Supplementary Figure 1. Differential Expression of Genes Confirmed by Qrt-PCR

Supplementary File 1

Complete lists of differentially expressed genes shown in Fig. 1 including fold change and p-values.

Supplementary Figure 1. Differential expression of genes confirmed by qRT-PCR

A, In order to validate the gene expression data, 10 genes with different patterns of medium-high expression were evaluated. Fold changes between the Illumina microarray and qRT-PCR data were highly consistent for all genes, R2 = 0.97 (p < 0.0001, Supplementary Fig 1). E-cadherin (CDH1), Hippostasin (KLK11) and p21 (CDKN1A) had greater expression with combination therapy. Interleukin 8 (IL8), transforming growth factor and beta-induced (TGFBI) were more down-regulated by the combination than trastuzumab alone. Ovalbumin (SERPINB5) was more highly expressed by either agent, whereas Stanniocalcin 1 (STC1) and protein tyrosine phosphatase, receptor type R (PTPRR) were reduced by pertuzumab only. ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 and matrix metalloproteinase 7 were up-regulated or down-regulated respectively upon combination therapy only. B, Correlation between microarray and qRT-PCR data. Scatter plot demonstrating highly consistent fold changes between Illumina microarray and qRT-PCR with a correlation coefficient (R2) of 0.97 (p < 0.0001).

Supplementary Figure 2. Comparison with previous microarray studies of trastuzumab response

A. Expression of 16 genes down regulated by trastuzumab in vitro in the breast cancer cell lines SKBR3 and BT474 (Le et al., 2005a) and Cyclin G1 highlighted in a follow-up study (Le et al., 2007).

B. Expression of genes used to distinguish responding and non-responding HER2+ breast cancer patients to trastuzumab+docetaxel treatment (Vegran et al., 2009) in the current dataset.

Heatmap colours, red=Up-regulated Green=down-regulated relative to untreated mean and values are fold changes. * Significant changes (students t-test p<0.05) Bold = significant changes in anticipated direction.

C. Expression of genes highlighted as responding to trastuzumab or pertuzumab alone or in combination in a dataset from a recent study of HER2 homodimers and HER2/HER1 or HER2/HER3 heterodimers in HER2-expressing MCF10A cells (Ghosh et al., 2011). Heatmap colours, red=Up-regulated Green=down-regulated relative to control mean.

D. Genes that were identified to be differentially expressed between HER2 homo- and heterodimers in a previous study (Ghosh et al., 2011). Red is high and blue is low relative expression. Genes on the right heatmap (this study) are ordered as in the previous one.

Supplementary Figure 3. HER2 FISH analysis of xenograft models.

Gene amplification of HER2 was assessed by FISH using the Pathvysion HER2 DNA Probe Kit (Vysis). HER2:CEP17 ratios>2.0 were considered amplified. The level of amplification identified in the SKOV-3 xenograft (2.15) is comparable to previous reports (Rhodes et al, 2002).