Monoclonal Antibody HMB-45 Research Articles

Markers of cell proliferation and expression of melanosomal antigen in lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM), a disease of young women, is characterized by proliferation of immature-appearing smooth-muscle cells (LAM cells) in the lungs and abdomen. LAM cells react with monoclonal antibody HMB45, which recognizes a 100-kD glycoprotein (gp100) originally found in human melanoma cells. We investigated the expression and the subcellular localization of gp100 in lung tissue from patients with LAM and in human melanoma cell lines (Malme-3M, A2058, and CHL-1), and the relationship between this expression and cellular proliferation. Binding sites for HMB45 antibody in melanoma and LAM cells were located in cytoplasmic granules resembling immature melanosomes. LAM cells reactive for proliferating-cell nuclear antigen (PCNA), a marker of cellular proliferation, were spindle-shaped, in contrast to the large, epithelioid cells reacting with HMB45 antibody. In accord with this finding, we observed an inverse relationship between the immunostaining for HMB45 antibody and PCNA in LAM and melanoma cells. Thus, LAM and melanoma cells are heterogeneous with respect to their stages of proliferation and their expression of melanoma antigens. PCNA-positive cells, which are more likely to be negative for reactivity with HMB45 antibody, may be more relevant to the progression of LAM than are HMB45-positive cells, which are the hallmark of LAM.

Morphologic and immunocytochemical characterization of four human uveal cell lines (melanoma- and melanocytes-derived)

PURPOSE. To characterize three cell lines from human uveal melanomas and one ocular melanocyte cell line to study the specificity of several antigens in the malignant transformation of melanocytic uveal cells. METHODS. Light microscopy (LM), transmission electron microscopy (TEM), and immunocytochemical techniques were used in the characterization of OCM-1, SP 6.5, and MKT-BR human uveal melanoma cell lines and UW-1 normal melanocyte cell line from human uvea. Several monoclonal antibodies (MoAbs) S-100, HMB-45, MNF-116, PAL-M1, NK1/C-3, IND-1, and MAAMA were used. RESULTS. All cell lines showed an epithelioid/spindle morphology with occasional multinucleated cells, and nuclear pleomorphism. TEM showed intracytoplasmatic premelanosomes. Incubation with HMB-45 MoAb was positive in all cell lines. PAL-M2, NK1/C-3, MAAMA, and IND-1 MoAbs stainings were positive with variable intensity. MNF-116 MoAb showed negative staining in the four lines, and S-100 MoAb was also negative except for the UW-1 cell line. CONCLUSIONS. Human uveal melanoma cell lines OCM-1, SP 6.5, and MKT-BR and the ocular melanocyte cell line UW-1 exhibited maintenance of some structural and ultrastructural characteristics of melanocytic cells. All four MoAbs, PAL-M2, NK1/C3, IND-1, and MAAMA against cutaneous melanoma-associated antigens stained positively all melanoma cell lines as well as the melanocytic cell line, suggesting that in vitro proliferation of melanocytes could modify their antigenic expression.

Anti-melanoma monoclonal antibody HMB-45 on enhanced chemiluminescence-western blotting recognizes a 30-35 kDa melanosome-associated sialated glycoprotein.

HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.

PMel17 is recognised by monoclonal antibodies NKI-beteb, HMB-45 and HMB-50 and by anti-melanoma CTL.

Recently, we cloned the cDNA encoding the melanocyte lineage-specific antigen gp100 and demonstrated that gp100 is recognised by three different monoclonal antibodies (MAbs) used to diagnose malignant melanoma. In addition, we showed that tumour-infiltrating lymphocytes (TIL 1200) from a melanoma patient reacted specifically with cells transfected with the gp100 cDNA. Molecular characterisation of the gp100 cDNA revealed that the gp100 antigen is highly homologous, but not identical, to another melanocyte-specific protein, pMel17. Here, we report that cells transfected with pMel17 cDNA also react with all three MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-45 and HMB-50. Moreover, pMel17 transfectants are specifically lysed by TIL1200. These data demonstrate that antigenic processing of both gp100 and pMel17 give rise to peptides seen by anti-melanoma cytotoxic T lymphocytes (CTL) and are therefore potential targets for immunotherapy of malignant melanoma.

A CASE OF ANORECTAL AMELANOTIC MALIGNANT MELANOMA

Anorectal amelanotic malignant melanoma is an extremely rare. There have been only 17 cases in the Japanese literature so far. We report a case of anorectal amelanatic malignant melanoma. A 58-year-old woman was seen at the hospital because of anal bleeding and anal pain. An elastic soft and elevated mass was palpated in the anal canal. The patient was diagnosed as the desease by histolagical examination of biopsy specimen which was stained by hematoxylin and eosin. Abdominoperitoneal resection was performed. The resected specimen showed a light grey tumor resembling Borrmann I type, sized 6.5×3.5×2.2cm just above the dentate line. The tumor stained positive for S-100 protein and HMB-45 monoclonal antibody. Adjuvant chemotherapy with dacarbazine, cisplatine, tamoxifen was done, but the patient died of cachexia seven months after the operation. To imorove the prognosis of the desease, we would emphasize the importance of preoperative diagnosis, especially our surgion's careful attitude to suspect the disease based on macroscopic findings.

Human recombinant stem-cell factor induces melanocytic hyperplasia in susceptible patients

Background: Recombinant human stem-cell factor (SCF), a cytokine acting on hematopoietic progenitor cells, has potential for the treatment of several hematologic and oncologic disorders. In a hematology-oncology phase I trial of SCF, several patients had cutaneous hyperpigmentation at the SCF subcutaneous injection sites. Objective: Our purpose was to investigate the pathogenesis of this hyperpigmentation phenomenon. Methods: Skin biopsy specimens were obtained before, at the completion of, and after SCF therapy and were processed for histology, immunohistology, and electron microscopy. Results: Skin at the site of SCF injection had an increased number of melanocytes, increased melanocytic dendrite extension, and melanin as compared with noninjected tissue. Immunohistochemical stains revealed an increase in staining with melanocyte-specific monoclonal antibodies HMB-45 and NKI/beteb, and a monoclonal antibody to the receptor for SCF, c-kit. Conclusion: Subcutaneous injection of SCF results in hyperplasia of melanocytes. SCF may be useful in the treatment of melanocytopenic disorders, but caution may be necessary in patients with disorders of melanocyte proliferation.

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

Solitary Renal Melanoma? A Case with Long Survival After Initial Treatment

A case of a solitary melanocytic renal tumor with no apparent primary lesion or metastasis is reported. Pathologically the tumor was composed of cells with brown pigmentation that strongly reacted to monoclonal antibody HMB-45 immunohistochemically. Few mitotic figures, mild nuclear atypia and nucleolar prominence were observed. These findings confirmed that the tumor cells were melanogenetic but the malignant potential of the tumor was low in comparison to that of typical malignant melanoma. The patient is doing well 44 months after partial resection of the renal tumor and postoperative chemotherapy. The interesting pathological feature may account for the unique clinical course.

HMB45: a specific marker for melanoma metastases in the central nervous system?

The monoclonal antibody HMB45 is used to detect an epitope specific for melanocytes, malignant melanomas and melanoma metastases. Using the PAP method, we observed consistent expression of HMB45 in 19 metastases of melanotic and amelanotic malignant melanomas of the central nervous system, while metastases of 32 adenocarcinomas, 10 squamous cell and 8 small cell carcinomas were negative except for 2 cases of breast cancer. Differential diagnosis between cancer and melanoma metastases can be made using cytokeratins as an additional immunocytochemical marker protein. Ten meningeomas and 5 pineocytomas were also negative. Even though it is not absolutely specific, we consider the HMB45 immunoreaction diagnostic for a metastasis of a malignant melanoma if the tumour is cytokeratin negative and HMB45 positive in a large number of tumour cells.

Characterization of nail matrix melanocytes with anti-PEP1, anti-PEP8, TMH-1, and HMB-45 antibodies.

The normal nail matrix contains quiescent melanocytes with a peculiar arrangement and behavior. Our purpose was to identify nail matrix melanocytes with antibodies that recognize melanocytic cells in tissue sections. We used the polyclonal antibodies anti-PEP1 and anti-PEP8 and the monoclonal antibody TMH-1, which recognize melanocytic enzymes, and the monoclonal antibody HMB-45, which reacts with melanoma cells and fetal melanocytes, but not with normal adult melanocytes. Nail matrix specimens were obtained from longitudinal specimens of eight white patients with ingrown toenails. Specimens from normal adult forearm skin were used as controls. All nail specimens gave similar results. Dendritic melanocytes were more numerous in the distal than in the proximal nail matrix. They were not restricted to the basal layer, but were also found in the suprabasal layers of the nail matrix epithelium. Melanocytes were seen both a single dendritic cells among the nail matrix keratinocytes and as small clusters that appeared irregularly distributed along the length of the nail matrix. Each cluster usually consisted of three to four cells. Even if normally quiescent, nail matrix melanocytes possess the key enzymes responsible for the formation of melanin. The suprabasal location of nail matrix melanocytes may be a consequence of the distribution of adhesion molecules in the nail epithelium. In fact, in the nail matrix alpha 2, alpha 3, and beta 1 integrins are not only expressed on the basal, but also on the fourth to fifth suprabasal layers, with suprabasal expression gradually decreasing from distal to proximal matrix. The behavior of nail matrix keratinocytes may cause the peculiar arrangement and behavior of nail matrix melanocytes.

Margin control for lentigo maligna.

Lentigo maligna is an in situ malignant melanoma for which the treatment of choice is surgical excision. The current recommendation is local resection with a 0.5 to 1.0 cm margin of normal skin. Because many lesions occur on the face, the narrowest possible margin reduces the amount of scarring. Controversy surrounds the use of Mohs micrographic surgery to preserve normal skin and resect the lentigo maligna. The purposes of this prospective study were to determine the narrowest possible margin of resection of lentigo maligna and the accuracy of frozen and fixed histologic specimens from those margins. In addition, the benefit of adjunctive immunoperoxidase staining with antibodies to S-100 protein and HMB-45 monoclonal antibody was examined retrospectively. A Wood's light was used to delineate the clinical margin in 16 cases of lentigo maligna that were resected with serial excisions 0.3, 0.6, 1.0, and 1.3 cm from the clinical border of the tumor. Frozen sections were confirmed by fixed histopathologic specimens. Subsequently these tissue blocks were examined with antibodies to S-100 protein and HMB-45 monoclonal antibodies. Patients were observed 5 to 9 years. One of the 16 patients had a recurrence 8 years after surgery. Although lesions with a diameter less than 2.0 cm had narrower margins of resection, the majority of lesions were resected with a margin of 0.6 to 1.0 cm. Lesions larger than 3.0 cm in diameter required a margin of resection greater than 1.0 cm. The antibody to S-100 protein was neither sensitive nor specific enough to assist with identification of the process. HMB-45 monoclonal antibody was sensitive and assisted in the identification of atypical melanocytes. The modifications of Mohs micrographic surgery including the use of fixed histopathologic specimens and the use of HMB-45 monoclonal antibody to help delineate atypical melanocytes offer the possibility of narrower margins of resection for lentigo maligna.

Immunocytochemical diagnosis of amelanotic metastatic melanoma using monoclonal antibodies HMB-45 and Ep1-3

The analysis of fine-needle aspirates and effusions has proved to be a valuable tool for the cytological identification of metastatic melanoma. Nevertheless, while malignant pigmented cells can be easily detected on cytological specimens, their identification may be very difficult in patients bearing amelanotic lesions. In the present study we have evaluated whether this limitation can be overcome using two monoclonal antibodies (mAbs) HMB45 and Ep1-3, which are highly specific for the melanocyte lineage. These reagents were assayed in immunocytochemical tests performed on cytological material obtained from 50 patients with a past history of melanoma and 463 bearing metastases from a cryptic primary tumour. These mAbs, employed in combination with a panel of monoclonal reagents with well-defined tumour specificity, may improve the accuracy of the conventional cytopathological diagnosis of melanoma metastases in the two groups of patients.

HMB-45 Staining in Angiomyolipoma, Cardiac Rhabdomyoma, Other Mesenchymal Processes, and Tuberous Sclerosis-associated Brain Lesions

HMB-45 monoclonal antibody is thought to be highly specific for melanocytic prolifer ations. In a recent study of two cases of hepatic and one case of renal angiomyolipoma, strong staining of lesional cells with this antibody was unexpectedly encountered. In follow-up evaluation, the authors studied 51 additional soft tissue, renal, and brain tumors and tumor-like processes, with emphasis on those with prominent vascular, myogenous, and fatty components or those known to be associated with tuberous sclerosis. Varying degrees of staining with HMB-45 were seen in 17 of the lesions tested. Strong staining with HMB-45 was demonstrated in four renal angiomyolipo mas. Clearly positive results were also obtained in three other processes associated with the tuberous sclerosis complex, including two cardiac rhabdomyomas and one tuberous lesion of the brain. Weak staining of several other mesenchymal and neural lesions was also observed. Electron microscopic studies showed a spectrum of granules within angiomyolipoma, some of which resembled melanosomes and others renin granules. Similar granules were observed in cardiac rhabdomyoma, cerebral tuberous lesions, and in subependymal giant cell astrocytoma. We propose that tuberous scleros is-associated tumors and tumor-like processes are interrelated, and that these and perhaps other mesenchymal proliferations may share components previously believed to be unique to melanocytic lesions. Int J Surg Pathol 1 (3):191-198, 1994

A Case of Superficial Spreading Melanoma In situ 2.5 mm in Diameter

A 60-year-old woman with superficial spreading melanoma in situ, measuring 2.5 mm in diameter, was examined. She had noticed a very small pigmented lesion 1.2 mm in diameter on her left lower leg in April of 1989. By April of 1990, it had grown to 2.5 mm in diameter. Its edge was irregular, and its color was variegated black to brown. Skin surface markings had disappeared in the center portion. Histopathologically, the lesion was asymmetrical. Atypical large cells nested in the lower epidermis and were scattered singly in the mid and upper epidermis, as seen in Paget's disease. At the periphery of the lesion, single large tumor cells were scattered in the mid epidermis. The tumor cells reacted to monoclonal anti-melanoma antibody HMB-45.

Renal angiomyolipomas and HMB-45 reactivity.

Renal angiomyolipomas (RAML) are mesenchymal hamartomas composed of varying amounts of blood vessels, smooth muscle, adipose tissue, and supporting connective tissue. Although most of these tumors are easy to recognize, some show unusual histologic features and may pose a diagnostic dilemma. Recent reports indicate that RAML are immunoreactive for HMB-45 antibody, which is directed against a premelanomasome-associated glycoprotein and is thought to be specific for melanocytic differentiation. To determine whether HMB-45 reactivity would differentiate RAML from other renal tumors, the authors immunostained 72 primary renal tumors, including 19 angiomyolipomas and 2 retroperitoneal liposarcomas extending into the kidney, with HMB-45 monoclonal antibody. Also, the immunohistochemical profile of 19 renal angiomyolipomas was investigated using a broad panel of immunostains, including cytokeratin (AE1/AE3), epithelial membrane antigen, vimentin, smooth muscle actin (SMA), desmin, muscle-specific actin (MSA), S-100 protein, and neuron-specific enolase (NSE). All tumors except RAML were negative for HMB-45 antibody. HMB-45 immunoreactivity was present in 17 of 19 RAML. Seventeen of 19 were positive for SMA and MSA (HHF 35), 8 for desmin, 17 for vimentin, and 9 for NSE. Based on this study, it was concluded that, along with SMA and MSA, HMB-45 reactivity is a useful tool to distinguish RAML, especially those with unusual morphological features, from other primary renal neoplasms, including liposarcomas that extend into the kidney.

Labial melanotic macule: A clinical, histopathologic, and ultrastructural study

The labial melanotic macule (LMM) is a recently described pigmentary anomaly that may simulate malignant melanoma. Our purpose was to define the LMM clinically, histologically, and immunohistochemically in a large group of patients. We describe the clinical features of 36 LMMs in 29 patients (aged from 4 to 79 years, 4 male, 25 female) seen during the past 4 years. Histopathologic findings in 21 of these patients are discussed. Seventeen lesions were immunostained with HMB-45 monoclonal antibody, and electron microscopy was performed on eight lesions. The majority of patients were women and had solitary lesions on the lower lip with the mean age of onset of 30 years. Histologically prominent basilar hyperpigmentation accentuated at the tips of the rete ridges was present without atypia or nevoid formation. Immunohistochemical studies showed that all intralesional melanocytes were HMB-45 negative, supporting their benign nature. Ultrastructurally, numerous stage III and IV melanosomes clustered within basal keratinocytes and papillary dermal melanophages were found. The LMM is a clinically and histologically distinctive benign pigmentary anomaly.

RELIABILITY OF A PRIMARY CULTURE SYSTEM TO TEST CYTOTOXIC DRUG ACTIVITY IN HUMAN-MALIGNANT MELANOMA

Optimal conditions have been defined to grow primary cultures of malignant melanoma suitable for in vitro pharmacological studies. A feasibility of primary cultures was observed in 60% of 62 clinical melanoma lymph node metastases. The neoplastic nature of the cells grown in culture, as assessed by the highly specific monoclonal antibodies anti-S100 and HMB45, was confirmed in 100% and 65% of the cases, respectively. Flow cytometric analysis showed a high stability of DNA content profiles observed in clinical samples through all the methodologic steps used to obtain in vitro cultures. The intertumor variability of the degree of the antiproliferative effect of melphalan and its relation with DNA interstrand cross-links (DNA ISC) provided preliminary evidence of the reliability of the experimental system for in vitro evaluation of anticancer drug activity.

Anti-melanoma monoclonal antibody HMB45 identifies an oncofetal glycoconjugate associated with immature melanosomes.

The anti-melanoma monoclonal antibody HMB45 is widely used in diagnostic pathology owing to its great specificity and sensitivity in identifying pigmented tumors such as malignant melanoma. However, little is known regarding the nature of the antigen(s) recognized by this antibody. In the observations reported here, the HMB45-defined antigen was identified in another pigmented tissue, the retinal pigment epithelium (RPE). A series of immunocytochemical studies demonstrated transient reactivity of the prenatal and infantile human RPE with antibody HMB45; adult RPE is non-reactive with the antibody. By immunoelectron microscopy, the antibody was demonstrated to react with immature melanosomes. Pre-treatment of deparaffinized tissue sections with neuraminidase completely eliminated HMB45 immunoreactivity, suggesting that the antigen(s) recognized is a sialated glycoconjugate. Mannosidase or N-acetylglucosaminidase pre-treatment had no effect on immunoreactivity. Thus, HMB45 may identify an oncofetal antigen present in cutaneous melanocytes, RPE, and melanoma cells, and changes in immunoreactivity with maturation or malignant transformation may be a function of post-translational modification.

Epithelial and melanoma antigens in gliosarcoma: An immunohistochemical study

Gliosarcomas are mixed tumors with malignant glial and mesenchymal elements. The number of GFAP-positive tumor cells decreases with the increase of sarcomatous components, until whole areas may be GFAP negative. These distinct differentiations may, however, lead to false interpretations in small tissue samples. In this connection, it is of interest that, according to other reports, glial tumors may be positive for different anti-keratin antibodies and this prompted us to undertake a systematic investigation of the immunoreactivity of gliosarcomas using a panel of well-characterized monoclonal antibodies against cytokeratins (KL1, AE 1/3, Lu-5, CK-19, CK MNF 116 and Ma-903). These cases were further studied with the anti-epithelial non-cytokeratin antibodies EMA, HEA 125, Ber-EP4, CEA as well as the melanoma-antibody HMB-45, Leu-M1, GFAP and vimentin. As screening study we examined 20 cerebral metastatic carcinomas, 21 malignant gliomas (including 6 gliosarcomas) and 3 metastatic melanomas with the monoclonal antibodies KL1 and HMB-45. All cerebral metastatic carcinomas and 4/6 gliosarcomas were positive for KL1, whereas all melanomas, 2 metastatic carcinomas and 3 gliosarcomas showed an immunostaining with HMB-45. All gliosarcomas were positive with at least one of the tested anti-cytokeratin antibodies. The gliosarcomas did not show an immunoreaction in any of the cases when CEA, HEA 125, Ber-EP4, EMA or Leu M1 were applied. In our opinion, the monoclonal antibodies HEA 125 and Ber-EP4 could obviously be helpful in differentiating gliosarcomas from metastatic carcinomas.

HMB‐45 staining of dysplastic melanocytic nevi in melanoma risk groups

To evaluate the hypothesis that reactivity of the intradermal component of melanocytic nevi to the monoclonal antibody HMB-45 correlates with melanoma risk, dysplastic compound melanocytic nevi were examined for expression of the HMB-45 epitope in three subject groups differing in epidemiological risk for melanoma. The study groups consisted of 10 subjects with dysplastic nevi and a personal history of melanoma, 25 subjects with dysplastic nevi and a history of melanoma in one or more first degree relatives, and 15 population control subjects with sporadic dysplastic nevi. For each case, sections from one lesion, immunohistochemically processed for HMB-45 binding, were evaluated by two pathologists without knowledge of the clinical data. Of all dysplastic nevi, 98% showed diffusely positive cytoplasmic staining of the junctional nevomelanocytes and 90% had such positive staining of those cells within the superficial dermis. Nevus cells within the deeper dermis did not stain positively in any case. Furthermore, the data showed no differences in frequency, pattern, or intensity of HMB-45 reactivity between the subject groups. These observations indicate that evaluation of dysplastic nevi with the monoclonal antibody HMB-45, an apparent marker of proliferative or otherwise stimulated melanocytes, has no discriminating value for identifying subjects at increased historical risk for melanoma. The data, however, support the concept that so-called dysplasia within nevi, as defined by histologic criteria, actually represents the active or proliferative phase of melanocytic nevi.