Extreme loss of immunoreactive phosphoproteins during routine fixation of primary breast cancer Isabel Pinhel, Fiona MacNeill, Margaret Hills, Simone Detre, Janine Salter, Ian E Smith, Mitch Dowsett.

1. Extreme loss of
immunoreactive
phosphoproteins during
routine fixation of primary
breast cancer

2. Background
• Following surgical resection, breast cancer specimens are routinely X-
rayed for evaluation of margin clearance and fixed in formalin.
• Very few studies have investigated whether the time elapsed between
surgical resection and actual tissue fixation impact on
immunohistochemically measured biomarkers, including phosphorylated
proteins.
• The impact of time to fixation on changes in biomarker expression needs
to be addressed, especially in pre-surgical trials (POETIC and MAPLE).
• AIM: To characterize the changes in commonly assessed biomarkers
before and after tumour specimen fixation.

3. Methods
• The variation in expression of Ki67, ER, PgR, HER2, p-Akt and p-Erk1/2 were
investigated by immunohistochemistry.
• H scores were used for all markers, except for Ki67 (percentage of cells
staining) and HER2 (IHC categories 0, 1+, 2+ and 3+).
Study Design

4. Results
• 28 patients were studied for which 21 pairs of samples A,B&C were available.
• Median time elapsed between core-cuts A and B was 30 minutes.
• No differences were found between samples A&B for expression of all markers
and there were no correlations with time elapsed (all p>0.20).
•Comparisons with resection made with mean expression values for core-cuts
A&B: no significant differences in expression of Ki67, ER, PgR and HER2.
p=0.19
p=0.01 p=0.89
p=0.06
n=1
n=2
n=3
n=10
n=3
n=2
n=1

5. Results
Resection C
Core-cut B
Core-cut A Core-cut A
Core-cut B
Resection C
p<0.0001
p<0.0001
R=0.68
p=0.0008
6/21 resections
virtually negative
R=0.52
p=0.015
8/21 resections
virtually negative

6. Conclusions
• Time from resection to core cut fixation had no significant impact on
expression of Ki67, HER2, ER, PgR, p-Akt or p-Erk1/2.
• Extreme loss of phospho-staining was noted after routine fixation of most
resection specimens.
• May be attributable to a more rapid penetration of formalin in core-cuts
(smaller volume). Further supported by greater phospho-staining loss in
mastectomy specimens.
• Resection specimens are unreliable for assessment of p-Akt and p-Erk1/2
(possibly other phosphoproteins) unless specific procedures to allow proper
preservation are in place.
• Implications for the measurement of these proteins in research studies and
may impact on clinical management of breast cancer patients.

7. THANK YOU