Validation of Tandem Mass Spectrometry Study Using Laser Micro-Dissected Melanoma and Nevus Cells: Increase of Silver and Fatty Acid Synthase Protein Level in Melanoma.
Josie Munkberg, Richard Owings, Stephanie Byrum, Nathan Avaritt, Alan Tackett, Wang Cheung. UAMS, Little Rock, AR
Background: The incidence of melanoma continues to rise every year, yet the ability to treat advanced disease has not changed. Previous studies from our group and others have recognized that proteins involved in tumorigenesis can be identified by mass spectrometry on formalin fixed paraffin embedded (FFPE) sections. Our study aims to identify melanoma markers on FFPE sections by comparing metastatic melanoma and benign nevus with improved protein extraction, gel separation and mass spectrometry analyses. We decided to study some of these significant proteins and determine if the mass spectrometric data can be validated and if this method can yield other novel biomarkers. Once this method is validated, additional proteins identified as significant using mass spectrometry can be analyzed on a large scale basis.
Design: Melanoma cells or melanocytes were harvested from laser micro-dissection and proteins were extracted and analyzed by Thermo-LTQ-XL mass spectrometer coupled to an Eksigent nanoLC-2D. Approximately 300 proteins were differentiately expressed between metastatic melanoma and nevi with high significance. We found two published possible melanoma markers, SILV and fatty acid synthase (FAS), to validate the mass spectrometry results using immunohistochemical stains. FFPE blocks were retrieved from our archives as follows: 12 benign nevi, 7 dysplastic nevi, and 13 melanoma. Stain patterns were scored based on extent (0-3) and intensity (0-3). The product of the extent and intensity was determined generating a score of 0-9 for each. These were then divided as follows: 0-1=0; 2-4=1+; 5-6=2+; 7-9=3+.
Results: Higher immunoreactivity for SILV was detected in the melanoma cases than in the benign and dysplastic nevi cases which was statistically significant (p<0.001). 8 out of the 13 melanoma cases had a score of 3+, in contrast 19 out of 20 benign or dysplastic nevi cases expressed minimal or absent immunoreactivity (< or = 1+). Similar findings were found for FAS. 8 of 9 melanoma cases had high expression (2+ or 3+) whereas only 2 of 14 benign nevus and dysplastic nevus had high expression (p < 0.0015).
Conclusions: This study showed that laser micro-dissected FFPE sections coupled with mass spectrometry can identify biomarkers when comparing metastatic melanoma with a benign nevus. Immunostains for two markers validated the mass spec findings. We will continue to examine proteins that were found to be significant by mass spectrometry in metastatic melanoma in a larger scale future study.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 68, Monday Morning