Toi et al. BMC Cancer 2010, 10:56 http://www.biomedcentral.com/1471-2407/10/56 These data suggest that patients can be characterized according to the degree of HER2 protein expression that their tumors exhibit, and that quantitative expression levels matter clinically. It is important to note that these data do not address the question of whether some patients treated with trastuzumab may not benefit clini- cally. Because we tested only patients who were treated with trastuzumab, the finding that some did not benefit as much as others does not equate to some did not ben- efit at all. Prospective studies or retrospective analyses of prospectively designed clinical trials where HER2-posi- tive patients were randomized to receive trastuzumab or not will need to be tested in order to answer that ques- tion. Nonetheless, data from the current study demon- strate that trastuzumab-treated patients with low HER2 expression levels appear to behave differently with respect to OS than those with high HER2 expression levels. Whether this subset of patients behaves differ- ently because they exhibit normal rather than elevated but low levels of HER2 is a matter for future study. In addition, we should have carried out the analyses to examine the relationship between HER2 expression levels and OS in patients with IHC 2+ or 3+. We could not obtain statistically reliable results, however, because the number of patients included in the subgroup was small (n = 50) and, in particular, the number of patients with brain metastases was only 5. This is also a matter for future study. What do these data imply about the impact of trastu- zumab on HER2-positive tumors that express different levels of the target? Previous data have shown that the amount of HER2 protein expressed correlates with dis- ease progression in MBC patients not treated with tras- tuzumab [3]. Data from the current study are restricted to patients that have been treated with trastuzumab, but nonetheless support the general conclusion that the amount of HER2 expression matters clinically. One potential explanation for such a finding is that the amount of HER2 expression is a marker for the degree of HER2 dependency that exists in the tumor. Tumors that massively over-express HER2 may be “addicted” to signaling through HER2, potentially explaining their association with rapid progression in the absence of trastuzumab as well as enhanced susceptibility to antag- onism by trastuzumab. Tumors which express lower levels of HER2 may be less vulnerable to trastuzumab because they are less dependent on HER2 alone, but rather derive at least some of their proliferative drive from protein-protein associations (HER1:HER2 or HER2:HER3 heterodimers) or variants (p95/HER2) that are not as susceptible to antagonism by trastuzumab [17-23]. Other explanations involving mutations in downstream components (PTEN) that affect PI3-kinase signaling activity as well as involvement of alternative Page 8 of 10 signaling pathways (eg. IGF-1R) are equally plausible [24,25]. Alternatively, the association between higher levels of HER2 expression and better outcomes on tras- tuzumab may reflect enhanced targeting of HER2 over- expressing cells by immune mechanisms triggered by trastuzumab binding, like antibody dependent cellular cytotoxicity (ADCC). Perhaps tumors at the lowest end of the HER2-expression distribution, that appear to have favored outcomes on trastuzumab, are simply expressing normal levels of HER2, and thus have a fundamentally different prognosis than their counterparts expressing significantly higher levels of HER2. These questions, as well as the general finding that, among tumors already classified as HER2 over-expressing or gene-amplified by standard assays, precise assessments of HER2 expression levels correlate with clinical outcomes following trastu- zumab treatment, demand further scrutiny in larger, well-controlled trials of patients with metastatic breast cancer. Although the technology enabling quantitative assess- ments of potential escape pathways for trastuzumab is still evolving, the opportunity to begin to sub-divide populations of patients into their constituent groups based on precise measurements of HER2 and its family members holds tremendous promise at a time when numerous targeted drugs are becoming available for inclusion in combination therapy regimens for patients with breast cancer. It is our hope that observations derived from the current study represent an early step in that direction. Conclusion It is concluded that the quantitive HER2 measurement by Hermark assay may be useful for identifying a more relevant target population to trastuzumab treatment in patients with MBC. It is warranted to study the signifi- cance of the quantitive HER2 measurement in the adju- vant situation with anti-HER2 therapy for primary breast cancer patients. Additional file 1: Figure S1. Correlation of HER2 expression Vera Tag and IHC in 170 breast tumors (FFPE). The correlation between the Vera Tag measure of HER2 expression and IHC in 170 FFPE breast tumor specimens. This is a reproduction of figure 6a from Shi, et al. (footnote 2) provided here for convenience. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2407-10- 56-S1.DOC ] Acknowledgements This study was partly funded by Japan’s Ministry of Health, Labour and Welfare research grant, a study on the construction of algorithm of multimodality therapy with biomarkers for primary breast cancer by a formulation of decision making process, led by Masakazu Toi (H19-3JIGAN- IPPAN-007).