Quantitative Tumor Apoptosis Imaging Using Technetium-99m–HYNIC Annexin V Single Photon Emission Computed Tomography By Christophe Van de Wiele, Christophe Lahorte, Hubert Vermeersch, D. Loose, Kris Mervillie, Neil D. Steinmetz, Jean-Luc Vanderheyden, Claude A. Cuvelier, Guido Slegers, and Rudi A. Dierck 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 ⴝ 19) or recurrent (n ⴝ 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99m Tc-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 ⴝ .012). Quantitative 99m Tc-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 99mTcHYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays. J Clin Oncol 21:3483-3487. © 2003 by American Society of Clinical Oncology.

POPTOSIS, 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 an-

nexin 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 99m Tc-HYNIC–labeled annexin V in oncology patients.

A

PATIENTS AND METHODS

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

From 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. Submitted December 11, 2002; accepted July 8, 2003. Authors’ disclosures of potential conflicts of interest are found at the end of this article. 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: [email protected]. © 2003 by American Society of Clinical Oncology. 0732-183X/03/2118-3483/$20.00

Journal of Clinical Oncology, Vol 21, No 18 (September 15), 2003: pp 3483-3487 DOI: 10.1200/JCO.2003.12.096

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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. 99m

CT Scan Spiral CT scans of the head and neck were obtained in all patients using a conventional CT scanner (Somaton 4⫹ , Siemens, Erlangen, Germany). Slice thickness was 3 mm. Contrast material enhancement was achieved by intravenous administration of 100 mL of nonionic contrast material (Ultravist, Schering, Brussels, Belgium) at a rate of 2 mL/sec. Matrix size was 512 ⫻ 512. Images were analyzed by a radiologist and a head and neck surgeon who were experienced in CT imaging. When a sample was judged positive for the presence of tumor tissue, measured tumor diameters in three dimensions were divided in half, yielding three radii, r1, r2, and r3, respectively. Tumor volumes were calculated using the volume equation of an ellipsoid: tumor volume ⫽ 4/3 ␲ r1r2r3 cm3.

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 ⫻ 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

Table 1. Patient

Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

M M M M F M M M M M M M M M M M F M M M

50 70 51 66 78 55 73 60 52 47 56 54 52 51 49 66 77 50 51 56

WIELE ET AL

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 ⫻ radioactivity of standard at time of SPECT scan)/(standard counts at time of SPECT scan ⫻ total radioactivity injected)] ⫻ 100%.

Tc-HYNIC Annexin V Planar and Tomographic Imaging

Age (years)

DE

Histopathologic Examination Tumor sections (5 mm thick) with paraffin removed were cut from the largest cross-sectional area of each tumor and processed by in situ endlabeling of DNA fragments (TUNEL) using a commercially available kit, including two positive and one negative control (In Situ Cell Death Detection Kit, AP, Roche, Nutley, NJ). Subsequently, the mean number of apoptotic cells per high-power field was assessed. To evaluate the percentage of necrosis, paraffin-embedded tumor sections were stained with hematoxylin and eosin, and estimated semiquantitatively.

Patient Data

Location of Suspected Lesion

Trigonum retromolare Epiglottis Tongue Trigonum retromolare Lip Tonsil, tongue basis, palatum mole Tongue Trigonum retromolare, mouth floor Adenopathy region II Trigonum retromolare, tonsil, palatum mole Epiglottis, plicca aryepiglottica, pharynx Tonsil, ramus tonsillaris Glottis Tongue, mouth floor Tongue, vallecula, epiglottis epiglottic fold, pharynx Pharynx, rhinopharynx, palatum mole Cheek Tongue Negative Sinus piriformis

Staging

Histology

Differentiation Degree

pT2pN0M0 pT1pN2M0 pT4pN1M0 pT2pN0M0 pT2pN0M0 pT4pN2bM0 pT1pN1M0 pT1cpN0M0 T0pN3M0 pT1pN0M0 pT4pN0M0 pT1pN0M0 pT4pN0M0 pT4pN2bM0 pT3pN2cM0 pT3pN0M0 pT2pN0M0 pT1pN0M0 NA pT1pN2bM0

Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Adenocarcinoma Spinocellular carcinoma Spinocellular carcinoma Spinocellular epithelioma (adenopathy) Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma Anaplastic carcinoma (Schmincke tumor) Spinocellular carcinoma Spinocellular carcinoma Spinocellular carcinoma NA Spinocellular carcinoma

Good Poor Moderate Good Good Poor Good Moderate Moderate Moderate Moderate Moderate Poor Moderate Poor Good Good Poor NA Moderate

Abbreviations: M, male; F, female; NA, not applicable.

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QUANTITATIVE TUMOR APOPTOSIS IMAGING Table 2.

Patient Results

% Uptake ID/cm3

Patient

Tumor Volume (cm3)

1 Hour

5-6 Hours

Mean No. of Apoptotic Cells/HPF

% Necrosis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

NA 10.0 4.5 2.4 9.4 6.9 0.6 17.3 7.3 — 14.3 0.3 8.9 6.2 4.4 13.2 2.4 1.2 4.1 —

0.0000220 0.001700 0.000150 0.000260 0.000340 0.000790 0.000300 0.000170 0.000029 — 0.000098 0.000330 0.000090 0.000080 0.000068 0.000015 0.000085 0.000180 0.000049 —

NA 0.0001700 0.0000340 0.0001100 0.0000360 0.0000140 0.0000680 0.0000098 0.0000120 — 0.0000560 0.0000600 0.0000110 0.0000640 0.0000136 0.0000230 0.0000850 0.0001600 0.0000240 —

0 228 7 150 7 10 7 6 30 3 41 24 39 8 24 NA 13 213 20 —

NA 0 0 2 0 40 1 5 90 0 15 5 10 10 2 NA 1 0 5 —

Abbreviations: ID, injected dose; HPF, high-power field; NA, not available.

Statistical Methods Statistical analysis was performed using the commercially available statistics package MedCalc (MedCalc Software, Mariakerke, Belgium). Normalcy was assessed by means of the Kolmogorov-Smirnov test. Differences between early and delayed absolute percentage uptake of the dose injected per cubic centimeter tumor volume was assessed using a two-sided Student’s t test. A possible relationship between the mean number of apoptotic cells per high-power field and the mean percentage uptake of the dose injected of 99mTc-HYNIC Annexin V per cubic centimeter tumor tissue derived from images taken 5 to 6 hours PI was assessed using Spearman rank correlation analysis. The significance level used was P ⬍ .05.

RESULTS

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

Fig 1.

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 99mTcHYNIC 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. The percentage of 99mTc-HYNIC annexin V taken up by tumors correlates well with the number of apoptotic cells if only tumor

Technetium-99m-HYNIC annexin V uptake in a lip tumor: ant, anterior; post, posterior; crn, coronal; caud, caudal; sin, left; dx, right.

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Fig 2.

Correlation coefficients with increasing necrosis.

samples with no or minimal amounts of necrosis are considered. Incremental inclusion of tumors with increasing amounts of necrosis resulted in a progressive decrease of the correlation coefficient values and a loss of statistical significance. DISCUSSION

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

DE

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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

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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 99mTcHYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells per high-power field as assessed by TUNEL assays. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The authors indicated no potential conflicts of interest.

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