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01.10.2010 | Research article | Ausgabe 5/2010 Open Access

Breast Cancer Research 5/2010

Extreme loss of immunoreactive p-Akt and p-Erk1/2 during routine fixation of primary breast cancer

Breast Cancer Research > Ausgabe 5/2010
Isabel F Pinhel, Fiona A MacNeill, Margaret J Hills, Janine Salter, Simone Detre, Roger A'Hern, Ashutosh Nerurkar, Peter Osin, Ian E Smith, Mitch Dowsett
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​bcr2719) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

IP coordinated sample collection, conducted immunohistochemical determination and scoring of markers Ki67, p-Akt and p-Erk1/2, acquired data on clinical-pathological parameters, conducted statistical analysis and interpretation of data for all markers and assisted in drafting the manuscript; FM selected eligible patients, collected samples in theatre and assisted in drafting the manuscript; MH sectioned tissue blocks and conducted immunohistochemical determination and scoring of markers ER, PgR and HER2; SD assisted in the immunohistochemical determination of phospho-markers; RA supervised the statistical analysis and assisted in drafting the manuscript; AN and PO supervised the histopathological processing of samples and contributed towards the histopathological discussions; JS provided training and supervision of the immunohistochemical tests; IS contributed to the design of the study and its interpretation; and MD conceived and designed the study, interpreted data and drafted the manuscript. All authors approved the final manuscript.



Very few studies have investigated whether the time elapsed between surgical resection and tissue fixation or the difference between core-cut and excision biopsies impact on immunohistochemically measured biomarkers, including phosphorylated proteins in primary breast cancer. The aim of this study was to characterise the differences in immunoreactivity of common biomarkers that may occur (1) as a result of tissue handling at surgery and (2) between core-cuts and resected tumours.


Core-cuts taken from surgical breast cancer specimens immediately after resection (sample A) and after routine X-ray of the excised tumour (sample B) were formalin-fixed and paraffin-embedded and compared with the routinely fixed resection specimen (sample C). The variation in immunohistochemical expression of Ki67, oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor 2 (HER2), p-Akt and p-Erk1/2 were investigated.


Twenty-one tissue sets with adequate tumour were available. Median time between collection of core-cuts A and B was 30 minutes (range, 20 to 80 minutes). None of the markers showed significant differences between samples A and B. Similarly, Ki67, ER, PgR and HER2 did not differ significantly between core-cuts and main resection specimen, although there was a trend for lower resection values for ER (P = 0.06). However, p-Akt and p-Erk1/2 were markedly lower in resections than core-cuts (median, 27 versus 101 and 69 versus 193, respectively; both P < 0.0001 [two-sided]). This difference was significantly greater in mastectomy than in lumpectomy specimens for p-Erk1/2 (P = 0.01).


The delay in fixation in core-cuts taken after postoperative X-ray of resection specimens has no significant impact on expression of Ki67, ER, PgR, HER2, p-Akt or p-Erk1/2. However, extreme loss of phospho-staining can occur during routine fixation of resection specimens. These differences are likely attributable to suboptimal fixation and may have major repercussions for clinical research involving these markers.
Authors’ original file for figure 1
Authors’ original file for figure 2
Authors’ original file for figure 3
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