Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Metabolic Profiling of Formalin-Fixed Paraffin-Embedded Tissues Discriminates Normal Colon from Colorectal Cancer.

Author(s): Arima K, Lau MC, Zhao M, Haruki K, Kosumi K, Mima K, Gu M, Väyrynen JP, Twombly TS, Baba Y, Fujiyoshi K, Kishikawa J, Guo C, Baba H, Richards WG, Chan AT, Nishihara R, Meyerhardt JA, Nowak JA, Giannakis M, Fuchs CS, Ogino S

Publication: Mol Cancer Res, 2020, Vol. 18, Page 883-890

PubMed ID: 32165453 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared metabolic profiles in matched snap-frozen and formalin-fixed paraffin-embedded (FFPE) tumor and normal adjacent colorectal tissue. Matched FFPE and fresh frozen specimens were collected from 11 patients undergoing radical colectomies (processing details not provided). Tissue blocks were sectioned and stained with hematoxylin and eosin (H&E) to assess tumor cell percentage (4 µm sections) and for metabolomics analysis (20 µm sections). Metabolites were extracted from frozen specimens by incubation in 80% methanol at room temperature for 4 hours and centrifugation at 14,000 x g for 10 minutes. Supernatants were collected and stored at -80°C until analysis. Metabolites were extracted from FFPE specimens by incubation in 80% methanol at 70°C for 45 minutes without deparaffinization, storage on ice for 15 minutes, and then centrifugation at 14,000 x g for 10 minutes at 4°C. Supernatants were transferred to new tubes and placed on ice for 10 minutes followed by centrifugation at 14,000 x g for 5 minutes at 4°C and storage at -80°C until analyses. Metabolite profiling was performed by ultrahigh performance liquid chromatography (UHPLC).

   Summary of Findings:

   Overall, a total of 539 metabolites were detected with 197 (37%) identified in both FFPE and snap-frozen specimens, 339 in only snap-frozen specimens, and three in only the FFPE specimens. There was a very weak negative correlation between metabolites molecular weight and amount detected in FFPE samples (r = -0.16) and a weaker correlation observed in snap-frozen tissues (r = -0.014). The detection rate of metabolites with a molecular weight >500 in FFPE samples was 9.5% compared to 21.5% in snap-frozen specimens (P<0.001). Principal component analysis of the 200 metabolites detected in the FFPE specimens and the 536 in the snap-frozen specimens revealed 193 metabolites that were differentially abundant between tumor and normal adjacent tissues (P<0.05), 15 of which were identified in both FFPE and snap-frozen specimens, 159 only in snap-frozen tissues and 19 only in FFPE tissues. Of the 15 metabolites differentially abundant between tumor and normal adjacent tissue, six were found at higher abundance in tumor specimens than normal adjacent tissue and nine at lower abundance. Metabolic pathway enrichment analysis of the differentially abundant metabolites in tumor tissues revealed four pathways (glyoxylate and dicarboxylate metabolism; arginine and proline metabolism; glycerophospholipid metabolism; and glycine, serine, and threonine metabolism) were enriched in both snap-frozen and FFPE specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	LC-MS or LC-MS/MS
   Lipid	LC-MS or LC-MS/MS
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen Formalin (buffered)
   Preaquisition	Diagnosis/ patient condition	Colorectal tumor tissue Normal adjacent tissue

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