Colon Cancer: Protein Biomarkers in Tissue and Body Fluids

Abstract

Purpose: The aim of the study was to validate the concept that potential biomarkers of colon cancer can be identified from body fluids. We concurrently tested multiple body fluid samples (urine, saliva, stool) and tumor tissue samples to identify concordant proteins. In our Institutional Review Board approved prospective pilot study, we explored the proteomic diagnosis of colon cancer by identification of concordant proteins in tumor and body fluids from the same patient. Methods: After informed consent, body fluids and tumor tissue samples were obtained from 20 patients who underwent colonoscopy for tumor biopsy at The Brooklyn Hospital. Tissue and body fluids from patients with negative biopsies (10 subjects) were controls. The samples were frozen and transported to New York University School of Medicine, Department of Pharmacology for analysis. Proteins were extracted from tissue and stool with high pressure, 35K PSI, (Barocycler, Pressure Biosciences, Woburn, MA). Protein separation from tumor, urine, saliva and stool was performed with either Two-dimensional gel electrophoresis or HPLC (high performance liquid chromatography). Control and tumor samples were reduced (DTT), alkylated (iodoacetamide) and trypsin digested. The protein digests were applied to MALDI (matrix-assisted laser desorption/ionization) target plates for MALDI MS (MALDI mass spectrometry) and MALDI MS MS (MALDI tandem) mass spectrometry (Axima TOF, Shimadzu Biotech, Columbia, MD). Protein were identified using NCBInr data base interrogation with Mascot© software (Matrix Science, London, UK). Results: The control sample MALDI MS spectra were all similar to each other as were the tumor sample spectra, however, there were clear differences between the MALDI MS control and sample spectra. Concordant proteins were identified from MALDI MS MS spectra in body fluids and tumor. Conclusion: The apparent tumor signature proteins present in the body fluids could allow us to develop non-invasive clinical diagnostic tests of colon carcinoma. To prove the validity of these findings a larger molecular epidemiolgy study will be undertaken.