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Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum; a nested case-control study

Annemieke WJ Opstal-van Winden12, Esmeralda JM Krop3, Monica H Kåredal4, Marie-Christine W Gast25, Christian H Lindh4, Marina C Jeppsson4, Bo AG Jönsson4, Diederick E Grobbee1, Petra HM Peeters16, Jos H Beijnen27, Carla H van Gils1* and Roel CH Vermeulen13

Author Affiliations

1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

2 Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands

3 Institute for Risk Assessment Sciences, Utrecht University, Jenalaan 18D, 3584 CK Utrecht, The Netherlands

4 Department of Occupational and Environmental Medicine, Lund University, SE- 221 85 Lund Sweden

5 Central Hospital Pharmacy, Escamplaan 900, 2547 EX The Hague, The Netherlands

6 Department of Epidemiology, Public Health and Primary Care, Faculty of Medicine, Imperial College Norfolk Place, London W2 1PG, UK

7 Faculty of Science, Department of Pharmaceutical Sciences, Division of Biomedical Analysis, Section of Drug Toxicology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

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BMC Cancer 2011, 11:381  doi:10.1186/1471-2407-11-381

Published: 26 August 2011



Serum protein profiles have been investigated frequently to discover early biomarkers for breast cancer. So far, these studies used biological samples collected at or after diagnosis. This may limit these studies' value in the search for cancer biomarkers because of the often advanced tumor stage, and consequently risk of reverse causality. We present for the first time pre-diagnostic serum protein profiles in relation to breast cancer, using the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort.


In a nested case-control design we compared 68 women diagnosed with breast cancer within three years after enrollment, with 68 matched controls for differences in serum protein profiles. All samples were analyzed with SELDI-TOF MS (surface enhanced laser desorption/ionization time-of-flight mass spectrometry). In a subset of 20 case-control pairs, the serum proteome was identified and relatively quantified using isobaric Tags for Relative and Absolute Quantification (iTRAQ) and online two-dimensional nano-liquid chromatography coupled with tandem MS (2D-nanoLC-MS/MS).


Two SELDI-TOF MS peaks with m/z 3323 and 8939, which probably represent doubly charged apolipoprotein C-I and C3a des-arginine anaphylatoxin (C3adesArg), were higher in pre-diagnostic breast cancer serum (p = 0.02 and p = 0.06, respectively). With 2D-nanoLC-MS/MS, afamin, apolipoprotein E and isoform 1 of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4) were found to be higher in pre-diagnostic breast cancer (p < 0.05), while alpha-2-macroglobulin and ceruloplasmin were lower (p < 0.05). C3adesArg and ITIH4 have previously been related to the presence of symptomatic and/or mammographically detectable breast cancer.


We show that serum protein profiles are already altered up to three years before breast cancer detection.

Biomarkers; Breast cancer; Early diagnosis; 2D-nanoLC-MS/MS; Prospective; Proteomics; SELDI-TOF MS