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

Abstract and Introduction


HMB45 is a mouse monoclonal antibody raised against Pmel17/gp100, a melanoma-specific marker, which is routinely used in the diagnosis of primary cutaneous malignant melanoma. The standard expression pattern for a positive HMB45 staining result on immunohistochemistry is based upon the results of chromogenic-based methods. We re-evaluated the patterns of HMB45 staining across the 480-core 'SPORE melanoma progression array' containing lesions representing the spectrum of melanocytic lesions ranging from thin nevus to visceral metastasis using the fluorescence-based staining technique and automated quantitative analysis (AQUA) of the obtained digital images. The methods validated the expected cytoplasmic HMB45 staining pattern in 70/108 malignant lesions and in the epithelial components of nevus specimens. However, the fluorescence-based approach revealed a nuclear gp100 localization present in the dermal component of all nevi that was not seen before. This nuclear localization could not be observed on routine chromogenic stains, because the standard hematoxylin nuclear counterstain overwhelms the weak nuclear HMB45 stain. The thin (0.450±0.253) and thick (0.513±0.227) nevi had strongly positive mean ln(nuclear/non-nuclear AQUA score ratios), which are significantly higher than those from the group of malignant lesions (P<0.0001). This finding was reproduced on a smaller but independent progression array composed of nevi and melanomas from the Yale Pathology archives (P<0.01). The odds ratio associated with a sample being a nevus was 2.24 (95% CI: 1.87–2.69, P<0.0001) for each 0.1 unit increase of the ln(nuclear/non-nuclear AQUA score ratio) to yield an ROC curve with 0.93 units of area and a simultaneously maximized sensitivity of 0.92 and specificity of 0.80 for distinguishing benign nevi from malignant melanomas. On the basis of this preliminary study, we propose that the ratio of nuclear to non-nuclear HMB45 staining may be useful for diagnostic challenges in melanocytic lesions.


The human homolog of the mouse silver protein (gp100 or Pmel17) is a melanocyte-specific type I membrane protein required for proper formation of melanosomal fibrils, which facilitates the maturation of stage I premelanosomes to stage II.[1,2] Gp100 has long been used as a melanocyte/melanosome marker in the diagnosis of primary cutaneous melanoma and in the identification of melanoma cells in sentinel lymph node biopsies.[3,4] The recognition of gp100 is commonly performed with HMB45, a mouse monoclonal antibody that specifically reacts with the glycosylated form of gp100 restricted to the fibrillar matrix of stage II premelanosomes.[5,6,7]

The distribution of HMB45 staining patterns for benign and malignant melanocytic lesions has been characterized by multiple groups using routine techniques that employ 3–3'-diaminobenzidine(DAB) or 3'-aminoethyl-carbazole (AEC) as a chromogen. HMB45 staining is observed in over 95% of epithelioid cutaneous melanomas with most lesions yielding over 50% positive staining of the cytoplasmic portion of the cells.[8,9,10,11] Spindle cell and desmoplastic melanomas, however, tend to be HMB45-negative under standard antigen retrieval and immunostaining techniques.[12] Aggressive antigen retrieval can induce HMB45 staining in spindle cell but not in desmoplastic melanomas.[12,13] Evaluation of a series of metastatic lesions from 121 individuals with stage IV disease revealed substantial heterogeneity in HMB45 staining patterns with no staining in 26 (21.5%) of the lesions and one-third of the remaining lesions demonstrating either weak, moderate or strong staining.[14]

Analysis of HMB45 staining patterns in benign melanocytic lesions is stratified by the histologic type of lesion and location of the specific nevoid cells relative to the dermal–epidermal junction. Compound melanocytic nevi are characterized by cytoplasmic HMB45 reactivity within the epidermal component of the lesion, focal positivity in the junctional regions and subsequent loss of stain in the dermal component.[9,12,15] This corresponds to the traditional conception of maturation with the depth of melanocytes in nevi. Ordinary dermal nevi are HMB45 negative,[16] whereas dysplastic nevi[17] and blue nevi[18,19] are positive.

Chromogenic stains, however, suffer from several drawbacks including a limited dynamic range, lack of robust standardization methods and the reliance mostly on the subjective interpretation of a human observer to classify staining patterns into discrete ordinal categories, although automated readers have been developed.[20] This limitation was addressed by the optimization of fluorescence-based immunohistochemistry techniques on tissue microarrays coupled with the development of automated methods of quantification of staining intensity as a continuous parameter. Here, we re-evaluate the patterns of HMB45 staining across a tissue microarray containing a series of 167 unique melanocytic lesions ranging from thin benign nevi to visceral melanoma metastases using fluorescence-based immunohistochemistry followed by automated quantitative analysis (AQUA) of protein expression.[21] This technique revealed, for the first time, the patterns of nuclear gp100 expression in benign nevi suggesting its potential diagnostic utility to determine the ratio of nuclear to non-nuclear HMB45 staining for discriminating benign from malignant melanocytic lesions.


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