The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

Tissue Factor Expression and Serum Level in Patients with Melanoma does not Correlate with Disease Progression

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

Interferon-gamma regulates the T cell response to precursor nevi and biologically early melanoma

To examine the potential regulatory role of interferon-gamma in the cellular immune response to melanoma and its precursor lesions, we have tested the capacity of this lymphokine to enhance HLA class II antigen-dependent T lymphocyte blastogenesis, its in vitro production by autologous T cells stimulated by melanoma, and its presence in melanocytic lesions in situ. Cell lines derived from a dysplastic nevus, a radial growth phase primary tumor, a vertical growth phase primary, and metastatic lesions were induced by recombinant interferon-gamma to express increased amounts of HLA class II antigens. Such cells were then examined in radioimmunoassay for expression of HLA-DR antigens and in co-culture for their ability to stimulate proliferation of autologous T cells. Interferon-gamma treatment of melanocytic cells increased their expression of HLA-DR antigens threefold to sixfold. In parallel with these findings, co-culture of T cells with interferon-treated cells of a dysplastic nevus and a radial phase melanoma led to augmented T cell incorporation of tritiated thymidine, and this stimulation was inhibited with a monoclonal antibody to HLA-DR antigens. Despite augmented expression of HLA class II antigens (HLA-DR, -DQ, and -DP), vertical growth phase and metastatic melanoma cells failed to stimulate autologous T cells. When T cells were co-cultured with stimulating melanoma cells, culture supernatants contained significantly increased amounts of interferon-gamma (12 U/ml) in comparison with supernatants of T cells alone (4 U/ml). No interferon was detectable in cultures of melanoma cells alone. To link these in vitro phenomena to in situ events, we used murine monoclonal antibodies to interferon-gamma, the interleukin 2 receptor, and HLA-DR antigens in an immunoperoxidase system to detect interferon production and lymphocyte activation in frozen sections of lesions representative of melanocytic tumor progression. In these studies, precursor dysplastic nevi and radial phase melanomas contained the highest numbers of activated lymphocytes and stained positively for interferon-gamma. These results suggest that interferon-gamma plays a central role in the regulation of the cellular immune response to melanoma. It is produced by T cells, likely activated by tumor antigens seen in the context of HLA class II antigens. In turn, interferon-gamma production enhances expression of HLA class II antigens by melanoma and precursor cells, and such enhancement is associated with additional T cell activation in a positive feed-back loop.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

Risk of melanoma arising in large congenital melanocytic nevi: a systematic review

Large congenital melanocytic nevi are cutaneous lesions regarded by many as premalignant; estimates of malignancy incidence range from 0 to 42 percent. Given the often complex and extensive nature of large congenital melanocytic nevi resection and reconstruction, the risk of malignant transformation is a crucial factor that surgeons and families must weigh when deciding whether or not to excise the lesion. The authors conducted a systematic analysis of data from the existing literature to critically evaluate the published studies and to establish a crude incidence rate for the risk of malignant melanoma transformation in large congenital melanocytic nevi. After a comprehensive literature search, they analyzed data from eight studies (containing a total of 432 large congenital melanocytic nevi patients) of sufficient scientific quality. Twelve patients (2.8 percent) in this sample developed cutaneous malignant melanoma during the reported follow-up periods. Using a subset of this data and comparing the incidence rates to those of the Surveillance, Epidemiology, and End Results population-based database using a standardized morbidity ratio, the authors found that the large congenital melanocytic nevi patients had an increased risk of melanoma (standardized morbidity ratio, 2599; 95 percent confidence interval, 844 to 6064) compared with the general population. Regarding treatment before developing melanoma in the 12 patients, 50 percent were observed before diagnosis, 17 percent had partial excision, 8.3 percent had dermabrasion, 8.3 percent had a chemical peel, and 17 percent did not have any treatment information. These combined data are clinically useful when consulting with the parents of children with large congenital melanocytic nevi and in the management of older patients with existing lesions. This study shows that there is a significantly increased risk of melanoma in large congenital melanocytic nevi patients. The data also reveal the need for a standardized definition of large congenital melanocytic nevi and a long-term, prospective outcomes study to determine the true lifetime risk of melanoma in patients with and without surgical excision.     

Recombinant gamma-interferon induces changes in expression and shedding of antigens associated with normal human melanocytes, nevus cells, and primary and metastatic melanoma cells

Recombinant gamma-interferon (rIFN-gamma) induced or augmented the expression of HLA-DR class II antigens on melanocytes isolated from newborn foreskin, from congenital, common acquired, and dysplastic nevi, and from primary and metastatic melanoma. The stimulatory effect of rIFN-gamma on HLA-DR antigen expression was suppressed by the addition of the phorbol ester TPA or its analog PDBu to the culture medium. Whereas rIFN-gamma did not significantly alter the expression of melanoma-associated, non-class II antigens on melanoma cells, there was a marked decrease in the expression of antigens associated with nevus cells. In addition, rIFN-gamma stimulated shedding of antigens. Increased antigen shedding was most apparent for an intracytoplasmic melanoma-associated protein of 80kd, followed by the ganglioside GD2 and by an alkali labile ganglioside. The simultaneous stimulation of class II antigen expression and shedding of melanoma-associated antigens as well as suppression of nevus-associated antigen expression could play an important role in the host immune response to premalignant and malignant melanocytic lesions.     

Comparison of immunohistochemical labelling of melanocyte differentiation antibodies melan-A, tyrosinase and HMB 45 with NKIC3 and S100 protein in the evaluation of benign naevi and malignant melanoma

A panel of three melanocyte differentiation antibodies has been compared with anti-S100 protein and NKIC3 in an assessment of benign and malignant melanocytic lesions.Anti-polyclonal S100 protein labelled all cases of primary cutaneous malignant melanoma, metastatic melanoma, desmoplastic melanoma and myxoid melanomas. In addition all benign and dysplastic naevi were positive. Conversely, HMB 45 was the least sensitive marker, labelling 24/31 primary cutaneous melanomas, 14/24 metastatic melanomas and only 1/6 desmoplastic melanomas. In the case of naevi, only junctional forms labelled consistently. Results for anti-melan-A and anti-tyrosinase were similar, although anti-tyrosinase proved slightly more sensitive in cases of malignant melanoma. NKIC3 revealed similar results to anti-tyrosinase, but had the disadvantage of reduced selectivity.It is concluded that anti-tyrosinase and anti-melan-A are useful additions to the panel of melanocytic monoclonal antibodies. In addition, both antibodies appear to have greater sensitivity for malignant melanoma than the conventionally used HMB 45 and could be considered as supportive markers to polyclonal anti-S100 protein in the diagnosis of malignant melanoma.     

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.     

Primary cilium depletion typifies cutaneous melanoma in situ and malignant melanoma

Cutaneous melanoma is a lethal malignancy that arises spontaneously or via in situ precursor neoplasms. While melanoma in situ and locally invasive malignant melanoma can be cured surgically, these lesions can sometimes be difficult to distinguish from melanocytic nevi. Thus, the identification of histolopathologic or molecular features that distinguish these biologically distinct lesions would represent an important advance. To this end, we determined the abundance of melanocytic primary cilia in a series of 62 cases composed of typical cutaneous melanocytic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma. Primary cilia are sensory organelles that modulate developmental and adaptive signaling and notably, are substantially depleted from the neoplastic epithelium of pancreatic carcinoma at a stage equivalent to melanoma in situ. In this series, we find that while nearly all melanocytes in 22 melanocytic nevi possessed a primary cilium, a near-complete loss of this organelle was observed in 16 cases of melanoma in situ, in 16 unequivocal primary invasive melanomas, and in 8 metastatic tumors, each associated with a cutaneous primary lesion. These findings suggest that the primary cilium may be used to segregate cutaneous invasive melanoma and melanoma in situ from melanocytic nevi. Moreover, they place the loss of an organelle known to regulate oncogenic signaling at an early stage of melanoma development.     

Immune reactions in benign and malignant melanocytic lesions: lessons for immunotherapy

Spontaneous regression of benign and malignant melanocytic lesions can be a visible sign of immunosurveillance. In this review, we discuss different immune reactions against melanocytic lesions: halo nevus, Meyerson's nevus, regression in melanoma and melanoma-associated depigmentation. These entities present with particular clinical aspects, histology and evolution. In all entities, a melanocyte-specific T-cell reaction has been assumed but a different degree of melanocyte destruction is present. A focus on the immune responses in melanocytic lesions reveals several aspects of an adequate skin immunity and may help to identify the key points in the immune destruction of melanocytes. These insights can add to the knowledge of how to optimize immunotherapeutic strategies in melanoma.     

Vitiligo-related pigment cell differentiation antigens are expressed on malignant melanoma cells following phenotypic reversion induced by contact inhibitory factor

Most vitiligo sera contain antibodies to surface antigens on pigmented human melanocytes but not to human or mouse amelanotic melanoma cells. A density-dependent line of hamster amelanotic melanocytic cells (FF) produces a diffusible factor (CIF) which restores contact inhibition of growth as well as several other normal phenotypic characteristics to hamster, murine, and human melanoma cells. The ability of CIF to induce the expression of a phenotypic characteristic of pigmented human melanocytic cells, i.e., the vitiligo-related surface antigens, on hamster and mouse amelanotic melanoma cells was investigated. Vitiligo and normal sera were reacted with CIF-treated and untreated hamster and mouse amelanotic melanoma cells for both indirect-immunofluorescence assays and ELISA. Immunofluorescence testing showed that about 80% of hamster and mouse melanoma cells had pigment-cell antigens (in the absence of pigmentation) in a granular surface pattern after, but not prior to, CIF-induced morphologic reversion and confluent growth. Less than 5% of the control hamster and mouse melanoma cells expressed such antigens at confluence. These results were confirmed by ELISA. Metabolic-labeling studies with 35S-methionine showed that the vitiligo antigens were synthesized by the CIF-treated melanoma cells. The slowing of melanoma cell proliferation in isoleucine-deficient medium failed to elicit the expression of vitiligo antigens. Since antigen appearance following phenotypic reversion occurred without pigment induction, it is concluded that vitiligo-related surface antigens and pigmentation are distinct aspects of a differentiated function which may be non-coordinately expressed. The expression of pigment-cell differentiation antigens on amelanotic melanoma cells is an additional feature of the pleiotypic trans-species response to CIF.     

Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.     

In vitro reconstruction of malignant melanoma

It is report here that three dimensional melanocytic tumors may be reconstituted when adding collagen to the cells harvested during the under covering lattice culture of malignant melanoma explants (cutaneous metastasis). The cells issued from these rebuilt malignant melanoma may be used following the same methods. Thus, the serial making of melanocytic tumors becomes possible. The problem of the relation between cellular differentiation and invasive potential of the cells may be studied using as an original model the rebuilt tumors and the cellular population which are issued from them.     

Histopathological prognostic factors of malignant melanoma

The incidence of melanocytic skin lesions, including malignant melanoma has increased in the past few years; histopathologists and dermatopathologists have to face them more often. The correct treatment of melanoma patients by the oncodermatologist and oncologist is based on the histopathological report containing the most important histological prognostic factors. However, the accurate interpretation of these factors may be difficult in the everyday practice, especially in reporting tumor thickness, the level of invasion, the type of exulceration and regression. It is important to standardize the content of the histopathological reports in a reproducible way.     

Oral desmoplastic melanoma mimicking inflammatory hyperplasia

doi: 10.1111/j.1741‐2358.2011.00482.x Oral desmoplastic melanoma mimicking inflammatory hyperplasia Introduction: Desmoplastic melanoma (DM) arising in the oral cavity is a rare neoplasm that may be confused with a variety of non‐melanocytic benign or malignant lesions. Objectives: To present a case of DM in the oral mucosa mimicking fibrous inflammatory hyperplasia, discusses the difficulties involved in the diagnosis and offers a literature review on the clinicopathologic and immunohistochemincal aspects of this neoplasm. Case report: A 62‐year‐old white male, smoker, was referred with a chief complaint of pain and swelling in the palate. The oral examination revealed multiple brown‐to‐black patches and a non‐pigmented sessile nodule located on the mucosa of the hard palate. The clinical diagnosis of the pigmented lesions was either oral melanosis or melanoma. The nodular lesion was clinically diagnosed as fibrous inflammatory hyperplasia. Incisional biopsy was performed on the pigmented lesion and the microscopic sections revealed a melanotic macule. The nodular lesions histologically revealed an amelanotic desmoplastic melanoma. Conclusions: Reactive lesions close to a pigmented area should be investigated with great care.     

Imprint cytology in the intraoperative diagnosis of malignant melanoma

Imprints were prepared from 73 pigmented skin lesions, 19 of which were diagnosed as malignant melanoma. The cytologic findings in malignant melanoma, large clusters of cells with loss of cellular cohesiveness and large pleomorphic nuclei, were positive in 17 of the 19 cases. In other malignant and benign pigmented lesions the few cells adherent to the glass showed characteristic cytologic features of the particular lesion. Imprint cytology seems to be a valuable adjunct to the examination of frozen sections for the intraoperative diagnosis of malignant melanoma.     

The dysplastic nevus: recognition and management

The recognition of atypical or dysplastic nevomelanocytic nevi potentially provides clinicians with another means of identifying individuals at increased risk for cutaneous malignant melanoma. However, a great deal of controversy still surrounds these lesions, their significance, and the clinical and histologic criteria needed for their diagnosis at present. In general, dysplastic nevi tend to be asymmetrical and larger (greater than 5 mm) than ordinary acquired nevi, have a macular component, irregular and ill-defined borders, and haphazard (variegate) coloration. A clinical diagnosis of dysplastic nevi must be confirmed by histopathology, since not all clinically atypical nevi are dysplastic. While precise histopathologic criteria for dysplastic nevi are lacking, most authorities agree that an abnormal nevomelanocytic proliferative pattern as manifested by increased numbers of basilar melanocytes and/or abnormal junctional nevomelanocytic nesting in the setting of lentiginous epidermal hyperplasia, variable degrees of nevomelanocytic nuclear atypia, and a lymphocytic host response are consistent with a histologic diagnosis of dysplastic nevi. Current data for individuals with dysplastic nevi and a family history of cutaneous malignant melanoma (at least two family members with cutaneous malignant melanoma) indicate a relative risk for cutaneous malignant melanoma about 148 times that of the general population. In comparison, cutaneous malignant melanoma risk seems lower for individuals with familial dysplastic nevi (but without familial cutaneous malignant melanoma) and "sporadic" dysplastic nevi. With respect to progression to melanoma, probably the vast majority of dysplastic nevi remain stable or possibly regress. Management of individuals with histologically confirmed dysplastic nevi involves periodic skin examinations. Regional overview and life-size photography are helpful in following these patients. Patients should also be instructed in the examination of their own skin. While a definite relationship between sun exposure and dysplastic nevi remains unproved, the use of sunscreens and avoidance of unnecessary sun exposure are advised. Examination of family members for atypical melanocytic lesions is also recommended.     

Discriminant analysis of image karyometric variables in benign and malignant melanocytic skin lesions

OBJECTIVE: To perform a quantitative analysis to identify which of 7 nuclear morphometry-related variables are of diagnostic value in distinguishing benign from malignant melanocytic skin lesions. STUDY DESIGN: At the Institute of Pathology, University of Nis, formalin-fixed, paraffin-embedded skin biopsies from 23 cases of benign nevi (18 intradermal and 5 junctional) and 25 cases of primary nodular malignant melanomas were retrieved. Specimens were routinely stained with hematoxylin and eosin and analyzed using a computer-assisted interactive image analysis system. Nuclear area, equivalent diameter, volume of equivalent sphere, perimeter, mean chord, circularity and integrated optical density were estimated after manual editing of binary images. RESULTS: In univariate analysis, 6 features were found to be significantly different between the benign and malignant groups (P < .0001); all measured nuclear variables (except circularity) were higher in malignant melanomas. No significant differences were found among lesions with respect to nuclear shape. Using discriminant function analysis, a correct diagnosis was achieved in 95.8% of benign nevi cases and 84.0% of malignant melanoma cases. The best discriminant variable was nuclear area. CONCLUSION: Image analysis is diagnostically relevant to the evaluation of melanocytic lesions of the skin. The area of the nucleus appeared to have potential for differentiating benign from malignant tumors and can be estimated in the course of routine histology.