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

Materials and Methods

Sample Selection

Two separate collections of melanoma samples were used for this analysis. The SPORE progression array was constructed by the Harvard tissue microarray facility using the standard method[22] and included a series of randomly-selected, formalin-fixed, paraffin-embedded melanocytic lesions retrieved from the archives at Harvard University, the University of Pennsylvania and the MD Anderson Cancer Center. The array includes 21 thin (<1 mm) nevi, 15 thick (>1 mm) nevi, 38 radial growth phase (<1 mm) primary melanomas, 20 vertical growth phase (>1 mm) primary melanomas, 28 lymph node metastases and 45 metastases to visceral organs. Duplicate 0.6 mm cores were obtained from each thin lesion as well as from the set of metastatic lesions. For the thick lesions, three depths of invasion relative to the skin edge were defined (superficial, mid-level and deep) and duplicate 0.6 mm cores were taken at each of these depths to produce a total of six cores from each tumor.

The Yale Melanoma Boutique array was constructed by the Yale tissue microarray facility using formalin-fixed, paraffin-embedded archival material. It included single 0.6 mm cores from 20 benign nevi, 20 vertical growth phase primary melanomas and 20 metastases, the latter representing lesions from subcutaneous, lymph node and visceral sites. In addition, the tissue microarray included as controls 0.6 mm cores from paraffin-embedded pellets of 16 melanoma and 18 nonmelanoma cell lines grown in culture to confluency and harvested into 10% neutral-buffered formalin.


Five micron sections were cut from each array master block, adhered to the recipient glass slide using an ultraviolet cross-linkable tape transfer system (Instrumedics Inc., Hackensack, NJ), dipped in paraffin and stored in a nitrogen chamber until use. Slides were deparaffinized using two xylene exchanges followed by rehydration in ethanol gradient. Antigen retrieval was performed by boiling the slides in buffer containing 6.5 mM sodium citrate (pH=6.0) in a sealed pressure cooker for 10 min. The slides were immersed in absolute methanol containing 0.75% hydrogen peroxide for 30 min to neutralize endogenous peroxidase activity, followed by a 30 min incubation in 0.3% bovine serum albumin (BSA) dissolved in 1 M tris-buffered saline (TBS; pH=8.0) to block nonspecific binding.

Fluorescence-based immunohistochemical staining was performed by multiplexing the prediluted HMB45 mAb (BioGenex, San Ramon, CA) with rabbit polyclonal anti-S100B (Dako Cytomation, Carpenteria, CA) at 1:200 dilution. The latter was used to define the 'tumor mask,' which discriminates the regions corresponding to melanoma from the surrounding tissue within the histospot in the absence of simultaneous H&E staining. Primary antibodies were incubated at 4°C overnight. Visualization was accomplished using fluorescent probes. The secondary fluorescent antibodies, Alexa-546-conjugated goat antirabbit (1:100 dilution, Molecular Probes, Eugene, OR) diluted into Envision antimouse (neat; DAKO) were applied for 1 h at room temperature. The slides were washed three times for 10 min with 1 M TBS, incubated with cyanine-5-conjugated tyramide (Perkin-Elmer, Wellesley, MA) for 10 min, and then with 0.01 mg/ml 4',6-diamidino-2-phenylindole (DAPI) for 20-min for nuclear compartment visualization. The slides were mounted with Prolong-Gold antifade reagent (Invitrogen), sealed with a nylon-based lacquer and stored in the dark until scoring.

For brown-stain-based immunohistochemistry, the prediluted HMB45 antibody was applied neat for 1 h at room temperature and visualized using Envision™ with a 3-3'-diaminobenzidine chromogen. Counterstaining was achieved using Meyer's hematoxylin. Evaluation of chromogen-stained arrays and conventional imaging was performed by a single pathologist (DLR).

Digital Image Capture and Automated Quantitative Analysis (AQUA®) of Protein Expression

The AQUA image acquisition and analysis was performed as described previously.[21] Briefly, stained slides were imaged on a modified computer-controlled epifluorescence microscope (Olympus BX-51 with xy-stage and z controller) illuminated by a high-pressure mercury bulb (Photonic Solutions, Missisauga, ON) with a high-resolution monochromatic camera (Cooke Corporation, Romulus, MI). Following user optimization of the focus, sets of monochromatic, high-resolution (1024 x 1024, 0.5 μm) images were captured for each histospot, for each of the DAPI, Alexa-546 and Cy5 fluorescent channels. Two images were captured for each channel: one in the plane of focus and the other 8 μm below it. The gp100 staining patterns were captured by a 65 ms exposure time in the cyanine-5 channel on the SPORE progression array and 57 ms for the Yale Melanoma Boutique array. The Alexa-546 signal representing S100B staining was binary gated to indicate whether a pixel is within the tumor mask ('on') or not ('off'). Further subcellular compartmentalization defined nuclear from non-nuclear regions within the tumor. The nuclear compartment was defined as the subset of pixels that demonstrated any DAPI staining within the plane of focus. This was required to compensate for the 3-dimensional thickness of the tumor sections, which can blur the discrimination of the nuclear boundary. The non-nuclear compartment was then defined as all pixels assigned to the tumor mask but are not included within the nuclear compartment. Finally, gp100 expression levels were determined in an automated fashion, blinded to any a priori clinical information, from the images acquired under the Cy5 channel to obtain a relative pixel intensity restricted to the signal emanating from the plane of focus. The final AQUA score for the entire tumor mask or any of its subcellular compartments was calculated as the average AQUA score for each of the individual pixels included in the selected compartment and was reported on a scale of 0–255.

Statistical Analysis

Cores whose tumor mask covered <5% of the total histospot area were dropped from further analysis. The raw AQUA scores for the total area under the tumor mask as well as each of the specific nuclear and non-nuclear compartments were used. To compare the intensity of nuclear to non-nuclear staining, the natural log of the ratio of nuclear to non-nuclear AQUA scores was calculated.

Evaluation of the AQUA scores associated with HMB45 staining in each of the three defined compartments and the ratio of nuclear to non-nuclear staining across the progression of melanocytic lesions was performed by the analysis of variance (ANOVA). To account for the redundant cores within the SPORE progression array, a mixed-effect ANOVA model was used which included a random effects parameter for sample. Testing for the significance of individual pairwise comparisons in each ANOVA was adjusted using the Tukey method for multiple comparisons. Assessment of a linear trend across lesion progression was accomplished by mixed model linear regression incorporating a random effects parameter for sample following ordinalization of the lesion type. Calculation of the receiver–operator characteristic curve was performed from the logistic regression model measuring the likelihood that a given sample is a benign nevus. For all analyses, significant associations were defined as P<0.05. All statistical analyses were performed using SAS version 9.1.3 and Statview (SAS Institute, Cary, NC).


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