Development of a PET Imaging Probe for Discrimination of Secondary Mutation in the Epidermal Growth Factor Receptor

Objectives Lung cancer is broadly divided into small-cell lung carcinoma and non-small-cell lung carcinoma (NSCLC). Approximately 10-30% of patients with NSCLC have a mutation in the epidermal growth factor receptor (EGFR) gene and receive molecular targeting therapy toward EGFR tyrosine kinase (EGFR-TK). The phosphorylation of EGFR-TK plays a key role in cell proliferation and angiogenesis in cancer cells. EGFR-TK inhibitors (EGFR-TKIs) , such as gefitinib (Iressa®), erlotinib (Tarceva®), and afatinib (Giotriff®) are used in therapy of NSCLC and bind to the ATP domain in EGFR-TK expressing the L858R mutation. The acquisition of resistance to EGFR-TKIs is inevitable; it is known that patients who initially respond typically develop resistance within 6-12 months from the initiation of therapy. The T790M mutation in EGFR is the most common factor, which accounts for resistance in approximately 50% of these patients. Thus, the ability to predict whether EGFR gains secondary mutation is extremely important. In clinical settings, genetic testing is performed by biopsy to find mutations and give a definitive diagnosis of cancer before starting the therapy. However, because of the invasiveness of this procedure, testing is rarely done when the effect of molecular targeting drugs reduces during treatment. Therefore, in order to perform effective treatment, the development of non-invasive diagnostics techniques has been desired. In this study, we designed and synthesized a series of HO compounds with a 4-(anilino)pyrido[3,4-d]pyrimidine scaffold, and evaluated their potential for use as a PET imaging probe for discriminating between L858R and L858R/T790M mutant EGFR in NSCLC.

Methods The inhibitory activity on EGFR was examined by the EGFR Kinase Enzyme System and an ADP-GloTM Kinase Assay Kit. Biodistribution studies were carried out in H3255 tumor-bearing nude mice. A blocking experiment was performed by co-injection of [18F]HO-J and an excess of AZD9291. [18F]HO-J was injected intravenously into the tails of H3255 (L858R) or H1975 (L858R/T790M) tumor-bearing nude mice and PET scanning was started after 3h.

Results We designed and synthesized a series of HO compounds with a 4-(anilino)pyrido[3,4-d]pyrimidine scaffold as novel PET imaging agents for discrimination between L858R and L858R/T790M mutant EGFR. When used in the EGFR-TK inhibition assay, HO-J exhibited the highest inhibition potency (IC50 = 15.6 nM) to the L858R mutant EGFR-TK, while also exhibiting low inhibition (IC50 = 326 nM) of the L858R/T790M mutant. Further assessment of biodistribution revealed that [18F]HO-J displays a high tumor-to-muscle (T/M), tumor-to-blood (T/B) and tumor-to-lung (T/L) ratio in H3255 tumor-bearing mice (T/M = 13, T/B = 3, T/L = 3 at 3h post injection). Co-injection of excess AZD9291 resulted in a significant decrease in tumor uptake by 54.3% at 3 h post-injection, and there was no significant decrease in other tissues, indicating that tracer uptake was specific for EGFR-TK. In addition, we were able to visualize the H3255 tumor more clearly than the H1975 tumor at 3h post injection by PET imaging.

Conclusions We conclude that [18F]HO-J has potential for use as a PET imaging probe for discriminating between L858R and L858R/T790M mutant EGFR in NSCLC.

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