myelodysplastic syndromes classification
TRANSCRIPT
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
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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
