High‐resolution MALDI‐FT‐ICR MS imaging for the analysis of metabolites from formalin‐fixed, paraffin‐embedded clinical tissue samples

Abstract

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.

Prognostic Metabolite Biomarkers for Soft Tissue Sarcomas Discovered by Mass Spectrometry Imaging

“…The more limited scope of a biomarker study, in which the goal is to find mass spectral features that differentiate between patient groups, in this instance between poor and good survival, does not require maintenance of the system’s original metabolic state, only that the mass spectral features consistently differentiate between the groups. This work and previous work using MALDI of FFPE tissues [27], and DESI based analyses [28, 29], have clearly demonstrated the utility of metabolic signatures for differentiating between patient groups. For clinical research in which metabolic integrity needs to be kept as close as possible to the original physiologic state flash frozen needle biopsies are recommended.…”

“…Within 2 ppm difference, the molecule at m/z 160.8417 was previously reported by Buck et al at m/z 160.8420 in colon cancer and assigned as carnitine [27]. The molecule observed at m/z 241.0118 was assigned as inositol cyclic phosphate using the Human Metabolome Database with an error of 0.32 ppm and matching the isotope pattern (Supplementary Figure 3).…”

“…Both matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) have been used to acquire metabolite MSI datasets of cancer tissues. For example MALDI MSI may be used to visualize metabolites and metabolic pathways in fresh frozen tumor tissues [26] and identify biomarkers, including in formalin fixed paraffin embedded tissues and tissue microarrays [27]; DESI has been used to identify metabolite biomarkers for discriminating between different brain and breast tumors [28, 29]. Combining tumor-specific metabolite signatures with patient follow-up data has revealed prognostic biomarkers [27].…”

“…For example MALDI MSI may be used to visualize metabolites and metabolic pathways in fresh frozen tumor tissues [26] and identify biomarkers, including in formalin fixed paraffin embedded tissues and tissue microarrays [27]; DESI has been used to identify metabolite biomarkers for discriminating between different brain and breast tumors [28, 29]. Combining tumor-specific metabolite signatures with patient follow-up data has revealed prognostic biomarkers [27]. Though less established for clinical research than protein MSI, these studies indicate that the metabolite signatures obtained via MSI can also be used to differentiate between different tumor entities and between patient groups.…”

Metabolic Profiling in Formalin-Fixed and Paraffin-Embedded Prostate Cancer Tissues

“…However, MALDI-MS imaging has very low metabolome coverage and requires a matrix for co-crystallization with analytes, which interferes in the low mass range (<500 Da). Moreover, the suboptimal resolution of the MALDI instruments (11) could limit the ability to delineate the tumor and normal tissue area in heterogeneous and multifocal tumors such as prostate cancer. These promising albeit incomplete feasibility studies prompted us to perform a controlled, comprehensive analysis of the classes of metabolites detectable in FFPE material.…”