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