Landscape of Epidermal Growth Factor Receptor Heterodimers in Brain Metastases.

HER2+ breast cancer patients have an elevated risk of developing brain metastases (BM), despite adjuvant HER2-targeted therapy. The mechanisms underpinning this reduced intracranial efficacy are unclear. We optimised the in situ proximity ligation assay (PLA) for detection of the high-affinity neuregulin-1 receptor, HER2-HER3 (a key target of pertuzumab), in archival tissue samples and developed a pipeline for high throughput extraction of PLA data from fluorescent microscope image files. Applying this to a large BM sample cohort ( = 159) showed that BM from breast, ovarian, lung and kidney cancers have higher HER2-HER3 levels than other primary tumour types (melanoma, colorectal and prostate cancers). HER2 status, and tumour cell membrane expression of pHER2(Y) and pHER3(Y) were positively, but not exclusively, associated with HER2-HER3 frequency. In an independent cohort ( = 78), BM had significantly higher HER2-HER3 levels than matching primary tumours ( = 0.0002). For patients who had two craniotomy procedures, HER2-HER3 dimer levels were lower in the consecutive lesion ( = 7; = 0.006). We also investigated the effects of trastuzumab and pertuzumab on five different heterodimers in vitro: HER2-EGFR, HER2-HER4, HER2-HER3, HER3-HER4, HER3-EGFR. Treatment significantly altered the absolute frequencies of individual complexes in SKBr3 and/or MDA-MB-361 cells, but in the presence of neuregulin-1, the overall distribution was not markedly altered, with HER2-HER3 and HER2-HER4 remaining predominant. Together, these findings suggest that markers of HER2 and HER3 expression are not always indicative of dimerization, and that pertuzumab may be less effective at reducing HER2-HER3 dimerization in the context of excess neuregulin.