Toi et al. BMC Cancer 2010, 10:56 http://www.biomedcentral.com/1471-2407/10/56 Table 4 Cox regression analyses in two subgroups divided by the median HER2 expression value. Cohort subset> Log10HER2 expression 1.799* (n = 37) Log10HER2 expression < 1.799* (n = 37) Variable Log10 HER2 expression Hormone receptor status Brain mets Log10 HER2 expression Hormone receptor status Brain mets Hazard ratio 0.06 0.39 0.14 16.0 0.20 0.04 95%C.I. 0.01 - 0.51 0.05 - 2.86 0.02 - 0.87 1.64 - 155.9 0.03 - 1.57 0.01 - 0.28 Page 7 of 10 P-value 0.010 0.354 0.034 0.017 0.125 0.001 * Median (50 percentile) value of Log10HER2 expression measurements effectively by trastuzumab. Such patients may be expected to experience fleeting responses when treated with trastuzumab, or even to show no evidence of clini- cal response at all. As the ability to accurately assess such escape pathways gradually evolves, we should be able to identify the mechanisms of resistance to trastu- zumab, so that effective strategies for combination ther- apy can be developed and tested in prospective clinical trials. That process will take time, but perhaps a first step could be to improve our ability to accurately quan- titate HER2 protein expression and try to gain a better understanding of how differences in expression levels among individual breast tumors affect the natural his- tory of HER2-positive breast cancer and, more impor- tantly, response to HER2-targeted agents like trastuzumab. Recently presented data at the 2007 ASCO annual meeting have raised questions about the accuracy of current testing methods [12-14]. In particular, a re- analysis of the central testing data from the NSABP B- 31 trial demonstrated that nearly 10% of the trastuzu- mab-treated population in the trial were IHC < 3+, FISH-negative on repeat testing, but benefited from trastuzumab therapy just as the IHC 3+ and FISH- positive groups did [14]. Perhaps this observation reflects fundamental differences between the pathophy- siology of breast cancer in the adjuvant setting com- pared with the metastatic setting, or perhaps we need better tests for HER2. Whatever the case, there appears to be a genuine need to re-examine the issue of patient selection for trastuzumab, with an eye toward new technologies and new approaches that have the potential not only to improve current stan- dards, but also to begin identifying alternative signaling complexes that can be targeted by combination therapy regimens including trastuzumab. In this study, we describe the relationship between quantitative measures of HER2 expression or HER2 homodimers and overall survival in a cohort of patients with MBC who were treated with trastuzumab. The assay system used to make these measurements is CLIA validated for accuracy and reproducibility, and by virtue of its ability to quantitate proteins and protein dimers, provides a more granular view of the HER2 landscape than either IHC or FISH [16]. The assay has previously been correlated with clinical outcomes following trastu- zumab therapy in a cohort of MBC patients who were very carefully selected for treatment using FISH at the Jules Bordet Institute in Belgium [15]. The current study represents the second clinical experience with the HERmark assay, involving a group of patients who were selected for trastuzumab treatment primarily by IHC (88%) performed in the course of rou- tine clinical care. The initial KM analyses and hazard function plots of the entire cohort suggested that two distinct populations were present, and the observed rela- tionships between HER2 expression and OS for the two sub-groups were going in opposite directions. Using a cutoff of the median value of log10 HER2 expression = 1.80, we then re-analyzed the patient sub-group that had HER2 expression values greater than the cutoff to see whether this sub-population showed any evidence that higher levels of HER2 expression yielded longer OS on trastuzumab. Data from the Bordet cohort described previously had shown such an association, and we wanted to see if we could confirm this relationship in the current study. Because the Bordet cohort was so stringently selected for HER2 over-expression or gene amplification, patients with low HER2-expression, such as those that represented approximately one-third of the current cohort, may have been excluded. Given the dif- ferences in the methodology used to select patients in the two cohorts (90% FISH in Bordet, 88% IHC in the current study), as well as the stringency applied (expanded access cohort with mandatory central confir- mation of HER2 status in Bordet, routine clinical care in the current study), it is perhaps not so surprising that we might observe this difference. These data appear to confirm the relationships that were seen in the Bordet cohort and also suggest that, within a population of patients that are identified as HER2-positive by the com- monly used methods currently available to clinical oncologists, precise quantitation of HER2 expression can define multiple sub-populations of patients that experience different clinical outcomes in response to trastuzumab treatment.