Molecular Monitoring of CML Organized by John Goldman International representation of 53 participants October 25-26,

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 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

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 • BCRandABL 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 (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

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 >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

8. Universal Standardization of Levels Your lab’s BCR-ABL1/ control gene Purchase commercial universal standards and run them in your lab’s 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 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-log reduction = 10 % X 2-log reduction =1 % Your lab’s BCR-ABL1/ control gene X MMR = 3-log reduction = 0.1 % Purchase commercial universal standards and run them in your lab’s 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 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

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 (>half 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@uhn.on.ca margaret_gulley@med.unc.edu

14. 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- Suzanne.Kamel-Reid@uhn.on.ca margaret_gulley@med.unc.edu braziel@ohsu.edu