Anticancer therapy and apoptosis imaging

Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discove...

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Опубліковано в: :Experimental Oncology
Дата:2012
Автори: Yalcin, T.J., Haimovitz-Friedman, A., Verheij, M.
Формат: Стаття
Мова:English
Опубліковано: Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України 2012
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Цитувати:Anticancer therapy and apoptosis imaging / T.J. Yang, A. Haimovitz-Friedman, M. Verheij // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 269-276. — Бібліогр.: 94 назв. — англ.

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Digital Library of Periodicals of National Academy of Sciences of Ukraine
id nasplib_isofts_kiev_ua-123456789-139046
record_format dspace
spelling Yalcin, T.J.
Haimovitz-Friedman, A.
Verheij, M.
2018-06-19T18:45:36Z
2018-06-19T18:45:36Z
2012
Anticancer therapy and apoptosis imaging / T.J. Yang, A. Haimovitz-Friedman, M. Verheij // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 269-276. — Бібліогр.: 94 назв. — англ.
1812-9269
https://nasplib.isofts.kiev.ua/handle/123456789/139046
Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discoveries of novel markers in functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging with various radiotracers as predictive tools for tumor response after anticancer therapy. Particular focus will be on annexin V imaging, a technique with the largest clinical experience to date. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
en
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
Experimental Oncology
Reviews
Anticancer therapy and apoptosis imaging
Article
published earlier
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
title Anticancer therapy and apoptosis imaging
spellingShingle Anticancer therapy and apoptosis imaging
Yalcin, T.J.
Haimovitz-Friedman, A.
Verheij, M.
Reviews
title_short Anticancer therapy and apoptosis imaging
title_full Anticancer therapy and apoptosis imaging
title_fullStr Anticancer therapy and apoptosis imaging
title_full_unstemmed Anticancer therapy and apoptosis imaging
title_sort anticancer therapy and apoptosis imaging
author Yalcin, T.J.
Haimovitz-Friedman, A.
Verheij, M.
author_facet Yalcin, T.J.
Haimovitz-Friedman, A.
Verheij, M.
topic Reviews
topic_facet Reviews
publishDate 2012
language English
container_title Experimental Oncology
publisher Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
format Article
description Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discoveries of novel markers in functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging with various radiotracers as predictive tools for tumor response after anticancer therapy. Particular focus will be on annexin V imaging, a technique with the largest clinical experience to date. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
issn 1812-9269
url https://nasplib.isofts.kiev.ua/handle/123456789/139046
citation_txt Anticancer therapy and apoptosis imaging / T.J. Yang, A. Haimovitz-Friedman, M. Verheij // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 269-276. — Бібліогр.: 94 назв. — англ.
work_keys_str_mv AT yalcintj anticancertherapyandapoptosisimaging
AT haimovitzfriedmana anticancertherapyandapoptosisimaging
AT verheijm anticancertherapyandapoptosisimaging
first_indexed 2025-11-26T01:39:37Z
last_indexed 2025-11-26T01:39:37Z
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fulltext Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��� ANTICANCER THERAPY AND APOPTOSIS IMAGING T.J. Yang1, A. Haimovitz-Friedman1, M. Verheij2,* 1Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York 10065, USA 2Department of Radiation Oncology and Division of Biological Stress Response, The Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital, 1066 CX, Amsterdam, The Netherlands Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discoveries of novel markers in functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging with various radiotracers as predic- tive tools for tumor response after anticancer therapy. Particular focus will be on annexin V imaging, a technique with the largest clinical experience to date. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. Key Words: apoptosis, cell death, imaging, anticancer therapy, annexin V. INTRODUCTION Apoptosis is an evolutionary highly preserved and well-orchestrated �iological process involved in �oth physiological and pathological conditions� and there- fore possi�ly the most a�undant form of programmed cell death [�]. Therapy-induced apoptosis in vivo has �een shown to significantly contri�ute to tumor response [�� �] and to correlate with su�sequent outcome [���]. After �� years of intense research� apoptosis is now considered not only as a fundamental process leading to disorders of normal tissues [����]� �ut also as a form of cell death in response to oncolytic therapies [�����]. Apoptosis can �e triggered �y exogenous and endogenous stimuli leading to the activation of the ex- trinsic and intrinsic pathways of apoptosis� respectively �Fig. ��. These pathways converge at the activation of a su�set of proteases� the executioner caspase-�� -� and -�� targeting specific intracellular proteins such as those involved in DNA damage repair and cellular cytoskeleton. The extrinsic pathway is engaged �y �inding of spe- cific death ligands to specific death receptors on the cell mem�rane� such as tumor necrosis factor a �TNF- a�� TNF-related apoptosis-inducing ligand �TRAIL�� Fas ligand �FasL� to TNF receptor � �TNFR��� death receptor � or death receptor 5 �DR� or DR5� and Fas/ CD�5 receptor� respectively. Death receptor-ligand �inding recruits an intracellular adaptor molecule� TNF-receptor-associated death domain �TRADD� or Fas-associated death domain �FADD� via its cy- toplasmic death domain �DD�� clustering to form the death-inducing signaling complex �DI�C� which in turn recruits and activates cytoplasmic pro-apoptotic cas- pase-8 �initiator caspase� via its death effector domain �DED�. This is followed �y the sequential activation of downstream executioner caspase-�� -�� and -�. Activated caspase-� translocates to the nucleus and activates poly-ADP-ri�ose polymerase �PARP-��� which facilitates the degradation of nuclear DNA into 5� to ��� kilo�ase-sized fragments. Anticancer drugs and ionizing radiation utilize the intrinsic pathway to trigger apoptosis. This process involves mitochondrial outer mem�rane permea�ili- zation and the su�sequent release of pro-apoptotic factors� including cytochrome c� into the cytosol. Cy- tochrome c interacts with Apaf-� �apoptotic activating factor-��� ATP� and procaspase-� to form a complex known as the apoptosome which in turn activates caspase-� and further activates the executioner cas- pase-�� -�� and -�� generating a variety of molecular damages in essentially every organelle. In terms of cell survival� however� it is the damage to DNA leading to the loss of proliferative and clonogenic capac- ity that is most important. Although chemotherapy and radiotherapy-induced apoptosis is a caspase- dependent process [�5��8]� it is unclear how cel- lular signals from DNA lesions lead to the execution of apoptosis. �ince Bcl-� is a�le to �lock cytochrome c release and prevent apoptosis� it has �een proposed that anticancer therapy-induced caspase activation is mitochondria dependent [��� �����]. This concept Received: June 25, 2012 *Correspondence: Phone: +31 20 512-21-20 Fax: +31 20 669-11-01 E-mail: m.verheij@nki.nl Abbreviations used: 99mTc – 99mTechnetium; Apaf-1 – apoptotic activating factor-1; BTAP – 4,5-bis(thioacetamido)pentanoyl; CT – computed tomography; DD – death domain; DED – death effector domain; DISC – death-inducing signaling complex; DR – death receptor; FADD – Fas-associated death domain; FasL – Fas ligand; HL – Hodgkin lymphoma; HNSCC – head and neck squamous cell carcinoma; HYNIC – hydrazinonicotinamide; MDR1 – multidrug resistance gene 1; MIBI – methoxyisobutyliso- nitrile; NHL – non-Hodgkin lymphoma; NSCLC – non-small cell lung cancer; PARA – pro-apoptotic receptor agonist; PARP-1 – poly-ADP-ribose polymerase 1; PET – positron emission tomog- raphy; PS – phosphatidylserine; SCLC – small cell lung cancer; SPECT – single photon emission computerized tomography; TAVS – 99mTc-annexin V scintigraphy; TNF-a – tumor necrosis factor a; TNFR1 – TNF receptor 1; TRADD – TNF-receptor-as- sociated death domain; TRAIL – TNF-related apoptosis-inducing ligand; TUNEL – terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling. Exp Oncol ���� ��� �� ������� INVITED REVIEW ��� Experimental Oncology ��� �������� ���� ��eptem�er� is further su�stantiated �y the o�servation that activa- tion of p5� �y anticancer therapy due to DNA damage is a direct transcriptional regulator of Bcl-�� Bax� Puma� Noxa and Bid and can act as an apoptogenic factor at the mitochondrial mem�rane [����5]. Furthermore� there are functional connections �etween p5� and death receptors genes �CD�5 and TRAIL receptor-�� that can �e upregulated in response to therapy [��� ��]� in turn leading to the activation of inducer cas- pase-8. It has also �een shown that pretreatment of cells with DNA damaging agents improves the ca- pacity of TRAIL-�ound receptors to recruit FADD and activate caspase-8 and -�� in the DI�C� irrespective of p5� status [�8]. As apoptosis has �een recognized as a major form of cell death after anticancer therapy� it is �eing increasingly evaluated as a prognostic marker of treat- ment outcome. For this purpose� a noninvasive method to analyze treatment-induced apoptosis is most at- tractive� as it can �e used to determine and predict the effectiveness of an anticancer regime. In this review� we will discuss apoptosis imaging modalities in �oth animal models and patients using annexin V� detection of apoptotic mem�rane imprint� methoxyiso�utylisoni- trile �MIBI�� and the novel caspase-� small-molecule inhi�itor� Isatin� and address their a�ility to improve patient treatment �see Fig. ��. Annexin V scintigraphy� an imaging technique for which we have the most clini- cal experience with� will especially �e detailed in this discussion. IMAGING OF APOPTOSIS USING ANNEXIN V Annexin V-�ased tracers are the most frequently used agents for in vitro detection and quantification of apoptotic cells. This is �ased on the high affinity of annexin V for the mem�rane �ound phospholipid �P��� which in via�le cells� resides in the inner leaflet of the plasma mem�rane. Upon exposure of cells to apoptotic stimuli g-scram�lase is activated result- ing in P� flipping to the outer leaflet of the plasma mem�rane� there�y allowing annexin V to �ind to P�. In addition� it has �een shown that mem- �rane �inding of proteins that recognize exposed P� on apoptotic cells is regulated �y the transmem- �rane potential [��]. A decreasing mem�rane potential in Jurkat T leukemia cells and K5�� promyelocytic leukemia cells undergoing apoptosis increases the extracellular �inding of annexin V in a dose-dependent manner. �tudies with P� vesicles also showed that the mem�rane potential increases the �inding affinity of annexin V for the P� cell surface molecules. Single photon emission computed tomography (SPECT) ��mTechnetium-linked annexin V has �een exten- sively used in apoptosis detection in patients� exploit- ing its optimal radionuclidic properties for �PECT Fig. 1. Imaging of apoptosis �y various radiotracers. While annexin V �ased compounds and Apo�ense agents work at the level of the cell plasma mem�rane� MIBI acts at the mitochondria and Isatin targets executioner caspase � �DR=Death Receptor; PARA=Pro-Apoptotic Receptor Agonist�. Inserts are examples of images acquired �y the indicated modalities. Modified from: Haimovitz-Friedman et al.� ���� [��] Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��� imaging� relative low costs and easy availa�ility [��]. In ���5� �tratton et al. [��] were the first to demon- strate ��mTc-annexin V’s value for the in vivo detection of mem�rane-associated P� exposure using �PECT �y injecting ��mTc-la�eled human annexin V intrave- nously and calculating the atrial throm�us/�lood ratio in throm�o-em�olic diseases. Other �PECT studies using ��mTc-annexin V derivatives provided feasi�i- lity and potential clinical utility of apoptosis imaging in various other medical disorders [��]. Multiple con- jugators of annexin V have �een developed� including ��5-�is�thioacetamido�pentanoyl �BTAP� for its rapid and extensive radioactivity accumulation in the gastro- intestinal tract. �ince this tracer was mainly excreted �y the liver and the kidneys� resulting in an increased radionuclide accumulation in these organs as well [��]� the role of ��mTc-BTAP annexin V for a�dominal imaging was limited. Blanken�erg et al. [��] su�sequently coupled annexin V to hydrazinonicotinamide and created ��mTc-HYNIC annexin V. In vivo studies using a CD�5L- induced hepatocyte apoptosis mouse model with intravenously administered anti-CD�5 anti�ody dem- onstrated that ��mTc-HYNIC annexin V can �e used to image apoptotic �and necrotic� cell death in vivo. Although the concentration of tracer in liver and kidneys were still high� and similar to ��mTc-BTAP an- nexin V� radioactivity accumulation in the �owel was eradicated� making it a suita�le candidate tracer for a�dominal examination [�5]. Following further optimization of image quality� ��mTc-annexin V proves to �e an effective modality for non-invasive evalua- tion of cell death and treatment response in allograft rejection� myocardial infarction� reperfusion injury and infectious disease [����8]. 99mTc-annexin V studies in combination with anticancer therapy The first in vivo demonstration of anticancer therapy- induced apoptosis involved the use of ��mTc-annexin V in an experimental mouse lymphoma model treated with cyclophosphamide. The animals treated with che- motherapy demonstrated a more than ���% increase in annexin V uptake �� h after treatment compared to untreated animals [��]. More recently� the value of ��mTc-annexin V imaging in response monitoring was evaluated in a mouse model for hereditary �reast cancer after docetaxel treatment [��]. The sensitive tumors showed an increase in ��mTc-annexin V uptake and immunohistochemical evidence of apoptosis one day post-treatment. On the other hand� resistant tumors showed neither an increase in ��mTc-annexin V uptake nor significant immunohistochemical changes �Fig. ��. Despite these encouraging findings� ��mTc-annexin V ima ging could not �e used to predict tumor response� due to large variations in uptake �etween animals. In addition to animal studies� annexin V imaging has also �een applied in various clinical protocols. In ����� ��mTc-annexin V was first used in clinical trials with pa- tients scheduled to receive chemotherapy for locally advanced or metastatic non-small cell lung cancer �N�CLC�� small cell lung cancer ��CLC�� Hodgkin �HL� and non-Hodgkin �NHL� lymphoma� and �reast cancer. Fifteen patients underwent ��mTc-annexin V scintigra- phy �efore and within � days after their first course of chemotherapy. Patients with lung cancer received platinum-�ased chemotherapy� lymphoma patients were treated with vincristine or cyclophosphamide- �ased chemotherapy and �reast cancer patients received taxane as their chemotherapy regime. Five patients had increased annexin V uptake ����8 hours after chemotherapy �� NHL� � HL� � �CLC� and � N�CLC�� and � patients showed increased uptake ����� hours after treatment �� N�CLC� � �CLC�. At the median follow-up of ��� days� while patients with no change in radiotracer uptake after the first cycle of chemotherapy had no su�sequent o�jective clinical response� patients demonstrating increased tracer uptake post-treatment had either a partial or complete tumor response. From these results� it was concluded that ��mTc-annexin V could �e used clini- cally for in vivo imaging of apoptosis after one course of chemotherapy [��]. a b T*23 sensitive tumor T*23 resistant tumor Fig. 2. Preclinical imaging of apoptosis. Quantitative whole ani- mal ��mTc-annexin V �PECT imaging and histology of a T*�� sen- sitive �a� and resistant tumor �b�� o�tained �efore and � day after docetaxel treatment. Right panel: tumors were stained for TUNEL at day �. Modified from: Beekman et al.� ���� [��] One of the first reports on the application of ��mTc- annexin V in patients receiving radiotherapy was �y Haas et al. [��] who applied ��mTc-annexin V scin- tigraphy �TAV�� to monitor radiation-induced apoptotic cell death in �� follicular lymphoma patients �Fig. ��. All patients underwent a �aseline scan within one week prior to the start of radiotherapy to detect �aseline levels of spontaneous tumor apoptosis or necrosis. Pa- tients were then irradiated to the involved lymph node areas to a total dose of � Gy in � fractions �8 h apart. At �� h after the second radiation fraction� TAV� was repeated. Fine needle aspiration cytologic analysis for apoptosis was also performed prior to� during and after irradiation. In �� patients� post-treatment TAV� ��� Experimental Oncology ��� �������� ���� ��eptem�er� matched the post-treatment cytology� confir ming TAV� as a valua�le non-invasive method to detect in vivo apoptosis caused �y radiation. In addition� the increase in ��mTc-annexin V uptake post-treatment in this type of malignancy correlated with clinical outcome: all patients with prominent cytologic and scintigraphic signs of apoptosis achieved complete remission within � week. In ����� Kartachova et al. [��] from the Nether- lands Cancer Institute conducted a study of �� patients with malignant lymphoma� leukemia� N�CLC� and head and neck squamous cell carcinoma �HN�CC� sched- uled for radiotherapy� platinum-�ased chemotherapy� or concurrent chemoradiation �see Fig. ��. Fig. 3. Typical examples of anticancer therapy-induced apop- tosis as demonstrated �y in vivo annexin V scintigraphy. �hown are � examples of �PECT �efore �left panel� and early during treatment �i.e. ����8 h after start of therapy; right panel�. Upper panel: NHL treated �y low dose ��x� Gy� involved-field radiation. Middle panel: HN�CC treated �y cisplatin-�ased chemora- diation. Lower panel: N�CLC treated �y cisplatin/gemcita�ine chemotherapy. Arrows indicate the target lesions. Note the physi- ologic uptake in �ones and salivary glands �Modified from: Haas et al.� ���� [��] and Kartachova et al.� ���� [��]� Kartachova et al.� ���� [��]� and Verheij et al.� ���8 [��] The investigators demonstrated increased ��mTc- annexin V accumulation in lesions early during treat- ment when compared to �aseline values in patients with complete or partial tumor remission� while there was no significant early increase in uptake in those pa- tients with sta�le or progressive disease. This study es- ta�lished ��mTc-annexin V scintigraphy as a predictive marker in tumor response. A su�sequent study from these authors evaluated the predictive value of TAV� in �� chemotherapy-naive patients with advanced stage N�CLC undergoing platinum-�ased chemo- therapy. Also under these conditions� a significant correlation �etween annexin V changes and treatment outcome was found [��]. In a ���8 update of their study� Kartachova et al. [�5] showed that visual evalu- ation of �PECT images� �PECT/�PECT and �PECT/ CT co-registered images correlated with quantitative analysis. Using �oth methods� all patients with early post-treatment increase in tumor uptake of ��mTc-an- nexin V either developed complete response or partial response� resulting in statistically highly significant correlations �etween changes of ��mTc-annexin V tu- mor uptake and therapeutic outcome for �oth visual and quantitative analysis. In addition to the monitoring of treatment-induced apoptosis in tumor cells� TAV� may also �e used to detect normal tissue toxicity. Hoe�ers et al. [��] applied ��mTc-annexin V scintigraphy to demonstrate apoptosis in patients with HN�CC� �oth in tumor and normal tissue. TAV� was performed �efore and within �8 h after the first course of cisplatin-�ased chemoradiation in �� patients. Already after a dose of ��8 Gy increased annexin V uptake was o�served in �� of the �� irradiated parotid glands. Glands� which received higher radiation dosages� showed more an- nexin V uptake. The authors concluded that within the dose range of ��8 Gy� TAV� showed a radiation- dose-dependent uptake in parotid glands� indicative of radiation-induced apoptosis. A similar pattern in the su�mandi�ular glands was o�served. Limitations of annexin V imaging Although TAV� appears very promising as an early predictor for tumor response to anticancer therapy� several limitations remain to firmly esta�lish its value as an imaging �iomarker of response. First of all� apoptosis is an acute event and contri�utes to early therapy-induced tumor shrinkage. Therefore� TAV� may �e less suita�le to predict long-term response to treatment [�����]. To determine whether this type of early cell death predicts long-term treatment out- come parameters in patients� such as disease-free survival and overall survival� changes in TAV� uptake should �e correlated with tumor response measure- ments after sufficiently long follow-up. �econdly� an- nexin V �inding to P� does not discriminate �etween apoptotic and necrotic cell death as disrupted plasma mem�ranes also make P� accessi�le at the inner leaflet of cells undergoing necrosis. Thirdly� in terms of �iodistri�ution� annexin V has a relatively slow clear- ing rate from non-targeted tissues� therefore creating a low signal-to-noise ratio [5�]. Fourthly� the optimal timing of apoptosis-imaging in vivo that yields most predictive information in terms of tumor response remains uncertain and should ideally �e determined for each specific tumor type and treatment moda- lity. Multiple TAV� measurements may �e necessary to define this tumor- and/or therapy-specific optimal timing. Finally� apoptosis represents only one aspect of the complex �iological response to therapy� and its relative contri�ution varies among different tumor entities. More studies are needed to demonstrate the applica�ility of TAV�� especially in �solid� tumors that are therapy-resistant. Com�ining TAV� with other anatomical and functional imaging modalities may �e helpful in o�taining a more complete� and perhaps more solid �iomarker. OTHER RADIOTRACERS IN APOPTOSIS IMAGING Detection of apoptotic membrane imprint The apoptotic mem�rane imprint is a complex of cel- lular changes occurring in the plasma mem�rane early during the apoptotic process. These include irreversi�le loss of mem�rane potential� permanent acidification of the external plasma mem�rane leaflet and cytosol� and activation of g-scram�lase while preserving the Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��� integrity of the plasma mem�rane. A set of novel small- molecule pro�es designated the Aposense compounds �Aposense Ltd.� Petach-Tikva� Israel� have �een devel- oped to detect these apoptosis-related plasma mem- �rane alterations �see Fig. � [5�]�. This family of small molecules �DDC� ML-��� ML-�� N�T-���� and N�T- ���� have demonstrated activities in a num�er of tumor models in response to anticancer agents [5��5�]� however there is no clear mechanism of uptake of these compounds. The positron emission tomography �PET� tracer �8F-ML-�� shows selective uptake �y apoptotic cells in tumors following radio- and chemotherapy� cor- relating with �reakdown of mitochondrial mem�rane potential� caspase activation and DNA degradation. As the signal is lost upon rupture of the plasma mem- �rane� �8F-ML-�� should �e capa�le of discriminating apoptotic from necrotic cells [5�]. Recently� the role of �8F-ML-�� was evaluated in the early detection of response of �rain metasta- ses to whole �rain irradiation ���x� Gy�. In this study �� patients underwent an �8F-ML-�� PET scan prior to treatment and a second scan after � or �� fractions of radiation. MRI was performed ��8 weeks after completion of treatment. In all �� patients� �oth MRI and the �8F-ML-�� PET scan detected all �rain lesions. A highly significant correlation was found �etween early changes on the �8F-ML-�� scan and later changes in tumor anatomical dimensions on MRI [55]. Methoxyisobutylisonitrile (MIBI) Technetium-��m methoxyiso�utylisonitrile ���mTc- MIBI� is a lipophilic cation isonitrile compound that crosses the cell mem�rane due to the negative trans- mem�rane potential and accumulates in mitochondria [5�� 5�]. Because of their higher meta�olic activity� tumor cells show large differences in mitochondrial mem�rane potential and high num�ers of mitochon- dria� making ��mTc-MIBI an attractive agent in tumor imaging �see Fig. ��. Indeed� studies have demon- strated that good responders of anti-tumor treatment display more MIBI accumulation as compared to poor responders in various tumor types� including small cell and non-small cell lung cancer [58���]� �reast cancer [�5]� malignant lymphoma [��]� osteosarcoma [��]� and nasopharyngeal carcinoma [�8]. One explanation for this phenomenon could �e the overexpression of the multidrug resistance gene �MDR��� which en- codes P-glycoprotein �Pgp�� a transmem�rane protein that acts as an efflux pump to a wide range of cyto- toxic drugs and ��mTc-MIBI [����5]� in chemotherapy resistant tumors. Another possi�ility is the inhi�ition of mitochondrial mem�rane permea�ility as a result of the overexpression of anti-apoptotic proteins such as Bcl-� [��� ��]. More recently� studies have esta�lished a correlation �etween ��mTc-MIBI tumor cell uptake and apoptosis after irradiation or che- motherapy [�8� ��]. In an unpu�lished study� Del Vecchio et al. [��] demonstrated that the overex- pression of Bcl-� which prevented ��mTc-MIBI uptake in untreated �reast carcinoma could �e counteracted �y the initiated drug therapy� allowing a transient in- crease in ��mTc-MIBI accumulation. The authors specu- lated that altered expression of proteins participating in the apoptotic process would affect mitochondrial permea�ility and transmem�rane potential� leading to a change in intracellular ��mTc-MIBI accumulation �oth prior to and after initiation of therapy. Further- more� high levels of early ��mTc-MIBI uptake after treatment [8�]� could potentially indicate therapeutic efficacy. These results suggest that ��mTc-MIBI scin- tigraphy has important clinical implications in pro- viding prognostic information prior to treatment and also in monitoring effectiveness of treatment after initiation of anticancer therapy. Caspase-3 tracers �everal groups have developed novel PET pro�es designed to non-invasively image caspase-� activa- tion� including the small-molecule caspase inhi�itor� Isatin� used with different radioisotopes �see Fig. ��. �everal of these ��C or �8F Isatins examined in vivo [8��8�] demonstrated high affinity to caspase-� and high uptake in the liver in response to cycloheximide or anti-CD�5 anti�ody� confirmed �y immunohistol- ogy of cell death. Although the radiola�eled Isatins appear to �ind specifically to caspase-�� their sen- sitivity is limi ted� indicating that further optimization is required for clinical application of these tracers. Coppola et al. [88] generated sta�le cell lines transfected with a hy�rid luciferase reported construct� which is activated �y caspase-� cleavage. Using this system� the apoptotic response of D5� glioma xenograft tumors to temozolomide and radiation was monitored �y detecting �ioluminescence emission. Apoptosis was detected within � h after � Gy of ra- diation in mice receiving the com�ination treatment. Although this method appeared to �e very sensitive and quantitative� the experience seemed to �e lim- ited to several animal models [85� 8�]. No pu�lished patient data are availa�le thus far. In addition� it has recently �een found that caspase-� activation is not unique to apoptosis [��� ��] and is involved in other physiological processes such as platelet aggregation and secretion of enzymes from pancreatic acinar cells. CONCLUDING REMARKS This review focuses on in vivo apoptosis imag- ing as a clinical �iomarker of response to treatment. In evaluating radiotracers for apoptosis imaging� there are a num�er of issues to consider. First� the tumor is heterogeneous and consists of a mixture of different cell types and no tracer discussed displays sufficient specificity to identify su�populations of tumor cells undergoing primary apoptosis. 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