Kostmann syndrome

eller“Severe congenital neutropenia”

Nordkalotten050526

Göran Carlsson

Neutropenia

• Definition– ANC < 1.5x109/L (– Mild 1-1.5x109/L– Moderate 0.5-1x109/L– Severe < 0,5x109/L– Agranulocytosis < 0,2x109/L– Chronic > 3 months– African Americans have 0.2-0.6x109/L lower

ANC than Caucasians

Rolf Kostmann

• Rolf Kostmann 1:e barnläkaren i Boden (1942-1952)

• 1949 inkom en 2 mån flicka med otit, mastoiditoch hudinfektioner.

• Blodstatus visade agranulocytos. I benmärgenmognadsstopp i myelopoesen påpromyelocyt/myelocyt nivå.

• Hon var 9:e barnet, 4 syskon hade dött i späd åldervarav 3 i feber o bölder.

• Ärftlig agranulocytos ej beskrivet

“In this work, a new disease, infantile genetic agranulocytosis, will be described.

As a rule, the outcome of this disease is fatal within the first year of life.

The children succumb to infections of various kinds, most commonly manifested as septic skin conditions.

The affected children have been discovered in a certain district in Norrbotten, the northernmost province of Sweden.

The disease appears to depend primarily on a single recessive gene difference” (Kostmann, 1956).

Patients with agranulocytosis reported from a small municipality in the north

of Sweden

• 1956 6 + 8 (2 later excluded) patients (Kostmann)• 1975 10 patients (Kostmann)• 2001 5 patients (Carlsson & Fasth, Paper 1)• Total 27 patients

Pedigree of ”Kostmann family”. The family tree is based on the pedigree published by Kostmann 1956 and 1975. The letters appoint the different subfamilies used by Kostmann. The patients in paper I are marked P1– 6

25/27 patients had been able to be connected to one extended family

Myelopoesen and mognadsstoppet vid Kostmanns sjukdom

Myeloblast Promyelo-cyte

Myelocyte Meta-myelocyte

Band cells

Segmented cells

Maturation stop

Background - Mb Kostmann 1

• Severe Congenital Neutropenia, SCN.• Rare disorder: 1-2/1.000.000 population.• Absolute Neutrophil Count < 0.2x109/L.• In the bone marrow typical maturation

arrest in the myelopoiesis at promyelocyte-myelocyte level.

• Severe bacterial infections.

Background - MB Kostmann 2

• Earlier fatal during the first year of life. • Treatment today: Granulocyte Colony-Stimulating

Factor (G-CSF). • G-CSF normalize ANC and life threatening

infections disappear.• Probably normal life span today if not develop

MDS/AML (10%).• Still though problems with bacterial infections

especially chronic gingivitis and periodontitis.

Kostmanns sjukdom• MDS/AML hos 13.7% (58/422, SCNIR)

– Är associerat med en eller flera förvärvade genetiska abnormiteter

• G-CSFReceptor mutation >80% • Monosomi 7 eller andra cytogenetiska abberationer

>90%• ras mutation 50%

– NE mutation 90%– G-CSF association?

Kostmanns sjukdom

• Osteoporos – Incidens oklar

• I hela SCNIRs patientmaterial ca 15%

– Bentäthets mätning av 236 pat visade patologi hos 113 (48%) men bara 67 (67/236=28%) av dessa hade kliniska problem

– G-CSF association?

Kostmanns sjukdom

• Behandling – G-CSF (Neupogen®, Granocyte®)

• Starta med 5 µg/kg och dygn– Sänka eller höja dosen efter 1-2 veckor

• Målsättning ANC 1.0-5.0x109/L– Högre nivåer kan krävas för att ha kontroll på

infektionerna

– Tandläkare/tandhygienist

KOSTMANN SYNDROME

A Clinical and Pathophysiological study

Göran Carlsson

Aim of the thesis

The general aim of this project was to improve our understanding of the clinical

course and the pathophysiology of the autosomal recessive Kostmann syndrome

The specific aims of the thesis

• Characterize the clinical presentation and course (I)• Investigate the neutrophils’ antibacterial capacity

(II)• Elucidate the role of apoptosis in the maturation

arrest (III)• Study the efficacy of different granulocyte colony-

stimulating factor (G-CSF) preparations (IV)• Characterize the oral presentation and course (V)

Paper I

Infantile genetic agranulocytosis, morbus Kostmann: presentation of six cases from

the original “Kostmann family” and a review

Carlsson G, Fasth A.

Acta Paediatr 2001;90:757-764

Clinical study

Why?• Patients from the “Kostmann family” and with

autosomal recessive inheritance• Pathophysiology still mainly unclear • Comparing the experience from Severe Chronic

Neutropenia International Registry (SCNIR)• Preleukemic illness? Osteoporosis?

• Remaining problems?• Suggestions of further studies

Six children with Kostmann syndrome diagnosed after 1970. Clinical data and laboratory findings.

PatientYear of

birth Sex

Age at diagnosis

(mo)ANC at

diagnosis Alive

Age at death/last follow up

(y)1 1973 F 2 0.1 No 122 1976 F 0,5 0.3 Yes 243 1977 M 0.2 0.2 No 0.54 1983 F 5 0.3 Yes 175 1984 M 2 0 Yes 166 1993 M 5 0.1 Yes 7

ANC = Absolute neutrophil count x109/L. Reference range 1.7-8.7 for infants.

Six children with Kostmann syndrome diagnosed after 1970. Clinical data and laboratory findings.

PatientYear of

birth Sex

Age at death/last follow up

(y)G-CSF

receptorCytogenetics

Bone density Other important clinical findings

1 1973 F 122 1976 F 24 Normal Normal Normal Severe periodontitis with AA3 1977 M 0.54 1983 F 17 Normal Normal Osteoporosis Chronic gingivitis and mild MeR5 1984 M 16 Normal Normal Normal BMT, ADHD and epilepsy6 1993 M 7 Normal Normal Normal Chronic gingivitis and delayed

psychomotor development

AA = Actinobacillus actinomycetemcomitans; MeR = Mental retardation; HSCT = Hematopoietic stem cell transplantation; ADHD = Attention deficit hyperactivity disorder

Summary of Paper I

• Prior to G-CSF:– Recurrent potential life-threatening bacterial

infections– Chronic periodontal disease– Surviving due to modern antibiotics and better

care– Similar presentation as reported of SCNIR

Summary of Paper I

• On G-CSF: – All patients respond with normal ANC– Marked reduction of bacterial infections– Much improved quality of life– Improved oral status for most patients, but still

a problem and for one patient deterioration to aggressive periodontitis

– No transformation to myelodysplastic syndrome/acute myeloid leukemia(MDS/AML) at that time

Paper II

Deficiency of antibacterial peptides in patients with morbus Kostmann: an

observation study

Katrin Pütsep, Göran Carlsson, Hans G Boman, Mats Andersson

Lancet 2002;360:1144-1149

The periodontal disease

• The pathogenesis is multifactorial and is a disturbed homeostasis between the host and the microbes

• It is mainly an opportunistic infection by oral bacteria that causes inflammation and tissue destruction

• The neutrophils are important to prevent infections in the gingiva by phagocytosis and killing of the microflora

Background – Antibacterial peptide

• All animals and plants produce antibacterial peptides

• They are important for the innate immunity and first line defence

• Potency comparable to classical antibiotics• In humans in epithelial cells, leukocytes especially

neutrophils• Cathelin-LL-37 (proform to the active peptide LL-

37) are stored in secondary granules and α-defensins (HNP1-3) in primary granules

Antibacterial peptides

– These peptides have microbial targets– Usually bacterial membranes, which are

sufficiently different from cell membranes in the host

– Bacterial lysis is the most common mechanism to kill the bacteria, either by formation of pores in the membrane or by destabilization of the entire membrane

Question?

• Are the neutrophils in Kostmann syndrome deficient in antibacterial peptides?

Methods• We studied 4 patients from the original Kostmann family and 2 other

patients with congenital neutropenia (1 with unknown inheritance and one with a dominant cyclic neutropenia)

• Controls were parents and siblings and 3 unrelated individuals (22 healthy persons)

• We studied samples of neutrophils, plasma and saliva • Western blot with antibody to LL-37 and cathelin-LL-37 for all

samples• High performance liquid chromatography (HPLC) and mass

spectrometry for α-defensins in neutrophils• Neutrophils were also tested for lactoferrin and ability to produce

oxidative burst

Characteristics of patients and controlsand some results

Western blot on samples from neutrophils and plasma

Western blot on saliva

Cel

lsMB PM MC MMC BC SeC

Maturation arrest in Kostmann syndrome

Gra

nule

s

Primary Secondary GelatinaseSecretoryvesicles

Defensins

Elastase

BPI

Proteinase-3

Cathelin-LL-37

Lactoferrin

Gelatinase Plasma proteins

In g

ranu

la m

atrix

Prot

ein

synt

hesi

s

Defensins

Cathelin-LL-37

mR

NA Defensins

Cathelin-LL-37

Conclusions

• Neutrophils G-CSF treated Kostmann patients have sufficient number of neutrophils in the blood

• Neutrophils from our patients lack cathelin-LL-37 and LL-37 and have reduced level of α-defensins

• Saliva also lack cathelin-LL-37 and LL-37 • Our patients have problems with chronic gingivitis

and periodontitis• Here we have found a correlation – and evidence

of in-vivo role of antibacterial peptides in humans

Paper IIIKostmann syndrome: severe congenital

neutropenia associated with defective expression of Bcl-2, constitutive

mitochondrial release of cytochrome c, and excessive apoptosis of myeloid progenitor

cells

Göran Carlsson, Andrew A.G. Aprikyan, Ramin Tehranchi, David C. Dale, Anna Porwit, Eva Hellström-Lindberg, Jan

Palmblad, Jan-Inge Henter and Bengt Fadeel

Blood 2004;103:3355-3361

Background Apoptosis

• The regulated destruction of a cell• Tissue hemostasis is the balance between

proliferation and apoptosis • Excessive apoptosis is showed in other BM

disorders as myelokathexis, cyclic neutropenia and MDS

• Question: Is the maturation arrest due to decreased survival of myeloid progenitors resulting from accelerated apoptosis?

cyt c

Cellular stress

Bcl-2

Death receptor Fas

Caspase-8

Bid

(“initiator”)

Caspase-3(“effector”)

Death

Apaf-1

Caspase-9

Bax

“PS exposure”

Bcl-2 family members regulate apoptosis

• Bcl-2 is anti-apoptotic– Blocks cytochrome c release

• Bax is pro-apoptotic– Induce cytochrome c release

cyt c

Bcl-2

Bax

Mitochondria

Apoptosis

Patients, material and methods

• 5 patients with SCN• Controls: 8 healthy adults • BM-biopsies before and during G-CSF treatment

and fresh BM-aspirations • Morphology• Immunohistochemical staining (Ki-67, Bcl-2 and

Bax)• Flow cytometry for Bcl-2• Apoptosis assays with isolated BM-cells• Cytochrome c release

Carlsson et al., Fig. 1

A

Bprior to G-CSF

prior to G-CSF

after G-CSF

after G-CSF

patient 2

Carlsson et al., Fig. 2

prior to G-CSF

after G-CSF

control

Bcl-2 Bax

A

B

0

20

40

60

80

100

Bcl

-2-F

ITC

MFI

patie

nt 1

patie

nt 3

patie

nt 4

patie

nt 5

dono

rs

patient 2

patient 3

Car

lsso

n et

al.,

Fig

. 4B

anti-F

as+z

VAD-fmk

anti-F

as+z

VAD-fmk

0

2

4

6

8

10

DEV

D-A

MC

cle

avag

e (p

mol

/min

)

spon

t.+z

VAD-fmk

anti-F

as

spon

t.+z

VAD-fmk

spon

t.+z

VAD-fmk

anti-F

as

patient 1 patient 5 donors

A+zVAD-fmkspont.

anti-Fas + zVAD-fmkanti-Fas

+zVAD-fmkspont.

patient 2

donor

annexin V fluorescence

prop

idiu

m io

dide

fluo

resc

ence

11% 4%

12% 4%

9% 6%

CD34+ cells

A

control no G-CSF G-CSF 4 hours

CD34+

CD33+

0

10

20

30

40B

% c

ytoc

hrom

e c

rele

ase

patient 1 patient 3 donor 1 donor 2patient 4 patient 5C

0

20

40

60

80

% c

ytoc

hrom

e c

rele

ase

patient 1 patient 3 donor 1 donor 2patient 4 patient 5

Carlsson et al., Fig. 5

CD33+

CD34+

patient 3

cyt c

Cellular stress

Bcl-2

Death receptor Fas

Caspase-8

Bid

(“initiator”)

Caspase-3(“effector”)

Death

Apaf-1

Caspase-9

BaxG-CSF

Conclusions 1

• An elevated degree of apoptosis was observed

• Selective decrease of anti-apoptotic Bcl-2 in myeloid progenitor cells

• Increased in vitro apoptosis and cytochrome c release in CD34+ and CD33+ cells

• G-CSF restored Bcl-2 expression and decreased in vitro apoptosis

Conclusions 2

These studies support a role for mitochondria-dependent apoptosis in the pathogenesis in Kostmann syndrome and an explanation of the maturation arrest. Further that G-CSF seems to have anti-apoptotic effects and inhibit cytochrome c release.

Paper IV

Efficacy and safety of two different rG-CSFpreparations in the treatment of patients with

severe congenital neutropenia

Göran Carlsson, Anders Åhlin, Göran Dahllöf, Göran Elinder, Jan-Inge Henter, Jan Palmblad.

Br J Haematology 2004;126:127-132

• G-CSF raise the ANC count over 1.0 x 109/L in more than 90 % of patients with SCN

• A higher biological activity has been reported for the glycosylated than for the non-glycosylated G-CSF in vitro and in healthy volunteers

• The rHuG-CSF preparations:– Glycosylated - lenograstim (Granocyte®)– Non-glycosylated - filgrastim (Neupogen®)

The aims were• Any difference in the biological activity

assessing ANC values between the two G-CSF preparations in equimolar doses?

• Any differences in the incidence and severity of infections, number of dose modifications and the state of gingivitis?

8 patients with SCN from 4 different hospitals in Sweden were

studied between Dec 1997 and June 2000.

Run in 12 weeks

Lenograstim12 weeks

Lenograstim12 weeks

Filgrastim12 weeks

Filgrastim12 weeks

Filgrastim12 weeks

Lenograstim12 weeks

Filgrastim12 weeks

Lenograstim12 weeks

Randomisation

Summary of resultsLenograstim Filgrastim

Mean of ANC x 109/L^ 4.17 (n=73) 5.14 (n=78)Median of ANC x 109/L 3.50 (n=73) 5.05 (n=78)Quartile for all ANC x 109/L 2.2-5.8 (n=73) 2.3-7.1 (n=78)Number of ANC < 1x109/L 2 2Number of ANC >10x109/L 2 4Number of dose changes 1 1Infections according to definition 2 3Number of antibiotic courses 9 9Number of hospital care 0 1*Median GBI% change from baseline -4.1 -2.0*EBV-infection and subsequent, suspected septicaemia^p = 0.042 (z-test)

0

5

10AN

C,x

10/L9

Lenograstim Filgrastim

Mean ANC per treatment period

0

2

4

6

8

10

Patient number and treatment period

AN

C x

10E9

/L

LenograstimFilgrastim

1/1 1/2 2/1 3/1 3/2 4/1 4/2 5/1 5/2 6/1 6/2 8/1 8/2

Wilcoxon’s signed rank test p=0.033

ANC values patient 4

ANC Patient 4

0

3

6

9

0 12 24 36 48

Week

x10E

9/l

Filgrastim Lenograstim Filgrastim Lenograstim

Conclusions

• Both drugs were efficient in maintaining ANC levels above 1.0x109/L

• There were no significant differences in the frequencies of infections, antibiotic courses, gingival bleeding or hospital admissions

• The non-glycosylated G-CSF was associated with higher ANC values

• Both drugs were well tolerated, clinically effective and safe

• No advantage of the glycosylation of G-CSF in the treatment of patient with SCN

CONCLUSIONS OF THE THESIS

• G-CSF normalize ANC in our patient group• Glycosylated G-CSF showed no advantage • Periodontal disease despite normal ANC• Patients investigated, with SCN and periodontal

disease, lack LL-37 in neutrophils and in saliva• Patients still need dental care• Increased apoptosis was observed in bone marrow and

in myeloid precursors, and was counteracted by G-CSF

• A role of mitochondria-dependent apoptosis in the pathogenesis in Kostmann syndrome and an explanation of the maturation arrest