West Midlands Regional Genetics Laboratory 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

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

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

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

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

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

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

Current Testing Strategy 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 28S rRNA 18S rRNA DNA Before After BCR ABL BCR F ABL R ABL F

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

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

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

Resistance profiles *unique mutation Mutation Imatinib Dasatinib Nilotinib M244V Partially sensitive Sensitive L248V Resistant ?Sensitive Partially Sensitive G250E Resistant/?Partially Sensitive Partially sensitive/?Sensitive G250E & A412V* Unknown Y253H E279K Unknown/?Sensitive V299L & K247R Neutral F317L F359I F359V E450Q 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

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

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

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

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.

Follow up form responses Mutation? Useful? Alter treatment? Comments G250E & A412V Y May change to dasatinib if BCR/ABL levels increase E279K Will change to dasatinib if can get funding F359V N Confirms clinical decision G250E Patient already on dasatinib E450Q Thought non-compliance an issue so changed to dasatinib Potentially Pt referred for psychiatric review for non-compliance Patient scheduled for BMT Would consider nilotinib/dasatinib Possible allograft depending on response to dasatinib Considering non-compliance Patient has died -

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%

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

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