Pei Lin, MDPei Lin, MDDepartment of Hematopathology Department of Hematopathology

UT M.D. Anderson Cancer Center, Houston, TXUT M.D. Anderson Cancer Center, Houston, TX

Monitoring of Minimal Residual Disease Monitoring of Minimal Residual Disease Principles and ApplicationsPrinciples and Applications

MRD studies in AML: Potential MRD studies in AML: Potential UtilityUtility

• Definition: Residual disease morphologic Definition: Residual disease morphologic complete remission (CR) (complete remission (CR) (≤ ≤ 5% blasts)5% blasts)

• Time points of testing: post induction, post Time points of testing: post induction, post consolidation, pre-transplant, during CR consolidation, pre-transplant, during CR • Prognostic in most studiesPrognostic in most studies• Effectiveness of therapy: quantitative, kineticsEffectiveness of therapy: quantitative, kinetics• Guidance for risk adjusted therapyGuidance for risk adjusted therapy• Distinguish early recovery from persistent AMLDistinguish early recovery from persistent AML• Predicting early relapsePredicting early relapse

Methods

• MRD by PCR• Leukemic fusion genes (PML-RARA)• Mutations (NPM1)• Gene overexpression (WT1)

• Multiparameter flow cytometry (FCM)

Partner 1 Partner 2

FluorescentTaqman Probe

Amplicon

Forward Primer

Reverse Primer

Break-Point

Translocation-specific Quantitative RT-Translocation-specific Quantitative RT-PCRPCR

t(8;21)RUNX1-RUNX1T1 Positive Negative

MRD by PCR: Leukemic Fusion MRD by PCR: Leukemic Fusion TranscriptsTranscripts

• Recurrent fusions, e.g. t(15;17), t(8;21), inv(16)Recurrent fusions, e.g. t(15;17), t(8;21), inv(16)• Together ~30% of AMLsTogether ~30% of AMLs• qRT-PCR assays qRT-PCR assays • Highly sensitive (1 in 10Highly sensitive (1 in 1055-10-1066))• Normalize to controlNormalize to control• Absolute copy number vs. degree of reductionAbsolute copy number vs. degree of reduction

• RNA instability, turn around timeRNA instability, turn around time• Limited applicabilityLimited applicability• Establish standardized assays and cut-offsEstablish standardized assays and cut-offs

Mutation detection – Mutation detection – NPM1NPM1• NPM1 NPM1 mutations in ~30% of overall AML, ~50% of mutations in ~30% of overall AML, ~50% of

AML with normal karyotypeAML with normal karyotype• Most are 4 bp insertions, two adjacent sitesMost are 4 bp insertions, two adjacent sites

• Allele-specific primers detect 1 in 10Allele-specific primers detect 1 in 1044-10-1055

• Post-therapy MRD is prognostic, can monitor kinetics Post-therapy MRD is prognostic, can monitor kinetics to predict relapseto predict relapse**

• Other potential markers: Other potential markers: FLT3, MLL-PTD, KIT, DMNT3AFLT3, MLL-PTD, KIT, DMNT3A

*Schnittger et al. 2009, Blood 114:2220

mutated

wild typeStd.RT-PCR

Detection of MRD by Detection of MRD by flow cytometry in AMLflow cytometry in AML

• Identify aberrant vs. normal myeloid precursorsIdentify aberrant vs. normal myeloid precursors• Leukemia-associated immunophenotypes Leukemia-associated immunophenotypes

[[LA(I)PsLA(I)Ps]]::• Aberrant lymphoid antigen (CD19, CD7, CD56…)Aberrant lymphoid antigen (CD19, CD7, CD56…)• Aberrant levels of normally expressed antigens Aberrant levels of normally expressed antigens

((↓,↑ ↓,↑ CD38, CD34…)CD38, CD34…)• Coexpression of early and later antigens Coexpression of early and later antigens

(CD34++CD15++)(CD34++CD15++)• Altered forward and side scatterAltered forward and side scatter

Approaches• Must know the patterns of Must know the patterns of normalnormal and and

recoveringrecovering bone marrow bone marrow • If available, compare MRD to the original If available, compare MRD to the original

phenotypephenotype• Need detailed description of antigen Need detailed description of antigen

expression or dot-plotsexpression or dot-plots• Rely on “LAIP” or deviation form normal Rely on “LAIP” or deviation form normal

to identify leukemic cellsto identify leukemic cells

Criteria for Dx and SensitivityCriteria for Dx and Sensitivity• LAIP vs “different-from normal” approachLAIP vs “different-from normal” approach• A: LAIP approach: A: LAIP approach:

• Find aberrant clusters of at least 20 cells, Find aberrant clusters of at least 20 cells, showing abnormal expression of at least 2 showing abnormal expression of at least 2 markers in the LAIP boxmarkers in the LAIP box

• Many Many ““LAIPLAIP” ” have a low frequency in have a low frequency in normal BMnormal BM

• B: “different-from normal” approach B: “different-from normal” approach (monocytic leukemia)(monocytic leukemia)

8-color MRD: Baseline study

Courtesy of Dr. Jeffrey Jorgensen

BM CD34+: Normal vs. AML MRDNo

rmal

AML

MRD

, 0.1

%

Courtesy of Dr. Jeffrey Jorgensen

Normal BM

CD45 V500-A

SS

C-A

-102102 103 104 1050

65536

131072

196608

262144

CD45dim gate

7.5%

CD34 PerCP-Cy5-5-AS

SC

-A-102 103 104 105

0

65536

131072

196608

262144

0.8%

CD34-Percp

CD34 PerCP-Cy5-5-A

CD

38 F

ITC

-A

-102 103 104 105

-102102

103

104

105

CD64 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102 103 104 105

-102102

103

104

105

CD14 APC-H7-AC

D64

PE

-Cy7

-A

-102 103 104 105

-102102

103

104

105

CD33 PE-AC

D34

Per

CP

-Cy5

-5-A

-102 103 104 105

-102102

103

104

105

CD13 APC-A

CD

33 P

E-A

-102 103 104 105

-102102

103

104

105

CD64 PE-Cy7-A

CD

38 F

ITC

-A

-102 103 104 105

-102102

103

104

105

Normal Bone Marrow

HLA-DR V450-A

CD

34 P

erC

P-C

y5-5

-A-102 103 104 105

-102102

103

104

105

CD123 APC-A

CD

34 P

erC

P-C

y5-5

-A

-102 103 104 105

-102102

103

104

105

HLA-DR V450-A

CD

123

AP

C-A

-102 103 104 105

-102102

103

104

105

CD2 FITC-AC

D34

Per

CP

-Cy5

-5-A

-102 103 104 105

-102102

103

104

105

HLA-DR V450-A

CD

117

PE

-A

-102 103 104 105

-102102

103

104

105

CD117 PE-A

CD

34 P

erC

P-C

y5-5

-A

-102 103 104 105

-102102

103

104

105

Normal BM

CD5 FITC-A

CD

38 A

PC

-A

-102 103 104 105

-102

102

103

104

105

CD19 PE-Cy7-AC

D38

AP

C-A

-102 103 104 105

-102102

103

104

105

CD7 PE-A

CD

38 A

PC

-A

-102 103 104 105

-102102

103

104

105

CD56 V450-AC

D38

AP

C-A

-102

103

104

105

-102

102

103

104

105

CD19 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102

103

104

105

-102

102

103

104

105

%

CD19 PE-Cy7-A

SS

C-A

-102

103

104

105

0

65536

131072

196608

262144

0.8%

CD19-PeCy7 gate

Normal BM

FG, 1-31-2013

CD45 V500-A

SS

C-A

-102 102 103 104 105

-400

65236

130872

196508

262144

CD45dim gate6.7%

CD33 PE-A

CD

34 P

E-C

y7-A

-102 103 104 105

-102

102

103

104

105

CD34 PE-Cy7-A

CD

117

PE

-A

-102102 103 104 105

-102101

103

104

105

%

CD34 PE-Cy7-A

CD

117

PE

-A

-102 103 104 105

-102

102

103

104

105

CD45 V500-A

SS

C-A

-102 102 103 104 105

-400

65236

130872

196508

262144

CD45dim gate57.7%

CD33 PE-A

CD

34 P

E-C

y7-A

-102102 103 104 105

-102101

103

104

105

%

FG, 11-9-2012

CD33 PE-A

CD

34 P

erC

P-C

y5-5

-A

-102 103 104 105

-102102

103

104

105

CD

117P

ECD34 PE-CY7

Normal

Patient 1

Patient 2: 21 days post induction

CD45 V500-A

SS

C-A

-102 102 103 104 105

-400

65236

130872

196508

262144CD45dim gate58.5%

CD34 PE-Cy7-AS

SC

-A-10

210

210

310

410

5-400

65236

130872

196508

262144CD34-PEcy711.2%

Morphology: 21% of blasts

Post therapy

CD56 V450-A

CD

34 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102100

103

104

105

CD64 PE-Cy7-A

CD

34 P

erC

P-C

y5-5

-A

-102 102 103 104 105

-102100

103

104

1051.5%

0.3% 16.8%

CD34 PE-Cy7-A

CD

38 P

erC

P-C

y5-5

-A

-102102 103 104 105

-102101

103

104

105

0.0%0.4%

28.1%71.5%

CD34 PerCP-Cy5-5-A

CD

38 F

ITC

-A

-102 102 103 104 105

-102100

103

104

10512.3%

1.1% 3.6%

CD56 PerCP-Cy5-5-A

CD

117

PE

-A

-102102 103 104 105

-102101

103

104

105

CD64 APC CD

34 P

E-C

y7

Original

Sensitivity

• To yield sensitivity of 0.01%, collect at To yield sensitivity of 0.01%, collect at least 200K cells per tube (20/200K = 1 least 200K cells per tube (20/200K = 1 in 10in 104 4 = 0.01%)= 0.01%)

• Sensitivity may be limited due to Sensitivity may be limited due to background normal cells, 0.1% or higher background normal cells, 0.1% or higher

• * 0.1% is commonly used threshold in the * 0.1% is commonly used threshold in the literatureliterature

Detection of MRD by Flow Detection of MRD by Flow CytometryCytometry

• Advantages: Advantages: • Widely applicable (90- 95% of cases) Widely applicable (90- 95% of cases) • Relatively rapid turn around timeRelatively rapid turn around time

• Disadvantages: Disadvantages: • Interpretation often challenging, requires Interpretation often challenging, requires

experienceexperience• Can be expensiveCan be expensive• Lack of standardization Lack of standardization

Potential challenges• LAIPs may not cover all leukemic blasts, partial LAIPs may not cover all leukemic blasts, partial

overlap with normaloverlap with normal• Antigen shift resulting from selection/emergence Antigen shift resulting from selection/emergence

of subclones of subclones • A complete change in LAIPs in about 20% of A complete change in LAIPs in about 20% of

AML, with 80% having at least one LAIP similar AML, with 80% having at least one LAIP similar to the original (Voskova et al)to the original (Voskova et al)

• Post therapy hypocellular samplePost therapy hypocellular sample• Use a comprehensive panel of antibodies to Use a comprehensive panel of antibodies to

establish baselineestablish baseline

Summary• MRD detection by FCM or/and PCR are promising MRD detection by FCM or/and PCR are promising

tools to guide therapy and to improve outcomestools to guide therapy and to improve outcomes• Each method has pros and consEach method has pros and cons• More studies are underway to better incorporate More studies are underway to better incorporate

the data into clinical decision making (dose the data into clinical decision making (dose intensification and/or ASCT)intensification and/or ASCT)

• Timing of MRD testing by FCM and the cut off Timing of MRD testing by FCM and the cut off levels for each time point that are significant are levels for each time point that are significant are being refinedbeing refined

Acknowledgement • Dr. Jeffrey Jorgensen MD Anderson Cancer

Center