Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues

J Natl Cancer Inst. 2012 Dec 5;104(23):1815-24. doi: 10.1093/jnci/djs438. Epub 2012 Oct 22.


Background: Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues.

Methods: A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided.

Results: We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series.

Conclusions: Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • A Kinase Anchor Proteins / analysis
  • Biomarkers, Tumor / analysis*
  • Biopsy, Large-Core Needle
  • Breast Neoplasms / chemistry*
  • Breast Neoplasms / surgery
  • Cold Ischemia*
  • Confounding Factors, Epidemiologic
  • False Negative Reactions
  • Female
  • Fixatives
  • Fluorescent Antibody Technique
  • Formaldehyde
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / analysis
  • Ki-67 Antigen / analysis*
  • Mastectomy, Segmental
  • Matched-Pair Analysis
  • Minor Histocompatibility Antigens
  • Prospective Studies
  • Proto-Oncogene Proteins / analysis
  • Receptor, ErbB-2 / analysis*
  • Receptors, Estrogen / analysis*
  • Receptors, Progesterone / analysis*
  • Research Design
  • Time Factors


  • A Kinase Anchor Proteins
  • AKAP13 protein, human
  • Biomarkers, Tumor
  • Fixatives
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Ki-67 Antigen
  • Minor Histocompatibility Antigens
  • Proto-Oncogene Proteins
  • Receptors, Estrogen
  • Receptors, Progesterone
  • Formaldehyde
  • ERBB2 protein, human
  • Receptor, ErbB-2