Transcript example - Association for Molecular Pathology (AMP)

Consensus Workshop in Bethesda
- Molecular Monitoring of CML
- Organized by John Goldman
- International representation of 53 participants
- October 25-26, 2005
(These slides will be posted on the AMP webpage)
Objectives of Bethesda Workshop
-
Harmonize methods and ways of expressing results of BCRABL1 Q-PCR assays in CML patients undergoing treatment
-
Discuss methods to detect and quantitate BCR-ABL1 kinase
domain mutations and their clinical significance
-
Discuss the frequency with which individual patients should be
monitored in the clinic
-
Make recommendations for international practice
(NOT evidence-based practice guidelines)
Sample Collection and Processing
Don’t switch back and forth between blood and marrow in a given patient over time
Example Q-PCR ratios over time in 4 patients:
EDTA is suitable,
Avoid Li-Heparin
Blood Volume = 10ml
(If WBC=5k/ul, this is
50 million cells)
Process within 24hrs
for MRD samples
Susan Branford and Timothy Hughes, October 2005
Reverse Transcription
- Use random primers at final concentration of at least 25M
- Superscript may be the optimal reverse transcriptase
- Run duplicate assays on extracted RNA for minimal residual
disease measurement
Susan Branford and Timothy Hughes, October 2005
Control Genes for BCR-ABL1 Q-PCR
BCR and ABL are suitable control genes.
Other genes are suitable only if:
- similar expression level as the target transcript
- target and control degrade at the same rate
- assay does not cross-react with DNA
Use control gene to identify samples with inadequate RNA
Use control gene to compensate for differences in:
- WBC count
- Efficiency of extraction, RT, and amplification
- Degradation
Susan Branford and Timothy Hughes, October 2005
Q-PCR
- Primer and probe design should avoid the polymorphic site in BCR
exon 13= exon b2 (T>C g.108,654, GenBank U07000)
- No cross-reactivity with DNA
- Standards = cells, RNA, armored RNA, cDNA, or plasmid
at 10-fold serial dilutions over dynamic range in every run; R2>0.99
- High, low and negative controls in every run
- Reproducibility should allow for 2-fold differences to be detected at
levels near the major molecular response (MMR)
- Anti-contamination measures are essential
Susan Branford and Timothy Hughes, October 2005
Q-PCR Standardization
A commercial standard is recommended so that all labs can
calibrate to the same BCR-ABL/control gene ratio that is
equivalent to the major molecular response (MMR)
established by Hughes et al in the IRIS trial*
(meantime log-fold reduction over “lab mean” seems a good way to
report data, with a >3-log reduction indicating success)
Should composition of the commercial standard be:
cells, RNA, cDNA, armored RNA, or plasmid?
* Hughes TP, Kaeda J, Branford S, et al. N Engl J Med 349:1423, 2003
Your lab’s BCR-ABL1/ control gene
Universal Standardization of Levels
1) Purchase commercial universal standards and run them in your lab’s Q-PCR
2) Ensure your assay is linear
3) Calculate a multiplier that adjusts your assay to the IRIS trial MMR of 0.1%
Susan Branford and Timothy Hughes, October 2005
Universal Standardization of Levels
Your lab’s BCR-ABL1/ control gene
X
X
X
1-log reduction
= 10 %
2-log reduction
=1 %
X
MMR = 3-log reduction
= 0.1 %
Baseline
=100 %
In this lab’s case,
multiply all results by 0.8
to shift curve down
MMR is thus expressed
on an international scale
of 0.1%
1) Purchase commercial universal standards and run them in your lab’s Q-PCR
2) Ensure your assay is linear
3) Calculate a multiplier that adjusts your assay to the IRIS trial MMR of 0.1%
Susan Branford and Timothy Hughes, October 2005
Reporting Q-PCR results
-
Report ratio of BCR-ABL1 to control gene, having standardized
your assay to the IRIS trial MMR of 0.1% (3-logs below 100%)
-
Until standardization to the IRIS trial MMR level is possible,
report as a log-reduction over “lab mean at diagnosis”
-
Report assay sensitivity for patients with undetectable levels;
sensitivity is based on control results in that run (including RT
step) and qualities of that patient sample (e.g. control gene level)
Susan Branford and Timothy Hughes, October 2005
Indications for Q-PCR testing
- At initial diagnosis, save sample in case needed to establish
lab mean or to later prove an amplifiable breakpoint is
present in that patient’s tumor (if karyotype or MRD negative);
Do karyotype to prove 9;22 and to detect secondary changes
suggestive of progression
- Every 3 months during therapy
Susan Branford and Timothy Hughes, October 2005
ABL1 Mutation Testing
A minimum panel tests for the G250E and T315I that are proven to
confer resistance to tyrosine kinase inhibitors
Sequencing and mutation-specific assays are suitable
Direct sequencing
ASO PCR
Pyrosequencing
Fluor. PCR & PNA clamp
Screen by DHPLC or DGGE
What analytic sensitivity is needed for clinical purposes?
Indications for mutation testing:
- Failure to respond by 3 months
- Rising Q-PCR levels of BCR-ABL (>half log)
- Loss of cytogenetic or hematologic response
- Routine quarterly monitoring of accelerated phase/ blast crisis
- After therapy change to assess effect
Your Opinion Counts
This slideshow is on the AMP Website
Your opinion and our AMP consensus will be
synthesized and transmitted through
Suzanne Kamel-Reid and Peggy Gulley
[email protected]
[email protected]
Discussion Points on MRD Testing
1. Blood volume required for MRD testing…NIH 10ml, we think 5 ml
2. Should duplicate testing be done on blood, extracted RNA, or cDNA?
3. What are suitable control-normalizer genes…BCR, ABL1, others?
4. Should we calibrate our assays to a single international standard? YES
If so, should standard be cells, RNA, armored RNA, cDNA, engineered plasmid,
or plasmid?
Meantime report as log-fold reduction over “lab mean at diagnosis”? YES
5. What level of reproducibility is acceptable…2-fold, 5-fold, 10-fold?
6. Is a standard curve needed in every run? YES
Feed back to- [email protected]
[email protected]
[email protected]