Converging or Crossing Curves: Untie the Gordian Knot or Cut it? Appropriate Statistics for Non-Proportional Hazards in Decitabine DACO-016 Study (AML).

INTRODUCTION: Among patients with acute myeloid leukemia (AML), the DACO-016 randomized study showed reduction in mortality for decitabine [Dacogen(®) (DAC), Eisai Inc., Woodcliff Lake, NJ, USA] compared with treatment choice (TC): at primary analysis the hazard ratio (HR) was 0.85 (95% confidence interval 0.69-1.04; stratified log-rank P = 0.108). With two interim analyses, two-sided alpha was adjusted to 0.0462. With 1-year additional follow-up the HR reached 0.82 (nominal P = 0.0373). These data resulted in approval of DAC in the European Union, though not in the United States. Though pre-specified, the log-rank test could be considered not optimal to assess the observed survival difference because of the non-proportional hazard nature of the survival curves. METHODS: We applied the Wilcoxon test as a sensitivity analysis. Patients were randomized to DAC (N = 242) or TC (N = 243). One-hundred and eight (44.4%) patients in the TC arm and 91 (37.6%) patients in the DAC arm selectively crossed over to subsequent disease modifying therapies at progression, which might impact the survival beyond the median with resultant converging curves (and disproportional hazards). RESULTS: The stratified Wilcoxon test showed a significant improvement in median (CI 95%) overall survival with DAC [7.7 (6.2; 9.2) months] versus TC [5.0 (4.3; 6.3) months; P = 0.0458]. CONCLUSION: Wilcoxon test indicated significant increase in survival for DAC versus TC compared to log-rank test. FUNDING: Janssen-Cilag GmbH.

Inflammatory proteins in plasma are associated with severity of Alzheimer's disease.

Markers of Alzheimer's disease (AD) are being widely sought with a number of studies suggesting blood measures of inflammatory proteins as putative biomarkers. Here we report findings from a panel of 27 cytokines and related proteins in over 350 subjects with AD, subjects with Mild Cognitive Impairment (MCI) and elderly normal controls where we also have measures of longitudinal change in cognition and baseline neuroimaging measures of atrophy. In this study, we identify five inflammatory proteins associated with evidence of atrophy on MR imaging data particularly in whole brain, ventricular and entorhinal cortex measures. In addition, we observed six analytes that showed significant change (over a period of one year) in people with fast cognitive decline compared to those with intermediate and slow decline. One of these (IL-10) was also associated with brain atrophy in AD. In conclusion, IL-10 was associated with both clinical and imaging evidence of severity of disease and might therefore have potential to act as biomarker of disease progression.

Plasma transthyretin as a candidate marker for Alzheimer's disease.

Diagnosis of the progressive neurodegenerative disorder Alzheimer's disease (AD) can only definitively be made postmortem. The most promising AD biomarkers identified to date are found in cerebrospinal fluid (CSF). Among these, one of the most interesting candidates is transthyretin (TTR), the carrier of thyroxine and retinol, which also binds with amyloid-ß (Aß), and it has been suggested that it protects against Aß deposition. A biomarker detectable in plasma would have great diagnostic value and could be of use for determining disease progression and the monitoring of therapeutic efficacy due to its greater accessibility over CSF-based markers. We aimed to validate TTR as a prognostic marker in AD and to determine its relation with cognitive measures. We examined the plasma protein levels of TTR in 90 people with late-onset AD and 50 age-matched non-demented controls (NDC) by immunoblotting and found lower plasma TTR levels in AD compared to NDC (p = 0.004). We then quantified plasma TTR by enzyme-linked immunosorbent assays in a larger independent cohort (n = 270) including subjects with mild to severe AD. Plasma TTR levels were significantly lower in AD cases with rapid cognitive decline and with severe cognitive impairment. Regression analyses showed plasma TTR levels also predicted cognitive decline over the ensuing 6 months. These data indicate that plasma TTR is a strong candidate AD biomarker that should be included in the development of blood based biomarker panels for disease diagnosis and also suggests that plasma TTR is a marker of disease severity and progression.

Plasma clusterin concentration is associated with longitudinal brain atrophy in mild cognitive impairment.

Recent genetic and proteomic studies demonstrate that clusterin/apolipoprotein-J is associated with risk, pathology, and progression of Alzheimer's disease (AD). Our main aim was to examine associations between plasma clusterin concentration and longitudinal changes in brain volume in normal aging and mild cognitive impairment (MCI). A secondary objective was to examine associations between peripheral concentration of clusterin and its concentration in the brain within regions that undergo neuropathological changes in AD. Non-demented individuals (N=139; mean baseline age 70.5 years) received annual volumetric MRI (912 MRI scans in total) over a mean six-year interval. Sixteen participants (92 MRI scans in total) were diagnosed during the course of the study with amnestic MCI. Clusterin concentration was assayed by ELISA in plasma samples collected within a year of the baseline MRI. Mixed effects regression models investigated whether plasma clusterin concentration was associated with rates of brain atrophy for control and MCI groups and whether these associations differed between groups. In a separate autopsy sample of individuals with AD (N=17) and healthy controls (N=4), we examined the association between antemortem clusterin concentration in plasma and postmortem levels in the superior temporal gyrus, hippocampus and cerebellum. The associations of plasma clusterin concentration with rates of change in brain volume were significantly different between MCI and control groups in several volumes including whole brain, ventricular CSF, temporal gray matter as well as parahippocampal, superior temporal and cingulate gyri. Within the MCI but not control group, higher baseline concentration of plasma clusterin was associated with slower rates of brain atrophy in these regions. In the combined autopsy sample of AD and control cases, representing a range of severity in AD pathology, we observed a significant association between clusterin concentration in the plasma and that in the superior temporal gyrus. Our findings suggest that clusterin, a plasma protein with roles in amyloid clearance, complement inhibition and apoptosis, is associated with rate of brain atrophy in MCI. Furthermore, peripheral concentration of clusterin also appears to reflect its concentration within brain regions vulnerable to AD pathology. These findings in combination suggest an influence of this multi-functional protein on early stages of progression in AD pathology.

Combinatorial markers of mild cognitive impairment conversion to Alzheimer's disease--cytokines and MRI measures together predict disease progression.

Progression of people presenting with Mild Cognitive Impairment (MCI) to dementia is not certain and it is not possible for clinicians to predict which people are most likely to convert. The inability of clinicians to predict progression limits the use of MCI as a syndrome for treatment in prevention trials and, as more people present with this syndrome in memory clinics, and as earlier diagnosis is a major goal of health services, this presents an important clinical problem. Some data suggest that CSF biomarkers and functional imaging using PET might act as markers to facilitate prediction of conversion. However, both techniques are costly and not universally available. The objective of our study was to investigate the potential added benefit of combining biomarkers that are more easily obtained in routine clinical practice to predict conversion from MCI to Alzheimer's disease. To explore this we combined automated regional analysis of structural MRI with analysis of plasma cytokines and chemokines and compared these to measures of APOE genotype and clinical assessment to assess which best predict progression. In a total of 205 people with MCI, 77 of whom subsequently converted to Alzheimer's disease, we find biochemical markers of inflammation to be better predictors of conversion than APOE genotype or clinical measures (Area under the curve (AUC) 0.65, 0.62, 0.59 respectively). In a subset of subjects who also had MRI scans the combination of serum markers of inflammation and MRI automated imaging analysis provided the best predictor of conversion (AUC 0.78). These results show that the combination of imaging and cytokine biomarkers provides an improvement in prediction of MCI to AD conversion compared to either datatype alone, APOE genotype or clinical data and an accuracy of prediction that would have clinical utility.

Association of plasma clusterin concentration with severity, pathology, and progression in Alzheimer disease.

CONTEXT: Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD). OBJECTIVE: To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach. DESIGN: Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model. SETTING: A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging. PARTICIPANTS: Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging. MAIN OUTCOME MEASURES: Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid. RESULTS: Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques. CONCLUSIONS: These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

Plasma gelsolin is decreased and correlates with rate of decline in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder where definite diagnosis can only be made postmortem, and for which the most promising peripheral markers of disease state and severity have been found in the cerebrospinal fluid. However, recent results suggest that differences in the levels of certain plasma proteins do exist between AD patients and non-demented controls (NDC). Herein, we undertook an untargeted discovery study using isobaric mass tagging to compare the plasma protein levels between slow cognitive declining AD patients, rapid cognitive declining AD patients (RCD) and NDC subjects. Subsequent relative quantification and statistical analysis identified a list of candidate proteins able to distinguish RCD from NDC groups based on multivariate analysis. Selected proteins were then validated by western blot analysis in an independent sample set of 60 AD and 35 NDC subjects. In this cohort, AD patients displayed significantly lower plasma gelsolin levels compared to NDC subjects. Additionally, gelsolin levels correlated with disease progression rate estimated by Mini-Mental Status Examination decline per year. In order to further investigate gelsolin expression, three different brain regions from an additional cohort of 23 subjects and their respective plasma samples were analysed. No significant change in brain gelsolin levels could be established between AD and control subjects. Interestingly, this study reveals yet another condition where plasma gelsolin levels are decreased and our findings, together with the reported interaction of gelsolin and amyloid-beta, makes plasma gelsolin an attractive candidate for further studies targeted at better understanding disease progression in AD.

Proteomics for brain disorders--the promise for biomarkers.

Biomarkers of brain disorders are urgently needed to aid diagnosis, monitor disease progression, and, as new medicines are introduced, detect the patient's response to treatment. Proteomics provides the opportunity to discover novel biochemical markers based on protein or peptide changes, either in concentration levels or post-translational modification status. There are many challenges associated with proteomics studies, and this article represents a review of the issues discussed during the proteomics breakout sessions held at the Biomarkers for Brain Disorders conference in Oxford in January 2009. Although to date, there are very few qualified biomarkers that have arisen as a result of proteomics efforts, we remain optimistic that proteomics will deliver biomarkers for brain disorders. To be successful, we need to recognize that such endeavors are likely to require multidisciplinary teams and continued collaboration between academia, the biotechnology industry, and the pharmaceutical sector.

Proteomics of Alzheimer's disease: understanding mechanisms and seeking biomarkers.

Alzheimer's disease is the scourge of the modern, aging world: a costly, damaging disease that robs the elderly of their ability to function as well as their memories. Three decades of progress have resulted in a deep understanding of the pathological processes and a range of targets for therapy, many of which have advanced to late-stage clinical trials. Proteomics has contributed greatly to these advances and will continue to have a growing role in determining the nature of the pathological lesions in the brain. In addition, proteomics (both gel based and gel free, mass spectrometry based), is likely to play an increasing role in identifying biomarkers that may assist in early diagnosis and in monitoring progression and, most importantly, response to therapy.

Quantification of the A beta peptide in Alzheimer's plaques by laser dissection microscopy combined with mass spectrometry.

The accumulation and aggregation of the beta-amyloid peptide (A beta) in the brain represents a key factor in the pathogenesis of Alzheimer's disease (AD). Many of the transgenic mouse models for AD exhibit an amyloid pathology with neuritic plaques but they typically vary by the type and abundance of plaques identified in their brains and by the onset and severity of cognitive impairment. Thus, an important consideration in the characterization of AD transgenic mouse models should be the quantitative evaluation of the amyloid load in the brain together with a detailed physico-chemical analysis of A beta from the deposited plaques. Here we present an analytical procedure to collect single amyloid plaques from anatomically defined brain regions by laser dissection microscopy that can be quantitatively assessed in their A beta isoforms composition by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Quantification was achieved by stable isotope dilution using calibrated 15N-labeled A beta standards that were spiked in the sample immediately after laser dissection. Using this method, we found that the amyloid loads in brain plaques isolated from the transgenic AD mouse model PS2APP or from human were similar. Total A beta composition was estimated at approximately 50-100 fmol per excised plaque disc, as confirmed by immunoblot analysis. N-Terminal truncated A beta isoforms were identified in both transgene and human amyloid plaques but with significantly elevated levels in human samples.