The differences and correlations of BCR-ABL transcripts between peripheral blood and bone marrow assays are associated with the molecular responses in the bone marrow for chronic myelogenous leukemia

1. Shivali Jasrotia
16 Nov 2012
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2. American Journal of Haematology
Article first published online: 11 Sep 2012
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3. Introduction
CML (Chronic Myeloid Leukemia)
Result of an acquired genetic abnormality.
20% of all leukemias
Primarily affects adults 25-60 years old, with a peak incidence at 40-50.
Clinical Manifestation:
▪ Asymptomatic (~30%)
▪ Fatigue, weight loss, fever
▪ Abdominal pain and/or early satiety due to splenomegaly (~ 50-90%)
▪ Easy bruising and purpura
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4. Introduction
A translocation first described by Nowell and Hungerford (1960).
and subsequently termed the Philadelphia (Ph) chromosome
after the city of discovery.
“one copy of chromosome 22 is extremely
short in CML patient”
Peter Nowell
CML: The Philadelphia Chromosome
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5. Multiple Breakpoints in Bcr-Abl
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6. Pathophysiologic Result of the Expression of Bcr-Abl
Introduction
Marley et al. Clinical Science, 2005;109
1. Deregulated cellular proliferation
2. Decreased adherence of leukemia
cells to the BM stroma
3. Prevent apoptosis of leukemic
cells.
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7. Clinical Course: Phases of CML
Chronic phase
Median 5–6
years
stabilization
Accelerated
phase
Median duration
6–9 months
Blast crisis
Median survival
3–6 months
Advanced phases
Introduction
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8. Test Target Tissue Sensitivity
(%)*
Use
Cytogenetics Ph chromosome BM 1-10 ▪ Confirm diagnosis of CML
▪ Evaluate karyotypic
 abnormalities other than Ph
 chromosome (ie, clonal
 evolution)
FISH Juxtaposition of
bcr and abl
PB/BM 0.5-5 ▪ Confirm diagnosis of CML
▪ Routine monitoring of
 cytogenetic response in
 clinically stable patients
▪ Routine measurement of
 MRD
RT-PCR bcr-abl mRNA PB/BM 0.0001-0.001 ▪ Routine measurement of
 MRD
▪ Determine the breakpoints of
 the fusion genes
*Number of leukemic cells detectable per 100 cells.
BM = bone marrow; FISH = fluorescence in situ hybridization; PB = peripheral blood;
MRD = minimal residual disease; RT-PCR = reverse transcriptase polymerase chain reaction.
Diagnostic Considerations in CML
Wang et al. Genes Chromosomes Cancer. 2001;32:97

9. Quantitative RT-PCR
for Bcr-Abl in CML
a. Real-time monitoring of the
amplification reaction.
b. Based on the detection and
quantification of a fluorescent
reporter.
c. Does not require a BM aspirate.
d. Can quantify the amount of disease
e. Allows for the identification of cryptic
translocations involving Bcr-Abl.
Diagnostic Considerations in CML
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10. To determine whether molecular monitoring of BCR-ABL
mRNA using PB is comparable to monitoring using BM.
Purpose of the study
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11. a. 330 patients’ samples (Ph positive and diagnosed with p210 transcript) b/w
may 2006 and July 2011.
b. Categorized according to WHO criteria as either in chronic phase (CP, n=319)
or accelerated phase (AP, n=11).
c. On treatment with imatinib at an initial dose of 400 mg (CP) or 600 mg (AP)
daily.
Patients and Methods
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Patients

12. Definitions of Responses to Treatments
Hematologic Response
Complete Hematologic response
1) Normal PB counts (WBC < 10 and plt < 450)
2) Normal WBC differential
3) Normalization of the size of the liver and spleen
Cytogenetic Responses: Ph+ Metaphases
1) complete: 0%
2) partial: 1% - 35%
3) minor: 36% - 65%
4) minimal: 66% - 95%
5) none: 96% - 100%
Molecular Responses: ratio of Bcr-Abl/Abl
Major Molecular Response
3-log10 reduction from initial diagnosis sample
(i.e. 25 →0.025)
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Patients and Methods
Response evaluation:
 The cytogenetic responses in
BM were analyzed every 3 or
6 months until a CCR was
achieved, and subsequently
analyzed every 12 or 18
months after CCR.
 The molecular responses in
PB were analyzed every 3 or
6 months.

13. Molecular studies:
PB (10ml) and BM (5ml) was collected.
Followed by RNA extraction, cDNA synthesis, and Q-PCR.
I. If BCR-ABL mRNA was detected, the sample was considered positive and the
number of transcripts was calculated as BCR-ABL/ABL %.
II. If BCR-ABL mRNA was undetected, the sample was regarded as negative and
BCR-ABL/ABL% was equal to zero.
The molecular responses in PB and BM samples were defined as the log-
reductions of BCR-ABL mRNA level from the baseline value of PB and BM,
respectively, which were the median levels from newly diagnosed CP CML
patients.
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Patients and Methods

14. Statistical Analysis
1. The Wilcoxon Signed Ranks test was used to compare the difference of
the values of BCR-ABL/ABL % between PB and BM assays
2. The Spearman rank order correlation coefficient was used to assess the
correlation between paired PB and BM BCR-ABL values.
3. The Pearson Chi-Squared test was used to compare the categorical
variables.
4. These calculations were performed using SPSS 13.0 software.
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Results
The differences and correlations of BCR-ABL transcripts in PB vs. BM
according to the cytogenetic responses

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Results
Median log-reduction value of BCR-
ABL mRNA from the baseline in PB
was comparable to those in BM in:
a. no MCR samples (0.004 vs. 0.084,
P =0.619)
b. PCR samples (1.263 vs. 1.065, P
=0.185)
c. CCR samples (2.610 vs. 2.903, P <
0.001).
The molecular responses in PB vs. BM associated
with the cytogenetic responses.

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Results
The differences, correlations and agreement of BCR-ABL transcripts in PB
vs. BM associated with the molecular responses in BM

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The dynamics of the molecular responses in PB vs. BM during imatinib therapy
Results
212 paired PB and BM samples (55 CP pts) who underwent sequential BCR-ABL mRNA
detections every 3 or 6 months, from the onset of imatinib therapy.
Median value of the BCR-ABL mRNA in PB for the 55 patients on imatinib treatment was
was lower than that in BM at 3 months (P < 0.001).
Strong correlations among the samples as a whole, at 3, 6, and 18 months.

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Results
Based on the time point of imatinib
therapy, the depth of the molecular
responses in PB and BM were
comparable at 3 months (P=0.975) and
6 months (P =0.076).
However, the log-reduction values of
BCR-ABL mRNA from the baseline in
PB were lower than those in BM at 12,
18, and 24 months (all P values <
0.05).

20. Discussion
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a. Although the amounts of BCR-ABL mRNA in PB and BM from the on
treatment samples strongly correlated as a whole, the differences and
correlations shifted when data were grouped based on the molecular
responses in BM.
b. Study showed that at:
Lower levels of molecular response: BCR-ABL mRNA level in PB was lower
than that in BM and correlates strongly,
High degree of molecular response: BCR-ABL mRNA level in PB was found
to be significantly higher than BM values and correlate only modestly or
poorly .

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c. Findings may suggest that Q-PCR monitoring for CML MRD using PB is not
as equally effective as monitoring using BM. The mechanisms are still not
clear.
d. Further studies focusing on the variation of BCR-ABL mRNA levels in
different cellular compartments in PB and BM might be warranted.
e. A comparative trial to internationally standardize the Q-PCR values of PB
and BM for use in monitoring the diagnostic and treatment responses in
CML patients is needed.
Discussion

22. Thankyou
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