Nuclear to Non-nuclear Pmel17/gp100 Expression (HMB45 staining) as a Discriminator Between Benign and Malignant Melanocytic Lesions

Bonnie E Gould Rothberg; Christopher B Moeder; Harriet Kluger; Ruth Halaban; David E Elder; George F Murphy; Alexander Lazar; Victor Prieto; Lynn McDivitt Duncan; and David L Rimm


Mod Pathol. 2008;21(9):1121-1129. 

In This Article


HMB45 reactivity is a cornerstone for the pathologic identification of melanocytic lesions. The cytoplasm of epithelioid cutaneous melanomas characteristically stains positively for HMB45, a diagnostic feature frequently used when pathologists evaluate malignancies of unknown primary.[4] Since HMB45 was first described in 1986,[23] the body of literature on the patterns of HMB45 staining among distinct classes of melanocytic lesions has been largely derived from nonfluorescent-based immunohistochemical techniques with brown or red colored chromogens. As such, their results are limited by the usual issues associated with any chromogen evaluation, including lack of dynamic range, lack of standardization, the reliance on subjective scoring methods and the categorization of scores into discrete categories. In this analysis, we reconsidered patterns of HMB45 reactivity through the application of immunofluorescence-based staining techniques. The increased sensitivity, combined with the lack of obscuring counterstains, revealed an interesting expression patterns that have not been previously described for this protein.

Several reports in the literature documented an inverse trend of gp100 expression in compound nevi as the lesion extends from epithelial to dermal surroundings.[9,12,15] In our analysis of 15 compound nevi, we did not detect a significant aggregate difference in the non-nuclear AQUA score across serial cores representing superficial, mid-level and deep regions of each lesion. In the light of this result, review of the S100 staining patterns among the set of superficial lesions revealed only one core with a clearly identifiable epidermal component of the nevus, which did demonstrate strong cytoplasmic HMB45 staining that was absent in the adjacent dermal component. Mid-level and deep sections from this same specimen did not reveal any cytoplasmic HMB45 staining. Interestingly, two other thick nevi had evidence of cytoplasmic staining in epithelial regions captured in their mid-level sections. As no previous assessment of HMB45 staining in compound nevi used either tissue microarray technology or immunofluorescent methods, a more targeted study designed to capture epidermal elements of compound nevi is needed to verify this result using AQUA methodology.

Our analysis, however, did reveal a previously undocumented finding of nuclear staining of dermal nevus cells with the HMB45 antibody in the absence of cytoplasmic staining. Interestingly, while faint HMB45 nuclear staining can be observed in nevi using conventional nonfluorescent chromogen stain methods in the absence of a nuclear counterstain, the application of standard nuclear stains such as Meyer's hematoxylin overwhelms the HMB45 staining. This observation highlights a specific niche for fluorescence-based immunohistochemical techniques where the detection of antigens expressed at low levels can be optimized by both leveraging the increased dynamic range associated with fluorescent staining compared with conventional brown stain methods[20] and using image capture techniques that allow for extended exposure times to capture light signals below the detection capabilities of the human eye. Our observation also highlights an underappreciated weakness of conventional techniques where application of the counterstain can hinder target visualization.

By using the ln(nuclear/non-nuclear AQUA score ratio), which maximizes the contrast between the nuclear staining pattern observed among the benign nevi and the predominantly cytoplasmic staining pattern observed in the malignant lesions, we were able to demonstrate an odds ratio of 2.24 for being a nevus vs a malignant lesion with each increase of 0.1 in the measured ratio. This logistic association was converted into a receiver–operator characteristic curve that demonstrated an area of 0.93 and simultaneously maximized sensitivity of 0.92 and specificity of 0.80, suggesting the potential utility of this ratio as an additional diagnostic tool in the characterization of melanocytic lesions. However, these cases were all selected from various institutions as classic examples of the histotype. The selective value of the assay and the true sensitivity and specificity of the test remains to be determined in tests of a wider range of serially collected melanocytic lesions.

One diagnostic dilemma that may benefit from this assay is the ability to distinguish a Spitz nevus from Spitzoid melanoma. Currently, no definitive criteria exist that can triage among the two types of lesions and a misdiagnosis can increase patient morbidity and mortality through either undertreatment of a melanoma or overtreatment of a benign lesion with unnecessary surgery and other systemic therapies in addition to the burden of psychological stress associated with the misdiagnosis and its sequelae.[24] Of greater concern is the estimate from one group that, among children, more than 40% of cases diagnosed as Spitz nevus are actually a malignancy that has been misdiagnosed.[25] Few studies have been published characterizing the differential expression of any marker between Spitz nevi and spitzoid melanomas with only CD99, a transmembrane protein associated with the epithelial-mesenchymal transformation, showing moderate discriminant activity between the two lesions.[26] Two immunohistochemical studies of HMB45 staining in Spitz nevi have been published, one reporting on 15 cases, the other on 30. Both studies describe heterogeneous staining patterns where half of the lesions were positive for HMB45 in the dermal cells and the remaining lesions divided between no staining and staining only in the epithelial component.[8,10] None of these studies commented on long-term follow-up of these cases. One report evaluating a small series of spitzoid melanomassuggested that these lesions may have more consistent dermal cytoplasmic HMB45 staining compared to ordinary benign nevi.[27] A targeted follow-up study testing the ability of HMB45 to discriminate benign and malignant Spitzoid lesions is planned.


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