Using fluorodeoxythymidine to monitor anti-EGFR inhibitor therapy in squamous cell carcinoma xenografts

Abstract

Background: 3'-18F-fluoro-3'-deoxy-fluorothymidine (18F-FLT), a nucleoside analog, could monitor effects of molecularly targeted therapeutics on tumor proliferation.

Methods: We tested whether (18)F-FLT positron emission tomography (PET) uptake changes are associated with antitumor effects of erlotinib in A431 xenografts or cetuximab in SCC1 xenografts.

Results: Compared with pretreatment FLT PET scans, 3 days of erlotinib in A431 reduced the standardized uptake value (SUV) by 18%, whereas placebo increased SUV by 1% (p = .005). One week of cetuximab in SCC1 reduced SUV by 62%, whereas placebo reduced SUV by 16% (p = .005). FLT uptake suppression following anti-epidermal growth factor receptor (EGFR) treatment was associated with reduced tumor thymidine kinase-1 (TK1) activity. In vitro TK1 knockdown studies confirmed the importance of TK1 activity on intracellular FLT accumulation suppression.

Conclusions: 18F-FLT PET imaging detects tumor responses to EGFR-inhibitors within days of starting therapy. This technique may identify patients likely to benefit from EGFR-inhibitors early in their treatment course.

Figures

FIGURE 1

Erlotinib treatment of A431 xenografts. (A) The schedule for FLT PET imaging relative to erlotinib treatment is shown. (B) The relative tumor volumes (mean ± SD) are shown for erlotinib or placebo-treated mice. (C) Western blotting with the indicated antibodies of tumors from 3 placebo- and 3 erlotinib-treated mice.

FIGURE 2

18F-FLT PET imaging of response to erlotinib. (A) A PET/CT fusion image of an animal imaged following 18F-FLT injection is shown. (B) Representative pretreatment and posttreatment axial PET images from mice treated with erlotinib or placebo. The tumor volume of interest on the image is outlined in blue. (C) Relative change in standard uptake value (Δ-SUV) for each animal imaged, and the median value for each treatment group is shown. (D) A similar analysis is shown for the relative change in tumor to muscle activity ratio (Δ-TMR).

FIGURE 3

Cetuximab treatment of SCC1 xenografts. (A) The schedule for FLT PET imaging relative to cetuximab treatment is shown. (B) The relative tumor volumes (mean ± SD) are shown for placebo and cetuximab-treated mice. (C) Western blotting with the indicated antibodies of tumors from 3 placebo- and 3 cetuximab-treated mice.

FIGURE 4

18F-FLT PET imaging of response to cetuximab. (A) Representative pretreatment and posttreatment coronal maximum-intensity projection PET images from mice treated with cetuximab or placebo are shown. (B) Relative change in standard uptake value (Δ-SUV) is plotted for each animal imaged and the median value for each treatment group is shown. (C) A similar analysis is shown for the relative change in tumor to muscle activity ratio (Δ-TMR).

FIGURE 5

The MIB-1 labeling index for placebo and treated mice (mean SD). A) Results from A431 xenografts treated with or without erlotnib and associated representative photomicrographs. B) Results from SCC1 xenografts treated with or without cetuximab and associated representative photomicrographs.

FIGURE 6

Evaluation of thymidine kinase-1 activity. (A) Western blotting for TK1 expression with actin loading control. (B). Western blotting with the indicated antibodies following A431 and SCC1 cell siRNA oligonucleotide transfection for TK1 or firefly luciferase (Luc). (C) Parallel 3H-FLT evaluation of TK1 knockdown on FLT accumulation (mean ± SEM of 3 independent experiments). (D) The effects of erlotinib treatment of A431 cells on relative TK1 protein levels by Western blot, and parallel 3H-FLT uptake. Mean TK1 protein levels, normalized to β-actin, and corresponding levels of 3H-FLT uptake are shown as mean ± SEM of 3 independent experiments.