Molecular Monitoring of CML

1
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)

2
Objectives of Bethesda Workshop

• Harmonize methods and ways of expressing results
  of BCR- ABL1 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)

3
Sample Collection and Processing Dont 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 WBC5k/ul, this is 50 million
cells) Process within 24hrs for MRD samples
Susan Branford and Timothy Hughes, October 2005
4
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
5
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
6
Q-PCR

• Primer and probe design should avoid the
  polymorphic site in BCR exon 13 exon b2 (TgtC
  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 R2gt0.99
• 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
7
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 gt3-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 3491423, 2003
8
Universal Standardization of Levels
Your labs BCR-ABL1/ control gene

• Purchase commercial universal standards and run
  them in your labs Q-PCR
• 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
9
Universal Standardization of Levels
X
Baseline 100
X
In this labs case, multiply all results by
0.8 to shift curve down MMR is thus expressed on
an international scale of 0.1
1-log reduction 10
X
2-log reduction 1
Your labs BCR-ABL1/ control gene
X
MMR 3-log reduction 0.1

• Purchase commercial universal standards and run
  them in your labs Q-PCR
• 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
10
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
11
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
patients tumor (if karyotype or MRD negative)
Do karyotype to prove 922 and to detect
secondary changes suggestive of progression -
Every 3 months during therapy
Susan Branford and Timothy Hughes, October 2005
12
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 (gthalf log)
• Loss of cytogenetic or hematologic response
• Routine quarterly monitoring of accelerated
  phase/ blast crisis
• After therapy change to assess effect

13
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
Suzanne.Kamel-Reid_at_uhn.on.ca margaret_gulley_at_med.u
nc.edu
14
Discussion Points on MRD Testing
1. Blood volume required for MRD testingNIH
10ml, we think 5 ml 2. Should duplicate testing
be done on blood, extracted RNA, or cDNA? 3.
What are suitable control-normalizer genesBCR,
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 acceptable2-fold, 5-fold, 10-fold? 6. Is a
standard curve needed in every run? YES
Feed back to- Suzanne.Kamel-Reid_at_uhn.on.ca
margaret_gulley_at_med.unc.edu

braziel_at_ohsu.edu