Bruno Rotoli MemorialJoint Educational Course

EBMSevere Aplastic Anaemia and Infectious Diseases Working Parties

29th Sept – 1st Oct 2014, Naples, Italy

The differential diagnosis of inherited aplastic anemiasCarlo Dufour

• Differential diagnosis & diagnostic algorythm of inheritedBone Marrow Failure Syndromes (IBMFS)

• Single lineageBlackfan Diamond AnemiaSevere Congenital Neutropenia Congenital Amegacaryocytic ThrombocytopeniaThrombocytopenia Absent Radii (TAR)

Contents

Thrombocytopenia Absent Radii (TAR)

• More than one lineageFanconi AnemiaPearson SyndromeSchwachman Diamond SyndromeVariants of Dyskeratosis Congenita with neonatal onset

Algorythm of most common Bone Marrow Failure Syndromes

Single lineageBlackfan Diamond Anemia

Severe Congenital Neutropenia Congenital Amegacaryocytic ThrombocytopeniaThrombocytopenia Absent Radii (TAR)

More than one lineageFanconi Anemia

Pearson SyndromeSchwachman Diamond SyndromeVariants of Dyskeratosis Congenita with neonatal onset

If FBC shows:Cytopeniaof one, two or three lineage

Bone marrow aspiration

Differential diagnosis, diagnostic algorythm

Bone marrow aspiration(morphology, cytogenetics and if possible colony assays & immunophenotype)

andBone marrow trephyne biopsy

Normal total cellularity,but reduced single lineage

Reduced total cellularityy

Normal total cellularity,but a reduced single lineage

�� Erythroid lineErythroid line

Blackfan-Diamond AnemiaAnemia(BDA)

Blackfan-Diamond Anemia

• Disorder of ribosomal synthesis.

• 14 genes identified so far

11 ribosomal

• About 35% of patients are gene orphan (Italian Registry)

• Macrocytic/normocytic anemia at birth or within first 6 mos birth (ca 60%)

• Reticulocytopenia.

• Elevated red cell ADA.

• Normocellular bone marrow with selective erythroid precursor deficiency.

Blackfan-Diamond Anemia(BDA)

Blackfan-Diamond Anemia

• Malfromations co-exist in 50% of patients (Italian Registry)

• Upper limb

• heart

• cranio-facial• cranio-facial

• uro-genital

• mental retardation

• low stature

•• Transient Erythroblastopenia of Childhood (TEC)

Differential Diagnosis

In active phase In remission

DBA TEC DBA TEC

Previous normal Previous normal blood countsblood counts

No Yes No Yes

MacrocytosisMacrocytosisHb F Hb F ↑↑

80%100%

5%20%

80%72%

0%0%Hb F Hb F ↑↑

Ag i Ag i ↑↑eADA eADA ↑↑

100%100%72%

20%20%0%

72%85%90%

0%0%0%

Permanent Permanent spontaneousspontaneous

remissionremission

yes(usually in 8

months)

•• Autoimmune and postAutoimmune and post--viral cytopeniasviral cytopenias

Culture studies ± autologus serum, ± T cellsViral studies (serology and DNA/RNA)

Quanti/qualitative characteriztion of RNA by capillary electrophoresis with

Agilent Bioanalyzer 2100 - NANO chip

DIAGNOSTIC TEST

Flu

ores

cenc

e

20

25

30

35

40

45

50

Distinct 28S ribosomal subunitDistinct 18S ribosomal subunit

28S

18S

DIAGNOSTIC TEST

18S

28S

Flu

ores

cenc

e

Time (seconds)

0

5

10

15

20

19 24 29 34 39 44 49 54 59 64 69

No well defined peaks between ribosomal peaks

Flat baseline throughout electropherogram

Marker

5S and 5.8S subunits, tRNAs, and small RNA fragments about 100bpMarker

18S

~100 bp

Normal Control

28S

18S28S

18S/28S Subunit ratio test

RPS mutation RPL mutation

18S

28S

Am J Hematol. 2014 Jul 15. doi: [Epub ahead of print]Exploiting pre-rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis.Farrar JE, Quarello P, et al.

Normal global hematopoietic cellularity,but a reduced single lineage

������������ Erythroid lineErythroid lineErythroid line maturation arrest of myelopoiesisat the promyelocyte/myelocyte stage

Severe CongenitalBlackfan-Diamond Severe CongenitalNeutropenia

(SCN)

Blackfan-Diamond Anemia(BDA)

Severe Congenital NeutropeniaSevere Congenital Neutropenia(SCN)(SCN)

Severe Congenital Neutropenia(SCN)

• Genes associated with maturative block

ELA-2HAX-1G6PC3WAS (exon 9)JAGN1

• Gene associated with no block + physical abnormalities• Gene associated with no block + physical abnormalities

VPS45, CSF3R

• Genes associated with dmmune deficiency

GFI 1WASCXCR4 (WHIM)COH (Cohen)BTK X linked , CD40 L (Iper IgM)

Normal global hematopoietic cellularity,Normal global hematopoietic cellularity,but a but a reduced single lineage reduced single lineage

������������ Erythroid lineErythroid lineErythroid line maturation arrest of myelopoesismaturation arrest of myelopoesismaturation arrest of myelopoesisat the promyelocyte/myelocyte stageat the promyelocyte/myelocyte stageat the promyelocyte/myelocyte stage

�/absent mega-karyocytes

Severe Severe Severe CongenitalCongenitalCongenitalNeutropeniaNeutropeniaNeutropenia

(SCN)(SCN)(SCN)

BlackfanBlackfanBlackfan---Diamond AnemiaDiamond AnemiaDiamond Anemia

(BDA)(BDA)(BDA)

Congenital AmegakaryocyticThrombocytopenia

(CAMT)

Thrombocytopenia absentradii

(TAR)

Congenital Amegacaryocytic Thrombocytopenia Congenital Amegacaryocytic Thrombocytopenia CAMTCAMT

• Severe thrombocytopenia in neonatal period associated with bleeding

• Normocellular bone marrow with selective deficiency/absence of megacaryocytes

• Normal MPV

• Progressive evolution to pancytopenia and AML.

• High TPOserum level.

• Mutation in the gene of thrombopoietin receptor- c-mpl -that impairsmega and other lineages developement .

Thrombocytopenia absent radiiThrombocytopenia absent radiiTARTAR

• Modertate/severe thrombocytopenia in neonatal period

• Not marked bleeding tendency

• Thrombocytopenia tends to improve over time

• No tenedency to develop leukemia and aplastic anemia

Normal global hematopoietic cellularity,but reduced single lineage

� Erythroid line maturation arrest of myelopoesisat the promyelocyte/myelocyte stage

�/absent megakaryocytes

Severe Congenital Blackfan-Diamond Congenital Amegakaryocytic

Summary 1Summary 1

Severe Congenital Neutropenia

(SCN)

Blackfan-Diamond Anemia(BDA))

Congenital AmegakaryocyticThrombocytopenia

(CAMT)

Thrombocytopenia absentradii

!Memo! A single lineage disesease can, overtime, become a global

marrow failure (DBA, cAMT, Pearson, SDS)

Sites of neonatal hematopoiesis

Normal hematological values in the neonate

Diagnostic difficulties and limits in the neonate

Algorythm of most common Bone Marrow Failure Syndromes

Single lineageSingle lineageBlackfan Diamond Anemia

Severe Congenital Neutropenia Congenital Amegacaryocytic ThrombocytopeniaThrombocytopenia Absent Radii (TAR)

More than one lineageFanconi Anemia

Pearson SyndromeSchwachman Diamond SyndromeVariants of Dyskeratosis Congenita with neonatal onset

If FBC shows:CytopeniaOof one, two, three lineages

Bone marrow aspiration

Differential diagnosis, diagnostic algorythm

Bone marrow aspiration (morphology, cytogenetics, immunophenotype, colony assays)

± Bone marrow trephyne biopsy

Normal total cellularity,but reduced single lineage

Reduced total cellularityy

TOTAL reduced hematopoietic cellularity

no major dysplasia+

normal cyogenetics+

blasts <5%

dysplasiadysplasia+/+/--

abnormalabnormal cytogeneticscytogenetics+/+/--

ALIPALIP+/+/--

fibrosisfibrosis+ +

blastsblasts >5%>5%++

+ + somaticabnormalities

-- Somaticabnormalities

•Fanconi Anemia (FA)•Pearson Syndrome (PS)•Shwachman Diamond Syndrome(SDS)•Variants of DKC with neonatal onset(Hoyerdaal-Hreidarsson Syndrome, ReveszSyndrome, Clericuzio Syndrome)

•Other genetic thrombocytopenias

•CAMT in aplastic phase•Reticular dysgenis•Asymptomatic DC

++increasedincreased CD 34+CD 34+

Fanconi Anemia

• Progressive pancytopenia that usually develops in the first decade of life+/-somatic malformations

• Somatic malformations diagnostic suspect,

esophagus,

GI-tract, genitourinary tract, GI-tract, genitourinary tract,

upper limbs,

hands/thumb,

VACTERL complex (vertebral, anal atresia, cardiac, tracheo-esophagealfistula, renal, limb)

• DNA-repair deficiency disease, cancer proness, bone marrow failure

• Diagnosis : Chromosomal fragility test (DEB/MMC)

• NGS

FA genes

16 known FA genes

FANCA

FANCB

FANCC

FANCD1 (BRCA2)

.10FANCD1 (BRCA2)

FANCD2

FANCE

FANCF

FANCG

FANCI

FANCJ (BRIP1)

FANCL

FANCM

FANCN (PALB2)

.59.14

NEW GENES: RAD51C (FANCO), SLX4 (FANCP) and ERCC4 (FANCQ)

IFAR 2008

80

100

Cell survival (%)

Control

Fanconi

Complementation analysis of FA patients

0

20

40

60

0 3 10 33 100 333

MMC (mM)

Cell survival (%)

Fanconi+FA-G

Fanconi+FA-A

Pearson Syndrome

• Macrocytic anemia in first months of life, neutropenia and thrombocytopenia,

• Characteristic bone marrow morphology:

vacuolated myeloid and erythroid precursors,

ringed sideroblasts.ringed sideroblasts.

• Deletion of mitochondrial DNA.

• Metabolic acidosis.

• Exocrine pancreas insufficiency, liver and renal failure

Vacuolated myeloyd cells

Pearson Syndrome

• Macrocytic anemia in first months of life, neutropenia and thrombocytopenia,

• Characteristic bone marrow morphology:

vacuolated myeloid and erythroid precursors,

ringed sideroblasts.ringed sideroblasts.

• Metabolic acidosis.

• Exocrine pancreas insufficiency, diarrhoea/steatorrhoea.

• Liver and renal failure.

• Deletion of mitochondrial DNA.

Shwachman-Diamond Syndrome

• Neutropenia associated with severe infections (skin, lungs) in first months of life. May fluctuate

• Anemia (Macrocytic) and thrombocytopenia develop in up to 80% of pts.

• Exocrine pancreas insufficiency.

• Skeletal abnormalities (long bone metaphyses, costochondraljunction).

• Others: liver, kidney, brain, immune system.

• Mutation analysis

10% of patients are mutation orphan,

monoallelic mutations,

variants with no phenotypical consequences.

Hoyeraal-Hreidarsson Syndrome (DKC1)IUGR,microcephaly, cerebellar hypoplasia, progressive pancytopenia and immunodeficiency

Revesz Syndrome (TNF 2)

DKC Variants with neonatal presentation

Revesz Syndrome (TNF 2)

Bilateral exudative rethinopaty

CNS calcifications,

cerebellar hypolasia

Clericuzio Syndrome (Neutropenia with Poikyloderma (c16orf 57)

PoikylodermaFacial dysmorphismsNail dysotrophyNeutropenia

Poichiloderma + nail dystrophy + facial dysmorphism

•Midollo ipocellulato senza blocco maturativo

Progenitori: CFU-MK normaliCFU-e/BFU-e lievemente ridottiCFU-GM marcatamente ridotti 3 (vn 33-100)

TOTAL reduced hematopoietic cellularity

no major dysplasia+

normal cyogenetics+

blasts <5%

+ somaticabnormalities

-- Somaticabnormalities

•Fanconi Anemia (FA•Pearson Syndrome (PS)•Shwachman Diamond Syndrome(SDS)•Variants of DKC with neonatal onset

• cAMT

•Reticular dysenesis (hypoplasia thymus)

•Asymptomatic DC.

TOTALTOTAL redduced hematopoietic cellularityredduced hematopoietic cellularity

no major dysplasiano major dysplasia++

normal cyogeneticsnormal cyogenetics++

NO ALIPNO ALIP++

No fibrosisNo fibrosis++

blasts <5% blasts <5% ++

reduced CD34+reduced CD34+

dysplasiadysplasia+/+/--

abnormalabnormal cytogeneticscytogenetics+/+/--

ALIPALIP+/+/--

fibrosisfibrosis+ +

blastsblasts >5%>5%++

increasedincreased CD 34+CD 34+

YES somatic YES somatic abnormalitiesabnormalities

NoNo ssomaticomaticabnormalitiesabnormalities

Hypocellular congenital Hypocellular congenital MDSMDS

Runx1, CeBPA, GATA1Runx1, CeBPA, GATA1

••Fanconi Anemia (FA) (MMC or DEB test)Fanconi Anemia (FA) (MMC or DEB test)••Dyskeratosis Congenita (DC)Dyskeratosis Congenita (DC)TERC, TERt, TNF2, DKC1 gene mutationsTERC, TERt, TNF2, DKC1 gene mutations

••Pearson Syndrome (Mithocondrila DNA)Pearson Syndrome (Mithocondrila DNA)••Shwachman Diamond Syndrome Shwachman Diamond Syndrome SDS gene muations)SDS gene muations)••BlackfanBlackfan--Diamond Anemia Diamond Anemia (BDA gene muations)(BDA gene muations)

••Other genetic thrombocytopeniasOther genetic thrombocytopenias

•• Aplastic AnemiaAplastic Anemia•• CAMTCAMT ((ccmpl gene mutationsmpl gene mutations))•• Asymptomatic DCAsymptomatic DC

(TERC, TERt, TNF2, DKC1 gene (TERC, TERt, TNF2, DKC1 gene mutations)mutations)

•• FAFA (MMC or DEB test)(MMC or DEB test)

CytopeniaCytopenia

Bone marrow aspirationBone marrow aspiration ++-- trephyne biopsytrephyne biopsy

single lineage reducedsingle lineage reduced

�������� Erythroid Erythroid line line

maturation maturation arrest of arrest of

myelopoesismyelopoesis

��MegakaryoMegakaryocytescytes

reduced hematopoietic cellularityreduced hematopoietic cellularity

no major dysplasia +no major dysplasia +normal cytogenetic +normal cytogenetic +

blasts <5%blasts <5%

dysplasiadysplasia+/+/--

abnormalabnormalcytogeneticscytogenetics

+/+/--

Summary 2 Summary 2

SCNSCNDBADBA CAMTCAMTThrombThrombocytopniocytopniaa--absent absent

radiiradii

+ somatic + somatic abnormalitiesabnormalities

-- SomaticSomaticabnormalitiesabnormalities

••FA FA (DEB test)(DEB test)••DKC VariantsDKC Variants••HHSHHS••PSPS••SDSSDS••BDABDA••Other genetic Other genetic thrombocytopeniasthrombocytopenias

••AAAA••CAMTCAMT••Reticular Reticular dysgenesisdysgenesis

eADAeADA

+/+/--fibrosisfibrosis

+ + blastsblasts >5%>5%

++increasedincreased CD 34+CD 34+

Congenital Congenital hypocellular hypocellular

MDSMDS

Take Home Message

• Diagnosis of IMFS may be challenging.

• NGS promising but not fully established at least in many europeancountries.

• Early diagnosis is important to address subsequent monitoring/ treatment strategies.

• Follow-up supported/driven by ped-hem team.