Immune-phenotypical markers for the differential diagnosis of melanocytic lesions

For specific subsets of melanocytic proliferations, there are morphologic limitations in the histological diagnosis, especially for borderline melanocytic tumors. In particular, Spitzoid proliferations can be difficult to diagnose. For these reasons, in the last years, clinic research has focusedattention on discovery of new diagnostic markers. Published gene expression and proteomic profiling data indicate new candidate molecules involved in melanoma pathogenesis, and useful in differential diagnosis of difficult melanocytic lesions. Recently, the diagnostic power of galectin-3 was demonstrated in series of melanocytic lesions, with a strong increasing of expression in malignant lesions compared with benign lesions. Similarly, the accumulation of Collagen XVII antibody was detected in vertical melanoma fronts and associated with invasive phenotype. Moreover, overexpression of cyclin D1 and p21 was detected in Spitz nevi compared with non-spitzoid melanomas; Ki-67 appears highly expressed in deep areas of non-spitzoid melanomas. In this review,werealizedan overviewofthe main molecular markersthat canbe a usefultoolfor the differential diagnosisofbenign, borderlineand malignant melanocytic lesions, related to their biological behavior, useful also for predicting the evolutionof the disease.     

Finding of conjunctival melanocytic pigmented lesions within pterygium

AIMS: Conjunctival pigmented lesions have characteristic clinical and histopathological appearances. Melanocytic pigmented lesions commonly occur in the conjunctiva, although they have not been previously reported in pterygium, a common lesion which originates from conjunctiva. Our aim was to evaluate the possibility of an association between pterygium and conjunctival melanocytic pigmented lesions. METHODS AND RESULTS: A total of 80 samples of pterygium excised from Ecuadorian patients in 2002 were collected. Clinical data were available regarding age, sex, race and place of residence. Histological sections were evaluated for the presence of melanocytic pigmented lesions. Nine cases of conjunctival melanocytic, pigmented lesions within pterygium were found and were classified according to the histopathological criteria previously published for pigmented lesions of the conjunctiva, as naevi and primary acquired melanosis (PAM) with varying degrees of atypia. Five of the nine cases showed primary acquired melanosis without atypia, while two cases had atypia; one case showed features of compound naevus and one lesion was designated as subepithelial naevus. CONCLUSIONS: Our findings suggest that conjunctival melanocytic, pigmented lesions occasionally occur in pterygium. All surgically removed pterygia should undergo careful histopathological examination.     

DEK expression in melanocytic lesions

The diagnosis of malignant melanoma presents a clinical challenge and relies principally on histopathological evaluation. Previous studies have indicated that increased expression of the DEK oncogene, a chromatin-bound factor, could contribute to the development of melanoma and may be a frequent event in melanoma progression. Here, we investigated DEK expression by immunohistochemistry in a total of 147 melanocytic lesions, including ordinary nevi, dysplastic nevi, Spitz nevi, melanoma in situ, primary invasive melanomas, and metastatic melanomas. Most benign nevi (ordinary, dysplastic, and Spitz nevi) were negative or exhibited weak staining for DEK, with only 4 of 49 cases showing strong staining. Similar to benign nevi, melanoma in situ also demonstrated low levels of DEK expression. In contrast, the expression of DEK in primary invasive melanomas was significantly higher than benign nevi (P < .0001). Moreover, DEK expression was significantly increased in deep melanomas (Breslow depth >1 mm) and metastatic melanomas as compared with superficial melanomas (Breslow depth ≤1 mm) (P < .05). Our findings indicate that DEK overexpression may be a frequent event in invasive melanomas, and further augmentation of DEK expression may be associated with the acquisition of ominous features such as deep dermal invasion and metastasis. These data suggest a role of DEK in melanoma progression.     

Differential immunoreactivity of melanocytic lesions of the conjunctiva

AIMS: To evaluate the expression of S100NKI/C3 and HMB45 antigens in melanocytic lesions of the conjunctiva and the ability of HMB45 to aid assessment of neoplasia. METHODS AND RESULTS: Stored formalin-fixed specimens of conjunctival melanomas and primary acquired melanosis were considered as participants and conjunctival naevi and racial melanosis as controls. Ninety-seven conjunctival melanocytic lesions were analysed using formalin-fixed paraffin-embedded material. These included 20 melanomas arising in the context of primary acquired melanosis (PAM), 22 melanomas arising without evidence of pre-existing PAM, seven cases of PAM with atypia, nine cases of PAM with no atypia, 35 conjunctival naevi and four cases of racial melanosis. S100 and NKI/C3 were similarly expressed in all lesions, with at least one of these markers positive in 100% of the lesions examined. HMB45 was expressed in 72.7% of primary melanomas and 85% of melanomas in the context of PAM; 42.8% of PAM with atypia expressed HMB45 while it was expressed in 11.1% of PAM without atypia and 8.5% of naevi. Racial melanosis cases did not express HMB45. S100 and NKI/C3 were expressed to a similar extent in all groups. CONCLUSIONS: S100 and NKI/C3 are useful markers to assess the extent of melanocytic lesions in the conjunctiva. HMB45 immunoreactivity can act as a useful aid to histopathology for the distinction of benign from malignant conjunctival lesions, particularly in the context of primary acquired melanosis.     

Expression of gamma-H2AX in melanocytic lesions

gamma-H2AX is a marker of activated DNA damage and is overexpressed in many malignancies and their precursor lesions. Previous studies have demonstrated the expression of gamma-H2AX in melanoma and dysplastic nevus, but its diagnostic and prognostic utility in a full range of melanocytic lesions has not been fully studied. In the current study, we investigated gamma-H2AX expression in a total of 162 melanocytic lesions. We found that gamma-H2AX was observed at higher levels (percentage and intensity of staining) in melanoma in situ (12/13), primary cutaneous melanoma (32/33; with the exception of desmoplastic melanoma), and metastatic melanoma (58/62), which was statistically different from that in benign nevus (7/9), dysplastic nevus (6/10), and Spitz nevus (5/9) considered together (P < .0001). Of note, desmoplastic melanoma (20/26) demonstrated weak or negative gamma-H2AX staining. The expression of gamma-H2AX did not show significant correlation with many melanoma prognostic factors, including Breslow depth, mitotic rate, and sentinel lymph node status. Except for desmoplastic melanoma, no difference in gamma-H2AX levels was observed among various melanoma subtypes. The overexpression of gamma-H2AX in melanoma as opposed to nevus indicates its possible role in melanomagenesis. Based on the overlap in subsets of nevi and melanomas, the potential clinical utility of this antibody remains uncertain until further studies have been carried out in a larger cohort of melanocytic lesions, including borderline cases.     

The role of immunohistochemical markers in the diagnosis of follicular-patterned lesions of the thyroid

Thyroid nodules are extremely common in the general population. The differential diagnosis includes numerous entities, non-neoplastic and neoplastic, benign and malignant. However, the diagnosis of follicular-patterned lesions remains an area fraught with controversy and diagnostic criteria are highly variable. It is, therefore, a field in need of objective, scientific markers that better characterize these lesions than has been possible by classical morphology. A number of candidates have been proposed. No single marker can identify all malignant follicular-patterned lesions, however, various combinations have been proposed. They include HBME-1, high molecular weight cytokeratins and ret, galectin-3 and TPO, galectin-3, fibronectin-1, CITED-1, HBME-1, and CK19. Advances in our understanding of the molecular basis of thyroid cancer will allow the identification of new markers and more accurate characterization of specific subtypes of neoplasia and malignancy. As new markers are characterized and validated, directed by molecular profiling of thyroid lesions with characteristic morphology, behavior, and outcome, they will become available as routine immunohistochemical markers that will provide a more accurate, scientific, and clinically relevant consultation report from the pathologist for cytology and surgical pathology procedures. Application of these markers will enhance the diagnosis of thyroid nodules and better guide the management of patients with these lesions.     

Cathepsin B, D and K expression in adamantinomatous craniopharyngiomas relates to their levels of differentiation as determined by the patterns of retinoic acid receptor expression

AIMS: To investigate the potential predictive value of cathepsins B, D and K in a series of 51 adamantinomatous craniopharyngiomas. While almost always benign, craniopharyngiomas exhibit a high propensity to recur postsurgically and biological markers are therefore needed to predict their recurrence. We have previously demonstrated the potential predictive value of retinoic acid receptors (RARs) (Lefranc et al., J. Neurosurg. 2003; 98; 145-153). METHODS AND RESULTS: Computer-assisted microscopy was used to determine quantitatively the immunohistochemical levels of expression of the alpha, beta and gamma RAR subtypes and cathepsins B, D and K. The levels of expression of cathepsin D and of cathepsin B correlated significantly with the levels of expression of RARbeta. The levels of expression of cathepsin K correlated significantly with the levels of expression of RARgamma. CONCLUSIONS: Recurrent adamantinomatous craniopharyngiomas are characterized by low levels of RARbeta and high levels of RARgamma. The tendency to recurrence seems, at least partly, to relate to the fact that (i) craniopharyngiomas with low levels of RARbeta express low levels of cathepsin D, and (ii) craniopharyngiomas with high levels of RARgamma express high levels of cathepsin K.     

Cathepsin D expression in normal,hyperplastic and malignant endometrial tissue: an immunohistochemical analysis

Cathepsin D (CathD), a lysosomal aspartyl protease secreted by normal and malignant cells, is considered to be involved in breakdown of the extracellular matrix. Aim of the present study was to determine the frequency and tissue distribution of CathD in normal, hyperplastic and malignant endometrium. Paraffin-fixed endometrial tissue was obtained from premenopausal women in the proliferative phase (n = 5), early secretory phase (n = 4) and late secretory phase (n = 4) as well as glandular-cystic hyperplasia (n = 5), endometrial polyps (n = 5), endometrial polyps from the use of tamoxifen (n = 5), adenomatous hyperplasia (AH) grade I (n = 5), grade II (n = 4), grade III (n = 5) and endometroid adenocarcinoma (n = 5). CathD expression was evaluated with the IRS score and ANOVA analysis was used for statistical evaluation. CathD was primarily localised in luminal and glandular epihelium with little staining in stromal cells. The expression of CathD was significantly higher during the late secretory phase than in the proliferative phase. Highest expression of CathD was observed in the late secretory phase and in glandular-cystic hyperplasia, whereas endometroid carcinoma showed no expression. A continuous increase in CathD expression was observed in AH, with a significant difference between AH grade I and III. In conclusion, CathD was found to be expressed in normal and hyperplastic endometrial tissue. CathD immunostaining in normal endometrial glands varied on the basis of the phase of the menstrual cycle, suggesting physiological functions of CathD in endometrial maturation and degradation. Adenocarcinomas did express significant lower amounts of CathD. Therefore, the prognostic value of this parameter remains uncertain. A continuous increase in CathD immunostaining was observed in AH. Since AH grade III can be considered as a precursor of endometrial cancer, CathD could be a possible parameter for assessing malignant transformation.     

The value of nucleolar organizer region staining in the differential diagnosis of borderline melanocytic lesions

The technique of silver (Ag) staining of nucleolar organizer region-associated proteins (AgNORs) has been shown to be of value in differentiating between benign and malignant cells. We have studied 33 borderline melanocytic lesions, in which a diagnosis of melanoma had been seriously considered, in order to assess the value of this technique in a commonly encountered diagnostic situation. We found that benign naevus cells possessed single compact or granular AgNORs, whereas some malignant melanocytes possessed large, often loosely arranged groups of AgNORs. However, the pattern of AgNORs observed in melanocytes of some atypical but benign lesions was also seen in some melanomas. The differential diagnosis of borderline melanocytic lesions is not clarified by use of the AgNOR technique.     

Melan-A/Mart-1 expression in various melanocytic lesions and in non-melanocytic soft tissue tumours

AIMS: The purpose of this study was to test different malignant non-melanocytic tumours with the commercially available antibody Melan-A to examine its diagnostic specificity and to compare the S100, Melan-A and HMB-45 reactivity in various melanocytic lesions. METHODS AND RESULTS: Seventy-three benign and malignant melanocytic lesions and 31 cases of non-melanocytic tumours, sarcomas, carcinomas and carcinoids, were selected. Immunohistochemical staining of paraffin sections, following a high temperature antigen unmasking technique, was performed. Melan-A stains junctional and dermal melanocytes in all benign melanocytic lesions with the exception of neuro-naevoid areas. The epithelioid and the spindle cells in malignant melanomas did not show considerable difference in their Melan-A reactivity. The predominantly spindle cell type mucosal melanomas contained more Melan-A-positive cells than HMB-45-positive cells and similar results were observed in metastatic malignant melanomas. In desmoplastic melanomas the positivity of Melan-A was not consistent. None of the sarcomas, carcinomas and carcinoids expressed Melan-A. Almost all soft tissue tumours, except for two malignant gastrointestinal stromal tumours, were unreactive for HMB-45. These two cases did not react with Melan-A antibody. CONCLUSIONS: Melan-A is a useful additional marker to differentiate non-melanocytic tumours from primary or metastatic melanoma. In melanocytic lesions the Melan-A staining pattern is similar to S100, but seems to be more specific. In desmoplastic melanomas, however, the variable Melan-A staining further necessitated detailed histological examination and the use of the S100 reaction.     

Primary small cell carcinoma of the maxillary sinus: a case report with immunohistochemical and molecular genetic study involving KIT and PDGFRA

Primary small cell carcinoma of the nose and paranasal sinuses is very rare; only a few reports are present in the English literature. The author herein reports a very rare case of primary small cell carcinoma of the maxillary sinus with an emphasis on immunohistochemistry and on KIT and PDGFRA. A 64-year-old man was admitted to our hospital because of left nasal obstruction. Endoscopy revealed three nasal polyps, and imaging modalities revealed an infiltrative tumor (45 x 45 mm) in the left maxillary sinus with invasion into nasal cavity. Multiple biopsies are taken from the nasal lesions. Histologically, the tumor consists of proliferation of malignant small epithelioid cells with hyperchromatic nuclei, fine chromatin, scant cytoplasm, molded nuclei, and absent nucleoli. Immunohistochemically, the malignant cells were positive for cytokeratin (CK) 18, synaptophysin, CD56, p53, Ki-67 (labeling=95%), bcl-2, KIT, and PDGFRA. However, they were negative for pancytokeratins, high molecular weight CK, CK5/6, CK7, CK 14, CK 19, CK20, vimentin, neuron-specific enolase, chromogranin, CD15, CD45, S100 protein, CEA, CA19-9, glial fibrillary acidic protein, neurofilaments, neuroblastoma, CD99, surfactant apoprotein A, melanosome, and TTF-1. The pathologic diagnosis was small cell carcinoma. A molecular genetic analysis using PCR-direct sequencing was performed using paraffin sections, and it showed no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. Imaging modalities including CT, MRI and PET did not reveal any tumors, including the lung, other than the maxillary sinus tumor. The present case is the first of small cell carcinoma of the maxillary sinus with a comprehensive immunohistochemical examination and a gene analysis of KIT and PDGFRA.     

Expression of stabilin-1 in M2 macrophages in human granulomatous disease and melanocytic lesions

Abstract: Stabilin-1 is an endocytotic scavenger receptor, specifically expressed by non-continuous sinusoidal endothelial cells in the liver, spleen and lymph nodes and by M2 or alternatively activated macrophages in human malignancies. We analysed paraffin-embedded tissue of melanocytic lesions and granulomatous diseases for stabilin-1 expression, using the human/murine RS1 antibody. The specificity of the RS1 staining was confirmed in a knockout model, as only M2-like tumor-associated macrophages and vessels of a B16F10 melanoma in wild type mice stained positive; while staining of tumor-associated macrophages and vessels originating from stabilin-1 deficient mice remained negative for stabilin-1 specific antibody RS1. In human specimens, the RS1 antibody stained tumor-associated macrophages in all pathological stages of melanoma. In addition, five cases of juvenile xanthogranulomas and one case of necrobiotic xanthogranuloma were strongly stabilin-1 positive, while Th-1 cytokine dominated granulomatous diseases such as sarcoidosis and granulomatous leprosy were negative. Stabilin-1 positive vessels were found in all analysed non-Langerhans cell histiocytoses and melanocytic lesions. No stabilin-1 positive vessels were present in any other granulomatous diseases.     

Simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of the thyroid

AIMS: The histological diagnosis is critical for the postsurgical management and follow-up of thyroid malignancies. The differential diagnosis between papillary carcinoma and hyperfunctioning lesions, either with papillary hyperplasia or with a follicular architecture, can create real diagnostic difficulty. The aim of this study was to evaluate the expression of several antibodies considered to be markers of malignancy in malignant and hyperfunctioning thyroid neoplasms and to include the most effective of them in a diagnostic panel. METHODS AND RESULTS: One hundred resected thyroid nodules--58 hyperfunctioning benign lesions and 42 papillary carcinomas (14 follicular variant, 14 macrofollicular variant and 14 classic type)--were immunohistochemically studied for HBME-1, galectin-3, cytokeratin (CK) 19 and RET-proto-oncogene. HBME-1 and galectin-3 showed 92.8% and 89% sensitivity, respectively, and their coexpression was present in 36 out of 42 papillary carcinomas (85.7%) and absent in non-malignant lesions. Their association increased sensitivity to 94.7% and the diagnostic accuracy to 97.9% and involved the highest number of cases (95%) in comparison with two other panels including, respectively, three (HBME-1, galectin-3, CK19) and all four antibodies. CONCLUSION: An immunohistochemical panel consisting of HBME-1 and galectin-3 can make a correct distinction between malignant and hyperfunctioning thyroid neoplasms with high diagnostic accuracy.     

Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.     

High prevalence of methicillin resistance and PVL genes amongStaphylococcus aureus isolates from the nares and skin lesions of pediatric patients with atopic dermatitis

Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmectyping, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureusisolates from skin infections. Methicillin-resistant S. aureus(MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.     

A histological and electronmicroscopic study of intracorneal melanin in benign and malignant melanocytic lesions

Intracorneal melanin is a feature quite frequently encountered in a range of melanocytic lesions, yet has rarely been described in the literature. We examined a variety of melanocytic lesions with increasing degrees of melanocytic atypia for this feature. The distribution and amount of intracorneal melanin is described and it was shown to be entirely within keratinocytes. In general, the amount of intracorneal melanin was related to increasing melanocytic atypia and was typically distributed in a disorderly fashion in malignant lesions.