LAL: significato clinico e biologico delle mutazioni di Bcr-Abl

Transcription

1 LAL: significato clinico e biologico delle mutazioni di Bcr-Abl Simona Soverini Dipartimento di Ematologia e Scienze Oncologiche L. e A. Seràgnoli Università di Bologna

2 The vast majority of Ph+ ALL patients treated with imatinib relapse with evidence of a Bcr-Abl KD mutation

3 Imatinib-resistant pts with Ph+ ALL have the highest incidence of Bcr-Abl KD mutations Data of the GIEA WP on CL on 655 I-resistant pts Phase at the time of I failure: CP (I first-line) AP/BC CL Ph+ ALL 30% 64% 76% resistant pts harbouring Abl KD mutations

4 is the single most frequent Bcr-Abl KD mutation in imatinib-resistant Ph+ ALL 66 mutations in 62/82 pts resistant to imatinib mutations: 17/66 (26%)* P-loop mutations: 26/66 (39%) P-loop catalytic domain activation loop 244V L248V Q252R/H Y253/H D276G T277A E255K/V E279K V289A E281A/K E292V 343T H396R/P L364I 351T L387/ L V/I S417Y/ E355G/D 382L E453G/K V379I 311L/I 317L E459K/Q A380T 486S * requency of mutations in imatinib-resistant CL: ~10-12%

5 Second-line TKIs in imatinib resistant Ph+ ALL patients have limited efficacy mcgr 311I V299L NEL CCgR 317L Y253H CCgR 100 E255K CCgR E255K, D276G PCgR L E255K CCgR Y253H CCgR, 317L 351T CCgR T, 317L 317I L387 L387, 317L months on dasatinib

6 Inhibiting Bcr-Abl in Ph+ ALL is like shooting at a moving target dasatinib imatinib 800 nilotinib months ale, 18 yrs, Ph+ ALL Y253H Y253H Y253H Y253H 3 CCgR, R relapse relapse relapse imatinib dasatinib nilotinib months ale, 69 yrs, Ph+ ALL 317L 317L 317L Y253H Y253H 317L 317L Y253H relapse relapse relapse

7 In ALL genetic instability is high and allows the Ph+ clone to rapidly escape inhibition Novel TKIs are active against the vast majority of I-resistant mutations, but they inexorably retain one or some insensitive mutations Disease burden IATINIB INHIBITOR 2 INHIBITOR 3?? time RELAPSE 1 RELAPSE 2 RELAPSE 3 BCR-ABL KD sequence (?) I-res mutation Accumulation of compound drug-resistant mutations

8 Are Bcr-Abl KD mutations already detectable at the time of diagnosis? In Ph+ ALL pts treated with imatinib, resistance-associated Bcr-Abl KD mutations arise more frequently and more rapidly than in CL pts Imatinib does not to induce mutations, but simply selects pre-existent mutated clones In how many newly diagnosed Ph+ ALL can we detect Bcr-Abl KD mutations at low levels?

9 Are Bcr-Abl KD mutations already detectable at the time of diagnosis? German experience: Bcr-Abl KD mutations detected by D- HPLC at the time of diagnosis in: - 9/22 (41%) cases in elderly pts (Pfeifer et al, Blood 2007) 6/34 (18%) cases in younger pts (Pfeifer et al, EHA 2008) Lower detection limit of D-HPLC is routinely 5-10% What happens if we increase the sensitivity of our screening method?

10 Patients (I) RNA samples collected at the time of diagnosis from 13 patients enrolled on a phase II study of the treatment of adult de novo Ph+ ALL with dasatinib (GIEA LAL1205) DASATINIB 70 mg BID PDN i.t. mtx i.t. mtx days

11 Patients (II) N Sex Age Bcr-Abl type Status (time from the start of dasatinib, mo) utation at diagnosis (direct seq) utation at relapse (direct seq) 1 49 Relapsed (10) 2 56 Relapsed (6) 3 56 Relapsed (7) 4 47 Relapsed (4) 5 74 Relapsed (8) 6 58 Relapsed (7) E255K 7 59 Relapsed (2) 8 30 Relapsed (6) 9 57 Remission (13) Remission (5) Remission (12) Remission (23) Remission (20), not applicable

12 Experimental design Y342Y E292G BCR KD ABL 1. Amplification of the BCR-ABL KD (a.a ) by nested RT-PCR and ligation into a plasmid lacz lacz pcr2.1topo vector AP R 4. Direct sequencing of 150 to 200 independent clones 2. Transformation of E. coli competent cells 3. Plating and overnight incubation at 37 C

13 Results (I) Aberrant Bcr-Abl KD sequences were detected in ALL the patients insertions deletions point mutations missense ( amino acid change) silent ( no amino acid change) nonsense ( premature stop codon) N

14 Results (II) Point mutations were randomly and uniformly distributed all over the KD ost of them were not likely to be clinically relevant P-loop C-helix SH3 contact SH2 contact A-loop P216P K245R Y257H E275E E292G E334E I360T T392I Q477stop K222E G249S Y264C E281K I293T 317L V335V A366A H396P D223N G250R A288T V304I/A T319A N336D R367stop I403T G227G Q252stop V289A R307Q Y326Y A337A K378E W405C V228A Y253Y L327L Y342Y A380A A407A S229T/P G254E A344T L384P C346stop T389T E352G/K Y353Y utations that have been reported in imatinib-resistant pts are highlighted

15 N Status (time from the start of dasatinib, mo) Relapsed (10) Relapsed (6) Relapsed (7) Relapsed (4) Relapsed (8) Relapsed (7) Relapsed (2) Relapsed (6) Remission (13) Remission (5) Remission (12) Remission (23) Remission (20) Results (III) Each In In two contrast, patients, had an the 317L evidence detected observed of two at to diagnosis at twelve relapse different failed could to already mutations expandbe detected at the time of diagnosis Sex Age Bcr- Abl type utations* at diagnosis (total no. of clones sequenced) L266P; A337A (150) K222E; V289A; V335V; K378E (180) V228A; R307Q; W405C (150) E275E; E281K; T319A; N336D (200) K245R; I293T; V304I; D363D; R367stop; K378E; 39 bp ins (180) G250R; L327L; I360T (150) G227G; I293T; V304I; ; C346stop; W405C (200) P216P; Y264C; ; A344T (150) D223N; S229T; Q252stop; L384P; T392I; A407A; del(cggga)1343 (200) S229P; K245R; A288T; 171 bp ins (200) G254E; Y326Y; E334E (150) E286E; E352K; A380A; T389T; Q477stop (150) G249S; Y253Y; Y257H; E292G; T304A; 317L; Y342Y; E352G; Y353Y; A366A; H396P; I403T (200) * Each mutation was detected by bidirectional sequencing in 2 to 5 independent clones utation at relapse (direct seq) E255K

16 Results (IV) 55/61(90%) nucleotide substitutions were transitions Transitions: G>A A>G T>C C>T Total Transversions: G>T A>T T>A T>G A>C Total Such a high prevalence of transitions (which normally occur 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is predominant

17 Take-home messages a) Bcr-Abl KD mutations can probably be found at the time of diagnosis in all Ph+ ALL pts b) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity c) It is essential to understand the molecular mechanism(s) sustaining this mutator phenotype as well as how to therapeutically interfere with them

18 Acknowledgments Department of Hematology and Oncological Sciences L. e A. Seràgnoli, University of Bologna, Italy ichele Baccarani Giovanni artinelli Alessandra Gnani Sabrina Colarossi Stefania Paolini Ilaria Iacobucci Cristina Papayannidis Annalisa Lonetti arilina Amabile Department of Hematology and Cellular Biotechnologies, La Sapienza University, Rome, Italy Robin oà Antonella Vitale arco Vignetti Loredana Elia Giovanna eloni Anna Guarini ranco andelli GIEA ALL WORKING PARTY