Monitoring afatinib treatment in HER2-positive gastric cancer with 18F-FDG and 89Zr-trastuzumab PET

Abstract

We evaluated the ability of the PET imaging agent (89)Zr-trastuzumab to delineate HER2-positive gastric cancer and to monitor the pharmacodynamic effects of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor afatinib.

Methods: Using (89)Zr-trastuzumab, (18)F-FDG, or 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT PET), we imaged HER2-positive NCI-N87 and HER2-negative MKN74 gastric cancer xenografts in mice. Next, we examined the pharmacodynamic effects of afatinib in NCI-N87 xenografts using (89)Zr-trastuzumab and (18)F-FDG PET and comparing imaging results to changes in tumor size and in protein expression as monitored by Western blot and histologic studies.

Results: Although (18)F-FDG uptake in NCI-N87 tumors did not change, a decrease in (89)Zr-trastuzumab uptake was observed in the afatinib-treated versus control groups (3.0 ± 0.0 percentage injected dose per gram (%ID/g) vs. 21.0 ± 3.4 %ID/g, respectively; P < 0.05). (89)Zr-trastuzumab PET results corresponded with tumor reduction, apoptosis, and downregulation of HER2 observed on treatment with afatinib. Downregulation of total HER2, phosphorylated (p)-HER2, and p-EGFR occurred within 24 h of the first dose of afatinib, with a sustained effect over 21 d of treatment.

Conclusion: Afatinib demonstrated antitumor activity in HER2-positive gastric cancer in vivo. (89)Zr-trastuzumab PET specifically delineated HER2-positive gastric cancer and can be used to measure the pharmacodynamic effects of afatinib.

Keywords: 18F-FDG; 89Zr-trastuzumab; HER2; afatinib; gastric cancer.

FIGURE 1

Effects of afatinib therapy in HER2-positive gastric cancer NCI-N87 xenograft. (A) Athymic nude mice bearing NCI-N87 tumors were treated (10 mice per group) with vehicle alone (intraperitoneally daily), afatinib (25 mg/kg orally daily), trastuzumab (20 mg/kg intravenously once a week), and combination of afatinib (25 mg/kg orally daily) with trastuzumab (20 mg/kg intravenously once a week). Tumor volume was measured twice a week after onset of treatment. (B) Western blot analyses of tumors harvested from HER2-positive NCI-N87 xenografts treated with afatinib (25 mg/kg) orally × 1 dose, specimen collected at 8, 24, 48, and 72 h after a single dose of afatinib. *P < 0.05, for afatinib vs. vehicle or trastuzumab alone.

FIGURE 2

Specificity of ⁸⁹Zr-trastuzumab for HER2-positive tumors. Coronal ⁸⁹Zr-trastuzumab, ¹⁸F-FDG, and ¹⁸F-FLT PET images of athymic nude mice bearing subcutaneous HER2-positive NCI-N87(left) and HER2-negative MKN-74 (right) are shown. ROIs (%ID/g) for ⁸⁹Zr-trastuzumab, ¹⁸F-FDG, and ¹⁸F-FLT are indicated. +ve = positive; −ve = negative.

FIGURE 3

Effects of afatinib therapy in HER2-positive xenograft models. (A) Timeline of imaging and treatment experiments. (B) Coronal ⁸⁹Zr-trastuzumab PET images of control (left) and afatinib (right)-treated mice bearing subcutaneous HER2-positive NCI-N87 tumors. Trans = transaxial; tras = trastuzumab.

FIGURE 4

Correlative analysis between ⁸⁹Zr-trastuzumab and ¹⁸F-FDG tumor uptake with tumor weight and HER2 levels. (A) Bar charts show ROIs (%ID/g) for ⁸⁹Zr-trastuzumab and ¹⁸F-FDG uptake in NCI-N87 tumors recorded and superimposed with tumor weight (graph) at baseline, 7 d, 14 d, and 21 d after treatment with afatinib (50 mg/kg daily). (B) Western blot analyses of tumors harvested from HER2-positive NCI-N87 xenografts treated with vehicle or afatinib (50 mg/kg orally daily); specimen collected at 1, 7, 14, and 21 d after treatment.

FIGURE 5

Autoradiographic determination of ⁸⁹Zr-trastuzumab distribution and HER2 histology of HER2-positive xenografts. (A) Frozen sections (10 µm) from NCI-N87 xenografts treated with afatinib (50 mg/kg) or vehicle for 7 d and removed 24 h after administration of ⁸⁹Zr-trastuzumab. Bar = 2 mm. (B) Representative HER2 immunostaining (brown) of FFPE NCI-N87 tumor sections demonstrating downregulation of HER2 in tumors administered with afatinib (50 mg/kg) at 7 d (middle) and 21 d (right) after treatment, compared with placebo-dosed tumors (left). Bar = 1 mm. H&E = hematoxylin and eosin.