evaluation of two commercial kits for flow cytometric minimal residual disease...
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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
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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
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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)
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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
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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)
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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
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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
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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
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MATERIALS AND METHODS
BM aspirates (n=50)
•
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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%)
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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
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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
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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
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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)
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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
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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
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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%
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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)
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21 21
1.149% 1.145% 1.144% 0.850% 1.144%
BCP-ALL-MRD
tube2
(D14) Singlets
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HEMATOGONES
(D90)
BM: COMPLETE
REMISSION
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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)
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25 25
0.007% 0.000% 0.000% 0.000% 0.007%
Singlets
BCP-ALL-MRD
tube2
(End of con-
solidation)
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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
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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
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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
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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.
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