Proto-oncogene c-kit expression in malignant melanoma: protein loss with tumor progression

The c-kit gene encodes a transmembrane receptor that has tyrosine kinase activity. c-kit plays a role in hematopoiesis, gametogenesis, and melanogenesis. c-kit is found in melanocytes, and there is evidence that expression is lost in melanoma. We studied 85 melanocytic lesions for c-kit by immunohistochemical techniques using a monoclonal antibody. The lesions included banal nevi, junctional and compound nevi with melanocytic dysplasia, nontumorigenic radial growth phase melanoma, tumorigenic vertical growth phase melanoma, and metastatic melanoma. We found intense membrane staining in normal melanocytes and mast cells. Staining in compound nevi was strongest in junctional and superficial dermal components, whereas dermal nevi showed weak reactivity. Dysplastic nevi stained strongly, particularly in junctional cells. In melanoma, strong reactivity was most prominent in radial growth phase disease, but there was little or no staining in vertical growth phase and metastatic melanomas. In summary, c-kit protein is expressed in normal melanocytes, benign nevi, dysplastic nevi and nontumorigenic melanoma, but expression is lost in tumorigenic primary melanomas and metastases. The role of c-kit loss in advanced melanoma requires additional investigation.     

Hepatocyte growth factor/scatter factor and its receptor c-Met are overexpressed and associated with an increased microvessel density in malignant pleural mesothelioma

The product of the p16/INK4a/CDKN2/MTS1 tumor-suppressor gene acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein. p16 is inactivated in a wide range of human malignancies, including familial melanoma. However, its expression and function in sporadic melanoma has not been extensively investigated. We studied p16 expression in 62 archival melanomas and 30 archival nevi and lentigines by immunohistochemistry. Eighteen of 26 (69%) superficial spreading melanomas, 17 of 28 (61%) nodular melanomas, all of three lentigo maligna melanomas, and all of five melanoma metastases were found to harbor less than 10% p16-positive tumor cells. In contrast, only six of 24 (25%) nevi had less than 10% positive cells. No correlation between tumor thickness and loss of p16 expression was found. Using DNA from micro-dissected tumor and matched normal tissues, five of seven (71%) p16-negative melanoma cases had 9p21 loss of heterozygosity (LOH), and one of these 9p21 LOH cases had promoter region hypermethylation of the remaining p16 allele. These data demonstrate that partial or complete loss of p16 expression is prevalent in sporadic melanoma and is frequently associated with 9p21 LOH.     

Effect of Pichia anomala killer toxin on Candida albicans

BACKGROUND: An acral lentiginous melanoma in situ on the sole is often difficult to differentiate with the naked eye from an acquired plantar melanocytic nevus. Recent technical advances in epiluminescence microscopy have contributed to the differentiation of these two pigmented skin lesions. OBJECTIVE: In this study, the correlation between dermatoscopic and histopathologic findings of acral lentiginous melanoma in situ on the sole are compared to those of acquired plantar melanocytic nevi. METHODS: Three acral lentiginous melanomas in situ on the sole, and two cases of acral lentiginous melanoma were compared with 50 acquired plantar melanocytic nevi by means of dermatoscopy and histopathology. Results: The dermatoscopic surface profiles of acquired melanocytic nevi were composed of linear pigmentation accentuated mainly on the sulcus superficialis. Histologically, some areas of the sulcus superficialis corresponded to rete ridges of the epidermis, and nests of nevus cells were also often located there. In contrast, the acral lentiginous melanomas in situ showed diffuse, irregularly shaped pigmentation distributed in a disorderly fashion over the entire surface. Histologically, isolated areas of proliferation and small nest formations of atypical melanocytes were irregularly distributed in the epidermis. CONCLUSION: A distinctive dermatoscopic feature of acral lentiginous melanoma in situ is diffuse and irregular pigmentation over the entire surface of the lesion. This feature is helpful for differentiating acral lentiginous melanoma in situ from acquired plantar melanocytic nevi.     

Heterogeneous expression of immunotherapy candidate proteins gp100, MART-1, and tyrosinase in human melanoma cell lines and in human melanocytic lesions

In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predictors of the success rate of such a treatment is the extent of positive (target) tumor cells within the lesions of the patient. First, we investigated the presence of these three proteins in 18 human melanoma cell lines using RT-PCR and immunohistochemistry. In 11 cell lines, mRNA and protein of all three markers could be detected; in one cell line, only two markers were present, and six melanoma cell lines showed no evidence for these markers. Secondly, we stained frozen sections of 105 human melanocytic lesions, 13 common nevocellular nevi, 13 atypical nevi, 13 early primary melanomas (Breslow < 1.5 mm), 25 advanced primary melanomas (aPM; Breslow > or =1.5 mm), and 41 melanoma metastases (MM) with antibodies against glycoprotein 100, melanoma antigen recognized by T cells, and tyrosinase. In addition, we used the 3,4-dihydroxy-L-phenylalanine reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results in a subset of the lesions. In the benign lesions, glycoprotein 100 was more prominently expressed in epidermal melanocytes, whereas melanoma antigen recognized by T cells was encountered in all or nearly all dermal melanocytes in all nevocellular nevi and atypical nevus lesions. Tyrosinase was found in a lower percentage of melanocytes, both in the epidermis and in the dermis within these lesions. With regard to heterogeneity of staining within the malignant lesions, we found that 54% (early primary melanomas), 48% (aPMs), and 56% (MM) of the lesions stained within the same staining category for all three proteins studied. Approximately 17% of the aPM and MM lesions did not show positive tumor cells for any of the three proteins. We conclude that a subgroup of patients with high expression should be selected for immunotherapeutic treatment approaches based on the presence of these proteins.     

Nursing staff perceptions of the use and reduction in the use of physical restraints

Substance P (SP) is a neuropeptide found in both the central and peripheral nervous system. In the skin, SP-containing neurons stimulate the release of histamine from connective tissue mast cells (MC). SP also can potentiate neoangiogenesis and induce dermal fibrosis. MC-derived histamine has potent vasoactive effects, is angiogenic, and promotes tissue fibroplasia. In addition to histamine, MC contain many other angiogenic factors and a variety of cytokines, growth factors, and proteolytic enzymes implicated in tissue remodeling, and normal as well as tumor-associated neoangiogenesis. Many MC-derived factors, including histamine, can enhance melanoma cell growth directly. MC often concentrate around cutaneous melanomas which also frequently are associated with angiogenesis and peritumoral fibrosis. The precise mediators of these responses have not been well defined. We evaluated by immunohistochemistry cutaneous lesions representing stages of progression of malignant melanoma and its precursor lesions for the expression of SP. SP was expressed in 17/25 (68%) primary invasive malignant melanomas, 2/5 (40%) metastatic melanomas, 6/10 (60%) melanomas in situ, 7/12 (58%) atypical (dysplastic) nevi, and 4/10 (40%) spindle and epithelioid cell (Spitz) nevi, but was not detected in any (0/11, 0%) acquired benign melanocytic nevi (p<0.05). Invasive melanomas were immunolabeled in both the intraepidermal and the dermal components of the lesions. For those atypical and Spitz nevi which expressed SP, most of the immunoreactive melanocytes were located at the dermal-epidermal junction overlying areas of papillary dermal fibrosis. The results show differential expression of SP among cutaneous melanocytic lesions and suggest that the expression of this neuropeptide together with other factors may contribute to some of the host responses associated with these lesions.     

Adhesion molecule expression in normal skin and melanocytic lesions. Role of UV-irradiation and architectural characteristics in nevi

Cell adhesion between surfaces of cells and to extracellular matrices represents a fundamental mechanism in tissue organization and influences the biological behaviour and the architecture of tumors. We investigated the expression of various adhesion molecules in normal skin (n=5), nevi (n=29), and malignant melanoma (n=10) by immunohistochemistry. Special attention was paid to the correlation between adhesion molecule expression and the respective architectural features, e.g. UV-induced morphological changes, and the arrangement of melanocytes in congenital nevi. In nevi, a single erythemagenic dose of UV-light did not influence the integrin expression of melanocytes, but results in an upregulation of alpha3 beta1- and alpha6 beta1-integrin within the suprabasal layers of the epidermis. This suprabasal labelling was associated with an increased number of suprabasal melanocytes in UV-irradiated nevi which were detected with HMB-45 antibody. Nine of 10 congenital nevi demonstrated a labelling of alpha4 beta1-integrin only in melanocytes of the deeper dermis. This integrin previously has been associated with high tumor thickness and the clinical outcome in melanomas. The integrin profile observed in melanomas differed in part from that seen in nevi with expression of beta2- and beta3-integrins in some cases. The results may indicate a correlation between adhesion molecule expression and histopathological findings in melanocytic lesions.     

Quantification of vascularity in nodular melanoma and Spitz's nevus

Spitz's nevi are acquired benign melanocytic skin tumors. Usually they are differentiated from nodular melanoma by clinical and histopathological criteria. Since Spitz's nevi are one of the most common simulators of nodular melanomas their bizarre histopathology may cause diagnostic confusion and make it difficult to differentiate these two melanocytic tumors. One of the histologic features shared by Spitz's nevus and nodular melanoma is prominent vascularity. The ability of malignant melanoma to induce angiogenesis is well established whereas benign melanocytic tumors do not have a prominent overall vascularity. The purpose of this study was to find out whether the degree of vascularity of nodular melanomas differs significantly from that of benign Spitz's nevi. In this study the number of microvessels and the vessel area were determined in 23 Spitz's nevi and 16 nodular melanomas. The number of microvessels and the vessel area were determined on Ulex Europaeus agglutinin I-stained sections by computer-assisted image analysis. Two methods of measurement were used, namely systematic and selective sampling. Measurement of the whole tumor specimen (systematic sampling) revealed a vessel count of 10.83/field (SD +/-5.97) for Spitz's nevi whereas nodular melanomas exhibited a significantly lower (p=0.04) vessel count of 6.44/field (SD +/-3.85). This difference was even more pronounced when the vessel area (Spitz's nevi: 17.85x10-4mm2, SD +/-10.32; nodular melanomas: 7.88x10-4mm2, SD +/-5.23) was investigated (p < 0.001). The difference in vessel area and vessel count was insignificant for areas exhibiting the greatest vascularity (selective sampling). Measurement of vessel count and vessel area lead us to conclude that Spitz's nevi have a significantly higher vascularity than do nodular melanomas. Our results thus indicate that angiogenesis in these pigmented lesions is not correlated with malignancy.     

Release of cell cycle constraints in mouse melanocytes by overexpressed mutant E2F1E132, but not by deletion of p16INK4A or p21WAF1/CIP1

Compared to normal melanocytes, melanoma cell lines exhibit overexpression of hyperphosphorylated retinoblastoma tumor suppressor protein (Rb) or a marked decrease in, or lack of, expression of Rb. Hyperphosphorylation of Rb results in increased E2F-mediated transactivation of target genes and cell cycle progression. Using a combination of gene disruption and ectopic expression in growth factor-dependent mouse melanocytes, we studied the roles of E2F1 and the p16INK4A and p21WAF1/CIP1 CKIs (cyclin dependent kinase inhibitors) in the acquisition of TPA (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in culture, a hallmark of melanomas. Surprisingly, melanocytes from p16INK4A- or p21WAF1/CIP1-null mice remained TPA-dependent, and disruption of p21WAF1/CIP1 accelerated cell death in the absence of this mitogen. Disruption of E2F1 had the most profound effect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA. Furthermore, enforced overexpression of the DNA-binding-defective E2F1E132 mutant conferred TPA-independence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21WAF1/CIP1. We conclude that neutralization of Rb by E2F1E132, but not the disruption of p16INK4A or p21WAF1/CIP1, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16INK4A or p21WAF1/CIP1 that activate E2F may play an important role in melanomas.     

In situ distribution of integrin alpha 2 beta 1 and alpha-actinin in melanocytic proliferations

Integrin alpha 2 beta 1 is a transmembrane protein receptor for collagen and laminin previously reported as a melanoma tumor progression antigen. alpha-Actinin is an actin-binding protein reported to interact with the cytoplasmic domain of the beta 1-integrin chain of alpha 2 beta 1. In vitro, both alpha 2 beta 1 and alpha-actinin play a role in melanoma cell motility. In turn, increased melanoma cell line motility (measured as mean migration rates), correlates with metastasis. To determine the in situ distribution of these proteins, we used monoclonal antibodies directed against the alpha 2-integrin subunit of alpha 2 beta 1 and alpha-actinin on frozen sections of 33 melanocytic proliferations, which included dermal nevi, primary melanomas, and metastatic melanomas. We found that the superficial portion of all of the melanocytic proliferations tested stained for alpha-actinin. In benign nevi and superficial spreading melanoma, there was a notable loss of staining for alpha-actinin in the cells in the deep reticular dermis. In contrast, alpha-actinin was present on almost all of the tumor cells in the nodular melanomas and the melanoma metastases. Tumors stained either uniformly positive or uniformly negative for alpha 2 beta 1; the expression of this protein correlated with the later stages of melanoma progression. Our findings suggest that alpha-actinin protein levels initially decrease and then increase during melanocytic tumor progression, whereas the alpha 2 subunit protein appears in the later stages of melanoma progression. The variable distribution of these proteins is evidence for the differential adhesive and motile properties of subpopulations of cells in melanocytic proliferations.     

Dynamics of cell-associated viremia and antibody response during the early phase of lentivirus infection in sheep

OBJECTIVE: To investigate the characteristic epiluminescent microscopic features of early lesions of malignant melanoma affecting glabrous skin, which is the most prevalent site of the neoplasm in nonwhite populations. DESIGN: The epiluminescent microscopic features of various kinds of melanocytic lesions affecting glabrous skin were investigated using a videomicroscope. All the diagnoses were determined clinically and histopathologically using the standard criteria. SETTING: A dermatology clinic at a university hospital. PATIENTS: The following 130 melanocytic lesions consecutively diagnosed at our department were examined: 16 lesions of acral lentiginous melanoma, 6 lesions of malignant melanoma in situ, and 108 lesions of benign melanocytic nevus (acquired or congenital). MAIN OUTCOME MEASURE: The incidence of each characteristic epiluminescent feature was compared among disease categories. RESULTS: On epiluminescent microscopy, malignant melanoma in situ and the macular portions of invasive malignant melanoma showed accentuated pigmentation on the ridges of the skin markings, which are arranged in parallel patterns on glabrous skin. This "parallel ridge pattern" was found in 5 (83%) of 6 lesions of malignant melanoma in situ and in 15 (94%) of 16 lesions of malignant melanoma. The parallel ridge pattern was rarely found in the lesions of benign melanocytic nevus. Most benign melanocytic nevi showed 1 of the following 3 typical epiluminescent patterns: (1) a parallel furrow pattern exhibiting pigmentation on the parallel sulci of [he skin markings (54%), (2) a latticelike pattern (21%), and (3) a fibrillar pattern showing filamentous or meshlike pigmentation (15%). The remaining 11 benign nevi (10%) showed a nontypical pattern. CONCLUSION: Because epiluminescent microscopic features of early malignant melanoma on glabrous skin are characteristic, we can effectively detect early lesions using this noninvasive method.     

Variants of melanoma

The current classification of malignant melanomas gives recognition to superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular types. In addition, neurotropic and desmoplastic types are recognized. The relativity inherent in the diagnosis of melanoma, provides the basis for the classification of melanomas on the basis of size. Lesions measuring 1 mm or less in vertical dimensions are unlikely to metastasize; they qualify as borderline melanocytic neoplasia of indeterminant malignant potential. The current classification has little relevancy to the category of variant nevi with the exceptions of malignant cellular blue nevus and melanoma arising in giant congenital nevi. A classification of variant melanomas as related to variant nevi is proposed. From a different perspective, a classification of melanomas with attention to nesting and cytological patterns in vertical growth is proposed: this alternate approach gives recognition to lesions that might otherwise be classified as "nevoid" melanomas. It also provides a default category for lesions that might otherwise be assigned to the Spitz nevus-like category. All of these tools for the manipulation of the real and virtual images of melanomas have been emphasized in the concept of minimal deviation melanoma.     

CDKN2A mutations in multiple primary melanomas

BACKGROUND: Germ-line mutations in the CDKN2A tumor-suppressor gene (also known as p16, p16INK4a, and MTS1) have been linked to the development of melanoma in some families with inherited melanoma. Whether mutations in CDKN2A confer a predisposition to sporadic (nonfamilial) melanoma is not known. In some patients with sporadic melanoma, one or more additional primary lesions develop, suggesting that some of these patients have an underlying genetic susceptibility to the cancer. We hypothesized that this predisposition might be due to germ-line CDKN2A mutations. METHODS: We used the polymerase chain reaction, single-strand conformation polymorphism analysis, and direct DNA sequencing to identify germ-line mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease. A quantitative yeast two-hybrid assay was used to evaluate the functional importance of the CDKN2A variants. RESULTS: Of 33 patients with multiple primary melanomas, 5 (15 percent; 95 percent confidence interval, 4 percent to 27 percent) had germ-line CDKN2A mutations. These included a 24-bp insertion at the 5' end of the coding sequence, three missense mutations (Arg24Pro, Met53Ile, and Ser56Ile), and a 2-bp deletion at position 307 to 308 (resulting in a truncated CDKN2A protein). In three families, CDKN2A mutations identical to those in the probands were found in other family members. In two families with mutations, we uncovered previously unknown evidence of family histories of melanoma. CONCLUSIONS: Some patients with multiple primary melanomas but without family histories of the disease have germ-line mutations of the CDKN2A gene. The presence of multiple primary melanomas may signal a genetic susceptibility to melanoma not only in the index patient but also in family members, who may benefit from melanoma-surveillance programs.     

Diagnosing and treating congenital melanocytic nevus simulating malignant melanoma

Many cases reported as malignant melanomas arising in benign congenital melanocytic nevi in the neonatal period have not shown evidence of metastases after several years of follow-up. These lesions were probably pathologically misdiagnosed, thus creating a controversy regarding the precise incidence. This article describes the case of an infant with a giant melanocytic nevus simulating malignant melanoma to illustrate the proper criteria for diagnosis of this condition so extensive and unnecessary therapy procedures can be avoided.     

Prognostic significance of allelic losses in primary melanoma

Loss of genetic material, including loss of loci on chromosome arms 6q, 9p, and 10q, occurs frequently in cutaneous melanoma but infrequently in benign melanocytic nevi or other melanocytic lesions, suggesting that these genetic alterations are important in the development and progression of melanoma. To examine whether allelic loss is of prognostic importance in melanoma, disease-free survival was related to loss of heterozygosity on 6q, 9p and 10q in 83 individuals with sporadic primary cutaneous melanoma. Loss of chromosome arms 6q and 10q were each significantly associated with a poorer clinical outcome (P=0.013 and P=0.001 respectively). In a subgroup of 41 subjects whose primary tumours were allelotyped, the fractional allelic loss (FAL) at 39 autosomal arms also significantly correlated with disease-free survival (P=0.013), with an increase in FAL associated with a poorer outcome; this association remained significant when controlled for tumour thickness (P=0.035). In addition, a greater proportion of cells were immunopositive for Ki67 antigen, p53 and p21WAF1 protein in the primary melanomas than in the benign melanocytic nevi, however, only p53 over-expression was significantly associated with improved survival (P=0.041).     

The ABCD rule in dermatoscopy: analysis of 500 melanocytic lesions

500 melanocytic lesions were examined by dermatoscopy using the ABCD rule prior to excision and histologic diagnosis. Regular nevi (n = 272) exhibited a score of 3.55 +/- 0.87, nevi with histologic signs of dysplasia (n = 190) a score of 4.0 +/- 0.68 and melanomas (n = 30) a score of 5.08 +/- 1.24. This study suggests that the ABCD rule of dermatoscopy greatly facilitates the evaluation of melanocytic lesions. When the dermatoscopic score is higher than 4.2, melanoma should be considered.     

Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis

It is still unclear whether the sporadic form of dysplastic nevi (SDN) represents a premalignant lesion of malignant melanoma and whether genetic alterations are involved in the development of SDN. To determine whether p16INK4a and p53 genetic abnormalities could be associated with development of SDN, nevus cell nests were procured selectively from H & E-stained slide sections by using a modified microdissection technique and were screened for the presence of mutations and loss of heterozygosity (LOH) of p16INK4a and p53 genes using a polymerase chain reaction-based LOH, single-strand conformation polymorphism, and direct DNA sequencing analyses. Hemizygous deletion was detected in 9 of 12 informative cases (75%) for 9p21-22 (p16INK4a) at one or more loci and 60% (6/10) for 17p13 (p53). As for mutation, we found 3 missense mutations and 1 mutation in the first intron in p16INK4a and 2 missense mutations in p53. Among these mutations in p16INK4a and p53, 5 of 6 mutations were of the C:G to T:A transitional type; this is known to be related to ultraviolet radiation as previously confirmed in other skin cancers. This indicates that p16INK4a and p53 genetic alterations may play an important role in the evolution of SDN and may represent an early event in the development of malignant melanoma. Furthermore, ultraviolet radiation might be the predominant etiologic agent in the development of SDN.     

Protein expression of the cell-cycle inhibitor p27Kip1 in malignant melanoma: inverse correlation with disease-free survival

In the present study we analyzed, by immunohistochemistry, a panel of human melanomas for protein expression of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 and evaluated whether deregulated expression correlates with clinical outcome for this type of cancer. We found that p27Kip1 was strongly expressed by normal melanocytes and benign nevi, whereas in malignant melanoma, a heterogeneous expression pattern was observed. In the case of nodular melanomas, the level of p27Kip1 was found to correlate significantly with the thickness of the tumor, with less protein expressed in thicker lesions. We also found that patients having tumors with fewer than 5% p27Kip1-staining cells had a significantly higher risk of early relapse of their disease compared with those expressing moderate or high levels. In contrast, the level of p27Kip1 did not correlate with tumor thickness or disease-free survival in patients with superficial spreading melanomas, suggesting that p27Kip1 may play different roles in these two major pathological subgroups of malignant melanoma. Furthermore, p27Kip1 did not appear to have an influence on overall survival for either subgroup. When we examined the combined effect of p21WAF1/CIP1 (another cdk inhibitor) and p27Kip1 on clinical outcome, we found that analysis of these two cdk inhibitors together may have greater prognostic potential than either alone. In conclusion, our results suggest that virtually complete loss of p27Kip1 protein expression has potential importance as a prognostic indicator of early relapse in patients with nodular melanoma The results, furthermore, underscore the value of analyzing multiple cell cycle regulatory proteins to obtain the most reliable indication of prognosis.