Abstract 4231: Analysis of FFPE treated clinical tissue sections obtained from human intraocular malignancy, uveal melanoma by mass spectrometry imaging (MSI)
Authors: Cole Laura M. , Mudhar Hardeep S. , Sisley Karen , Peck Andrew , Batey Mike , Claude Emmanuelle , Clench Malcolm 
Published: July 2016

Abstract
Introduction
Clinical research into human intraocular disease UM FFPE tissue sections is described. UM remains the most common intraocular malignancy in adults, with poor prognosis for UM within the choroid region and distant sites UM metastasis. Two imaging MS techniques Matrix Assisted Laser Desorption/Ionisation (MALDI) and Desorption Electrospray Ionisation (DESI) MSI were applied. Molecular profiles were obtained and analysed by multivariate statistical approaches, providing insight into the biochemical and biological differences/similarities within the patient sample cohort (n = 15).
Methods
Enucleations were collected over 6 years and subjected to the standard fixation and paraffin embedding protocols. In preparation for MS, 5 μm sections were produced with removal of the paraffin followed by heat induced antigen retrieval.
MALDI MSI tissue sections were prepared by applying a matrix solution onto the tissue sections to help ionization of molecules directly from the tissue. All MSI experiments (MALDI and DESI) were carried out using a SYNAPT mass spectrometer (Waters Corporation, Manchester, UK). Multivariate analyses were performed using MATLAB (MathWorks, Inc., Natick, MA, US) and the Eigenvector PLS_Toolbox.
Results
Initial MALDI MSI images acquired with a spatial resolution of 100 μm x 100 μm in positive ion mode showed distinct spatial distribution of many molecular species throughout the choroid, cornea, retina, lens and UM tumor regions. Further MALDI MSI experiments using consecutive tissue sections at 50 μm x 50 μm spatial resolution show substantial variation in the spatial distribution of species within the low molecular mass range. DESI MSI experiments were carried out at 200 μm x 200 μm in negative ionization mode. Deprotonated molecular ions were detected from a variety of lipid related species, localized to specific regions within the eye including the tumor region.
Multivariate analysis classified UM samples (good vs. poor prognosis). Using unsupervised PCA, an unbiased representation of the data was generated, with clear sample grouping and differentiation observed based upon tumor status. Use of the supervised PLS-DA technique provided even clearer separation between the selected samples, with tumor profiles displaying dominant discriminatory peaks. These peaks could be identified as sphingolipids and Lyso-phosphocholine, a phosphatidylcholine degradation product.
Conclusions
It was possible to analyze clinical research FFPE tissue sections by MALDI and DESI MSI, illustrating that specific small molecular species remained localized to certain tissue types including the UM tumor. Initial correlation with tumor status was determined from the statistical analysis of the MALDI MSI datasets.
Citation Format: Laura M. Cole, Hardeep S. Mudhar, Karen Sisley, Andrew Peck, Mike Batey, Emmanuelle Claude, Malcolm Clench. Analysis of FFPE treated clinical tissue sections obtained from human intraocular malignancy, uveal melanoma by mass spectrometry imaging (MSI). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4231.