Date of Award

10-30-2009

Document Type

Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Deepak Narayan

Second Advisor

David Rimm

Third Advisor

John Geibel, Rossitza Lasova

Abstract

SUBCELLULAR LOCALIZATION OF NEDD9 AND HMB45 WITH AQUA TECHNOLOGY TO DISTINGUISH SPITZ NEVI FROM MELANOMA. Matthew C. McRae, Rossitza Lasova, Bonnie Gould-Rothberg, David Rimm (Sponsored by Deepak Narayan). Section of Plastic Surgery, Department of Surgery, Yale University School of Medicine, New Haven, CT. Our hypothesis is that the expression level and subcellular localization of HMB45 and NEDD9 as demonstrated by the ln(nuclear/non-nuclear) Automated Quantitative Analysis (AQUA) score, defined as the subcellular AQUA ratio, will be consistently altered between benign nevi and melanoma and between Spitz nevi and Spitzoid melanoma. Our specific aims are to assess quantitative expression and subcellular localization of HMB45 and NEDD9 to aid in the diagnosis of benign Spitz nevi and malignant Spitzoid melanoma. This remains a vexing clinical problem with important implications for treatment and patient care. The quantitative expression and subcellular AQUA ratio will be assessed in the following samples: benign derived versus malignant derived cell lines, human benign nevi, human primary melanoma, human metastatic melanoma, typical Spitz nevi, atypical Spitz nevi and Spitzoid melanoma. AQUA was used to quantify protein expression levels in subcellular compartments using fluorescence-based immunohistochemistry. Tissue Microarrays (TMA) analysis was used for cell line, benign nevi and malignant melanoma while whole section analysis was used for Spitz nevi and Spitzoid melanoma. NEDD9 subcellular AQUA ratio was significantly reduced in primary melanoma (mean=-0.645, std dev=0.29) versus benign nevi (mean = -0.429, std dev=0.108) on YTMA98-2 (p=0.0086), significantly reduced in melanoma metastases (mean=-0.482, std dev=0.149) versus benign nevi (mean= -0.342, std dev=0.159) on YTMA66A (p<0.0001), and significantly reduced in primary melanoma (mean= -0.435, std. dev.=0.185) and melanoma metastases (mean= -0.42, std. dev.= 0.188) versus benign nevi (mean= -0.319, std. dev.= 0.141) in SPORE84 array (p=0.0003, Tukey/Kramer post-hoc significance p<0.05). HMB45 subcellular AQUA ratio was significantly reduced in primary melanoma (mean=-0.463, std. dev.=0.264) versus benign nevi (mean=-0.159, std. dev.=0.158) on YTMA 98-2 array (p=0.0001). On whole section analysis, the HMB45 and NEDD9 subcellular AQUA ratio shared a similar distribution between Spitz nevi, atypical Spitz nevi and Spitzoid melanoma. Subcellular localization using the subcellular AQUA ratio of HMB45 and NEDD9 defines benign nevi from melanoma on TMA but is not useful in discriminating between benign Spitz nevi and melanoma with Spitzoid features. The maximum HMB45 AQUA score in the tumor mask in a single 20X high-powered field of a whole tissue section was deemed promising on discovery analysis at differentiating between Spitz nevi and melanoma with Spitzoid features (p=0.007, receiver operating characteristic area under curve 0.711) but requires validation on an independent cohort.

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