Multiplexed assessment of the Southwest Oncology Group-directed Intergroup Breast Cancer Trial S9313 by AQUA shows that both high and low levels of HER2 are associated with poor outcome

Abstract

Assessment of key breast cancer tissue biomarkers is often done using nonquantitative methods. We hypothesized that use of continuous analysis of expression with the AQUA method of automated quantitative analysis will provide prognostic information beyond that attainable with conventional methods. A tissue microarray was made from 2123 of 3122 patients accrued to SWOG 9313, in which sequential doxorubicin (A) and cyclophosphamide (C) was compared with combination AC and in which all patients except premenopausal estrogen receptor (ER)-negative patients received tamoxifen. Multiplexed assays of 1) HER2 and estrogen receptor and 2) progesterone receptor (PgR) and p53 were performed on the two slides using the immunofluorescence-based AQUA method of automated quantitative analysis. Both ER and PgR showed unimodal distributions and significantly predicted disease-free survival when tested as continuous variables and adjusted for node status, tumor size, treatment, and menopausal status (P = 0.005 and P < 0.001, respectively). HER2, measured as a continuous variable, showed a biphasic effect on disease-free survival. Both high and low expressers of HER2 have worse outcomes (when low levels are equivalent to that seen in normal breast ducts). In patients who were uniformly treated with AC chemotherapy and tamoxifen (when indicated), both ER and PgR, assessed as continuous variables, were highly prognostic, whereas p53 expression was not. This assay method may provide a new companion diagnostic approach for targeted therapies.

Figure 1

Distribution of expression of ER (A) and HER2 (B) in the study population is shown with log transformation of the data in the insets. C: Validation of multiplexing data is shown by comparison of single ER versus multiplexed (double) HER2 with ER on a control TMA using tissues from the Yale Pathology Archives. D: Single HER2 versus multiplexed (double) HER2 with ER. Similar results were seen for PgR and p53 (not shown). AQUA levels for each protein are presented on the y axis as arbitrary units.

Figure 2

Kaplan-Meier survival plot for DFS by AQUA ER (A), PR (B), and HER2 (C), all split into quartiles.

Figure 3

A: Relationships between the log AQUA scores of ER, PR, and HER2 show that ER and PR are moderately highly correlated (Pearson r = 0.43, P < 0.0001; Spearman r = 0.48). B: The relationship between HER2 and ER is more complete, showing a correlation for low ER and HER2 that is lost at higher levels. The dotted line shows the lowest decile of HER2 AQUA scores. C: No relationship is seen between PgR and HER2.

Figure 4

A: Kaplan-Meier analysis of HER2 AQUA shows that both the top and bottom deciles have worse DFS than the middle 80% (log rank P = 0.012). B: Modeling the continuous hazard ratio against the log of the HER2 AQUA score shows a U-shaped relationship.