MRD in CML (BCR-ABL1)

MRD in CML (BCR-ABL1) Moleculaire Biologie en Cytometrie cursus Barbara Denys LAbo Hematologie UZ Gent 6 mei 2011 2008 Universitair Ziekenhuis Gent 1

Myeloproliferative Neoplasms o WHO classification 2008: - Chronic myelogenous leukaemia, BCR-ABL1 positive - Chronic neutrophilic leukaemie (CNL) - Polycythemia vera (PV) - Primary myelofibrosis (PMF) - Essential thrombocythaemia (ET) - Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) - Mastocytosis - Myeloproliferative neoplasm, unclassifiable o MDS/MPD: CMML, JMML, atypic CML (BCR-ABL1 negative), MDS/CMD unclassifiable

Chronic myeloid leukemia or CML CML accounts for 15-20% of all adult leukaemias (incidence: 1-2 cases per 100.000) Median patient age at diagnosis: 55-60 years 3 phases: chronic accelerated blast crisis Majority (>80%) of cases of CML diagnosed in chronic phase (CML-CP) CML is the first cancer to be shown to be caused by an underlying genetic abnormality

CML pathogenesis: Philadelphia (Ph) chromosome Specific translocation in > 95% of CML patients: t(9;22)(q34.1;q11.2) Constitutively active tyrosine kinase

BCR-ABL1 genome/dna ALL CML mrna Nashed et al., J. Mol. Diag. 5:63-72 (2003) M-Bcr = major breakpoint cluster region m-bcr = minor breakpoint cluster region

BCR-ABL1 detection BCR ABL der(9) der(22) (Ph) 22 9

Tyrosine kinase inhibitoren (TKI): Imatinib mesylate (Gleevec, Glivec, STI-571)

Rationale for molecular monitoring Response to treatment (imatinib); most patients in CP have an excellent response to imatinib (CCyR) Some patient, however, still progress Early detection of relapse, progression or treatment failure provides an opportunity for alternative therapy (second generation TKI) Prognostic information: MMR Clinical need for the additional risk stratification is necessary

Molecular monitoring «real-time RT-PCR» RT: mrna cdna PCR: amplification (specific primers in fusion region) RQ: quantification - sensitivity = 10-5 10-4 >> FISH - faster - more accurate - less contamination

Goals of CML therapy / MRD

Schematic overview for BCR-ABL qpcr

Rationale for molecular monitoring Response to treatment (imatinib); most patients in CP have an excellent response to imatinib (CCyR) Some patient, however, still progress Early detection of relapse, progression or treatment failure provides an opportunity for alternative therapy (second generation TKI) Prognostic information: MMR Clinical need for the additional risk stratification is necessary

BCR-ABL mrna levels are an independent prognostic marker of disease progression A best molecular response of at least a 3-log reduction in BCR- ABL mrna levels predicts better progression-free survival. Kaplan-Meier survival curve is shown for patients achieving a best RQ-PCR level (at any time) above various thresholds. The panel compares patients with a best molecular response of at least 3 versus those below 3. These are landmark analyses starting at the time of CCR. RD Press et al, Blood, June 2006 (4250)

Progression-free Survival on 1 st -line Imatinib by Molecular Response (MR) at 12 months MMR estimated rate of survival without progression at 42 months: n=138 75% PFS n=94 90% PFS n=136 98% PFS IRIS Study,update 2004

Standardisation/interntional scale (IS) An International Scale (IS) was proposed, identical to that used in the International Randomized Study of Interferon versus STI571 trial = IRIS trial Measurements were related to a standardized baseline MMR is standardised as 0.1% BCR-ABL/ ABL (normalised ratio, %)

IRIS study The evidence obtained with the IRIS study is that the absolute and not the relative amount is important

Event-free Survival on 1 st -line Imatinib by Molecular Response (MR) at 18 months Hughes et al, Blood 2010

< 10%: EFS >85% 56% > 1%: EFS <65% Hughes et al, Blood 2010

Overall respons Imatinib first-line in CML-CP Recommendation of European LeukemiaNet: incorporating molecular responses at 6, 12 and 18 months based on analysis ot the IRIS PCR data Evaluation Time Optimal Suboptimal Failure 3 months CHR and at least minor CyR (Ph+ 65%) No CHR 6 months At least partial CyR (Ph+ 35%) No partial CyR (Ph+ > 35%) and/or BCR-ABL (IS) > 10% 12 months CCyR No CCyR (Ph+ > 1%) and/or BCR-ABL (IS) > 1% No HR No CHR No CyR (Ph+ > 95%) No partial CyR (Ph+ > 35%) and/or BCR-ABL (IS) > 10% 18 months MMR ( 0.1%) No MMR ( 0.1%) No CCyR (Ph+ > 1%) and/or BCR-ABL (IS) > 1% Baccarani et al., J Clin Oncol 2009

Recommendation of European LeukemiaNet (ELN): Monitoring the response to imatinib Hematologic response Cytogenetic response Molecular response Dx / 2-weekly CHR Dx / 3 m / 6 m then every 6 m CCyR Every 3 months Every 12 months Baccarani et al., J Clin Oncol 2009

Recommendation of European LeukemiaNet (ELN): Monitoring the response to imatinib Molecular monitoring Every 3 months until MMR has been achieved and confirmed, then at least every 6 months NO MMR at 18 months 5/10-fold rise Loss of MMR 5/10-fold rise in < 0.1% - Consider patient compliance - Mutation analysis - Dose escalation imatinib - Switch to second generation TK (dasatinib, nilotinib) - Allo-HSCT warning

BCR-ABL1 mutations Acquired resistance Point mutations in the BCR-ABL kinase domain More than 50 different mutations have been described Early identification of mutations may prevent disease progression

Mutational analysis Imatinib failure or suboptimal response: " never achieve MMR (cfr. ELN recommandations) Loss of MMR Rising BCR-ABL1 transcript levels: optimal increase that should trigger mutation testing? 1-log increase, repeated in 1-3 months (Kantarjian et al, J Clin Oncol 2009 / NCCN guidelines v.2.2009) 5-fold rise and loss of MMR certainly warrants mutational screening (Branford et al, Curr Opin Hematol 2011) Range > MMR (0.1%): optimal cut-off = 2.6 fold or 0.4 log rise (Press et al, Blood 2009) in range < MMR: rise is NOT clinical relevant (warning), to avoid unnecessary screening (Branford et al, Curr Opin Hematol 2011) Significance of a rise is highly dependent on the inherent variability of the local RQ-PCR assay!

Need for standardization of MRD assessment MMR is NO log-reduction but fixed value MMR IRIS trial = 3-log lower (0,1%) than mean baseline level of 30 pre-treatment samples in newly diagnosed CML patients (100%) = standardized baseline value Problem: different standardized baseline level of individual laboratories Standardized reporting of BCR-ABL measurements is useful for: Optimal clinical management Comparison of measurements from different study groups Pooling results from different studies

The international Scale (IS) for BCR-ABL

Conversion Factor (CF) Conversion to IS with laboratory-specific conversion factors Pyramidal standardization: Pionneer centre (Adelaide, Australia) European Ref Lab (Mannheim Germany) National Ref Lab (Leuven and Hôpital Erasme ULB) different local labs complex, time-consuming, expensive Local centers can continue to use their existing assay conditions Modifications in the routine process (RT protocol, master mix,.) the whole process need to be restarted!

Problem CF: calculations After application of CF NO improvement of comparability! UK Neqas Definition MMR in own lab Define own baseline BCR-ABL/ABL transcript level = mean baseline level of 30 pre-treatment samples in newly diagnosed CML-CP patients in your lab (100%) MMR lab = 3-log reduction of your own baseline Baseline BCR-ABL/ABL transcript level MMR UZG PB (n = 16) 100.2% ~0.1% BM (n = 16) 108.1% ~0.11%

Primary reference material Better to use universal reference material in order to simplify the process Primary ref. material should be As close as possible to the real samples Cover the entire analytical process (including RNA extraction) Applicable to all/most methods in use Stable of a period of several years

Blood 2010 Calibrated, acredited primary reference reagent developed (released) Freeze-dried K562 cells, 4 dilution points in HL60 cell line Proven stability, homogeneity Limiting factor is the large amount of cells required secondary reference reagents calibrated to primary reagent (widely available)

Complete molecular response (CMR) The new golden standard? Undetectable BCR-ABL mrna levels Depends on how hard you look Variability of sample quality within centres Variability if assay sensitivity beween centres There is a need for a robust definition of CMR Possible clinical significance of CMR: very low probability of progression with ongoing TKI therapy Indicator that TKI can be stopped with reasonable expectation of durable remission? limited info in the outcome after cessation of imatinib treatment of CML patients in CMR

Definitions of CMR

Conclusion / Future perspectives BCR-ABL mrna MRD levels are important predictors of response and relapse in patients undergoing treatment with imatinib Molecular monitroing will also be important for predicting response with second-generation TKIs Standardised measurement of MMR is achievable and ongoing Standardised measurement of CMR is the next major challenge