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.     

Differentiating benign nevi from malignant melanoma using DNA microdensitometry and karyometry and maturation: a zonal comparison,correlation and multivariate analysis

OBJECTIVE: To evaluate the diagnostic effectiveness of cytometric features of DNA microdensitometry, karyometry (nuclear morphometry) and maturation and their combinations in separating benign nevi from malignant melanomas. STUDY DESIGN: Tumor cells were measured from each of the superficial, middle and deep zones of 81 melanocytic lesions using video image analysis for nuclear DNA content, chromatin compactness, and nuclear size and shape variables. There were 27 banal compound melanocytic nevi, 20 dysplastic compound nevi, 10 Spitz nevi and 24 malignant melanomas (MM). Maturation of cells with depth into the dermis was also studied by comparing cells from superficial to deep zones. RESULTS: MM showed distinct characteristics of DNA microdensitometry, karyometry and maturation as compared to all groups of benign nevi. There were overall close correlations between nuclear DNA content variables and nuclear size parameters in the total group of 81 lesions. However, there were fewer significant correlations between the various indices in the group of melanomas alone. Using multivariate discriminant analysis, up to 97% of the lesions could be correctly separated as benign or malignant by a combination of five key microdensitometric, karyometric and maturation parameters. CONCLUSION: DNA microdensitometry, karyometry and maturation parameters have independent abilities in identifying individual malignant melanomas. Coevaluation of various cytometric features and maturation profiles offers better diagnostic ability in separating benign nevi from MM.     

Karyometry of melanocytic lesions: quantitative assessment of the 'maturation to the depth'

40 cases each of malignant melanoma. Spitz's nevus and benign intradermal nevus were examined using an interactive image analysis system. 60 consecutive nuclei were evaluated in the upper and lower portion of the melanocytic lesions. Besides basic karyometric data, a 'maturation parameter' (MP) expressing the previously described 'maturation to the depth' was assessed in each individual case, by calculating the ratio of the nuclear area in the deep portion and in the superficial portion. When the three parameters (superficial nuclear area, deep nuclear area and maturation parameter) were evaluated separately (k-nearest neighbour method), the efficiency of the superficial nuclear area was only 19%, compared with 72% for the deep nuclear area and 94% for the maturation parameter. Combination of the maturation parameter and the deep nuclear area provided an efficiency of 98% with a sensitivity of 98% and a specificity of 97%. The results indicate that the maturation parameter is superior to conventional karyometric data in the differentiation between benign and malignant melanocytic lesions.     

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.     

Novel three‐way complex rearrangement of TRPM1‐PUM1‐LCK in a case of agminated Spitz nevi arising in a giant congenital hyperpigmented macule

The genetic anomalies associated with the agminated variant of Spitz nevus have so far been limited to HRAS G13R mutations, especially when arising within a nevus spilus. A previous report exposed the case of a man with a giant pigmented macule involving his upper right limb and trunk. Since childhood, Spitz nevi have been periodically arising, within the pigmented area. The histopathology of several lesions displayed the usual criteria of junctional, compound, or intradermal Spitz nevi with a diversity of cytomorphological and architectural features. Some lesions spontaneously regressed. Genetic studies confirmed in three lesions an identical translocation involving TRPM1, PUM1, and LCK. No mutations in HRAS, NRAS, BRAF, or other known fusion genes linked to Spitz nevus were detected. LCK break‐apart fluorescence in situ hybridization confirmed the rearrangement was present not only in the melanocytic proliferation but also in the surrounding non‐spitzoid melanocytes. This report expands the list of genetic alterations involved both in giant congenital macules and in agminated Spitz nevi, and also extends the concept of mosaicism in melanocytes to gene translocations.     

Morphometric analysis of AgNORs in tubular and papillary parts of canine mammary gland tumors

OBJECTIVE: To test the value of the silver staining nucleolar organizer regions (AgNORs) technique on canine mammary gland tumors using image analysis and to estimate differences in AgNOR parameters in structurally different parts of canine mammary gland tumors. STUDY DESIGN: Analysis was performed on 13 complex type and 10 simple type malignant canine mammary gland tumors containing tubular and/or papillary structures. Ten normal mammary glands were used as controls. Morphometric analysis was done by a computer-assisted image analysis system and consisted of evaluation of nuclear area, number and area of AgNORs per nuclear area, ratio of nuclei with five or more AgNORs, nuclear perimeter, area fraction between nuclear area and area of AgNORs, and area, equivalent diameter, volume equivalent sphere, perimeter and circularity of a singular AgNOR. RESULTS: Distinct differences were detected between normal and malignant mammary gland tissue for all measured parameters. There were no significant differences between the tubular and papillary parts of the same tumor or between the tubular and papillary parts of complex and simple type tumors. CONCLUSION: Despite the fact that no significant differences were found for AgNOR parameters between papillary and tubular structures of mammary gland tumors, the results of grouping tumors by the number of AgNORs indicate that this might help with classification of canine mammary gland tumors.     

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.     

Transformation of nuclear morphology during cellular maturation.

Using in vivo labeling of mouse leukocytes with tritiated thymidine, cells in the neutrophilic series were studied to determine the change in their nuclear shape as a function of maturation level. Several morphologic shape parameters including perimeter and bending energy were used to quantify the distribution of the nuclear morphology in a given age cohort. The change in these distributions as a function of calender age level was determined. The two parameters named above were used to test the possibility of inferring the age from the quantitative morphology.     

Congenital nevocellular nevus: a review of the treatment controversy and a report of 46 cases

Because congenital nevocellular nevi can be distinguished clinically and histologically from acquired nevi, and because of their apparent increased potential for malignant degeneration, we favor complete one-stage excision of these nevi, regardless of the size of the lesion or the age of the patient, at the earliest opportunity, whenever such surgery is feasible and practical. If there is a question about the clinical diagnosis, a cutaneous punch biopsy can help determine the true nature of the lesion. Significantly, Walton et al. and Rhodes and coworkers found discrepancies in the literature concerning the level of nevus cells in neonates. They concluded that until these differences are reconciled, nevus cells in the deep reticular dermal collagen may be a sufficient, but not a necessary criterion for the diagnosis of congenital melanocytic nevus. We currently favor complete one-stage excision of congenital nevocellular nevi and feel that treatment by tangential excision or dermabrasion require further study. Finally, we present this paper as "advice" not only to the three authors who, in a recent issue of the British Journal of Plastic Surgery, requested it, but also to all clinicians. Hopefully, with time and further study, better criteria will be determined and a more definitive approach to this problem will be established.     

不同类型甜瓜成熟过程中果肉质地及其细胞显微结构的变化

采用质地剖面分析(TPA)和穿刺方法,测定3种不同口感质地的5个甜瓜材料(梗硬果肉的P10和3-6、脆酥果肉的417和20-5以及软果肉的Charentais)不同成熟时期果肉硬度、咀嚼性和黏着性以及脆性和平均硬度,评价甜瓜果肉的质地特性;采用组织切片法观察果肉组织细胞的显微结构,并通过Image-pro plus 6.0软件测定细胞大小参数(细胞面积、周长、长度及宽度)和形状参数(细胞纵横比及圆度),明确不同果肉质地类型甜瓜果实成熟过程中果肉细胞显微结构的变化特征,探讨甜瓜细胞形态参数与果肉质地的关系,为甜瓜品质育种提供理论依据。结果显示:(1)梗硬果肉甜瓜(P10和3-6)的果肉细胞较小,排列紧密;脆酥果肉甜瓜(417和20-5)的细胞较大,排列较疏松;软果肉甜瓜(Charentais)的细胞最大,排列极不规则。(2)不同口感甜瓜果肉在成熟期的细胞面积和周长差异显著,与口感呈显著负相关关系;在成熟过程中,甜瓜果肉细胞面积、周长和长度等表现出不同程度的增大,而细胞纵横比和圆度总体表现为下降趋势,即细胞越来越圆。(3)甜瓜果肉的口感与质地及细胞大小参数呈显著或极显著相关关系;细胞大小与黏着性呈显著或极显著正相关关系(0.951*~0.983**),细胞面积与TPA硬度及脆性呈显著负相关关系(分别为-0.910*和-0.926*),长度和宽度与脆性呈显著负相关关系(分别为-0.884*和-0.894*);细胞形状参数中的圆度与黏着性具有显著相关性(0.936*)。研究表明,口感不同的甜瓜果肉具有显著不同的质地和细胞显微结构,且甜瓜果肉口感与其果肉质地及细胞大小密切相关,即细胞越小,甜瓜果肉质地越硬。     

The 'Abtropfung phenomenon' revisited: Dermal nevus cells from congenital nevi cannot activate matrix metalloproteinase 2 (MMP-2)

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro-matrix metalloproteinase (MMP)-2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP-2 was not in its active form, we used Western blot to study the expression of members of the MMP-2 activation pathway (membrane type 1-MMP and tissue inhibitor of metalloproteinase-2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol-12-myristate-13-acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP-2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.     

An Ex Vivo Study of Congenital Pigmented Nevi in Epidermal Reconstructs

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

The ‘Abtropfung Phenomenon’ Revisited: Dermal Nevus Cells from Congenital Nevi Cannot Activate Matrix Metalloproteinase 2 (MMP‐2)

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro‐matrix metalloproteinase (MMP)‐2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP‐2 was not in its active form, we used Western blot to study the expression of members of the MMP‐2 activation pathway (membrane type 1‐MMP and tissue inhibitor of metalloproteinase‐2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol‐12‐myristate‐13‐acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP‐2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.     

Pigmented Nevi—Induced Changes in the Junctional Component

The pigmented nevus represents a potentially more dynamic lesion than has been indicated by most published studies. New nevus cell clusters frequently appear in the epidermis over the residual portion of a nevus that remains after partial surgical excision. Even in relatively inactive nevi in adults, new junctional nevus cells may be induced by surgical trauma. This stimulated growth usually regresses by the time one year or more has elapsed. The growth of nevus cells is probably comparable to that induced in other cells by traumatic injury. There is no evidence to suggest that it is related to the development of melanoma in pigmented nevi.     

An ex vivo study of congenital pigmented nevi in epidermal reconstructs.

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

Cytomorphometry of large cell carcinoma of the lung

A computerized morphometry system was used to evaluate criteria for the cytologic diagnosis of large cell carcinoma (LCC) and poorly differentiated adenocarcinoma of the lung. There were 143 cells measured in six cases of LCC (five sputums and one bronchial washing) and 61 cells in four cases of adenocarcinoma (all sputum samples). Cellular and nuclear areas were significantly larger in adenocarcinoma whereas nucleolar area was greater in LCC, producing a higher nucleolar/nuclear area ratio in LCC. Cellular and nuclear form factors were smaller in LCC while the minor axis was longer in adenocarcinoma, resulting in a smaller axial ratio in adenocarcinoma. These data indicate that adenocarcinoma cells are larger and have a more rounded shape and less nucleolar material, as compared to the smaller, more ellipsoid and convoluted cells of LCC, which have more nucleolar area. A logistic regression identified cellular area, nucleolar/nuclear area ratio and cellular and nuclear form factors as significant contributors to the discrimination of LCC from adenocarcinoma, with a positive predictive value of 92%. Morphometry may therefore be helpful in the differential cytologic diagnosis of adenocarcinoma and LCC.     

The role of morphometry in the cytology of well-differentiated hepatocarcinoma and cirrhosis with atypia

A morphometric study was performed on 200 nuclei per case in six well-differentiated hepatocarcinomas and in six cirrhoses with cytologic atypia, using samples obtained by fine needle aspiration (FNA) biopsy of the liver. The parameters measured were the nuclear area, the nuclear perimeter and the maximum nuclear diameter. The nuclei of well-differentiated hepatocarcinomas could be distinguished from those of cirrhoses on the basis of the larger size and greater anisonucleosis of the former. A statistical analysis (using a two-sided t-test) of the means of the parameters showed significant differences between the two diagnostic groups. These results suggest that morphometric analysis can help in the differential diagnosis between well-differentiated hepatocellular carcinoma and cirrhosis with cytologic atypia in FNA biopsy samples.     

Cultured epithelial autografts for giant congenital nevi

Eight pediatric patients with giant congenital nevi confluent over 21 to 51 percent body surface area were treated by excision and grafting. The nevus was excised to the muscle fascia, and the open wound was grafted with cultured epithelial autografts and split-thickness skin grafts. The patients have been followed from 17 to 56 months. Seventeen operations were performed in the eight patients, excising a mean of 6.9 percent body surface area at each procedure. The mean duration of anesthesia was 3.7 hours, and the mean operative blood loss was 12.3 percent estimated blood volume. The mean "take" for the cultured epithelial autografts was 68 percent, and for the split-thickness skin grafts, 84 percent. Epithelialization of open wound areas adjacent to the grafts was somewhat slower for the cultured epithelial autografts than for the split-thickness skin grafts, but it led to a healed wound in all patients except one. Ten of the 17 areas grafted with cultured epithelial autografts resulted in small open wounds that required regrafting. Wound contraction under the cultured epithelial autografts and under split-thickness skin grafts was similar and depended more on the anatomic site grafted than on the type of graft employed. in 16 of 17 operations, the cultured epithelium remained as a permanent, durable skin coverage. The use of cultured epithelial autografts allowed a larger area of excision than would have been possible with split-thickness skin grafts alone and, therefore, a more rapid removal of nevus. Cultured epithelial autograft are an important new technique in the care of patients with giant congenital nevi.     

NRAS and BRAF Mutations in Melanoma-Associated Nevi and Uninvolved Nevi

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF^V600E-mutation specific antibody VE1. The BRAF^V600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF^V600E-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.