Participants

Table 1. Table 1. Characteristics of the Participants.

The participants were 26 former NFL players who reported cognitive, mood, and behavioral dyscontrol symptoms and 31 asymptomatic control participants who reported no history of traumatic brain injury (Table 1). The inclusion criteria for the former players were male sex, age 40 to 69 years, a minimum of 2 years playing football in the NFL, a minimum of 12 years of total tackle football experience, and cognitive, behavioral, and mood symptoms reported by the participant through telephone screening. Quarterbacks, kickers, and special-teams players were excluded, as were former players with a reported concussion or traumatic brain injury within 1 year before study entry. The inclusion criteria for the controls were male sex, age 40 to 69 years, no cognitive symptoms, and no history of traumatic brain injury. Descriptions of the exclusion criteria and information on the enrollment of participants, including those specific to the use of the flortaucipir PET radioligand, are provided in the Supplementary Appendix, available with the full text of this article at NEJM.org. The study builds on a phase 2a pilot study (ClinicalTrials.gov number, NCT02079766), the details of which are provided in the Supplementary Appendix.

The 26 former NFL players were recruited through word of mouth, social media posts, and e-mail notifications. A total of 18 of the former players were participants in an investigator-initiated CTE study that was supported by Avid Radiopharmaceuticals and the National Institutes of Health13,14 and was conducted at Boston University and Brigham and Women’s Hospital, Boston. The other 8 former players were consecutively enrolled in an associated investigator-initiated CTE study that used similar enrollment criteria, assessments, and imaging protocols, supported by Avid Radiopharmaceuticals and the State of Arizona, and was conducted at Mayo Clinic, Scottsdale, and at Banner Alzheimer’s Institute, Phoenix.

The control group included 31 asymptomatic male research volunteers from four clinical research studies (details are provided in the Supplementary Appendix). Nine were consecutively enrolled control participants in the CTE study conducted in Boston, and 1 was in the associated CTE study conducted in Arizona. Data were also acquired from 21 additional controls, including 18 participants who were enrolled in previous Avid-sponsored Alzheimer’s disease case–control studies15 and 3 participants from Alzheimer’s Disease Neuroimaging Initiative studies (ClinicalTrials.gov numbers, NCT01231971 and NCT01687153; http://adni.loni.usc.edu/data-samples/adni-data-inventory/). This group of additional controls included every participant from the four other studies who was male, was in the same age range as the former-player group, had a score of 29 or higher on the Mini–Mental State Examination (MMSE; scores range from 0 to 30, with lower scores indicating greater cognitive impairment),16 reported no history of traumatic brain injury, and, at the time of our image analysis, had available flortaucipir and florbetapir PET and magnetic resonance imaging (MRI) scans obtained with the use of the same imaging protocols as those used for the former-player group. These participants were not asked about their participation in collision or contact sports. Participants in all control groups were selected without our knowledge of their flortaucipir PET findings.

The study procedures were approved by the relevant institutional review board at each study site, and participants provided written informed consent for participation in this study or, if they participated in one of the other studies, for their data to be shared and combined with data in other studies. Imaging studies and other assessments were performed for research purposes and at no cost to the research participants, who were not informed of the tau or amyloid-beta PET scan results. Avid Radiopharmaceuticals provided funding for the study and provided the PET ligands, and Avid investigators were involved in study design, data analysis, and preparation of the manuscript. There were no confidentiality agreements between the authors and Avid, any other commercial entity, or the NFL. The authors vouch for the accuracy and completeness of the data and adverse-events reporting.

Clinical Measures

All participants were administered the MMSE16 as a measure of cognitive status and, in the case of the controls, as one of the screening criteria for inclusion in the studies from which they were drawn. The former NFL players were also administered neuropsychological tests and neuropsychiatric assessments as reported in a previous study.17 A subset of six tests of executive functioning (Trail Making Test Part B, Category [Animal] Fluency), episodic memory (Neuropsychological Assessment Battery List Learning Test), mood (Beck Depression Inventory II and Beck Hopelessness Scale), and behavioral regulation (Barratt Impulsiveness Scale) was used for the analyses, since these tests assess functions affected by CTE.6,7 (Detailed explanations of the scores are provided in Table S1 in the Supplementary Appendix.)

Brain Imaging

Each participant underwent flortaucipir PET (for the detection of tau), florbetapir PET (for the detection of amyloid-beta), and T 1 -weighted volumetric MRI of the head. Flortaucipir PET was performed on PET–computed tomography (CT) systems with an intravenous bolus of 370 MBq (10 mCi [±10%]) of flortaucipir and a CT scan to correct the PET scan for radiation attenuation and scatter. Dynamic emission PET data collected over a 20-minute period beginning 80 minutes after radiotracer administration were used for analysis in this study. Florbetapir PET was performed on the same systems with an intravenous injection of approximately 370 MBq (10 mCi) of florbetapir, a 50-minute radiotracer uptake period, a 10-minute dynamic emission scan, and a CT scan to correct the PET scan for radiation attenuation and scatter. As part of site qualification for participation in the study, a priori procedures were used to adjust scanner parameters to harmonize images across investigative sites. (Details are provided in the Supplementary Appendix.) All PET images were smoothed with an 8-mm gaussian kernel as a filter to harmonize images acquired on different PET–CT systems and to mitigate noise.

Statistical Analysis

Between-group comparisons of age, years of education, and MMSE scores were analyzed with Mann–Whitney U tests. Group differences in race were analyzed with the use of chi-square tests. For between-group comparisons of amyloid-beta plaque burden, chi-square tests were used to compare the proportion of participants with a positive florbetapir PET, and t-tests were used to compare the mean cortical:cerebellar florbetapir standard uptake value ratio (SUVR, the ratio of radioactivity in a cerebral region to that in the cerebellum as a reference) between the groups. Because several of the controls from previous studies had negative florbetapir PET findings, these group comparison analyses for florbetapir were also conducted with the subset of 10 control participants enrolled in the CTE study, for whom the availability of florbetapir results was not an entry criterion.

For the initial analysis of flortaucipir PET images, automated brain-mapping algorithm software (Statistical Parametric Mapping, version 12 [SPM12] [www.fil.ion.ucl.ac.uk/spm/software/]) was used to coregister each participant’s flortaucipir PET image to his MRI, transform the coregistered images into the coordinates of a standard brain atlas, control for age, and generate statistical parametric maps of the between-group differences at each voxel in regional cerebral:cerebellar gray-matter flortaucipir SUVRs, with the use of a prespecified threshold of a P value less than 0.005, uncorrected for multiple comparisons, to select regions that differed between groups. Early analyses in this study used a qualitative ordinal scale and visual interpretation of scans. This method was replaced by the automated analysis. (Details are provided in the Supplementary Appendix.)

To address possible type I error in the examination of group differences in an entire voxel-based PET image, a post hoc Monte Carlo simulation procedure (see the Supplementary Appendix) was performed with 1000 iterations, testing the hypothesis that the number of flortaucipir SUVR differences observed in the postulated direction (i.e., SUVR higher in the former-player group than in the control group at P<0.005) was significantly greater than the number of voxels with elevations in the opposite direction (i.e., SUVR higher in the control group than in the former-player group).

The initial exploratory analysis was of voxel-based flortaucipir SUVR, which was used to create regional SUVR values for individual participants. These data were used to select voxels that had higher SUVR values in the former-player group than in the control group (with P<0.005 for the between-group difference) in clusters of at least 100 contiguous voxels and to characterize these regions as having either elevated or not-elevated values in order to identify regions of interest. Between-group differences in each of the resulting regional flortaucipir uptake values were examined by analysis of covariance, with age as the covariate.

Multivariate mixed-effect models were used to examine associations between the regional flortaucipir SUVRs and the six neuropsychological and neuropsychiatric measures. To reduce type I error and to account for possible nonnormality due to the small sample size, 95% confidence intervals and P values were calculated with the use of bias-corrected bootstrapping from 1000 resamples.

In post hoc analyses, partial correlations, controlling for age, were used to explore the extent to which regional flortaucipir SUVRs in the former-player group were associated with total years playing tackle football. In addition, to account for possible effects of outliers, post hoc Spearman correlation coefficients were calculated from the age-adjusted residuals.

Flortaucipir has off-target binding to melanocytes in the choroid plexus, such that flortaucipir PET measurements in the adjacent hippocampi are higher in black than in white research participants as a result of the effects of partial-volume averaging.18-21 Since there was a higher percentage of black participants in the former-player group than in the control group, a post hoc analysis with t-tests as well as nonparametric Mann–Whitney U tests was performed to examine racial differences in the regional flortaucipir SUVRs and determine whether these measurements were confounded by race.

For the analysis of florbetapir PET images, the mean cortical:cerebellar florbetapir SUVRs were generated from each participant’s florbetapir image with the use of SPM12 software and the above-described cerebral and whole cerebellar regions of interest. A positive amyloid-beta PET scan, corresponding to postmortem evidence of moderate-to-frequent neuritic plaques (consistent with a neuropathological diagnosis of Alzheimer’s disease), was defined as an SUVR greater than or equal to 1.10, in accordance with previous prospective cohort studies.22