Molecular imaging of nonsmall cell lung carcinomas expressing active mutant EGFR kinase using PET with [(124)i]-morpholino-IPQA

Abstract

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) have high levels of basal receptor phosphorylation and are associated with clinical responsiveness to Iressa in patients with nonsmall cell lung cancer (NSCLC). This study aimed to assess the feasibility of morpholino-[(124)I]IPQA derivative as an in vivo PET imaging tool for the expression of different EGFR mutants in NSCLC. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention after washout of morpholino-[(131)I]IPQA derivative in high EGFR-expressing H1299 NSCLC derivative cell lines (L858R and E746-A750 del cell lines), but not in EGFR-transfected H1299 cell line and vector-transfected H1299 cell line. Using the morpholino-[(124)I]IPQA derivative, we obtained noninvasive microPET images of EGFR activity in L858R and E746-A750 del subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown form control cell line. Different EGFR mutant (activity) tumors have a different morpholino-[ (∗) I]IPQA derivative uptake. However, it still needs to modify the structure of IPQA to increase its water solubility and reduce hepatobiliary clearance. Morpholino-[(124)I]IPQA derivative may be a potential probe for selection of the candidate patients suffering from NSCLC for the small molecule tyrosine kinase inhibitor therapy (e.g., Iressa) in the future.

Figures

Figure 1

The chemical structure of the (E)-But2-enedioic acid [4-(3-[124I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-ylpropyl)-amide, termed as morpholino-[124I]IPQA.

Figure 2

Assessment of irreversible binding of morpholino-[131I]IPQA to wild-type and mutant EGFR kinase in four types of human nonsmall cell lung carcinoma cell lines. (a) The autoradiography demonstrates the irreversible and covalent bindings of morpholino-[131I]IPQA to the EGFR kinase domain in different cell lines. (b) The same membrane was stained with anti-EGFR kinase antibody. A single band of radiolabeled protein corresponds to the predominate band of ~170 kDa.

Figure 3

In vitro uptake and washout phases of morpholino-[131I]IPQA in L858R (a), E746-A750 del (b), wild-type EGFR-transfected (c), and EGFR-vector (d). Liner fits of radioactivity accumulation time points show the washout rate at short duration (20–80 minutes, blue dot line) and long duration (60–120 minutes, red dot line) after initial accumulation (from (e) to (g)). Coefficient B (slope) of the exponential washout = A × exp⁡(−BX). Panels (a)–(d) are shown in full scale, and panels (e)–(g) are from the same data in panels (a)–(c) but are shown in smaller scale to visualize differences in washout results.

Figure 4

Reprehensive coronal and axial PET images obtained 24 hours after morpholino-[124I]IPQA administration in mice bearing L858R EGFR (a), E746-A750 del (b), wild-type EGFR-transfected (c), and EGFR-vector ((a)–(c)) subcutaneous tumor xenografts (dot circle). Color coding in the image is set to maximize the visualization of tumor in each projection. The different scale was used in (c). L: L858R EGFR; V: vector; D: E746-A750 del; E: EGFR-vector.

Figure 5

Quantification of accumulation of [124I]IPQA in different tumor xenografts at baseline (blue) and pretreated with Iressa (red), respectively. Error bars represent standard deviation; statically significant difference is indicated by an asterisk (*P < 0.05) or double asterisks (**P < 0.01).