MolecularDiagnostic.be Third Scientific Meeting Molecular Diagnostics.be t(9;22) CML: definition Management of CML patients treated with TKI: the place of molecular monitoring Antwerp, December 13 th 11 C. Graux CHU Mont-Godinne CML epidemiology CML diagnosis CML accounts for 14% of all leukemias The incidence is 1.6 per 1. /year +/- new cases/ year in Belgium 1 CML = 2 AML = 3 MM = 12 NHL = 37 CLL Increases with age(median 67 y) Male predominance: 1.4/1 The only known risk factor is ionizing radiations (high doses) Exposure to atomic bomb in Nagasaki and Hiroshima induced CML Symptoms - Fatigue, anorexia, weight loss Clinical examination - Splenomegaly Biology - Hyperleucocytosis - Circulating bone marrow myeloid precursors (left shift) - Increased basophilia -Thrombocytosis Cytogenetics: t(9;22) = Philadelphia (Ph) chromosome Molecular biology: BCR-ABL1 CML: peripheralbloodsmear Cytogenetic abnormality of CML Normal Chronic phase of CML 1 2 3 4 5 6 7 8 1 11 9 12 13 14 15 16 17 18 19 21 22 x Y Ph chromosome 1
Ph chromosome and BCR-ABL1 gene BCR-ABL1: types of transcripts 9 9 q+ Chromosome 22 Chromosome 9 1a BCR ABL1 m-bcr 55 kd 1b 22 Ph(or 22q-) P21 BCR-ABL1 M-bcr 2,9 kb a2 a3 P19 BCR-ABL1 ABL1 BCR BCR-ABL1 Chimeric protein with tyrosine kinase activity Exons Introns CML Breakpoints ALL Breakpoint µ-bcr t(9;22) translocation BCR-ABL1 gene structure Multiplex-PCR for BCR-ABL1 transcripts Constitutively activated tyrosine kinase BCR BCR-ABL1 Bcr-Abl1 signal transduction pathways Clinical evolution : CML phases BCR-ABL1 JAK/STATs DNA repair GRB2 CRKL CBL(p1 CBL ) Actin Adhesion Chronic phase Accelerated phase Advanced phases Blastic crisis RAS Paxillin Adhesion PI3 kinase AKT Variable Median time 6 9 months Median survival 3 6 months RAF-MEK-MAPK cascade MYC BCL-2 Regulates cell cycle progression and differentiation Inhibition of apoptosis Adapted from Pasternak G et al. J Cancer Res ClinOncol. 1998;124:643-66 2
What are the therapeutic goals in CML? Criteria for response to R/ Disappearance of the symptoms Modify the natural evolution of the disease blastic phase Cure Hematologic response Complete -Plateletcount < 45 x 1 9 /L -WBC count < 1 x 1 9 /L -Differential: no immature granulocyte -Basophils< 5% -Non palpable spleen Cytogenetic response Complete(CCgR) = No Ph+ metaphases Partial(PCgR) = 1-35% Ph+ metaphases Minor= 36-65% Ph+ metaphases Minimal= 66-95% Ph+ metaphases None = > 95% Ph+ metaphases Molecular response Allogeneic SCT Interferon α National Marrow Donor Program (NMDP) overview slide presentation. Available at http://www.marrow.org/nmdp/slideset/sld31.htm#slide. Accessed 17 June 2. P =.1 Survival by disease stage, June 1, based on transplants 1987 Feb 1. Proportion surviving 1..9.8.7.6.5.4.3.2.1 P <.1 Major cytogenetic response Minor or no response The only known cure but is associated with high morbidity and mortality rates in CML.. 12 24 36 48 6 Months after treatment with IFN-α Guilhot F et al. N Engl J Med. 1997;337:223-229. Oral cytotoxic agents Chemotherapy BCR-ABL1: the ideal target for molecular therapy Hydroxyurea Busulfan Hematological responses in up to 9% of patients Major cytogenetic responses are rare (1% 5%) 1-4 Palliative care: no effect on disease progression Present in the majority of patients with CML (95%) The cause of the disease Necessary for the initiation of the disease (primary event) ABL1 is non essential for normal cellular functions - Abl1 neg mice are viable targets BCR-ABL1 1. The Italian Cooperative Study Group on Chronic Myeloid Leukemia. N Engl J Med. 1994;33:8-825. 2. Hehlmann R et al. Blood. 1994;84:464-477. 3. Allan NC et al. Lancet. 1995;345:1392-1397. 4. Ohnishi K et al. Blood. 1995;86:96-916. 3
Mechanism of action of IRIS Study versus IFN-α + Ara-C 116 patients enrolled from June to January 1 S R IF: Loss of MCyR or CHR Increasing WBC count Intolerance of treatment Failure to achieve MCyR Cross-over S = screening R = randomisation IFN-a+ Ara-C Progression Death Accelerated phase or blast crisis Loss of MCR or CHR Increasing WBC count Goldman JM, Melo JV. N Engl J Med. 344 :184-186 Complete hematological responses Major cytogenetic responses % Responding 1 94% 9 8 7 6 55% 5 4 3 1 IFN-α + Ara-C 3 6 9 12 15 18 21 Months Since Randomisation % Responding 1 9 8 7 6 5 4 83% 3 % 1 IFN-α + Ara-C 3 6 9 12 15 18 21 Months Since Randomisation Superiority of to IFN-α+ Ara-C 1 8 IRIS Study : Summary of the 12-Month Data 94 83 IFN- α+ Ara-C Is CCyR the best surrogate endpoint? 6 55 4 23 8,7 1,5,7 CHR MCyR PD to AP/BC Intolerance CHR = complete haematological response; MCR = major cytogenetic response; PD = progressive disease; AP = accelerated phase; BC = blast crisis. Baccarani, M. et al. Blood 6;18:189-18 4
Progression to AP/BP Towards cure under TKI? IRIS study Is MMR the best surrogate endpoint? No proven effect on survival Variability of the assay Data on benefit of MMR based on good responders But MMR = very low progression rate Loss of MMR signals relapse/progression Early MMR predicts complete molecular response cure? MMR underscores the basic oncology principle that less disease is better Less is probably more Molecular monitoring: difficulties RQ-PCR is technically challenging Issues concerning comparability of results between centres International standardisation of molecularmonitoring for CML to enable testing laboratories to accurately mesure key therapeutic molecular milestones in CML (MMR and CMR) International scale for BCR-ABL1 International scalefor BCR-ABL1 Historically (IRIS trial; ), the mean BCR-ABL1 levels of 3 CML patients was defined as 1% in each of the three participating laboratories using BCR as a control The value corresponding to MMR in each laboratory has been defined as,1% (reduction of 3 log from IRIS baseline) International Scale (IS) fixed to these key points The absolute and not the relative amount is important MMR is defined as,1% IS = - 3 log reduction of BCR-ABL1 from IRIS standardised baseline, NOT 3 log reduction from individual pretreatment levels 5
Second generation TKI Nilotinibvs. in CML-CP (ENESTnd Trial) Nilotinib vs. in CML-CP (ENESTnd Trial, NEJM 1, Lancet, 11) More frequent and faster MMR Decreased progression to accelerated or blastic phase More frequent CMR room for cure? Dasatinib vs. in CML-CP (NEJM 1) More CCyR More frequent and faster MMR Effect on long term outcome? BCR-ABL1 kinase domain mutants BCR-ABL1 kinase domain mutants Are associated with various degrees of TKI insensitivity Select resistant clones = most important mechanism of resistance Can precede or accompany progression to advanced-phase disease KD mutations above a certain level should be identified as early as possible to reconsider the therapeutic strategy 9 KD mutations known to date Martinelli. The Hematology Journal, 5 Activity of Nilotinib on -Resistant BCR-ABL1 Mutants Spectrum of Kinase Inhibition for and Novel Compounds 3 IC5 (nm) on proliferation 25 15 1 5 Gleevec AMN17 maternal + IL3 bcr-abl wt M237I M244V L248V G25A G25E G25V Q252H Y253H E255D E255K E255V E255R E275K E276G E281K K285N E292K F311V T315I F317C F317L F317V D325N S348L M351T E355A E355G F359C F359V A38S L387F M388L F468S 72-hour proliferation assay with BCR-ABL expressing Ba/F3 cells (ATPLite; Perkin Elmer). Trough levels at a dose of 4 mg twice daily (BID) (1.7 µm) exceed the IC 5 determined in vitro for 32/33 BCR-ABL mutants (exception T315I) Weisberg et al. Br J Cancer. 6;94:1765. Melo J, Hematology 9 6
Management of CML Recommendations from the European LeukemiaNet Conclusion Time Optimal response Suboptimal response Failure Warnings Diagnosis N/A N/A N/A High risk CCA/Ph+ 3 months CHR, atleast Minor CgR No CgR LessthanCHR N/A The next step is - to better define CMR (EUTOS project) 6 months Atleast PCgR LessthanPCgR No CgR N/A 12 months CCgR PCgR LessthanPCgR LessthanMMR 18 months MMR LessthanMMR LessthanCCgR N/A Anytime (during treatment) Stable or improving MMR Lossof MMR Mutations (IM-sensit) Lossof CHR, lossof CCgR, mutations (IM-insensit) CCA/Ph+ Increasein transcript levels CCA/Ph- - to identify patients cohorts not relapsing after TKI withdrawal New recommendations 1 are marked in red. 7