Formaldehyde-induced fluorescence in melanomas and other lesions. Diagnostic significance and mechanism of fluorescence

We have investigated 40 cases of malignant melanoma (MM) and 81 other lesions including benign melanocytic neoplasms, other malignancies with a potential for confusion with MM, and tissues with biogenic amine-synthesizing apparatus for induction of formaldehyde-induced fluorescence (FIF). Unstained sections were examined with an ultraviolet light source for yellowish fluorescence. Twenty-four of 27 primary cutaneous MMs and 11 of 13 metastatic MMs were positive. Formaldehyde-induced fluorescence was quenched by prior sodium borohydride treatment, suggesting monoamine origin. None of the ten intradermal, five junctional, or three blue nevi showed FIF, but two of eight compound nevi and two of six spindle cell nevi were positive for FIF. One of six large-cell undifferentiated carcinomas was positive; none of six mesenchymal sarcomas were positive; one of seven histiocytic lymphomas showed weak, granular, cytoplasmic fluorescence. Two of five carcinoid tumors and one case of medullary carcinoma of thyroid showed FIF. We conclude that FIF is a simple, reproducible, technique for aiding in the diagnosis of amelanotic melanomas.     

CD40 is a prognostic marker in primary cutaneous malignant melanoma.

CD40 is a receptor at the surface of B lymphocytes with important functions in the immune response. CD40 has also been found on a variety of carcinoma and melanoma cell lines where it has been suggested to serve as a possible receptor for mitogenic signals. We studied the expression and distribution of CD40 in paraffin sections of 71 uniformly treated malignant melanomas (MMs) with a long clinical follow-up using well known monoclonal antibodies. For comparison, 71 benign nevi were also studied. Common acquired nevi occasionally expressed CD40 in nests or single cells at the dermo-epidermal junction; no immunoreactivity was observed in the dermal part of acquired nevi, and all Spitz' nevi were entirely negative. One-third of large congenital nevi expressed CD40 in small clusters of heavily pigmented, epithelioid cells, corresponding to so-called proliferative nodules. In 41 of 71 MMs, CD40 was expressed in single or clustered neoplastic melanocytes; 9 cases showed CD40 expression only in the radial growth phase, and in 32 cases, the vertical growth phase showed CD40 expression. The same staining pattern was obtained with other anti-CD40 monoclonal antibodies, directed to different epitopes of the CD40 molecule. In 29 of 32 MMs showing CD40 in the vertical growth phase, expression of the CD40 ligand (CD40L) was studied; in 13 of these 29, CD40L was found in the same tumor areas that expressed CD40. Analysis of 28 metastases from 24 MM patients showed in the majority of cases a similar, scattered or nodular staining pattern as observed in the primary tumor. Patients expressing CD40 in the vertical growth phase of their MM did not differ significantly from CD40-negative patients with respect to any of the known prognostic parameters but showed a significantly shorter tumor-free survival. Patients with CD40+ CD40L+ MM tended to have a shorter tumor-free survival than those lacking CD40L. We conclude that CD40 represents a novel prognostic parameter in primary cutaneous MM. The co-localization of CD40 and CD40L suggests an autocrine growth loop in the vertical growth phase of MM.     

Progression to invasive melanoma from malignant melanoma in situ, lentigo maligna type

We have previously hypothesized that lesions that have been termed lentigo maligna can be divided into 2 categories: 1 represents a pigmented lesion that is a precursor to melanoma, and the other melanoma in situ. We and others have hypothesized that there is a progressive acquisition of attributes in pigmented lesions that results in malignant melanoma. Based on these 2 hypotheses, we have predicted that the intraepidermal component of invasive malignant melanomas, lentigo maligna type, should be similar to those lesions that we have termed malignant melanoma in situ, lentigo maligna type rather than lentigo maligna. The intraepidermal component of 42 consecutive cases of invasive malignant melanoma, lentigo maligna type was evaluated by all of the authors. Malignant melanoma in situ, lentigo maligna type is characterized by pagetoid spread, confluence, and nesting of atypical melanocytes. All of the cases evaluated showed features diagnostic of malignant melanoma in situ, lentigo maligna type, in the epidermis overlying the invasive dermal component. We conclude that invasive lentigo maligna melanoma arises in association with those lesions that we have termed malignant melanoma in situ, lentigo maligna type, which may represent a step in the progression between atypical melanocytic hyperplasia (lentigo maligna) and invasive melanoma. This finding supports the distinction of these entities and may have therapeutic implications.     

A retrospective histological study of 669 cases of primary cutaneous malignant melanoma in clinical stage I. The consequences of a reclassification of the original group of lentigo maligna melanomas.

A selected series of primary malignant melanoma of the skin, clinical stage I, was originally classified according to Clark's system. The consistency of this classification was tested by two Brisbane pathologists who indicated that we had misinterpreted some cases of superficial spreading malignant melanoma as lentigo maligna melanoma. We have therefore reclassified the original group of 86 lentigo maligna melanomas. This resulted in a total series of 37 (5.5%) lentigo maligna melanomas, 301 (45%) superficial spreading malignant melanomas, 194 (29%) nodular malignant melanomas (unchanged) and 137 (20.5%) unclassifiable malignant melanomas. The diagnosis of lentigo maligna melanoma was not made unless the epidermis was atrophic and dermal solar elastosis was present. The new group of lentigo maligna melanomas is dominated by cases on the head among patients over 50 years of age (especially women). This is in better agreement with other studies than our previous findings. The relationship with tumour cell type, pigmentation, mitotic count, atypia, transsectional profile, level of invasion, ulceration, vascular invasion, lymphocyte infiltration and prognosis shown by the new groups of lentigo maligna melanoma and superficial spreading malignant melanoma indicates that the cases by which the diagnosis has been changed are relatively benign. Our previous conclusions are still valid. The lentigo maligna melanoma is still the most benign type and nodular malignant melanoma still the most malignant type of melanoma. The superficial spreading malignant melanoma still represents an intermediate tumour type, although it has deviated in the benign direction.     

Concurrent Ki-67 and p53 immunolabeling in cutaneous melanocytic neoplasms: an adjunct for recognition of the vertical growth phase in malignant melanomas?

Ki-67 labeling of paraffin sections has been correlated with the number of cells in non-G(o) phases of the replicative cell cycle, and this immunohistochemical technique has been applied to the evaluation of a variety of human neoplasms. Similarly, immunolabeling for p53 protein has been used to detect mutations in the corresponding gene, as a reflection of possible cellular transformation in the same context. Both of these techniques were applied to 253 melanocytic tumors of the skin to assess their possible utility in the diagnosis and subcategorization of such lesions. They included 76 banal (common) nevi (CN), 39 Spitz nevi (SN), 62 superficial spreading malignant melanomas in radial growth (SSMMs), 32 nodular malignant melanomas (NMMs), 21 lentigo maligna melanomas in radial growth (LMMs), and 23 melanomas arising in association with preexisting compound nevi (MCN). One hundred cells were counted randomly in each tumor, and dark, exclusively nuclear reactivity was scored as positive labeling; results were recorded as percentages. Negligible Ki-67 and p53 labeling was seen in CN and SN, at a level that was similar to that obtained in cases of LMM and MCN. The largest proportion of Ki-67-positive and p53-positive cells was observed in NMMs, followed by SSMMs. Radial growth-phase SSMMs and LMMs demonstrated immunoprofiles that were similar to those of melanocytic nevi, and MCN did so as well. The prototypical malignant melanocytic tumor representing the vertical growth phase-nodular melanoma--demonstrated a statistically significant difference from all other lesions in this study with respect to Ki-67 index (P = .008, chi2) and p53 reactivity (P < .000001, chi2). Subsequent concurrent use of a Ki-67 threshold index of 10% and a p53 index of 5% correctly indicated the presence of vertical growth in 75% of NMMs, whereas only 8% of radial growth phase melanomas of other types were colabeled at the same levels of reactivity for the two markers (P < .00001, chi2). Thus, although the distinction between benign and malignant melanocytic tumors could and should not be based on immunohistology for Ki-67 and p53, these results suggest that the latter determinants may, in fact, be used as an adjunct to morphology in the recognition of the vertical growth phase in cutaneous malignant melanomas.     

Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.

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.     

Lentigo maligna and malignant melanoma in situ, lentigo maligna type.

Some authors have considered lentigo maligna to be an atypical melanocytic proliferation, whereas others have considered it to be melanoma in situ. We reviewed 50 cases of lentigo maligna. We have identified two subsets of lesions. The first has atypical melanocytic hyperplasia, which we postulate to be correctly designated lentigo maligna. The second subset has the following features in addition to the melanocytic hyperplasia: individual and nests of cells at varying layers of the epidermis, confluence of the melanocytes replacing the basilar region, uniformity of the cytological atypia, and nesting of uniformly atypical melanocytes. These lesions we designate as malignant melanoma in situ, lentigo maligna type. We are proposing that the lesions that have been termed lentigo maligna represent a spectrum of atypia and that the application of some of the traditional features for the diagnosis melanoma may permit the segregation of more and less aggressive lesions.     

Subepidermal cleft formation as a diagnostic marker for cutaneous malignant melanoma

Cleft formation has been postulated as a clue to the histopathological diagnosis of malignant melanoma (MM). The frequency and reliability of clefts as a diagnostic criterion remain to be determined. We reviewed 503 cases of histologically proven MM searching for clefting between the epidermal layer and underlying MM. Cleft was defined as a separation of at least 0.3 mm in length. Conspicuous cleft formation was present in 120 (24%) of 503 MMs. The presence of clefts was not associated with age or sex of the patients, but showed a slight predilection for the back, a slightly higher prevalence in superficial spreading type of MM and for tumors with a Breslow thickness between 1 and 2 mm. Morphologically, clefts could be separated in 3 different types: linear (37.5%), single-nest (10.9%), and multi-nest (51.6%). In comparison, among 939 benign melanocytic lesions including 100 Spitz or Reed nevi, only 9 exhibited clefts longer than 0.3 mm (< 1%). One was an atypical compound nevus; all others were Spitz nevi, with the majority exhibiting an arched morphology above 1 or 2 large round nests. The relative frequency of cleft formation allowed a highly significant differentiation between MM and benign melanocytic lesions. Clefts are a reliable diagnostic criterion in favor of MM.     

IMP-3 is a novel progression marker in malignant melanoma

Insulin-like growth factor-II messenger RNA (mRNA)-binding protein-3 (IMP-3), also known as K homology domain-containing protein overexpressed in cancer (KOC) and L523S, is a member of the insulin-like growth factor-II mRNA-binding protein family and is expressed during embryogenesis and in some malignancies. IMP-3 expression in melanocytic neoplasms has not been investigated. Fifty-six melanocytic neoplasms from 48 subjects were immunohistochemically studied using a monoclonal antibody against L523S/IMP-3. IMP-3 expression in melanoma was significantly higher than in Spitz nevi (P<0.05), and the staining intensity in the Spitz nevi was weak. IMP-3 expression in metastatic melanoma was significantly higher than in primary cutaneous melanoma with a Breslow depth 相似文献   

Biological behaviour of cutaneous malignant melanomas

The classification proposed by Clark et al. of cutaneous malignant melanoma into 3 groups: lentigo maligna melanoma (LMM), superficial spreading melanoma (SSM) and nodular melanoma (NM) was based on a correlation between histology and biological behaviour. Many studies have shown different growth rates, prognosis and incidence of antecedent benign nevi for each of these groups, confirming that each is a separate entity with its own biological behaviour. The recent proposal that acral lentiginous melanoma (ALM) is a distinct clinico-histological entity, however, is more difficult to accept. Many melanomas on acral locations are histologically intermediate between SSM and ALM and the epidemiological value of classifying ALM as an entity separate from SSM is doubtful.     

CD44 expression in benign and malignant nevomelanocytic lesions

CD44 is an integral membrane glycoprotein that is a principal receptor for hyaluronan and plays a role in cell-extracellular matrix interactions. Recent studies of melanomas in mouse models have suggested that increased CD44 expression by these tumors may relate to metastatic potential. Immunohistochemical expression of CD44 (standard [s] and variant [v6]) in benign and malignant nevomelanocytic lesions was assessed in formalin-fixed, paraffin-embedded tissue and was correlated with histological parameters and prognostic factors. Cases included benign nevi (three junctional, four compound, five intradermal, five blue, six Spitz, one deep penetrating), architecturally disordered (dysplastic) nevi (three, and primary (22) and metastatic melanomas (eight). All of the benign lesions showed diffuse and essentially uniform membrane staining of CD44s in nevomelanocytic cells, regardless of lesion size, depth, or extent of dermal involvement. In contrast, semiquantitative analysis (0 to 3+) of the primary melanomas showed heterogeneous and decreased staining of CD44s, which inversely correlated with lesion size (−0.569) and depth of invasion (−0.622 and −0.617 for Breslow's depth and Clark's level, respectively). These results were significant at P < .05. CD44s expression in metastases paralleled that of their respective primaries. None of the benign nevomelanocytic lesions showed CD44v6 staining. In contrast, all of the malignant nevomelanocytic lesions showed cytoplasmic staining of the tumor cells. Pretreatment with chondroitinase did not alter CD44s staining. CD44s expression by immunohistochemical determination is uniform in benign nevomelanocytic lesions. Malignant melanomas show decreased, heterogeneous staining that inversely correlates with increasing size, depth, and level of invasion. CD44 expression may be a prognostic indicator in malignant melanomas. Tumor staining with anti-chondroitin sulfate monoclonal antibodies suggests that CD44s may be expressed as a chondroitin sulfate proteoglycan in primary melanomas.     

Expression of protease-activated receptors 1 and 2 in melanocytic nevi and malignant melanoma

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.     

Bcl-2 expression in human melanocytes and melanocytic tumors.

During an immunohistochemical study of the distribution of the Bcl-2 proto-oncogene product in frozen sections of normal human skin, a hitherto unrecognized strong reactivity with melanocytes was observed. This prompted us to study Bcl-2 expression in a variety of pigment lesions. In nevocellular nevi, immunoreactivity gradually diminished or even disappeared toward the deeper dermal component. In malignant melanomas of all stages and histological subtypes, the neoplastic cells expressed Bcl-2 oncoprotein, the most intense positivity being restricted to cells in the radial growth phase. Cutaneous and lymph node metastases of malignant melanomas were negative or showed only weak and focal reactivity. The specificity of the staining was confirmed by Western blotting of tissue lysates. The loss of Bcl-2 expression in the deeper parts of nevi may offer an explanation for the "maturation" and final disappearance of dermal nevocellular nevi. The expression of Bcl-2 oncoprotein by malignant melanomas adds these neoplasms to a growing list of tumors expressing this oncoprotein. Bcl-2 in malignant melanoma may play a role in tumor development by sparing the cells from apoptotic death (and thereby exposing them to secondary events) or through cooperation with other oncogenes. The lack of reactivity in metastatic melanoma suggests that mechanisms other than Bcl-2 are involved in the survival and growth of metastatic melanoma cells.     

Differential expression of basic fibroblast growth factor (bFGF) in melanocytic lesions demonstrated by in situ hybridization. Implications for tumor progression.

Basic fibroblast growth factor (bFGF) is an angiogenic and mitogenic polypeptide produced by diverse cell types including cell lines derived from malignant melanomas but not from normal melanocytes. However, there is no consensus concerning in vivo expression of bFGF in melanocytic lesions due in part to the small numbers of cases studied to date. To evaluate further the possible differential expression of bFGF in melanocytic lesions, we examined 110 formalin-fixed, paraffin-embedded metastatic and primary invasive melanomas, melanomas in situ, nevi with architectural disorder and cytological atypia, and ordinary benign melanocyte nevi by nucleic acid in situ hybridization. All metastatic and primary invasive melanomas studied expressed bFGF mRNA, whereas melanomas in situ and benign melanocyte nevi were negative. Melanomas in situ with features of tumor regression and a majority of nevi with architectural disorder and cytological atypia also contained bFGF mrNA. The results suggest that in vivo bFGF expression is not requisite for malignant transformation per se, but appears to correlate more with invasion or fibroblastic reactions adjacent to the melanocyte lesions.     

Blue nevus of the colorectal mucosa

The blue nevus is a well-described benign melanocytic proliferation that generally occurs on the skin. Infrequently, blue nevi are found on mucosal surfaces. The most common location for mucosal blue nevi is the oral mucosa, with reported cases in the sinonasal mucosa and genital tract, as well as in other locations. To our knowledge, blue nevi of the rectal mucosa have not been described. Here, we describe a case of blue nevus arising in the rectal mucosa. Blue nevi are benign melanocytic proliferations with the potential for malignant transformation and should be included in the differential diagnosis of pigmented mucosal lesions of the rectum.     

Evaluation of melanocytic neoplasms: application of a pan-melanoma antibody cocktail

Incidence of malignant melanoma (MM) is rising rapidly throughout the Western world, and the number of melanocytic lesions removed for histological assessment has increased. MM can present with a myriad of histological appearances that make diagnosis problematic, particularly when dealing with metastatic deposits. Immunohistochemical diagnosis relies on a panel of antibodies comprising polyclonal S100 protein and the monoclonal antibodies HMB 45, MART-1, tyrosinase and, to a lesser extent, NKIC3. Confirmation of problematic cases relies on the use of polyclonal S100 protein, as its sensitivity has yet to be matched by any monoclonal antibody. The introduction of a potentially valuable pan-melanoma cocktail, composed of HMB 45, MART-1 and tyrosinase, is examined in 50 primary cutaneous malignant melanomas, five desmoplastic malignant melanomas (DMM), 35 benign naevi, 20 metastatic malignant melanomas, 10 basal cell carcinomas (BCC) and 10 squamous cell carcinomas (SCC) and compared to individual immunolabelling with S100 protein, HMB 45, MART-1 and tyrosinase. All BCCs and SCCs were negative with all antibodies. S100 protein, MART-1, tyrosinase and the pan-melanoma cocktail were positive for all cases of benign naevi. HMB 45 labelled all junctional and compound naevi, five of the eight intradermal naevi and five of the seven blue naevi. All 50 primary cutaneous MMs were positive with S100 protein, 49/50 with the pan-melanoma cocktail and tyrosinase, 47/50 with MART-1 and 46/50 with HMB 45. Of the five cases of DMM, all were positive with S100 protein and three of the five were positive with HMB 45, MART-1, tyrosinase and the pan-melanoma cocktail. In the case of metastatic MM, all 20 cases were positive with S100 protein, the pan-melanoma cocktail and tyrosinase. MART-1 was positive in 19/20 cases and HMB 45 in 17/20 cases. The pan-melanoma cocktail showed a high sensitivity for all forms of MM and should be considered a complementary marker to polyclonal S100 protein. Results confirmed that currently there is no alternative antibody available to match the sensitivity of polyclonal S100 protein for immunolabelling DMM.