Metabolite Analysis and Histology on the Exact Same Tissue: Comprehensive Metabolomic Profiling and Metabolic Classification of Prostate Cancer

Abstract

We report a method of metabolomic profiling of intact tissue based on molecular preservation by extraction and fixation (mPREF) and high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). mPREF extracts metabolites by aqueous methanol from tissue biopsies without altering tissue architecture and thus conventional histology can be performed on the same tissue. In a proof-of-principle study, we applied dansylation LC-MS to profile the amine/phenol submetabolome of prostate needle biopsies from 25 patient samples derived from 16 subjects. 2900 metabolites were consistently detected in more than 50% of the samples. This unprecedented coverage allowed us to identify significant metabolites for differentiating tumor and normal tissues. The panel of significant metabolites was refined using 36 additional samples from 18 subjects. Receiver Operating Characteristic (ROC) analysis showed area-under-the-curve (AUC) of 0.896 with sensitivity of 84.6% and specificity of 83.3% using 7 metabolites. A blind study of 24 additional validation samples gave a specificity of 90.9% at the same sensitivity of 84.6%. The mPREF extraction can be readily implemented into the existing clinical workflow. Our method of combining mPREF with CIL LC-MS offers a powerful and convenient means of performing histopathology and discovering or detecting metabolite biomarkers in the same tissue biopsy.

Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status

“…Altogether, 136 metabolites including different lipid species were identified (summary see Additional file 1: Table S1). We observed, as others [24], variation in the total amount of metabolites in the extracted tissues most likely related to sample size and composition of tissues. Therefore, we used relative intensities of metabolites in all data sets.…”

“…Like in those two studies we observed an increase in levels of uracil in PC. Another interesting approach of metabolomic profiling of intact tissue was presented by Huan et al [24]; an approach based on molecular preservation by extraction and fixation and high-performance chemical isotope labeling LC-MS. They proposed a subset of five metabolites, including, adenosine monophosphate, uracil and spermidine, significant in comparison between PC and normal samples.…”

Metabolomics Biomarkers of Prostate Cancer: A Systematic Review

“…A relatively stringent validation approach using three independent sample sets was employed by Huan and colleagues, although sample sizes were small [42]. For biomarker discovery, they used a set of 12 non-cancerous tissue samples and 13 PCa tissue samples (10 samples with >30% cancer proportion) obtained ex vivo by core biopsy from 16 subjects after prostatectomy.…”

“…Some metabolites were independently reported by different groups. In regard to PCa diagnosis, uracil [28,42,45,47] and proline [28,44,46,47] were most frequently reported as tissue biomarkers and uracil was independently validated in one study. In addition, there was some evidence for glycine, kynurenine, glycerol-3-phosphate, leucine, choline and citrate as potential diagnostic markers of PCa.…”

Small-Molecule (Micro)arrays for Sensitive Multiplex Metabolites Detection by Surface Plasmon Resonance Imaging

“…For example, the measurement of small-molecule metabolites is of great importance due to the promise of linking the metabolite profiles to disease states. 6 Till now, the identification and quantification of metabolites are being mainly driven by the development of nuclear magnetic resonance [7][8][9] (NMR) and mass spectrometry 10 (MS) methodologies. These powerful tools are promising and will continue to generate metabolite profiles that can benefit biomarker discovery and medical diagnostic.…”