Landscape of Epidermal Growth Factor Receptor Heterodimers in Brain Metastases

SIMPLE SUMMARY: HER2+ breast cancer patients are treated with agents that tag HER2+ tumour cells for elimination by the immune system, down-modulate HER2 activity and/or block the formation of HER2 dimers, including the neuregulin-1 receptor, HER2-HER3. HER2-targeted therapies prolong survival by lowering the risk of relapse, but do not prevent brain metastases. The reasons for this are not fully understood. We quantified HER2-HER3 dimers in 203 brain metastases, and 34 primary breast tumour samples. Dimer frequency was relatively high in brain metastases from breast, ovarian, lung and kidney cancers, and in brain metastases compared to patient-matched breast tumours; but did not reliably correlate with HER2/HER3 expression or activation. In in vitro experiments, pertuzumab failed to suppress HER2-HER3 dimers in HER2+ breast cancer cells provided with a saturating concentration of neuregulin-1. These findings may provide insights about the differences in intracranial versus extracranial efficacy of HER2-targeted therapies. ABSTRACT: 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 (n = 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(1221/1222)) and pHER3(Y(1222)) were positively, but not exclusively, associated with HER2-HER3 frequency. In an independent cohort (n = 78), BM had significantly higher HER2-HER3 levels than matching primary tumours (p = 0.0002). For patients who had two craniotomy procedures, HER2-HER3 dimer levels were lower in the consecutive lesion (n = 7; p = 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.