In vivo detection of resistance to EGFR tyrosine kinase inhibitors by 18F-FLT PET/CT and its reversal in non small cell lung cancer.

Multiple molecular mechanisms may underlie the resistance to EGFR tyrosine kinase inhibitors (TKIs), including the occurrence of secondary mutations such as T790M in the kinase domain of EGFR, redundant lateral signaling or alterations of apoptotic program mainly due to dysregulation of Bcl-2 family members. Aim. To test whether 18F-Fluorothymidine (18F-FLT) could detect EGFR TKI refractory tumors and identify the mechanisms underlying such resistance so that specific therapeutic strategies can be adopted in patients with non-small cell lung cancer (NSCLC). Materials and Methods. EGFR TKI sensitive and resistant NSCLC cells (HCC827, H1975, and H1650 cells) were preliminarily evaluated for erlotinib sensitivity and growth arrest. Cells were also tested for erlotinib-induced inhibition of EGFR signaling and levels of Bcl-2 family members. NSCLC cells were subcutaneously injected into flanks of individual female BALB/c (nu/nu) mice and cells were then allowed to grow until tumors reached a diameter ranging between 0.3-1 cm. The synthesis of 18F-FLT was performed using the commercially available TRACERlab FX F-N synthesis module (GE Healthcare) and (5’-O-DMTr-2’-deoxy-3’-O-nosyl-β-D-threo-pentofuranosyl)-3-N-BOC-thymine as a precursor. The resulting labeled products had >99% radiochemical purity as assessed by high-performance liquid chromatography. Tumor-bearing mice were injected with 7.4 MBq of 18F-FLT through the tail vein and were subjected to microPET/CT (eXplore Vista Pre-Clinical PET Scanner GE Healthcare) 1 h post-injection. Each animal was subjected to 18F-FLT microPET/CT study before and after treatment with reversible (erlotinib, 50 mg/kg/d p.o. for 3 days) or irreversible (CL-387,785, 50 mg/kg/d p.o. for 3 days) EGFR TKIs. Results. We found that the uptake of 18F-FLT in sensitive tumors was dramatically reduced after treatment with both reversible and irreversible EGFR TKIs. Resistant NSCLC bearing T790M mutation showed a persistent high uptake of 18 F-FLT after treatment with reversible inhibitors indicating lack of growth arrest. Conversely, treatment with irreversible inhibitors caused a reduction of 18F-FLT uptake in resistant tumors indicating the reversal of T790M mutation-dependent resistance. Furthermore, NSCLC cells that were resistant due to dysregulation of Bcl-2 family members became sensitive to EGFR TKIs when treatment included Bcl-2 inhibitors. Conclusions. Resistance to EGFR TKI may be caused by multiple mechanisms. PET/CT with 18F-FLT may contribute to the selection of patients that may benefit from treatment with irreversible EGFR TKI inhibitors.