Re-evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis

Excessive amounts of melanin pigments may hamper histopathological assessments of melanocytic lesions by obscuring cellular morphology and hindering antibody-antigen interactions. To determine the optimal melanin-bleaching conditions for histopathological examination, heavily pigmented melanomas were treated with warm hydrogen peroxide (H2O2) diluted with various diluents (1% disodium hydrogen phosphate 12H2O (Na2 HPO4); phosphate buffer 0.05 M, pH 7.4 (PB); and PBS 0.05 M, pH 7.4) at varying temperatures (50°C, 55°C, and 60°C) and for varying incubation times (0.5, 1, 2, and 3 h). The effect of the sequential order of antigen retrieval and bleaching on preserving tissue morphology was then evaluated. Additionally, the effect of melanin bleaching using warm diluted H2O2 on the antigenicity of melanoma-related markers (HMB-45, MART-1, and S-100) and other markers used for histopathology was examined in amelanotic melanomas and tonsil tissue. Optimal and complete bleaching was achieved using warm 3% H2O2 in PB treatment at 55°C for 2 h following antigen retrieval with microwaving or digestion with trypsin. Under these conditions, the tissue morphology and antigenicity of various immunohistochemical markers were also well preserved. Bleaching with warm 3% H2O2 PB is a fast and efficient method of bleaching melanin pigments and performing immunohistochemical examination in heavily melanin-pigmented lesions.     

Immunohistochemical study of melanocytic differentiation antigens in cutaneous malignant melanoma. A comparison of six commercial antibodies and one non-commercial antibody in nodular melanoma, superficially spreading melanoma and lentigo maligna melanoma

In certain primary and metastatic malignant melanomas diagnostic problems may arise due to their cytologic features and/or absence of synthesis of melanin. As the "classic" combination of S-100 protein and HMB-45 may occasionally fail to stain cells of malignant melanoma, we have tested a series of commercially accessible antibodies which were so far not compared by other authors in the three most frequent subtypes of this tumor. In surgical specimens from 104 cutaneous malignant melanomas (40 nodular melanomas, 46 superficially spreading malignant melanomas and 18 lentigo maligna melanomas) the staining intensity and the proportion of neoplastic cells stained with antibodies to S-100 protein, HMB-45, NKI/C3, NKI/beteb, MART 1 (Melan A), KBA 62 and Mitf was semiquantitatively analysed. The use of this group of antibodies against melanoma-associated antigens revealed it to be a favourable supplement for the bioptical or cytological diagnosis of malignant melanoma in case the traditional/conventional combination of S-100 protein and HMB-45 antibody fails. According to the authors' experience the antibody against KBA 62 has shown to be the most effective antibody followed by the antibodies against MART-1 (Melan A) and NKI/C3.     

Primary malignant melanoma of the ovary: case report and review of the literature

Ovarian malignant melanomas are extremely rare tumors. Most of them are secondary tumors and disseminated metastases are recognized at the time of diagnosis. Primary tumors are even more rare and usually associated with a teratoma. A 67-year-old female had a pelvic mass that was recognized on ultrasonography (USG) and physical examination. Intraoperative pathological consultation was reported as "pigmented solid ovarian tumor, probably compatible with malignant melanoma". Paraffin sections, and histochemical (Masson Fontana and Prussia blue) and immunohistochemical examination (S-100 and HMB-45) were also consistent with "malignant melanoma". This case was accepted as "Probably primary ovarian malignant melanoma" in lack of any other tumor focus on detailed clinical and radiological investigation, skin biopsies or pigmented lesions in medical history. It is reported for being an extremely rare tumor and its distinctive characteristics for differential diagnosis are emphasized.     

Heavily pigmented melanocytic neoplasms: comparison of two melanin-bleaching techniques and subsequent immunohistochemical staining

The effect of melanin bleaching using permanganate/oxalate and dilute hydrogen peroxide on subsequent immunohistochemical staining of heavily pigmented melanocytic neoplasms is investigated. Permanganate/oxalate precluded the use of some antibodies but allowed much faster bleaching times, whereas dilute hydrogen peroxide enabled a full range of antibodies to be used, yet bleaching times were far longer. Each technique has advantages; however, the choice of method should be determined by the nature of the information needed to make a diagnosis and the speed at which a report is required.     

A case of mucoepidermoid carcinoma with melanin pigmentation manifested in the palate

This paper describes the first documented case of mucoepidermoid carcinoma (MEC) with melanin pigmentation manifested in the palate. Histopathological sections showed a neoplasm composed of epidermoid, mucous-producing and intermediate cells. Numerous large cells contained dark pigmented materials. Fontana Masson staining revealed dendritic melanocytes and melanin granules. HMB-45, Melan A and S-100 protein were all positive for melanocytes. Histopathological examination was not typical for malignant melanoma; the lesion was diagnosed as a low-grade MEC with melanin pigmentation.M. Vieth received a research grant from the Japanese Society of Pathology at the Institute of Pathology, Shiga University of Medical Science, Ohtsu, Japan     

Heavily melanotic perivascular epithelioid clear cell tumor of the kidney

A black-colored and well-circumscribed renal tumor in a 71-year-old woman is reported. The tumor was unique in that it was rich in vasculature and exclusively composed of perivascular epithelioid clear cells. Morphological features were reminiscent of conventional renal cell carcinoma (RCC). However, immunohistochemical examinations showed that the tumor cells did not express any epithelial markers, but diffusely and intensely expressed a melanocytic marker, gp-100/HMB-45. Another striking feature of the tumor was a large amount of cytoplasmic pigment that made the tumor wholly black. The pigment was not stained with Berlin-blue, completely bleached with potassium permanganate, and stained with Fontana-Masson staining, which suggests that the pigment was melanin. Morphological features and immunohistochemical findings indicated that the present tumor was an extreme example of a perivascular epithelioid clear cell tumor with a large amount of melanin, which has not been previously reported. One should be aware of the pure form of perivascular epithelioid clear cell tumor of the kidney because it is sometimes very difficult to differentiate this tumor from conventional RCC. Immunohistochemical examinations and the presence of cytoplasmic melanin can help the differentiation.     

Comparison of HMB-45 monoclonal antibody and S-100 protein in the immunohistochemical diagnosis of melanoma

The value of HMB-45 mouse monoclonal antibody in the immunohistochemical diagnosis of melanoma was compared with that of two antibodies to S-100 protein. Tissue from 32 (91.4%) of the 35 melanomas studied reacted with HMB-45, however, none of the 98 nonmelanoma tumors stained with this antibody. In contrast, 31 (88.5%) melanomas and 24 (24.4%) of the nonmelanoma neoplasms expressed S-100 protein. These results indicated that HMB-45 monoclonal antibody has a higher specificity for melanoma cells than did S-100 protein and that, because of its reactivity in routinely processed tissues, it may be helpful in surgical pathology.     

Melanin production in medullary thyroid carcinoma

A case of melanin-producing medullary thyroid carcinoma is reported in a 51-year-old man. Histologically, the tumour had a typical pattern of medullary thyroid carcinoma with numerous scattered pigmented cells which contained large amounts of melanin pigment as confirmed by bleached Fontana-Masson stain. Immunohistochemical staining revealed positivity of almost all tumour cells for calcitonin and chromogranin, whereas S-100 protein and HMB-45 staining was positive only in the pigmented tumour cells. This finding confirms the ability of medullary thyroid carcinoma cells to have multidirectional differentiation, although melanocytic differenciation remains an exceptional phenomenon.     

PNL2, a new monoclonal antibody directed against a fixative-resistant melanocyte antigen.

We report the production of a new monoclonal antibody, PNL2, directed against a fixative resistant melanocyte antigen. The analysis of PNL2 immunostaining on a broad range of normal or malignant human tissues and on various melanocytic lesions revealed its high specificity. PNL2 gave a strong cytoplasmic staining of skin and oral mucosae melanocytes, and staining of granulocytes when used at high concentration. PNL2 stained all intra-epidermal nevi irrespective of their histologic type, but common intradermal nevi and the dermal component of compound nevi were largely non-reactive as only scattered nevus cells in the papillary dermis were labeled. PNL2 labeled more than 70% of the neoplastic cells in all primary melanomas irrespective of their histologic type. However, PNL2 did not label desmoplastic melanomas. All metastatic melanomas were also stained but the percentage of labeled cells was occasionally lower than the primary tumor. PNL2, as anti-Melan A and HMB-45 antibodies, stained most of the clear cell sarcoma cells, and a few cells in angiomyolipomas and lymphangioleiomyomatosis. None of the other non-melanocytic lesions tested were labeled. Proteomic approaches showed that the immunoaffinity purified PNL2-binding complexes isolated from melanoma cell lines comprise at least TAP1, Clathrin 17 and prealbumin proteins, but not the gp100 recognized by HMB-45. In conclusion, this new monoclonal antibody, PNL2, is directed against a new fixative resistant melanocyte associated antigen. This antigen is chemically resistant and thus allows immunostaining after melanin bleaching or decalcification. We also demonstrate that it is different from Melan A and from gp100, even if PNL2 and HMB-45 staining patterns are sometimes similar.     

A primary amelanotic melanoma of the vagina, diagnosed by immunohistochemical staining with HMB-45, which recurred as a pigmented melanoma

Usually, malignant melanoma is readily diagnosed by the presence of melanin granules. Although amelanotic melanoma contains a few melanin granules, it is often difficult to differentiate from non-epithelial malignant tumours. This report describes a case of amelanotic melanoma of the vagina, which was originally suspected to be a non-epithelial malignant tumour, but was subsequently correctly diagnosed by immunohistochemical staining with the HMB-45 antibody and for the S-100 protein. A light grey tumour with superficial ulceration was located in the upper third of the vagina. The patient was treated with irradiation followed by chemotherapy. Subsequently, the tumour disappeared and cytology was negative; thus, she achieved complete remission. However, 20 months after complete remission, the tumour recurred locally: the site had a grossly black appearance, which was pathognomonic for a malignant melanoma. Thus, HMB-45 and S-100 protein immunohistochemistry confirmed the diagnosis of amelanotic melanoma.     

溃变纤维Fink-Heimer Ⅱ法镀银染色后漂白复染技术

Fink-Heiller Ⅱ法能较好地分辨溃变纤维和溃变终末,广泛它用于神经束略的追踪。但该法常因正常组织中有许多金属银颗粒不能全部去除而造成切片背景不清,又固不能区分溃变来末梢周围的细胞构筑关系而致评价溃变终末的范围造成困难。本实验将Fink-Heimer Ⅱ法染色后的切片作漂白处理,而后作焦油紫复染。结果表明,经漂白处理的切片正常组织完全漂白而使背景清晰,而黑色的溃变纤维不受影响,完全保留。经复染的切片,复染的神经细胞显示较佳,能很好地反映出溃变终末的分布范围。因而是一种较为理想的溃变染色技术。     

Immunohistochemical study of malignant melanoma

Fifty-two malignant melanoma cases were divided morphologically into round, spindle and mixed-cell types and were studied immunohistochemically on the localization and staining intensity of S-100 protein and neuron specific enolase (NSE). Most of malignant melanomas were positively stained for S-100 protein and NSE. There were no correlation between the localization of S-100 protein and three cell types. However S-100 protein and degree of melanin production seemed to have an inverse relationship. On the other hand, for NSE, there were some differences on immunostaining intensity among the three cell types. Especially, deeply invasive melanomas showed strong reactivities for NSE and it may clinicopathologically indicate their poorer prognosis.     

Melanin containing cells of the uterine cervix and a possible histogenesis--a case report

A 30 year old nulligravidfemale attended gynaecological OPD for investigation of primary infertility. Local examination revealed presence of a dark pigmented area in the posterior lip of the cervix. The biopsy from cervix showed, squamous metaplasia of the lining epithelium with presence of granules of melanin pigment in the basal layer. Schmorl's stain for melanin and immunohistochemical staining for S-100 and HMB-45 showed strong positivity in these cells. Melanosis of the uterine cervix is usually an incidental finding in females with uterine prolapse in their fifth and sixth decade. The origin of melanin containing cells in the uterine cervix is debatable till date. Amongst the various possibilities for the origin of these cells in the uterine cervix, neural origin is probably more acceptable than epithelial cell origin. The combined expression of melanocytic and Schwanian markers in the index case, suggest a biphasic differentiation of melanin containing cells in the uterine cervix. Although the exact histogenesis and clinical significance of these are still unknown, a long term follow-up is needed to study the nature of these lesions to look for any precursor lesion for development of malignant melanoma.     

IMMUNOHISTOCHEMICAL STUDY OF MALIGNANT MELANOMA

Fifty-two malignant melanoma cases were divided morphologically into round, spindle and mixed-cell types and were studied immunohistochemically on the localization and stainig intensity of S-100 protein and neuron specific enolase (NSE). Most of malignant melanomas were positively stained for S-100 protein and NSE. There were no correlation between the localization of S-100 protein and three cell types. However S-100 protein and degree of melanin production seemed to have an inverse relationship. On the other hand, for NSE, there were some differences on immunostaining intensity among the three cell types. Especially, deeply invasive melanomas showed strong reactivities for NSE and it may clinicopathologically indicate their poorer prognosis.     

Morphologic diversity in malignant melanomas

A review was conducted of 335 malignant melanomas to identify variant morphologic patterns that might be confused with other tumors. In all, 27 predominantly amelanotic neoplasms with unusual histologic features were selected for additional study. These included nine with an adenoid or pseudopapillary pattern, seven small cell neoplasms, five with prominent myxoid stroma, four with a hemangiopericytoma-like appearance, and two composed of neoplastic cells with a signet-ring configuration. A diagnosis of melanoma was confirmed in all cases by Fontana-Masson strains for melanin pigment, electron microscopic examination, or the results of immunohistochemical analyses for cytokeratin, vimentin, S-100 protein, and the HMB-45 antigen. One tumor was associated with a congenital hairy melanocytic nevus, five were vulvovaginal lesions, four arose in the sinonasal tract, and one occurred in the rectum. Four of the specified microscopic patterns were observed in both primary and secondary neoplasms; the two signet-ring cell melanomas were recurrent lesions. The authors conclude that malignant melanomas may assume the histologic guise of adenocarcinomas, small cell carcinomas, and sarcomas, in a variety of tissue sites. Special studies designed to detect melanocytic differentiation are therefore appropriate in diverse differential diagnostic settings.     

Blue nevus and melanosis of the prostate. Electron-microscopic and immunohistochemical studies

Two cases of blue nevus and one case of melanosis of the prostate were studied with ultrastructural and immunohistochemical methods. All patients complained of urinary obstruction, and the clinical impression in all was benign prostatic hyperplasia. Melanin was present in the stroma of the prostate in all cases. In one, pigment was also demonstrated both in benign and malignant epithelial cells. Electron microscopically, melanosomes in different stages were present in the two white patients, but only mature stage IV melanosomes were demonstrated in the black patient. The melanin in epithelial cells consisted only of mature melanosomes. Immunohistochemically, the stromal cells that contained melanin stained with S-100 protein. The evidence suggests that the pigmented cells in the prostatic stroma are melanocytes and the melanin in the glandular epithelium is a result of the transfer of pigment from the stromal melanocytes.     

Melanoma-associated antigens in tumours of the nervous system: an immunohistochemical study with the monoclonal antibody HMB-45

Summary The aim of this study was to determine the specificity and sensitivity of the commercially available, monoclonal anti-melanoma antibody HMB-45 in brain tumours and peripheral nerve sheath tumours. Hence, a series of 155 different non-melanotic tumours of the central and peripheral nervous system were examined immunohistochemically. The brain lesions consisted of primary tumours and metastases from various carcinomas. Twenty melanotic tumours (cerebral metastases of malignant melanomas, meningeal melanomatosis, meningeal melanocytomas) and dermal blue cell naevi served as controls. All melanotic tumours stained positive. Furthermore, a positive immunohistochemical reaction was observed in the following non-melanotic tumours: gliosarcomas, primitive neuroectodermal tumours, ependymoma, malignant schwannomas and different intracranial hamartomas. Two plasmocytomas and 4 metastatic carcinomas also revealed positive staining for HMB-45. Our results confirm the necessity for cautious interpretation of HMB-45 immunoreactivity as a tool in the immunohistochemical characterization of nervous system tumours.     

Microwave Methods for Demonstrating Melanin

Abstract

Microwave modifications of Churukian's ammoniacal silver, Lillie's melanin bleach, Lillie's ferrous ion uptake, Lillie's Nile blue A, Schmorl's for reducing substances, and the Giemsa stain are described and recommended for use in demonstrating melanin. The use of the microwave oven reduces the time required to perform these methods without compromising the quality of the staining results. In fact, the results obtained with these methods are usually better than those obtained with the conventional nonmicrowave techniques. Even though these methods are not as specific or sensitive as the monoclonal antibodies HMB-45 and melanin A and the polyclonal antibody S-100, they are still useful for demonstrating melanin in malignant melanomas. (The J Histotechnol 26:239, 2003)     

Immunohistochemical detection of melanoma-specific antigens in spontaneous canine melanoma

Twenty-five formalin-fixed, paraffin wax-embedded canine melanomas were examined immunohistochemically by an immunoperoxidase method to assess their reactivity with three human melanoma-specific monoclonal antibodies (HMB-45, MEL-1, NK1/C3). HMB-45 and MEL-1 reacted with 22/25 (88%) and 18/25 (72%) of canine melanomas, respectively, but only after microwave antigen retrieval and pretreatment with potassium permanganate and oxalic acid (KMnO(4)/OA). Positive reactivity to HMB-45 and MEL-1 was as follows: oral melanomas, 13/16 and 9/16, respectively; cutaneous melanomas, 8/8 and 8/8; melanoma of the digit, 1/1 and 1/1; all pigmented melanomas, 16/18 and 14/18; all amelanotic tumours, 6/7 and 4/7. HMB-45 immunolabelling was characterized by a diffuse granular cytoplasmic pattern within the tumour cells, and MEL-1 labelling by a cytoplasmic pattern with sporadic nuclear localization. In most tumours the labelling was homogeneous, but some showed a multifocal distribution. Generally, a higher percentage of canine melanomas was labelled by HMB-45 than by MEL-1. NK1/C3 failed to label any of seven melanomas tested, regardless of KMnO(4)/OA-pretreatment. Of 16 non-melanocytic tumours (specificity controls), 15 showed no significant reactivity with HMB-45, the exception being one of three plasmacytomas. Immunolabelling by MEL-1 on non-melanocytic tumours was not pursued, due to the poor sensitivity of this antibody as compared with that of HMB-45.     

Anorectal malignant melanoma: morphologic and immunohistochemical features

We reviewed 17 cases of primary anorectal malignant melanoma. Morphologic features evaluated included junctional change, pigmentation, morphology, and mitotic rate. Immunohistochemical stains were performed for/with S-100 protein, HMB-45, MelanA, tyrosinase, vimentin, KIT, and pankeratin. Morphologic subtypes were as follows: epithelioid, 12 cases; spindle cell, 7 cases; lymphoma-like, 10 cases; and pleomorphic, 6 cases. Pigmentation was present in 9 cases. Junctional change was present in 6 cases. The mitotic rate was 3 or more per high-power field in 8 cases. S-100 protein was present in all cases, HMB-45 stained 16 of 17, MelanA was present in 14 of 15, tyrosinase in 12 of 14, vimentin in 13 of 14, and KIT in 12 of 16. Pankeratin was absent in all cases. The mean length of follow-up was 25.6 months (range, 8-96 months), and the average survival with disease was 32.3 months (range, 8-96). No morphologic or immunohistochemical features were predictive of survival. Anorectal malignant melanoma shows considerable morphologic variability. Immunohistochemical staining is similar to cutaneous melanomas. Expression of KIT was present frequently, including cases with spindle cell morphologic features, in which it may lead to confusion with gastrointestinal stromal tumors.