Identification of progenitor cancer stem cell in lentigo maligna melanoma

ABSTRACT:  The potential role of stem cells in neoplasia has aroused considerable interest over the past few years. A number of known biologic characteristics of melanomas support the theory that they may originate in a mutated stem cell. Melanocytic stem cell markers have been described recently. Moreover, the CD133 cells that show surface markers for CD34 are stem cells primitive. These stem cells are capable of differentiating into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. The identification of cancer stem/initiating cells with a crucial role in tumor formation may open up new pharmacologic perspectives. The purpose of this study is to detect the expression of CD133 and CD34, two putative markers of cancer stem cells in the lentigo maligna melanoma. Thirty cases of lentigo maligna melanoma were analyzed using indirect immunohistochemical staining. The vast majority of the samples analyzed showed the presence of rare cells, which were clearly positive for CD133 and CD34. Strong CD133 and CD34 staining was found in the outer root sheath of the mid-lower hair follicles, intermixed with atypical melanocytes extending along layers of the hair follicles. A number of these staminal cells were adjacent and intermixed with melanoma cells. This study supports the stem cell origin of this tumor and suggests that the precursor of the melanoma in question is a stem-like cell rather than the primitive melanoblast committed to be exclusively involved in melanocytic differentiation.     

Immunohistochemistry of pigmented actinic keratoses, actinic keratoses, melanomas in situ and solar lentigines with Melan-A

Distinguishing lentigo maligna from solar lentigo, and pigmented actinic keratosis can sometimes be problematic. Melan-A is an immunohistochemical marker which that can be helpful in decorating the melanocytes of pigmented lesions. A recent report has suggested that Melan-A may spuriously label nests of junctional keratinocytes, potentially leading to the misdiagnosis of melanoma in situ. We compared Melan-A immunohistochemical staining in pigmented actinic keratosis , non-pigmented actinic keratoses , melanoma in situ of lentigo maligna type and solar lentigines. We found a statistically significant increase of Melan-A staining in melanoma in situ, but no statistical difference in the number of junctional Melan-A positively staining cells, in solar lentigines, pigmented actinic keratoses, and non-pigmented actinic keratoses, respectively. In the non non-melanoma samples, the Melan-A A-positive cells located at the dermal-epidermal junction were interspersed and not observed in clusters. Increased staining with Melan-A, in an actinic keratosis, or solar lentigo should raise the possibility of a contiguous melanoma in situ.     

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.     

CD10 and CD34 in fetal and adult human hair follicles: dynamic changes in their immunohistochemical expression during embryogenesis and hair cycling

Background  CD10 and CD34 have been detected in both epithelial and mesenchymal components of anagen human hair follicles.
Objectives  To analyse the expression of CD10 and CD34 in human hair follicle development as well as in different phases of the hair cycle.
Methods  Fetal and adult hair follicles at different stages of the hair cycle were examined by immunohistochemistry for CD10 and CD34.
Results  In fetal follicles, CD10 is expressed by the cells of the placodes, and CD34 by the mesenchymal cells of the dermal condensate. As the follicle matures, CD10 can be seen in the matrix cells, inner root sheath and dermal sheath. In adult follicles, the expression of CD10 in the follicular epithelium is present in anagen follicles, but tends to disappear in catagen, and is not detected in telogen. The CD10 positivity of the dermal sheath is more intense in catagen than in anagen follicles. CD34 immunostaining of the external root sheath was seen in adult anagen follicles but not in fetal follicles. This staining of the anagen outer sheath tends to disappear in catagen and is not detected in telogen.
Conclusions  CD10 and CD34 are not proteins constantly present in a specific cell type of the hair follicle, but are proteins that can be expressed by both epithelial and mesenchymal cells depending on the stage of development and hair cycle. The distribution of the immunoreactivity to CD10 in the placode and CD34 in the dermal condensate suggests a role of these proteins in initial stages of hair formation.     

Microinvasive lentigo maligna melanoma

Ninety-one skin biopsy specimens previously identified as lentigo maligna were examined for the presence of microinvasion, using the demonstration of S100 protein within atypical cells as the means for locating these superficial foci. In 14 cases, atypical melanocytes were identified, most often in the papillary dermis. The mean depth of invasion in this group was 0.23 mm with a range of 0.10 mm to 0.75 mm. In these cases, atypical cells were difficult if not impossible to identify in routinely processed sections, either because the invasive cell was a spindle cell variant and indistinguishable from a fibrohistiocytic cell, because the invasive cells were occasionally solitary or in small groups, or because there was an inflammatory infiltrate that obscured the tumor cells. Recent studies of lentigo maligna melanoma have revealed no better prognosis when compared to that of other forms of malignant melanoma after normalization for depth and body location. We therefore advocate close examination of lentigo maligna with the use of appropriate immunohistochemical techniques if there are areas of dermal fibrosis or inflammation that might obscure invasion.     

Lentigo Maligna: Review of Salient Characteristics and Management

Lentigo maligna is a melanocytic neoplasm, often regarded as ‘melanoma in situ,’ which may progress to lentigo maligna melanoma. Lentigo maligna clinically presents as a pigmented, asymmetric macule that originates on the head and neck and spreads slowly. The preferred method for diagnosing lentigo maligna is excisional biopsy. Histology shows proliferation of atypical melanocytes at the epidermal–dermal junction in small nests or single cells. The differential diagnosis includes solar lentigo, seborrheic keratosis, lichen planus-like keratosis, pigmented actinic keratosis, and melanocytic nevus. Stains used in diagnosis include hematoxylin and eosin, HMB-45, MART-1/Melan-A, Mel-5, and S-100. Surgical excision is the preferred treatment for lentigo maligna. Second-line techniques include medical (topical imiquimod) and destructive therapy.     

Differential expression of proliferation- and apoptosis-related markers in lentigo maligna and solar keratosis keratinocytes

Keratinocytes influence the number, morphology, and proliferation of melanocytes. An interference in the melanocyte-keratinocyte relationship may contribute to melanoma development. This study examined the expression of apoptotic and proliferative markers in keratinocytes in lentigo maligna to characterize the epidermis permissive to these lesions. Formalin-fixed and paraffin-embedded tissues from 25 samples of lentigo maligna, 20 samples of solar keratoses, and 5 samples each of normal sun-exposed and non-sun-exposed skin (controls) were immunostained with antibodies directed against the proapoptotic markers bax and p53, the antiapoptotic marker bcl-2, and the proliferation marker ki-67. Eight percent of the lentigo maligna samples were positive for keratinocyte expression of bcl-2, 24% were positive for p53, and 76% were positive for bax; respective findings for solar keratoses were 35%, 85%, and 90%. Comparison with normal sun-exposed skin yielded lower rates of keratinocyte proliferation in 56% of the lentigo maligna samples, similar rates in 36%, and higher rates in 8%; for solar keratoses, proliferation was higher than controls in 60% of samples, similar in 35%, and lower in 5%. All these differences were statistically significant. These findings indicate that there are variable patterns of epidermal reaction to chronic sun exposure. The epidermis in lentigo maligna shows overall low proliferation and an apparently low apoptotic tendency. The dysfunctional epidermis may be permissive to aberrant melanocyte proliferation in the early stages of melanoma development.     

Development of a new mouse model (xeroderma pigmentosum a-deficient, stem cell factor-transgenic) of ultraviolet B-induced melanoma

It is well established that exposure to sunlight or ultraviolet radiation (UVR) is the major environmental risk factor for the development of skin neoplasms. To date, however, there have been few appropriate mouse models available for studying the role of UVR in melanoma carcinogenesis, mainly because of the murine lack of the epidermal melanocyte, which is a major source of origin of human melanoma. In this study, we established xeroderma pigmentosum group A gene-deficient, stem cell factor-transgenic mice, which are defective in the repair of damaged DNA and do have epidermal melanocytes. The mice were exposed to UVR three times a week for 10 wk. More than 30% of the irradiated mice developed tumors of melanocyte origin that metastasized to the lymph nodes. Histologically, proliferated cells exhibited lentigo maligna melanoma or nodular melanoma. Immunohistochemistry confirmed that the tumor cells were characteristic of melanoma. Non-irradiated mice did not develop skin tumors spontaneously. The newly generated model mouse might be useful for studying the photobiological aspects of human melanoma, because the mice developed melanoma from epidermal melanocytes only after UVR exposures.     

干细胞标志物CD133、nestin和CD44在恶性黑素瘤皮损中的表达及意义

目的 探讨干细胞标志物CD133、nestin和CD44在恶性黑素瘤中的表达及意义.方法 采用免疫组化SP法检测3种标记物在30例恶性黑素瘤及30例皮内痣皮损中的表达.结果 CD133、nestin和CD44在恶性黑素瘤组织中的阳性表达率分别是53.33%,80.00%和20.00%,在皮内痣分别是23.33%,53.33%和0,两组各标志物阳性率差异比较均有统计学意义(P＜0.05).CD133、nestin和CD44在恶性黑素瘤组织中阳性细胞率平均值分别为2.98%±5.62%,34.92%±34.89%和1.28%±3.26%,在皮内痣分别为0.10%±0.21%,7.26%±13.13%和0,两组各标志物差异比较均有统计学意义(P＜0.05).两组中阳性细胞率与患者性别、年龄、病程比较,差异无统计学意义(P＞0.05).结论 干细胞标志物CD133、nestin和CD44在恶性黑素瘤中高表达,在皮内痣中低表达或不表达,提示恶性黑素瘤中可能存在肿瘤干细胞.     

Environmental modification of the shape of pigment-producing cells

With fluorescence-histochemical methods (FIF method) pigment-producing cells were investigated in benign and malignant skin lesions. Here, pigment cells appear predominantly dendritic, whilst they are separated from each other by keratinocytes, cells of hair follicles, or cells of sebaceous glands. In lentigo maligna melanoma, superficial spreading melanoma as well as in nodular melanoma and some metastases of these melanomas, the tumour cells can show a dendritic pattern under special environmental situations. It is, therefore, assumed that the shape of tumour cells in melanomas is more closely dependent on their particular environment and other modifying factors than on a different histogenesis.     

Following lentigo maligna may not prevent the development of life-threatening melanoma.

BACKGROUND--Management of lentigo maligna (Hutchinson's melanotic freckle, in situ lentigo maligna melanoma) by regular observation relies on the detection of invasive melanoma before it has developed significant life-threatening potential. Recent studies indicate that lentigo maligna melanoma does not have a better prognosis than other forms of melanoma. OBSERVATIONS--A case is reported of an amelanotic lentigo maligna that evolved from a macular lesion to a deeply invasive, amelanotic, lentigo maligna melanoma within 6 months. The melanoma was Clark level IV and measured 3.0 mm in maximum tumor thickness. CONCLUSIONS--Observation of lentigo maligna at 6-month intervals would not seem to be sufficiently reliable in detecting the development of invasive lentigo maligna melanoma before it becomes a life-threatening disease. Early surgical excision is the treatment of choice.     

Primary invasive signet-ring cell melanoma

The histopathological variants of malignant melanoma include the common type (lentigo maligna, superficial spreading melanoma, nodular melanoma, acrolentiginous melanoma), spindle cell, desmoplastic, balloon cell, pleomorphic (fibrohistiocytic), myxoid, small cell melanoma and malignant blue nevus. Recently, signet-ring cell melanoma was introduced as an additional cytologic variant. We describe a 72-year-old patient with a primary signet-ring cell melanoma of the skin located on the upper arm. Histopathologic examination disclosed a melanocytic tumor extending from the epidermis to the deep reticular dermis. Numerous pleomorphic tumor cells showed large, intracellular vacuoles and oval to spindle-shaped nuclei at their periphery. Mitotic figures and multinucleated melanocytes were also observed. Some of the signet-ring cells exhibited cytoplasmatic periodic acid-Schiff (PAS)-positivity. Immunohistochemistry showed positive reaction of the tumor cells for S-100, HMB-45 protein and vimentin, confirming their melanocytic differentiation. Tumor cells were negative for cytokeratins, epithelial membrane antigen (EMA), and carcinoembryonic antigen (CEA). The signet-ring cell melanoma disclosed an invasion to Clark Level IV and tumor thickness of 2.2 mm. Signet-ring cell melanoma is a rare morphologic variant of melanoma. Its recognition is important for differentiation from other tumors featuring signet ring cells.     

Cellular fine structure in the invasive nodules of different histogenetic types of malignant melanoma

Ultrastructural studies were carried out on the invasive nodule of forty malignant melanomas. The findings support the concept that the fine structure of lentigo maligna melanoma is often characteristic, and differs from that of superficial spreading and nodular melanoma. The melanosomes in lentigo maligna melanoma are usually ellipsoidal and resemble those of normal melanocytes, whereas the melanosomes in superficial spreading and nodular melanoma are most often spheroidal and abnormal in appearance. Superficial spreading and nodular melanomas cannot be distinguished reliably by their ultrastructure. Melanosomal appearances could not be related to the presence of a pre-existing naevus or the depth of invasion of the tumour nodule.     

Folliculotropism in pigmented facial macules: Differential diagnosis with reflectance confocal microscopy

Pigmented facial macules are common on sun damage skin. The diagnosis of early stage lentigo maligna (LM) and lentigo maligna melanoma (LMM) is challenging. Reflectance confocal microscopy (RCM) has been proven to increase diagnostic accuracy of facial lesions. A total of 154 pigmented facial macules, retrospectively collected, were evaluated for the presence of already‐described RCM features and new parameters depicting aspects of the follicle. Melanocytic nests, roundish pagetoid cells, follicular infiltration, bulgings from the follicles and many bright dendrites and infiltration of the hair follicle (ie, folliculotropism) were found to be indicative of LM/LMM compared to non‐melanocytic skin neoplasms (NMSNs), with an overall sensitivity of 96% and specificity of 83%. Concerning NMSNs, solar lentigo and lichen planus‐like keratosis resulted better distinguishable from LM/LMM because usually lacking malignant features and presenting characteristic diagnostic parameters, such as epidermal cobblestone pattern and polycyclic papillary contours. On the other hand, distinction of pigmented actinic keratosis (PAK) resulted more difficult, and needing evaluation of hair follicle infiltration and bulging structures, due to the frequent observation of few bright dendrites in the epidermis, but predominantly not infiltrating the hair follicle (estimated specificity for PAK 53%). A detailed evaluation of the components of the folliculotropism may help to improve the diagnostic accuracy. The classification of the type, distribution and amount of cells, and the presence of bulging around the follicles seem to represent important tools for the differentiation between PAK and LM/LMM at RCM analysis.     

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.

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.     

Induction of cellular immunity against hair follicle melanocyte causes alopecia

Alopecia areata (AA) is generally regarded as an organ-specific autoimmune disease. Although it has been hypothesized that the autoimmunity is mediated by T cells and that hair follicle melanocyte is one of the targets, definitive evidence is lacking. We here demonstrate that AA-like lesions can be induced in mice by inducing CD8⁺ T-cell-mediated immunity to hair follicle melanocytes. We found that hair loss was induced in mice-bearing interleukin-12-producing B16 melanoma cells by the depletion of CD4⁺ T cells, accompanied by vitiligo-like coat color change. The alopecic lesions varied in size from pachy to extensive. In many instances, hair loss developed and was followed by the regrowth of white hairs. Histological analysis revealed that mononuclear cells infiltrated in and around the bulb region of hair follicles. Furthermore, immunohistochemical examination clearly showed the intra-follicular infiltration of CD8⁺ T cells. Neither the vitiligo-like coat color nor AA-like lesions were induced when CD8⁺ T cells were codepleted. These observations indicate that the induction of CD8⁺ T-cell-mediated immunity against hair follicle melanocytes causes alopecia. It is thought that there are many types of AA with different mechanisms, targets etc. Although hair follicle melanocytes have long been thought to be one of the targets of AA, evidence to support the hypothesis is sparse. Therefore, we believe that our observation is significant to support the hypothesis.     

Expression of the proliferation and apoptosis-associated CAS protein in benign and malignant cutaneous melanocytic lesions

We have examined the expression of the cellular apoptosis susceptibility protein, a nuclear transport factor that plays a role in apoptosis and cell proliferation, in benign and malignant melanocytic lesions. Tissue samples of 55 formalin-fixed, paraffin-embedded melanoma (primary n=32, metastatic n=23) and of 27 control cases (junctional dermal, compound, Spitz, Reed, blue nevi, balloon-cell nevus, lentigo maligna) were analyzed by immunohistochemistry with anti-cellular apoptosis susceptibility antibodies. The percentage of cellular apoptosis susceptibility-positive cells as well as the intensity on a four-point scale was evaluated. In normal skin, expression of cellular apoptosis susceptibility was primarily found in the basal cell layer of the epidermis. Benign melanocytic lesions that stained positive for cellular apoptosis susceptibility (13 of 27) showed a homogeneously distributed staining pattern with a mean of 5+/-12% cellular apoptosis susceptibility positive cells. Five out of 7 lentigo maligna melanoma, 11 out of 12 superficial spreading melanoma and all acrolentiginous (n=7) and nodular (n=6) melanoma showed immunoreactivity of medium (++) to high ( ) intensity. Vertical growth phases of primary cutaneous melanoma stained stronger than horizontally growing cell clusters. All metastases (n= 23) stained strongly positive, the staining pattern being inhomogeneous. Cellular apoptosis susceptibility detection in clinical stages according to UICC showed an increase from 43+/-34% cellular apoptosis susceptibility positive cells in stage I, to 53+/-26% in stage II, 68+/-24% in stage III and 72+/-24% in stage IV, respectively. Because the expression of cellular apoptosis susceptibility correlates predominantly with advanced stages of melanoma, staining with anti-cellular apoptosis susceptibility antibodies may be useful for diagnosis of melanoma and possibly as an immunohistochemical prognostic factor in cutaneous melanocytic lesions.     

PLANTAR MALIGNANT MELANOMA

A case of a 51 year old man with a plantar malignant melanoma is reported. The tumor had clinical and histologic features of lentigo maligna melanoma, but its biologic activity was that of a more potently malignant tumor. Electron microscopic observation reveales features similar to that of lentigo maligna melanoma, but not inconsistent with that of superficial spreading melanoma. A consideration of our case and of previous reports suggests that there is a relationship between the biologic potency and the site of involvement of lentigo maligna melanoma and that such lesions on relatively non-pigmented areas such as the palms, soles, mucous membranes, and nail beds should be treated as having a highly malignant potential.     

Recurrent lentigo maligna as amelanotic lentigo maligna melanoma

Amelanotic lentigo maligna and lentigo maligna melanoma are extremely rare tumours. Even rarer is a recurrent amelanotic lentigo maligna or amelanotic lentigo maligna melanoma at the site of a previously removed pigmented lentigo maligna. We describe two cases of recurrent amelanotic lentigo maligna melanoma manifesting as erythematous plaques evolved from previously excised pigmented lentigo maligna.