Comprehensive metabolite profiling of FFPE tissue to analyze metabolic changes in colorectal cancer.

Abstract

e15716 Background: Although fresh-frozen samples are currently the gold standard when performing mass spectrometry-based metabolomics, clinical workflows commonly produce formalin-fixed paraffin-embedded (FFPE) tissues. Fixation in formalin and embeddement in paraffin offers a number of advantages, such as mitigating the risks of infectious agents and preserving the architectural components of the tissue. The latter is important for pathological assesement of cancer, where changes in tissue architecture can be used for diagnosis and to guide treatment decisions. Methods: A concern of using FFPE for metabolomics is that it chemically modifies metabolites and lipids. Thus, while metabolomics data can be generated from FFPE specimens, there remains a major question about its reliability. In this study, we optimized a sample preparation method for use in FFPE metabolomics, then validated the approach on 12 tissue samples from colon cancer patients comparing the results to healthy nearby adjacent tissue (NAT). The analysis was performed on matched fresh frozen and FFPE tissues. By comparing the data, we establish a panel of metabolites and lipids that can be reliably profiled from FFPE tissues by using our workflow. Results: A total of 946 unique metabolites and lipids were measured from FFPE samples using our next-generation metabolomics platform. Across all assays, the coefficient of variation (CV) values were less than 10%. More than 50% of the unique metabolites and lipids from FFPE samples were also identified in matched fresh-frozen tissues. Molecules measured from both tissue types spanned multiple chemical classes ranging from fatty acids to central carbon metabolites. Next, changes in metabolite abundance across cancer and NAT tissues were used to assess the reliability of our FFPE metabolomics workflow in producing biologically relevant findings. Among the metabolites and lipids that were measured in both platforms, a large fraction showed consistent fold changes between fresh frozen and FFPE specimens. As an example, metabolites in glycolysis and the TCA cycle were found to be altered with a similar statistical magnitude in both fresh frozen and FFPE samples. These results are consistent with expected changes associated with the Warburg effect. Conclusions: This study reveals that metabolite profiling in FFPE tissues can effectively identify biologically significant compounds and pathways, offering a new tool for discovery research in cancer.