AIRC.6 – A RISK ADAPTED, MRD-DRIVEN STRATEGY FOR THE TREATMENT MYELOID LEUKEMIA
Transcription
AIRC.6 – A RISK ADAPTED, MRD-DRIVEN STRATEGY FOR THE TREATMENT MYELOID LEUKEMIA
AIRC.6 – A RISK ADAPTED, MRD-DRIVEN STRATEGY FOR THE TREATMENT OF NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA Responsabile scientifico del progetto FRANCESCO LO-COCO Università di Roma Tor Vergata – Fondazione Santa Lucia Associazione Italiana per la Ricerca sul Cancro – Finanziamento 2011 Sezione III: Attività per progetti BACKGROUND AND CURRENT STATE OF ART Acute myeloid leukemia (AML) is one of the most common forms of blood cancer. It affects 5-20/100,000 individuals/year and accounts for ~75% of all the acute leukemias [Fey, 2009]. Available treatments produce complete remission (CR) in up-to 80% of patients. However, ~60% of them will eventually relapse, due to the emergence of chemotherapy-resistant disease, and die of leukemia [Dohner, 2010]. Relapse is thought to result from residual chemoresistant leukemic cells that while left behind following achievement of CR, are below the limits of detection using conventional morphologic assessment. Sensitive techniques are now available to detect subclinical levels of residual leukemia, termed minimal residual disease (MRD). Investigation of MRD has proven to be a valuable tool in patients with acute lymphoblastic or promyelocytic leukemia and chronic myeloid leukemia for predicting impending relapses and improving patient stratification, including treatment reduction or intensification [Campana, 2009; Grimwade, 2009; Baccarani, 2009]. As to AML, a recent study on MRD-driven, risk adapted treatment conducted in children at St. Jude’s Hospital (Memphis, USA) showed a striking improvement in patient outcome using this strategy [Rubnitz, 2010]. By contrast, treatment strategies in adult AML still rely on upfront risk stratification, regardless of subsequent MRD evaluation [Dohner, 2010]. MRD in AML can be quantified by polymerase chain reaction (RQ-PCR) analyses of AML-associated transcripts or by immunophenotypic analysis of AML cells. The former are based on molecular detection of translocationderived fusion transcripts (e.g. RUNX1-RUNX1T1, CBFB-MYH11) [Gabert, 2003], transcripts of genes carrying somatic mutations (e.g. nucleophosmin gene NPM1) [Gorello, 2006; Schnittger, 2009] or overexpressed genes (Wilm’s tumor gene WT1) [Cilloni 2009]. Immunophenotypic analyses are based on the multiparametric flow cytometry (MPC) detection of aberrant (asynchronous or cross-lineage) marker-expression on leukemic cells at diagnosis (so-called leukemia-associated immunophenotypes - LAIP) [Venditti, 2003]. These LAIPs are highly sensitive (MPC can identify one leukemic cell in 104-105 cells) and can be applied to ~90% of AML patients. Contributions from our groups include discovery of novel AML genetic markers (mutated NPM1) [Falini, 2005] and development of several approaches for MRD monitoring (NPM1 mutations, WT1 overexpression and LAIPs) [Gorello, 2006; Cilloni, 2009; Maurillo, 2008]. To date, no studies have been conducted to investigate in parallel and prospectively distinct methodologic approaches to detect MRD in AML. We aim in this project to increase the long term survival rate of AML through a risk-adapted treatment approach and to investigate the clinical value of these MRD-detection methods. Feasibility here is guaranteed by the execution of this clinical trial in the context of GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto), an ltalian cooperative group involving >80 clinical centres with a proven ability (more than 180 publications) to conduct clinical-biological studies in hematological malignancies. 486 2013 AIRC.6 – A risk adapted, MRD-driven strategy... GENERAL STRUCTURE OF THE STUDY The proposed project is centred on a (Phase 2) multicentric and prospective clinical trial, which uses up-front conventional chemotherapy followed by patient stratification based on MRD detection. The trial is designed to determine whether an MRD-based risk-adapted approach improves treatment stratification and overall prognosis in adult AML. This will be the first prospective study of MRD-based treatment in adult AML. The clinical trial also includes sampling of leukemia cells at different time-points during treatment, which will provide the biological material for a series of laboratory investigations aimed at prospective evaluation of MRD. The design of the clinical trial is based on a number of specific considerations: 1. The traditional chemotherapy regimens used to treat adult AML have not changed significantly. Large randomized trials failed to demonstrate the superiority of adding new cytotoxic agents to the traditional cytarabine and anthracycline-based regimens [Dohner, 2010]. Thus, all patients enrolled in our clinical trial will be offered one of the so-called standard induction and post-remission (or consolidation) therapies available. It is expected that this regimen will induce ~70% of CR and ~40% of cure. 2. Cytogenetic and molecular features of AML (as determined at diagnosis) are critical determinants of outcome and allow stratification of ~40% of patients in good-risk (based on the presence of mutated NPM1, t(8;21) or inv 16) or poor-risk (mutated FLT3 or poor karyotype) groups (WHO 2008 classification of myeloid neoplasms) [Dohner, 2010]. Good-risk patients achieve high survival and disease free survival rates with standard treatments, while the high-risk patients do poorly without intensified therapy with allogeneic transplant [Dohner, 2010]. Accordingly, we plan to treat good-risk and high-risk patients with low- or high-intensity regimens after consolidation, respectively, regardless of the presence of MRD (autologous or allogeneic stem cell transplantation). 3. There are no available criteria to assist treatment choices after induction/consolidation for the remaining patients (intermediate risk group; ~60%). These patients will be stratified according to post-consolidation MRD level: MRD-positive patients will receive allogeneic stem cell transplantation; MRD-negative patients will receive autologous stem cell transplantation. 4. There are scarce data derived from prospective studies on the clinical significance of MRD detection after consolidation, and no information on the best approach to be used for MRD quantification (among those available: translocation-derived fusion transcripts, NPM1 mutations, WT1 expression, LAIP), as these approaches have never been compared prospectively. We will measure MRD in all patients using all the available methods (regardless of the risk-assignment at diagnosis) and stratify the intermediate risk group after consolidation using the LAIP method. We have decided to use LAIP based MRD evaluation on our recent findings showing that use LAIP based MRD evaluation significantly improves outcome prediction in AML [Maurillo, 2008; 2013 487 Sezione III: Attività per progetti Buccisano, 2010]. In addition, the LAIP method is the only tested and validated monitoring approach in MRD-oriented therapy in prospective clinical trials for childhood ALL and AML [Campana, 2009; Rubnitz, 2010]. The conventional morphological, and immunophenotypic diagnostic characterization of the enrolled AML patients will be conducted by the GIMEMA participating Institutions. Though this initial sample-characterization is part of the standard diagnostic evaluation of AMLs, it is critical for the success of the entire project. The participating Institutions have been previously screened by GIMEMA for their ability to execute high-quality standard evaluation of AMLs at diagnosis. We plan to collect leukemia samples (peripheral blood and bone marrow) at diagnosis and at different time-points during and after treatment. Samples will be centralized in Rome at the PI laboratory (FLC) to define LAIP and for part of molecular investigations, as described in the next sections. The logistic of sample collection is guaranteed by the GIMEMA working structure. Cytogenetic (conventional karyotyping) and molecular tests (NPM1, FLT3, c-KIT mutational status) will be carried out by a network of experienced reference laboratories already operating in the GIMEMA as described [Falini, 2005; Lo-Coco, 2008]. AML offers the unique opportunity to define the biological and clinical significance of persistent disease after treatment. Detection of MRD after treatment does not necessarily correlate with impeding relapse. This has been clearly shown, for example, for AML carrying the RUNX1-RUNX1T1 gene rearrangements, where cured patients have been identified who still express RUNX1-RUNX1T1 transcripts as MRD [Miyamoto, 1996]. These findings are in keeping with our observations showing that residual leukemia cells are found in every AML patient after front-line treatment, while clinical relapse correlates with residual leukemia cells exceeding 3.5x10 -4 using LAIP based MRD evaluation [Maurillo, 2008]. Thus, the presence of residual disease after treatment is not a feature, per se, of the impeding relapse. Comparative characterization of LAIP cells isolated from patients above or below the threshold of 3.5x10-4 (MRD+ or MRD- patients) will allow investigations on the biological significance of residual disease with respect to the probability of clinical relapse. AML offers the unique opportunity to investigate the immunophenotypic features of MRD and their correlation with genetic heterogeneity. AMLs are a highly heterogeneous disease genetically and among the best-known forms of cancer (the primary genetic lesion has been identified in ~60% of cases). Yet, AML patients are all treated with identical therapeutic regimens. Recently introduced 9-color immunophenotypic technologies will permit better dissection of this heterogeneity and, combined to cell sorting, will offer the unique opportunity to better assess the correlation between phenotypic and genetic determinants of MRD. GENERAL AIMS 1) To optimize AML management-choices at diagnosis and after induction, based on risk-adapted patient-stratification. 488 2013 AIRC.6 – A risk adapted, MRD-driven strategy... 2) To evaluate in parallel and prospectively immunophenotypic and molecular technologies and distinct disease markers to detect MRD. MILESTONES (M), TASKS (T) AND DELIVERABLES (D) M1: Clinical value of LAIP for the stratification of AML patients after induction therapy We will test the clinical value (in terms of OS and RFS) of MRD detection by LAIP for treatment patient-stratification. T1: The GIMEMA MRD-refined risk-adapted phase II clinical trial – The planned clinical trial will start in late 2011 and continue for 3 years. It is structured as a Phase II study and will be conducted by the GIMEMA. The GIMEMA includes over 80 Hematology Units in Italy, including the most advanced National academic centres. It functions since 1982, possesses a centralized structure for the collection and statistical analysis of clinical data and has a proven ability to conduct clinical-biological studies on hematological malignancies. Based on previous GIMEMA studies we anticipate an accrual of ~140 AML patients/year. Diagnostic tests and procedures in the initial work-up of AML patients will be carried out as recommended by the European LeukemiaNet expert panel [Dohner, 2010]. For patients with normal cytogenetics AML (NC-AML), mutational screening at diagnosis will include FLT3, NPM1, IDH1 and IDH2, DNMT3 genes [Kottaridis, 2002; Gorello, 2006; Boissel, 2010; Ley, 2010]. All patients will receive induction and consolidation chemotherapy according to the standard arm of the GIMEMA LAM99P trial [Lo Coco, 2008]. After the first consolidation, MRD will be evaluated in all patients. At this step, patients in the intermediate-risk group will be stratified into MRD+ or MRD- using LAIP based MRD evaluation by MPC and will receive risk-adapted treatment (autologous vs. allogeneic stem cell transplantation). All patients failing to attain CR with the scheduled induction course will be included in the high-risk category for subsequent allogeneic transplant. They will receive salvage reinduction therapy with one course of the FLA-IDA regimen and, after achievement of CR, consolidation followed by an allotran-splant. Those not attaining CR after salvage therapy will be prioritized to directly proceed to allogeneic transplant. All patients will undergo transplant based on donor availability. In this clinical trial, allogeneic transplant will represent a therapeutic option offered to all patients who meet the established criteria independently from the availability of an HLA identical sibling. For patients lacking a family, HLA-compatible donor, all other sources of hematopoietic stem cells (matched unrelated donor from international registry, unrelated cord blood, family haploidentical donor) will be considered. The choice of the alternative donor will follow the most recent recommendations related to each hematopoietic stem cell source and will take into account the limit of 3 months from complete remission as time to transplant. 2013 489 Sezione III: Attività per progetti T2: LAIP based MRD evaluation using MPC – Standard immunophenotypic studies will be performed as part of the diagnostic work-up by the GIMEMA Participating Institutions using standard techniques [Buccisano 2006; Maurillo, 2008]. Bone marrow and peripheral blood samples will be sent to the central laboratory and re-analyzed by MPC to assess the “leukemia immunophenotypic fingerprint” or LAIP (we predict LAIP detection in ~90% of cases), which will then be used to track residual leukemic cells post-induction, post-consolidation and every 3 months during the 2-year follow-up. We will use the threshold of >3.5 x 10-4 to define MRD positivity, as a predictor of relapse [Venditti, 2000 and 2003; Buccisano, 2006; Maurillo, 2008]. MRD negative (<3.5x10-4 ) (MRD-) patients will receive autologous transplant, while the MRD positive (>3.5x10-4 ) (MRD+) will undergo allogeneic transplant. T3: Analysis of the relative frequency of leukemia SCs in LAIPs – Leukemia SCs at diagnosis, remission and relapse will be identified phenotypically, by MPC analysis. This approach will enable us to recognize leukemia cells, regardless of their phenotypic profile, thus allowing the unambiguous distinction of leukemia vs. normal SCs. T4: Statistical analysis – In order to demonstrate a significant difference between the historic control [Lo Coco at al., Prognostic impact of genetic characterization in the GIMEMA LAM99P multicenter study for newly diagnosed acute myeloid leukemia (2008) Haematologica] and the present trial, an estimated number of 213 subjects is required. This sample size achieves 90% power to detect a difference of 10% between the null hypothesis that the OS at two years is 50% and the alternative hypothesis that the OS is 60%, using a Single-Stage Phase II design with a 5% significance level. Considering that approximately 70% of the observed patients will fall into the intermediaterisk category with a historical CR rate of 67%, a number of 213 subjects will also permit to detect a 15% difference in intermediate-risk patients with a 90% power, between the null hypothesis that relapse rate (cumulative incidence of relapse) at one year is 30% and the alternative hypothesis that the relapse rate is 15%, using a Single-Stage Phase II design with a 5% significance level. Calculations will be implemented in PASS2008. D1 – Analysis of 2 year OS and relapse rates with a 10% improvement in survival and relapse rates at this time point based on statistical analysis. M2: Clinically validated MRD-monitoring protocols In parallel to LAIPs, we will evaluate MRD prospectively, using all the other available protocols: RQ-PCR for fusion genes, WT1 expression, NPM1 mutations (each of these protocols has been previously standardized in retrospective clinical studies). Moreover, we will explore new and less-sophisticated tools for MRD monitoring of AML, such as anti NPM1-mutated antibodies [Gruszka, 2010]. Each patient will be investigated at diagnosis, post-induction, post-consolidation and during follow up (at 3 months interval for the first 2 years). We will use bone marrow samples and (in selected cases) paired marrow and peripheral blood samples. 490 2013 AIRC.6 – A risk adapted, MRD-driven strategy... T5: Prospective evaluation of MRD-monitoring protocols – We will evaluate MRD by RQ-PCR analyses of: a) WT1 expression; b) the most common AML fusion-transcripts (RUNX1-RUNX1T1, CBFB-MYH11, BCR-ABL); c) NPM1 mutations. Methods – 1. WT1 expression. WT1 is over-expressed in up-to 90% of AMLs at diagnosis. A 2-10g reduction after induction therapy is the clinically validated threshold that predicts relapse in a retrospective study [Cilloni, 2009]. For quantification of WT1 expression, we will use the European LeukemiaNet standardized quantitative WT1 assay, as recently reported [Cilloni, 2008]. 2. NPM1 mutations. NPM1 mutational status at diagnosis will be screened using capillary gel electrophoresis, as reported [Noguera, 2005]. Direct sequencing of mutated samples will be used to characterize the type of mutation. For patients with demonstrated NPM1 mutation, RQ-PCR will be used to determine copies of the NPM1-mutant transcript. We have recently developed an RQ-PCR assay that recognizes 17 different NPM1 mutations employing 17 specific primers and one common reverse primer [Schnittger, 2009]. 3. Fusion transcripts. Diagnostic samples will be screened for the most common AML fusion genes (i.e. RUNX1RUNX1T1, CBFB-MYH11, BCR-ABL) by standardized reverse transcriptase RT-PCR assays, as reported [van Dongen, 1999]. Once a fusion transcript is identified, we will use the RQ-PCR assays that have been standardized by the “Europe Against Cancer” consortium [Gabert, 2003]. T6. Clinical correlations and statistical analysis – Results of the above mentioned standard MRD assays will be correlated with clinical outcome, specifically relapse rate, and RFS. Comparative analysis of the different MRD assays will be conducted at month 33-39 after collection of data from ~280 patients. For NPM1 and fusion genes, we will first identify the most informative time-points and threshold values for each assay, following the same methodology used and recently reported for WT1 [Cilloni, 2008]. In brief, identified threshold values adjusted for known prognostic variables will be evaluated by Cox regression for relapse rates, to define the most significant threshold. Thresholds will be calculated as 1, 2, 3 or 4 log-reductions, as compared to pre-treatment levels, or as absolute levels of the tested genes after normalization (against 104 ABL copies). Results obtained by each MRD monitoring protocol will be correlated with patient outcome in comparison to our established method (LAIP by MPC), to evaluate sensitivity and specificity, and to optimize methods for distinct AML subtypes, when applicable. MPC analysis of LAIP will be also be prospectively validated for its value as patient stratification tool, by its ability to improve survival rates and reduce relapse rates (Kaplan-Meier analysis), as compared to historic controls of previous GIMEMA trials (employing the same treatment schedules). D2 – Prospectively validated protocols for MRD monitoring using WT1. D3 – Prospective evaluation of RQ-PCR NPM1 gene mutations and RQ-PCR for tested fusion genes. D4 – Novel monitoring protocols using the anti NPM1 mutant A MoAb. D5: Clinical value of each of the tested MRD protocols. 2013 491 Sezione III: Attività per progetti – – – – – – – – – – – – – – – – – – – – – – – – – Baccarani M, et al. (2009) J Clin Oncol 27:6041-6051. Boissel N, et al. (2010) J Clin Oncol 28(23):3717-3723. Buccisano F, et al. (2006) Leukemia 20(10):1783-1789. Buccisano F, et al. (2010) Blood 116(13):2295-2303. Campana D (2009) Hematol Oncol Clin North Am 23:1083-1098. Cilloni D, et al. (2008) Haematologica 93:921-924. Cilloni D, et al. (2009 ) J Clin Oncol 27:5195-5201. Dohner H, et al. (2010) Blood 115:453-474. Falini B, et al. (2005) N Engl J Med 352:254-266. Fey M, Dreyling M (2009) Ann OncoI 20(Suppl4):100-101. Gabert J, et al. (2003) Leukemia 17:2318-2357. Gorello P, et al. (2006) Leukemia 20:1103-1108. Grimwade D, et al. (2009) J Clin Oncol 27:3650-3658. Gruszka AM, et al. (2010) Blood 116(12):2096-2102. Kottaridis PD, et al. (2002) Blood 100:2393-2398. Ley TJ, et al. (2010) N Engl J Med 363:2424-2433. Lo-Coco F, et al. (2008) Haematologica 93:1017-1024. Maurillo L, et al. (2008) J Clin Oncol 26:4944-4951. Miyamoto T, et al. (1996) Blood 87:4789-4796. Noguera NI, et al. (2005) Leukemia 19:1479-1482. Rubnitz JE, et al. (2010) Lancet Oncol 11(6):543-552. Schnittger S, al. (2009) Blood 114:2220-2231. van Dongen JJ, et al. (1999) Leukemia 13:1901-1928. Venditti A, et al. (2000) Blood 96:3948-3952. Venditti A, et al. (2003) Leukemia 17:2178-2182. FEASIBILITY AND PITFALLS OF THE PROJECT Available expertise. Since the early ‘90s the involved investigators have made important contributions in various aspects of AML, including: molecular characterization and diagnostics, MRD studies, and treatment of AML. These include the identification of new prognostic factors in AML (including the NPM1 mutated/FLT3-ITD-ve genotype); assessment of the clinical role of MRD studies in APL and AML (through studies on disease monitoring using PML/RARA, WT1 and LAIP as markers of MRD; production and validation of MoAbs against proteins delocalized in AML which are routinely used to implement disease diagnosis. Available technologic platforms. All necessary equipment, material and services needed for genetic, immunofluorescence and cell sorting technologies are available at Tor Vergata University and Santa Lucia Foundation (Rome) as well as through the experienced GIMEMA centers for initial AML characterization. Collaborations with clinical centers and access to patient samples. Continuous collaborations with many clinical centers and access to a large number of patient samples is guaranteed through the GIMEMA Cooperative group. The strength of 492 2013 AIRC.6 – A risk adapted, MRD-driven strategy... the GIMEMA in patient accrual, sample centralisation and successful network for biological studies (also supported by AIRC) is witnessed in several publications [see for example Falini et al. (2005) NEJM]. Additional institutional supports. a) Personnel support: the majority of expertise and personnel working in the project are paid by their Institutions and no support is requested except for personnel working 100% in this project. b) The costs for running the clinical trial are completely covered by the GIMEMA. In addition, the non-profit GIMEMA organization will be the sponsor of the study which involves therefore no commitment of pharma industry (conventional chemotherapic drugs will be used, as discussed above). Furthermore, the GIMEMA will cover expenses related to initial immuno-phenotypic and genetic characterization of AML for patient stratification and conventional MRD assessment (3-color MPF). Pitfalls and caveats 1 - In the clinical trial: a) Poor accrual, although we are expecting 120 patients/year. If this happens we will stimulate more GIMEMA centres to participate or call centres outside the GIMEMA and invite them to join the study. b) No significant improvement in OS. In this case we will extend the accrual period to overcome low number effect. c) New drugs approved for therapy of AML during our investigation. Although we are not expecting this to happen within the coming 2 years, in such instance we will incorporate the new drug or treatment protocol but within the same design of refined risk oriented strategy, even if it will cause patient heterogeneity. We believe it will not affect the approval of the trial hypothesis. 2 - Comparison of MRD results and risk-stratification, although our preliminary results support LAIP in MRD oriented therapy (see feasibility results), WT1 (or NPMI RQ-PCR) may prove better outcome prediction. In this case we will modify the design and use WT1 or (when appropriate, NPM1) for MRD-refined risk adapted therapy. 3 - Poor amount of patient material at remission. To counteract this limitation we will allocate sequentially a certain number of paired samples to investigate a specific objective. SIGNIFICANCE AND IMPACT OF THE PROJECT AML affects approximately 5-20/100,000 in Italy and worldwide. More than 60% of patients relapse after achieving remission and die within 4 years from diagnosis. In addition, the economic burden of the disease is extremely high due to long hospital stays and the necessity of expensive supportive care measures especially when using allogeneic transplant. This latter represents the only curative option in many instances and particularly in relapsing patients. Unfortunately, because of the few predictive markers and limited availability of targeted therapies, the majority of AML patients are offered inadequate front-line therapy with excessive toxicity and/or insufficient treatments being frequently 2013 493 Sezione III: Attività per progetti delivered. As a first output of our project we expect an initial 10% improvement in survival with the proposed refined risk stratification (without changing the current treatment options). A further significant improvement in survival of AML patients would derive from improved diagnostics and risk stratification strategies plus potential to targeting chemoresistance pathways emerging from our investigation. Novel biomarkers will in fact allow to better predicting treatment outcome thus achieving our ultimate goal for avoiding over/undertreatment. PRELIMINARY DATA 1- MRD monitoring. We have analysed MRD in 20 AMLs by tracking simultaneously LAIP and WT1 expression. The two methods equally correlated with CIR rates (using a Spearman rho coefficient (ρ= 0.50, p = 0.010) and the Kendall tau coefficient (τ= 0.35, p = 0.017). 2 - Clinical trial design. We found (in retrospective analyses) that a combined upfront (genetic markers) plus delayed (MRD status at CR by MPC) prognostic evaluation significantly improves outcome prediction in AML [Buccisano et al., 2010]. 494 2013