1

Progressive Multifocal Leukoencephalopathy in the Era of 

Monoclonal Antibodies 

Joseph R. Berger, M.D.

Department of Neurology

University of Pennsylvania

Conflicts of InterestJoseph R. Berger, M.D.

• Grants: • BiogenIdec, PML Consortium

• PML Adjudication Committees:• Amgen; Astra Zeneca; Bristol Myers Squibb;

Eisai; Janssen; Millennium; Parexel; Pfizer; Roche; Takeda

• Consultancies: • Genentech; Genzyme; Incyte; Inhibikase;

Johnson & Johnson; Novartis

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History of PML and Discovery of JC Virus

PML – The Early Years

71 year old woman• CLL 52 years• L hemiparesis• Death in 4 months73 year old woman• CLL 68 years• Unable to concentrate • Clumsy, stupor • Death 4 months42 year old man• Hodgkins disease 42• Aphasia, hemiparesis, stupor• Death in 10 weeks

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PML - JC virus

• 40 nm non-enveloped DS DNA icosahedron

• Full length sequence of Mad-1 (1984)• Three regions of genome - early, late and

transcriptional control (noncodingregulatory region)

• 6 regulatory and 3 structural proteins• Minor regulatory proteins T’135, T’136, T’165

• Responsible for all PML• 8 types of JCV based on VP1

• Potential ↑ PML with type 2 JCV• No animal models for PML

• Astroglial and neuroectodermal tumors in hamsters and New World monkeys

• Hemagglutination of type O erythrocytes permits serological study

Saribas et al: Future Virol 2010

Seroprevalence of JCV Antibody

Brooks and Walker Neurol Clin 1984

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JCV Antibody Testing

• 2 Step ELISA Antibody Assay

• 1096 patients tested

• Prevalence 56%• Males (64.3%); Females (53.4%)

• Increase with age

• <30 42.3%

• 30-39 51.1%

• 40-49 56%

• 50-59 61.6%

• >60 69.3%

• Prior immunosuppressive Rx w/o effect

• False negative 2.5%-4.5%

• Conversion from sero+ to sero-: 3-4.7%

Gorelik et al: Ann Neurol 2010 Trampe et al: Neurology ;2012

JCV Antibody Testing

• Over 18 months, testing serostatus q 6 months • 38% of subjects remained consistently anti-JCV Ab negative

• 52% remained consistently anti-JCV Ab positive

• 10% changed serostatus

• If JCV negative at baseline • 16% became seropositive

• 31% had intermittently positive results

• Probability of consistently remaining anti-JCV Ab negative after 3 negative tests performed at 6 month intervals is 93.5%

Plavina: AAN Mar 2013; Lee et al: J Clin Virol 2013 epub

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JCV Antibody Testing • Urine and PBMC of 239 patients tested for JCV by qPCR• 67 of 239 enrolled in Stratify study

• Falsely negative JCV seronegatives had significantly lower mean copy viral numbers (2.657) than JCV seropositives (5.968) with detectable urine JCV

Berger et al: Ann Neurol 2013 Mar 22. doi: 10.1002/ana.23893. [Epub ahead of print]

Proposed Pathogenesis of PML

JCV NCCR archetype in

tonsils

Gene rearrangement

of NCCR

Kato: J Neurovirol 2004

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Multiple High Barriers for the Development of PML

No PML PML

Immunological Barriers

VirologicalBarriers

Impaired Immune Response

to JCV

Transformation of Archetype to

Neurotropic Virus

Host Factors

PML

Inter-related Factors Leading to PML

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Genesis of Neurotropic Strain

Jensen and Major: J Neurovirol 2001;7:280

Genetic Mutation in VP1 Capsid Protein

Sunyaev et al: PLOS Genetics Feb 2009, e1000368

JCV VP1 sequences from 55 PML and 253 healthy urinesSer269 codon substitution subsets seen only in PMLReduces hemagglutination properties of viral particles

Structural model of JCV VP1 – tetrasaccharide complex

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PML – A Stochastic Event

1. Initial infection (typically <20 years)2. Establishment of low level persistent or latent infection3. Conversion of archetype to prototype JCV

a) Gene rearrangement in NCCRb) Other gene rearrangements ? (point mutation in VP1)

4. JCV expression and replication in PBMC5. Entry of JCV into brain 6. Establishment of productive oligodendrocyte infection7. Failure of immunosurveillance in the brain

Predisposing Causes for PML

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Primary – no cause

Immune deficiency disorders

Carcinoma

Myeloproliferative diseases

Lymphoproliferative diseases

PML in the Pre –AIDS ERAPredisposing Illnesses

62.2%

6.5%2.2%

16.1%

7.4%

5.6%

Granulomatous/Inflammatory disorders

230 cases published and unpublished cases (1958-1984)69 pathologically confirmed and 40 virologically and pathologically confirmed

Brooks and Walker Neurol Clin 1984

Immunologically healthy persons

•9,800,000 cancer survivors

•2,100,000 RA treated with immunotherapy

•200,000 organ transplants

•>100,000 bone marrow transplantsHIV/AIDS

Abnormal CMI

1,200,000 HIV Infected

Until recently HIV-PML represented 80-90% of all PML cases

- PML Cases

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July 28, 2005Volume 353 Number 4

Brief Report: Progressive Multifocal Leukoencephalopathy after Natalizumab Therapy for Crohn's Disease

G. Van Assche and Others

Brief Report: Progressive Multifocal Leukoencephalopathy Complicating Treatment with Natalizumab and Interferon Beta-1a for Multiple Sclerosis

B.K. Kleinschmidt-DeMasters and K. L. Tyler

Brief Report: Progressive Multifocal Leukoencephalopathy in a Patient Treated with Natalizumab

A. Langer-Gould and Others

Progressive Multifocal Leukoencephalopathy and Natalizumab — Unforeseen ConsequencesJ. R. Berger and I. J. Koralnik

Patients at RiskJ. M. Drazen

PML Risk with Natalizumab

• 541 cases (538 MS and 3 Crohn’s) as of March 3, 2015

• Duration of natalizumab prior to PML ranged from 8 to 92 months as of May 2014

• Mean duration was 42 0. months

• 15% had 8-24 doses and 85% > 24 doses

• Overall incidence 3.87/1000 (95% C.I. 3.55-4.21/1000)

• Factors increasing risk include • JCV exposure

• prior immunosuppressive therapy

• longer treatment duration

• 30/372 patients (8.1%) asymptomatic at time of diagnosis

Biogen US Tysabri Update April 2015

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Estimates of PML Incidence by Treatment Epoch

Biogen US Tysabri Update April 2015

Estimates Global PML Risk by Treatment Epoch

Biogen US Tysabri Update April 2015

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Blood CNS

Natalizumab Primary MOA

Blocking the VLA4 receptor, prevents T cell entry into CNS

AdhesionXT cell

LFA-1

AVLA4 or 41 integrin

Natalizumab: VLA4 or 41 integrin antibody

End

othe

lial

Lin

ing

ICAM-1

Natalizumab

• In brain, prevents entry of 80% of T cells• B cells and other mononuclear cells• PMNs, e.g., basophils, to a lesser degree

• Entry of these cells into other tissues is not as complete

• Time after administration: • 1 mos: 80% of binding sites remain occupied [Rudick 2004]

• 3 mos: biological activity for MS wanes [Tubridy 1999]

• 6 mos: alteration in CSF lymphocytes [Stuve 2005]

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Impairment of CNS Immunosurveillance

Hematopoietic Progenitor Cells and B Cells in Bone Marrow

Major NEJM 2009 after Von Andrian and Englehart

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High frequency of JCV laden CD34 and CD19 with natalizumab

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• 13 of 26 MS patients (50%) had detectable JCV DNA in at least 1 cell compartment at ≥1 time points

• 10 of 23 patients (44%) receiving treatment for >24 months also had detectable JCV DNA in 1 or more cell compartment

• 15 of 49 MS patients (31%) harbored JCV in CD34+ cells, and 12 of 49 (24%) in CD19+ cells

• 9 patients viremic but were seronegative for JCV antibodies

Frohman et al. JAMA Neurol, March 24, 2014

Natalizumab mobilizes CD34+ cells in bone marrow

JCV-infected CD34+ cells enter the peripheral circulation, then cross the blood-brain barrier into the CNS to cause PML

Drugs reported with PMLTreatment Drug(s)1

Oral glucocorticoids All

Alkylating agents• Cyclophosphamide • Camstine

• Dacarbazine (DTIC-Dome®)

Purine analogs• Fludarabine (Fludara®)• Cladribine (Leustat®)

• Azathioprine (Imuran®)• Nelarabine (Arranon®)

Antimetabolite • Methotrexate (Trexall™)

Monoclonal antibodies

• Rituximab (Rituxan®)• Infliximab (Remicade®)• Natalizumab (Tysabri)• Basiliximab (Simutect®)• Belimumab (Benlysta®)

• Daclizumab (Zenapax®)• Muromonab-cd3• Efalizumab (Raptiva®)• Alemtuzumab

(Campath®)

Fusion Proteins • Etanercept (Enbrel®)• Belatacept

• Brentuximab vedotin

Immunosuppressants • Cydosporin• Cyclosporine

(Sandimmune®)4

• Tacrolimus (Prograf®)

• Sirolimus• Mycophenolate

(CellCept®)

ImmunomodulatoryAgents •Dimethyl fumarate

• Fingolimod

Modified from B. Kieseier and D. Leppert

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PML with Therapeutic AgentsTherapeutic Agent Underlying condition Latency from

initiation to PMLFrequency of PML development

CLASS I No risk of PML Long High

Natalizumab MS and Crohn’s disease >8 months with peak at 24 months

1/1000 to 24 months to >1/100 with JCV Ab+, prior

IS, duration

Efalizumab Psoriasis >3 years 3/166 at >3 years

CLASS II Risk of PML No latency Infrequent

Rituximab LPD, RA, SLE, AIDS None 1:30,000

Mycophenolate mofetil Solid organ transplant, SLE, other autoimmune disease

None ?

Brentuximab vedotin Hodgkin’s disease None ?

Alemtuzumab Lymphoproliferativedisorders

None ?(Rare)

Modified from Zaheer, Berger: Therap Adv Drug Safety 2012

PML and Fumarate• Dimethyl fumarate used in treatment of psorasis since

1950s

• Approved by FDA for MS on March 27, 2013

• Lipophilic molecule that is immunosuppressive but precise MOA is unknown

• Three sources of cases of PML• FUMADERM database ( 2011-1993 from Germany has (6 cases (1

removed and double reporting may account for 2 cases)

• NEJM from April 25, 2013

• 74 yr old man with 3 yrs of fumaderm and prior IS and corticosteroid Rx

• 42 yr woman with 5 years of fumaderm and no prior immunosuppressive Rx or underlying illnesses

• NEJM from April 9, 2015

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PML with Lymphopenia and DMF• 54 year old woman • MS diagnosed in 1996

• Treated with glatiramer acetate

• Enrolled in ENDORSE study • Severe prolonged lymphopenia over 3.5 years

• Lymphopenia within 12 months of DMF start• Lymphocyte counts 290-580 cells/µL

• Developed severe gait disorder, speech abnormality, and left sided incoordination

• PML diagnosis based on MRI and CSF JCV PCR+ • Death October 2014 after 54 months of DMF

Rosenkranz et al, NEJM 2015;372:1476

PML with Lymphopenia and DMF

Rosenkranz et al, NEJM 2015;372:1476

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PML without Lymphopenia and DMF

• 64 year old woman with psoriasis• On compounded delayed release DMF since June

2012• Lymphocyte and WBC nadirs – 792 and 4000

cells/cu mm (subsets not reported)• Developed progressive apraxia → somnolence

and hemiparesis

• August 2014 - PML diagnosed by CSF, MRI and brain biopsy

Nieuwkamp et al, NEJM 2015;372:1474

PML without Lymphopenia and DMF

Nieuwkamp et al, NEJM 2015;372:1474

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PML Cases in MS Patients Switching from Natalizumab to Fingolimod

• Fingolimod treatment is not increasing the risk of PML• The 11 reported cases were attributable to natalizumab

No PML cases were reported in FTY-treated pts in the absence of previous NTZ exposure

5 cases prior to 1st

FTY dose

4 cases with time to onset 2 wks-6mos; unknown in 2 cases

1 reported PML without prior NTZ (consistent with NMO)

Putzki et al. ACTRIMS/ECTRIMS 2014; abstract FC3.1.

PML with Fingolimod (#1)• Patient with complicated and atypical c/w NMOSD• Patient received 1 month of IFN β1b and azathioprine for

1 month • Also received nearly every month steroids before and

after fingolimod• Fingolimod thereafter for 8 months• PML diagnosed based on MRI and CSF low copy #s of

JCV • Subsequent review suggested that patient had NMO• MRI most consistent with NMO rather than PML

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PML with Fingolimod (#2)• 49 year old man with MS sxs from Oct 2009• Dx’d with RRMS Nov 2009 and started on IFN-β1a SQ• IFN-β1a sq dc’d Sept 2010; Gilenya initiated Oct 5, 2010• Last MRI was 2009; 1st repeat MRI on Jan 23, 2015 showed 2 new

large T2 lesions (right cerebellar hemisphere and left juxtacorticaltemporal region) with slight DWI+ and no CE

• CSF Jan 27, 2015: 43 copies of JCV and increased JCV IgG in CSF (JCV Ab specificity index 23 with normal of <1.5)

• Lymphs 240-890 cells/µL• Repeat MRI 5 weeks after fingolimod dc’d shows lesion larger and

no CE• Repeat CSF JCV 12 copies/mL (NIH)• Patient remains asymptomatic

PML and Rituximab• Rituximab carries a “Black Box” warning• About 80 cases of PML with Rituximab

• Almost all are CLL or other lymphoproliferative disease • Incidence 1:/30,000 in LPD population

• All received chemotherapy and/or stem cell transplant

• Other illnesses SLE (14); RA (8); ANCA+ vasculitis (1); cryoglobulinemia (1); ITP (1)

• All but ITP at risk for PML

• All received other immunosuppressive therapy

• No cases of MS or other neurological disease

Nived: Lupus 2008 ;17:1036 Carson et al: Blood ;2009 ;113:4834 ; http://www.fda.gov/bbs/topics/NEWS2006/NEW01432.html.l; W. Rigby personal communication

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PML and Rituximab

• Median previous or concomitant therapies was 4 months (1-14 months)

• Median time from rituximab initiation to PML was 16months (range 1-90 months)

• Median time from rituximab last dose to PML was 5.5months (range 0.3-66 months)

• Median time to death after PML diagnosis was 2.0months

• 90% case fatality

• FDA includes black box warning on PML in Dec 2006

Nived: Lupus 2008; Carson et al: Blood 2009;113:4834; http://www.fda.gov/bbs/topics/NEWS2006/NEW01432.html.l; W. Rigby personal communication

July 2009: 42 woman dx with MS and natalizumab initiatedDec 2011: JCV seropositive for 1st timeFeb 2012: cranial MRI without evidence of PMLNov 2012: diplopia attributed to breakthrough MS; natalizumab dc’dJan 2013: rituximab initiated; CD19 and 20 remains 0 for next 15 monthsMar 2013: worsening neurological symptoms; PML-IRIS dx’d; CSF JCV 630Dec 2013: no JCV DNA in CSF with clinical improvementDec 2014: independent in most ADLs

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PML and Alemtuzumab

• Anti-CD52 Ab expressed on 95% of B and T cells

• Depletes both B and T cells• B cells recover in 27 months

• CD4 T cells recover in 61 months

• CD8 T cells recover in 30 months

• Indications: Organ transplantation, LPD and MS

• Handful of cases reported to date • 5 5 in CLL (at least 3 with fludarabine as well)

• 2 following lung transplant

D’Souza et al: Lymphoma and Myeloma 2010 Martin et al: Clin Infect Dis ;2006 Wagner et al: J Heart Lung ;2009

PML and Mycophenolate Mofetil• CellCept and Myfortic• Inhibits inosine monophosphate synthesis

• ↓ de novo synthesis of purine • ↓T cell and B cell proliferation

• PML developed in 18 patients :

• Solid organ transplant recipients ( 10(• Men between 33 and 62

• Kidney, heart and lung

• Systemic lupus erythematosus ( 7(• Women between 40 and 53

• Dermatomyositis ( 1(• 44 year old man with 2.5 years of DM• 1 of 5 cases of DM with PML

http://www.fda.gov/cder/drug/earlycomm/mycophenolate.htm; Vulliemoz et al: JNNP 77:1079;2006

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PML Cases Reported with DMDs or Biosimilars

Risk Factors for Natalizumab-associated PML

Anti-JCV Antibody Status

Negative Positive

NTZExposure NO YES

1–24 months 0.6/1,000 patients 2/1,000 patients

25-48 months

3/1,000 patients 13/1,000 patients

<1/1,000

.

Prior IS Use

49-72 months 7/1000 patients 9/1000 patients

Tysabri product Information. December 2013. http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm355780.htm.

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JCV Antibody Index Titer Correlates with PML

Plavina et al: Ann Neurol 2014

• Association of JCV Ab index and PML

• No correlation in those receiving prior IS

• High sensitivity, but low specificity as many patients not developing PML had high JCV Ab index

The Anti-JCV-Ab index may provide further information regarding PML riskAnti JCV Ab index

1-24 months 25-48 months 49-72 months

< 0.9 0.06 / 1000 0.51 / 1000 0.58 / 1000

< 1.1 0.11 / 1000 0.76 / 1000 0.98 / 1000

< 1.3 0.14 / 1000 1.06 / 1000 1.30 / 1000

< 1.5 0.17 / 1000 1.13 / 1000 1.37 / 1000

> 1.5 1.17 / 1000 8.83 / 1000 10.12 / 1000

Risk estimates for patients receiving natalizumb who have never received prior immune suppressive therapy

JCV Ab index does not appear to be predictive in those receiving prior immunosuppressive therapy

Kuesters G. American Academy of Neurology 67th annual meeting. April 18-25, 2015. Washington,DCP4.031

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Risk of PML by Treatment Duration in JCV Ab+

Natalizumab Exposure

1-24 months (99% C.I.)

25-38 months(99% C.I.)

49-72 months (99% C.I.)

PML Risk 0.6/1000 (O.42-0.88)

1/1667

5.2/1000 (4.28-6.19)

1/192

5.4/1000 (4.30-7.14)

1/185

Plavina et al: Ann Neurol 2014

L-Selectin (CD62L) Expression by CD4 Cells and PML Risk

For natalizumab associated PML, threshold of 15% exhibited specificity of 97.32% and sensitivity 100%

Schwab et al: Neurology 2013

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Diagnosis of PML

• Biopsy• characteristic histopathology• EM or immunocytochemistry for JCV

• CSF• PCR for JCV highly specific

• sensitivity varies (75% to >95%) depending on primer and methods)

• NTL-PML – 50% with <500 JCV copies/ml

• criteria must include • compatible clinical picture • typical brain MRI • absence of other illnesses

Berger, et al: Neurology, 2013

0 10 20 30 40 50 60 70

Cognitive and behavioral

Motor symptoms

Speech difficulties

Visual disturbances

Cerebellar symptoms

Sensory symptoms

Seizure

Other symptoms

No clinical symptoms

Natalizumab PML Heralding Symptoms (n = 144 patients)

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Radiographic Characteristics of PML

CT Scan• hypodense lesions• rare contrast enhancement (<10%)

MRI• increased signal on T2WI and FLAIR• Hypointense on T1WI• no mass effect (present occasionally with IRIS)• often parieto-occipital and frontal lobes • atypical locations

• cerebellum, brainstem, basal ganglia, temporal lobe• rare faint Gd enhancement (<15% AIDS associated PML; 30-40%

natalizumab associated PML)

Whiteman et al: Radiology 1992; Berger JNV 1998

CT and MRI in PML

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Comparison of T2WI and T1WI MRIs

FLAIR DWI T1 with Contrast

MRI of PML

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MRI of PML: Abnormality on Susceptibility Weighted Imaging

Miyagawa et al: J Neurol Sci 2014:344:198-202

Radiographically Isolated PML• 23 year old man with MS on natalizumab for 24 months shows new

lesion. One month later he is recognized to have inappropriate behavior on routine visit.

Langer-Gould A et al: N Engl J Med 2005;353:375-81.

October 2003 October 2004

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Pathological Criteria of PML

• Demyelination

• Bizarre astrocytes

• Enlarged oligodendroglial nuclei

Demyelination in PML

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Classic Histopathological Triad of PML

“Puffballs” of demyelination

Axonal preservation

Bizarre astrocytes

Enlarge oligodendroglial nuclei

Demonstration of JCV in Tissue

Biotinylated stain

Fluorescein label

Electron microscopy

Immunohistochemistry

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Other CNS Diseases Caused by JCV

Tan and Koralnik: Lancet Neurol 2010

Disease course

Lesions

Clinical manifestations

Diagnosis

Pathology

Treatment

Granular cell degeneration with JCV

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Common Themes to Improved Outcome • Outcome is, in large measure, predicted

by the nature of the underlying immunological defect • Reversible or irreversible

• For PML due to reversible immunosuppression, i.e., monoclonal antibodies• Early detection of PML• Immediate removal of offending agent

Potential Strategies for the Treatment of PML

• Antiviral therapies• Topoisomerase I inhibition• Cytosine arabinoside• 5HT2a receptor blockade• Inhibition of clathirin dependent endocytosis• Cyclosporin A• Interferon-α and IFN-β• Recombinant IL-2• Small interfering RNA• Antimalarials - mefloquine

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Features of PML IRIS in HIV

• Clinical worsening• MRI progression

• Extension of lesion on T2WI and FLAIR

• Contrast enhancement (may be transient)

• Brain edema

Initial MRI July 2004

Follow-up MRI Oct 2004Martinez JV et al: Neurology 2006; 67:1692-4

Intense perivascular inflammation with CD8+ cells

Acute perivenular demyelination and inflammation

Vendrely A et al: Acta Neuropath 2005; 109:449-55.

Pathology of PML-IRIS

Travis J et al: PML IRIS Neurologist 2008;14:321-6

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Recommended Treatment for PML-IRIS

• No controlled trials to date• Suggested therapies

• 1 g IVMP for 3-5 days followed by oral taper over 6-8 weeks1

• 1 g IVMP for 5 days followed by oral taper over 2 weeks2

• If symptoms during or after taper worsen, re-treatment with the same dose or IVMP 2 g for 5 days with subsequent taper

1. Johnson T and Nath A: Curr Opin Neurol 2011;24:284-90. 2. Hartung H-P, Berger JR, et al: Actuelle Neurologie 2011;38:2-11.

PML Summary

• PML is extremely rare disease due to a ubiquitous polyoma virus, JC virus

• Develops as stochastic event• Impaired cell mediated immunity, CNS

immunosurveillance and B cell activation frequently evident predisposing factors

• Results in demyelination from death of productively infected oligodendrocyte

• Reversing immunosuppression is most important treatment

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There are known knowns. These are things we know that we know. There are 

known unknowns. That is to say, there are things that we know we don't know. But there are also unknown 

unknowns. There are things we don't know we don't 

know.

Donald RumsfieldFormer U.S. Secretary of Defense