title: Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens.
abstract: BACKGROUND
  The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content.
  
  
  OBJECTIVE
  It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens.
  
  
  METHODS
  Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist's reading.
  
  
  RESULTS
  We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results were obtained with normalization by both the computer estimated and pathologist estimated epithelium percentage.
  
  
  CONCLUSIONS
  Our results show that estimation of tissue epithelium percentage using our color-based segmentation method correlates well with pathologists' estimation of tissue epithelium percentages. The epithelium contents estimated by color-based segmentation may be useful in immuno-based analysis or clinical proteomic analysis of tumor proteins. The codes used for epithelium estimation as well as the micrographs with estimated epithelium content are available online.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Shadi
authors.initials: S
authors.lastname: Toghi Eshghi
authors.forename: George Steven
authors.initials: GS
authors.lastname: Bova
authors.forename: Qing Kay
authors.initials: QK
authors.lastname: Li
authors.forename: Xingde
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University, 1550 Orleans Street, Cancer Research Building II, Room 3M-03, Baltimore, MD 21231, USA. hzhang32@jhmi.edu.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang