Transcript Converting BCR-ABL1 to the International Scale

Converting BCR-ABL1 to
the International Scale:
Standardizing the
Measurement of Relative
Gene Expression
Charles E. Hill
Emory University School of Medicine
Outline
Measuring BCR-ABL1
The International Scale
Approaches for calibrating to the IS
Challenges of relative gene expression
testing
Disclosures
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No financial interests/compensation from
any of the companies discussed
Assay kits were provided for evaluation by
both Asuragen and Ipsogen
Chronic Myelogenous
Leukemia
1.6 cases per 100,000
1.75:1 male:female
BCR-ABL1 translocation (Ph chromosome)
Molecular monitoring of BCR-ABL1 has
become standard of care
SEER data 2012
Why test?
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BCR-ABL1 targeted by small molecules
Prognosis
Monitoring response
Prediction of relapse
Monitoring BCR-ABL1
Karyotype
Fluorescence in-situ Hybridization
PCR
RT-PCR
Competitive RT-PCR
Nested RT-PCR
qRT-PCR
Sensitivities
Karyotype – 1:20
FISH – 1-2:200
RT-PCR – 1:100,000
Response to Therapy
Hughes, NEJM, 2003, 349:1423
Prediction of Relapse
Press, Clin Cancer Res, 2007, 13:6136
NCCN Guidelines
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From NCCN 2.2013, qPCR should be
performed:
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At diagnosis
Every 3 months when responding to
therapy. After CCyR, every 3 months for
3 years, then every 3-6 months
For rising transcript levels (1 log) with
MMR, repeat in 1-3 months
The IRIS Trial
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The International Randomized Study of
Interferon versus STI571
Major Molecular Response = 3 log
decrease from average level at diagnosis
Three laboratories harmonized results
(Australia, UK, and USA)
BCR-ABL1 in Practice
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Many labs test for BCR-ABL1
Testing and reporting are variable
Many report change from diagnostic (local)
baseline, but not calibrated the same
Results not generally comparable between
labs
Why is testing variable?
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Sample volume and integrity
Processing and extraction
Reverse transcription
Control gene (BCR, ABL, GUSB, G6PDH, β2M)
Primers
Quantification standards
Instrument and analysis
Harmonizing Results
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Median measurement of 30 shared
baseline samples established as 100%
3 log decrease from baseline (0.1%) is
Major Molecular Response
For example, if median ratio BCR-ABL/BCR
is 0.75, MMR = 0.00075
International Scale defined by these two
points
Getting to the IS
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3 IRIS trial labs use IS by definition (Adelaide,
Seattle, London)
IRIS trial samples used to define 100% IS are
not available to individual labs
Sample exchange with a laboratory calibrated
to the IS?
Pre-WHO Standard
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Branford, et al., Blood, 112:3330, 2008, “Desirable
performance characteristics for BCR-ABL measurement
on an international reporting scale to allow consistent
interpretation of individual patient response and
comparison of response rates between clinical trials,”
Exchange of patient samples with IS calibrated
reference lab to determine Conversion Factor
Requires many samples and periodic re-assessment
Very burdensome for few reference labs
Calibration Pre-WHO
Standard
Branford, Blood, 2008, 112:3330
What does an IS do?
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An International Scale helps standardize
quantification
An IS does not improve pre-analytic issues
An IS does not reduce inherent assay
variability
An IS does improve reporting
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BCR-ABL1 WHO
Standard
1st WHO International Genetic Reference Panel
for quantitation of BCR-ABL translocation by RQPCR
Intended for manufacturers/labs to develop
secondary standards
4 ampules of freeze dried cells
K562 cells (e14a2) diluted to different ratios in
HL60 cells
Spanning relevant range of %IS values
WHO BCR-ABL1
Standard
Secondary Standards
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Asuragen – ARQ IS Calibrators and
BCR\ABL1 Quant
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Ipsogen – BCR-ABL Mbcr-IS assay
Asuragen ARQ IS
Calibrator Panel
http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/ARQ_IS_CAL/arq_is_cal.aspx
Asuragen Calibrators
http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/BCR_ABL1/
Ipsogen Calibrator
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Single point calibrator
Tied to WHO Standard
Plasmids
Set to approximate 0.1% IS (MMR cutoff)
Specific for Ipsogen assay
Correlation of two IS
based assays
Asuragen IS %
Patient Correlation
100
90
80
70
60
50
40
30
20
10
0
y = 1.0582x + 2.5081
0
20
40
Ipsogen IS %
60
80
100
Comparison to Predicate
Test
1.4
Difference (IS - predicate)
0.9
0.4
-1.5
-1
-0.5
-0.1 0
0.5
-0.6
-1.1
-1.6
Mean Log %
1
1.5
2
2.5
Comparison to Predicate
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Very similar results for both calibrated to
the IS
IS tests are 0.4 and 0.35 lower than LDT
Very important to communicate this to
clinicians when changing reports
Alternatively, report old result and IS
Advantages of
Secondary Standards
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Allow calibration to WHO standard
Simpler than exchanging many samples
with reference lab
Manufacturer’s QC and lot-to-lot control
Periodic checks for drift are more feasible
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Control Genes and
Quantification
ABL1 is the most commonly used control
gene
BCR was used as control gene for IRIS trial
labs
GUSB used by some
EAC found ABL1, GUSB, and B2M suitable
(Beillard, Leukemia, 2003, 17:2474)
BCR was not reported in study by EAC
Measured % IS
ABL1 as Control
Log BCR-ABL1 Positive Cells
Measured % IS
BCR as Control
Log BCR-ABL1 Positive Cells
Other genes as Controls
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Do not participate in the translocation
Should be expressed constitutively and not
vastly over/under expressed compared to
transgene
GUSB and B2M
Limit of Detection
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Depends on transgene copies and control
gene level
What is minimum control gene for
acceptable sensitivity?
Calculate minimum control gene level to
detect MMR?
“Complete” Molecular
Response
CMR = no detectable
BCR-ABL transcripts
Press et al, Clin Cancer Res 13, 6136 (2007)
Practical LOD Issues
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Balance sensitivity with not rejecting too
many specimens
For LOD=5 copies BCR-ABL, need 50,000
copies of control gene to get 4.0 log
For 5.0 log need 500,000 copies control
gene
LOD is affected by total RNA input (5
copies in isolation easier to detect than 5
copies with background nucleic acid)
Summary
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The WHO Standard and development of IS
have better standardized testing and
reporting of BCR-ABL1
Development of assays/calibrators tied to
the IS make transition much simpler
Are more sensitive assays needed?
Thank You
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Ruan Ramjit, Kaiser Permanente
Karen Mann, Emory
International BCR-ABL Standardization
Group
Emmanuel Labourier, Asuragen
Mary Christopher, Ipsogen