charles e. hill emory university school of medicine
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
Converting BCR-ABL1 to the International Scale:
Standardizing the Measurement of Relative Gene
Expression
Charles E. HillEmory University School of
Medicine
Outline
Measuring BCR-ABL1
The International Scale
Approaches for calibrating to the IS
Challenges of relative gene expression testing
Disclosures
• 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?
• BCR-ABL1 targeted by small molecules
• Prognosis
• Monitoring response
• Prediction of relapse
Monitoring BCR-ABL1Karyotype
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
• From NCCN 2.2013, qPCR should be performed:
• 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
• 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
• 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?
• Sample volume and integrity
• Processing and extraction
• Reverse transcription
• Control gene (BCR, ABL, GUSB, G6PDH, β2M)
• Primers
• Quantification standards
• Instrument and analysis
Harmonizing Results
• 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
• 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
• 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?
• 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
BCR-ABL1 WHO Standard
• 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR
• 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
• Asuragen – ARQ IS Calibrators and BCR\ABL1 Quant
• 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
• 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
Patient Correlation
y = 1.0582x + 2.5081
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100Ipsogen IS %
Asu
rag
en I
S %
Comparison to Predicate Test
-1.6
-1.1
-0.6
-0.1
0.4
0.9
1.4
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Mean Log %
Dif
fere
nce
(IS
- p
red
icat
e)
Comparison to Predicate
• 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
• 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
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
ABL1 as Control
Me
asu
red
% I
S
Log BCR-ABL1 Positive Cells
BCR as Control
Me
asu
red
% I
S
Log BCR-ABL1 Positive Cells
Other genes as Controls
• 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
• 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
Press et al, Clin Cancer Res 13, 6136 (2007)
CMR = no detectableBCR-ABL transcripts
Practical LOD Issues
• 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
• 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
• Ruan Ramjit, Kaiser Permanente
• Karen Mann, Emory
• International BCR-ABL Standardization Group
• Emmanuel Labourier, Asuragen
• Mary Christopher, Ipsogen