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

Abstract

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.

Figure 1

Study design. (a) Murine breast cancer specimens were collected. The reference samples were immediately fixed in standard formalin (room temperature) (SF) or cold formalin (4 °C) (CF) and paraffin embedded. The ischemia samples were stored at room temperature or under vacuum at 4 °C during the ischemia time up to 24 h and were fixed in either standard formalin or cold formalin before paraffin embedding. (b) Human breast cancer specimens were collected. Biopsies were immediately fixed in standard formalin before paraffin embedding. The matched resection specimens were stored under vacuum at 4 °C for 20 h before preservation and fixed either in standard formalin or cold formalin before paraffin embedding. Proteins were analyzed by reverse phase protein array and/or western blot analysis.

Figure 2

Breast cancer specimens fixed alternatively in cold formalin (a, c) or in neutral-buffered formalin at room temperature and stained with hematoxylin & eosin. Figures a and b: breast cancer from BALB-neuT mice. (a) Time 0 specimens, fixed in cold formalin. (b) Specimen with cold ischemia time of 5 h at room temperature, followed by standard neutral-buffered formalin fixation at RT. Please notice that morphology is similar while, as shown in Figure 3, the preservation of phosphoprotein markers is dramatically different. (c and d) Human breast carcinoma (same case), fixed alternatively in cold formalin (c) or in formalin at room temperature (d). The histological features are not affected by the temperature of the fixative (magnification: 200 × ; insets in figures a and b: 400 × ).

Figure 3

Loss of phosphoprotein signal due to delayed preservation (murine samples). Murine breast cancer specimens were either fixed in standard formalin or cold formalin immediately after resection or after different ischemia time points with storage until preservation at room temperature or under vacuum at 4 °C. Protein levels were either determined by reverse phase protein arrays (a and b) or by western blot analysis (c) with the depicted antibodies. The reverse phase protein arrays data in (a) are shown as normalized signal intensities that were calculated by normalization to total protein (SYPRO Ruby staining). Each bar in the reverse phase protein arrays study reflects n=3 data points, besides setting ‘cold formalin, ischemia room temperature', time point 5 h reflects n=4 and time point 24 h reflects n=1. For the setting ‘standard formalin, the ischemia room temperature' time point 24 h is missing because it was not analyzable. In (b), two examples (pAKT and pHER2) are highlighted showing the advantage of cold formalin if specimens are kept at 4 °C during the cold ischemia duration. Data are shown as signal intensities in percent relative to the time point zero. Western blot data were normalized to the reference protein beta-Actin before quantification.

Figure 3

Loss of phosphoprotein signal due to delayed preservation (murine samples). Murine breast cancer specimens were either fixed in standard formalin or cold formalin immediately after resection or after different ischemia time points with storage until preservation at room temperature or under vacuum at 4 °C. Protein levels were either determined by reverse phase protein arrays (a and b) or by western blot analysis (c) with the depicted antibodies. The reverse phase protein arrays data in (a) are shown as normalized signal intensities that were calculated by normalization to total protein (SYPRO Ruby staining). Each bar in the reverse phase protein arrays study reflects n=3 data points, besides setting ‘cold formalin, ischemia room temperature', time point 5 h reflects n=4 and time point 24 h reflects n=1. For the setting ‘standard formalin, the ischemia room temperature' time point 24 h is missing because it was not analyzable. In (b), two examples (pAKT and pHER2) are highlighted showing the advantage of cold formalin if specimens are kept at 4 °C during the cold ischemia duration. Data are shown as signal intensities in percent relative to the time point zero. Western blot data were normalized to the reference protein beta-Actin before quantification.

Figure 4

Loss of phosphoprotein signal due to delayed preservation (human samples). Human breast cancer biopsies from six patients were immediately fixed in standard formalin, and matched resection specimens were either fixed in standard formalin or cold formalin. Protein expression was determined by reverse phase protein arrays with depicted antibodies. The reverse phase protein arrays data are depicted as normalized signal intensities, which were calculated by normalization to total protein (SYPRO Ruby staining).