Nuclear to non-nuclear Pmel17/gp100 expression (HMB45 staining) as a discriminator between benign and malignant melanocytic lesions

Abstract

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.

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).

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.

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.

Figure 4

A) Box plot describing the distributions of ln(nuclear/non-nuclear compartment AQUA scores) for the 6 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).

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.