Purpose: Many anticancer therapies exert their therapeutic effect by inducing apoptosis in target tumors. We evaluated in a Phase I study the safety and the feasibility of 99mTc-Annexin V for imaging chemotherapy-induced apoptosis in human cancers immediately after the first course of chemotherapy.

Experimental Design: Fifteen patients presenting with lung cancer (n = 10), lymphoma (n = 3), or breast cancer (n = 2) underwent 99mTc-Annexin V scintigraphy before and within 3 days after their first course of chemotherapy. Tumor response was evaluated by computed tomography and 18F-fluoro-2-deoxy-d-glucose positron emission tomography scans, 3 months in average after completing the treatment. Median follow-up was 117 days.

Results: In all cases, no tracer uptake was observed before treatment. However, 24–48 h after the first course of chemotherapy, 7 patients who showed 99mTc-Annexin V uptake at tumor sites, suggesting apoptosis, had a complete (n = 4) or a partial response (n = 3). Conversely, 6 of the 8 patients who showed no significant posttreatment tumor uptake had a progressive disease. Despite the lack of tracer uptake after treatment, the 2 patients with breast cancer had a partial response. Overall survival and progression-free survival were significantly related to tracer uptake in treated lung cancers and lymphomas (P < 0.05). No serious adverse events were observed.

Conclusions: Our preliminary results demonstrated the feasibility and the safety of 99mTc-Annexin V for imaging apoptosis in human tumors after the first course of chemotherapy. Initial data suggest that early 99mTc-Annexin V tumor uptake may be a predictor of response to treatment in-patients with late stage lung cancer and lymphoma.

The molecular basis of cancer is now widely believed to involve mutations that lead to deregulated cellular proliferation and suppression mechanisms controlling programmed cell death (1, 2). For instance, mutations of the powerful apoptosis-inducing myc protein or the loss of p53 protein function have been shown to play a crucial role in carcinogenesis (3). Tumor sensitivity to any given therapeutic regimen commonly is mediated by the initiation of programmed cell death via available active apoptotic pathways. Many therapeutically effective anticancer drugs act to interfere with DNA synthesis and cell division, thereby inducing apoptosis in susceptible target tumors (4, 5). Thus, it may be possible to determine the effectiveness of a proposed anticancer regimen on a patient-by-patient basis by assessing the degree of apoptosis in target tumors soon after the initial treatment.

Recombinant human Annexin V (rh-Annexin V)3 has been shown to bind with high avidity to PS, a membrane-associated intracellular phospholipid invariably expressed on the external cell membrane surface early in the apoptotic cascade (6). Fluorescein-labeled rh-Annexin V has been widely used as a histopathological marker of apoptosis. More recently, radiolabeled rh-Annexin V has been shown to provide noninvasive imaging of programmed cell death in animal models associated with Fas administration, organ transplant rejection, neonatal hypoxic brain injury, terminal differentiation of WBCs associated with inflammation, and cytoxan treatment of murine lymphoma (7, 8, 9, 10, 11, 12, 13, 14). Similarly, radiolabeled rh-Annexin V was successfully used for localizing apoptosis in human diseases such as myocardial infarction and cardiac allograft rejection (15, 16).

To assess the potential of such an imaging agent to demonstrate treatment-induced apoptosis as an early predictor to anticancer treatment, patients initiating chemotherapy for stage III or IV SCLC and NSCLC, breast cancer or lymphoma was prospectively studied with a newly developed nuclear medicine imaging agent, Technetium Tc-99m rh-Annexin V (Apomate). Subjects were imaged before initiation of chemotherapy and immediately after one course of chemotherapy for evaluating the ability of the radiopharmaceutical to visualize apoptosis posttreatment in comparison to baseline. In addition, patients were followed up for 1 year to correlate the occurrence of detectable increases in tumor cell death 24 to 48 h after completing the first course of chemotherapy with tumor response to treatment, time to progression of disease, and survival time.

Patients.

Fifteen patients (11 male and 4 female; mean age = 60 years) scheduled for chemotherapy of histologically confirmed NSCLC (n = 7), SCLC (n = 3), NHL (n = 2), HL (n = 1), or disseminated breast cancer (n = 2) were enrolled in this study. All patients were at least 18 years of age and clinically stable with a baseline Karnofsky Performance Status score of at least 70 and a minimum estimated life expectancy of 16 weeks. Patients were considered eligible if they had one or more extra-abdominal bidimensionally measurable lesions on an imaging study (X-ray, CT, ultrasonography, or magnetic resonance imaging) at least 1 cm in the longest diameter. All patients signed an informed consent form at moment of recruitment. Patient characteristics are summarized in Table 1.

Biochemical Basis for the Imaging of Apoptosis.

The externalization of PS from the inner leaflet to the outer leaflet of the cell membrane is an universal feature occurring within 90–120 min of apoptotic signalizing “in vitro,” before membrane bleb formation and DNA degradation (6, 17). Annexin V, a human protein with a molecular weight 36,000 (18, 19), which is physiologically found in the cytoplasm of a wide variety of cell types, including placental, endothelial and smooth muscle cells, is known to have a high affinity for cells with exposed PS in vitro and in vivo(20, 21).

Preparation of 99mTc rh-Annexin V.

rh-Annexin V produced in Escherichia coli was labeled using a kit (Apomate; Theseus Imaging Corporation, Boston, MA) based on the preformed 99mTc phentioate ligand method described by Kasina and Fritzberg (22). A radiochemical purity of ≥85% determined by instant thin layer chromatography was required before injecting the radiolabeled tracer.

99mTc rh-Annexin V Imaging Procedure.

All patients enrolled in the study were evaluated with physical examination and laboratory studies (chemistries, hematology, coagulation, routine analysis, and vital signs) before injection of the imaging agent as well as after administration of 99mTc rh-Annexin V. The protocol design is summarized in Fig. 1.

Fifteen to 30 mCi of 99mTc rh-Annexin V were administered i.v. slowly over 3–5 min. According to the study protocol, all patients underwent 99mTc rh-Annexin V scintigraphy, including planar anterior and posterior thoracic views of all measurable tumor masses at 3–6 and 24 h after tracer injection (before and after the first course of chemotherapy). Eleven patients had also dynamic sequences and whole body images immediately and 3–6 h, respectively, after 99mTc rh-Annexin V administration. Two patients had an optional SPECT acquisition 4 h after tracer injection.

Annexin V imaging was performed using a large field of view camera fitted with a low energy, parallel hole, high resolution collimator. Dynamic images were collected for 10 min after injection (20 s/frame); planar studies were acquired in a 256 × 256 matrix; anterior and posterior whole body images were obtained at a scan speed of 10 cm/min; SPECT data acquisition was obtained using a 128 × 128 matrix with a complete rotation of 360°. A minimum number of counts/images and 250,000 counts/image were collected for the planar image performd at the 3–6-h and 24-h, respectively. SPECT images were reconstructed using an iterative method based on an Ordered Subset–Expectation Maximization principle.

Image Interpretation.

Qualitative assessment of tumor uptake was performed using visual reading of the Apomate images by two nuclear physicians blinded to tumor response to chemotherapy. Except for physiological distribution of tracer, any Annexin V-Tc99m uptake detected immediately after chemotherapy at tumor sites and not seen on the pretreatment images was considered as a positive result. Otherwise, in the absence of posttreatment tumor uptake of the apoptosis agent, the case was then interpreted as a negative result.

Semiquantitative evaluation of tumor uptake was performed in terms of TBR as well as by calculating the relative tumor uptake posttreatment compared with baseline (TR). For this purpose, several circular ROIs with similar pixel size were automatically drawn using a computerized processing (Sophy NXT, Sopha Medical). The ROIs were initially defined on the posttreatment images when an apoptotic signal was localized at the tumor sites. In a second step, these ROIs were translated on the same anatomical sites on the pretreatment images. Practically, in cases of increased Annexin V uptake after chemotherapy, the number of counts obtained for tumor-bearing Annexin V was successively noted for each ROI [i.e., ROI1 (tumor signal posttreatment) = 16,000 counts and ROI2 (similar tumor site pretreatment) = 8,000 counts]. Thereby, allowing us to calculate the TR (i.e., ROI1/ROI2 = 2). Additional ROIs were also defined in areas free of tracer uptake [i.e., ROI3 (tumor signal posttreatment) = 16,000 counts and ROI4 (lung background posttreatment) = 4,000 counts]. The TBR was then determined (i.e., TBR = ROI3/ROI4 = 4). Otherwise, for the negative studies, no ROI was drawn. Overall, the change in tumor uptake of Apomate on posttreatment as compared with pretreatment images were graded as: grade 0: no change in uptake or a decrease in uptake posttreatment; grade 1: 10–50% increase in uptake posttreatment; grade 2: 50–100% increase in uptake posttreatment; grade 3: 100–200% increase in uptake post treatment; and grade 4: >200% increase in uptake posttreatment.

Conventional Anatomical Imaging.

All patients were explored before and after chemotherapy by thoracic CT with contrast agent. CTs were performed using a PQ 2000 fourth generation (Picker, Cleveland, OH). In accordance with study enrollment criteria, all patients demonstrated at least one bidimensionally measurable tumor lesion (primary site and/or metastases) with diameters ≥1 cm.

Metabolic 18FDG PET Imaging.

Whole body PET was performed 1 h after i.v injection of 4–6 mCi of 18FDG in 14 patients before chemotherapy and in all patients (n = 15) during follow-up. PET scans were performed using either a Penn Pet 240H scanner (UGM, Philadelphia, PA) or a C-Pet scanner (ADAC; Philips Medical Systems, Milpitas, CA).

Efficacy Assessment and Response Criteria.

In all cases, tumor response to chemotherapy was evaluated in routine clinical fashion, including the CT and PET evaluations. In particular, the longest diameters of tumors measurable on posttreatment CT were compared with baseline pretreatment measurement. Patients were classified as having complete or PR and PD as indicated in Table 3. The median follow-up in this prospective study was 117 days (47–356 days).

Statistical Analysis.

Qualitative results, suggesting or not an apoptotic signal after chemotherapy at tumor sites, were inferred from the visual comparison of the Annexin V-Tc99m images immediately pre- and posttreatment (as detailed above). Quantitative results (TBR, TR) were expressed as mean and SD. Mean values were compared using the Student’s t test when the distribution was normal and by Wilcoxon signed rank test otherwise.

The relationship of survival time to the 99mTc-Annexin V uptake (positive or negative) was estimated using the Kaplan-Meier method. The log rank test was used to compare the equality of survival curves. The degree of response to chemotherapy (CR, PR, and PD) was also appreciated in comparison to nuclear image data grading (Grade 0 to Grade 4) using Fisher’s exact test for contingency tables and for assessing the dependence between categorical variables. In addition, a linear effect between the nuclear image grading and the response to tumor therapy was analyzed using Mantel-Haenszel’s χ2 test. All statistical results were considered to be significant at the 5% critical level (P < 0.05). All calculations were performed using SAS (version 6.12 for Windows) and S-PLUS 2000.

Thirteen patients received two doses of 99mTc-Annexin V immediately before chemotherapy and within 3 days after completing the first course of treatment. Two patients received only one injection of Apomate after chemotherapy because of the urgency of treatment.

On prechemotherapy images, no 99mTc-Annexin V uptake was noted in tumors. Immediately before treatment, the patients only showed a similar nonpathologic biodistribution of radiotracer with uptake in salivary glands, liver, spleen, bone marrow, colon, kidneys, and bladder. On images obtained within 72 h after completing the first course of antitumor therapy, 7 patients (1 NHL, 1 HL, 2 SCLC, and 3 NSCLC) demonstrated a 99mTc-Annexin V uptake at the sites of primary or metastatic tumors. These sites were predominantly detected in regions of metastatic lymph nodes (cervical, mediastinal, and hilary nodes). Additionally, 3 patients also had a lung localization (1 NHL and 2 SCLC) and a rib uptake (1 NSCLC). Overall, 5 patients (1 NHL, 1HL, 1 SCLC, and 2 NSCLC) had a significant uptake 24 h after the radiopharmaceutical injection (P = 0.02), corresponding to the 48th h after completion of the first course of chemotherapy, whereas 2 patients (1 NSCLC and 1 SCLC) presented with a more intense 99mTc-Annexin V uptake 4 h after injection. The quantitative evaluation of tumor uptake in terms of TBR and TR confirmed the qualitative evaluation of the images in 6 of 7 patients having obvious tracer uptake at their tumor sites after chemotherapy. In 1 case of HL presenting with a large cervical mass, the visual interpretation showed diffuse faint uptake, whereas the TBR and TR ratios significantly differed before and after chemotherapy.

Among the 7 patients presenting with an increased Annexin V-Tc99m uptake early after chemotherapy, 4 of them had CR (1 NHL, 1 HL, 1 NSCLC, and 1 SCLC) and the other 3 patients (2 NSCLC and 1 SCLC) had PR. On the basis of the CT and PET evaluations, all tumor sites with grade 3 and grade 4 99mTc rh-Annexin V uptake completely disappeared after chemotherapy (Fig. 2), whereas the subjects with grade 1 and grade 2 uptake had partial response to treatment. On the other hand, 6 of 8 patients without Annexin V tumor uptake (grade 0) after chemotherapy (4 NSCLC, 2 BC, 1 SCLC, and 1 NHL) had PD (Fig. 3). Of them, 4 patients (4 of 6) died after a median follow-up of 3 months (64–138 days). Despite the lack of significant tracer uptake after chemotherapy, two cases of breast cancer had, however, complete and partial response to Taxol, respectively. Statistically, the tumor uptake of 99mTc-Annexin V was significantly correlated with the patient outcomes in terms of tumor response to chemotherapy and survival (Figs. 4,5,6). The results of the Annexin V imaging are detailed in Table 2.

And last but not least, no serious adverse events associated with 99m Tc-Annexin V administration were observed, with a median follow-up of 4 months. In particular, no physical, biological, and hematological changes, including the coagulation tests related to the 99m Tc-Annexin V administration, were noted. However, 1 patient (case 6 in Table 2) presented a mild reaction 15 min after the second injection of 99m Tc-Annexin V (after chemotherapy) corresponding to a facial rash, which regressed spontaneously 1 h later without any treatment. This patient was still in CR at his last follow-up (76 days).

Most anticancer drug agents as diverse as topoisomerase inhibitors, alkylating agents, antimetabolites, and hormone antagonists generate apoptosis in sensitive cells (23, 24, 25). The genetic measurement of individual components of the apoptotic pathway does not necessarily reflect the functional ability of a tumor cell to commit to apoptosis in response to chemotherapy triggering. Mutations of p53, for instance, block the induction of apoptosis by various chemotherapeutic drugs in many cell types, whereas newer anticancer agents such as topoisomerase poisons, particularly topoisomerase I inhibitors, are able to induce apoptosis in many cells that lack functional p53(26, 27). In this study, we evaluated the technical feasibility and the clinical interest of 99mTc rh-Annexin V for noninvasively assessing the apoptotic functional capacity of treated tumors on a patient-by-patient basis.

Preclinical studies have shown that Annexin V binds tightly to PS. PS is a phospholipid normally expressed on the inner leaflet of the bilamellar cell membrane and is invariably translocated to the outer surface as an early event in apoptosis. i.v. administered 99mTc rh-Annexin V has been shown in preclinical and clinical studies to bind to externalized PS on apoptotic and necrotic cells with high avidity. Thus, 99mTc rh-Annexin V uptake suggests cellular apoptosis or necrosis. Blankenberg and Strauss (7, 8, 9, 10, 11, 12, 13, 14) showed the potential of 99mTc rh-Annexin V for in vivo imaging of Fas-mediated fulminant hepatic apoptosis, chemotherapy-induced apoptosis in normal bone marrow and treated murine lymphoma, as well as in association with cardiac and lung allograft rejection and in hypoxic-ischemic cerebral reperfusion. Recently, in 7 patients presenting with documented acute myocardial infarction, Hofstra et al.(15) reported the feasibility of the radiolabeled Annexin V for the in situ imaging of necrosis and/or apoptosis. Similarly, in a series of 18 cardiac allograft recipients, Narula et al.(16) demonstrated the capability of the apoptosis imaging agent for noninvasive detection of transplant rejection confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling and/or caspase-3 (an apoptosis-specific proteolytic enzyme) immunohistochemical staining.

In oncology patients, the ability of tumor cells to respond apoptotically to chemotherapy varies from one tissue to another. In lymphomas, for instance, various treatments have shown to be efficient via chemotherapy-induced apoptosis in target tumor cells (28, 29, 30, 31). This is one reason why this group of patients should be a priori good candidates for Annexin V imaging to assess the early apoptotic response of individual lymphomas to treatment. Indeed, 2 of 3 lymphoma patients studied in this series showed uptake of the imaging agent, suggesting their tumors had apoptotic capacity and both demonstrated objective clinical response to treatment. The third patient with a NHL showed no tracer uptake after treatment and had PD.

On the other hand, in untreated primary lung cancer, a previous work has indicated that the incidence of apoptosis known as the AI can vary widely (32). Histological analysis of 134 cases of NSCLC showed a mean AI of 0.3% (range, 0.02–1.4%), which was not correlated with the stage of disease, the degree of nodal involvement, the grade of tumor differentiation, or the differences in ploidy. Thus, the lack of significant pretreatment 99mTc rh-Annexin V uptake in NSCLC suggests that the planar imaging technique used in this study cannot routinely detect only 0.3% apoptosis. Nonetheless, in our series, pretreatment images were obtained as a control to compare with posttreatment uptake of the imaging agent.

Similar AI data on untreated primary tumors exist for patients with breast cancer. Studies from 105 women with invasive breast cancer have shown most of the specimens from untreated patients had <1% apoptosis (33). Although no uptake of 99mTc rh-Annexin V was seen in the 2 breast cancer patients enrolled in this series, both patients had partial clinical responses. The reason for the lack of tracer uptake visualized on imaging is not clear but may be related to failure to optimize the time of imaging after treatment when AI would have been elevated. The limited spatial resolution of currently used gamma cameras must also be considered.

Despite initial promise, in vitro sensitivity testing of tumors has not proved practical for routine clinical applications. Immunohistological and/or cytological apoptotic indexes determination on needle biopsy specimens showed promising results but are invasive (34). Morphological imaging procedures such as CT or magnetic resonance imaging provide accurate topographical information about the tumor changes after treatment, but they are unable to predict the response to chemotherapy. Metabolic changes usually precede gross tumor changes. PET imaging has been proposed to assess tumor viability by using either a glucose analogue (18FDG) or radiolabeled amino acids (11C-methionine, 11C-tyrosine) (35, 36, 37). However, PET performances for early imaging of the chemotherapy response can be impaired in some clinical situations by inherent biochemical and technical limitations. For instance, the possibility of cellular stunning in the first few weeks after chemotherapy, resulting in false negative results, must be considered (38).

Our preliminary results demonstrated the ability of 99mTc rh-Annexin V to localize at tumor sites immediately after the first course of chemotherapy in lung cancer and lymphoma. Biopsies were not performed in this series, and the mechanism of localization of the imaging agent must be inferred from: (a) the preclinical and clinical data showing histological evidence of 99mTc rh-Annexin V localization at sites of apoptosis and necrosis; (b) the lack of 99mTc rh-Annexin V tumor uptake immediately before treatment; (c) the objective clinical responses seen in all patients whose tumors demonstrated posttreatment 99mTc rh-Annexin V uptake; and (d) the lack of objective clinical response in 6 of 8 patients whose tumors failed to demonstrate posttreatment 99mTc rh-Annexin V uptake. For these reasons, the observations are consistent with the hypothesis that 99mTc rh-Annexin V localizes at regions of apoptosis and necrosis immediately after anticancer treatment in patients whose tumors are able to respond apoptotically to such treatment. The optimal timing for scintigraphic imaging of apoptosis in this study was about 20–24 h after the second injection (48 h after chemotherapy). By taking in account the 6-h half-time of 99mTc, the blood clearance of the 99mTc rh-Annexin V, as well as the counting statistics required for an adequate quality of the images, the acquisition protocol that appears the most flexible for the patient and the most efficient for imaging apoptosis after one course of treatment includes: static spot views of the thorax (anterior and posterior views) at 3–6 and 24 h after injection. Also, SPECT acquisition should probably be performed at 3–6 h after i.v. injection to improve the accuracy of detection.

This study demonstrated an excellent positive predictive value of 99mTc rh-Annexin V imaging in NSCLC and lymphoma that warrants additional larger multicentric studies to assess in vivo the apoptotic capacity of tumors by using the apoptosis imaging agent and, consequently, tumor response to therapy on patient-by-patient basis. Although promising, the results must be, however, interpreted cautiously. The heterogeneity of the histological nature of the tumors explored, as well as the heterogeneity of the drugs administrated, could explain some of the differences of tumor behavior observed. Prospective studies based on more homogeneous groups of patients in terms of histology and drugs protocol will be useful to evaluate more objectively the value of 99mTc-Annexin V for imaging apoptosis in human tumors.

In conclusion, the preliminary results of a Phase I study demonstrated the feasibility and the safety of 99mTc-Annexin V for localizing apoptosis in treated human tumors, particularly in lymphomas and late stage lung cancers. The selective in situ detection of the apoptotic signal is possible, as early as 1 day after the first course of chemotherapy. The determination of apoptotic competence of human tumors via their actual apoptotic response to therapy using a noninvasive and reproducible imaging tool could have important clinical implications in cancer management.

Additional clinical trials based on larger multicentric series remain, however, necessary before the introduction of the technique in oncology practice.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by research grant from Theseus Imaging Corporation (Boston, MA). Presented in part at the 47th annual meeting of the Society of Nuclear Medicine (St. Louis, MO, June 2000).

3

The abbreviations used are: rh-Annexin V, recombinant human Annexin V; PS, phosphatidylserine; CT, computed tomography; SPECT, single-photon emission computed tomography; TBR, tumor-to-background ratio; ROI, regions of interest; TR, tumor ratio; 18FDG, 18F-fluoro-2-deoxy-d-glucose; PET, positron emission tomography; NHL, non-Hodgkin’s lymphoma; HL, Hodgkin’s lymphoma; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; AI, apoptotic index; CR, complete remission; PR, partial remission; PD, progressive disease.

Fig. 1.

Protocol design. ∗, the follow-up evaluation included physical examination, thoracic CTs, and whole body 18FDG PET scans. ∗∗, day 0 = the day of injection of the apoptosis agent; day 1 = the 24th h after the injection of the apoptosis agent.

Fig. 1.

Protocol design. ∗, the follow-up evaluation included physical examination, thoracic CTs, and whole body 18FDG PET scans. ∗∗, day 0 = the day of injection of the apoptosis agent; day 1 = the 24th h after the injection of the apoptosis agent.

Close modal
Fig. 2.

A case of NHL (stage IV) treated by cyclophosphamide-doxorubicine-vincristine-prednisone protocol with a positive 99mTc-Annexin V study. A, the CTs of the neck and of the thorax (left) and the 18FDG PET scan (middle and right) performed before treatment showed a lymph node dissemination at the cervical and axillary levels. B, the Annexin V imaging performed immediately before (left) and 48 h after chemotherapy (right) demonstrated an increased uptake of the apoptosis agent at the tumor sites (arrows). C, the posttreatment evaluation by the CTs and PET showed complete disappearance of disease.

Fig. 2.

A case of NHL (stage IV) treated by cyclophosphamide-doxorubicine-vincristine-prednisone protocol with a positive 99mTc-Annexin V study. A, the CTs of the neck and of the thorax (left) and the 18FDG PET scan (middle and right) performed before treatment showed a lymph node dissemination at the cervical and axillary levels. B, the Annexin V imaging performed immediately before (left) and 48 h after chemotherapy (right) demonstrated an increased uptake of the apoptosis agent at the tumor sites (arrows). C, the posttreatment evaluation by the CTs and PET showed complete disappearance of disease.

Close modal
Fig. 3.

A case of NSCLC (stage IV) treated by mitomycin-ifsofamide-cis-platinum protocol with a negative 99mTc-Annexin V study. A, thoracic CT and 18FDG PET pretreatment (1 and 2) compared with posttreatment evaluation (3 and 4) showing an obvious progression of tumor. B, first Annexin V imaging performed immediately before chemotherapy with dynamic sequences (1, top left) and static anterior and posterior views at 15 min (2, top right), 4 h (3, bottom left), and 24 h (4, bottom right) after i.v. injection of the apoptosis agent. C, second Annexin V imaging performed immediately after chemotherapy with the same protocol showing no tumor uptake but the physiological distribution of tracer (liver, spleen, and colon).

Fig. 3.

A case of NSCLC (stage IV) treated by mitomycin-ifsofamide-cis-platinum protocol with a negative 99mTc-Annexin V study. A, thoracic CT and 18FDG PET pretreatment (1 and 2) compared with posttreatment evaluation (3 and 4) showing an obvious progression of tumor. B, first Annexin V imaging performed immediately before chemotherapy with dynamic sequences (1, top left) and static anterior and posterior views at 15 min (2, top right), 4 h (3, bottom left), and 24 h (4, bottom right) after i.v. injection of the apoptosis agent. C, second Annexin V imaging performed immediately after chemotherapy with the same protocol showing no tumor uptake but the physiological distribution of tracer (liver, spleen, and colon).

Close modal
Fig. 4.

Nuclear image grading correlated with the tumor response to chemotherapy. A statistical significance was observed in both Fisher’s exact test (P = 0.043) and Mantel-Haenszel’s χ2 test (P = 0.007).

Fig. 4.

Nuclear image grading correlated with the tumor response to chemotherapy. A statistical significance was observed in both Fisher’s exact test (P = 0.043) and Mantel-Haenszel’s χ2 test (P = 0.007).

Close modal
Fig. 5.

Overall survival time of patients correlated with the Apomate results using the Kaplan-Meier method. P < 0.01 in log rank test.

Fig. 5.

Overall survival time of patients correlated with the Apomate results using the Kaplan-Meier method. P < 0.01 in log rank test.

Close modal
Fig. 6.

Progression-free survival time of patients correlated with the Apomate results using the Kaplan-Meier method. P < 0.01 in log rank test.

Fig. 6.

Progression-free survival time of patients correlated with the Apomate results using the Kaplan-Meier method. P < 0.01 in log rank test.

Close modal
Table 1

Patient characteristics

CasesGenderAge (yr)Tumor histology (stage)Chemotherapy drugs
Male 65 SCLC (IV) MIPa-VP16 
Male 69 NSCLC (IV) MIP 
Male 67 NHL (IV) CHOP 
Male 69 NSCLC (IV) MIP 
Male 80 NSCLC (IV) MIP 
Female 69 BC (III) 
Female 68 NSCLC (IV) MIP 
Female 61 BC (III) 
Male 68 NSCLC (IV) MIP 
10 Male 42 NSCLC (IV) MIP 
11 Male 33 NSCLC (IV) MIP 
12 Male 56 NHL (IV) MCE 
13 Female 45 HL (II) ABVD 
14 Male 56 SCLC (III) C-VP16 
15 Male 53 SCLC (III) P-VP16 
CasesGenderAge (yr)Tumor histology (stage)Chemotherapy drugs
Male 65 SCLC (IV) MIPa-VP16 
Male 69 NSCLC (IV) MIP 
Male 67 NHL (IV) CHOP 
Male 69 NSCLC (IV) MIP 
Male 80 NSCLC (IV) MIP 
Female 69 BC (III) 
Female 68 NSCLC (IV) MIP 
Female 61 BC (III) 
Male 68 NSCLC (IV) MIP 
10 Male 42 NSCLC (IV) MIP 
11 Male 33 NSCLC (IV) MIP 
12 Male 56 NHL (IV) MCE 
13 Female 45 HL (II) ABVD 
14 Male 56 SCLC (III) C-VP16 
15 Male 53 SCLC (III) P-VP16 
a

MIP, mitomycin-ifosfamide-cis-platinum; VIP, vepeside; CHOP, cyclophosphamide-doxorubicine-vincristine-prednisone; T, taxane; MCE, melphalan-cyclolal-endoxan; ABVD, adriamycin-bleomycin-vincristine-doxorubicine; C, carboplatine; BC, breast cancer.

Table 2

Results from the Apomate study

CasesApomate gradingΔTBRa 4 h/24 hΔTR 4 h/24 hAnnexin V-rh-Tc99m uptakeClinical responseFollow-up (days)
Grade 0 No uptake No uptake None Died 64 
Grade 3 1.7/2.0 0.6/2.8 Neck, mediastinum, hilum CR 356 
Grade 4 1.4/1.7 1.6/3.2 Neck, axilla, hilum CR 345 
Grade 0 No uptake No uptake None Died 70 
Grade 0 No uptake No uptake None Died 105 
Grade 0 No uptake No uptake None PR 76 
Grade 1 1.7/18.6 0.9/8.9 Mediastinum PR 85 
Grade 0 No uptake No uptake None PR 124 
Grade 2 1.6/0.9 1.8/1.2 Right rib PR 85 
10 Grade 0 No uptake No uptake None Died 138 
11 Grade 0 No uptake No uptake None PD 99 
12 Grade 0 No uptake No uptake None PD 60 
13 Grade 1 0.7/1.5 1.1/2.9 Neck (left area) CR 58 
14 Grade 2 2.4/1.7 NA Hilum, mediastinum PR 51 
15 Grade 3 2.3/2.0 3.7/0.6 Hilum, mediastinum CR 47 
CasesApomate gradingΔTBRa 4 h/24 hΔTR 4 h/24 hAnnexin V-rh-Tc99m uptakeClinical responseFollow-up (days)
Grade 0 No uptake No uptake None Died 64 
Grade 3 1.7/2.0 0.6/2.8 Neck, mediastinum, hilum CR 356 
Grade 4 1.4/1.7 1.6/3.2 Neck, axilla, hilum CR 345 
Grade 0 No uptake No uptake None Died 70 
Grade 0 No uptake No uptake None Died 105 
Grade 0 No uptake No uptake None PR 76 
Grade 1 1.7/18.6 0.9/8.9 Mediastinum PR 85 
Grade 0 No uptake No uptake None PR 124 
Grade 2 1.6/0.9 1.8/1.2 Right rib PR 85 
10 Grade 0 No uptake No uptake None Died 138 
11 Grade 0 No uptake No uptake None PD 99 
12 Grade 0 No uptake No uptake None PD 60 
13 Grade 1 0.7/1.5 1.1/2.9 Neck (left area) CR 58 
14 Grade 2 2.4/1.7 NA Hilum, mediastinum PR 51 
15 Grade 3 2.3/2.0 3.7/0.6 Hilum, mediastinum CR 47 
a

ΔTBR (mean values), tumor-to-background ratios posttreatment (4 versus 24 h); ΔTR (mean values), relative tumor ratios posttreatment versus pretreatment (4 and 24 h); NA, not available because the patient had a single posttreatment Annexin V imaging because of the urgency of treatment.

We thank Marybeth Mallett for helpful assistance.

1
Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br. J. Cancer
,
26
:
239
-257,  
1972
.
2
Evan G. I., Vousden K. H. Proliferation, cell cycle, and apoptosis in cancer.
Nature (Lond.)
,
411
:
342
-348,  
2001
.
3
Harrington E. A., Fanidi A., Evan G. I. Oncogenes and cell death.
Curr. Opin. Genet. Dev.
,
4
:
120
-129,  
1994
.
4
Darzynkiewicz Z. Apoptosis in antitumor strategies: modulation of cell cycle or differentiation.
J. Cell. Biochem.
,
58
:
151
-159,  
1995
.
5
Lennon S. V., Martin S. J., Cotter T. G. Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli.
Cell Prolif.
,
24
:
203
-214,  
1991
.
6
Martin S. J., Reutelingsperger C. P., McGahon A. J., Rader J. A., van Schie R. C., LaFace D. M., Green D. R. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by over expression of Bcl-2 and Abl.
J. Exp. Med.
,
182
:
1545
-1556,  
1995
.
7
Ogura Y., Krams S. M., Martinez O. M., Kopiwoda S., Higgins J. P., Esquivel C. O., Strauss H. W., Tait J. F., Blankenberg F. G. Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation.
Radiology
,
214
:
795
-800,  
2000
.
8
Vriens P. W., Blankenberg F. G., Stoot J. H., Ohtsuki K., Berry G. J., Tait J. F., Strauss H. W., Robbins R. C. The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection.
J. Thorac. Cardiovasc. Surg.
,
116
:
844
-853,  
1998
.
9
Blankenberg F. G., Robbins R. C., Stoot J. H., Vriens P. W., Berry G. J., Tait J. F., Strauss H. W. Radionucleide imaging of acute lung transplant rejection with annexin V.
Chest
,
117
:
834
-840,  
2000
.
10
Blankenberg F. G., Ohtsuki K., Tait J., Strauss H. W. Apoptosis: the importance of nuclear medicine.
Nucl. Med. Commun.
,
21
:
241
-250,  
2000
.
11
Ohtsuki K., Akashi K., Aoka Y., Blankenberg F. G., Kopiwoda S., Tait J. F., Strauss H. W. Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in vivo detection of apoptosis.
Eur. J. Nucl. Med.
,
26
:
1251
-1258,  
1999
.
12
Blankenberg F. G., Katsikis P. D., Tait F., Davis R. E., Naumovski L., Ohtsuki K., Kopiwoda S., Abrams M. J., Strauss H. W. Imaging of apoptosis (programmed cell death) with 99mTc annexin V.
J. Nucl. Med.
,
40
:
184
-191,  
1999
.
13
D’Arceuil H., Rhine W., de Crespigny A., Yenari M., Tait J. F., Strauss W. H., Engelhorn T., Kastrup A., Moseley M., Blankenberg F. G. 99mTc annexin V imaging of neonatal hypoxic brain injury.
Stroke
,
31
:
2692
-2700,  
2000
.
14
Blankenberg F. G., Naumovski L., Tait J. F., Post A. M., Strauss H. W. Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc-radiolabeled annexin V.
J Nucl Med.
,
42
:
309
-316,  
2001
.
15
Hofstra L., Liem I. H., Dumont E. A., Boersma H. H., van Heerde W. L., Doevendans P. A., De Muinck E., Wellens H. J., Kemerink G. J., Reutelingsperger C. P., Heidendal G. A. Visualisation of cell death in vivo in patients with acute myocardial infarction.
Lancet
,
356
:
209
-212,  
2000
.
16
Narula J., Acio E. R., Narula N., Samuels L. E., Fyfe B., Wood D., Fitzpatrick J. M., Raghunath P. N., Tomaszewski J. E., Kelly C., Steinmetz N., Green A., Tait J. F., Leppo J., Blankenberg F. G., Jain D., Strauss H. W. Annexin-V imaging for noninvasive detection of cardiac allograft rejection.
Nat. Med.
,
7
:
1347
-1352,  
2001
.
17
Zwaal R. F., Schroit A. J. Pathophysiologic implications of membrane phopholipid asymmetry in blood cells.
Blood
,
89
:
1121
-1132,  
1997
.
18
Cookson B. T., Engelhardt S., Smith C., Bamford H. A., Prochazka M., Tait J. F. Organization of the human annexin V (ANX5) gene.
Genomics
,
20
:
463
-467,  
1994
.
19
Huber R., Berendes R., Burger A., Schneider M., Karshikov A., Luecke H., Romisch J., Pâques E. Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.
J. Mol. Biol.
,
223
:
683
-704,  
1992
.
20
Pigault C., Follenius-Wund A., Schmutz M., Freyssinet J. M., Brisson A. Formation of two-dimensional arrays of annexin V on phosphatidylserine-containing liposomes.
J. Mol. Biol.
,
236
:
199
-208,  
1994
.
21
Meers P., Mealy T. Relationship between annexin V tryptophan exposure, calcium, and phospholipid binding.
Biochemistry
,
32
:
5411
-5418,  
1993
.
22
Kasina S., Rao T. N., Srinivasan A., Sanderson J. A., Fitzner J. N., Reno J. M., Beaumier P. L., Fritzberg A. R. Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent.
J. Nucl. Med.
,
32
:
1445
-1451,  
1991
.
23
Hickman J. A. Apoptosis induced by anticancer drugs.
Cancer Metastasis Rev.
,
11
:
121
-139,  
1992
.
24
Dive C., Evans C. A., Whetton A. D. Induction of apoptosis: new targets for cancer chemotherapy.
Semin. Cancer Biol.
,
3
:
417
-427,  
1992
.
25
Lowe S. W., Lin A. W. Apoptosis in cancer.
Carcinogenesis (Lond.)
,
21
:
485
-495,  
2000
.
26
Murakami Y., Hayashi K., Hirohashi S., Sekiya T. Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas.
Cancer Res.
,
51
:
5520
-5525,  
1991
.
27
Lowe S. W. Cancer therapy and p53.
Curr. Opin. Oncol.
,
7
:
547
-553,  
1995
.
28
Weiss L. M., Warnke R. A., Sklar J., Cleary M. L. Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas.
N. Engl. J. Med.
,
317
:
1185
-1189,  
1987
.
29
Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.
Nature (Lond.)
,
335
:
440
-442,  
1988
.
30
Maloney D. G., Smith B., Appelbaum F. R. The anti-tumor effect of monoclonal anti-CD20 antibody therapy includes direct anti-proliferative activity and induction of apoptosis in CD20 positive non-Hodgkin’s lymphoma cell lines (Abstract).
Blood
,
88 (Suppl. 1)
:
637a
1996
.
31
Demidem A., Lam T., Alas S., Hariharan K., Hanna N., Bonavida B. Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs.
Cancer Biother. Radiopharm.
,
12
:
177
-186,  
1997
.
32
Ghosh M., Crocker J., Morris A. Apoptosis in squamous cell carcinoma of the lung: correlation with survival and clinicopathological features.
J. Clin. Pathol.
,
54
:
111
-115,  
2001
.
33
Gonzalez-Palacios F., Sancho M., Martinez J. C., Bellas C. Microvessel density, p53 overexpression, and apoptosis in invasive breast carcinoma.
Mol. Pathol.
,
50
:
304
-309,  
1997
.
34
Allen R. T., Hunter W. J., III, Agrawal D. K. Morphological and biochemistry characterization and analysis of apoptosis.
J. Pharmacol. Toxicol. Meth.
,
37
:
215
-228,  
1997
.
35
Warburg O. On the origin of cancer cells.
Science (Wash. DC)
,
123
:
309
-314,  
1956
.
36
Pauwels E. K., Ribeiro M. J., Stoot J. H., McCready V. R., Bourguignon M., Maziere B. FDG accumulation and tumor biology.
Nucl. Med. Biol.
,
25
:
317
-322,  
1998
.
37
Delbeke D. Oncological applications of FDG PET imaging.
J. Nucl. Med.
,
40
:
1706
-1715,  
1999
.
38
Bar-Shalom R., Valdivia A. Y., Blaufox M. D. PET imaging in oncology.
Semin. Nucl. Med.
,
30
:
150
-185,  
2000
.