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 Staining Trends Across Core Depth in Thick Lesions

AQUA was performed on the SPORE array with 334 histospots representing 136 samples available for analysis. To determine the trend of gp100 expression across core depth among the thick nevi, the distributions of AQUA scores for superficial, mid-level and deep cores from each thick nevus were compared. No significant difference in the distribution of AQUA was observed among the three tissue depths for either the nuclear (P=0.42), the non-nuclear compartments (P=0.75), or the aggregate AQUA score when the entire area under the tumor mask was considered (P=0.76; Figure 1a-b).

Figure 1.

Box plots showing the relative distributions of AQUA scores for (a) nuclear compartment, (b) non-nuclear compartment and (c) for total area under the tumor mask for the superficial, mid-level and deep regions of the thick nevi (as indicated). No significant differences were observed using mixed model ANOVA (P>0.20).

gp100 Expression Across Melanocytic Lesion Progression

Representative HMB45 staining patterns for each class of lesion spotted on the tissue microarray are shown in Figure 2a-d. Among the specimens from primary and metastatic lesions, HMB45 most strongly stained the cytoplasmic regions (non-nuclear compartment) of the cells although some degree of nuclear staining could be observed. Setting an AQUA score of 20 as the maximal cutoff level designating moderate to strong HMB45 staining in the non-nuclear compartment, cytoplasmic staining was observed in 16/25 radial growth phase primaries, 14/19 vertical growth phase primaries, 16/24 lymph node metastases and 24/40 visceral metastases. Cytoplasmic staining was present in none of the thin nevi and only in one thick nevus. Interestingly, definitive nuclear staining was observed in the dermal component of all thin and thick nevi, whereas only three metastatic melanoma lesions demonstrated any nuclear staining and weak cytoplasmic HMB45 staining. Focal staining was observed in two of these cases, with the third lesion displaying a punctuated nuclear staining pattern throughout the histospot.

Figure 2.

Representative images of a thin nevus (a), thick nevus at mid-level (b), a thick primary (c) and a visceral metastasis (d). For each image series, the first image captures S100B staining in the Alexa-546 channel and the remaining two images represent a magnification of the inset region. The solid inset represents the conversion of S100B staining into the binary-gated tumor mask, which differentiates cells from the melanocytic lesion from surrounding stroma and matrix. The composite of the DAPI stain-positive nuclear (blue) and DAPI stain-negative non-nuclear (green) compartments within the tumor mask (aii–dii), and the distribution of HMB45 within these compartments (images aiii–diii) is presented. In both thin and thick nevi, HMB45 is concentrated in the nuclear compartment, whereas in the malignant lesions, HMB45 distribution is predominantly cytoplasmic.

Examination HMB45 reactivity of nevi using conventional immunohistochemistry with a DAB chromogen and hematoxylin counterstain of the Yale Melanoma Boutique array shows no appreciable staining (Figure 3a). We hypothesize that the low levels of expression are obscured by the hematoxylin counterstain. To verify that the fluorescence-based staining described above (Figure 2) was not an artifact of the visualization method, a second, serial section of the Yale Melanoma Boutique array was stained using DAB as the chromogen but without the hematoxylin counterstain. Faint DAB stain was observed in the nuclei of nevoid melanocytes in the absence of hematoxylin (Figure 3b), but the intensity of the nuclear counterstain overwhelms the faint HMB45-produced DAB stain (Figure 3a). Figure 3c and d show a more typical example of HMB-45 staining of a malignant melanoma with strong cytoplasmic staining. The absence of counterstain in this case (Figure 3d) does not reveal the pale nuclear staining seen in the nevus in Figure 3b.

Figure 3.

HMB45 staining of melanocytic lesions using conventional immunohistochemistry with a DAB chromogen. In the presence of hematoxylin counterstain, a nevus from the Yale Melanoma Boutique collection (a) shows no appreciable HMB45 staining. In the absence of counterstain, faint nuclear DAB stain can be observed in a serial section (b). A representative primary melanoma lesion from the same collection shows intense cytoplasmic staining that can be visualized in the presence (c) or absence (d) of hematoxylin counterstain, but no nuclear reactivity is seen, even in the absence of counterstain.

The Ratio of Nuclear/Non-Nuclear AQUA Scores is a Significant Discriminator of Benign Melanocytic Lesions

To extend our observation defining a distinct, predominantly nuclear HMB45 staining pattern for benign melanocytic lesions that is significantly different from the predominantly cytoplasmic distribution seen in the malignant lesions, the ratio of nuclear to non-nuclear AQUA scores was evaluated as a possible discriminator of benign melanocytic lesions. To normalize the distribution of ratios, the natural log transformation of the ratio was selected as the preferred parameter. Using linear mixed models, both the thin (0.450±0.253) and thick (0.513±0.227) nevi had strongly positive mean ln(nuclear/non-nuclear ratios) that were significantly different (P<0.0001) from the group of malignant lesions whose means±s.d. range from 0.128±0.285 to 0.055±0.186 (Figure 4a). To determine the reproducibility of this finding on an independent collection of benign and malignant melanocytic lesions, the Yale Melanoma Boutique array was stained with the HMB45 antibody using fluorescence-based methods. Twenty benign nevi, 20 vertical growth phase primaries and 18 metastases yielded histospots with sufficient material for analysis. The mean ln(nuclear/non-nuclear ratio) for benign nevi of 0.68±0.22 was significantly higher than those from vertical growth phase primaries (0.16±0.32; P<0.0001) and metastases (0.42±0.37; P=0.01; Figure 4b).

Figure 4.

(a) Box plot describing the distributions of ln(nuclear/non-nuclear compartment AQUA scores) for the six levels of disease spotted on the melanoma SPORE progression array. Thin and thick nevi display elevated ratios that are significantly different from each category of malignant lesions (mixed model ANOVA pairwise comparison P-values all <0.0001). (b) Box plot describing the distribution of ln(nuclear/non-nuclear compartment AQUA scores) for nevi, thick primaries and metastatic lesions in the Yale Boutique Array (fixed effects ANOVA pairwise comparison P-values all <0.01).

To evaluate the predictive capability of the ln(nuclear/non-nuclear ratio), logistic regression was executed to determine the likelihood of a sample being a nevus, yielding an odds ratio of 2.24 (95% CI: 1.87–2.69; P<0.0001) for each 0.1 unit increase in ln(nuclear/non-nuclear ratio). A receiver–operator characteristic curve was constructed from these data across all possible dichotomizing cut points covering the full range of the ln(nuclear/non-nuclear ratio) scores, which produced 0.93 units of area and, at the shoulder of the curve, created by designating an ln(nuclear/non-nuclear HMB45 AQUA score ratio) >0.154 as positively identifying a nevus, a maximized simultaneous sensitivity of 0.92 and specificity of 0.80 for discriminating benign nevi from malignant melanocytic lesions (Figure 5).

Figure 5.

Receiver–operator characteristic curve describing the predictive capabilities of the ln(nuclear/non-nuclear compartment AQUA score ratio) for distinguishing a nevus from a malignant lesion. The shoulder of the curve (green dot) identifies the simultaneously maximized sensitivity=0.92 and specificity=0.80.


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