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Use of a Rapid Genetic Assay to Confirm

the Diagnosis of Complement-Mediated

Thrombotic Microangiopathy:

A Preliminary Report

JC Hofmann, MD, MPH; DD Kiprov, MD Apheresis Care Group,

Division of Immunotherapy,

Department of Medicine,

California Pacific Medical Center,

San Francisco, CA

May 8, 2015

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Disclosure of Conflicts of Interest

“Use of a Rapid Genetic Assay to Confirm the

Diagnosis of Complement-Mediated Thrombotic

Microangiopathy: A Preliminary Report ”

Jan Hofmann, MD has reported the following financial

relationships with commercial interests related to the

content of this educational activity:

Consulting Fees; Honoraria: Fresenius Medical Care,

Alexion Pharmaceuticals

Background

• While the ability to rapidly diagnose primary TTP (patients with

ADAMTS13 activity <5-10%) has significantly improved, atypical

HUS (aHUS) remains a diagnosis of exclusion due to the high cost,

lack of sensitivity, and long turnaround time of current aHUS genetic

assays.

• Since April, 2014, availability of a Next-Gen sequencing aHUS

genetic panel which allows rapid detection (2-5 days) of >230 known

or suspected mutations, deletions, or polymorphisms reported as

potentially associated with aHUS (including 12 of the most common

genetic mutations, sensitivity of 75-84%1) has enabled faster

assessment of challenging TMA cases.

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TMA patients (4/14-11/14)

• 4/14-11/14: 41 TMA patients (evaluate 62-66 TMA pts/year)

• Primary TTP (ADAMTS13 activity <5-10%): 27/41 (66%) pts

• Shiga-toxin ecoli (STEC) HUS: 2/41 (5%) pts

• Non-TTP/non-STEC TMA (TMA): 12/41 (29%) pts

5 5 5 5 5 5

Primary TTP vs TMA vs STEC-HUS (Admission Labs)

Selected Admission

Labs (mean)

hemoglobin (g/dl)

platelet ct (X 109/L)

LDH (100-220 U/L)

creatinine (mg/dl)

Primary TTP (ADAMTS13 <5-10%)

(n=27) (66%)

7.3 (5.6-11.9)

14 (5-27)

2318 (844-4386)

0.9 (0.5-2.1)

TMA (non-TTP/non-STEC)

(n=12) (29%)

10.3 (8.2-12.1)

62 (19-153)

813 (457-1336)

3.8 (2.0-17.7)

STEC-HUS (+ stool STEC ELISA)

(n=2) (5%)

8.4 (7.7-9.1)

43 (39-47)

532 (390-674)

3.4 (2.7-4.1)

5 5

6 6 6 6 6 6

Primary TTP vs TMA vs STEC-HUS (CNS abnormalities & renal insufficiency)

Clinical Presentation CNS

abnormalities

Renal

insufficiency

(RI) (Cr >1.5)

ADAMTS13

activity (mean %)

Primary TTP (ADAMTS13 <5-10%)

(n=27) (66%)

6/27 (22%) 2/27 (7%) 1.5% (<0.5-4%)

TMA (non-TTP/non-STEC)

(n=12) (29%)

2/12 (7%)

9/12 (75%)

71% (15-125%)

STEC-HUS (+ stool STEC ELISA)

(n=2) (5%)

0/2 (0%)

2/2 (100%)

62% (44-79%)

6 6

7

Coppo P et al. Cardiovasc Hematol

Disord Drug Targets 2009; 9 (1): 36-50.

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Thrombotic Microangiopathy (TMA): (pathologic process micro-occlusion +/- inflammation of small

blood vessels)

TMA* (25-35%)

• ADAMTS13 >10-40%

• +++ immune triggers

• utility of PE less defined

• ASFA Cat. II-IV

HUS (5-15%)

• ADAMTS13 nl

• ASFA Cat. I-IV

Primary TTP§ (55-70%)

• ADAMTS13 <5-10%

• relapse rate: 30-50%

• rituximab very useful

• ASFA Cat. I

• infx-assoc. HUS

• aHUS#

Evolving Towards a Pathophysiology-Based

Classification of TMAs (a simplified approach)

*Possible complement-mediated TMA #Complement-mediated TMA §Congenital and acquired TTP European Paediatric Research Group for HUS

ADAMTS13 normal range 40-130%

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Conditions Associated with TMA (partial list)

TMA* (no clear etiology) (25-35%): ADAMTS13 >10-40%:

• Systemic autoimmune disorders

• SLE

• Scleroderma

• Infectious diseases

• GI or GU infection

• HIV infection

• Pregnancy

• Post-partum TMA

• Neoplastic diseases

• Breast, lung, prostate CA

• Hematologic

• Malignant HTN

• CAPS

• DIC

• Medications • Ticlopidine (I), Clopidogrel (III)

• Quinine (IV)**

• Cyclosporine, Tacrolimus (III)

• Mitomycin C

• Gemcitabine (IV)

• Bleomycin, Cisplatin

• Bevacizumab, Sunitinib

• Sepsis syndrome (TAMOF)

• Surgeries • Cardiovascular

• Intestinal

• HPSC transplantation (III)

• Ionizing radiation

*Possible complement-mediated TMA (in some cases) I-IV = 2013 ASFA indication categories

** Defective cobalamine metabolism

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TMA Diagnostic Workup (r/o Primary TTP)

From Pentad to Triad

• Thrombocytopenia

• MAHA

• CNS abnormalities

• Renal insufficiency

• Fever

TMA confirmatory lab studies: • haptoglobin (<< lower limits of normal)

• Negative direct coombs (positive coombs = AIHA)

• retic. count (nl or low retic. count = BM suppr.)

• normal PT/PTT/fibrinogen (elevated PT or PTT,

or low fibrinogen may be DIC).

• indirect >> direct bilirubin (if total bili elevated)

• ++ schistocytes (smear): indicates hemolysis (seen in

TTP, DIC, AIHA, sepsis, etc).

• ADAMTS13 < 5-10% (1º TTP); normal (HUS)

• Thrombocytopenia

• MAHA

• LDH elevation

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Primary TTP vs TMA vs STEC-HUS (Treatment Approaches)

• Primary TTP: 27/41 (66%) pts

• Daily PE & corticosteroid (CS) therapy (prednisone 1 mg/kg/d or equiv.)

• 24/27 (89%) received plasma infusion (PI) (over first 8-12 hrs)

• Mean 14.8 PE txs (5-43 txs); 23/27 (85%) had PE taper (QD 2X/wk)

• 20/27 (74%) had refractory response received rituximab (weekly X 4)

• TMA: 12/41 (29%) pts

• Mean 6.1 PE txs (0-12 txs); 4/12 (33%) received no PE tx (3/4 of these pts received daily PI instead, 4-5 units FFP/day for 3-5 days).

• 3/12 (25%) received CS therapy

• 7/12 (58%) had persistent RI requiring hemodialysis (hemo)

• 2/12 (17%) had RI which resolved

• STEC-HUS: 2/41 (5%) pts

• Supportive care (including mean 5.5 PE txs (5-6 txs); no CS therapy

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Breakdown of TMA Patients (aHUS Genetic Testing Threshold)

• TMA: 12/41 (29%) pts

• Mean 4.1 PE txs (0-12 txs); 4/12 (33%) received no PE tx (3/4 of these pts received daily FFP infusion instead).

• 7/12 (58%): persistent RI +hemo; 2/12 (17%): RI resolved

--------------------------------------------------------------------------------------

• 5/12 (42%) with unexplained, persistent RI [4 pts requiring hemo] (and/ or other systemic thrombotic symptoms) aHUS genetic testing (GT): - mean age 41 y.o. (20-59); 80% female

• 7/12 (58%) did not get aHUS GT and were treated for assoc. conditions:

- sepsis/DIC (2 pts)

- SLE flare, resolved (1 pt)

- malignant hypertension, resolved (1 pt)

- gemcitabine-associated TMA, comfort care (1 pt)

- pre-eclampsia, resolved RI (1 pt)

- HIV TMA, HAART tx (1 pt)

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TMA Patients (aHUS Genetic Testing Results)

• TMA: 5/12 (42%) TMA pts aHUS GT:

• 3/5 (60%) aHUS GT: positive (2 pts), equivocal (1 pt):

- CFH and MCP

- thrombomodulin (and 3 CFH polymorphisms)

- variant CFI gene (unknown significance)

• 2/5 (40%) aHUS GT: negative

• 4/5 (80%) TMA pts (with GT) diagnosed with aHUS

Summary of TMA cases (aHUS GT)

• 49 yo male, h/o colitis sepsis/ARF (Cr 7.0, on hemo); dx TMA s/p PI

(4d), AT13 51% (HD 4), aHUS GT (HD 6 HD 9): +THMD mutation,

eculizumab (HD 11) off hemo (3+ mos).

• 20 yo female, prior pre-eclampsia, 2nd pregnancy s/p TAB, ARF (Cr 17.7,

on hemo), dx TMA s/p PI (3d) + PE (3 txs), AT13 90% (HD 4), aHUS GT

(HD 5 HD 9): +CFH/MCP mutation; eculizumab (HD 14) on hemo

& kidney transplant list (8+ mos).

• 59 yo female, h/o hypothyroidism, progressive RI (Cr 2.72, no hemo), dx

TMA s/p PE (11 txs), AT13 79% (HD 4), aHUS GT (HD 10 HD17):

CFI positive variant (equivocal); ecul. (HD 21) RI resolved (2+ mos).

• 33 yo female, malignant HTN (237/146), ARF (Cr 7.9 10.6, on hemo);

dx TMA s/p PE (6 txs), AT13 105% (HD 6), aHUS GT (HD 7 HD 10):

negative; serologies: +scleroderma (SRC); ecul. (HD 12) still on hemo.

• 42 yo female with 10 yr h/o SLE SLE flare/ARF (Cr 2 4.87, brief

hemo), dx mild TMA s/p PI (2d), AT13 86% (HD 3); aHUS GT (HD 6

HD 11): negative; final dx: SLE flare with nephritis, no aHUS. 14

“New” Complement Inhibitor Genetic Panel

(aHUS Genetic Panel: 2-5 day turnaround time)

• CFH (1994, 1996)

• MCP/CD46 (2003)

• CFI (2004)

• C3 (1973)

• CFB

• CFHR1

• CFHR3

• CFHR4

• CFHR5

• Thrombomodulin (THBD) (2009)

• Plasminogen (PLG) (first reported 2013)

• DGKE (first reported 2013)

• current literature suggests that up to 75-84% of aHUS cases could be genetically

confirmed by sequencing the genes within this proposed panel. Multiple studies

linking first 10 genes to aHUS.

15 2Le Quintrec M et al. Semin Thromb Hemost 2010; 36: 641-665.

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1Bu et al. J Am Soc

Nephrol 2014; 25: 55-64.

Summary

• While current aHUS genetic assays lack the ability to “rule out”

complement-mediated TMA, these assays can assist with “ruling in” the

diagnosis.

• With the development of improved genetic testing (ie, increased

sensitivity and speed), aHUS genetic assays may represent a “real-time”

diagnostic tool enabling more rapid assessment of selected TMA cases

leading to more targeted and effective treatment.

• TMA patients in whom severe ADAMTS13 deficiency and infection-

associated HUS are ruled out, and who have persistent renal failure

despite appropriate therapies to treat any associated conditions, should

be considered for aHUS genetic testing.

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References

Complement Inhibitor Genetic Assays:

• 1Bu F et al. J Am Soc Nephrol 2014; 25 (1): 55-64.

• 2Le Quintrec M et al. Semin Thromb Hemost 2010; 26 (6): 641-652.

• Mele C et al. Clin J Am Soc Nephrol 2015 Apr 8. pii: CJN.08520814.

• Lemaire M et al. Nature Genetics 2013; 45: 531-534

• Broeders EN et al. BMJ Case Rep 2014 Dec 23; 2014. pii: bcr2014207630.

• Valoti E et al. J Am Soc Nephrol 2015; 26(1): 209-219.

• Ermini L et al. Mol Immunol 2012; 49 (4): 640-648.

• Bu F et al. Clin Dev Immunol 2012; 2012: 370426.

• Noris M et al. Clin J Am Soc Nephrol 2010; 5: 1844.

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Thank you for your attention Questions?

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