HER3 PET Imaging Predicts Response to Pan-Receptor Tyrosine Kinase Inhibtion Therapy in Gastric Cancer

1203 Objectives: Receptor tyrosine kinase (RTK) inhibitors have become an integral part of gastric cancer (GCa) treatment, as approximately 50% of these tumors primarily depend on RTK signaling for their growth. Despite the favorable initial response to specific RTK inhibition therapy in GCa, the overall survival of patients with advanced GCa remains suboptimal due to developed resistance to specific RTK inhibition. Newer pan-RTK inhibitors alleviate this problem by inhibition of downstream signaling from multiple RTKs. In this study, we hypothesize that the response to pan-RTK inhibition can be predicated by upregulation of HER3 shortly after treatment initiation, as an indicator of addiction to RTK oncogenes for tumor growth. Methods: A GCa cell line panel was assessed for baseline level of total and phosphorylated RTKs including EGFR, HER2, and HER3, and their downstream signaling nodes. Cell viability was assessed by WST assay in the cell panel treated with 0.01 - 1M of Afatinib over 72 hours. HER3-expressing afatinib-sensitive (NCI-N87) and afatinib-resistant (SNU16) cell lines were selected for evaluation of quantitative changes in RTKs, and their downstream signaling pathways following treatment with afatinib (a pan-RTK inhibitor) in vitro. Murine xenograft models of GCa were developed by subcutaneous injection of NCI-N87 and SNU16 in nude mice (n=20 per group). PET/MR imaging was performed with a previously developed HER3 targeting peptide (68Ga-HER3P1) 1 hour after intravenous injection of the probe at baseline and 3 days after treatment with Afatinib (10 mg/kg, PO, Daily, n=10 per group) or vehicle (water, PO, daily). The temporal quantitative changes in HER3 PET uptake (SUVmax and SUVmean) between baseline and day 3 of treatment were assessed. HER3 PET quantitation was correlated with the changes in HER3 expression in tumor tissue by immunofluorescence microscopic evaluation. The tumor growth curves were measured in tumors for each group over 3 weeks. Results: In the afatinib-sensitive NCI-N87 cells, afatinib treatment at 100nM concentration resulted in markedly increased expression of total HER3 and reduction of pHER3 and all other RTKs and downstream signaling nodes over 72 hours, while in afatinib-resistant SNU16 cells a complete opposite profile was observed (total HER3 at 72 h afatinib / baseline: 3.7 ± 0.22 in NCI-N87 vs. 0.48 ± 0.72 in SNU16, respectively; p<0.001).68Ga-HER3P1 PET/MR imaging showed significant increase in NCI-N87 tumor SUVmean on day 3 of treatment with afatinib compared to baseline, while in resistant SNU16 xenografts the difference in SUVmean was not statistically significant between the time points (SUVmean in NCI-N87 tumors was 1.6 ± 0.56 vs. 3.85 ± 0.69, p<0.05 and in SNU16 was 1.72 ± 0.73 vs. 1.5 ± 0.69, p=0.09 at baseline vs. 72h afatinib, respectively). Quantitative changes of HER3 expression level was confirmed with the immunofluorescence microscopic imaging of tumor tissue slices at baseline and 3 days post treatment. Tumor growth trajectory over 3 weeks of treatment with afatinib was concordant with changes in HER3 expression (mean tumor volume of NCI-N87: 11.2 ± 16.6 mm3 vs. 293 ± 79.35 mm3p< 0.05, SNU16: 178.3 ± 91.2 mm3vs. 281.9 ± 34.9 mm3p= 0.2 treated vs. control group, respectively). Conclusions: HER3 PET imaging could provide a rapid and non-invasive assessment of temporal quantitative changes in HER3 expression in gastric cancers treated with RTK inhibition therapy. The paradigm of pre- and intra-therapy HER3 PET imaging could predict response to pan-RTK inhibitors within few days after initiation of treatment and help with personalizing the treatment of gastric cancer patients in advanced stages.This proposed quantitative HER3 PET imaging approach could also be applied to a wide range of other cancers in which the HER3 feedback loop is an important mechanism of therapy resistance, including non-small cell lung, breast and thyroid cancer, among others.