Disorders of Neutrophils

Home , Agranulocytosis, Autoimmune neutropenia, Cyclic neutropenia, Leukocytosis

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. MYELOPOIESIS

myeloblast promyelocyte myelocyte metamyelocyte band segmented neutrophil

HSC PMN ▪phagocytosis ▪chemotaxis ▪respiratory burst ▪NETs

▪O2 independent killing

10 granule 20 granule mRNA mRNA NETosis

Neutrophil extracellular traps form NETS

Activates Histone Cell membrane Stimulation granules processing ruptures of receptors and expelling DNA adhesion J. Cell Biol 2012;198(5):773-83 CYTOKINES GOVERN MYELOPOIESIS

G-CSF/GM-CSF are produced by endothelium and HSC macrophages Proliferate myeloid progenitors Induce myeloid maturation Protect from apoptosis Enhance neutrophil function G-CSF DEFICIENCY GM-CSF DEFICIENCY - Relative neutropenia - No neutrophil defect - Pulmonary alveolar PMN proteinosis Dranoff G and Mulligan RC. Stem Cells;1994;12 Suppl 1:173-82 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 ILLNESSES Infection Acute: Demargination/release storage pool Chronic: Granulomatous dx (leukoerythroblastic) Stress, obesity Smoking Drug-induced (steroids, b-agonists, lithium, G-CSF) Chronic inflammation Post-splenectomy Non-hematologic malignancy Marrow stimulation (ITP, hemolysis) PRIMARY HEMATOLOGIC DISEASE CML Other myeloproliferative disease Lymphoproliferative 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

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

Curr Opin Hematol 2002;9:30-35 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 NEUTROPENIA

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 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 neutropenia Constitutional neutropenia: Duffy Ag Receptor Chemokine (DARC) SNP Benign neutropenia (familial, idiopathic) Congenital Severe congenital neutropenia (Kostmann’s syndrome) Cyclic neutropenia Chediak-Higashi Schwachman-Diamond CONGENITAL NEUTROPENIA SYNDROME INHERITANCE GENES Ethnic neutropenia ? DARC SNPS Benign familial neutropenia AD Unknown AD ELANE (55-60%) AR HAX1 and G6PC3 (<5%) Severe congenital neutropenia 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 X-linked DKC1 (80%) Dyskeratosis Congenita 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 SEVERE CONGENITAL NEUTROPENIA

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 SEVERE CONGENITAL NEUTROPENIA

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; ?SLE Age 26: Fatigue, adenopathy, oral ulcers, arthritis. Leukopenia, thrombocytopenia, +ANA, +ACA Age 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

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

Ann Hematol 1991;63:249-252 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, Sjogren syndrome) • 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

Procainamide, propranolol, Cardiac medications 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 500 Neutropenia 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

Fever, Drug or Yes Discontinue drug ANC <500/µL? Toxin? or toxin Yes No Abnormal growth and development, Admit for IV skin, hair, bone, appendage, antibiotics; consider nail changes? G-CSF Family history of neutropenia, recurrent Infection, sudden death? No Yes

Evaluate for Yes No Serial follow-up Evaluate for congenital of clinical genetic syndromes neutropenia features and assoc. with neutropenia Appropriate cell counts No ethnic group? Stable/Improved Progressive Yes Bone marrow biopsy, Ethnic or benign familial Immune or Idiopathic aspirate, and flow neutropenia neutropenia cytometry Jacobson and Berliner. Wintrobe’s Clinical Hematology. Lippincott Williams and Wilkins, 13th Edition, 2011 EVALUATION OF THE NEUTROPENIC ADULT

Neutropenia

Yes Fever, Drug or Discontinue drug or toxin ANC <500/µL? Toxin?

Yes No Admit for IV No Ethnic or benign familial neutropenia, antibiotics; consider New onset? or cyclic neutropenia G-CSF Yes Pancytopenia Low B12 or folate, Single or multi- or autoimmune lineage cytopenia? disease? MDS Abnormal Isolated neutropenia cytogenetics No Bone marrow biopsy, aspirate, and flow Idiopathic Yes Normal cytometry neutropenia Low B12, Autoimmune folate disease Megaloblastic Immune LGL anemia cytopenia LGL by flow Jacobson and Berliner. Wintrobe’s Clinical Hematology. Lippincott Williams and Wilkins, 13th Edition, 2011 MANAGEMENT OF NEUTROPENIA

Diagnosis: Stop potential offending drugs Bone marrow aspiration/biopsy Serologic studies: ANA, viral titers R/O Primary malignancy: Chromosome analysis Flow cytometry Molecular testing Treatment: Aggressive treatment of infections Immune neutropenia: Steroids, IVIG LGL leukemia: Low-dose MTX G-CSF: SCN, recovery from drugs Stem-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 subtherapeutic dose 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 / hemophagocytic lymphohistiocytosis (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 engraftment Insertional 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 morphology Bone 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 Gene or genomic sequencing

Vasculitis, thrombosis, NETs (neutrophil extracellular traps) autoimmune, etc. 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 Rapid Heme Panel

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