Cerebrospinal fluid biomarkers in Alzheimer's Disease

321 Pages·2017·4.76 MB·English

by  Ross Paterson

University College London
Institute of Neurology

Cerebrospinal fluid biomarkers in
Alzheimer’s Disease: from bedside
to bench and back

A thesis submitted for the Degree of Doctor of Philosophy

Ross William Paterson

August 2016

1
In memory of Margaret Mackinnon

2
Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in
cognitive impairment and death. The pathological hallmarks are extracellular
cortical amyloid plaques and intraneuronal tangles composed of
hyperphosphorylated tau. Although environmental and genetic factors contribute to
the development of AD, the sequence of pathophysiological events that lead to
Alzheimer’s dementia is not yet completely clear. The clinical diagnosis of AD
during life can be challenging and factors that explain clinical phenotypic
heterogeneity and variability in rates of disease progression are not well understood.
Biomarkers, objective measures of biological function, can be employed to support a
clinical diagnosis of AD and may be abnormal before the onset of clinical symptoms.
Imaging and cerebrospinal fluid biomarkers (CSF) are now incorporated into clinical
and research diagnostic criteria. CSF, which is in direct contact with the brain, is a
promising source of biomarkers and has the potential to differentiate AD from other
neurodegenerative dementias, explain clinical heterogeneity within AD and
elucidate the role of other pathobiological pathways. Ultimately CSF biomarkers
might facilitate diagnosis of AD in its pre-clinical phase and allow for treatment
responses to be measured.
In this thesis CSF samples from clinical cohorts of individuals with AD, other
neurodegenerative diseases and healthy controls are analysed using an extended
panel of enzyme-linked immunosorbent assays (ELISA) and a novel mass
spectrometry based assay. For the established CSF biomarkers, the practical issues
related to collection, transportation and storage of CSF are investigated. Amyloid
positron emission tomography (PET) imaging is investigated as a means of
validating clinical cutpoints. An extended panel of established and emerging ELISAs
is used to determine the diagnostic utility of biomarkers for differentiating AD from
other neurodegenerative dementias and for explaining phenotypic heterogeneity
within AD. The role of CSF biomarkers as predictors of disease progression is
investigated employing robust measures of brain atrophy as surrogate measures of
rates of neurodegeneration. Finally CSF samples are probed for new AD biomarkers
using a novel mass spectrometry based assay.
A number of practical conclusions are drawn from this work: aliquot storage volume
is identified as an important confounder in measured CSF b-Amyloid concentration.
CSF laboratory transportation methods are shown not to have a significant impact
3
on measured biomarker concentration. Amyloid PET is a valuable means of
validating clinical diagnostic cutpoints of core CSF biomarkers. Tau/Ab1-42 ratio,
Ab40/42 ratio, P-tau and NFL emerge as having diagnostic utility for
differentiating AD from other neurodegenerative diseases, and have high sensitivity
and specificity for distinguishing AD from bvFTD, SD and healthy controls.
Important differences in T-tau, P-tau and neurofilament light distinguish different
AD atypical phenotypes and may help to elucidate underlying biological differences
between these syndromes: individuals with the visual variant of AD (posterior
cortical atrophy) have the lowest levels of CSF Tau and lowest rates of cognitive
decline while the frontal executive cases have highest levels of NFL and highest
rates of cognitive decline indicating more rapid neurodegeneration. Several novel
biomarkers including trefoil factor 3 and several markers involved in vascular
remodeling, amyloid processing and neuroinflammation are identified as predictors
of increased atrophy rates in amyloid positive individuals suggesting possible
independent mechanisms driving differing rates of neurodegeneration between
individuals. Other novel AD biomarkers including malate dehydrogenase are
identified as distinguishing AD from controls using a novel mass spectrometry
based assay. Moreover, this assay demonstrates how mass spectrometry might be
used for biomarker discovery and rapid development of a high throughput
multiplexed clinical CSF assay.
Taken together these results address some of the unanswered questions about how
CSF should be collected, handled and stored to optimize analytical standardization,
and how clinical results might be validated using amyloid PET. This work
establishes the clinical utility of established biomarkers for differentiating AD from
other neurodegenerative diseases and identifies established and novel biomarkers
that might explain clinical heterogeneity and rates of progression between
individuals. Finally a method for rapidly developing new biomarkers is tested and
validated.

4
Acknowledgements
I am greatly indebted to my supervisors Jonathan Schott, Henrik Zetterberg and
Nick Fox for their immense enthusiasm, unwavering support and for generously
sharing their knowledge of the field.

I have been privileged to share my time at the Dementia Research Centre with
Catherine Slattery and I am grateful for her support, ideas and friendship.

I am grateful to have collaborated with Jamie Toombs, Martha Foiani, Wendy
Heywood and Kevin Mills. I am particularly indebted to Amanda Heslegrave for her
time, patience and dedication in the laboratory.

Finally, I am thankful to Michael and to my parents and family for their love and
support.

5
Contents
Contents ......................................................................................................................... 6
Abbreviations............................................................................................................... 13
List of Tables ............................................................................................................... 15
List of Figures ............................................................................................................. 17
Chapter 1. Introduction ............................................................................................. 20
1.1 Alzheimer’s Disease ......................................................................................... 20
The Problem ........................................................................................................ 20
1.1.2 History and Epidemiology........................................................................ 22
1.1.3 Clinical features......................................................................................... 23
1.1.4 Differential Diagnosis of AD .................................................................... 25
1.1.5 Neuropathology of AD ............................................................................. 28
1.1.6 AD pathophysiology: The Amyloid Cascade Hypothesis ........................ 36
1.1.7 Genetic determinants and risk factors ..................................................... 38
1.1.8 Alzheimer’s disease progression ............................................................... 40
1.1.9 Current hypothetical models of biomarker changes ................................ 43
1.1.10 Diagnostic Criteria ................................................................................. 45
1.2 Biomarkers for Alzheimer’s disease................................................................. 46
1.2.1 Currently Available Biomarkers............................................................... 48
1.3 Cerebrospinal Fluid ......................................................................................... 55
1.3.1 Normal physiology and function of CSF .................................................. 55
1.3.2 Why is CSF of interest in Alzheimer’s disease?....................................... 56
1.3.3 Sampling CSF ........................................................................................... 57
1.3.4 CSF Analysis............................................................................................. 59
1.3.5 Confounders in CSF biomarker measurement ......................................... 61
1.3.6 Currently available CSF biomarkers ........................................................ 63
1.3.7 Practical Application of CSF biomarkers ................................................. 73
1.3.8 Biomarkers in revised diagnostic criteria................................................. 79
1.3.9 Limitations of currently available biomarkers ......................................... 81
1.3.10 Developing new biomarkers ................................................................... 81
1.4 Aims and objectives ......................................................................................... 83
1.4.1 Aims .......................................................................................................... 83
1.4.2 Objectives .................................................................................................. 83
1.5 Publications arising from this chapter ............................................................ 84
6
Chapter 2. General Methods ..................................................................................... 85
2.1 Cohorts............................................................................................................. 85
2.1.1 Retrospective cohort ................................................................................. 88
2.1.2 Prospective Cohort ................................................................................... 93
2.1.3 Healthy Control Cohort............................................................................ 99
2.1.4 Alzheimer’s Disease Neuroimaging Initiative (ADNI) Cohort ............. 101
2.1.5 Young Onset AD (YOAD) cohort.......................................................... 103
2.1.6 Swedish Cohort ....................................................................................... 106
2.2 Cerebrospinal Fluid Analysis ........................................................................ 107
2.2.1 Enzyme-linked immunosorbent assays (ELISA) ................................... 107
2.2.2 Mass Spectrometry ................................................................................. 114
2.3 Imaging .......................................................................................................... 120
2.4 Genetic Testing ............................................................................................. 120
2.5 Data Management ......................................................................................... 121
2.6 Statistical Analysis......................................................................................... 121
2.6.1 Testing for normality of data ................................................................. 121
2.6.2 Handling missing values......................................................................... 122
2.6.3 Handling truncated values...................................................................... 122
2.6.4 Adjusting for multiple comparisons ....................................................... 122
Chapter 3. CSF collection, handling and processing: Aliquot volume ................... 123
3.1 Introduction ................................................................................................... 123
3.2 Contributions and collaborations .................................................................. 124
3.3 Materials and Methods.................................................................................. 125
3.3.1 Sample pools ........................................................................................... 125
3.3.2 Sample treatment .................................................................................... 126
3.3.3 Schedule and CSF analysis ..................................................................... 126
3.3.4 Statistical analysis................................................................................... 126
3.3.5 Surface area calculation .......................................................................... 126
3.4 Results............................................................................................................ 127
3.4.1 Aβ1-42..................................................................................................... 127
3.4.2 T-tau ....................................................................................................... 127
3.4.3 P-tau ........................................................................................................ 127
3.4.4 Tube surface area .................................................................................... 127
3.5 Discussion ...................................................................................................... 128
3.5.1 Surface area .............................................................................................. 128
7
3.5.2 Tween 20................................................................................................. 129
3.5.3 Temperature ........................................................................................... 130
3.5.4 pH ............................................................................................................ 131
3.6 Further Work ................................................................................................ 131
3.7 Conclusions .................................................................................................... 132
3.8 Publications arising from this chapter .......................................................... 133
3.9 Publications related to this chapter............................................................... 133
Chapter 4. Transferring CSF from bedside to laboratory; does careful pre-analytical
handling of CSF affect measured Aβ1-42, T-tau, P-tau in clinical practice? ........... 134
4.1 Introduction ................................................................................................... 134
4.2 Contributions and collaborations .................................................................. 135
4.3 Methods ......................................................................................................... 135
4.3.1 Subjects ................................................................................................... 135
4.3.2 Methods .................................................................................................. 135
4.3.3 Statistics .................................................................................................. 136
4.4 Results............................................................................................................ 137
4.5 Discussion ...................................................................................................... 142
4.6 Conclusions .................................................................................................... 144
4.7 Publications related to this chapter................................................................. 144
Chapter 5. Using Amyloid PET to determine clinical cutpoints for established CSF
biomarkers ................................................................................................................. 145
5.1 Introduction ................................................................................................... 145
5.2 Contributions and collaborations .................................................................. 147
5.3 Subject and Methods ..................................................................................... 148
5.3.1 Ethics statement ....................................................................................... 148
5.3.2 Subjects and CSF collection ................................................................... 148
5.3.3 Amyloid PET .......................................................................................... 149
5.3.4 Statistical Analysis.................................................................................. 150
5.4 Results............................................................................................................ 150
5.5 Discussion ...................................................................................................... 153
5.6 Conclusion...................................................................................................... 154
5.7 Publications arising from this chapter .......................................................... 154
Chapter 6. CSF in the differential diagnosis of Alzheimer’s disease: clinical utility of
a panel of biomarkers in a specialist cognitive clinic ................................................ 155
6.1 Introduction ................................................................................................... 155
8
6.2 Contributions and Collaborations ................................................................. 156
6.3 Methods ......................................................................................................... 156
6.3.1 Ethics statement ..................................................................................... 156
6.3.2 Test (‘retrospective’) cohort ................................................................... 157
6.3.3 Validation (‘prospective’) cohort............................................................. 158
6.3.4 Healthy controls ..................................................................................... 158
6.3.5 Sample treatment and analysis ............................................................... 158
6.3.6 Statistical analysis................................................................................... 159
6.4 Results............................................................................................................ 161
6.4.1 Subject demographics ............................................................................. 161
6.4.2 Diagnostic concordance .......................................................................... 161
6.4.3 CSF biomarker concentrations ............................................................... 162
6.4.4 Diagnostic accuracy of CSF biomarkers ................................................ 168
6.4.5 Diagnostic utility of combinations of biomarkers to predict diagnosis . 171
6.4.6 Validation ................................................................................................ 172
6.5 Discussion ...................................................................................................... 174
6.6 Conclusions .................................................................................................... 179
6.7 Publications arising from this chapter .......................................................... 179
Chapter 7. CSF in the differentiation of AD clinical phenotypes ........................... 180
7.1 Introduction ................................................................................................... 180
7.2 Contributions and collaborations .................................................................. 181
7.3 Subjects and methods .................................................................................... 181
7.3.1 Ethics statement ..................................................................................... 181
7.3.2 Subjects ....................................................................................................... 181
7.3.3 Cerebrospinal fluid Collection and Biomarker Analysis ........................ 183
7.3.4 Statistical Analysis.................................................................................. 184
7.4 Results............................................................................................................ 184
7.4.1 Comparison of IWG2 typical and atypical Alzheimer’s disease ............ 184
7.4.2 Comparing atypical Alzheimer’s disease subtypes................................. 186
7.4.3 Comparing atypical Alzheimer’s disease subgroups to amnestic
Alzheimer’s disease ............................................................................................ 188
7.4.4 Behavioural (frontal variant) AD cases .................................................. 191
7.5 Discussion ...................................................................................................... 192
7.6 Conclusion...................................................................................................... 199
7.7 Publications arising from this chapter .......................................................... 200
9
Chapter 8. CSF biomarkers of disease progression ................................................. 201
8.1 Introduction ................................................................................................... 201
8.2 Contributions and collaborations .................................................................. 203
8.3 Methods ......................................................................................................... 203
8.3.1 Subjects ................................................................................................... 203
8.3.2 Cerebrospinal fluid.................................................................................. 204
8.3.3 Image acquisition ..................................................................................... 205
8.3.4 Volume loss measurement ...................................................................... 205
8.3.5 Statistical Analysis.................................................................................. 206
8.4 Results............................................................................................................ 207
8.4.1 Subject demographics ............................................................................... 207
8.4.2 Analytes predicting atrophy ................................................................... 209
8.3.4 Analytes predicting cognitive decline .................................................... 217
8.3.5 Predictive Value ...................................................................................... 217
8.4 Discussion ...................................................................................................... 218
8.4.1 Trefoil Factor 3 (TFF3) ......................................................................... 219
8.4.2 Cystatin C (CysC), Vascular Endothelial Growth Factor (VEGF), and
Chromogranin A (CgA) ..................................................................................... 220
8.5 Conclusions .................................................................................................... 223
8.6 Publication arising from this chapter ............................................................ 223
Chapter 9. Exploring New AD biomarkers ............................................................ 224
9.1 Introduction ................................................................................................... 224
9.1.1 Limitations of current biomarkers ......................................................... 224
9.1.2 CSF Biomarker Discovery ...................................................................... 224
9.1.3 Existing targeted mass spectrometry assays ......................................... 226
9.2 Contributions and Collaborations ................................................................. 226
9.3 Methods ......................................................................................................... 227
9.3.1 Subjects and Cerebrospinal Fluid Collection ......................................... 227
9.3.2 Cerebrospinal fluid Collection and Routine Biomarker Analysis .......... 228
9.3.3 Neurochemical Classification.................................................................. 228
9.3.4 Mass Spectrometry ................................................................................. 229
9.3.5 Statistical Analysis.................................................................................. 234
9.4 Results............................................................................................................ 236
9.4.1 Comparing Neurochemical AD and Non-AD subjects .......................... 236
10