MSc Verena Heise

PhD Nicola Filippini

MD Klaus P Ebmeier

PhD Clare E Mackay
email: clare.mackay@psych.ox.ac.uk

The APOE ε4 allele modulates brain white matter integrity in healthy adults

Keywords: APOE, diffusion tensor imaging, neuroimaging, white matter, healthy subjects, aging

The Apolipoprotein E (APOE) ε4 allele is the best-established genetic risk factor for sporadic Alzheimer's disease (AD) and is also associated with structural gray matter (GM) and functional brain changes in healthy young, middle-aged and elderly subjects. Because APOE is implicated in brain mechanisms associated with white matter (WM) development and repair, we investigated the potential role played by the APOE polymorphism on WM structure in healthy younger (age range: 20-35 years) and older (aged 50-78 years) adults using diffusion tensor imaging (DTI). General reduction of fractional anisotropy (FA) and increase in mean diffusivity (MD) values was found in carriers of the APOE ε4 allele relative to non-carriers. No significant interactions between genotype and age were observed, suggesting that differences in WM structure between APOE ε4-carriers and non-carriers do not undergo significant differential changes with age. This result was not explained by differences in brain morphology or cognitive measures. The APOE ε4 allele modulates brain WM structure before any clinical or neurophysiological expression of impending disease.

Introduction

Apolipoprotein E (apoE, protein; APOE, gene) is a very-low-density lipoprotein that has a key role in coordinating the mobilization and redistribution of cholesterol, phospholipids, and fatty acids. [START_REF] Mahley | Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS[END_REF] In the central nervous system, apoE is implicated in mechanisms such as neuronal development, brain plasticity, and repair functions. [START_REF] Mahley | Apolipoprotein E: far more than a lipid transport protein[END_REF][START_REF] Bu | Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy[END_REF] The human APOE gene has 3 allelic variants (ε2, ε3, and ε4). The ε4 allele is associated with higher risk of developing both early-onset [START_REF] Okuizumi | ApoE-epsilon 4 and early-onset Alzheimer's[END_REF] and late-onset [START_REF] Corder | Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families[END_REF] Alzheimer's disease (AD), poor outcome from traumatic brain injury [START_REF] Teasdale | Association of apolipoprotein E polymorphism with outcome after head injury[END_REF] and age-related cognitive impairment. [START_REF] Deary | Cognitive change and the APOE epsilon 4 allele[END_REF] Neuroimaging studies have shown that the APOE ε4 allele is associated with modification of brain function and gray matter (GM) structure, both in AD patients [START_REF] Lehtovirta | Volumes of hippocampus, amygdala and frontal lobe in Alzheimer patients with different apolipoprotein E genotypes[END_REF][START_REF] Cosentino | APOE epsilon 4 allele predicts faster cognitive decline in mild Alzheimer disease[END_REF][START_REF] Filippini | Anatomically-distinct genetic associations of APOE epsilon4 allele load with regional cortical atrophy in Alzheimer's disease[END_REF] and in healthy subjects. [START_REF] Bookheimer | Patterns of brain activation in people at risk for Alzheimer's disease[END_REF][START_REF] Small | Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer's disease[END_REF][START_REF] Reiman | Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia[END_REF][START_REF] Bondi | fMRI evidence of compensatory mechanisms in older adults at genetic risk for Alzheimer disease[END_REF][START_REF] Wishart | Increased brain activation during working memory in cognitively intact adults with the APOE epsilon4 allele[END_REF] GM volume reduction in AD patients carrying the ε4 allele relative to non-carriers has been found mainly in the hippocampus and entorhinal cortex. [START_REF] Lehtovirta | Volumes of hippocampus, amygdala and frontal lobe in Alzheimer patients with different apolipoprotein E genotypes[END_REF][START_REF] Filippini | Anatomically-distinct genetic associations of APOE epsilon4 allele load with regional cortical atrophy in Alzheimer's disease[END_REF][START_REF] Frisoni | Hippocampal and entorhinal cortex atrophy in frontotemporal dementia and Alzheimer's disease[END_REF][START_REF] Du | Age effects on atrophy rates of entorhinal cortex and hippocampus[END_REF] Similarly, reductions in MTL volumes have been reported among healthy middle-aged/ elderly, [START_REF] Barboriak | Hippocampal sulcal cavities on MRI: relationship to age and apolipoprotein E genotype[END_REF][START_REF] Den Heijer | Hippocampal, amygdalar, and global brain atrophy in different apolipoprotein E genotypes[END_REF][START_REF] Wishart | Regional brain atrophy in cognitively intact adults with a single APOE epsilon4 allele[END_REF][START_REF] Burggren | Reduced cortical thickness in hippocampal subregions among cognitively normal apolipoprotein E e4 carriers[END_REF] and adolescent [START_REF] Shaw | Cortical morphology in children and adolescents with different apolipoprotein E gene polymorphisms: an observational study[END_REF] APOE ε4-carriers, although not all studies replicate this finding. [START_REF] Schmidt | Apolipoprotein E e4 allele in the normal elderly: neuropsychologic and brain MRI correlates[END_REF][START_REF] Reiman | Hippocampal volumes in cognitively normal persons at genetic risk for Alzheimer's disease[END_REF][START_REF] Cherbuin | Total and regional gray matter volume is not related to APOE*E4 status in a community sample of middle-aged individuals[END_REF] Positron emission tomography (PET) studies have consistently shown resting glucose metabolism reduction in healthy young and middleaged APOE ε4-carriers in brain regions known to be affected by AD pathology, such as the parietal, temporal and prefrontal cortices. [START_REF] Small | Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer's disease[END_REF][START_REF] Reiman | Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia[END_REF] Taskbased functional magnetic resonance imaging (fMRI) studies have been less consistent, showing increased [START_REF] Bookheimer | Patterns of brain activation in people at risk for Alzheimer's disease[END_REF][START_REF] Bondi | fMRI evidence of compensatory mechanisms in older adults at genetic risk for Alzheimer disease[END_REF][START_REF] Wishart | Increased brain activation during working memory in cognitively intact adults with the APOE epsilon4 allele[END_REF] , decreased [START_REF] Mondadori | Better memory and neural efficiency in young apolipoprotein E epsilon4 carriers[END_REF][START_REF] Lind | Reduced functional brain activity response in cognitively intact apolipoprotein E epsilon4 carriers[END_REF] or even no difference [START_REF] Burggren | Specificity of brain activation patterns in people at genetic risk for Alzheimer disease[END_REF] in task-related BOLD signal between healthy APOE ε4-carriers and non-carriers.

Methodological differences may explain inconsistencies in the studies.

Because of the link between APOE and AD, to date structural imaging studies have largely concentrated on GM changes, especially in the entorhinal cortex and hippocampal regions, which are the first structures to show AD pathology. [START_REF] Braak | Pattern of brain destruction in Parkinson's and Alzheimer's diseases[END_REF] Very few studies have investigated the potential effects of the APOE genotype on white matter (WM) structure. This is surprising considering that the major role of apoE in the brain is the transport of lipid components which contribute to building up the myelin sheath. [START_REF] Mahley | Apolipoprotein E: cholesterol transport protein with expanding role in cell biology[END_REF]31,[START_REF] Han | Potential mechanisms contributing to sulfatide depletion at the earliest clinically recognizable stage of Alzheimer's disease: a tale of shotgun lipidomics[END_REF] Moreover, AD pathology not only involves GM atrophy but also changes in WM [START_REF] Rose | Loss of connectivity in Alzheimer's disease: an evaluation of white matter tract integrity with colour coded MR diffusion tensor imaging[END_REF] , which have been shown to correlate with disease severity. [START_REF] Duan | White matter damage of patients with Alzheimer's disease correlated with the decreased cognitive function[END_REF][START_REF] Ferrarini | MMSE scores correlate with local ventricular enlargement in the spectrum from cognitively normal to Alzheimer disease[END_REF] Diffusion tensor imaging (DTI) is an imaging technique that is increasingly used for WM analysis in vivo. DTI-derived measurements, such as fractional anisotropy (FA) and mean diffusivity (MD), have proved to be sensitive for detection of disease-related WM changes. [START_REF] Johansen-Berg | Diffusion MRI: From quantitative measurement to in vivo neuroanatomy[END_REF] A recent meta-analysis of DTI measurements in AD and MCI reported widespread increases in MD and decreases in FA with large effect sizes. [START_REF] Stebbins | Diffusion tensor imaging in Alzheimer's disease and mild cognitive impairment[END_REF] To date studies using DTI to investigate the effects of APOE on FA and MD have focused on specific age groups [START_REF] Nierenberg | Abnormal white matter integrity in healthy apolipoprotein E epsilon4 carriers[END_REF] or were limited to preselected regions-of-interest. [START_REF] Persson | Altered brain white matter integrity in healthy carriers of the APOE epsilon4 allele: a risk for AD?[END_REF] The aim of this study is to investigate the relationship between APOE genotype, WM structure and age in healthy individuals using an unbiased whole-brain analysis (tract-based spatial statistics -TBSS 40 ) using DTI. This will allow us to investigate whether the ε4 allele selectively modulates well-defined WM pathways or rather has a more general effect. In particular, we will test whether the APOE ε4 allele alters WM integrity both in distinct groups of younger (age range = 20 to 35 years old) and older (age range = 50 to 78 years old) participants.

Based on previously reported studies showing age-related WM changes in healthy subjects [START_REF] Head | Differential vulnerability of anterior white matter in nondemented aging with minimal acceleration in dementia of the Alzheimer type: evidence from diffusion tensor imaging[END_REF][START_REF] Salat | Age-related alterations in white matter microstructure measured by diffusion tensor imaging[END_REF][START_REF] Ardekani | Exploratory voxel-based analysis of diffusion indices and hemispheric asymmetry in normal aging[END_REF][START_REF] Pagani | Voxel-based analysis derived from fractional anisotropy images of white matter volume changes with aging[END_REF] , we hypothesize that older subjects will have increased MD and reduced FA. As the APOE ε4 allele is associated with an earlier onset of AD [START_REF] Okuizumi | ApoE-epsilon 4 and early-onset Alzheimer's[END_REF] and cognitive decline at a younger age [START_REF] Deary | Cognitive change and the APOE epsilon 4 allele[END_REF] , we expect that the trajectories of WM changes with age will differ between APOE ε4-carriers and non-carriers. Moreover, we will study GM volumes to investigate whether whole brain GM or WM analyses are more sensitive to changes associated with the APOE ε4 allele. Based on previous studies [START_REF] Wishart | Regional brain atrophy in cognitively intact adults with a single APOE epsilon4 allele[END_REF] , we hypothesize that GM volume will be reduced in older APOE ε4-carriers. Lastly, we will compare the data between younger and older groups to study a possible interaction between age and genotype. We will test alternative hypotheses: either brain structure of APOE ε4-carriers already differs from non-carriers in young adulthood, or structural modifications associated with the APOE ε4 allele only manifest at a certain age, perhaps preceding AD development in healthy adults.

Materials and methods

Participants: Imaging data for 73 right-handed subjects (35 males) aged 20 to 78 years were acquired. Thirty-four subjects aged 20 to 35 years comprised the "younger" group (mean age 28.6 years ± 4.20), and thirty-nine subjects aged 50 to 78 years (mean 64.9 years ± 7.19) comprised the "older" group.

A priori exclusion criteria were: current or past history of neurological or psychiatric disorders, memory complaints, head injury, substance abuse (including alcohol), corticosteroid therapy, and youth diabetes therapy. A posteriori (after MRI scan) exclusion criteria included presence of brain vascular insults and two or more hyperintense lesions equal or larger than 10 mm diameter, or more than eight hyperintense lesions with a diameter from 5 Image Analysis: Data analysis was carried out using FMRIB Software Library (FSL) tools (www.fmrib.ox.ac.uk/fsl).

Diffusion Tensor Imaging (DTI):

Raw images were pre-processed using "Eddy Current correction", in order to correct for distortions due to the gradient directions applied. Fractional anisotropy (FA) and mean diffusivity (MD) maps were generated using DTIFit, part of FMRIB's Diffusion Toolbox (http://www.fmrib.ox.ac.uk/fsl/fdt), that fits a diffusion tensor model at each voxel [START_REF] Behrens | Characterization and propagation of uncertainty in diffusion-weighted MR imaging[END_REF] . The magnitude and direction of tissue water mobility is measured in the three dimensions [START_REF] Beaulieu | The basis of anisotropic water diffusion in the nervous system -a technical review[END_REF] and the diffusion tensor is modelled as an ellipsoid with symmetry across any one of its three axes. The largest axis or eigenvalue is denoted λ1 or axial diffusivity. The two minor axes (λ2, λ3) are usually averaged to compute radial diffusivity. The average of all three eigenvalues is called mean diffusivity (MD). Fractional anisotropy (FA) is a ratio of axial to radial diffusivity, and thus provides a measure of the directionality of diffusion.

In axons, net intravoxel water diffusion perpendicular to the fibres is reduced due to the myelin sheath but unrestricted parallel to the axon. This directionality of water diffusion gives rise to a higher FA in WM, whereas less restricted diffusion, e.g. in GM or cerebrospinal fluid leads to lower FA. GM and WM have similar MD values.

The FA output images were used as input for tract-based spatial statistics (TBSS), a voxelwise approach for analysis of FA data. [START_REF] Smith | Tract-based spatial statistics: voxelwise analysis of multi-subject diffusion data[END_REF] All subjects' FA data were aligned into a common space using the nonlinear registration tool FNIRT. The mean FA image was generated and thinned to create a mean FA skeleton, which represents the centres of all tracts common to the group. Each subject's aligned FA data was then projected onto this skeleton and the resulting data fed into voxelwise GLM cross-subject statistics. A voxel by voxel permutation nonparametric test (5000 permutations) was used to assess group-related differences using threshold-free cluster enhancement (TFCE), which avoids using an arbitrary threshold for the initial cluster-formation. [START_REF] Smith | Tract-based spatial statistics: voxelwise analysis of multi-subject diffusion data[END_REF] All results are shown at p < 0.05 corrected for multiple comparisons across space. In addition to FA data, mean diffusivity (MD), axial diffusivity (DA) and radial diffusivity (DR) were also compared using TBSS in an analogous fashion. Separate analyses were performed for the younger and older group, then data from both groups were pooled together. GM volume, WM volume and cognitive scores were added to the TBSS analysis as covariates of no interest.

Structural MRI: A voxel-based morphometry (VBM) approach was used to study brain morphology and to relate it to the APOE polymorphism. Total brain volume, GM and WM and cerebrospinal fluid (CSF) measurements were calculated using FMRIB's Automated Segmentation Tool (FAST). [START_REF] Zhang | Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm[END_REF] Wholebrain analysis was carried out with FSL-VBM [START_REF] Douaud | Anatomically related grey and white matter abnormalities in adolescent-onset schizophrenia[END_REF] , using default settings. In brief, brain extraction and tissue-type segmentation were performed, and resulting GM or WM partial volume images were aligned to standard space using FMRIB's Linear Image Registration Tool (FLIRT) and then nonlinear (FNIRT) registration tools. The resulting images were averaged, modulated, and smoothed with an isotropic Gaussian kernel of 3mm (~7 mm FWHM) to create a study-specific template. Finally, voxelwise general linear modelling (GLM) was applied using permutation nonparametric testing (5000 permutations) and p < 0.05 correcting for multiple comparisons across space. GM volume and total brain volume were used as covariates of no interest in the VBM analysis.

ROI analysis of hippocampal volume:

Individual hippocampal measures were obtained using FMRIB'S Integrated Registration and Segmentation Tool (FIRST), an automatic subcortical segmentation program. [START_REF] Patenaude | Bayesian statistical models of shape and appearance for subcortical brain segmentation[END_REF] Boundary correction was used for the classification of the boundary voxels. ROIs were visually inspected in the coronal plane to ensure accuracy.

White matter lesions: Measurement of WM lesions in the older group was manually performed on FLAIR by a trained neuroscientist (VH) blind to the genotypic profile using the Jim 4.0 software (Xinapse Medical Systems, Thorpe Waterville, UK). All axial slices of each subject were investigated for WM hyperintensities and periventricular WM lesions. After marking all hyperintensities and periventricular lesions as Regions-of-interest (ROIs), total ROI volume was calculated for each subject.

Statistics: Statistical analyses of non-imaging variables were carried out using SPSS software (SPSS Inc., Chicago, USA). T-tests comparison were used for continuous variables (sociodemographic, cognitive performance and brain volumes). Exact Fisher's continuity correction was used for categorical variables (gender and family history of dementia). An alpha of p < 0.05 was considered significant.

Results

Participants: Seventy-three subjects completed the MRI protocol. Two of the older subjects (one APOE ε4-carrier, one non-carrier) were excluded because they showed an abnormally high volume of white matter lesions (more than 3 standard deviations from each group mean), leaving a total of 71 subjects (Table 1). There was no effect of genotype on white matter lesion volume (F 1,34 = 2.40, p = 0.13). Within the younger and older group, APOE ε4-carriers and non-carriers did not differ for age, sex, years of education, number of individuals with a family history of dementia and ACE-r results. Similarly, memory performance and reaction times in an encoding memory task as described in Filippini et al. [START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-epsilon4 allele[END_REF] were not different between APOE ε4-carriers and non-carriers (Table 1).

Diffusion tensor imaging (DTI):

In the younger group whole brain WM analysis with TBSS revealed decreased FA values in APOE ε4-carriers relative to noncarriers in widespread areas, including the cingulum, corona radiata, corpus callosum, external capsule, internal capsule and superior longitudinal fasciculus (Figure 1a). There were no regions in which FA values were higher in APOE ε4-carriers relative to non-carriers. The decreased FA in APOE ε4carriers was not associated with significant differences in either MD, DA or DR.

In the older group, whole brain analysis with TBSS showed increased MD in APOE ε4-carriers relative to non-carriers in widespread areas including the cingulum, corona radiata, corpus callosum, external capsule, internal capsule and superior longitudinal fasciculus (Figure 1b) but there were no significant differences in FA, DR or DA. There were no regions in which MD values were lower in APOE ε4-carriers relative to non-carriers. TBSS analysis with the data from both age groups combined was performed to study a possible interaction between AGE (younger or older) and GENOTYPE (APOE ε4-carrier and non-carrier) factors. No significant interaction between the two factors was detected for FA or MD. However, a main effect of GENOTYPE was found for FA values in areas including the cingulum, corona radiata, corpus callosum, external capsule, internal capsule and superior longitudinal fasciculus (Figure 1c). Additionally, DR was significantly higher in APOE ε4-carriers compared to non-carriers (Figure 1d) but there were no significant effects on MD or DA.

As we expected, a widespread main effect of AGE was observed for FA. FA was significantly increased in the younger compared to the older group in WM tracts including the cingulum, corona radiata, corpus callosum, external capsule, internal capsule and superior longitudinal fasciculus (Figure 4a). This was associated with significantly increased DA and DR in the older group (Figure 4c andd). The same structures showed significantly increased MD values in the older group (Figure 4b). There were no regions in which FA values were higher or MD, DA or DR values were lower in the older group compared to the younger group.

Structural MRI: No differences between APOE ε4-carriers and non-carriers in both age groups were found for total brain volume, GM volume, WM volume, cerebrospinal fluid (CSF) and hippocampal volumes (Table 1). Moreover, no differences between APOE ε4-carriers and non-carriers were observed in GM or WM using a whole brain Voxel-Based-Morphometry approach.

Discussion

We investigated the effect of the APOE genotype on WM structure in healthy individuals using Diffusion Tensor Imaging (DTI) and found that the APOE ε4 allele affects WM integrity. In detail, we observed significantly reduced FA values in younger APOE ε4-carriers and increased MD in older ε4-carriers relative to matched non-carriers in widespread areas of the brain. Reduced FA values in APOE ε4-carriers relative to non-carriers were also found when the data from both groups were combined. No significant interactions between AGE and GENOTYPE were observed, suggesting that APOE has an effect on WM that is evident even in early adulthood and remains relatively stable throughout adulthood. Increased MD and decreased FA are generally markers of pathology, thus our observation may provide clues to the basis of the vulnerability of APOE ε4-carriers to late-life pathology.

EFFECT OF APOE GENOTYPE ON WM

This is the first study to describe an effect of the APOE ε4 allele on WM structure in a group of healthy young adults. Whole-brain analysis showed that young APOE ε4-carriers had reduced FA values in widespread brain areas.

Therefore carrying the APOE ε4 allele influences WM integrity even in young adulthood. A genotype effect on WM structure was also found in the older group. Although FA was not altered, widespread significant increases in MD were observed in APOE ε4-carriers. This is in accordance with several other studies that reported effects of the APOE ε4 allele on WM integrity in brain tracts such as parahippocampal WM [START_REF] Nierenberg | Abnormal white matter integrity in healthy apolipoprotein E epsilon4 carriers[END_REF][START_REF] Honea | Impact of APOE on the Healthy Aging Brain: A Voxel-Based MRI and DTI Study[END_REF] and the corpus callosum [START_REF] Persson | Altered brain white matter integrity in healthy carriers of the APOE epsilon4 allele: a risk for AD?[END_REF][START_REF] Smith | White matter diffusion alterations in normal women at risk of Alzheimer's disease[END_REF] whereas a recent study did not replicate these results [START_REF] Bendlin | White matter is altered with parental family history of Alzheimer's Disease[END_REF] .

Although the APOE genotype affects different measures of WM integrity in the two age groups, this is probably caused by age differences in FA and MD measures. It is possible that FA is more sensitive to detect differences in younger adults because we found that FA values decrease with age independent of genotype. The opposite effect would be found for MD.

AD and amnesic MCI are associated with an increased volume of WM lesions compared to healthy controls [START_REF] Appel | Association of White Matter Hyperintensity Measurements on Brain MR Imaging with Cognitive Status, Medial Temporal Atrophy, and Cardiovascular Risk Factors[END_REF][START_REF] Burton | Progression of white matter hyperintensities in Alzheimer disease, dementia with lewy bodies, and Parkinson disease dementia: a comparison with normal aging[END_REF] , which predicts the rate of cognitive decline. [START_REF] Groot | Periventricular cerebral white matter lesions predict rate of cognitive decline[END_REF] We did not find a significant effect of the APOE ε4 allele and this is in concordance with two studies reporting an effect of APOE genotype on volume of WM lesions only for homozygous individuals and no gene-dose effect. [START_REF] Godin | Apolipoprotein E Genotype Is Related to Progression of White Matter Lesion Load[END_REF][START_REF] Paternoster | Genetic determinants of white matter hyperintensities on brain scans: a systematic assessment of 19 candidate gene polymorphisms in 46 studies in 19,000 subjects[END_REF] Although APOE genotype effects were observed within WM regions in both the younger and the older group, we found that GM structure did not differ between APOE ε4-carriers and non-carriers. This was interesting because we expected that at least in the older group the higher risk of AD for APOE ε4carriers would show in GM changes, especially in regions affected early by AD [START_REF] Mcdonald | Regional rates of neocortical atrophy from normal aging to early Alzheimer disease[END_REF] , as was shown in two studies [START_REF] Wishart | Regional brain atrophy in cognitively intact adults with a single APOE epsilon4 allele[END_REF][START_REF] Honea | Impact of APOE on the Healthy Aging Brain: A Voxel-Based MRI and DTI Study[END_REF] . However, other studies also failed to observe APOE-related structural GM differences in healthy subjects. [START_REF] Schmidt | Apolipoprotein E e4 allele in the normal elderly: neuropsychologic and brain MRI correlates[END_REF][START_REF] Reiman | Hippocampal volumes in cognitively normal persons at genetic risk for Alzheimer's disease[END_REF][START_REF] Cherbuin | Total and regional gray matter volume is not related to APOE*E4 status in a community sample of middle-aged individuals[END_REF] We therefore suggest that brain function [START_REF] Bookheimer | Patterns of brain activation in people at risk for Alzheimer's disease[END_REF][START_REF] Small | Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer's disease[END_REF][START_REF] Reiman | Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia[END_REF][START_REF] Bondi | fMRI evidence of compensatory mechanisms in older adults at genetic risk for Alzheimer disease[END_REF][START_REF] Wishart | Increased brain activation during working memory in cognitively intact adults with the APOE epsilon4 allele[END_REF][START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-epsilon4 allele[END_REF] and WM-related measurements are more sensitive than GM-related measurements to detect the physiological effects of carrying an APOE ε4 allele.

EFFECT OF AGE ON WM INTEGRITY

As expected, we found age-related changes in FA and MD. FA was significantly lower in the older than in the younger group independent of APOE genotype. In addition, a widespread increase in MD was found in the older group. Several studies also observed reductions in FA with age mainly in prefrontal structures [START_REF] Head | Differential vulnerability of anterior white matter in nondemented aging with minimal acceleration in dementia of the Alzheimer type: evidence from diffusion tensor imaging[END_REF][START_REF] Salat | Age-related alterations in white matter microstructure measured by diffusion tensor imaging[END_REF][START_REF] Ardekani | Exploratory voxel-based analysis of diffusion indices and hemispheric asymmetry in normal aging[END_REF] as well as changes in WM volume unevenly distributed across brain regions. [START_REF] Pagani | Voxel-based analysis derived from fractional anisotropy images of white matter volume changes with aging[END_REF] Therefore, our results are similar to age-related changes in WM integrity observed by other groups and our unbiased whole-brain approach strengthens these results.

INTERPRETATION

The main goal of our study was to investigate whether differences in WM structure between APOE ε4-carriers and non-carriers change across the lifespan. As TBSS revealed no significant AGE by GENOTYPE interactions for FA or MD we showed that genotype-dependent differences in WM structure are already present in young adulthood and do not undergo significant differential changes with age. Previously, we have shown functional differences between APOE ε4-carriers and non-carriers in the same group of younger subjects. [START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-epsilon4 allele[END_REF] It is possible that WM differences might underlie these functional differences found in the default-mode network (DMN). Indeed, it has been shown that structural connectivity between the cingulum, superior frontal occipital fasciculus and genu of the corpus callosum determine the functional links of the DMN. [START_REF] Van Den Heuvel | Functionally linked resting-state networks reflect the underlying structural connectivity architecture of the human brain[END_REF] We found that FA was significantly decreased in young APOE ε4-carriers in the cingulum and genu of the corpus callosum. However, the relationship between structural and functional differences between APOE ε4-carriers and non-carriers require further investigation.

The effect of APOE on brain structure and function and the associated risk of AD remains poorly understood. The major role of apoE in the brain is the transport of lipid components which contribute to building up the myelin sheath. [START_REF] Mahley | Apolipoprotein E: cholesterol transport protein with expanding role in cell biology[END_REF]31,[START_REF] Han | Potential mechanisms contributing to sulfatide depletion at the earliest clinically recognizable stage of Alzheimer's disease: a tale of shotgun lipidomics[END_REF] In-vitro and in-vivo studies have shown that the apoE isoforms differentially modulate neuritic outgrowth, sprouting and branching [START_REF] Nathan | Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro[END_REF][START_REF] Mahley | Apolipoprotein E: impact of cytoskeletal stability in neurons and the relationship to Alzheimer's disease[END_REF][START_REF] Nathan | The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule depolymerization[END_REF] and the apoE4 isoform has been specifically associated with microtubule depolymerisation [START_REF] Nathan | The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule depolymerization[END_REF] and axonal degeneration 66 thus potentially affecting WM integrity. Although these studies point to a differential role for the apoE isoforms on white matter structure, the molecular mechanisms require further investigation.

LIMITATIONS AND FUTURE DIRECTIONS

Our cross-sectional study did not cover the entire lifespan but instead concentrated on distinct age groups of younger (20-35 years) and older (50-78 years) volunteers. The age-related changes in WM that we observed are unlikely to be caused by a cohort effect, but longitudinal studies would be required to rule this out. Given our observation of white matter changes even in young adulthood, it would be particularly interesting to investigate differences in brain development of children and adolescents carrying the APOE ε4 allele.

It is important to note that the changes in WM that we observe in APOE ε4carriers may not relate to AD risk because there are other genes in linkage disequilibrium with APOE ε4 that might affect WM structure. [START_REF] Yu | Comprehensive analysis of APOE and selected proximate markers for late-onset Alzheimer's disease: patterns of linkage disequilibrium and disease/marker association[END_REF] For example APOC1 and TOMM40 have been associated with increased risk of AD development [START_REF] Scacchi | Plasma levels of apolipoprotein E and genetic markers in elderly patients with Alzheimer's disease[END_REF] or earlier age of AD onset. [START_REF] Roses | A TOMM40 variable-length polymorphism predicts the age of late-onset Alzheimer's disease[END_REF] Large population samples would be needed to study the contribution of other genes to WM structure and AD.

The link between APOE, WM structure and AD risk would be strengthened if evidence of a gene-dose effect in WM integrity that mirrored the gene dose effect in AD risk could be found. WM changes might prove to be a useful imaging marker for early detection of subjects at increased risk of developing AD. The methods for including imaging markers in genome wide association studies are now being worked out. [START_REF] Potkin | Hippocampal atrophy as a quantitative trait in a genome-wide association study identifying novel susceptibility genes for Alzheimer's disease[END_REF]

CONCLUSION

In conclusion, this study demonstrated that individuals carrying the APOE ε4 allelic variant have different WM-related measurements even at a young age but show no differences in GM structure. No significant interactions of genotype and age group were found indicating that differences in WM structure between APOE ε4-carriers and non-carriers remain relatively stable during adulthood. The WM changes we observed in young adults occur decades before any impending cognitive or clinical manifestations of disease, and are thus probably not caused by preclinical AD pathology. Nevertheless, the changes we see mirror those found in early AD and MCI and may therefore inform our understanding of the increased risk of disease in APOE ε4-carriers.

to 9 mm 45 ,

 45 on a Fluid Attenuated Inversion Recovery (FLAIR) image. In order to exclude possible confounds due to cognitive complaints older subjects underwent a pre-screening cognitive test [Addenbrooke's Cognitive Examination-revised version (ACE-R) 46 ]. Subjects were pre-screened for APOE genotype (using a cheek swab sample) and selected for the study on the basis of either having an APOE ε4 allele (APOE ε4-carrier) or being an ε3 homozygote (non-carrier). Three APOE ε4carriers were homozygous for the ε4 allele (one in the younger and two in the older group). The genotyping process was conducted at the Wellcome Trust Centre for Human Genetics in Oxford. DNA was extracted from cheek swab samples of subjects according to standard procedures to allow PCR for the characterization of APOE genotype. The study was approved by the local Ethics Committee, and written informed consent was signed by all participants. Image Acquisition: Scans were performed at the University of Oxford Centre for Clinical Magnetic Resonance Research (OCMR) using a 3-T Siemens Trio scanner (Siemens AG, Erlangen, Germany) with a 12-channel head coil. The neuroimaging protocol included a 3D T1-weighted structural scan (TR = 2040 ms, TE = 4.7 ms, flip angle = 8°, field of view = 192 mm, voxel dimension = 1 mm isotropic, acquisition time = 12 min), diffusion weighted imaging (echo planar imaging, TR = 9300 ms, TE = 94 ms, field of view = 192 mm, voxel dimension = 2 mm isotropic, B-value = 1000, gradients applied = 60 isotropically distributed, acquisition time = 21 min) and fluid attenuated inversion recovery (FLAIR) imaging (TR = 9000 ms, TE = 89 ms, field of view = 220 mm, voxel dimension = 1.1x0.9x3 mm, acquisition time = 5 min 8 sec).

Figure 1

 1 Figure 1Mean FA images with mean FA skeleton used for TBSS (green).

Figure 2

 2 Figure 2Mean FA images with mean FA skeleton used for TBSS (green).

Table 1

 1 Sociodemographic features and general brain morphology of both age groups. Values denote mean (±Standard Deviation) or number of subjects, *Values are expressed as percentage of whole-brain volume; pvalues refer to t-tests (parametric data) and chi-square tests (categorical data).

	Younger group	APOE ε4 carriers (N = 17)	APOE ε4 non-carriers (N = 17)	P
	Age [years]	28.88 (±4.69)	28.24 (±3.77)	0.66
	Sex [Male / Female]	10 / 7	10 / 7	1.00
	Education [years]	19.53 (±2.35)	19.41 (±1.91)	0.87
	Family history of dementia	2	2	1.00
	Encoding memory task (max 83)	70.88 (±8.17)	69.24 (±8.12)	0.56
	Reaction time, familiar blocks [s]	0.78 (±0.21)	0.76 (±0.15)	0.70
	Reaction time, novel blocks [s]	1.07 (±0.42)	0.99 (±0.27)	0.56
	Whole brain volume [cm 3 ]	2038.99 (±168.25)	2016.80 (±205.80)	0.73
	Gray Matter*	43.63 (±0.54)	43.78 (±0.93)	0.59
	White Matter*	32.76 (±1.11)	32.90 (±0.86)	0.68
	CSF*	23.61 (±1.07)	23.32 (±1.41)	0.51
	Left Hippocampus*	0.19 (±0.02)	0.20 (±0.01)	0.24
	Right Hippocampus*	0.22 (±0.03)	0.23 (±0.02)	0.24
	Older group	APOE ε4 carriers (N = 16)	APOE ε4 non-carriers (N = 21)	P
	Age [years]	65.13 (±8.45)	64.85 (±6.27)	0.92
	Sex [Male / Female]	6 / 10	8 / 13	1.00
	Education [years]	15.69 (±3.79)	16.66 (±3.54)	0.43
	Family history of dementia	3	6	0.70
	ACE-r results (max 100)	98.81 (±0.98)	99.29 (±1.27)	0.21
	Encoding memory task (max 83)	68.20 (±9.76)	68.90 (±6.57)	0.81
	Reaction time, familiar blocks [s]	0.75 (±0.09)	0.72 (±0.11)	0.44
	Reaction time, novel blocks [s]	0.99 (±0.15)	0.96 (±0.16)	0.55
	Whole brain volume [cm 3 ]	1900.07 (±173.27)	1872.54 (±184.07)	0.65
	Gray Matter*	41.50 (±1.05)	41.77 (±0.55)	0.35
	White Matter*	33.39 (±1.27)	32.95 (±1.18)	0.29
	CSF*	25.12 (±1.62)	25.28 (±1.34)	0.74
	Left Hippocampus*	0.19 (±0.03)	0.20 (±0.03)	0.37
	Right Hippocampus*	0.20 (±0.04)	0.21 (±0.03)	0.35

Acknowledgements

We thank Prof. Jonathan Flint and Amarjit Bhorma, Wellcome Trust Centre for Human Genetics, for genotyping APOE data. VH was supported by the Alzheimer's Research Trust (English Charity Register: 1077089) and the German National Academic Foundation (Studienstiftung des deutschen Volkes), NF by the Gordon Edward Small's Charitable Trust (Scottish Charity Register: SC008962).

Conflict of Interest

The authors declare no conflict of interest.


MD J Michael Dixon

Lorna Renshaw

Carolyn Langridge

Oliver E Young

Mary Mchugh

Linda Williams

Juliette Murray

E Jane Macaskill

Fiona Mccaig

Oliver M Dixon

; E Jane Macaskill

Lesley J Fallowfield

Anastrozole and letrozole: an investigation and comparison of quality of life and tolerability

Keywords: Anastrozole, Breast cancer, Letrozole, Quality of life, Tamoxifen

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Third-generation aromatase inhibitors (AIs) are approved as adjuvant endocrine therapy for postmenopausal women (PMW) with hormone-sensitive breast cancer [START_REF] Cazzaniga | on behalf of the NORA Study Group (2007) Adjuvant treatment of early breast cancer: do the St. Gallen recommendations influence clinical practice? Results from the NORA study[END_REF]. The AIs include two broad categories, the reversible nonsteroidal agents, anastrozole and letrozole, and the irreversible steroidal inhibitor, exemestane [START_REF] Eisen | Aromatase inhibitors in adjuvant therapy for hormone receptor positive breast cancer: a systemic review[END_REF]. These endocrine therapies have been developed to increase efficacy while decreasing the toxicities associated with more traditional estrogen receptor modulators (e.g., tamoxifen). AIs have been shown to improve disease-free survival (DFS) compared with tamoxifen [START_REF] Eisen | Aromatase inhibitors in adjuvant therapy for hormone receptor positive breast cancer: a systemic review[END_REF]. Given this obvious benefit, the question of which AI to use is important.

Anastrozole and letrozole are now both widely used as adjuvant therapy in the treatment of breast cancer [START_REF] Thürlimann | The Breast International Group (BIG) 1-98 Collaborative Group (2005) A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer[END_REF][START_REF] Forbes | Tamoxifen, Alone or in Combination (ATAC) Trialists' Group (2008) Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 100-month analysis of the ATAC trial[END_REF]. While previous studies have demonstrated that both of these agents are well tolerated [START_REF] Thürlimann | The Breast International Group (BIG) 1-98 Collaborative Group (2005) A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer[END_REF][START_REF] Coates | Five years of letrozole compared with tamoxifen as initial adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer: update of study BIG 1-98[END_REF][START_REF] Buzdar | Tamoxifen, Alone or in Combination Trialists' Group (2006) Comprehensive side-effect profile of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: long-term safety analysis of the ATAC trial[END_REF][START_REF] Goss | Randomized trial of letrozole following tamoxifen as extended adjuvant therapy in receptor-positive breast cancer: updated findings from NCIC CTG MA.17[END_REF], there are reported differences in the potency of estrogen suppression between these agents. Studies have consistently demonstrated that letrozole reduces plasma estradiol and estrone sulfate levels to a significantly greater extent than does anastrozole [START_REF] Geisler | Influence of letrozole and anastrozole on total body aromatization and plasma estrogen levels in postmenopausal breast cancer patients evaluated in a randomized, cross-over study[END_REF][START_REF] Dixon | Letrozole suppresses plasma estradiol and estrone sulphate more completely than anastrozole in postmenopausal women with breast cancer[END_REF][START_REF] Geisler | Letrozole is superior to anastrozole suppressing breast cancer tissue and plasma estrogen levels[END_REF].

Direct head-to-head trials of AIs are ongoing and will determine the extent to which these differences in potency translate into differences in clinical efficacy. The MA.27 trial is comparing event-free survival in PMW with receptor-positive primary breast cancer treated with anastrozole or exemestane following surgery (ClinicalTrials.gov identifier: NCT00066573), and the Femara Anastrozole Clinical Evaluation trial compares DFS in PMW with hormone receptorpositive (HR+), node-positive breast cancer treated with anastrozole or letrozole following surgery (ClinicalTrials.gov identifier: NCT00248170).

Indirect information from available large-scale trials comparing AIs with tamoxifen indicates that there are differences in clinical efficacy between AIs. Initial adjuvant therapy with AIs has been examined in the Arimidex, Tamoxifen Alone or in Combination (ATAC), Breast International Group 1-98, and the Tamoxifen, Exemestane Adjuvant Multicenter (TEAM) trials.

In terms of efficacy, both anastrozole and letrozole have been shown to improve DFS compared with tamoxifen in patients with HR+ disease, although only letrozole significantly reduced early (at 2 years) distant metastases (DM) events in this population (hazard ratio [HR] 0.73, P = 0.001) [START_REF] Thürlimann | The Breast International Group (BIG) 1-98 Collaborative Group (2005) A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer[END_REF]. A significant reduction in DM events was seen with anastrozole in the HR+ patients but only at 100 months of follow-up [START_REF] Forbes | Tamoxifen, Alone or in Combination (ATAC) Trialists' Group (2008) Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 100-month analysis of the ATAC trial[END_REF]. The early improvement in DM events with letrozole has resulted in what appears to be an improvement in overall survival in patients taking letrozole compared with those on tamoxifen [11]. No improvement in survival has emerged from the ATAC study (HR 0.97, P = 0.7) [START_REF] Forbes | Tamoxifen, Alone or in Combination (ATAC) Trialists' Group (2008) Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 100-month analysis of the ATAC trial[END_REF]. In the ongoing TEAM study, the first planned analysis at 2.75 years failed to demonstrate a significant difference between exemestane and tamoxifen with respect to DFS, although there was a reduction in breast cancer events (exemestane:tamoxifen, HR 0.89, P = 0.12) [START_REF] Jones | Results of the first planned analysis of the TEAM (tamoxifen exemestane adjuvant multinational) prospective randomized phase III trial in hormone sensitive postmenopausal early breast cancer[END_REF].

Patient tolerability and its potential influence on adherence and therapeutic outcome are an important concern [START_REF] Fallowfield | There's many a slip twixt cup and lip: adherence issues in cancer therapy[END_REF]. As a class, all AIs are reported to have similar toxicity profiles [START_REF] Thürlimann | The Breast International Group (BIG) 1-98 Collaborative Group (2005) A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer[END_REF][START_REF] Coates | Five years of letrozole compared with tamoxifen as initial adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer: update of study BIG 1-98[END_REF][START_REF] Buzdar | Tamoxifen, Alone or in Combination Trialists' Group (2006) Comprehensive side-effect profile of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: long-term safety analysis of the ATAC trial[END_REF][START_REF] Goss | Randomized trial of letrozole following tamoxifen as extended adjuvant therapy in receptor-positive breast cancer: updated findings from NCIC CTG MA.17[END_REF]; however, the adjuvant AI trials are not directly comparable because of differences in trial design, particularly with regard to the time at which AI treatment commenced and the variable methods used to collect safety and tolerability data. In the absence of direct comparative data, it is not clear whether the greater activity/potency of letrozole might translate into greater effects on a patient's quality of life (QOL). Differences between agents in terms of QOL and tolerability are likely to be important when selecting the optimal therapy for individual patients. QOL assessments are useful for the clinician to make informed treatment decisions and to identify supportive interventions that improve tolerability and adherence. The aim of this study was to compare the effects of anastrozole and letrozole on patient tolerability and QOL as assessed by the Functional Assessment of Cancer Therapy -Breast -Endocrine Symptoms (FACT-B-ES, version 4) questionnaire and to determine if there is a patient preference for one agent over the other. Other aims, which are reported elsewhere, were to assess the effects of these two drugs on hormone levels and on markers of bone metabolism.

Methods

Study design

The Anastrozole versus Letrozole: Investigation into Quality of Life and Tolerability (ALIQUOT) study was an open-label crossover study of PMW with estrogen receptor-positive breast cancer receiving adjuvant AI therapy [START_REF] Dixon | Letrozole suppresses plasma estradiol and estrone sulphate more completely than anastrozole in postmenopausal women with breast cancer[END_REF]. The ALIQUOT study examined the tolerability and side-effect profile associated with anastrozole and letrozole. One hundred eighty-five patients were randomized as part of their adjuvant endocrine therapy to receive either 12 weeks of letrozole followed by 12 weeks of anastrozole or 12 weeks of anastrozole followed by 12 weeks of letrozole. Assignment to treatment order was by randomized permuted blocks, undertaken manually using tables of random permutations. This crossover strategy was designed to allow direct comparison of anastrozole and letrozole in the same patient. Three patients withdrew before taking any drugs, and one patient withdrew because she was found to be premenopausal; therefore, a total of 181 patients were enrolled in the study and took one or the other drug. The current analysis was a limited-duration study comparing QOL, tolerability, and morbidity. The study was approved by the Lothian Research Ethics Committee in August 2003 and was conducted to comply with the principles laid down in the Declaration of Helsinki.

Assessments

Patient self-report FACT-B-ES (version 4) QOL questionnaires were completed at trial entry, after 4 weeks, and after 12 weeks of each drug. FACT-B is a 38-item questionnaire with six subscales assessing physical, social, emotional, and functional well-being, relationship with doctor, and additional concerns more specific to women with breast cancer. The FACT-B has a 5-point Likert-type response scale ranging from 0 to 4 (0 = not at all, 1 = a little bit, 2 = somewhat, 3 = quite a bit, and 4 = very much). The ES assesses endocrine side effects and comprises 18 items designed for use with the FACT-B [START_REF] Brady | Reliability and validity of the Functional Assessment of Cancer Therapy-Breast quality-of-life instrument[END_REF][START_REF] Fallowfield | Assessment of quality of life in women undergoing hormonal therapy for breast cancer: Validation of an endocrine subscale for the FACT-B[END_REF]. FACT-B-ES is the combined total of the FACT-B and the ES subscale scores.

Nurse interview

Graded adverse events (AEs) were collected using Common Terminology Criteria for Adverse Events (version 3.0) as reported directly by patients to study nurses at 1 month (by telephone), 2 months (by telephone), 3 months (at follow-up for trial), 4 months (by telephone), 5 months (by telephone), and 6 months (at trial follow-up), and for those receiving extended adjuvant therapy, at 9 months (at trial follow-up). At the end of the 24-week study period, each patient completed a patient preference form, which indicated drug preference and reasons for preference.

Statistical methods

Planned sample size

The typical standard deviation between patients using the FACT-B-ES QOL scale (the primary outcome) is 18. In order to have 80% power to detect a difference of 4 units between paired treatments, a sample size of 160 was required. As there was likely to be patient dropout, the sample size was increase by another 20 (12.5%) to allow for this.

Statistical testing

The primary outcome of QOL (FACT-B-ES) was analyzed by Student's t-test for the comparison of baseline data, and by paired t-test for the change from baseline at 3 and 6 months. Both tests were two-sided. The secondary outcomes of patient-reported side effects and individual patient preference were compared by McNemar's test of paired data and chi-squared test, respectively.

Patients who withdrew following the first period were included in the comparisons of change to 3 months but excluded from the 6-month analysis and the paired analyses of side effects.

Sufficient additional patients were recruited to ensure that the number available for analysis would still exceed 160, at a level of attrition of 5% every 3 months.

Results

Patients/demographics

A total of 181 women (excluding a woman subsequently found to be premenopausal) participated in the study and received at least one dose of trial medication (Table 1). Patients

Withdrawal rates

Although both drugs were well tolerated, there were withdrawals for drug-associated reasons; these included joint pain and nausea associated with anastrozole treatment and hot flushes, joint pain, tiredness, headache, hair thinning, and blurred vision associated with letrozole treatment. A total of 21 patients withdrew before study end. Ten of 179 (5.6%) withdrew while taking letrozole, and 4/173 (2.3%) withdrew while taking anastrozole (P = 0.12). Withdrawals were not related to drug sequence. Two women withdrew following vaginal bleeding after finishing tamoxifen and before starting letrozole. Similarly, two women withdrew when bleeding developed after the completion of tamoxifen and before the start of anastrozole (Fig. 1). When using the FACT-B questionnaire, if there is a missing item in a scale, then an overall score cannot be produced. Endocrine Subscale scores for patients were subdivided by whether they had previously taken 5 years of tamoxifen. Tamoxifen-naïve patients had a mean ES score at entry of 66.0, compared with a score of 61.9 for those patients who had received prior tamoxifen (P = 0.001).

The mean changes in ES scores from baseline are shown in Table 3 (an increase in score corresponds to fewer endocrine-related symptoms). There was a small but non-significant reduction in score in tamoxifen-naïve patients and a small but non-significant increase in score in patients who received prior tamoxifen therapy (A clinically meaningful score on the ES subscale is approximately 0.3 per item; with 18 items, this will be approximately 5). Overall, regardless of the sequence or prior tamoxifen, neither anastrozole nor letrozole significantly affected ES scores.

Side effect/symptom analysis

The percentages of patients with any reported side effect at any time are shown in Table 4.

Reporting of side effects was similar for both drugs after the initial 12 weeks of treatment. There were no significant differences in the frequency or range of side effects between anastrozole and letrozole. Nearly 80% of patients complained of one or more side effects with either anastrozole or letrozole. Joint pain occurred with the highest frequency (from 40-52% depending upon the agent and previous tamoxifen exposure). Of the reported events of joint pain, 19-29% of patients experienced grade 2 pain. Hot flushes, lack of energy, and night sweats were also commonly reported by patients in both treatment arms. Table 5 reports the numbers of women with either no side effects or minimal (grade 1)/none on anastrozole or letrozole. Prior tamoxifen treatment did not significantly influence the reporting of side effects. When minimal (grade 1) side effects were excluded, this result did not change (Table 5).

Given the possibility that symptoms may increase or decrease over time irrespective of drug treatment, comparisons of first-and second-period side effects were performed. Only the number of hot flushes (more in the first period; P = 0.0009), joint problems (more in the second period; P < 0.0001), and rashes (more in the first period; P = 0.003) were influenced by time period. This result was found whether letrozole or anastrozole data were analyzed individually. These differences and the direction of the differences were still present when the subgroup of patients who had taken anastrozole first were analyzed (P = 0.0164, 0.0009, and 0.0253, respectively) or when the subgroup of patients who had taken letrozole first were analyzed (P = 0.02, < 0.0001, and = 0.046, respectively). This implies that the time period rather than the drug treatment is more of an influence on the presence of side effects and indicates that certain side effects take time to develop or improve.

Patient preference questionnaires

One hundred sixty patients completed a drug preference questionnaire. Forty-nine (30.6%) preferred letrozole, 57 (35.6%) preferred anastrozole, and 54 (33.8%) had no preference. These results indicate that more than two thirds of women have a preference for one or the other AI.

Drug order and previous tamoxifen exposure had no effect on preference. Figure 2 (online) presents the comparison of patient preferences according to previous tamoxifen use. There was no statistically significant preference in any group for any drug. Preferences were based on factors such as generally felt better, fewer hot flushes, less joint pain, less nauseated, less stomach upset, less tired, less depression, less emotional, less flatulence, less vaginal dryness, less abdominal bloating, less hair thinning, less weight gain, and less increased appetite. Table 6 gives patients' reasons for preferring letrozole or anastrozole.

Discussion

The impact on patient QOL is an important consideration in the treatment of breast cancer. Since current treatment recommendations are based upon efficacy results from 5 years of therapy, it is vital for the clinician to appreciate the long-term impact on QOL parameters over the same timeframe. Deterioration in these parameters may affect adherence and, in turn, have a negative impact on overall treatment outcome. Patients should be able to actively participate in the selection of therapy, weighing the clinical benefit against the side-effect profile and impact on QOL. Regular and standardized QOL assessments will ensure that both the clinician and patient have enough information to make informed decisions about treatment intervention.

Sub-protocol assessments of patients' QOL are being incorporated into increasing numbers of breast cancer trials. To date, QOL sub-analyses have been included in trials comparing AI therapy with tamoxifen (the Intergroup Exemestane Study [IES], ATAC, the National Surgical Adjuvant Study of Breast Cancer [NSAS BC] 03, and the TEAM trial NSAS BC 01 sub-study) and in trials comparing AIs with placebo (MA.17) [START_REF] Fallowfield | Quality of life in the intergroup exemestane study: a randomized trial of exemestane versus continued tamoxifen after 2 to 3 years of tamoxifen in postmenopausal women with primary breast cancer[END_REF][START_REF] Fallowfield | Quality of life of postmenopausal women in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) Adjuvant Breast Cancer Trial[END_REF][START_REF] Cella | Quality of life of postmenopausal women in the ATAC ("Arimidex", tamoxifen, alone or in combination) trial after completion of 5 years' adjuvant treatment for early breast cancer[END_REF][START_REF] Ohsumi | Healthrelated quality-of-life and psychological distress of postmenopausal breast cancer patients after surgery during the randomized trial, N-SAS BC 03, comparing further tamoxifen with switching to anastrozole after adjuvant tamoxifen for 1 to 4 years: the final results[END_REF][START_REF] Whelan | Assessment of quality of life in MA.17: a randomized, placebo-controlled trial of letrozole after 5 years of tamoxifen in postmenopausal women[END_REF][START_REF] Abetz | Letrozole did not worsen quality of life relative to placebo in post-menopausal women with early breast cancer: results from the US subjects of the MA-17 study[END_REF][START_REF] Muss | Efficacy, toxicity, and quality of life in older women with early-stage breast cancer treated with letrozole or placebo after 5 years of tamoxifen: NCIC CTG Intergroup Trial MA.17[END_REF][START_REF] Takei | Health-related quality of life and psychological distress of breast cancer patients after surgery during phase III randomized trial comparing tamoxifen, exemestane, and anastrozole: N-SAS BC 04[END_REF]. In general, the majority of results from these trials suggest that the superior efficacy benefits of AIs over tamoxifen are achieved without compromising overall QOL in the short term [START_REF] Fallowfield | Quality of life in the intergroup exemestane study: a randomized trial of exemestane versus continued tamoxifen after 2 to 3 years of tamoxifen in postmenopausal women with primary breast cancer[END_REF][START_REF] Cella | Quality of life of postmenopausal women in the ATAC ("Arimidex", tamoxifen, alone or in combination) trial after completion of 5 years' adjuvant treatment for early breast cancer[END_REF][START_REF] Whelan | Assessment of quality of life in MA.17: a randomized, placebo-controlled trial of letrozole after 5 years of tamoxifen in postmenopausal women[END_REF][START_REF] Abetz | Letrozole did not worsen quality of life relative to placebo in post-menopausal women with early breast cancer: results from the US subjects of the MA-17 study[END_REF][START_REF] Muss | Efficacy, toxicity, and quality of life in older women with early-stage breast cancer treated with letrozole or placebo after 5 years of tamoxifen: NCIC CTG Intergroup Trial MA.17[END_REF][START_REF] Takei | Health-related quality of life and psychological distress of breast cancer patients after surgery during phase III randomized trial comparing tamoxifen, exemestane, and anastrozole: N-SAS BC 04[END_REF][START_REF] Coombes | Intergroup Exemestane Study (2007) Survival and safety of exemestane versus tamoxifen after 2-3 years' tamoxifen treatment (Intergroup Exemestane Study): a randomised controlled trial[END_REF].

It has been well established that tamoxifen is associated with some potentially lifethreatening AEs, while the third-generation AIs are associated with more predictable, manageable, and preventable side effects [3, 4, 16-18, 20, 21, 25]. This more favorable tolerability profile should translate into an improved impact on a patient's overall QOL, but there are adherence issues with AIs, and significant numbers of patients discontinue therapy because of side effects. This is potentially very worrying, because some symptoms, especially vasomotor symptoms and arthralgias, have been shown to be associated with better outcomes [START_REF] Cuzick | Treatment emergent endocrine symptoms and the risk of breast cancer recurrence: a retrospective analysis of the ATAC trial[END_REF]. Given the lack of direct head-to-head comparisons of different AIs and their impact on QOL, it is difficult for clinicians to make evidence-based decisions when choosing an AI. The results presented here compare anastrozole and letrozole directly in the same patients and assess the effects of these AIs on QOL. This design has significant advantages over comparing groups of women, as it removes a major variable, the patient. Despite the reported greater potency of letrozole, it was as well tolerated as anastrozole, and neither drug had a significant impact on overall QOL. Although patients who completed treatment had little if any measured impact on QOL, side effects with both drugs were common and were reported by 80% of women. These side effects were identical with both drugs and represent a class effect of AIs rather than effects that are limited/restricted to a specific drug. Most patients had a definite preference for either anastrozole or letrozole, with similar numbers of patients preferring each agent.

A similar crossover study by Thomas et al. comparing anastrozole and letrozole in women with breast cancer (N = 72) showed that a patient's preference for either anastrozole or letrozole was related to the patient's adverse events and QOL while taking the drug [START_REF] Thomas | Giving patients a choice improves quality of life: a multi-centre, investigator-blind, randomised[END_REF]. In contrast to the current study, which showed similar results for both drugs, the study by Thomas et al. showed that significantly more women chose letrozole, which was associated with significantly fewer adverse events and a significantly better QOL compared with anastrozole [START_REF] Thomas | Giving patients a choice improves quality of life: a multi-centre, investigator-blind, randomised[END_REF].

This study has several limitations. It was an open-label, single-institution design with short follow-up and limited patient numbers, and patient reporting is potentially subject to influence from family/friends, support groups, and publically available medical information.

Anastrozole is the most widely and frequently used AI in the United Kingdom, and potential social influences for preference must be considered. Despite these limitations and potentially confounding variables, the data reported here provide a useful framework within which physicians and patients can make informed decisions about initial adjuvant or extended adjuvant AI therapy. Larger-scale analyses in direct comparator trials will be required to confirm the current findings, but such studies have their own limitations. While treatment decisions cannot be made solely on the basis of QOL and reported side-effect profiles, such assessments are an important consideration, as adherence is likely to be significantly affected by a drug's impact on these parameters.

  were recruited into the QOL study fromNovember 24, 2003 to April 11, 2006 from the Edinburgh Breast Unit and Queen Margaret Hospital Breast Unit, Dunfermline. One hundred six received immediate adjuvant AIs following surgery, and 75 received extended adjuvant AI therapy following the completion of 5 years of adjuvant tamoxifen treatment. Thirty-eight patients received chemotherapy post surgery before receiving tamoxifen. Eighty-nine patients received letrozole therapy for 3 months followed by anastrozole therapy for 3 months. Ninetytwo patients received the reverse sequence: 3 months of anastrozole followed by 3 months of letrozole. The mean age of patients was 63 years (range, 40 to 87 years), the mean height was 161 cm (range, 128 to 183 cm) and the mean weight was 72.8 kg (range, 48.0 to 140.0 kg).

  QOL scores and endocrine subscale results of the FACT-B-ES scores Baseline scores for FACT-B-ES (overall quality of life score which is an additive of the FACT-B and the Endocrine Subscale Score) were 183.6 (n = 92; 95% confidence interval [CI], 179.4-187.8) for tamoxifen-naïve patients and 185.4 (n = 74; 95% CI, 181.6-189.3) (P = 0.54) for patients on prior tamoxifen. Mean changes in FACT-B-ES scores from baseline are shown in Table2. Mean changes from baseline were recorded at 3 and 6 months. An increase in score indicates a better overall QOL. There was no significant change in overall FACT-B-ES score or endocrine symptoms subscale (ES) score while patients were taking anastrozole or letrozole, and no significant differences between the two drugs were observed. Neither AI had any measureable detrimental effect on overall QOL. Neither the drug sequence nor prior tamoxifen treatment had any influence on the results.

*Table 3

 3 Abbreviation: CI, confidence interval.

Fig 2 .

 2 Fig 2. Results from the drug preference questionnaire (online only).

Table 2

 2 Mean changes from baseline for the Functional Assessment of Cancer Therapy with Endocrine Subscale scores (FACT-B-ES) (overall quality of life score)

	Patient population Drug sequence	Mean change from	Mean change from
			baseline at 3 months	baseline at 6 months
	All patients	Letrozole to	1.3 (n = 67; 95% CI,	0.2 (n = 67; 95% CI,
	(N = 181)	anastrozole	-2.2-4.8)*	-3.5-3.9)
		Anastrozole to	1.6 (n = 75; 95% CI,	1.7 (n = 74; 95% CI,
		letrozole	-1.8-4.9)	-1.8-5.2)
	Tamoxifen-naïve	Letrozole to	0.7 (n = 39; 95% CI,
	patients (n = 106)	anastrozole

Table 6

 6 Patients' stated reason for drug preference

		Patient preference
	Reason for preference	Letrozole	Anastrozole
	Generally felt better	67% (16/24)	94% (30/32)*
	Less tired	29% (7/24)	69% (22/32) †
	Less sick	29% (7/24)	38% (12/32)
	Less tummy upset	29% (7/24)	31% (10/32)
	Fewer hot flushes	33% (8/24)	53% (17/32)
	Fewer joint pains	42% (10/24)	50% (16/32)

*P = 0.009.

† P = 0.003.

Acknowledgments

Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals.

We thank Maria Soushko, PhD, of Phase Five Communications Inc. for medical editorial assistance with this manuscript.

Financial support for this study was provided by Novartis Oncology.

Prior