doi:10.1093/brain/awl178 Brain (2006), 129, 2856–2866
Two-year follow-up of amyloid deposition in
patients with Alzheimer’s disease
Henry Engler,1Anton Forsberg,5Ove Almkvist,6,7Gunnar Blomquist,1,3Emma Larsson,5
Irina Savitcheva,2Anders Wall,1Anna Ringheim,1Bengt La ˚ngstro ¨m1,4and Agneta Nordberg5,6
1Uppsala Imanet AB, Imanet, GE Healthcare,2Uppsala University Hospital,3Department of Oncology,
Radiology and Clinical Immunology,4Department of Biochemistry and Organic Chemistry, Uppsala University, Uppsala,
5Division of Molecular Neuropharmacology, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet,
6Department of Geriatric Medicine, Karolinska University Hospital Huddinge, Stockholm and7Department of Psychology,
Stockholm University, Stockholm, Sweden
Correspondence to: Prof. Agneta Nordberg, MD, PhD, Karolinska Institutet, Department of Neurobiology, Care Sciences
and Society, Division of Molecular Neuropharmacology, Karolinska University Hospital Huddinge, Novum, S-141 86
Beta amyloid is one of the major histopathological hallmarks of Alzheimer’s disease. We recently
reported in vivo imaging of amyloid in 16 Alzheimer patients, using the PET ligand N-methyl[11C]2-
metabolic rate for glucose (rCMRGlc) at follow-up. Sixteen patients with Alzheimer’s disease were re-
examined by means of PET, using PIB and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) after 2.0 6 0.5 years. The
patients were all on cholinesterase inhibitor treatment and five also on treatment with the N-methyl-D-
aspartate (NMDA) antagonist memantine. In order to estimate the accuracy of the PET PIB measurements,
four additional Alzheimer patients underwent repeated examinations with PIB within 20 days (test–retest).
Relative PIB retention in cortical regions differed by 3–7% in the test–retest study. No significant difference
in PIB retention was observed between baseline and follow-up while a significant (P < 0.01) 20% decrease in
rCMRGlc was observed in cortical brain regions. A significant negative correlation between rCMRGlc and PIB
retention was observed in the parietal cortex in the Alzheimer patients at follow-up (r = 0.67, P = 0.009). A non-
significant decline in Mini-Mental State Examination (MMSE) score from 24.3 6 3.7 (mean 6 standard devia-
tion) to 22.7 6 6.1 was measured at follow-up. Five of the Alzheimer patients showed a significant decline in
MMSE score of >3 (21.4 6 3.5 to 15.6 6 3.9, P < 0.01) (AD-progressive) while the rest of the patients were
cognitively more stable (MMSE score = 25.6 6 3.1 to 25.9 6 3.7) (AD-stable) compared with baseline. A positive
correlation (P = 0.001) was observed in the parietal cortex between Rey Auditory Verbal Learning (RAVL) test
score and rCMRGlc at follow-up while a negative correlation (P = 0.018) was observed between RAVL test
and PIB retention in the parietal at follow-up. Relatively stable PIB retention after 2 years of follow-up in
patients with mild Alzheimer’s disease suggests that amyloid deposition in the brain reaches a plateau by the
early clinical stages of Alzheimer’s disease and therefore may precede a decline in rCMRGlc and cognition.
It appears that anti-amyloid therapies will need to induce a significant decrease in amyloid load in order for
of cerebral metabolism caused by therapy administered to patients with a clinical diagnosis of Alzheimer’s
Keywords: Alzheimer’s disease; amyloid; PET; PIB; FDG; follow-up
6-hydroxy-benzothiazole; rCMRGlc = regional cerebral metabolic rate for glucose; RAVL = Rey Auditory Verbal Learning;
ROIs = regions of interest; SD = standard deviation
Received March 18, 2006. Revised May 26, 2006. Accepted June 7, 2006. Advance Access publication July 19, 2006.
#The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: firstname.lastname@example.org
The pathology of Alzheimer’s disease has been coupled to an
abnormal deposition of amyloid in the brain (the amyloid
cascade hypothesis) (Hardy and Higgins, 1992). The regional
evolution of Alzheimer’s disease pathology in terms of
amyloid deposition (amyloid plaques) and neurofibrillary
tangles at several stages of the disease was described by
Braak and Braak (1991), using post-mortem brain tissue.
The distribution of amyloid plaques shows high variability
in patients and has been difficult to correlate with the stage
of the disease (Braak and Braak, 1991). Owing to the limita-
tions of post-mortem brain studies, several attempts have
been made to develop tracers for in vivo imaging of amyloid,
and three compounds have been applied in PET studies in
Alzheimer patients (Shoghi-Jadid et al., 2002; Klunk et al.,
2004; Verhoeff et al., 2004). The finding that certain
benzothiazol derivatives bind to amyloid with high affinity
and cross the brain–blood barrier (BBB) led to the deve-
hydroxy-benzothiazole (PIB) (Klunk et al., 2001; Mathis
et al., 2002; Wang et al., 2002; Mathis et al., 2003). PIB
binds amyloid with sufficiently high affinity to be detected
in PET examinations. Animal studies have shown that PIB
crosses the BBB and is cleared from normal brain tissue
(Bacskai et al., 2003). The results of binding studies in
autopsy-derived human brain tissue suggested that PIB
might be a relevant ligand for the measurement of amyloid
deposition but not for the detection of neurofibrillary tan-
gles, since PIB mainly binds to aggregated fibrillar Ab depos-
its (Klunk et al., 2003). In 2002–2003, the first study on
measurement of amyloid in humans, based on the retention
of PIB, was performed in Uppsala, Sweden, among 16
patients with mild Alzheimer’s disease from Karolinska
University Hospital Huddinge, Stockholm, Sweden (Klunk
et al., 2004). PIB showed high retention in the frontal and
temporal–parietal association cortices and striatum in these
patients versus controls. The retention of PIB was similar in
Alzheimer patients and healthy controls (HC) in brain
regions known to be relatively unaffected by amyloid deposi-
tion, such as the pons, cerebellum and subcortical white
matter (Klunk et al., 2004). A strong negative correlation
was found between cerebral glucose metabolism [measured
using 2-[18F]fluoro-2-deoxy-D-glucose (FDG)] and PIB
retention in the parietal areas, suggesting a relationship
between deficits in neural function and amyloid deposition
(Klunk et al., 2004). In order to explore PIB further as a
tracer, we have now performed a follow-up study with PIB,
and FDG, in these 16 Alzheimer patients 1.5–2.5 years after
the baseline study.
recruited at the Department of Geriatric Medicine, Karolinska
University Hospital Huddinge, Stockholm (Klunk et al., 2004),
and fulfilling the diagnosis of probable Alzheimer’s disease accord-
ing to the criteria of the National Institute of Neurological and
Communication Disorders, Alzheimer’s Disease and Related
Disorders Association (NINCDS–ADRDA) were clinically followed
for a time period of 2.0 6 0.5 years (1.5–2.5 years) and then
re-examined, using PIB and FDG, at Uppsala Imanet, Uppsala,
Sweden. The demography of the Alzheimer patients is presented
in Table 1.
patientswith mild Alzheimer’sdiseasepreviously
Table 1 Demography
Age at baseline
Age range at baseline
MMSE at baseline
MMSE at follow-up
RAVL at follow-up (Z-score)
Duration of disease (year)
Time from diagnosis to baseline (year)
CSF Ab (pg/ml) at baseline
CSF tau (pmol/ml) at baseline
Between scans (m)
Treatment at baseline
Treatment at follow-up
12.3 6 3.7
24.3 6 3.7
22.7 6 6.1
?2.60 6 1.45
4.3 6 1.5
2.4 6 1.8
372 6 129
599 6 311
23.9 6 5.6
11 on ChEls
16 on ChEls 5 on memantine
13.6 6 2.5
25.6 6 3.1
25.9 6 3.7
?1.55 6 1.36
4.4 6 1.5
2.4 6 1.8
401 6 131
441 6 223
23.2 6 6.1
7 on ChEls
11 on ChEls 3 on memantine
9.6 6 4.8
21.4 6 3.5
15.6 6 3.9#,**
?3.35 6 0.68***
4.2 6 1.6
2.3 6 1.9
326 6 125
883 6 238**
25.6 6 4.4
3 on ChEls
5 on ChEls 2 on memantine
stable patients (MMSE decline <3); AD-P = patients with disease progression (MMSE decline >3); RAVL = Rey Auditory Verbal Learning
test; *significant difference in age at baseline between AD-S and AD-P (Student’s t-test; P < 0.05);#significant difference in MMSE score
between baseline and follow-up (paired t-test; P < 0.01); **significant difference between AD-S and AD-P (Student’s t-test; P < 0.05);
***significant difference between AD-S and AD-P (Student’s t-test, P = 0.01).
Follow-up of amyloid in Alzheimer’s disease Brain (2006), 129, 2856–28662857
Mini-Mental State Examination (MMSE) (Folstein et al., 1975)
was used as measurement of global cognitive status and Rey Audi-
tory Verbal Learning (RAVL) (Lezak, 2004) as test of episodic
memory. For statistical analysis Z-scores of RAVL were generated
on the basis of the raw scores, by comparing data with the mean
value and standard deviation (SD) of a large control group.
Data from six healthy, age-matched controls (HC), studied at
baseline, were utilized for comparisons (Klunk et al., 2004). The
oldest healthy control (OHC) from the baseline study, who showed
high PIB retention but normal cognitive performance in neuro-
psychological tests (Klunk et al., 2004), was also reinvestigated in
the follow-up study.
The Alzheimer patients and the OHC gave written consent
to participate in the study. The Ethics Committees of Uppsala
University, the Karolinska Institute and the Isotope Committee
at Uppsala Academic Hospital approved the study.
In order to study the variability in PIB retention, four additional
Alzheimer patients (T1–T4) (3 females and 1 male) (58–79 years of
age) (MMSE 9–28) were recruited from the Department of Geriatric
Medicine, Karolinska University Hospital Huddinge, Stockholm,
and they underwent repeated PET investigations with PIB. For
three of the Alzheimer patients the two PIB studies were performed
within 12 h and for the fourth patient, after a 20-day interval. The
studies were performed under a separate study protocol with
corresponding authorization as regards ethics. The changes in
PIB retention were calculated for each patient by means of the
formula %Difference = [(R?T)/(R+T)] · 200; (T = test, R = retest).
The mean absolute percentage difference [Mean abs (%Diff)] was
calculated for all four patients, yielding an interval of expected
variance in the analysis of PIB retention (Price et al., 2005).
Production of FDG and PIB was carried out according to
the standard good manufacturing process at Uppsala Imanet.
Synthesis of N-methyl[11C]2-(40-methylaminophenyl)-6-hydroxy-
benzothiazole (PIB) was performed by means of the method
described previously (Mathis et al., 2003; Klunk et al., 2004).
The PET scans were performed using Siemens ECAT EXACT HR+
scanners (CTI PET-systems Inc.), with an axial field of view of
155 mm, providing 63 contiguous 2.46-mm slices with 5.6-mm
transaxial and 5.4-mm axial resolution. The patients were scanned
after fasting for 4 h under resting conditions in a dimmed room.
The orbitomeatal line was used to centre the heads of the subjects.
The data were acquired in three-dimensional mode. The doses of
PIB and FDG, and the scanner protocol for transmissions, emis-
sions and reconstructions were the same as used in the
previous study (Klunk et al., 2004). The subjects were given
287 6 65 (mean 6 standard deviation) MBq of11C-PIB in the
baseline study and 254 6 86 MBq in the follow-up study. They
received 231 6 40 MBq of18F-FDG in the baseline study and 226 6
33 MBq in the follow-up study.
Regions of interest (ROIs)
The set of ROIs applied for statistical analyses in the follow-up
and the test–retest study was the same as in the previous study,
described in detail earlier (Engler et al., 2003; Klunk et al., 2004).
The following areas were included in the analyses: the frontal,
parietal, temporal, occipital and cerebellar cortices, pons, white
matter and striatum and posterior cingulum.
A computerized reorientation procedure applied in the first
study was used to align consecutive PET images for accurate
intra- and inter-individual comparisons (Andersson and Thurfjell,
1997). The FDG images obtained in the follow-up study were rea-
ligned to the FDG images from the previous study, and the PIB
images from both studies were co-realigned using the respective
FDG images as templates. In addition, FDG images were analysed
according to a clinical routine set of ROIs (Engler et al., 2003). In
three of the Alzheimer patients the calculation of FDG uptake could
not be performed according to the protocol in the follow-up study.
In one patient this was a result of technical problems during acqui-
sition and in the other two patients, a result of the fact that blood
samples could not be obtained. For one of these patients, regional
mean uptake values could be obtained, whereas no data concerning
glucose uptake were available for the two other patients. MR images
were not used to delineate ROIs, nor were such data used to do any
partial volume correction.
For the FDG examinations, venous arterialized blood samples were
obtained and parametric maps of the regional cerebral metabolic
rate for glucose (rCMRGlc) were generated by means of the Patlak
method, using the time course of the tracer in the arterialized
venous plasma as an input function (Patlak et al., 1983). The
rCMRGlc values were normalized to the pons value (ROI/ref) to
allow inter- and intra-individual comparisons (Minoshima et al.,
1995). In the earlier baseline PIB study (Klunk et al., 2004), PIB
retention data were given as standard uptake values (SUVs). In the
present follow-up study the mean uptake values of the ROI
obtained in a late time interval (40–60 min) were normalized to
the corresponding uptake in a reference region (ROI/ref) (Lopresti
et al., 2005). The cerebellar cortex was chosen as reference because
of its previously reported lack of Congo red- and thioflavin-S-posi-
tive plaques (Yamaguchi et al., 1989; Mirra et al., 1994). Among
several different PIB evaluation methods the late scan reference
method has shown a large size effect (Lopresti et al., 2005).
The statistical method used to compare the Alzheimer patients
and the HC was a two-sample, unequal variance, two-tailed
Student’s t-test. Paired t-tests were used to study changes in PIB
retention and rCMRGlc in the Alzheimer patients over the time
period. Analysis of correlation between PIB retention and rCMRGlc
parameters was conducted, yielding Pearson’s product moment
correlation coefficient r.
Role of the funding source
No funding source had a role in the preparation of this article or the
decision to submit it for publication. The authors had full access to
all data in the study and final responsibility for the decision to
submit for publication.
Four Alzheimer patients were recruited for a test–retest PIB
study within 20 days. The individual results are shown in
2858Brain (2006), 129, 2856–2866 H. Engler et al.
Table 2. Low variability in PIB retention was observed in
the frontal cortex (7.3%), parietal cortex (4.1%), temporal
cortex (3.2%) and occipital cortex (3.7%), while the highest
variability was observed in the striatum (12.7%). Variability
was also low in the pons (3.4%), white matter (6.4%) and the
Clinical follow-up of Alzheimer patients
The 16 Alzheimer patients who had undergone baseline
studies with PIB (Klunk et al., 2004) were clinically followed
for 1.5–2.5 years and then they underwent the follow-up PET
studies with PIB and FDG (Table 1). Eleven Alzheimer
patients were being treated with cholinesterase inhibitors
at the baseline PET scans, while all 16 patients were on
cholinesterase inhibitor treatment at the time of the follow-
up studies. In addition, 5 of the 16 patients were also being
treated with the N-methyl-D-aspartate (NMDA) antagonist
memantine at the time of follow-up (Table 1).
Cognitive testing showed a non-significant decrease in
MMSE score of 1.6 points at follow-up compared with base-
line. Five of the 16 patients showed a very clear clinical
deterioration at follow-up, with a decrease of 3 points or
more (3–9) in the MMSE test. In these five Alzheimer
patients the mean MMSE score was significantly lower
(P < 0.01) at follow-up [15.6 6 3.9 (SD)] compared with
baseline (21.4 6 3.5). This group of patients was considered
to comprise those who had clinically deteriorated (AD-P;
progression) (Table 1). The change in MMSE score
among the other 11 patients was <3 (?2 to +3) at follow-
up versus baseline. This group of Alzheimer patients was
considered to be clinically relatively stabile at follow-up
(AD-S) (Table 1). The AD-S group (n = 10) differed
significantly in RAVL test at follow-up (Z-score) compared
with the AD-P group (n = 4), P < 0.01. The AD-P group was
significantly younger than the AD-S group (P < 0.05). They
showed significantly higher CSF tau values (P < 0.05) while
there was no difference in CSF Ab 1-42 values between the
two groups (Table 1). Three of the 5 patients in the AD-P
group carried two APO e4 alleles, while 5 out of 10 carried
e4 alleles in the AP-S group (Table 1).
Regional PIB retention at follow-up
Significantly greater retention of PIB (expressed as ROI/ref)
(P < 0.006) was observed at follow-up in the frontal, parietal,
temporal and occipital cortices as well as in the striata of
the Alzheimer patients versus age-matched HC (Fig. 1). No
significant changes in PIB retention were observed between
baseline and follow-up in any brain region of the Alzheimer
patients as an overall group (Fig. 1). Retention of PIB was
somewhat higher at both baseline and follow-up in the cor-
tical brain regions and striata of the AD-P group versus the
AD-S group. This difference was of statistical significance in
the posterior cortex cinguli at baseline (P < 0.05) (Table 3).
Owing to error in the scanning procedure at baseline, data
for one Alzheimer patient (02) are not included in the
Regional FDG uptake at follow-up
Analysis of rCMRGlc in the entire cohort according to
routine clinical standard assessment revealed significant
decreases in rCMRGlc (ROI/ref) in several cortical brain
regions (Table 4). Both the AD-P group and the AD-S
group showed significant decreases in rCMRGlc in various
Table 2 Changes in PIB retention (ROI/ref) in four Alzheimer patients undergoing repeated PET scans
GroupFr Par TempOcc StriatumPonsSWMCb
Mean abs (% Diff)
Three of the patients underwent two PET investigations within 12 h (T1–3) while one Alzheimer patient (T4) underwent a second PET
study after 20 days; AD 1 to 3 were retested within 12 h; AD 4 was retested after 20 days; percentage difference calculated:
[(R?T)/(R+T)] · 200; ROI = region of interest; ref = reference; SD = standard deviation.
Follow-up of amyloid in Alzheimer’s diseaseBrain (2006), 129, 2856–2866 2859
cortical brain regions at 2.0 6 0.5 years of follow-up versus
baseline (Table 4). Cortical rCMRGlc values were in general
the AD-S group (Table 4). Owing to technical problems at
follow-up data are missing for two of the patients (08, 20).
For one Alzheimer patient (09) SUV values were generated.
analysis were based upon 13 Alzheimer patients.
Comparison of changes in regional PIB
retention and FDG uptake at follow-up
In order to compare the changes over time in PIB retention
and rCMRGlc, we focused our analysis on the parietal
region, where a significant correlation between rCMRGlc
and PIB was present both at baseline (Klunk et al., 2004)
(Fig. 2A) (P = 0.002) and at follow-up (Fig. 2B) (P = 0.009).
Low rCMRGlc values were defined as those >1 SD below the
mean value for HC and high PIB values as those >1 SD above
the mean value for the HC subjects.
At follow-up (Fig. 2B), most of the Alzheimer patients
showing changes of <3 in the MMSE score, and high
PIB retention at baseline, showed only slight changes in
PIB ROI/ref values at follow-up, and a slight decrease in
rCMRGlc. The five patients with low MMSE scores at base-
line and a decrease in MMSE score of 3 or more at follow-up
showed clearly lower rCMRGlc values (Fig. 2B, filled circles).
Parametric images of one of these five Alzheimer patients
Fig. 1 Comparison of PIB retention in the frontal cortex (Fr), parietal cortex (Par), temporal cortex (Temp), occipital cortex (Occ),
striatum, pons, subcortical white matter (SWM) and cerebellum (Cb) between healthy controls (HC) and Alzheimer patients at baseline
(AD 1) and follow-up (AD 2). Mean 6 standard deviation. Number of Alzheimer patients = 15 and healthy age-matched controls = 6.
Significant difference between groups (HC versus AD 1 and AD 2, respectively) indicated with *P < 0.05; **P < 0.01; ***P < 0.001;
#significant difference between AD 1 and AD 2 (paired t-test; P < 0.05). Missing data for one patient with Alzheimer’s disease
owing to errors in the scanning procedure at baseline.
Table 3 Changes in PIB retention (ROI/ref) in the frontal cortex (Fr ctx), parietal cortex (Par ctx), temporal cortex (Temp
ctx), cingulum posterior (Cing post), occipital cortex (Occ ctx), striatum, pons, subcortical white matter (SWM) and
cerebellum cortex (Cb ctx) in 10 Alzheimer’s patients cognitively relatively stable at follow-up changes of less than 3 MMSE
scores (AD-S) and 5 Alzheimer patients with progression in cognitive decline (change of 3 MMSE scores or more at
AD-S (n= 10) AD-P (n= 5)
Baseline Follow-up (P-value)Baseline Follow-up (P-value)
1.86 6 0.74
1.81 6 0.55
1.51 6 0.55
1.84 6 0.65*
1.49 6 0.41
1.97 6 0.69
1.81 6 0.24
1.73 6 0.31
1.09 6 0.08
1.90 6 0.72 (0.346)
1.85 6 0.55 (0.344)
1.52 6 0.37 (0.740)
1.88 6 0.63 (0.480)
1.54 6 0.45 (0.291)
1.92 6 0.69 (0.292)
1.83 6 0.32 (0.828)
1.69 6 0.26 (0.642)
1.07 6 0.12 (0.668)
2.38 6 0.35
2.21 6 0.27
1.82 6 0.22
2.43 6 0.33*
1.89 6 0.33
2.23 6 0.30
1.64 6 0.22
1.72 6 0.29
1.06 6 0.08
2.32 6 0.25 (0.684)
2.18 6 0.26 (0.790)
1.78 6 0.19 (0.737)
2.29 6 0.32 (0.441)
2.02 6 0.39 (0.057)
2.35 6 0.34 (0.271)
1.65 6 0.22 (0.940)
1.71 6 0.21 (0.923)
1.08 6 0.08 (0.051)
Mean values 6 standard deviation; probabilities were calculated by using Student’s paired t-test; S = stable/normally progressing Alzheimer
patients; P = faster progressing Alzheimer patients; *significant difference between AD-S and AD-P group (P < 0.05); missing data for
one patient owing to errors in the scanning procedure at baseline.
2860 Brain (2006), 129, 2856–2866 H. Engler et al.
(02), based on PIB (ROI/ref) and rCMRGlc (ROI/ref) ratios
at baseline and follow-up, are presented in Fig. 3.
Three Alzheimer patients (04, 12, 14; Fig. 2B) with high
MMSE scores and low PIB retention at baseline (Klunk et al.,
2004) showed unchanged MMSE scores and low PIB reten-
tion at follow-up, although some increase in cortical PIB
retention was observed in all three patients. Patient 14
showed a slight increase in PIB retention in the frontal
and parietal cortices and Patient 04 showed a slight increase
in PIB retention in the frontal and temporal areas with some-
what decrease in rCMRGlc but within the range for HC.
A fourth Alzheimer patient (05, an 82-year-old man,
APOE «2/4 carrier) showed relatively unchanged PIB reten-
tion, at the lower level for these patients (Fig. 2B). His
MMSE score (20 out of 30) at follow-up was similar to
that at baseline although his relatives were complaining of
increasing problems in coping with daily activities of living.
The OHC, who showed high PIB retention at baseline
(Klunk et al., 2004), showed unchanged PIB retention and
rCMRGlc in the frontal cortex and striatum at follow-up. A
slight increase in PIB retention was observed in the temporal
and parietal cortices at follow-up. The glucose uptake was in
these brain regions unchanged or slightly increased, respec-
tively. The observed changes in PIB retention and rCMRGlc
were <1 SD compared with test–retest data. The OHC
showed quite normal cognitive performance in neuropsy-
chological test at follow-up.
Correlation between cognition, PIB
retention and rCMRGlc
Correlation analysis was performed to investigate the rela-
tionship between cognitive status, measured by MMSE score,
RAVL test, PIB retention and rCMRGlc. The MMSE score
showed significant negative correlation with PIB retention
at baseline in three areas: the frontal cortex (r = ?0.64;
P = 0.010), parietal cortex (r = ?0.60; P = p.018) and occi-
pital cortex (r = ?0.56; P = 0.028). There were, however, no
significant correlations between MMSE score and PIB reten-
tion at follow-up. Changes in MMSE score and PIB retention
expressed as percentages of baseline values did not show
Significant correlation was observed between MMSE score
and rCMRGlc both at baseline and at follow-up. Two brain
regions showed significant positive correlations at baseline,
the parietal cortex (r = 0.56; P = 0.005) and temporal cortex
(r = 0.51; P = 0.044). Four areas showed significant positive
correlation at follow-up, namely the frontal cortex (r = 0.74;
P = 0.002), parietal cortex (r = 0.79; P = 0.001), temporal
cortex (r = 0.56; P = 0.036) and cerebellar cortex (r = 0.64;
P = 0.013). A significant positive correlation was also
observed between percentage change in MMSE score
and percentage change in rCMRGlc in the parietal cortex
(r = 0.59; P = 0.027).
The RAVL test score (expressed as Z-score) showed
significant negative correlation with PIB retention at
Table 4 Changes in regional cerebral glucose metabolism (rCMRGlc; mmol/min/100 ml) in different cortical brain regions, caudate nucleus, whole brain and pons in
nine Alzheimer patients cognitively relatively stable at follow-up and in four patients showing cognitive decline
AD-stable (AD-S) n = 9
AD-progressive (AD-P) n = 4
Cing post dx
33.98 6 10.51
27.65 6 10.28 (0.0019)
38.29 6 9.50
31.30 6 10.27 (0.0145)
24.29 6 4.56**
19.44 6 3.42** (0.0448)
Cing post sin
35.67 6 11.13
29.45 6 9.38 (0.0019)
40.05 6 10.63
33.25 6 9.27 (0.0556)
25.81 6 2.70**
20.69 6 3.10** (0.0564)
Fr ctx dx
35.48 6 10.76
28.71 6 7.74 (0.0081)
37.75 6 12.09
30.85 6 7.32 (0.0500)
30.36 6 4.78
23.91 6 7.23 (0.0700)
Fr ctx sin
36.16 6 8.44
30.36 6 7.56 (0.0069)
38.42 6 9.30
33.18 6 4.25 (0.0614
31.67 6 2.11*
24.02 6 6.87 (0.0592)
Par ctx dx
31.49 6 8.76
25.67 6 8.87 (0.0091)
35.31 6 6.43
29.26 6 8.22 (0.0486)
22.90 6 7.38*
17.61 6 3.09** (0.0964)
Par ctx sin
33.67 6 7.38
27.41 6 8.10 (0.0041)
36.65 6 6.48
30.65 6 7.66 (0.0430)
26.96 6 4.36**
20.11 6 1.83** (0.0964)
Par temp dx
30.44 6 6.47
24.43 6 8.96 (0.0058)
32.81 6 6.11
27.77 6 8.75 (0.0720)
25.09 6 3.54*
16.90 6 2.81** (0.0273)
Par temp sin
32.84 6 7.18
26.01 6 9.01 (0.0037)
35.08 6 7.56
29.29 6 8.71 (0.0503)
27.78 6 2.06*
18.62 6 4.12** (0.0356)
Caudate nucleus dx
41.55 6 8.49
36.50 6 6.18 (0.0941)
43.47 6 9.59
37.36 6 5.87 (0.1417)
37.25 6 2.50
34.58 6 7.34 (0.5180)
Caudate nucleus sin
42.39 6 6.89
33.93 6 6.59 (0.0042)
43.92 6 7.75
35.05 6 7.69 (0.0320)
38.95 6 2.66
31.41 6 1.90 (0.0342)
32.47 6 4.57
27.25 6 4.65 (0.0051)
35.42 6 4.96
28.31 6 5.01 (0.0453)
30.34 6 3.04
24.85 6 2.93 (0.0400)
24.20 6 4.03
22.54 6 3.32 (0.2336)
24.63 6 4.66
22.16 6 3.90 (0.2138)
23.26 6 2.30
23.41 6 1.85 (0.9030)
There were technical problems with the18F-FDG investigations in two subjects in the AD-S group and one subject in the AD-P group (data not included); mean values 6 standard deviation;
P-values (paired t-test) indicate difference between baseline and follow-up; *significant difference between AD-S and AD-P groups (Student’s t-test); P < 0.05; **P< 0.01.
Bold indicates significant P values of 0.05 and less.
Follow-up of amyloid in Alzheimer’s diseaseBrain (2006), 129, 2856–28662861
follow-up in four areas: the frontal cortex (r = 0.67; P =
0.009), parietal cortex (r = 062; P = 0.018), cingulum poster-
ior (r = 0.64; P = 0.014) and striatum (r = 0.62; P = 0.018)
Significant positive correlation was observed between
RAVL test score (expressed as Z-score) and rCMRGlc
at follow-up in seven areas, the frontal cortex (r = 0.77;
P = 0.004), parietal cortex (r = 0.85; P = 0.001), temporal
cortex (r = 0.76; P = 0.005), cingulum posterior (r = 0.90;
P = 0.001), occipital cortex (r = 0.68; P = 0.015), striatum
(r = 0.69; P = 0.012) and cerebellum (r = 0.62; P = 0.031)
the amyloid PET ligand PIB, and FDG, in a group of 16
Alzheimer patients. When re-examined, the Alzheimer
representsa 1.5–2.5-year follow-upwith
0.4 0.6 0.81.0 1.2 1.41.6 1.82.0 2.2 2.4
PIB SUV (ROI/ref)
0.4 0.6 0.81.0 1.2 1.41.6 1.82.02.22.4
PIB SUV (ROI/ref)
Fig. 2 Correlation between rCMRGlc (ROI/ref) and PIB retention (ROI/ref) in the parietal cortex of individual Alzheimer patients at
baseline (A) and at follow-up (B). Dotted lines indicate mean values for the healthy controls plus 1 SD for PIB (mean = 1.35) and minus 1 SD
for rCMRGlc (mean = 1.47). Open circles represent Alzheimer patients cognitively relatively stable, with changes in MMSE score of <3.0 at
follow-up (AD-S). Filled circles represent Alzheimer patients who deteriorated cognitively with a decrease in MMSE score of >3.0 at
follow-up (AD-P). Only SUV rCMRGlc values were available for one patient (09) indicated with filled squares. Activity in the parietal
cortex was normalized to that in the pons as regards glucose uptake.
2862 Brain (2006), 129, 2856–2866H. Engler et al.
patients, as earlier, showed significantly higher retention of
PIB in several cortical brain regions and the striatum com-
pared with age-matched HC. No significant change in regio-
nal PIB retention was observed at follow-up versus baseline.
The group, however, was somewhat heterogeneous, showing
small variations (both increase/decrease) in PIB retention.
The number of patients examined in the test–retest study is
too small to give a definitive answer as to the accuracy of
the method, and studies with larger numbers are needed.
Similar variations in test–retest PIB retention, using the
reference Logan model, have recently been reported (Price
et al., 2005).
Interestingly, PIB retention remained unchanged despite
the fact that the Alzheimer patients showed a decrease
in rCMRGlc and a subgroup showed significant clinical dete-
rioration (AD-P) at follow-up. This PIB retention was gen-
erally higher in the AD-P group than in the Alzheimer
patients with less progression of disease at follow-up
(AD-S group). The cortex cinguli posterior, which showed
the highest PIB retention in the AD-P group, significantly
differed from the corresponding brain area in the AD-S
group. It seems also that the patients with the highest parietal
PIB retention (both at baseline and follow-up) showed
the largest decrements in rCMRGlc. A larger number of
Alzheimer patients are needed to estimate the significance
of these observations.
The results of the present study reveal relatively stable PIB
retention in patients with mild Alzheimer’s disease over a
mean time period of 2 years (1.5–2.5 years) despite progres-
sive deterioration in rCMRGlc as well as a clear decrease in
cognitive function in some cases. This relatively stable PIB
retention, with only minor variations (increase/decrease),
might reflect a dynamic process in amyloid deposition reach-
ing an equilibrium. The results are in agreement with those
in earlier human post-mortem studies showing a dynamic
balance between amyloid deposition and resolution in senile
plaques, or amyloid burden (Hyman et al., 1993). An in vivo
multiphoton microscopy study of thioflavin-S-positive
senile plaques in the Tg2576 transgenic mouse model of
Alzheimer’s disease did not reveal detectable changes in
plaque size over extended periods (Christie et al., 2001).
Nonetheless, the authors described rare examples of growth
or shrinkage of individual plaques, and the appearance of
new plaques between imaging sessions, and they suggested
that glial interaction with amyloid stabilizes the size of
plaques and prevents continued enlargement. The dynamic
changes over time of the amyloid depositions in the brain
may produce variation in the binding properties of amyloid
to the tracer. Structural changes in the evolution of the
plaques, with an increment in the number of neuritic
plaques, could, for example, result in a slight decrease in
the binding of PIB. Furthermore, concomitant inflammatory
processes also influence the binding properties (Su et al.,
1996). Our results suggest that the accumulation of
fibrillar amyloid may increase to a certain level and then
become relatively stable, while degeneration of the neurons
continues. It is quite possible that the amyloid plaques might
be growing but that only a relatively stable part is accessible
to exogenous tracer binding. It was recently suggested
that there are three distinct binding sites for thioflavin com-
pounds on b-amyloid peptide fibrils (Lockhart et al., 2005).
Further PET multi-tracer binding studies involving visuali-
zation of not only amyloid but also inflammatory processes,
microglial activation and perhaps also neurotransmitter
activity might provide further insight into these ongoing
processes in the brains of patients with Alzheimer’s disease.
The fact that high cortical PIB retention can be observed in
patients with only mild cognitive impairment (Lopresti et al.,
2005) indicates that further studies with PIB should now be
performed among subjects at a genetically high risk of
developing Alzheimer’s disease.
Since all patients with Alzheimer’s disease in this
study were on cholinesterase inhibitor treatment (and
some also on memantine treatment) at the follow-up PET
studies we cannot exclude the possibility of interactions
between cholinesterase inhibitor treatment and amyloid
deposition (Ballard et al., 2005; Inestrosa et al., 2005).
The results of several PET studies have revealed stabilizing
effects on cognition, rCMRGlc and cerebral blood flow
(CBF) after long-term treatment with cholinesterase inhibi-
tors (Nobili et al., 2002; Stefanova et al., 2003; Tune et al.,
2003; Stefanova et al., 2005). Experimental data also suggest
Fig. 3 Parametric images concerning PIB (ROI/ref) and rCMRGlc
(ROI/ref). The Alzheimer patient showed deterioration in
cognition at follow-up (7 points in MMSE). The images show
only slightly increased PIB retention (upper) but a pronounced
decrease in glucose uptake (lower).
Follow-up of amyloid in Alzheimer’s diseaseBrain (2006), 129, 2856–28662863
that cholinesterase inhibitors might influence amyloid
deposition (Francis et al., 2005). Only a placebo-controlled
study analysing the effect of cholinesterase inhibitor treat-
ment on PIB retention can rule out this possibility. The
relatively low decrease in MMSE score measured at follow-
up might be expected to be larger if the Alzheimer patients
had not been on cholinesterase inhibitor treatment (Birks,
2005). The patients in this study represented those with mild
Alzheimer’s disease, for whom it is known that the MMSE is
a relativelyinsensitive measure
An extensive evaluation of the outcome of consecutive
neuropsychological tests in this patient group is ongoing
and will be presented later. In the present study we presented
the outcome of the episodic memory test, RAVL test, at
The three patients with low PIB retention and normal
rCMRGlc presented in the baseline study (Klunk et al.,
2004) still showed low PIB retention and normal rCMRGlc
at follow-up. Their MMSE scores at follow-up were still
high and the RAVL test Z-scores were low. These patients
may have very slow development of the disease with a
slight or no increment in amyloid deposition over time.
PIB SUV (ROI/ref)
-5-4 -3 -2-1012
-5-4 -3 -2-1012
Fig. 4 Correlation between RAVL and PIB retention (ROI/ref) (A) and RAVL and rCMRGlc (ROI/ref), respectively (B), in the parietal
cortex of individual Alzheimer patients at follow-up. Open circles represent Alzheimer patients cognitively relatively stable, with
changes in MMSE score of <3.0 at follow-up (AD-S). Filled circles represent Alzheimer patients who deteriorated cognitively with a
decrease in MMSE score of >3.0 at follow-up (AD-P) only SUV rCMTglc values were obtained for patient 9. Patient 9 is indicated as filled
squares in both A and B. Activity in the parietal cortex was normalized to that in the pons as regards glucose uptake. Z-scores of RAVL were
generated from raw scores by comparing data with the mean value and standard deviation of a large control group.
2864 Brain (2006), 129, 2856–2866H. Engler et al.
In addition to showing impairment in neuropsychological
testing, these subjects also showed deficits in cortical
rCMRGlc before initiation of cholinesterase inhibitor treat-
ment. One of the subjects showed a pathologically low CSF
Ab 1-42 level (365 pmol/ml) but a low CSF tau value (75
pmol/ml) This illustrates the complexity in findings, as also
recently described in CSF data by Iqbal et al. (2005). These
three patients should today be considered as mild cognitive
impairment patients. We can conclude that in two consecu-
tive PIB PET studies we have measured consistently low
cortical PIB retention and unchanged MMSE values in
these subjects. Their clinical symptoms may have a different
pathological basis than Alzheimer’s disease.
The OHC who showed high PIB retention and relatively
well-preserved rCMRGlc at baseline (Klunk et al., 2004) still
showed high PIB retention at follow-up but normal cognitive
performance in testing. This finding illustrates and supports
the assumption that amyloid deposition can be detected in
the brains of elderly subjects without cognitive deficits
(Schmitt et al., 2000). Such amyloid deposition might solely
be related to ageing. We cannot exclude the possibility that it
may lead to cognitive symptoms in future years.
The PIB retention data in this study are presented as
ROI/ref values for 60 min of scanning time. Sixty minutes
were chosen in order to be able to compare follow-up data
with baseline data. The cerebellum was chosen as reference
region. The influence of the time interval chosen for late scan
ratio has recently been investigated by Lopresti et al. (2005).
PIB data from Alzheimer patients as well as control subjects
were used to compare the intervals 40–60 and 60–90 min.
The 60–90 min interval gave slightly better values than the
40–60 min interval in the frontal cortex, slightly worse values
in the mesial-temporal cortex and the same values in the
posterior cingulate gyrus (Lopresti et al., 2005). In the same
study, several methods of analysis of PIB data were com-
pared, and no method was better than the late scan ratio.
An important question is whether changes in CBF in
patients with Alzheimer’s
the measured PIB retention in cortical brain regions of
Alzheimer patients. We tested this crucial question recently
in a monkey model (Blomquist et al., 2005). The effect of
changes in CBF on the late target to cerebellum ratio of PIB
was tested. An increase in CBF of 50–80% caused an increase
in target to reference of ?10% in the monkey. Since PIB
redistribution has already taken place at 60 min, when PIB
retention is measured, CBF changes in Alzheimer patients
will probably have only a minor influence on the late scan
ratio of PIB.
The results of this study show, for the first time, that
amyloid deposition in vivo as measured by using the PET
ligand PIB remains high but stable despite further decreases
in cortical rCMRGlc and cognitive function. It is possible
that the increase in amyloid deposition in the brains of
Alzheimer patients represents a dynamic pathological brain
process reaching equilibrium or plateau very early in the
course of Alzheimer’s disease. The time course for increasing
PIB retention might be different from ongoing processes
regarding cortical hypometabolism, which might be more
related to cognitive function. This was also reflected in
the observation of a significant correlation between MMSE
score and rCMRGlc both at baseline and follow-up. This
indicates a link between these two parameters that seems
to be stronger than with PIB. This assumption was also
strengthened by the stronger positive correlation between
RAVL Z-score and rCMRGlc in the parietal cortex compared
with the negative correlation between RAVL and PIB
retention in the parietal cortex at follow-up. The combina-
tion of PIB and rCMRGlc in a dual tracer application might
increase understanding and interpretation of the underlying
pathological mechanisms in Alzheimer’s disease. The small
changes in PIB retention observed in patients with mild
Alzheimer’s disease over 2 years strongly suggest that PIB
might be a suitable tracer in studies involving evaluation of
the outcome of drug treatments with a focus on amyloid
clearance. Further studies are necessary to enlarge the patient
material and to study the time course of Alzheimer’s disease,
including pre-symptomatic carriers. In addition, other sui-
table PET tracer combinations must be explored in order
to understand better the in vivo pathological changes in
Financial support from the Swedish Medical Research
Council (project 05817, A.N.), the Foundation for Old
Servants (A.N.), the Stohnes foundation (A.N.), the KI
foundation (A.N.), the Swedish Brain Power (A.N., B.L.) and
the EC-FP5-project NCI-MCI, QLK6-CT-2000-00502 (A.N.)
is gratefully acknowledged. We thank all the patients who
have participated in this study as well as their relatives. We
thank the staff at the Department of Geriatric Medicine,
Karolinska University Hospital Huddinge and Uppsala
Imanet for their dedication and high level of professionalism
in performing these studies and Mrs Marianne Grip for
professional help with preparation of the manuscript.
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