Depicting Changes in Tumor Biology in Response to Cetuximab Monotherapy or Combination Therapy by Apoptosis and Proliferation Imaging Using ¹⁸F-ICMT-11 and ¹⁸F-FLT PET.

Abstract

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7-specific isatin-5-sulfonamide ¹⁸F-ICMT-11 (¹⁸F-(<i>S</i>)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluoro-phenoxymethyl)-pyrrolidine-1-sulfonyl)isatin) and ¹⁸F-FLT (3'-deoxy-3'-¹⁸F-fluorothymidine), were used to detect early treatment-induced changes in tumor biology and determine whether any of these changes indicate a response to cetuximab, administered as monotherapy or combination therapy with gemcitabine. <b>Methods:</b> In mice bearing cetuximab-sensitive H1975 tumors (non-small lung cancer), the effects of single or repeated doses of the antiepidermal growth factor receptor antibody cetuximab (10 mg/kg on day 1 only or on days 1 and 2) or a single dose of gemcitabine (125 mg/kg on day 2) were investigated by ¹⁸F-ICMT-11 or ¹⁸F-FLT on day 3. Imaging was also performed after 2 doses of cetuximab (days 1 and 2) in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer). For imaging-histology comparison, tumors were evaluated for proliferation (Ki-67 and thymidine kinase 1 [TK1]), cell death (cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and target engagement (epidermal growth factor receptor expression) by immunohistochemistry, immunofluorescence, and immunoblotting, respectively. Tumor and plasma were analyzed for thymidine and gemcitabine metabolites by liquid chromatography-mass spectrometry. <b>Results:</b> Retention of both tracers was sensitive to cetuximab in H1975 tumors. ¹⁸F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab (75%) and combination treatment (46%). Although a single dose of cetuximab was insufficient to induce apoptosis, it did affect proliferation. Significant reductions in tumor ¹⁸F-FLT uptake (44%-50%; <i>P</i> < 0.001) induced by cetuximab monotherapy and combination therapy were paralleled by a clear decrease in proliferation (Ki-67 decrease, 72%-95%; <i>P</i> < 0.0001), followed by a marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly reduced. Neither imaging tracer depicted the gemcitabine-induced tumor changes. However, cleaved caspase-3 and Ki-67 staining did not significantly differ after gemcitabine treatment whereas TK1 expression and thymidine concentrations increased. No cetuximab-induced modulation of the imaging tracers or other response markers was detected in the insensitive model of HCT116. <b>Conclusion:</b>¹⁸F-ICMT-11 and ¹⁸F-FLT are valuable tools to assess cetuximab sensitivity depicting distinct and time-variant aspects of treatment response.