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results were not optimal as the study reported no
signicant differences in P2X7 receptor binding
between the two groups; consequently, it was not
tested anymore in AD patients [80]. In the eld of
monoamine oxidases (MAO), developing radiotracers with afnity for only B isoform has proven
challenging. [11C]L-deprenyl, the radiolabelled
L-deprenyl (selegiline)—a MAO-B inhibitor—
was rst used in human clinical applications
[81]. Nevertheless, because of the irreversible
nature of the binding that limits quantitative studies, a deuterium analogue, [11C]L-deprenyl-D2,
with more favourable pharmacokinetics, has
been developed and it is now considered the better MAO-B radioligand [82, 83]. As a part of
the inammation mechanisms, arachidonic acid
(AA) has been tested as a target for invivo PET
studies using the radiolabelled AA itself,[11C]
AA.However, the involvement in several functions does not make its altered bindings easily
interpretable [84–86]. The only applied PET
radioligand for COX is [11C]ketoprofen and its
methyl ester, which binds selectively to COX-1
[87]. In a study, the authors concluded that it cannot be useful as a potential diagnostic biomarker
for MCI/AD [87]. Regardingcox-2, none of the
tested radiotracers has been considered to be useful for the study of neuroinammation [88, 89].
Acknowledgements We thank Prof. Daniela Perani from
Vita-Salute University of Milan and her research group
for the remarkable work on the knowledge of neurodegenerative diseases, for mentoring so many nuclear medicine
physicians and for advising us on how to dene clinical
case research for this chapter.
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11-

Hypoxia Pathway: 18F-FAZA
https://t.me/med1917
PierpaoloAlongi, PaolaMapelli, ViolaVultaggio,
AnnachiaraArnone, andMariaPicchio
20
20.1 Introduction
Hypoxia plays a crucial role in various cellular
events, including proliferation, survival, angiogenesis, immuno-surveillance, metabolism, as
well as in tumour invasion and metastasis [1, 2].
In the eld of the tumorigenesis, the rapid growth
exceeding the vascular supply and the need for
oxygen as an energy substrate determine a poorly
vascularized microenvironment in which
hypoxia and acidosis, as well as the lack of nutrients, take place [3, 4]. Tumours react to this hostile system through a series of compensatory
mechanisms: turning to a glycolytic metabolism,
expressing various growth factors (e.g. EGF,
insulin, IGF-1, IGF-2, and PDGF, known to promote cell proliferation), producing a number of
angiogenic factors that promote blood vessel
formation (e.g. VEGF, IL-8, angiogenin, FGF,
and PDGF), secreting various immunosuppres-
P. Alongi (*) · V. Vultaggio
Nuclear Medicine Unit, A.R.N.A.S.Ospedale Civico
Di Cristina Benfratelli, Palermo, Italy
P. Mapelli · M. Picchio
Vita-Salute San Raffaele University, Milan, Italy
Nuclear Medicine Department, IRCCS San Raffaele
Scientic Institute, Milan, Italy
picchio.maria@hsr.it
e-mail:
A. Arnone
Nuclear Medicine Unit, Department of Experimental
and Clinical Biomedical Sciences “Mario Serio”,
University of Florence, Florence, Italy
sive factors by hypoxia-inducible factor (HIF)dependent or -independent pathways aiming to
escape immune surveillance [5–9]. In the role of
one of the most pervasive microenvironmental
stresses that can impact the cancer progression,
hypoxia can be recognized in almost every solid
tumour and be widely considered as a potential
reason of treatment failure and poor outcome for
a variety of malignancies and consequently
needs to be taken into account during the evaluation of prognostic and therapeutic options for
cancer patients [10–12].
This chapter will describe the current knowledge on technical procedures of radiosynthesis,
features of biodistribution, and current research
on potential clinical application of 18F-FAZA
PET as a radiopharmaceutical agent able to
assess the hypoxia expression in neoplastic cells.
20.2 Synthesis
The overall radiosynthesis time of 18F-FAZA is
about 70 min. After, quality controls are performed to determine the chemical, radiochemical, and radionuclidic purity of the nal product
[13].
The nucleophilic substitution reaction of nocarrier- added 18F-uoride with 1-(2,3-di-O-
acetyl-5-O-tosyl-d-arabinofuranosyl)-2nitroimidazole as precursor has to be performed
[14]. 18F-uoride has to be produced using a
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
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cyclotron for irradiation of approximately 2mL
of enriched 18O water through the 18O(p,n) 18F
nuclear reaction. The radiosynthesis of 18F-FAZA
is automatically performed with a synthesis module. In the rst step of the synthesis, 18F-uoride
is separated from 18O water using an anion
exchange cartridge. To elute the 18F-uoride
absorbed on the resin, a mixture of 0.5mL of a
solution of K2CO3 in water (6mg/mL) and 1mL
of Kryptox 222 (20mg/mL; Merck) in CH3CN
was used. The solution in the reactor is then dried
under vacuum at 60 °C for 5 min and then at
120°C for another 5min. Labelling is carried out
using 5mg of the precursor dissolved in 1mL of
anhydrous dimethyl sulfoxide under stirring at
100°C for 5min. To remove acetyl groups, basic
hydrolysis is achieved with 1mL of 0.1N NaOH
at 35°C in about 2 min. Neutralization is performed by adding 0.5 mL of 0.5 N aqueous
NaH2PO4. The reaction mixture has to be combined with 2 mL of high-performance liquid
chromatography eluent (H2O:EtOH 98:2 [v/v])
and then passed through an Al2O3 cartridge
(Waters), where unreacted uorine was adsorbed.
Then, the mixture is injected into a semipreparative high-performance liquid chromatography
column to separate the reaction product from
other impurities. At a ow rate of 4mL/min, the
product has a retention time of about 34 min,
detected with an ultraviolet detector in series
with a Geiger–Müller radiodetector. The collected radiopharmaceutical has to be ltered
using a 0.22-μm sterile lter and then diluted
with 0.9% NaCl in order to have 12mL.
18
F-FAZA
is obtained with an overall radiochemical yield of
20–25% (not decay-corrected), radiochemical
purity of more than 99%, and specic activity of
more than 37GBq/μmol [14].
sumption (steady state) and (2) modelling of
tracer diffusion and retention depending on its
specic diffusion and clearance properties as
well as the previously simulated oxygen distributions [15].
Oxygen distribution is described as the steady
state of oxygen diffusion and consumption, as
each pixel on the vessel map was regarded as a
separate oxygen source of a size of approximately 2.5×2.5μm2 and thus much smaller than
the distance of hypoxic regions from the vessels,
this was judged to be a valid approximation.
Therefore, if the initial pO2 distribution is known,
pure diffusion at any point in time can be determined by convolving the initial oxygen distribution with the diffusion kernel. The parameters
considered are clearance rate, tracer retention in
viable hypoxic tissue areas, in fact variations in
the vascularization patterns of tissue sections
with the same median pO2 can affect overall
tracer retention in that section. Tracer uptake in
hypoxic cell culture suggests nitroreductase
afnity for FAZA; however, overall activity is
lower for FAZA than for other hypoxia PET tracers in pre-clinical studies, indicating that mechanisms other than enzyme activity, such as tracer
clearance and diffusion, are responsible for this
observation.
Pharmacokinetic analysis of 18F-FAZA
dynamic PET imaging acquisitions have also
been used to discriminate different tumour proles through visual imaging and quantication of
blood and tumour time–activity curves, as evidenced by Provost etal. In this work, the interval
between injection and image acquisition normally ranges from 2 to 4h, considered the best
time to capture images and a compromise
between radiopharmaceutical accumulation in
hypoxic cells and clearance from the blood [
16].
20.3 Pharmacokinetics
Dedicated hypoxia PET tracers are bound to cells
depending on the local oxygen concentration.
Therefore, to simulate hypoxia PET signals, a
two-step process can be used: (1) Simulation of
the diffusive oxygen supply from blood vessels
that is in equilibrium with cellular oxygen con-
20.4 Physiological Distribution
Preliminary biodistribution data are indicative of
the potential utility of FAZA for studies of
hypoxia in oncology and other pathologies. Its
lower lipophilicity may produce higher tissue
perfusion and more rapid clearance from blood,

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thereby providing higher hypoxia (tissue):blood
differentials at short time intervals after injection
that are more compatible with the relatively short
half-life of 18F (109 min). Blood clearance is
characterized by a rapid distribution followed by
rst-order elimination [13].
The highest uptake is in muscle and liver and
the critical organ, with the highest absorbed radiation doses, is the urinary bladder wall, because
radioactivity is excreted through the kidneys,
with a fast clearance and hepatic metabolism, so
clearance is therefore also dependent on patient
kidney function and may hence affect the suitability or patients treated with (concurrent) nephrotoxic systemic agents. This adds variability to
the total rate at which the tracer is cleared from
the system, resulting in larger absolute ranges of
clearance rates [15].
20.5 PET/CT Acquisition Protocols
andImage Interpretation
The acquisition protocol of 18F-FAZA PET/CT
scans usually starts 120min after the injection of
approximately 370 MBq of radiotracer and
includes the acquisition of PET images in 3D
mode and CT images used for attenuation correction [17]. Regarding scan acquisition time, some
authors investigated the possibility to acquire
images at different time points. Available data
suggest that the image quality that is gained at
120min is almost the same as for later acquisitions and it is higher compared to images acquired
less than 120min [
differences between the biodistribution at 1, 2,
and 4h after injection are reported.
PET/CT images performed to assess hypoxia
usually rely on the measurement of tumour-tobackground (T/B) or tumour-to-muscle (T/M)
ratio in order to dene the hypoxic fraction within
a tumour. In particular, the majority of published
literature use a threshold varying between 1.2
and 1.4 to dene hypoxic fraction, regardless of
the adopted radiotracer or the types of investigated tumour [18, 19, 21–23]. Few data are currently available regarding the different approaches
used to evaluate hypoxic fraction. However, it
14, 18–20]. In Fig.20.1, the
has been reported that the T/M uptake ratio might
be used as reliable alternative to T/B ratio in
those situations where no great vessels (e.g.
aorta) or the heart itself is included in the eld of
view [17].
20.6 Clinical Indications
18
F-FAZA have shown very promising results in
different oncological clinical settings.
A potential application of hypoxia tracers with
possible relevant impact on treatment decisions is
related to the radiotherapy eld. In this scenario,
Grosu et al. outlined the gross tumour volume
(GTV) on 18F-FAZA PET in 17 patients with
head and neck cancers [24]. The delineated GTV
was obtained by applying a threshold of 50%
with regard to background, leading to the inclusion of PET-positive areas with a T/M ratio≥1.5.
Interestingly, in all cases GTV-FAZA was inside
the GTV outlined on CT images, thus suggesting
the feasibility of a dose-painting approach based
on 18F-FAZA PET images.
Later on, Mortensen etal. investigated the role
of 18F-FAZA PET in 40 head and neck cancer
before and during radiotherapy [24]. In 63% of
patients, PET showed a hypoxic volume with a
median T/M of 1.5, having prognostic signicance; in fact, at a median follow-up of 19months
patients with non-hypoxic tumours had a diseasefree survival of 93%, compared to 60% observed
in patients presenting hypoxic tumours [
An extremely interesting oncological clinical
scenario of possible application of 18F-FAZA
PET/CT is represented by gliomas. Several studies have investigated the potentiality of this imaging technique in the assessment of hypoxic
fraction within this aggressive brain tumours.
One of the largest cohorts investigating the
role of 18F-FAZA for hypoxia assessment has
been reported by Postema etal. who rstly evaluated the safety and general biodistribution of this
radiotracer [18]. In a cohort of 50 patients with
different types of cancers, including head and
neck squamous cell carcinoma, small cell lung
cancer (SCLC), non-SCLC, malignant lymphoma, and high-grade gliomas, the authors
25].

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One-hour post injection
Two-hours post injection
Fig. 20.1 Biodistribution of 18F-FAZA at 1, 2, and 4h post-injection
reported a high uptake of 18F-FAZA in gliomas,
with variable uptake in the remaining tumours.
Correlations between 18F FAZA and histological parameters assessing hypoxia have been
recently reported. In a series of 17 patients with
high-grade glioma, Mapelli et al. reported an
inverse correlation between tumour
vascularization, SUVmax, and SUVmean, thus
suggesting that highly vascularized tumours
might present more oxygen supply than hypoxia
[17].
The same group reported the potentiality of
18
F-FAZA PET/CT in guiding tailored radiotherapy treatment in high-grade glioma. The possibility to use an additional boost on the more hypoxic
regions by using 18F-FAZA PET images in a simulated radiotherapy scheme has been reported,
along with the possible use of this molecular
imaging technique to assess tumour response
after radiotherapy, with promising implications
for clinical practice [26]. The authors also
reported the ability of 18F-FAZA in guiding stereotactic biopsy by identifying the tumour areas
with the highest aggressive potential, as dened
by the higher degree of hypoxia. In this report, a
co-registration between MRI and 18F-FAZA PET/
CT images has been performed and the areas
with high-perfusion MRI markers and high
18
F-FAZA uptake have been identied to be most
representative regions to be sampled [21].

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a
Fig. 20.2 18F-FAZA PET/CT and MRI: Tumor Hypoxic evaluation in the suspicion of glioma
a
b
b
c
Fig. 20.3 18F-FAZA PET/CT: Tumor Hypoxic evaluation of lung lesion
20.7 Clinical Cases
Departments, Ospedale San Raffaele, Milan,
Italy.
20.7.1 18F-FAZA PET/CT
inGlioblastoma
20.7.2 18F-FAZA inLung Cancer
See Fig.20.2.
Fifty-seven year-old patient who underwent a
brain MRI scan for behavioural disorders and
headache. A large edematous cystic necrotic
enhancing frontal lesion suggestive for highgrade glioma is seen on MRI image (A:
T1weighted image). In the suspicion of glioma,
the patient underwent 18F-FAZA PET/CT to identify tumour hypoxic regions, showing tracer
uptake (hypoxic volume, 16.69) in the left frontal
brain lesion (B: PET transaxial image; C: fused
PET/CT transaxial image); a central photopenic
area, corresponding to necrosis, has been also
identied. Glioblastoma was conrmed at histology. From Database of Nuclear Medicine
See Fig.20.3.
A 76-year-old man underwent CT scan showing a pulmonary mass and ipsilateral hilar lymph
adenopathy. 18F-FDG PET/CT performed to
characterize the lung lesion and to stage the disease showed intense uptake in correspondence of
the right pulmonary lesion (A: transaxial PET/
CT). The patient underwent 18F-FAZA PET/CT
as part of a clinical research protocol (EudraCT:
2011–002647-98).
18
F-FAZA PET/CT showed moderate heterogeneous radiotracer uptake in correspondence of
the right pulmonary lesion (B: transaxial PET/
CT). The lung mass presented a hypoxic volume

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a
b
Fig. 20.4 18F-FAZA PET/CT and 18F-FDG PET/CT: Tumor Hypoxic of brain metastasis of lung origin
(T/B ratio > 1.2) 115 cc. From Database of
Nuclear Medicine Departments, Ospedale San
Raffaele, Milan, Italy.
cd
e
(C: major intensity projection; D: transaxial PET/
CT) and of the pathological adenopathy (E: transaxial PET/CT).
Images from database of Nuclear Medicine
Department, IRCCS San Raffaele Scientic
20.7.3 Cerebral Metastases
18
Identied by
F-FAZA PET/CT
Institute, Milan, Italy.
See Fig.20.4.
A 72-year-old patient presented severe neurological symptoms and underwent a brain MRI
scan showing a brain mass in the right parietal
lobe, suggestive for high-grade glioma. The
patient underwent 18F-FAZA PET/CT as part of
a clinical protocol (EudraCT: 2015–000679-28).
The scan showed 18F-FAZA with a photopenic
area of necrosis (A: transaxial PET; B: transaxial
PET/CT). The patient underwent surgical intervention; histological examination revealed a metastatic lesion of a poorly differentiated carcinoma
compatible with lung origin and immunohistochemical analysis related to hypoxic factors was
performed. A CT scan was performed to identify
the primary tumour and revealed a left lung mass
with ipsilateral adenopathy. Disease staging was
completed with 18f-FDG PET/CT scan showing
tracer uptake in correspondence of the left lung
20.7.4 18F-FAZA PET/CT asaGuide
forTumour Biopsy
Fig. 20.5.
A 77-year-old patient underwent brain MRI
was admitted for progressive left leg hypertonia. MRI showed an enhancing, edematous
lesion in the right basal ganglia and corona
radiata on T1-weighted images (A), suggestive
for high- grade glioma. The lesion also showed
high values of transfer constant (b: yellow
arrow). 18F-FAZA PET/CT was performed to
assess tumour hypoxia and to identify the
tumour regions more likely to be resistant to
chemo and/or radiotherapy. 18F-FAZA PET
images (C) showed uptake in correspondence
of the brain lesion with a central photopenic
area. A co-registration of MRI and 18F-FAZA
PET/TC images was performed and an area of
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