Acute Leukemia - Present and Future - GBCFLUX · Wood and Borowitz (2006) Henry’s Laboratory...

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

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

Normal T cell Maturation

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

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

Blasts - Abnormal Antigen Intensity

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

Blasts - Aberrant Lymphoid Antigens

Blasts - Aberrant Maturation

Normal Granulocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Granulocytic Maturation

Acute Promyelocytic Leukemia

Normal Monocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Monocytic Maturation

Acute Monocytic Leukemia

Acute Myelomonocytic 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-

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-

A594 APC APC-

Tube 1 CD16 cCD3 CD7 CD56 CD5 CD38 CD3 CD45

Tube 2 CD8

CD48 CD5 CD34 CD16+

CD3 CD4 CD7 CD45

0.1% abnormal immature B cells

ALL MRD

06-01469

T-ALL MRD

12-03268

12-02987

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

Roshal, et al (2009) submitted

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

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

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)

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

(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

Flow Cytometry

TCRBSEQ

0.00001

0.0001

Individual

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

Acute Leukemia - Present and Future - GBCFLUX · Wood and Borowitz (2006) Henry’s Laboratory...

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