Introduction

High rates of cellular proliferation are a key component of aggressive breast cancers. Markers of proliferation, such as Ki67, are widely used to identify cancers with high proliferative indices and have the potential to alter treatment rationale [1–3]. In addition, determining cellular proliferation rates provides an insight into the intrinsic biology of a tumour, indicating the degree of growth stimulation which is contributing to increasing tumour size. The Human Epithelial Growth Factor Receptor (HER) family of receptor tyrosine kinases form part of a complex signal cascade modulating cell proliferation, survival, adhesion, migration and differentiation. The family is comprised of 4 homologous receptors HER1 (EGFR/ErbB1), HER2 (ErbB2/neu), HER3 (ErbB3) and HER4 (ErbB4) [4]. Over-expression of all 4 receptors has been observed in breast cancer [5–8]. We have recently demonstrated that patients with overexpression of HER1-3 exhibit markedly reduced survival whilst patients with overexpression of HER4 demonstrate increased survival when compared with patients not expressing high levels of any of these receptors [9]. There is evidence, within the literature, that suggests that HER4 is less able to recruit proliferative (RAS/RAF) signal transduction pathways than other HER family members [10, 4]. Therefore, we have postulated that the apparent protective effect of HER4 may be reflected by lower proliferative indices, whilst cases with higher expression of HER1-3 would demonstrate higher proliferative indices.

HER1 and HER2 are both linked to poor prognostic factors in breast cancer [11–14, 8]. Conflicting evidence is available on the prognostic significance of HER3 with some studies demonstrating a relationship between HER3 and some markers of poor prognosis [9, 15, 16]. However others have shown HER3 overexpression is consistent with a good prognostic outlook [17, 18]. HER4 has more consistently been linked to good prognostic factors and a longer disease free interval [19, 20, 9]. We have therefore studied HER1-4 expression in a cohort of patients in whom we have previously performed BrdU labelling to determine tumour proliferative indices and potential tumour doubling times [21] in order to further investigate the biological significance of HER1-4 overexpression with respect to cellular proliferation.

Materials and methods

Patients

89 patients who had a breast cancer of more than 2 cm in diameter were recruited in the original study between 1989–1992. This size criteria was used to ensure that adequate tissue was available for analysis once diagnostic needs had been met. Only postmenopausal patients were entered in view of the unknown embryotoxicity of bromodeoxyuridine. The relevant ethics committees gave approval. Patients who had either breast conservation or mastectomy were included as were those having adjuvant radiotherapy, chemotherapy or tamoxifen as part protocols at that time. Follow up status was obtained by review of patients records with further clarification from data held by the 'Scottish Cancer Intelligence Unit' when there was any doubt over the cause of death. Calculation of BLI was possible in 84 patients. 4 were lost to follow-up and 2 patients were excluded from survival analysis because they had metastasis at the time of diagnosis. 2 patients had bilateral tumours; in these cases the tumour with the highest BLI value was included. Thus there are 78 cases in whom BLI and follow up were available with calculation of T pot present for 55 of them. Material for evaluation of HER1-4 status was available for 55 of the labelled cases.

Bromodeoxyuridine labelling

200 mg of BrdU was administered as an intravenous bolus 4–10 hours prior to surgery. Tumour sample specimens were subjected to flow cytometric analysis and the subsequent calculation of BLI and T pot values has been described previously [21]. The BLI (BrdU labelling index) is presented as a percentage of tumour cells labelling with BrdU (i.e. within the DNA synthetic "S" phase of the cell cycle during exposure to BrdU). The tumour potential doubling time (T pot ) is a measure of the minimum time to doubling of tumour size based on calculation of the length of S phase (Ts) and is expressed in days.

HER 1-4

HER 1-4 status was determined in 55 patients by immunohistochemistry with specific antibodies as described by Witton et al [9] previously. Scoring of membrane staining was performed with excellent agreement between observers (inter-class correlation coefficient ICCC = 0.892).

Statistics

All statistical analysis was performed using the SPSS statistical package (version 9.0.0). Kaplan-Meier life-table analysis was undertaken with log-rank testing of differences in breast cancer-related survival (end-point breast cancer specific death). BLI values ≥ 75th percentile were stratified as 'high' values and Tpot values ≤ 25th percentile were classified as 'low' values for the purpose of survival calculations. These cut-offs were used to identify the relevant 'high risk' populations with regard to their proliferation indices. Multivariate analysis using Cox's multiple regression analysis was performed with the inclusion of known prognostic values such as grade, nodal status, and tumour size. Mann-Whitney test using mean ranks was used to evaluate any correlation between BLI values and HER 1-4 status.

Results

Patients

The median age of patients (n = 78) included in the analysis was 64.6 years. Median follow up available was 6.6 years (range 0.1–13 years). There were 35 breast cancer specific deaths and a further 15 deaths from unrelated causes (mainly cardiac/lung disease with 4 deaths from other cancers). There were 69 ductal tumours, 4 lobular tumours and 5 other or non-specified type. There were 44 ER positive and 30 ER negative cases (4 unknown). The mean size of the tumours is 32.1+/- 20.9 S.D. mm (median 27 mm with interquartile range 20–40 mm). The nodal status was known on 74 patients, with 40 having no nodal involvement, 23 with 1–3 nodes and 11 having over 3 nodes involved (Table 1). 44 patients received a mastectomy and 34 patients had a wide local excision. For the patients with known adjuvant treatment details 93% (69/74) received tamoxifen and only 4% (3/71) received chemotherapy.

Table 1 Tumour size, grade, nodal and ER status of cohort
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BLI and Tpot

The mean BLI value is 4.0 % +/- 3.1 S.D. with the median at 3.2% (interquartile range 1.3–6.4 %). The mean T pot value is 28.3 days +/- 29.3 S.D. with the median at 14.6 days (interquartile range 8.0–37.5 days). BLI and T pot are inversely related in a non-linear fashion with a Spearman rank correlation coefficient of -0.938 (at 0.01 significance level). For this reason and because of the smaller numbers in the T pot group; regression analysis and correlations with the HER group were performed using BLI only.

HER 1-4

HER overexpression was defined as described by Witton et al [9]. Thirteen percent (7/55) of patients were HER1 positive, 19% (10/54) HER2 positive, 20% (10/51) HER3 positive and 11% (6/54) HER4 positive (Table 2). Eighteen (48%) patients were positive for HER1, 2 and or 3 (HER1-3 positive).

Table 2 Relationship between BLI values and HER 1-4 status. *p-values from Mann-Whitney test
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HER1 positivity was significantly correlated with increased BLI values (p = 0.024); median BLI values for HER1 overexpressing tumours were 2.5 fold higher than for HER1 negative cases. BLI values for HER2 & HER3 positive tumours were 42% and 69% higher than tumours not expressing these proteins at high levels. Neither value reached significance levels (p = 0.216 and p = 0.313). However when considering patients positive for one or more of HER1, HER2 or HER3 (n = 18) this group had significantly higher BLI values (p = 0.041) than tumours which did not express these proteins.

Conversely, tumours with high HER4 expression showed BLI values significantly lower than non HER4 expressing cases (p = 0.013) with a median reduction in BLI of 65%.

Survival

Patients with higher BLI values (≥ 75th percentile, n = 20) demonstrated a significantly poorer prognosis in terms of survival (10 yr survival 35%) from breast cancer when compared to patients with values < 75th percentile (n = 58, 10 yr survival 60%) (p = 0.0174; log-rank test, Figure 1).

Figure 1
figure 1

Survival curves (Kaplan-Meier) for BLI. Cumulative survival differences between patients whose tumours had high BLI values (≥ 75th percentile ie 6.4%) and low BLI values (<75th percentile). p-values represent log-rank differences in cumulative survival between the two groups.

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Patients with low T pot times (≤ 25th percentile, n = 14) had significantly reduced survival (10 yr survival 29%) compared to patients with T pot times > 25th percentile (n = 41, 10 yr survival 58%, p = 0.0258: log-rank test, Figure 2). Further stratification of BLI and T pot into other quartile groups did not show improved distinction in terms of survival (Data not shown).

Figure 2
figure 2

Survival curves (Kaplan-Meier) for T pot . Cumulative survival differences between patients whose tumours had low T pot values (≤ 25th percentile i.e 8.0 days) or high T pot values (> 25th percentile). p-values represent log-rank differences in cumulative survival between the two groups.

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When BLI and HER status were included in Cox's multiple regression analysis along known prognostic variables (Table 3); size and HER 1-3 status were the only independent predictors of survival.

Table 3 Results of Cox's multivariate regresssion analysis for BLI, HER status and known prognostic variables
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Discussion

Studies of tumour labelling index have significant correlations between high proliferation indices and poor outcome [22, 23] although this is not always the case in the context of a multivariate analysis [24, 25]. More recently the proliferation marker Ki67, a nuclear antigen expressed in the G1, S, G2 and M cell phases but not in G0 [26], has been used as a proliferation marker. These methods provide "static" information about the cell cycle in a single point in time, but do not give any information about the rate of progress of the cells through the cycle. Providing dynamic information about the rate of cell cycling may be a more accurate reflection of tumour growth as it allows estimation of parameters such as the potential doubling time. Using flow cytometry and in vivo labelling with the thymidine analogue 5-bromo-2-prime-deoxyuridine (BrdU) it is possible to obtain the labelling index for a tumour (BLI) as well as the potential doubling time of a tumour (T pot ) [27, 28].

We have demonstrated that tumours with either high BLI values or low T pot times have significantly poorer breast cancer specific survival rates. However along with other known prognostic factors BLI was not shown to be an independent prognostic factor in multivariate analysis. This may reflect the small size of the current study, rather than any failure of the method itself, since stage, grade and ER status were also not significant indices of survival in this study. Cutress et al [29] followed-up 75 patients with invasive breast cancer but did not show any significant correlation between proliferation data and outcome measures. A larger study of 129 patients demonstrated a significant correlation between low proliferation indices and survival which persisted in multivariate analysis when known prognostic factors such as node status, age and size of the tumour were included [3].

Tumour growth, which we have sought to measure by calculating T pot , is a balance between cell growth and cell loss and is therefore impacted by factors including oxygenation, apoptosis etc. BrdU labelling does not provide any information about cell loss, which is now recognised to be a significant factor in determining the actual growth of a tumour. Future measurements, which include an estimate of both proliferation and cell loss, may provide more accurate prognostic indices. The use of molecular profiling to identify tumours with differing proliferative and apoptotic signatures may provide valuable information in the future [30].

The use of proliferation indices has however provided us with a method of clarifying the varying effects of the HER4 family at a biological level. Our study demonstrates a significant correlation between high BLI values and HER1 positivity. This is in keeping with previous published results [31]. It is also consistent with other studies using the proliferation marker Ki67 [32]. Interestingly HER1 expression in bladder cancer is also associated with high BrdU indices [33]. We demonstrate a non-significant trend for HER2 positive tumours to have a higher BLI. This is consistent with previously published results by ourselves and others [21, 34]. However there is evidence of a significant correlation between Ki67 and HER2 expression [35, 36].

There is no previous evidence published examining the relationship between HER3, HER4 and BLI. However one study using Ki-67 showed a significant relationship between HER3 and HER4 positivity and decreasing Ki-67 levels [19], arguing that neither of these receptors are involved in directing proliferation. In terms of HER3 in this study we show a non significant trend towards higher BLI values. In addition when tumours positive for one or more of HER 1-3 are taken as a group they show significantly higher proliferation rates. This is in contrast to the findings demonstrated using Ki67 [19] but is more in keeping with studies which have identified a poor prognostic role for HER3 [9, 16]

We demonstrate a significant inverse correlation between HER4 positivity and BLI values. This suggests that HER4 expression in breast cancer may be involved in an anti-proliferative role. Although a proliferative role for HER4 (in keeping with the other HER family members) has been suggested [37, 38] there is other evidence to the contrary. Our previous results suggest that HER4 is very rarely co-expressed with other HER receptors [9]. Data from cell lines has shown that activation of HER4 receptor homodimers results in a weak biological response in comparison to other receptor combinations [10]. Other cell line work has shown that HER4 is necessary to mediate the anti-proliferative and differentiation responses elicited by neuregulin [39]. Indeed in normal breast tissue HER4 appears to play a critical role in the late differentiation of mammary gland function [40]. Thus this evidence appears to support the association HER4 has with improved breast cancer survival times [9]. Some of the conflicting evidence may be explained by the various isoforms of HER4 [41] or because of the inherent difficulty of separating HER4 responses from those of the other family members.

Conclusions

We have provided a biological correlate to substantiate the deleterious effect of HER 1-3 and the protective role for HER4. This possibility of differing roles within the HER4 family in driving proliferation has an implication in the choice of future anti-HER treatment options. Currently the HER2 monoclonal antibody Herceptin (trastzumab) is being used successfully to treat patients with strongly HER2 positive metastatic breast cancer [42]. Our results suggest that the use of pan-HER inhibitors may not be advised in tumours that are positive for HER4 and that more individually directed therapies are needed.