HER2-targeting agents are standard of care for the treatment of HER2+ breast cancer (BC), but resistance inevitably occurs and the underlying molecular mechanisms remain poorly characterized. The comparison between mutational profiles of primary (treatment-naïve) and metastatic (who will mostly have received multiple courses of HER2-targeting agents) tumors can inform the search for resistance-inducing genetic alterations to be modeled in vitro and in vivo.
We analyzed the mutational spectrum of metastatic vs primary HER2+ BC by retrieving data from ERBB2-amplified cases in the MSKCC-IMPACT and the TCGA datasets, containing almost exclusively samples from metastatic sites and primary tumors, respectively. Multiple in vitro (HER2+ cell lines with stable RNA interference of candidate genes, 2D- and 3D-culturing) and in vivo models (NOD-SCID xenografts and the MMTV-neu transgenic mice) are used for validation.
Mutations in NF1, TSC2 and HER2 were significantly enriched in metastatic cases. All are plausible mediators of resistance, being associated with signalling pathways downstream of HER2 or the HER2 receptor itself. NF1 in particular is a tumor suppressor gene which encodes a RAS-GTPase that negatively regulates Ras/MEK/ERK and PI3K/AKT/mTOR pathways. NF1 alterations were the most significant (Odds Ratio 11.7, p = 0.00018, FDR=0.23) and the most prevalent in metastatic cases (∼10%). As NF1 mutations lead to protein loss of function, we modeled their effect in vitro by RNA interference and found that it induces an increase in mTOR activation. Stably-interfered HER2+ cell lines will be used to explore synergistic drug combinations in vitro and in vivo.
We provide proof of concept that secondary resistance to anti-HER2 agents can be mapped to specific genetic alterations in a sizeable fraction of patients. Ongoing efforts are aimed at investigating if NF1 loss instructs HER2+ BC with a metastasis-promoting phenotype as well as metabolic and transcriptional changes.
European Institute of Oncology.
Fondazione IEO-CCM University of Milan.
G. Curigliano: Advisor: Roche, Pfizer, Novartis, Lilly. All other authors have declared no conflicts of interest.