Technical Report

Laboratory Investigation (2015) 95, 561–571; doi:10.1038/labinvest.2015.37; published online 2 March 2015

Critical roles of specimen type and temperature before and during fixation in the detection of phosphoproteins in breast cancer tissues

Sibylle Gündisch1,5, Laura Annaratone2,5, Christian Beese1, Enken Drecol1, Caterina Marchiò2,3, Elena Quaglino4, Anna Sapino2,3, Karl-Friedrich Becker1 and Gianni Bussolati2

  1. 1Institute of Pathology, Technische Universität München, Trogerstrasse, Munich, Germany
  2. 2Department of Medical Sciences, University of Turin, Via Santena, Turin, Italy
  3. 3Pathology Service, Azienda Ospedaliera Città della Salute e della Scienza di Torino, Via Santena, Turin, Italy
  4. 4Department of Molecular Biotechnologies and Health Sciences, University of Turin, Via Nizza, Turin, Italy

Correspondence: Professor Dr K-F Becker, PhD, Technische Universität München, Institute of Pathology, Trogerstrasse18, Munich, D-81675, Germany. E-mail:; Professor G Bussolati, MD, Department of Medical Sciences, University of Turin, Via Santena 7, Turin 10126, Italy. E-mail:

5These authors contributed equally to this work.

Received 9 October 2014; Revised 9 December 2014; Accepted 31 December 2014
Advance online publication 2 March 2015



The most efficient approach for therapy selection to inhibit the deregulated kinases in cancer tissues is to measure their phosphorylation status prior to the treatment. The aim of our study was to evaluate the influence of pre-analytical parameters (cold ischemia time, temperature before and during tissue fixation, and sample type) on the levels of proteins and phosphoproteins in breast cancer tissues, focusing on the PI3 kinase/AKT pathway. The BALB-neuT mouse breast cancer model expressing HER2 and pAKT proteins and human biopsy and resection specimens were analyzed. By using quantitative reverse phase protein arrays (RPPA), 9 proteins and 16 phosphoproteins relevant to breast cancer biology were assessed. Cold temperatures before and during fixation resulted in a marked improvement in the preservation of the reactivity of biological markers (eg, ER, HER2) in general and, specifically, pHER2 and pAKT. Some phosphoproteins, eg, pHER2 and pAKT, were more sensitive to prolonged cold ischemia times than others (eg, pS6RP and pSTAT5). By comparing the phosphoprotein levels in core needle biopsies with those in resection specimens, we found a marked decrease in many phosphoproteins in the latter. Cold conditions can improve the preservation of proteins and phosphoproteins in breast cancer tissues. Biopsies≤1 mm in size are the preferred sample type for assessing the activity of deregulated kinases for personalized cancer treatments because the phosphoprotein levels are better preserved compared with resection specimens. Each potential new (phospho)protein biomarker should be tested for its sensitivity to pre-analytical processing prior to the development of a diagnostic assay.