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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.
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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;
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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.
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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.
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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.
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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.
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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
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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
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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].
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