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© 2003 American Society for Clinical Oncology Quantitative Tumor Apoptosis Imaging Using Technetium-99m–HYNIC Annexin V Single Photon Emission Computed TomographyFrom the Division of Nuclear Medicine and Department of Head and Neck Surgery, University Hospital Ghent; Department of Radiopharmacy and Department of Pathology, Ghent University, Ghent, Belgium; and Theseus Imaging Corp, Cambridge, MA. Address reprint requests to Christophe Van de Wiele, MD, PhD, Division of Nuclear Medicine, University Hospital Ghent, De Pintelaan 185B, Ghent 9000, Belgium; e-mail: christophe.vandeweile{at}rug.ac.be.
Purpose: Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m– (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. Patients and Methods: Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. Results: Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P = .012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. Conclusion: In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.
APOPTOSIS, A form of programmed cell death, is a natural, orderly, energy-dependent process that causes cells to die without inducing an inflammatory process.1 The apoptotic process is either triggered by a decrease in factors required to maintain the cell in good health or by an increase in factors that causes cells to die without inducing an inflammatory process. The notion that apoptosis represents a critical element in cell number control in various physiologic and pathologic situations has been well reviewed and its role in oncogenesis is now well established.2–5 Various xenografted tumor models support the notion that many of the effects of chemotherapy and radiotherapy are mediated by rapid induction of apoptosis.6–10 In the clinical setting, however, the occurrence of apoptosis after chemotherapy or radiation therapy of solid tumors is less well documented, likely reflecting the difficulty in obtaining sequential biopsy material at appropriate times after therapy. In addition, the appropriate timing for serial biopsies, even if they were feasible, is not clear because different agents and treatment options induce apoptosis with different kinetics. Annexin V binds to membrane-bound phosphatidyl serine, a constitutive anionic membrane phospholipid that is normally restricted to the inner leaflet of the plasma membrane lipid bilayer but is selectively exposed on the surfaces of cells as they undergo apoptosis. Annexin V labeled with a fluorescent tag is routinely used for histologic and cell-sorting studies to identify apoptotic cells.11 In analogy, annexin V can be radiolabeled with an appropriate radionuclide tag, such as technetium-99m (99mTc) or iodine-123, for gamma camera imaging.12 Thus, radiolabeled annexin V could be used for repetitive and selective in vivo identification of the site and extent of apoptotic cell death, and for monitoring cell death kinetics without the need for invasive biopsy. This study, approved by the Ethics Committee of the Ghent University Hospital (Ghent, Belgium), reports on the relationship between quantitative 99mTc–6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake measurements and the related number of tumor apoptotic cells derived from histologic tumor analysis. This study is the first to report on the use of 99mTc-HYNIC–labeled annexin V in oncology patients.
We prospectively included 20 patients (18 men and two women; mean age, 58.2 years; range, 47 to 78 years) clinically suspected to have primary head and neck carcinoma (n = 19) or local recurrence (n = 1) of a formerly biopsy-proven head and neck carcinoma. These 20 patients were enrolled onto the study after written informed consent was received. Patient characteristics are shown in Table 1  . All patients underwent a spiral computed tomography (CT) scan, which allowed estimation of lesion size in three dimensions, and a 99mTc-HYNIC annexin V scintigraphy within 1 week from the CT scan. Surgical resection of the suspected primary tumor or local recurrence for gold standard histopathologic analysis was performed on all patients within a period of 10 days after 99mTc-HYNIC annexin V scintigraphy. All resected tissues were exactly localized and documented at each level to allow comparison between histopathologic findings and preoperative imaging results. The percentage uptake of the injected dose of 99mTc HYNIC annexin V in visible tumor lesion on scintigrams divided by the tumor volume (derived from CT) was related to the number of apoptotic cells, derived from terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays, per high-power field in tumors.
99mTc-HYNIC Annexin V Planar and Tomographic Imaging Radiolabeling. 99mTc-HYNIC annexin V in kit formulation was prepared according to the guidelines provided by the manufacturer (Theseus Imaging Corp, Cambridge, MA). Data acquisition. Each patient received a volume of approximately 1.6 mL of reconstituted injectate containing approximately 550 MBq (standard deviation [SD], 110 MBq) of 99mTc-HYNIC annexin V. A standard of 4 MBq 99mTc HYNIC annexin V in 5 mL saline inside a syringe, put in a Perspex cylindrical phantom, was positioned aside the head of the patients for single photon emission computed tomography (SPECT) imaging. Planar whole-body images (sweep rate 15 cm/min), SPECT images of the head and neck region, and standard images (step and shoot mode, one step per 3 degrees, 30-second frame time, matrix size 128 x 128) were acquired 1 hour and 5 to 6 hours postinjection (PI) in all patients using a dual- or triple-headed gamma camera (Marconi, Cleveland, OH) equipped with low-energy, high-resolution collimators. The energy peak was centered at 140 keV with a 20% window. Acquired images were transferred to a Hermes system (Nuclear Diagnostics Ltd, Stockholm, Sweden; Solaris operating system) for processing. Data processing and reconstruction. Raw SPECT data were reconstructed using a commercially available ordered subset expectation maximization algorithm (four iterations, six subsets) and were postfiltered using a Butterworth filter (cutoff frequency, 0.8; order, 7.0). Transaxial, coronal, and sagittal slices were visually assessed for the presence of foci of increased tracer accumulation by two experienced nuclear medicine physicians. For the purpose of quantification, tomographic slices were summed to incorporate the entire dimensions of the tumor. Percentage uptake was obtained using the following equation: [(total lesion radioactivity counts at time of SPECT x radioactivity of standard at time of SPECT scan)/(standard counts at time of SPECT scan x total radioactivity injected)] x 100%.
CT Scan
Histopathologic Examination To evaluate the percentage of necrosis, paraffin-embedded tumor sections were stained with hematoxylin and eosin, and estimated semiquantitatively.
Statistical Methods
Clinical patient data and results are shown in Tables 1  and 2, respectively.
Final diagnosis on the basis of histopathology was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary in one patient, and the absence of recurrence in one patient. Tumor 99mTc-HYNIC annexin V activity was identified in 18 patients on SPECT, corresponding to pathologic regions identified by CT. In the two other patients, both CT and 99mTc-HYNIC annexin V SPECT proved negative, although the result was a false-negative on one occasion. Figure 1 shows uptake of 99mTc-HYNIC annexin V on the corresponding transaxial, sagittal, and coronal slices in a tumor of the lower lip of patient 5.
Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (SD, 0.0004%) at 1 hour PI and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P = .012).
TUNEL assay results were available in 16 of 18 99mTc-HYNIC annexin V–positive patients. Correlation coefficients and their corresponding P values between the number of apoptotic cells and the percentage of 99mTc-HYNIC annexin V taken up by tumors, including incremental inclusion of tumor samples with increasing percentages of necrosis, are shown in Figure 2
Several gold standards for measuring apoptosis have been described.13 Although historically, morphologic methods (including ultrastructural methods) have been favored, the recognition that apoptosis was associated with DNA fragmentation and the cleavage of DNA between nucleosomes led to the development of techniques using enzymes, either the terminal deoxynucleotidyl (used in TUNEL assays) or the Klenow fragment of the DNA polymerase (used for in situ end labeling), that are able to add labeled nucleotides to the DNA ends. The labeled nucleotides can then be identified by immunologic methods akin to immunohistochemistry. DNA fragments generated during apoptosis may be 3' recessed, 5' recessed, or blunt ended. In head and neck carcinoma, 3'-recessed fragments are predominant and can be identified using both TUNEL or in situ end-labeling assays. TUNEL assays, however, can also identify blunt and 5'-recessed ends and are thus more sensitive.14 Although the number of apoptotic cells is most often expressed as a percentage of the total number of tumor cells present or an apoptotic index, in this series the severity of ongoing apoptosis was expressed as the cumulative mean number of apoptotic cells per high-power field to allow for direct comparison with the 99mTc-HYNIC annexin V SPECT findings. The signal intensity generated by SPECT findings reflects the total number of apoptotic events, which is not so for apoptotic indices; in fact, low apoptotic indices can occur in the presence of a high number of total apoptotic cells in highly cellular tumors. Similar to previous reports on baseline apoptosis in head and neck tumors, apoptotic cells were present to a variable degree in all tumor samples studied.15 The presence of baseline apoptosis in head and neck tumors reflects the internal functioning of the death machinery of the neoplastic cells but also of normal lymphocytes. Squamous head and neck carcinoma cells express Fas ligand, which may associate with the Fas death receptor on invasion of normal T lymphocytes, which in turn triggers apoptosis in the cells.15,16 Thus, generated apoptotic lymphocytes are difficult to discern from apoptotic neoplastic cells.13 In concordance with the TUNEL assay results, 99mTc-HYNIC annexin V was taken up in head and neck tumors identified by CT. Tumor volume–normalized absolute percentage uptake changed significantly from early to delayed imaging because of ongoing clearance of nonspecific intratumor blood pool activity. The contribution of the latter on the absolute measurements on late tomographic images is likely to be low given the absence of intracardiac tracer activity on either planar or tomographic late images. As such, uptake values derived from late SPECT images rather than those from the early images were used for correlation analysis. 99mTc-HYNIC annexin V can also gain access to inner plasma membrane leaflet phosphatidylserine after the onset of irreversible membrane failure (poly-adenosine diphosphate-ribose polymerase–mediated cell death) as seen in necrosis (eg, myocardial infarction).17,18 Furthermore, the molecular weight of 35.8 kd of annexin V allows for rapid penetration of tumor tissues despite high interstitial intratumor pressures that oppose the influx of macromolecules, which leads to aspecific tumor uptake.19 As shown by Weissleder et al20 in an MCF-7 breast tumor model, leakage of macromolecules across tumor neovasculature into tumor interstitium is dependent on molecular mass with minimal extravasation occurring at and above 120 kd. Thus, the relationship between 99mTc-HYNIC annexin V uptake and the number of apoptotic cells as evidenced in this series is likely to be flawed by aspecific tumor uptake as well as by the inclusion of tumor samples containing significant amounts of necrosis. Correlation analysis indeed showed that whereas exclusion of tumor samples containing 5% or less necrosis resulted in good, statistically significant correlations between 99mTc-HYNIC annexin V uptake and the number of apoptotic cells, incremental inclusion of tumors with increasing amounts of necrosis resulted in a progressive decrease of correlation coefficient values and a loss of statistical significance. Consistent with the evidence that most chemotherapeutic agents cause tumor cell death primarily by induction of apoptosis, resistance to anticancer treatment is widely believed to involve mutations (eg, loss of p53 or overexpression of bcl-2) that lead to deregulated cellular proliferation and loss of mechanisms that control apoptosis.21 However, although these mutations may block the induction of apoptosis to many conventional chemotherapeutics, some new anticancer agents such as topoisomerase inhibitors can induce apoptosis in many cells that lack functional p53. Therefore, it may not be possible to predict the functional ability of human malignancies to undergo apoptosis solely by evaluation of the individual genetically determined components of the apoptotic pathway. Preclinical data and to a lesser degree clinical data derived from patients suffering from breast carcinoma and receiving adjuvant therapy suggest that chemotherapy-induced apoptosis significantly increases and peaks between 10 and 24 hours after administration of the first course of chemotherapy in responders but not in nonresponders.22–25 Accordingly, a noninvasive technique such as 99mTc-HYNIC annexin V scintigraphy that is able to monitor baseline and therapy-induced apoptosis in real time could allow for assessment of response to treatment within the first 24 hours after instigation of chemotherapy. If the treatment schedule used does not include the use of biologic response modifiers that are able to increase aspecific diffusion-related tumor uptake of peptides (eg, interferon), the amount of aspecific diffusion-related uptake will be fairly constant from pretherapy to posttherapy scans.26 Thus, by subtracting pretherapy from posttherapy tumor uptake values, diffusion-related bias on measurements will be minimized. Support in favor of the potential of radiolabeled annexin-V to monitor treatment response derives from both preclinical and ongoing clinical studies.27,28 In preclinical animal studies, 99mTc–ethylene cysteine annexin V and 99mTc-HYNIC annexin V were shown to allow for identification of tumor response to chemotherapy within 24 hours after therapy was started in animal models. Yang et al28 treated breast tumor xenografts with paclitaxel and performed planar 99mTc–ethylene cysteine annexin V imaging at 0.5, 2, and 4 hours. A significant increase was found in uptake among groups before paclitaxel treatment and groups treated with paclitaxel at 2 and 4 hours postinjection. In an experimental model of lymphoma in mice treated with cyclophosphamide at a dose of 100 mg/kg, therapy-induced apoptosis was readily visible with 99mTc-HYNIC annexin V imaging, with noted uptake increases of 360% when compared with pretreatment scans. In rats implanted with allogeneic hepatoma cells, tumor uptake of the imaging agent markedly increased after the administration of cyclophosphamide. Finally, preliminary results from human clinical trials have demonstrated the ability of 99mTc-N2S2 annexin V to localize to sites of tumor immediately after the first course of chemotherapy, particularly in lung cancer and lymphoma. In those patients who showed increased uptake as compared with baseline studies, a significant correlation was observed between changes in uptake results (positive or negative) and response to treatment. In conclusion, in the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells per high-power field as assessed by TUNEL assays.
The authors indicated no potential conflicts of interest.
1. White E: Life, death and the pursuit of apoptosis. Genes Dev 10:1–15, 1996
2. Ashkenazi A, Dixit VM: Death receptors: Signaling and modulation. Science 281:1305–1308, 1998
3. Evan G, Littlewood T: A matter of life and cell death. Science 281:1317–1322, 1998
4. Green DR, Reed JC: Mitochondria and apoptosis. Science 281:1309–1312, 1998
5. Thornberry NA, Laebnik Y: Caspases: Enemies within. Science 281:1312–1316, 1998 6. Dive C, Hickman JA: Drug-target interactions: Only the first step in the commitment to a programmed cell death? Br J Cancer 64:192–196, 1991[Medline] 7. Schmitt CA, Lowe S: Apoptosis and therapy. J Pathol 187:127–137, 1999[CrossRef][Medline] 8. Zhivotovsky B, Joseph B, Orrenius S: Tumor radiosensitivity and apoptosis. Exp Cell Res 248:10–17, 1999[CrossRef][Medline] 9. Mesner P, Rudthardjo I, Kaufman SH: Chemotherapy induced apoptosis. Adv Pharmacol 41:461–499, 1997 10. Kaufman SH, Earnshaw WC: Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256:42–49, 2000[CrossRef][Medline] 11. Vermes I, Haanen C, Steftensnakken H, et al: A novel assay for apoptosis: Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein-labelled annexin V. J Immunol Methods 184:39–51, 1995[CrossRef][Medline] 12. Blankenberg FG, Tait JF, Strauss W: Apoptotic cell death: Its implications for imaging in the next millennium. Eur J Nucl Med 27:359–367, 2000[CrossRef][Medline] 13. Hall PA: Assessing apoptosis: A critical survey. Endocr Relat Cancer 6:3–8, 1999[Abstract] 14. Mundle SD, Gao XZ, Khan S, et al: Two in situ end labeling techniques reveal different patterns of DNA fragmentation during spontaneous apoptosis in vivo and induced apoptosis in vitro. Anticancer Res 15:1895–1904, 1995[Medline] 15. Gastman BR: Apoptosis and its clinical impact. Head Neck 23:409–425, 2001[CrossRef][Medline]
16. Gastman BR, Atarashi Y, Reichert TE, et al: Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes. Cancer Res 59:5356–5364, 1999
17. Dumont EAWJ, Hofstra L, van Heerde WL, et al: Cardiomyocyte death induced by myocardial ischemia and reperfusion measurement with recombinant human annexin V in a mouse model. Circulation 102:1564–1568, 2000 18. Hofstra L, Lien IH, Dumont EA, et al: Visualization of cell death in vivo in patients with acute myocardial infarction. Lancet 356:209–212, 2000[CrossRef][Medline] 19. Jain RK: Vascular and interstitial physiology of tumors: Role in cancer detection and treatment, in Bicknell R, Lewis CE, Ferrara N (eds): Tumour Angiogenesis (ed 1). Oxford, UK, Oxford University Press, 1997, pp 45–49 20. Weissleder R, Bogdanov A, Tung CH, et al: Size optimization of synthetic graft copolymers for in vivo angiogenesis imaging. Bioconjug Chem 12:213–219, 2001[CrossRef][Medline] 21. Evan GI, Vousden KH: Proliferation, cell cycle and apoptosis in cancer. Nature 411:342–348, 2001[CrossRef][Medline] 22. Milas L, Hunter RN, Kurdoglo B, et al: Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol. Cancer Chemother Pharmacol 35:297–303, 1995[Medline] 23. Meyn RE, Stephens LC, Hunter NR, et al: Induction of apoptosis in murine tumors by cyclophosphamide. Cancer Chemother Pharmacol 33:410–414, 1994[Medline] 24. Ellis PA, Smith IE, McCarthy K, et al: Preoperative chemotherapy induces apoptosis in early breast cancer. Lancet 349:849, 1997[CrossRef][Medline] 25. Chang J, Ormerod M, Powles IJ, et al: Apoptosis and proliferation as predictors of chemotherapy response in patients with breast carcinoma. Cancer 89:2145–2152, 2000[CrossRef][Medline] 26. Thakur ML, Defulvio J, Tong J, et al: Evaluation of biological response modifiers in the enhancement of tumor uptake of 99mTc-labeled macromolecules: A preliminary report. J Immunol Methods 15:209–216, 1992 27. Green AM, Steinmetz ND: Monitoring apoptosis in real time. Cancer J 8:82–93, 2002[Medline] 28. Yang DJ, Azkdarinia A, Wu P, et al: In vivo and in vitro measurement of apoptosis in breast cancer cells using Tc-99m-EC-annexinV. Cancer Biother Radiopharm 16:73–83, 2001[CrossRef][Medline] Submitted December 11, 2002; accepted July 8, 2003.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
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