Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens

Jing Chen, Shadi Toghi Eshghi, George Steven Bova, Qing Kay Li, Xingde Li, Hui Zhang

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number18
JournalClinical Proteomics
Volume10
Issue number1
DOIs
StatePublished - 2013

Fingerprint

Epithelium
Tissue
Cathepsin L
Cell Adhesion Molecules
Proteins
Enzyme-Linked Immunosorbent Assay
Staining and Labeling
Prostate
Neoplasms
Color
Tumors
Histology
Proteomics
Reading
Assays
Throughput
Epithelial Cell Adhesion Molecule
Epithelial Cells
Pathologists

Keywords

  • Cancer
  • Computer-aided classification
  • Epithelium
  • Stroma

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine
  • Clinical Biochemistry

Cite this

Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens. / Chen, Jing; Eshghi, Shadi Toghi; Bova, George Steven; Li, Qing Kay; Li, Xingde; Zhang, Hui.

In: Clinical Proteomics, Vol. 10, No. 1, 18, 2013.

Research output: Contribution to journalArticle

@article{87082ceca60c412685b90e421c67b6f3,
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.",
keywords = "Cancer, Computer-aided classification, Epithelium, Stroma",
author = "Jing Chen and Eshghi, {Shadi Toghi} and Bova, {George Steven} and Li, {Qing Kay} and Xingde Li and Hui Zhang",
year = "2013",
doi = "10.1186/1559-0275-10-18",
language = "English (US)",
volume = "10",
journal = "Clinical Proteomics",
issn = "1542-6416",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens

AU - Chen, Jing

AU - Eshghi, Shadi Toghi

AU - Bova, George Steven

AU - Li, Qing Kay

AU - Li, Xingde

AU - Zhang, Hui

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - Cancer

KW - Computer-aided classification

KW - Epithelium

KW - Stroma

UR - http://www.scopus.com/inward/record.url?scp=84903713636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84903713636&partnerID=8YFLogxK

U2 - 10.1186/1559-0275-10-18

DO - 10.1186/1559-0275-10-18

M3 - Article

C2 - 24289299

AN - SCOPUS:84903713636

VL - 10

JO - Clinical Proteomics

JF - Clinical Proteomics

SN - 1542-6416

IS - 1

M1 - 18

ER -