myelodysplastic syndromes, aplastic anemia, & other bone marrow

Myelodysplastic Syndromes,

Aplastic Anemia, & Other Bone

Marrow Failure States

ASCP 10-22-11

Friederike Kreisel, MD

Washington University

Faculty/Author/Speaker Disclosure

The faculty/speakers for this live session do

not have relevant relationships with

commercial interests to disclose

Credit type: HEME, SURGCredit type: HEME, SURG

Myelodysplastic Syndromes (MDS)

• Clonal proliferation combined with

increased apoptosis

– Cytopenia(s)– Cytopenia(s)

– Dysplasia in one or more myeloid lines

– Ineffective hematopoiesis

– Increased risk of transformation to acute

myeloid leukemia (~30% of cases)

Myelodysplastic Syndromes (MDS)

• Incidence has increased from 3.3 per 100,000 in

2001 to 3.8 per 100,000 in 2006

– ~10% of MDS patients are <50 years old

– Exceedingly rare in children (0.01 per 100,000)– Exceedingly rare in children (0.01 per 100,000)

• The increase has been attributed to

– Enhanced awareness

– Aging population

– Availability of more effective therapies, making

hematologists more likely to pursue the diagnosis

Pathophysiology of MDS• Genetic alterations

– del(5q) is best understood MDS-associated molecular abnormality: RPS14 (codes for a ribosomal subunit) has been shown to induce a MDS phenotype when experimentally inhibited and to correct the MDS phenotype when resupplied to cells with del(5q) phenotype when resupplied to cells with del(5q)

• Epigenetic alterations– DNA hypermethylation – silences gene transcription by hypermethylation of CpG islands in promotor regions – silencing of tumor suppressor genes – may to some degree be reversed by cytidine analogues (i.e. decitabine) that irreversibly bind DNA methyltransferases – decreased methylation throughout the genome

Case #1

• 69-year old female with pancytopenia:

– WBC: 2.2 (4.5-11 K/cumm)

– Hemoglobin: 10.6 (13.0-17.0 g/dl)– Hemoglobin: 10.6 (13.0-17.0 g/dl)

– Platelets: 103 (140-400 K/cumm)

Patient #1 Peripheral Blood

Neutrophils

Monocyte

Pseudo-Pelger Huet

Normal

Neutrophils

Patient #1

Pseudo-Pelger Huet

Pseudo-Pelger Huet

Patient #1 Bone Marrow

Normal Patient #1

Patient #1, LederPatient #1, H&E

Normal, H&E Normal, Leder

Spicules

Normal

Patient #1

Spicules

Promyelocyte

Pronormoblast Myelocyte

Normal

Megakaryocyte

Promyelocyte

Erythroid precursors

Neutrophils

Erythroid precursors

Patient #1

Megakaryocytes

Patient #1

Erythroid precursor

with nuclear budding

Metamyelocyte

Myeloblast

Megakaryocyte

Patient #1

Ring sideroblast

Patient #1

Ring sideroblasts

Patient #1

Cytogenetics

• Fluorescence in situ hybridization for MDS

panel:

– -7/del7q (D7S486): negative

– -5/del5q (EGR1): negative

– del20q (D20s108): negative

– 12p13 (ETV6): negative

– +8 (CEP8): negative

– del13q (D13S319): negative

• Normal conventional karyotype

DiagnosisHypercellular marrow for age with multilineage

dysplasia; no excess blasts seen

Comment: Dyspoiesis is a morphologic change that

can be due to myelotoxic / myelosuppressive

drugs, nutritional deficiency, chronic viral infection, drugs, nutritional deficiency, chronic viral infection,

severe or sustained inflammatory conditions, or

primary myelodysplastic syndrome (MDS).

Diagnosis of MDS, specifically, refractory

cytopenia with multilineage dysplasia may be

considered if all secondary causes are excluded.

Dysplasia

(≥≥≥≥10% of lineage)

Increased blasts or Auer rods?

(PB>1% or BM>5%; <20% and no

recurrent cytogenetics of AMLN

1 lineage ≥≥≥≥2 lineageRefractory cytopenia with

multilineage dysplasia

≤≤≤≤ bicytopenia pancytopenia

Refractory cytopenia with

unilineage dysplasia

Y

1-4%PB

blasts or

5-9% BM

blasts +

≥≥≥≥15% ringed

sideroblasts

Refractory anemia with

ring sideroblastsY

Isolated del(5q) Myelodysplastic syndrome

with isolated del(5q)

Refractory anemia, refractory

neutropenia, refractory

thrombocytopenia

YN

N

blasts +

cytopenias

Auer rods

5-19%PB

blasts or

10-19% BM

blasts +

cytopenias

RAEB-2RAEB-1

Myelodysplasia Algorithm. Adapted from K. Faucar, 3rd ed. p. 343

Approach to the Diagnosis of MDS

• Correlate with CBC (cytopenia or cytosis?)

• Perform an iron stain on aspirate, core or clot section– Ringed sideroblasts can be found in

• Myelodysplastic syndrome• Myelodysplastic syndrome

• Sideroblastic anemia

• Alcoholism

• Hyposplenism

• Lead poisoning

• Cooper deficiency

• Zinc intoxication

• Drugs (isoniazid, chloramphenicol)

From Color Atlas

of Hematology,

E. Glassy


• Check laboratory values for

– Reticulocytes

– Cooper

– Vit. B12 and folate

• Correlate with clinical presentation • Correlate with clinical presentation

(splenomegaly?, HIV status?, age, sudden or

insidious onset of symptoms, medications:

cotrimoxazole, zinc containing cold remedies)

• Perform flow cytometry to assess for clonal B-cell

population (hairy cell leukemia?) or abnormal T-

cells (T-cell LGL leukemia?)


• Conventional cytogenetics: Essential diagnostic test; requires adequate BM aspirate specimen

• Fluorescence in situ hybridization for MDS panel (fast turn-around-time 24-48hrs):– -7/del7q (D7S486); 5/del5q (EGR1); del20q

(D20s108); 12p13 (ETV6); +8 (CEP8); del13q (D20s108); 12p13 (ETV6); +8 (CEP8); del13q (D13S319)

• Flow cytometry: Not required for either blast enumeration or assessment of dysplasia

• Diagnostic interpretation: Requires integration of clinical, hematologic, morphologic, and genetic findings– Provide WHO 2008 subtype

Cytogenetic Abnormalities in MDS Abnormality MDS Frequency

(%)

Therapy related MDS

Frequency (%)

Comments

del(5q) 10-25 40 Median survival 80 months

-7, del(7q) 10-50 50 Median survival 14 months

del(20q)

+8

-Y

5-8

10-20

1-5

As sole cytogenetic

abnormality in the absence

of morphological criteria not

considered MDS

del(11q) 3-7 Median survival 16 months

+21 1-5 Median survival 100 months

-5 1-5 Median survival 15 months

Complex 15 Median survival 9 months

Balanced

translocation

<1 2-3 t(11;16)(q23;p13.3),

t(3;21)(q26.2;q22.1),

t(1;3)(p36.3;q21.2),

t(2;11)(p21;q23),

inv(3)(q21q26.2),

t(6;9)(p23;q34)

Adapted from K. Faucar, 3rd ed. p. 341

Case #2

• 31-year old male who was transferred to

the hematology/oncology service with

chief complaint of pancytopenia, fatigue,

and dyspnea on exertion:and dyspnea on exertion:

• Complete blood count:

– WBC: 2.9 (4.5-11 K/cumm)

– Hemoglobin: 7.7 (13.0-17.0 g/dl)

– Platelets: 62 (140-400 K/cumm)

Patient #2

Patient #2

Leder

H&E

Patient #2

Sideroblast

Diagnosis

Hypercellular marrow with erythroid

hyperplasia and megaloblastic changes;

no excess blasts seen

Review of laboratory data revealed a B12

deficiency of 77 (211-911 pg/ml).

Diagnosis (without clinical or

laboratory information)

Hypercellular marrow with erythroid hyperplasia and megaloblastic changes; no excess blasts seen

Comment: Dyspoiesis is a morphologic change that can be due to myelotoxic / that can be due to myelotoxic / myelosuppressive drugs, nutritional deficiency, chronic viral infection, severe or sustained inflammatory conditions, or primary myelodysplastic syndrome (MDS). Diagnosis of MDS may be considered if all secondary causes are excluded, and established if a MDS associated genetic abnormality is present .

Conditions Mimicking MDS

Clinically and Morphologically

– Viral infections (HIV, Parvovirus B19)

– Vit. B12 and folate deficiency

– Toxic agents (lead, chemotherapy, arsenic,

zinc)

– Cooper deficiency

– T-cell large granular lymphocytic leukemia

– Hairy cell leukemia

– Medication (e.g. Cotrimoxazole)

Copper Deficiency (normal 0.75-1.45 pg/ml)

H&ELeder

Erythroid cluster

Copper Deficiency (normal 0.75-1.45 pg/ml)

• Anemia and

neutropenia

• Hypercellular bone

marrow is raremarrow is rare

• Vacuolization of

erythroid and

myeloid precursors

• Ringed

sideroblasts

Groups of ringed

sideroblasts

HIV-Infection Related BM Changes

Leder

MegakaryocytesH&E

HIV-Infection Related BM Changes

• Plasmacytosis

• Lymphocytosis

• Lymphohistiocytic Plasma cells

Megakaryocyte

• Lymphohistiocytic

infiltrate

• Dysplastic

features are not

clonal!

Hairy Cell Leukemia

• Splenomegaly,

cytopenias (mono-

cytopenia) and “hairy

cells” in bloodH&E cells” in blood

• BM with interstitial

infiltrate of CD20+ B-

cells causing fibrosis

CD20

PB

Hairy cellsH&E

T-Cell Large Granular Lymphocytic

Leukemia

T-Cell Large Granular Lymphocytic

Leukemia

CD8

CD4

CD7

• Splenomegaly

• Anemia and leukopenia,

often with severe neutropenia

• ?History of rheumatoid arthritis

• PB with increased LGLs

Unique Types of Myelodysplasia

Subtype Comments

Hypocellular MDS 5-10% of MDS cases, more

prevalent in pediatric MDS and

females

MDS with fibrosis 10% of MDS, 50% of therapy MDS with fibrosis 10% of MDS, 50% of therapy

related MDS, adverse prognosis

MDS with acquired α thalassemia Mutation of ATRX → down-

regulation in α-globin gene ex-

pression

MDS with normal morphology and

MDS-type clone

Presumptive evidence of MDS in

patients with sustained cytopenias

Therapy-related MDS Complex cytogenetic

abnormalities, poor outcome,

abnormal karyotype in 90%,

fibrosis common

Helpful Clues in the Diagnosis of MDS

• Poor specimen quality causes diagnostic errors

• The diagnosis of MDS requires integration of clinical, hematologic, morphologic, and cytogenetic features

• Exclude non-neoplastic causes of cytopenia →megaloblastic anemia, cooper deficiency, and chronic viral infections show the most overlap megaloblastic anemia, cooper deficiency, and chronic viral infections show the most overlap with MDS

• Megakaryoblasts, monoblasts, and promonocytes should be included in the blast percentage

• Conventional cytogenetics is the most valuable specialized test in MDS, perform routinely!

Helpful Clues in the Diagnosis of MDS

• MDS with associated monocytosis of ≥ 1X109/L are included in MDS/MPN category

• MDS in children is rare may be linked to underlying constitutional disorders or prior chemotherapy

• ~10% of MDS in adults are either hypocellular or fibroticfibrotic

• Exclude low blast count AML from RAEB-2 by cytogenetic assessment for AML-defining translocations

• CD34 immunohistochemistry is useful in assessing overall number and localization of blasts in the core

References

1. David Bowen et al. Guidelines for the diagnosis and therapy of adult

myelodysplastic syndromes. British Journal of Haematology, 2003, 120:187-200.

2. Roos J Leguit et al. The pathology of bone marrow failure. Histopathology, 2010,

DOI:10.1111/j.1365-2559.2010.03612.x

3. Afsaneh Barzi et al. Myelodysplastic syndromes: A practical approach to diagnosis

and treatment. Cleveland Clinic Journal of Medicine, 2010, 77:37-44.

4. Mikkael A Sekeres. The epidemiology of myeodysplastic syndromes.

Hematology/Oncology Clinics of North America, 2010, 24:287-294.

5. Rami S. Komrokji et al. Myelodysplastic syndromes: classification and Risk 5. Rami S. Komrokji et al. Myelodysplastic syndromes: classification and Risk

Stratification. Hematology/Oncology Clinics of North America, 2010, 24:443-457.

6. John R. Feussner et al. Arsenic-induced bone marrow toxicity: ultrastructural and

electron-probe analysis. Blood, 1979, 53:820-827.

7. Monte S. Willis et al. Zinc-induced copper deficiency. American Journal for Clinical

Pathology, 2005, 123:125-131.

8. Steven H. Swerdlow et al. WHO classification of tumours of haematopoietic and

lymphoid tissues. 4th Edition IARC, Lyon, 2008

9. Kathryn Foucar et al. Bone Marrow Pathology. 3rd Edition ASCP Press, Chicago,

2010

Bone Marrow Failure Syndromes

Michele Paessler, DO

Children’s Hospital of Philadelphia

University of Pennsylvania School of Medicine

Bone Marrow Failure • Definition

• Criteria

• Classification

• Work up

• Differential Diagnosis including

important diagnoses to exclude

• Inherited bone marrow failure

syndromes that should be considered

• Other Important tests to consider

Bone Marrow Failure

• Definition:

– Inability of the marrow to produce circulating

mature cells.

– This may result from:

• Reduction in number of progenitors and • Reduction in number of progenitors and

subsequently paucity of differentiated precursors

in the periphery:

– Aplastic anemia

• Increased number of differentiated precursors

but reduction of mature products in the periphery

– Ineffective hematopoiesis (ie myelodysplastic

syndrome)

Picture from Clinical Hematology, Young NS, Gerson SL, High KA, Mosby, Philadelphia 2006


Picture from Clinical Hematology, Young NS,

Gerson SL, High KA, Mosby, Philadelphia

2006

Diagnostic Algorithm for

Aplastic Anemia• Step 1: Establish criteria

• Step 2: Exclude Malignancy

• Step 3: Etiology

• Step 4: Prognostic Data• Step 4: Prognostic Data

Criteria for Diagnosis of Aplastic

AnemiaMust have two of the following in the peripheral blood:

• ANC <0.5 x 10 9/L

• Platelet count <20 x10 9/L

• Reticulocyte count< 20 x 10 9/L

Bone marrow must show:• Biopsy with <25% of normal cellularity for age OR

• 25-50% of normal cellularity for age, with <30% of the cells being hematopoietic

Bone Marrow Pathology, 3rd Edition, Volume 1. Foucar 2010

pg 138

What defines a hypocellular

marrow?• Pancytopenia and a hypocellular/aplasticmarrow– Hypocellular is considered <25% of the NORMAL cellularity for AGE.• Newborn 80-100% cellularity

• 1-3 month 80-100% cellularity• 1-3 month 80-100% cellularity

• Child (>1 year) 60-80% cellularity

• Adult (30-70 years) 40-70% cellularity

• Adult (>70 years) ~25% cellularity

• Requires bone marrow biopsy and aspirate– Core biopsy is recommended > 2 cm for accurate determination of cellularity

• Cellularity can be variable

• Subcortical marrow is hypocellularAdapted from Foucar, Bone Marrow Pathology,3 rd Edition, ASCP Press 2010 pg 32

Classification of Aplastic Anemia

ACQUIRED (80-90%)

Idiopathic (~70%)

Secondary (~10-20%)Drugs/toxins

Viruses (HIV, Hepatitis,

EBV)

Autoimmune (SLE)

INHERITED (10-20%)

Fanconi anemia

Dyskeratosis congenita

Shwachman-Diamond Autoimmune (SLE)

Hepatits (non viral)

Nutritional deficiencies

(Vit B 12/Folate)

PNH

Malignant infiltration (

tumors)

Nonmalignant

infiltration (storage

disorders)

Other (anorexia

nervosa, pregnancy)

Shwachman-Diamond

syndrome

Amegakaryocytic

thrombocytopenia

Familial aplastic

anemias

Pearson syndrome

Adapted from Clinical

Hematology, Young NS,

Gerson SL, High KA,

Mosby, Philadelphia

2006

Bone Marrow Pathology, 3rd Edition, Volume 1. Foucar 2010 pg 138

Hematopoietic Malignancies that

Masquerade as Aplastic Anemia

• Acute Leukemia CD34

– Acute lymphoblastic leukemia (TdT)

– Acute myeloid leukemia (MPO)

• Myelodysplastic syndrome CD34

Immunohistochemical Panel

– Dysplasia, Auer rods

• Hairy cell leukemia CD20

• T-cell LGL CD3

2 year old female with

pancytopenia

No lymphadenopathy

History of viral illness and fever

CD34CD34 highlights

clusters of

blasts. CD79a,

CD19 and TdT

were also

positive

Diagnosis:

Hypocellular Precursor B Acute Lymphoblastic

Leukemia (B-ALL)

:

55 year old male with cytopenias and

splenomegaly

CD20 Immunohistochemistry

Hypocellular hairy cell leukemia

CD20

Diagnosis:

Hypocellular Hairy Cell Leukemia

Positive for:

CD19, CD20,CD103, CD25, CD11C

Lambda restricted

Inherited Forms Associated with

Multilineage Bone Marrow Failure

• Primarily (but not always!!!!) associated with pediatric and young

adult

– Reports of patients being diagnosed in 5th-8th decades

• Can affect single lineage or multilineage

• Associated with increased risk of developing hematopoietic and

other malignanciesother malignancies

– Important to identify these patients

• Two disorders classically present with aplastic or hypoplasticbone marrow failure involving all hematopoietic lineages:

– Fanconi anemia

– Dyskeratosis congenita

Alogorithm for Congenital

Aplastic AnemiasHypocellular/Aplastic

Marrow

Fanconi Anemia

Screen :

Dyskeratosis

CongenitaScreen : Chromosome

Breakage Test

CongenitaTelomere length

assessment:

Flow-FISH

Fanconi

Anemia

positive

Non Fanconi

Anemia AA

negative

Dyskeratosis

Genetic Testing

TERC,

TERT,Dyskerin

sequencing

Assess other

BMF if

necessary

short normal

Acquired AA

Assess other BMF if

necessary

Acquired AA

negative

negative

• Most common inherited bone marrow failure syndrome

• Heterogeneous disorder with various mutations in genes encoding DNA repair proteins

• Present with a spectrum of clinical findings

Fanconi Anemia

clinical findings

• Patient’s get progressive cytopenias/pancytopenia

• Marrow progresses to an aplastic marrow


Fanconi Anemia

Fanconi Anemia Test

Chromosome Breakage Test

• Best screening

method:

• Chromosome

Breakage test

• Genetic testing is • Genetic testing is

usually performed

if there is a

positive

chromosome

breakage test

Shimamura, A. Hematology; 2006:63-71

Case History

• 14 year old male

• Pancytopenia

• Mild immunodeficiency

• Small stature• Small stature

• Family history:

– Mother small stature and history of anemia

– Father small(ish)

– 3 brothers normal size

Diagnosis:

• Aplastic marrow, no leukemia seen.

• Recommendations:• Recommendations:

– Flow fish for telomere length

– Fanconi anemia testing

– PNH flow cytometry

Leukoplakia

Case #2

Hyper/hypo-pigmentation

Nail dystrophy

Bone marrow failure©

Dyskeratosis Congenita: Rare bone marrow failure syndrome

Classic triad:abnormal skin pigmentationnail dystrophyoral leukoplakia

Pancytopenia is the hematologic hallmark of DC


Median age ~10 years

50% of patients develop severe AA and ~90% develop a cytopenia by 40 years

Age dependent Manifestations of Dyskeratosis Congenita

AA PF OPHH Classic DC Cancer

Age (years)

Dyskeratosis Congenita

Shimamura, A. Hematology 2006;2006:63-71

12

14

16

18

20

Re

lati

ve

Te

lom

ere

Le

ng

th (

% 4

N c

ell

lin

e)

99%

90%

75%

Among Patients with BMF Telomere Length Measurement

is a Sensitive Method to Identify


Patients with DC N=23

Age (years)

0

2

4

6

8

10

12

0 10 20 30 40 50 60 70 80 90

Re

lati

ve

Te

lom

ere

Le

ng

th (

% 4

N c

ell

lin

e)

50%

25%

10%

1%

Telomere length ≤1% N=23, (100%)

Du et al. BLOOD 2009


http://www.expertreviews.org/

Myelodysplastic Syndrome/ Acute Myeloid Leukemia

(MDS/AML)

in Dyskeratosis Congenita

Who Should Be Screened for Inherited

Bone Marrow Failure Syndromes?

• Pediatric, adolescent or young adult

– Primarily occur in this age group including young adult

• Any congenital abnormalities

– Congenital abnormalities (including cardiac and renal)

are important clues

– 30-50% of IBMFS patients have NO associated – 30-50% of IBMFS patients have NO associated

abnormalities

• Family history of aplastic anemia, cytopenias,

pulmonary fibrosis, AML or epithelial malignancies

• Pre-transplant workup for AA

– IBMFS patients cannot undergo standard conditioning

and undergo reduced intensity conditioning therapy

pretransplant

Aplastic Anemia vs. Hypoecellular

MDS• Can be very difficult to distinguish!!!

• Aplastic anemia

– Can see compensatory cellularity

– Can be associated with macrocytic anemia

– Can show “stress” dyserythropoiesis

– Cytogenetic abnormalities can also be seen

• Monosomy 7 – associated with progression MDS/AML****

• Trisomy 8 – usually respond to immunsuppressive therapy

• Factors that favor MDS

– Increase in blasts

– Dysplasia in non-erythroid lineages (myeloid and

megakaryocytic)

- Ringed sideroblasts in erythroid dysplasia

Case Presentation:

15 year old with pancytopenia15 year old with pancytopenia

May 2005

Normocellular marrow, no blasts,

no dysplasia, cytogenetics normal

October 2005

No increase in blasts

Erythroid Dysplasia

Diagnosis: Variably cellular marrow with

dyserythropoiesis, see note.

• Note: The marrow markedly hypocellular with areas of

hypercellularity. Dyserythropoiesis is present. No

megakaryocytic or myeloid dysplasia is seen. No blasts

are present. These findings may represent an evolving

aplastic anemia, but an underlying MDS cannot be aplastic anemia, but an underlying MDS cannot be

excluded. However, given that the dysplasia is limited to

the erythroid lineage and no blasts are present, an

evolving aplastic anemia is favored.

• Factors favoring Aplastic Anemia:

– No blasts

– No myeloid or megakaryocytic dysplasia

– Dysplasia limited to erythroid series with no

ringed sideroblasts (stress dyseryhropoiesis)

December 2005

January 2006

Bone Marrow Aspirate: Hypocellular with

myeloid dysplasia and scattered blasts (3-

5%)

Case History22 year old with fatigue and found to have pancytopenia

Case #4 22 year old with fatigue and found to

have pancytopenia

CD117

Clusters of CD117

positive

mononuclear cells

Dilemma??????

• Reason for consultation:

– Aplastic anemia OR

– Hypocellular MDS

• Factors that favor MDS• Factors that favor MDS

– Myeloid dysplasia

– Increase in blasts (~5-10%)

• Blasts seen on aspirate

• Confirmed CD34 and CD117

• Flow cytometry showed 5% myeloid blasts

• FISH showed monosomy 7

Diagnosis….

• Consistent with hypocellular MDS with

increase in blasts, best classified as

refractory anemia with excess blasts

(RAEB1)(RAEB1)

• Recommended tests based on the

patients age

– Telomere length testing

– Fanconi testing

– PNH

Case History:

46 year old woman with fatigue and

bruising

• Clinical history:

• Diagnosed with acquired idiopathic

aplastic anemia on immunosuppression

• Developed hemolysis and venous

thrombosis

– Coombs negative anemia

Bone Marrow Aspirate and

Biopsy

Small clusters of

erythroid precursors

and lymphocytes

Diagnosis: PNH in setting of

Aplastic Anemia• PNH is an ACQUIRED clonal disorder of

hematopoiesis

• Clone arises by somatic mutation in X-

linked phosphatidylinositol glycan class A linked phosphatidylinositol glycan class A

gene (PIG-A gene)

• Leads to deficiency in GPI anchor,

deficiency of GPI linked proteins

• Results in hemolytic anemia,

hypercoaguable state, and bone marrow

failure.

New Techniques for PNH & Flow

Cytometry• High sensitivity

– Sensitivity 0.01%

– Traditional PNH flow cytometry sensitivity 1%

• Antibodies targeting GPI linked proteins (CD55 and CD59)

• Use of FLAER

– (proaerolysin variant) that directly binds GPI-anchor and allows

direct assessment of GPI anchor expression and is more direct assessment of GPI anchor expression and is more

accurate assessment of GPI deficitNormal

neutrophil

FLAER

expression Neutrophil FLAER

Expression in PNH

pattern in PNH

PB Flow Cytometry for PNH

Normal RBC CD59

expression pattern

RBC CD59 expression

pattern in PNH

RBC CD59 expression

pattern in PNH

PB Flow Cytometry for PNH

Normal neutrophil

CD59

expression pattern

Neutrophil

CD59 expression

pattern in PNH

When to test for PNH• Recommended to test PNH at diagnosis of AA and then

yearly

– This is recommended for patients with inherited bone

marrow failure syndromes too not just acquired AA

– ?PNH clones may have a selective advantage in

marrow failure

• Small clones can be seen in AA in up to 40-80% of • Small clones can be seen in AA in up to 40-80% of

cases

• Clone sizes are followed and not treated any differently

than AA without a clone unless clinical symptoms

develop and there is progression to classical PNH or

clone size begins to increase

AplasticAplastic Anaemia Patient With Anaemia Patient With

Increasing PNH Clone SizesIncreasing PNH Clone SizesRichards 2005Richards 2005

40

50ALGALG CyACyA

% PNH Clone Size

0

10

20

30

11-Mar-97 24-Jul-98 6-Dec-99 19-Apr-01 1-Sep-02 14-Jan-04 28-May-05

Abs Granulocyte

Hb g/dl

Gran PNH clone %

RBC PNH clone %

biochemical biochemical

haemolysishaemolysis

% PNH Clone Size

Significance of PNH clones

• AA associated with PNH clones may have

a better prognosis and show a better

response to immunosuppression.

• MDS has been associated with PNH

clones (20% of cases)

• Prognosis of these clones in MDS is

controversial although some have shown a

better response other groups have not.

Summary• Hypocellular /aplastic marrows require a

vigorous work up

• Hypocellular presentations of

malignancies should be excluded

–ALL,AML,MDS, hairy cell leukemia, T –ALL,AML,MDS, hairy cell leukemia, T

cell LGL

– Immunohistochemical panel CD34,

CD20 and CD3

• Secondary causes of acquired AA should

be ruled out

–Clinical history, laboratory studies

Summary • Inherited bone marrow failures should be

considered

– pediatric/young patients and those with family

history and congenital anomalies

– Screening tests available – Screening tests available

• Fanconi – chromosome breakage test

• Dyskeratosis Congenita – telomere length

assessment FLOW-FISH

Prognostic data

– PNH clone

– Monitor clone in AA at diagnosis and yearly

for progression to clinical PNH

Thank You!!!

• [email protected]

•

• Acknowledgements:

• Monica Bessler, MD, PhD• Monica Bessler, MD, PhD

Selected References• Foucar K, Reichard K, CzuchlewskiD. Bone Marrow Pathology, 3rd edition. 2010.

ASCP Press: Chicago.

• Young NS, Gerson SL, High KA, Clinical Hematology, 2006. Mosby, Philadelphia

• Shimamura A, Alter BP. Pathophysiology and management of inherited bone marrow

failure syndromes.Blood Reviews.2010;24:101-22.2010;584:3831-3838.

• Bessler M, Mason P, Link D and Wilson D. Nathan and Oski’s Hematology of Infancy

and Childhood. Pp 307-39, Saunders , Philadelphia.

• Bessler M,Wilson B, Mason P. Dyskeratosis Congenita. Federation of European

Biochemical Societies (FEBS) Letters.

• Garcia CK, Wrights WE, Shay JW. Human diseases of telomerase dysfunction: • Garcia CK, Wrights WE, Shay JW. Human diseases of telomerase dysfunction:

insights into tissue aging. Nucleic Acids Research. 2007;35(22):7406-7416.

• O’Sullivan RJ and Karlseder J. Telomeres: protecting chromosomes against genome

instability. Nat Rev Mol Cell Biol. 11, 171-181.

• De Winter JP, Joenje H. The genetic and molecular basis of Fanconi anemia. Mutat

Res. 2009;668:11-19.

• Green AM, Kupfer GM. Fanconi Anemia. Hematol Oncol Clin North AM 2009.;

23:193-214.

• Moldovan GL, D’Andrea AD. How the Fanconi anemia pathway guards the genome.

Ann Rev Genet 2009;43:223-49.

• Gluckman E, Wagner JE, Hematopoietic stem cell transplantation in childhood

inherited bone marrow failure syndrome. Bone Marrow Transplant. 2008;41:127-32.

• Gupta V, Brooker C, Tooze JA, et al. Clinical relevance of cytogenetic abnormalities

at diagnosis of acquired aplastic anemia in adults. Br J Hematol. 2006;143(1):95-9.

Selected References• Soland E. Hypocellular myelodysplasia. Hematol Oncol Clin North America.

2009;23:347-60.

• Greenberg P, CoxC, LeBeau MM, et al. International scoring system for evaluating

prognosis in myelodysplastic syndromes. Blood. 1997;89)6):2079-88.

• Kojima S, Ohara A, Tsuchida M. et al. Risk factors for evolution of acquired aplastic

anemia into myelodysplastic syndrome and acute myeloid leukemia after

immunosuppressive therapy in children. Blood.2002;100(3):P786-90.

• Wang H, Chuhjo T, Yasue S et al. Clinical significance of a minor population of

paroxysmal nocturnal hemoglobinuria-type c ells in bone marrow failure syndromes.

Blood. 2002;100:3897-3902.

• Wang SA, Pozdyakova O, Jorgensen JL et al. Detection of paroxysmal nocturnal

hemoglobinuria clones in patients with myelodysplastic syndromes and related bone

marrow diseases, with emphasis on diagnostic pitfalls and caveats. Haematologica.

2008;941:29-37.

• Young NS. Paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes:

clonal expansion of PIG-A mutant hematopoietic cells in bone marrow failure.

Haematologica. 2009;94:3-7.

• Sugimori C, Chuhjo T, Feng X, et al. Minor population of CD55 –CD59- blood cells

predicts response to immunosuppressive therapy and prognosis in patients with

aplastic anemia. Blood 2006:107:1308-14.

• Borowitz et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal

hemoglobinuria and related disorders by flow cytometry. Cytometry B 2010; B.

• Rachidi S, Musallam K,Taher AT. A closer look at paroxysmal nocturnal

hemoglobinuria. Eur J of Int Med 2010;21:260-7.

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