Imaging Inflammation in Elders With Different Clinical and Biomarker Profiles of Alzheimer's Disease
Phase: PHASE2
Conditions: Alzheimer's Disease
Sponsor: Patrick Lao

Imaging Inflammation in Elders With Different 
Clinical and Biomarker Profiles of Alzheimer's 
Disease 
NCT02831283
Protocol Version: June 3, 2022

1 Introduction 
This document is a protocol for a human research study. This study is to be conducted according 
to US and international standards of Good Clinical Practice (FDA Title 21 part 312 and 
International Conference on Harmonization guidelines), applicable government regulations and 
Institutional research policies and procedures.  
1.1 Background 
1.1.1. Background on Alzheimer’s disease 
While amyloid plaques are a pathological hallmark of Alzheimer’s disease (AD), whether 
β-amyloid is the causal agent of neurodegeneration in AD remains unclear. Evidence supporting 
the hypothesis that amyloid is a primary contributor to AD pathogenesis (the amyloid-first 
hypothesis) include the finding that genetic mutations responsible for inherited forms of AD 
cause increased amyloid production. Studies using positron emission tomography (PET) to 
measure amyloid plaque burden in vivo have demonstrated amyloid positivity before obvious 
atrophy or cognitive decline in healthy older controls. While tau positive neurofibrillary tangles 
represent the second hallmark of AD, tau mutations are seen in patients with variants of 
frontotemporal dementia, not AD. The propagation of tau pathology is thought to follow 
amyloidosis, suggesting amyloid in some way induces tau mediated degeneration. However, the 
link between amyloid and tau not clear as there are spatial and temporal distinctions. For 
example, bulk of amyloid burden in medial parietal cortex and frontal cortex early on, while tau 
burden begins in medial temporal cortex.  
 While β-amyloid has been shown to have direct neurotoxic effects in vitro, these effects 
are seen at concentrations of β-amyloid much higher than found in human brain[1].Therefore, 
amyloid may not confer significant direct toxicity. Rather, amyloid may induce secondary injury 
via activation of immune response. At physiologic concentrations, β-amyloid activates 
microglia[2], triggering a pro-inflammatory cascade as these resident neuroimmune cells attempt 
to clear β-amyloid from the brain[3]. This inflammatory response may be more toxic than β-
amyloid itself, as the resulting release of cytokines, activation of complement, and phagocytosis 
cause loss of neurons[4]. This inflammatory response may be the link between amyloid and tau, 
as inflammation can result in hyper-phosphorylation of tau, leading to the destabilization of tau 
filaments and aggregation of the neurofibrillary tangles that make up the second pathological 
hallmark of AD[5]. In turn, extracellular tau activates microglia[6], which could create a positive 
feedback loop of inflammation and tau pathology that propagates, independent of amyloid 
burden. The inflammation/tau relationship could also occur independently of the presence of 
amyloid. Indeed, autopsy findings of pre-tangle pathology occurring in midlife prior to 
amyloidosis[7] suggest that tauopathy may begin prior and independent to significant amyloid 
plaque deposition. Since amyloid burden correlates poorly with cognition[8], inflammation may 
be a more important mediator of neurodegeneration in AD. 
Measuring inflammation in vivo may also have an important role in determining prognosis 
and evaluating progression of disease in AD patients. Up to 30% of cognitively normal elders are 
amyloid positive on PET. Determining predictors of future cognitive decline among amyloid-
positive elders would allow us to distinguish incidental amyloid-positivity from that due to 
incipient Alzheimer’s disease in an elderly population. Furthermore, identifying the relative 
impact of inflammation on cognition may justify a more strategic approach for the development 
of anti-inflammatory therapeutics for AD. 
 

1.1.2. Background on PET imaging of inflammation 
Inflammation can be quantified in vivo using PET imaging with radioligands that bind to 
the 18 kDa translocator protein (TSPO, formerly called the peripheral benzodiazepine receptor). 
Brain inflammation activates microglia and causes them to over-express TSPO[9]. Measurement 
of microglia activation, defined by the number of available TSPO binding sites, is used as a 
surrogate bio-marker of active brain inflammation[10].  PET imaging can quantify TSPO density 
in vivo using radioligands that cross the blood-brain barrier and bind to TSPO sites. 11C-(R)-PK 
11195, the prototypical TSPO radioligand, has been used to measure neuroinflammatory changes 
in several diseases, including Alzheimer’s disease[10] and 11C-(R)-PK 11195 binding has been 
shown to correlate with Mini-Mental State Examination score in patients with Alzheimer’s 
disease[11]. However, there have been conflicting reports about whether 11C-(R)-PK 11195 
binding is increased in Alzheimer’s disease or not[10, 12, 13]. Whether 11C-(R)-PK 11195 
binding is increased in MCI appears even less conclusive. For instance, [14] found a small 
increase in 11C-(R)-PK 11195 in PIB-positive patients with MCI relative to controls, but other 
studies found no increase in patients with MCI, even those who later progressed to dementia[12, 
13]. 
Limitations of 11C-(R)-PK 11195 have led to development of second generation TSPO 
radioligands. One such radioligand is PBR28, which has high in vitro affinity (~ 1 nM) for 
TSPO. In nonhuman primates, [11C]PBR28 has high brain uptake and fast washout[15], which 
allows reliable PET quantification. In addition, more than 80% of brain uptake is displaceable by 
nonradioactive TSPO ligands in nonhuman primates[15]. This means that over 80% of uptake is 
specifically bound to TSPO. [11C]PBR28 has high enough signal-to-noise ratio to localize 
inflammation to relatively small areas in humans with lacunar stroke[16]. Animal toxicology 
studies and early clinical PET studies have shown safety and tolerability of [11C]PBR28. In 
addition, the radiation exposure after injection of activity needed for reliable PET measurement 
(10-20 mCi) is well within limits for human research studies.  
The main limitation of 11C-PBR28, shared by all tested second generation TSPO 
radioligands, is differential affinity for the target protein[17]. This differential affinity is caused 
by the rs6971 polymorphism on the TSPO gene that causes a non-conservative amino acid 
substitution, resulting in three patterns of TSPO binding. Subjects without the polymorphism 
have high affinity binding for PBR28 and are referred to as high affinity binders (HABs). 
Homozygotes have low affinity binding and are referred to as low affinity binders (LABs). 
Heterozygotes express both high and low affinity TSPO and are referred to as mixed affinity 
binders (MABs). LABs are easily identified by PET due to negligible 11C-PBR28 binding in 
vivo; however, PET cannot easily resolve the difference between HABs and MABs, and MABs 
have, on average, 22% less total 11C-PBR28 binding than HABs[18]. Previous work from our 
laboratory demonstrated that correcting in vitro binding data for rs6971 genotype improves the 
ability of 3H-PBR28 to detect differences in TSPO density in schizophrenia and control brain 
tissue[18]. This strategy of TSPO genotype correction can be applied to PET imaging to remove 
confounding effects of differences in TSPO affinity. 
We recently demonstrated that 11C-PBR28 binding is greater in AD patients than in MCI 
patients or older controls[19]. Areas with greatest binding included parietal and temporal 
cortices, regions with high amyloid burden. No difference in binding was seen in cerebellum, a 
region typically spared of pathology in AD. However, 11C-PBR28 binding did not correlate with 
PIB binding, suggesting a disassociation between inflammation and amyloid burden, particularly 

in more severe patients. Instead, 11C-PBR28 binding correlated with brain atrophy and lower 
cognitive scores, suggesting inflammation is associated with neurodegeneration. This study used 
arterial sampling to calculate 11C-PBR28 binding using kinetic modeling and required a 
relatively large sample size (19 AD patients, 10 MCI patients, and 13 controls) due to relatively 
high variance of the PET data to see the reported effect. While kinetic modeling is considered the 
gold standard method of PET quantification, a less invasive method with less variance would be 
preferred to improve subject tolerability and increase statistical power. 
We developed a simplified ratio method using the cerebellum as a pseudo-reference 
region[20]. This method calculates standardized uptake value ratios (SUVR) similar to methods 
used for other radioligands such as PIB. This method removes the need for arterial sampling and 
increases sensitivity for detecting differences in 11C-PBR28 binding among AD and MCI 
patients and healthy controls by reducing variance. For example, using the SUVR method we 
were able to detect significantly increased 11C-PBR28 binding in the hippocampus in MCI 
patients, while this difference was only at the trend level using the gold standard method. 
We already have preliminary data suggesting that 11C-PBR28 binding increases with AD 
progression. Eleven patients (7 AD, 4 MCI) and 7 older controls had 11C-PBR28 binding at 
baseline and then returned for repeat imaging after mean follow up of 2 years. The patients 
showed greater binding in the entorhinal cortex at follow up, while there was no difference 
between baseline and follow up scan in the controls. Since the entorhinal cortex is a region with 
high tau burden, these data suggest that inflammation may be increasing in parallel with 
neurodegeneration in the progression of AD. 
1.1.3. Background of PET imaging of amyloid. 
While post-mortem examination is required to definitively demonstrate the presence of 
amyloid plaques in suspected cases of AD, PET imaging allows measurement of amyloid burden 
in vivo. 18F-florbetaben (FBB, formerly known as BAY94-9172) binds to fibrillar amyloid 
plaques in vivo[21]. FDA approved for commercial use to exclude AD. Dosimetry studies have 
shown that radiation exposure from this radioligand is similar to that from other 18F radioligand 
commonly used in human PET studies and clinical practice.  
Amyloid PET imaging allows in vivo detection of amyloid plaques to improve risk 
assessment and diagnosis of AD. Therefore, clinical evaluation using current neuroimaging 
techniques can be used to identify the following four groups:  
1) Amyloid-positive with cognitive impairment (AD). Amyloid-positive patients who 
already have cognitive impairment are most likely to be clinically expressing AD pathology[22, 
23], either in the prodromal stage (MCI) or dementia stage (AD dementia). Patients with MCI 
who are amyloid-positive on PET convert to AD dementia at reported rates as high as 80% 
within 3 years[24, 25]. 
2) Amyloid-positive without impairment (preclinical AD). Cognitively normal subjects 
who are amyloid-positive show greater rates of memory decline than those who are amyloid-
negative[26]. These subjects represent a preclinical stage of AD[27]. 
3) Amyloid-negative without impairment (normal aging). Cognitively normal subjects 
who are amyloid-negative on PET, and lack signs of neurodegeneration from other biomarkers, 
have no discernible evidence of AD pathology[28].  While such subjects may develop 
pathological changes in the future, at the time of investigation they represent normal aging. 
4) Amyloid-negative with cognitive impairment (impairment due to suspected non-
AD pathophysiology). Notably, 29% of elders with MCI in a recent population-based study had 

evidence of neurodegeneration without amyloidosis[29], and longitudinal PET studies have 
shown that amyloid-negative MCI patients progress to dementia at rates ranging from 0 - 23% 
per year[8, 24, 30, 31]. Evidence of neurodegeneration (i.e., hippocampal atrophy on MRI or 
hypometabolism on FDG PET) without amyloidosis has been termed “suspected non-AD 
pathophysiology”[32], and subjects with this biomarker profile are thought to mostly likely 
represent patients with non-Alzheimer’s diagnoses[28]. 
Taken together, these four groups may be considered different categories of disease along 
the aging spectrum.  
1.2 Investigational Agent 
11C-PBR28 is a PET radioligand that binds to the 18 kDa translocator protein (TSPO), a 
marker of inflammation. 11C-PBR28 has previously been administered in humans.  
18F-Florbetaben (Neuraceq) has FDA approval for human use in evaluation of Alzheimer’s 
disease. We will use 18F-Florbetaben in this study for research purposes. 
Both radioligands will be administered in tracer doses. 11C-PBR28 will be administered at 
activity of up to 20 mCi per injection. 18F-Florbetaben will be administered at activity up to 8.1 
mCi per injection. mCi per injection. 
1.3 Preclinical Data 

1.5 
Dose Rationale and Risk/Benefits 
Based on prior human experience and dosimetry data, we expect the proposed injected activity of 
11C-PBR28 (up to 20 mCi) to be safe without expected toxicity. 18F-florbetaben is an FDA-
approved radiopharmaceutical used here for research purposes at injected activity recommended 
by the manufacturer (up to 8.1 mCi). 
2 
Study Objectives 
2.1. The primary objective is to determine the extent and spatial distribution of inflammation at 
different stages along the cognitive spectrum of aging. 
2.2. Secondary objectives are to determine how 11C-PBR28 binding in brain relates to cognitive 
performance, amyloid binding, atrophy on MRI, and CSF concentrations of markers of 
inflammation and neurodegeneration. 
3 Study Design 
3.1 General Design 
3.1.1. Subject recruitment and screening procedures 
Men and women age 50 and older will be recruited from the CUMC Washington 
Heights-Inwood Community Aging Project (known locally as WHICAP, R01 AG037212, PI 
Richard Mayeux), the CUMC Alzheimer's Disease Research Center (ADRC), and Dr. Davangere 
Devanand’s Questionable Dementia 2 (QD2) cohort, and CUMC clinics. Subjects may also be 
co-enrolled in Columbia University Medical Center protocols AAAO9758 (PI Adam Brickman), 
AAAI2752 (PI Yaakov Stern), AAAQ8249 (PI Yaakov Stern), AAAQ8096 (PI Yaakov Stern), 
AAAR4352 (PI James Noble) and AAAR6570 (PI Edward Huey). Informed consent will be 
obtained prior to study enrollment. This protocol will encourage data sharing to reduce repeat 
procedures and thus participant burden. De-identified neuroimaging data may be shared with 
IRB-approved collaborators. 
PET Imaging has ended for this study. Please note that anywhere in the protocol that 
makes mention of PET imaging, and its corresponding drugs/biologics (18F-florbetaben 
and 11C-PBR28) is only in reference to previous study design. PET imaging is no longer a 
part of the protocol but is kept in for study design comprehension. 
For subjects with cognitive impairment, prior to obtaining informed consent, a licensed physician 
who is not listed as personnel on this protocol will determine whether the subjects has capacity to 
provide their own consent. If the subject lacks consent, a legally authorized representative will be 
required to provide consent for the subject. 
Up to 150 subjects will undergo screening including history, physical examination, routine 
laboratory studies, neuropsychological testing, brain MRI, and amyloid PET imaging. If any of 

the screening procedures have been performed with in one year of the 11C-PBR 28 scan visit, 
then those procedures will not need to be repeated under this protocol. Rather the previously 
obtained results will be used to reduce participant burden. Subjects will be categorized as either 
amyloid-positive or amyloid-negative on PET. Subjects will be identified as either cognitively 
normal or cognitively impaired in consensus conference. Subjects must have Clinical Dementia 
Rating scale score of 0.5 or 1 and meet clinical criteria for either amnestic MCI (single or 
multiple-domain)[22] or Alzheimer’s disease[23] to be included in the cognitively impaired 
category. Therefore, subjects who fulfill screening criteria will be stratified into four categories: 
1) amyloid-positive with cognitive impairment, 2) amyloid-positive without impairment, 3) 
amyloid-negative with cognitive impairment, and 4) amyloid-negative without cognitive 
impairment. Hippocampal atrophy on MRI will be inclusionary criteria for group 3 and 
exclusionary for group 4, as neurodegeneration is a prerequisite for suspected non-AD 
pathophysiology and inconsistent with normal aging. Screening will include genetic analysis to 
exclude subjects homozygous for the rs6971 (Ala147Thr) SNP on the TSPO gene. Homozygous 
subjects have negligible binding on 11C-PBR28 PET imaging and must be excluded; however, 
heterozygotes may be included[18]. We request to perform screening procedures, including 18F-
florbeteben PET, on up to 150 subjects in anticipation of33% screen failure/dropout rate.
Target sample size for completers is 25 amyloid-positive elders with impairment, 25 
amyloid-positive elders without impairment, 25 amyloid-negative elders without impairment, 
and 25 amyloid-negative elders with impairment (100 subjects total). Results from a population-
based study showed that 43% of MCI patients are amyloid-negative on PET[29], and in a phase 
3 bapineuzimab trial in mild-to-moderate AD patients, 36% of ApoE4 non-carriers were negative 
on baseline amyloid imaging[44]. Therefore, significant oversampling is not anticipated to be 
necessary for the impaired amyloid-negative group. Our enrollment goal is 25 subjects per group 
to account for a 30% general screen failure/drop-out rate and a 9% prevalence of low affinity 
binders. Using subjects from WHICAP with pre-determined TSPO affinity status from GWAS 
will reduce the screen failure rate.  
3.1.2 Neuropsychological data analysis 
Selected neuropsychological tests scores will be combined into four composite scores 
(memory, language, executive/speed, and visuospatial)[45]. Memory testing will include 
Selective Reminding Test and Benton Visual Retention Test (BVRT), Recognition Memory 
Multiple Choice version. Z-scores for each cognitive measure will be calculated and averaged to 
create a composite z-score for each domain. These factor domain scores will be subsequently 
averaged to produce a composite cognitive z-score to indicate mean cognition. Higher z-score 
indicates better cognitive performance.  
3.1.3. Imaging procedures 
One brain MRI will be performed on each subject using a 3 T Philips scanner. Sequences 
performed will include 3D T1 (MPRAGE, 180 slice 1 mm resolution, 256 x 256 voxel count) for 
volumetric analysis and clinical sequences to exclude subjects with visible evidence of cerebral 
amyloid angiopathy (microbleeds on GRE), more than moderate white matter disease on T2, or 
other intracranial pathology unrelated to AD. PET scans will take place on a Biograph mCT PET 
scanner (Siemens Healthcare) at the CUMC Kreitchman PET Center. Subjects will have one 18F-
florbetaben (Neuraceq) PET scan (up to 8.1 mCi) to determine amyloid status[46]. Subjects will 
have one PET scan with 11C-PBR28 (injected activity 10-20 mCi). PET imaging will be 

performed without arterial sampling. 11C-PBR28 will be synthesized by the CUMC PET 
Department Radiochemistry Laboratory. 18F-florbetaben will be purchased from Piramal 
Imaging. Cognitively normal subjects will not be informed of 18F-florbetaben PET scan results, 
as we do not know the clinical significance of a positive amyloid scan in cognitively normal 
people. Subjects will not be informed of 11CPBR28 PET scan results, as this scan is used only in 
research and have not yet been validated for clinical use. Subjects will be informed if a clinically 
important abnormality is detected on PET imaging (e.g., brain tumor). 
Subjects will be allowed to take low dose lorazepam (0.5 - 1 mg orally one time dose) or 
equivalent dose of alternative benzodiazepine prior to the MRI, Florbetaben PET scan and/or 
PBR28 PET scan in the case that the subject is claustrophobic or otherwise unable to lay still 
without pre-medication. Low-dose one-time administration of anxiolytic medication is 
commonly done in clinical practice when patients have anxiety about undergoing the MRI and/or 
PET scan. Subjects will be informed of the risks associated with one-time administration of low-
dose benzodiazepine, including sedation, lightheadedness, motor incoordination, difficulties with 
balance, and confusion. Subjects who receive benzodiazepine pre-medication will be 
accompanied during the appointment by a member of the study staff and the PI will be available 
in the case of an adverse event. Subjects who receive benzodiazepine pre-medication will be 
instructed not to drive, handle machinery, or drink alcohol until the next day, and will be 
required to bring a companion to escort them home. Standard doses of benzodiazepines, 
particularly lorazepam, are not expected to affect 11C-PBR28 binding to TSPO[47]. 
3.1.4 Image processing 
FreeSurfer (http://surfer.nmr.mgh.harvard.edu/), the MRI software package comprising a suite of 
automated tools for segmentation, reconstruction, and derivation of regional volumes and 
surface-based rendering, will be used for derivation of regions-of-interest (ROIs). Eleven ROIs 
will be extracted from the structural T1 image: entorhinal cortex, hippocampus, inferior temporal 
cortex, combined superior and middle temporal cortex, superior parietal lobule, inferior parietal 
lobule, precuneus, occipital cortex, prefrontal cortex, striatum, and thalamus. Age-adjusted 
hippocampal volumes will be used to define the presence or absence of neurodegeneration[28]. 
A single static 18F-florbetaben image acquired 50-70 or 90-110 min post-injection will be read 
by a trained investigator blinded to the subject’s diagnosis. A visual binary read (positive or 
negative) will be determined according to criteria established by Piramal imaging to stratify 
subjects as amyloid-positive or amyloid-negative. Semi-quantitative measurement of 18F-
florbetaben binding will be used as an outcome measure for baseline amyloid burden (see 
below). 
18F-Florbetaben and 11C-PBR28 PET images will be analyzed according to previously 
published methods for 11C-PBR28[19]. In brief, Freesurfer based ROIs will be applied to 
coregistered PET images. Correction for partial volume effects using a region-based voxel-wise 
method[48] will be applied. Regional time-activity curves using 50-70 or 90-110 min of scan 
data for 18F-florbetaben and 60-90 min for 11C-PBR28 will be extracted from the PET scans, 
including cerebellum which will be used as a reference region. Standardized uptake value ratio 
(SUVR) values will be calculated by dividing SUV values for each target region by that of the 
cerebellum 
3.1.5. Blood and cerebrospinal fluid sampling and analysis 

Subjects not recruited from WHICAP will undergo venipuncture to collect blood to test TSPO 
genotype, determined by SNP analysis using polymerase chain reaction with a pre-existing 
Taqman assay[17-19] in the Biomarkers Shared Resource of CUMC. Subjects will have the 
option of having one lumbar puncture (LP) for CSF collection. Subjects may refuse the LP and 
still participate in the other study procedures. CSF analysis will be performed by Dr. Honig to 
determine CSF concentrations of total tau, phospho-tau, β-amyloid, and markers of inflammation 
such as IL-1β, TNF-α, glial fibrillary acidic protein, S100B, and YLK-40. 
3.1.6. Procedures performed in WHICAP 
Procedures that are performed under WHICAP include screening interview, history and physical 
examination, neurological examination, neuropsychological testing, genome-wide association 
study and diagnosis made at consensus conference. To date, over 500 WHICAP subjects have 
had brain MRI and 30 have had 18F-florbetaben PET. GWAS data will be interrogated to screen 
out subjects homozygous for the rs6971 SNP.  
3.2 Primary Study Endpoints 
Because the drugs used in this study are radioligands given at tracer doses, there are no clinical 
endpoints of the study.  
The primary outcome measures are: 
1. Amount of 11C-PBR28 binding in each of the four groups.
Secondary outcome measures are: 
1. CSF concentration of inflammatory markers
2. CSF concentration of tau and phospho-tau
3.3 Secondary Study Endpoints 
N/A 
3.4 Primary Safety Endpoints 
N/A 
4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
1. Age 50 and older
2. Meet criteria for either a) amnestic mild cognitive impairment (single or mixed domain)
or mild Alzheimer’s disease, or b) have no cognitive impairment based on history, exam,
neuropsychological testing, and consensus diagnosis. MCI and mild AD patients must
have Clinical Dementia Rating scale score of 0.5 or 1. Unimpaired subjects must have
Clinical Dementia Rating scale score of 0.
3. Subjects unable to provide informed consent must have a surrogate decision maker

4. Written and oral fluency in English or Spanish
5. Able to participate in all scheduled evaluations and to complete all required tests and
procedures.
6. In the opinion of the investigator, the subject must be considered likely to comply with
the study protocol and to have a high probability of completing the study.
4.2 Exclusion Criteria 
1. Past or present history of certain brain disorders other than MCI or AD.
2. Certain significant medical conditions, which make study procedures of the current study
unsafe.
3. Contraindication to MRI scanning
4. Conditions precluding entry into the scanners (e.g., morbid obesity, claustrophobia, etc.).
5. Exposure to research related radiation in the past year that, when combined with this
study, would place subjects above the allowable limits.
6. Low affinity binding on TSPO genetic screen
7. Participation in the last year in a clinical trial for a disease modifying drug for AD.
8. Inability to have a catheter in subject’s vein for the injection of radioligand.
9. Inability to have blood drawn from subject’s veins.

6 Study Procedures 
This study will involve up to 5 outpatient visits. See schedule below. 
At the first screening visit, subjects will sign informed consent, undergo history and physical and 
neurological examination, neuropsychological testing, and have blood drawn routine safety 
laboratories and TSPO genotyping. Additional screening visit is required for brain MRI. 
Flexibility is allowed in screening procedures such that procedures may be performed in any 
order and may be performed on the same day if schedule allows for subject convenience. 
Subjects who already had screening procedures performed under WHICAP, or other CUMC 
studies listed, do not need to have them repeated unless performed more than 12 months prior to 
inclusion in this study. Up to 3 screening visits are anticipated. However, additional visits may 
be necessary if certain tests must be scheduled on different days due to scheduling. 
One visit will be for the optional lumbar puncture. Subjects may decline the lumbar puncture and 
still participate in other parts of the study. LP must be completed within 12 months of the brain 
MRI.  

Study Schedule 
Study Procedures 
Screen 
a
Study b 
Day -
60 to -
1 
Visit 1 
Visit 2 
Informed consent 
X 
Medical/psychiatric history 
X 
Inclusion/exclusion criteria 
X 
X 
X 
Blood draw for TSPO genotyping 
X 
Neuropsychological testing 
X 
MRI 
X 
Lumbar puncture c, d 
X 
Laboratory determinations e 
X 
Neurological examination 
X 
Vital signs 
X 
Height and weight 
X 
X 
Adverse events f 
X 
X 
X 
a. Screening procedures may be performed in any order and may be performed on the same day
if schedule allows for subject convenience. Subjects who already had screening procedures
performed under a different protocol do not need to have them repeated unless performed more
than 12 months prior to inclusion in this study. Up to 3 screening visits are anticipated. However,
additional visits may be necessary if certain tests must be scheduled on different days due to
scheduling.
b. Study procedures must be completed within 12 months of MRI.
c. Lumbar puncture is optional.
d. Laboratory determinations here include complete blood count, basic metabolic panel, liver
functions tests, thyroid stimulating hormone, and urinalysis.
e. 18F-florbetaben and 11C-PBR28 PET Scans have concluded for this study.
7 Statistical Plan 
7.1 Sample Size Determination 
Power analysis was performed using preliminary data from a study using 11C-PBR28 PET 
in patients with AD and MCI and healthy controls.  
For the main effects, the minimal detectable effect size is Cohen d=0.74 with the sample 
size N=30 per group, 80% of power and a two-sided test at the 5% significance level. Based on 
my preliminary data using 11C-PBR28, the effect size for the group difference between MCI and 
controls is d=1.3 (hippocampal SUVR mean (SD) of MCI: 0.98 (0.18); HC: 0.82 (0.06)), and it 
is similar (d=1.3) after adjusting for TSPO genotype. Although these preliminary results were 
not controlled for amyloid, with the assumption of additive effect of amyloid I would be able to 
 

detect the group difference between amyloid-positive MCI and amyloid-positive without 
impairment with more than enough power. For the interaction between diagnosis and amyloid,  if 
the mean differences between positive/negative amyloid among the subjects with and without 
impairment differ by d=1.06, I would be able to detect such effect size with 80% power at 5% 
significance level for a two-sided test. 
We also plan to compare 11C-PBR28 binding with the amount and distribution of binding 
with 18F-MK-6240, a tau radioligand that will be used under a separate IND (#135,864). Because 
some subjects who have 11C-PBR28 PET may not wish to also undergo 18F-MK-6240 PET, we 
need to increase the number of subjects so that a total of 60 subject receive both 11C-PBR28 and 
18F-MK-6240 PET. 
7.2 Statistical Methods 
Subjects will be stratified using a two factor design based on diagnosis (impaired vs. 
normal) and amyloid status (positive vs. negative). Target sample size is 25 subjects per group 
(100 subjects total). 11C-PBR28 binding will be the primary outcome measures. Between-group 
differences in SUVR values from 11C-PBR28 PET images will be compared in each of the 11 
ROIs. Analysis will be performed using a two-way MANCOVA with diagnosis (MCI vs. 
normal) and amyloid status (positive vs. negative) as independent variables and TSPO genotype 
status (high affinity vs. mixed affinity binder) as covariate. I will additionally test the interaction 
between diagnosis and amyloid status. This model will be fitted using PROC GLM in SAS.  
Regression models will be fit for 11C-PBR28 binding as dependent variable and cognitive 
scores, voxel count on MRI, 18F-florbetaben binding, and CSF concentrations of markers of 
inflammation and neurodegeneration as independent variables. 
7.3 Subject Population(s) for Analysis 
Subjects will be elders age 50 and older with normal cognition, mild cognitive impairment, or 
mild AD. 
 

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