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The introduction of ABL kinase domain mutation testing in CML patients showing resistance to 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 n n n Introduction to CML Methods for AKD analysis Mutations identified Talk Outline n n n Introduction to CML Methods for AKD analysis Mutations identified to date WMRGL database Follow up Future work

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

Developing resistance n n n Glivec has revolutionised treatment for CML but resistance is Developing resistance n n n 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 n n n 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 n Identification of mutations can direct patient management Setting up an AKD mutation service n Identification of mutations can direct patient management n n n Increase dose of imatinib Provide evidence that an alternative drug may be more suitable Work currently funded by Novartis for 18 months n Monitoring user satisfaction with a view to obtaining local NHS funding

Monitoring of CML patients n n Samples received at regular intervals usually every 3 Monitoring of CML patients n n 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 1. 2. Clinical request Internal Activation of samples for ABL kinase domain mutation testing 1. 2. Clinical request Internal activation based on ELN guidelines n n n treatment failure at 6 or more months sub-optimal treatment response loss of response

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

ABL kinase domain mutations T 315 I/A/D F 311 L/I/V F 317 L C ABL kinase domain mutations T 315 I/A/D F 311 L/I/V F 317 L C 305 S V 299 L L 248 V L 298 V G 250 E/A/F Q 252 H/R P 296 H G 321 E M 237 I K 285 N E 281 A E 279 K T 277 A L 384 M A 344 V L 387 F/M S 348 L A 350 V Imatinib binding site M 244 V D 241 G G 383 D M 343 T K 357 R E 292 V Y 253 H/F E 255 K/V V 289 A/I P-loop F 382 L L 324 Q M 388 L C M 351 T/L E 355 G/D A 397 P E 453 G/K/A/V E 450 Q H 396 R/P F 486 S Q 447 R Activation loop V 379 I L 3641 F 359 V/C/D/I A 412 V S 438 C E 459 K/Q Adapted from Melo et al. , Cancer Lett 2007 249(2): 121 -32 E 276 G E 275 K n n n Over 100 mutations identified in the literature T 315 I is most common 16 mutations account for 86% reported cases

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

WMRGL Database n Compiled a mutation databased on an extensive literature search consisting of WMRGL Database n Compiled a mutation databased on an extensive literature search consisting of 3 types of data n n Mutations which emerge in response to treatment and the ability of each drug to overcome a particular mutation IC 50 values In vitro mutagenesis data Resistance profiles are created for each mutation and added to the report

Resistance profiles Mutation Imatinib Dasatinib Nilotinib M 244 V Partially sensitive Sensitive L 248 Resistance profiles Mutation Imatinib Dasatinib Nilotinib M 244 V Partially sensitive Sensitive L 248 V Resistant ? Sensitive Partially Sensitive G 250 E Resistant/? Partially Sensitive Partially sensitive/? Sensitive G 250 E & A 412 V* Resistant/? Partially Sensitive Unknown Partially sensitive/? Sensitive Unknown Y 253 H Resistant Sensitive Resistant/? Partially Sensitive E 279 K Resistant Unknown/? Sensitive V 299 L & K 247 R Sensitive Neutral Resistant/? Partially Sensitive Neutral Unknown Neutral F 317 L Partially sensitive Resistant Sensitive F 359 I Unknown F 359 V Partially sensitive Sensitive Partially sensitive E 450 Q Unknown ? Sensitive Unknown IC 50 value: the concentration of drug required to inhibit 50% of enzymatic activity Imatinib: Nilotinib: Dasatinib: Sensitive <4, Partially Sensitive ≥ 4, <9 Resistant ≥ 9 Sensitive <4, Partially Sensitive ≥ 4, <30, Resistant ≥ 30 Sensitive <4, Partially Sensitive ≥ 4, <6, Resistant ≥ 6 Cut-offs: Numbers refer to fold increases in IC 50 compared to “wild-type” BCR-ABL *unique mutation

Case report - Patient VK n n n n r. 730 A>G p. Met Case report - Patient VK n n n n r. 730 A>G p. Met 244 Val; M 244 V n 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 Resistance cytopaenias Profile Imatinib: Partially sensitive Mutation analysis requested Dasatinib: Sensitive Nilotinib: Sensitive

Case report – Patient SH n n Patient diagnosed with CML Oct 04 Previous Case report – Patient SH n n 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. 742 C>G p. Leu 248 Val r. 742_822 del 81

Patient SH cont… n n n Resistance Profile Imatinib: Resistant Dasatinib: ? Sensitive Nilotinib: Patient SH cont… n n n Resistance Profile Imatinib: Resistant Dasatinib: ? Sensitive Nilotinib: Partially sensitive 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

Follow up form This patient has been tested for BCR/ABL kinase domain mutations, either 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 G 250 E & A Follow up form responses Mutation? Useful? Alter treatment? Comments G 250 E & A 412 V Y Y May change to dasatinib if BCR/ABL levels increase E 279 K Y Y Will change to dasatinib if can get funding F 359 V Y N Confirms clinical decision G 250 E Y N Patient already on dasatinib E 450 Q Y N Thought non-compliance an issue so changed to dasatinib N Y Potentially Pt referred for psychiatric review for non-compliance N Y N Patient scheduled for BMT N Y N Would consider nilotinib/dasatinib N Y N Possible allograft depending on response to dasatinib N Y Y Considering non-compliance N N N Patient has died N - N

n n n Limitations in quantification of mutations Sequencing cannot easily be used to n n n Limitations in quantification of mutations Sequencing cannot easily be used to quantify the level of mutation n 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 n n n BCR/ABL wild type clone decreases and another Kinetics of AKD mutations n n n 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 n n n n n Mike Griffiths Joanne Mason Julian Borrow Elizabeth Perrott Acknowledgements n n n n n Mike Griffiths Joanne Mason Julian Borrow Elizabeth Perrott Elizabeth Ormshaw Sue Rose Susanna Akiki Molecular Oncology team Molecular Genetics Sequencing team § Work funded by Novartis