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In vivo imaging of radiation-induced apoptosis in follicular lymphoma patients
Int. J. Radiation Oncology Biol. Phys., Vol. 59, No. 3, pp. 782–787, 2004 Copyright © 2004 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/04/$–see front matter
doi:10.1016/j.ijrobp.2003.11.017
CLINICAL INVESTIGATION
Lymphoma
IN VIVO IMAGING OF RADIATION-INDUCED APOPTOSIS IN FOLLICULAR LYMPHOMA PATIENTS RICK L. M. HAAS, M.D.,* DAPHNE DE JONG, M.D., PH.D.,† RENATO A. VALDE´ S OLMOS, M.D., PH.D.,‡ CEES A. HOEFNAGEL, M.D., PH.D.,‡ IRIS VAN DEN HEUVEL,‡ SHURAILA F. ZERP,* HARRY BARTELINK, M.D., PH.D.,* AND MARCEL VERHEIJ, M.D., PH.D.* Departments of *Radiotherapy, †Pathology, and ‡Nuclear Medicine, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Purpose: To evaluate 99mTc-Annexin-V (TAV) scintigraphy in monitoring radiation-induced apoptotic cell death in follicular lymphoma (FL) patients. Patients and Methods: Eleven FL patients (7 female and 4 male; median age, 58 years; range, 42– 80 years) with recurrent disease underwent TAV imaging before and 24 hours after the last fraction of the 2 ⴛ 2 Gy involved field radiotherapy regimen. Fine-needle aspiration cytology was performed on 5 consecutive days to determine the optimal time window for apoptosis detection and to confirm the apoptotic nature of the response. The TAV scintigraphy (total body studies and SPECT of the irradiated sites) was performed 4 hours after the administration of the radiopharmaceutical. Tumor uptake was scored in a semiquantitative manner as absent (ⴚ) weak (ⴞ), present (ⴙ), or intense (ⴙⴙ) with corresponding categories for the cytologic slides. Response evaluation was performed after 1 week and 4 weeks both in terms of completeness and speed of remission. Results: Baseline TAV uptake was absent in 6 and weak in 5 patients. Sequential cytology indicated that the optimal time period for apoptosis assessment was between 24 and 48 hours after the last fraction of the 2 ⴛ 2 Gy regimen. Baseline cytology was concordant with baseline TAV in all patients. Apoptotic feature appearance (nuclear chromatin condensation, margination and apoptotic body formation) after low-dose irradiation matched the irradiation response in all patients. In all but 1 patient the posttreatment TAV uptake matched the posttreatment cytology. In these 10 patients the cytology and TAV results correlated with the type and onset of the clinical response. Conclusion: Tumor 99mTc-Annexin-V uptake can be increased after 2 ⴛ 2 Gy involved field radiotherapy. This increase was concordant with the appearance of apoptotic morphology as determined by cytology, and correlated with the clinical outcome. Apoptotic cell death can be observed on Day 4 of this regimen and if so predicts a complete remission within 1 week. © 2004 Elsevier Inc. 99m
Tc-Annexin-V, Radiotherapy, Follicular lymphoma, Apoptosis.
INTRODUCTION
total dose of 36 – 40 Gy in about 50% of cases (2– 4). In advanced stage disease (Ann Arbor Stage III and IV) prolonged remissions are seen after low-dose total body irradiation fractionated in daily doses of 10 –15 cGy to a total of 1.5–2.5 Gy (5–7). Follicular lymphoma patients responding to radiotherapy usually do so already during treatment. This suggests that apoptotic cell death might play a role in the clinical outcome. Dubray et al. (8, 9) were the first to show in vitro that the high response rates after radiotherapy in FL might be due to radiation-induced apoptosis. Apoptosis is a distinct mode of cell death and represents an important regulatory mechanism to remove abundant and unwanted cells during many physiologic processes. Failure
Characteristic for follicular non-Hodgkin’s lymphomas (FL) is the t(14;18) translocation. This cytogenetic feature involves a translocation of the bcl-2 gene on chromosome 18 to the immunoglobulin heavy-chain locus (IgH) on chromosome 14, resulting in an overexpression of Bcl-2, a well-known inhibitor of apoptosis (1). It is hypothesized that in this disease expansion is not primarily caused by proliferation but rather by the lack of apoptotic cell loss. These lymphomas are one of the most radiosensitive tumors. Patients in early-stage (Ann Arbor Stage I and II) can be cured by conventionally fractionated irradiation to a Reprint requests to: Rick L. M. Haas, M.D., Department of Radiotherapy, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel: (⫹31) 205122124; Fax: (⫹31) 206691101; E-mail: [email protected]
Acknowledgments—Theseus Imaging Corporation, Boston, MA, kindly provided the kits for 99mTc-Annexin-V preparation. Received Aug 4, 2003, and in revised form Nov 11, 2003. Accepted for publication Nov 13, 2003. 782
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Table 1. Pretreatment and posttreatment apoptosis scores listed according to radiotherapy outcome Patient characteristics Age (years)
Sex
72 45 66 80 56 60 42 58 60 54 42
F F F M M F F F M F M
FL FL FL FL FL FL FL FL FL FL FL FL
I II II II I I II II I II II
Pretreatment apoptosis scores
Posttreatment apoptosis scores
Radiotherapy outcome
Node ⭋ (cm)
Cytology
TAV scan
Cytology
TAV scan
Cytology vs TAV
Response
Onset
6 4 3 13 5 4 7 8 4 6 5
⫺ ⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺ ⫺ ⫺ ⫹/⫺
⫺ ⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺ ⫺ ⫺ ⫹/⫺
⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹ ⫹/⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹ ⫹/⫺ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹
Concordant Discordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant
SD PR PR CR CR CR CR CR CR CR CR
⫺ Slow Slow Slow Slow Slow Slow Fast Fast Fast Fast
Abbreviations: F ⫽ female; M ⫽ male; FL I ⫽ follicular lymphoma grade I; FL II ⫽ follicular lymphoma grade II; Node ⭋ (cm) ⫽ largest lymph node diameter in centimeters; TAV ⫽ 99mTc-Annexin V scintigraphy; vs ⫽ versus; SD ⫽ stable disease; PR ⫽ partial remission; CR ⫽ complete remission. Note: A negative pretreatment TAV scintigram indicates that there was no uptake in lymphoma localizations; however, physiological uptake elsewhere was noted.
to eliminate cells that have been exposed to mutagenic agents may contribute to the development of cancer and resistance to anticancer therapy. Apoptosis is defined by specific morphologic criteria and by the requirement for active participation of the dying cell. For malignancies of lymphoid, myeloid, or germinal lineage, apoptosis appears to be a major mechanism of radiation-induced cell death (10). The exposure of phosphatidylserine at the outer leaflet of the membrane lipid bilayer is one of the first events during the apoptotic process. The human endogenous protein Annexin V has a high affinity for phosphatidylserine (10 –13). For flow cytometric analysis purposes Annexin V can be labeled to analyze apoptosis in vitro (14, 15). Recently, the radiopharmaceutical compound 99mTc-Annexin V (TAV) has become available for in vivo scintigraphic imaging both in animals (12, 16 –18) and in humans (19, 20 –24). In this study we investigated the role of apoptosis and the predictive value of in vivo TAV scintigraphy in the radiation response in FL patients. The FL patient group was chosen as a model for initial study, because of the high radiation sensitivity to the low-dose 2 ⫻ 2 Gy regimen and the usual rapid onset of response.
and Grade II in 7. Before and after irradiation all patients were thoroughly studied for FL localizations and irradiation outcome by history and physical examination, ultrasonography of neck nodes, and contrast-enhanced computed tomography (CT) scans of chest and abdomen. The investigation procedure was as follows (Fig. 1). All patients underwent baseline TAV scintigraphy within 1 week before radiotherapy to study the presence of spontaneous apoptosis and to detect sites of aspecific TAV uptake. Patients were irradiated only to the involved lymph node areas. No irradiation to adjacent nonpathologic lymph node areas was performed. Radiation was administered on Days 1 and 3. Fine-needle aspiration cytology was taken on Days 1 through 5. Involved field irradiation, to a dose of 4 Gy in two equal fractions, was performed without any concurrent chemotherapy or corticosteroid drug prescription. All patients had recurrent disease and were previously treated by several regimens: chlorambucil (n ⫽ 3) or fludarabin (n ⫽ 3) single-agent regimens; cyclophosphamide-vincristineprednisone polychemotherapy (n ⫽ 2); involved field irra-
PATIENTS AND METHODS This prospective study was approved by the local Ethical Committee. All patients agreed to participate with written informed consent. A total of 11 FL patients (7 female and 4 male; median age, 58 years; range, 42– 80 years) were included in this study (Table 1). Median lymph node size was 5 cm (range, 3–13 cm). All histologic and cytologic materials were reviewed and the diagnosis of FL was confirmed (by D.d.J.). Follicular lymphoma Grade I was diagnosed in 4 patients
Fig. 1. Flow sheet of study. TAV ⫽ 99mTc-Annexin V scintigraphy; RT ⫽ involved field radiotherapy to a dose of 2 Gy per fraction; FNAC ⫽ fine-needle aspiration cytology.
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diation (n ⫽ 2); and anti-CD20 immunotherapy (n ⫽ 1). Interval since last treatment was 16 months (range, 7–34 months). Posttreatment TAV scintigraphy was performed on Day 4 (24 hours after the second 2 Gy fraction). The TAV scintigraphy was performed according to the manufacturer’s guidelines. Planar and single-positron emission computed tomography (SPECT) images using 99mTc Phentiotate-rhAnnexin V (the first 5 patients) and 99mTc Hynic-rh-Annexin V (the last 7 patients) were performed 4 hours after i.v. administration of approximately 800 MBq TAV (range, 482–1013 MBq). Thin-layer chromatography was used to assess radiochemical purity of the radiopharmaceutical. A dual-head gamma camera (Genesys, Philips ADAC) equipped with high-resolution, low-energy collimators was used. The TAV images were semiquantitatively evaluated for uptake, ranging from absent (⫺), weak (⫾), present (⫹), to intense (⫹⫹). Images were evaluated by two observers masked from cytologic results. Cytologic assessment for apoptosis was performed on routinely prepared Giemsa-stained smear preparations and scored using the same categories as mentioned above. Involved field radiotherapy by megavoltage photon or electron beams to a dose of 4 Gy in two fractions of 2 Gy with 48-hour interval was administered to all patients. Radiation response was assessed 4 weeks afterward according to four generally accepted qualities; complete remission (CR; in this setting defined as local control), partial remission (PR), stable disease (SD), and progressive disease (PD). Because in general FL patients respond very promptly to radiotherapy, we also assessed whether the treatment response occurred within 1 week after irradiation (designated as fast response) or beyond (designated as slow response).
Fig. 2. (A) Total body, partially shown, (B) single-positron emission computed tomography (SPECT) transverse, (C) sagital, and (D) coronal images are typical examples of pretreatment scans in a 42-year-old man: physiologic uptake in bones and salivary glands and weak (⫾) uptake in right-sided high neck nodes. Note that the weak tumor uptake in the malignant nodes is only clearly appreciated on the SPECT images. (E) Total body, partially shown, (F) SPECT transverse, (G) sagital, and (H) coronal imaging performed after irradiation shows intense (⫹⫹) tumor uptake (arrows) in the irradiated right-sided high neck nodes. Again, SPECT more clearly shows the uptake than total body studies.
RESULTS
Posttreatment investigations In 1 patient the TAV imaging results were concordant with cytology in the pretreatment evaluation, but discordant after radiotherapy. There was a weak increase of apoptotic
Radiochemical analysis resulted in 93% purity (range, 88%–98%). None of the TAV procedures was complicated by any adverse side effect like allergic reactions, coagulation disorders, or any other blood hematology or chemistry abnormalities, or patient well-being. If present, the first detection of apoptotic features by cytology was on Day 4 or 5. Therefore, cytologic scoring of apoptosis (Table 1) was performed on Day 4. Pretreatment investigations In all patients physiologic TAV uptake in bones, salivary glands, kidneys, liver, colon, and bladder was observed. This phenomenon was earlier described by Blankenberg and Belhocine (13, 24). On preirradiation images, weak TAV uptake was seen in FL sites in 5 patients and correlated with low levels of morphologically recognisable spontaneous apoptosis. Uptake of TAV was undetectable in FL sites in 6 patients and correlated with absence of spontaneous apoptosis. There were no false-positive or false-negative pretreatment scans in 11 evaluable patients.
The last patient in the Table (male, 42 years old) is illustrated in Figs. 2 and 3. Figures 2A (total body, partially shown), 2B (SPECT transverse), 2C (sagital), and 2D (coronal) show typical examples of pretreatment images: physiologic uptake in bones and salivary glands and weak (⫾) uptake in right-sided high neck nodes. Note that the weak tumor uptake in the malignant nodes is only clearly appreciated on the SPECT images. Figure 3A shows a cytologic Giemsa-stained smear preparation of viable lymphoma cells before irradiation.
Fig. 3. (A) Cytologic Giemsa-stained smear preparation of viable lymphoma cells is shown before irradiation in the 42-year-old male patient. (B) Massive nuclear chromatin condensation, margination, and apoptotic body formation are shown, 24 hours after the second fraction of 2 Gy (Day 4) in the same patient, designated as ⫹⫹.
In vivo imaging of radiation-induced apoptosis
cell death seen in the biopsies, but no increased radiopharmaceutical uptake could be noted. The other 10 patients showed concordant results. In 4 patients abundant apoptotic cell death (⫹⫹) was seen. Intense hotspots (⫹⫹) were seen in 3 of them and in 1 a (⫹) hotspot. Clear induction of apoptotic cell death, marked as ⫹, was cytologically diagnosed in 2 patients and correlated with hotspots in both. In 3 patients there was a weak (⫾) increase in apoptotic cell death seen in the cytology, and at the same time a weak increase in local radiopharmaceutical uptake was found. In 1 patient both cytology and imaging remained negative (⫺) for apoptotic cell death after irradiation. The last patient of the Table (male, 42 years old) is illustrated in Figs. 2 and 3. Figures 2E (total body, partially shown), 2F (SPECT transverse), 2G (sagital), and 2H (coronal), performed after irradiation, show intense (⫹⫹) tumor uptake (arrows) in the irradiated right-sided high neck nodes. Again, SPECT more clearly shows the uptake than total body studies. Figure 3B shows massive nuclear chromatin condensation, margination and apoptotic body formation 24 hours after the second fraction of 2 Gy (Day 4) in the same patient, designated as ⫹⫹. Radiotherapy treatment response Four different radiation outcome categories were identified in this patient population: SD, PR, CR of slow onset, and CR of rapid onset (Table 1). Stable disease. The 1 patient who did not reveal apoptotic cell death either in the cytologic or in the scintigraphic investigations did not respond to irradiation and maintained stable disease. Partial remission. Patients with either weak (⫾) or absent (⫺) signs of apoptotic cell death responded slowly and partially on their involved field irradiation. Complete remission of slow onset. A CR of slow onset was seen in 4 patients; 2 of them had weak signs (⫾) of apoptotic cell death and 2 had present signs (⫹) of apoptosis in both modalities. Complete remission of rapid onset. The patients with intense signs (⫹⫹) of apoptosis in one or both modalities all achieved a CR within 1 week. In summary, pathology scoring of apoptosis correctly correlated with irradiation outcome both in quality and in onset in all 11 patients. Scintigraphic assessment was only incorrect in predicting outcome in the second patient in the Table (female, 45 years old).
DISCUSSION This is, to the best of our knowledge, the first study to show that radiation-induced apoptosis in FL patients can be visualized at early stages by in vivo 99mTc-Annexin V (TAV) scintigraphy. The optimum timing for pathologic assessment (fine-needle aspiration cytology or biopsy) in the setting of this radiation regimen appears to be on Day 4 or 5. No false-positive results were seen. Furthermore, TAV
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scintigraphy as a noninvasive procedure is accurate in predicting irradiation outcome. Radiation-induced apoptosis has been suggested as an early event in the response of FL cells after radiotherapy. This hypothesis is supported by performing fine-needle aspiration cytology repeatedly in the first week of the 2 ⫻ 2 Gy involved field radiotherapy regimen. Up to now, the gold standard to investigate apoptotic cell death is by cytologic or histologic examination. Typical features like nuclear chromatin condensation, margination and apoptotic body formation can be easily appreciated. These features have been seen to various extents in 10 of our 11 evaluable patients (Fig. 3B). These tests, however, rely on invasive and in vitro procedures. The results obtained by in vivo TAV scintigraphy are highly concordant with the cytologic results. Therefore, this imaging technique might be an attractive alternative to pathology. Apoptotic cell death is a rapid phenomenon from a biologic point of view. In case of an efficient apoptotic stimulus (like radiation in FL patients), the cascade of events evolves within hours to at most a few days. The time frame in which the cells have exposed phosphatidylserine at the outer leaflet of the bilipid cell membrane but have not yet been phagocytosed by macrophages is short. It is only in this limited time period, that cells destined to undergo apoptotic death can bind TAV. We found this to be on Days 4 and 5 of this radiotherapy regimen in FL patients. Therefore, this may be the optimal time point to perform postirradiation TAV. Belhocine et al. (24) and Blankenberg (25) perform posttreatment scintigraphy at the same day of or at the latest within 72 hours after the first chemotherapy course, because they anticipate apoptotic cell death to be an early event. This was based on the fact that phosphatidylserine exposure occurs within 90 –120 min after the apoptotic stimulus. In the present study two different 99mTc-Annexin V compounds have been used. The second generation apoptosis marker 99mTc Hynic-rh-Annexin V showed a more favorable biodistribution improving documentation of pathology in thorax and abdomen. Evaluation of the intensity of tumor uptake was limited to a visual analysis. Owing to the difficulties in identifying tumor contour on Annexin-V images of some patients, tumor uptake quantification by drawing the region of interest was not applied. At present, coregistration and matching of SPECT and CT is being used to solve this limitation. Tumor delineation is first effectuated on CT, and subsequently region of interest uptake is obtained from SPECT. Until now, clinical studies on in vivo imaging of apoptotic cell death in humans have mainly focused on cardiac allograft rejection and acute myocardial infarction (26 –31). The clinical relevance of these studies exists in initiating the appropriate antirejection measurements in the right patient at the right time. In oncology, it has been evaluated by many groups whether treatment outcome could be predicted on the basis of pretreatment levels of apoptosis (apoptotic index) (10). Whereas certain studies on lymphomas indeed showed a correlation between a high baseline apoptotic
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index and good prognosis (32), others reached the opposite conclusion (33, 34). Interestingly, a correlation has been established between radiation-induced apoptosis measured in vitro in cells obtained by cytology and early responses to in vivo radiotherapy in patients with low-grade nonHodgkin’s lymphoma (8). Therefore, early noninvasive assessment of apoptosis, indicative of the effectiveness of the initiated treatment, might result in adaptation of further therapeutic actions on a patient-by-patient decision. Ineffective treatments could then be stopped at an early stage to prevent further adverse side effects. Belhocine (24) showed that TAV scintigraphy was able to image apoptosis in lymphoma, lung and breast cancer patients and indicated a predictive value to chemotherapy response. There is one potential pitfall in TAV scintigraphy: necrotic cells are accessible for TAV to bind phosphatidylserine. Hence, extensive necrosis may result in false-positive
Volume 59, Number 3, 2004
tests. In our pathology controlled study, however, we have not observed necrosis in the irradiated lymph nodes. Further research is needed to expand the validation of TAV scintigraphy in daily oncologic practice. The follicular lymphoma was chosen as a model for initial study, because of the high radiation sensitivity to the low-dose 2 ⫻ 2 Gy regimen and the usual rapid onset of response. The next step will be to investigate tumors with worse response rates to conventional treatments and to assess whether TAV scintigraphy is predictive of treatment outcome as well. Current investigations are focusing on other lymphoma subtypes treated by radiotherapy and induction gemcitabin and cisplatin polychemotherapy in non–small cell lung cancer. In conclusion, TAV scintigraphy may prove an easy and reliable predictive assay for radiation treatment outcome in follicular lymphoma patients. Further investigations in other tumor types are ongoing.
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radiolabeled monocyte chemotactic peptide-1 and annexin V. Eur J Nucl Med 2001;28:1384–1393. Blankenberg FG, Tait JF, Strauss HW. Apoptotic cell death: its implications for imaging in the next millennium. Eur J Nucl Med 2000;27:359–367. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserineexpression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184:39–151. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994;84:1415–1420. Blankenberg FG, Naumovski L, Tait JF, Post AM, Strauss HW. Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc-radiolabeled annexin V. J Nucl Med 2001;42:309–316. Ogura Y, Krams SM, Martinez OM, et al. Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation. Radiology 2000; 214:795–800. Post AM, Katsikis PD, Tait JF, et al. Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis. J Nucl Med 2002;43:1359–1365. Kemerink GJ, Boersma HH, Thimister PW, et al. Biodistribution and dosimetry of 99mTc-BTAP-annexin-V in humans. Eur J Nucl Med 2001;28:1373–1378. Yang DJ, Azhdarinia A, Wu P, et al. In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTcEC-annexin V. Cancer Biother Radiopharm 2001;16:73–83. Kemerink GJ, Liem IH, Hofstra L, et al. Patient dosimetry of intravenously administered 99mTc-annexin V. J Nucl Med 2001;42:382–387. Ohtsuki K, Akashi K, Aoka Y, et al. Technetium-99m HYNIC-annexin V: A potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med 1999;26:1251– 1258. Blankenberg FG, Katsikis PD, Tait JF, et al. Imaging of apoptosis (programmed cell death) with 99mTc annexin V. J Nucl Med 1999;40:184–191. Belhocine T, Steinmetz N, Hustinx R, et al. Increased uptake of the apoptosis-imaging agent (99m)Tc recombinant human Annexin V in human tumors after one course of chemotherapy
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30. Vriens PW, Blankenberg FG, Stoot JH, et al. The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Cardiovasc Surg 1998;116:844–853. 31. Thimister PWL, Hofstra L, Liem IH, et al. In vivo detection of cell death in the area at risk in acute myocardial infarction. J Nucl Med 2003;44:391–396. 32. Czader M, Mazur J, Pettersson M, et al. Prognostic significance of proliferative and apoptotic fractions in low grade follicle center cell-derived non-Hodgkin’s lymphomas. Cancer 1996;77:1180–1188. 33. Leoncini L, Del Vecchio MT, Megha T, et al. Correlations between apoptotic and proliferative indices in malignant nonHodgkin’s lymphomas. Am J Pathol 1993;142:755–763. 34. Logsdon MD, Meyn RE, Jr, Besa PC, et al. Apoptosis and the Bcl-2 gene family—patterns of expression and prognostic value in stage I and II follicular center lymphoma. Int J Radiat Oncol Biol Phys 1999;44:19–29.
doi:10.1016/j.ijrobp.2003.11.017
CLINICAL INVESTIGATION
Lymphoma
IN VIVO IMAGING OF RADIATION-INDUCED APOPTOSIS IN FOLLICULAR LYMPHOMA PATIENTS RICK L. M. HAAS, M.D.,* DAPHNE DE JONG, M.D., PH.D.,† RENATO A. VALDE´ S OLMOS, M.D., PH.D.,‡ CEES A. HOEFNAGEL, M.D., PH.D.,‡ IRIS VAN DEN HEUVEL,‡ SHURAILA F. ZERP,* HARRY BARTELINK, M.D., PH.D.,* AND MARCEL VERHEIJ, M.D., PH.D.* Departments of *Radiotherapy, †Pathology, and ‡Nuclear Medicine, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Purpose: To evaluate 99mTc-Annexin-V (TAV) scintigraphy in monitoring radiation-induced apoptotic cell death in follicular lymphoma (FL) patients. Patients and Methods: Eleven FL patients (7 female and 4 male; median age, 58 years; range, 42– 80 years) with recurrent disease underwent TAV imaging before and 24 hours after the last fraction of the 2 ⴛ 2 Gy involved field radiotherapy regimen. Fine-needle aspiration cytology was performed on 5 consecutive days to determine the optimal time window for apoptosis detection and to confirm the apoptotic nature of the response. The TAV scintigraphy (total body studies and SPECT of the irradiated sites) was performed 4 hours after the administration of the radiopharmaceutical. Tumor uptake was scored in a semiquantitative manner as absent (ⴚ) weak (ⴞ), present (ⴙ), or intense (ⴙⴙ) with corresponding categories for the cytologic slides. Response evaluation was performed after 1 week and 4 weeks both in terms of completeness and speed of remission. Results: Baseline TAV uptake was absent in 6 and weak in 5 patients. Sequential cytology indicated that the optimal time period for apoptosis assessment was between 24 and 48 hours after the last fraction of the 2 ⴛ 2 Gy regimen. Baseline cytology was concordant with baseline TAV in all patients. Apoptotic feature appearance (nuclear chromatin condensation, margination and apoptotic body formation) after low-dose irradiation matched the irradiation response in all patients. In all but 1 patient the posttreatment TAV uptake matched the posttreatment cytology. In these 10 patients the cytology and TAV results correlated with the type and onset of the clinical response. Conclusion: Tumor 99mTc-Annexin-V uptake can be increased after 2 ⴛ 2 Gy involved field radiotherapy. This increase was concordant with the appearance of apoptotic morphology as determined by cytology, and correlated with the clinical outcome. Apoptotic cell death can be observed on Day 4 of this regimen and if so predicts a complete remission within 1 week. © 2004 Elsevier Inc. 99m
Tc-Annexin-V, Radiotherapy, Follicular lymphoma, Apoptosis.
INTRODUCTION
total dose of 36 – 40 Gy in about 50% of cases (2– 4). In advanced stage disease (Ann Arbor Stage III and IV) prolonged remissions are seen after low-dose total body irradiation fractionated in daily doses of 10 –15 cGy to a total of 1.5–2.5 Gy (5–7). Follicular lymphoma patients responding to radiotherapy usually do so already during treatment. This suggests that apoptotic cell death might play a role in the clinical outcome. Dubray et al. (8, 9) were the first to show in vitro that the high response rates after radiotherapy in FL might be due to radiation-induced apoptosis. Apoptosis is a distinct mode of cell death and represents an important regulatory mechanism to remove abundant and unwanted cells during many physiologic processes. Failure
Characteristic for follicular non-Hodgkin’s lymphomas (FL) is the t(14;18) translocation. This cytogenetic feature involves a translocation of the bcl-2 gene on chromosome 18 to the immunoglobulin heavy-chain locus (IgH) on chromosome 14, resulting in an overexpression of Bcl-2, a well-known inhibitor of apoptosis (1). It is hypothesized that in this disease expansion is not primarily caused by proliferation but rather by the lack of apoptotic cell loss. These lymphomas are one of the most radiosensitive tumors. Patients in early-stage (Ann Arbor Stage I and II) can be cured by conventionally fractionated irradiation to a Reprint requests to: Rick L. M. Haas, M.D., Department of Radiotherapy, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel: (⫹31) 205122124; Fax: (⫹31) 206691101; E-mail: [email protected]
Acknowledgments—Theseus Imaging Corporation, Boston, MA, kindly provided the kits for 99mTc-Annexin-V preparation. Received Aug 4, 2003, and in revised form Nov 11, 2003. Accepted for publication Nov 13, 2003. 782
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Table 1. Pretreatment and posttreatment apoptosis scores listed according to radiotherapy outcome Patient characteristics Age (years)
Sex
72 45 66 80 56 60 42 58 60 54 42
F F F M M F F F M F M
FL FL FL FL FL FL FL FL FL FL FL FL
I II II II I I II II I II II
Pretreatment apoptosis scores
Posttreatment apoptosis scores
Radiotherapy outcome
Node ⭋ (cm)
Cytology
TAV scan
Cytology
TAV scan
Cytology vs TAV
Response
Onset
6 4 3 13 5 4 7 8 4 6 5
⫺ ⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺ ⫺ ⫺ ⫹/⫺
⫺ ⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺ ⫺ ⫺ ⫹/⫺
⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹ ⫹/⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫺ ⫺ ⫹/⫺ ⫹/⫺ ⫹ ⫹/⫺ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹
Concordant Discordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant
SD PR PR CR CR CR CR CR CR CR CR
⫺ Slow Slow Slow Slow Slow Slow Fast Fast Fast Fast
Abbreviations: F ⫽ female; M ⫽ male; FL I ⫽ follicular lymphoma grade I; FL II ⫽ follicular lymphoma grade II; Node ⭋ (cm) ⫽ largest lymph node diameter in centimeters; TAV ⫽ 99mTc-Annexin V scintigraphy; vs ⫽ versus; SD ⫽ stable disease; PR ⫽ partial remission; CR ⫽ complete remission. Note: A negative pretreatment TAV scintigram indicates that there was no uptake in lymphoma localizations; however, physiological uptake elsewhere was noted.
to eliminate cells that have been exposed to mutagenic agents may contribute to the development of cancer and resistance to anticancer therapy. Apoptosis is defined by specific morphologic criteria and by the requirement for active participation of the dying cell. For malignancies of lymphoid, myeloid, or germinal lineage, apoptosis appears to be a major mechanism of radiation-induced cell death (10). The exposure of phosphatidylserine at the outer leaflet of the membrane lipid bilayer is one of the first events during the apoptotic process. The human endogenous protein Annexin V has a high affinity for phosphatidylserine (10 –13). For flow cytometric analysis purposes Annexin V can be labeled to analyze apoptosis in vitro (14, 15). Recently, the radiopharmaceutical compound 99mTc-Annexin V (TAV) has become available for in vivo scintigraphic imaging both in animals (12, 16 –18) and in humans (19, 20 –24). In this study we investigated the role of apoptosis and the predictive value of in vivo TAV scintigraphy in the radiation response in FL patients. The FL patient group was chosen as a model for initial study, because of the high radiation sensitivity to the low-dose 2 ⫻ 2 Gy regimen and the usual rapid onset of response.
and Grade II in 7. Before and after irradiation all patients were thoroughly studied for FL localizations and irradiation outcome by history and physical examination, ultrasonography of neck nodes, and contrast-enhanced computed tomography (CT) scans of chest and abdomen. The investigation procedure was as follows (Fig. 1). All patients underwent baseline TAV scintigraphy within 1 week before radiotherapy to study the presence of spontaneous apoptosis and to detect sites of aspecific TAV uptake. Patients were irradiated only to the involved lymph node areas. No irradiation to adjacent nonpathologic lymph node areas was performed. Radiation was administered on Days 1 and 3. Fine-needle aspiration cytology was taken on Days 1 through 5. Involved field irradiation, to a dose of 4 Gy in two equal fractions, was performed without any concurrent chemotherapy or corticosteroid drug prescription. All patients had recurrent disease and were previously treated by several regimens: chlorambucil (n ⫽ 3) or fludarabin (n ⫽ 3) single-agent regimens; cyclophosphamide-vincristineprednisone polychemotherapy (n ⫽ 2); involved field irra-
PATIENTS AND METHODS This prospective study was approved by the local Ethical Committee. All patients agreed to participate with written informed consent. A total of 11 FL patients (7 female and 4 male; median age, 58 years; range, 42– 80 years) were included in this study (Table 1). Median lymph node size was 5 cm (range, 3–13 cm). All histologic and cytologic materials were reviewed and the diagnosis of FL was confirmed (by D.d.J.). Follicular lymphoma Grade I was diagnosed in 4 patients
Fig. 1. Flow sheet of study. TAV ⫽ 99mTc-Annexin V scintigraphy; RT ⫽ involved field radiotherapy to a dose of 2 Gy per fraction; FNAC ⫽ fine-needle aspiration cytology.
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diation (n ⫽ 2); and anti-CD20 immunotherapy (n ⫽ 1). Interval since last treatment was 16 months (range, 7–34 months). Posttreatment TAV scintigraphy was performed on Day 4 (24 hours after the second 2 Gy fraction). The TAV scintigraphy was performed according to the manufacturer’s guidelines. Planar and single-positron emission computed tomography (SPECT) images using 99mTc Phentiotate-rhAnnexin V (the first 5 patients) and 99mTc Hynic-rh-Annexin V (the last 7 patients) were performed 4 hours after i.v. administration of approximately 800 MBq TAV (range, 482–1013 MBq). Thin-layer chromatography was used to assess radiochemical purity of the radiopharmaceutical. A dual-head gamma camera (Genesys, Philips ADAC) equipped with high-resolution, low-energy collimators was used. The TAV images were semiquantitatively evaluated for uptake, ranging from absent (⫺), weak (⫾), present (⫹), to intense (⫹⫹). Images were evaluated by two observers masked from cytologic results. Cytologic assessment for apoptosis was performed on routinely prepared Giemsa-stained smear preparations and scored using the same categories as mentioned above. Involved field radiotherapy by megavoltage photon or electron beams to a dose of 4 Gy in two fractions of 2 Gy with 48-hour interval was administered to all patients. Radiation response was assessed 4 weeks afterward according to four generally accepted qualities; complete remission (CR; in this setting defined as local control), partial remission (PR), stable disease (SD), and progressive disease (PD). Because in general FL patients respond very promptly to radiotherapy, we also assessed whether the treatment response occurred within 1 week after irradiation (designated as fast response) or beyond (designated as slow response).
Fig. 2. (A) Total body, partially shown, (B) single-positron emission computed tomography (SPECT) transverse, (C) sagital, and (D) coronal images are typical examples of pretreatment scans in a 42-year-old man: physiologic uptake in bones and salivary glands and weak (⫾) uptake in right-sided high neck nodes. Note that the weak tumor uptake in the malignant nodes is only clearly appreciated on the SPECT images. (E) Total body, partially shown, (F) SPECT transverse, (G) sagital, and (H) coronal imaging performed after irradiation shows intense (⫹⫹) tumor uptake (arrows) in the irradiated right-sided high neck nodes. Again, SPECT more clearly shows the uptake than total body studies.
RESULTS
Posttreatment investigations In 1 patient the TAV imaging results were concordant with cytology in the pretreatment evaluation, but discordant after radiotherapy. There was a weak increase of apoptotic
Radiochemical analysis resulted in 93% purity (range, 88%–98%). None of the TAV procedures was complicated by any adverse side effect like allergic reactions, coagulation disorders, or any other blood hematology or chemistry abnormalities, or patient well-being. If present, the first detection of apoptotic features by cytology was on Day 4 or 5. Therefore, cytologic scoring of apoptosis (Table 1) was performed on Day 4. Pretreatment investigations In all patients physiologic TAV uptake in bones, salivary glands, kidneys, liver, colon, and bladder was observed. This phenomenon was earlier described by Blankenberg and Belhocine (13, 24). On preirradiation images, weak TAV uptake was seen in FL sites in 5 patients and correlated with low levels of morphologically recognisable spontaneous apoptosis. Uptake of TAV was undetectable in FL sites in 6 patients and correlated with absence of spontaneous apoptosis. There were no false-positive or false-negative pretreatment scans in 11 evaluable patients.
The last patient in the Table (male, 42 years old) is illustrated in Figs. 2 and 3. Figures 2A (total body, partially shown), 2B (SPECT transverse), 2C (sagital), and 2D (coronal) show typical examples of pretreatment images: physiologic uptake in bones and salivary glands and weak (⫾) uptake in right-sided high neck nodes. Note that the weak tumor uptake in the malignant nodes is only clearly appreciated on the SPECT images. Figure 3A shows a cytologic Giemsa-stained smear preparation of viable lymphoma cells before irradiation.
Fig. 3. (A) Cytologic Giemsa-stained smear preparation of viable lymphoma cells is shown before irradiation in the 42-year-old male patient. (B) Massive nuclear chromatin condensation, margination, and apoptotic body formation are shown, 24 hours after the second fraction of 2 Gy (Day 4) in the same patient, designated as ⫹⫹.
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cell death seen in the biopsies, but no increased radiopharmaceutical uptake could be noted. The other 10 patients showed concordant results. In 4 patients abundant apoptotic cell death (⫹⫹) was seen. Intense hotspots (⫹⫹) were seen in 3 of them and in 1 a (⫹) hotspot. Clear induction of apoptotic cell death, marked as ⫹, was cytologically diagnosed in 2 patients and correlated with hotspots in both. In 3 patients there was a weak (⫾) increase in apoptotic cell death seen in the cytology, and at the same time a weak increase in local radiopharmaceutical uptake was found. In 1 patient both cytology and imaging remained negative (⫺) for apoptotic cell death after irradiation. The last patient of the Table (male, 42 years old) is illustrated in Figs. 2 and 3. Figures 2E (total body, partially shown), 2F (SPECT transverse), 2G (sagital), and 2H (coronal), performed after irradiation, show intense (⫹⫹) tumor uptake (arrows) in the irradiated right-sided high neck nodes. Again, SPECT more clearly shows the uptake than total body studies. Figure 3B shows massive nuclear chromatin condensation, margination and apoptotic body formation 24 hours after the second fraction of 2 Gy (Day 4) in the same patient, designated as ⫹⫹. Radiotherapy treatment response Four different radiation outcome categories were identified in this patient population: SD, PR, CR of slow onset, and CR of rapid onset (Table 1). Stable disease. The 1 patient who did not reveal apoptotic cell death either in the cytologic or in the scintigraphic investigations did not respond to irradiation and maintained stable disease. Partial remission. Patients with either weak (⫾) or absent (⫺) signs of apoptotic cell death responded slowly and partially on their involved field irradiation. Complete remission of slow onset. A CR of slow onset was seen in 4 patients; 2 of them had weak signs (⫾) of apoptotic cell death and 2 had present signs (⫹) of apoptosis in both modalities. Complete remission of rapid onset. The patients with intense signs (⫹⫹) of apoptosis in one or both modalities all achieved a CR within 1 week. In summary, pathology scoring of apoptosis correctly correlated with irradiation outcome both in quality and in onset in all 11 patients. Scintigraphic assessment was only incorrect in predicting outcome in the second patient in the Table (female, 45 years old).
DISCUSSION This is, to the best of our knowledge, the first study to show that radiation-induced apoptosis in FL patients can be visualized at early stages by in vivo 99mTc-Annexin V (TAV) scintigraphy. The optimum timing for pathologic assessment (fine-needle aspiration cytology or biopsy) in the setting of this radiation regimen appears to be on Day 4 or 5. No false-positive results were seen. Furthermore, TAV
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scintigraphy as a noninvasive procedure is accurate in predicting irradiation outcome. Radiation-induced apoptosis has been suggested as an early event in the response of FL cells after radiotherapy. This hypothesis is supported by performing fine-needle aspiration cytology repeatedly in the first week of the 2 ⫻ 2 Gy involved field radiotherapy regimen. Up to now, the gold standard to investigate apoptotic cell death is by cytologic or histologic examination. Typical features like nuclear chromatin condensation, margination and apoptotic body formation can be easily appreciated. These features have been seen to various extents in 10 of our 11 evaluable patients (Fig. 3B). These tests, however, rely on invasive and in vitro procedures. The results obtained by in vivo TAV scintigraphy are highly concordant with the cytologic results. Therefore, this imaging technique might be an attractive alternative to pathology. Apoptotic cell death is a rapid phenomenon from a biologic point of view. In case of an efficient apoptotic stimulus (like radiation in FL patients), the cascade of events evolves within hours to at most a few days. The time frame in which the cells have exposed phosphatidylserine at the outer leaflet of the bilipid cell membrane but have not yet been phagocytosed by macrophages is short. It is only in this limited time period, that cells destined to undergo apoptotic death can bind TAV. We found this to be on Days 4 and 5 of this radiotherapy regimen in FL patients. Therefore, this may be the optimal time point to perform postirradiation TAV. Belhocine et al. (24) and Blankenberg (25) perform posttreatment scintigraphy at the same day of or at the latest within 72 hours after the first chemotherapy course, because they anticipate apoptotic cell death to be an early event. This was based on the fact that phosphatidylserine exposure occurs within 90 –120 min after the apoptotic stimulus. In the present study two different 99mTc-Annexin V compounds have been used. The second generation apoptosis marker 99mTc Hynic-rh-Annexin V showed a more favorable biodistribution improving documentation of pathology in thorax and abdomen. Evaluation of the intensity of tumor uptake was limited to a visual analysis. Owing to the difficulties in identifying tumor contour on Annexin-V images of some patients, tumor uptake quantification by drawing the region of interest was not applied. At present, coregistration and matching of SPECT and CT is being used to solve this limitation. Tumor delineation is first effectuated on CT, and subsequently region of interest uptake is obtained from SPECT. Until now, clinical studies on in vivo imaging of apoptotic cell death in humans have mainly focused on cardiac allograft rejection and acute myocardial infarction (26 –31). The clinical relevance of these studies exists in initiating the appropriate antirejection measurements in the right patient at the right time. In oncology, it has been evaluated by many groups whether treatment outcome could be predicted on the basis of pretreatment levels of apoptosis (apoptotic index) (10). Whereas certain studies on lymphomas indeed showed a correlation between a high baseline apoptotic
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index and good prognosis (32), others reached the opposite conclusion (33, 34). Interestingly, a correlation has been established between radiation-induced apoptosis measured in vitro in cells obtained by cytology and early responses to in vivo radiotherapy in patients with low-grade nonHodgkin’s lymphoma (8). Therefore, early noninvasive assessment of apoptosis, indicative of the effectiveness of the initiated treatment, might result in adaptation of further therapeutic actions on a patient-by-patient decision. Ineffective treatments could then be stopped at an early stage to prevent further adverse side effects. Belhocine (24) showed that TAV scintigraphy was able to image apoptosis in lymphoma, lung and breast cancer patients and indicated a predictive value to chemotherapy response. There is one potential pitfall in TAV scintigraphy: necrotic cells are accessible for TAV to bind phosphatidylserine. Hence, extensive necrosis may result in false-positive
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tests. In our pathology controlled study, however, we have not observed necrosis in the irradiated lymph nodes. Further research is needed to expand the validation of TAV scintigraphy in daily oncologic practice. The follicular lymphoma was chosen as a model for initial study, because of the high radiation sensitivity to the low-dose 2 ⫻ 2 Gy regimen and the usual rapid onset of response. The next step will be to investigate tumors with worse response rates to conventional treatments and to assess whether TAV scintigraphy is predictive of treatment outcome as well. Current investigations are focusing on other lymphoma subtypes treated by radiotherapy and induction gemcitabin and cisplatin polychemotherapy in non–small cell lung cancer. In conclusion, TAV scintigraphy may prove an easy and reliable predictive assay for radiation treatment outcome in follicular lymphoma patients. Further investigations in other tumor types are ongoing.
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