title: Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach.
abstract: BACKGROUND
  Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.
  
  
  METHODS
  We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).
  
  
  RESULTS
  Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.
  
  
  CONCLUSIONS
  Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.
authors.affiliation: Cancer Care Centre, St George Hospital, Kogarah, Australia.|St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, Australia.|SEALS, Anatomical Pathology, St George Hospital, Kogarah, Australia.
authors.forename: Julia
authors.initials: J
authors.lastname: Beretov
authors.affiliation: Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW, Kensington, Australia.|School of Medical Sciences, UNSW, Kensington, Australia.
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Wasinger
authors.affiliation: SEALS, Anatomical Pathology, St George Hospital, Kogarah, Australia.|School of Medical Sciences, UNSW, Kensington, Australia.|Cancer Research Program, Kinghorn Cancer Centre and Garvan Institute of Medical Research, Darlinghurst, Australia.|School of Medicine and Health Sciences, University of Western Sydney, Campbelltown, Australia.
authors.forename: Ewan K A
authors.initials: EK
authors.lastname: Millar
authors.affiliation: Breast Surgery, St George Private Hospital, Kogarah, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Schwartz
authors.affiliation: Cancer Care Centre, St George Hospital, Kogarah, Australia.|St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, Australia.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Graham
authors.affiliation: Cancer Care Centre, St George Hospital, Kogarah, Australia.|St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, Australia.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000378:metabolism / Q000652:urine*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism* / Q000473:pathology / Q000652:urine*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D055088:Early Detection of Cancer; D005260:Female; D000091344:Filaggrin Proteins; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000652:urine; D008875:Middle Aged; D009367:Neoplasm Staging; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000652:urine; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D008869:Microtubule-Associated Proteins; C515549:MAST4 protein, human; D017346:Protein Serine-Threonine Kinases