Toi et al. BMC Cancer 2010, 10:56 http://www.biomedcentral.com/1471-2407/10/56 Table 1: Clinical characteristics of the cohort (Continued) 1 2 or 3 N.A. Tumor stage Recurrence Advanced Number of met sites 1 or 2 3 or 4 unknown Brain mets yes no Treatment Herceptin + chemo Herceptin only 15 (20.0) 7 (9.3) 12 (16.0) 66 (88.0) 9 (12.0) 49 (65.3) 25 (33.3) 1 (1.3) 7 (9.3) 68 (90.7) 63 (84.0) 12 (16.0) 9 (23.7) 3 (7.9) 8 (21.1) 32 (84.2) 6 (15.8) 25 (65.8) 12 (31.6) 1 (2.6) 5 (13.2) 33 (86.8) 31 (81.6) 7 (13.5) Page 4 of 10 6 (16.2) 4 (10.8) 4 (10.8) 34 (91.9) 3 (8.1) 24 (64.9) 13 (35.1) 0 2 (5.4) 35 (94.6) 32 (86.5) 5 (18.4) * For the 66 patients with recurrence, the number of pre-chemotherapy received after recurrence was analyzed. Correlation of HERmark measurements with IHC We have previously shown the relationship between HERmark measures of HER2 expression and IHC per- formed under standard conditions by a single patholo- gist in 170 FFPE breast tumors [16]. The results demonstrated a general correlation between the two assays, however the HERmark assay provided a more quantitative assessment of HER2 expression distributed over a 3-log dynamic range compared to the semi- quantitative readout of IHC (0-3+ scale) (see Addi- tional file 1: Figure S1). We attempted to make the IHC measurement more quantitative by considering the histo-score (H score), where staining intensity is corrected for percent tumor involvement. The HER- mark assay still demonstrated a greater ability to quan- titate HER2 expression over a wider dynamic range. In this cohort, we initially compared H2T to the screening IHC calls provided by the treating hospitals. The distribution of H2T measurements ranged over a 3-log dynamic range, and there was no significant dif- ference between the H2T distributions of those sam- ples scored as 2+ or 3+ by the screening IHC assays (Figure 1a). We repeated the IHC assays on all speci- mens. The repeat assays were performed centrally and read by a single pathologist. Approximately one-third of the specimens were scored as 0 or 1+ upon repeat. Comparison of the repeat IHC results with the HER2 expression levels by HERmark yielded a stair-step correlation (Figure 1b) reminiscent of previous com- parisons with centralized IHC results (see Additional file 1, Figure S1). Kaplan-Meier, hazard function plots, and multivariate Cox analyses As a whole, two-year survival rate of the cohort was 60.8% (95% C.I., 48.4 - 73.2%). In the univariate ana- lyses, patients were classified into four subgroups defined by the 25th, 50th, and 75th percentiles for each of the three variables, H2T, H2D, and their ratio H2D/ H2T, in order to examine the assumption of linearity for the continuous variables used in the subsequent Cox regression analysis. A significant difference in OS was found between the four subgroups with respect to H2T (Log rank test, p = 0.003; Figure 2), but not for the others (p = 0.371 and p = 0.573). The 25th, 50th, and 75th percentiles for the log-transformed values of H2T were 1.41, 1.80, and 2.25, respectively. The patients’ characteristics by H2T subgroup (< the median value of H2T: n = 37 v.s. the median value of H2T: n = 37) is summarized in Table 1. Higher proportion of patients with greater H2T value than its median value had IHC 2+ or 3+ (p < 0.001) and higher HER2 score on repeat testing (p < 0.001). No significant difference in OS was found between the IHC subgroups (log-rank test: p = 0.610). Table 2 summarizes the other univariate analyses for OS. Figure 3 shows the hazard function plots esti- mated in the four H2T subgroups. The subgroups with the 25% highest and lowest H2T values had substantially lower risk of death than the middle two subgroups. These univariate analyses revealed a parabolic relation- ship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of lin- earity for the HER2 expression measurements may be