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Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings

Hiroyuki Katayama12, Sophie Paczesny13, Ross Prentice4, Aaron Aragaki4, Vitor M Faca1, Sharon J Pitteri1, Qing Zhang1, Hong Wang1, Melissa Silva1, Jacob Kennedy1, Jacques Rossouw5, Rebecca Jackson6, Judith Hsia7, Rowan Chlebowski8, JoAnn Manson9 and Samir Hanash1*

Author Affiliations

1 Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Fairview Avenue North, Seattle, WA 98109, USA

2 Laboratory of Core Technology, Eisai Co. Ltd, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan

3 Department of Pediatrics, University of Michigan, Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA

4 Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Fairview Avenue North, Seattle, WA 98109, USA

5 Women's Health Initiative Branch, National Heart, Lung, and Blood Institute, Rockledge Dr., Bethesda, MD 20892, USA

6 Division of Endocrinology, Ohio State University, Dodd Dr., Columbus, OH 43210, USA

7 AstraZeneca LP, Concord Pike, Wilmington, DE 29850, USA

8 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, W. Carson Street, Torrance, CA 90502, USA

9 Brigham and Women's Hospital, Harvard Medical School, Boylston Street, Boston, MA 02215, USA

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Genome Medicine 2009, 1:47  doi:10.1186/gm47

Published: 29 April 2009

Abstract

Background

The availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.

Methods

Five independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.

Results

Of 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.

Conclusions

CEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and fracture.

Clinical Trials Registration

ClinicalTrials.gov identifier: NCT00000611