[2089] Metabolites and Histology on Exactly the Same Biopsy Tissue

Dean A Troyer, Jeffrey R Shuster, Raymond Lance. Eastern Virginia Medical School, Norfolk, VA; Biomarker Factory, Durham, NC

Background: An increasing number of molecular biomarker assays are performed that frequently compete with histopathology for the same tissue. Small molecule metabolites are increasingly recognized as potential biomarkers of cancer and other diseases. We have developed a method to assay metabolites and perform histology on the exact same tissue called molecular preservation by extraction and fixation (mPREF).
Design: 18 gauge core-needle biopsies taken ex vivo from human prostatectomy samples were immediately placed in 1 mL of 80% aqueous methanol. After 12-24 hours, biopsy cores were transferred to formalin and processed by standard histologic methods. The methanol is retained for metabolite analysis. From more than 200 small molecules identified on a metabolomics platform, we have selected nine for quantification by liquid chromatography/mass spectroscopy (LC/MS): betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, and alanine. The molecules were chosen in part because their physical and chemical properties allows them to be run on a single LC/MS run.
Results: Preliminary results showed trends in tumor vs. no tumor for betaine, proline, uracil, cysteine, and N-acetylglucosamine when normalized to the biopsy surface area. Shown in the attached table are results for a total of 15 normal/tumor pairs.

Metabolite Quantitation
MetaboliteNo Tumor µM/cm2Tumor µM/cm2
betaine2.044.71
malate30.2333.92
proline42.9447.54
N-acetylaspartate2.792.99
uracil41.1350.56
xanthine3.904.38
cysteine877.901209.76
alanine948.75927.44
N-acetylglucosamine20.8628.97
Legend: µM/Cm2 = data normalized to surface area of the biopsy core

Histopathology using routinely processed paraffin embedded tissue following methanol treatment was satisfactory. IHC for basal keratin and p63 also performed well.
Conclusions: The results indicated that quantitation of metabolite molecules is feasible in 18 gauge core needle biopsies of prostate tissue. Studies are underway to determine the best means of normalizing metabolite results to overall size of individual biopsies and to volumes of tumor, non-tumor stroma, and normal glands. mPREF aligns well with existing histology workflows as the tissue is moved from alcohol to formalin. Once immersed in formalin, the tissue can be processed by any histology laboratory. The literature indicates that alcohol fixed tissues are suitable for FISH, IHC, and for extraction of RNA and DNA.
Category: Techniques

Tuesday, March 5, 2013 2:15 PM

Proffered Papers: Section E, Tuesday Afternoon

 

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