Analysis of FFPE treated clinical tissue sections obtained from Human Intraocular Malignancy, Uveal Melanoma by Mass Spectrometry Imaging (MSI)

Significance of the Topic

Mass spectrometry imaging (MSI) of formalin-fixed, paraffin-embedded (FFPE) clinical tissue offers a unique, label-free approach to map biomolecular distributions at histological resolution. In uveal melanoma (UM), the most common intraocular malignancy in adults, MSI provides spatially resolved chemical insights that complement conventional pathology, assisting in diagnosis, prognostic assessment and biomarker discovery.

Objectives and Study Overview

This study evaluated the feasibility and analytical performance of two MSI modalities—MALDI (matrix-assisted laser desorption/ionization) and DESI (desorption electrospray ionization)—applied to FFPE sections from human UM enucleations (n=15). The goals were to:

• Demonstrate spatial mapping of key molecular species across ocular compartments including cornea, choroid, lens, retina and tumour.
• Compare resolution and detection capabilities of MALDI and DESI MSI workflows on FFPE tissue.
• Use multivariate statistical models to differentiate tumour profiles and explore potential prognostic markers.

Methodology and Instrumentation

Clinical samples collected over six years were processed by standard fixation and paraffin embedding, then sectioned at 5 µm. For MALDI MSI, a chemical matrix solution was applied to tissue sections to facilitate laser desorption/ionization. DESI MSI was conducted directly on the unstained sections without additional sample preparation. All experiments used a SYNAPT mass spectrometer (Waters Corporation, Manchester, UK) configured for positive-ion MALDI and negative-ion DESI. Data processing and multivariate analyses (PCA, PLS-DA) were performed in MATLAB with the Eigenvector PLS_Toolbox.

Key Results and Discussion

MALDI MSI at 100 µm spatial resolution revealed distinct distributions of multiple lipid species across ocular structures. Increasing resolution to 50 µm provided finer detail of low-mass compounds within the tumour region. DESI MSI in negative mode (200 µm) detected deprotonated lipid-related ions localized to the melanoma. Unsupervised PCA clearly separated samples by tumour status, while PLS-DA yielded enhanced discrimination with dominant loadings corresponding to sphingolipids and lyso-phosphocholine species, highlighting their potential as tumour-associated markers.

Benefits and Practical Applications

• Enables direct histochemical analysis of routine FFPE clinical specimens without additional staining or labeling.
• Provides high-resolution spatial maps of metabolites and lipids, supporting more informed pathological evaluation.
• Facilitates identification of molecular signatures linked to tumour biology and prognosis.

Future Trends and Potential Uses

Advancements in MSI instrumentation and sample preparation are expected to yield subcellular resolution, extended metabolome coverage and faster acquisition speeds. Integration with digital pathology and machine learning algorithms will enhance biomarker discovery and clinical decision support. Combined multimodal imaging approaches could further refine molecular phenotyping of ocular tumours and other FFPE specimens.

Conclusion

This study demonstrates that both MALDI and DESI MSI can be successfully applied to FFPE uveal melanoma tissues, delivering spatially resolved molecular profiles that distinguish tumour from normal ocular tissue. The approach holds promise as a complementary pathology tool for diagnosis and prognostic evaluation in clinical oncology.