Monitoring of minimal residual disease in acute myeloid leukemia

Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Monitoring of minimal residual disease in acute myeloid leukemia Wolfgang Kern ∗ , Claudia Schoch, Torsten Haferlach, Susanne Schnittger Laboratory for Leukemia Diagnostics, Ludwig-Maximilians-University, University Hospital Grosshadern, Department of Internal Medicine III, 81366 Muenchen, Germany Accepted 24 June 2004

Contents 1.

2.

3.

4. 5.

∗

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 1.1. Prognostic factors in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 1.2. Clinical use of monitoring of minimal residual disease in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Assessment of minimal residual disease in acute myeloid leukemia by multiparameter flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . 285 2.1. Immunophenotype of normal bone marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2.2. Classification of leukemia-associated aberrant immunophenotypes in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2.3. Applicability and sensitivity of immunologic monitoring in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.4. Stability of LAIP at relapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 2.5. Prognostic impact of immunologically quantified MRD in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 2.6. Improvement by application of CD45 gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Assessment of minimal residual disease in acute myeloid leukemia by quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.2. Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.2.1. Sample material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.2.2. Technique of real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.2.3. Technical equipment for RQ-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 3.2.4. PCR-targets for MRD detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 3.2.5. Quantification of a target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 3.2.6. Quality control and standardization of QRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 3.3. Quantification of fusion genes in AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 3.3.1. Transcript ratios at diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 3.3.2. Transcription reduction during therapy and its impact on prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 3.3.3. New score for risk stratification of AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 3.3.4. Prediction of relapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 3.4. New targets for PCR-based MRD detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.4.1. FLT3-LM as marker for MRD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.4.2. MLL-PTD as marker for MRD detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 3.4.3. WT1 overexpression as marker for MRD detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 3.4.4. EVI1 overexpression as marker for MRD detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Comparison of multiparameter flow cytometry and quantitative RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Corresponding author. Tel.: +49 89 990 17200; fax: +49 89 990 17111. E-mail addresses: [email protected] (W. Kern), [email protected] (S. Schnittger).

1040-8428/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2004.06.004

284

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Abstract Monitoring minimal residual disease (MRD) becomes increasingly important in the risk-adapted management of patients with acute myeloid leukemia (AML). The two most sensitive and quantitative methods for MRD detection are multiparameter flow cytometry (MFC) and real-time polymerase chain reaction (QRT-PCR). Fusion gene-specific PCR in AML is based on the RNA level, and thus in contrast to MFC expression levels rather than cell counts are assessed. For both methods independent prognostic values have been shown. The strong power of MFC has been shown mainly in the assessment of early clearance of the malignant clone. MRD levels in AML with fusion genes have the strongest prognostic power after the end of consolidation therapy. In addition, with QRT-PCR highly predictive initial expression levels can be assessed. With both methods early detection of relapse is possible. So far, validated PCR-based MRD was done with fusion genes that are detectable in only 20–25% of all AML MFC is superior since it is applicable for most AML. However, QRT-PCR is still more sensitive in most cases. Thus, it is desirable to establish new molecular markers for PCR-based studies. Large clinical trials will determine the role and place of immunologic and PCR-based monitoring in the prognostic stratification of patients with AML. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Minimal residual disease; Acute myeloid leukemia; Multiparameter flow cytometry

1. Introduction Antileukemic therapy is applied to patients with acute myeloid leukemia (AML) in order to eradicate all leukemic cells and to thus achieve cure from the disease. The use of modern intensive induction chemotherapies results in a complete remission (CR) rate of 60% to 80% of all patients [1–4] with CR being defined as the cytomorphologically assessed absence of leukemic cells in peripheral blood and bone marrow and full recovery of peripheral blood values. However, without the application of additional chemotherapies in nearly all cases AML would relapse within weeks or few months. These comprise intensive consolidation therapies [5,6], less intensive maintenance therapies [7–10], and autologous or allogeneic stem cell transplantations [11–14]. The need for these multiple therapeutic elements is due to leukemic cells persisting within the patient after achievement of a CR. These cells have the potential to form a regrowing leukemic population, which clinically occurs as relapse of AML. Due to the lack of sensitivity of the method even large amounts of residual cells are not detectable by cytomorphology. In fact, about 1012 leukemic cells are present in a patient at diagnosis and are reduced by an induction therapy resulting in a CR. However, at this stage up to 1010 leukemic cells may still persist [15,16]. Different courses of this minimal residual disease (MRD) are depicted in Fig. 1. Consecutive elements of antileukemic therapy may lead to repeated reductions of the amount of residual leukemic cells until eventually cure from the disease is achieved. In contrast, residual leukemic cells may become resistant to antileukemic therapy in other cases leading to increasing levels of MRD while the patient still is in complete remission as assessed cytomorpholgically. In most of the latter cases, the regrowth of the leukemic population results in the occurrence of clinical relapse. Taking these issues into account, the level of MRD is anticipated not only to have substantial impact on the course of the disease and to become a valuable therapy-dependent prognostic factor but also to be increasingly used for the selection of risk-adapted treatment strategies.

Along this line, the recently “Revised Recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia” now include the definition of treatment failure based on the reappearance of molecular and cytogenetic abnormalities [17]. In addition, the flow cytometric evaluation of AML at diagnosis is recommended in order to identify leukemia-associated aberrant immunophenotypes (LAIP) useful for the quantification of MRD. 1.1. Prognostic factors in AML Today the karyotype of the leukemic cells is considered the most important parameter indicating the prognosis in patients with AML [18–23]. Although further pre-therapeutically defined prognostic parameters have been identified like age of the patient, AML occurring as secondary disease, and WBC

Fig. 1. Leukemia treatment outcome. The red line indicates a sequential reduction of the leukemic cell mass with the exception of a slight increase before the second consolidation therapy. Eventually, cure is achieved. The green lines indicate cases with relapses of AML in which increasing MRD levels are present before relapse. The light blue area refers to the cytomorphologic finding of 1–5% bone marrow blasts which is compatible with complete remission. The intermediate blue area refers to a 0% bone marrow blasts count with MRD levels detectable by MFC or QRT-PCR. The dark blue area refers to a 0% bone marrow blasts count with MRD levels below the sensitivity of MFC and QRT-PCR.

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

count the prognosis of patients within the respective subgroups defined by these parameters is still heterogeneous. As a consequence, the implementation of therapy-dependent parameters into stratification systems has been approached. Along this line, the degree of reduction of the leukemic cell mass following the first course of induction therapy as well as the time to achievement of complete remission have been demonstrated to independently impact the prognosis [24–26]. These studies thus have proved the concept of prognostication based on therapy-dependent factors in AML. To overcome the limitation of these parameters, i.e. the rather low sensitivity of 5% achievable by cytomorphology, further studies dealt with the use of quantitative polymerase chain reaction (Q-PCR) and of multiparameter flow cytometry (MFC) in detecting MRD at significantly lower levels. These studies will be discussed in detail in this review. 1.2. Clinical use of monitoring of minimal residual disease in AML The two most important aspects of monitoring of MRD in AML are its use as a stratification parameter and as a tool for the early detection of impending relapse. In the first setting, patients are divided based on distinct levels of MRD into two or more groups with different risks of relapse and probabilities of long-term remission. The resulting stratification models can be used in randomized clinical trials to delineate the relative efficacy of investigational treatment approaches within prognostically homogeneous subgroups of AML. In the second setting, the aim of monitoring of MRD is the identification of cases with a very high risk of relapse who then can be treated much earlier and more effectively by salvage protocols as compared to patients in overt relapse. A further possible application is the use of the reduction of MRD as a surrogate marker for long-term efficacy of newly developed and evaluated treatment approaches. 2. Assessment of minimal residual disease in acute myeloid leukemia by multiparameter flow cytometry The detection and quantification of MRD by multiparameter flow cytometry MFC relies on the presence of leukemiaassociated aberrant immunophenotypes on leukemic cells in patients with AML [27]. These LAIP are present in all of or in a subset of the leukemic cells and are present at very low frequencies or even absent in normal bone marrow cells. Given that at diagnosis of AML the LAIP present in an individual case has been identified by using MFC with a comprehensive panel of combinations of monoclonal antibodies MRD during the course of treatment and follow-up can be assessed by the quantification of the frequencies of these cells by MFC. 2.1. Immunophenotype of normal bone marrow Extensive flow cytometric analyses of normal bone marrow samples has consistently demonstrated that normal bone

285

marrow cells display a reproducible sequence of the expression of distinct antigens at different time points during the myeloid, monocytic, lymphoid, and erythroid differentiation [28–32]. The identification of lineage-specific antigens in CD34-positive bone marrow stem cells revealed a coexpression of the progenitor cell marker CD38 and of CD71, CD33, CD10, and CD5, respectively, in parallel to the cytomorphologically observed development of primary blasts into erythroblasts, myeloblasts, and probably also into lymphoblasts although the development of T-lymphocyte still is a matter of debate [33]. In fact, the lack of lineage-specific antigens has been observed in only 1% of the CD34-positive cells. A co-expression of CD15 which is expressed mainly on myelomonocytically differentiated cells has been observed in only 10% to 20% of cells expressing CD34 and HLADR. Of particular interest for the identification of LAIP in AML a co-expression of CD11b, CD14, CD65, or CD56 in CD34-positive normal bone marrow cells has not been present at all [34]. These analyses demonstrated that AML cells which virtually always display an aberrant expression of antigens cover an area within the flow cytometrically defined multi-dimensional space, which does not overlap with normal bone marrow cell populations. However, with regard to the application of MFC for the detection and quantification of MRD these analyses also raise the question of the validity of quantifying very small cell populations since some of the immunophenotypes useful for monitoring of MRD are not absent but present at very low frequencies in normal bone marrow. Thus, leukemic cells at the stage of MRD may be present at similarly low frequencies and normal bone marrow cell populations may interfere with the quantification of leukemic cell populations. As a consequence, each individual LAIP identified in a patient with AML must be compared to the immunophenotypes of normal bone marrow cells and the precise definition of gate combinations which describe the LAIP (see below) has to aim at maximum differences in the frequencies of leukemic cells and normal bone marrow cells covered by the respective LAIP. 2.2. Classification of leukemia-associated aberrant immunophenotypes in AML Following the pivotal description of the aberrant expression of lymphoid markers on AML cells [35] further evaluations led to the classification of leukemia-associated aberrant immunophenotypes into four groups (Table 1): (a) “crosslineage” expression (expression of lymphoid antigens on AML cells), (b) lack of expression of an antigen (an antigen which is expressed during normal granulocytic or monocytic differentiation is not expressed), (c) overexpression (expression of antigens with an expression intensity higher than observed in normal bone marrow or expression of antigens normally not expressed in bone marrow), and (d) asynchronous expression of antigens (co-expression of antigens which during normal granulocytic and monocytic differentiation are expressed sequentially but not simultaneously). In

286

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Table 1 Classification of leukemia-associated aberrant immunophenotypes (LAIP) in AML LAIP class

Examples

Cross-lineage expression of lymphoid antigens

CD33+CD2+CD34+

Table 2 Distribution of LAIP and of LAIP classes in 1400 patients with newly diagnosed and untreated AML (Laboratory for Leukemia Diagnostics, Munich, Germany) LAIP

Asynchronous

Total CD11b+CD117+CD34− CD11b+CD117+CD34+ CD11b+CD117−CD34+ CD34+CD116+CD33+ CD34+CD15+CD33+ CD65+CD87+CD34+ CD65+CD87−CD34+

652 156 92 36 113 193 12 50

20.6 4.9 2.9 1.1 3.6 6.1 0.4 1.6

Cross-lineage

Total CD34+CD13+CD19+ CD34+CD2+CD33+ CD34+CD56+CD33+ CD34−CD13+CD19+ CD34−CD2+CD33+ CD34−CD56+CD33+ CD4+CD13+CD14− CD7+CD33+CD34− CD7+CD33+CD34+

742 48 51 83 21 33 189 87 75 155

23.5 1.5 1.6 2.6 0.7 1.0 6.0 2.8 2.4 4.9

Lack of expression

Total CD15+CD13+CD33− CD15+CD13−CD33+ CD34−CD135+CD117+ CD38−CD133+CD34+ CD4+CD13−CD14+ CD9−CD34+CD33+ CD9−CD34−CD33+ HLA-DR+CD33−CD34+ HLA-DR−CD33+CD34− HLA-DR−CD33+CD34+ MPO+LF−cCD15− MPO+LF−cCD15+ MPO−LF+cCD15+

625 6 7 17 10 7 30 34 12 143 37 315 4 3

19.8 0.2 0.2 0.5 0.3 0.2 0.9 1.1 0.4 4.5 1.2 10.0 0.1 0.1

Overexpression

Total CD11b−CD117++CD34+ CD13++CD34++ CD15++CD13++CD33++ CD34++CD135+CD117++ CD34++CD33++ CD34−7.1++CD33+ CD36++CD235a++CD45(+) CD38++CD133++CD34++ CD4++CD64++CD45++ CD4+CD13++CD14++ CD61++CD14−CD45+ CD65++CD87++ CD90++CD117++CD34+ HLA-DR++CD33++CD34++ TdT(+)cCD33++cCD45++

1139 9 163 52 35 65 53 25 16 144 19 5 162 23 41 327

36.1 0.3 5.2 1.6 1.1 2.1 1.7 0.8 0.5 4.6 0.6 0.2 5.1 0.7 1.3 10.4

3158

100.0

CD34+CD13+CD19+ Overexpression

HLA-DR++CD33++CD34++ CD64++CD4++CD45++

Lack of expression of an antigen

HLA-DR−CD33+CD34+

Asynchronous expression of antigens

CD15+CD33+CD34+ CD65+CD33+CD34+

(+) Expression; (++) overexpression; (−) no expression; the respective LAIP is displayed on all of or on a subset of leukemic cells in AML.

addition, leukemic cells frequently display an aberrant light scatter pattern, which reflects mainly the size (forward-light scatter; FLS) and the granularity (orthogonal-light scatter; OLS) of the cells. Table 2 gives an overview on the frequencies of different LAIP and their distributions between classes. From this overview it becomes clear that most of the LAIP identified in patients with AML are present in less than 5% of all cases. In order to be able to identify each of these LAIP and, most importantly, at least one LAIP in each patient with AML it is necessary to apply a large and comprehensive panel of antibody combinations. The presented approach applying triple combinations of monoclonal antibodies has substantially increased the portion of AML patients in which a LAIP can be identified, which was the case applying double staining in only 24–51% [36,37]. 2.3. Applicability and sensitivity of immunologic monitoring in AML Besides the determination of the prognostic impact of MRD levels in AML in general one of the key issues in immunologic monitoring in AML is the balance between maximizing the applicability to all patients and the sensitivity and specificity of the method. Due to the phenotypic heterogeneity of AML within individual cases, which occurs as more than one population or, in most cases, as a wide range of expression intensity of various antigens, the inclusion of all AML cells into one LAIP is possible in some cases only (e.g. CD34+CD56+CD33+, Fig. 2) while in other cases only a part of the total population of AML cells can be included into one LAIP (e.g. CD64++CD4++CD45++, Fig. 2). Clearly, the portion of AML cells, which can be covered by a LAIP, is dependent on the degree of aberration in the leukemiaassociated immunophenotype as compared to the respective immunophenotype in normal bone marrow cells. Thus, in AML patients with leukemic cells displaying a less aberrant LAIP the choice with regard to immunologic MRD monitoring is (a) to include only a portion of all cells into one LAIP which is absent in normal bone marrow cells, (b) to include all leukemic cells into one LAIP but at the same time to lose sen-

n

LAIP class

Total

%

sitivity and specificity due to the overlap between leukemic and normal immunophenotypes, and (c) to not include these AML patients with less aberrant immunophenotypes into the monitoring program. From the technical point of view the latter approach is the most preferred one and has been adopted in a variety of analyses on the prognostic impact of immunologically quantified MRD in AML. Thus, the AML cells in

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

287

Fig. 2. Four different classes of leukemia-associated aberrant immunophenotypes. First and second plots are leukemic bone marrow; third and fourth plots are normal bone marrow; second and fourth plots display only cells gated in first and third plots (green cells); green dots represent cells with immunophenotype as defined in AML by first two antigens, red dots represent cells with immunophenotype as defined in AML by third antigen and OLS-signal; A (crosslineage expression): CD33+CD34+ and CD7+SSClow; B (overexpression): CD45++CD64++ and CD4+SSClow; C (lack of expression): CD34+CD33+ and HLA-DR−SSClow; D (asynchronous expression): CD34+CD117+ and CD11b+SSClow.

more than 25% of all patients analyzed at diagnosis were considered not to carry a LAIP [38–40]. Although these studies consistently demonstrate the high prognostic impact of immunologically determined MRD levels in AML they do not address the issue of optimizing the applicability of this approach by improving the panel of antibodies, and thus the capability of identifying LAIP in a larger number of patients. New data in this regard suggest that maximizing this applicability is possible even to 100% of all AML patients with the resulting sensitivity as analyzed by normal bone marrow samples amounting to a median of 0.05% [41]. In fact, data are available to prove that MRD levels determined in this way

have significant impact on the patients’ prognosis [42,43] (see Section 2.5). 2.4. Stability of LAIP at relapse A prerequisite for the validity of immunologic monitoring in AML is the stability of the LAIP between diagnosis and relapse. Along this line, analyses of changes in chromosomal aberrations between diagnosis and relapse indicated that in about one-third of all cases the karyotype is different at relapse [44,45]. Since these genetic lesion are considered to determine the biology of the disease changes

288

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

in the immunophenotype and in the LAIP in particular are expected to occur in a similar way and frequency. Obviously, these changes are most relevant for monitoring late in the course of the disease, i.e. following consolidation therapy or during follow-up. At these checkpoints, the early identification of clinically still inapparent relapses is the main focus of the approach, and thus in individual cases in which the LAIP changes between diagnosis and relapse prediction of relapse would be hampered. In contrast, evaluations as early as directly following induction therapy or after achievement of complete remission may be prone to this problem insignificantly only since in this context changes are expected to occur at a much lower frequency. This is important with regard to the clinical use of prognostication by this approach since a risk-based stratification is most valuable in the early course of the disease in order to best take advantage of salvage regimens and procedures. Published data in this regard suggest that with a few exceptions the LAIP is detectable also at relapse, however, the populations covered by the LAIP at relapse in some cases represent only less than 10% of all leukemic cells and the intensity of aberrantly expressed antigens may have changed [37,46–50]. In addition, phenotypic changes may occur due to the expansion at relapse of small cell subsets that have been undetected at diagnosis. The total numbers of analyzed patients in these reports range between 12 and 66. Thus, the frequencies of significant changes of LAIP cannot be estimated accurately yet. Along this line, however, we have observed in 53 patients analyzed both at diagnosis and at relapse applying a large panel of triple combinations of monoclonal antibodies that in 72% of cases at least one of the LAIP present at diagnosis is present at relapse in the majority of leukemic cells [204]. Out of the remaining cases (=28%) 17% were shown to carry the LAIP present at diagnosis in a minority of the leukemic cells while in another 11% it was absent. Overall, the data available indicate that in the majority of patients with AML the LAIP present at diagnosis will also be present at relapse, and thus are useful for MRD monitoring. However, in a minority of patients the early detection of relapse may be hampered by a change in immunophenotype. 2.5. Prognostic impact of immunologically quantified MRD in AML Data on the sensitivity of MFC used for detection of MRD are significantly better than for the current standard method used to evaluate the response to therapy in AML, i.e. cytomorphology with a sensitivity of 5% bone marrow blasts. Depending on the selection of applied antibodies and on the aberrantly expressed genes the sensitivity in most AML cases ranges between 0.1% and 0.01% of all nucleated bone marrow cells [51,52]. The studies conducted in patients after achievement of a complete remission indicate that the amount of residual disease for the majority of patients are in the same range, and thus assure the feasibility of using MFC for the quantification of MRD [38–40,53].

Published data on the detection of MRD by MFC suggest that in a portion of all patients an estimation of the prognosis is possible based on the amount of residual disease. While in both childhood and adult acute lymphoblastic leukemia an independent prognostic relevance has been demonstrated for the MRD levels in complete remission as well as during induction therapy [54–57], the current data on AML are less complete yet [38–40,53,58,59]. A total of four clinically relevant studies have been published dealing with adult AML. In all of them, a prognostic impact of MRD levels determined by MFC has been shown. San Miguel et al. demonstrated in a subgroup of 53 of a total of 89 initially immunophenotyped patients in whom a LAIP has been identified and who achieved a complete remission that an MRD level of 0.5% following induction therapy and an MRD level of 0.2% following consolidation therapy allowed to separate patients into two groups with significantly different relapse-free survival [40]. In a similar study Venditti et al. could demonstrate that an MRD level of 0.045% following induction therapy and of 0.035% following consolidation therapy had an independent impact on both event-free survival (EFS) and overall survival (OS) [38]. Also in this study, the number of patients in whom MRD levels were analyzed for prognostic impact was 56 out of a total of 93 patients initially immunophenotyped at diagnosis. A different approach was attempted in another study on 22 patients in whom an asynchronous co-expression of CD15 and CD117 has been detected in at least 5% of all leukemic cells [53]. An association between the amount of these cells at 10 months after start of induction therapy on the one hand and the duration of the remission on the other hand was shown. An extension of one of these studies revealed a prognostic impact of MRD level between 0.01% and 0.1%, between 0.1% and 1%, and higher than 1% at the time of achievement of complete remission after induction therapy. The separation of patients according to these limits resulted in significant differences in the overall survival and identified flow cytometrically quantified MRD level as an independent prognostic marker in a multivariate analysis [39]. Furthermore, a study performed in childhood AML demonstrated the applicability of flow cytometric quantification of MRD in 85% of all cases and identified the MRD levels after both induction 1 and induction 2 as independent prognostic markers with regard to overall survival in multivariate analyses [59]. Taken together, these studies suggest that the prognostication in AML based on MRD levels determined by MFC is feasible, however, some of the most important aspects still need to be clarified in more detail yet. In the studies discussed above the total number of patients analyzed for prognosis is about 100. This relatively small number limits the strength of the performed multivariate analyses. A valid comparison of the prognostic impact of immunologically determined MRD levels and the parameters today considered the most important prognostical indicator, i.e. chromosome abnormalities, is not yet possible. Another aspect that potentially allows an improvement of the applicability of these analyses is the

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

selection of earlier evaluation time points. While in the analyses cited above the earliest evaluation was performed after achievement of complete remission recently published data show that as soon as one week after completion of the first course of induction therapy, i.e. during aplasia, the amount of cytomorphologically identified residual leukemic blasts has an independent prognostic impact [24]. The most important limitation of the studies on immunologic MRD assessment cited above, however, is the inclusion of a portion of all patients only which limits the usefulness of this method as a stratification parameter in clinical trials. Two recent reports from our group, however, indicate that prognostication by flow cytometric assessment of MRD is possible in the large majority of patients with AML [42,43]. Thus, the MRD levels both during aplasia after induction therapy as well as during CR (before and after consolidation therapy) were shown to strongly correlate with the prognosis of the patients. Thus, in all of 106 unselected patients analyzed at diagnosis and at day 16 following the start of induction therapy MRD levels at day 16 were significantly related to the achievement of CR (p = 0.0001), event-free survival (p < 0.0001) (Fig. 3), overall survival (p = 0.003), and relapse-free survival (p = 0.0003). Importantly, the prognostic impact of day 16 MRD was independent of cytogenetics and other prognostic parameters with regard to achievement of CR, event-free survival, and relapse-free survival. An additional analysis of 58 patients before and 62 patients after consolidation therapy revealed similar results with independent prognostic impact of MRD levels at both checkpoints (Fig. 4). These data are in line with previous reports an in addition demonstrate that the flow cytometric assessment of MRD (a) may be extended to virtually all patients with AML and (b) yield powerful prognostic parameters applicable as stratification parameters in clinical trials. However, this approach may be improved even further if the accuracy of the quantification of very low-level MRD can

289

Fig. 4. Prognostic impact of MRD after consolidation therapy as quantified by multiparameter flow cytometry. Cases with an MRD level after consolidation therapy lower than the 75%-percentile after consolidation therapy have a better event-free survival than patients with an MRD level higher than the 75%-percentile (event-free survival at 2 years: 83.3% vs. 25.7%, p = 0.0034).

be augmented. In this regard, it is anticipated that the application of MFC using four colors and using the pan-leukocyte antigen CD45 a significant improvement of the capability to distinguish between leukemic cells and normal bone marrow cells, and thus an optimization of the applicability of this method for the quantification of MRD can be achieved. A conceptionally different approach has been followed by not defining an aberrant immunohenotype at diagnosis and detect cells displaying this immunohenotype during follow-up but rather compare cells of different compartments analyzed during follow-up directly with the corresponding compartments in normal bone marrow samples [60]. Multivariate analyses proved for both relapse-free and overall survival the flow cytometrically detected levels of leukemia the most important prognostic factor. 2.6. Improvement by application of CD45 gating

Fig. 3. Prognostic impact of MRD at day 16 as quantified by multiparameter flow cytometry. Cases with an MRD level at day 16 lower than the median at day 16 have a better event-free survival than patients with an MRD level higher than the median (53% at 2 years vs. a median of 2.8 months, p < 0.0001).

The studies published so far on the detection of MRD used MFC applied three fluorochromes and different triplecombinations of monoclonal antibodies. This implies that the basic parameters used in all combinations for the identification of the blast population are the light scatter characteristics, i.e. forward-light scatter (FLS) and orthogonal-light scatter (OLS). Fig. 5 demonstrates the limitations of this approach: the blast cells do not form a distinct population but they overlap with the lymphocyte and the monocyte populations. A significant step forward in this regard has been the introduction of the simultaneous detection of four fluorochromes allowing the detection of one further antigen. The prerequisite of the use of a further antibody to uniformly identify

290

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Fig. 5. Improvement of separation of populations by CD45 gating. (A) Normal bone marrow (left: FLS-OLS plot, right: CD45-OLS plot) and (B) AML bone marrow (left: FLS-OLS plot, right: CD45-OLS plot). G, granulocytes; M, monocytes; L, lymphocytes; E, erythrocytes; B, blasts. CD45-OLS gating allows to isolate bone marrow blasts from all other populations which is not possible by FLS-OLS gating.

the blast cell population in all combinations of antibodies applied is the availability of a gene which is expressed in different intensities on all of the evaluated cells and which thus can be used globally just like the light scatter characteristics. The pan-leukocyte antigen CD45 meets these criteria. Differences in the expression are present not only between different lineages of hematopoietic differentiation but also within these lineages during the process of maturation. The strongest expression is observed in mature T-lymphocytes and in NK-cells. In B-lymphocytes and in monocytes the expression of CD45 becomes stronger in parallel to the maturation while CD45 is expressed only lightly in granulocytes during all phases of maturation [61]. The expression of CD45 in erythroid precursors is weak and absent after full maturation. The combination of the differential expression of CD45 with the also differential OLS signal results in a much better separation of all relevant cell populations. In particular, the blast cell population covers a separate area, overlaps with other populations are not present [62]. A further advantage of

this CD45 gating approach is the possibility to exclude from the analysis erythroid cells based on their very low CD45 expression levels while for the application of the FLS/OLSplot a significant interference between lymphocyte and blasts cell populations is observed (Fig. 5). The CD45 gating has not yet been used for the analysis of MRD in AML. For its application, in the quantification of lymphocyte subpopulations, an improvement of the accuracy has been achieved [63]. Furthermore, an analysis of 50 patients with acute leukemias a high degree of correlation between a manually performed differential count of the bone marrow on the one hand and the portion of the respective cell populations using CD45 gating on the other hand was found [64]. The anticipated improvement of the specificity of immunologic MRD assessment in AML by the application of CD45 gating has not yet been analyzed. Further improvements are anticipated by the extension of parameters novel cytometers are capable of detecting, i.e. by the application of as much as eight channels for the detection different of fluorescence dyes [65].

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

3. Assessment of minimal residual disease in acute myeloid leukemia by quantitative PCR 3.1. Introduction A large number of studies have shown that the presence of very low levels of disease-specific markers detectable by PCR significantly correlates with clinical outcome in many hematologic malignancies [66–74]. In analogy during the last years, an accumulating number of studies have shown that detection of MRD in AML also is of high prognostic value [75–80]. However, in AML the molecular detection of MRD was mainly restricted to patients with defined chromosomal rearrangements and the corresponding fusion genes. Of the PCR-based methods for MRD detection quantitative reverse transcription polymerase chain reaction (QRT-PCR) was the one most frequently used for highly sensitive minimal disease detection in AML during the last 5 years. The application of QRT-PCR is based on genetic aberrations, i.e. mutations and fusion genes, which occur exclusively in the respective subentities of AML but not in normal bone marrow. The three most common reciprocal rearrangements producing fusion genes in AML are t(15;17), inv(16)/t(16;16), and t(8;21). These aberrations occur in about 20% of all AML and are associated with a favorable prognosis [81–83]. In these entities, most patients achieve a complete remission (CR), however, 10–30% finally suffer from relapse [84–86]. The corresponding leukemia-specific fusion transcripts PML-RARA, CBFB-MYH11, and AML1-ETO, can be targeted by PCR-based methods at diagnosis and at followup to detect MRD [87–89]. The most extensively studied fusion gene in AML is PML-RARA. Early reports applying non-quantitative PCR indicate that long-term remissions in patients with acute promyelocytic leukemia are associated with PCR negativity [90–98] while persistingly positive and recurring PCR results identify patients at very high risk of relapse [90,93–95,97–101]. In many of the cases with acute promyelocytic leukemia salvage therapy is initiated before overt relapse of the disease [102]. It has been shown that the presence of residual PML-RARA-positive cells strongly predicts relapse and therefore is an important parameter for treatment decisions [103–109]. Although this non-quantitative MRD information can be highly significant in APL, it only gives limited information and does not allow precise analysis of tumor load kinetics. In addition, the prognostic impact of the mere detection of residual CBFB-MYH11 or AML1-ETO transcripts in complete remission has not been clarified yet. Data on MRD levels determined by conventional and nested PCR results in AML with t(8;21) consistently have shown that in many patients long-term remissions are associated with a persistence of the molecular marker [110–117]. Now data are cumulating on the prognostic impact of MRD levels in AML determined by QRT-PCR that enables accurate assessment of the number of leukemic transcripts at consecutive follow-up time points [114,118,119].

291

Quantification of MRD by PCR can be performed by comparing the PCR signal (often after blotting and hybridization) with serial dilutions of a standard with known amounts of target DNA or RNA [120], by limited dilution experiments until negative PCR results are obtained [121,122], and by competitive PCR [123–125]. These methods are labor intensive and require a lot of sample material. However, real-time quantitative PCR (RQ-PCR) methods have recently been developed [126], applied to hematologic malignancies and together with recent GeneScan technology [127–130] may replace the complex and time consuming (semi)quantitative PCR analysis [131]. Also promising with regard to the highly sensitive and accurate detection of transcription levels in PML-RARA-, AML1-ETO- and CBFB-MYH11-positive AML is the application of RQ-PCR for the quantification of fusion transcript levels before, during, and after therapy. The use of RQ-PCR for AML-specific fusion gene started in 1998 [132]. Most previous studies were focussed on the applicability of the method per se and the validation of longitudinal monitoring MRD during the clinical course of the disease by real-time RT-PCR [133–135]. Since then an increasing number of studies have shown the clinical importance of PCR-based MRD detection in AML [76–80,106]. With QRT-PCR at least three different aspects can be assessed: (1) the transcription ratio at diagnoses, (2) the kinetics of the reduction of the leukemic clone, and (3) the possibility of early detection of the recurring clone. 3.2. Material and methods 3.2.1. Sample material Although it has been shown that peripheral blood may be used for follow-up studies in ETV6-AML1-positive ALL [136], little and controversial data have been reported for peripheral blood samples in AML. Thus, standard material for MRD analysis in AML is bone marrow (BM). The use of peripheral blood as sample material is under discussion, especially with respect to more frequent sampling time points. However, few studies with acceptable numbers of paired BM/PB samples are available. Peripheral blood may be discussed in APL as has been suggested [79]. For AML1-ETO, it has been shown that fusion gene ratios are 1–2 orders of magnitude lower in the PB than in the BM [137]. This reduced detection limit in PB may be compensated by the use of more frequent probe sampling and analysis. At least 5 ml BM and 10–20 ml PB should be analyzed per time points. Isolation of mononucleated cells is being done by a ficoll density gradient in standard analysis. 3.2.2. Technique of real-time PCR RQ-PCR permits accurate quantitation of PCR products during the exponential phase of the PCR amplification process, which is in full contrast to the classical PCR end point analysis (Fig. 6). The detection and acquisition of fluorescent signals during and/or after each subsequent PCR cycle

292

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

an acceptor fluorochrome at its 5 end. Both probes hybridize in close vicinity to the target sequence during the annealing phase of the PCR. Upon excitation of the donor fluorochrome, light with a longer wavelength will be emitted. The emitted light of the donor fluorochrome will excite the acceptor fluorochrome, a process referred to as fluorescence energy transfer.

Fig. 6. Exponential amplification during PCR. In a standard or nested PCR reaction the detection is performed after completion of the reaction when PCR has reached the plateau-phase (endpoint analysis). Real-time PCR allows the quantitative detection during the logarithmic amplification phase (log-phase analysis).

can be obtained during a short period of time. An additional advantage is that no post-PCR processing is needed, thereby reducing the risk of PCR product contamination. At present, three main types of RQ-PCR techniques are available (Fig. 7). In addition to these three main approaches that are described in detail below further types of probes are coming up, e.g. Molecular beacons [138], Scorpions [139], Minor groove binding probes [140], ResonSense, Hy-beacon, and Light-up probes [141]. 3.2.2.1. RQ-PCR using the hydrolysis probe format. RQPCR with the hydrolysis probe (“TaqMan probe”) format makes use of the 5 –3 exonuclease activity of the Thermus aquaticus (Taq) polymerase to detect and quantify the PCR product. The hydrolysis probe is positioned within the target sequence and is conjugated with a reporter fluorochrome at the 5 end and a quencher fluorochrome at the 3 end. The quencher avoids the reporter from emission of a fluorescence signal as long as the probe is intact and both fluorochromes are in close vicinity. Upon amplification of the target sequence, the hydrolysis probe is displaced from the DNA strand by the Taq polymerase and subsequently hydrolysed by the 5 –3 exonuclease activity of the Taq polymerase. This results in displacement of the reporter from the quencher. Consequently, the fluorescence of the reporter becomes detectable. During each consecutive PCR cycle, this fluorescence will increase because of the exponential accumulation of free reporter fluorochromes. 3.2.2.2. RQ-PCR using the hybridization probe format. RQPCR with the hybridization probe format makes use of two juxtaposed sequence-specific probes, one labelled with a donor fluorochrome at the 3 end and the other labelled with

3.2.2.3. RQ-PCR using SYBR Green Dye. This RQ-PCR method is based on the intercalation of a SYBR Green Dye. This dye can bind to the minor groove of double-stranded DNA, which greatly enhances its fluorescence. During the consecutive PCR cycles, the amount of double-stranded PCR product will exponentially increase, and therefore more SYBR Green Dye can bind and emit its fluorescence. The fluorescence signal has is maximum at the end of each extension phase were it is acquired. This is the simplest method of RQ-PCR but has its major drawbacks in the reduced specificity compared with hybridization and hydrolysis probe formats. With SYBR green also non-specifically amplified PCR products and primer dimers will be detected. To evaluate the specificity of the assay melting curve analysis can be performed. This allows the detection but not the elimination of unspecific signals. 3.2.3. Technical equipment for RQ-PCR At present seven different RQ-PCR instruments are commercially available. These are the ABI PRISM 7000, ABI PRISM 7700, ABI PRISM 7900 (Applied Biosystems), LightCycler (Roche), Smartcycler (Cepheid), i-Cycler (Biorad), and MX-4000 (Stratagene). Some of these are highly flexible with small numbers of sample positions but with high speed (LightCycler, Smartcycler), the others offer the possibility to process up to 96 or even 386 samples (ABI prism 7900) and have a lower speed. The choice of the appropriate machine will depend on the requirements of the user. Additional parameters that may play a role in the selection may be the light source, type of detection channels, and sample volume. The maximal sensitivity is more dependent on the choice of the assay than on the instrument type. In general, different RQ-PCR techniques can be applied on all available machines. A comparison of the TaqMan and LightCycler using hydrolysis probes for consensus regions of immunoglobulin (Ig) and T cell receptor (TCR) genes gave comparable results [142]. 3.2.4. PCR-targets for MRD detection Targets that are available for MRD detection in hematologic malignancies are, for example (1) rearrangements of Ig and/or TCR genes, (2) fusion-gene transcripts and (3) breakpoint regions around chromosomal or molecular rearrangements, and (4) aberrantly expressed genes. With the exception of aberrantly expressed genes most of these targets are highly specific and have no background signal in

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

293

Fig. 7. Schematic presentation of the three main types of RQ-PCR techniques. The four most relevant PCR phases (annealing; early, late, and end of extension) for fluorescence detection are depicted. Arrows indicate forward and reverse primers of the reaction. Yellow jagged arrows indicate the time point of fluorescence detection. By use of the hydrolysis and SYBR green format the detection is in the end of the annealing phase. By use of the hybridization probe format detection is performed in the annealing phase. R, reporter dye; Q, quencher dye; D, fluorescence donor dye; A, fluorescence acceptor dye; FRET, fluorescence resonance energy transfer.

normal cells. The applicability of these different targets varies per disease. Whereas the rearrangements of Ig and/or TCR genes played no role in AML, here the fusion transcripts where the most frequently applied targets so far. However, the use of breakpoint-specific assays and aberrantly expressed genes as targets for MRD detection in AML are of growing importance, especially to increase the number of cases to be monitorable by sensitive PCR techniques (see below, in new markers).

3.2.5. Quantification of a target Whereas with flow cytometry the real number of malignant cells in a population can be assessed, the QRT-PCR measures the mRNA expression level of an AML-specific gene relative to one or more other reference genes. The RNA gain per cell may greatly vary due to the time from sampling to RNA preparation, stabilisation of sample, cell count per microliter, and other factors. In addition, the cDNA yield may vary mainly due to differences in daily used enzyme lots

294

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Fig. 8. Quantification by real-time PCR. (a) Amplification of defined plasmid dilutions containing the target to be quantified. (b) Standard curve that is constructed from the plasmid dilution. (c) Limited dilution series of a fusion gene positive in a fusion gene negative sample, indicating the sensitivity of the assay. (d) Assessment of the ABL control gene in the same samples as depicted in “c”.

and inhibitors in the sample. To normalize for such quality variation each transcript to be quantified has to be compensated with the expression of one or more reference genes (Fig. 8). As reference gene usually so-called housekeeping genes are being used that are believed to be expressed at similar levels in all different cell types. An attractive feature of this implementation of reference genes in comparison to standard RT-PCR is that the final sensitivity of each individual assay can be documented and poor quality samples can be eliminated. It is still a matter of debate which housekeeping gene is the most stable and most suitable one in AML. The Europe Against Cancer (EAC) program initiated a comparison of 14 different potential control genes. Based on the absence of pseudogenes and the level and stability of expression, three genes were finally selected: Abelson (ABL), beta-2-micoglobulin (B2M), and beta-glucuronidase (GUS) [143]. Out of these three, ABL was the only one that did not differ significantly between normal and leukemic samples at diagnosis and therefore was proposed to be used as reference in leukemic patients. In addition, in a different single centre study ABL was reported to be the best out of four different reference genes for AML1-ETO-positive AML [144,145]. In conclusion, the use of control genes for QRT-PCR detection of aberrantly expressed genes or fusion genes in leukemic patients is mandatory for different reasons: (i) to evaluate

the RNA quality and quantity (degradation or presence of an inhibitor); (ii) to correct for such variations; (iii) to exclude poor quality samples; (iv) to calculate theoretical sensitivity of the transcript detection. 3.2.6. Quality control and standardization of QRT-PCR One of the major drawbacks of QRT-PCR is the incomparability of the fusion gene/housekeeping gene ratios assessed in different laboratories. Variables that may influence these ratios are many pre-PCR and PCR options that are handled differently between laboratories. The most important pre-PCR variables are the kind of sample (blood or bone marrow), stabilization of the sample during shipment, ficoll separation or lysis of sample, method of RNA preparation, use of total RNA or mRNA, and method of cDNA synthesis especially with respect to cDNA priming (random, oligo-dT, or gene-specific priming). Variables during PCR are selection of primers and probes, selection of puffer, enzymes and nucleotides, selection of PCR machine, selection of labelling with hydrolysis probes, hybridization probes or SYBR green, and selection of standard-curve or

CT method. As has been suggested the interpretation of QRT-PCR needs standardization and for reporting of MRD data international uniformity is highly desirable [146,147]. Several European networks have now been established and common guidelines

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

for data analysis are being developed (e.g. Europe Against Cancer, European Leukemia Network). These networks also include standardization of technology as well as regular quality control rounds, both being essential for the introduction of QRT-PCR-based MRD detection in multicentre clinical treatment protocols. 3.3. Quantification of fusion genes in AML The PML-RARA, AML1-ETO, and CBFB-MYH11 fusion genes are the three most common ones in AML. All are associated with a favorable subtype of AML [81,82,103]. However, even in these subtypes 10–30% of all patients are prone to treatment failure and early identification of these high risk patients is important to adjust treatment strategies [104]. A number of studies dealing with the quantification of these fusion transcripts have shown the importance of MRD detection in these AML subtypes [75–79,135,148,149]. Also the value of MLL-AF9 [150] and other MLL-Fusiontranscripts [151] already has been shown in single studies. 3.3.1. Transcript ratios at diagnosis Like for all MRD analyses the first time point to measure the expression of a fusion gene must be the primary diagnosis of an AML before application of any therapy. This is necessary primarily to assess the molecular subtype of the fusion gene as this may vary tremendously at the molecular level (Fig. 9). For the different molecular subtypes different assays should be applied to guarantee for maximum sensitivity. In addition, this value is mandatory as the basis value for all following MRD time points. It has been shown in a number of studies using different housekeeping genes that transcript ratios may vary up to two orders of magnitude at diagnosis [75,76,80,148,149]. Most studies did not find any prognostic impact of the level of these initial ratios at diagnosis [80,148,149]. On the basis 14 patients studied in a further study it was suggested that high transcript rates in CBFB-MYH11-positive AML correlate with high blast count and shorter survival [75]. A recent large study from our group based on 349 patients for the first time showed on large numbers of AML1-ETO-, CBFBMYH11-, and PML-RARA-positive patients that transcription levels at diagnosis are strongly correlated with clinical outcome in all three AML subtypes [76] (Fig. 10). With a range of 2.5 orders of magnitude these variations were most prominent in AML1-ETO-positive cases. The percentage of blast cells within the bone marrow (15–90%; median: 70%) or the percentage of blast cells type I and II carrying the respective fusion genes as determined by FISH (52–99%; median: 90%) could not account for the high variation of expression levels which can only be explained by high differences in expression activity of the malignant cells between individual patients. The expression ratios of fusion genes (normalized to ABL as control gene) as a continuous variable in all three AML subtypes were shown to have a significant impact on overall survival as well as on event-free survival. In contrast

295

to another study [75], in this study, the initial ratio had no impact on CR rates, which may be more substantiated due to the higher samples number. By separation of the patients into two groups (high and low expressers) significant differences in the EFS was found for all three AML subgroups while differences in the OS were found only in the AML1-ETO group. This is most probably due to effective salvage therapies in the other two groups but may also due to the median being not the optimum separator to identify clinically relevant differences. In multivariate analyses (variables: age, blast percentage, absolute blast percentage, leukocyte counts, platelet counts, FISH positivity, secondary etiology, M3/M3v, fusion type) the transcript levels as a continuous variable were shown to be the only significant parameter for OS and EFS. Response rates were not affected by initial transcript levels, which most probably was due to the overall high complete remission rates in these three subtypes of AML. The only other parameter carrying prognostic importance was the kind of transcript in PMLRARA- and CBFB-MYH11-positive AML. In PML-RARApositive AML bcr3 was associated with a worse prognosis than bcr1, although this association was not independent but rather reflected the correlation between M3v and high leukocyte counts. Similarly, in AML M4eo the rare CBFB-MYH11 fusion type was correlated to secondary etiology but not independently to a worse prognosis. 3.3.2. Transcription reduction during therapy and its impact on prognosis For childhood ALL, it was shown that the level of MRD at 5 weeks after induction therapy [69] and even at day 15 [68] was highly correlated with risk of relapse. In contrast, in AML the kinetics of reduction of the transcript ratio after the first and second induction therapy was found to have no impact on OS or EFS whereas in the subgroup of patients with transcription ratios at diagnosis of less than the 75%-percentile the transcript level after consolidation treatment had impact on EFS and OS [76] (Fig. 11). Interestingly, while in AML in general early response to treatment as assessed by cytomorphology is of major prognostic impact this is not true for AML with AML1-ETO and CBFB-MYH11 [152–154]. These data suggest that early assessment of MRD in these AML subtypes may not have prognostic impact due to the overall very good response to therapy and to a rather slow decline of the leukemia cell mass. This is in accordance with a recent paper which shows the significance of transcription levels at later time points in patients who achieved CR in CBFBMYH11 positive AML [78]. Krauter et al. [80] suggested a minimum of 2 log reduction directly after induction therapy to be a prerequisite for ongoing remission. They suggest to consider CBF patients with less than 2 log reduction during ongoing therapy as high risk patients. Also Guerrasio et al. [78] found significantly higher CBFB-MYH11 transcript numbers in patients in CR that later relapsed compared to patients in ongoing CR. In this study, all patients with CCR ultimately obtained PCR negativity and the authors suggested

296

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Fig. 9. Molecular fusion subtypes. Molecular fusion subtypes of (a) PML-RARA and (b) CBFB-MYH11 due to different exon usage.

that the “cure” of M4eo is strictly associated with CBFBMYH11 negativity. In addition, they defined a threshold of one CBFB-MYH11 copy/10,000 ABL copies in CR to discriminate high and low risk patients. In a further report focussing on inv(16)/CBFB-MYH11-positive AML the authors suggested based on a limited number of patients a critical transcript ratio of 0.25% at any time in remission to be predictive for relapse [77]. In a recent study on CBF leukemias that included

37 AML1-ETO- as well as CBFB-MYH11-positive AML a cut of level of 1% of the initial ratio at all time points after induction therapy discriminated two prognostically different groups [80]. For PML-RARA-positive AML it was shown that patients with lower than a 5 log reduction had a four-fold risk of relapse [79]. A further recent study showed that even lower levels than 0.25% have been detected in three patients after previous negativity all of whom subsequently relapsed

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

297

Fig. 10. Prognostic relevance of fusion gene expression levels at diagnosis. Overall survival (OS) and event-free survival (EFS) for patients separated according to the 75%-percentile of the expression level of the respective fusion transcript (PML-RARA, AML1-ETO, CBFB-MYH11) at diagnosis. Patients with a lower level (blue lines) have a better prognosis than those with a higher level (red lines).

shortly thereafter [76]. Therefore, the different thresholds defined by different groups may be too insensitive and due to the high interpatient variability of transcript levels general cut-off levels may not be appropriate. It seems more informative to monitor the kinetics in single patients and to use an increase of transcript levels as predictor for relapse. Due to very variable time points of MRD testing that were applied in different studies the most predictive time point and the time span between follow-up assessments are still unknown and must be defined in prospective and multicentre trials.

Fig. 11. Prognostic impact of MRD after consolidation therapy as quantified by quantitative RT-PCR. Patients with MRD levels after consolidation therapy lower than the median MRD level after consolidation therapy (blue line) have a better prognosis than patients with MRD levels higher than the median (red line).

3.3.3. New score for risk stratification of AML Based on a multicentre study (central reference laboratory) with 131 patients (34 with AML1-ETO, 42 with CBFBMYH11, and 55 with PML-RARA) evaluated at diagnosis as well as after consolidation therapy a prognostic score was established by combining the transcript ratio of both checkpoints [76]. In all three AML subtypes a good risk group was identified by a transcription level at diagnosis of less than the 75%-percentile and an MRD level of less than the median in which no case with treatment failure was observed. In contrast, patients with either a higher transcription level at diagnosis or a higher MRD level after consolidation therapy did significantly worse (Fig. 12). 3.3.4. Prediction of relapse In a limited number of cases, a molecular relapse was demonstrated to precede the hematologic relapse by some weeks. For example, in a study reported by Krauter et al. [80] in 7/10 patients who relapsed, a good initial reduction was observed and some even obtained PCR negativity. In these patients, an increase in MRD levels was observed in CR after the end of therapy. This confirms the study by Marcucci et al. [75], who found low CBFB-MYH11 copy numbers during intensive chemotherapy whereas relapse was preceded by an increase of the fusion gene expression. The largest study focussing on the predictability of relapse by an increase of a fusion gene detectable by PCR analyzed 142 patients at three or more different time points during and after therapy [76]. Fifteen of these cases (10.6%) relapsed. In eight patients, increasing transcript ratios indicated molecular relapses 1–5 months before hematologic and cytogenetic relapses occurred. It was shown that an increase even from

298

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Fig. 12. Combined prognostic impact of expression levels at diagnosis and MRD levels. Patients with both a low transcript level at diagnosis and a low MRD level after consolidation therapy (blue lines) have a better prognosis than patients not fulfilling both criteria (red lines).

the undetectable to a marginal transcription level significantly predicts relapse. Stressing the importance of small followup intervals, in all seven relapsing patients with follow-up intervals larger than 6 months relapses were not detected beforehand by PCR. As in all eight cases with follow-up intervals of 3 months or less a clinical relapse was predictable by increasing PCR-levels, follow-up sampling at least every 3 months is mandatory to predict relapses at the molecular basis, at least during the first year of follow-up. In addition, it could be shown that higher levels at the time of increase during follow-up are correlated with a more rapid manifestation of the clinical relapse. Whereas the most feasible follow-up time points for risk assessment are still under debate, it has to be challenged to do follow-up assessments at least every 3 months since in all patients monitored at 3 months intervals an increasing MRD level was detectable before occurrence of relapse but not in those with larger follow-up time intervals [75,76,80]. In conclusion, the different studies discussed above are relatively incomparable because different AML subgroups were analyzed, different therapies were applied, different time points during and after therapy were analyzed, different housekeeping genes were used for normalization, and different kinds of threshold levels for risk assessment were defined. To define overall MRD risk parameters standardization of PCR based methods, housekeeping genes, and calculation methods have to be defined [143,146,147] and prospective as well as multicentre analyses have to be performed.

3.4. New targets for PCR-based MRD detection So far, the application of PCR to quantify MRD was limited on the presence of fusion genes or their respective fusion transcripts, which occur exclusively in the respective subentities of AML but not in normal bone marrow. Thus, in AML with normal karyotype or non-reciprocal chromosome aberration the application of PCR-based MRD detection was not possible. In AML with normal karyotype or other prognostically intermediate aberrations like trisomies 8 or 11 and del(9q) two molecular mutations, the length mutations within the FLT3-gene (FLT3-LM) and partial tandem duplications of the MLL gene (MLL-PTD) can frequently be detected and are promising markers for MRD detection. In addition, surrogate markers like aberrant expression of the WT1 gene or the EVI1 gene have been used in a limited number of studies. Thus, the portion of cases assessable to sensitive PCR based detection may now be widely extended (Table 3). 3.4.1. FLT3-LM as marker for MRD FLT3-LM can be detected in about 20–25% of all AML, and thus is the most frequent genetic marker in AML [155–158] (Fig. 13). As the frequency of this mutation with approximately 40% is especially high in AML with normal karyotype or AML with other prognostically intermediate aberrations like trisomies 8 or 11 and del(9q), i.e. AML subgroups in which PCR was not applicable due to the lack of fusion genes, it was suggested to use this marker as a

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

299

Table 3 Applicability of different PCR-markers for MRD detection PCR-marker

Positivity in newly diagnosed AML (%)

PML-RARA AML1-ETO CBFB-MYH11 MLL-AF6 MLL-AF9 MLL-AF10 MLL-ENL MLL-ELL MLL-PTD FLT3-LM WT1 EVI1

7–8 7–8 7–8

5

6 23 100 20

follow-up marker [159]. However, many studies reported on the instability of this marker during the course of the disease [160–163]. To more intensively evaluate FLT3-LM as follow-up marker a preliminary study was performed on 97 paired diagnostic/relapse samples and a total of 174 followup samples of 45 patients [164]. The sensitivity as estimated by limited dilution series of FLT3-LM positive patients RNA or DNA from the time of diagnosis in samples negative for the mutation was dependent on the strength of the initial mutation status and was shown to range between 1:100 and 1:1000. It was found that this marker was detectable in 93/97 (95.9%) of all paired diagnostic/relapse samples and was newly detected or could not be redetected in only 4/97 (4.1%) of all samples. This is in the range of other well characterized AML relapsing with a different geno- and/or phenotype [165]. In contrast, a

Fig. 13. Semiquantitative and quantitative analysis of FLT3-LM. (a) Semiqualitative conventional agarose gel electrophoresis. The wildtype allele (WT) is the smallest fragment. All that are larger than this fragment indicate FLT3-LM. Patients P1, P5, and P6 are positive for a FLT3-LM. P1 in addition has a loss of the WT-allele. –C, H2 O control; M, molecular weight marker. (b) Fragment analysis for quantification ot the FLT3-LM. In red the standard; in blue the FLT3-WT and the FLT3-LM.

Positivity in AML with normal and other intermediate karyotypes (%) – – –

Positivity in AML with complex aberrant karyotypes (%) – – –

–

10 40 100 10

–

2 2 100 10

change in the ratio of the mutated allele in comparison to the wildtype allele (WT) was observed frequently. In detail, the FLT3-LM showed a tendency to accumulate during disease progression and was found more frequently at relapse than at diagnosis. In addition, 45 patients were analyzed at different time points during and after therapy. Using conventional PCR it clearly could be shown that for most of the patients positive at presentation FLT3-LM is a reliable PCR marker for monitoring treatment response. Even early detection of relapse was possible in some cases. By a comparison of conventional one step PCR with FISH it was shown that for patients with fusion genes, fusion genespecific PCR or even FISH clearly are superior to FLT3-PCR. However, in cases with other aberrations like trisomy 8, 5q-, or 7q- FLT3-LM PCR seems to be a good and more reliable follow-up marker. FLT3-LM can be analyzed at the genomic as well as at the expression level. This offers the possibility to compare real cell counts with expression. Libura et al. [166] suggested to use RNA for more sensitive FLT3-LM detection. In contrast, Schnittger et al. [159] did not find any differences between both sample materials at diagnosis. However, in follow-up samples of patients in complete clinical remission, it was not always possible to obtain an amplification product of the wildtype FLT3 by PCR [164]. This is in accordance with previous studies that have shown FLT3 expression on early haematological and leukemic cells but not on differentiated haematological cells [167]. Thus, for follow-up controls DNA is highly recommended as diagnostic material. Because of these controversial results this issue needs further evaluation. In six cases, it was possible to detect FLT3-LM positivity after previous PCR-negativity 1–3 months before cytomorphological relapse. In additional four cases, a pending relapse was detectable by persistent PCR positivity of the FLT3-LM [164]. Partial or complete loss of the wildtype allele can be found in 17% at diagnosis and 84% at relapse [159]. This WT-loss was found to be of tremendous adverse prognosis [157,168,169]. Several studies have been published inves-

300

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

tigating FLT3 mutations in paired presentation and relapse samples [159–162,170]. In some of these studies, 7–15% of all AML revealed a gain of the FLT3-LM at relapse [161,162]. Like in the presented study many of the patients lost their WT at relapse. This accumulation of the FLT3-LM implies a role of FLT3-LM in leukemia progression and onset of relapse. Taking the results of these previous studies together [159–162,164,170] only 88% of the analyzed patients maintained the same FLT3 status (FLT3-LM positive or negative) at both time points. Consequently, as was suggested previously [163] that FLT3-LM should be regarded with caution with respect to its usefulness as follow-up marker. Patients that were positive for an FLT3-LM at presentation often showed an increased mutant level at relapse, usually with evidence for the loss of WT alleles. If more than one mutation had been detected at presentation, usually only one was dominant at relapse [159,161,162]. Thus, accumulation in the sense of progression seems to be the most common direction of the instability of the FLT3-LM and does not interfere with the applicability for MRD diagnostics. However, in some studies a significant proportion of patients either gained (8.3%) or lost (4%) a FLT3-LM at relapse, and some patients have been reported with loss of the presentation LM and gain of a completely new one at relapse [160–162]. Data from our group [164] suggest to regard in cases with only a low initial mutation ratio at diagnosis the results of follow-up analysis with special care. To make the FLT3-LM assessment truly semiquantitative, we suggest to use GeneScan analysis at diagnosis for better prognostification [157]. During follow-up this method also would improve the estimation of the reduction of the leukemic cells in comparison to standard PCR and gel electrophoresis. It could be shown that real-time quantification with patient-specific primers for individual FLT3-LM is applicable and highly specific and sensitive, however, it also is timeconsuming and expensive and therefore required reference laboratories to be carried out on a large-scale basis [171–173]. Using patient-specific primers, the sensitivity was increased to 1:100,000 compared to 1:1000 for the conventional PCR and GeneScan analysis [173] and is in the same range as sensitivity for fusion genes. Thus, in the future highly sensitive and quantitative PCR may still improve the use of FLT3-LM as follow-up marker. However, this approach is time consuming and expensive and for prospective assessment of the FLT3-LM in clinical studies it does not seem to be feasible for most of the diagnostic laboratories. As FLT3-LM characterizes an unfavourable subset of the intermediate group with an increased risk for relapse it is of high importance to monitor especially this group. As the marker is stable or even accumulating at relapse in 88% of AML carrying this marker at diagnosis the chance should not be missed to follow-up these patients. 3.4.2. MLL-PTD as marker for MRD detection The MLL-PTD can be detected at low levels in almost every PB or BM sample [174]. However, high expression that

can also be identified at the genomic level by Southern blot analyses indicative for a clone of at least 10% of total cells is specific for AML or high risk MDS [175–178]. The frequency of the MLL-PTD in unselected AML at diagnosis is 6.5% and in AML with normal karyotype it is 10% [175,179]. It has been shown by many study groups that this marker is indicative for an unfavourable outcome [175,179–181]. Thus, the MLL-PTD-positive AML are an especially interesting subgroup to be monitored during follow-up. A real-time PCR assay has been developed and applied to a high number of patients during follow-up. It is sensitive and quantitative and thus a very feasible marker for MRD studies that exactly reflects treatment response and early detects relapse [173] (Figs. 14 and 15). However, since the amount of naturally positive cells in healthy PB and BM ranges between 0.01% and 0.05%, the sensitivity is 1–2 orders of magnitude less that that of fusion genes and more in the range of MFC. 3.4.3. WT1 overexpression as marker for MRD detection The Wilms’s tumor gene (WT1) encodes a zinc-finger transcription factor that functions as a potent transcriptional repressor of several growth factors [182]. Its expression is strongly regulated in a time- and tissue-specific manner. In AML, it is overexpressed in almost all cases. It is thought to play a role in maintaining the viability of leukemic cells [183–185]. Overexpression of WT1 therefore can be regarded as a specific feature of the malignant cells and consequently can be used as a PCR target to detect MRD [186–194]. The sensitivity that can be reached with WT1 as a target was reported to be up to 1 cell in 10,000 [193]. This marker is applicable in almost all AML. However, as the attainable sensitivity is approximately two orders of magnitude lower than that of fusion genes this WT1 should be used only in cases that lack one of these markers. In addition, caution is indicated when interpreting the results obtained by this marker since presently available data are discrepant to each other [195]. 3.4.4. EVI1 overexpression as marker for MRD detection Ectopic expression of the EVI1 gene in AML has been associated with rearrangements of 3q26, the chromosomal location where EVI1 resides [196–198]. However, it has previously been shown that EVI1 expression is not strictly correlated with 3q26 rearrangements [199,200]. Recently it could be shown that in patients with initially high expression of EVI1 at diagnosis this marker was applicable as a follow-up marker [201]. The sensitivity for this assay was estimated by limited dilution series of highly expressing samples and was up to 1 in 10,000 cells. The analysis of 270 newly diagnosed AML samples of 13 defined cytogenetic subgroups showed that AML can be divided in three different subtypes with respect to EVI1 expression (Fig. 16) [201]. High expressers are all subtypes with EVI1 activation due to breakpoints in 3q26: inv(3)/t(3;3); t(3;12)/ETV6-EVI1, and t(3;21)/AML1-EVI1. Also the “other 3q-aberrations” (except t(1;3)(p36;q21) have

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

301

Fig. 14. Dilution series of MLL-PTD positive AMLs in normal control samples. (a) exon9/exon3 fusion type and (b) exon11/exon3 fusion type. The sensitivity is 1: 1000 to 1:10,000. In the highly diluted samples, the MLL-PTD expression is like in normal controls.

an elevated median level of EVI1-expression, indicating that also in less well characterized 3q aberrations EVI1 expression is accelerated by the chromosomal rearrangement. Lowexpressers are all reciprocal translocations: t(8;21), inv(16), t(15;17), t(1;3)(p36;q21), and all others. The third group is a mixed group, in which high, low, and intermediate EVI1expression levels can be found. These are AML cases with t(11q23)/MLL, monosomy 7, normal, and complex aberrant karyotypes. Out of the cytogenetic subgroups with mixed EVI1 expression (normal, complex aberrant, t(11q23)/MLL,

-7) 166 were uniformly treated within the AMLCG study. Cases with more than the median expression ratio a shorter OS (p = 0.051) was found, indicating that EVI1 expression at diagnosis is of prognostic significance. Based on the analysis of 319 patients Barjesteh et al. [202] also reported poor survival in patients with high initial EVI1 expression. Follow-up data were reported on 147 samples of 36 individual patients [201]. The EVI1 expression between days 21 and 60 after start of therapy was shown to be predictive for OS, EFS, and RFS (p = 0.049, 0.049, and 0.029, respectively). As high EVI1 expression can be found in approximately 20% of all unselected AML cases and predominate in AML without reciprocal translocation this marker may add a considerable subset of AML patients capable of being monitored by QRTPCR. Additional studies are needed, however, to delineate the exact value of this marker.

4. Comparison of multiparameter flow cytometry and quantitative RT-PCR

Fig. 15. Examples of follow-up with MLL-PTD in three patients. In pink, a resistant patient. In blue, a case with bad response to standard chemotherapy that underwent bone marrow transplantation. In yellow, a good responder.

Given the powerful applicability of both MFC-based and QRT-PCR-based quantifications of MRD to estimate the patients prognosis it becomes increasingly important to assess the respective value and superiority of either method

302

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

Fig. 16. Scattergram of EVI1 expression in different cytogenetic AML subtypes. Scattergram of EVI1 expression in different AML subtypes.

for distinct clinical settings. However, data published on this issue are scarce. Preliminary data from our group indicate that for the majority of cases both methods reveal comparable results [203]. Thus, out of 372 follow-up assessments from 144 patients with AML analyzed by both methods in parallel both at diagnosis and at follow-up 61% were found to give concordant results with regard to positivity and negativity, respectively. Another 19% were found positive by QRT-PCR and negative by MFC and 20% vice versa demonstrating differences in the sensitivity between both methods for different targets. QRT-PCR was superior to MFC in most cases with CBF leukemias due to the very good sensitivity of the fusion transcripts CBFB-MYH11 and AML1-ETO. Conversely, MFC was superior to QRT-PCR in many cases with acute promyelocytic leukemia due to the highly sensitive LAIP HLA-DR−CD33+CD34+ which, however, occurs in only 20% of all cases with acute promyelocytic leukemia. With regard to the quantitative comparison of data obtained by both methods there were also highly significant correlations (Fig. 17), which is also true for the comparison of the courses of MRD as determined by both methods in individual patients (Fig. 18). The performed evaluations in addition indicate that MRD levels determined at different checkpoints by both methods correlate with the prognosis, however, additional analyses are needed to get conclusive results on the topic which method to apply at which checkpoint for which subentity of AML. Overall, QRT-PCR due to the high sensitivity is anticipated to be most important in CBF leukemias while MFC due to its applicability to virtually all patients with

AML in anticipated to be more important in the remaining cases. Additional studies will have to be analyzed to substantiate these data [59]. It should be aimed to consider and include into these analyses also technical aspects like the stability of RNA during transportation with regard to QRT-PCR analysis and like the application of flow cytometric markers

Fig. 17. Correlation of MRD quantification as determined by MFC and QRTPCR in AML cases with t(11q23). The logarithmic difference between measurements at diagnosis and at follow-up for both multiparameter flow cytometry (FC log-difference) and quantitative RT-PCR (PCR log-difference) are plotted against each other. Pearson correlation (r = 0.719, p = 0.003) indicates a very good quantitative agreement between both methods.

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

303

Fig. 18. Correlation of courses of MRD. MRD monitoring by flow cytometry and PCR. Green lines show measurements by multiparameter flow cytometry, red lines show measurements by quantitative RT-PCR. The respective markers are given.

with a limited sensitivity. Clearly, approaching these issues should be led by the search for the clinically most informative and therefore most useful application in order to help optimizing the therapeutic management of patients with AML.

5. Conclusions Based on published data it is anticipated that the quantification of MRD will significantly improve the estimation of the prognosis in patients with AML and as a consequence will play a major role as a stratification parameter to guide the risk-adapted therapy of the disease. In cases with CBF leukemia, i.e. AML with t(8;21) and inv(16)/t(16;16), QRTPCR is more sensitive as compared to MFC, and thus will be used for MRD quantification preferably in these cases. In contrast, MFC offers an applicability with a clinically relevant sensitivity to virtually all patients with AML, and thus is considered an ideal tool for the use of MRD quantification in large AML populations including all subtypes of the disease. Technical improvement like the use of five or more colors in MFC and the identification of additional leukemia-specific genetic targets which can be quantified by QRT-PCR will lead to a further increase of the applicability and sensitivity of both methods. Future studies will define which methods at which checkpoint is most useful for the management of patients with AML.

Reviewers Jesus F. Miguel, Professor, Head, Department of Hematology, University Hospital of Salamanca, Paseo de San Vicente, 58, 37007 Salamanca, Spain. Dairo Campana, M.D., Ph.D., Member, Hematology– Oncology and Pathology, St. Jude Children’s Research Hospital, Professor of Pediatrics, University of Tennessee College of Medicine, 332 N. Lauderdale, Memphis, TN 38105, USA.

References [1] Buchner T, Hiddemann W, Wormann B, et al. Double induction strategy for acute myeloid leukemia: the effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine: a randomized trial by the German AML Cooperative Group. Blood 1999;93:4116–24. [2] Kern W, Haferlach T, Schoch C, et al. Risk-adapted therapy of AML: the AMLCG experience. Ann Hematol 2004;83:59–61. [3] Lowenberg B, van Putten W, Theobald M, et al. Effect of priming with granulocyte colony-stimulating factor on the outcome of chemotherapy for acute myeloid leukemia. N Engl J Med 2003;349:743–52. [4] Kern W, Estey EH. High-dose cytosine arabinos ide in induction treatment of acute myeloid leukemia: meta-analysis of three trials involving 1691 randomized patients. Blood 2002;100:155a. [5] Fopp M, Fey MF, Bacchi M, et al. Post-remission therapy of adult acute myeloid leukaemia: one cycle of high-dose versus standarddose cytarabine. Leukaemia Project Group of the Swiss Group for Clinical Cancer Research (SAKK). Ann Oncol 1997;8:251–7.

304

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309

[6] Mayer RJ, Davis RB, Schiffer CA, et al. Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B. N Engl J Med 1994;331:896–903. [7] Ohno R, Kobayashi T, Tanimoto M, et al. Randomized study of individualized induction therapy with or without vincristine, and of maintenance-intensification therapy between 4 or 12 courses in adult acute myeloid leukemia. AML-87 Study of the Japan Adult Leukemia Study Group. Cancer 1993;71:3888–95. [8] Cassileth PA, Lynch E, Hines JD, et al. Varying intensity of postremission therapy in acute myeloid leukemia. Blood 1992;79:1924–30. [9] Buchner T, Urbanitz D, Hiddemann W, et al. Intensified induction and consolidation with or without maintenance chemotherapy for acute myeloid leukemia (AML): two multicenter studies of the German AML Cooperative Group. J Clin Oncol 1985;3: 1583–9. [10] Rai KR, Holland JF, Glidewell OJ, et al. Treatment of acute myelocytic leukemia: a study by cancer and leukemia group B. Blood 1981;58:1203–12. [11] Burnett AK, Goldstone AH, Stevens RM, et al. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukaemia in first remission: results of MRC AML 10 trial. UK Medical Research Council Adult and Children’s Leukaemia Working Parties. Lancet 1998;351:700–8. [12] Zittoun RA, Mandelli F, Willemze R, et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) Leukemia Cooperative Groups. N Engl J Med 1995;332:217–23 [see comments]. [13] Cassileth PA, Harrington DP, Appelbaum FR, et al. Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission. N Engl J Med 1998;339:1649–56 [see comments]. [14] Harousseau JL, Cahn JY, Pignon B, et al. Comparison of autologous bone marrow transplantation and intensive chemotherapy as postremission therapy in adult acute myeloid leukemia. The Groupe Ouest Est Leucemies Aigues Myeloblastiques (GOELAM). Blood 1997;90:2978–86. [15] Campana D, Pui CH. Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 1995;85:1416–34. [16] Adriaansen HJ, Jacobs BC, Kappers-Klunne MC, Hahlen K, Hooijkaas H, van Dongen JJ. Detection of residual disease in AML patients by use of double immunological marker analysis for terminal deoxynucleotidyl transferase and myeloid markers. Leukemia 1993;7:472–81. [17] Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003;21:4642–9. [18] Schoch C, Haferlach T. Cytogenetics in acute myeloid leukemia. Curr Oncol Rep 2002;4:390–7. [19] Schoch C, Kern W, Schnittger S, Hiddemann W, Haferlach T. Karyotype is an independent prognostic parameter in therapy-related acute myeloid leukemia (t-AML): an analysis of 93 patients with tAML in comparison to 1091 patients with de novo AML. Leukemia 2004;18:120–5. [20] Haferlach T, Schoch C, Loffler H, et al. Morphologic dysplasia in de novo acute myeloid leukemia (AML) is related to unfavorable cytogenetics but has no independent prognostic relevance under the conditions of intensive induction therapy: results of a multiparameter analysis from the German AML Cooperative Group studies. J Clin Oncol 2003;21:256–65.

[21] Schoch C, Schnittger S, Klaus M, Kern W, Hiddemann W, Haferlach T. AML with 11q23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood 2003;102:2395–402. [22] Schoch C, Haferlach T, Haase D, et al. Patients with de novo acute myeloid leukaemia and complex karyotype aberrations show a poor prognosis despite intensive treatment: a study of 90 patients. Br J Haematol 2001;112:118–26. [23] Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998;92:2322–33. [24] Kern W, Haferlach T, Schoch C, et al. Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 Trial. Blood 2003;101:64– 70. [25] Estey EH, Shen Y, Thall PF. Effect of time to complete remission on subsequent survival and disease-free survival time in AML, RAEB-t, and RAEB. Blood 2000;95:72–7. [26] Wheatley K, Burnett AK, Goldstone AH, et al. A simple, robust, validated and highly predictive index for the determination of riskdirected therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council’s Adult and Childhood Leukaemia Working Parties. Br J Haematol 1999;107:69–79. [27] Kern W, Schnittger S. Monitoring of AML by flow cytometry. Curr Oncol Rep 2003;5:405–12. [28] Lucio P, Parreira A, van den Beemd MW, et al. Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL. Leukemia 1999;13:419–27. [29] Terstappen LW, Huang S, Picker LJ. Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood 1992;79:666–77. [30] Terstappen LW, Loken MR. Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multidimensional flow cytometry. Anal Cell Pathol 1990;2:229–40. [31] Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. I. Normal erythroid development. Blood 1987;69:255–63. [32] Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development. Blood 1987;70:1316–24. [33] Terstappen LW, Huang S, Safford M, Lansdorp PM, Loken MR. Sequential generations of haematopoietic colonies derived from single nonlineage-committed CD34+. Blood 1991;77:1218–27. [34] Macedo A, Orfao A, Ciudad J, et al. Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease. Leukemia 1995;9:1896–901. [35] Smith LJ, Curtis JE, Messner HA, Senn JS, Furthmayr H, McCulloch EA. Lineage infidelity in acute leukemia. Blood 1983;61:1138–45. [36] Campana D, Coustan-Smith E, Behm FG. The definition of remission in acute leukemia with immunologic techniques. Bone Marrow Transplant 1991;8:429–37. [37] Drach D, Zhao S, Drach J, et al. Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood 1992;80:2729–34 [see comments]. [38] Venditti A, Buccisano F, Del Poeta G, et al. Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 2000;96:3948–52.

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309 [39] San Miguel JF, Vidriales MB, Lopez-Berges C, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood 2001;98:1746–51. [40] San Miguel JF, Martinez A, Macedo A, et al. Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients. Blood 1997;90:2465–70. [41] Kern W, Danhauser-Riedl S, Ratei R, et al. Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry to define leukemia-associated immunophenotypes and determine their frequencies in normal bone marrow. Haematologica 2003;88:646–53. [42] Kern W, Voskova D, Schnittger S, Schoch C, Hiddemann W, Haferlach T. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in patients with acute myeloid leukemia. Blood 2003;102:100a. [43] Kern W, Voskova D, Schoch C, Schnittger S, Hiddemann W, Haferlach T. Prognostic impact of early response to induction therapy as assessed by multiparameter flow cytometry in acute myeloid leukemia. Blood 2003;102:876a. [44] Kern W, Schoch C, Hiddemann W. Prognostic significance of cytogenetics in relapsed acute myeloid leukaemia. Br J Haematol 2000;109:671–2. [45] Kantarjian HM, Keating MJ, Walters RS, McCredie KB, Freireich EJ. The characteristics and outcome of patients with late relapse acute myelogenous leukemia. J Clin Oncol 1988;6:232–8. [46] Oelschlagel U, Nowak R, Schaub A, et al. Shift of aberrant antigen expression at relapse or at treatment failure in acute leukemia. Cytometry 2000;42:247–53. [47] Macedo A, San Miguel JF, Vidriales MB, et al. Phenotypic changes in acute myeloid leukaemia: implications in the detection of minimal residual disease. J Clin Pathol 1996;49:15–8. [48] Reading CL, Estey EH, Huh YO, et al. Expression of unusual immunophenotype combinations in acute myelogenous leukemia. Blood 1993;81:3083–90. [49] Thomas X, Campos L, Archimbaud E, et al. Surface marker expression in acute myeloid leukaemia at first relapse. Br J Haematol 1992;81:40–4. [50] Campana D, Yokota S, Coustan-Smith E, Hansen-Hagge TE, Janossy G, Bartram CR. The detection of residual acute lymphoblastic leukemia cells with immunologic methods and polymerase chain reaction: a comparative study. Leukemia 1990;4:609–14. [51] Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999;38:139–52. [52] Coustan-Smith E, Behm FG, Hurwitz CA, Rivera GK, Campana D. N-CAM (CD56) expression by CD34+ malignant myeloblasts has implications for minimal residual disease detection in acute myeloid leukemia. Leukemia 1993;7:853–8. [53] Nakamura K, Ogata K, An E, Dan K. Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia. Br J Haematol 2000;108:710–6. [54] Ciudad J, San Miguel JF, Lopez-Berges MC, et al. Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 1998;16:3774–81. [55] Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 1998;351:550–4. [56] Roberts WM, Estrov Z, Ouspenskaia MV, Johnston DA, McClain KL, Zipf TF. Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N Engl J Med 1997;336:317–23.

305

[57] Coustan-Smith E, Sancho J, Behm FG, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood 2002;100:52–8. [58] Sievers EL, Lange BJ, Buckley JD, et al. Prediction of relapse of pediatric acute myeloid leukemia by use of multidimensional flow cytometry. J Natl Cancer Inst 1996;88:1483–8. [59] Coustan-Smith E, Ribeiro RC, Rubnitz JE, et al. Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia. Br J Haematol 2003;123:243–52. [60] Sievers EL, Lange BJ, Alonzo TA, et al. Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children’s Cancer Group study of 252 patients with acute myeloid leukemia. Blood 2003;101:3398–406. [61] Shah VO, Civin CI, Loken MR. Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis. J Immunol 1988;140:1861–7. [62] Borowitz MJ, Guenther KL, Shults KE, Stelzer GT. Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three- color analysis. Am J Clin Pathol 1993;100:534–40. [63] Gelman R, Wilkening C. Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%. Cytometry 2000;42:1–4. [64] Rainer RO, Hodges L, Seltzer GT. CD 45 gating correlates with bone marrow differential. Cytometry 1995;22:139–45. [65] Roederer M, De Rosa S, Gerstein R, et al. 8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 1997;29:328–39. [66] Mensink E, van de LA, Schattenberg A, et al. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. Br J Haematol 1998;102:768–74. [67] Hochhaus A, Weisser A, La Rosee P, et al. Detection and quantification of residual disease in chronic myelogenous leukemia. Leukemia 2000;14:998–1005. [68] Panzer-Grumayer ER, Schneider M, Panzer S, Fasching K, Gadner H. Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia. Blood 2000;95:790–4. [69] Brisco MJ, Condon J, Hughes E, et al. Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction. Lancet 1994;343:196– 200. [70] van Dongen JJM, Seriu T, Panzer-Gr¨unmayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998;352:1731–8. [71] Szczepanski T, Orfao A, van der Velden V, San Miguel JF, van Dongen JJ. Minimal residual disease in leukaemia patients. Lancet Oncol 2001;2:409–17. [72] Schultze JL, Gribben JG. Minimal residual disease in nonHodgkin’s lymphoma. Biomed Pharmacother 1996;50:451–8. [73] Anderson KC. Novel biologically based therapies for myeloma. Cancer J 2001;7(Suppl. 1):S19–23. [74] van Dongen JJ, Seriu T, Panzer-Grumayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998;352:1731–8. [75] Marcucci G, Caligiuri MA, Dohner H, et al. Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 2001;15:1072–80. [76] Schnittger S, Weisser M, Schoch C, Hiddemann W, Haferlach T, Kern W. New score predicting for prognosis in PMLRARA-, AML1-ETO-, or CBFB-MYH11-positive acute myeloid leukemia based on quantification of fusion transcripts. Blood 2003;102:2746–55. [77] Buonamici S, Ottaviani E, Testoni N, et al. Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid

306

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309 leukemia may indicate risk for clinical relapse and may identify patients in a curable state. Blood 2002;99:443–9. Guerrasio A, Pilatrino C, De Micheli D, et al. Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts. Leukemia 2002;16:1176–81. Gallagher RE, Yeap BY, Bi W, et al. Quantitative real-time RTPCR analysis of PML-RAR{alpha} mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood 2003;101:2521–8. Krauter J, Gorlich K, Ottmann O, et al. Prognostic value of minimal residual disease quantification by real-time reverse transcriptase polymerase chain reaction in patients with core binding factor leukemias. J Clin Oncol 2003;21:4413–22. Grimwade D, Walker H, Harrison G, et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 2001;98:1312–20. Mrozek K, Heinonen K, Bloomfield CD. Clinical importance of cytogenetics in acute myeloid leukaemia. Best Pract Res Clin Haematol 2001;14:19–47. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325–36. Plantier I, Lai JL, Wattel E, Bauters F, Fenaux P. Inv(16) may be one of the only ‘favorable’ factors in acute myeloid leukemia: a report on 19 cases with prolonged follow-up. Leuk Res 1994;18:885–8. Larson RA, Williams SF, Le Beau MM, Bitter MA, Vardiman JW, Rowley JD. Acute myelomonocytic leukemia with abnormal eosinophils and inv(16) or t(16;16) has a favorable prognosis. Blood 1986;68:1242–9. Martinelli G, Ottaviani E, Testoni N, et al. Molecular remission in PCR-positive acute myeloid leukemia patients with inv(16): role of bone marrow transplantation procedures. Bone Marrow Transplant 1999;24:694–7. Claxton DF, Liu P, Hsu HB, et al. Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia. Blood 1994;83:1750–6. van Dongen JJ, Macintyre EA, Gabert JA, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:1901–28. Miyamoto T, Nagafuji K, Harada M, Niho Y. Significance of quantitative analysis of AML1/ETO transcripts in peripheral blood stem cells from t(8;21) acute myelogenous leukemia. Leuk Lymphoma 1997;25:69–75. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalphapositive acute promyelocytic leukemia. PETHEMA group. Blood 1999;94:3015–21. Avvisati G, Lo Coco F, Diverio D, et al. AIDA (all-trans retinoic acid + idarubicin) in newly diagnosed acute promyelocytic leukemia: a Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) pilot study. Blood 1996;88:1390–8. Perego RA, Marenco P, Bianchi C, et al. PML/RAR alpha transcripts monitored by polymerase chain reaction in acute promyelocytic leukemia during complete remission, relapse and after bone marrow transplantation. Leukemia 1996;10:207–12. Koller E, Karlic H, Krieger O, et al. Early detection of minimal residual disease by reverse transcriptase polymerase chain reaction

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

predicts relapse in acute promyelocytic leukemia. Ann Hematol 1995;70:75–8. Korninger L, Knobl P, Laczika K, et al. PML-RAR alpha PCR positivity in the bone marrow of patients with APL precedes haematological relapse by 2–3 months. Br J Haematol 1994;88:427–31. Laczika K, Mitterbauer G, Korninger L, et al. Rapid achievement of PML-RAR alpha polymerase chain reaction (PCR)-negativity by combined treatment with all-trans-retinoic acid and chemotherapy in acute promyelocytic leukemia: a pilot study. Leukemia 1994;8:1–5. Diverio D, Pandolfi PP, Biondi A, et al. Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long-term remission. Blood 1993;82:3556–9. Huang W, Sun GL, Li XS, et al. Acute promyelocytic leukemia: clinical relevance of two major PML-RAR alpha isoforms and detection of minimal residual disease by retrotranscriptase/polymerase chain reaction to predict relapse. Blood 1993;82:1264–9. Miller Jr WH, Levine K, DeBlasio A, Frankel SR, Dmitrovsky E, Warrell Jr RP. Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA. Blood 1993;82:1689–94. Jurcic JG, Nimer SD, Scheinberg DA, DeBlasio T, Warrell Jr RP, Miller Jr WH. Prognostic significance of minimal residual disease detection and PML/RAR-alpha isoform type: long-term follow-up in acute promyelocytic leukemia. Blood 2001;98:2651–6. Burnett AK, Grimwade D, Solomon E, Wheatley K, Goldstone AH. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the Randomized MRC Trial. Blood 1999;93:4131–43. Ikeda K, Sasaki K, Tasaka T, et al. PML-RAR alpha fusion transcripts by RNA PCR in acute promyelocytic leukemia in remission and its correlation with clinical outcome. Int J Hematol 1994;60:197–205. Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood 1999;94:12–22. Lengfelder E, Reichert A, Schoch C, et al. Double induction strategy including high dose cytarabine in combination with alltrans retinoic acid: effects in patients with newly diagnosed acute promyelocytic leukemia. German AML Cooperative Group. Leukemia 2000;14:1362–70. Tallman MS, Nabhan C, Feusner JH, Rowe JM. Acute promyelocytic leukemia: evolving therapeutic strategies. Blood 2002;99:759–67. Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood 1999;94:12–22. Grimwade D. The significance of minimal residual disease in patients with t(15;17). Best Pract Res Clin Haematol 2002;15:137–58. Ikeda K, Sasaki K, Tasaka T, et al. PML-RAR alpha fusion transcripts by RNA PCR in acute promyelocytic leukemia in remission and its correlation with clinical outcome. Int J Hematol 1994;60:197–205. Korninger L, Knobl P, Laczika K, et al. PML-RAR alpha PCR positivity in the bone marrow of patients with APL precedes haematological relapse by 2–3 months. Br J Haematol 1994;88:427–31. Diverio D, Rossi V, Avvisati G, et al. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion genes in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP Multicenter “AIDA” Trial. GIMEME-AIEOP Multicenter “AIDA” Trial. Blood 1998;92:784–9.

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309 [110] Inokuchi K, Iwakiri R, Futaki M, et al. Minimal residual disease in acute myelogenous leukemia with PML/RAR alpha or AML1/ETO mRNA and phenotypic analysis of possible T and natural killer cells in bone marrow. Leuk Lymphoma 1998;29:553–61. [111] Chang KS, Fan YH, Stass SA, et al. Expression of AML1-ETO fusion transcripts and detection of minimal residual disease in t(8;21)-positive acute myeloid leukemia. Oncogene 1993;8:983–8. [112] Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in longterm remission. Blood 1993;82:712–5. [113] Marcucci G, Caligiuri MA, Bloomfield CD. Defining the “absence” of the CBFbeta/MYH11 fusion transcript in patients with acute myeloid leukemia and inversion of chromosome 16 to predict long-term complete remission: a call for definitions. Blood 1997;90:5022–4. [114] Tobal K, Newton J, Macheta M, et al. Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood 2000;95:815–9. [115] Costello R, Sainty D, Blaise D, et al. Prognosis value of residual disease monitoring by polymerase chain reaction in patients with CBF beta/MYH11-positive acute myeloblastic leukemia. Blood 1997;89:2222–3. [116] Tobal K, Johnson PR, Saunders MJ, Harrison CJ, Liu Yin JA. Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission. Br J Haematol 1995;91:104–8. [117] Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in longterm remission. Blood 1993;82:712–5. [118] Evans PA, Short MA, Jack AS, et al. Detection and quantitation of the CBFbeta/MYH11 transcripts associated with the inv(16) in presentation and follow-up samples from patients with AML. Leukemia 1997;11:364–9. [119] Laczika K, Novak M, Hilgarth B, et al. Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study. J Clin Oncol 1998;16:1519–25. [120] Pongers-Willemse MJ, Seriu T, Stolz F, et al. Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:110–8. [121] Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA. Quantitation of targets for PCR by use of limiting dilution. BioTechniques 1992;13:444–9. [122] Vescio RA, Han EJ, Schiller GJ, et al. Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapheresis autografts. Bone Marrow Transplant 1996;18:103– 10. [123] Hochhaus A, Reiter A, Saussele S, et al. Molecular heterogeneity in complete cytogenetic responders after interferon-alpha therapy for chronic myelogenous leukemia: low levels of minimal residual disease are associated with continuing remission. German CML Study Group and the UK MRC CML Study Group. Blood 2000;95:62–6. [124] Tobal K, Yin JA. Monitoring of minimal residual disease by quantitative reverse transcriptase-polymerase chain reaction for AML1-MTG8 transcripts in AML-M2 with t(8; 21). Blood 1996;88:3704–9. [125] Cave H, Guidal C, Rohrlich P, et al. Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes. Blood 1994;83:1892–902. [126] Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–94.

307

[127] Galimberti S, Brizzi F, Mameli M, Petrini M. An advantageous method to evaluate IgH rearrangement and its role in minimal residual disease detection. Leuk Res 1999;23:921–9. [128] Evans PA, Short MA, Owen RG, et al. Residual disease detection using fluorescent polymerase chain reaction at 20 weeks of therapy predicts clinical outcome in childhood acute lymphoblastic leukemia. J Clin Oncol 1998;16:3616–27. [129] Luthra R, McBride JA, Hai S, Cabanillas F, Pugh WC. The application of fluorescence-based PCR and PCR-SSCP to monitor the clonal relationship of cells bearing the t(14;18)(q32;q21) in sequential biopsy specimens from patients with follicle center cell lymphoma. Diagn Mol Pathol 1997;6:71–7. [130] Delabesse E, Burtin ML, Millien C, et al. Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations. Leukemia 2000;14:1143–52. [131] Wattjes MP, Krauter J, Nagel S, Heidenreich O, Ganser A, Heil G. Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia. Leukemia 2000;14:329–35. [132] Marcucci G, Livak KJ, Bi W, Strout MP, Bloomfield CD, Caligiuri MA. Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia 1998;12:1482–9. [133] Krauter J, Wattjes MP, Nagel S, et al. Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients. Br J Haematol 1999;107:80–5. [134] Krauter J, Hoellge W, Wattjes MP, et al. Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)positive acute myeloblastic leukemia by real-time RT-PCR. Genes Chromosomes Cancer 2001;30:342–8. [135] Cassinat B, Zassadowski F, Balitrand N, et al. Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia 2000;14:324–8. [136] Pallisgaard N, Clausen N, Schroder H, Hokland P. Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. Genes Chromosomes Cancer 1999;26:355–65. [137] Tobal K, Newton J, Macheta M, et al. Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood 2000;95:815–9. [138] Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 1996;14:303–8. [139] Thelwell N, Millington S, Solinas A, Booth J, Brown T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res 2000;28:3752–61. [140] de Kok JB, Wiegerinck ET, Giesendorf BA, Swinkels DW. Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum Mutat 2002;19:554–9. [141] Isacsson J, Cao H, Ohlsson L, et al. Rapid and specific detection of PCR products using light-up probes. Mol Cell Probes 2000;14:321–8. [142] Eckert C, Landt O, Taube T, et al. Potential of LigthCycler technology for quantification of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 2000;14:316–23. [143] Beillard E, Pallisgaard N, van der Velden V, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)—a Europe Against Cancer Program. Leukemia 2003;17:2474–86. [144] Weisser M, Schoch C, Haferlach T, Hiddemann W, Schnittger S. Quantitative analysis of AML1-ETO fusion transcripts in t(8;21)

308

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

[158]

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309 positive AML using real-time RT-PCR. In: Dietmaier W, Wittwer C, Sivasubramanian N, editors. Genetics and oncology. SpringerVerlag; 2002. p. 1–10. Weisser M, Schoch C, Haferlach T, Hiddemann W, Schnittger S. The choice of house keeping genes in MRD-quantification of AML1-ETO positive acute myeloid leukemia. In: Wittwer C, Hahn M, Kaul K, editors. Rapid cycle real-time PCR, methods and application, quantification. Springer; 2003. p. 1–10. Gabert J, Beillard E, van der Velden V, et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer Program. Leukemia 2003;17:2318–57. van der Velden V, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 2003;17:1013–34. Krauter J, Wattjes MP, Nagel S, et al. Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients. Br J Haematol 1999;107:80–5. Krauter J, Hoellge W, Wattjes MP, et al. Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)positive acute myeloblastic leukemia by real-time RT-PCR. Genes Chromosomes Cancer 2001;30:342–8. Scholl C, Breitinger H, Schlenk RF, Dohner H, Frohling S, Dohner K. Development of a real-time RT-PCR assay for the quantification of the most frequent MLL/AF9 fusion types resulting from translocation t(9;11)(p22;q23) in acute myeloid leukemia. Genes Chromosomes Cancer 2003;38:274–80. Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S. Risk assessment by monitoring expression levels of partial tandem duplications in the MLL gene in acute myeloid leukaemia. Haematologica 2005;90:881–9. Wheatley K, Burnett AK, Goldstone AH, et al. A simple, robust, validated and highly predictive index for the determination of riskdirected therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council’s Adult and Childhood Leukaemia Working Parties. Br J Haematol 1999;107:69–79. Kern W, Haferlach T, Schoch C, et al. Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 Trial. Blood 2003;101:64– 70. Nguyen S, Leblanc T, Fenaux P, et al. A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): a survey of 161 cases from the French AML Intergroup. Blood 2002;99:3517–23. Kottaridis PD, Gale RE, Frew ME, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001;98:1752–9. Schnittger S, Kohlmann A, Dugas M, Schoch C, Kern W, Hiddemann W. Acute myeloid leukemia (AML) with FLT3-length mutations (FLT3-LM) can be discriminated from AML without FLT3-LM in distinct AML-subtypes based on specific gene expression profiles. Blood 2002;100(11). Thiede C, Steudel C, Mohr B, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002;99:4326–35. Frohling S, Schlenk RF, Breitruck J, et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years)

[159]

[160]

[161]

[162]

[163] [164]

[165]

[166]

[167]

[168]

[169]

[170]

[171]

[172]

[173]

[174]

[175]

with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 2002;100:4372–80. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002;100:59–66. Hovland R, Gjertsen BT, Bruserud O. Acute myelogenous leukemia with internal tandem duplication of the Flt3 gene appearing or altering at the time of relapse: a report of two cases. Leuk Lymphoma 2002;43:2027–9. Kottaridis PD, Gale RE, Langabeer SE, Frew ME, Bowen DT, Linch DC. Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors. Blood 2002;100:2393–8. Shih LY, Huang CF, Wu JH, et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002;100:2387–92. Gilliland DG. Murky waters for MRD detection in AML: flighty FLT3/ITDs. Blood 2002;100:2277. Schnittger S, Schoch C, Kern W, Hiddemann W, Haferlach T. FLT3-length mutations as marker for follow up studies in acute myeloid leukaemia. Acta Haematol 2004;112:68–78. Kern W, Haferlach T, Schnittger S, Ludwig WD, Hiddemann W, Schoch C. Karyotype instability between diagnosis and relapse in 117 patients with acute myeloid leukemia: implications for resistance against therapy. Leukemia 2002;16:2084–91. Libura M, Asnafi V, Tu A, et al. FLT3 and MLL intragenic abnormalities in AML reflect a common category of genotoxic stress. Blood 2003;102:2198–204. Lyman SD, Jacobsen SE. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 1998;91:1101–34. Whitman SP, Archer KJ, Feng L, et al. Absence of the wildtype allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study. Cancer Res 2001;61:7233–9. Kottaridis PD, Gale RE, Linch DC. Prognostic implications of the presence of FLT3 mutations in patients with acute myeloid leukemia. Leuk Lymphoma 2003;44:905–13. Nakano Y, Kiyoi H, Miyawaki S, et al. Molecular evolution of acute myeloid leukaemia in relapse: unstable N-ras and FLT3 genes compared with p53 gene. Br J Haematol 1999;104:659– 64. Nakao M, Janssen JW, Erz D, Seriu T, Bartram CR. Tandem duplication of the FLT3 gene in acute lymphoblastic leukemia: a marker for the monitoring of minimal residual disease. Leukemia 2000;14:522–4. Stirewalt DL, Willman CL, Radich JP. Quantitative, real-time polymerase chain reactions for FLT3 internal tandem duplications are highly sensitive and specific. Leuk Res 2001;25:1085–8. Schnittger S, Schoch C, Kern W, Haferlach T, Hiddemann W. FLT3-LM and MLL-PTD as markers for PCR-based detection of minimal residual disease (MRD) in AML with normal karyotype. Blood 2001;98:581A. Schnittger S, Wormann B, Hiddemann W, Griesinger F. Partial tandem duplications of the MLL gene are detectable in peripheral blood and bone marrow of nearly all healthy donors. Blood 1998;92:1728–34. Schnittger S, Kinkelin U, Schoch C, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia 2000;14:796–804.

W. Kern et al. / Critical Reviews in Oncology/Hematology 56 (2005) 283–309 [176] Caligiuri MA, Schichman SA, Strout MP, et al. Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations. Cancer Res 1994;54:370–3. [177] Caligiuri MA, Strout MP, Schichman SA, et al. Partial tandem duplication of ALL1 as a recurrent molecular defect in acute myeloid leukemia with trisomy 11. Cancer Res 1996;56:1418– 25. [178] Schichman SA, Caligiuri MA, Gu Y, et al. ALL-1 partial duplication in acute leukemia. Proc Natl Acad Sci USA 1994;91:6236–9. [179] Schnittger S, Thiede C, Kern W, et al. Partial tandem duplication of the MLL-gene (MLL-PTD): a metaanalysis of 2885 AML patients enrolled into the German AMLCG99 and AML96 SHG trials. Blood 2003;102 [Abstract]. [180] Steudel C, Wermke M, Schaich M, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer 2003;37:237–51. [181] Dohner K, Tobis K, Ulrich R, et al. Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol 2002;20:3254–61. [182] Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GA. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature 1990;343:774–8. [183] Bergmann L, Miething C, Maurer U, et al. High levels of Wilms’ tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood 1997;90:1217–25. [184] Bergmann L, Maurer U, Weidmann E. Wilms tumor gene expression in acute myeloid leukemias. Leuk Lymphoma 1997;25:435–43. [185] Niegemann E, Wehner S, Kornhuber B, Schwabe D, Ebener U. wt1 gene expression in childhood leukemias. Acta Haematol 1999;102:72–6. [186] Weisser M, Kern W, Rauhut S, Schoch C, Hiddemann W, Haferlach T, Schnittger S. Prognostic impact of RT-PCR based quantification of WT1 gene expression during MRD monitoring of acute myeloid leukaemia. Leukemia 2005;19:1416–23. [187] Hosen N, Sonoda Y, Oji Y, et al. Very low frequencies of human normal CD34+ haematopoietic progenitor cells express the Wilms’ tumour gene WT1 at levels similar to those in leukaemia cells. Br J Haematol 2002;116:409–20. [188] Maurer U, Weidmann E, Karakas T, Hoelzer D, Bergmann L. Wilms tumor gene (wt1) mRNA is equally expressed in blast cells from acute myeloid leukemia and normal CD34+ progenitors. Blood 1997;90:4230–2. [189] Maurer U, Brieger J, Weidmann E, Mitrou PS, Hoelzer D, Bergmann L. The Wilms’ tumor gene is expressed in a subset of CD34+ progenitors and downregulated early in the course of differentiation in vitro. Exp Hematol 1997;25:945–50. [190] Baird PN, Simmons PJ. Expression of the Wilms’ tumor gene (WT1) in normal hemopoiesis. Exp Hematol 1997;25:312–20. [191] Inoue K, Sugiyama H, Ogawa H, et al. WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia. Blood 1994;84:3071–9. [192] Inoue K, Ogawa H, Sonoda Y, et al. Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia. Blood 1997;89:1405–12. [193] Kreuzer KA, Saborowski A, Lupberger J, et al. Fluorescent 5 -exonuclease assay for the absolute quantification of Wilms’ tumour gene (WT1) mRNA: implications for monitoring human leukaemias. Br J Haematol 2001;114:313–8. [194] Menssen HD, Siehl JM, Thiel E. Wilms tumor gene (WT1) expression as a panleukemic marker. Int J Hematol 2002;76:103–9. [195] Elmaagacli AH, Beelen DW, Trenschel R, Schaefer UW. The detection of wt-1 transcripts is not associated with an increased leukemic relapse rate in patients with acute leukemia after allogeneic bone

[196]

[197]

[198] [199]

[200] [201]

[202]

[203]

[204]

309

marrow or peripheral blood stem cell transplantation. Bone Marrow Transplant 2000;25:91–6. Suzukawa K, Parganas E, Gajjar A, et al. Identification of a breakpoint cluster region 3 of the ribophorin I gene at 3q21 associated with the transcriptional activation of the EVI1 gene in acute myelogenous leukemias with inv(3)(q21q26). Blood 1994;84:2681–8. Morishita K, Parganas E, Matsugi T, Ihle JN. Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor. Mol Cell Biol 1992;12:183–9. Nucifora G. The EVI1 gene in myeloid leukemia. Leukemia 1997;11:2022–31. Schnittger S, Schoch C, Mellert G, Haferlach T, Hiddemann W. Expression of EVI1 is not specific for AML with 3q21q26 aberrations. Blood 1999;94:497a. Langabeer SE, Rogers JR, Harrison G, et al. EVI1 expression in acute myeloid leukaemia. Br J Haematol 2001;112:208–11. Schnittger S, Kern W, Schoch C, Hiddemann W, Haferlach T. Analysis of EVI1 expression in 270 cases with AML at diagnosis and evaluation as a marker for follow up analysis using quantitative RT-PCR in 36 cases. Blood 2003;102 [Abstract]. Barjesteh van Waalwijk van Doorn, Erpelinck C, van Putten WL, et al. High EVI1 expression predicts poor survival in acute myeloid leukemia: a study of 319 de novo AML patients. Blood 2003;101:837–45. Kern W, Schoch C, Haferlach T, Voskova D, Hiddemann W, Schnittger S. Complemental roles for multiparameter flow cytomety and quantitative RT-PCR for the quantification of minimal residual disease in patients with acute myeloid leukemia. Blood 2003:102. Voskova D, Schoch C, Schnittger S, Hiddemann W, Haferlach T, Kern W. Stability of leukaemia-associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: comparison with cytomorphologic, cytogenetic, and molecular genetic findings. Clin Cytom 2004;62B:25–38.

Biographies Wolfgang Kern, M.D., since 12 years involved in the field of management of patients with acute and chronic leukemias. More than 5 years experience in leukemia diagnostics and monitoring using multiparameter flow cytometry. PD Dr. med. Claudia Schoch, M.D., since 15 years involved in the field of hematology and genetics in leukemia. More than 13 years experience in leukemia diagnostics using chromosome banding analysis, FISH including multicolor FISH and gene expression analysis with microarrays. Prof. Dr. med. Dr. phil. Torsten Haferlach, M.D., since 21 years involved in the field of management of patients with acute and chronic leukemias. More than 18 years experience in leukemia diagnostics especially in cytomorphology and cytochemistry. Author of several books in this field. Susanne Schnittger, Ph.D. in human genetics. Since 12 years involved in the field of genetics in leukemia. Experience in positial cloning in human inborn disorders and leukemias. More than 7 years experience in leukemia diagnostics and mutational screening using FISH, Southern blot, PCR, RT-PCR, real-time PCR, fragment analysis, sequencing.