Acute Leukemia -

Present and Future

Brent Wood MD, PhD

Department of Laboratory Medicine

University of Washington

Acute Leukemia

• Diagnosis

• 20% blasts by morphology

• Classification

• Lineage assignment

• Cytogenetic/Molecular lesions

• Prognostication

• Age, white count

• Cytogenetic/Molecular lesions

• Post-therapeutic Monitoring

Abnormal population identification

• Normal

– Antigens expressed in consistent and reproducible patterns with maturation

• Neoplastic

– Increased or decreased normal antigens

– Asynchronous maturational expression

– Aberrant antigen expression

– Homogeneous expression

Normal B cell Maturation

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Normal B cell Maturation

Wood (2004) Methods Cell Biology 75:559-576

Precursor B cell

Lymphoblastic Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Rothenberg, et al (2008) Nat Reviews 8:9-21

Normal T cell Maturation

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Precursor T-cell Lymphoblastic Leukemia

Cherian and Wood (2012) Flow Cytometry in Evaluation of Hematopoietic Neoplasms: A Case-Based Approach

T-ALL Outcome

Schrappe, et al (2011) Blood 2077-2084

T-ALL

Schrappe, et al (2011) Blood 2077-2084

Precursor T-cell Lymphoblastic Leukemia

Cherian and Wood (2012) Flow Cytometry in Evaluation of Hematopoietic Neoplasms: A Case-Based Approach

Early Thymic Precursor T-ALL

• CD1a and CD8 negative

• CD5 < 75% positive

• HLA-DR, CD13, CD33, CD34 or CD117 positive

• Overlap with MPAL

– Dependent on cyto CD3

– Absence of MPO or monocytic antigens

ETP T-ALL Pretreatment

09-01859

Coustan-Smith et al (2009) Lancet Oncology 10:147-156

Zhang, et al (2012) Nature 481:157-163

Acute Myeloid Leukemia

• Heterogeneous group of diseases

– Accumulation of non-lymphoid progenitors

– Poor maturation

– Stem cell-like characteristics (subset)

• Molecular basis

– Large number of abnormalities described

– Multiple mutations per case

• Adult > Pediatric

Cell Type Identification

Borowitz et al (1993) AJCP 100:534-40.

Steltzer et al (1993) Ann NY Acad Sci 667:265-280

Normal Blast Maturation

Wood (2004) Methods Cell Biology 75:559-576

Blasts - Abnormal Antigen Intensity

Wood (2007) Clinics in Lab Medicine 27:551-575

Blasts - Aberrant Lymphoid Antigens

Wood (2007) Clinics in Lab Medicine 27:551-575

Blasts - Aberrant Maturation

Wood (2007) Clinics in Lab Medicine 27:551-575

Acute Myeloid Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Normal Granulocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Granulocytic Maturation

Wood (2004) Methods Cell Biology 75:559-576

Acute Promyelocytic Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Normal Monocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Monocytic Maturation

Wood (2004) Methods Cell Biology 75:559-576

Acute Monocytic Leukemia

Acute Myelomonocytic Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Acute Myeloid Leukemia

• Immunophenotypic classification

– Loose relationship to genetic abnormalities

– Largely irrelevant clinically

• Molecular classification is the future

From Kern et al (2005) CRC Rev Onc/Hem 56:283-309

Methods for MRD Detection

• Antigenic abnormalities

– Alterations in protein expression

– Integrates effect of molecular abnormalities

– Flow cytometry commonly used

• Molecular mutations

– Specific molecular lesions

• No single target

– Most assays use PCR of single genes

• Next generation sequencing in development

COG MRD Panels

• B lineage ALL

• T lineage ALL

FITC PE PerCP-

Cy5.5

PE-Cy7 APC APC-H7

Tube 1 CD20 CD10 CD38 CD19 CD58 CD45

Tube 2 CD9 CD13/33 CD34 CD10 CD10 CD45

PB FITC PE PE-

TR

PE-

Cy5

PE-

Cy7

A594 APC APC-

H7

Tube 1 CD16 cCD3 CD7 CD56 CD5 CD38 CD3 CD45

Tube 2 CD8

BV421

CD48 CD5 CD34 CD16+

56

CD3 CD4 CD7 CD45

0.1% abnormal immature B cells

ALL MRD

06-01469

T-ALL MRD

12-03268

12-02987

From Kern et al (2005) CRC Rev Onc/Hem 56:283-309

Average 2.3 LAIP per patient

AML Informative Antigens

AML MRD stem cells

Van Rhenan (2007) Leukemia 21:1700-7

Residual Acute Leukemia

0.007% Wood (2007) Clinics Lab Med 27:551-575

Immunophenotypic Stability

• ALL

762510

Immunophenotypic Stability• ALL

– 30 consecutive patients with MRD detectable

– All had some change in immunophenotype

– CD10 and CD34 down-modulation, CD19 and CD20 upmodulation

– Associated with use of steroid in induction therapy

From Giapa et al (2005) Leukemia 19:49-56

Immunophenotypic Stability

T-ALL

Roshal, et al (2009) submitted

Immunophenotypic Stability

T-ALL

Roshal, et al (2009) submitted

56.3%

8.8%

0.2%

Diagnosis

Day 16

Day 32

HP06-05604

Population Identification

• Leukemia-associated immunophenotype

– At diagnosis

• Region containing infrequent number of events

• Relative to specific reagent combination

– Follow-up

• Enumerate number of events in predefined region

– Focus not on population identification

• Does not account for shifts in immunophenotype

• Increased dependence on background noise

• Uncertain what is being detected

LAIP

From Feller et al (2004) Leukemia 18:1380-1390

LAIP

From Kern et al (2005) CRC Rev Onc/Hem 56:283-309

Focus is immunophenotype, not population

Prognostic significance of MRD in pediatric

ALL (flow cytometry)

Borowitz et al. Blood 111:5477-85 (2008)

Prognostic significance of MRD in pediatric

ALL (flow cytometry)

Borowitz et al. Blood 111:5477-85 (2008)

Prognostic significance of MRD in pediatric

ALL (flow cytometry)

Borowitz et al. Blood 111:5477-85 (2008)

Prognostic significance of MRD in

pediatric ALL (flow cytometry)

Borowitz et al. Blood 111:5477-85 (2008)

San Miguel et al, (2001) Blood 98:1746-51

Large portion of adult AML patients have

occult leukemia at achievement of remission

• 61 of 82 (74%) with

> 0.1% LAP cells

had high relapse risk

• 21 of 82 (26%) had

much lower relapse

risk

(APL patients

excluded)

AML MRD Transplant

Walter, et al (2011) JCO 29:1190-7

OS EFS

CR1 prior to transplant HR = 7.06 DFS, 8.49 Relapse

MRD by Next Gen Sequencing

• Determine unique sequence at diagnosis

– TCR gamma

– TCR beta

• Deep sequence locus and count number

of unique sequences present

– ? Number of normal sequences present

• TCR gamma insufficiently diverse

• TCR beta is better

– ? Tumor heterogeneity

PT

_44

PT

_63

PT

_48

PT

_9

PT

_7

PT

_15

PT

_59

PT

_36

PT

_33

PT

_53

PT

_52

PT

_5

PT

_50

PT

_1

PT

_32

PT

_8

PT

_30

PT

_49

PT

_66

PT

_39

PT

_65

PT

_41

PT

_61

PT

_51

PT

_6

PT

_47

PT

_4

PT

_58

PT

_2

PT

_40

PT

_14

PT

_55

PT

_57

PT

_3

PT

_12

PT

_54

PT

_45

PT

_60

PT

_68

PT

_56

PT

_13

PT

_42

PT

_11

Flow Cytometry

TCRBSEQ

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Wu, et al (2012) Sci Trans Med 4: 1-7

Conclusions

• Flow cytometry is capable of minimal residual detection

– Rapid

– Cost effective

– Quantitative

– Applicable to wide range of disorders

• Requires

– Consistent flow cytometric technique

– Informative immunophenotype

– Knowing normal patterns of antigenic expression

• Immunophenotypic change is common

– Leukemia-associated immunophenotype may be misleading

• Residual disease strongly associated with poor outcome

– Post-induction, post-consolidation, pre-transplant, post-transplant