HMB-45 staining for cytology of primary melanoma of the vagina. A case report

BACKGROUND: Malignant melanoma in the vagina is very rare, but its diagnosis is usually easy if a melanin pigment is present. With cytodiagnosis, however, it is difficult to differentiate amelanotic melanoma or scantily pigmented melanoma from other conditions. In the present case, monoclonal antibody HMB-45, the efficacy of which has been established in histologic studies, was used in the cytodiagnosis of amelanotic melanoma in the vagina. CASE: A woman, aged 78 years, presented with a brownish, nodular tumor, diameter 3 cm, in the vagina. Scraping smears with Papanicolaou staining showed nonepithelial malignant cells without granules suggesting melanin. Smears stained with HMB-45 showed positive immunoreactivity. The diagnosis underwent histologic confirmation of amelanotic melanoma on the initial biopsy. CONCLUSION: Cytodiagnosis was made with HMB-45, which proved very effective in the differential cytodiagnosis of amelanotic melanoma and scantily pigmented melanoma, particularly because it obviated the need for tissue invasion.     

Usefulness of silver intensification of immunostaining for cytologic diagnosis of primary melanoma of the female genital organs

OBJECTIVE: To improve the procedure for diagnosing vaginal melanoma with cytopathologic analysis of HMB-45. STUDY DESIGN: The study examined silver intensification of immunostaining of HMB-45 in nine cases of primary melanoma of the vagina and vulva using archival Papanicolaou-stained smears. RESULTS: All nine samples showed positive staining for HMB-45. Five cases showed intensive staining, two moderate and two weak. The positive staining was black in the cytoplasm of melanoma cells but was detected in neither the background nor normal squamous cells. Though destaining of Papanicolaou stain was not performed before immunostaining, the positivity of immunostaining was easily judged. CONCLUSION: After morphologic observation, immunocytochemical study of HMB-45 is possible even though time has passed since the cytologic specimen was obtained. When there is a suspicion of amelanotic melanoma or scantily pigmented melanoma of the vagina and vulva, cytogenesis with HMB-45 is helpful, especially because it involves little invasion.     

Bowes human melanoma cell line. An immunocytochemical study

Bowes human melanoma cell line was investigated immunocytochemically to localize S-100 protein, HMB-45 and intermediate filament proteins. The majority of the cells showed strong positive staining with antibodies directed against S-100 protein, HMB-45 and vimentin filaments. Antibodies directed against the other cytoskeletal proteins failed to show any reactivity. These findings suggest that this transformed cell line does not dedifferentiate in culture and continues to express the specific antigens of human melanoma cells. Thus, Bowes cell line provides a useful model for studying the cellular biology and pathology of malignant melanoma.     

Inhibition of human tumor growth by IgG2A monoclonal antibodies correlates with antibody density on tumor cells

Eight monoclonal antibodies (MAb) of IgG2a isotype that were produced against human melanomas were tested for tumor growth-inhibiting properties in nude mice injected with human melanoma cells of various origins. Four of the eight MAb inhibited growth of these tumors, and all four of these antibodies reacted in antibody-dependent macrophage-mediated cytotoxicity (ADMC) assays in vitro. The MAb that were inactive in vivo also did not react in these assays in vitro. The number of antibody-binding sites per cell on the tumor cell surface was significantly higher for tumoricidal MAb as compared to unreactive MAb. On the other hand, the percentage of tumor cells binding the MAb and the binding affinity to these cells were the same for the two groups of MAb. Also, tumoricidal and nontumoricidal MAb bound with similar affinity and antibody density to Fc receptors on macrophages. The importance of the number of antibody sites on the tumor cell surface for tumor destruction by MAb was confirmed by the demonstration of tumoricidal effects of mixtures of MAb that were by themselves not tumoricidal. MAb binding to different molecules on melanoma cells were complementary in ADMC, whereas MAb directed to the same molecule but to different epitopes were not.     

Amelanotic malignant melanoma arising in an ovarian cystic teratoma: a case report

BACKGROUND: Malignant melanoma arising in a cystic teratoma is extremely rare. We report the clinicopathologic and cytopathologic features of an amelanotic malignant melanoma arising in an ovarian cystic teratoma. CASE: A 55-year-old woman presented with an asymptomatic right ovarian mass, showing features of cystic teratoma according to preoperative computed tomography and magnetic resonance imaging. The resected teratoma was suspected to include a nonepithelial malignancy in a touch preparation from a solid component. The tumor showed immunoreactivity for Melan-A, S-100 and HMB-45 in the absence of melanin granules, which established the diagnosis of amelanotic malignant melanoma arising in an ovarian cystic teratoma. CONCLUSION: Cytopathologic findings from touch preparations and immunohistochemical staining are useful for the diagnosis of amelanotic malignant melanoma arising in an ovarian cystic teratoma.     

Cytologic detection of penile malignant melanoma of soft parts in pleural effusion using monoclonal antibody HMB-45

A case of penile malignant melanoma of soft parts ("clear cell sarcoma") with pulmonary metastases and malignant effusions is reported. The tumor cells in the pleural effusion were scattered singly and admixed with reactive mesothelial cells. They had abundant granular cytoplasm and round nuclei with prominent nucleoli. Although staining for S-100 protein was positive in sections from the penile lesion, it was negative in sections of a cell block prepared from the effusion; however, the effusion tumor cells demonstrated immunoreactivity with HMB-45, an antimelanoma monoclonal antibody.     

Metastatic balloon cell melanoma: a case report

BACKGROUND: The protean morphology of malignant melanoma is diagnostically challenging. Balloon cell melanoma is a histologic variant composed predominantly or entirely of large cells with abundant, vacuolated cytoplasm. It shares the cytologic features of the other subcategories of malignant melanoma, such as discohesion, nuclear pleomorphism and intranuclear cytoplasmic pseudoinclusions, but generally lacks melanin pigment and, as the name would suggest, is characterized by the presence of numerous cytoplasmic vacuoles. CASE: A 55-year-old man presented with an enlarged right cervical lymph node. Clinically and radiographically this mass was considered to be metastatic; however, the patient had no known primary neoplasm. Fine needle aspiration biopsy (FNAB) and cytologic examination showed numerous discohesive, variably sized, malignant cells with abundant, vacuolated cytoplasm and pleomorphic nuclei with irregular nuclear contours, macronucleoli and frequent intranuclear cytoplasmic pseudoinclusions. Pigment was not identified. These features, along with strong immunohistochemical positivity for S-100, HMB-45 and Melan-A, suggested the diagnosis of balloon cell melanoma. A right parotidectomy and lymph node dissection were performed, and histologic tissue evaluation confirmed the diagnosis. CONCLUSION: This case of lymph node balloon cell melanoma metastasis was diagnosed by FNAB.     

Identification of a structure on human melanoma cells recognized by CTL exhibiting anomalous killer cell function

The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.     

Comparative histopathology of grey-horse-melanoma and human malignant melanoma

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.     

A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection

Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC(50)], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.     

Therapeutic Use of Antimelanoma Monoclonal Antibodies

Monoclonal antibodies (MAb) to tumor-associated antigens are attracting much attention for tumor therapy. Melanomas belong to the tumors most studied in this respect, and several melanoma-associated antigens have been studied in great detail. These include the melanoma-associated glycoprotein p97, the melanoma-associated proteoglycan, and glycolipid antigens. Although none of the antigens is absolutely specific for tumor, the degree of relative specificity appears to be sufficient to use several of the melanoma antigens as therapeutic “targets”. Antimelanoma MAb can be applied therapeutically in several ways. The most straightforward approach is use of MAb without further modification. MAb which kill melanoma cells in the presence of human serum as the source of complement or mediate antibody-dependent cellular cytotoxicity with human natural killer (NK) cells or macrophages as effectors are logical choices for this. Some cases of partial or even complete regression of metastatic melanoma have been observed in patients treated with such MAb. Combinations of such MAb with interleukin 2 (IL-2) or other immunological response modifiers are of great interest. Alternatively, one may use antimelanoma MAb (or fragments prepared from MAb) as carriers of antitumor agents, including radioactive isotopes, toxins, or chemotherapeutic drugs. Although it is premature to make any conclusions about the efficacy of such conjugates, we are optimistic that it will be feasible by using the right combination of MAb and antitumor agent to achieve therapeutic benefit. Another approach is to develop therapeutic “vaccines” for active immunization, once an antigen characterized by using a MAb has proven to have a relatively high level of tumor selectively. Anti-idiotypic antibodies and live recombinant viruses inducing tumor antigen expression in infected cells provide alternative strategies to this approach.     

Preparation and characterization of immunoconjugates for antibody-targeted photolysis.

Monoclonal antibody (MAb)-dextran-tin(IV) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fc oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDS-PAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)     

Melanoma metastatic to the breast: utility of fine needle aspiration and immunohistochemistry

OBJECTIVE: To describe the morphologic spectrum of metastatic malignant melanoma (MM) cells involving the breast and to explore the diagnostic utility of HMB45, Mart-1, Melan-A and T311 (antityrosinase) antibodies in fine needle aspiration material of MM metastatic to the breast. STUDY DESIGN: Cytologic material from 21 cases (18 women) was reviewed for cytomorphology (epithelioid, spindled, mixed) and immunocytochemical staining attributes for Mart-1, HMB45, T311, Melan-A and cytokeratin based on tissue availability. RESULTS: Seventeen cases (81%) demonstrated epithelioid cell morphology, with 14% exhibiting mixed and 5% spindled morphologies. All 21 cases (100%) were immunoreactive with Mart-1 antibody, with 81% (17/21) immunoreactive for HMB45. In 38% of cases there was a similar percentage of cells immunoreactive for Mart-1 and HMB45, while 48% showed a higher percentage of cells immunoreactive for MART-1 than HMB45. Immunoreactivity with T311 was seen in 8 of 11 cases tested (73%). All six cases tested (100%) were immunoreactive with Melan-A. Staining for cytokeratin was negative in all eight cases tested. CONCLUSION: Because the majority of MM metastatic to the breast shows epithelioid cell morphology, it may mimic primary breast carcinoma. Mart-1 should be part of the immunocytochemical panel utilized to confirm the diagnosis of MM metastatic to the breast.     

Preclinical and phase I studies of monoclonal antibodies in melanoma: application to boron neutron capture therapy of melanoma

Monoclonal antibodies (MAbs) provide an attractive method of selectively localizing sufficient boron atoms around tumour cells to capture neutrons. Assuming that 10(8)-10(10) 10B atoms are needed for one capture event and that 10(3)-10(4) atoms can be coupled to each antibody molecule, then 10(5)-10(6) antibody molecules gathered on an individual cell will destroy that cell. Binding to normal tissues, on the other hand, would need to be at least 20-fold less than that to tumour tissues to avoid toxic effects of neutrons on surrounding tissues. Preclinical studies in animals show that several MAbs may bind to melanoma cells in sufficient quantities in vitro to localize the required amount of Boron per cell. Whether this will occur in vivo, however, may depend not only on antigen density but a variety of other properties of the tumour cells and MAbs. These include the Ig class and affinity of the antibody and whether the antibody is internalized into the tumour cell. The ratio of uptake between tumour and normal tissue is governed by such factors as the percentage of tumour cells within a tumour expressing the antigen and whether the MAb react with normal tissues. Use of Fab or F(ab)2 preparations of the MAb may increase the uptake ratio by preventing uptake of MAb by cells with Fc receptors. In contrast to preclinical animal studies, tumour/normal tissue uptake ratios in phase I studies in humans have been disappointingly low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EGF) receptor, the type I insulin-like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti-EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell-cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin- and IGF-I-induced growth stimulation of melanoma cells was inhibited by MAb alpha IR-3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF-I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.     

Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.     

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.