New Approaches to

Myeloid Disorders in

Blood and Bone

Marrow

Kathryn Foucar, MD

kfoucar@salud.unm.edu

July 2013

Colorado Society

for Clinical

Pathologists

How to get from

2

to

and

Avoid

becoming

Goals • Master use of conventional

“tools” to sub-categorize myeloid

neoplasms; reorganize clues to

specific myeloid neoplasms

• Embrace the ever-expanding role

of molecular genetics in both

diagnosis and prognosis 3

4

Clinician to Pathologist

“What genetic tests do I

need to order in this case?”

“What do these results

mean?”

5

Old Expectations/Tools of the Trade

Good Old Days

Document and classify advanced disease

Tools of the Trade

Clinicopathologic correlation > 150yrs

Autopsy > 120yrs

Romanosky/cytochem stains ~ 100 – 120 yrs ago

Cytogenetics (1960) ~ 50yrs

Immunophenotype ~ 25 – 30 yrs

6

New Expectations/Tools of the Trade Today

• Provide comprehensive assessment of neoplasm

including molecular/genetic features

• Provide comprehensive prognostic factors

• Determine optimal treatment

• Predict risk of therapy

• Detect and quantify minimal residual disease

Tools of the Trade

• All traditional “tools”

• 8+ color Flow cytometry

• Molecular/genomic “tools” including Next

Generation Sequencing

Maintain all

“Good Old Days”

skills and

Expand Diagnostic

“Horizons” 7

Encode 2012

8

In the world of

Myeloid Neoplasms,

CML has always lead

the way

9

Battle

between

Bennett

and

Virchow

Leukemia First Described in 1845

10

Clinico-Pathologic

Correlation

Blood:

Buffy Coat

CML:

WBC > 900,000

WBC’s

11

Unstained

130 years ago 150 years ago 12

1960

13

Nowell and Hungerford

Philadelphia Chromosome

Courtesy J. Anastasi

1973

14

15

Courtesy J. Anastasi David Baltimore, 1980’s

1980’s; different groups

Ph1: reciprocal translocation

BCR-ABL1 fusion gene

1982-1985

Translocation results in

constitutive tyrosine kinase

activity CML 16

CML

•1st genetically defined

leukemia

•Must document BCR-ABL1

fusion gene for diagnosis

17

Source: Kalidas, et al.

NEJM 2001; 286:895-898

Leukemogenic

Effects of

Constitutive Non-

Receptor Tyrosine

Kinase Activation

18

Source: Kalidas, et al. NEJM 2001;286:895-898

Therapy to Block Tyrosine Kinase Activity (1987-1998 )

19

Blast-Phase in CML: 1983-present

Source: Hehlmann, R. How I treat CML blast crisis. Blood 2012;120:737.

20

Who 2008 Myeloid Neoplasm

• Myeloproliferative (MPN) (11)

• Myelodysplasia (MDS (8)

• MDS/MPN (4)

• Acute myeloid leukemia (24)

• Myeloid neoplasm with eos and

PDGFRA, etc (3) 21

Myeloid Neoplasms

• 4 broad categories

• Typical blood and BM features

• Systemic approach

• CBC clues, blast %, dysplasia,

megakaryocytes, degree of

maturation 22

Comparison of blood features

MDS MPN AML

23

MDS CML AML

Comparison of bone marrow features 24

25

Usual Features of Myeloid Neoplasms (at diagnosis)

Disorder Bld

Counts

BM

Cellularity

%

BM

Blasts

Maturation Morphol ↑

Spl/L

MPN ↑↑ Nl - ↑↑↑ Nl Present Nl

(megas)

Yes

MDS ↓↓ ↑ (usu) Nl –

19%

Present Dyspl. No

MDS/

MPN

↑, ↓ ↑↑ Nl –

19%

Present Dyspl. Yes

AML ↑, ↓ ↓ - ↑↑ (usu) 20% Minimal

(usu)

Dyspl.

(usu)

No

(usu)

26

Diagnosis requires integration of

• Clinical (e.g. spleen size)

• Hematologic (sequential CBC data)

• Other laboratory data (e.g. LDH)

• Morphologic (blood and bone marrow)

• Genetic features

Disorders are characterized by

• Uncontrolled cell proliferation (usually multiple lineages)

• Intact maturation (mature cells predominate)

Myeloproliferative Neoplasms

27 Erythrocytosis, thrombocytosis

74 yr-old Female Hgb 17 Hct 51% Plt 950,000

28 BM aspirate: ↑ cell, ↑ megakaryocytes

74 yr-old Female Hgb 17 Hct 51% Plt 950,000

29 BM biopsy: megs, dilated sinuses, EMH

74 yr-old Female Hgb 17 Hct 51% Plt 950,000

Likely Diagnosis?

P vera

30

31

71b22

32

71b26

Bone marrow core: osteosclerosis

33

•60b29, 62b33 inset

BM: pleomorphic megas; Bld: LER

Likely Diagnosis?

Primary

myelofibrosis

(PMF) (Or: Post polycythemic PV-history is key!)

34

What additional tests

are needed?

•JAK2 mutation analysis

including Exon 12

•Exclude BCR-ABL1

•Possible karyotype 35

36

Janus Kinases in Cytokine Signal Transduction

Source: Goldman,

J. NEJM 352;17,2005

Receptor

tyrosine kinase

negative

transmembrane

receptors

include EPO,

TPO, G-CSF

receptors

37

Why Does Excess, Unregulated Cell

Production Occur?

CML

Ph1 t(9;22) results in BCR-ABL1 fusion gene

with constitutive tyrosine kinase activity

Other MPN

Point mutation in regulatory region of JAK2

results in constitutive tyrosine kinase activity

38

MPN: Key Tips

• Recognize blood clues

• Molecular genetic assessment

essential (BCR-ABL1, JAK2)

• Assess for mast cell infiltrates

• Recognize features of disease

progression

Myelodysplasia

• Ineffective HP results in cytopenias

despite hypercellular BM

• Hallmark of cytopenias with

dysplasia

• Variable % blasts in blood and BM

• Dysplasia may involve 1, 2, or 3 HP

lineages 39

40 Neutropenia; assess qual/quant all lineages

Elderly Patient with

Unexplained Cytopenia(s)

• Common problem

• All the usual causes of anemia excluded

• Must distinguish MDS from non-

neoplastic

• Low grade MDS very challenging

–Minimal (so-so) dysplasia

–Normal cytogenetics 41

Blood Features in MDS

42 Blasts < 20% Dysplasia

Bone Marrow Aspirate in MDS

43 Megakaryocyte abnormalities; small, pawn ball

MDS – Look Alike • Zinc induces

copper

deficiency

• Cytopenias

• Vacuoles

• Normalizes

when zinc

discontinued

44

MDS: Key Tips

• Exclude lookalikes

• Low grade MDS dx of exclusion

• Count blasts in blood and BM

• Assess architecture

• Conventional cytogenetics key

(better than FISH)

• Exclude low blast count AML 45

Acute Myeloid Leukemia

• Many molecular genetic subtypes

• Key AML-defining genotypes

• Usual threshold 20% blasts

in blood and BM (exceptions)

46

Blasts and Immature Myeloid

Elements

• Myeloblasts

• Promyelocytes*

• Monoblasts

• Promonocytes

• Erythroblasts†

• Megakaryoblasts

47

* Blast equivalent in some AML’s † Not typically included in blast % except for acute erythroid leukemia

Myeloid Blasts

49

Acute megakaryoblastic leukemia

50 Acute megakaryoblastic leukemia

51

NSE

AMoL, t(9;11)(p22;q23)

Forward Scatter

Sid

e S

catt

er

CD45

Sid

e S

catt

er

Sid

e S

catt

er

CD45 CD14 CD4

CD

8

CD45

HL

A-D

R

AML with multilineage dysplasia 52

AML: Morphologic Classification*

*Source: J Clin Oncol 21:256, 2003

Years from Start of Therapy

53

Acute Promyelocytic Leukemia t(15;17)(q22;q11-12)

Clinical: Constant rate over lifetime Profound thrombocytopenia; coagulopathy Medical emergency!

54

Morphology: Major: Hypergranular promyelocytes (low WBC) Microgranular: 1) folded nuclei (high WBC) 2) inconspicuous granules Little, if any, maturation beyond promyelocyte

Cytogenetics/ Molecular:

t(15;17) PML/RARα fusion gene

IP: My antigen + , HLA/DR - , CD34 -, MPO+, High side scatter!

Outcome: Manage coagulopathy; favorable risk

APL with t(15;17) - Note cells rare in blood 55

Elevated WBC: Microgranular APL; looks Monocytic 56

WHO 2001: Acute Myeloid Leukemia

57

I. AML with recurrent cytogenetic abnormalities

• AML with t(8;21) (AML1/ETO)

• AML with inv(16) (CBFβ/MYH11)

• APL with t(15;17) (PML/RARα)

• AML with 11q23 (MLL) abnormalities

II. AML with multilineage dysplasia

III. Therapy – related AML/MDS: 2 types

IV. AML, NOS (lineage based)

AML: Cytogenetic Prognostic Groups*

Favorable: t(8;21), t(15;17), inv(16),

t(16;16), other

Intermediate: Normal karyotype, +8, -4, +6

Poor: -5/del(5q) , -7/del(7q) , t(11q23),

other, complex karyotype

( 3 abnormalities)

*Independent prognostic variable, multivariate analysis

Source: Haferlach, et al. J Clin Oncol 21:256, 2003

58

59

AML: Overall Survival by Karyotype*

Years from Start of Therapy

*Source: J Clin Oncol 21:256, 2003

P < 0.0001

AML Classification: Biologic Groups

2001 2008

AML with

recurrent genetic

abnormalities

4 types 9 types

t(1;22), NPM1, CEBPA,

inv(3), t(6;9)

AML with MDS-

related changes

AML after MDS AML after MDS, MDS/MPN

AML with multi. dysplasia

AML with MDS karyotypes

Therapy-related

AML

Alkylating Agent

Topo II inhibitor

T-AML, MDS, MPN

T-AML with balanced tx

T-AML w/o translocations

Down Syndrome None Transient abnormal

myelopoiesis, other

leukemias 60

AML: Overall Survival

Source: Grimwade, Hematology 2009 61

Combined Cytogenetic and Molecular

Stratification

Favorable

t(8;21), inv(16), t(15;17)

Mutated NPM1 without FLT3

Mutated CEBPA (normal CC)

Intermediate-1

(normal CC)

Mutated NPM1 and FLT3-ITD

Wild-type NPM1 with /without FLT3-ITD

Intermediate-2

t(9;11); other cytogenetics

Adverse inv(3), t(6;9), other MLL, -5, del (5q,) -7,

complex

62

63

AML: Class I and Class II Mutations

Class I Mutations

(Proliferation)

Class II Mutations

(Impaired differentiation)

FLT3 PML-RARA

KIT RUNX1-RUNX1T1

RAS CBFB-MYH11

PTPN11 MLL fusions

JAK2 CEBPA

* The mechanism of leukemogenesis for NPM1

mutations remains somewhat unclear 64

65

Source: NEJM 366(12):1079-89, 2012

400 cases; patients <60 massive parallel sequencing

Acute Myeloid Leukemia

Source: Patel, et al. NEJM 366:1079, 2012

Gene Overall

Frequency (%)

FLT3 (ITD, TKD) 37 (30, 7)

NPM1 29

DNMT3A 23

NRAS 10

CEBPA 9

TET2 8

WT1 8

IDH2 8

IDH1 7

KIT 6

RUNX1 5

MLL-PTD 5

ASXL1 3

PHF6 3

KRAS 2

PTEN 2

TP53 2

HRAS 0

EZH2 0

A Total Cohort

67 Source: Patel, et al. NEJM 366:1079, 2012

68 Source: NEJM 366(12):1079-89, 2012

• Dramatic expansion of

realm of pathology

expertise

• Dramatic pace of new

discoveries

• “Survival of the fittest” 69