Myelodysplastic Syndromes Classification

Myelodysplastic Syndromes Classification

Carlos E. Bueso-Ramos, M.D., Ph.D

Department of Hematopathology The University of Texas M. D. Anderson Cancer Center

Houston, Texas.

Outline

• Definition • Principles and limitations of the current

Classification of MDS • Cytogenetic/Genetic studies • Molecular studies • Prognosis

FAB classification 1976

Myelodysplastic Syndromes (MDS)

• Group of clonal neoplasms of marrow stem/progenitors

• Cytopenias

• Dysplasia w/ineffective hematopoiesis

• Increased risk of blastic transformation

AML with less than 20%

blasts MDS with PNH

features

AML FAB M6 when erythroblasts

are > 50%

MDS with hypocellular marrow

MDS with fibrosis

MDS with thrombocytosis

PNH

MPN AA

MDS AML

SHORT COURSE PROGRAM

2 ml EDTA for molecular (RNA, DNA NGS) studies

2 Touch imprints, 4 Wright-Giemsa, 5 unstained

BM biopsy, aspirate smears, clot

10% buffered formalin

2 ml BM aspirate in 0.5 ml of heparin for cytogenetics

1-2 ml PB or BM aspirate in EDTA for Flow cytometry

Fresh tissue for Tissue bank

Workflow of a BM Specimen suspected to be Hematopoetic Malignancy

Blood Smear Rouleaux, cytopenia, dysplasia, lymphoma, plasma cells, blasts

H&E sections x2 from each paraffin block; Reticulin Trichrome

xx

SHORT COURSE PROGRAM

Adequate Inadequate

Adequacy • Iliac crest trephine

biopsy • Biopsy 1.5 cm • Fix 3 hrs in 10%

buffered formalin • Decal Stat 1 hr; or 5%

formic acid 12 hrs • B5 fix/EDTA decal • Embedded in paraffin,

cut sections 3 microns thick

XXXXXXXXXXXXXXXX

Cazzola, et al. Blood, 2013

SHORT COURSE PROGRAM

Axis 1. Morphogenetic Differentiation type

Axis 2. Genetic progression Axis 3. Pathways & Targets

Axis 5. Host genetics Axis 4. Etiologic agents

Multidimensional model for tumor classification

XXXXXXXXXXXXXXXX

Discordance in the diagnosis was documented in 109 (12%) patients, with a majority reclassified as having higher-risk disease

Implications of discrepancy in morphologic diagnosis of MDS between referral and tertiary care centers

Kiran Naqvi , et al. Blood. 2011;118(17):4690-4693

Minimal Diagnostic Criteria in MDS

Prerequisite criteria

• Constant cytopenia in one or more of the following

cell lineages erythroid (WHO hemoglobin <10 g dL)

• Neutrophilic (ANC <1800 µL) or megakaryocytic (platelets

<100,000 µL)

Greenberg PL, et al. J Natl Compr Canc Netw 2017;15(1):60-87

• Reticulocyte counts • Chemistries • Transaminases • Bilirubin • Hepatitis serologies • PNH aerolysin assay

Exclude hematopoietic and non-hematopoietic disorders as reason

for cytopenia/dysplasia:

• Medications • Exposures • Transfusions • Splenomegaly • Hepatomegaly • Lymphadenopathy • Family history

Greenberg PL, et al. J Natl Compr Canc Netw 2017 Valent P, et al. Oncotarget. 2017

Minimal Diagnostic Criteria in MDS

MDS-related (decisive) criteria

• Dysplasia in at least 10% of all cells in one of the following

lineages in the bone marrow smear; erythroid; neutrophilic; or

megakaryocytic or > 15% ringed sideroblasts

• 5-19% Blast of all nucleated cells in bone marrow

• MDS-associated karyotype

Co-criteria: • Typical clinical features, macrocytic transfusion-dependent

anemia

• Abnormal phenotype by flow cytometry of BM cells indicative of a monoclonal population

• Abnormal BM histology & IHC (abnormal CD34, fibrosis, dysplastic megs, abnormal localization of immature progenitors)

• Molecular: Monoclonal myeloid population Greenberg PL, et al. J Natl Compr Canc Netw 2017

Valent P, et al. Oncotarget 2017

Minimal Diagnostic Criteria in MDS

Ring sideroblast

Haematologica 2008; 93(11)

Perinuclear Siderotic Granules

Type 1: Fewer than 5 siderotic granules in the cytoplasm Type 2: ≥ 5 siderotic granules, but NOT in a perinuclear distribution Type 3 or ring sideroblasts:

5 or more granules in a perinuclear position

Ring sideroblast

SF3B1 mutations in MDS

• SF3B1 mutations in 20% MDS • High frequency among patients with RS (65%)

Papaemmanuil, et al. NEJM 2011 Yoshida, et al. Nature 2011

Della Porta MG, et al. Leukemia 2015

Morphologic Features of MDS

Dysplasia Score

• 1150 patients with peripheral blood cytopenia

• Sensitivity and specificity >90%

• Acceptable reproducibility and was independently validated

• Useful in cases without a clearly objective myelodysplastic phenotype and myeloid disorders with fibrosis and hypocellularity

Della Porta, et al. Leukemia 2015

Morphological abnormalities

Cutoff values

Variable weighted

score

Megaloblastoid changes > 5% 2

Bi- or multinuclearity > 3% 1

> 5% 2

Nuclear lobulation or irregular contours > 3% 1

Pyknosis > 5% 1

Cytoplasmic fraying ≥ 7% 1

Ring sideroblasts > 5% 2

≥ 15% 3

Ferritin sideroblasts ≥ 30% 1

Megaloblastosis

Multinuclearity

Nuc. Lobulation

Pyknosis

Cyt. Fraying

Ring sideroblast

Erythroid lineage

Della Porta MG, et al. Leukemia 2015

Granulocytic lineage

Morphological abnormalities

Cutoff values

Variable weighted

score

Myeloblasts > 3% 1

> 5% 3

Auer rods ≥ 1% 3

Pseudo Pelger–Hüet anomaly

> 3% 1

> 5% 2

Abnormal nuclear shape ≥ 7% 1

Neutrophil hypogranulation

> 3% 1

> 5% 2

Myeloblasts

Auer rods

Hypolobulation

Abnormal nuclear shape

Hypogranulation

Morphological abnormalities

Cutoff values

Variable weighted

score

Micromegakaryocytes > 5% 3

Small binucleated megakaryocytes > 5% 1

Megakaryocytes with multiple separated nuclei

> 5% 2

Hypolobated or monolobar megakaryocytes

> 5% 2

Micromegakaryocytes Monolobar Megs.

Small binucleated Separated nuclei

Della Porta MG, et al. Leukemia 2015

Megakaryocytic lineage

Dysplasia Score

Erythroid dysplasia: Score value ≥ 3 (a minimum of 10% of dysplastic erythroid cells)

Granulocytic dysplasia Score value ≥ 3 (a minimum of 10% of dysplastic granulocytic cells) Exception

• > 5% blasts • Auer rods

Megakaryocytic dysplasia Score value ≥ 3 (a minimum of 10% of dysplastic megakaryocytes) Exception:

• > 5% micromegakaryocytes

Trephine biopsy

• Cellularity (hypoplastic MDS)

• Myelofibrosis (reticulin, MDS with fibrosis)

• Report estimated % CD34+ blasts & cluster

• Dysmegakaryopoiesis (CD61)

Morphologic Features of MDS

Della Porta MG, et al. Leukemia 2014, 1-10

Dysplasia Score and molecular correlation

Erythoid score: • Association RS and SF3B1 (P<0.001)

Granulocytic score: • Higher score in poor/ very poor risk cytogenetics

(P=0.001) • Higher score in ASXL1, RUNX1, TP53 and SRSF2 (P from

0.03 to 0.001)

Granulocytic and megakaryocytic score: • Negative correlation with ANC (P<0.001) and platelets

counts (P=0.001) respectively

Morphological Features of MDS in PB

Features of MDS in BM

WHO 2008 Classification of MDS • 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 blasts (RAEB-1, -2)

• Myelodysplastic syndrome with isolated del(5q)

• Myelodysplastic syndrome, unclassifiable (MDS,U)

• Childhood myelodysplastic syndrome

• Refractory cytopenia of childhood (RCC)

WHO 2016 nomenclature changes of MDS

• MDS with single lineage dysplasia (MDS-SLD) • MDS with multilineage dysplasia (MDS-MLD) • MDS with ring sideroblasts (MDS-RS)

• MDS-RS-SLD • MDS-RS-MLD

• MDS with isolated del(5q) • MDS with excess blast (MDS-EB)

• MDS-EB-1 • MDS-EB-2

• MDS unclassified • 1% blast • SLD and pancytopenia • Defining cytogenetic abnormality

MDS with Deletion of Chromosome 5q:Persistent Malignant Stem cells in

Remission

NEJM 2010;363:1025

2016 Update of MDS

Isolated del(5q)

MDS-U

MDS-MLD

MDS-RS-MLD

MDS SLD RA, RN, RT MDS-RS-SLD SF3B1mut

Unilineage dysplasia, pancytopenia dysplasia < 10% with abnormal CG*

Blast:1% in PB

Multilineage dysplasia cytopenia (uni/bi/pan)

unilineage dysplasia Uni/bicytonenia

dysplasia, cytopenia

MDS-EB-1 MDS-EB-2

RAEB >5% in PB or >9% in BM or AR

No Yes

AR >1 % in PB or

>5 % in BM

• R/o Hypoplastic AML • R/o MDS/MPN • MPO, butyrate • Immunophenotype

Including: Ring sideroblasts, SF3B1, JAK2 mut One additional cytogenetic abnormality NOT -7 or del(7q);

Arber, et al. Blood 2016

Blast ≥20% YES

No

YES No

Monocyte > 1,000 YES MDS/MPN

No

Recommendations & Definitions in MDS

• Recommendations • Differential: 500 in BM, 200 cells in PB • Number: 200 for G and E, 30 cells for meg • Ring sideroblasts: ≥ 5 iron granules encircling ≥ 1/3 of the nucleus

• Minimal dysplastic changes (good quality of smear) • > 10% in one single cell line* • or < 10% with recurrent abnormal cytogenetics

• Cytopenia (≥ 6 month), Transfusion-dependent, macrocytic anemia • Hgb < 10g/dL • ANC < 1.5 x 109/L • PLT < 100 x 109/L • Constant blast count 5-19%

• Blasts • Myeloblasts

• Dysmyelopoiesis • Dyserythropoiesis • Dysgranulopoiesis • Dysmegakaryopoiesis

• Trephine biopsy • Cellularity (hypoplastic MDS) • Myelofibrosis (reticulin, MDS with fibrosis) • Report estimated % of CD34+ blasts • Dysmegakaryopoiesis (CD61)

Morphologic Features of MDS

Bone Marrow Histology in MDS

• Separation from AML when smears are contaminated with blood cells (CD34-IHC)

• Separation from hypoplastic AML (CD34-IHC) • Separation from Aplastic Anemia • Multifocal accumulations of (CD34+) progenitor cells (CD34-IHC)

• Abnormal distribution/localization of CD34+ cells (CD34-IHC)

• Abnormal accumulation and morphology of megakaryocytes (IHC: CD31, CD42, or CD62)

• Demonstration of bone marrow fibrosis (Gomori’s silver impregnation) • Diagnosis of a second neoplasm • Diagnosis of hypocellular MDS • Diagnosis of MDS-U and SM-MDS • Demonstration of cytogenetic markers by in situ-FISH when no

karyogram is available

Valent P, et al. Oncotarget 2017

Morphological and Immunohistochemical Features of BM Biopsy in MDS

Markers Cell type(s) (A) Minimal panel

CD34 CD31 or CD42 or CD62 Tryptase

Blast cells, progenitors, endothelial cells Megakaryocytes Mast cells, basophils, myeloid progenitors

(B) Extended panel – according to the cell lineage to be examined CD3 CD15

CD20 CD25 CD38 CD68,CD68R

Lysozyme CD117 2D7, BB1

T cells Monocytes, granulocytes B cells T and B cell subset, atypical mast cells Plasma cells Monocytes, macrophages, myeloid cells Monocytes, macrophages Progenitor cells, mast cells Basophils

Recommended Immunohistochemical Markers in MDS

Valent P, et al. Oncotarget. 2017

HR Kim, Z Zuo, MJ Martinez, C Bueso-Ramos, ASH 2014

Validation of the 2016 Revisions to the WHO Classification in Lower-Risk MDS

(mo)

Kanagal-Shamanna, Am J Hematol. 2017

• Low incidence of abnormal cytogenetic

abnormalities • Low level of dysplasia seen in BM from

normal individual • Non-specific morphologic dysplasia • Cytogenetic abnormalities found in BM

with no overt dysplasia • Overlapping features between the

hypoplastic MDS and aplastic anemia (AA)

Difficulties in diagnosis of low grade MDS

Comprehensive Approach to Low Grade MDS

• A careful evaluation/review of − Morphology: dysplasia, blasts − Cytogenetics − Clinical history − Immunophenotype by flow cytometry & IHC

• To monitor the patient over time and repeat blood, marrow examination and other studies periodically, as dictated by clinical circumstances.

Comprehensive Approach to Diagnose Low Grade MDS

Figure 1

Nybakken & Bagg. Journal of Molecular Diagnostics, 2014; 16:145-158

Cytogenetic findings in MDS

ASH Education Book December 5, 2015 vol. 2015 no. 1 299-307

• Abnormalities in ~50% de novo MDS, ~ 90% t-

MDS • Biological distinct entity: isolated del(5q) • Prognostic stratification; basis for selection of

drug • Clues for molecular pathogenesis of MDS • FISH 5q31, CEP7, 7q31, CEP8, 20q, CEPY, p53

(17p)

Cytogenetics

MDS Cytogenetic Scoring System

Prognostic subgroups % of patients

Cytogenetic Abnormalities

Median survival,

*y

Median AML evolution,

25%,*y

Hazard ratios

OS/AML*

-Y, del (11q) 5.4 NR 0.7/0.4 0.5/0.5

Normal, del (5q), del (12p), del (20q), double including

del(5q)

4.8 9.4 1/1 1/1

Del(7q), +8, +19, i(17q), any other single or double independent

clones

2.7 2.5 1.5/1.8 1.6/2.2

-7, inv(3)/t(3q), double including -7/del(7q),

complex: 3 abnormalities

1.5 1.7 2.3/2.3 2.6/3.4

Complex: >3 abnormalities

0.7 0.7 3.8/3.6 4.2/4.9

Blood. 2012;120(12):2454-2465

Prognostic Variable Categories and Associated Scores

Cytogenetics

Very good Good Intermediate Poor Very Poor

0 0.5 1 1.5 2 3 4

BM blast ≤2 >2%-<5% 5%-10% >10%

Hemoglobin ≥10 8-<10 <8

Platelets =>100 50-

<100 <50

ANC

≥0.8 < 0.8

IPSS-R prognostic score values

Blood. 2012;120(12):2454-2465

Risk Category Risk group

Very low ≤1.5

Low >1.5-3 Intermediate >3-4.5

High >4.5-6 Very high >6

Table

IPSS-R prognostic risk categories/score

Blood. 2012;120(12):2454-2465

The Value of Adding FISH to the Diagnosis and Prognosis of MDS

• Single-institution experience of 62 patients with MDS who have had both cytogenetic analysis and FISH performed simultaneously

• FISH panel designed to detect abnormalities in chromosomes 5, 7, 8, 11, and 20

• Concordance between the 2 methods in 43 patients (69%) • FISH identified additional clones in 10 patients:

– 7/26 patients with normal karyoptype – 3/36 patients with abnormal karyotype – 3 had no change in their IPSS scores and 7 had an upgrade

Shammo et al. ASH 2008, Abstract 3630

Diagnostic algorithm for evaluating cytopenia patients to establish or exclude a diagnosis of MDS

New Cytogenetic Scoring System in MDS

(Schanz et al., JCO, 2012)

• Increasing recognition of the role of flow cytometry in the diagnosis of MDS

• Increase in CD34+ blasts with aberrancies • Abnormal maturation patterns of myeloid cells • Decrease in maturing B-lymphoid precursors

(hematogones)

• Ruling out other causes for cytopenia such as lymphoma

Immunophenotype by Flow Cytometry

Wells D, et al. Blood 2003; 102:394-403

Correlation of FCI Score in MDS

FCI Positive for MDS

• Expression of Lymphoid antigens CD2, CD5, CD7, CD56, CD10, CD19

• Lack of Hematogones • Discrete population • Alteration of CD45, CD117, CD123, CD38,

CD33/CD13 • Hypogranulation • Myelomonocytic maturation alterations

MDS Features Mapped to Molecular Defects

• Abnormal Signaling Pathways

• Loss of p53 Function (17p)

• Epigenetic Silencing of Genes: Aberrant DNA methylation, aberrant histone code

Mutations and bone marrow blasts

MDS working group

Mutations and thrombocytopenia

MDS working group

TP53 mutant MDS phenotype

Karyotype

SF3B1 SRSF2

NPM1 IDH1/2

ETV6 TK pathway

EZH2 ASXL1

TET2

U2AF1

TP53

DNMT3A

RUNX1

Years

Ove

rall

Surv

ival

Complex karyotype, TP53 mutation present (n = 26) Complex karyotype, TP53 mutation absent (n = 31) Non-complex karyotype, TP53 mutation absent (n = 368)

P < 0.0001 *

P = 0.83

TP53 mutant phenotype: Complex karyotype Elevated blast count Thrombocytopenia Poor survival

Bejar et al., NEJM 2011

Modified from Blood 2013;122(25):4021-4034

SF3B1 mutation: MDS with ring

sideroblasts

SF3B1/JAK2 or SF3B1/MPL co-

mutation: MDS/MPN with ring

sideroblast & thrombocytosis

Miscellaneous driver mutations: MDS with unilineage dysplasia (refractory anemia)

MDS with multlilineage

dysplasia

MDS with excess blasts

Various combinations of founding driver mutations involving genes of RNA splicing (SRF2, U2AF1) or DNA methylation (TET2, DNMT3A), and subclonal driven mutations involving genes like ASXL1, EZH2,

RUNX1, or TP53

SETBP1 mutation ETNK1 mut: atypical chronic myeloid leukemia

TET2/ /SRSF2 co- mutation: chronic

myelomonocytic leukemia

MDS/ MPN with isolated i(17) (q10)

with ASXL1 mut, SETBP1 mut

AML

Activating CSF3R mutation: chronic neutrophillic leukemia

MDS: Classification process based on morphologic & genetic criteria

Malcovati et al. Blood.124(9):1513-1521

International Prognostic Scoring System (IPSS)

Scores for risk groups: Low: 0/ INT-1: 0.5-1.0/ INT-2: 1.5-2.0/ High, ≥ 2.5

Cytopenia: Neutrophils<1,800/ μl, hemoglobin<10 g/dl, Platelets <100,000/ μl.

Cytogenetics Good: normal, -Y, del(5q), del(20q) Poor: complex (≥ 3 abnormalities) or chromosome 7 anomalies Intermediate: other abnormalities

Greenberg et al., Blood 1997

Variable 0 0.5 1 1.5 2.0

BM blasts (%) <5 5-10 - 11-20 21-30

Karyotype Good Intermediate Poor

Cytopenias 0/1 2/3

International Prognostic Scoring System (IPSS)

Scores •Low: 0 •INT-1: 0.5 – 1 •INT-2: 1.5 – 2 •High: ≥ 2

•Cytogenetics •Good: normal, -Y, del(5q), del(20q) •Poor: complex (≥ 3 abnormalities) or chromosome 7 anomalies •Intermediate: other abnormalities

Greenberg et al., Blood 1997

WPSS Score Variable 0 1 2 3

WHO RA, RARS, 5q-

RCMD, RCMD-RS

RAEB-1 RAEB-2

Cytogenetics1 Good Int. Poor __

RBC Transfusions No Regular2 __ __

1)Cytogenetics as per IPSS; 2)Regular transfusions: ≥RBC unit q8 weeks for 4 months

Malcovati et al. JCO 2007;25:3503-3510

WPSS Score

WPSS Risk (score) % Patients Median OS (months)

AML progression

(2 years)

Very low (0) 10-23 103-141 0-0.03

Low (1) 22-28 66-72 0.06-0.11

Int (2) 19-23 40-48 0.21-0.28

High (3 or 4) 23-33 21-26 0.38-0.52

Very High (5 or 6) 7-12 9-12 .80

Malcovati et al. JCO 2007;25:3503-3510

MDACC Model - Simplified MDS Risk Score

Prognostic Factor Coefficient Points Performance status ≥2 0.267 2 Age (y) 60-64 0.179 1 ≥65 0.336 2 Platelets (x109/L) <30 0.418 3 30-49 0.270 2 50-199 0.184 1 Hemoglobin < 12 g/dL 0.274 2 Bone marrow blasts (%) 5-10 0.222 1 11-29 0.260 2 WBC >20 x 109/L 0.258 2 Karyotype: Chromosome 7 0.479 3 abnormality or complex ≥ 3 abnormalities Prior transfusion, yes 0.107 1

Score points were obtained by dividing the coefficients by 0.15 and rounding to the nearest integer.

No. of Median No. of Score Patients (%) (Months) Score Patients (%)

0-4 157 (16) 54 63 38

5-6 227 (24) 25 34 13

7-8 233 (24) 14 16 6

≥ 9 341 (36) 6 4 0.4

SURVIVAL

Adapted, with permission, from Kantarjian H, O’Brien S, Ravandi F, et al. Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System. Cancer 2008:113(6):1351-1361.

MDACC Model – Estimated Overall Survival by 4 Levels of Prognostic Score Points

Courtesy of Dr. Garcia-Manero

Conclusion

Classification: Defining more homogeneous MDS subtypes

MDS with characteristic genetic events: • del(5q): haploinsufficiency of RPS14, SPARC • RARS: perturbation of genes involved in mitochondrial metabolism • trisomy 8: genes involved in inflammatory and immune responses

The impact of these new developments on targeted therapy

• Epigenetic silencing, hypomethylating agents, histone deacetylase inhibitors

• N-RAS, FLT3 and JAK2 mutations: tyrosine kinase inhibitors, farnesyl transferase inhibitors

• MDS with del(5q): immune modulating agent, lenalidomide

• MDS with trisomy 8: immunosuppression

Conclusion

Conclusion • Increased role of genomic annotation in MDS

• Newer agents: antiCD33, antiCD123,ABT-119, TGF-β inhibitors, TLR inhibitors

• Lower dose HMAs for lower risk MDS

• Potent oral forms of HMAs: CC-486, ASTX727

• Second generation HMAs: SGI-110

• Combinations: + PD1/PDL1 inhibitors

• Potential role for cytotoxic therapy in diploid HMA failure

• 3 ongoing Phase III trials: CC-486, Rigosertib, ACE-536

THANK YOU

cbuesora@mdanderson.org