Attilio Orazi, M.D., FRCPath. (Engl.)

Weill Medical College of Cornell University

New York, NY

Congresso Nazionale SIE - Società Italiana di Ematologia - MICMilano Convention Centre . Milano 18-21 Ottobre, 2009

Myelodysplastic Syndromes: WHO 2008

MDS are clinically heterogeneous: Classification/Scoring Systems

FAB plus dysplasia (10% rule; uni vs. multi), and cytogenetics (5q-)

Based on % blasts,karyotype,

# of cytopenias

Based onWHO classificationand Transfusion

FAB1976, 1982

IPSS1997

WHO2001

WPSS 2007

Based on morphologicdifferences including

% blasts

WHO2008

Table courtesy of Dr. John Bennett

Myelodysplastic syndromes

Key diagnostic points andupdates

Guidelines: recommended levelsfor defining cytopenias:

• Hemoglobin <10 g/dL• Neutrophil count <1.8 x109/L• Platelet count <100 x109/L

Values higher than these are not exclusionary of adiagnosis of MDS, if definitive morphologic and/orcytogenetic findings consistent with MDS are present

Morphologic Guidelines for theDiagnosis and Classification of MDS:

• Dyserythropoiesis, dysgranulopoiesis:>10% of cells on the BM aspirate smear

• Dysmegakaryopoiesis >10% of cellsevaluated on 30 megakaryocytes at least(BM aspirate smear or biopsy)

• Ring sideroblasts (iron stain of aspirate)• Blast percentage• Correlate the findings with marrow biopsy

Erythroid precursors - multinucleation/asymmetrical nuclei and/ornuclear bridging

GranulocytesPelger-Huet likenuclei,hypogranularcytoplasms

MegakaryocytesDwarf, non/hypolobatedforms includingmicromegakaryos

Definition of Ring Sideroblasts (updated from 3rd Edition) > 15% erythroblasts with 1/3 or more of the nucleus encircled by >5 iron granules (was >10)

Blast Count• PB 200 / BM 500

cell differential• Cells counted as

“blasts” include: - Myeloblasts - Monoblasts - Mk-blastsBut not erythroblasts

or promyelocytes!Mufti et al. Haematologica. 2008; 93:1712-7

• Quality check for the adequacy of BM aspirate• Provides information on stroma and cellularity

(e.g. fibrosis, fatty marrow)• Yields tissue for a number of studies: - immunohistochemistry

- in-situ hybridization

- molecular procedures (e.g. microdissection)

The role of BM biopsy in MDS is betterdescribed in the 4th edition

Blast count is poorly reproducible particularly in thickparaffin sections. Clusters of blasts more specific.

CD34 pos. cell clusters: 3-5 cells

Blasts and blast clusters identification made easier by immunohistology(e.g. CD34, CD117). Micromegakaryocytes by CD61 or CD42b

CD42b pos. dwarf- / micro- megs.

• Cases with inconclusive morphologic andcytogenetic findings and >3 or more aberrantfeatures by flow cytometry should be re-evaluated over several months for definitivemorphologic or cytogenetic evidence of MDS

• Utility of CD14/CD56 in CMML• Substitution of blast % determined by flow

cytometry for a visual blast count is discouraged

Flow cytometry to demonstrate anabnormal myeloid maturation

(abnormalities in light scatter and antigenic expression)

Interlaboratory variability issues:still work in progress

• Ogata K, et al. Diagnostic utility of flow cytometryin low-grade myelodysplastic syndromes: aprospective validation study. Haematologica. 2009Aug;94(8):1066-74. Epub 2009 Jun 22.

• van de Loosdrecht AA et al. Standardization offlow cytometry in myelodysplastic syndromes:report from the first European LeukemiaNetworking conference on flow cytometry inmyelodysplastic syndromes. Haematologica. 2009Aug;94(8):1124-34.

MDS: WHO 2008

Subtypes and changes

Refractory Cytopenia with Unilineage Dysplasia (RCUD)

Refractory Anemia (RA)

Refractory Neutropenia (RN)

Refractory Thrombocytopenia (RT)

Refractory Anemia with Ring Sideroblasts (RARS)

Refractory Cytopenia with Multilineage Dysplasia (RCMD)

Refractory Anemia with Excess of Blasts (RAEB)

Subtypes: RAEB - 1, RAEB – 2

Myelodysplastic Syndrome with isolated del(5q) chrom.abnormality

Myelodysplastic syndrome, Unclassifiable (MDS,U)

2008 WHO Classification of MDS

Refractory cytopenia with Mono-/bi-cytopenia1 Dysplasia (≥10%) unilineageunilineage dysplasia No or <1% blasts2,3 <5% blasts

<15% ring sideroblasts

Refractory anemia with Anemia Erythroid dysplasia onlyring sideroblasts No blasts <5% blasts

≥15% ring sideroblasts

Refractory cytopenia with Cytopenia(s) Dysplasia in ≥10% of themultilineage dysplasia No or <1% blasts2,3 cells of two or more lineages

<5% blasts< or >15% ring sideroblasts

PB BM

[Note: Monocytes <1 × 109/L; No Auer rods]

(RA, RN, RT)

1 Pancytopenia= MDS,u2 If 1% blasts in PB (<5% blasts in BM)= MDS,u 3 If 2-4% blasts in PB (< 5% blasts in BM)= RAEB-1

RAEB-1 Cytopenias<5% blasts*No Auer rods

Uni- or multi-lineage dysplasia5–9% blasts*No Auer rods

RAEB-2 Cytopenias5–19% blasts

Uni- or multi-lineage dysplasia10–19% blasts

Auer rods +/- Auer rods +/-

MDS with isolateddel(5q)

Anemia, usuallynormal or mildlyincreased platelets

Normal to increasedmegakaryocytes withhypolobated nuclei

No or <1% blasts <5% blastsNo Auer rods No Auer rods

PB BM

*Also RAEB-1 if 2-4% PB blasts in cases of RCUD or RCMD with < 5% blasts in BM

[Note: Monocytes <1 × 109/L]

Step 1:Type of Cytopenia

Step 2: P.B. BlastPercentage

No or <1%

UD = RCUD UD = RARS*

MD = RCMD

1%: MDS,u

2-4%: RAEB-1

5-19%: RAEB-2

No or <1%

UD = MDS,u MD = RCMD

*no blasts, > 15% RS

Diagnostic algorithm for MDS: P.B. smear (200 cell differential)

Mono-bi-cytopenia Pancytopenia

P.B. diagnostic algorithm for MDS with BM <5% blasts (P.B. smear 200 cell differential)

Step 2: P.B. BlastPercentage

MDS with isolated 5q-

• Frequent erythroidhypoplasia

• Lenalidomideefficacy; potentialleukemogenic effect

• A subset of patientwith JAK2 mutationis mentioned (i.e.the so calledproliferative variant)

Isolated 5q- associated with JAK2 mutation(6.2% of isolated 5q- cases)

• Thrombocytosis plusgranulocytic proliferation inthe BM and/or PB

• JAK2 positive cases had atrend towards:– Higher platelet count (475

vs 250 x 109/L; P=0.15)– Higher WBC (5.21 vs

4.45x109/L; P=0.012)– No difference in median HB

(9.0 vs 8.7 g/dL; P=0.272)Ingram et al: Leukemia 2006;20:1319-1321

MDS, unclassifiable (3 settings)

1. Patients with refractory cytopenia withunilineage dysplasia (RCUD) or refractorycytopenia with multilineage dysplasia(RCMD) but with 1% blasts in theperipheral blood

2. Cases of MDS with unilineage dysplasiawhich are associated with pancytopenia.RCUD (in contrast with RCMD) only allowsfor a single cytopenia or bicytopenia

3. Persistent (6 mo.) cytopenia(s) lacking morphologicfeatures of MDS, but with cytogenetic abnormalities

presumptive for MDS*• +8*• -7 or del(7q)• -5 or del(5q)• del(20q)*• -Y*• i(17q) or t(17p)• -13 or del(13q)• del(11q)• del(12p) or t(12p)• del(9q)• idic(X)(q13))

• 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)

* As single cytogenetic abnormality, inthe absence of morphologic findings,is insufficient evidence for MDS

Subtypes of MDS difficult todistinguish from other myeloid

neoplasms (discussed in the 4th edition)• Hypoplastic MDS from acquired

aplastic anemia

• MDS with fibrosis from other fibroticmyeloid neoplasms (e.g. PMF)

• MDS with > 50% erythroblasts fromacute erythroid leukemia

hypo-MDS

AA

CD34: h-MDS vs. AA: p=.001

Maschek H,et al. Ann Hematol 1993; 66: 117–122. Orazi et al. Am J Clin Pathol 1997; 107:268-74. Bennet & Orazi. Haematologica 2009; 94:264-8

MDS, hypoplastic(Cellularity <30% in patients <70 years old, and <20% in >70 years old)

hypo-MDS

• Lim ZY, et al. Low IPSS score and bone marrowhypocellularity in MDS patients predict hematologicalresponses to antithymocyte globulin. Leukemia.2007;21:1436-41.

• Sloand EM, et al. Factors* affecting response andsurvival in patients with myelodysplasia treated withimmunosuppressive therapy. J Clin Oncol.2008;26:2505-11.

• Sloand EM. Hypocellular myelodysplasia. HematolOncol Clin North Am. 2009;23:347-60. Review.

Hypocellularity favors response toimmunosuppressive therapy ?

*Younger age & HLA-DR15 were the most significant factors favoring response totherapy

MDS with fibrosis (>2+ fibrosis)

100

80 % MDS fibrosis <2

Survival 60

40 p < .0005

20

0 1 6 12 18 24 30 36 42 mo

I

III

II

Lambertenghi-Deliliers G, Orazi A, Lucks R, et al.: Br J Haematol, 1991

MDS fibrosis > 2

• Scott BL, et al. Marrow fibrosis as a risk factor forposttransplantation outcome in patients withadvanced myelodysplastic syndrome or acutemyeloid leukemia with multilineage dysplasia. BiolBlood Marrow Transplant 2007;13:345-54

• Buesche G, et al. Marrow fibrosis predicts earlyfatal marrow failure in patients withmyelodysplastic syndromes. Leukemia 2008;22:313-22

• Della Porta MG, et al. Clinical relevance of bonemarrow fibrosis and CD34-positive cell clusters inprimary myelodysplastic syndromes. J Clin Oncol2009;27:754-62

Erythroid-rich MDS: when the erythroidcells are > 50% of all the nucleated cells

• A second count for the non-erythroidcells:

– AML, if the blasts are > 20% of the non-erythroid cells (erythroleukemia)

– MDS, if the blasts are less than 20% of thenon-erythroid cells

• If MDS, then use the original blast %for further MDS subtyping

• Chronic myelomonocytic leukemia (CMML)

• Atypical chronic myeloid leukemia* (aCML)

• MDS/MPN, unclassified (MDS/MPN,u)

–– Refractory anemia with ring sideroblastsRefractory anemia with ring sideroblastsand thrombocytosis (RARS-T)and thrombocytosis (RARS-T)

Myelodysplastic/MyeloproliferativeNeoplasms (MDS/MPN)

(Hematopoietic malignancies with hybrid features both of MDS and MPN)

*BCR-ABL1 negative

*May resemble CMML or aCML.Include cases formerly termed CMMLwith t(5;12)

In all cases the presence of• Ph’ chromosome/BCR/ABL1 fusion gene

plus, in cases with eosinophilia• PDGFR Alpha abnormalities

• PDGFR Beta abnormalities*

Diagnosis of MDS/MPN: WHO requiresto exclude

CMML: definition• Monocytes

– >1 x109/L– >10% of WBC

• Dysplasia (mono or multi-lineage) is not required if allother criteria are met and– there is a cytogenetic

abnormality– or reactive causes are

excluded andmonocytosis has lastedfor at least 3 months

ANBE

Monoblasts

Promonocytes

Abnormal monocytes (immature) Adapted from WHO Classification of Tumors of Haematopoieticand Lymphoid Tissues. IARC: Lyon 2008. Fig.1-04 (p. 21).

MonoblastsLarge cells with abundantcytoplasm minimallyvacuolated round nuclei withfine chromatin and nucleoli

PromonocytesIrregular or folded nuclei, smallindistinct nucleoli, finechromatin and a morevacuolated and/or granularcytoplasm

Abnormal monocytesMore condensed chromatin,convoluted nuclei and moregranulated

RARS-T• Its incidence is rare (underdiagnosed?)• JAK2 positive in approx. 60% of cases• MPL W515K/L (very rare)• RARS-T has a worse outcome than ET• Is controversial

Wardrop D, Steensma DP. Is RARS-T a necessary or usefuldiagnostic category? Br J Haematol. 2009;144:809-17

BM Biopsy

• Massachusetts General Hospital, Boston, MA1

• U. Mass Memorial Med. Center, Worcester, MA2

• Brigham and Women’s Hospital, Boston, MA3

• Weill Cornell College of Medicine, New York, NY4

• Genzyme, Inc, New York, NY5

• Baystate Med. Center, Springfield, MA5

Hasserjian RP1, Soupir CP1, Wang Sa A2, VergilioJoA3, Orazi A4 , Dong H5

RARS-T study (44 cases)

Karyotype (40 patients)

• 31 (78%) had a normal karyotype

• 9 cases (22%) with abnormal karyotypewhich included:

isolated +8 (4 cases)isolated del(20)(q11.2) (1 case)isolated –Y (1 case)isolated del(11)(q23) (1 case)+1p,-6 (1 case)del(7)(q11.2)(1 case)

Morphology: comparative analysisof RARS-T, RARS and MPN

RARS-T & RARS:have similar

– Erythroidhyperplasia

– Left shift erythrop.

– Dyserythropoiesis

RARS-T & MPNboth have:– Megs. equally

numerous

– Similarly large andpleomorphic

– Similarly variablefibrosis

Fibrosis in RARS-T

• 5 of 32 cases(16%) in ourseries

• 12 of 28 cases*(43%) hadfibrosis:– MF-1: 7 cases– MF-2: 5 cases

*Schmitt-Graeff AH, et al. Haematologica 2008

Morphology of JAK2 (-) vs. JAK2 (+)

• JAK2(+) cases had a significantly higher PLTand WBC counts, and a trend towards a higherHB level

• JAK2(+) cases had more prevalent large Megsoften with staghorn or bulbous nuclearmorphology while small and normal-sized Megs.were predominant in the JAK2(-) cases

• No differences in reticulin fibrosis andmorphologic dysplasia in other lineages

Additional issues

• Prognosis

• Molecular pathogenesis

Overall survival of RARS-Tpatients

0 100 200 3000

20

40

60

80

100

Time (months)

Perc

ent s

urvi

val

The median follow-up for all patients was 59 months; at the time of study analysis(9/20/09), 26 patients had died..Median survival for all pts. 74 months

44 cases

Transformation to AML• Our series: 4/27 (15%)

cases with follow up

• Pich A, Godio L. RARS-T associated withJAK2V617F mutationtransformed to AML withchromosomal evolutionincluding monosomy 7.Leuk Res (2009). Inpress.

Schmitt-Graeff A et al. Haematologica 2008

23 cases

Prognostic role for JAK2

Prognostic role for JAK2?

Suggested by: Atallah E, et al. Leukemia. 2008; 22:1295-8

Not confirmed by: Wang SA, et al. Leukemia. 2006;2 0:1641-4

Raya JM, et al. Int J Hematol. 2008; 88:387-95

Hasserjian RP, et al. our current study

Molecular pathogenesis: Malcovati L, et al.Blood. 2009 Aug 19. [Epub ahead of print]

• 19 RARS-T defined according to WHO 2008• Like in RARS, up-regulation of ALAS2 and down-

regulation of ABCB7 in CD34+ cells, i.e. RARS-T hasdistinctive “sideroblastic” features at the molecularlevel.

• X-chromosome inactivation shows that JAK2 pos. cellsare only a fraction of clonal granulocytes

• 3 RARS pts. with low or normal plts. progressed toRARS-T (2/3 acquired JAK2). It may develop fromRARS through the acquisition of somatic mutations ofJAK2, MPL or other as-yet-unknown genes

Summary• Morphology is still very important for diagnosis• Perform always iron and reticulin stains• Look out for monocytes in PB and BM and use

cytochemistry and flow to confirm monocytosis• Bone marrow biopsy supplemented by

immunohistology (particularly useful in cases withfibrosis or fatty BM)

• Karyotype is mandatory. FISH may supplement• BCR-ABL1 and, in cases with eosinophilia, PDGFR

rearrangements• Test for JAK2 when appropriate (e.g. RARS-T, 5q-)