Disclosures (last 12 months) - WordPress.com · 9/17/2017 · relationship between the two. ... D-dimer Fibrinogen Ferritin Triglyceride ... Renal failure 13 (5.6) 2.5 3.6 (2.9) 0.9

1

MacrophageActivationSyndrome–KillorBeKilled

RandyQ.Cron,MD,PhD

UniversityofAlabamaatBirmingham

September22,2017UniversityofMissouri

5th AnnualJames&NancyCassidyRheumatologySymposium

Disclosures(last12months)

RandyQ.Cron,M.D.,Ph.D.

SOBI– investigatorinitiatedclinicaltrialofanakinra totreatMAS

CaseofPediatricMAS

•14yo girlwithCHPsince6mo.(dailyfeversto104,totalbodypanniculitis &lipoatrophy)•Presentswithabdominalpain&hepatomegaly,sizeof7-yearold® CsA &steroids•Onemonthlater,abdominalpain,seizure,GCSof7,CSFprotein, D-dimers,pancytopenia, ferritin, IFNg,IL-12•Multi-organsystemfailure(heart,lungs,kidney,liver,pancreas,bonemarrow,coagulopathy,CNS)

Behrens EM, Kreiger PA, Cherian S, Cron RQ. J Rheumatol. 2006 Oct;33(10):2081-4.

2

CaseofPediatricMAS- Pathology


Bone marrow

CD68 stain for skin -histiocytes

Skin biopsy at 9 mo.

CaseofPediatricMAS- Therapy


Hemophagocytic Lymphohistiocytosis(HLH)criteria

3

BoneMarrowHemophagocytosis

Filipovich

ShuttingDownanImmuneResponse

Verbsky

IneffectiveCD8killingleadstohemophagocytosisbymacrophages

Adapted from Jordan MB, et al. Blood. 2004;104:735-43

4

HLHAssociatedMutations

MPRSTX 11?

Courtesy: Dr. Alexei Grom

WhathavewelearnedfromthePrimarymodels?

• Requirementforinfection– EBVinSAP-/- mice(murineg-herpesvirus68)– LCMVinpfp -/-,MUNC-13-4-/-,Rab27a-/- mice–NeitherlisterianorMCMVinMUNC-13-4-/- mice–Animalsdeficientincytotoxicexocytosisareessentiallynormaluntilchallengedwithcertaininfections.

5

A“gradient”ofcytotoxicty determinestheseverityofHLH

Ehl

Courtesy of Dr. Ed Behrens (UPenn)

Hemophagocytic Lymphohistiocytosis (HLH)andMacrophageActivationSyndrome(MAS)

Activated MF

Hemophagocytosis

A rose thorn by any other name … Shakespeare

MAS/HLH

•Primary– Geneticmutationthatresultsindiseaseusuallywithinfirstyearoflife–Perforin–MUNC13-4–Rab27a

•Secondary– Associatedwithanunderlyingillnesspresentingatonsetofillnessoranytimeduringthechroniccourseofadisease–Rheumatologic–Infectious–Malignant

6

SystemicOnsetJuvenileRheumatoidArthritis(soJRA)

• soJRA=ACRcrtieria• 6weeksofarthritis• Age<16• Fever• LargelysupplantedbytheILARcriteriaforsystemicJuvenileIdiopathicArthritis(sJIA)–arthritis&quotidianfever–1ofthefollowing:

• Rash• HSM• LAD• Serositis

Behrens EM, Beukelman T, Cron RQ.J Rheumatol. 2007;34:234.

MASinsJIA

• Estimatedtobeacomplicationin7%ofallsJIAcases• Maytakeafulminantcourse,canbefatal• Earlydetectioncanbedifficult,particularlybecauseMASsymptomsmaybesimilartoansJIAflare

PASOJAR

• InternetbasedregistryofpatientswithsoJRAfromthelast15yearsinthestateofPennsylvania• Currentlyholdsdatafrominitialpresentationanddiseaseactivityfromlastvisit• 136patientsfrom3centersinPA• Dataanalyzedforpresentingcharacteristics

Behrens EM, et al. J Rheumatol. 2008;35:343-8.

7

LabsatPresentation

Lab Test Patients evaluated (% of total)

Abnormal Cut-off value % Above Cut-off

Aldolase 26 (19%) >9.9 Units/L 73%

Triglycerides 17 (12.5%) >200 mg/dL 18%

D-Dimers 29 (21%) >1X normal for reporting lab 83%

Ferritin 66 (48.5%) >500 ng/mL 70%

Sedimentation Rate 135 (99%) >20 mm/hr 95%

C-Reactive Protein 56 (41%) >1X normal for reporting lab 96%

Behrens EM, et al. J Rheumatol. 2008;35:343-8.

IsMASa“complication”ofsJIAorisitmoreintegraltodiseasepathogenesis?

•ThesimilarityofHLHtosJIA suggestsamoreintimaterelationshipbetweenthetwo.•WelookedatbonemarrowbiopsiesdoneaspartofthesJIA workupforevidenceof“subclinicalMAS”todeterminetheprevalenceofhemophagocytosis insJIA.

Behrens EM, Beukelman T, Paessler M, Cron RQ.J Rheumatol. 2007;34:1133-8.

TheOccultMacrophageActivationSyndromeinSystemicJuvenileIdiopathicArthritis(JIA)

BehrensCHOP

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Patient ID Cytopenia ESR CRP D-

dimer Fibrinogen Ferritin Triglyceride Initial

BM Read

BM Review

1 None 83 10.4 Normal Normal

2 None 77 28.9 1421 Normal HPC

3 Anemia 98 43.08 497 Normal HPC

4 None 96 Normal Unavailable

5 None 38 4.25 47 Normal Normal

6 Anemia 80 18.7 428 97 Normal Normal

7 Anemia 72 8.12 Normal Normal

8 None 92 4.19 6.36 98 Normal Normal

9 Anemia 50 7.7 2.39 419 Normal Normal


11 Anemia 4.3 10.22 530 51 Activated MФ

Activated MФ

12 Anemia 135 15.5 5.47 427 Frank HPC

Frank HPC

13 Anemia 56 Normal Activated MФ

14 Normal HPC

15 None 89 12.5 10.29 568 8810 207 Frank HPC

Frank HPC

16 None 90 4.6 1.00 609 Frank HPC

Frank HPC

Control 1 None 131 3.0 Normal Normal


Control 3 None 56 4.6 14.9 968 Normal Normal


13% were diagnosed clinically with MAS

2/3 patients with “frank hemophagocytosis” were not diagnosed clinically

No controls had evidence of HPC

50% had either HPC or activated MF

6/16 (37%) of patients have “Occult MAS”



BM Read

BM Review












Activated MФ

12 Anemia 135 15.5 5.47 427 Frank HPC

Frank HPC


14 Normal HPC

15 None 89 12.5 10.29 568 8810 207 Frank HPC

Frank HPC

16 None 90 4.6 1.00 609 Frank HPC

Frank HPC





Bleesing

Arthritis Rheum. 2007 Mar;56(3):965-71.

9

ConceptofOccultMASinsJIA• BehrensEM,etal. JRheumatol.2007;34:1133-8.

– 37%haveoccultMASbybonemarrowexam

• BlessingJ,etal. ArthritisRheum.2007;56:965-71.

– 31%haveoccultMASbysCD25andsCD163

– 2/5developedfulminantMAS

Grom

IL-18 >47,750 pg/ml > MASGreen = MAS

Not active

Shimizu

Clin Immunol. 2015 Oct;160(2):277-81.

arthritis

MAS

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MASDiagnosticGuidelinesforsJIA

Ravelli A, et al. J Pediatr. 2005;146:598-604.

Ravelli

HLH vs. MAS criteria• Primary HLH• Fever• Splenomegaly• Peripheral cytopenia (2 or more

lineages)– Hgb < 10.0 g/dl (<9.0 in <4 wks of age)– Platelets <100E9/L– Neutrophils <1.0E9/L

• Hypertriglyceridemia or hypofibrinogenemia– Triglycerides >265 mg/dL– Fibrinogen <150 mg/dL

• Hemophagocytosis– Bone marrow, spleen, LN, CSF

• Low or absent NK cell activity• Ferritin >500 mg/L• Soluble CD25 >2,400 U/ml

• HLH = 5 or more criteria OR molecular diagnosis c/w HLH

• (Pediatr Blood Cancer. 2007;48:124-31.)

• sJIA MAS• Labs

– Platelets < 262E9/L– AST > 59 U/L– WBC < 4.0E9/L– Fibrinogen < 2.5 g/L

• Clinical– CNS dysfunction (irritable, seizure,

coma, lethargy, disorientation)– Hemorrhages (purpura, easy

bruising, mucosal bleeding)– Hepatomegaly (>3 cm below costal

arch)

• MAS = ≥2 lab OR 2 or 3 clinical and/or lab (bone marrow showing hemophagocytosis only when in doubt)

• (J Pediatr. 2005;146:598-604.)

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ANINTERNATIONALCONSENSUSSURVEYOFDIAGNOSTICCRITERIAFORMACROPHAGEACTIVATIONSYNDROMEINSYSTEMICJUVENILEIDIOPATHIC

ARTHRITIS(JRheumatol.2011Apr;38(4):764-8)SergioDavì,MD,AlessandroConsolaro,MD,DinaraGuseinova,MD,AngelaPistorio,MD,PhD,AlbertoMartini,

MD,RandyQ.Cron,MD,PhD,andAngeloRavelli,MD

•Table1.Frequencyofselectionofthe28MASfeaturesbythe232questionnairerespondents,medianandmeanranksoffeatures,andpercentageofrespondentsattributinghighranktoeachfeature.

Feature

No. (%) of respondents who selected the

feature Median rank Mean (SD) rank

% of respondents giving rank 8-

10 to the feature

% of respondents giving rank 5-

10 to the feature

Falling platelet count 201 (86.6) 6.5 6.1 (2.3) 24.6 61.6

Hyperferritinemia 194 (83.6) 7 6.5 (3.0) 39.2 53.9

Bone marrow hemophagocytosis 188 (81.0) 9 6.9 (3.6) 44.8 55.2

Increased liver enzymes 174 (75) 5 5.0 (2.4) 13 40.9

Falling leukocyte count 172 (74.1) 5 5.6 (2.5) 20.3 46

Persistent continuous fever ≥ 38°C 158 (68.1) 7 6.0 (3.4) 30.2 40.1

Falling erythrocite sedimentation rate 142 (61.2) 6 5.5 (2.7) 15.9 38.8

Hypofibrinogenemia 142 (61.2) 5 5.4 (2.4) 12.9 36.6

Hypertrigliceridemia 135 (58.2) 5 5.1 (2.7) 16.8 31

Central nervous system dysfunction 104 (44.8) 5 5.0 (2.9) 11.6 23.7

Falling hemoglobin level 100 (43.1) 5 4.8 (2.3) 4.3 23.3

Prolongation of clotting times 81 (34.9) 4.5 4.5 (2.3) 4.7 17.2

Increased D-dimer 76 (32.8) 5 5 (2.6) 7.3 19.4

Hemorragic manifestations 72 (31.0) 5 5.3 (3.0) 10 17.7

Liver enlargement 71 (30.6) 4 4.8 (2.8) 7.3 14.2

Spleen enlargement 57 (24.6) 4 4.5 (2.8) 4.3 10.3

Increased lactic dehydrogenase 45 (19.4) 6 5.5 (2.6) 4.7 11.6

Increased soluble IL-2 receptor α 39 (16.8) 6 5.1 (3.1) 4.7 9.1

Increased soluble CD163 27 (11.6) 5 5.2 (3.0) 4.3 6

Lymphoadenopathy 22 (9.5) 4 4.4 (2.9) 1.3 3.4

Decreased albumin 19 (8.2) 3 4.3 (2.9) 1.3 2.6

Hyponatremia 16 (6.9) 5 5 (3.2) 1.7 3.9

Arthritis improvement 14 (6.0) 2 3.1 (2.4) 0.9 0.9

Renal failure 13 (5.6) 2.5 3.6 (2.9) 0.9 1.7

Jaundice 9 (3.9) 5 5.9 (3.3) 1.7 2.2

Increased bilirubin 9 (3.9) 2 3.4 (2.0) 0 1.7

Respiratory failure 6 (2.6) 3 4.4 (3.0) 0.4 0.9

Cardiac failure 5 (2.2) 3 3.8 (2.6) 0 0.9

Datacollection– Finalresults

NsJIAwithMAS 362ActivesJIAwithoutMAS 404Acutefebrileconditions 346

Investigatorswhoincludedpatients 95Countriesinvolved 33

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Clinical,laboratoryandhistopathologic characteristics,currenttreatmentandoutcomeofmacrophageactivationsyndromecomplicatingsystemicjuvenile

idiopathicarthritis:amultinational,multicenterstudyof362patients

No of available data N (%)

Fever 355 341 (96.1)

Hepatomegaly 350 245 (70)Splenomegaly 347 201 (57.9)Lymphadenopathy 346 178 (51.4)

Active arthritis 354 230 (65)CNS involvement

Lethargy SeizuresIrritabilityConfusionHeadacheMood changesComaOther

349 122 (35)35 (10)32 (9.1)17 (4.9)13 (3.7)11 (3.1)

7 (2)6 (1.7)30 (8.6)

Heart involvementPericarditisPericardial effusionArrhythmiaHeart failureCardiomegalyOther

353 90 (25.5)31 (8.8)26 (7.3)5 (1.4)4 (1.1)4 (1.1)20 (5.6)

Minoia F, Davi S, Horne A, et al. Arthritis Rheumatol. 2014;66(11):3160-9.

Horne, Minoia, Davi

Minoia F, et al. J Rheumatol. 2015;42:994-1001.

InternationalconsensusconferenceonMASclassificationcriteria

Villa Quartara della Castagna, Genoa, Italy, 21-22 March 2014

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Finaldefinition(>80%consensus)– selectedfrom982computermodeled

Afebrile patientwithknownorsuspectedsJIAis classified as havingMASif thepatient has:

Ferritin>684ng/L

ANDatleast2ofthefollowing4laboratoryvariables:

• Platelets≤181x109/mL• AST>48U/L• Triglycerides>156mg/dL• Fibrinogen≤360mg/mL

Ravelli A, et al. Arthritis Rheumatol. 2016;68:566-76.Ravelli A, et al. Annals Rheum Dis. 2016;75:481-9.

Conference Participants

Horne

14

1

Arthritis Care Res (Hoboken). 2017 May 12. doi: 10.1002/acr.23277. [Epub ahead of print]

Schulert

Hirsch

The ferritin to ESR ratio is a simple measure to distinguish macrophage activation syndrome from systemic arthritis flare

Eloseily, E.M.A., Minoia, F., Beukelman, T., Ravelli, A., and Cron, R.Q(upcoming talk at the American College of Rheumatology Annual

Scientific Meeting, San Diego, CA, November 6, 2017)

MAS vs sJIA flare MAS vs infection

Eloseily

2

CD163Haptoglobinreceptor

•MorespecificmarkerforMF thanCD68•Abletohighlight“overstuffed”MF thatmightbemissedonroutinestaining

3/4 = dead

Unknown MAS

Mono. Leishmania

?? CMV

EBV

CD163higherpowerimages

Initial read - Frank HPC Initial read - NormalInitial read - NormalClinical dx of MAS

Normal

Behrens E, Beukelman T, Paessler M, Cron RQ.

J Rheumatol. 2007;34:1133-8.

3



BM Read

BM Review












Activated MФ

12 Anemia 135 15.5 5.47 427 Frank HPC

Frank HPC


14 Normal HPC

15 None 89 12.5 10.29 568 8810 207 Frank HPC

Frank HPC

16 None 90 4.6 1.00 609 Frank HPC

Frank HPC





CD163 staining•4+/HPC

•2+

•1+

Paessler

FerritinasaScreenforMAS

.

High Sensitivity & Specificity of Elevated Ferritin for HLH Diagnosis

Allen CE, et al.

4

Grom

MUNC13-4

• MUNC13-4genesequencedinadditional16MAS/SJIApatients–Asetof12SNPs(inheritedasahaplotype)in9ofthe16(57%)MASpatients,allCaucasian

– Presentin27/229controls(12%)–AlsoincreasedfrequencyinReHLHw/ounderlyinginflammatorydisease

5

Wulffraat

Pt Age Sex Diagnosis Trigger Gene Mutation AminoAcidChange

PriorAssociation

Outcome

1 15 M leukemia ? UNC13D 2896C>T R966W Pt.6only Died2 1.5 M uveitis ? UNC13D 847A>G I283V 4%AA Alive3 18 F uveitis ? RAB27A 259G>C A87P Griscelli-2 Alive4 9 F ? ? PRF1 272C>T A91V 3%USA Alive5 16 F gastroparesis CMV STXBP2 1298C>T A433V none Died6 12 M systemicJIA HSV1 UNC13D 2896C>T R966W Pt.1only Alive7 7 M polyarteritis ? STXBP2 1782G>A 3’UTR 6others Alive8 1.5 F systemicJIA ? UNC13D 2368C>T splicesite none Alive9 3 M ?immune

deficiency? UNC13D

PRF1869C>T272C>T

S290LA91V

oneprior3%USA

Alive

10 1 F chronicHepB HepB PRF1 272C>T A91V 3%USA Alive11 17 M ? EBV STXPB2 822G>A A274A none Died12 8 M ? HHV6 STXBP2

STXBP2STXBP2STXBP2

1782G>A1782G>A1026C>G1357G>A

3’UTR3’UTRnon-codingnon-coding

nonenonenonenone

Died

13 14 F Sjogren ? STXBP2STXBP2

1590G>A-37delete

splicesite5’UTR

3otherslinked

Alive

14 1.5 M systemicJIA ? UNC13D 610A>G M204V seeninHLH Alive

15 1.5 F systemicJIA ? PRF1 272C>T A91V 3%USA Alive

HLH Genetics of Recent MAS Patients at Children’s Hospital of Alabama

OurHypothesis

• Theheterozygousmutationsfoundincytolytic pathwaygenes(e.g.RAB27A,PRF1,STXBP2,MUNC13-4)inMASpatientsmaycausefunctionalimpairmentsofcytolytic/naturalkiller(NK)cellsviaadominantnegativeeffectand,therefore,contributetoMAS.

6

Approach

1. Make cDNA by reverse transcription from the human NK-92 cell line total RNA;

2. Generate patient mutation by site-directed mutagenesis of wild-type cDNA;

3. Generate lentiviral expression vectors;

4. Transduce human NK-92 cell line to express wild-type and mutant genes;

5. Measure transduced NK cell functions: cytolysis of K562 (NK target cell), CD107a mobilization/cell surface expression, IFNgexpression, granzyme B polarization during immunologic/cytolytic synapse formation (by confocal microscopy), & interaction with other cytolytic pathway proteins by PLA and co-immunoprecipitation.

Zhang Funded by Kaul Pediatric Research Institute

(STXBP2)

7

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

1.11

66.5

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

12.3MFI = 2994

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

2.21

51.8

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

18MFI = 2797

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

4.17

33.9

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

23.2MFI = 2651

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

7.1

18.4

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

26.8MFI = 2625

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

1.56

48.4

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

28.4MFI = 2489

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

2.14

45

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

28MFI = 2527

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

4.81

27.8

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>31.9

MFI = 2524

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<GFP

-A>

9.08

15

0 102 103 104 105<eFLUOR450-A>

0102

103

104

105

<7AA

D-A

>

35.1MFI = 2629

STXBP2 mSTXBP2

0.5:1

1:1

2:1

4:1

E:T

STXBP2 (C1298T, A433V) mutation also leads to decreased NK-92 cell cytotoxicity

Zhang M, Behrens EM, Atkinson TP, Shakoory, B, Grom, AA, Cron RQ. Curr. Rheumatol. Rep. 2014;16:439-446.

Cytotoxicity assays: 1. Labeling K562

cells;2. Mixing NK-92

effectors and K562 targets at different E:T ratios;

3. Incubating 5 hours;

4. Measuring cell death by 7AAD.

Blood. 2015;125:1566..

Giraudo

(STXBP2)

8

MASPatientPresentation•18-year-oldwhitefemalepresentedwith2weeksoffever (>102oF)andabdominalpain.•Examrevealedfebrile,semi-coherentfemalewithhepatosplenomegaly.• Labfindingsincluded:pancytopenia,severehepatitis,coagulopathy,ferritin8,446ng/ml,triglycerides,sCD25,sCD163,CD163stainingofbonemarrow,¯¯NKcellfunction,andESRof10mm/hr.

ResponsetoImmunosuppression

•Rxwithcyclosporin,•highdosecorticosteroids•andanakinra.

Zhang M, Bracaglia C, Prencipe G, Bemrich-Stolz CJ, Beukelman T, Dimmitt RA, Chatham WW, Zhang K, Li H, Walter MR, De Benedetti F, Grom AA, Cron RQ.J Immunol. 2016;196:2492-503.

Rab27aA87PmutationdecreasesNKcelllyticactivity,whichisexacerbatedinthe

presenceofIL-6

Zhang M, Bracaglia C, Prencipe G, Bemrich-Stolz CJ, Beukelman T, Dimmitt RA, Chatham WW, Zhang K, Li H, Walter MR, De Benedetti F, Grom AA, Cron RQ.J Immunol. 2016;196:2492-503.

9

RAB27A mutation decreases expression of CD107a on NK-92 cells

0

50K

100K

150K

200K

250K

SS

C-A

0.67

0

50K

100K

150K

200K

250K

SS

C-A

26.9

0

50K

100K

150K

200K

250K

SS

C-A

39.9

0

50K

100K

150K

200K

250K

SS

C-A

44.2

0

50K

100K

150K

200K

250K

SS

C-A

22.7

0 102 103 104 1050

50K

100K

150K

200K

250K

SS

C-A

4.81

0.83

23.6

32.6

31.7

13.4

0 102 103 104 105

2.79

0.75

22.3

27.2

21.4

9.6

0 102 103 104 105

1.67

0.61

23.4

32

24.2

8.97

0 102 103 104 105

1.58

0.83

22.9

27.8

18.9

6.88

0 102 103 104 105

1.1

0.68

20.6

28.2

19.9

6.52

0 102 103 104 105

1.18

CD107a

Med

0.5h

1h

2h

4h

8h

RAB27A mRAB27A PRF1 mPRF1 STXBP2 mSTXBP2RAB27a mutant Zhang M, et al. J Immunol.

Rab27aA87PmutationpredictsdisruptionofbindingwithWTMunc13-4

Zhang M, et al. J Immunol. 2016;196:2492-503..

DecreasedBindingtoMUNC13-4byCo-Immunoprecipitation


10

DecreasedBindingtoMUNC13-4

http://www.sigmaaldrich.com

Proximity ligation assay


RAB27A mutation leads to decreased NK-92 cell granzyme B polarization to the cytolytic synapse with K562 target cells

WT RAB27A transduced NK-92 mRAB27A transduced NK-92

Zhang M, Bracaglia C, Prencipe G, Bemrich-Stolz CJ, Beukelman T, Dimmitt RA, Chatham WW, Zhang K, Li H, Walter MR, De Benedetti F, Grom AA, Cron RQ.J Immunol. 2016;196:2492-503..


Better definition of the plasma membrane demonstrating RAB27A mutation leading to decreased NK-92 cell granzymeB polarization to the cytolytic synapse with K562 target cells

11

DelayedPolarizationofCytolytic Granules


DelayedPolarizationofCytolytic GranulesTimeCourse


J Exp Med. 2015;212:307 Jenkins

QVD inhibits caspases required for target cell death

Increased synapse dwell time results in cytokine production

12

IncreasedIFNg ExpressionbyRab27aA87PMutantNKCells


Rab27aA87PinanotherpatientdecreasesNKcelldegranulation&cytolytic activity


Conclusion

• Theheterozygous(A87P)RAB27AmutationlikelycontributestodecreasedNKcellactivityandsubsequentclinicalMASinthepatientsbearingthemutationviaapartialdominantnegativeeffect.

13

ANovelUNC13D Intronic VariantDisruptingaNFkBEnhancerinaPatientwithRecurrentMASandsJIA

GrantSchulert,MingceZhang,AmmarHusami,NdateFall,HermineBrunner,KejianZhang,RandyCron,&

AlexeiGromsubmitted

TheNFkBsiteintheUNC13D 1stintronfunctionsasatranscriptionalenhancer,andthepatientmutationrepressestranscriptionalactivity.

14

De Benedetti

1

Extentoftheproblem?

HLH/MAS

MODSSIRSCulture negativesepsis

Fever & ferritin

Carcillo

Harms

GenesAssociatedwithHLH/MAS

Cron RQ, et al. Expert Rev Clin Immunol. 2015;11:1043-53.

2

Sample Gene Variant Identifier Variant type

PredictionSIFT/Polyphen

1000 Genomes MAF

ExAC MAF

Conservation (phyloP)

7,11

PRF1 c.272C>T(p.A91V) rs35947132 Missense Damaging/Probably damaging

0.013 0.031 3.6

4 LYST c.8913T>G(p.N2971K) rs34702903 Missense Tolerated/Possibly damaging

0.0006 0.002 0.77

1,11,14

LYST c.5945C>T(p.T1982I) rs146591126 Missense Tolerated/Benign 0.0016 0.006 3.03

7 LYST c.11086G>A(p.V3696I) rs147221131 Missense Tolerated/Benign 0.0002 0.0008 2.87

Two have PRF1 A91V mutations and 5 have LYSTmutations of the 14 analyzed (36%). Schulert

Schulert G, et al. J Infect Dis. 2016;213:1180-8.

PRF1(C272T,A91V)mutationalsodecreasesNK-92cellcytotoxicity

Schulert G, et al. J Infect Dis. 2016;213:1180-8.

3

MASandhemorrhagicfevers:EbolaVirus

Markers of inflammation associated with a fatal outcome (black bars) or hemorrhagic manifestations (red bars). *P ≤ .05.

McElroy AK, et al. J Infect Dis. 2014;210:558-566.

McElroy

FeaturesofMAS/HLHinHemorrhagicFeverVirusDisease

Cron RQ, Behrens EM, Shakoory B, Ramanan AV, Chatham WW. Does ViralHemorrhagic Fever Represent Reactive Hemophagocytic Syndrome?J. Rheumatol. 2015;42:1078-1080.

HLH-04 Criteria Features of VHF

Fever routinely

Splenomegaly sometimes

Bicytopenia or pancytopenia frequently

Hypertriglyceridemia or hypofibrinogenemia both have been reported for

some hemorrhagic fever viruses

Hemophagocytosis on biopsy yes, for the viruses studied

Hyperferritinemia (>500 ng/ml) yes, often >10,000 ng/ml

Elevated soluble CD25 yes, for the viruses studied

Absent of decreased natural killer cell function poorly studied to date

Chatham

CommontriggersofMAS&MODS

4

High Prevalence of MODSin the PICU

Pediatric MODS has a High Mortality Rate

MASRxOutline

• Highdosecorticosteroids• Cyclosporin A• Cyclophosphamide• ReviewofHLH-94&HLH-2004• Plasmaexchange• Biologictherapies:

–IVIg–ATG–Anti-CD20–TNFinhibition–IL-1antagonism–IL-6RmAb–Others

5

PulseSolumedrolRxforMAS

• Hadchouel M,Prieur AM,Griscelli C. Acutehemorrhagic,hepatic,andneurologicmanifestationsinjuvenilerheumatoidarthritis:possiblerelationshiptodrugsorinfection.JPediatr.1985;106:561-6.

• Syndromeofhemorrhagic,neurologic,hepatic,hematologic,andmetabolicmanifestations– distinctfromReyesyndrome.

• FallingESRandplateletcountandfibrinogenlevels

• 7JIA(6systemic)childrentreatedwithhighdosesteroids

• 2deaths

“Any sufficiently advanced technology is indistinguishable from magic.” - Arthur C. Clarke

24 patients with MAS (18 sJIA, 2 poly JIA, 2 SLE, 2 unclassified)High spiking fevers: 24 Hepatosplenomegaly: 14Hemorrhagic diathesis: 6 Pulmonary involvement: 12

Neurologic involvement (coma or seizures): 12 HLH on bone marrow: 14/17

Treatment (29 episodes) – 2 fatalitiesIVIg failed in 4/4 pulse steroids 1st line rx success: 15/21CsA was rapidly effective all who failed steroids and in 5 patients as 1st line rx

CyclosporinAtoTreatMASinJIA

Stéphan JL,etal. Rheumatology(Oxford).2001;40:1285-92.

Prieur

HLH-2004Protocol

6

“Success”ofHLH-94[VP-16,CsA,Dexamethasone,BMT]

Blood. 2002;100:2367-73.

Henter

IVIgforMAS

•MaybebetterforinfectionassociatedMAS–HotA,MadouxMH,Viard JP,Coppéré B,Ninet J. Successfultreatmentofcytomegalovirus-associatedhemophagocytic syndromebyintravenousimmunoglobulins.AmJHematol.2008;83:159-62.[CMV]

–AsciG,Toz H,OzkahyaM,Cagirgan S,Duman S,Sezis S,OkE. High-doseimmunoglobulintherapyinrenaltransplantrecipientswithhemophagocytichistiocyticsyndrome.JNephrol.2006;19:322-6.[multipledifferentinfections]

– Larroche C,Bruneel F,AndréMH,Bader-Meunier B,Baruchel A,Tribout B,Genereau T,Zunic P;Comité d'Evaluation etdeDiffusiondesInnovationTechnologiques (CEDIT).[Intravenouslyadministeredgamma-globulinsinreactivehemaphagocytic syndrome.Multicenterstudytoassesstheirimportance,bytheimmunoglobulinsgroupofexpertsofCEDIToftheAP-HP]AnnMedInterne(Paris). 2000;151:533-539.[variousviralinfections]

– ChenRL,LinKH,LinDT,SuIJ,HuangLM,LeePI,Hseih KH,LinKS,LeeCY.Immunomodulationtreatmentforchildhoodvirus-associatedhaemophagocyticlymphohistiocytosis.BrJHaematol.1995;89:282-90.[EBVandHHV-6]

Anti-thymocyteGlobulinforMAS

Fischer

7

PlasmaExchangeforMAS

Anti-CD20forMAS

Sano T, Sakai H, Takimoto K, Ohno H.Rituximab alone was effective for the treatment of a diffuse large B-cell lymphoma associated with hemophagocytic syndrome. IntJ Clin Oncol. 2007;12:59-62.

Balamuth NJ, Nichols KE, Paessler M, Teachey DT.Use of rituximab in conjunction with immunosuppressive chemotherapy as a novel therapy for Epstein Barr virus-associated hemophagocytic lymphohistiocytosis. J PediatrHematol Oncol. 2007;29:569-73.

Nichols

ProposedPathophysiologyofMAS

Jordan Behrens

8

TNFinhibition&MAS

• CASEREPORTS

•MASfollowingetanercept therapy:– JRheumatol.2007;34:241-2– JClin Rheumatol.2001;7:252-6.– JRheumatol.2003;30:401-3.

•MASresolvedbyetanercept therapy:– Pediatr BloodCancer.2008;50:419-21.–ModRheumatol.2008;18:72-5.–AmJHematol.2006;81:59-61.[infliximab]– JRheumatol.2001;28:2120-4.– SwissMedWkly.2002;132:414-22. Prahalad

Successful treatment of severe pediatric rheumatic disease associated macrophage activation syndrome with IL-1 inhibition following conventional immunosuppressive

therapy: case series with 12 patients.

Miettunen PM, Aru N, Jayanthan A, Behrens EM, Cron RQ.Rheumatology (Oxford). 2011 Feb;50(2):417-9.

Miettunen

IL-1ImbalanceinMAS

IL-1b

IL-1b (red) complexed with IL-1Ra

Anakinra (rhIL-1Ra)

9

ProposaltoTreatEbolaHemorrhagicFeverwithIL-1Blockade

Chatham WW, Cron RQ. The Rheumatologist. 8(12):1, 21-23.

Opal Dinarello Shakoory

Crit Care Med. 2016 44:275-81.

Anakinra forMASClinicalTrialatUAB/CoA

• ClinicalTrials.govIdentifier:• NCT02780583

OfficialTitle: RandomizedPlaceboControlledTrialofSubcutaneousrhIL-1Ra(Anakinra)intheManagementofHospitalizedPediatricandAdultPatientsWithMacrophageActivationSyndrome

Chatham

10

First4patientsamplesforWGS

33 yo WM with fevers, pancytopenia, AST/ALT elevations, AKI, very high ferritin (108,000) coagulopathy (d-dimer >20,000). No identified infectious/viral triggers. Screen fail for protocol due to AKI. Probable AOSD. Complete response to pulse steroids, CsA, anakinra--off steroids, off CsA, now on q.o.d. anakinra without relapse.

24 yo AA female with SLE presented with flare of rash, arthritis, altered mental status, pancytopenia, LFT elevations, coagulopathy, very high ferritin. No infectious trigger. Better with pulse steroids, anakinra, brief course of CsA x 2 weeks, Now back on SoC SLE meds, no relapse off CsA/off anakinra.

21 yo AA female arthralgias, malar rash, alopecia and oral ulcers March 2017. Diagnosed SLE(positive ANA, anti-SSA Ab, anti-Smith Ab at that time) and started on pred 60mg, Plaquenil 200mg in addition to levothyroxine for hypothyroidism. As steroids tapered developed headache, fever, aseptic meningitis CSF: (40 WBCs, 61% segs, 39% lymphs/monos, protein 89, glucose 39); blood CSF cultures negative. Despite 80 mg/d Solumedrol, HCQ has ongoing fever, progressive pancytopenia (1.86/24/85k), increased ferritin (2,600), coagulopathy w/ high d-dimer (8557), elevated LDH (841) elevated TG (321), elevated CRP (50).

9 yo AA male with 2 weeks of fever, cervical LN, malar rash, hepatomegaly, and parotitis. Diagnosed with SLE (+ANA, +Sm, +dsDNA, low complements, ferritin 13K) and MAS (pancytopenia, coagulopathy, elevated transaminases, altered mental status). Started on clinical trial. Doing well as an outpatient, currently on mycophenolate mofetil and tapering corticosteroids.

PotentialPathogenicVariants

1. All 4 patients have at least one heterozygous (mostly missense) mutation in a known HLH gene.

2. Still disease patient has a LYST mutation, same mutation as found in 3 patients who died from H1N1 influenza (J Infect Dis. 2016 ;213:1180-8).

3. First adult SLE patient has a STXBP2 mutation, and pediatric SLE patient has AP3B1 mutations.

4. Second adult SLE patient has mutations (mostly missense) in 5 different HLH genes: AP3B1 (same mutations as pediatric patient), LYST, PRF1, STX11, & UNC13D.

5. This same SLE patient also has a rare 1st intronicmutation in UNC13D.

Anakinra forMASTrialPatientMutations

Pt #

Age Sex Diagnosis Trigger Gene Mutation Amino acid change

Freq-uency

Outcome

1 33 M AOSD present LYST 5945 C>T Thr 1982 Ile 0.6% Alive

2 24 F SLE present STXBP2 646 G>A Val 216 Ile 0.4% Alive

3 9 M SLE present AP3B1AP3B1

1038 T>C3023_3025del

Asn 346 AsnAla 1008 del

1.1%18%

Alive

4 21 F SLE EBV AP3B1AP3B1LYSTPRF1STX11UNC13DUNC13D

1038 T>C3023_3025del7793 T>A403 G>A829 A>G904 C>Tc.118-174 C>T

Asn 346 AsnAla 1008 delPhe 2598 TyrVal 135 MetThr 277 AlaLeu 302 Phe1st intron

1.1%18%0.8%0.1%1.1%0.3%?

Alive

mut. likely deleterious

11

PolygenicmutationsinthecytotoxicitypathwayincreasesusceptibilitytodevelopHLHimmunopathologyinmice

SepulvedaFE,etal.Blood.2016;127:2113-21.

AS=Rab27+/- x STX11+/-; AP=Rab27+/- x PRF1+/-; ASP=Rab27+/- x STX11+/- x PRF1+/-

UNC13D(Munc13-4)1st IntronVariants

c.118-174 C>T

9 patients with severe cytokine-release syndrome benefited from anti-IL-6R monoclonal antibody therapy. N=30

Grupp

12

BlockadeofIL-33and18

Control (n = 10)IL-33RB (n = 9)

Survival

0 2 4 6 8 10 12 14 16 180

20

40

60

80

100 Control

IL-33RB

Days post-infection

Perc

ent s

urviv

al

p=0.0005

0

100000

200000

300000

0

50

100

150

To

tal I

L-1

8 a

nd

IL-1

8B

P (p

g/m

L)

Above Upper Limit of Detection

IL-18BP

Free IL-18

Total IL-18

Fre

e IL

-18

(pg

/mL

)

Ferritin

Courtesy of Dr. Ed Behrens

fHLH mice

NLRC4 inflammasome mutant patient

J Allergy Clin Immunol. 2017;139:1698

Blood. 2016;127:426

13

Prf1-/-

In Press

In Press

Anti-interferon-gammatrial

NI-0501: A Study to Investigate the Safety and Efficacy of an Anti-IFNγ mAb in Children Affected by Primary Hemophagocytic Lymphohistiocytosis

Children and adolescents, up to and including 18 years old at diagnosis of primary HLH, who, after having received conventional therapy, continue to have active disease, are potentially eligible. Children or adolescents with intolerance to HLH conventional treatment can also participate in the study.

“AllWeareSayingisGiveCytokineInhibitionaChance”

• Corticosteroids– sure,ofcourse• Cyclosporin A– youbetcha• Cytotoxicdrugs– canyouwait?• CytokineAntagonism– worthatry

– TNFa (probablynot)– IL-1(lookspromising)– IL-6,IL-18,IL-33,IFNg (???)– JAKinhibition(???)

"Imagination is more important than knowledge..." - Albert Einstein

Expert opinion.

14

Future Possibilities:Anakinra Treatment of

Hospitalizable Fever with Elevated Ferritin

ferritin ~$30 per testanakinra ~$130 per 100 mg dose

Lives saved - priceless

EarlyAggressiveTherapy

“Evendoctorswithgreatknowledgeandtechnicalskillcanhavemediocreresults;morenebulousfactorslikeaggressivenessandconsistencyandingenuitycanmatterenormously.”–Atul Gawande,MD,MPH (HarvardSchoolofPublicHealth)

• http://www.newyorker.com/fact/content/?041206fa_fact• (in:The New Yorker)

Funding

KAUL PEDIATRIC RESEARCH INSTITUTE

15

Questions??

Documents

Disclosures (last 12 months) - WordPress.com · 9/17/2017 · relationship between the two. ... D-dimer Fibrinogen Ferritin Triglyceride ... Renal failure 13 (5.6) 2.5 3.6 (2.9) 0.9