Abstract 3203: Modulation of cellular phospholipids correlates with tumor regression grade and radio resistance in rectal cancer
Authors: Poynter Liam Robert , Mirnezami Reza , Soares Renata , McKenzie James , Patel Pranav , Peppa Nadia , Mirnezami Alexander , Kinross James , Darzi Ara , Takats Zoltan 
Published: August 2018

Abstract
Objectives
Progression through the adenoma-adenocarcinoma sequence has been shown to demonstrate hallmark shifts in the tissue lipidome. The incorporation of certain phospholipid subclasses into cellular membranes has previously been found to correlate with susceptibility to external stressors through alteration of membrane biodynamics. On this basis, it is hypothesized that modulation of cellular lipid modulation may influence radioresistance in rectal cancer. A desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform was utilized to demonstrate lipidomic shifts reflective of tumor heterogeneity and modulation of cellular lipids as a radioresistance mechanism.
Methods
26 specimens of fresh, human rectal cancer tissue were harvested at surgical resection from matched cohorts; 13 of each from those who had received RT, and those who proceeded straight to surgery (STS). DESI-MSI was performed using an Orbitrap Fourier transform mass spectrometer in negative ion mode (mass range 150-2000m/z). Lipid spectra acquired were spatially aligned to subsequent H&E staining of the tissue section in a chemometric toolbox in MatLab, annotating morphologically distinct regions of the tumour microenvironment. Statistical modeling was performed incorporating clinicopathological metadata. ANOVA identified spectral peaks with discriminating power between both RT and STS groups, and between clusters of patients across the range of Mandard tumor regression grades (TRG). Lipid classes and putative structures were determined using an existing MS/MS data reference library (MetLin).
Results
Significant clustering of lipid species was demonstrated in both RT and STS groups, for both adenocarcinoma and stroma. Leave-One-Out, all-pixels cross-validation (LOOCV) yielded high predictive values (&gt;97.9%) for RT vs. STS adenocarcinoma pixels. Cancers subjected to RT demonstrated significantly increased abundance of phospholipids compared to the STS group, notably phosphatidylglycerols (PG), phosphatidylethanolamines (PE) and phosphatidylserines (PS) on univariate analysis of all spectra, with a defined p/q value of 0.05 and negative log2 fold change of 0.1. Three-class PCA and discriminant analysis demonstrated clustering of samples according to Mandard TRG 1-2, TRG 3, and TRG 4-5. Clear separation of spectra was observed on LOOCV between all three classes. Compounds in the range 240-450m/z were implicated in this shift, in addition to the phospholipids as described.
Conclusion
This work has demonstrated a putative role for phospholipids and fatty acids in radioresistance, in a human rectal cancer cohort. DESI-MSI provides a unique foundation from which to continue ‘bottom-up' pathway analysis - utilizing both immunohistochemistry and tissue genotyping – which will assist in elucidating the possible mechanisms of response to radiotherapy in rectal cancer.
Citation Format: Liam Robert Poynter, Reza Mirnezami, Renata Soares, James McKenzie, Pranav Patel, Nadia Peppa, Alexander Mirnezami, James Kinross, Ara Darzi, Zoltan Takats. Modulation of cellular phospholipids correlates with tumor regression grade and radio resistance in rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3203.