Sources/Clones
Axcel/Accurate, Biodesign, Biogenesis, Biogenex, Dako, Enzo and Immunotech.
Fixation/Preparation
The antibody is immunoreactive in paraffin-embedded tissue as well as frozen sections. Immunoreactivity is stronger in ethanol-fixed tissues than following formalin fixation, with immunoreactivity diminishing significantly following prolonged fixation in the latter. Sensitivity is enhanced following heat-induced antigen retrieval. Mercury-based fixatives result in a high degree of non-specific staining.
Background
The HMB-45 monoclonal antibody was generated to a whole-cell extract of a heavily pigmented lymph node deposit of human melanoma and has been shown to be a highly specific and sensitive reagent for the identification of melanoma. The designation HMB is derived from the immunogen employed, i.e. Human Melanoma, Black. The antigen is intracytoplasmic and ultrastructural studies suggest that the antibody reacts with melanosomes before melanin deposition with HMB-45 binding to stage 1 and 2 melanosomes and to the non-melanized portion of stage 3, whereas stage 4 melanosomes and melanosome complexes found in macrophages and keratinocytes have been negative. The antibody appears to label premature and immature melanosomes in retinal pigment epithelium from fetuses and neonates but not from adults, leading to the suggestion that this "oncofetal" pattern of expression may indicate a role in melanocytic cell proliferation. This thesis has not been confirmed and the sequential expression of the HMB-45 antigen in melanocytes may relate to the activation by specific growth factors, resulting in alterations in protein glycosylation during various ontogenic and pathologic states of melanocytes. The epitope recognized by HMB-45 appears to be, in part, the oligosaccharide side chain of a sialated glycoconjugate as the immunoreactivity can be abolished with neuraminidase treatment (Bacchi et al, 1996).
The gene corresponding to the HMB-45 defined proteins has recently been cloned and designated gp 100-cl. This gene encodes the melanocyte lineage-specific antigens recognized by HMB-45 and HMB-50 (one of two other monoclonal antibodies to melanocytes initially obtained with HMB-45) as well as another monoclonal antibody, NKI-beteb. These three antibodies appear to recognize different epitopes of the same antigen, the melanosomal matrix protein or pmel 17 gene product defined by them being apparently related by differential splicing.
Applications
Immunoreactivity for HMB-45 is seen in normal fetal and neonatal melanocytes but not in adult resting melanocytes. Reactive or proliferating melanocytes in inflamed adult skin, wound healing, increased vascularity and in skin overlying certain dermal neoplasms may label for HMB-45 as a result of activation and stimulation by growth factors and "reexpression" of the antigen. HMB-45+ melanocytes have been demonstrated in the anal squamous zone and transitional zone but not in the colorectal zone. Increased numbers of such melanocytes can be present adjacent to primary anal melanomas.
The staining for HMB-45 in melanocytic nevi depends on their location within the skin. Junctional nevi and the junctional components of compound nevi are HMB-45+. In contrast, intradermal nevi and the dermal components of compound nevi are consistently negative. Thus, HMB-45 does not provide distinction between benign and malignant melanocytic proliferations and the difference in reactivity supports the concept, based on differences in morphology, enzyme activity and other immunological reactivity, that junctional and dermal cells are not identical. Junctional nevus cells are in an activated or proliferative state compared to their quiescent dermal counterparts and their immunoreactivity with HMB-45 is analogous to the proliferating fetal melanocytes which are positive for the antigen whilst quiescent, adult melanocytes are non-reactive.
Dysplastic nevi, in contrast, usually express HMB-45 in both the junctional nevus cells as well as in the dysplastic cells in the superficial dermis. Nevus cells within the deeper dermis do not usually react with HMB-45. In one study, minimally dysplastic nevi displayed intense immunolabeling of the junctional melanocytes but no staining of dermal nevus cells whereas, with the moderately and severely dysplastic nevi, the dermal melanocytes showed focal cytoplasmic immunoreactivity. The likelihood of expression of HMB-45 paralleled the degree of dysplasia of the nevi. Common blue nevi and cellular blue nevi are generally HMB-45+, as are malignant blue nevi. Other nevi such as spindle and epithelioid cell nevi, congenital nevi and other nevi occurring in hormonally reactive sites show immunostaining in nevus cells in the deep dermis as well as those near the dermoepidermal junction. Less common benign melanocytic proliferations such as plexiform spindle cell nevi, Spitz nevi and atypical melanocytic hyperplasias are also HMB-45+ (Bacchi et al, 1996).
Malignant melanoma show strong cytoplasmic positivity for HMB-45 in the majority of cases (65-95%), with the proportion of positive tumor cells ranging from a few to 100%. When the expression of the antigen is weak, staining may appear as a fine granularity similar to that seen in cytologic preparations. The positivity for HMB-45 is seen in almost all types of primary and metastatic melanoma including amelanotic melanoma, spindle cell melanoma and acral lentiginous melanoma (Appendix 1.10). One important exception is desmoplastic malignant melanoma, which consistently displays a much lower rate of positivity and may be completely negative. When positive, reactivity is usually seen in the superficial epithelioid cell rather than the dermal spindle cells, which only rarely stain for HMB-45 (Leong & Milios, 1989).
Attesting to the specificity of the antigen, HMB-45 reactivity has been demonstrated in malignant melanomas of diverse morphology such as signet ring melanoma, myxoid melanoma, small cell melanoma, balloon cell melanoma and in melanomas of different anatomic sites such as the gallbladder, urinary bladder, anorectal region, vulva, sinonasal region, uterine cervix, other mucosal sites and bone. Melanomas and melanocytic proliferations occurring in complex tumors such as pulmonary blastoma have also been HMB-45+ (Ordonez et al, 1988; Bacchi et al, 1996).
HMB-45 staining also has application in the separation of melanin-containing macrophages from melanoma cells, allowing the accurate determination of tumor thickness and depth of invasion. Similarly, labeling for the antigen helps the distinction of recurrence or residual spindle melanoma cells from desmoplastic fibroblasts at resection sites.
As HMB-45 immunoreactivity is melanocyte specific, positivity can be encountered in lesions with melanin production such as adrenal pheochromocytoma, melanotic neuroectodermal tumor of infancy (progonoma), melanin-containing hepatoblastoma, malignant epithelioid schwannoma of the skin, pigmented carcinoid tumor and esthesioneuroblastoma.
More recently, HMB-45 positivity has been reported in a variety of lesions, which may have implications for their differentiation or histogenesis. These include angiomyolipoma, lymphangiomyomatosis and sugar tumor of the lung. While these tumors consistently manifest HMB-45 immunoreactivity, they do not display obvious pigmentation. However, recent ultrastructural studies confirm the presence of premelanosomes and all three lesions also manifest evidence of smooth muscle differentiation. The reactivity for HMB-45 can be a useful diagnostic discriminant, especially in the case of clear cell or sugar tumor which resembles metastatic renal cell carcinoma and clear cell carcinoma of the lung (Appendix the lung (Appendix 1.9). Similarly, immunoreactivity for HMB-45 can be helpful in the identification of lymphangiomyomatosis in transbronchial biopsies, obviating the need for an open biopsy for definitive diagnosis.
The apparent expression of this antigen in angiomyolipoma and lymphangiomyomatosis, both manifestations of the tuberous sclerosis complex, has been linked by the recent demonstration of HMB-45 immunoreactivity in cardiac rhabdomyoma, brain lesions and other mesenchymal as well as neural lesions found in the tuberous sclerosis complex (Weeks et al, 1994). These lesions have also shown ultrastructural granules suggestive of melanosome formation and are in agreement with previous suggestions that a smooth muscle cell with unusual features links the various lesions of tuberous sclerosis. HMB-45 immunoreactivity in these lesions now provides another common denominator.
Comments
Immunoreactivity of formalin-fixed tissue is enhanced following heat-induced antigen retrieval. Enzyme pretreatment does not significantly improve immunostaining for HMB-45. As in other diagnostic situations, heavily pigmented melanocytic lesions may pose a problem in differentiating melanin in other cells, such as macrophages, from tumor cells with true brown immunoreactivity when 3,3'-diaminobenzidine (DAB) is used as the chromogen. This problem can be simply eliminated by employing azure B as a substitute for hematoxylin as the counterstain. Azure B renders melanin granules blue-green, contrasting against the brown granules resulting from positive immunoreactivity.
The HMB-45 antibody has been reported to rarely show false-positive staining in non-melanomatous tumors and some normal tissues. These include breast carcinoma and normal breast epithelium, sweat gland tumors and normal counterparts, pheochromocytomas, hepatocellular carcinoma, chordoma, adenocarcinomas, lymphoma, plasmacytoma and plasma cells (Leong & Milios, 1989; Bonetti et al, 1991). This spurious staining is usually apical or perinuclear in location and granular in nature. This false positivity has been attributed to contamination of commercial ascites fluid preparations with non-specific antibodies and the culture supernatant fluid of the hybridoma cell line, now available from Dako, has been shown to eliminate this falsepositivity with HMB-45 (Bonetti et al, 1991; Bacchi et al, 1996).
Mercury-based fixatives such as B5 should be avoided as they result in extensive false-positive staining of mesenchymal cells including vessels, fibroblasts and inflammatory cells.
References
•Bacchi CE, Bonetti, Pea M, Martignoni G, Gown AM 1996. HMB-45. A review. Applied Immunohistochemistry 4:73-85.
•Bonetti F, Pea M, Martignoni G et al 1991. False-positive immunostaining of normal epithelia and carcinomas with ascites fluid preparations of antimelanoma monoclonal antibody HMB-45. American Journal of Clinical Pathology 95: 454-459.
•Leong AS-Y, Milios J 1989. An assessment of a melanoma-specific antibody (HMB-45) and other immunohistochemical markers of malignant melanoma in paraffin-embedded tissues. Surgical Pathology 2: 137-145.
•Ordonez NG, Sneige N, Hickey RC, Brooks TE 1988. Use of monoclonal antibody HMB-45 in the cytologic diagnosis of melanoma. Acta Cytologica 32: 684-688.
•Weeks DA, Chase DR, Malott RL et al 1994. HMB-45 staining in angiomyolipoma, cardiac rhabdomyoma, other mesenchymal processes and tuberous sclerosis-associated brain lesions. Journal of Surgical Pathology 1:191-198.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
DESCARGAR INFORMACION TRADUCIDA AL IDIOMA ESPAÑOL
Axcel/Accurate, Biodesign, Biogenesis, Biogenex, Dako, Enzo and Immunotech.
Fixation/Preparation
The antibody is immunoreactive in paraffin-embedded tissue as well as frozen sections. Immunoreactivity is stronger in ethanol-fixed tissues than following formalin fixation, with immunoreactivity diminishing significantly following prolonged fixation in the latter. Sensitivity is enhanced following heat-induced antigen retrieval. Mercury-based fixatives result in a high degree of non-specific staining.
Background
The HMB-45 monoclonal antibody was generated to a whole-cell extract of a heavily pigmented lymph node deposit of human melanoma and has been shown to be a highly specific and sensitive reagent for the identification of melanoma. The designation HMB is derived from the immunogen employed, i.e. Human Melanoma, Black. The antigen is intracytoplasmic and ultrastructural studies suggest that the antibody reacts with melanosomes before melanin deposition with HMB-45 binding to stage 1 and 2 melanosomes and to the non-melanized portion of stage 3, whereas stage 4 melanosomes and melanosome complexes found in macrophages and keratinocytes have been negative. The antibody appears to label premature and immature melanosomes in retinal pigment epithelium from fetuses and neonates but not from adults, leading to the suggestion that this "oncofetal" pattern of expression may indicate a role in melanocytic cell proliferation. This thesis has not been confirmed and the sequential expression of the HMB-45 antigen in melanocytes may relate to the activation by specific growth factors, resulting in alterations in protein glycosylation during various ontogenic and pathologic states of melanocytes. The epitope recognized by HMB-45 appears to be, in part, the oligosaccharide side chain of a sialated glycoconjugate as the immunoreactivity can be abolished with neuraminidase treatment (Bacchi et al, 1996).
The gene corresponding to the HMB-45 defined proteins has recently been cloned and designated gp 100-cl. This gene encodes the melanocyte lineage-specific antigens recognized by HMB-45 and HMB-50 (one of two other monoclonal antibodies to melanocytes initially obtained with HMB-45) as well as another monoclonal antibody, NKI-beteb. These three antibodies appear to recognize different epitopes of the same antigen, the melanosomal matrix protein or pmel 17 gene product defined by them being apparently related by differential splicing.
Applications
Immunoreactivity for HMB-45 is seen in normal fetal and neonatal melanocytes but not in adult resting melanocytes. Reactive or proliferating melanocytes in inflamed adult skin, wound healing, increased vascularity and in skin overlying certain dermal neoplasms may label for HMB-45 as a result of activation and stimulation by growth factors and "reexpression" of the antigen. HMB-45+ melanocytes have been demonstrated in the anal squamous zone and transitional zone but not in the colorectal zone. Increased numbers of such melanocytes can be present adjacent to primary anal melanomas.
The staining for HMB-45 in melanocytic nevi depends on their location within the skin. Junctional nevi and the junctional components of compound nevi are HMB-45+. In contrast, intradermal nevi and the dermal components of compound nevi are consistently negative. Thus, HMB-45 does not provide distinction between benign and malignant melanocytic proliferations and the difference in reactivity supports the concept, based on differences in morphology, enzyme activity and other immunological reactivity, that junctional and dermal cells are not identical. Junctional nevus cells are in an activated or proliferative state compared to their quiescent dermal counterparts and their immunoreactivity with HMB-45 is analogous to the proliferating fetal melanocytes which are positive for the antigen whilst quiescent, adult melanocytes are non-reactive.
Dysplastic nevi, in contrast, usually express HMB-45 in both the junctional nevus cells as well as in the dysplastic cells in the superficial dermis. Nevus cells within the deeper dermis do not usually react with HMB-45. In one study, minimally dysplastic nevi displayed intense immunolabeling of the junctional melanocytes but no staining of dermal nevus cells whereas, with the moderately and severely dysplastic nevi, the dermal melanocytes showed focal cytoplasmic immunoreactivity. The likelihood of expression of HMB-45 paralleled the degree of dysplasia of the nevi. Common blue nevi and cellular blue nevi are generally HMB-45+, as are malignant blue nevi. Other nevi such as spindle and epithelioid cell nevi, congenital nevi and other nevi occurring in hormonally reactive sites show immunostaining in nevus cells in the deep dermis as well as those near the dermoepidermal junction. Less common benign melanocytic proliferations such as plexiform spindle cell nevi, Spitz nevi and atypical melanocytic hyperplasias are also HMB-45+ (Bacchi et al, 1996).
Malignant melanoma show strong cytoplasmic positivity for HMB-45 in the majority of cases (65-95%), with the proportion of positive tumor cells ranging from a few to 100%. When the expression of the antigen is weak, staining may appear as a fine granularity similar to that seen in cytologic preparations. The positivity for HMB-45 is seen in almost all types of primary and metastatic melanoma including amelanotic melanoma, spindle cell melanoma and acral lentiginous melanoma (Appendix 1.10). One important exception is desmoplastic malignant melanoma, which consistently displays a much lower rate of positivity and may be completely negative. When positive, reactivity is usually seen in the superficial epithelioid cell rather than the dermal spindle cells, which only rarely stain for HMB-45 (Leong & Milios, 1989).
Attesting to the specificity of the antigen, HMB-45 reactivity has been demonstrated in malignant melanomas of diverse morphology such as signet ring melanoma, myxoid melanoma, small cell melanoma, balloon cell melanoma and in melanomas of different anatomic sites such as the gallbladder, urinary bladder, anorectal region, vulva, sinonasal region, uterine cervix, other mucosal sites and bone. Melanomas and melanocytic proliferations occurring in complex tumors such as pulmonary blastoma have also been HMB-45+ (Ordonez et al, 1988; Bacchi et al, 1996).
HMB-45 staining also has application in the separation of melanin-containing macrophages from melanoma cells, allowing the accurate determination of tumor thickness and depth of invasion. Similarly, labeling for the antigen helps the distinction of recurrence or residual spindle melanoma cells from desmoplastic fibroblasts at resection sites.
As HMB-45 immunoreactivity is melanocyte specific, positivity can be encountered in lesions with melanin production such as adrenal pheochromocytoma, melanotic neuroectodermal tumor of infancy (progonoma), melanin-containing hepatoblastoma, malignant epithelioid schwannoma of the skin, pigmented carcinoid tumor and esthesioneuroblastoma.
More recently, HMB-45 positivity has been reported in a variety of lesions, which may have implications for their differentiation or histogenesis. These include angiomyolipoma, lymphangiomyomatosis and sugar tumor of the lung. While these tumors consistently manifest HMB-45 immunoreactivity, they do not display obvious pigmentation. However, recent ultrastructural studies confirm the presence of premelanosomes and all three lesions also manifest evidence of smooth muscle differentiation. The reactivity for HMB-45 can be a useful diagnostic discriminant, especially in the case of clear cell or sugar tumor which resembles metastatic renal cell carcinoma and clear cell carcinoma of the lung (Appendix the lung (Appendix 1.9). Similarly, immunoreactivity for HMB-45 can be helpful in the identification of lymphangiomyomatosis in transbronchial biopsies, obviating the need for an open biopsy for definitive diagnosis.
The apparent expression of this antigen in angiomyolipoma and lymphangiomyomatosis, both manifestations of the tuberous sclerosis complex, has been linked by the recent demonstration of HMB-45 immunoreactivity in cardiac rhabdomyoma, brain lesions and other mesenchymal as well as neural lesions found in the tuberous sclerosis complex (Weeks et al, 1994). These lesions have also shown ultrastructural granules suggestive of melanosome formation and are in agreement with previous suggestions that a smooth muscle cell with unusual features links the various lesions of tuberous sclerosis. HMB-45 immunoreactivity in these lesions now provides another common denominator.
Comments
Immunoreactivity of formalin-fixed tissue is enhanced following heat-induced antigen retrieval. Enzyme pretreatment does not significantly improve immunostaining for HMB-45. As in other diagnostic situations, heavily pigmented melanocytic lesions may pose a problem in differentiating melanin in other cells, such as macrophages, from tumor cells with true brown immunoreactivity when 3,3'-diaminobenzidine (DAB) is used as the chromogen. This problem can be simply eliminated by employing azure B as a substitute for hematoxylin as the counterstain. Azure B renders melanin granules blue-green, contrasting against the brown granules resulting from positive immunoreactivity.
The HMB-45 antibody has been reported to rarely show false-positive staining in non-melanomatous tumors and some normal tissues. These include breast carcinoma and normal breast epithelium, sweat gland tumors and normal counterparts, pheochromocytomas, hepatocellular carcinoma, chordoma, adenocarcinomas, lymphoma, plasmacytoma and plasma cells (Leong & Milios, 1989; Bonetti et al, 1991). This spurious staining is usually apical or perinuclear in location and granular in nature. This false positivity has been attributed to contamination of commercial ascites fluid preparations with non-specific antibodies and the culture supernatant fluid of the hybridoma cell line, now available from Dako, has been shown to eliminate this falsepositivity with HMB-45 (Bonetti et al, 1991; Bacchi et al, 1996).
Mercury-based fixatives such as B5 should be avoided as they result in extensive false-positive staining of mesenchymal cells including vessels, fibroblasts and inflammatory cells.
References
•Bacchi CE, Bonetti, Pea M, Martignoni G, Gown AM 1996. HMB-45. A review. Applied Immunohistochemistry 4:73-85.
•Bonetti F, Pea M, Martignoni G et al 1991. False-positive immunostaining of normal epithelia and carcinomas with ascites fluid preparations of antimelanoma monoclonal antibody HMB-45. American Journal of Clinical Pathology 95: 454-459.
•Leong AS-Y, Milios J 1989. An assessment of a melanoma-specific antibody (HMB-45) and other immunohistochemical markers of malignant melanoma in paraffin-embedded tissues. Surgical Pathology 2: 137-145.
•Ordonez NG, Sneige N, Hickey RC, Brooks TE 1988. Use of monoclonal antibody HMB-45 in the cytologic diagnosis of melanoma. Acta Cytologica 32: 684-688.
•Weeks DA, Chase DR, Malott RL et al 1994. HMB-45 staining in angiomyolipoma, cardiac rhabdomyoma, other mesenchymal processes and tuberous sclerosis-associated brain lesions. Journal of Surgical Pathology 1:191-198.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
DESCARGAR INFORMACION TRADUCIDA AL IDIOMA ESPAÑOL
