Extended Data Fig. 4 |. Treatment-associated changes and model performance assessed in cases treated with trastuzumab.

a. Volcano plot demonstrating treatment-associated changes based on comparison of pre-treatment versus on-treatment protein marker expression levels in pancytokeratin-enriched (PanCK-E) regions in the trastuzumab-treated cases (arms 1 and 3, n = 23 biologically independent tumors). Significance, −log10(FDR adjusted p-value), is indicated along the y-axis. Analyses based on the discovery cohort. b. Receiver operating characteristic (ROC) curves for On- plus Pre-treatment DSP protein L2-regularized classifier in the discovery cohort using the subset of cases with data available at both timepoints that were treated with trastuzumab or trastuzumab+lapatinib (n = 19 biologically independent tumors). Model performance was assessed via cross-validation using the 40 DSP protein markers profiled in both cohorts c. Correlation plot comparing the marker coefficients for the On- plus Pre-treatment DSP protein trained using all cases in the discovery cohort (n = 23 tumors with both timepoints versus using only those cases treated with trastuzumab (arms 1 and 3, n = 19 tumors with both timepoints). The grey shading indicates the 95% confidence interval of the correlation coefficient. P value is assessed via a one-sided t-test (correlation coefficient not equal to 0). d. Coefficients for each marker in the L2-regularized On- plus Pre-treatment DSP protein model, trained using only those cases treated with trastuzumab (arms 1 and 3, n = 19 tumors with both timepoints). e. OC curves for classifiers trained using either DSP on-treatment CD45 levels or IHC CD45 % positive. These models were evaluated on the subset of cases on which CD45 IHC was performed that were treated with trastuzumab or trastuzumab+lapatinib (n = 20 biologically independent tumors).