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

Tao Huan,Liang Li,Dean A Troyer

doi:10.1038/srep32272

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.

Highlights

We report a method of metabolomic profiling of intact tissue based on molecular preservation by extraction and fixation 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 conventional histology can be performed on the same tissue
The addition of an analytical step for quantifying the metabolite biomarker(s) is needed, which can be carried out using liquid chromatography multiple-reaction monitoring mass spectrometry (LC-MRM-MS), a technique routinely used for targeted metabolite quantification[6]
This proof-of-principle study illustrates that the combination of mPREF and CIL LC-MS can be a powerful tool for discovery of potential metabolite biomarkers of tumors or other diseases using clinical tissue samples that undergo conventional processing for histology

Summary

Introduction

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 conventional histology can be performed on the same tissue. One is related to the small sample amount available for analysis, limiting the detection of less abundant metabolites, the small diameter of these biopsies allows for consistent and complete extraction of the methanol extractable metabolites[7] Another challenge is normalizing the amount of different samples with varying sizes and compositions for comparative metabolite quantification[8]. We identified seven metabolites to distinguish normal and tumor samples with high sensitivity and specificity This proof-of-principle study illustrates that the combination of mPREF and CIL LC-MS can be a powerful tool for discovery of potential metabolite biomarkers of tumors or other diseases using clinical tissue samples that undergo conventional processing for histology

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Conclusion