disorders of neutrophils · case presentation. 1-month-old boy with elevated wbcs • 1 month old...

DISORDERS OF NEUTROPHILS

Aric Parnes, M.D.

Division of Hematology

Brigham and Women’s Hospital

Cancer Medicine and Hematology

September 24, 2018

Disclosure Information: Aric Parnes

I have no financial relationships to disclose.

AND

I will NOT include discussion of off-label or investigational use of any products in my

presentation.

▪O2 independent killing

▪respiratory burst▪phagocytosis ▪chemotaxis

10

granule

mRNA20 granule mRNA

myeloblast promyelocyte myelocyte metamyelocyte band segmented

neutrophil

MYELOPOIESIS

HSC PMN▪NETs

NETosis

Neutrophil extracellular traps form NETS

Activates

granules

and

adhesion

Histone

processingStimulation

of receptors

Cell membrane

ruptures

expelling DNA

J. Cell Biol 2012;198(5):773-83

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&ved=2ahUKEwji3afg6fbcAhVGPN8KHdBbCSoQjRx6BAgBEAU&url=https://openi.nlm.nih.gov/detailedresult.php?img%3DPMC3432757_JCB_201203170_Fig3%26req%3D4&psig=AOvVaw0XBD9qdLPsMF4jSLKMEr8L&ust=1534689258697666

HSC

PMN

CYTOKINES GOVERN MYELOPOIESIS

G-CSF/GM-CSF are produced by endothelium and macrophages

Proliferate myeloid progenitors

Induce myeloid maturation

Protect from apoptosis

Enhance neutrophil function

Dranoff G and Mulligan RC. Stem Cells;1994;12 Suppl 1:173-82

G-CSF DEFICIENCY GM-CSF DEFICIENCY

- Relative neutropenia - No neutrophil defect- Pulmonary alveolar proteinosis

LIFE SPAN OF THE NEUTROPHIL

Maturation in the bone marrow: 7-10 days

Circulation in the peripheral blood: 3-24 hours

Duration in the tissues: 2-3 days

PERIPHERAL WBC COUNT

Myeloid Precursors 20%Storage Pool 75%Marginating Pool 3%Circulating Pool 2%

Peripheral neutrophil count reflects <5% of total WBCs and ~2% of the total WBC lifespan

Elevation of WBC counts:Acute: Changes in distribution (demargination)Chronic: Changes in production and release from storage pool

Decreased WBC counts: Defect in WBC production, increased destruction or increased margination (sequestration)

CASE PRESENTATION

43-year-old woman with elevated WBCs

• Previously healthy

• Seen for routine office visit

• WBC 12K with normal differential

• Repeat CBC three weeks later: No change

• HCT 42%, PLTs 230K

LEUKOCYTOSIS: DIFFERENTIAL DIAGNOSIS

SECONDARY TO OTHER ILLNESSESInfection

Acute: Demargination/release storage poolChronic: Granulomatous dx (leukoerythroblastic)

Stress, obesitySmokingDrug-induced (steroids, b-agonists, lithium, G-CSF)Chronic inflammationPost-splenectomyNon-hematologic malignancyMarrow stimulation (ITP, hemolysis)

PRIMARY HEMATOLOGIC DISEASECMLOther myeloproliferative diseaseLymphoproliferative disease

EVALUATION OF LEUKOCYTOSIS

EVALUATION OF LEUKOCYTOSIS

Neutophilia is usually reactive, indicative of a normal functioning bone marrow. Bone marrow evaluation is often unnecessary

•Repeat CBC: Artifact, factitious

•Evaluation for acute/chronic infection or inflammation

•LAP score: Replaced by BCR-ABL testing

•PCR or FISH for BCR-ABL

•Bone marrow biopsy: Granulomatous disease, fungus

CASE PRESENTATION

1-month-old boy with elevated WBCs

• 1 month old infant with delayed umbilical cord separation

• High-grade fever, MRSA infection, poorly healing skin lesions, otitis, failure to thrive, poor response to antibiotics

• WBC 90k

What to do??

Adapted from Pediatr Transplantation 2007;11:453-5

LEUKOCYTOSIS: DIFFERENTIAL DIAGNOSIS

PRIMARY LEUKOCYTOSIS:

Down syndrome: Transient myeloproliferative disorder

Hereditary neutrophilia: CSF3R activating mutation

Leukocyte adhesion deficiency

ADHESION MOLECULES AND LAD

Curr Opin Hematol 2002;9:30-35

Pathogenesis:

LAD I: Defective b2 integrin (CD18)

LAD II: Defective L-selectin ligand (Lewis X)

LAD III: Other integrin defects

LAD IV: Defective integrin in cystic fibrosis

Failure of chemotaxis

WBCs do not extravasate

LEUKOCYTE ADHESION DEFICIENCY

Clinical manifestations:

Elevated WBC

Recurrent bacterial and fungal infections, such as cutaneous abscesses, gingivitis, pneumonia

Delayed wound healing, delayed umbilical cord separation

Many die before age 2

Treatment:Antibiotics

Stem cell transplant: Treatment of choice

G-CSF does not help

ANC Clinical Significance

1000-1500 Not clinically significant

500-1000 Slight predisposition to infection

200-500 Significant predisposition to infection;

IV antibiotics for febrile illness

<200 Very high risk of infection;

decreased signs of inflammation;

aggressive in-patient treatment for febrile

illness

NEUTROPENIA

In chronic neutropenia, patients frequently have little or no manifestations of neutropenia with counts of 50-100

CASE PRESENTATION

2-month-old girl with agranulocytosis• Fever, purulent otitis, and skin abscesses

• FHx: 1 of 9 children; 4 had died at a young age

• Blood culture: S. aureus

• Treated with streptomycin

• Peripheral smear: No granulocytes

• Marrow: Maturation arrest at the promyelocyte stage

• Subsequent course: Died at age of 6 months from sepsis

Adapted from Kostmann, Acta Paediatr Scand 1956

NEUTROPENIA: DIFFERENTIAL DIAGNOSIS

CONGENITAL NEUTROPENIAS:

Benign neutropeniaConstitutional neutropenia: Duffy Ag Receptor Chemokine (DARC) SNPBenign neutropenia (familial, idiopathic)

Congenital Severe congenital neutropenia (Kostmann’s syndrome)Cyclic neutropeniaChediak-HigashiSchwachman-Diamond

CONGENITAL NEUTROPENIA

SYNDROME INHERITANCE GENES

Ethnic neutropenia ? DARC SNPS

Benign familial neutropenia AD Unknown

Severe congenital neutropenia

AD ELANE (55-60%)

AR HAX1 and G6PC3 (<5%)

X-linked WASP (<5%)

Unknown Unknown (40%)

Cyclic neutropenia AD ELANE (95-100%)

IMMUNODEFICIENCY SYNDROMES ASSOCIATED WITH NEUTROPENIA

Schwachman-Diamond Syndrome AR SBDS (100%)

Fanconi Anemia AR and X-linked FANC A-P genes

Dyskeratosis CongenitaX-linked DKC1 (80%)

AD TERC (0-20%)

AR TERT (0-20%)

Glycogen storage disease Ib AR SLC37A4 (100%)

Myelokathexis AD CXCR4 (100%)

Chediak-Higashi syndrome AR LYST (100%)

Griscelli syndrome II AR RAB27A (100%)

Hermansky-Pudlak syndrome II AR AP3B1 (100%)

Cartilage-hair hypoplasia AR RMRP (100%)

SEVERE CONGENITAL NEUTROPENIA

Congenital agranulocytosis•Rare •Autosomal dominant, recessive, and sporadic cases reported

•Severe infections•Survival dramatically changed with G-CSF•High incidence (30% over 10 years) of evolution to AML


Autosomal dominant form of SCN:

•Linked to mutations in the neutrophil elastase gene (ELANE)

•Mutant ELANE accumulates in the cytoplasm, and activates the “unfolded protein response,” a cellular stress response that results in apoptosis

•AML associated with a truncation mutation of the G-CSF receptor of uncertain pathogenic significance


Autosomal recessive SCN:

•Original syndrome described 60 years ago by Kostmann

•Linked to mutations in HAX1, a mitochondrial protein associated with signal transduction

•Disruption of HAX1 in myeloid cells destabilizes the mitochondrial membrane and leads to apoptosis

CYCLIC NEUTROPENIA

Dominantly inherited disorder

•Cycle of neutropenia q15-35 days

•Marrow during neutropenia: Myelocyte arrest with hypoplasia

•Usually benign

•Patients with severe infections may require G-CSF

CYCLIC NEUTROPENIA

Linked to neutrophil elastase mutation, like SCN

ELANE (ELA2) mutations found in essentially 100% of cyclic neutropenia

NOT associated with an increased risk of AML

CASE PRESENTATION

38-year-old woman with SLE and neutropenia

HPI:Age 14: Pericarditis, Raynaud’s with prolonged period of bedrest. ?JRA; ?SLEAge 26: Fatigue, adenopathy, oral ulcers, arthritis. Leukopenia, thrombocytopenia, +ANA, +ACAAge 30: Miscarriage. Documented ACLA

MEDS: Hydroxychloroquine, ASA 81mg, prednisone 5mg recently tapered from 50mg

EXAM: Malar rash; no active joint disease

LABS: WBC 1.8

AUTOIMMUNE NEUTROPENIA

Ann Hematol 1991;63:249-252

Primary AIN:

•Primarily in children

•Associated with

antibodies against

common antigens

Secondary AIN:

•Primarily in adults

•Associated with

autoimmune diseases

•Associated with LGL

leukemia

•Associated (rare) with

leukemia/lymphoma

PRIMARY AUTOIMMUNE NEUTROPENIA

A disease of childhood caused by a-neutrophil antibodies

•Average age of onset: 6-12 months

•Moderate to severe neutropenia

•Spontaneous remission over 2 yrs: 95%

•Treatment:

• Prophylactic antibiotics

• G-CSF only with severe/recurrent infection

SECONDARY AUTOIMMUNE NEUTROPENIA

• Associated with autoimmune disease: SLE, RA

• Mostly adults

Associated diseases in 42 patients

Underlying disease Patients (n)

Autoimmune thrombocytopenia 12

Evans’ syndrome 3

Autoimmune hemolytic anemia 1

Systemic lupus erythematosus 9

Rheumatoid arthritis 2

Felty’s syndrome 2

Leukemia 3

Non-Hodgkin’s lymphoma 3

Adapted from Ann Hematol 1991;63:249-252

AUTOIMMUNE NEUTROPENIA IN SLE

Occurs in approximately 50% of SLE patients

•Marker of disease activity

•Little impact on the course of the disease

•Infectious complications correlate with immunosuppressive therapy rather than height of neutrophil count

Pathophysiology:

•Neutrophil-specific antibodies

•Immune-complex mediated destruction

•Increased apoptosis of neutrophils

•Decreased marrow neutrophil production

CASE PRESENTATION

58-year-old man admitted with fever and cellulitis

PMHx: Hypercholesterolemia, NIDDM, arthritis

Medications: Naproxen, glucosamine, simvastatin

PE: Multiple joint deformities, splenomegaly, no adenopathy

CBC: HCT 40%, PLT 200K; WBC 5900 with 90% lymphs, 1% polys

AUTOIMMUNE NEUTROPENIA IN RA

Felty syndrome• Typically in patients with longstanding RA• Associated with end-organ RA manifestations (pulmonary

fibrosis, vasculitis, rheumatoid nodules, Sjogrensyndrome)

• Splenomegaly• Considerable morbidity from bacterial infection

LGL-associated neutropenia• Shares many features with Felty syndrome• Monoclonal neoplastic disorder (vs. Felty is polyclonal)

Both have a very high (90%) incidence of HLADR4, suggesting they are a spectrum of the same disease

WHEN DO I CHECK ANTI-NEUTROPHIL ANTIBODIES IN ADULTS WITH

NEUTROPENIA?

NEVER

CASE PRESENTATION

65-year-old man with sore throat and fever

PMHx:

Chronic CHF

Started new cardiac drugs 2 months ago

CBC: Hb 12, PLT 190K, WBC 0.7, ANC 50

DRUGS CAUSING AGRANULOCYTOSIS

Anti-thyroid medications Carbamizole

Methimazole

Propylthiouracil

Antibiotics Cephalosporins

Penicillins

Sulfonamides

Chloramphenicol

Anticonvulsants Carbamazapine

Valproic Acid

Antipsychotics Clozapine

Cardiac medications Procainamide, propranolol, ACE-I, flecainide

DRUG-INDUCED NEUTROPENIA

• Idiosyncratic drug reaction leading to profound neutropenia or agranulocytosis

• Pathogenesis poorly understood, and studies are difficult

since it is rare, sporadic, and transient

• Treatment:

• Stop the drug ASAP

• Consider G-CSF (e.g. in sepsis)

• Associated with significant morbidity and a mortality of 5-10% (UNLIKE CHRONIC NEUTROPENIA)

CASE PRESENTATION

31-year-old woman referred for neutropenia

HISTORY:Age 16: Episodic abdominal pain, fever, and vomiting → diagnosed with appendicitis after multiple episodes

Appendectomy → symptoms resolved and WBCs fell to 2000 with ANC 500Neutropenia persisted ever since

PMHx: In retrospect: Frequent upper respiratory illnesses as a child, including several episodes of pneumonia Age 30: Started on weekly G-CSF

ROS: LUQ pain, nausea and vomiting 1-2 days after taking G-CSF

NON-IMMUNE CHRONIC NEUTROPENIA

Chronic neutropenia• Normal marrow cytogenetics; variable cellularity• No evidence of autoimmune disease, nutritional

deficiency, myelodysplasia• Benign clinical course, often diagnosed by routine blood

tests in asymptomatic patients• Variable need for G-CSF support

Pathophysiology

• Probably a heterogeneous disorder

• Patients with myeloid hypoplasia: Selective decrease in CD34+/CD33- progenitors and increased TNF

Papadaki et al. Blood 2003;101:2591

Papadaki et al. AJH 2000;65:271

EVALUATION OF THE NEUTROPENIC CHILD

Neutropenia

Drug or

Toxin?

Yes Discontinue drug

or toxin

Abnormal growth and development,

skin, hair, bone, appendage,

nail changes?

Yes

Evaluate for

genetic syndromes

assoc. with neutropenia

Yes

No

Family history of

neutropenia, recurrent

Infection, sudden death?

Evaluate for

congenital

neutropenia

Appropriate

ethnic group?

Ethnic or benign familial

neutropenia

No

Yes

Fever,

ANC <500/µL?

Yes

Admit for IV

antibiotics; consider

G-CSF

No

No

Serial follow-up

of clinical

features and

cell counts

Stable/Improved Progressive

Immune or Idiopathic

neutropenia

Bone marrow biopsy,

aspirate, and flow

cytometry

Jacobson and Berliner. Wintrobe’s Clinical Hematology.

Lippincott Williams and Wilkins, 13th Edition, 2011

EVALUATION OF THE NEUTROPENIC ADULT

Neutropenia

Fever,

ANC <500/µL?

Yes

Admit for IV

antibiotics; consider

G-CSF

Drug or

Toxin?

Yes

Discontinue drug or toxin

No

Ethnic or benign familial neutropenia,

or cyclic neutropeniaNew onset?

No

MDS

LGL

Idiopathic

neutropenia

Abnormal

cytogenetics

Normal

LGL by flow

Isolated neutropenia

Bone marrow biopsy,

aspirate, and flow

cytometry

No

Low B12 or folate,

or autoimmune

disease?

Yes

Single or multi-

lineage cytopenia?

Pancytopenia

Yes

Megaloblastic

anemia

Immune

cytopenia

Low B12,

folateAutoimmune

disease

Jacobson and Berliner. Wintrobe’s Clinical Hematology.

Lippincott Williams and Wilkins, 13th Edition, 2011

MANAGEMENT OF NEUTROPENIA

Diagnosis:Stop potential offending drugsBone marrow aspiration/biopsySerologic studies: ANA, viral titersR/O Primary malignancy:

Chromosome analysisFlow cytometryMolecular testing

Treatment:Aggressive treatment of infectionsImmune neutropenia: Steroids, IVIGLGL leukemia: Low-dose MTXG-CSF: SCN, recovery from drugsStem-cell transplant: SCN

G-CSF OR NO G-CSF?

For neonatal, autoimmune, and non-immune chronic idiopathic neutropenia in adults:

• Treatment is frequently unnecessary

• Reserve G-CSF for recurrent or serious infections

• G-CSF may cause flare of RA

For drug-induced neutropenia/agranulocytosis:

• Evidence-based data lacking: Only randomized trial was negative, but had only 24 patients, and used a subtherapeuticdose of G-CSF

• Meta-analyses & retrospective analyses suggest shorter time to WBC recovery, reduced cost, ? reduced mortality

• 5-10% mortality rate → safety and efficacy justify G-CSF use

CASE PRESENTATION

5-year-old boy responding poorly to antibiotics

HPI:• 3days PTA: Fever, cheek pain

• X-ray: Opacification of right maxillary sinus

• Admitted for poor response to oral antibiotics

PMH:• Multiple episodes of otitis media in first two years of life,

requiring tube placement

• Two episodes of pneumonia requiring hospitalization

• S. aureus abscess of the thigh at age 3

CBC:• WBC 22K, 88% polys, 5% bands

• Plts 608K

• Hb 9.9

MECHANISMS OF NEUTROPHIL FUNCTION

Receptor function/chemotaxis/phagocytosis• Leukocyte adhesion deficiency

• Hyper IgE syndrome (Job syndrome)

Degranulation

• Chédiak-Higashi syndrome

• Specific granule deficiency

Oxygen-dependent killing• Chronic granulomatous disease

• Neutrophil G6PD deficiency

• Glutathione reductase/synthase deficiency

Oxygen-independent killing• Specific granule deficiency

• Myeloperoxidase deficiency

PHAGOCYTE NADPH OXIDASE (NOX2)

Chronic Granulomatous Disease (CGD) Etiology:

• Decreased activity of NADPH oxidase → failure of the respiratory burst

• Heterogeneous disorder

• X-linked (p91 phox = CYBB): 70% of CGD cases

• Others: Autosomal recessive (p47, p67, p22)

CHRONIC GRANULOMATOUS DISEASE

Recurrent infections, with onset usually (not always!) early in life. Oldest patient at diagnosis: Age 69

Infections with opportunistic organisms

Chronic inflammation:

Granulomatous colitis

Restrictive lung disease

Obstruction of gastric outlet, ureters

Immune-mediated disease

Discoid lupus

Macrophage activation syndrome / hemophagocyticlymphohistiocytosis (HLH)

CHRONIC GRANULOMATOUS DISEASE

Treatment:

•IV antibiotics for infections•Interferon gamma

Multicenter trial of IFN showed 70% reduction in infections despite failure to demonstrate increased production of O2

•Stem cell transplantation•Gene therapy?

Poor long-term engraftmentInsertional mutagenesis

CASE PRESENTATION

3-year-old boy with fever, sore throat, poor response to abx

HISTORY:

• PTA: Sore throat and high spiking fever despite antibiotics

• 10 days later, diffuse adenopathy and hepatosplenomegaly

• Cervical lymph node bx: Malignant lymphoma

• Spontaneous remission over next 3 months

• 1 year later, recurrent adenopathy responsive to corticosteroids

• Subsequently relapsed with adenopathy, respiratory distress, died

• Autopsy: Infiltration of lung, liver, nodes, spleen, kidneys with immature lymphoid cells and histiocytes

PMHx: • Recurrent ulcerations of buccal mucosa from early age

• Light coloring, photophobia, nystagmus

FHx: Sister with photophobia, nystagmus

Adapted from Blood 1962;20:330

CHÉDIAK-HIGASHI SYNDROME

Etiology:• Generalized defect of granule morphogenesis

• Neutrophils show multiple functional abnormalities, including impaired granule release

• LYST gene mutation → impaired membrane fusion and granule trafficking (lysosomes and others)

Clinical manifestations: • Oculocutaneous albinism

• Recurrent bacterial infections

• Neuropathies, spinal cord and cerebellar anomalies

• Accelerated phase: Hepatosplenomegaly, pancytopenia, and death, perhaps from EBV → HLH

Disorders of Neutrophil Function: Lab tests

Chronic granulomatous disease Phagocyte oxidase activity by NBT or DHR

Chédiak–Higashi syndrome Leukocyte morphologyBone marrow better than blood

Chemotactic disorders IgE(In vitro leukocyte migration)(In vivo Rebuck skin window)

Leukocyte adhesion deficiency Flow cytometry (CD18, CD11b)

All of the above

Vasculitis, thrombosis, autoimmune, etc.

Gene or genomic sequencing

NETs (neutrophil extracellular traps)

Rapid Heme Panel

• Next generation sequencing of 95 oncogenes• 2 week return• ABL1, ASXL1, ATM, BCL11B, BCOR, BCORL1, BRAF, BRCC3,

CALR, CBL, CBLB, CD79B, CEBPA, CNOT3, CREBBP, CRLF2, CSF1R, CSF3R, CTCF, CTNNB1, CUX1, CXCR4, DNMT3A, DNMT3B, EED, EGFR, EP300, ETV6, FANCL, FBXW7, EZH2, FLT3, GATA1, GATA2, GATA3, GNAS, GNB1, IDH1, IDH2, IKZF1, IKZF2, IKZF3, IL7R, JAK1, JAK2, JAK3, KIT, KRAS, LUC7L2, MAP2K1, MEF2B, MPL, MYD88, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, PAX5, NT5C2, PDS5B, PHF6, PDGFRA, PIGA, PIM1, PRPF40B, PIK3CA, PRPF8, PTEN, PTPN11, RAD21, RET, RIT1, RPL10, RUNX1, SETBP1, SF3B1, SF1, SF3A1, SETD2, SH2B3, SMC1A, SMC3, SRSF2, STAG2, TET2, STAT3, TLR2, TP53, U2AF1, U2AF2, WHSC1, WT1, XPO1, ZRSR2

CLONAL HEMATOPOIESIS:The Road to Leukemogenesis

Blood 2015;126(1):9-16

TAKE-HOME MESSAGES

• Leukocytosis is usually reactive signaling a healthy

marrow

• Peripheral smear and gene sequencing can usually

provide answers to the less common primary marrow

disorders associated with elevated neutrophils. Don‘t

miss CML!

• Neutropenia is more commonly a manifestation of a

primary marrow problem

• Congenital neutropenia and disorders of neutrophil

function have provided key insights into neutrophil

biology

• Next generation sequencing has emerged as a

powerful diagnostic, therapeutic, and research tool

disorders of neutrophils · case presentation. 1-month-old boy with elevated wbcs • 1 month old...

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