. Despite its simplicity, a high sensitivity, specificity, and diagnostic accuracy was achieved. This simplified approach in dermoscopic diagnostics may contribute to further spread and enable to learn and use this method more easily.
Early melanoma detection: Nonuniform dermoscopic features and growth.Lucas CR, Sanders LL, Murray JC, Myers SA, Hall RP, Grichnik JM.
Department of Medicine, Duke University Medical Center.
J Am Acad Dermatol 2003 May;48(5):663-71 Abstract quote BACKGROUND: Dermoscopy alone is not sufficient to detect all early melanomas. Total body photos reveal growth of melanomas but also reveal growth of melanocytic nevi.
OBJECTIVE: We set out to determine whether a simplified algorithm on the basis of nonuniform dermoscopic features combined with growth noted from baseline total body photos targeted the early melanocytic neoplasms most likely to be malignant.
METHODS: Lesions removed during follow-up of patients with total body photographs were reviewed and 169 melanocytic lesions were identified for which both gross clinical and dermoscopic photos were available. The images were evaluated separately by 3 academic dermatologists, without knowledge of the given pathologic diagnosis, for uniformity (consistent gradient of features from center to edge) and change (specifically, that which could indicate melanoma growth in normal skin or within a nevus).
RESULTS: Using a minimum of 2 out of 3 agreement for uniformity and change, 12 of 16 melanomas (including all 5 superficially invasive tumors) were graded as nonuniform and changed. The 4 melanomas not included in this category were in situ. The predicted odds of melanoma for lesions scored as both nonuniform and changed was 4.06 (P >.0195). If 3 out of 3 agreement was used, the odds ratio increased to 6 (P >.0010).
CONCLUSION: An algorithm on the basis of dermoscopic nonuniformity and change suggestive of growth as determined by total body photography segregates melanocytic neoplasms most likely to be malignant.
Preoperative Melanoma Thickness Determination by 20-MHz Sonography and Digital Videomicroscopy in Combination.Pellacani G, Seidenari S.
Department of Dermatology, University of Modena and Reggio Emilia, 41100 Modena, Italy.
Arch Dermatol 2003 Mar;139(3):293-8 Abstract quote OBJECTIVE: To identify accurately thick melanomas preoperatively by means of a combined approach based on sonography and clinical-videomicroscopic evaluation.
DESIGN: Ultrasonographic thickness measurement, obtained by means of a 20-MHz B-scanner, and identification of clinical and videomicroscopic variables useful in distinguishing between thick and thin melanomas were performed on a training set of 40 melanomas. An algorithm based on echographic, clinical, and videomicroscopic criteria was constructed to develop a method for preoperative evaluation of melanoma thickness and was validated on a test set of 48 melanomas.
SETTING: University medical department.
PATIENTS: Eighty-eight patients affected by primary cutaneous melanoma.
MAIN OUTCOME MEASURES: Sensitivity and specificity of the algorithm, with the use of sonographic, clinical, and videomicroscopic data, in thick melanoma identification.
RESULTS: Echographic thickness was calculated for each lesion. On the training set, 2 clinical and 7 videomicroscopic features were identified for distinction between thick and thin melanomas: nonpalpability, central pigment network, central brown globules, and blotches were characteristic of thin melanomas; clinical regression, localized peripheral pigment network, veil, grayish polygonal areas, and blood vessels were characteristic of thick ones. A coefficient was attributed to each variable and a score was obtained for each lesion. The algorithm, developed for preoperative thickness prediction, was validated on the test set, enabling the distinction of thick melanomas with an 86.7% sensitivity and a 100% specificity.
CONCLUSION: The correct classification of all thin melanomas as such renders this approach suitable in clinical practice.
J Am Acad Dermatol 2001;44:207-18
At 4 clinical centers, images were taken of pigmented lesions suspected of being melanoma before biopsy. Ten gray-level (MelaFind) images of each lesion were acquired, each in a different portion of the visible and near-infrared spectrum
The images of 63 melanomas (33 invasive, 30 in situ) and 183 melanocytic nevi (of which 111 were dysplastic) were processed automatically through a computer expert system to separate melanomas from nevi. The expert system used either a linear or a nonlinear classifier.
The “gold standard” for training and testing these classifiers was concordant diagnosis by two dermatopathologists
On resubstitution, 100% sensitivity was achieved at 85% specificity with a 13-parameter linear classifier and 100%/73% with a 12-parameter nonlinear classifier. Under leave-one-out cross-validation, the linear classifier gave 100%/84% (sensitivity/specificity), whereas the nonlinear classifier gave 95%/68%. Infrared image features were significant, as were features based on wavelet analysis.
Automatic differentiation of invasive and in situ melanomas from melanocytic nevi is feasible, through multispectral digital dermoscopy.
Detection of Clinically Amelanotic Malignant Melanoma and Assessment of Its Margins by In Vivo Confocal Scanning Laser Microscopy
Klaus J. Busam, MD; Katherine Hester, MD; Carlos Charles, MD; Dana L. Sachs, MD; Cristina R. Antonescu, MD; Salvador Gonzalez, MD; Allan C. Halpern, MD
Arch Dermatol. 2001;137:923-929 Abstract quote
Background Near-infrared confocal scanning laser microscopy (CSLM) represents a novel imaging technique for in vivo microscopic analysis of skin lesions, including pigmented lesions.
Objectives To investigate the feasibility of detecting a clinically amelanotic malignant cutaneous melanoma using CSLM and to explore the use of this technique for assessing its margins.
Patients and Methods Two lesions from 2 patients were imaged and analyzed using CSLM. Sites suspected to represent melanoma or benign skin on CSLM were marked as such; then, biopsy specimens were obtained for diagnosis using conventional histological analysis. Both lesions were stained for melanin pigment and analyzed immunohistochemically for the expression of melanosomal markers. In 1 case, a biopsy specimen was also examined with electron microscopy.
Results The images obtained using CSLM allowed recognition of an abnormal intraepidermal melanocytic proliferation that was distinctly different from normal skin. Comparison of the sites examined using CSLM and subsequently using conventional histological methods revealed that CSLM correctly identified intraepidermal melanoma and benign skin. Fontana-Masson stains and immunohistochemical and ultrastructural studies showed that clinically amelanotic melanoma cells contained melanosomes and rare melanin granules.
Conclusions We demonstrated, for the first time, the detection of clinically amelanotic melanoma using CSLM. This technique may aid in the early detection of clinically barely visible or nonpigmented melanomas and may facilitate preoperative noninvasive assessment of their margins.
GENE EXPRESSION MICROARRAY
UCLA Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095-1732, USA.
Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology.
To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes.
In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology.
Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.IN VIVO EXAMINATION
Instruments and new technologies for the in vivo diagnosis of melanoma.
Marghoob AA, Swindle LD, Moricz CZ, Sanchez Negron FA, Slue B, Halpern AC, Kopf AW.
J Am Acad Dermatol. 2003 Nov;49(5):777-97. Abstract quote
The principal objective of screening individuals at risk for melanoma is detection of cutaneous melanoma during the curable stages of its early evolution. Unaided visual inspection of the skin is often suboptimal at diagnosing melanoma. Improving the diagnostic accuracy for melanoma remains an area of active research. These research efforts have focused on both the detection of early melanoma and the in-depth evaluation of suspicious pigmented lesions for the presence or absence of melanoma.
Numerous instruments are under investigation to determine their usefulness in imaging and ascertaining a correct in vivo diagnosis of melanoma. It is anticipated that some of these tools, alone or in combination, will improve our ability to differentiate, in vivo, melanoma from its simulators.Ultimately, these advances may prevent unnecessary biopsies (increased specificity) while increasing the sensitivity for diagnosing melanoma. This article reviews the current instruments and new technologies for the in vivo diagnosis of melanoma.
At the conclusion of this learning activity, participants should be acquainted with the instruments designed to facilitate the early detection of melanoma. They should also be familiar with the basic technology behind these instruments and should recognize the potential benefits and limitations inherent in each.In vivo examination of lentigo maligna and malignant melanoma in situ, lentigo maligna type by near-infrared reflectance confocal microscopy: Comparison of in vivo confocal images with histologic sections
Zeina S. Tannousa
Martin C. Mihm
Thomas J. Flotte
Salvador González
Boston, MassachusettsJ Am Acad Dermatol 2002;46:260-3 Abstract quote
In vivo confocal microscopy can noninvasively image thin en face sections within living intact human tissue with high resolution and contrast.
This evolving technique may provide clinicians with tools to help detect lentigo maligna lesion progression in vivo and may be important in defining tumor margins, thus providing a more definitive surgical eradication of lentigo maligna and malignant melanoma in situ, lentigo maligna type.
We present a case of malignant melanoma in situ, lentigo maligna type, and we describe the images seen with confocal microscopy in correlation with routine histopathology.
LABORATORY CD95
Increased soluble CD95 (sFas/CD95) serum level correlates with poor prognosis in melanoma patients.Ugurel S, Rappl G, Tilgen W, Reinhold U.
Department of Dermatology, The Saarland University Hospital, 66421 Homburg/Saar, Germany.
Clin Cancer Res 2001 May;7(5):1282-6 Abstract quote Functional impairment of the Fas/CD95 receptor-ligand system is associated with the development and progression of malignancies. One possible cause might be the inhibition of the formation of a functional Fas/CD95-FasL complex by soluble Fas/CD95 molecules (sFas/CD95).
In the present study we determined sFas/CD95 serum concentration in 125 melanoma patients of different clinical stages of disease compared with 30 healthy controls using an ELISA. sFas/CD95 serum level was significantly elevated (P < 0.0005) in melanoma patients (mean +/- SE = 8.60 +/- 0.26 ng/ml) compared with healthy controls (mean +/- SE = 6.27 +/- 0.25 ng/ml). Univariate analysis revealed a correlation of sFas/CD95 serum concentration with advanced stages of disease (P = 0.009). Only a slight increase in sFas/CD95 serum level (P = 0.057) could be observed in regard to the tumor burden. Patients undergoing current treatment with cytostatics (n = 18) revealed a strong increase in sFas/CD95 serum level (P < 0.0005), whereas treatment with IFN-alpha alone or combined with cytostatics (n = 19) showed no change in serum sFas/CD95 concentration. According to univariate analysis, elevated sFas/CD95 serum levels were associated with a poor overall (P < 0.005) and a progression-free (P < 0.0005) survival. Multivariate analysis revealed sFas/CD95 serum concentration as an independent predictive factor for progression-free (P = 0.011), but not overall (P = 0.078), survival.
Our results show a prognostic relevance of serum sFas/CD95 in melanoma patients, indicating that the evaluation of sFas/CD95 serum level may be important for the selection of therapeutic strategies.
DETECTION OF CIRCULATING MELANOMA CELLS IN PERIPHERAL BLOOD Detection of tyrosinase mRNA by RT-PCR in the peripheral blood of patients with advanced metastatic melanoma.
Alao JP, Mohammed MQ, Slade MJ, Retsas S.
Department of Medical Oncology, Charing Cross Hospital, London, UK.
Melanoma Res 1999 Aug;9(4):395-9 Abstract quote
Detection of melanoma cells in the peripheral blood has been facilitated by the reverse transcriptase-polymerase chain reaction (RT-PCR), but their presence is of uncertain importance in the evolution of the disease. We studied the detection of melanoma cells using RT-PCR in the peripheral blood of 21 patients, four with regional lymph node metastases (American Joint Committee on Cancer [AJCC] stage III) and 17 with disseminated disease (AJCC stage IV). RNA was extracted from 10 ml of heparinized blood following density gradient centrifugation and converted into cDNA for PCR analysis. Assay sensitivity of 10 cells in 10(7) mononuclear cells and granulocytes obtained from 10 ml of peripheral blood was achieved using the G361 and C32 melanoma cell lines. Tyrosinase mRNA was not detected in control samples from healthy volunteers or patients with non-malignant disease. Six patients (one stage III, five stage IV) tested positive for tyrosinase mRNA (28.6%); with one exception, all patients were receiving chemotherapy at the time of sampling. Of the six positive results, three were from patients who initially tested negative but were subsequently positive after a 3-4 week interval. The low detection rates of melanoma cells in the peripheral blood of patients with widely disseminated disease is consistent with recent reports and correlates poorly with the clinical stage of melanoma. This may be partly explained by the clinically observed intermittent and random evolution of melanoma metastases.
Prognostic significance of the detection of circulating malignant cells by reverse transcriptase-polymerase chain reaction in long-term clinically disease-free melanoma patients.
Mellado B, Gutierrez L, Castel T, Colomer D, Fontanillas M, Castro J, Estape J.
Medical Oncology Department, Institut de Investigacions Biomediques August Pi i Sunyer, Hospital Clinic, University of Barcelona, Spain.
Clin Cancer Res 1999 Jul;5(7):1843-8 Abstract quote
The purpose of this study was to assess the prognostic significance of the detection of circulating melanoma cells by reverse transcriptase-PCR in long-term clinically disease-free melanoma patients.
Patients with melanoma who were free of clinical relapse for at least 6 months after primary tumor diagnosis were included and prospectively followed. Tyrosinase mRNA in peripheral blood from these patients was assayed by reverse transcriptase-PCR at the time of their inclusion in the study. One hundred six blood samples from 57 melanoma patients were analyzed. The median time between melanoma diagnosis and inclusion in the study was 24 months (range, 7-51 months). The median follow-up time calculated from the time of inclusion in the study was 27 months (range, 11-36 months). Tyrosinase mRNA in blood was detected in 10 (17.5%) of 57 patients: 2 (18%) of 11 stage I patients, 6 (19%) of 33 stage II patients, and 2 (15%) of 13 stage III patients. Actuarial 2-year DFS was 89% for the tyrosinase-negative patients versus 30% for the positive patients (P = 0.003). Actuarial 2-year OS was 97% for the tyrosinase-negative patients versus 72% for the positive patients (P = 0.001). Tyrosinase mRNA could be detected in the blood of a proportion of long-term disease-free melanoma patients, regardless of their initial clinical stage.
The presence of late circulating melanoma cells in this selected group of clinically disease-free patients was significantly associated with a subsequent high risk of relapse and death.
Arch Dermatol. 2001;137:325-330
Objective:
To systematically review the use of reverse transcriptase–polymerase chain reaction (RT-PCR) for tyrosinase messenger RNA as a molecular serum marker for metastatic melanoma.Data Sources:
Computerized searches (1966-1999) of the PubMed and MDConsult databases and a manual search of retrieved article references.Study Selection:
Cohort studies containing test subjects and negative controls were reviewed.Data Extraction:
Three investigators independently screened abstracts for relevant studies and 2 investigators independently reviewed all eligible studies.Data Synthesis:
Of 127 identified studies, 50 were reviewed in detail and 23 met all inclusion criteria. From these 23 studies, the PCR methods, the total number of patients, the number of control subjects, and the number of RT-PCR–positive patients per stage were analyzed. Results of RT-PCR for tyrosinase messenger RNA were positive in 18% (95% confidence interval [CI], 3%-22%) patients for stage I disease, 28% (95% CI, 23%-34%) for stage II disease, 19% (95% CI, 16%-21%) for stage I/II localized disease, 30% (95% CI, 26%-34%) for stage III disease, and 45% (95% CI, 41%-50%) for stage IV disease. Specificities were 100% in all but 1 study. Results of RT-PCR were positive in only 0.4% of healthy controls and patients with nonmelanoma cancer.Conclusions:
The lack of data on the outcome of stage I, II, and III patients who were RT-PCR positive and the low prevalence of RT-PCR positivity in patients with known stage IV disease limit the applicability of this test at this time. Ongoing and future studies on a quantitative RT-PCR, amplification of multiple melanoma-associated antigens, and use of the test as a prognostic indicator might improve the utility of this molecular serologic tool.Facts and pitfalls in the detection of tyrosinase mRNA in the blood of melanoma patients by RT-PCR.
Seiter S, Rappl G, Tilgen W, Ugurel S, Reinhold U.
Department of Dermatology, Saarland University Hospital, Homburg/Saar, Germany.
Recent Results Cancer Res 2001;158:105-12 Abstract quote
Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear.
We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells.
Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood.
Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.
HYPERCALCEMIA Hypercalcaemia of melanoma: incidence, pathogenesis and therapy with bisphosphonates.
des Grottes JM, Dumon JC, Body JJ.
Supportive Care Clinic and Clinic of Endocrinology/Bone Diseases, Department of Medicine and Laboratory of Endocrinology and Breast Cancer Research, Institut Jules Bordet, Universite Libre de Bruxelles, Rue Heger-Bordet 1, 1000 Brussels, Belgium.
Melanoma Res 2001 Oct;11(5):477-82 Abstract quote
Tumour-induced hypercalcaemia (TIH) is a frequent complication of advanced cancer but has been rarely reported in patients with malignant melanoma, and its pathogenesis remains unexplored.
We studied eight patients with TIH and melanoma. We determined the incidence and pathogenesis of this complication and the effects of bisphosphonate therapy. The incidence of TIH in 751 patients with melanoma was 1.1%. All patients had liver and bone metastases at the time of hypercalcaemia. All patients had osteolytic lesions, most often multiple. The median survival was 30 days (range 4-136 days). After rehydration, the mean (+/- SEM) corrected calcium was 3.42 +/- 0.17 mmol/l.
Parathyroid hormone levels were adequately suppressed and vitamin D concentrations were normal. Serum osteocalcin, a marker of bone formation, was low, except in the two patients with renal insufficiency, whereas fasting urinary calcium and hydroxyproline were increased, indicating inhibition of bone formation and stimulation of bone resorption. Increased parathyroid hormone-related protein secretion was noted in only one patient. Three of four patients became normocalcaemic after bisphosphonate therapy for a median duration of 2 weeks.
In conclusion, hypercalcaemia is a rare complication of melanoma. It occurs in the context of far advanced disease and is essentially due to aggressive lytic bone metastases with an uncoupling in bone turnover. Bisphosphonates can offer short-term palliation.
DNA MICROARRAY
Gene expression profiling of melanocytic lesions.Seykora JT, Jih D, Elenitsas R, Horng WH, Elder DE.
Am J Dermatopathol 2003 Feb;25(1):6-11 Abstract quote DNA microarrays, microscopic grids of DNA, can be used to assess gene expression within a particular cell or cell population. Since DNA from thousands of genes can be hybridized and analyzed in one experiment, researchers can globally characterize genes expressed in normal and various pathologic states.
To accurately assess the differences between normal and pathologic states, one derives cDNA from control and diseased tissue specimens for genes expression profiling. For these reasons, microarray technology may be of particular interest to dermatopathologists and dermatologists interested in understanding cutaneous disease because these physicians have access to tissue specimens. In addition, microarray technology is an efficient way of identifying molecules expressed in a cell population; therefore, it can be used to search for unique immunohistologic markers.
To this end, we have used microarray technology to define differences in the gene expression profile of nevi and melanomas. In this manuscript, we discuss the results of our study, which confirm previously known differences in gene expression between melanoma and nevi. While a few genes appear slightly overexpressed in nevi, a number of genes involved in regulating cell proliferation were upregulated in melanoma, such as cyclin D1, cdc2-related protein kinase, c-Myc binding protein, early growth response protein 1, and pleiotrophin. "Housekeeping" genes such as glyceraldehyde 3-phosphate dehydrogenase were expressed at similar levels in melanoma and nevi.
Surprisingly, a majority of genes were expressed at similar levels in both nevi and melanoma. Based on this study, DNA microarray technology appears to be a valuable tool for identifying genes that may be specifically expressed in cutaneous lesions.
GROSS APPEARANCE/
CLINICAL VARIANTSCHARACTERIZATION MELANOSIS ASSOCIATED WITH METASTATIC DISEASE Am J Dermatopathol 1999;21:28-30
Arch Derm Venereal (Stackh) 1993;73:241-250
Diffuse melanosis after chemotherapy-induced tumor lysis syndrome in a patient with metastatic melanoma
Klaus J. Busam, Jedd Wolchok, Achim A. Jungbluth and Paul Chapman
Journal of Cutaneous Pathology
Volume 31 Issue 3 Page 274 - March 2004 Abstract quote
Abstract: Diffuse melanosis is a rare event associated with advanced metastatic malignant melanoma. A 35-year-old woman with stage IV melanoma is presented, who developed slate bluish-gray to brown discoloration of her skin after chemotherapy-induced tumor lysis syndrome. A number of studies were performed to re-evaluate possible mechanisms of melanosis.
Skin tissue was examined on routine hematoxylin-and-eosin-stained sections, Fontana stains, immunohistochemical studies with antibodies for Melan-A, gp100, tyrosinase, FXIIIa, and CD68, and by electron microscopy. The main cell types found to contain melanin pigment were histiocytes and dendritic cells. In the dermis, they were distributed mainly around venules. In the subcutaneous fat, they were scattered throughout the fat lobule. Melanin pigment was not only seen within cells but also extracellularly. No melanoma cells were seen in the skin. No increase in melanin pigment or number of melanocytes was seen in the epidermis. A bone marrow biopsy contained melanophages but no melanoma cells.
Ultrastructural examination of the patient's serum revealed the presence of melanosomes. Sequence analysis of the tumor's cDNA failed to identify any mutations in the tyrosinase gene, and no tyrosinase protein was detected in non-melanocytic cells, indicating that it was unlikely that a mutation had resulted in a secretory form of the protein.
These findings document that diffuse melanosis may result from tumor lysis, with release of melanosomes into the bloodstreamDiffuse melanosis arising from metastatic melanoma: Pathogenetic function of elevated melanocyte peptide growth factors
Markus Böhm, etal.
J Am Acad Dermatol 2001;44:747-54 Abstract quoteThe origin of diffuse melanosis resulting from metastatic melanoma is unknown. We examined the light microscopic and ultrastructural changes in the skin of an affected 35-year-old woman and determined the peripheral blood levels of melanocyte growth factors. A total of 7 biopsy specimens were examined by light and electron microscopy and immunohistology (S-100, HMB45, MART1, CD68, MAC387). Serum/plasma levels of melanocyte growth factors of the patient were determined by enzyme-linked immunosorbent assays and compared with those of normal volunteers (n = 10) and amelanotic patients with metastatic melanoma (n = 10), matched to the UICC stage of the affected patient. Hyperpigmented but otherwise apparently normal skin of the patient displayed epidermal melanocyte hyperplasia, increased melanogenesis, and dermal pigment stored in histiocytes and other cells along with extracellular deposits. Blood levels of -melanocyte stimulating hormone, hepatocyte growth factor, and endothelin-1 were significantly elevated in the affected patient.
Aberrant production of these factors may not only be responsible for activation of the pigment system in diffuse melanosis of metastatic melanoma, but also for increased proliferation, motility, and pigment incontinence of normal and malignant melanocytes.
LEPTOMENINGEAL MELANOMATOSIS
Extensive metastatic leptomeningeal melanomatosis as the first clinical sign of a cutaneous melanoma: Morphological correlations between magnetic resonance imaging and autopsy findings. A case report.Bussani R, Cova M, Pozzi-Mucelli R, Camilot D, Silvestri F.
Hum Pathol. 2003 Jun;34(6):625-8. Abstract quote In recent years, the diagnosis and management of leptomeningeal carcinomatosis have gained increased attention as patients with neoplasms live longer and the condition becomes more common. Conclusive hallmarks of this disease have yet to be identified.
We report and discuss a case of massive invasion of the cerebral leptomeninges by neoplastic cells from a malignant melanoma in a shoulder. Symptoms of cerebral dysfunction were the first indication of neoplasm. The onset of symptoms, magnetic resonance imaging, and patient death all occurred within a brief time span.
To our knowledge, this is the first case of meningeal melanomatosis in a patient not treated with chemotherapeutic drugs in which the radiologic evidence is virtually synchronous with direct anatomic observation. Thus the images provided with this report may be of use in the radiographic diagnosis of cerebrospinal metastatic colonization.MULTIPLE PRIMARY
- Multiple primary melanoma: two-year results from a population-based study.
Titus-Ernstoff L, Perry AE, Spencer SK, Gibson J, Ding J, Cole B, Ernstoff MS.
Department of Community and Family Medicine, Dartmouth Medical School, Lebanon, NH 03756, USA.
Arch Dermatol. 2006 Apr;142(4):433-8. Abstract quote
OBJECTIVE: To assess the frequency of occurrence and risk factors for multiple primary melanoma.
DESIGN: Population-based, case-control study.
SETTING: New Hampshire.
PARTICIPANTS: Three-hundred fifty-four New Hampshire residents with a confirmed first diagnosis of cutaneous melanoma.
MAIN OUTCOME MEASURE: Diagnosis of a subsequent primary cutaneous melanoma.
RESULTS: An additional melanoma occurred in 27 individuals (8%) within 2 years of their initial diagnosis, including 20 (6%) within the first postdiagnosis year. In 9 (33%) of these 27 cases, at least 1 subsequent melanoma was deeper than the first tumor. The 27 individuals with a subsequent melanoma diagnosis were classified as "cases" and were compared on the basis of risk factors to the 327 "controls" with a single melanoma diagnosis. The data indicate an inverse relation of risk of multiple primary melanomas with multiple blistering sunburns (P = .01 for the trend); the odds ratio (OR) was 0.32 (95% confidence interval [CI], 0.11-0.93) for 2 or more sunburns compared with none. The number of atypical moles was significantly related to increased risk (P = .004 for the trend). The presence of 3 or more atypical moles compared with none was associated with more than a 4-fold risk of multiple primary melanomas (OR, 4.29; 95% CI, 1.51-12.16).
CONCLUSIONS: Additional melanomas occur more frequently than previously shown. Our study confirms that atypical moles are strongly associated with risk of multiple primary melanomas but provides little evidence that risk is influenced by pigmentary characteristics, hours of sun exposure, or benign moles. The inverse association with blistering sunburn may reflect the influence of an unmeasured covariate.
- Clinicopathological features of and risk factors for multiple primary melanomas.
Ferrone CR, Ben Porat L, Panageas KS, Berwick M, Halpern AC, Patel A, Coit DG.
Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
JAMA. 2005 Oct 5;294(13):1647-54. Abstract quote
CONTEXT: The incidence of multiple primary melanomas ranges from 1.3% to 8.0% in large retrospective reviews; however, the impact of certain risk factors is not understood.
OBJECTIVES: To determine the incidence of multiple primary melanomas (MPM) from a prospective, single-institution, multidisciplinary database, and to describe the clinical and pathological characteristics and risk factors specific to these patients.
DESIGN AND SETTING: Review of a prospectively maintained database at Memorial Sloan-Kettering Cancer Center in New York, NY.
PATIENTS: A total of 4484 patients diagnosed with a first primary melanoma between January 1, 1996, and December 31, 2002.
MAIN OUTCOME MEASURES: Incidence of and risk factors for MPM.
RESULTS: Three hundred eighty-five patients (8.6%) had 2 or more primary melanomas, with an average of 2.3 melanomas per MPM patient. Seventy-eight percent had 2 primary melanomas. For 74% of patients, the initial melanoma was the thickest tumor. Fifty-nine percent presented with their second primary tumor within 1 year. Twenty-one percent of MPM patients had a positive family history of melanoma compared with only 12% of patients with a single primary melanoma (SPM) (P<.001). Thirty-eight percent of MPM patients had dysplastic nevi compared with 18% of SPM patients (P<.001). The estimated cumulative 5-year risk of a second primary tumor for the entire cohort was 11.4%, with almost half of that risk occurring within the first year. For patients with a positive family history or dysplastic nevi, the estimated 5-year risk of MPM was significantly higher at 19.1% and 23.7%, respectively. The most striking increase in incidence for the MPM population was seen for development of a third primary melanoma from the time of second primary melanoma, which was 15.6% at 1 year and 30.9% at 5 years.
CONCLUSIONS: The incidence of MPM is increased in patients with a positive family history and/or dysplastic nevi. These patients should undergo intensive dermatologic screening and should consider genetic testing.Multiple primary melanomas J Am Acad Dermatol 2001;44:22-27
Controversial topic but most studies indicate about 1.2-8.2% increased risk for a second melanoma after the first diagnosisRetrospective review of 56 patients with 157 melanomas found the following clinical features:
Early age of onset <40 years in 58.9%
Clinically diagnosed dysplastic nevi (82%)
Histologically diagnosed dysplastic nevi (64%)
Family history of clinicaly diagnosed dysplastic nevi (70.8%) or melanoma (64.7%)
Histologically diagnosed dysplastic nevius with a family history of melanoma (48%)Mean interval between first and second melanoma was 34.3 months
76.8% of second melanomas developed in different anatomic location
Mean tumor thickness decreased from 1.11 mm for first melanoma to 0.90mm for secondOverall, suggests that genetic factors may play a role in certain subset of patients who develop melanoma early and successively
UNKNOWN PRIMARY Melanoma of unknown primary Ann Surg 1981;191:98-104
Ann Plast Surg 1992;28:81-84
Pathology 1975;7:91-99
Am J Surg Pathol 1987;11:140-146
Estimated to occur in 5% of patients
More common in men
Peak 4-5th decades
Prognosis appears to be similar to patients with comparable to individuals with comparable stage disease and a known primaryImportant to consider regression in a primary melanoma as the possible source
Other possibilities include:
Transformation of nodal melanocytes
Primary melanomas in unusual sites which are clinically silent but still generate metastasesEXTRACUTANEOUS MELANOMA SMALL MELANOMAS Small melanomas: a clinical study on 270 consecutive cases of cutaneous melanoma. Bono A, Bartoli C, Moglia D, Maurichi A, Camerini T, Grassi G, Tragni G, Cascinelli N.
Unit for Cutaneous Oncology, Istituto Nazionale Tumori, Milan, Italy.
Melanoma Res 1999 Dec;9(6):583-6 Abstract quote
The ABCD (asymmetry, border, colour, dimension) criteria represent a commonly used clinical guide for the diagnosis of early cutaneous melanoma (CM). This guide stipulates that CMs usually are more than 6 mm in diameter. The purpose of this retrospective study was to establish the frequency of occurrence of small (< or =6 mm) melanomas in a clinical context. Our series consisted of 270 consecutive CMs (39 in situ and 231 invasive) in 267 patients. Of these 270 lesions, 47 (17%) were small lesions, ranging from 2 to 6 mm in maximum linear extent, with a median value of 5 mm. Of these small lesions, 14 were in situ and 33 Invasive CMs. The median thickness of the 33 small invasive lesions was 0.31 mm. The clinical features of CMs were sufficiently distinctive to suggest a diagnosis of CM in half of the cases, irrespective of the invasiveness or not of the lesions. Dermatoscopy was performed on 36 of the small lesions and achieved a correct diagnosis in 72% of the cases. The combination of simple visual examination with dermatoscopy allowed a higher rate of recognition (86%) than when the two methods were considered separately.Results of our study show that small CMs represent a considerable clinical subset of all CMs. Clinicians must be aware of this fact in their diagnostic activity.
Macpherson and Pincus. Clinical Diagnosis and Management by Laboratory Methods. Twentyfirst Edition. WB Saunders. 2006.
Rosai J. Ackerman's Surgical Pathology. Ninth Edition. Mosby 2004.
Sternberg S. Diagnostic Surgical Pathology. Fourth Edition. Lipincott Williams and Wilkins 2004.
Robbins Pathologic Basis of Disease. Seventh Edition. WB Saunders 2005.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 6th Edition. McGraw-Hill. 2003.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fifth Edition. Mosby Elesevier 2008
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