[Lani]

DNA amplification of HER2 was readily evident in this study (Supplementary Fig. 9) together with overexpression of multiple HER2-amplicon-associated genes that in part define the HER2E mRNA subtype (Supplementary Fig. 5). However, not all clinically HER2+ tumours are of the HER2E mRNA subtype, and not all tumours in the HER2E mRNA subtype are clinically HER2+. Integrated analysis of the RPPA and mRNA data clearly identified a HER2+ group (Supplementary Fig. 12). When the HER2+ protein and HER2E mRNA subtypes overlapped, a strong signal of EGFR, pEGFR, HER2 and pHER2 was observed. However, only ~50% of clinically HER2+ tumours fall into this HER2E-mRNA-subtype/HER2-protein group, the rest of the clinically HER2+ tumours were observed predominantly in the luminal mRNA subtypes.

These data indicate that there exist at least two types of clinically defined HER2+ tumours. To identify differences between these groups, a supervised gene expression analysis comparing 36 HER2E-mRNA-subtype/HER2+ versus 31 luminal-mRNA-subtype/HER2+ tumours was performed and identified 302 differentially expressed genes (q-value = 0%) (Supplementary Fig. 18 and Supplementary Table 7). These genes largely track with ER status but also indicated that HER2E-mRNA-subtype/HER2+ tumours showed significantly higher expression of a number of RTKs including FGFR4, EGFR, HER2 itself, as well as genes within the HER2 amplicon (including GRB7). Conversely, the luminal-mRNA-subtype/HER2+ tumours showed higher expression of the luminal cluster of genes including GATA3, BCL2 and ESR1. Further support for two types of clinically defined HER2+ disease was evident in the somatic mutation data supervised by either mRNA subtype or ER status; TP53 mutations were significantly enriched in HER2E or ER-negative tumours whereas GATA3 mutations were only observed in luminal subtypes or ER+ tumours.

Analysis of the RPPA data according to mRNA subtype identified 36 differentially expressed proteins (q-value <5%) (Supplementary Fig. 18G and Supplementary Table 8). The EGFR/pEGFR/HER2/pHER2 signal was again observed and present within the HER2E-mRNA-subtype/HER2+ tumours, as was high pSRC and pS6; conversely, many protein markers of luminal cancers again distinguished the luminal-mRNA-subtype/HER2+ tumours. Given the importance of clinical HER2 status, a more focused analysis was performed based on the RPPA-defined protein expression of HER2 (Supplementary Fig. 19)—the results strongly recapitulated findings from the RPPA and mRNA subtypes including a high correlation between HER2 clinical status, HER2 protein by RPPA, pHER2, EGFR and pEGFR. These multiple signatures, namely HER2E mRNA subtype, HER2 amplicon genes by mRNA expression, and RPPA EGFR/pEGFR/HER2/pHER2 signature, ultimately identify at least two groups/subtypes within clinically HER2+ tumours (Table 1). These signatures represent breast cancer biomarker(s) that could potentially predict response to anti-HER2 targeted therapies.

Many therapeutic advances have been made for clinically HER2+ disease. This study has identified additional somatic mutations that represent potential therapeutic targets within this group, including a high frequency of PIK3CA mutations (39%), a lower frequency of PTEN and PIK3R1 mutations (Supplementary Table 6), and genomic losses of PTEN and INPP4B. Other possible druggable mutations included variants within HER family members including two somatic mutations in HER2, two within EGFR, and five within HER3. Pertuzumab, in combination with trastuzumab, targets the HER2–HER3 heterodimer49; however, these data suggest that targeting EGFR with HER2 could also be beneficial. Finally, the HER2E mRNA subtype typically showed high aneuploidy, the highest somatic mutation rate (Table 1), and DNA amplification of other potential therapeutic targets including FGFRs, EGFR, CDK4 and cyclin D1.