Talk Outline

1. The introduction of ABL kinase domain mutation testing in CML patients showing resistance to Imatinib Davina Clavering, Elizabeth Perrott, Julian Borrow, Joanne Mason, Susanna Akiki and Mike Griffiths West Midlands Regional Genetics Laboratory

2. Talk Outline • Introduction to CML • Methods for AKD analysis • Mutations identified to date • WMRGL database • Follow up • Future work

3. Chronic Myeloid Leukaemia • Characterised by the Philadelphia chromosome t(9;22) • Results in fusion of BCR and ABL genes • Imatinib mesylate is the frontline therapy

4. Developing resistance • Glivec has revolutionised treatment for CML but resistance is a problem in a minority of patients • Point mutations in the ABL kinase domain are the most frequently reported cause of resistance • Other mechanisms • BCR-ABL amplification • Over expression of LYN, changes in drug influx and efflux proteins • To overcome resistance: increase dose / try different targeted therapy (new generation kinase inhibitors e.g. dasatinib, nilotinib), bone marrow transplant

5. Setting up an AKD mutation service • Identification of mutations can direct patient management • Increase dose of imatinib • Provide evidence that an alternative drug may be more suitable • Work currently funded by Novartis for 18 months • Monitoring user satisfaction with a view to obtaining local NHS funding

6. Monitoring of CML patients • Samples received at regular intervals usually every 3-6 months • Quantitative RT-PCR carried out and disease levels compared to ABL “housekeeping gene” values Good response to imatinib Good response post transplant

7. Activation of samples for ABL kinase domain mutation testing • Clinical request • Internal activation based on ELN guidelines • treatment failure at 6 or more months • sub-optimal treatment response • loss of response

8. ABL F ABL R BCR F Current Testing Strategy 28S rRNA • RNA clean-up using DNase • Semi-nested RT-PCR followed by direct sequencing • Analysis of sequencing using Mutation Surveyor • Send report and follow-up form to clinician Before 18S rRNA DNA After BCR ABL

9. T315I/A/D F311L/I/V F317L C305S G321E F382L V299L L324Q G383D L298V M343T K357R L384M P296H A344V L387F/M E292V S348L M388L A350V V289A/I M351T/L V379I E355G/D L3641 F359V/C/D/I Adapted from Melo et al., Cancer Lett 2007 249(2):121-32 K285N E281A E279K T277A E276G E275K L248V G250E/A/F Q252H/R P-loop Imatinib binding site C Activation loop Y253H/F E255K/V A397P M244V H396R/P D241G M237I E453G/K/A/V F486S E450Q Q447R E459K/Q S438C ABL kinase domain mutations A412V • Over 100 mutations identified in the literature • T315I is most common • 16 mutations account for 86% reported cases

10. Mutations identified to date • 51 patients reported so far • 13 mutations identified in 12 patients • Pick up rate of 24%

11. WMRGL Database • Compiled a mutation database based on an extensive literature search consisting of 3 types of data • Mutations which emerge in response to treatment and the ability of each drug to overcome a particular mutation • IC50 values • In vitro mutagenesis data • Resistance profiles are created for each mutation and added to the report

12. Resistance profiles IC50 value: the concentration of drug required to inhibit 50% of enzymatic activity Imatinib: Sensitive <4, Partially Sensitive ≥4, <9 Resistant ≥9 Nilotinib: Sensitive <4, Partially Sensitive ≥4, <30, Resistant ≥30 Dasatinib: Sensitive <4, Partially Sensitive ≥4, <6, Resistant ≥6 Cut-offs: Numbers refer to fold increases in IC50 compared to “wild-type” BCR-ABL *unique mutation

13. Case report - Patient VK • Patient diagnosed June 05, aged 18 • On imatinib for 15 months • Underwent bone marrow transplant Oct 06 • Tested 1 month later; low disease levels • 4 weeks later loss of response • Mutation analysis unavailable in 2006 • Jan 07 – clinical relapse, drug dose restricted due to cytopaenias • Mutation analysis requested Resistance Profile Imatinib: Partially sensitive Dasatinib: Sensitive Nilotinib: Sensitive r.730A>G p.Met244Val; M244V

14. Case report – Patient SH • Patient diagnosed with CML Oct 04 • Previous ALL in early childhood • No response to treatment after 32 months on imatinib • Mutation analysis initiated upon clinician request r.742C>G p.Leu248Val r.742_822del81

15. Patient SH cont… • Reported in the literature in 2 patients • 2 forms • Activates a cryptic 5’ donor splice site within exon 4 • Yields a splice variant with a deletion • Important to be aware of this mutation due to the mixed isoforms present, producing a messy sequence trace Resistance Profile Imatinib: Resistant Dasatinib: ?Sensitive Nilotinib: Partially sensitive

16. Follow up form This patient has been tested for BCR/ABL kinase domain mutations, either at your request or because of treatment failure, sub-optimal response, or loss of response as defined by ELN guidelines. In order to audit the effectiveness of this service, with implications for future commissioning (as the service is currently only grant funded for 18 months), we would be grateful if you could provide feedback on the usefulness of the enclosed result.

17. Follow up form responses

18. Limitations in quantification of mutations • Sequencing cannot easily be used to quantify the level of mutation • Report 100% or less than 100% • Currently testing other samples to identify when mutation emerges • Pyrosequencing to be used in the future 100% <100%

19. Kinetics of AKD mutations • BCR/ABL wild type clone decreases and another resistant clone emerges in response to treatment • After time resistant transcripts constitute all mutant transcripts • Pyrosequencing will be able to assess whether this mutant transcript decreases when alternative drugs are used Leukemia (2006) 20, 658–663

20. Acknowledgements • Work funded by Novartis • Mike Griffiths • Joanne Mason • Julian Borrow • Elizabeth Perrott • Elizabeth Ormshaw • Sue Rose • Susanna Akiki • Molecular Oncology team • Molecular Genetics Sequencing team