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Role of neurofilaments & other biomarkers in blood and CSF
Gavin GiovannoniBarts and The London
Why MS biomarkers?
• Diagnostic testing
• Positive & negative predictive testing
• Pathogenesis
• Immunology
• Aetiology
• Disease progression & recovery
• Disease heterogeneity
• Pharmacovigilance
• Monitor disease processes
• Prognosis (high vs. low risk patients)
• Monitoring effect of therapeutic
interventions
Diagnostic & pathogenic markers
The evolving clinical definition of MS
1. Schumacher, et al. Problems of Experimental Trials of Therapy in
Multiple Sclerosis: Report by the Panel on the Evaluation of Experimental
Trials of Therapy in Multiple Sclerosis. Ann N Y Acad Sci 1965;122:552-68.
2. Poser, et al. New diagnostic criteria for multiple sclerosis: guidelines for
research protocols. Ann Neurol 1983;13:227-31.
3. McDonald, et al. Recommended diagnostic criteria for multiple sclerosis:
guidelines from the International Panel on the diagnosis of multiple
sclerosis. Ann Neurol 2001;50:121-7.
4. Polman, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to
the "McDonald Criteria". Ann Neurol 2005;58:840-6.
5. Polman, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to
the McDonald criteria. Ann Neurol. 2011;69:292-302.
Will Rogers Phenomenon in Multiple Sclerosis
1879 - 1935
“When the Okies left Oklahoma and moved to California, they raised the average intelligence level in both states.”
Will Rogers Phenomenon in Multiple Sclerosis
Sormani et al. Ann Neurol 2008;64:428–433.
Poser
McDonald
Inactive CIS Active CIS RRMS
MS diagnosed according the old Poser Criteria
Inactive CISLess activeRRMS
More ActiveRRMS
MS diagnosed according the New McDonald Criteria
Intrathecal synthesis of IgG
Images courtesy of Alastair Compston and Ed Thompson.
Kabat et al. J Clin Invest. 1942 Sep;21(5):571-7.
Carl Lange – Colloidal Gold Curve
Isoelectric focusing with immunfixation
Diagnostic criteria for Primary Progressive MS
Polman et al. Ann Neurol 2005;58:840-6.
Accumulation of disability in PPMS:stratified by intrathecal IgG abnormalities
Proportion Progressing as Percent
Epoch CSF- CSF+
6 mo 7.3 9.8
12 mo 15.0 20.4
18 mo 22.8 28.1
24 mo 25.4 34.3
Years to Progression
2.43 2.26
Based on data from a second meeting of the DSMB and assume no therapeutic effect
Slide courtesy of Jerry Wolinsky
0 1 2 3Years
0.0
0.2
0.4
0.6
0.8
1.0
Prop
ortio
n Pr
ogre
ssin
g
PositiveNegative
CSF
P =0.03
Dobson R, et al. J Neurol Neurosurg Psychiatry 2013;0:1–6.
What constitutes a useful diagnostic test or set of criteria?
TARGET DISORDER
PRESENT ABSENT
DIAGNOSTIC TEST RESULT
+ a b a + b
- c d c + d
a + c b + d a + b + c + d
From these we determine the sensitivity and specificity as follows:
SENSITIVITY = a/(a+c) > 80%SPECIFICITY = d/(b+d) > 80%
Neurobiol Aging 1998; 19:109-116.
A clinico-pathoanatomical study of multiple sclerosis diagnosis
SENSITIVITY = True+ve /(True+ve + False-ve)
Eye Department, Hvidovre Hospital, Denmark.
• Neuropathological examination of 518 consecutive patients with clinically definite MS revealed a correct diagnosis in 485 cases (94%).
• Clinical diagnosis had been established by a neurologist in all cases. • Erroneous diagnosis included a variety of other neurological
disorders. • Also investigated was a randomly selected series of 33 patients with a
clinical diagnosis of probable MS: – post mortem confirmation of MS was obtained in circa 66%. – The remainder the error pattern was similar to the above.
Engell T. Acta Neurol Scand. 1988 Jul;78(1):39-44.
Lennon et al. Lancet 2004;364:2106-12.
NMO
Kleinschmidt-DeMasters,et al. N Engl J Med. 2005 Jul 28;353(4):369-74.
PML complicating treatment with natalizumab and IFNb-1a for MS
Pathogenic markers
“Inflammation”
“Oligodendrocyte Toxicity & Demyelination”
Axonal Toxicity (conduction block)
Axonal & Neuronal Loss
Gliosis
Remyelination & Axonal Recovery
“Inflammation”
Central Adaptation & Plasticity
Key pathological processes in MS
Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with
early onset of disease and severe cortical pathology
Magliozzi et al. Brain 2007; 130:1089-1104.
Increased urinary free immunoglobulin light chain excretion in MS
Petzold et al. J Neurol Neurosurg Psychiatry. 2005 Feb;76(2):206-11.
Spinal fluid neurofilament levels
Gunnarsson et al. Ann Neurol 2010; Epub.
CSF NFL
-1.0%
-0.8%
-0.6%
-0.4%
-0.2%
0.0%Years 0-2
-0.82%
-0.80%
P=0.822
Placebo (N=315) Natalizumab (N=627)
Year 0-1* Year 1-2
-0.40%
-0.56%
-0.43%
-0.24%
P=0.004
P=0.002
Miller DH et al. Neurology 2007;68:1390-1401.
Natalizumab and brain atrophyMean (SE) percentage change in BPF
Very low risk
ageplace of residence
outdoor activity / sun exposure / sun screendiet / vitamin D supplements
age of exposure to EBVsmoking
At risk High Risk
Low risk
RIS CIS MS
family historygenetics
sexmonth of birthplace of birth
Unfavourable disease-modifying factorsdynamic risk factorsstatic risk factors
dynamic protective factorsstatic protective factors
MRI / evoked potentials changes
Peripheral immunological changesT-regs (), NK cells, CD8 ()
Clinical disease
In utero childhood Adolescence / early adulthood adulthood
1. Declining Physiology – “peripheral immunological endophenotype”2. Biological disease threshold – “CNS endophenotype”3. Asymptomatic disease – RIS (abnormal MRI and/or evoked potentials)4. Clinical disease
a. Clinically isolated syndrome (CIS)b. Relapsing MSc. Relapsing secondary progressive MSd. Non-relapsing secondary progressive MS
Favourable disease-modifying factors
protective HLA haplotypes
CNS changes(OCBs and microscopic pathology)
2
3
24b 24c 24d
24a
1
The MS ‘Endophenotype’
Vitamin D as an early predictor of MS activity and progression
Ascherio JAMA Neurol. 2014 Mar;71(3):306-14.
Vitamin D as an early predictor of MS activity and progression
Ascherio JAMA Neurol. 2014 Mar;71(3):306-14.
Vitamin D as an early predictor of MS activity and progression
Ascherio JAMA Neurol. 2014 Mar;71(3):306-14.
P=0.007
Multivariate International CIS risk factor study - 25-OH D3
Conversion to CDMS] HR 95% CI P value
25-OH D3 0.996 0.993-0.999 0.01
P=0.008
Median Survival: 935 days vs. 1262 days
Kuhle et al. submitted 2014.
Higher 25-OH vD is associated with lower relapse risk
Simpson et al. Ann Neurol. 2010;68:193–203.
vD status predicts new brain MRI activity in MS
Mowry et al. ANN NEUROL 2012;72:234–240.
• EPIC is a 5-year longitudinal MS cohort study at the UCSF. • 469 subjects annual clinical evaluations, brain MRI, and biomarkers.
• Each 10ng/ml higher vitamin D level was associated with lower
subsequent disability (-0.047; 95% CI = -0.091 to -0.003; p = 0.037).
Chicken or Egg
Causation?
Association?
The effect of the systemic inflammatory response on plasma vitamin 25 (OH) D concentrations adjusted for albumin
Ghashut et al. PLoS One. 2014 Mar 25;9(3):e92614.
Vitamin D3
CRP
Albumin
Hypothesis
“Hypovitaminosis D3 is a consumptive vitaminopathy.”
Therefore, the association between low vD levels and disease is due to reverse causation.
Causation?
Association?
Immunomodulatory effects of vD in MS
Correale et al. Brain 2009: 132; 1146–1160.Vitamin D3
Immune response
Seasonal Effects
Seasonal patterns in optic neuritis and MS: a meta-analysis
Jin et al. J Neurol Sci 2000:181;56–64.
Seasonal prevalence of MS disease activity
Meier et al. Neurology 2010;75:799–806.
vD and disease activity in MS before and during IFN-beta treatment
Løken-Amsrud et al. Neurology. 2012 Jul 17;79(3):267-73.
Treatment effects
The effect of vitamin D-related interventions on multiple sclerosis relapses: a meta-analysis
James et al. Mult Scler. 2013 Oct;19(12):1571-9.
The effect of vitamin D-related interventions on multiple sclerosis relapses: a meta-analysis
James et al. Mult Scler. 2013 Oct;19(12):1571-9.
Pharmacovigilance markers
What is the diagnosis?
Take special care with Interferon-beta-1b:
If you might have a disorder of the immune system in which abnormal proteins are found in the blood (monoclonal gammopathy), you must check this with your doctor before you use interferon beta-1b. Patients who have the rare condition known as monoclonal gammopathy may develop problems with their small blood vessels (capillaries) leading to shock (collapse) which can be fatal, when they use medicines like interferon-beta-1b.
See also 4. Possible side effects.
?
Natalizumab
Progressive multifocal leukoencephalopathy (PML)
Kleinschmidt-DeMasters,et al. N Engl J Med. 2005 Jul 28;353(4):369-74.
207 cases -1st February 2012
44 (21%) died
163 (79%) alive
Mild disability – 10%Moderate disability – 50%Severe disability – 40%
5% NAbs – infusion reactions
Natalizumab PML risk stratification tool
Anti-JC virus antibody status
Negative Positive
Prior immunosuppressant use
Natalizumab treatment>2 Years
Natalizumab treatment>2 Years
No Yes
No Yes No Yes
Lowest HighestRelative PML Risk
< 1 in 10,000 1 in 941 in 256 1 in 6681 in 1887
Mitoxantrone AzathioprineMethotrexateCyclophosphamideMycophenolate CladribineRituximabEtc.
PML risk estimates for anti-JCV antibody index thresholds were calculated based on the current PML risk stratification algorithm (September 2012) and predicted probabilities (using data from patients with no prior IS use: 2242 non-PML patients and 51 patients who developed PML using all available anti-JCV antibody index data at least 6 months prior to PML diagnosis) for the population at or below that particular index (0.9−1.5) and for the population above an index of 1.5. For index thresholds below 0.9, patient numbers were insufficient to allow for calculation of risk estimates.
Anti-JCV antibody index values may differentiate PML risk for those with no prior
immunosuppression
Index 1−24 months
≤0.9 0.1(0, 0.41)
≤1.1 0.1(0, 0.34)
≤1.3 0.1(0.01, 0.39)
≤1.5 0.1(0.03, 0.42)
>1.5 1.0(0.64, 1.41)
63
PML risk estimates (95% CI) per 1000 patients with no prior IS use
Plavina T et al. Poster DX51, CMSC May 29–June 1 2013, Orlando, USA; Ticho B. et al, Presented at ENS, June 8–11 2013, Barcelona, Spain O228.
63
25−48 months 49−72 months
0.3(0.04, 1.13)
0.4(0.01, 2.15)
0.7(0.21, 1.53)
0.7(0.08, 2.34)
1.0(0.48, 1.98)
1.2(0.31, 2.94)
1.2(0.64, 2.15)
1.3(0.41, 2.96)
8.1(6.64, 9.80)
8.5(6.22, 11.38)
Predicting autoimmunity following treatment of MS with alemtuzumab
• 30% of alemtuzumab-treated pts develop autoimmune side-effects (primarily thyroid disease and idiopathic thrombocytopenia)
• Aim: To define predictive factors for autoimmune side-effects
• Sera of 141 pts screened at baseline for 8 different cytokines/chemokines
A combined IL-21/IL-7 test on pre-treatment serum may be useful to identify patients at low risk of developing autoimmunity following treatment with alemtuzumab
Jones JL, et al. ECTRIMS 2011, Amsterdam. P1009
Sensitivity NPV Specificity PPV
IL-21 alone 81 84 70 66
IL-7 alone 76 76 54 54
CCL21 alone 63 65 49 47
IL-21 or IL-7 98 97 41 55
IL-21 OR IL-7OR CCL21 98 91 12 45
Given that pts may elect to receive treatment based on results of this test – most weight given to minimizing false negative results. Combining IL-21 and IL-7 into a single test offers improved test accuracy over IL-21 alone. CCL21 did not improve test accuracy
0
10
20
30
40
IL-7
Autoimmunity No autoimmunity
0
500
1000
1500
IL-2
1
1.0
Se
ns
itiv
ity 0.8
0.6
0.4
0.2
0.00.0 0.2 0.4 0.6 0.8 1.0
1.0
Se
ns
itiv
ity 0.8
0.6
0.4
0.2
0.00.0 0.2 0.4 0.6 0.8 1.0
1-Specificity
IL-21 and IL-7 levels in sera of pts who did or did not develop autoimmunity
Receiver operating characteristic (ROC) curves
Neurology 2012;78(Suppl.): [S41.006]
Anti-natalizumab Antibodies
Number of Patients at Risk
PlaceboAntibody NegativeTransiently PositivePersistently Positive
3155682037
2965501932
2835381826
2645261625
2485061624
2404871622
2294801522
2164701419
2084601416
2004491415
Weeks
0.0
0.1
0.2
0.3
0.4
0.5
0 12 24 36 48 60 72 84 96 108 120
29%
Placebo
17%
Antibody Negative
17%
Transiently Antibody Positive
34%Persistently Antibody Positive
Cu
mu
lati
ve P
rop
ort
ion
of
Pa
tie
nts
w
ith
Su
sta
ine
d D
isa
bil
ity
Pro
gre
ssio
n (
ED
SS
) *,†
*p ≤0.05 vs. antibody-negative patients†p=0.66 vs. placebo
Number of Patients at Risk
PlaceboAntibody NegativeTransiently PositivePersistently Positive
3155682037
2965501932
2835381826
2645261625
2485061624
2404871622
2294801522
2164701419
2084601416
2004491415
Weeks
0.0
0.1
0.2
0.3
0.4
0.5
0 12 24 36 48 60 72 84 96 108 120
29%
Placebo
17%
Antibody Negative
17%
Transiently Antibody Positive
34%Persistently Antibody Positive
Cu
mu
lati
ve P
rop
ort
ion
of
Pa
tie
nts
w
ith
Su
sta
ine
d D
isa
bil
ity
Pro
gre
ssio
n (
ED
SS
) *,†
*p ≤0.05 vs. antibody-negative patients†p=0.66 vs. placebo
0.73
0.220.16
0.48*
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ad
jus
ted
An
nu
ali
zed
Rel
apse
Ra
te (
95%
CI)
Placebo (n=315)
Antibody Negative(n=568)
TransientlyAntibody Positive
( n=20)
PersistentlyAntibody Positive
(n=37)
*p=0.009 vs. antibody-negative patients
0.73
0.220.16
0.48*
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ad
jus
ted
An
nu
ali
zed
Rel
apse
Ra
te (
95%
CI)
Placebo (n=315)
Antibody Negative(n=568)
TransientlyAntibody Positive
( n=20)
PersistentlyAntibody Positive
(n=37)
*p=0.009 vs. antibody-negative patients
Calabresi et al, Neurol 2007
Impact of anti-natalizumab antibodies on . . . . .
Annualized relapse rate Progressive disability
Natalizumab infusion reactions
• Acute hypersensitivity reactions are well-recognized• Generalized urticaria, dizziness, fever, rash, rigors, pruritus,
nausea, flushing, dyspnea, chest pain
• Onset generally during or within 1 hour of second infusion
• Incidence ~4%• severe anaphylactic/anaphylactoid reactions <1%
• Most reactions are associated with anti-natalizumab antibodies
• Treatment: • immediate and permanent cessation of natalizumab
• antihistaminesRudick et al, NEJM 2006
Monitoring effect of therapeutic interventions
Reduced efficacy due to NAbs – systematic review
Farrell & Giovannoni, Multiple Sclerosis 2007; 13: 567-577.
Clinical importance of neutralising antibodies against interferon
beta in patients with relapsing-remitting multiple sclerosis
Sorensen et al. Lancet 2003; 362: 1184–91.
Jacob Elkins, James Sheridan, Lakshmi Amaravadi, Katherine Riester, Gilmore O’Neill
Neurology 2012;78(Suppl.): S31.004
Prognostic markers
Intrathecal synthesis of IgG
Images courtesy of Alastair Compston and Ed Thompson.
Kabat et al. J Clin Invest. 1942 Sep;21(5):571-7.
Carl Lange – Colloidal Gold Curve
Isoelectric focusing with immunfixation
Petzold, J Neurol Sci. 2005 Jun 15;233(1-2):183-98.
Conclusion• Diagnostic/prognostic biomarkers
• Intrathecal OCBs• IgG Index
• Pharmacovigilance• Baseline screening
• Monoclonal gammaopathy (IFNbeta)• Serology – VZV, JCV (immunosuppression)
• Monitoring• FBC, LFTs, U&E, TFTs• Monthly platelets and possibly urine
(alemtuzmab)• Serology – JCV (natalizumab)• CD56-bright cells (daclizumab)• NABs (IFNbeta and natalizumab)
• Potential surrogate treatment markers• CSF neurofilament levels
• Potential future baseline response markers• Type 1 interferon signature• PBMC transcriptomic profiles
Acknowledgements
• Giovannoni
• Sharmilee Gnanapavan
• David Baker
• Gareth Pryce
• Sarah Al-Izki
• Sam Jackson
• Katie Lidster
• Yuti Chernajovsky
• Alex Annenkov
• Anne Rigby
• Michelle Sclanders
• Larry Steinman
• Peggy Ho
• Charles ffrench-Constant• Robin Franklin
• Siddharthan Chandran• David Hampton
• Ian Duncan• Sam Jackson
• Peter Calabresi• Avi Nath
• Raj Kapoor• John Zajicek• Doug Brown• UK MS Clinical Trial Network• BioMS
• Co-investigators• NABINMS• Affirm study• Care MS 1 & 2 studies• Select trial