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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Hematology, University Hospital;
Department of Cytometry, University of Salamanca; Centro de
Investigación del Cancer, Salamanca; Department of Hematology,
San Carlos University Hospital, Madrid; Department of Hematology,
Complejo Hospitalario, León; and Department of Hematology,
General Hospital, Segovia, Spain.
Early response to therapy is one of the most important prognostic
factors in acute leukemia. It is hypothesized that early immunophenotypical evaluation may help identify patients at high risk
for relapse from those who may remain in complete remission (CR). Using
multiparametric flow cytometry, the level of minimal residual disease
(MRD) was evaluated in the first bone marrow (BM) in morphologic CR
obtained after induction treatment from 126 patients with acute myeloid
leukemia (AML) who displayed aberrant phenotypes at diagnosis. Based on
MRD level, 4 different risk categories were identified: 8 patients were
at very low risk (fewer than 10 Investigation of minimal residual disease (MRD) has
proven to be a valuable tool for predicting impending relapses before clinical and hematologic manifestations, and establishing different risk categories in patients with acute leukemia (AL).1-5
The detection of residual leukemic cells is usually based on either molecular or immunophenotypical markers present in leukemic but not in
normal cells, allowing for their specific discrimination. Most
available information on large series of patients with AL correlates
with childhood acute lymphoblastic leukemia (ALL). Thus, studies based
on semiquantitative polymerase chain reaction (PCR)1,6 or
immunophenotyping2,5 have demonstrated the clinical
relevance of MRD investigation; both methods provide similar results.
However, information on acute myeloid leukemia (AML) is scarce and is
mostly restricted to the investigation of AML subgroups with molecular
markers involved in chromosomal translocations, particularly t(15;17).
In fact, monitoring of PML/RAR gene rearrangement is
used for assessing molecular relapse and subsequent therapeutic
intervention in acute promyelocytic leukemia (APL).4,7,8
By contrast, few data have been reported on MRD analysis of AML by
immunophenotyping.3,9,10 This is probably because of the
technical difficulties related to the immunologic characterization of
myeloid leukemias given that large panels of monoclonal antibodies are
needed to cover all the different myeloid lineages and that several
blast cell subpopulations frequently coexist at
diagnosis.11-13 Altogether, these technical problems complicate the identification of phenotypic aberrancies in AML to be
used as leukemia-associated markers for MRD
detection.12-15 Nevertheless, in a preliminary study, our
group has shown that immunophenotypical investigation of MRD predicts
outcome in patients with AML and that the number of residual leukemic
cells correlates with multidrug resistance.3
Most of the reported MRD studies have been based on sequential analysis
of follow-up samples.1,2,5,6 However, it would also be of
interest to exploit MRD techniques for the assessment of initial
response to treatment, as a single time-point evaluation. Early
response to therapy is one of the most, if not the most, important
prognostic factor in AL. In childhood acute lymphocytic leukemia (ALL),
it has been shown that morphologic evaluation of blast cells in
peripheral blood (PB) after 1 week of steroid treatment16
and molecular analysis of the bone marrow (BM) on day 1517
help to identify different groups of patients at risk. In AML, the
relevance of early response is also well established; thus, patients
who do not enter into morphologic complete remission (mCR) after one
cycle of induction therapy have poor prognoses.18 Moreover, Estey et al19 have shown that patients requiring
more than 51 days to achieve mCR also have lower survival rates.
However, most of these latter data emerge from a negative
indicator Patients
The following variables collected at diagnosis were included in the
database: age, white blood cell (WBC) count, platelet count, hemoglobin
(Hb) level, percentage of blast cells in BM and absolute number in PB;
number of cycles to achieve mCR, type of treatment, morphologic FAB
classification, and cytogenetics. Karyotypic findings were grouped
according to Grimwade et al28 with the following
distribution: (1) favorable cytogenetic markers [t(15;17), t(8;21),
inv 16)] in 49 patients; (2) intermediate (no abnormalities, +8,
11q23, del (9q), +22, and other abnormalities not included in the other
groups) in 35 patients; and (3) adverse cytogenetics (complex, Immunophenotypical investigation of MRD
Our aim was to define within leukemic cells those phenotypes that are
absent or extremely infrequent in normal BM samples by using a
5-dimensional space formed by the 2 light scatter parameters (forward
scatter [FSC] and side scatter [SSC]) and the 3 fluorescence-associated characteristics. The existence of 2 or more
blast cell populations was established on the basis of a clearly
differentiated antigen expression, as previously
defined.23 Based on our previous
experience,3,13,14,23 4 main types of aberrant phenotypes
were considered: cross-lineage antigen expression, asynchronous antigen
expression, antigen overexpression, and abnormal light scatter pattern.
As a second step, once the immunophenotype of the leukemic cells for
the above-mentioned panel of mAbs was established, the need for
additional mAb combinations that could help identify the aberrant
phenotypes was evaluated and, if appropriate, carried out
to define a custom-built phenotypic pattern for follow-up studies.
Table 1 presents the frequencies of
the most common aberrant phenotypes detected in the present series.
Although we have previously reported that phenotypic changes involving
aberrant phenotypes may occur in 16% of patients with AML, these
changes usually take place during long-term clonal evolution.14 Here, we have focused on MRD detection during
an early phase of the disease (after induction therapy).
Interestingly, at this stage, the only phenotypic change observed was
the selection, in some patients, of a minor blast cell subpopulation,
but these abnormalities had been identified at diagnosis. This
represents an additional advantage of the early investigation of
MRD.
For the investigation of MRD in the mCR BM sample obtained after induction treatment, only those mAb combinations selected at diagnosis as informative for MRD studies were used. Additionally, to increase the sensitivity of the analysis, data acquisition in the flow cytometer was performed in 2 consecutive steps. Briefly, in the first step, all cells in the sample were acquired, and, at this point, at least 15 000 events per tube were measured. Subsequently, in a second step, a multiparametric live-gate was used to acquire more data on leukemic cells that might have been present in low numbers within the original BM sample. For that purpose, acquisition through an SSC/antigen live-gate was performed, and information was collected for at least 106 BM nucleated cells. Data analysis was based on the identification of cells with aberrant phenotypic features identical to those of leukemic cells at diagnosis. For data acquisition, the Lysis II or Cell Quest software programs (Becton Dickinson) were used. The Paint-a-Gate Pro software program (Becton Dickinson) with the polynomial SSC transformation capability was used for further data analysis. Analysis was performed on gated blast cells according to previously defined methods.3,5,13-15,23 Statistical analysis The 2 and the Mann-Whitney U tests
were used to estimate the statistical significance of differences
between groups. Survival curves were plotted according to the method of
Kaplan and Meier, and comparison between the curves was performed using
the log-rank and Breslow tests.24 Relapse rates are
reported as % ± 1 SE and overall survival rates as median ± 1
SE. In univariate analysis for relapse-free survival, the following
variables were tested: age, WBC, platelet count, proportion of blast
cells in BM, absolute number of blast cells in PB, number of cycles to
achieve CR (1 or 2 courses), type of treatment, FAB classification,
cytogenetics, and MRD level at the end of induction therapy.
Subsequently, multivariate analysis stepwise regression25 was performed to explore the independent effect of variables that showed a significant influence on disease-free survival found by univariate analysis. Statistical analysis was performed using the SPSS software (version 8.0.1S; SPSS, Chicago, Illinois).
As mentioned, this study is based on 126 consecutive patients with
de novo AML in whom blast cells displayed antigenic phenotypic aberrancies at diagnosis and who achieved mCR after induction therapy.
Immunophenotypic analysis of the first BM in mCR demonstrated that,
based on the level of residual phenotypically aberrant cells displaying
a leukemia-associated phenotype (LAP+), it was
possible to identify 3 different groups of patients at risk
(P < .0001). Seventeen patients were in the high-risk category (MRD greater than 10
The prognostic impact of MRD levels was also observed once M3 and
non-M3 leukemias were separately analyzed. Because of the reduced
number of M3 patients (40 patients uniformly treated with all-trans retinoic acid), only 2 risk categories were
established for M3, low risk (less than or equal to
2 × 10
Univariate analysis of prognostic factors revealed that, in addition to
MRD levels, 4 other disease characteristics had significant impact on
relapse-free survival: (1) cytogenetic characteristics (median survival
times, 18 months, 40 months, and not reached for the adverse,
intermediate, and favorable cytogenetic subgroups; P = .005); (2) number of chemotherapy cycles needed to
achieve response (1 vs 2; median survival time, 77 vs 15 months,
respectively; P = .003); (3) WBC (less than or greater
than or equal to 50 × 109/L; median survival time, 67 vs
11 months, respectively; P = .003); and (4) absolute PB
blast cells count (less than or equal to or greater than
50 × 109/L; median survival time, 67 vs 11 months, respectively; P = .009). By contrast, neither the
type of consolidation treatment (chemotherapy vs autologous or
allogeneic transplantation) nor the FAB classification or age showed
significant influence on relapse-free survival. Subsequently, we
analyzed the level of MRD in the patient subgroups according to the
above-mentioned prognostic factors (Table
2). Thus, on analyzing whether MRD level
was different in patients who achieved mCR with one cycle of
chemotherapy compared with those patients who needed 2 cycles, our
results show that in the first group, MRD levels were lower than in the
second (median, 1.9 × 10
To explore whether MRD level was an independent prognostic factor for relapse-free survival of patients with AML, multivariate analysis was conducted. In 32 of 126 patients, either cytogenetic information or mitosis was not obtained. As a result, the number of patients for multivariate analysis was reduced to 94. Therefore, we decided to perform analyses with and without the cytogenetic information. In the latter, all 126 patients were included in the regression analysis. Only the MRD levels proved to have consistent, independent prognostic influence in the Cox model (P = .0003), whereas the number of cycles of chemotherapy (1 vs 2) needed to achieve morphologic response was on the limit of statistical significance (P = .058). Because M3 leukemia is sometimes considered a distinct AML subtype, we repeated the multivariate analysis for the non-M3 patients. Once again, the most significant variable for predicting relapse-free survival was MRD followed by response to one cycle of chemotherapy (P = .01). When cytogenetic information was included in the Cox model, the parameters selected as having independent prognostic influence on relapse-free survival were the MRD levels (P = .002) and cytogenetics (P = .03).
Investigation of MRD has proven to be of relevant clinical value in the decision-making process for the most appropriate treatment of several hematologic malignancies, among them ALL, APL, and chronic myeloid leukemia.3,4,6-8,11 In AML, most MRD studies have been restricted to APL4,8 because the proportion of molecular targets available from non-M3 AML is still relatively small; in fact, only approximately 40% of patients with AML can be monitored by PCR.26 An alternative to molecular studies is the use of multiparametric flow cytometry immunophenotyping to monitor patients with AL who have aberrant phenotypic features. We have previously shown13 that on using multiparametric immunophenotyping, 70% to 80% of patients with AML have phenotypic aberrancies appropriate for MRD detection. However, in contrast to ALL, little information is available on the clinical value of MRD investigation by immunophenotyping of AML.3,9-11 In most therapeutic strategies for patients with AML, the treatment,
including the possibility of transplantation, is administered within
the first 6 months of diagnosis. Therefore, stratification of patients
according to risk group should be based on parameters either already
available at diagnosis or derived from response to initial treatment.
The most relevant factors for risk stratification of AML, and probably
the only ones used, are cytogenetics and morphologic evaluation of
response to induction therapy.18,27-29 However, the latter
parameter is of limited value because it identifies only a subgroup of
patients with poor prognoses: those who do not achieve mCR. Among
patients who do achieve CR, a high relapse rate persists that cannot be
predicted using conventional morphology. Accordingly, more sensitive
techniques, such as PCR or flow cytometry immunophenotyping, may be
useful for the evaluation of MRD level in patients with AML in mCR.
Although most MRD studies have focused on sequential follow-up samples,
we hypothesize that early investigation of MRD in BM samples obtained
immediately after induction treatment could help to subdivide AML
patients who have achieved mCR into different risk categories. Our
results show that 4 different patient risk groups can be clearly
identified based on the number of residual cells displaying
leukemia-associated phenotypes (LAP+ cells). Thus, none of
the patients with fewer than 10 As previously mentioned, the strategy of monitoring MRD based on aberrant phenotypes can be used in 70% to 80% of patients with AML. For the remaining patients, new approaches must be developed. One possibility, recently explored in ALL,30 is based on the investigation of myeloid progenitors. In these precursor cells, the detection of deviations from the normal maturation pathways could reflect the presence of abnormal hemopoiesis that might signal impending relapse. Different treatment strategies are available for consolidation therapy in AML patients, including autologous and allogeneic transplantation with the mini-allografting variants, and novel therapeutic weapons, such as monoclonal antibodies and immunotherapy. Some of these treatment modalities should still be considered experimental, and it would be desirable to test them in uniform groups of patients whose risk is defined by objective prognostic factors. Otherwise, identifying patients who could benefit from a particular treatment strategy would be extremely difficult, and progress in the therapeutic outcome of AML would be delayed. Our results show that early immunophenotypical evaluation of MRD in patients with AML identifies different risk groups and may contribute to postinduction treatment stratification.
We thank all members of the BIOMED-1 Concerted Action (BMH-CMT 94-1675), particularly Jacques van Dongen for fruitful discussion during the standardization of MRD studies, and M. Anderson and M. J. Rodrigo for their help with the English language.
Submitted February 12, 2001; accepted May 16, 2001.
Supported in part by national grants from Spain (FISS 95/1640,CICYT-SAF 94-038,AECC-95,FIS 00/0023-03) and integrated in the European BIOMED-1 Concerted Action (BMH-CMT 94-1675).
Accepted for oral presentation at the 2000 American Society of Hematology Meeting, San Francisco, CA, December 1-5, 2000.
J.F.S.M., M.B.V., M.G., and A.O. are members of the European BIOMED-1 Concerted Action (BMH-CMT 94-1675).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jesús F. San Miguel, Hematology Department, Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca. Spain; e-mail: sanmigiz{at}gugu.usal.es.
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  B. Paiva, M.-B. Vidriales, J. Cervero, G. Mateo, J. J. Perez, M. A. Montalban, A. Sureda, L. Montejano, N. C. Gutierrez, A. G. de Coca, et al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation Blood, November 15, 2008; 112(10): 4017 - 4023. [Abstract] [Full Text] [PDF]
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 L. Maurillo, F. Buccisano, M. I. Del Principe, G. Del Poeta, A. Spagnoli, P. Panetta, E. Ammatuna, B. Neri, L. Ottaviani, C. Sarlo, et al. Toward Optimization of Postremission Therapy for Residual Disease-Positive Patients With Acute Myeloid Leukemia J. Clin. Oncol., October 20, 2008; 26(30): 4944 - 4951. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
4 cells), and none have
relapsed thus far; 37 were at low risk (10
the failure to achieve early mCR
7,
abnormalities in 3q, del (5q) and 
