Early detection of erlotinib treatment response in NSCLC by 3'-deoxy-3'-[F]-fluoro-L-thymidine ([F]FLT) positron emission tomography (PET)

Roland T Ullrich, Thomas Zander, Bernd Neumaier, Mirjam Koker, Takeshi Shimamura, Yannic Waerzeggers, Christa L Borgman, Samir Tawadros, Hongfeng Li, Martin L Sos, Heiko Backes, Geoffrey I Shapiro, Jürgen Wolf, Andreas H Jacobs, Roman K Thomas, Alexandra Winkeler, Roland T Ullrich, Thomas Zander, Bernd Neumaier, Mirjam Koker, Takeshi Shimamura, Yannic Waerzeggers, Christa L Borgman, Samir Tawadros, Hongfeng Li, Martin L Sos, Heiko Backes, Geoffrey I Shapiro, Jürgen Wolf, Andreas H Jacobs, Roman K Thomas, Alexandra Winkeler

Abstract

Background: Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking.

Methodology/principal findings: We performed a systematic comparison of 3'-Deoxy-3'-[(18)F]-fluoro-L-thymidine ([(18)F]FLT) and 2-[(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [(18)F]FLT uptake after only two days of treatment, [(18)F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [(18)F]FLT PET but not [(18)F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [(18)F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [(18)F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR.

Conclusions: [(18)F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [(18)F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Erlotinib treatment induces down-regulation of…
Figure 1. Erlotinib treatment induces down-regulation of EGFR/EGFR-coupled signaling pathways and cell cycle arrest with subsequent induction of apoptosis in EGFR sensitive tumor cells.
The erlotinib sensitive cell lines HCC827 and PC9 and the erlotinib-resistant cell line H1975 were treated with the indicated doses of erlotinib for 24 hours. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies (A). PC9, HCC827 and H1975 cells were treated with erlotinib (0.5 µM) for 24h, 48h and 72h; nuclei were prepared, stained with propidium iodide and analyzed by flow cytometry. Results are shown for the G1 and S phases of the cell cycle (A). Apoptotic effects of erlotinib on EGFR-sensitive cell lines in comparison to the T790M mutant H1975 (B). Annexin V FACS was performed 12h, 24h, 36h, 48h, 72h and 96h after 0.5 µM erlotinib treatment. Images show Annexin V-positive cells after 48h in the different cell lines.
Figure 2. [ 18 F]FLT PET indicates…
Figure 2. [18F]FLT PET indicates response to therapy after 2 days of erlotinib treatment.
In (A) a representative [18F]FLT PET image of a mouse bearing the sensitive PC9, HCC827 and the resistant H1975 xenografts before beginning of treatment, 48h and 96h after daily erlotinib treatment (Tarceva, 50mg/kg). (B) Quantitative analysis of changes in [18F]FLT and [18F]FDG uptake ratios after 48h and 96h daily erlotinib treatment vs. vehicle only as control (PC9: n = 8; vehicle, n = 2; HCC827: n = 7; vehicle, n = 2; H1975: n = 8; vehicle, n = 2).
Figure 3. Immunohistochemistry of tumor tissue for…
Figure 3. Immunohistochemistry of tumor tissue for Ki-67 expression and TUNEL, relation of [18F]FLT and [18F]FDG uptake to Ki-67 expression, and measurement of tumor volume for the assessment of treatment response.
(A) Frozen tissue was stained for Ki-67 and TUNEL (magnification 10×). Columns, average number of TUNEL positive cells (green cells) were counted in three randomly selected field (area 0.625mm2) in two tumor samples for each cell line. The Ki-67 labeling index as assessed by the percentage of nuclei stained with MIB-1 per total number of nuclei was compared to uptake ratios of [18F]FLT and [18F]FDG (B). Effects of daily Erlotinib treatment on the tumor size of the xenografts for the assessment of tumor response (C).

References

    1. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350:2129–2139.
    1. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304:1497–1500.
    1. Pao W, Miller V, Zakowski M, Doherty J, Politi K, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004;101:13306–13311.
    1. Thomas RK, Nickerson E, Simons JF, Janne PA, Tengs T, et al. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat Med. 2006;12:852–855.
    1. Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet 2007
    1. Pieterman RM, van Putten JW, Meuzelaar JJ, Mooyaart EL, Vaalburg W, et al. Preoperative staging of non-small-cell lung cancer with positron-emission tomography. N Engl J Med. 2000;343:254–261.
    1. Weber WA, Dietlein M, Hellwig D, Kirsch CM, Schicha H, et al. [PET with (18)F-fluorodeoxyglucose for staging of non-small cell lung cancer]. Nuklearmedizin. 2003;42:135–144.
    1. Weber WA, Petersen V, Schmidt B, Tyndale-Hines L, Link T, et al. Positron emission tomography in non-small-cell lung cancer: prediction of response to chemotherapy by quantitative assessment of glucose use. J Clin Oncol. 2003;21:2651–2657.
    1. Su H, Bodenstein C, Dumont RA, Seimbille Y, Dubinett S, et al. Monitoring tumor glucose utilization by positron emission tomography for the prediction of treatment response to epidermal growth factor receptor kinase inhibitors. Clin Cancer Res. 2006;12:5659–5667.
    1. Moyer JD, Barbacci EG, Iwata KK, Arnold L, Boman B, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 1997;57:4838–4848.
    1. Shields AF, Grierson JR, Dohmen BM, Machulla HJ, Stayanoff JC, et al. Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat Med. 1998;4:1334–1336.
    1. Toyohara J, Waki A, Takamatsu S, Yonekura Y, Magata Y, et al. Basis of FLT as a cell proliferation marker: comparative uptake studies with [3H]thymidine and [3H]arabinothymidine, and cell-analysis in 22 asynchronously growing tumor cell lines. Nucl Med Biol. 2002;29:281–287.
    1. Buck AK, Schirrmeister H, Hetzel M, Von Der Heide M, Halter G, et al. 3-deoxy-3-[(18)F]fluorothymidine-positron emission tomography for noninvasive assessment of proliferation in pulmonary nodules. Cancer Res. 2002;62:3331–3334.
    1. Ullrich R, Backes H, Li H, Kracht L, Miletic H, et al. Glioma proliferation as assessed by 3′-fluoro-3′-deoxy-L-thymidine positron emission tomography in patients with newly diagnosed high-grade glioma. Clin Cancer Res. 2008;14:2049–2055.
    1. Solit DB, Santos E, Pratilas CA, Lobo J, Moroz M, et al. 3′-deoxy-3′-[18F]fluorothymidine positron emission tomography is a sensitive method for imaging the response of BRAF-dependent tumors to MEK inhibition. Cancer Res. 2007;67:11463–11469.
    1. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–216.
    1. Hamacher K, Coenen HH, Stocklin G. Efficient stereospecific synthesis of no-carrier-added 2-[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported nucleophilic substitution. J Nucl Med. 1986;27:235–238.
    1. Machulla H, Blocther A, Kuntzsch M, Piert M, Wei R, et al. Simplified labeling approach for synthesizing 3-deoxy-3-[F-18]fluorothymidine ([F-18]FLT). J Radiochem Nucl Chem. 2000;243:843–846.
    1. Fueger BJ, Czernin J, Hildebrandt I, Tran C, Halpern BS, et al. Impact of animal handling on the results of 18F-FDG PET studies in mice. J Nucl Med. 2006;47:999–1006.
    1. Schiffer WK, Mirrione MM, Dewey SL. Optimizing experimental protocols for quantitative behavioral imaging with 18F-FDG in rodents. J Nucl Med. 2007;48:277–287.

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