The present study investigated the prognostic value of the activated HER1, HER2 and HER3 receptors, the total HER4 expression and the activated form of the downstream kinases Akt and Erk. We addressed this issue in a series of postmenopausal patients presenting with primary hormone receptor-positive breast cancer, who had all received adjuvant tamoxifen therapy.
Total HER2 expression is an independent predictor of poor prognosis [2
] and is also a clinical target for treatment [22
]. In this study, 8% of the patients had HER2-positive tumors in accordance with this being a hormone receptor-positive population, and HER2 positivity was associated with shorter disease-free survival. Fourteen percent of cases were recorded with strong membrane expression of HER2 phosphorylated at tyrosine 1221/1222, however, and this HER2 phosphorylation was associated with high tumor grade and with shorter disease-free survival and overall survival. We speculate that the reason for this difference is due to the fact that moderate or even low levels of HER2 may be sufficient to elicit a potent mitogenic signal, upon activation by dimerization with ligand activated HER1 or HER3. This hypothesis is not new [23
], and especially the importance of the HER ligands for activation of the HER receptors has been substantiated by several reports [3
]. In agreement, we find that the hormone receptor-positive breast cancer cell line MCF-7, which we score HER2-negative, has weak to moderate levels of HER2 mRNA and protein [27
], and addition of the HER3/HER4 ligand heregulin 1β clearly abrogated the inhibitory effect of antiestrogen treatment equally well in both wild-type and HER2/HER3 overexpressing MCF-7 cells – suggesting that activation of even a low level of HER2 via dimerization of ligand-activated HER3 may suffice to protect against antiestrogen therapy [27
A recent study has compared HER2 mRNA expression in MCF-7 cells with mRNA expression in tumors classified as HER2-negative or HER2-positive, and the HER2 mRNA level in MCF-7 cells was lower than in most HER2-negative tumors [28
]. This supports tumors classified as HER2-negative perhaps having sufficient HER2 protein to elicit signal transduction upon activation, and thereby may explain why also some patients with HER2-negative tumors respond to trastuzumab treatment [28
It is noteworthy that 82% of cases with strong pHER2 staining without HER2 overexpression were scored positive for pHER1 and/or pHER3, clearly supporting that HER2 may be activated via dimerization with another HER family member. This is further supported by the finding that pHER2 was often expressed in the same tumor cells that were also positive for pHER1 and/or pHER3 (Figure ). Our multivariate analysis clearly revealed that IHC evaluation of tyrosine 1221/1222 pHER2 was significantly better than the current HER2 tests with respect to select patients with both poor disease-free survival and overall survival. Other studies have also indicated that pHER2 expression may provide additional survival information [23
]. In two of these studies, IHC detection of pTyr 1248 was measured and only a small fraction of the HER2-positive cases (12%) was positive for pHER2 [23
]. In the study by Cicenas and colleagues [30
], the same antibody against pTyr1248 was applied in a highly sensitive chemoluminescence-linked immune assay. In that study, pHER2 expression was found in both HER2-positive and HER2-negative tumors (68% and 27%, respectively). In concert with our finding, the multivariate analysis showed that pHER2 was a marker of poor prognosis independent of HER2. We have tested the antibody against pTyr 1248, but found only weak staining in our positive controls, indicating that this antibody may not be sensitive enough for IHC analysis as also suggested by Cicenas and colleagues [30
]. We therefore selected the antibody against pTyr 1221/1222 directed against phosphotyrosines, which like pTyr 1248 is related to HER2 receptor activation [20
]. Our data support that the pTyr1221/1222 antibody is a good antibody for IHC detection of activated HER2.
Future studies shall validate the clinical usefulness of the pTyr 1221/1222 antibody with respect to predict response to endocrine therapy and also in relation to predict response to therapy directed against the activated HER2 receptor. Our finding that activated HER2 is expressed also in HER2-negative tumors indicates that these tumors may utilize HER2 receptor signaling to promote growth, and thus may be potential responders to treatment targeting the activated HER2. Besides trastuzumab, which has been found to be beneficial also in a fraction of the patients with HER2-negative tumors [28
], targeted therapy may involve the monoclonal antibody Pertuzumab, which targets HER2 receptor dimerization [31
], or treatment with the tyrosine kinase inhibitor Lapatinib, which targets the kinase activity of both HER2 and HER1 [32
In the present series, membrane expression of phosphorylated HER3 at tyrosine 1289 was found in 15% of cases and was associated with shorter disease-free survival. No previous work has investigated the importance of phosphorylated HER3 in primary breast cancers, but most studies of total HER3 expression have found HER3 to correlate with adverse clinicopathological properties [2
]. Contrary results have also been observed, however; for example, inverse association with local recurrence [2
] and association with longer survival [13
]. In breast cancer cell lines, HER3 is a promoter of cell growth and is required for HER2-mediated proliferation [35
]. More recently, HER3 activation has also been shown to be important for both tamoxifen-resistant and fulvestrant-resistant cell growth [10
], and a significant role for HER3 in resistance towards HER-directed therapy is also evident [37
]. In the present patient series, membrane expression of pHER3 did not add prognostic information to the current clinical parameters, hence substantiating that HER2 is the dominant HER receptor and that the function of HER3 is primarily to act as a co-activator of HER2. Moreover, nuclear expression of pHER3 was found in 10% of the patient tumors, but it was not significantly associated with the parameters investigated in the present series.
We observed membrane expression of HER1 phosphorylated at tyrosine 1173 in 18% of cases, and to the best of our knowledge only two other studies have measured pHER1 in breast cancer biopsies. These investigations were carried out on 225 and 154 cases of advanced breast cancer, and 7% and 36% of cases displayed membrane staining, respectively [39
]. In agreement with these studies, our work showed that pHER1 was significantly correlated to shorter disease-free survival. Moreover, high total HER1 expression is also generally related to poor prognosis [2
]. Overall, however, the literature on the prognostic value of total HER1 expression in breast cancer is not completely clear [41
]. Our analyses show that membrane expression of pHER1 did not add prognostic value to the current clinical parameters, thus indicating that HER1, like HER3, also mainly acts as a co-activator of HER2.
In contrast to the predominantly membrane staining of total HER2, pHER1, pHER2 and pHER3, the HER4 staining was primarily cytoplasmic but also membranous. The overall intensity scoring method was therefore applied. HER4 expression was found in 97% of cases. The high percentage of positive cases is in concert with the data from Abd El-Rehim and coworkers, who found 80% HER4-positive cases in a series of more than 1,500 patients [2
], and another study recorded 82% HER4-positive cases [18
]. When only reactivity in the membrane is scored, however, the observed frequencies are much lower: 21% [17
], 14% [5
], and 12% [6
]. In agreement, we found 15% of cases to express HER4 in the membrane. In this series, high HER4 expression was inversely associated with pHER2 and tumor grade, and had a positive effect on disease-free survival and overall survival. Furthermore, high HER4 expression independently predicted for longer disease-free survival and overall survival, compared with the currently used parameters. This result is much in line with earlier data from both protein and mRNA analyses, where HER4 predicted prolonged survival in multivariate analysis [6
]. Moreover, others have found associations between high HER4 expression and longer disease-free survival and/or overall survival compared with low or negative cases [17
We also looked at the activated levels of the HER downstream kinases Erk and Akt – we found that pAkt was correlated with pHER2, pHER3 and pErk, whereas pErk was not correlated with any of the HER receptors. Our data therefore suggest that pAkt is likely to be an important downstream mediator of HER2/HER3 signaling, which is very much in line with the current knowledge [8
]. We did not find associations, however, between pAkt or pErk and clinicopathological parameters or survival. For pAkt this is in agreement with a study of 691 cases where the authors did not find an association with survival [42
]. In contrast, others have observed pAkt expression in 54% of 93 patients analyzed and have shown that pAkt was an independent marker for disease-free survival [43
]. Also, another study of 399 cases found pAkt to be associated with decreased overall survival in univariate analysis [44
]. Few studies have been conducted for pErk, and also here conflicting results have been obtained. Using both immunoblotting and IHC, it has been shown by multivariate analysis that pErk was a marker for prolonged survival [45
], while others have reported that pErk independently predicted for reduced survival [46
]. In our patient cohort, there was a trend for pErk to be associated with expression of HER4 (P
= 0.07) and this would indicate a preferable effect of pErk, but this was not evident in the Cox analysis. Moreover, the number of pErk and pAkt targets is currently sought to be around 160 and 70 proteins, respectively [48
]. This fact is likely to, at least partly, explain the discrepancies between the studies.
The comparison of the expression levels of the six proteins in 30 distant metastases and their corresponding primary tumors disclosed a significant increase in expression of pHER1 and pHER3 in the metastases. This observation fits well with the poor prognosis of cases with primary tumors expressing the phosphorylated receptors and, worthy of note, it indicates that pHER1 and pHER3 may be important activators of HER2 in metastatic tumor cell growth. We did not find other reports investigating pHER1, pHER2 and pHER3 levels in this setting, but eight other studies have, in agreement with our finding, consistently found that the total HER2 expression, evaluated by IHC, is unaltered between the primary and metastatic lesions [50
]. Analysis of activated HER1 and HER3 in distant metastasis may therefore be helpful in the clinical setting. Lastly, we observed a significant increase in the number of cases with nuclear expression of pHER3 in the metastasis. No data on nuclear HER3 expression exist in breast cancer, but a recent report showed that nuclear HER3 was absent in nonmalignant prostate tissue, whereas it was highly expressed in the cancerous prostate tissue and associated with increased tumor grade [51
]. Studies with more statistical power should therefore investigate whether increased nuclear expression of HER3 in metastatic lesions may be associated with an adverse prognosis.