Evaluation of Two Commercial Kits

for Flow Cytometric Minimal Residual

Disease Detection

in B-Acute Lymphoblastic Leukemia

Ari Ahn1, Chan-Jeoung Park

1, Young-Uk Cho

1, Seongsoo Jang

1,

Eul-Ju Seo1, Hyery Kim

2, Kyung-Nam Koh

2, Ho-Joon Im

2, Jong-Jin Seo

2

DEPARTMENT OF LABORATORY MEDICINE1, DEPARTMENT OF

PEDIATRICS2, UNIVERSITY OF ULSAN, COLLAGE OF MEDICINE

AND ASAN MEDICAL CENTER

Selected Short Talk II

I have no personal or financial interests to declare:

I have no financial support from an industry source at the current

presentation.

대한혈액학회 Korean Society of Hematology

COI disclosure

Name of author ： Ari Ahn

INTRODUCTION

ALL risk stratification →risk-based therapy

Minimal residual disease (MRD)

• Analytical methods with better sensitivity that allow the detection of leukemic cells in lower proportions than those achieved by morphological evaluation

2

Pediatric Annals. 2015;44(7):e168-e174.

Risk Factor Favorable Unfavorable

NCI risk group Age: 1-10 years

WBC

MRD is an independent prognostic factor and highly predictive

factor of relapse

1. The end of induction therapy

– MRD negativity is the main favorable outcome predictor independent of

other risk factors

⃰ MRD after 7 or 14 days of therapy: additional benefit in risk stratification

2. The end of consolidation therapy

– the most informative for relapse risk stratification

3 Blood. 2015;126(8):964-971)

INTRODUCTION

Flow cytometric MRD detection is based on the identification of leukemia-associated immunophenotypes (LAIPs)

• Differences in antigen expression between leukemic cells and normal B-cell precursor

1. Asynchronous antigen expression

– Coexpression of antigens normally expressed in a different maturation stage

Coexpression CD34 and CD20

2. Cross-lineage antigen expression

– Myeloid, B-lymphoid, or T-lymphoid

3. Changes in the usual intensity of antigen expression

– Overexpression, Underexpression, or even loss of expression

CD34, CD58, CD10, CD123 ↑/ CD45, CD38, CD24 ↓

4

INTRODUCTION

B-cell precursors (Hematogones)

• Morphologically resemble the neoplastic

lymphoblasts of precursor B-ALL

• Often increase in regenerating marrow following chemotherapy or HSCT,

especially after more than 28 days

5 Leukemia & Lymphoma 2004;45:277–85.

Hematogones

Mature B cells

Early hematogone

(~17%)

Late hematogone

(~17%)

Intermediate hematogone

(mean ~65%)

(dim)

INTRODUCTION

Hematogone always exhibit a continuous and complete maturation spectrum of antigen expression

• Flow cytometry cytograms represent the “Hematogone’s pattern” of sequence and intensity of antigen expression

The neoplastic lymphoblasts show immunophenotypic deviation from the hematogone’s pattern

6

early stage

hematogone

mature B-cell

late stage hematogone

Intermediate stage

hematogone

CD34 CD45

CD

10

CD19+

gating

Blood. 2001;98:2498-2507.

SS

C

Mature B-cell

early stage

hematogone

late stage

hematogone

CD

10

CD20

Intermediate stage

hematogone

INTRODUCTION

MRD detection by flow cytometry

Advantages

1. Broad applicability

• > 95% of patients present identifiable LAIPs

2. Rapid turnaround time of results →risk-based therapy

3. Ability to distinguish between viable and apoptotic cells

4. Relatively less expensive

Disadvantages

1. False positive results

• Phenotypic similarities between leukemic lymphoblasts and hematogones

2. False negative results

• Phenotypic changes in residual leukemic cells throughout treatment

3. Limited standardization

→There are ongoing efforts to standardize MRD quantification and commercial kits are emerging to improve accuracy and reproducibility

7

MATERIALS AND METHODS

BM aspirates (n=50)

•

MATERIALS AND METHODS

Multiple time points of MRD monitoring according to

treatment protocol in B-ALL

9

End of consolidation

(n=18, 36%)

End of intensification

(n=3, 6%)

End of maintenance

(n=4, 8%)

After HSCT

(n=8, 16%)

Chemo off (n=9, 18%)

D7(0%), D14 (n=3, 6%)

COMPLETE REMISSION

End of induction

(n=5, 10%)

MATERIALS AND METHODS

Flow cytometric MRD

• measured by two commercial kits and analyzed using each analysis

instrument by manual serial gating according to our protocol

10

DuraClone RE ALB Tube

(Beckman Coulter, Miami, USA)

BCP-ALL-MRD

(Cytognos SL, Salamanca, Spain)

Analysis instrument Kaluza (Beckman Coulter, Miami, USA) FacsDIVA software (BD, CA, USA)

Infinicyt (Cytognos SL, Salamanca, Spain)

Acquisition cell

numbers 2.0x106 2.0x106

Detection of the limit 0.001% 0.001%

Main LAIP Antigen over/ under expression Aberrant expression

Antibodies

CD20/CD45/CD38/CD58/

CD34/ CD19/CD10

tube1: D20/CD45/CD81/CD66c+CD123/

CD34/CD19/CD10/CD38,

tube2: D20/CD45/CD81/CD73+CD304/

CD34/CD19/CD10/CD38

LAIP, leukemia-associated immunophenotypes

RESULTS

Pediatric patients with B-ALL (n=30)

• The median patients age was 7 years (range, 3-20)

• The ratio of boy to girl was 1:1

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Table 1. Molecular subtypes of 30 pediatric patients with B-ALL

Molecular subtype Number

B-lymphoblastic leukemia with hyperdiploidy 11

B-lymphoblastic leukemia with t(12;21)(p13;q22); ETV6-RUNX1 6

B-lymphoblastic leukemia with t(9;22)(q34;q11.2); BCR-ABL1 2

B-lymphoblastic leukemia with t(1;19) (q23;p13.3); TCF3-PBX1 2

B-lymphoblastic leukemia, NOS 9

RESULTS

The immunophenotypes of leukemic cells at diagnosis

• CD10 positive (cutoff ≥20%) in all patients and variable expression of CD34 and CD20

• All positive for CD58 but variable for CD38 from negative to intermediate positive

• One or more aberrant expressions were present in 91.7% (22/24) of pediatric patients with B-ALL

12

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CD66c CD123 CD73 CD304 CD13 CD33 CD56

Figure 1. Frequency of aberrant expression of leukemic cells at diagnosis of

pediatric patients with B-ALL.

54.2 %

(13/24) 50.0 %

(12/24)

33.3 %

(8/24)

4.2 %

(1/24)

17.2 %

(5/29) 10.3 %

(3/29) 3.4 %

(1/29)

Flow Cytometric MRD

• The leukemic cell% (mean±SD) were 1.4±1.2% by conventional morphology, 0.166±0.268% by DuraClone and 0.034±0.143% by Cytognos BCP-ALL-MRD (P=0.001 in difference)

• MRD% by DuraClone and Cytognos BCP-ALL-MRD were well correlated (P

CASE (5/M)

B-lymphoblastic leukemia with t(9;22)(q34.1;q11.2);BCR-ABL1

• A total of 5 follow up BM studies were performed after diagnosis

• The residual leukemic cells were measured by conventional morphology,

DuraClone, Cytognos BCP-ALL-MRD, and RQ-PCR

All variables showed good correlation (r= 0.9~1.0, P=0.005~0.023)

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Time point Morphology Duraclone Cytognos

BCP-ALL-MRD

BCR-ABL1,

normalized copy

number

Diagnosis 90.8 62.612 63.889 111.67

D7 after induction 36.4 29.723 38.567 75.57

D14 after induction 2.0 1.318 1.145 13.41

End of induction 1.6 0.044 NA 1.25

End of consolidation 0.0 0.004 0.000 0.25

End of maintenance 0.4 0.005 0.000 0.0046

CASE (5/M)

B-lymphoblastic leukemia with t(9;22)(q34.1;q11.2);BCR-ABL1

• Immunophenotypes of leukemic cells at diagnosis

Acute Lymphoblastic Leukemia, common cell, group III

CD19+/CD10+/CD34+/CD20- (cutoff ≥20%)

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CD10: 98.5% CD34: 98.5% CD20: 6.7% CD19: 98.4%

76.3%

16

DuraClone

(Dx)

+

and and

17

32.030% 31.937% 32.014% 31.707% 32.010%

BCP-ALL-MRD

tube1

(Dx.) Singlets

18

63.924% 63.889% 63.918% 63.251% 63.877%

Singlets

BCP-ALL-MRD

tube2

(Dx.)

19

+

DuraClone

(D14)

and and

20

0.435% 0.409% 0.433% 0.330% 0.432%

Singlets

BCP-ALL-MRD

tube1

(D14)

21 21

1.149% 1.145% 1.144% 0.850% 1.144%

BCP-ALL-MRD

tube2

(D14) Singlets

HEMATOGONES

(D90)

BM: COMPLETE

REMISSION

22

Hematogones

(End of

consolidation)

early stage

hematogone

mature B-cell

late stage hematogone

Intermediate stage

hematogone

CD34

CD

10

Mature B-cell

early stage

hematogone late stage

hematogone

CD

10

CD20

Intermediate stage

hematogone

23

+

DuraClone

(End of con-

solidation)

and and

24

0.007% 0.000% 0.001% 0.000% 0.007%

Singlets

BCP-ALL-MRD

tube1

(End of con-

solidation)

25 25

0.007% 0.000% 0.000% 0.000% 0.007%

Singlets

BCP-ALL-MRD

tube2

(End of con-

solidation)

DISSCUSSIONS

DuraClone RE ALB Tube (Beckman Coulter, Miami, USA)

• It is two-dimensionally displayed, intuitive and easy to analyze

• No aberrant expression markers of the leukemic blasts

Phenotypic similarities between leukemic lymphoblasts and

hematogones→ false positive MRD

26

Fixed backbone of lymphoid precursor

cells

Aberrant antigen

expression of the leukemic

blasts

MRD evaluation by

flow cytometry

Haematologica 2009;94(6):870-874.

Figure. The recommended antibodies panel of MRD evaluation by flow cytometry

BCP-ALL-MRD (Cytognos SL, Salamanca, Spain)

• More antibodies, including aberrant expression markers

• Infinicyt™, software recommended by Cytognos, is not available

– Automated population separator

– Revolutionary approach for data integration and multidimensional analysis

Two-dimensional analysis instead of Infinicyt™→false negative MRD

27

SUMMARY AND CONCLUSIONS

Detection of MRD in patients with B-ALL is limited by

conventional morphology.

For the flow cytometric detection of MRD in B-ALL,

the kit detecting antigen over or under expression was

adequate, but the kit detecting aberrant expression showed

significantly lower level of MRD.

It is very helpful for analysis of MRD to know the

immunophenotype of leukemic cells at diagnosis and to

confirm the presence of hematogones by flow cytometric

cytograms pattern.

28