Imaging the Molecular Signatures of Apoptosis and
Injury with Radiolabeled Annexin V
Francis G. Blankenberg1
1Department of Radiology/Division of Pediatric Radiology, Lucile Salter Packard Children’s Hospital, Stanford, California
Annexin V is a ubiquitous intracellular protein in humans that has
avarietyof intriguing characteristics,includingananomolaraffinity
for the membrane-bound constitutive anionic phospholipid known
of apoptotic or physiologically stressed cells. As such, radiolabeled
forms of annexin V have been used in both animal models and
human Phase I and Phase II trials to determine if this tracer can be
employed as an early surrogate marker of therapeutic efficacy in
NSCLC and non-Hodgkin’s lymphoma. Many other pulmonary
imaging applications of radiolabeled annexin V are also possible,
including the detection and monitoring of active pulmonary in-
flammation and other pathophysiologic stressors in a variety of
diseases. In this article, the salient molecular features of apoptosis
(and other forms of cell death) that permits imaging with radio-
labeled annexin V will be discussed. The latest results from Phase II
the imaging of other pulmonary pathologies will be outlined.
Keywords: apoptosis; SPECT; annexin V; imaging, pulmonary
Despite over a decade of intense investigation, there is still no
fully validated method for the imaging of apoptosis (irreversible
injury) or physiologic cellular stress (potentially reversible
injury) in humans. There have been multiple tracers proposed,
but none as of yet has received FDA approval. Radiolabeled
annexin V is one of the few radiotracers that has been widely
used in Phase II trials in NSCLC and is still under development.
In this article we will first outline the signaling pathways of
apoptotic cell death that form the basis of radiolabeled annexin
V imaging. Then the imaging applications of radiolabeled
annexin V in animal models and clinical studies of several
common cardiopulmonary pathologies will be examined. Lastly,
the possible future directions of radiolabeled annexin V imaging
will be described.
THE INTRINSIC AND EXTRINSIC PATHWAYS OF
APOPTOTIC CELL DEATH
Apoptosis, or type I cell death, is the organized, energy-
dependent self disassembly of unneeded or senescent cells (1,
2). When triggered by appropriate internal and/or external
signals, these cells undergo pre-programmed cytoplasmic
shrinkage, membrane blebbing, and budding off of intracellular
called ‘‘apoptotic bodies.’’ Apoptotic bodies are subsequently
ingested by adjacent cells and phagocytes without provoking an
Before these morphologic changes there is an initiation
sequence called the ‘‘lag or trigger phase’’ (3, 4). There are many
triggers of apoptosis, such as withdrawal of growth factors,
radiation, chemotherapy, and ischemic injury. The lag time
between exposure to the trigger(s) and the time of observable
morphologic signs of apoptosis is highly variable depending
heavily on cell type, type of trigger(s), its intensity and duration,
as well as the local environmental conditions of the cell. Most
apoptotic pathways, however, converge on a family of cysteine
aspartate–specific proteases known as the ‘‘caspases’’ (5). When
activated, each caspase, whether as an initiator (8–10) or execu-
tioner (3, 6, 7), cross-links and cleaves specific intracellular
proteins involved with apoptosis.
Activation of the executioner set of caspases occurs via the
extrinsic (i.e., Type I Apoptosis, Figure 1 in red) and/or intrinsic
(i.e., Type II Apoptosis, Figure 1 in blue) pathways of apoptosis.
Type I and II caspase-dependent apoptosis are categorized more
generally as Type I cell death (as opposed to Type II cell death,
known as ‘‘autophagy’’ and defined later in this article). The
extrinsic pathway is mediated by death receptors that bind
specific molecules, including tumor necrosis factor (TNF) that
binds to the TNF receptor (TNFR), TNF-related apoptosis-
inducing ligand (TRAIL) that binds to the DR4 and DR5 death
receptors, or Fas ligand (FasL) that binds to the Fas receptor.
After binding to a given receptor, adapter molecules such as Fas-
associated death domain (FADD) or tumor-associated death
domain (TRADD) are recruited from the cytoplasm along with
caspase-8 to form what is known as death-inducing signaling
DISC propagates the ‘‘extrinsic’’ death signal by proteolytic
activation of caspase 10 that in turns cleaves and activates the
downstream executioner caspases. The final enzyme activated is
caspase-3. Activated caspase-3 then travels to the nucleus and in
turn activates poly-ADP-ribose polymerase (PARP-1), an en-
zyme that facilitates the degradation of nuclear DNA into 50- to
by gel electrophoresis). Spliced or fragmented DNA can also be
readily detected in situ and in vitro by the application of terminal
deoxynucleotidyl transferase-mediated dUTP-biotin nick end
labeling (TUNEL)-based immunohistochemical or fluorescent
methods. Activated caspase-3 also triggers other processes that
orderly breakdown and self packaging of cellular proteins, in-
cluding the cytoskeleton, and nuclear matrix (chromatin clump-
ing and condensation that can be seen by bright light or fluo-
Alternatively, DISC can provoke the translocation of trun-
cated Bcl interacting domain (BID), a pro-apoptotic Bcl 2 family
protein, to mitochondria. BID induces the oligomerization of the
pro-apoptotic proteins, BAK and BAX (Bcl-2 antagonist/Killer
required to form holes/channels in the outer mitochondrial
membrane inaprocess knownasmitochondrialouter membrane
permeabilization (MOMP). These channels permit the escape of
intermembrane space to the cytoplasm. The release of cyto-
chrome c into the cytoplasm is the hallmark of the intrinsic
(Received in original form January 22, 2009; accepted in final form May 22, 2009)
Supported in part by NIH Grant # EB000898 (to F.G.B.).
Correspondence and requests for reprints should be addressed to Francis Gerard
Blankenberg, M.D., 725 Welch Road, Palo Alto, CA 94304. E-mail: blankenb@
Proc Am Thorac Soc
Internet address: www.atsjournals.org
Vol 6. pp 469–476, 2009
loss of the normally high negative mitochondrial membrane
factor-1 (Apaf-1), ATP, and pro-caspase 9 to form a structure
known as the apoptosome. The apoptosome then cleaves and
activates caspase9,whichinturnleadstothe activation of caspase
3, 6, and 7. Activation of caspase 9 without involvement of the
apoptosome has also been described (6).
After caspase-3 activation, the terminal step of caspase-
dependent apoptosis, there is a rapid redistribution and expo-
sure of the anionic phospholipid phosphatidylserine (PS) on the
cell surface (7, 8) (Figure 2). PS is normally restricted to the
inner surface (inner leaflet) of the lipid bilayer by an ATP-
dependent enzyme called ‘‘flippase (translocase).’’ Flippase, in
concert with a second ATP-dependent enzyme, ‘‘floppase,’’ that
pumps cationic phospholipids such as phosphatidylcholine (PC)
pathways of apoptosis.
Extrinsic and intrinsic
phatidylserine exposure with apo-
ptosis. Reprinted by permission
from Reference 72.
Mechanisms of phos-
470 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETYVOL 62009
and sphingomyelin to the cell surface, maintains an asymmetric
distribution of different phospholipids between the inner and
outer leaflets of the plasma membrane (9). The rapid redistri-
bution of across the cell membrane (measured in minutes) is
facilitated by a calcium-dependent deactivation of flippase and
the activation of a third enzyme called ‘‘scramblase.’’ It is this
selective exposure of PS that forms the basis of annexin V
binding to apoptotic cells both in vitro and in vivo. In fact, the
most widely used in vitro assay for apoptotic cells involves the
use of fluorescent or biotinylated annexin V.
Annexin V is a ubiquitous intracellular human protein (MW ?
36,000, 319 amino-acid residues) that has a nanomolar affinity
for membrane bound PS (10). The protein is folded into a planar
cyclic arrangement of four repeats, with each repeat composed of
five a-helical segments. The binding to membrane-bound PS is
quite complex (beyond the scope of this article), and may involve
the binding of eight PS molecules per molecule annexin V at low
levels of membrane occupancy (with respect to protein) (11). The
function(s) of annexin V, despite its widespread intracellular
distribution are still unknown but a variety of in vitro and in vivo
properties have been described (12, 13).
OTHER SIGNALING PATHWAYS THAT CAN INDUCE
APOPTOTIC CELL DEATH
Under certain circumstances the inner mitochondrial mem-
brane can also be permeabilized along with MOMP, releasing
other proteins within the intercristal space including apoptosis-
inducing factor (AIF), Omi, and EndoG (14) (Figure 3 in
black). These factors are then translocated to the nucleus and,
once there, induce the autodigestion of a cell’s DNA, resulting
in a noncaspase form of apoptosis.
The endoplasmic reticulum (ER) can also trigger apoptosis
(i.e., ER stress–induced cell death) (15) (Figure 3 in green).
Normally, the ER is the site of secretory protein synthesis,
conformational maturation, and quality control for correctly
folded proteins. Proteins failing to adopt a stable conformation
are dislocated into the cytosol, where they are targeted for
ubiquitylation (a tag to identify a protein for elimination) and
proteosomal degradation. Certain conditions and/or drugs can
lead to the abnormal accumulation of unfolded proteins,
resulting in ER stress. During ER stress, cells can re-achieve
homeostasis by initiating a series of orchestrated events known
as the unfolded protein response (UPR). If unsuccessful, ER
stress can directly initiate a specific ubiquitin E3 ligase that tags
anti-apoptotic Bcl-2 family members with ubiquitin. Subse-
quently, the proteosome degrades these anti-apoptotic mole-
cules, thereby tipping the balance between pro- and anti-
apoptotic factors toward apoptosis. Subsequently, mitochondria
are activated, and pro-apoptotic factors such as cytochrome c,
AIF, Omi, and others are released. As a result, both caspase-
dependent and -independent signaling cascades can be activated
and eventually lead to apoptotic cell death.
AUTOPHAGY AND ITS RELATIONSHIP(S) WITH
APOPTOTIC CELL DEATH
While apoptotic cells externalize PS, other forms of cell death
can also demonstrate this same feature, including necrosis/
PARP-1–mediated cell death, and autophagy (16). Autophagy
(Type II cell death as opposed to apoptosis), otherwise known
as ‘‘self-eating,’’ is a highly regulated form of cell death in
a fashion that has considerable overlap with apoptosis (17)
(Figure 4 in light blue). Autophagy is initiated by derepression
of mTOR Ser/Thr kinase (mammalian target of rapamycin)
quickly followed by the formation of the Beclin-1–class III
phosphatidylinositol 3-kinase complex. This complex mediates
the formation of isolation membranes that engulf targeted
cytoplasmic material (or organelles) resulting in double-mem-
braned vesicles called autophagosomes (autophagic vacuoles).
Bcl-2 and Bcl-XL are regulators of beclin-1 and can be a link
with apoptotic (Type I) cell death (18). Vesicle elongation
requires the conjugation of phosphatidylethanolamine (PE) to
LC3. Lipid conjugation leads to the conversion of the soluble
form of LC3 (named LC3-I) to the autophagic vesicle–associ-
ated form (LC3-II). LC3-II is used as a marker of autophagy.
Autophagosomes then undergo maturation by fusion with
lysosomes to create autolysosomes. In the autolysosome, the
inner membrane and the luminal contents of the autophagic
vacuole are degraded without inciting inflammation.
As opposed to apoptosis, autophagy normally serves a house-
keeping function by removing unneeded, senescent, or damaged
cytoplasmic contents. It also permits a cell to survive periods of
cellular famine through the autodigestion of intracellular DNA/
RNA, proteins, and lipids into free nucleotides, amino acids,
and fatty acids, respectively. These free nucleotides, amino
acids, and fatty acids can then be reused by a cell to maintain
vital functions, such as macromolecular synthesis and energy
production. Autophagic cell death, however, can be analterna-
tive to apoptosis if the classical apoptotic mechanisms are
damaged or are inhibited. There can also be a massive induction
of autophagy to such an extent that a cell can literally eat itself
to death. In this circumstance PS is again exposed on the cell
surface or the cell particles (autophagic vesicles) and can
therefore be detected by radiolabeled annexin V.
A related form of cell death involves lysosomal stress
(Figure 4 in pink) followed by LMP (lysosomal membrane
permeabilization) and cathepsin releases that causes general-
ized proteolysis. Details of when and how this process occurs
are still poorly understood, but again result in the accessibility
of PS to extracellular radiolabeled annexin V.
PARP-1–MEDIATED CELL DEATH
PARP-1 normally functions as a DNA damage sensor, and its
activation serves to repair low levels of DNA damage (19). With
high levels of DNA damage, however, PARP-1 activation
promotes cell death by consuming all available stores of NAD1,
its primary substrate. As NAD1can only be regenerated by
cleavage of ATP, the cell literally runs out of energy and dies.
Once the cell can no longer maintain its ATP-dependent
membrane functions, it swells, resulting in necrosis/oncosis
(i.e., irreversible membrane injury). With the loss of plasma
membrane integrity, PS becomes accessible to relatively large
impermeable molecules such annexin V and therefore detect-
able by radiolabeled annexin V imaging. Similarly necrotic cell
death (by any noxious stimuli), a process characterized by the
primary irreversible loss of membrane integrity, can also be
detected by radiolabeled annexin V.
PS EXPOSURE AND THE COMMITMENT TO APOPTOSIS
While the set of events outlined above are believed to be largely
correct, Balasubramanian and coworkers (20) have recently
found that PS externalization can be reversibly induced in
a process independent of cytochrome c release, caspase activa-
tion, or DNA fragmentation. Reversible PS externalization,
however, does require a sustained elevation in cytosolic ionized
calcium; an event that can be inhibited by calcium channel
blockers in vitro. PS exposure, whether related to apoptosis
(irreversible) or associated with reversible cellular events
Blankenberg: Annexin V Imaging of Apoptosis471
(physiologic stress), has also been found to be necessarily
preceded by cell shrinkage and increased lipid mobility (de-
creased packing) (21).
Reversible PS externalization demonstrates far lower levels
of PS exposure as compared with apoptosis and other forms of
cell death (22, 23). The relatively low levels of PS exposure
observed with reversible PS externalization can be readily
counteracted by the prompt removal the offending physiologic
stressor such as nitric-oxide, p53 activation, allergic mediators,
or growth factor deprivation. However, if the stress remains
uncorrected, a cell may undergo apoptosis.
Reversible uptake of radiolabeled annexin V has not only
been observed in vitro but also in human models of forearm
muscle exercised induced ischemia (24–26). The ability of
annexin V to bind to cells with low but potentially reversible
autophagy (Type II cell death).
Major steps involved in
Figure 3. Alternate pathways of pro-
grammed cell death.
472 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 62009
annexin V outside regions of apoptosis (as seen histologically by
TUNEL staining) in patients with hypoxic-ischemic reperfusion
injury in the heart (27, 28) or brain (29–31).
In fact, even in regions with apoptosis/ischemic injury, there
are far more annexin V–positive cells after the administration of
radiolabeled annexin Vthan apoptotic nuclei as seen byTUNEL
staining (28). These observations suggest that much (if not most)
to large numbers of stressed cells (not necessarily committed to
apoptosis) with relatively low levels of PS expression in contrast
to the relatively fewer cells with high levels of PS exposure that
are irreversibly committed to apoptosis.
The ability of radiolabeled annexin V to bind to ‘‘stressed’’
cells with relatively low levels of PS exposure also implies that
annexin V imaging maybe far more sensitive than what would
be expected if the tracer only localized to the relatively few
apoptotic cells observed histologically. The ability of annexin V
to localize cells that are stressed but not necessarily committed
to apoptotic cell death suggests that this radiotracer can be used
to identify tissues or organs at risk for irreversible injury, such
as seen in hypoxic-ischemic injury (32) or chronic heart failure
(33), or sites of active disease that are seen in infection (34–36),
unstable atherosclerotic plaques (37), allograft rejection (38), or
autoimmune disorders (39, 40).
Annexin V imaging could therefore be useful in the serial
assessment of acute and chronic disorders in organs or tissues at
risk for permanent damage in which prompt treatment with
effective drug or surgical intervention may prevent irreversible
cellular injury and cell death.
RADIOLABELED FORMS OF ANNEXIN V FOR
In an alternate clinical paradigm, annexin V imaging can also be
used to noninvasively assess chemotherapeutic and radiation
efficacy, treatments in which the induction of tumor cell
apoptosis/necrosis is the major goal of therapy.
The first human trials with annexin V were conducted in
patients with primary and metastatic lung tumors using human
rh-annexin V labeled with
99mTc-pertechnetate by the pen-
thioate radioligand (N2S2) method (41). This method begins
with the chelation of
99mTc in the presence of stannous
gluconate to yield99mTc-gluconate, which is then reacted with
acidified phenthioate ligand under heating to form a stable
99mTc–N2S2 complex. The99mTc–N2S2-TFP ester complex is
then randomly conjugated to the N-H groups of lysine of the
protein at basic pH. Despite this cumbersome labeling method
and nonspecific excretion of radiolabeled ligand into bile,
Belhocine and colleagues (42) successfully conducted a study
of 15 patients with cancer with pulmonary disease before and
after chemotherapy. In this trial, a negative annexin V study
after therapy (i.e., no change in tumor uptake of tracer from
pretreatment baseline SPECT scans) correlated well with a lack
of treatment response in six of eight patients. The remaining
two patients (with metastatic breast cancer) actually had
a clinically significant response to Taxol-based chemotherapy.
All seven patients with increased tumor uptake over baseline
(positive annexin V study), however, had an objective tumor
response (shrinkage of tumor). Five of these patients showed
increased annexin V uptake at the 40–48th hour after chemo-
therapy (1 NHL, 1HL, 1 SCLC, and 2 NSCLC), and two
patients had increases in annexin V uptake observed at the
20–24th hour after treatment (1 NSCLC and 1 SCLC). To-
gether, these preliminary results suggested a significant vari-
ability of the optimal timing with regard to the cancer type and
therapeutic regimen (43).
An improved labeling method based on the bifunctional
agent hydrazino nicotinamide (HYNIC) was selected for fur-
ther clinical trials (44). Similar to the penthioate radioligand
(N2S2method, i.e., BTAP-Anx V kit),99mTc-HYNIC–annexin
V showed the greatest uptake in the kidneys, liver, and urinary
bladder but demonstrated no biliary excretion (45). Multiple
trials have confirmed the potential clinical utility of HYNIC–
annexin V in determining the efficacy of chemotherapy (46–49).
Figure 5. Early detection of tumor response
in non–small cell lung cancer (NSCLC) with
radiolabeled Annexin V. A 70-year-old man
with stage IV NSCLC presenting with a new
right humeral metastasis as demonstrated on
bone scan (black arrow). 99mTc-HYNIC-
Annexin V assessment (in the coronal plane)
metastasis 2 hours before the first course of
chemotherapy (yellow arrows), which signifi-
cantly increased ‘‘1 day’’ after the start of
treatment; in addition, increased tracer up-
take was detected within the primary lung
tumor as early as 1 day after the initiation of
cisplatinum-based chemotherapy (black ar-
site of left arm. Chest computed tomography
(axial or transverse plane) showed a response
to treatment 1 month after treatment with
a decrease of the greatest tumor diameter
from 7.5 cm to 6.5 cm. This patient was still
alive after 126 days. This clinical case, which
was part of a Phase II/III clinical trial (NAS
2021, Middelheim Hospital, Antwerp, Bel-
gium), illustrates the capability of 99mTc-
HYNIC-annexin V to localize at tumor sites
undergoing spontaneous and chemotherapy-
induced apoptosis. Reprinted by permission
from Reference 73.
Blankenberg: Annexin V Imaging of Apoptosis473
(See Figure 5.) Kartachova and coworkers (50) found that
degree of tumor response to platinum-based chemotherapy
(i.e., % decrease in tumor size 4 to 8 wk after therapy) in
patients with non–small cell lung carcinoma directly correlated
with the percentage increase in annexin V tumor uptake (as
compared with pretreatment baseline) at 48 hours after the first
injection of cisplatin, whereas a less successful treatment (i.e.,
stable disease) was associated with a slightly increased, un-
changed, or even a slightly decreased annexin V tumor uptake
(r25 0.86; P , 0.001). In patients with progressive disease (i.e.,
PD) a marked decrease of annexin V tumor uptake was noted
as compared with baseline tumor uptake.
Kartachova and colleagues (51) also systematically examined
how best to measure chemotherapy induced increases in
annexin V uptake as seen by SPECT in a study of 38 patients
with lymphoma (n 5 31), non–small cell lung cancer (n 5 4),
and head and neck squamous cell carcinoma (n 5 3). Maximal
counts per pixel in the tumor volume (Cmax) were calculated for
every target lesion in addition to grading on a visual four-grade
score (i.e., Cmax/expressed as percentages of baseline values:
grade 21, decrease . 25%; grade 0, 1–25% decrease; grade 11,
1–25% increase; grade 12, . 25% increase; visual analysis: 0 5
absent, 1 5 weak, 2 5 moderate, 3 5 intense). Both the
quantitative and visual assessments of increases in annexin V
uptake after treatment correlated well with therapeutic out-
come as determined by RECIST criteria (r 5 0.99 [P , 0.0001]
and r 5 0.97 [P , 0.0001], respectively]. Excellent intra-
observer reproducibility, with high kappa values (0.82–0.90)
and an inter-observer variability of 0.82, suggest that chemo-
therapy-induced increases in annexin V uptake seen at SPECT
can be consistently and routinely applied to the early (24–48 h
after initiation of therapy) noninvasive assessment of anticancer
treatment efficacy, weeks before actual tumor shrinkage.
Another recent clinical finding is that the uptake of annexin
V in normal tissues such as the spleen and bone marrow, organs
that are susceptible to drug-induced injury, is not significantly
changed with chemotherapy or prior administration of radio-
labeled annexin V within a 48-hour period (52). Furthermore,
these investigators also found that the biodistribution of radio-
labeled annexin V in the kidneys, liver, and whole body
remained unchanged after chemotherapy or prior administra-
tion of tracer.
MUTANT FORMS OF RADIOLABELED ANNEXIN V FOR
There are alternative methods to radiolabel annexin V, including
the use of self-chelating annexin V mutants; V-117 (53) and V-128
(54, 55). These proteins have an endogenous site for
followed by amino acids 1–320 of wild-type annexin V, while the
amino acid Cys-316 is also mutated to serine.99mTc chelation is
terminal cysteine and the immediately adjacent amino acids. The
purified protein is then reduced and stored for later labeling with
99mTc using glucoheptonate as an exchange reagent.
Both V-117 and V-128 have major advantages over HYNIC–
annexin V, including a 50 to 75% decrease renal uptake of
99mTc and a markedly improved in vivo localization to sites of
apoptosis in animal models (56). Using related annexin mutants,
it has been found that all four calcium-binding sites are needed
for full in vitro and in vivo binding of annexin V. Mutation (loss
of function) of any one the four calcium-binding sites decreased
in vivo location of tracer by 25% and any two site mutations
resulted in a 50% decline. Further work also has established
that random modification of the lysine residues of annexin V
with HYNIC, mercaptoacetyltriglycine (MAG3), fluorescein
isothiocyanate, and biotin-labeled annexin V showed a 50%
decrease in liver uptake of tracer as compared with self-
chelating (site-specific) protein. The adverse effects of the
random modification of annexin V have also been observed
with111In-DTPA-PEG–annexin V (57). Annexin V has also
been proven to be quite heat labile and loses most of its activity
even with heating at 568C for 10 minutes (58) (while being quite
stable at 378C) precluding the use of many different types of
As compared with SPECT, PET has major advantages for
quantitative imaging, and has spurred the development of
several approaches to label annexin V with fluorine 18 (18F)
(59). One method has used N-succinimidyl 4-fluorobenzoate to
synthesize F–annexin V. The fluorine-labeled agent has lower
uptake in the liver, spleen, and kidney compared with HYNIC–
annexin V. Another method involves site-specific derivatization
with an18F-maleimide–labeled compound to mutant annexin V-
117 or annexin V-128 (60). Both these methods however, need
more preclinical study before further development as imaging
markers of apoptosis.
FDG PET AND ANNEXIN V SPECT IMAGING
The relationship between18F-FDG uptake as seen by PET as
compared with annexin V SPECT imaging has not been
systematically compared in clinical trials. Tumor models, how-
ever, have demonstrated that that an enhanced apoptotic
reaction (increased radiolabeled annexin V uptake) correlated
with suppressed tumor glucose utilization (decreased FDG
uptake) 48 hours after the start of cytotoxic chemotherapy
(61). In a trial of 45 patients with breast cancer receiving three
cycles of neoadjuvant chemotherapy before and after therapy
(before surgical removal of primary tumor), PET scans with18F-
FDG showed significant decreases in tracer uptake coupled to
marked increases in TUNEL-positive (apoptotic) tumor cells
(62). These data suggested that neoadjuvant chemotherapy may
effectively induce apoptosis in breast tumors and decrease their
Decreases in FDG uptake have also been confirmed in several
studies, including: (1) human gastric tumor cells treated with
epirubicin, cisplatin, and 5-fluorouracil (63); (2) effective anti-
cancer therapy for patients with GIST (gastrointestinal stromal
tumor) with the selective tyrosine kinase inhibitor, imatinib
mesylate (STI571,Gleevec) (64); and (3) and EGFR kinase
inhibition of non–small cell lung cancer with gefitinib (65).
glucose demand may increase temporarily in some clinical
situations (66). One example appears to be the ‘‘metabolic flare’’
often observed on18F-FDG PET images after hormonal therapy
administrated for estrogen receptor–positive human breast can-
also be a useful indicator of responsiveness to anti-estrogen
therapy as opposed to nonhormonal chemotherapy.
In summary, while apoptosis is an energy requiring process,
it appears that outside hormonal therapy for breast cancer,
short-term tumor response is directly correlated with a signifi-
cant decrease in FDG uptake.
ANNEXIN V DEVELOPMENT AND IMPLICATIONS FOR
With the current lack of GMP-grade HYNIC–annexin V kits
for clinical imaging trials due to the closure of the Theseus
Imaging Corporation (Cambridge, MA), there is a great unmet
need to complete not only the studies of patients with NSCLC
474PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 62009
and NHL but to pursue new imaging trials for other pulmonary
diseases in which apoptosis and cellular stress play important
pathophysiologic roles (68, 69). These diseases include acute
respiratory distress syndrome (ARDS), respiratory distress
syndrome (RDS) (70), chronic obstructive pulmonary disease
(COPD), bronchial asthma, usual interstitial pneumonitis (UIP),
idiopathic pulmonary fibrosis (IPF) (71), bronchiolitis obliterans
with organizing pneumonitis (BOOP), and a variety of viral,
bacterial, and fungal infections. Common to all these important
disease entities is the increased rates of apoptosis of alveolar cells
with or without proliferation of myofibroblasts in response to
a variety of ongoing stresses. These include reactive oxygen
species (ROS, NO, etc.), overexpression of Fas (death) receptors,
increased presence of inflammatory cells and protease activity,
and in many cases decreased levels of VEGF and VEGF re-
ceptor expression (i.e., loss of normal growth factors for the
vascular endothelial cells of the lung vessels/capillaries). The
basis of therapy for these pulmonary diseases, as opposed to
cancer treatment, is the prevention of apoptosis (irreversible
tissue injury) and the reversal of physiologic stress or neutrali-
zation of an infectious/inflammatory agent(s).
As radiolabeled annexin V has the demonstrated ability to
identify both stressed and apoptotic cells in the lung and other
sites in the body, it should prove to be a very helpful agent in
determining presence, extent, and severity of active disease, as
well as the serial monitoring of the effect(s) of therapy in the
pulmonary disorders listed above.
Conflict of Interest Statement: F.G.B. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript.
1. Ameisen JC. On the origin, evolution, and nature of programmed cell
death: a timeline of four billion years. Cell Death Differ 2002;9:367–
2. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic
description of dead and dying eukaryotic cells. Infect Immun 2005;73:
3. Chan A, Reiter R, Wiese S, Fertig G, Gold R. Plasma membrane
phospholipid asymmetry precedes DNA fragmentation in different
apoptotic cell models. Histochem Cell Biol 1998;110:553–558.
4. Martin SJ, Reutelingsperger CPM, McGahon AJ. Early redistribution of
plasma membrane phosphatidylserine in a general feature of apopto-
sis regardless of the initiating stimulus: inhibition by overexpression
of Bcl-2 and Abl. J Exp Med 1995;182:1545–1556.
5. Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH. Screening and
detection of apoptosis. J Surg Res 2007;139:143–156.
6. Sperandio S, de Belle I, Bredesen DE. An alternative, nonapoptotic
form of programmed cell death. Proc Natl Acad Sci USA 2000;97:
7. Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane
phospholipid asymmetry in blood cells. Blood 1997;89:1121–1132.
8. Zwaal RFA, Comfurius P, Bevers EM. Surface exposure of phosphati-
dylserine in pathological cells. Cell Mol Life Sci 2005;62:971–988.
9. Wood BL, Gibson DF, Tait JF. Increased phosphatidylserine exposure
in sickle cell disease: flow cytometric measurement and clinical
associations. Blood 1996;88:1873–1880.
10. Boersma HH, Kietselaer BL, Stolk LM, Bennaghmouch A, Hofstra L,
Narula J, Heidendal GA, Reutelingsperger CP. Past, present, and
future of annexin A5: from protein discovery to clinical applications.
J Nucl Med 2005;46:2035–2050.
11. Meers P, Mealy T. Calcium-dependent annexin V binding to phospho-
lipids: stoichiometry, specificity, and the role of negative charge.
12. Lahorte CMM, Vanderheyden J-L, Steinmetz N, Van de Wiele C,
Dierckx RA, Slegers G. Apoptosis-detecting radioligands: current
state of the art and future perspectives. Eur J Nucl Med Mol Imaging
13. Munoz LE, Frey B, Pausch F, Baum W, Mueller RB, Brachvogel B,
Poschl E, Ro ¨del F, von der Mark K, Herrmann M, Gaipl US. The role
of annexin A5 in the modulation of the immune response against
dying and dead cells. Curr Med Chem 2007;14:271–277.
14. Green DR, Kroemer G. The pathophysiology of mitochondrial cell
death. Science 2004;305:626–629.
15. Egger L, Madden DT, Rheme C, Rao RV, Bredesen DE. Endoplasmic
reticulum stress-induced cell death mediated by the proteasome. Cell
Death Differ 2007;14:1172–1180.
16. Verheij M. Clinical biomarkers and imaging for radiotherapy-induced
cell death. Cancer Metastasis Rev 2008;27:471–480.
17. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell
18. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-
killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell
19. Aguilar-Quesada R, Mun ˜oz-Ga ´mez JA, Martı ´n-Oliva D, Peralta-Leal
A, Quiles-Pe ´rez R. Rodrı ´guez-Vargas JM, Ruiz de Almodo ´var M,
Conde C, Ruiz-Extremera A, Oliver FJ. Modulation of transcription
by PARP-1: consequences in carcinogenesis and inflammation. Curr
Med Chem 2007;14:1179–1187.
20. Balasubramanian K, Mirnikjoo B, Schroit AJ. Regulated externalization
of phosphatidylserine at the cell surface: implication for apoptosis.
J Biol Chem 2007;282:18357–18364.
21. Elliott JI, Sardini A, Cooper JC, Alexander DR, Davanture S, Chimini
G, Higgins CF. Phosphatidylserine exposure in B lymphocytes: a role
for lipid packing. Blood 2006;108:1611–1617.
22. Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to
loss of membrane asymmetry can be reversible and precede commit-
ment to apoptotic death. Exp Cell Res 1999;251:16–21.
23. Geske FJ, Lieberman R, Strange R, Gerschenson LE. Early stages of p53-
induced apoptosis are reversible. Cell Death Differ 2001;8:182–191.
24. Riksen NP, Oyen WJ, Ramakers BP, Van den Broek PH, Engbersen R,
Boerman OC, Smits P, Rongen GA. Oral therapy with dipyridamole
limits ischemia-reperfusion injury in humans. Clin Pharmacol Ther
25. Rongen GA, Oyen WJ, Ramakers BP, Riksen NP, Boerman OC,
Steinmetz N, Smits P. Annexin A5 scintigraphy of forearm as a novel
in vivo model of skeletal muscle preconditioning in humans. Circu-
26. Riksen NP, Zhou Z, Oyen WJ, Jaspers R, Ramakers BP, Brouwer RM,
Boerman OC, Steinmetz N, Smits P, Rongen GA. Caffeine prevents
protection in two human models of ischemic preconditioning. J Am
Coll Cardiol 2006;48:700–707.
27. Thimister PW, Hofstra L, Liem IH, Boersma HH, Kemerink G,
Reutelingsperger CP, Heidendal GA. In vivo detection of cell death
in the area at risk in acute myocardial infarction. J Nucl Med 2003;44:
28. Sarda-Mantel L, Michel JB, Rouzet F, Martet G, Louedec L,
Vanderheyden JL, Hervatin F, Raguin O, Vrigneaud JM, Khaw
BA, et al. (99m)Tc-annexin V and (111)In-antimyosin antibody
uptake in experimental myocardial infarction in rats. Eur J Nucl
Med Mol Imaging 2006;33:239–245.
29. Lorberboym M, Blankenberg FG, Sadeh M, Lampl Y. In vivo imaging of
apoptosis in patients with acute stroke: correlation with blood-brain
barrier permeability. Brain Res 2006;1103:13–19.
30. Blankenberg FG, Kalinyak J, Liu L, Koike M, Cheng D, Goris ML,
Green A, Vanderheyden JL, Tong DC, Yenari MA.99mTc-HYNIC-
annexin V SPECT imaging of acute stroke and its response to
neuroprotective therapy with anti-Fas ligand antibody. Eur J Nucl
Med Mol Imaging 2006;33:566–574.
31. Tang X-N, Wang Q, Koike M, Cheng D, Goris ML, Blankenberg FG,
Yenari MA. Monitoring the protective effects of minocycline treat-
ment with radiolabeled annexin V in an experimental model of acute
focal cortical ischemia. J Nucl Med 2007;48:1822–1828.
32. Taki J, Higuchi T, Kawashima A, Fukuoka M, Kayano D, Tait JF,
Matsunari I, Nakajima K, Kinuya S, Strauss HW. Effect of post-
conditioning on myocardial 99mTc-annexin-V uptake: comparison
with ischemic preconditioning and caspase inhibitor treatment. J Nucl
33. Kietselaer BL, Reutelingsperger CP, Boersma HH, Heidendal GA,
Liem IH, Crijns HJ, Narula J, Hofstra L. Noninvasive detection of
programmed cell loss with 99mTc-labeled annexin A5 in heart failure.
J Nucl Med 2007;48:562–567.
34. Lorberboym M, Feldbrin Z, Hendel D, Blankenberg FG, Schachter P.
The use of 99mTc-recombinant human annexin V imaging for
differential diagnosis of aseptic loosening and low-grade infection in
hip and knee prostheses. J Nucl Med 2009;50:534–537.
Blankenberg: Annexin V Imaging of Apoptosis475
35. Rouzet F, Dominguez Hernandez M, Hervatin F, Sarda-Mantel L, Lefort A,
Duval X, Louedec L, Fantin B, Le Guludec D, Michel JB. Technetium
99m-labeled annexin V scintigraphy of platelet activation in vegetations
of experimental endocarditis. Circulation 2008;117:781–789.
36. Kietselaer BL, Narula J, Hofstra L. The Annexin code: revealing
endocarditis. Eur Heart J 2007;28:948.
37. Tahara N, Imaizumi T, Virmani R, Narula J. Clinical feasibility of
molecular imaging of plaque inflammation in atherosclerosis. J Nucl
38. Narula J, Acio ER, Narula N, Samuels LE, Fyfe B, Wood D, Fitzpatrick
JM, Raghunath PN, Tomaszewski JE, Kelly C, et al. Annexin-V
imaging for noninvasive detection of cardiac allograft rejection. Nat
39. Peker C, Sarda-Mantel L, Loiseau P, Rouzet F, Nazneen L, Martet G,
Vrigneaud JM, Meulemans A, Saumon G, Michel JB, et al. Imaging
apoptosis with (99m)Tc-annexin-V in experimental subacute myocar-
ditis. J Nucl Med 2004;45:1081–1086.
40. Tokita N, Hasegawa S, Maruyama K, Izumi T, Blankenberg FG, Tait JF,
Strauss HW, Nishimura T. 99mTc-Hynic-annexin V imaging to
evaluate inflammation and apoptosis in rats with autoimmune myo-
carditis. Eur J Nucl Med Mol Imaging 2003;30:232–238.
41. Boersma HH, Liem IH, Kemerink GJ, Thimister PW, Hofstra L, Stolk
LM, van Heerde WL, Pakbiers MT, Janssen D, Beysens AJ, et al.
Comparison between human pharmacokinetics and imaging proper-
ties of two conjugation methods for 99mTc-annexin A5. Br J Radiol
42. Belhocine T, Steinmetz N, Hustinx R, Bartsch P, Jerusalem G, Seidel L,
Rigo P, Green A. Increased uptake of the apoptosis-imaging agent
(99m)Tc recombinant human Annexin V in human tumors after one
course of chemotherapy as a predictor of tumor response and patient
prognosis. Clin Cancer Res 2002;8:2766–2774.
43. Blankenberg F. To scan or not to scan, it is a question of timing:
technetium-99m-annexin V radionuclide imaging assessment of treat-
ment efficacy after one course of chemotherapy. Clin Cancer Res
44. Kemerink GJ, Liu X, Kieffer D, Ceyssens S, Mortelmans L, Verbruggen
AM, Steinmetz ND, Vanderheyden J-L, Green A, Verbeke K. Safety,
biodistribution, and dosimetry of 99mTc- HYNIC-annexin V, a novel
human recombinant annexin V for human application. J Nucl Med
45. Van den Brande JM, Koehler TC, Zelinkova Z, Bennink RJ, te Velde
AA, ten Cate FJ, van Deventer SJ, Peppelenbosch MP, Hommes
DW. Prediction of antitumour necrosis factor clinical efficacy by real-
time visualisation of apoptosis in patients with Crohn’s disease. Gut
46. Haas RL, de Jong D, Valdes Olmos RA, Hoefnagel CA, van den Heuvel
I, Zerp SF, Bartelink H, Verheij M. In vivo imaging of radiation-
induced apoptosis in follicular lymphoma patients. Int J Radiat Oncol
Biol Phys 2004;59:782–787.
47. Vermeersch H, Ham H, Rottey S, Lahorte C, Corsetti F, Dierckx R,
Steinmetz N, Van de Wiele C. Intraobserver, interobserver, and day-
to-day reproducibility of quantitative 99mTc-HYNIC annexin-V
imaging in head and neck carcinoma. Cancer Biother Radiopharm
48. Kartachova M, Haas RL, Olmos RA, Hoebers FJ, van Zandwijk N,
Verheij M. In vivo imaging of apoptosis by 99mTc-Annexin V
scintigraphy: visual analysis in relation to treatment response. Radio-
ther Oncol 2004;72:333–339.
49. Rottey S, Slegers G, Van Belle S, Goethals I, Van de Wiele C.
Sequential 99mTc-hydrazinonicotinamide-annexin V imaging for
predicting response to chemotherapy. J Nucl Med 2006;47:1813–1818.
50. Kartachova M, van Zandwijk N, Burgers S, van Tinteren H, Verheij M,
Valde ´s Olmos RA. Prognostic significance of 99mTc Hynic-rh-
annexin V scintigraphy during platinum-based chemotherapy in
advanced lung cancer. J Clin Oncol 2007;25:2534–2539.
51. Kartachova MS, Valde ´s Olmos RA, Haas RL, Hoebers FJ, van Herk M,
Verheij M. 99mTc-HYNIC-rh-annexin-V scintigraphy: visual and
quantitative evaluation of early treatment-induced apoptosis to pre-
dict treatment outcome. Nucl Med Commun 2008;29:39–44.
52. Rottey S, Van den Bossche B, Slegers G, Van Belle S, van de Wiele C.
Influence of chemotherapy on the biodistribution of [(99m)Tc]
hydrazinonicotinamide annexin V in cancer patients. Q J Nucl Med
Mol Imaging 2009;53:127–132.
53. Tait JF, Brown DS, Gibson DF, Blankenberg FG, Strauss HW. De-
velopment and characterization of annexin V mutants with endoge-
nous chelation sites for (99m)Tc. Bioconjug Chem 2000;11:918–925.
54. Jin M, Smith C, Hsieh HY, Gibson DF, Tait JF. Essential role of B-helix
calcium binding sites in annexin V-membrane binding. J Biol Chem
55. Tait JF, Smith C, Blankenberg FG. Structural requirements for in vivo
detection of cell death with 99mTc-annexin V. J Nucl Med 2005;46:
56. Tait JF, Smith C, Levashova Z, Patel B, Blankenberg FG, Vanderheyden
JL. Improved detection of cell death in vivo with annexin V radio-
labeled by site-specific methods. J Nucl Med 2006;47:1546–1553.
57. Ke S, Wen X, Wu QP, Wallace S, Charnsangavej C, Stachowiak AM,
Stephens CL, Abbruzzese JL, Podoloff DA, Li C. Imaging taxane-
induced tumor apoptosis using PEGylated, 111In-labeled annexin V.
J Nucl Med 2004;45:108–115.
58. Van den Eijnde SM, Boshart L, Reutelingsperger CPM, De Zeeuw CI,
Vermeij-Keers C. Phosphatidylserine plasma membrane asymetry
in vivo: a pancellular phenomenon which alters during apoptosis.
Cell Death Differ 1997;4:311–316.
59. Murakami Y, Takamatsu H, Taki J, Tatsumi M, Noda A, Ichise R, Tait
JF, Nishimura S. 18F-labelled annexin V: a PET tracer for apoptosis
imaging. Eur J Nucl Med Mol Imaging 2004;31:469–474.
60. Li X, Link JM, Stekhova S, Yagle KJ, Smith C, Krohn KA, Tait JF. Site-
specific labeling of annexin V with F-18 for apoptosis imaging.
Bioconjug Chem 2008;19:1684–1688.
61. Takei T, Kuge Y, Zhao S, Sato M, Strauss HW, Blankenberg FG, Tait
JF, Tamaki N. Enhanced apoptotic reaction correlates with sup-
pressed tumor glucose utilization after cytotoxic chemotherapy: use of
99mTc-Annexin V, 18F-FDG, and histologic evaluation. J Nucl Med
62. Li D, Yao Q, Li L, Wang L, Chen J. Correlation between hybrid 18F-
FDG PET/CT and apoptosis induced by neoadjuvant chemotherapy
in breast cancer. Cancer Biol Ther 2007;6:1442–1448.
63. Suttie SA, Park KGM, Smith TAD. [18F]-2-Fluoro-2-deoxy-D-glucose
incorporation by AGS gastric adenocarcinoma cells in vitro during
response to epirubicin, cisplatin and 5-fluorouracil. Br J Cancer 2007;
64. Trent JC, Ramdas L, Dupart J, Hunt K, Macapinlac H, Taylor E, Hu L,
Salvado A, Abbruzzese JL, Pollock R, et al. Early effects of imatinib
mesylate on the expression of insulin-like growth factor binding
protein-3 and positron emission tomography in patients with gastro-
intestinal stromal tumor. Cancer 2006;107:1898–1908.
65. Su H, Bodenstein C, Dumont RA, Seimbille Y, Dubinett S, Phelps ME,
Herschman H, Czernin J, Weber W. Monitoringtumor glucose
utilization by positron emission tomography for the prediction of
treatment response to epidermal growth factor receptor kinase
inhibitors. Clin Cancer Res 2006;12:5659–5667.
66. Haberkorn U, Bellemann ME, Brix G, Kamencic H, Morr I, Traut U,
Altmann A, Doll J, Blatter J, Kinscherf R. Apoptosis and changes in
glucose transport early after treatment of Morris hepatoma with
gemcitabine. Eur J Nucl Med 2001;28:418–425.
67. Mortimer JE, Dehdashti F, Siegel BA, Trinkaus K, Katzenellenbogen
JA, Welch MJ. Metabolic flare: indicator of hormone responsiveness
in advanced breast cancer. J Clin Oncol 2001;19:2797–2803.
68. Tang PS, Mura M, Seth R, Liu M. Acute lung injury and cell death: how
many ways can cells die? Am J Physiol Lung Cell Mol Physiol 2008;
69. Kuwano K. Epithelial cell apoptosis and lung remodeling. Cell Mol
70. Bem RA, Bos AP, Matute-Bello G, van Tuyl M, van Woensel JB. Lung
epithelial cell apoptosis during acute lung injury in infancy. Pediatr
Crit Care Med 2007;8:132–137.
71. Thannickal VJ, Horowitz JC. Evolving concepts of apoptosis in idio-
pathic pulmonary fibrosis. Proc Am Thorac Soc 2006;3:350–356.
72. Blankenberg F. In vivo imaging of apoptosis. Cancer Biol Ther 2008;7:
73. Blankenberg F. In vivo detection of apoptosis. J Nucl Med 2008;49:81S–
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