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.     

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.     

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.     

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.     

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.     

Aid to diagnosis of melanoma in primary medical care.

OBJECTIVE--To evaluate an intervention designed to reduce the number of benign melanocytic lesions excised from the skin. DESIGN--A randomised controlled field trial based in the medical practices of two cities. Examination of histopathological reports of 5823 melanocytic skin lesions excised over the intervention period and in the preceding six months. INTERVENTION--Medical practitioners were offered an algorithm and use of an instant developing camera. SETTING AND SUBJECTS--Over 50 medical practitioners, mostly in general practice, in each of two cities in tropical Queensland, Australia. MAIN OUTCOME MEASURES--Percentages of benign (neither malignant nor potentially malignant) melanocytic lesions excised during the two year intervention period. RESULTS--There were no significant differences in the percentages of benign lesions reported in the intervention and control cities before the intervention started (93.6% and 94.0%, respectively), but there was a significant difference afterwards (88.8% and 93.8%, P < 0.001). There was no difference in the percentage of invasive melanomas excised per month in the intervention city (3.4%) compared with control city (3.4%). CONCLUSION--Clinical diagnostic accuracy may be enhanced by offering to clinicians managing suspicious melanocytic skin lesions a simple algorithm and a camera with which to record the appearance of lesions objectively.     

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.     

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.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

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.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

Black lesions of the skin

Benign melanocytic lesions include lentigo, ephelid (freckle), pigmented nevus, sacral spot, blue nevus, and combined nevus and blue nevus. Malignant melanocytic lesions are melanomas, which arise from melanocytes at the epidermodermal junction, or, rarely, from blue nevi. They usually originate in brown plaques known as lentigo maligna, in pigmented nevi, or in normal skin. Melanoma is diagnosed clinically in less than 50 per cent of instances. Biopsy is therefore of great importance, since practically all melanoma can be cured by adequate early resection.     

Morphometry of nuclei of the normal and malignant prostate in relation to DNA ploidy.

Fine needle biopsies from 70 patients with prostate carcinoma and 10 patients with benign hyperplasia were used to study area, variation in size and form factors of the nuclei by image analysis. The results were related to DNA ploidy of the cell populations as measured by flow cytometry, cytologic grade and patients' survival. Nuclear area differed significantly between benign lesions and tumors. It increased in diploid low-grade tumors from a normal value of 54.2 +/- 3.1 microns2 to 75.6 +/- 5.3 microns2. In aneuploid tumors with an increase in the chromosome number, the nuclear size further increased to about twice that of benign nuclei. Variation in size also differed between benign and malignant epithelium, with a further increase between diploid and gross aneuploid tumors. While nuclear size and variation in nuclear size made it possible to discriminate malignant from benign lesions, form factor did not differ between benign and malignant lesions. In follow-up, however, none of these factors reached significance for predicting survival.     

"Halo nevi" and UV radiation

Halo nevi, also termed Sutton nevi, are defined as benign melanocytic nevi that are surrounded by an area of depigmentation resembling a halo. Halo nevi are common in children and young adults, with a mean age at onset of 15 years. The incidence in the population is estimated to be approximately 1%. Affected individuals frequently have multiple lesions which are usually localized on the back. A familial tendency for halo nevi has been reported. The etiology of halo nevi is unknown. It is an autoimmune response and T lymphocytes are considered to play a key role in the progressive destruction of nevus cells. Halo nevi may be associated with autoimmune disorders such as vitiligo, Hashimoto thyroiditis, alopecia areata, celiac disease, atopic dermatitis and others. It has been proved that halo nevi are detected after an intense sun exposure especially after sunburns. The etiology of halo nevi, association with malignant melanoma and the role of sun exposure in the development of halo nevi are discussed.     

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.     

Automated analysis of rat breast tumors. Comparison of four methods

Chemically-induced malignant rat breast tumors pose diagnostic dilemmas since the majority are well-differentiated, noninvasive papillary lesions that are barely distinguishable from benign papillary lesions. This study compared several automated modalities to see which best separated benign from malignant breast tumors. Thirty-three carcinogen-induced rat breast tumors (13 adenomas, 10 papillary carcinomas and 10 invasive carcinomas) were evaluated by static (image) cytometry (ICM) of integrated optical density, by flow cytometry (FCM) and by two automated morphometric protocols, contextual analysis and single-gland analysis. DNA ploidy analysis, by either ICM or FCM, did not discriminate between the benign and malignant tumors. Contextual analysis correctly identified 11 of 13 benign and 17 of 20 malignant lesions (P less than .01). Single-gland analysis correctly identified all 13 benign and 17 of 20 malignant lesions (P less than .01). No method distinguished invasive from noninvasive carcinomas. The data suggest that architectural features are more important than nuclear features in differentiating benign from malignant rat breast tumors.     

Thyroid neoplasms. Can we do any better with quantitative cytology?

The differential diagnosis of thyroid neoplasms by routine cytology presents major difficulties. We therefore looked for measurable nuclear parameters that could be generated from Feulgen-stained smears obtained by fine needle aspiration biopsy (FNAB). These parameters would then be used to differentiate between benign and malignant lesions. Seventy-six patients whose cold thyroid nodule was surgically excised after FNAB and examined by a pathologist were used in this study: 56 benign, 18 malignant and 2 atypical adenomas. A set of 3,662 cells from the 33 benign cases was compared with the set of 1,712 cells from the 11 malignant nodules. Discrimination between the two populations was based on four nuclear features ranked according to their discriminating power. The first ranked was a textural parameter, followed by a densitometric and finally two other textural parameters. The rate of nuclei that could be regarded as benign was computed for each case using the ranked parameters. The average rate for the 33 benign cases was 85% (50.2 = 100%, set at the 95% confidence interval). In a prospective study of the 32 remaining patients, sensitivity and specificity were, respectively, 92% and 88%, which were higher than those obtained by conventional cytology.     

Accuracy in clinically evaluating pigmented lesions.

OBJECTIVE--To determine the ability of three doctors experienced in managing melanocytic lesions to diagnose correctly melanoma, dysplastic naevi, and various benign pigmented lesions. DESIGN--Independent clinical evaluation and histopathological assessment. SETTING--Pigmented lesion clinic, which patients attend without an appointment for early diagnosis of melanoma. PATIENTS--86 Patients with lesions that were judged to be benign by at least one of the three doctors. INTERVENTIONS--The lesions were excised under local anaesthesia and sent for histopathological examination in coded bottles without clinical details. MAIN OUTCOME MEASURE--Comparison of clinical with histopathological diagnosis for each lesion. RESULTS--A total of 120 lesions were evaluated by at least two of the three doctors. The histopathological diagnoses were made by the same pathologist. The overall sensitivity (diagnostic accuracy) for the three doctors for all types of lesion was 50%. Of the 39 dysplastic naevi, only 19 were identified correctly by all observers, and a further 24 banal lesions were wrongly diagnosed as dysplastic by at least one doctor. Particular difficulty was experienced with small (less than 5 mm), flat lesions, which can be banal or potentially malignant. CONCLUSIONS--Critical diagnosis and management decisions concerning pigmented lesions should always be based on a combination of clinical and histopathological assessments and the history of the patient.     

BLACK LESIONS OF THE SKIN

Benign melanocytic lesions include lentigo, ephelid (freckle), pigmented nevus, sacral spot, blue nevus, and combined nevus and blue nevus.Malignant melanocytic lesions are melanomas, which arise from melanocytes at the epidermodermal junction, or, rarely, from blue nevi. They usually originate in brown plaques known as lentigo maligna, in pigmented nevi, or in normal skin.Melanoma is diagnosed clinically in less than 50 per cent of instances. Biopsy is therefore of great importance, since practically all melanoma can be cured by adequate early resection.