5/17/2013

MYELODYSPLASTIC SYNDROMES:

Myelodysplastic Syndrome: Part 1 – Example MDS cases coming into our a pathologist’s perspective practice over the last several weeks

Kajal Sitwala, MD, PhD

Part 2 – Review of MDS definition, features, morphology, and biology

Case 1:

-64 y.o. man with longstanding low back pain (unrelated)

-MRI showed abnormal bone marrow signal  referred to Hematology

-Had normocytic anemia dating back 15 years

-Bone marrow biopsy showed fairly normal overall cellularity, but relative expansion in erythropoiesis. Iron stain showed many ring sideroblasts

-Diagnosis of RARS (low-grade MDS) -Refractory Anemia with Ring Sideroblasts

-Interval treatment supportive (erythropoietin to boost RBC production)

-Follow-up bone marrow

Dyserythropoiesis in aspirate smear

Ring sideroblasts (special stain of aspirate smear) Hypercellularity; odd megakaryocytes, but not typical for MDS

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Diagnosis:

Persistent RARS Case 2:

-No increase in blasts -62 y.o. man with anemia and thrombocytopenia

-Frank dysplasia still limited to the erythroid lineage -Bone marrow showed RAEB-1 (high-grade MDS)

-Refractory Anemia with Excess Blasts -Cytogenetic analysis showed same abnormality as before (+8) -Trisomy 8 can be seen in myeloid disorders including MDS and AML -For RARS, it’s worse prognosis to see it (versus normal karyotype) -Treatment/management course complicated by cold agglutinin disease and transfusion refractoriness -Flow cytometry: main contribution is confirming no increase in blasts -Mild aberrancy of CD56 co-expression on maturing granulocytes and monocytes -Recently, transfusion requirements became too severe to manage -(supportive finding, not enough to definitively diagnose MDS) supportively, hospitalized for aggressive chemotherapy

-Bone marrow performed to assess response -Recently, MDS-RARS shown to have strong association with mutation: -Haploinsufficiency of SF3B1

Peripheral blood: Granulocyte with mature, clumped chromatin but lack of granulation or nuclear lobation (Pseudo-Pelger-Huet) Increased blasts in aspirate smear

Diagnosis:

Persistent MDS, now best classified as RAEB-2

-In subsequent bone marrows, I only reclassify MDS if worse -improvements are described, but disease isn’t “downgraded” as a new MDS subtype

-14% blasts in differential count of aspirate smear

-Reticulin stain confirmed the presence of fibrosis

-Historically normal karyotype in leukemia cells; not repeated with this specimen

-Flow cytometry with 15% blasts, similar phenotype as prior cells

-Patient given even more aggressive treatment (AML induction), however, disease is persisting

Hypercellular but loose marrow (fibrosis), Dysplastic megakaryocytes

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Case 3:

-83 y.o. woman with history of MDS dating back to 2008 -When seen here in 2010, classified as RCMD -Refractory Cytopenia with Multilineage Dysplasia

-2010 bone marrow with similar cytogenetic abnormalities, still low-grade (no increase in blasts

-Since then, CBC counts have held pretty steady with lenolidamide treatment -uniquely efficacious in cases involving chromosomal deletions on 5q

-Some concern with low hemoglobin values, so bone marrow reassessed

Basophilic stippling in peripheral blood

Dyserythropoiesis in aspirate smear Small and hypolobated megakaryocytes in aspirate smear

Diagnosis:

Persistent RCMD

-Cytogenetics unchanged over past 5 years -46,XX,del(5)(q13q33),add(11)(q23)[19]/46,XX[1] -doesn’t fit for isolated 5q deletion (‘’5q-minus syndrome’’) but some features overlap -often, dramatic worsening (e.g. increase in blasts) corresponds with clonal evolution

-Flow cytometry: increased basophils, light scatter changes (but no aberrant phenotype) -No increase in blasts

-Later: discuss role of lenolidamide in 5q deleted cases

Ring sideroblasts in aspirate smear

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1953 – Knudson hypothesis: cancer results from accumulated DNA mutations MDS 1990’s – 2-hit model of leukemia

Class I mutations Class II mutations A group of clonal hematopoietic stem cell diseases characterized by cytopenias, FLT3-ITD PML-RARA FLT3-TKD AML1-ETO dysplasia, ineffective hematopoiesis, and JAK2-V617F CBF -MYH11 increased risk of developing acute KIT mutations CEBPA mutations myeloid leukemia. RAS mutations NPM1 mutations?

Proliferation advantage Differentiation arrest Survival advantage clonogenicity -Principally a disease of older adults -Disorder of HSCs and their microenvironment

AML

Adapted from WHO 2008

General (simplified) model of myeloid malignancies So MDS is a stem cell neoplasm: but what does “immortalization” look like?

Clonogenic, but not rapid, proliferation of stem cells (There is also increased apoptosis) Inability to complete differentiation and leave marrow

Hallmark of MDS – peripheral blood cytopenias PLUS bone marrow hypercellularity

In contrast to MPDs… Rapid proliferation but complete differentiation – cells accumulate in both places (clinical presentation from tumor burden rather than loss of function)

CLASS I CLASS II Or acute leukemia… Proliferation and lack of differentiation: blasts in bone marrow and blood Proliferation advantage Differentiation arrest Survival advantage clonogenicity Programs of self-renewal and Myeloproliferative disorders Myelodysplastic syndromes differentiation use some of the same molecules (transcription factors)

AML

-Clonogenicity: (Can’t see whether a cell will keep dividing or not)

-Hypercellularity

-Maturation arrest: (Marrow is full of precursors anyhow)

-Skew toward immaturity

As Hematopathologists, what we SEE is morphologic abnormalities that result From these molecular processes

Normal Erythroid Maturation (ASH image Bank)

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Pictures of normal hematopoietic elements

A blood smear and a marrow, or just a single marrow picture

Normal Megakaryocyte making platelets (ASH image Bank) Normal Leukocytes in peripheral blood (ASH image Bank)

Predictions about dysplasia, based on concept of “maturation arrest” – 1a. Immaturity 1. immaturity of any kind a. cells that are more like a precursor form Excess blasts b. failure to develop characteristics of final state Erythroid immaturity (increase in early forms)

2. dyssynchrony between different elements/split Small megakaryocytes that have not divided the nucleus yet personality as far as further cell division

3. just plain weird

Patient with RCMD (including 5q minus but with additional abnormalities) Patient with RCMD (including 5q minus but with additional abnormalities)

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Patient with RCMD (including 5q minus but with additional abnormalities) Patient with RCMD (including 5q minus but with additional abnormalities)

1b. Failure to finalize

-lack of granulation in circulating neutrophils

-lack of nuclear lobation in circulating neutrophils *mimic of harmless genetic state (Pelger-Huet anomaly)

Patient with RAEB-1)

2. Dyssynchrony, or confusion about further division

-Hemoglobinized cytoplasm with still immature nucleus

-Nuclear budding in erythroid precursors

-Megakaryocytes with completely separated nuclear lobes

Pseudo-Pelger Huet neutrophils with hypogranulation (ASH image Bank)

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Patient with RAEB-1 Dysplastic erythroid precursor with hemoglobinization and large nucleus (ASH image Bank)

Dysplastic megakaryocytes with separation of nuclear lobes (ASH image Bank) Nuclear budding in erythroid precursor (ASH image Bank)

3. Just plain weird

-Ring sideroblasts

-Megaloblastoid chromatin in erythroid precursors

-Basophilic stippling in red blood cells

-Dimorphic circulating red cell population

-Vacuoles (especially erythroid precursors) *beware of MDS mimic – copper deficiency (sometimes caused by zinc toxicity)

Patient with RCMD (core biopsy touch imprints)

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Patient with RCMD (including 5q minus but with additional abnormalities) Megaloblastoid chromatin (ASH image Bank)

That “classification” was purely speculative, a tool to mentally account for dysplasia

Are there any cases where we have pinpointed the connection between and dysplasia?

Ring sideroblasts (ASH image Bank)

5q minus syndrome 5q minus syndrome Anemia – can be quite severe Normal to elevated platelet count; characteristic megakaryocytes Hypercellular marrow, variable erythroid dysplasia Female predominance – middle to older age 1974 – Nature – clinical syndrome with chr 5 long arm deletion reported

2001 – WHO classification recognizes 5q- as distinct subtype of MDS

2002 – Blood – commonly deleted region narrowed to 40 genes by FISH/Southern

2007 – ASH plenary abstract – identification of candidate gene

no biallelic deletions or point mutations in 40 genes – likely haploinsufficiency

therefore reduction in could be exploited: RNA interference

findings published: Ebert et al., Nature, January 2008 (Golub laboratory – Harvard)

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Haploinsufficiency of ribosomal : Megakaryocyte:erythroid ratio using markers CD41, glycophorin A Phenotype of macrocytic anemia shows dependence -Cell cycle arrest, failure to complete erythroid differentiation RPS14 participates in ribosomal synthesis -Interestingly, 5q minus syndrome differs from other MDS by exhibiting RPS19 mutations found in 25% Diamond-Blackfan Anemia cases erythroid hypoplasia

Studies in mice prove the genotype:phenotype correlation for anemia

Further research showed increased RPS14 levels after lenolidamide therapy

But, doesn’t answer the whole question…megs are normal in those mice

Also, DBA patients (RPS19) don’t have platelet or megakaryocyte problems

Ebert et al., Nature, January 2008

microRNA: from non-coding parts of genome -regulate mRNA (coding parts of genome) However, another gene on 5q-, in fact the genes for two microRNAs, have recently been shown to recapitulate the phenotype of small megs….

From Identification of miR-145 and miR-146a as mediators of the 5q– syndrome phenotype; From Identification of miR-145 and miR-146a as mediators of the 5q– syndrome phenotype; Daniel T Starczynowski, et al Nature Medicine 16, 49–58 (2010) (obtained with permission) Daniel T Starczynowski, et al Nature Medicine 16, 49–58 (2010) (obtained with permission)

The genes used to regulate hematopoiesis are the same set of genes that determine body patterning during embryogenesis!

Targets of these microRNAs are transcription factors (TiRAP, TRAF6), and these in turn Regulate level of interleukin-6

Increased IL-6 can recapitulate megakaryocyte phenotype

Lenolidamide* reduces IL-6 levels

But wait…didn’t they think lenolidamide worked by increasing RPS14 expression?

But wait…there’s also the SPARC gene on 5q-, necessary for mouse hematopoiesis, lenolidamide increases its expression as well!

*Thalidomide analog with many anti-cancer mechanisms in vitro and in vivo WikipediaFrom

Genotype:phenotype correlations are real, but messy and redundant

Hard to untangle, because of complexity of hematologic pathway, evolved in parallel to general embryogenic cascade

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The genes used to regulate hematopoiesis are the same set of genes that determine body patterning during embryogenesis!

Human genes:

cluster genes

HOXA chromosome 7 HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13

HOXB chromosome 17 HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13

HOXC chromosome 12 HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13

HOXD HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13

-Used once to make our body (in utero), then over and over for the rest of our lives to make blood cells

-HOXA9 is most upregulated gene in AML -Potent mediator of leukemia

>-8-fold no change >8-fold Meis1

72 96 120 h 72 96 120 h

Slc18a1 Csf2ra Sox4 Lhfpl2 Control Auts2 Myo1e Hoxa9 Flt3 Ier3 Dnajc10 4631426J05 A930001M12 Per2 Vertebrate Cd34 Vcl conservation Pctk2 Ctsc Hoxa9 Amot Cpne2

2SD Conservation Score Conservation2SD Inpp5a gene

Tgm3 Ifngr1 - Cradd Dach1 exon Rora Dfna5h Foxp1 Bpil2 B4galt4 Klf5 Mean+/ Man1a Crem Meis1 0 1 2 3 4 5 6 Lmo2 2900024C23 Peak-widths from center of peak 5830405N20 A130090K04 Pcnx Hist1h1c Pdcd4 Niban Pde7a Ebi2 MLL Polycomb Fut8 Plxnd1 Tox P2ry1 Tcf4 Trps1 12000 Fndc3b - 4-OHT Bcar3 Hox Loci TALE factors - 4931406I20 Msr1 10000 + 4-OHT + Map4k5 Frmd4b Bmp2k Ccl4 8000 9330182L06 Osbpl3 Aff3 Mrvi1 Common developmental Camk2d Gpr84 genes (direct targets) 6000 Il10rb Ch25h 1110028C15 Rgs1 4000 Pdk1 Gca cells x 10000 Usp12 Mgll Body patterning/ Hematopoiesis/ Zfp36l1 Cd34 Csf2ra Embryonic development Leukemogenesis 2000 Hgf Flt3 B2Galt6 Id2 Context-specific Gpr56 Tgfbr3 Thbs1 0 Tlr4 downstream targets

day 0 2 4 6 8

WHO 2008 Myelodysplastic Syndromes:

Refractory cytopenia with unilineage dysplasia: Refractory anemia Summary – A pathologist’s perspective on MDS Refractory neutropenia Refractory thrombocytopenia Refractory anemia with ring sideroblasts -WHO classification is our guide to approaching the diagnosis Refractory anemia with multilineage dysplasia -Threshold is key Refractory anemia with excess blasts -CBC information is the predictor Myelodysplastic syndrome associated with isolated del(5q) -This is how patients come to attention of hematology, then to us Myelodysplastic syndrome, unclassifiable -Morphology and cytogenetics make up our toolset Childhood myelodysplastic syndrome -Flow cytometry can sometimes contribute provisional category: Refractory cytopenia of childhood -FISH plays a more minor role than conventional karyotype -New studies support possible role for point mutations In the separate category of AML and Related Precursor Neoplasms:

Therapy-related myeloid neoplasms (includes cases meeting morphologic MDS criteria) -The biology guides our understanding, and contextualizes morphology

Transformation from MDS: AML with myelodysplasia-related changes -And genetics holds (some of) the answers

Also a separate category: Myelodysplastic/Myeloproliferative Neoplasms

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