Anemie emolitiche

autoimmuni

Wilma Barcellini

Classificazione e diagnosi

Meccanismi patogenetici

Terapia

Anemie emolitiche

autoimmuni

Wilma Barcellini

Classificazione e diagnosi

Meccanismi patogenetici

Terapia

Autoantibody characteristics

DAT positivity

In vivo

hemolysis (RBC

sequestration)

Clinical associationClass

Optimal T of

reaction

(range)

Specificity

1. Warm AIHA

IgG

(possible

complement

fixation)

37°C

(0-40)

Rh

systemIgG or IgG+C

Extravascular

(spleen)

primary or secondary to

autoimmune,

lymphoproliferative, or

neoplastic diseases

2. Cold AIHA

2a. Cold agglutinin

disease (CAD)

IgM

(common

complement

fixation)

4°C

(4-34)

I/i

systemC

Intravascular

and

extravascular

(liver/spleen)

primary or secondary to

infections (acute

transitory) or to

lymphoproliferative

diseases (chronic)

2b. Paroxysmal Cold

Hemoglobinuria

(PCH)

IgG

(common

complement

fixation)

Reacts at

4°C and

hemolyses

at 37°C

P

antigen

Positive

Donath-

Landsteiner

test

Intravascular

and

extravascular

(liver/spleen)

primary or secondary to

syphilis (chronic) or to

other infections (acute

transitory)

3. Mixed AIHA warm IgG and

cold IgM

4°C and

37°C

IgG+high titer

cold IgM

Extravascular/

Intravascular

(spleen /liver)

primary or secondary to

lymphoproliferative or

autoimmune diseases

W. Barcellini, Current treatment strategies in autoimmune hemolytic disorders. Expert Rev Hematol. 2015

60-70%

20-25%

5-10%

1-5%

• estimated incidence 1-3 per 105/year, prevalence of 17:100,000

• heterogeneous condition (from fully compensated to life-threatening )

• major determinants of clinical severity: autoantibody class, thermal amplitude, efficiency in activating complement,

activity of the reticuloendothelial system (in spleen, liver and lymphoid organs), and the efficacy of the bone marrow

compensatory response

Autoimmune hemolytic anemia (AIHA)

• associated infections(Mycoplasma EBV, CMV,HIV, HCV, HBV, etc)

• Autoantibodies for systemic autoimmune

diseases (ANA, anti-DNA, ENA, LAC, ACA, anti-b2).

• careful anamnesis to identify

• drug-associated immune hemolysis (rare but

frequently undiagnosed),

• recent transfusions (DHR) or

• solid organ transplant (passenger

lymphocyte syndrome)

• Bone marrow biopsy and flow cytometry

and TC scan to exclude an underlying

lymphoproliferative disorder

• in all CAD cases prior to therapy

• In warm and mixed cases relapsed after

steroid therapy

• In case of DAT negativity, other causes of

hemolysis should be considered

(membrane and enzyme defects, PNH, HUS, TTP,

mechanical hemolysis, toxic noxae, Gaucher disease)

Diagnostic workup in AIHA include investigation to identify:

Represent 11% of AIHA (Petz e Garratty (Immune hemolytic anemias. New York

Churchill Livingstone, 2004)

Causes of false negative results Possible solutions

IgA auto Abs Use of monospecyfic anti-IgA antisera

low-affinity IgG Abs test with cold washed RBC

test with low-ionic solutions (LISS)

polybrene or PEG

low sensitivity of the tests Use of more sensitive techniques

Microcolumn agglutination technique (CAT)

Solid phase agglutination technique

Immunoenzymatic assay

Flow cytometry

Mitogen-stimulated (MS)-DAT

Positive DAT not specific for AIHA0.007–0.1% of healthy population and 0.3–8% of hospitalized patients

without AIHA have a +DAT

False positive DAT (hypergammaglobulinemia, high dose immunoglobulin therapy) Valent P, Lechner K,Wien Klin Wochenschr. 2008

Positive DAT for alloantibodies (in recently trasfused patients)

DAT negative AIHA

Yamada, Transfusion 2008, Bencomo et al, Immunohematology 2003, Lin et al, Transfusion 2009,

Barcellini et al, BJH 2000, Int J Hematol 2010

• McCann EL et al. IgM autoagglutinins in warm AIHA: a poor prognostic feature. Acta Hematol 1992;82:120-5.

• Garratty G et al. Severe AIHA associated with IgM warm autoantibodies directed against determinants on or

associated with glycophorin A. Vox Sang 1997;72:124-30.

• Friedman AM et al. Fatal AIHA in a child due to warm-reactive immunoglobulin M antibody. J Pediatr

Hematol Oncol 1998;20:502-5.

• Nowak-Wegrzyn A et al. Fatal warm AIHA resulting from IgM autoagglutinins. J Pediatr Hematol Oncol

2001;23:250-2.

• Shirey RS et al. Fatal immune hemolytic anemia and hepatic failure associated with a warm-reacting IgM

autoantibody. Vox Sang 1987; 52:210-22.

• McCann EL et al. IgM autoagglutinins in warm AIHA: a poor prognostic feature. Acta Hematol 1992;82:120-5.

• Garratty G et al. Severe AIHA associated with IgM warm autoantibodies directed against determinants on or

associated with glycophorin A. Vox Sang 1997;72:124-30.

• Friedman AM et al. Fatal AIHA in a child due to warm-reactive immunoglobulin M antibody. J Pediatr

Hematol Oncol 1998;20:502-5.

• Nowak-Wegrzyn A et al. Fatal warm AIHA resulting from IgM autoagglutinins. J Pediatr Hematol Oncol

2001;23:250-2.

• Shirey RS et al. Fatal immune hemolytic anemia and hepatic failure associated with a warm-reacting IgM

autoantibody. Vox Sang 1987; 52:210-22.

Severe and lethal AIHA caused by “warm” IgM autoantibodiesSevere and lethal AIHA caused by “warm” IgM autoantibodies

�“...AIHAs associated with IgM warm auto Abs are often severe with more fatalities than other

types of AIHA….” (Garratty et al, Vox Sang. 1997,72:124-30).

�Can appear to be DAT negative by routine DAT or to have only C3 on RBCs by routine DAT

�Spontaneous RBCs agglutination due to crosslinking of IgM autoabs

�Difficult and delayed diagnosis

Additional factors that influence the clinical characteristics of AIHA

The major determinants of the clinical severity of AIHA are rate and type of hemolysis which depend

on

• the autoantibody class, thermal amplitude, and efficiency in activating complement

• the activity of the reticuloendothelial system (in spleen, liver and lymphoid organs)

• the efficacy of the bone marrow compensatory response

• AIHA with reticulocytopenia. A Medical emergency(Conley CL et al, JAMA, 1980;244:1688)

• Reticulocytopenia is present in 20% adults and 39% children(Liesveld et al, 1987; Aladjidi et al, 2011: Barcellini et al, 2014)

• Reticulocytopenia is present in severe cases

• Erythropoietin useful to overcome the temporary bone

marrow failure in compensating hemolysis

• Erythropoietin may reduce transfusion need and avoid

hemolysis related to overtransfusion

• Due to autoimmune reaction against BM erythroid

precursors?

• the reticulocyte index was lower in cases with Hb<6 g/dL

(65 vs 98, p<0.001), along with more frequent inadequate

reticulocytosis (87 vs 70%, p=0.01)

Clinical follow-up of 378 patients with AIHAfrom 9 italian centers: prognostic impact of Hb levels,

autoAb class, and reticulocytopenia at onset on the relapse risk and outcome

Absolute reticulocyte counts as a

function of Hb at onset

05

10

15

hb

1 2 3 5

Hb as a function of autoAb class and

thermal characteristics

CAD wIgG wIgG+C /Mix Atypical

(n=109) (n=159) (n=90) (n=20)

Anemia severity at onset is associated with

an increased risk of relapse

HR 1.98 for Hb<6 g/dL,

HR 1.74 for Hb 6.1-8 g/dL

HR 1.61 for Hb 8.1-10 g/dL

HR 1.21 for forms other than warm IgG AIHA

HR 1.84 for Evans association

The association of 3 conditions led to a ~4-fold

increased risk of multiple relapses

/refractoriness to therapy (multivariate Cox regression analysis)

Anemie emolitiche

autoimmuni

Wilma Barcellini

Classificazione e diagnosi

Meccanismi patogenetici

Terapia

Barcellini W. New Insights in the Pathogenesis of Autoimmune Hemolytic Anemia. Transfus Med Hemother. 2015;42:287-93

Role of the spleen in the pathogenesis of AIHA

• Major organ for antibody synthesis

• Major site of immune destruction of IgG-coated erythrocytes

• Splenectomy usually ineffective in CAD since most RBC

destruction occurs in liver

• Effective in about 70% of warm AIHA

• Ineffective in about 30% of cases, presumably because there is

still an elevated synthesis of autoantibodies in other lymphoid

organs, and the liver replaces the spleen’s function of RBC

destruction

• The rate of splenectomy is about 10-15% of all AIHA cases, but

is nowadays decreasing given the availability of equally

effective second line medical treatments, primarily rituximab

• No reliable predictors of its effectiveness, such as the extent of

splenic sequestration assessed by radiolabeled erythrocytes,

disease duration and the response to steroids

• The emergence of long-lived autoreactive plasma cells in the

spleen of primary warm AIHA patients relapsed after

rituximab. Spleens (obtained after splenectomy) showed the

presence of non-proliferative memory B-cells and plasma cells,

possibly sustained by increased BAFF (new therapeutic

options?)

Mahévas M et al, J Autoimmun. 2015 Aug;62:22-30

hemolysis

thrombosis

thrombocytopenia

renal failure

hemolysis

thrombosis

thrombocytopenia

renal failure

Close integration between the complement cascade (grey) and the coagulation cascade (green)Close integration between the complement cascade (grey) and the coagulation cascade (green)

Hill A et al. Blood 2013;121:25

•Thrombotic risk in Autoimmune hemolytic anemia

•There is increasing awareness of the thrombotic risk in hemolytic conditions, mainly coming from the experience

on paroxysmal nocturnal hemoglobinuria (PNH)

• In the retrospective multicenter GIMEMA study a thrombotic event was recorded in 34/308 (11%) of patients and

included 11 cases of pulmonary embolisms, 13 deep venous thrombosis, 5 splanchnic thrombosis, 1 disseminated

intravascular coagulation, 3 strokes, 2 transient ischemic attacks and 3 cardiac ischemic events (with 5 patients

experiencing more than one event). (Barcellini, Blood 2014)

•In a case-control single-center study a venous thromboembolism (VTE) was reported in 8/40 patients (20%), all

pulmonary embolus associated with a deep venous thrombosis in 4 (Lecouffe-Desprets , Autoimmun Rev. 2015 ).

•By using a USA cohort of commercial insurance enrolees (Truven Health MarketScan® Databases) the risk of VTE in

AIHA was 10-fold increased (19 per 1,000 person-years, versus 1.9 in the control group) with an adjusted hazard

ratio of 6.30 (95% confidence interval: 4.44-8.94) (Yusuf H, Thromb Res. 2015 ).

•Severity of anemia, high median LDH levels, and previous splenectomy have been associated with the occurrence

of thrombotic events, whereas the role of anti-cardiolipin antibodies or LAC is still controversial

• Recommendations:

• The risk of thrombotic events (both venous and arterial) should be taken into account, mostly during acute

hemolytic flares and in splenectomized patients

• Assessment of a general thrombotic risk factor is advisable in AIHAs (age >70 years, active cancer, previous VTE,

reduced mobility, already known thrombophilic condition, recent trauma and/or surgery, heart and/or

respiratory failure, acute infection, etc)

• VTE prophylaxis should be considered in patients with marked hemolysis and associated risk factors

• Recommendations:

• The risk of thrombotic events (both venous and arterial) should be taken into account, mostly during acute

hemolytic flares and in splenectomized patients

• Assessment of a general thrombotic risk factor is advisable in AIHAs (age >70 years, active cancer, previous VTE,

reduced mobility, already known thrombophilic condition, recent trauma and/or surgery, heart and/or

respiratory failure, acute infection, etc)

• VTE prophylaxis should be considered in patients with marked hemolysis and associated risk factors

Anemie emolitiche

autoimmuni

Wilma Barcellini

Classificazione e diagnosi

Meccanismi patogenetici

Terapia

Treatment algorithm for warm AIHA in adults

Zanella A & Barcellini W,

Haematologica, 2014

• Response to steroids in 75-80% of

patients

• estimated cure rate 20-30% only

• 50% require maintenance doses

• 20-30% need additional 2nd line

therapies

• Side effects vary with length of

administration

(hypertension, diabetes, weight gain,

anxiety, osteoporosis, gastro-

intestinal ulcers, immune-

suppression, psychosis, cataract)

•Transfusions, never deny, but avoid if

unnecessary

•Decide on patient’s clinical status and

comorbidities rather than on the Hb

level

•Generally, Hb<6 g/dL, acuteness of

onset, and hemodynamic instability

deserve support

•RBCs should be administered slowly,

possibly not exceeding 1 ml/kg/h

(avoid overtransfusion!)

•Attention to alloantibodies, known to

occur in 12%-40% of AIHA patients,

that may be responsible for severe

hemolytic reactions

22ndnd line treatmentline treatment

Rituximab SplenectomyNR

Zanella & Barcellini, Haematologica, 2014, Barcellini, Expert Rev Hematol, 2015

Warm AIHA

• Increasingly preferred among 2nd line options

• unfitted for surgery, refuse splenectomy

• Predictors of response • younger age

• early administration as second-line therapy

• Response 70-80% (half of cases CR)

• Median duration of response of 1-2 years

• DFS ~70% at one and ~55% at two years

• Low-dose equally effective

• Median time to response is 4-6 weeks

(responses after 3-4 months possible)

• Response in primary and secondary AIHA,

independent from prior treatment, re-

treatment and combination possible

• Good safety profile (infectious events in roughly

7%, rare cases of PML, HBV reactivation)

• Age < 65-70 y, women that wish to become

pregnant, good performance status

• response ∼∼∼∼70%, presumed curative rate ∼∼∼∼20%

• current rate 10-14%, but progressively decreasing

• The drawbacks • overwhelming sepsis (3-5% of cases, mortality rate 50%,

even after the recommended vaccination against

pneumococci, meningococci, and hemophilus, and re-

vaccination every 5 years)

• thrombotic events

• lack of reliable predictors of outcome

• surgical complications

• patient education (early administration of oral antibiotics,

prompt referral to hospital for suspected sepsis)

• discouraged age >65-70 years or comorbidity (cardiopulmonary disorders, previous history or serious risk of

thrombosis, hepatitis C, underlying immunodeficiency,

lymphoproliferative, and systemic autoimmune conditions

• laparoscopic splenectomy preferred but should

be performed by experienced operatorsRituximab subcutaneous formulation (SC-R) is safe,

effective and cost-Saving in Patients with AIHA, Francesco Iuliano, ASH 2016

Treatment of cold agglutinin disese (CAD)

Treatment Algorithm

Primary CAD Secondary CAS

W & W

Cold agglutinin mediated AIHA

Symptomatic (anemia, transfusion, circulatory symptoms)

Asymptomatic

Supportive

Care/Therapy

Rituximab(375 mg/m2 weekly x 4)

(Rituximab + Benda-

mustine for fit patients)

Clinical Trial (if available)

Emergency Situation Eculizumab

Plasmapheresis

Rituximab

+ Bendamustine

No Response/

Relapse

Clinical Trial /

Experimental TreatmentEculizumab, Ibrutinib,

Venetoclax ...

Rituximab

+ Fludarabine or

Bortezomib

Relapse

Treat underlying

disease(if possible)

Supportive

Care/Therapy

• Ab anti-platelets

• Ab anti-

megakaryocyte

• T Cytotoxic

• ↓↓↓↓ Tregs

• APC activity

• ↑↑↑↑IL-2, TNF-αααα

• TGF-1ββββ, ↓ Tregs

Increased spleen

destruction

↓↓↓↓ megakaryocyte maturation

↑↑↑↑ megakaryocyte apoptosis

TPO deficiency

IL-2IFN-γγγγ

ITP has a compound and heterogeneous pathophysiology with 3 main drivers

• Ab anti-platelets

• Ab anti-

megakaryocyte

• T Cytotoxic

• ↓↓↓↓ Tregs

• APC activity

• ↑↑↑↑IL-2, TNF-αααα

• TGF-1ββββ, ↓ Tregs

Increased spleen

destruction

↓↓↓↓ megakaryocyte maturation

↑↑↑↑ megakaryocyte apoptosis

TPO deficiency

IL-2IFN-γγγγ

TPO-RA

Splenectomy

Immunesuppressors

(targeting T and B

lymphocytes)

ITP has a compound and heterogeneous pathophysiology with 3 main drivers

Increased spleen

destruction

ITP and AIHA second cousins not brothers…AIHA has a compound and heterogeneous pathophysiology with various main

drivers

The complement system

Bone marrow erythroid hyperplasia

Displasia ?

Apoptosis ?

Fibrosis ?

EPO ? ?

• five paradigmatic cases of

refractory/relapsing autoimmune

cytopenias, namely autoimmune hemolytic

anemia (AIHA), immune thrombocytopenia

(ITP), and chronic idiopathic neutropenia

(CIN), that evolved to IDUS/bone marrow

failure syndromes over time

• Idiopathic cytopenia of uncertain

significance (ICUS) and idiopathic dysplasia

of uncertain significance (IDUS) are two

recently recognized provisional conditions

characterized by isolated/unexplained

cytopenia and/or dysplasia in <10% bone

marrow cells

• The relationship between myelodysplasia

and autoimmunity is supported by their

epidemiologic association, the existence of

common immune-mediated

physiopathologic mechanisms, and the

response to similar immunosuppressive

therapies

STUDY GROUP MF0 N=30 MF>1 N=17

Hb, g/dL, 7.4 (3.5-13.1) 8 (2-11)

LDH x UNL 1.27 (0.4-7) 1 (0-3)

Ret x103/mmc 113 (13-275) 157 (58-284)*

Bone marrow characteristics

Cellularity, %

Normo, N (%)

Hypo, N (%)

Hyper, N (%)

52.5 (25-90)

12 (40)

2 (7)

16 (53)

65(20-99)**

2 (12)

1 (6)

14 (82)*

Diserythro N (%) 16 (53) 14 (82)*

Lymphoid infiltrate

T-cell, N (%)

B-cell, N (%)

Mixed, N (%)

8 (26)

4 (13)

10 (30)

2 (12)

3 (18)

10 (59)

(A) loose network of reticulin fibers

with many intersections (MF-1),

(B) The hyperplastic erythropoietic

series shows some grade of

dyserytropoiesis

(C) centro-lacunar lymphoid aggregate predominantly

composed of CD20-positive small B lymphocytes,

(D) with scattered small CD3-positive T lymphoid cells.

(E) focal and interstitial lymphoid infiltrate composed

mainly of CD3+ small T-lymphoytes

Cytokine serum levels in patients with (black)

and without (white) bone marrow fibrosis

• Reticulinic fibrosis (MF1) is present in 36% of AIHA cases and

correlates with the presence of a hypercellular dyserythropoietic

bone marrow , increased levels of TGF-ββββ , , , , and a higher rate of

relapse and treatment requirement

• These chronic refractory cases may show clinical/pathologic

features similar to low-risk myelodysplastic syndromes and

recently described idiopathic cytopenia/dysplasia of unknown

significance (ICUS/IDUS)

• MF0 AIHA patients seems to present with a more florid

hemolytic disease, increased immune activation and cytokine

release, more frequent thrombotic events, and a better response

to immunesuppression

T

Complement

IL2R

CD20

C1

MACC3C5

FcγγγγR

Syk

MMF

Rituximab

CD52

CD52BCR

BCRi

EPO EltrombopagLuspatercet

EculizumabC3i

C1i

Bortezomib

FostamatinibSYNT001

IL2

CD38

Bone marrow

Daratumumab

B

Alemtuzumab

Spleen

Activated lymphocytes, phagocytes

Liver

IgG

IgM

Proteosome

Phagocytes

AIHA Clinical Studies 2017 – clinical trials.gov (N=18)

Study Title Conditions Interventions Locations Recruitment

Efficacy and safety of BIVV009 open in

transfusion- dependent (Cardinal) and vs

placebo in non-transfusion dep (Cadenza)

CAD BV009 (C1 inhibitor) US- Europe

Bioverativ

recruiting

Safety, tolerability, efficacy of APL-2 in

patients with wAIHA or CAD

WAIHA

CAD

APL-2 (C3 peptide inhibitor) Lakes Research, Miami Lakes, FL, US

Apellis

recruiting

A safety and efficacy study of R935788 in the

treatment of warm AIHA

WAIHA Fostamatinib 150mg bid UCSD, Insitute of Hope and innovation,

Downey, Arizona Oncology Assoc.-US

recruiting

SYNT001 in subjects with chronic, stable

WAIHA

WAIHA SYNT001

(biologic with FcRn affinity)

US (incl. Mass. General) recruiting

Interleukin-2 in WAIHA resistant to

conventional treatment

WAIHA IL-2 Bordeaux, Hopital Haut Leveque, Pessac,

FR

recruiting

Take home messages

•AIHA may be challenging (severe and refractory), particularly IgG+C wAIHA, mixed, and

atypical forms (warm IgM and DAT-negative)

•Prompt therapy with rituximab (and possibly PEX) is recommended in severe cases

•Rituximab is becoming the preferred second line therapy for steroid-refractory wAIHA

and is recommended as first line in CAD

•Splenectomy, when feasible, is still a valid option for wAIHA but its exact timing is

difficult to establish

•Early diagnosis/prevention of thrombotic events (occurring in 10-20% of AIHA),

particularly after splenectomy and in cases with severe onset and intravascular

hemolysis is mandatory

•Careful follow-up to monitor infections, particularly after splenectomy, acute renal

failure, and Evans’ syndrome are recommended. All this complications and multi-

treatment are predictors of fatal outcome

•New therapeutic drugs directed at “pathogenic” mechanism (complement or Fc

blocking) are in progress

•Possible evolution of autoimmune cytopenias to idiopathic cytopenia of uncertain

significance (ICUS) and idiopathic dysplasia of uncertain significance (IDUS) should be

taken into account to avoid improper therapy