Toi et al. BMC Cancer 2010, 10:56 http://www.biomedcentral.com/1471-2407/10/56 tumor specimens may have the potential to identify responders more precisely. We have previously reported that quantitative mea- sures of HER2 expression or HER2 homodimers in FFPE specimens from MBC patients enrolled in a trastuzumab expanded access program identified sub-populations of patients with different clinical outcomes, such that patients whose tumors expressed higher levels of HER2 or HER2 homodimers lived longer [15]. Those patients had been stringently selected for trastuzumab treatment primarily by FISH (90%). Here we describe the correla- tion of quantitative measurements of HER2 expression (H2T) or HER2 homodimers (H2D) with OS following trastuzumab exposure in a cohort of patients with MBC who were selected for treatment by IHC (88%) or FISH (12%) in a routine clinic setting. Quantitative HER2 mea- surements were made by the HERmark assay (Monogram Biosciences) using FFPE breast tumor specimens. HER2 homodimers are defined as HER2:HER2 associations that are sufficient to lead to HER2 phosphorylation and sub- sequent activation of downstream signaling pathways. These could include HER2:HER2 dimers or HER2 mono- mers that are in close proximity (< 80 nm) as a result of pathological over-expression. Methods The VeraTag technology The VeraTag technology is a proximity-based method designed to accurately and reproducibly quantitate pro- tein expression and protein-protein complexes, includ- ing cell surface-expressed protein dimers, in FFPE specimens. The first assays developed for clinical testing using the VeraTag platform measure HER2 protein expression (H2T) and HER2 homodimer (H2D) levels accurately and reproducibly, and are referred to as HER- mark. These assays have undergone a formal validation at Monogram Biosciences and are regulated by the Col- lege of American Pathologists under the specifications established by CLIA (Clinical Laboratory Improvement Amendment) [16]. IHC (Herceptest, DAKO) and FISH assays were performed according to standard protocols at the local institutions where the patients were treated. Description of the clinical cohort This cohort is composed of 75 patients drawn from six oncology clinics in Japan. Patients were included if they had metastatic breast cancer, had been treated with at least one chemotherapy regimen prior to receiving tras- tuzumab either as single agent or in combination with chemotherapy, and had FFPE specimens available for testing by the HERmark assay. Assessments of HER2 over-expression by IHC or FISH were performed by the pathologist at the local hospital. All clinical follow-up was performed at the local hospital, and clinical Page 2 of 10 outcome data were drawn from medical chart review. This study protocol was approved by the ethical com- mittee of each institution and informed consent from the patients was taken Statistical methods Measurements of H2T and H2D were tested for associa- tion with OS, defined as the time from start of trastuzu- mab treatment to cancer-associated death or the end of the follow-up period. The log-transformed values of HER2 expression and HER2 homodimers were used for analyses. Deaths not attributed to breast cancer were considered as censored at the time of death. One patient was excluded from the analyses because she died merely 20 days after her first dose of trastuzumab, and the duration of treatment was considered insufficient to include the patient as “trastuzumab-treated.” Univariate analyses were performed using the Kaplan and Meier (KM) method, hazard function plots were estimated using the life-table method, and multivariate models were generated using Cox proportional hazards regression analysis. Cox proportional hazards models were fitted to examine the associations of H2T, H2D, or their ratio H2T/H2D with outcome, with adjustment for other potential prognostic factors. The adjustment fac- tors included treatment group (trastuzumab-only or trastuzumab plus chemotherapy), estrogen and proges- terone receptor status, and a composite of them, hor- mone receptor status, screening Herceptest score performed at the treating hospital, Herceptest score repeated at Monogram, Histoscore performed on repeat IHC at Monogram, tumor stage, and presence of metas- tases in the liver, lung, skin, bone, lymph nodes, brain, chest wall, or other viscera. Metastatic sites occurring in less than 10% of the total cohort were not considered as individual variables. The stepwise variable selection method was used to select the “best” models. Statistical analyses were performed at Yokohama City University Medical Center using the statistical software packages SAS and R. Results and Discussion The median duration of patient follow-up period was 18.2 months. The median duration of trastuzumab treat- ment was 14.6 months. A description of the clinical characteristics of the cohort is provided in Table 1. Of the 75 patients with HER2 test +2 or +3 assessed at the local hospitals, 25 had IHC 0 or +1 by the central pathological review. Nine patients had advanced disease, while 66 had recurrence. The number of pre-che- motherapy before trastuzumab treatment was also shown in Table 1. For the 66 patients with recurrence, the number of pre-chemotherapy received after recur- rence was analyzed.