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The diagnostic utility of cerebrospinal fluid alpha-synuclein analysis in dementia with Lewy bodies – A systematic review and meta-analysis
Parkinsonism and Related Disorders 19 (2013) 851e858
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Review
The diagnostic utility of cerebrospinal fluid alpha-synuclein analysis in dementia with Lewy bodies e A systematic review and meta-analysis Xuxin Lim a, Jing Ming Yeo a, Alison Green b, Suvankar Pal c, d, e, * a
College of Medicine and Veterinary Medicine, University of Edinburgh, UK CJD Research & Surveillance Unit, Western General Hospital, Edinburgh, UK c Department of Neurology, Forth Valley Royal Hospital, NHS Forth Valley, UK d Division of Clinical Neurosciences, Western General Hospital, Edinburgh, UK e Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2013 Received in revised form 30 May 2013 Accepted 15 June 2013
Background: Dementia with Lewy Bodies (DLB) can be difficult to distinguish clinically from other dementias. Objective: To investigate the diagnostic utility of CSF alpha-synuclein in differentiating between DLB and other dementias. Methods: Electronic databases were systematically searched for studies investigating reproducible alpha synuclein quantification methods. Random effects model was used to calculate weighted mean difference (WMD) and 95% confidence intervals between DLB and other groups. Results: A total of 13 studies, comprising 2728 patients were included. Mean CSF alpha-synuclein concentration was significantly lower in DLB patients compared to those with Alzheimers disease (AD) [WMD 0.24; 95% CI, 0.45, 0.03; p ¼ 0.02]. No significant difference was found between patients with DLB compared to Parkinsons disease [WMD 0.05; 95% CI, 0.17, 0.28; p ¼ 0.65] or other neurodegenerative conditions. Conclusion: CSF alpha synuclein may be of diagnostic use in differentiating between DLB and AD. We propose several recommendations to guide better design of future studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Alpha-synuclein Cerebrospinal fluid Dementia with Lewy bodies Biomarker Dementia
1. Introduction Dementia with Lewy Bodies (DLB) is a neurodegenerative condition characterized by progressive fluctuating cognitive and behavioral disturbance, visual hallucinations, and Parkinsonism [1]. It is the second most common form of dementia after Alzheimer’s disease (AD) [1]. The prevalence of DLB has been reported to be as high as 5% in the general population and 30.5% amongst patients with dementia [2]. Together with Parkinsons disease (PD) and multiple system atrophy (MSA), the disorder is part of the synucleinopathy spectrum characterized by the deposition of fibrillary aggregates of alpha-synuclein protein in the cytoplasm of selective populations of neurons and glia [3]. Clinical diagnosis of DLB is often challenging due to heterogeneity in clinical presentation and phenotypic overlap with other dementia syndromes, particularly AD and other degenerative
* Corresponding author. Division of Clinical Neurosciences, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. E-mail addresses: [email protected] (X. Lim), [email protected] (S. Pal). 1353-8020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parkreldis.2013.06.008
parkinsonian syndromes. Moreover, characteristic features of DLB often present several months into disease course resulting in delayed or missed diagnosis, prolonged uncertainty for patients and caregivers and delay in initiating effective symptomatic treatments. Currently, the diagnosis of DLB is based on fulfilment of international diagnostic criteria which are sensitive (80e100%) but not very specific (20e60%) for DLB [4]. Additional structural and functional information derived from brain magnetic resonance imaging (MRI) and dopamine transporter scans (DaTSCAN) are useful diagnostic adjuncts. Brain biopsy or post-mortem histopathological confirmation of alpha-synuclein aggregates in Lewy bodies constitutes the diagnostic gold standard, although this is rarely undertaken. There is a need to establish an accurate, less invasive and replicable means of diagnosing DLB early for diagnostic, prognostic and therapeutic purposes. Quantification of several biomarkers for DLB has been proposed, one of which is cerebrospinal fluid (CSF) alpha-synuclein. Changes in levels of CSF alpha-synuclein are proposed to reflect accumulation of alpha-synuclein in Lewy bodies, although the precise mechanism by which this occurs is unclear. One hypothesis is that lower alpha-synuclein levels observed in CSF from patients with
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synucleinopathies may be attributed to lower levels of the cleaving enzyme neurosin [5]. This may lead to slower degradation of alphasynuclein and promote formation of insoluble alpha-synuclein fibrillary aggregates following gradual accumulation of alphasynuclein oligomers. Several tests based on enzyme-linked immunosorbant assays (ELISA) have been developed to determine if CSF alpha-synuclein is a useful marker for distinguishing DLB from other synucleinopathies. However, reports on the correlation between CSF alphasynuclein levels and presence of DLB pathology have been mixed. Hence, the aim of this systematic review and meta-analysis is to determine the diagnostic utility of CSF alpha-synuclein analysis in distinguishing DLB from other neurodegenerative dementias. 2. Methods 2.1. Literature search Electronic databases including MEDLINE OVID (1948 onwards), EMBASE OVID (1974 onwards), PsychINFO OVID (1806 onwards) and Web of Knowledge (1900 onwards) to March 2013 were systematically searched using search terms dementia with Lewy body, Lewy body disease, Lewy body, cerebrospinal fluid and alphasynuclein. No language restrictions were applied and only human studies were included. Citation tracing, bibliographic review of references in relevant studies and hand searching were also performed in order to ensure all relevant studies were included.
Syntax Reference, SPSS Inc., Chicago and Review Manager (RevMan) [Computer program]. Version 5.1. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2011.
3. Results 3.1. Search outcomes Fig. 1 shows a flow diagram of all studies identified and the number of studies which were subsequently included or excluded. A total of 417 studies were identified from the databases searched. 23 duplicates were removed. 394 individual studies remained. A further 381 papers were excluded after screening by title, abstract and full-article reviews. Title and abstract screening eliminated most irrelevant studies, duplicates and publications such as case reports, abstracts and narrative reviews. Full-text review of the remaining studies rejected those which did not quantify concentration of CSF alpha-synuclein through replicable assays, used postmortem samples from patients or measured other CSF biomarkers such as tau or amyloid instead of alpha-synuclein. A total of thirteen studies, including 2728 patients, met stringent search criteria and were included in the final review. A summary of data extracted from these studies is compiled in Table 1. Only seven of the thirteen studies were included in the meta-analysis due to incomplete data reporting in the remaining six.
2.2. Inclusion and exclusion criteria Studies were included if they, 1) Involved reproducible methods of quantification of CSF alpha-synuclein, 2) Included a representative spectrum of patients encountered in clinical practice and 3) Classified patient groups using internationally agreed consensus criteria such as the McKeith criteria for DLB [6e9], the National Institute of Neurological, Communicative Diseases and Stroke e Alzheimers Disease and Related Disorders Association (NINCDS-ADRDA) criteria for AD [10], United Kingdom Parkinsons Disease Society (UKPDS) Brain Bank criteria for PD [11] and Lund-Manchester criteria for fronto-temporal dementia [12]. Studies were excluded if they measured CSF alpha-synuclein from post-mortem samples, measured plasma alpha-synuclein, included patients with mixed pathologies or uncertain diagnosis, or made no attempt of excluding other structural causes of cognitive decline. 2.3. Data collection and analysis All studies identified from the electronic database search were independently screened by title and abstract (XL and JMY). Irrelevant or duplicate publications were excluded and the remaining studies were reviewed thoroughly to assess for eligibility. All reasons for exclusion were documented and disagreements were settled by discussion with AG and SP. Data extracted included author and year of publication, patient demographics, Mini Mental State Examination (MMSE) scores, international consensus criteria (reference standard) used, type of assay (index test) used, mean and standard deviation or inter-quartile ranges of CSF alpha-synuclein concentration as reported.
3.2. Methodological quality of included studies Fig. 7 provides diagrammatic representation of the QUADAS item checklist for all studies included in the review. Overall, patients included in the studies were drawn from a representative spectrum of patients encountered in outpatient memory and neurology clinics. All studies included acceptable reference standards for clinical diagnosis. Despite minor variation between studies, partial and differential verification was avoided as
417 studies identified from databases
23 duplicates removed
394 studies remaining after duplicates removed
2.4. Assessment of methodological quality and reporting bias All studies were marked against Quality Assessment for Diagnostic Accuracy Studies (QUADAS) criteria to assess for quality and risk of bias using a modified checklist. Each criteria was marked as ‘Yes’, ‘No’ or ‘Unclear’. For studies in which there was a lack of information or uncertainty, authors of the original paper were contacted for clarification of methodology to aid assessment of methodological quality or potential for reporting bias. 2.5. Statistical analysis and data synthesis Summary statistics including mean, standard deviations, medians and interquartile ranges of CSF alpha-synuclein concentrations were extracted from each study. Where CSF data was not explicitly stated in papers or only represented graphically, the authors of the original papers were contacted directly to obtain necessary data. The means and standard deviations of concentrations of CSF alphasynuclein were compiled into Forest plots and analyzed as continuous variables. Statistical significance was set at p < 0.05 and confidence intervals at 95% were determined. We also attempted to estimate degree of heterogeneity not due to chance alone by deriving I2 test statistic. Random effects model analysis was performed to take into account the effect of heterogeneity in meta-analysis. Statistical analysis was performed using SPSS Inc. (2009) PASW STATISTICS 17.0 Command
381 studies excluded by screening abstract, title and/or full-text review 13 studies included in qualitative analysis (systematic review)
7 studies included in quantitative analysis (meta-analysis) Fig. 1. Flow diagram of study selection.
Table 1 Summary of studies included in the systematic review. MMSE (score)
Assay
Conc CSF a-sync (pg/ml)
Author’s conclusion
(50e82) (65e88) (60e80) (53e74) (21e81)
4.2 (2e6) 3.5 (1e6) 3.8 (1e8) 6 (3e10) NA
19.5 17.8 26.6 22.6 NA
In-house solid-phase sandwich ELISA (antihuman SNCA antibody by Invitrogen)
0.0976 (0.0581) 0.1841 (0.0711) 0.0942 (0.0277) 0.108 (0.069) 0.1378 (0.0312)
CSF a-syn differentiates synucleinopathies from AD. CSF a-syn does not differ between synucleinopathies.
71 69 73 67 54
(7) (10) (7) (7) (21)
2.25 (1e7.5) 2.5 (1e5) 12.2 (2e26) 2.9 (1e7) NA
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
1.42 1.85 1.19 1.24 1.73
(1.26) (1.47) (0.81) (0.99) (1.83)
CSF a-syn levels and tau distinguishes synucleinopathies from other neurological diseases.
72 72 70 72.3 77 73
(7.2) (6.8) (7.1) (6) (6.4) (8.5)
3.83 (1.1e13.7) 6.17 (1e24.7) 5 (1.1e12.8) 5 (2e6.5) 5.3 (1.2e19) 5.2 (1.3e19.8)
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
1.32 1.34 1.11 1.78 1.72 2.22
(0.62) (0.81) (0.45) (0.91) (1.12) (1.31)
CSF a-syn levels and tau distinguishes synucleinopathies from other neurological diseases.
e
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
0.3 (0.2) 0.8 (0.9) 0.7 (0.5)
CSF a-syn levels discriminate between DLB and AD.
e
Tokuda sandwich ELISA with modifications (antibody 211/FL-140)
18.1 34.8 43.1 15.3 68.9
(16.0) (54.0) (47) (18) (71)
CSF a-syn levels lower in dementia groups compared to controls. CSF tau/a-syn ratio helps discriminate PD from OND.
Van Geel sandwich ELISA method (antibody 211/FL-140)
38.0 37.0 49.1 44.3 30.4 NA
(29.0) (36.1) (41.5) (40.3) (19.1)
CSF a-syn does not discriminate between dementia disorders.
11.2 (9.7) 14.0 (9.3) 12.4 (11.1)
In house modified sandwich ELISA (antibody syn-1/FL-140)
8.2 (4.2) 12.2 (5.8) 13.9 (7.5)
CSF a-syn significantly lower in DLB patients compared to AD and other dementias. CSF a-syn correlates with Ab42 levels in DLB patients.
4.25 (3) 2.2 (2.4) 8.3 (4.8) 0.083 (0.25) NA
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
6.2 (4.2) 3.8 (3.3) 3.0 (1.3) 300 (248) 6.0 (5.7)
CSF a-syn lower in synucleinopathies compared to non-synucleinopathies. CSF a-syn markedly elevated in CJD patients.
74.5 (5.2) 73.3 (4.8)
4.2 (3.0) 2.9 (1.4)
19.5 (5.8) 20.0 (5.6)
Tokuda sandwich ELISA with modifications (antibody 211/FL-140)
44.7 (24.9) 50.2 (24.1)
CSF a-syn not significantly different between DLB and AD patients. Lower CSF a-syn levels in DLB patients with longer disease duration
72.7 (9.5)
1.9 (1.7)
22.7 (3.1)
e
29 23 20 30
No of patients
Gender (M:F)
Tateno et al. 2012 [13]
6 DLB 9 AD 11 PD 11 MSA 11 Controls
2:4 6:3 6:5 6:5 7:4
73 75 71 66 49
Mollenhauer et al. 2011 (training cohort) [14]
55 62 51 29 76
27:28 22:40 32:19 16:13 33:43
Mollenhauer et al. 2011 (validation cohort) [14]
66 DLB 273 PD 15 MSA 8 PSP 22 NPH 23 Controls
47:19 184:89 10:5 6:2 19:3 17:6
Mollenhauer et al. 2011 (autopsy cohort) [14]
13 DLB 21 AD 7 Controls
7:6 11:10 5:2
Parnetti et al. 2011 [15]
32 48 38 31 32
18:14 19:29 22:16 17:14 18:14
71.4 68.7 69.3 64.3 61.9
(6) (9.7) (7.2) (6.4) (17.8)
3.9 4.0 3.5 4.2 NA
Spies et al. 2009 [16]
40 DLB 131 AD 39 FTD 28 VaD 57 Control A 55 Control B
27:13 54:77 24:15 18:10 30:27 26:29
74.0 71.7 66.4 75.4 61.3 61.1
(8.3) (8.8) (8.6) (8.4) (8.8) (8.9)
e
18.9 19.6 20.3 18.5 30.4 NA
Kasuga et al. 2010 [17]
34 DLB 31 AD 21 OD
13:21 13:18 9:12
75.8 (10.7) 67.9 (12.3) 70.0 (11.9)
e
Mollenhauer et al. 2008 [18]
13 AD 38 DLB 8 PD 8 CJD 13 Controls
4:9 23:15 5:3 3:5 7:6
69.9 71 76 71 64
NoguchiShinohara 2009 [19]
16 DLB 13 DLB (matched) 21 AD
6:10 6:7 8:13
15 15 66 55
4:15 6:15 47:19 30:25
Ohrfelt 2009 [20]
DLB AD PD MSA Controls
DLB AD PD FTD Controls
PD DLB AD Controls
Mean age (years)
72 (12) 67 (16) 61 (6)
71 79 77 65
(9) (8) (3) (7) (15)
(63e76) (76e80) (73e82) (62e70)
(2.7) (2.9) (2.3) (2.2)
(5e27) (10e25) (22e30) (21e30)
(8.2) (3.9) (6.1) (2.9) (19.1)
(27e30) (19e23) (14e26) (29e30)
X. Lim et al. / Parkinsonism and Related Disorders 19 (2013) 851e858
Mean duration of disease (years)
Studies (Author, Year)
49.3 (44.5) In-house ELISA (antibodies Syn1b, Syn3b, Syn 5d/ LO-MG1-13, LO-MG-2)
0.417 0.334 0.296 0.395
(0.246e0.522) (0.220e0.406) (0.234e0.372) (0.298e0.452)
CSF a-syn lower in AD patients compared to controls. CSF a-syn levels similar in PD and DLB patients. a-syn possible general marker for synapse loss
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854
Table 1 (continued ) Mean duration of disease (years)
MMSE (score)
Assay
Conc CSF a-sync (pg/ml)
Author’s conclusion
(5) (8) (8) (67)
e
e
Van Geel sandwich ELISA method (antibody 211/FL-140)
18 23 20 16
CSF a-syn does not discriminate DLB from AD
(10.8) (7.8) (6.9) (6.5) (8.8) (8.8)
3.21 (2.72) 3.21 (2.31) 5.0 (0.083) 4.74 (2.6) 3.07 (1.67) NA
28.4 27.9 22.7 26.6 27.2 NA
Van Geel sandwich ELISA method (antibody 211/ FL-140)
26.0 25.0 24.0 24.0 30.5 25.0
71 (7) 73 (6) 72 (8)
e
23 (4) 21 (5) 29 (1)
Commercial Invitrogen ELISA
0.378 (0.166) 0.464 (0.233) 0.500 (0.178)
Female DLB patients have lower a-syn levels than AD patients and controls
42 59 25 48 13 20 22 7
70 63 76 74 78 70 64 71
(63e76) (56e71) (73e80) (69e81) (74e80) (64e74) (59e72) (66e76)
e
29 (28e30) 29 (27e29) 24 (18e25) 21 (17e25) 21 (19e23) 27 (24e28) 29 (28e29) NA
Luminex Multiplex Assay
67 55 59 59 94 70 56 56
Cerebrospinal fluid a-syn were decreased in patients with PD, PDD, DLB and MSA but increased in patients with AD
50:50 76:24 55:45 47:53 27:73
64 78 74 64 75
(10) (6) (6) (9) (6)
e
28.5 20.6 20.2 28.5 23.1
Commercial Invitrogen ELISA
AD > controls > DLB > PD > PDD
No of patients
Gender (M:F)
Reesink et al. 2010 [21]
34 18 35 63
18:16 10:8 29:6 29:34
67 67 71 69
Aerts et al. 2012 [22]
58 PD 47 MSA 3 DLB 22 VaP 12 PSP/CBD 57 Controls
40:18 28:19 3:0 15:7 5:7 30:27
56.6 62.9 62.5 68.7 66.2 61.3
Wennstrom et al. 2012 [23]
18 DLB 26 AD 24 Controls
9:9 15:11 16:8
Hall et al. 2012 [24]
107 Controls 90 PD 33 PDD 70 DLB 48 AD 45 PSP 48 MSA 12 CBD
Wennstrom 2013 [5]
52 46 33 38 22
SC PD DLB AD
Controls AD DLB PD PDD
Mean age (years)
(1.6) (2.5) (6.4) (2.7) (1.8)
(1.1) (4.3) (5.6) (1.2) (4.4)
(14e26) (18e32) (15e27) (13e23) (20.5e32.5) (17.0e32.0) (23.0e34.0) (16.0e30.5) (19.5e38.0) (18.0e42.0)
(51e85) (40e70) (49e68) (44e74) (76e121) (51e86) (43e66) (50e84)
CSF a-syn does not differentiate between parkinsonian disorders
Patients with synucleinopathies have lower CSF levels of a-syn compared to controls and AD patients.
All values are expressed as mean standard deviation or p25e75 inter-quartile ranges unless otherwise stated. Abbreviations: DLB ¼ Dementia with Lewy Bodies, PD ¼ Parkinsons Disease, AD ¼ Alzheimers Disease, MSA ¼ Multiple System Atrophy, VaP ¼ Vascular Parkinsonism, VaD ¼ Vascular Dementia, FTD ¼ Frontotemporal Dementia, PSP ¼ Progressive Supranuclear Palsy, NPH ¼ Normal Pressure Hydrocephalus, CJD ¼ CreuzfeldteJakob Disease, CBD ¼ Corticobasal Degeneration, OD ¼ Other Dementia, SC ¼ Patients with Subjective Complaints, OND ¼ Other Neurological Disease, PDD ¼ Parkinsons Disease Dementia.
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Studies (Author, Year)
X. Lim et al. / Parkinsonism and Related Disorders 19 (2013) 851e858
855
Fig. 2. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs AD patients.
diagnoses were made using the same diagnostic criteria within each study. Only two of the thirteen studies attempted to carry out blinding of alpha-synuclein assay results [21,22]. Only one of the thirteen studies made efforts to blind the reference standard [16]. It was unclear if there was an acceptable delay between time of diagnosis based on international consensus criteria and time of performing CSF alpha-synuclein assay, as the duration from clinical diagnosis to lumbar puncture was not reported across all studies. Furthermore, clinical interventions, including symptomatic treatments introduced were not clearly described. None of the studies highlighted ambiguous immunoassay results and no withdrawals were reported. The methodology of several studies was not clearly described, raising some doubts over the replicability and validity of the assays. 3.3. Concentration of CSF alpha-synuclein in DLB vs other neurodegenerative conditions Fig. 2 shows a Forest plot which demonstrates a significantly lower mean concentration of CSF alpha-synuclein in patients with DLB patients compared to those with AD [weighted mean difference 0.24; 95% CI, 0.45, 0.03; p ¼ 0.02]. Fig. 3 shows that there was no significant difference in mean concentration of CSF alphasynuclein between patients with DLB and PD [weighted mean difference 0.05; 95% CI, 0.17, 0.28; p ¼ 0.65]. Similarly, no significant difference was found in the mean concentration of CSF alphasynuclein between DLB and other neurodegenerative conditions including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), fronto-temporal dementia (FTD) and vascular dementia (VaD) (data not shown). Meanwhile, Fig. 4 shows no significant difference in mean CSF alpha-synuclein concentration between DLB patients and normal/neurological controls [weighted mean difference 0.12; 95% CI, 0.29, 0.04; p ¼ 0.13] although the trend was toward lower CSF alpha-synuclein concentration in DLB patients compared to normal/neurological controls. One study [18] reported significantly higher mean CSF alpha-synuclein concentrations in Creutzfeldt-Jakob disease compared to DLB. CSF alpha synuclein levels were also compared between DLB and non-synucleinopathies (AD, PSP, FTD and VaD) as well as synucleinopathies (PD and MSA). Mean CSF alpha-synuclein
concentration in the DLB group was found to be significantly lower compared to non-synucleinopathies [weighted mean difference 0.25; 95% CI 0.44, 0.06; p ¼ 0.01] (Fig. 6). In contrast, mean CSF alpha-synuclein concentration in the DLB group was not significantly different compared to synucleinopathies [weighted mean difference 0.05; 95% CI, 0.17, 0.27; p ¼ 0.65] (Fig. 5). 3.4. Sources of heterogeneity and sensitivity analysis The degree of heterogeneity (I2) ranged from 60% to 73% which suggests that there were significant differences in experimental protocol between studies. Factors which may contribute to this include administration of interventions or treatments which modify physiological levels of CSF alpha-synuclein, differences in reference standards used to define patient populations, variation in sample processing and storage methods, differences in assays used, blood contamination in CSF samples and variation in diagnostic threshold of index test. 4. Discussion Dementia with Lewy Bodies is a condition which poses significant diagnostic challenges due to similarities with a spectrum of other degenerative dementias and its highly variable presentation. This study aims to evaluate evidence for the diagnostic utility of CSF alpha-synuclein assays in differentiating between DLB and other degenerative dementias. A number of studies have reported lower mean CSF alpha-synuclein levels in DLB compared to AD [4,14,17,24] whilst others have shown no difference [16,19,21] between the two dementias. Furthermore, some groups have reported a difference in CSF alpha-synuclein concentrations between synucleinopathies and AD [13,14,18] whilst others report no such difference [16,22]. Correlation between CSF alpha-synuclein and other biomarkers such as total tau, phosphorylated tau, betaamyloid 42 have also been reported in several studies [14e16,19e 21,23]. The correlation between CSF alpha-synuclein levels in patients with DLB and age, gender, cognitive function as well as duration of disease has additionally been the subject of much debate. Reesink et al. [21] suggested patients with worse cognitive function in DLB
Fig. 3. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs PD patients.
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Fig. 4. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs neurological/normal controls.
Fig. 5. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB patients vs patients with synucleinopathies.
have lower levels of CSF alpha-synuclein. However, this has not been replicated in findings from other studies [16,17]. Ohrfelt et al. [20] reported lower CSF alpha-synuclein levels in AD (particularly in patients with MMSE score less than 20) compared to controls. They also found CSF alpha-synuclein concentrations in DLB and PD patients to be similar [20]. Wennstrom et al. [23] reported lower CSF alpha-synuclein concentrations in female DLB patients compared to AD patients and controls. However, this was again not replicated in four other studies [4,16,17,19]. Noguchi-Shinohara et al. [19] commented that CSF alpha-synuclein concentration was lower in DLB patients with longer disease duration while other groups have reported no correlation between disease duration and CSF alpha-synuclein levels [15,17]. Our results suggest a trend toward lower CSF alpha-synuclein concentrations in DLB compared with AD, regardless of type of assay used. The lower CSF alpha-synuclein levels observed in DLB may be explained by sequestration of the protein as fibrillary aggregates in Lewy bodies. This is similar to the proposed mechanism underlying the reduced CSF concentrations of beta-amyloid in AD. Various other factors may also influence the balance between production, clearance and solubility of CSF alpha-synuclein. Mutations affecting protein structure may also increase the propensity of forming fibrillary aggregates [26]. Further studies are warranted to clarify specific molecular mechanisms underlying this observation. The difference in CSF alpha-synuclein concentrations in patients with DLB compared with AD is of potential clinical utility as AD is
one of the key differentials for DLB. Nevertheless, this difference must be interpreted with caution due to significant heterogeneity in study design, which may be caused by variation in factors such as patient characteristics and type of assays used. Several groups have developed in house assays making comparison between assays challenging. Lack of transparency in reporting of methodology and laboratory protocols makes attempts to reproduce assay results problematic. Furthermore, several groups have attempted to modify pre-defined assays without validating them independently. There is also a need to standardize pre-analytical confounding factors to ensure consistency of results across studies. Several recommendations have been made by Del Campo et al. [25] to address this issue. No difference in CSF alpha-synuclein concentration was identified between patients with DLB and Parkinsons disease. This is perhaps of little surprise as both disorders are synucleinopathies on a continuum of the same clinical spectrum. When comparing CSF alpha-synuclein concentrations in DLB with neurological controls, a trend toward lower CSF alpha-synuclein in DLB patients emerged, although this did not reach statistical significance. It is possible this may be attributed to the heterogeneity in subjects included in control groups. Several studies failed to adequately define control groups as normal, but instead used neurological controls with nonneurodegenerative pathologies. Only a minority of studies included post-mortem verification of the absence of co-existent neurodegenerative disease in controls which may have influenced observed CSF alpha-synuclein levels. Future studies should include
Fig. 6. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB patients vs patients with non-synucleinopathies.
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Fig. 7. Methodological quality summary based on modified QUADAS (Quality Assessment of Diagnostic Accuracy Studies) criteria.
appropriately matched neurologically normal controls with adequate post-mortem verification. Numerous methodological problems were identified in many of the studies such as assay variability, poor study design, use of outdated diagnostic reference standards, and poor standards of reporting. In terms of assays used, it was unclear which type of alpha-synuclein was being measured by the assays. Three molecular forms of alpha-synuclein have been identified as expressed in the brain: alpha-synuclein 140 (full length), alpha-synuclein 126 (lacks exon 3) and alpha-synuclein 112 (lacks exon 5) [26]. These isoforms are formed by variation in alternative splicing of alpha-synuclein, and each may be subjected to further post-translational modifications such as phosphorylation and ubiquitination. These posttranslational changes are thought to promote the aggregation of the small soluble monomeric forms of alpha-synuclein into diseaseassociated aggregated oligomers [26]. Therefore, the standardization of alpha-synuclein assays is hampered by the presence of heterogenous forms of the protein within the brain, uncertainty of which forms are present in the CSF and ambivalence regarding antibody specificity for the disease-associated form of alphasynuclein. It is possible that the assays involved in this study were measuring different components of the alpha-synuclein mixture present within the CSF resulting in such wide variations in levels of CSF alpha-synuclein reported. Type of study (prospective or cross-sectional) and whether the reference standard and index test were blinded or not, was often unclear. Patient recruitment and selection were sometimes vague. Inclusion and exclusion criteria were not always clearly stated and there were differences in the use of probable or possible clinical diagnostic criteria between studies. None of the studies defined the time period between clinical diagnosis and CSF sampling or mentioned therapeutic interventions the patients were subjected to which might confound interpretation of assay results as CSF
Table 2 Proposed recommendations for future studies on CSF biomarker assays in DLB. Study design Prospective cohort studies Ensure study is sufficiently powered Patients Recruit consecutive patients referred with suspected degenerative dementia from representative population Make diagnosis based on reference standard before performing index test Define time period between clinical diagnosis and CSF sampling State if clinical intervention or treatment is performed on patients between time of reference test and index test Reference standard Based on expert clinical opinion supplemented with clinical and imaging results Post-mortem confirmation of diagnosis whenever possible Define if possible, probable or definite diagnosis made Blind reference standard Index test Clear description of assay quantification Account for inter- and intra-observer reliability of test Validate biomarker in an independent cohort if using novel assay or in-house assays Provide mean and standard deviation values of alpha-synuclein concentration Include comparison with other biomarkers such as total tau, p-tau, betaamyloid and alpha-synuclein Blind index test Standard of reporting Use STARD guidelines for reporting results Perform ROC analysis on data Provide numerical threshold/cut-off values Provide values such as true positives, true negatives, false positives, false negatives, cut-off/threshold values Provide ratio between biomarkers where applicable
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alpha-synuclein levels may vary with progression of disease and treatment. Summary receiver operating characteristic (SROC) analysis on the index test was not feasible due to insufficient information available in papers or supplementary information provided by authors. Moreover, most studies failed to perform receiver operating characteristic (ROC) analysis on available dataset. Consequently, values for sensitivity, specificity, true positive, true negative, false positive and false negative of the assay were not reported. Only Mollenhauer et al. [14] attempted to characterize and validate their assays in an independent cohort. Only two studies reported efforts to blind assay results [21,22] and clinical data [16] while none of the studies attempted to blind the reference standard using an external clinical reviewer. Mean and standard deviation of concentrations of alpha-synuclein between patient subgroups were sometimes not reported as well. Power studies to guide robust sample sizes for future studies are problematic due to the heterogeneity of available assays and patient populations studied. Moreover, the difference in means between studies and within studies varied enormously which makes calculation of effect size challenging. Whilst investigation of statistically significant numbers of CSF samples is clearly of importance, of greater relevance perhaps, is the necessity for patients in studies to be well phenotyped clinically with postmortem neuropathological confirmation of disease. We propose several recommendations to ensure quality of future studies (Table 2).
5. Conclusion We conclude that CSF alpha-synuclein analysis is potentially useful in distinguishing DLB from AD but not from other synucleinopathies. However, there is a need for more adequately powered studies with standardized assays for CSF alpha-synuclein measurements, long-term follow-up, appropriately matched controls, post-mortem histopathological confirmation of patient groups, sensitivity and specificity analysis as well as good reporting standards (see STARD criteria) to yield more conclusive results in future reviews. Therefore, although CSF alpha-synuclein analysis is a promising diagnostic tool in distinguishing DLB from AD, significant further validation needs to be undertaken before it can be recommended for routine use in clinical practice. Contributions of authors All authors made substantial contributions to the conception, design, data analysis and interpretation of this paper. Each authors also made significant contributions in drafting and critically appraising the final version of this paper for publication.
Declarations of interest All authors report no conflict of interest.
Acknowledgments The authors would like to thank the authors of papers included in this systematic review for kindly sharing their data and expert advice. Dr Suvankar Pal is funded by a NHS Research Scotland Career Researcher Fellowship.
References [1] McKeith I. Dementia with Lewy bodies. Dialog Clin Neurosci 2004;6(3): 333e41. [2] Zaccai J, McCracken C, Brayne C. A systematic review of prevalence and incidence studies of dementia with Lewy bodies. Age Ageing 2005;34(6): 561e6. [3] Marti MJ, Tolosa E, Campdelacreu J. Clinical overview of the synucleinopathies. Mov Disord 2003;18(Suppl. 6):S21e7. [4] McKeith I, Mintzer J, Aarsland D, Burn D, Chiu H, Cohen-Mansfield J, et al. International Psychogeriatric Association Expert Meeting on DLB: dementia with Lewy bodies. Lancet Neurol 2004;3:19e28. [5] Wennstrom M, Surova Y, Hall S, Nilsson C, Minthon L, Bostrom F, et al. Low CSF levels of both alpha-synuclein and the alpha-synuclein cleaving enzyme neurosin in patients with synucleinopathy. PLoS ONE 2013;8(1):e53250. [6] McKeith I, Dickson D, Lowe J, Emre M, O’Brien J, Feldman H, et al. Diagnosis and management of dementia with Lewy bodies: third report of the DLB consortium. Neurology 2005;65(12):1863e72. [7] McKeith I, Galasko D, Kosaka K, Perry E, Dickson D, Hansen L, et al. Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop. Neurology 1996;47(5):1113e24. [8] McKeith I, O’Brien JT, Ballard C. Diagnosing dementia with Lewy bodies. Lancet 2003;354(9186):1227e8. [9] Mckeith I. Dementia with Lewy bodies and Parkinson. Parkinsonism 2004;10: S15e8. [10] McKhann GM, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimers disease: a report of the NINCDS-ADRDA work group. Neurology 1984;34:934e42. [11] Hughes A. UK Parkinson’s disease society brain bank clinical diagnostic criteria. J Neurol 1992;55:1992. [12] The Lund and Manchester Groups. Statement C. Clinical and neuropathological criteria for fronto-temporal dementia. J Neurol, Neurosurg Psychiatry 1994;57(4):416e8. [13] Tateno F, Sakakibara R, Kawai Takayuki, Kishi M, Murano T. Alpha-synuclein in the cerebrospinal fluid differentiates synucleinopathies (Parkinson’s disease, dementia with Lewy body, multiple system atrophy) from Alzheimer disease. Alzheimer Dis Assoc Disord 2012;26(3):213e6. [14] Mollenhauer B, Locascio JJ, Schulz-Schaeffer W, Sixel-Doring F, Trenkwalder C, Schlossmacher MG. alpha-Synuclein and tau concentrations in cerebrospinal fluid of patients presenting with parkinsonism: a cohort study. Lancet Neurol 2011;10(3):230e40. [15] Parnetti L, Chiasserini D, Bellomo G, Giannandrea D, De Carlo C, Qureshi MM, et al. Cerebrospinal fluid Tau/alpha-synuclein ratio in Parkinson’s disease and degenerative dementias. Mov Disord 2011;26(8):1428e35. [16] Spies PE, Melis RJ, Sjogren MJ, Rikkert MG, Verbeek MM. Cerebrospinal fluid alpha-synuclein does not discriminate between dementia disorders. J Alzheimers Dis 2009;16(2):363e9. [17] Kasuga K, Tokutake T, Ishikawa A, Uchiyama T, Tokuda T, Onodera O, et al. Differential levels of alpha-synuclein, beta amyloid 42 and tau in CSF between patients with dementia with Lewy bodies and Alzheimer’s disease. J Neurol Neurosurg Psychiatry 2010;81(6):608e10. [18] Mollenhauer B, Cullen V, Kahn I, Krastins B, Outeiro TF, Pepivani I, et al. Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp Neurol 2008;213(2):315e25. [19] Noguchi-Shinohara M, Tokuda T, Yoshita M, Kasai T, Ono K, Nakagawa M, et al. CSF alpha-synuclein levels in dementia with Lewy bodies and Alzheimer’s disease. Brain Res 2009;1251:1e6. [20] Ohrfelt A, Grognet P, Andreasen N, Wallin A, Vanmechelen E, Blennow K, et al. Cerebrospinal fluid alpha-synuclein in neurodegenerative disorders e a marker of synapse loss? Neurosci Lett 2009;450:332e5. [21] Reesink FE, Lemstra AW, van Dijk KD, Berendse HW, van de Berg WD, Klein M, et al. CSF alpha-synuclein does not discriminate dementia with Lewy bodies from Alzheimer’s disease. J Alzheimers Dis 2010;22(1):87e95. [22] Aerts MB, Esselink RA, Abdo WF, Bloem BR, Verbeek MM. CSF alpha-synuclein does not differentiate between parkinsonian disorders. Neurobiol Aging 2012;33(2):430e1e3. [23] Wennstrom M, Londos E, Minthon L, Nielsen H. CSF levels of orexin and asynuclein are linked in DLB patients and elderly controls. J Alzheimers Dis 2012;29(1):125e32. [24] Hall S, Ohrfelt A, Constantinescu R, Andreasson U, Surova Y, Bostrom F, et al. Accuracy of a panel of 5 cerebrospinal fluid biomarkers in the differential diagnosis of patients with dementia and/or parkinsonian disorders. Arch Neurol 2012 Nov;69(11):1445e52. [25] Del Campo M, Mollenhauer B, Bertolotto A, Engelborghs S, Hampel H, Simonsen AH, et al. Recommendations to standardize preanalytical confounding factors in Alzheimers and Parkinsons disease cerebrospinal fluid biomarkers: an update. Biomark Med 2012;6(4):419e30. [26] Beyer K. Alpha-synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers. Acta Neuropathol 2006;112(3): 237e51.
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Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Review
The diagnostic utility of cerebrospinal fluid alpha-synuclein analysis in dementia with Lewy bodies e A systematic review and meta-analysis Xuxin Lim a, Jing Ming Yeo a, Alison Green b, Suvankar Pal c, d, e, * a
College of Medicine and Veterinary Medicine, University of Edinburgh, UK CJD Research & Surveillance Unit, Western General Hospital, Edinburgh, UK c Department of Neurology, Forth Valley Royal Hospital, NHS Forth Valley, UK d Division of Clinical Neurosciences, Western General Hospital, Edinburgh, UK e Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2013 Received in revised form 30 May 2013 Accepted 15 June 2013
Background: Dementia with Lewy Bodies (DLB) can be difficult to distinguish clinically from other dementias. Objective: To investigate the diagnostic utility of CSF alpha-synuclein in differentiating between DLB and other dementias. Methods: Electronic databases were systematically searched for studies investigating reproducible alpha synuclein quantification methods. Random effects model was used to calculate weighted mean difference (WMD) and 95% confidence intervals between DLB and other groups. Results: A total of 13 studies, comprising 2728 patients were included. Mean CSF alpha-synuclein concentration was significantly lower in DLB patients compared to those with Alzheimers disease (AD) [WMD 0.24; 95% CI, 0.45, 0.03; p ¼ 0.02]. No significant difference was found between patients with DLB compared to Parkinsons disease [WMD 0.05; 95% CI, 0.17, 0.28; p ¼ 0.65] or other neurodegenerative conditions. Conclusion: CSF alpha synuclein may be of diagnostic use in differentiating between DLB and AD. We propose several recommendations to guide better design of future studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Alpha-synuclein Cerebrospinal fluid Dementia with Lewy bodies Biomarker Dementia
1. Introduction Dementia with Lewy Bodies (DLB) is a neurodegenerative condition characterized by progressive fluctuating cognitive and behavioral disturbance, visual hallucinations, and Parkinsonism [1]. It is the second most common form of dementia after Alzheimer’s disease (AD) [1]. The prevalence of DLB has been reported to be as high as 5% in the general population and 30.5% amongst patients with dementia [2]. Together with Parkinsons disease (PD) and multiple system atrophy (MSA), the disorder is part of the synucleinopathy spectrum characterized by the deposition of fibrillary aggregates of alpha-synuclein protein in the cytoplasm of selective populations of neurons and glia [3]. Clinical diagnosis of DLB is often challenging due to heterogeneity in clinical presentation and phenotypic overlap with other dementia syndromes, particularly AD and other degenerative
* Corresponding author. Division of Clinical Neurosciences, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. E-mail addresses: [email protected] (X. Lim), [email protected] (S. Pal). 1353-8020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parkreldis.2013.06.008
parkinsonian syndromes. Moreover, characteristic features of DLB often present several months into disease course resulting in delayed or missed diagnosis, prolonged uncertainty for patients and caregivers and delay in initiating effective symptomatic treatments. Currently, the diagnosis of DLB is based on fulfilment of international diagnostic criteria which are sensitive (80e100%) but not very specific (20e60%) for DLB [4]. Additional structural and functional information derived from brain magnetic resonance imaging (MRI) and dopamine transporter scans (DaTSCAN) are useful diagnostic adjuncts. Brain biopsy or post-mortem histopathological confirmation of alpha-synuclein aggregates in Lewy bodies constitutes the diagnostic gold standard, although this is rarely undertaken. There is a need to establish an accurate, less invasive and replicable means of diagnosing DLB early for diagnostic, prognostic and therapeutic purposes. Quantification of several biomarkers for DLB has been proposed, one of which is cerebrospinal fluid (CSF) alpha-synuclein. Changes in levels of CSF alpha-synuclein are proposed to reflect accumulation of alpha-synuclein in Lewy bodies, although the precise mechanism by which this occurs is unclear. One hypothesis is that lower alpha-synuclein levels observed in CSF from patients with
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synucleinopathies may be attributed to lower levels of the cleaving enzyme neurosin [5]. This may lead to slower degradation of alphasynuclein and promote formation of insoluble alpha-synuclein fibrillary aggregates following gradual accumulation of alphasynuclein oligomers. Several tests based on enzyme-linked immunosorbant assays (ELISA) have been developed to determine if CSF alpha-synuclein is a useful marker for distinguishing DLB from other synucleinopathies. However, reports on the correlation between CSF alphasynuclein levels and presence of DLB pathology have been mixed. Hence, the aim of this systematic review and meta-analysis is to determine the diagnostic utility of CSF alpha-synuclein analysis in distinguishing DLB from other neurodegenerative dementias. 2. Methods 2.1. Literature search Electronic databases including MEDLINE OVID (1948 onwards), EMBASE OVID (1974 onwards), PsychINFO OVID (1806 onwards) and Web of Knowledge (1900 onwards) to March 2013 were systematically searched using search terms dementia with Lewy body, Lewy body disease, Lewy body, cerebrospinal fluid and alphasynuclein. No language restrictions were applied and only human studies were included. Citation tracing, bibliographic review of references in relevant studies and hand searching were also performed in order to ensure all relevant studies were included.
Syntax Reference, SPSS Inc., Chicago and Review Manager (RevMan) [Computer program]. Version 5.1. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2011.
3. Results 3.1. Search outcomes Fig. 1 shows a flow diagram of all studies identified and the number of studies which were subsequently included or excluded. A total of 417 studies were identified from the databases searched. 23 duplicates were removed. 394 individual studies remained. A further 381 papers were excluded after screening by title, abstract and full-article reviews. Title and abstract screening eliminated most irrelevant studies, duplicates and publications such as case reports, abstracts and narrative reviews. Full-text review of the remaining studies rejected those which did not quantify concentration of CSF alpha-synuclein through replicable assays, used postmortem samples from patients or measured other CSF biomarkers such as tau or amyloid instead of alpha-synuclein. A total of thirteen studies, including 2728 patients, met stringent search criteria and were included in the final review. A summary of data extracted from these studies is compiled in Table 1. Only seven of the thirteen studies were included in the meta-analysis due to incomplete data reporting in the remaining six.
2.2. Inclusion and exclusion criteria Studies were included if they, 1) Involved reproducible methods of quantification of CSF alpha-synuclein, 2) Included a representative spectrum of patients encountered in clinical practice and 3) Classified patient groups using internationally agreed consensus criteria such as the McKeith criteria for DLB [6e9], the National Institute of Neurological, Communicative Diseases and Stroke e Alzheimers Disease and Related Disorders Association (NINCDS-ADRDA) criteria for AD [10], United Kingdom Parkinsons Disease Society (UKPDS) Brain Bank criteria for PD [11] and Lund-Manchester criteria for fronto-temporal dementia [12]. Studies were excluded if they measured CSF alpha-synuclein from post-mortem samples, measured plasma alpha-synuclein, included patients with mixed pathologies or uncertain diagnosis, or made no attempt of excluding other structural causes of cognitive decline. 2.3. Data collection and analysis All studies identified from the electronic database search were independently screened by title and abstract (XL and JMY). Irrelevant or duplicate publications were excluded and the remaining studies were reviewed thoroughly to assess for eligibility. All reasons for exclusion were documented and disagreements were settled by discussion with AG and SP. Data extracted included author and year of publication, patient demographics, Mini Mental State Examination (MMSE) scores, international consensus criteria (reference standard) used, type of assay (index test) used, mean and standard deviation or inter-quartile ranges of CSF alpha-synuclein concentration as reported.
3.2. Methodological quality of included studies Fig. 7 provides diagrammatic representation of the QUADAS item checklist for all studies included in the review. Overall, patients included in the studies were drawn from a representative spectrum of patients encountered in outpatient memory and neurology clinics. All studies included acceptable reference standards for clinical diagnosis. Despite minor variation between studies, partial and differential verification was avoided as
417 studies identified from databases
23 duplicates removed
394 studies remaining after duplicates removed
2.4. Assessment of methodological quality and reporting bias All studies were marked against Quality Assessment for Diagnostic Accuracy Studies (QUADAS) criteria to assess for quality and risk of bias using a modified checklist. Each criteria was marked as ‘Yes’, ‘No’ or ‘Unclear’. For studies in which there was a lack of information or uncertainty, authors of the original paper were contacted for clarification of methodology to aid assessment of methodological quality or potential for reporting bias. 2.5. Statistical analysis and data synthesis Summary statistics including mean, standard deviations, medians and interquartile ranges of CSF alpha-synuclein concentrations were extracted from each study. Where CSF data was not explicitly stated in papers or only represented graphically, the authors of the original papers were contacted directly to obtain necessary data. The means and standard deviations of concentrations of CSF alphasynuclein were compiled into Forest plots and analyzed as continuous variables. Statistical significance was set at p < 0.05 and confidence intervals at 95% were determined. We also attempted to estimate degree of heterogeneity not due to chance alone by deriving I2 test statistic. Random effects model analysis was performed to take into account the effect of heterogeneity in meta-analysis. Statistical analysis was performed using SPSS Inc. (2009) PASW STATISTICS 17.0 Command
381 studies excluded by screening abstract, title and/or full-text review 13 studies included in qualitative analysis (systematic review)
7 studies included in quantitative analysis (meta-analysis) Fig. 1. Flow diagram of study selection.
Table 1 Summary of studies included in the systematic review. MMSE (score)
Assay
Conc CSF a-sync (pg/ml)
Author’s conclusion
(50e82) (65e88) (60e80) (53e74) (21e81)
4.2 (2e6) 3.5 (1e6) 3.8 (1e8) 6 (3e10) NA
19.5 17.8 26.6 22.6 NA
In-house solid-phase sandwich ELISA (antihuman SNCA antibody by Invitrogen)
0.0976 (0.0581) 0.1841 (0.0711) 0.0942 (0.0277) 0.108 (0.069) 0.1378 (0.0312)
CSF a-syn differentiates synucleinopathies from AD. CSF a-syn does not differ between synucleinopathies.
71 69 73 67 54
(7) (10) (7) (7) (21)
2.25 (1e7.5) 2.5 (1e5) 12.2 (2e26) 2.9 (1e7) NA
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
1.42 1.85 1.19 1.24 1.73
(1.26) (1.47) (0.81) (0.99) (1.83)
CSF a-syn levels and tau distinguishes synucleinopathies from other neurological diseases.
72 72 70 72.3 77 73
(7.2) (6.8) (7.1) (6) (6.4) (8.5)
3.83 (1.1e13.7) 6.17 (1e24.7) 5 (1.1e12.8) 5 (2e6.5) 5.3 (1.2e19) 5.2 (1.3e19.8)
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
1.32 1.34 1.11 1.78 1.72 2.22
(0.62) (0.81) (0.45) (0.91) (1.12) (1.31)
CSF a-syn levels and tau distinguishes synucleinopathies from other neurological diseases.
e
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
0.3 (0.2) 0.8 (0.9) 0.7 (0.5)
CSF a-syn levels discriminate between DLB and AD.
e
Tokuda sandwich ELISA with modifications (antibody 211/FL-140)
18.1 34.8 43.1 15.3 68.9
(16.0) (54.0) (47) (18) (71)
CSF a-syn levels lower in dementia groups compared to controls. CSF tau/a-syn ratio helps discriminate PD from OND.
Van Geel sandwich ELISA method (antibody 211/FL-140)
38.0 37.0 49.1 44.3 30.4 NA
(29.0) (36.1) (41.5) (40.3) (19.1)
CSF a-syn does not discriminate between dementia disorders.
11.2 (9.7) 14.0 (9.3) 12.4 (11.1)
In house modified sandwich ELISA (antibody syn-1/FL-140)
8.2 (4.2) 12.2 (5.8) 13.9 (7.5)
CSF a-syn significantly lower in DLB patients compared to AD and other dementias. CSF a-syn correlates with Ab42 levels in DLB patients.
4.25 (3) 2.2 (2.4) 8.3 (4.8) 0.083 (0.25) NA
e
In-house sandwich ELISA (antibody mSA1/Syn1-BB)
6.2 (4.2) 3.8 (3.3) 3.0 (1.3) 300 (248) 6.0 (5.7)
CSF a-syn lower in synucleinopathies compared to non-synucleinopathies. CSF a-syn markedly elevated in CJD patients.
74.5 (5.2) 73.3 (4.8)
4.2 (3.0) 2.9 (1.4)
19.5 (5.8) 20.0 (5.6)
Tokuda sandwich ELISA with modifications (antibody 211/FL-140)
44.7 (24.9) 50.2 (24.1)
CSF a-syn not significantly different between DLB and AD patients. Lower CSF a-syn levels in DLB patients with longer disease duration
72.7 (9.5)
1.9 (1.7)
22.7 (3.1)
e
29 23 20 30
No of patients
Gender (M:F)
Tateno et al. 2012 [13]
6 DLB 9 AD 11 PD 11 MSA 11 Controls
2:4 6:3 6:5 6:5 7:4
73 75 71 66 49
Mollenhauer et al. 2011 (training cohort) [14]
55 62 51 29 76
27:28 22:40 32:19 16:13 33:43
Mollenhauer et al. 2011 (validation cohort) [14]
66 DLB 273 PD 15 MSA 8 PSP 22 NPH 23 Controls
47:19 184:89 10:5 6:2 19:3 17:6
Mollenhauer et al. 2011 (autopsy cohort) [14]
13 DLB 21 AD 7 Controls
7:6 11:10 5:2
Parnetti et al. 2011 [15]
32 48 38 31 32
18:14 19:29 22:16 17:14 18:14
71.4 68.7 69.3 64.3 61.9
(6) (9.7) (7.2) (6.4) (17.8)
3.9 4.0 3.5 4.2 NA
Spies et al. 2009 [16]
40 DLB 131 AD 39 FTD 28 VaD 57 Control A 55 Control B
27:13 54:77 24:15 18:10 30:27 26:29
74.0 71.7 66.4 75.4 61.3 61.1
(8.3) (8.8) (8.6) (8.4) (8.8) (8.9)
e
18.9 19.6 20.3 18.5 30.4 NA
Kasuga et al. 2010 [17]
34 DLB 31 AD 21 OD
13:21 13:18 9:12
75.8 (10.7) 67.9 (12.3) 70.0 (11.9)
e
Mollenhauer et al. 2008 [18]
13 AD 38 DLB 8 PD 8 CJD 13 Controls
4:9 23:15 5:3 3:5 7:6
69.9 71 76 71 64
NoguchiShinohara 2009 [19]
16 DLB 13 DLB (matched) 21 AD
6:10 6:7 8:13
15 15 66 55
4:15 6:15 47:19 30:25
Ohrfelt 2009 [20]
DLB AD PD MSA Controls
DLB AD PD FTD Controls
PD DLB AD Controls
Mean age (years)
72 (12) 67 (16) 61 (6)
71 79 77 65
(9) (8) (3) (7) (15)
(63e76) (76e80) (73e82) (62e70)
(2.7) (2.9) (2.3) (2.2)
(5e27) (10e25) (22e30) (21e30)
(8.2) (3.9) (6.1) (2.9) (19.1)
(27e30) (19e23) (14e26) (29e30)
X. Lim et al. / Parkinsonism and Related Disorders 19 (2013) 851e858
Mean duration of disease (years)
Studies (Author, Year)
49.3 (44.5) In-house ELISA (antibodies Syn1b, Syn3b, Syn 5d/ LO-MG1-13, LO-MG-2)
0.417 0.334 0.296 0.395
(0.246e0.522) (0.220e0.406) (0.234e0.372) (0.298e0.452)
CSF a-syn lower in AD patients compared to controls. CSF a-syn levels similar in PD and DLB patients. a-syn possible general marker for synapse loss
(continued on next page) 853
854
Table 1 (continued ) Mean duration of disease (years)
MMSE (score)
Assay
Conc CSF a-sync (pg/ml)
Author’s conclusion
(5) (8) (8) (67)
e
e
Van Geel sandwich ELISA method (antibody 211/FL-140)
18 23 20 16
CSF a-syn does not discriminate DLB from AD
(10.8) (7.8) (6.9) (6.5) (8.8) (8.8)
3.21 (2.72) 3.21 (2.31) 5.0 (0.083) 4.74 (2.6) 3.07 (1.67) NA
28.4 27.9 22.7 26.6 27.2 NA
Van Geel sandwich ELISA method (antibody 211/ FL-140)
26.0 25.0 24.0 24.0 30.5 25.0
71 (7) 73 (6) 72 (8)
e
23 (4) 21 (5) 29 (1)
Commercial Invitrogen ELISA
0.378 (0.166) 0.464 (0.233) 0.500 (0.178)
Female DLB patients have lower a-syn levels than AD patients and controls
42 59 25 48 13 20 22 7
70 63 76 74 78 70 64 71
(63e76) (56e71) (73e80) (69e81) (74e80) (64e74) (59e72) (66e76)
e
29 (28e30) 29 (27e29) 24 (18e25) 21 (17e25) 21 (19e23) 27 (24e28) 29 (28e29) NA
Luminex Multiplex Assay
67 55 59 59 94 70 56 56
Cerebrospinal fluid a-syn were decreased in patients with PD, PDD, DLB and MSA but increased in patients with AD
50:50 76:24 55:45 47:53 27:73
64 78 74 64 75
(10) (6) (6) (9) (6)
e
28.5 20.6 20.2 28.5 23.1
Commercial Invitrogen ELISA
AD > controls > DLB > PD > PDD
No of patients
Gender (M:F)
Reesink et al. 2010 [21]
34 18 35 63
18:16 10:8 29:6 29:34
67 67 71 69
Aerts et al. 2012 [22]
58 PD 47 MSA 3 DLB 22 VaP 12 PSP/CBD 57 Controls
40:18 28:19 3:0 15:7 5:7 30:27
56.6 62.9 62.5 68.7 66.2 61.3
Wennstrom et al. 2012 [23]
18 DLB 26 AD 24 Controls
9:9 15:11 16:8
Hall et al. 2012 [24]
107 Controls 90 PD 33 PDD 70 DLB 48 AD 45 PSP 48 MSA 12 CBD
Wennstrom 2013 [5]
52 46 33 38 22
SC PD DLB AD
Controls AD DLB PD PDD
Mean age (years)
(1.6) (2.5) (6.4) (2.7) (1.8)
(1.1) (4.3) (5.6) (1.2) (4.4)
(14e26) (18e32) (15e27) (13e23) (20.5e32.5) (17.0e32.0) (23.0e34.0) (16.0e30.5) (19.5e38.0) (18.0e42.0)
(51e85) (40e70) (49e68) (44e74) (76e121) (51e86) (43e66) (50e84)
CSF a-syn does not differentiate between parkinsonian disorders
Patients with synucleinopathies have lower CSF levels of a-syn compared to controls and AD patients.
All values are expressed as mean standard deviation or p25e75 inter-quartile ranges unless otherwise stated. Abbreviations: DLB ¼ Dementia with Lewy Bodies, PD ¼ Parkinsons Disease, AD ¼ Alzheimers Disease, MSA ¼ Multiple System Atrophy, VaP ¼ Vascular Parkinsonism, VaD ¼ Vascular Dementia, FTD ¼ Frontotemporal Dementia, PSP ¼ Progressive Supranuclear Palsy, NPH ¼ Normal Pressure Hydrocephalus, CJD ¼ CreuzfeldteJakob Disease, CBD ¼ Corticobasal Degeneration, OD ¼ Other Dementia, SC ¼ Patients with Subjective Complaints, OND ¼ Other Neurological Disease, PDD ¼ Parkinsons Disease Dementia.
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Fig. 2. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs AD patients.
diagnoses were made using the same diagnostic criteria within each study. Only two of the thirteen studies attempted to carry out blinding of alpha-synuclein assay results [21,22]. Only one of the thirteen studies made efforts to blind the reference standard [16]. It was unclear if there was an acceptable delay between time of diagnosis based on international consensus criteria and time of performing CSF alpha-synuclein assay, as the duration from clinical diagnosis to lumbar puncture was not reported across all studies. Furthermore, clinical interventions, including symptomatic treatments introduced were not clearly described. None of the studies highlighted ambiguous immunoassay results and no withdrawals were reported. The methodology of several studies was not clearly described, raising some doubts over the replicability and validity of the assays. 3.3. Concentration of CSF alpha-synuclein in DLB vs other neurodegenerative conditions Fig. 2 shows a Forest plot which demonstrates a significantly lower mean concentration of CSF alpha-synuclein in patients with DLB patients compared to those with AD [weighted mean difference 0.24; 95% CI, 0.45, 0.03; p ¼ 0.02]. Fig. 3 shows that there was no significant difference in mean concentration of CSF alphasynuclein between patients with DLB and PD [weighted mean difference 0.05; 95% CI, 0.17, 0.28; p ¼ 0.65]. Similarly, no significant difference was found in the mean concentration of CSF alphasynuclein between DLB and other neurodegenerative conditions including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), fronto-temporal dementia (FTD) and vascular dementia (VaD) (data not shown). Meanwhile, Fig. 4 shows no significant difference in mean CSF alpha-synuclein concentration between DLB patients and normal/neurological controls [weighted mean difference 0.12; 95% CI, 0.29, 0.04; p ¼ 0.13] although the trend was toward lower CSF alpha-synuclein concentration in DLB patients compared to normal/neurological controls. One study [18] reported significantly higher mean CSF alpha-synuclein concentrations in Creutzfeldt-Jakob disease compared to DLB. CSF alpha synuclein levels were also compared between DLB and non-synucleinopathies (AD, PSP, FTD and VaD) as well as synucleinopathies (PD and MSA). Mean CSF alpha-synuclein
concentration in the DLB group was found to be significantly lower compared to non-synucleinopathies [weighted mean difference 0.25; 95% CI 0.44, 0.06; p ¼ 0.01] (Fig. 6). In contrast, mean CSF alpha-synuclein concentration in the DLB group was not significantly different compared to synucleinopathies [weighted mean difference 0.05; 95% CI, 0.17, 0.27; p ¼ 0.65] (Fig. 5). 3.4. Sources of heterogeneity and sensitivity analysis The degree of heterogeneity (I2) ranged from 60% to 73% which suggests that there were significant differences in experimental protocol between studies. Factors which may contribute to this include administration of interventions or treatments which modify physiological levels of CSF alpha-synuclein, differences in reference standards used to define patient populations, variation in sample processing and storage methods, differences in assays used, blood contamination in CSF samples and variation in diagnostic threshold of index test. 4. Discussion Dementia with Lewy Bodies is a condition which poses significant diagnostic challenges due to similarities with a spectrum of other degenerative dementias and its highly variable presentation. This study aims to evaluate evidence for the diagnostic utility of CSF alpha-synuclein assays in differentiating between DLB and other degenerative dementias. A number of studies have reported lower mean CSF alpha-synuclein levels in DLB compared to AD [4,14,17,24] whilst others have shown no difference [16,19,21] between the two dementias. Furthermore, some groups have reported a difference in CSF alpha-synuclein concentrations between synucleinopathies and AD [13,14,18] whilst others report no such difference [16,22]. Correlation between CSF alpha-synuclein and other biomarkers such as total tau, phosphorylated tau, betaamyloid 42 have also been reported in several studies [14e16,19e 21,23]. The correlation between CSF alpha-synuclein levels in patients with DLB and age, gender, cognitive function as well as duration of disease has additionally been the subject of much debate. Reesink et al. [21] suggested patients with worse cognitive function in DLB
Fig. 3. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs PD patients.
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Fig. 4. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB vs neurological/normal controls.
Fig. 5. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB patients vs patients with synucleinopathies.
have lower levels of CSF alpha-synuclein. However, this has not been replicated in findings from other studies [16,17]. Ohrfelt et al. [20] reported lower CSF alpha-synuclein levels in AD (particularly in patients with MMSE score less than 20) compared to controls. They also found CSF alpha-synuclein concentrations in DLB and PD patients to be similar [20]. Wennstrom et al. [23] reported lower CSF alpha-synuclein concentrations in female DLB patients compared to AD patients and controls. However, this was again not replicated in four other studies [4,16,17,19]. Noguchi-Shinohara et al. [19] commented that CSF alpha-synuclein concentration was lower in DLB patients with longer disease duration while other groups have reported no correlation between disease duration and CSF alpha-synuclein levels [15,17]. Our results suggest a trend toward lower CSF alpha-synuclein concentrations in DLB compared with AD, regardless of type of assay used. The lower CSF alpha-synuclein levels observed in DLB may be explained by sequestration of the protein as fibrillary aggregates in Lewy bodies. This is similar to the proposed mechanism underlying the reduced CSF concentrations of beta-amyloid in AD. Various other factors may also influence the balance between production, clearance and solubility of CSF alpha-synuclein. Mutations affecting protein structure may also increase the propensity of forming fibrillary aggregates [26]. Further studies are warranted to clarify specific molecular mechanisms underlying this observation. The difference in CSF alpha-synuclein concentrations in patients with DLB compared with AD is of potential clinical utility as AD is
one of the key differentials for DLB. Nevertheless, this difference must be interpreted with caution due to significant heterogeneity in study design, which may be caused by variation in factors such as patient characteristics and type of assays used. Several groups have developed in house assays making comparison between assays challenging. Lack of transparency in reporting of methodology and laboratory protocols makes attempts to reproduce assay results problematic. Furthermore, several groups have attempted to modify pre-defined assays without validating them independently. There is also a need to standardize pre-analytical confounding factors to ensure consistency of results across studies. Several recommendations have been made by Del Campo et al. [25] to address this issue. No difference in CSF alpha-synuclein concentration was identified between patients with DLB and Parkinsons disease. This is perhaps of little surprise as both disorders are synucleinopathies on a continuum of the same clinical spectrum. When comparing CSF alpha-synuclein concentrations in DLB with neurological controls, a trend toward lower CSF alpha-synuclein in DLB patients emerged, although this did not reach statistical significance. It is possible this may be attributed to the heterogeneity in subjects included in control groups. Several studies failed to adequately define control groups as normal, but instead used neurological controls with nonneurodegenerative pathologies. Only a minority of studies included post-mortem verification of the absence of co-existent neurodegenerative disease in controls which may have influenced observed CSF alpha-synuclein levels. Future studies should include
Fig. 6. Forest plot comparing mean CSF alpha-synuclein concentrations of DLB patients vs patients with non-synucleinopathies.
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Fig. 7. Methodological quality summary based on modified QUADAS (Quality Assessment of Diagnostic Accuracy Studies) criteria.
appropriately matched neurologically normal controls with adequate post-mortem verification. Numerous methodological problems were identified in many of the studies such as assay variability, poor study design, use of outdated diagnostic reference standards, and poor standards of reporting. In terms of assays used, it was unclear which type of alpha-synuclein was being measured by the assays. Three molecular forms of alpha-synuclein have been identified as expressed in the brain: alpha-synuclein 140 (full length), alpha-synuclein 126 (lacks exon 3) and alpha-synuclein 112 (lacks exon 5) [26]. These isoforms are formed by variation in alternative splicing of alpha-synuclein, and each may be subjected to further post-translational modifications such as phosphorylation and ubiquitination. These posttranslational changes are thought to promote the aggregation of the small soluble monomeric forms of alpha-synuclein into diseaseassociated aggregated oligomers [26]. Therefore, the standardization of alpha-synuclein assays is hampered by the presence of heterogenous forms of the protein within the brain, uncertainty of which forms are present in the CSF and ambivalence regarding antibody specificity for the disease-associated form of alphasynuclein. It is possible that the assays involved in this study were measuring different components of the alpha-synuclein mixture present within the CSF resulting in such wide variations in levels of CSF alpha-synuclein reported. Type of study (prospective or cross-sectional) and whether the reference standard and index test were blinded or not, was often unclear. Patient recruitment and selection were sometimes vague. Inclusion and exclusion criteria were not always clearly stated and there were differences in the use of probable or possible clinical diagnostic criteria between studies. None of the studies defined the time period between clinical diagnosis and CSF sampling or mentioned therapeutic interventions the patients were subjected to which might confound interpretation of assay results as CSF
Table 2 Proposed recommendations for future studies on CSF biomarker assays in DLB. Study design Prospective cohort studies Ensure study is sufficiently powered Patients Recruit consecutive patients referred with suspected degenerative dementia from representative population Make diagnosis based on reference standard before performing index test Define time period between clinical diagnosis and CSF sampling State if clinical intervention or treatment is performed on patients between time of reference test and index test Reference standard Based on expert clinical opinion supplemented with clinical and imaging results Post-mortem confirmation of diagnosis whenever possible Define if possible, probable or definite diagnosis made Blind reference standard Index test Clear description of assay quantification Account for inter- and intra-observer reliability of test Validate biomarker in an independent cohort if using novel assay or in-house assays Provide mean and standard deviation values of alpha-synuclein concentration Include comparison with other biomarkers such as total tau, p-tau, betaamyloid and alpha-synuclein Blind index test Standard of reporting Use STARD guidelines for reporting results Perform ROC analysis on data Provide numerical threshold/cut-off values Provide values such as true positives, true negatives, false positives, false negatives, cut-off/threshold values Provide ratio between biomarkers where applicable
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alpha-synuclein levels may vary with progression of disease and treatment. Summary receiver operating characteristic (SROC) analysis on the index test was not feasible due to insufficient information available in papers or supplementary information provided by authors. Moreover, most studies failed to perform receiver operating characteristic (ROC) analysis on available dataset. Consequently, values for sensitivity, specificity, true positive, true negative, false positive and false negative of the assay were not reported. Only Mollenhauer et al. [14] attempted to characterize and validate their assays in an independent cohort. Only two studies reported efforts to blind assay results [21,22] and clinical data [16] while none of the studies attempted to blind the reference standard using an external clinical reviewer. Mean and standard deviation of concentrations of alpha-synuclein between patient subgroups were sometimes not reported as well. Power studies to guide robust sample sizes for future studies are problematic due to the heterogeneity of available assays and patient populations studied. Moreover, the difference in means between studies and within studies varied enormously which makes calculation of effect size challenging. Whilst investigation of statistically significant numbers of CSF samples is clearly of importance, of greater relevance perhaps, is the necessity for patients in studies to be well phenotyped clinically with postmortem neuropathological confirmation of disease. We propose several recommendations to ensure quality of future studies (Table 2).
5. Conclusion We conclude that CSF alpha-synuclein analysis is potentially useful in distinguishing DLB from AD but not from other synucleinopathies. However, there is a need for more adequately powered studies with standardized assays for CSF alpha-synuclein measurements, long-term follow-up, appropriately matched controls, post-mortem histopathological confirmation of patient groups, sensitivity and specificity analysis as well as good reporting standards (see STARD criteria) to yield more conclusive results in future reviews. Therefore, although CSF alpha-synuclein analysis is a promising diagnostic tool in distinguishing DLB from AD, significant further validation needs to be undertaken before it can be recommended for routine use in clinical practice. Contributions of authors All authors made substantial contributions to the conception, design, data analysis and interpretation of this paper. Each authors also made significant contributions in drafting and critically appraising the final version of this paper for publication.
Declarations of interest All authors report no conflict of interest.
Acknowledgments The authors would like to thank the authors of papers included in this systematic review for kindly sharing their data and expert advice. Dr Suvankar Pal is funded by a NHS Research Scotland Career Researcher Fellowship.
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