Kinetic analysis of dynamic 18F-fluoromisonidazole PET correlates with radiation treatment outcome in head-and-neck cancer
Kinetic analysis of dynamic 18F-fluoromisonidazole PET correlates with radiation treatment outcome in head-and-neck cancer
Daniela Thorwarth 1 3
Susanne-Martina Eschmann - 1 2
Jutta Scheiderbauer 0 1
Frank Paulsen 0 1
Markus Alber 1 3
0 Department of Radiation Therapy, Clinic for Radiation Oncology, University Hospital Tubingen , Germany
1 cancer (HNC) treatment. Even with an optimal combina- tion of radio- and chemotherapy , local recurrences are
2 Department of Nuclear Medicine, Radiological Clinic, University Hospital Tubingen , Germany
3 Section for Biomedical Physics, Clinic for Radiation Oncology, University Hospital Tubingen , Germany
Background: Hypoxia compromises local control in patients with head-and-neck cancer (HNC). In order to determine the value of [18F]-fluoromisonidazole (Fmiso) with regard to tumor hypoxia, a patient study with dynamic Fmiso PET was performed. For a better understanding of tracer uptake and distribution, a kinetic model was developed to analyze dynamic Fmiso PET data. Methods: For 15 HNC patients, dynamic Fmiso PET examinations were performed prior to radiotherapy (RT) treatment. The data was analyzed using a two compartment model, which allows the determination of characteristic hypoxia and perfusion values. For different parameters, such as patient age, tumor size and standardized uptake value, the correlation to treatment outcome was tested using the Wilcoxon-Mann-Whitney U-test. Statistical tests were also performed for hypoxia and perfusion parameters determined by the kinetic model and for two different metrics based on these parameters. Results: The kinetic Fmiso analysis extracts local hypoxia and perfusion characteristics of a tumor tissue. These parameters are independent quantities. In this study, different types of characteristic hypoxia-perfusion patterns in tumors could be identified. The clinical verification of the results, obtained on the basis of the kinetic analysis, showed a high correlation of hypoxia-perfusion patterns and RT treatment outcome (p = 0.001) for this initial patient group. Conclusion: The presented study established, that Fmiso PET scans may benefit from dynamic acquisition and analysis by a kinetic model. The pattern of distribution of perfusion and hypoxia in the tissue is correlated to local control in HNC.
observed in up to 50% of the treated patients [1,2]. Up to
now, no reliable parameter could be established that
would account for this high rate of local failures.
Tumor hypoxia has been known to be associated with
poor radiation response for several decades. Recent
publications suggested that hypoxia in tumors had a direct
influence on treatment success [3,4] by a variety of
mechanisms [5,6]. A prognostic impact of tumor hypoxia for
therapy outcome in head and neck cancer (HNC) has
been shown by different investigators [7-9]. Hypoxia has
also been related to lower survival probability and higher
risk of recurrence in patients with cervix cancer [4,10]. In
these studies, hypoxia was assessed invasively by
polarographic Eppendorf electrodes.
Positron emission tomography (PET) with appropriate
radiotracers enables non-invasive assessment of the
presence and distribution of hypoxia. The radiotracers in
frequent use are 18F-fluoromisonidazole (Fmiso) [11-13]
and chemically similar markers such as
18F-fluoroazomycin (Faza)  or, with a different binding mechanism,
60Cu-ATSM . Some investigations report an unclear
correlation between Eppendorf measurements and
standardized uptake values (SUV) determined on the basis of
Fmiso PET ; even though a tumor-to-blood ratio of
1.4 was defined as diagnostic of hypoxia . Thus, the
predictive value of Fmiso SUV even several hours after
tracer injection remains unclear. Based on their chemical
structure, nitroimidazoles are trapped inside hypoxic
cells. This feature makes these agents ideal markers for
hypoxia in in-vitro cell systems . However,
transforming this into larger scale biological systems is problematic
and the interpretation of Fmiso PET images remains
unclear. An advantage of PET compared to Eppendorf
measurements is the ability to display spatial
distributions, which is necessary for the integration of hypoxia
information into adaptive treatments such as hypoxia
dose painting [18-20]. For immunohistochemical
investigations, the marker pimonidazole is well established
 to stain hypoxic tumor cells. As the functional binding
mechanisms of pimonidazole and Fmiso are similar,
Fmiso should be specific to hypoxia to a similar degree.
However, the immunohistochemical staining patterns are
very complex and reveal a highly heterogeneous
distribution of perfused blood vessels and hypoxic patches,
sometimes interspersed with necrotic islands, all occurring on a
microscopic scale. This may hint as to why Fmiso tracer
uptake alone is not a reliable diagnostic quantity, and
indicates the requirement of an analysis of dynamic Fmiso
PET which takes into account the structural complexity of
hypoxic tumor tissues. The study described here was
designed to develop a kinetic model in order to
understand the spatial and temporal distribution of Fmiso in
the tumor tissue. Since the predictive character of Fmiso
SUV remains unclear in literature [13,16], the time course
of tracer accumulation in the tumor was investigated. This
analysis delivers patient specific values for perfusion,
kinetic constants and the concentration of tracer retaining
cells. Furthermore, the relation between these parameters
and radiation therapy (RT) treatment outcome for HNC
was investigated in a group of 15 HNC patients who were
examined with dynamic Fmiso PET prior to treatment
with primary radiotherapy.
After informed consent, sixteen patients (mean age: 57.2
years old, range: 46 69; 14 male, 2 female) with
advanced stage head and neck cancer (HNC) were
examined between November 2001 and March 2004. The
Fmiso examinations were performed prior to radiation
therapy (RT) treatment. All patients were treated with
primary RT to 70 Gy. Three of these patients were treated
with Intensity Modulated Radiotherapy (IMRT) in 35
fractions, 5 fractions a week with a daily dose of 2 Gy. The
other 13 patients received conventional RT, 5 fractions
with 2 Gy per week until 30 Gy. This first phase was
followed by a hyperfractionation composed of a dose of 1.4
Gy applicated twice per day until the end of treatment. In
addition, concomitant chemotherapy was prescribed for
14 patients. Seven patients received
5-Fluorouracil/Mitomycin chemotherapy, whereas for six patients Cisplatin/
Mitomycin was prescribed; one patient had
Paclitaxel/Cisplatin chemotherapy. Whenever possible (n = 12), an
additional [18F]-fluorodeoxyglucose (FDG) PET was taken
a few days (1 3) before or after the Fmiso PET scan. For
each patient, additional computed tomography (CT)
image data was available. These CT scans, on which
delineation of target volumes and organs at risk was performed,
were used for RT treatment planning.
After the end of therapy, patients were reviewed regularly
every three months with clinical examination, flexible
endoscopy and computed tomography (CT) when
recurrent disease was suspected. Routine CT scans were also
acquired six weeks and one year after therapy was
finished. Failure was defined as CT proven tumor
The Fmiso PET examinations were performed on a
wholebody scanner (Advance, GE Medical Systems, Milwaukee,
US) after automatic bolus injection of 400 MBq Fmiso.
PET data acquisition was started at the time of tracer
injection. During the first 15 (9 patients) to 60 min (7
patients), a dynamic image acquisition of 31 (40) frames
was performed. Additional static emission scans were
taken 2 h and 4 hour post injection (p.i.). Concerning the
LFeifgt:uSrceat1ter plot for one patient based on tracer retention and perfusion parameters resulting from a kinetic Fmiso analysis
Left: Scatter plot for one patient based on tracer retention and perfusion parameters resulting from a kinetic Fmiso analysis.
Schematically shown are typical regions for characteristic perfusion-hypoxia patterns: (1) High perfusion without hypoxia, (2)
well perfused and simultaneously hypoxic, and (3) severe hypoxia, low vessel density. Right: Corresponding types of
characteristic Fmiso Time-Activity Curves.
FDG PET acquisition, a static emission scan was taken 1 h
after injection of approximately 400 MBq FDG.
tion matrices were used to determine a time-activity curve
(TAC) for each tumor voxel.
For the delineation of the tumor volume relevant in the
context of this study, the FDG PET image data was used.
The tumor volume was defined as the volume including
all voxels with at least 40% of the maximum intensity.
This delineation technique was combined with a 12 mm
margin (3 PET voxels). The tumor volume variable V used
in the current study refers to the described FDG PET
volume. It is determined as V = nv, where n is the number of
tumor voxels. v represents the volume of a single voxel, in
our case v = (0.420.425) cm3 = 0.068 cm3. In order to
match the FDG-defined tumor volume onto the three
different Fmiso data sets (dynamic, 2 and 4 h p.i.), an
automatic coregistration  was performed, which achieved
a matching accuracy of 2 mm. The resulting
The voxel-by-voxel TACs were analyzed using a
pharmaco-kinetic model which is described in detail elsewhere
. Briefly, the kinetic model consists of two
compartments, one corresponding to the irreversible binding of
the tracer in hypoxic cells, the other representing freely
diffusive Fmiso. This two-compartment system is
combined with an input function which is individually
determined by a reference tissue approach for lack of a blood
signal in the field of view of the scanner (see  for
details). The voxel-by-voxel analysis of the Fmiso TACs
was done by fitting the five-parameter analytical model
function for the tracer concentration in the tissue
compartments to the measured Fmiso curves. This approach
SFcigatuterer p2lots of all 15 patients with increasing M-value
Scatter plots of all 15 patients with increasing M-value.
tumor volume V [cm3]
*T: tumor; N: node.
FOM: floor of mouth; BOT: base of tongue.
yields for each tumor voxel one characteristic value for
tracer retention and perfusion. The perfusion value, i.e.
the density of perfused blood vessels in the respective
tumor voxel, is mainly guided by the shape of the TAC
during the first few minutes after injection. In contrast, the
amount of tracer retention potential (TRP) in the voxel is
related to the properties of the curve several hours after
tracer injection. TRP takes into account the number of
viable hypoxic cells as well as their grade of hypoxia. In other
words, TRP is a measure of the concentration of
specifically bound tracer in the considered area. Fluctuations in
perfusion states are taken into account by construction of
the model . In order to visualize TRP and perfusion
characteristics of the whole tumor simultaneously, a
scatter plot is introduced (see appendix and fig. 1). In this plot,
the TRP in a voxel is plotted along the x-axis, while the
contribution to the signal from perfused blood vessels is
plotted along the y-axis. The variety of scatter patterns in
the patient group leads to the hypothesis that TRP and
perfusion are independent and spatially variable
parameters of a tumor tissue (see figure 2).
Data analysis and statistics
Tumor control was defined on the basis of computed
tomography (CT) scans as complete and persistent regression
of the primary tumor and failure was defined as local
recurrence of the tumor in the irradiated fields. Follow up
time was determined from the end of RT treatment until
the day of the last CT. Different variables that might
influence treatment outcome were compared using the
Wilcoxon-Mann-Whitney (Wilcoxon signed rank) U-test
between patient groups showing no local relapse and
failure. In all cases, a two-sided significance level of 0.05 was
used. Correlation of different variables with was assessed
using a Pearson correlation coefficient.
The impact on treatment outcome was checked for
different classes of variables: tumor volume and patient age,
SUV related factors and variables derived from the kinetic
analysis. The SUV related factors were the maximum
standardized uptake value (SUVmax) and the fractional
hypoxic volume (FHV) 4 h after Fmiso injection. FHV is
defined as the fraction of tumor volume presenting a
tumor-to-blood ratio larger than 1.4. Both variables
SUVmax and FHV have been correlated with tumor hypoxia in
earlier studies [11,13]. Finally, a number of parameters
derived from the compartmental analysis were checked
for a statistically significant influence on therapy
outcome. These parameters were the mean value of TRP, the
mean value of perfusion, and two metrics involving both
TRP and perfusion parameter values. A first metric was
defined intuitively as the volume integral of the
TRP-toperfusion ratio (HPR). A second metric, which was
derived from a model of tumor dose-response and
reoxygenation, is the malignancy value M as described in more
detail in the appendix.
Kinetic Fmiso data
The voxel-by-voxel Fmiso TACs showed a variety of
different tracer uptake patterns. Perfusion and hypoxia status of
the tissue area can be differentiated by means of the Fmiso
TAC shape. The former is determined by the part of the
TAC corresponding to time points only a few minutes p.i,
whereas the latter is linked to the shape of the curve
several hours after tracer injection. The TACs observed for the
group of 16 patients showed mainly three different types
of perfusion-hypoxia patterns which correspond to (1)
tissue areas with a high vessel density, (2) well perfused but
also hypoxic, and (3) severely hypoxic tumor areas (see
The presented compartment model allows us to derive
patient specific perfusion-hypoxia patterns. The model is
able to describe the different observed types of Fmiso time
curves. Characteristic TACs are associated to distinct areas
in the scatter plot (figure 1), which indicates high stability
of the model. The patterns for the whole group of patients
are displayed in scatter plots in figure 2 (appendix). The
ultimate purpose of the kinetic model is to subtract the
background of unbound tracer from the signal intensity.
Characteristics of the group of 15 patients are summarized
in table 1. For the examined patient group, the follow up
time was in the range of 2 21 months (median: 12.8
months). Patients were 46 to 68 years old (median: 59
years). FDG-tumor volumes ranged from 32.4 to 287.6
cm3 with a median volume of 114.5 cm3. Overall, 7 of the
15 patients had local recurrences. All observed failures
occurred in the first 8 months after the end of therapy.
Image analysis of the Fmiso PET scans taken 4 h p.i.
revealed maximum SUVs in the tumor volume between
1.36 and 4.02. The median SUVmax was 2.25. The FHV
ranged from 0 to 72.5% with a mean of 19.7%. Due to the
chosen tumor volume definition strategy, which implies
the addition of a margin, the determined FHV can never
Examination of the scatter plots showed very different
patterns of hypoxia and perfusion. All possible combinations
of hypoxia and perfusion parameters were observed: well
perfused tumors which were not at all hypoxic, tumors
showing at the same time a quite high vascular density
and hypoxic subareas, and finally also tumors that were
badly perfused and severely hypoxic. These two variables
represent physiological tumor characteristics that are not
correlated (r = -0.096). As a first result, it has to be stated
that hypoxia occurs independently from the degree of
perfusion in tumor tissues.
The Wilcoxon-Mann-Whitney U-test with respect to the
age of the patients showed no difference (P = 0.3) between
the subgroups with and without relapse. In contrast, there
was a significant difference in tumor volume between the
two subgroups (P = 0.014). This corroborates the findings
of earlier studies that correlated tumor size with treatment
outcome . Also, SUVmax, determined 4 h after
injection separated patients according to failure and
progression free survival (PFS). The significance for SUVmax was
only weak P = 0.041, whereas the U-test for the FHV
showed no significance at all (P = 0.13).
Regarding the variables derived from the kinetic analysis,
mean tumor perfusion and HPR discriminated between
the group without recurrence and the failure group (P =
0.05 and 0.008, respectively). The mean TRP value
showed no significance (P = 0.18). Finally, the
malignancy value M was highly significant, with P = 0.0013
(table 2). The prognostic value of this model based metric
M is higher than the value of tumor size or SUVmax after 4
Recent publications revealed contradictory results
concerning the correlation of static Fmiso PET data and tumor
hypoxia [11-13,16]. As the irregular architecture of
tumors complicates Fmiso uptake, a kinetic model was
developed in order to analyze spatial and temporal
distribution of the tracer in head-and-neck tumors. The
presented model enables to differentiate between tumor
perfusion and hypoxia. This feature of the model
constitutes the link between Fmiso distribution and retention
and the structural architecture of the tumor tissue.
The results of this study showed, that SUVmax alone even at
late time points has limited predictive value. These
findings are in line with results of other investigators  who
found that SUV 2 h p.i. and Eppendorf did not correlate
A limiting factor for the retention of Fmiso in the tumor is
that binding of the tracer can only take place in viable
hypoxic cells which may be few if the tumor is largely
necrotic. In other words, a low level of the Fmiso TAC
several hours after tracer injection is not necessarily due to
non-hypoxic tissue. This might also be caused by largely
necrotic tumor areas which contain only a very low
number of strongly hypoxic cells. In this case, the low
intensity of the PET signal would lead to an
underestimation of the extent of hypoxia by the SUV-method. A
kinetic analysis subtracts the non-specific background
signal and hence enables to determine the local TRP of the
tumor. Still, the classical hypoxic tumor core may only
give a weak signal due to the low density of tracer
retaining cells. Hence, a second parameter is needed to give a
more complete picture of the abnormalities of the tissue
The analysis of the parameters derived from the kinetic
model demonstrated, that TRP and perfusion values alone
do not predict treatment outcome. Additionally, hypoxia
occurred independent of degree of perfusion, since no
correlation was found between the two variables. Recent
immunohistochemical investigations of simultaneous
pimonidazole and blood vessel staining of tissue sections
[21-23] revealed the co-existence of hypoxic areas and
perfused blood vessels. These results were corroborated in
our study. Taking both parameters together proved to be
reliable predictors for treatment outcome. The
malignancy metric M, which involves these two physiological
characteristics of the tissue, was found to be the strongest
Most essential for the design of new adaptive treatment
strategies is the time until reoxygenation takes place after
the beginning of RT. The malignancy metric M involves an
estimate of this characteristic time. The worst
physiological setting in a tumor seems to be the combination of low
perfusion and severe hypoxia, as reoxygenation then
appears to be very slow. In contrast, a high degree of
perfusion co-existing with hypoxic areas may favor fast
reoxygenation. Hence, this setting might be associated with an
intermediate level of risk. This interpretation can be
supported by follow-up scans during RT, which will be
reported in a future publication.
Fmiso uptake kinetics are quite slow due to long diffusion
distances and for lack of active transport mechanisms. PET
scans several hours after injection of the radiotracer are
therefore essential. Nevertheless, dynamic scans at short
times p.i. cannot be abandoned, as they are needed to
determine the degree of perfusion of the tumor.
There is no possibility in Fmiso PET to distinguish
between acute and chronic hypoxia . On one hand,
this is due to a quite large size of the image voxels ( (4
mm)3). On the other hand, the slow kinetics of tracer
retention do not allow a distinction of fast re-perfusion.
Since both effects are a consequence of the deficient
vasculature, they may co-exist anyway.
The results of this study demonstrate that Fmiso PET has
prognostic value for therapy outcome, but only when
perfusion and retention are both taken into consideration. A
higher predictive value was associated to the malignancy
value M derived form kinetic analysis than to tumor
volume or SUV based variables. Hence, Fmiso PET might in
the future be used to individually select patients for an
adapted radiotherapy treatment as e.g. dose painting
. Furthermore, variables derived from a kinetic analysis
 may serve to determine individual dose escalation
factors in order to overcome hypoxia related treatment
The interpretation of Fmiso PET examinations with
respect to hypoxia benefits greatly from a kinetic analysis.
The presented kinetic analysis determines hypoxia and
perfusion parameters, which were shown to be able to
stratify patient groups according to RT treatment
outcome. The results of this explorating, hypothesis
generating study require validation in a larger group of patients.
By virtue of the kinetic model analysis of the time-activity
curves of tracer uptake, it is possible to eliminate the
nonspecific background activity in the signal. The model has
five fit parameters, which are determined for each voxel of
the tumor volume. Two parameters are of special interest:
the relative contribution of the perfused blood vessels,
short WP, which dominates the signal during the first few
minutes after injection. Further, the tracer retention
potential R, which is a combination of the concentration
of retaining cells and the kinetic constants of the reaction.
In a scatter plot, the values of R are plotted along the
xaxis, and the values of WP are plotted along the y-axis for
all voxels of the tumor volume. The scatter plots of all 15
patients in figure 2 show clearly that both values are
independent variables and vary widely in a population.
aTFtiCegdPurwceuitr3hvea ftitotteadl dtosteheofo7u0tcGomye data of 15 patients
irradiTCP curve fitted to the outcome data of 15 patients
irradiated with a total dose of 70 Gy.
The resistance of a hypoxic tumor to RT is governed,
among others, by two factors: the initial magnitude of the
hypoxic subpopulation of clonogenic cells, and the rate
with which these cells are reoxygenated. We assume that
the former is related to R, while the latter is related to WP.
The rationale for the second assumption is, that in areas
where the blood vessel density is high, hypoxic cells have
a greater chance to become oxic quickly. Conversely, if the
vasculature is severely deficient, reoxygenation is delayed.
The common Poisson model of tumor control probability
ln TCP (D) = n exp(0D)
where the sum runs over all voxels i of the tumor and n is
the number of cells per voxel. D is the total dose and 0
the radiation sensitivity of a non-hypoxic cell. We modify
this to include hypoxic subpopulations to read
ln TCP(D) = dh nhi(h )exp((0 h )D)
= n exp(0D) dh hi(h )exp(hD).
Here, h(h) is the frequency with which an average
reduction of the cell sensitivity by h occurs over the course of
the treatment. The integral is the malignancy M. We define
a phenomenological relation between the kinetic model
parameters and the malignancy by:
M = 1 + exp(bR/(WP + c)),
where b and c are fit parameters. Finally, we obtain
ln TCP(D) = a[1 + exp(bRi /(WP,i + c))]
with a = n exp(-0D) as an additional fit parameter. This
sum can be computed as a sum over the points of a scatter
The parameters a, b and c were determined by a maximum
log-likelihood fit of TCP to the group of 15 patients in this
study. Their values obtained as a = 5.710-5, b = 198.6 and
c = 0.565. The goodness of fit was estimated by evaluation
of the deviance . The deviance is defined as twice the
difference between the current and the full log-likelihood
= -2(Lc - Lf), which is supposed to follow a 2 distribution.
In our case, the deviance confirmed an acceptable fit ( =
2.75, p > 0.05).
Figure 3 shows the the fitted TCP curve as a function of the
malignancy value M together with the grouped data
points obtained from outcome data for the group of 15
patients in this study.
DT developed the kinetic model, the parameter analysis
strategy, and the TCP model, performed all data analysis,
and drafted the manuscript. SE carried out all PET
examinations and acquired the data. FP was involved in the
design of the study and its coordination. JS participated in
the design of the study and performed the statistical
analysis. MA conceived the study, developed the kinetic model
and the TCP model, has made substantial contributions
for data interpretation and was participated in drafting the
manuscript. All authors read and approved the final
This project has been financially supported by the German Research
Foundation (DFG), grant no. AL 877/1. The authors would like to thank Prof.
HJ Machulla and his team (Radiopharmacy Section, University Hospital
Tbingen) for excellent [18F]-Fmiso production. We also thank Dr. C Belka for
careful manuscript revision.
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