A prospective comparison of ER, PR, Ki67 and gene expression in paired sequential core biopsies of primary, untreated breast cancer

To re-evaluate the consistency of staining between core biopsy and breast cancer resection specimens, 41 Caucasian women with histologically proven stage I or II primary breast cancer gave written, informed consent to participation under the auspices of the Tayside Local Research Ethics Committee (Fig. 1). Patients taking hormone replacement therapy (HRT) or oral contraception were excluded; 26 women were postmenopausal and 15 women premenopausal. FFPE paired biopsies at the time of diagnosis (core biopsy) and 2 weeks later at resection (from the surgical resected specimen taken at pathology cut up) were examined. The resected tumor was delivered fresh to the pathology laboratory (in under 30 min), the margins inked, the specimen sliced at 5–10 mm intervals and fixed overnight in neutral buffered formalin prior to final dissection and block selection. Core biopsies taken at the time of diagnosis were compared with tissue microarrays (TMA) made from the resected specimen. For the TMA, 6 × 0.6 mm cores of invasive disease were selected to avoid prior biopsy sites by a specialist breast pathologist. No therapeutic intervention occurred between the two sampling time points.

Immunohistochemistry was performed on 4 μm sections of FFPE tissues using standard methodologies [27] using primary antibodies for estrogen receptor alpha (ER) antibody 6 F11 (1:200; Novocastra Laboratories Ltd), progesterone receptor (PR) antibody clone 16 (1:800; Novocastra Laboratories Ltd) and NCL-L-Ki67-MM1 (Anti-Ki67, monoclonal antibody, Leica Microsystems). Negative controls (lacking primary antibody) were performed for all staining runs.

Samples were scored independently to agreement by two authors (PGR and LBJ) for an average of the cores scored- usually all six on the TMA- using the Quickscore method assessing intensity and proportion (hence for example 6 × 2 reflects % cells staining x intensity) for ER, PR [28] and using a cut off of 20 % for Ki67 [9].

To eliminate potential tissue handling, fixation and processing differences, core biopsies were taken 2 weeks apart (n = 24) from consenting patients under a separate Tayside Local Research Ethics Committee permission as control tissues from a pre-surgical metformin trial [12]. All tissues were placed immediately in neutral buffered formalin and following overnight fixation processed to paraffin blocks at a single laboratory.

For the paired cores, immunohistochemistry for ER and PR was performed as described above and scored using the Quickscore method [28] and independently by the Allred method [29]. Immunohistochemistry was conducted blinded to the clinical data and scored by a single specialist breast pathologist (LBJ). Following light microscopy review, slides were scanned into a virtual microscopy format using an Aperio ScanScope XT TM (Aperio Technologies, Vista, Ca., USA) at the x40 objective utilizing standard compression methodology.

The Ki67 index (percentage of nuclear positive cells) per invasive tumor was calculated using manual annotation of the virtual microscopy slide by means of a Wacom Bamboo Pen & Touch tablet device (Wacom Corporation, Saitama Japan) within the WebScope environment (version 10.2.0.2319) of the Aperio Spectrum Plus system version 10.2.2.2317. The annotations were assessed by the Aperio IHC nuclear Algorithm version 10. Only invasive tumor cells were assessed; great care was taken to exclude normal epithelial, in situ epithelial, stromal and inflammatory elements. A mean 5600 nuclei (range 601–39,788) per invasive tumor was assessed to obtain the Ki67 index. A minimum of 1000 invasive tumor cells was examined except for one pre-treatment and one post-treatment core (601 and 825 cells respectively).

For RNA microarray analysis, FFPE core biopsy samples from 12 otherwise unselected patients from the control arm of a preoperative clinical trial [12] were examined. These represent 12 pairs of the 24 paired samples from the immunohistochemistry comparison between paired core biopsies where there was sufficient tumour material in the core for RNA extraction and analysisconfirmed on a Haematoxylin and Eosin slide was confirmed by a specialist breast pathologist (LBJ). RNA extraction and Breast Cancer Disease-Specific Array (DSA) gene expression profiling was performed as previously described [12].

Data were corrected for background noise, summarized and normalized using RMA in Partek® Genomics Suite™ software, 6.5 beta © 2009 (Partek Inc., St. Louis, MO, USA). Principle component analysis (PCA) revealed that the main variance associated with the first principle component was array quality. An additional transformation based in singular value decomposition was performed to remove this technical variation. The data was subsequently log2 transformed.

In tumor samples from 41 women (Table 1) there was a clinically significant change (loss) of ER between the diagnostic core and the resection specimen in cancers from 4/41 (10 %) women across the threshold for adjuvant endocrine therapy of a Quickscore of 4/18, although the ER score changed in a further 18 women, but would not change the clinical impact (Fig. 2 and Table 2). Loss of ER was identified in 3/15 (20 %) premenopausal women and PR changes occurred in both premenopausal and postmenopausal women. For Ki67 (Fig. 3), there was also a loss of staining in assessable samples to below 20 % in 1/15 (7 %) premenopausal and 4/25 (16 %) postmenopausal women and a rise above 20 % in 2/15 (14 %) premenopausal and 1/25 (4 %) postmenopausal women; Ki67 was not assessable on one core.

In paired core biopsies from 17 women, using the Quickscore method, in 2/17 (12 %) there was reduced expression of ER in the second core biopsy and in 3/17 (18 %) increased expression of ER in the second core (Fig. 2). In none of these five patients would the change in ER have led to a therapeutically important switch whether the Quickscore or Allred score was applied.

For PR in 6/17 (35 %) women there was reduced expression of PR in the second core biopsy and in 3/17 (18 %) increased expression of PR in the second core. In none of these nine patients would the change in PR have led to a therapeutically important switch whether the Quickscore or Allred score was used.

Ki67 was available on 23 paired core biopsies (including the 17 for ER and PR pairs). Using 20 % as a cut off [9], 5/23 (22 %) tumor samples would have crossed the 20 % threshold between the paired samples: 2/23 (9 %) patients would have crossed from above to below 20 % and tumor samples from a further 3/23 (13 %) patients from below to above 20 %. However, using 13.25 % as the cut off [10], only 1/23 (4 %) tumors would have crossed the 13.25 % boundary comparing the two cores (Fig. 3).

By GSA (Fig. 4), the differences between the two biopsies suggested changes in pathways involving myc, apoptosis and p53 amongst others in the second biopsy compared with the first. Several elements of cellular metabolism and immunological pathways were identified as overexpressed (Fig. 5a) in the second biopsy as compared with the first whereas, the Rho, integrin and potentially significantly the ER pathways were relatively underexpressed (Fig. 5b) in the second core biopsy.

IPA set in context a number of gene expression changes among which pathways involving PI3K, MEKK and IGF-1 may be of particular relevance in the setting of breast cancer.

To date there have been multiple comparisons of core biopsies and surgical resections for ER, PR, Ki67 for tumor grade and HER2 (Table 3) demonstrating a mean concordance of 92.4 % for ER (Fig. 6a), 84 % for PR (Fig. 6b) and 67.4 % for Ki67 (Fig. 6c), comparable to the data presented here. Reporting comparisons between ER, Ki67 and other biomarkers in this setting may be potentially misleading for well-rehearsed reasons [1, 5, 30] minimised by the use of (paired) core biopsies and consistent tissue handling. We revisited whether the changes in ER might be secondary to changes in circulating estradiol, confirming plausible evidence for premenopausal women [23], but likely due to tissue handling and processing at least in postmenopausal women [1, 5, 25].

Paired core biopsies of primary breast cancer before/after drug therapy has become popular [12, 13, 16], although quality standards for Ki67 have been of concern [9, 10]. In a trial setting [12], variations in specimen processing, specimen handling, laboratory processing and immunohistochemical staining and scoring were minimised, although patient selection (ER positive T1c and T2 cancers) occurred.

Slight variation of immunohistochemical scoring of ER and PR between paired cores, potentially attributable to geographic targeting differences over time, rarely crossed the boundary for clinical decision making. For Ki67, the cut point was key: at 20 % [9], 5/23 (22 %) paired tumor samples would have crossed the threshold, compared with only 1/23 (4 %) tumors using 13.25 %, in concordance with expert opinion [10] confirming a Ki67 boundary of 13.25 % is appropriate when seeking evidence of a drug effect.

While intra-tumoral heterogeneity has been considered elsewhere [26], the single cores at each time point may reflect clinical reality in small cancers for window of opportunity, pre-operative or neoadjuvant trials. Given the consensus, for a number of tumor types, that needle biopsy specimens result in reliable immunohistochemistry [1, 31], this study provides reassurance that immunohistochemical measurement of ER, PR and Ki67 from core biopsy pairs is consistent over 2 weeks.

By GSA, the changes expression of genes integral to cell cycle and apoptosis (Fig. 4), overexpression of cellular metabolism and immunological pathways (Fig. 5a) and underexpression of cell motility and cell adhesion (Fig. 5b) suggest that in the time frames of the biopsy, perturbation of such pathways remains several days after the initial wounding effect of the first core biopsy. The reduction in mRNA expression of the ER pathway (Fig. 5b) following the first biopsy holds potential concern and is in contrast to the only other published study of eight patients where no change was noted [32]. However, mRNA changes do not exactly reflect semiquantiative immunohistochemistry and ER mRNA imperfectly correlates with the level of ER protein expression [33]. The immunohistochemical studies on the same series of samples reported here provide comfort that for the technology most widely used in clinical practice (immunohistochemistry), ER on a second core biopsy may not be compromised.

IPA set in context a number of gene expression changes among which pathways involving PI3K, MEKK and IGF-1 [34, 35] may be of particular relevance in the setting of breast cancer.

These microarray data, within the limits of the experimental design, sample numbers and analytical techniques employed, suggest that core biopsy of primary breast cancer may generate a “wounding” effect evident on subsequent mRNA analysis. The time course, duration and variations in gene expression as a consequence of tumor and patient variability were not assessed within this study and are clinically challenging to obtain [25]. However, core biopsy may influence the mRNA expression profile of sequential clinical samples used in clinical trials and requires careful evaluation.