Automated Quantitative Analysis of HDM2 Expression in Malignant Melanoma Shows Association with Early-Stage Disease and Improved Outcome

Abstract

The incidence of cutaneous malignant melanoma continues to increase every year, and this disease remains the leading cause of skin cancer death in industrialized countries. Despite the aggressive nature of advanced melanoma, there are no standard biological assays in clinical usage that can predict metastasis. This may be due, in part, to the inadequacy of reproducible assessment of protein expression using traditional immunohistochemistry. We have previously described a novel method of quantitative assessment of protein expression (AQUA) with the continuity and accuracy of an ELISA assay but with maintenance of critical spatial information. Here, we modify this technology for the evaluation of protein expression in melanoma. Using a tissue microarray cohort of 405 melanoma lesions and 17 normal skin samples, we analyzed expression of HDM2, the human homologue of murine double minute 2 with automated quantitative analysis. We show that expression levels in the nucleus are significantly higher in primary melanomas than in metastatic lesions. Furthermore, high levels of expression are predictive of better outcome. This study demonstrates that quantitative assessment of protein expression is useful in melanoma to validate potential tissue biomarkers and suggests that human homologue of murine double minute 2 may be a valuable prognostic tool for management of malignant melanoma.

Suppressor role of activating transcription factor 2 (ATF2) in skin cancer

“…In contrast, however, previous studies suggested a tumor-promoting role of ATF2 in melanoma (12)(13)(14)(15)(16)(17). Similarly, Notch1 functions as a tumor suppressor in mouse skin, as opposed to its function as an oncogene in other organs (44).…”

“…Consistent with this possibility are earlier studies from our laboratory that have suggested an important role of ATF2 in melanoma development and progression. Nuclear localization of ATF2 coincides with poor prognosis in melanoma patients (13). In addition, peptides derived from the N-terminal region of ATF2 efficiently repressed ATF2 function and reduced growth and metastasis of melanoma tumor cells in mouse models (14)(15)(16)(17).…”

Expression of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptors 1 and 2 in Melanoma

“…The melanoma tissue microarrays were constructed as previously described (30). A total of 232 primary melanomas, 15 local recurrences, and 299 metastatic cores, each measuring 0.6 mm in diameter, were spaced 0.8 mm apart on glass slides.…”

“…Staining was done for automated analysis of melanoma specimens as previously described (29,30). Briefly, slides were deparaffinized in xylene and transferred through two changes of 100% ethanol.…”

Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137

“…Expression of ATF-2, which belongs to the ATF͞CREB (cAMP-response element-binding protein) family, within the nucleus rather than the cytoplasm is associated with poor prognosis of melanoma (8). SW1-P2 cells express less ATF-2 in the nucleus and more in the cytoplasm than SW1-C cells, and this is correlated with decreased malignancy and increased ability to undergo apoptosis after exposure to radiation or certain chemicals (6).…”