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
Isabel Pinhel1,2, Fiona MacNeill1, Margaret Hills1, Simone Detre1, Janine Salter1,3, Ian E Smith1, Mitch Dowsett1.
1The Royal Marsden Hospital, 2The Institute of Cancer Research and 3Breakthrough Breast Cancer Research Centre, London, United Kingdom.
Figure 1. POETIC Trial Pilot study design
RESULTS
Figure 3. Expression of p-Akt (panel A) and p-Erk1/2 (panel B) by IHC in core-cuts (mean samples A,B)
and resection (sample C) according to type of surgical specimen.
n=1
n=2
n=3
n=10
n=3
n=2
p=0.19
p=0.06
p=0.89
p=0.01
Figure 2. Expression of Ki67 (panel A), HER2 (panel B), ER (panel C) and PgR (panel D) in core-cuts
(mean samples A,B) and Resections (Samples C) by IHC.
Twenty-nine primary breast cancer specimens were available from 28 patients. Of
these 21 sets of paired samples (A, B & C) had sufficient tumour for biomarker
assessment. Median time between core-cuts A and B was 30 minutes (range 20-80
minutes).
No differences were found between samples A&B for Ki67, ER, PgR, p-Akt, p-Erk1/2
and HER2 expression and there were no correlations with time elapsed between
core-cuts (all p>0.20). Comparisons with resection (sample C) were therefore made
with mean expression values for core-cuts A&B: no significant difference in
expression for Ki67, ER and PgR was noted, although a trend to a lower ER value in
sample C was seen (Figure 2). For HER2, 12/15 cases categorised as 1+ on at least
one of core-cuts A or B were scored 0 in the resection but did not impact on HER2
positive/negative status.
However, p-Akt and p-Erk1/2 H scores were highly significantly lower in sample C than core-
cuts A/B (Figures 3A and 3B, respectively; p<0.0001). Near complete absence of staining (H
score <5) occurred in 6/21 resections for p-Akt and 8/21 for p-Erk1/2, despite core-cuts A/B
values for some of these being among the highest. Nonetheless there was a significant
correlation between values in samples A/B and C for both phosphoproteins (R=0.68,
p=0.0008; R=0.52, p=0.015, respectively).
The mean difference in phosphoprotein staining between core-cuts A/B and sample C was
greater in mastectomy than lumpectomy specimens (Figures 4A and 4B) but only
significantly so for p-Erk1/2 (p=0.01).
Figure 4. Percent (%)
difference between
resection (sample C)
and core-cuts (mean
samples A,B) scores
for p-Akt (panel A)
and p-Erk1/2 (panel
B) according to type
of surgical specimen.
% difference defined
as (sample C – mean
samples A,B) x
100/mean samples
A,B.
Very few studies have investigated
whether the time between surgical
resection and tissue fixation impacts
on immunohistochemically
measured biomarkers including
phosphorylated proteins (1, 2).
These biomarkers are subject to
intense research scrutiny and it is
therefore important to investigate
the impact of time to fixation on
changes in biomarker expression
especially with the advent of
presurgical trials such as POETIC and
MAPLE.
Aim
To characterize the changes in
commonly assessed biomarkers
before and after tumour specimen
fixation.
Background
Core-cuts (14-gauge) were taken from the breast cancer immediately after resection (sample A) and after lumpectomy specimen X-ray or a
random time lapse for mastectomy specimens (sample B). Cores were formalin-fixed, paraffin-embedded and compared to the main resection
specimen (sample C) which was only fixed after sample B cores were taken (Figure 1).
Methods
The variation in expression of Ki67, ER, PgR, HER2, p-
Akt and p-Erk1/2 were investigated by
immunohistochemistry (IHC) using the following
antibodies: Clone MIB-1 (Dako), Clone 6F11,
(Novocastra), Clone 16 (Novocastra), HercepTest
(Dako), p-Akt Ser473 (Cell Signalling) and p-Erk1/2
Thr202/Tyr204 (Cell Signalling), respectively.
H scores were used for all markers, except for Ki67
(percentage of invasive cells staining) and HER2 (IHC
categories 0, 1+, 2+ and 3+ as per ASCO/CAP
guidelines). Comparisons of IHC scores between
samples A, B & C were by Wilcoxon Signed Rank test.
Spearman regression analyses were conducted
between the difference in expression between
samples A&B and the time elapsed between their
collection. Data from samples placed in RNAlater will
be published separately. P-values were two-sided and
significant if <0.05. Figure 1. Study design.
References
1. Baker AF et al. Clin Cancer Res. 2005; 11(12):4338-
4340.
2. Cecco LD et al. BMC Cancer 2009; 9(409):1-14.
3. Wolff AC et al. J Clin Oncol 2007;25(1):118-145.
4. Dowsett M et al. Cancer Epidemiol Biomarkers
Prev. 2001; 10(9):961-966.
5. Cuzick J et al. Cancer Res. 2009; 69(24),suppl:503s.
6. Gutierrez MC et al. J Clin Oncol. 2005;
23(11):2469-2476.
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. However
extreme loss of phospho-staining was noted after routine fixation of most main resection specimens.
• The differences between core-cut and main resection specimen are most likely attributable to rapid penetration of fixative: fixation
of small volume core-cuts will initiate rapidly after immersion in formalin whilst penetration of the larger volume main specimen will
be slower resulting in greater loss of biomarker expression. This is supported by the reduction in phospho-staining between cores and
main resection being more noticeable in mastectomy specimens. 5-10mm slicing is already recommended to obtain good quality
fixation for larger resections (3).
• For ER, the trend towards a difference (a significant difference has been reported previously) between cores and main resection
specimen are unlikely to lead to erroneous exclusion of patients from endocrine therapy (4). However if quantitative levels of ER
become important for prognosis or predicting treatment response (5) then optimal standardised fixation procedures are crucial.
• These findings have profound implications for the measurement of these important proteins in research studies as well as the
potential to impact on clinical management of breast cancer patients.
A B
C D A B
Acknowledgements
The authors would like to thank the contribution of
Dr. Ash Nerurkar and Dr. Peter Osin in the
histopathological discussions and Roger A’Hern for
support in the statistical analyses.
A
Core-cut A
Core-cut B
Resection C
B
Core-cut B
Resection C
Core-cut A
p<0.0001 p<0.0001