EXTREME LOSS OF IMMUNOREACTIVE

PHOSPHOPROTEINS DURING ROUTINE

FIXATION OF PRIMARY BREAST CANCERIsabel 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=0.06p=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 resectionspecimen 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