Subcellular Localization of NEDD9 and HMB45 with AQUA Technology to Distinguish Spitz Nevi from Melanomas Matthew C. McRae MD, MHS 1; Rossitza Lasova MD 2; Deepak Narayan MD 3; David L. Rimm MD, PhD 4. 1 University of Toronto Division of Plastic and Reconstructive Surgery. 2 Yale University School of Medicine. Division of Dermatopathology. 3 Yale University School of Medicine. Division of Plastic Surgery. 4 Yale University School of Medicine. Division of Pathology.

Author Disclosures: No other disclosures Dr. David Rimm is a founder, stockholder and consultant for HistoRx, the exclusive licensee of the Yale-own patent on the AQUA technology. Supported in part by the American Society of Plastic Surgery, Plastic Surgery Education Foundation Basic Research Grant (Dr. Matthew C. McRae) No other disclosures

Objective Clinical problem: Benign Spitz Nevi or Melanoma with Spitz features? Assess the quantity and subcellular location of biomarkers HMB45 (Marker of pre- melanosome glycoprotein gp100) and NEDD9 (Neural precursor cell expressed developmentally regulated expression pattern in brain 9) in tumor samples. Our hypothesis was that the expression level and subcellular localization of HMB45 and NEDD9 would be consistently altered in melanoma with Spitz features when compared to benign Spitz nevi.

AQUA™ : Automated quantitative assay for protein expression in subcellular compartments Full section sample of tumor divided into grid. Area of actual tumor separate from stroma by “tumor mask” defined by S- 100 staining. Nuclear compartment defined by DAPI stain. Non-nuclear compartment defined as nuclear compartment subtracted from the tumor mask. AQUA score defined as signal intensity per pixel area of defined compartment. Subcellular AQUA ratio is a normalized score defined as the natural log of AQUA score in the nuclear compartment/ AQUA score in the non-nuclear compartment. DAPI stained nuclear compartment (Blue) Non nuclear compartment (Red)

Tissue Microarrays used for proof of principle: NEDD9 and HMB45 expression and subcellular localization consistently altered in Nevi compared to Primary Melanoma NEDD 9 AQUA score ratio: In(AQUA-nuclear/AQUA-non-nuclear) HMB 45 AQUA score ratio: In(AQUA-nuclear/AQUA-nonnuclear) Boxplot HMB45 YTMA 98-2 Cut 60 These results were replicated on Yale Nevi array (220 benign nevi and 37 primary melanomas), and the SPORE 84 array (54 benign nevi and 112 primary melanomas)

20 Typical Spitz Nevi 20 Atypical Spitz Nevi 9 Spitzoid Melanoma Tumor samples derived from the Yale Spitz Neoplasm Repository included 20 Spitz nevi, 20 atypical Spitz nevi and 9 Spitzoid melanomas. They were compared using the subcellular AQUA ratio for NEDD9 and HMB45 biomarkers.

NEDD 9 expression and subcellular localization by AQUA ratio was not useful diagnostically to distinguish between Spitz, Atypical Spitz and Melanoma tumor samples.

HMB45 expression and subcellular localization by AQUA ratio was not useful diagnostically to distinguish between Spitz, Atypical Spitz and Melanoma tumor samples.

Conclusion Subcellular localization using the ratio of the natural log of HMB45 and Nedd9 expression, which defines benign nevi from melanoma was not useful in discriminating between benign Spitz nevi and melanoma with Spitz features in our cohort. The Yale Spitz Neoplasm Repository and novel technology such as AQUA™ promise for rapid validation of biomarkers for possible future diagnostic application.