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Methods
Results
Discussion Monitoring and early prediction of patient response to therapy is increasingly important, particularly because of heterogeneity of response to expensive or toxic agents, and missed opportunity of effective therapy by early switching. Many drugs, although targeted to highly specific processes, are not targeted to genetic lesion, so it increased is difficult to select patients on baseline genetic criteria. For anti-angiogenic treatment, this is even more important as effectiveness is small overall but likely to have substantial benefit in minority of patients. It is notable that median progression-free interval in many trials of bevacizumab is often only 4–6weeks, and 1–2weeks overall survival benefit, suggesting this early pattern is what sets agenda for final benefit. Our aim was to link macroscopic heterogeneity of response to molecular pathways that are targetable. We combined monitoring by DCE-MRI and molecular profiling with short-term first-line treatment in primary breast cancer to measure early response to bevacizumab. Clearly when analysing whole tumor by imaging we can never sample to the same extent, but only conduct small number of biopsies. Furthermore, previous work from our (Ah-See et al., 2008) and other groups (Othman et al., 2016; Beuzit et al., 2016; Wang et al., 2015) has demonstrated DCE-MRI parameter estimation can depend on a variety of factors including normal physiological variability, MRI set-up and acquisition protocols, and different image processing methods. Strikingly, in spite of this, we observed strong correlations of functional imaging with molecular changes, regardless of the DCE-MRI method used to categorize the patients (binary response, continuous median Ktrans and total Ktrans), reflecting heterogeneity of response and providing added molecular information. DCE-MRI showed initial response to bevacizumab in most patients. Specifically, 91% (32/35) patients showed decrease in median Ktrans, and 77% (27/35) had a >30% decrease. This indicates a general initial vascular normalisation, and downregulation of angiogenic factors ESM1 and FLT1. Interestingly, another family member, FLT4, was predictive for bevacizumab response in ovarian cancer (Collinson et al., 2013). Whilst this work was submitted, a study was published (Tolaney et al., 2015) with similar window design, but a lower dose of bevacizumab. Decreased microvessel density [MVD] at 2weeks was observed. We, however, reveal the complex response to anti-bevacizumab therapy at an early timepoint, when this response can be evaluated alone. Notwithstanding initial response, our study revealed heterogeneity ranging from tumours developing central necrosis to continuous growth. In fact, it is clear that a subgroup of patients experienced vessels normalisation, with decreased proliferation. Equally, there is another group of patients where HIF1α and regulated genes (e.g. CA9), necrosis and angiogenesis increase. So we showed that vascular normalisation is likely to be a mechanism of response, but there is a subgroup of patients where changes are induced in response to hypoxia which can actually induce further vessel production and resistance, such as induction of CXCR4 and metabolism. This suggests a feedback loop in these patients where an initial response potentially shuts down, rather than normalize, blood supply with consequent induction of hypoxia. These resistance mechanisms offer great opportunities for combination therapy as we have already demonstrated in preclinical models (McIntyre et al., 2012; Favaro et al., 2012; Li et al., 2007). Importantly, immune checkpoint genes were induced on treatment, including CTLA4, and its ligand CD86, which could antagonize CD28 T-cell activation. Although CTLA4 has greater CD86 affinity than CD28 (Linsley et al., 1991); co-presence of activating/inhibiting signaling suggests co-existence of recruited versus non-recruited T-cell pockets in these tumours. Recently, Programmed Death Ligand 1, PD-L1, not showing significant changes in this study but member of the same CD28/CTLA4 family of receptors, has been shown to be direct target of HIF1A, and when blocked under hypoxia it enhanced myeloid-derived suppressor cells-mediated T-cell activation (Noman et al., 2014). We cannot ascertain at this stage whether this is to specific antibody effects or interaction with hypoxia; however, these findings support benefit from combination of bevacizumab with novel immune checkpoint inhibitors to restore and boost T-cell immune response.