1. Converting BCR-ABL1 to the International Scale: Standardizing the Measurement of Relative Gene Expression Charles E. Hill Emory University School of Medicine

2. Outline Measuring BCR-ABL1 The International Scale Approaches for calibrating to the IS Challenges of relative gene expression testing

3. Disclosures No financial interests/compensation from any of the companies discussed Assay kits were provided for evaluation by both Asuragen and Ipsogen

4. 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

5. Why test? BCR-ABL1 targeted by small molecules Prognosis Monitoring response Prediction of relapse

6. Monitoring BCR-ABL1 Karyotype Fluorescence in-situ Hybridization PCR RT-PCR Competitive RT-PCR Nested RT-PCR qRT-PCR

7. Sensitivities Karyotype – 1:20 FISH – 1-2:200 RT-PCR – 1:100,000

8. Response to Therapy Hughes, NEJM, 2003, 349:1423

9. Prediction of Relapse Press, Clin Cancer Res, 2007, 13:6136

10. 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

11. 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)

12. 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

13. 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

14. 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

15. 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?

16. 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

17. Calibration Pre-WHO Standard Branford, Blood, 2008, 112:3330

18. 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

19. 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

20. WHO BCR-ABL1 Standard

21. Secondary Standards Asuragen – ARQ IS Calibrators and BCR\ABL1 Quant Ipsogen – BCR-ABL Mbcr-IS assay

22. Asuragen ARQ IS Calibrator Panel http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/ARQ_IS_CAL/arq_is_cal.aspx

23. Asuragen Calibrators http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/BCR_ABL1/

24. Ipsogen Calibrator Single point calibrator Tied to WHO Standard Plasmids Set to approximate 0.1% IS (MMR cutoff) Specific for Ipsogen assay

25. Correlation of two IS based assays

26. Comparison to Predicate Test

27. 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

28. 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

29. 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

30. ABL1 as Control

31. BCR as Control

32. 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

33. 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?

34. “Complete” Molecular Response Press et al, Clin Cancer Res 13, 6136 (2007) CMR = no detectable BCR-ABL transcripts

35. 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)

36. 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?

37. Thank You Ruan Ramjit, Kaiser Permanente Karen Mann, Emory International BCR-ABL Standardization Group Emmanuel Labourier, Asuragen Mary Christopher, Ipsogen