Appendix E-1: Methods

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Appendix e-1: Methods

Patient selection

Patients had been referred for the diagnostic evaluation of cognitive impairment by general practitioners, neurologists, or psychiatrists, or from other institutions and underwent a standardized diagnostic protocol. All examinations were part of their routine check-up in the course of the evaluation of the patient’s neurodegenerative disorders. Patients who fulfilled inclusion criteria for AD (see below) were systematically selected from this pool of patients. The selection process of AD-patients for the study was performed on the basis of clinical standard criteria alone, and was blinded for the results of the [11C]PIB PET imaging examinations. All patients provided a written informed consent regarding the scientific evaluation of their data. The study concept was approved by the ethics committee of the Technical University of Munich and the local radiation protection authorities.

The baseline diagnostic work-up included an interview with the patient and an informant, medical, psychiatric and neurological examinations (performed by an experienced psychiatrist), routine blood screening and APOE genotyping. Cranial magnetic resonance imaging (MRI) was performed to assess structural brain abnormalities. [11C]PIB PET was performed to asses cerebral amyloid-plaque load in vivo.

Inclusion criteria: Participants had to meet the established NINCDS-ARDRA diagnostic criteria for probable Alzheimer’s disease (1), including memory loss and impairment in at least one further cognitive domain, e.g. language or executive functions. As performed in previous studies, we also determined that MRI-data in both patient groups should be compatible with the clinical diagnosis (2).

Exclusion criteria: Patients were not included in the study, if they met criteria for any other neurological or functional psychiatric disorder, including major depression, or if they showed any characteristic symptoms of diseases or abnormalities sufficient to cause memory impairment, such as normal pressure hydrocephalus, Parkinson’s disease, or progressive supranuclear palsy. Patients were also excluded if they showed any major structural abnormalities or signs of major vascular pathology in MRI, such as status post infarction, extensive leucoencephalopathy or atrophy, intracerebral aneurysm or AV malformation. The NINDS-AIREN criteria for vascular dementia were used to exclude relevant ischemic processes causing cognitive impairment in the patients (3). Furthermore, patients with other possible causes of neuropsychological dysfunction, such as current psychotropic medication (e.g. antidepressants, neuroleptics) or substance abuse were excluded.

Neuropsychological Evaluation

All patients underwent neuropsychological evaluation including subtests of the Consortium to Establish a Registry for Alzheimer’s disease neuropsychological assessment battery (CERAD-NAB), performed by an experienced neuropsychologist (4). For this study a German version of the CERAD-NAB was used for which normative data have been published (5). The Mini-Mental State Examination (MMSE) was used to assess the overall severity of cognitive decline. In addition to age and gender, the onset of the disease was assessed in an interview with an informant, and disease duration was calculated. Educational levels were assessed as years of education, defined as years attending school plus years of apprenticeship, technical school, college, and university.

Routine Blood Screening and APOE Genotype

Routine blood screening including a standard serologic chemistry group, full blood cell count, blood glucose, vitamin B12 and folic acid levels, thyroid hormone levels as well as serological tests for syphilis and Lyme borreliosis and revealed no major abnormalities. For determination of APOE genotype DNA was extracted according to standardized procedures and apolipoprotein polymorphism was assessed via PCR-based essay, simultaneously utilizing two distinct restriction enzymes, as described previously (6). For subsequent group analyses, subjects were divided into APOEε4-allele carriers and noncarriers; i.e. all ε4 allele–positive subjects were pooled together, as done in previous studies(7).

Every patient underwent [11C]PIB PET of the brain for assessment of cerebral amyloid-plaque load and MRI of the brain for exclusion of structural anomalies such as brain tumors, anatomic abnormalities or vascular pathology within one month of the baseline visit:

MRI

Structural Magnetic Resonance Imaging (MRI) was performed on a 1.5T Siemens Symphony system as recently described (8). A 3D T1-weighted anatomical dataset was obtained from each subject by using a magnetization-prepared rapid acquisition gradient echo sequence (TE = 3.93 ms, TR = 1500 ms, TI = 760 ms, flip angle = 5°, FoV = 256 mm2, matrix = 256 × 256, 160 slices, voxel size = 1 × 1 ×1 mm3). Furthermore, axial T2 weighted turbo-spin-echo images (TR 4510 ms, TE 104 ms, 19 slices, voxel dimensions 0.6 x 0.5 x 6.0 mm), coronal T1 weighted spin echo images (TR 527 ms, TE 17 ms, 19 slices, voxel dimensions 0.9 x 0.9 x 6.0 mm) and T2 weighted gradient echo images (TR 725, TE 29, 19 slices, voxel dimensions 0.7 x 0.7 x 6.0 mm) were acquired.

[11C]PIB Imaging

Imaging with [11C]PIB was performed as previously described in a recent publication (2). All patients were injected with 370 MBq [11C]PIB at rest outside the scanner. Thirty minutes later, patients were placed in the scanner and at 40 minutes post injection, three 10-minute frames of data acquisition were started and later summed into a single frame (40-70 minute). Acquisition was performed using a Siemens ECAT HR+ PET scanner (CTI, Knoxville, Tenn., USA) in 3D mode and a transmission scan was carried out subsequently to allow for later attenuation correction.

Image Analysis [11C]PIB PET

All image processing and statistical procedures applied to [11C]PIB PET imaging data were carried out following established protocols that have been previously validated and published in the literature (2, 9). The first step of the analysis consisted of a visual analysis of all individual [11C]PIB scans, followed by statistical group analysis. Image reconstruction, correction of dead time, scatter and attenuation was performed on a sun workstation (Sun Microsystems Inc., Mountain View, CA). Statistical parametric mapping software (SPM 5, Wellcome Department of Cognitive Neurology, London, UK) was used for image realignment, transformation into standard stereotactic space (MNI PET-template), smoothing and statistical analysis (2, 10). For spatial transformation of PIB-data, standardized uptake value (SUV) images (40-70 min p.i.) were co-registered to each individual’s volumetric MRI and then automatically spatially normalized to the MNI-template in SPM5 using warping parameters derived from previous individual MRI-normalization, as previously described (2, 11). For quantitative normalization of cerebral PIB uptake values, the cerebellum was used as reference region. As previously described, the uptake in all pixels of the subjects’ PIB-scans was divided by the individual mean value in the cerebellum to calculate SUVR40-70min images. Subsequently, images were smoothed (12x12x12mm) and voxel-based statistical group comparisons (unpaired t-tests) were performed between the patient groups (APOEε4-allele carriers versus noncarriers) and between patient groups and a preexisting age-matched healthy control population (n = 8, 5 female, 3 male, mean age (y): 65 ± 16, (72, 83, 70, 45, 39, 69, 76, 65 years), mean MMSE: 29,5 ± 0,7 ), which has been described in detail previously (2, 9). Only voxels surviving FDR-correction (false-discovery-rate) for the entire volume at a p-value < 0.05 were accepted in the statistical analysis between patient groups and between patients and healthy controls, in order to avoid false-positive results (12). The anatomical localization of the significant coordinates was determined in the SPM5 Anatomy toolbox (13) (

Correction of Partial Volume Effect (PVE)

The aim of partial volume correction is to get an undestorted activity measurement for a certain tissue of interest by removing the contributions from the other tissues. To correct the PET data for a potentially different influence of regional cortical atrophy in the two groups of patients, a correction of [11C]PIB PET data for partial volume effects was carried out with an algorithm implemented in the PMOD software package (PMOD Technologies Ltd., Adliswil, Switzerland) as described previously(2).This PVE-correction method is based on the method published by Giovacchini et. al (14). In short, it assumes that the uptake in white matter is homogeneous, and no activity is present in the cerebrospinal fluid. Following coregistration of individual MRI and PET data, segmentation of the MR-information into grey and white-matter is carried out. The segmented images are first adjusted to the PET resolution by smoothingthem with a three-dimensional Gaussian filter. An estimate of pure white matter uptake is obtained and removed by subtracting a synthetic white matter image. Subsequently, the subtraction result is divided by the smoothed gray matter segment. If the assumptions are met, the resulting image represents the undistorted gray matter activity. All PVE-corrected PET datasets were stereotactically normalized to the standard template and voxel-based statistical analysis was carried out following the identical strategy as described above.In general, it may be mentioned that PVE-correction of PET data does not guarantee completely unbiased results and in some cases may even attenuate true effects. For that reason we included corrected and un-corrected data in the manuscript.

Image analysis MRI-data

Visual rating of the individual MRI-scans was performed by experienced clinical readers. Images were screened for the predefined exclusion criteria such as structural abnormalities, tumors or signs of major vascular pathology in MRI (status post infarction, extensive leucoencephalopathy intracerebral aneurysm or AV malformation). It was verified that MRI-data were compatible with the original clinical diagnosis on the basis of all acquired MRI-sequences. In addition to visual assessment, MRI-data of every single patient was also used for stereotactic transformation and partial volume correction of individual [11C]PIB-data (see above). For this purpose the MRI-data was automatically spatially normalized to the MRI MNI-template in SPM5 as previously shown, to collect warping parameters for later normalization of PET data (2).For preprocessing and statistical VBM-analysis of the anatomical T1-weighted MRI images,default SPM5-software was used using the unified segmentation process as previously published (15). This approach has bee previously applied in comparable form for VBM-analysis of patients with dementia (16). Following spatial normalization of all images into the stereotactic space of the Montreal Neurological Institute (MNI), images were segmented into gray and white-matter and cerebrospinal fluid. Of the resulting images, the modulated gray-matter images were selected for further analysis and smoothed with an 8 × 8 × 8 mm Gaussian kernel. Voxel-by-voxel t-tests using the general linear model (17) were applied to detect gray-matter differences between patient groups and between patients and an age-matched preexisting population of healthy controls, which has been described in detail previously (8). We included only voxels with a GM value greater than 0.1 (maximum value, 1) to avoid possible edge effects around the border between gray-matter, white-matter and cerebrospinal fluid and to include only voxels with sufficient gray-matter. To correct for effects of global brain volume we performed Ancova global normalization. As for PIB PET analyses we predefined threshold of p < 0.05, corrected for multiple comparisons for all VBM data analyses.

Multiple regression analyses

In addition to group comparisons, SPM-based multiple regression analyses were carried out, to identify the relation between APOEgene-dose and cerebral amyloid-plaque deposition or between APOE gene-dose andcerebral atrophy. For this purpose patients were classified into 4 APOEgene-dosecategories: 1: carriers of one ε2-allele and one ε3-allel (ε2/ε3), 2: carriers of two ε3-alleles (ε3/ε3), 3: carriers of a single ε4-allele (ε3/ε4) or (ε2/ε4) and 4: carriers of two ε4-alleles (ε4/ε4). Using SPM5, a multiple regression analysis was performed with the APOEgene-dose as the covariate of interest. The variables age of the patients, degree of cognitive impairment, years of education, gender and duration of disease were included into this analysis as covariates of no interest. As published in several previous studies, for this type of voxel-based multiple regression analysis, a threshold of p < 0.001, uncorrected was applied (18, 19). In the case of significant findings, a scatterplot between the APOEgene-dose and the cerebral amyloid-plaque load was generated at the cluster with the strongest statistical correlation, and a regression line was fitted into the plot.In addition to these voxel-based multiple regression analyses, we calculated the mean global cortical PIB-binding values as established in previous studies and performed a correlation analysis between these values and the APOE4-allele frequency(20). For this analysis a p-value <0.05 was regarded significant.

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