Modern diagnostics in acute leukemias

Critical Reviews in Oncology/Hematology 56 (2005) 223–234

Modern diagnostics in acute leukemias Torsten Haferlach ∗ , Wolfgang Kern, Susanne Schnittger, Claudia Schoch Laboratory for Leukemia Diagnostics, Medical Department III, University Hospital Grosshadern, Ludwig-Maximilians-University, Marchioninistreet 15, 81377 Munich, Germany Accepted 15 April 2004

Contents 1. 2.

3.

4. 5.

6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample collection and preanalytic procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Processing of samples for different methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Cytomorphology and cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Multiparameter flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Fluorescence in situ hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5. Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification in acute leukemias with respect to diagnostic procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cytomorphology and cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Multiparameter flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Cytogenetics and FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New definition of remission and relapse in acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important aspects at diagnosis of specific subtypes in acute leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Acute myeloid leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Acute lymphoblastic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Problems and pitfalls in the diagnosis and at follow-up of acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future developments in the diagnosis of acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

224 224 225 225 225 226 226 227 227 227 228 228 228 228 229 229 230 230 230 231 232 234

Abstract Acute leukemias are a heterogeneous group of diseases. The different subtypes are characterized by certain clinical features and specific laboratory findings. Large clinical trials have confirmed the important impact of the underlying biology of each subtype for clinical outcome. Improvements in patient’s treatment resulting in better survival rates are closely linked to the biological understanding of the disease subtypes, which is assessed by specific diagnostic approaches. Thus, several diagnostic techniques are mandatory at diagnosis for classification and for individual therapeutic decisions. Furthermore they are also needed for follow up studies focusing especially on minimal residual disease (MRD) to guide further treatment decisions based on the response of the disease to given treatment protocols. Only by using a comprehensive diagnostic panel including cytomorphology, cytochemistry, multiparameter flow cytometry (MFC), cytogenetics, fluorescence in situ hybridization (FISH) and molecular genetic methods the correct diagnosis in acute leukemias can be established today. The results serve as a mandatory prerequisite for individual treatment strategies and for the evaluation of treatment response using especially newly defined and highly specific MRD markers. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Diagnosis of leukemia; Cytomorphology; Immunophenotyping; Cytogenetics; Fluorescence in situ hybridization; Polymerase chain reaction ∗

Corresponding author. Tel.: +49 89 99017 0; fax: +49 89 99017 111. E-mail address: [email protected] (T. Haferlach).

1040-8428/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2004.04.008

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1. Introduction Acute myeloid and acute lymphoblastic leukemia describe a heterogeneous group of clonal hematopoietic progenitor cell disorders. During the last 20 years, the diagnosis of acute leukemias emerged from cytomorphology alone to a comprehensive bundle of different methods that are necessary not only for the diagnosis and classification but also for individual treatment decisions. All these therapeutic aspects will be further outlined in other articles published in this issue. For diagnosis, however, an algorithm that combines cytomorphology and cytochemistry with immunophenotyping accompanied by cytogenetics and molecular genetic methods has to be established in a laboratory setting [1]. As methods not only add important information at diagnosis but more and more also define markers for minimal residual disease (MRD) studies, this has to be considered at the first time point of any analysis in every single patient. Furthermore, diagnostic results are equivalent to the most important prognostic parameters in acute leukemias. As new techniques have been established within the last decade and were brought to routine use it seems necessary to define a comprehensive global approach in the diagnosis of acute leukemias. This has to include a step-wise procedure in the lab flow in order to save time and money without loosing important and detailed information. Starting from peripheral blood smears and bone marrow cytomorphology, it is mandatory to further perform multiparameter flow cytometry (MFC), and metaphase cytogenetics in every case, in which acute leukemia is suspected. The latter has to be accompanied by FISH and also by PCR analysis or even screening for specific molecular markers. Although MFC is informative in AML only for the former FAB subtypes AML M0 and AML M7, it leads to very important information in ALL for classification and stratification to adapted therapy. In 50–70% of patients with acute leukemias acquired clonal chromosome aberrations can be observed after metaphase analyses. The cytogenetic results at diagnosis provide the most important single parameter for prognostication so far. Numerous recurrent karyotype abnormalities have been described. These findings on the chromosomal level are followed and complemented in some parts by molecular studies that have identified genes involved in leukemogenesis. Even more, molecular markers, such as MLL partial tandem duplications (MLL-PTD) or FLT3 length mutations (FLT3-LM) in AML, or BCR-ABL in ALL were found to characterize specific subtypes and complete the panel of genetic markers. The identification of specific chromosomal abnormalities or molecular markers and their correlation to cytomorphologic features as well as to clinical outcome led to a new understanding of acute leukemias as a heterogeneous group of different biological entities. The importance of cytogenetic and molecular genetic findings for the classification and for the understanding of pathogenetic mechanisms

is increasingly appreciated in the clinical context and was translated also into the new WHO classification that uses cytogenetic abnormalities as a major criterion in AML and in combination with MFC also in ALL [2]. After introducing the different techniques in the diagnosis of acute leukemia and their respective results this paper will suggest an algorithm for laboratory work-up with respect to a comprehensive diagnosis that can be the basis for classification, therapeutic decisions, MRD studies, leads to prognostic markers and is cost effective if applied in a step-wise workflow.

2. Sample collection and preanalytic procedures As methods at the diagnosis of acute leukemia and during follow-up studies have to be applied in parallel it is mandatory to receive optimally prepared samples into the laboratory at each time point. Therefore, several prerequisites have to be fulfilled. If possible, in all cases with suspected acute leukemia the investigation should include blood as well as bone marrow samples in parallel [3]. A trephine biopsy in acute leukemia is not necessary and should be performed only if the aspirate is dilute, very hypocellular or inaspirable (punctio sicca) [4]. In these circumstances, peripheral blood should be analysed and for cytomorphology smears from trephine cylinders can be produced. It is also possible to investigate morphology and immuno-histopathology on paraffin embedded bone marrow histologies as well as to perform a cytogenetic analysis after the trephine biopsy was cultivated in cytogenetic medium and processed afterwards to metaphase analysis. Different methods rely on different sources. It is necessary to realize that cytomorphology is hampered by heparin and that metaphases can not be cultivated after EDTA was added to a sample. All further details are listed in Table 1. Overall, a comprehensive investigation at diagnosis needs at best 3–5 ml EDTA anticoagulated bone marrow and 10 ml EDTA peripheral blood in parallel as well as additional 5–10 ml heparinized bone marrow and 10–20 ml heparinized peripheral blood. We are aware that investigation can be performed also with much less cells but with respect to the definition of markers for further MRD studies the first approach should not be hampered due to insufficient numbers of leukemia cells in a sample. The material should reach the laboratory at latest within 24 h after biopsy and should be shipped at room temperature without adding cool packs or dry ice. If this is guaranteed a successful investigation is possible in over 98% of cases including also metaphase cytogenetics, which is indeed the most susceptible technique [5]. But also for measurements of protein expression by MFC or gene expression by PCR of microarrays these preconditions should be considered [6–9]. This also means that for the diagnosis of acute leukemias a central reference laboratory has to be available every day for optimal service [1].

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Table 1 Techniques applied in the diagnosis of acute leukemias and specific cell material needed for optimal investigations Methods

EDTA aspirate (2–10 ml)

Heparin aspirate (10–20 ml)

Trephine biopsy#

Cytomorphology (MGG) Cytochemistry (MPO, NSE) Cytogenetics FISH (metaphase/interphase) Immunophenotyping PCR, real-time PCR Other molecular techniques

Yes* Yes No Yes (IP) Yes Yes Yes

No Yes Yes* Yes Yes Yes Yes

Yes Yes If needed Difficult No Yes No

* #

Mandatory, otherwise technique will be very artificial or will fail. Only if no aspirate is possible (punctio sicca).

2.1. Processing of samples for different methods 2.1.1. Cytomorphology and cytochemistry For cytomorphology and cytochemistry at least five peripheral blood smears and five bone marrow smears should be available. After they have been air-dried without any further fixation, Pappenheim or May Gr¨unwald Giemsa (MGG) staining should be performed and accompanied by myeloperoxidase (MPO) and non-specific esterase (NSE) in all cases (Figs. 1–3) [4,10,11]. In some cases, an iron staining may be helpful but normally will not be performed in the diagnostic set-up of acute leukemias but in myelodysplastic syndromes (MDS). Stainings, such as perjod acid Schiff’s reaction (PAS), acid phosphatase or chloro-acetate esterase (CE) are not further needed as they did not add important information if MFC is performed [3,10]. Exceptions may be special cases, for instance for the demonstration of glycogen in the erythroid lineage (PAS), or CE in histological sections where it is the best method for the demonstration of neutrophilic granulocytic lineage. 2.1.2. Multiparameter flow cytometry For multiparameter flow cytometry cells can be processed after lysis or after ficoll hypaque gradiation [7,12]. If in a

Fig. 1. Pappenheim staining of an AML M5a (according to FAB classification) with 11q23 aberration (according to WHO classification, step 1) (630×).

Fig. 2. Myeloperoxidase reaction in an AML M2 with Auer rods (according to FAB classification) with t(8;21) aberration (according to WHO classification, step 1) (630×).

work-flow the same syringe (EDTA- or if send over night better heparinized bone marrow or blood) is processed also for molecular techniques, many laboratories use ficoll gradiation before MFC. We would also recommend to prefer bone marrow if available.

Fig. 3. Non-specific esterase staining of an AML M5a (according to FAB classification) with 11q23 aberration (according to WHO classification, step 1). Blasts are strongly positive (630×).

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Fig. 4. Metaphase after G-banding in a c-ALL with t(9;22) so called Philadelphia translocation.

2.1.3. Cytogenetics For cytogenetics metaphases have to be generated. Therefore, an optimal number of 5 × 107 cells from the bone marrow (or blood, if no bone marrow is available) have to be cultivated in several parallel short-term cultures (16–24, 48 h) [13]. Usually colchecine is added for 1–2 h before harvesting the cells. In order to obtain a higher number of metaphases, especially in cases with low cell counts, the time of cultivation after adding colchecine for mitotic arrest can be increased to 24 h [14]. In many cases, a cocktail of cytokines is added to the medium. Doing so, myeloid or lymphoid blasts are intended to be specifically stimulated to increased proliferation in vitro. After several procedures of banding and staining (Giemsa (G-) or R-banding) the metaphases have to be analysed, mostly supported by digital picture capture systems (Fig. 4) (i.e., MetaSystems, Altlussheim, Germany). At least 25 metaphases should be investigated in acute leukemias [4]. If a clonal aberration is detected reproducibly 20 metaphases seem to be sufficient. If complex aberrations or marker chromosomes are detected, further fluorescence in situ hybridization (FISH) studies should be performed for clarification and for future MRD analyses (Fig. 5a and b) [15]. 2.1.4. Fluorescence in situ hybridization For fluorescence in situ hybridization metaphases as well as interphase nuclei from cytomorphological smears of bone marrow or peripheral blood can be used. After fixation 2 h time for hybridization in cases with suspected APL detecting PML-RARA fusion signals in interphase-FISH is sufficient (Fig. 6) in our hand [16]. All other probes for interphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24color FISH or comparative genomic hybridization (CGH) are usually hybridized in an overnight procedure and are available for analysis 24 h after the sample had reached the laboratory [16].

Fig. 5. (a and b) Metaphase before and after analysis by 24-color FISH in a case with AML and complex aberrant karyotype (software Isis® and hybridization probes by MetaSystems, Altlussheim, Germany).

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Table 3 The first hierarchical step in the WHO classification for AML refers to AML with recurrent balanced chromosomal aberrations AMLM2(1) AML M3(v) AML M4eo AML M4/5

t(8;21) t(15;17) inv(16), t(16;16) 11q23

Most of them correlate clearly with morphological subtypes as formerly defined by the FAB classification.

3.1. Cytomorphology and cytochemistry

Fig. 6. Interphase FISH with PML/RARA probe showing one PML/RARA positive cell with co-localisation of a red and a green signal and two independent signals, and one negative cell with two independent red and green signals each, respectively.

2.1.5. Molecular methods For molecular methods, such as PCR, real-time PCR (see Kern et al. in this issue), sequencing or even gene expression profiling using microarrays samples should be processed after Ficoll Hypaque density gradient centrifugation for DNA or RNA preparation. This seems to be required for diagnostic and for follow-up samples with respect to MRD also [17–19].

3. Classification in acute leukemias with respect to diagnostic procedures

The first step for the diagnosis of acute leukemias is still cytomorphology and cytochemistry. It is quick and cheap and the results allow to draw up an optimal work flow for the other much more labour-intensive and expensive techniques. Thus, the investigation of MGG, MPO and NSE at least should define if a respective blast population has myeloid or monocytic lineage commitment or fails to show these characteristics and therefore may lead to the diagnosis of FAB AML M0, M7 or even ALL [1,3,10]. In most cases of AML including the APL (M3 and its variant M3v) the diagnosis and classification based on cytomorphology is already sufficient to start treatment. However, the WHO classification for AML

Table 4 The WHO classification for AML introduces secondary AML after preceding MDS and secondary AML after pretreatment with chemotherapy or radiation, so called therapy-related AML • 10% of AML and MDS are t-AML • Two groups can be distinguished After Alkylating agents Often MDS prephase

Today the classification of acute myeloid leukemias should follow the WHO proposal published in 2001 (see Tables 2–5) and will only in small parts rely further on the FAB classification [2,20–25]. Furthermore, some specific classification systems (see Table 6 for EGIL classification) [26] and specific terminological aspects for cytogenetic and FISH as defined by the ISCN nomenclature [27] have to be considered. In detail, the following aspects are relevant for the diagnosis and for classification of acute leukemias.

Long latency period (4–6 years) −5/5q−, −7/7q−, complex aberrant karyotype Poor response to therapy

Topoisomerase II-inhibitors No MDS prephase, often M4/M5 Short latency period (0.5–3 years) Balanced aberrations, often 11q23, 21q22 Better response to therapy

Table 5 The WHO classification for AML Table 2 The hierarchy of the WHO classification for AML with five steps of subclassifications 1.

2. 3. 4. 5.

AML with recurrent genetic abnormalities (diagnosis of AML is independent of blast count) AML with multilineage dysplasia Therapy-related AML and MDS AML not otherwise categorized Acute leukemia of ambiguous lineage

New definition: blasts > 20% = AML, no RAEB-T.

• AML minimally differentiated • AML without maturation • AML with maturation • Acute myelo-monocytic leukemia • Acute monocytic and monoblastic leukemia • Acute erythroid leukemia • Acute megakaryoblastic leukemia • Acute basophilic leukemia • Acute panmyelosis with myelofibrosis • Myeloid sarcoma Most terms correlate to morphological subtypes as formerly defined by the FAB classification.

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Table 6 The EGIL classification is used to define acute leukemias of ambiguous lineage and is also included in the WHO classification Score

B-lymphoid

T-lymphoid

Myeloid

2 2 2

CD79a cyIgM cyCD22

cy/sCD3 Anti-TZR␣␤ Anti-TZR␥␦

Anti-MPO

1 1 1 1

CD19 CD10 CD20

CD2 CD5 CD8 CD10

CD13 CD33 CD65 CD117

0.5 0.5 0.5

TdT CD24

TdT CD7 CD1a

CD14 CD15 CD64

Leukemia of ambiguous lineage: if score >2 for myeloid and also for B- or T-lymphoid.

and ALL pays less attention to these morphological aspects. Only for the second step of the hierarchical approach in the WHO classification of AML the cytomorphologic perspective with respect to dysplastic features is needed (Table 2) [2,28]. However, we do not think that this parameter should be included in future classification systems as outlined in much more detail before by our group [5,29,30]. In conclusion, in AML and ALL cytomorphology classified according to FAB criteria or analogeously in the WHO (see Table 5) is warranted at diagnosis in acute leukemias. But more importantly, morphology is mandatory to direct all other techniques and to process samples in shortest time. In addition, this approach saves money and avoids unnecessary investigations. 3.2. Multiparameter flow cytometry MFC using three, four or nowadays even five color staining should be applied to all cases with the suspected diagnosis of acute leukemia. This is absolutely warranted at diagnosis and achieves increasing importance for MRD also (see Kern in this issue) [31–35]. It is quicker and in some cases even cheaper in comparison to other MRD techniques, such as real time PCR for fusion genes or patient specific markers, such as FLT3-LM, or IgVH-mutations (see the respective paper in this issue by Kern et al. for AML and by Hoelzer et al. for ALL) [18,36]. These two aspects of MFC lead to a broad spectrum of antibodies to be evaluated at diagnosis in all acute leukemias. MFC is not only mandatory for the FAB subtypes AML M0 and AML M7 as defined by FAB or WHO (very immature, or megakaryoblastic AML) [2,24,25], but leads to the most important subclasses in ALL: B-lineage versus Tlineage and further subclasses as defined by WHO and EGIL classification (see Table 6) [2,26]. Thus, MFC gives important information for diagnosis and leads to treatment decisions but also add important prognostic parameters in ALL (see the paper by Hoelzer et al. in this issue). 3.3. Cytogenetics and FISH The first hierarchical step in the WHO classification of AML is based on cytogenetics and includes the balanced

translocations t(15;17), inv(16), t(8;21) and 11q23 aberrations (see Table 3). This is a very important step for a more biologically defined approach in classification. Therefore, also in ALL cytogenetic markers are considered: ALL with t(9;22), 11q23 aberrations and t(8;14) are considered also as unique, biologically defined entities which need specific treatment approaches. Several additional cytogenetic abnormalities with reproducible prognostic impact have been defined within large clinical trials in AML as well as in ALL in childhood and in adults [37–43]. Subentities like the APL in AML or the BCR/ABL positive ALL were the first ones to demonstrate clearly that their recognition at diagnosis not only leads to specific treatment circumstances including drugs like ATRA or imatinib, respectively, but also point to the mission of future diagnostic and classification approaches in acute leukemia: define unique entities with specific biology, prognosis and hopefully individual treatment. 3.4. Molecular methods Reciprocal transloctions that lead to fusion genes can also be detected by PCR. This is needed for validation of cytogenetic results but even more, serves as a base line for MRD studies in the respective patient. Furthermore, the most often detectable molecular defect in AML, FLT3-LM or point mutations [17,44–49] and MLL-PTD [50–52] are only detectable on the molecular level. Mutations of other genes, such as NRAS [46,53–56], CKIT [57,58] or CEBP/A [59] demonstrate increasing importance in AML and will have to be screened for at diagnosis in every single case in the near future. In ALL the most important molecular markers are BCR/ABL, all MLL translocations with their respective partner genes, and c-MYC aberrations that have to be detected at diagnosis and for MRD studies [19,36,60,61]. Furthermore, for more than 90% of ALL patients specific markers can be detected by IgH mutations and serve as the most important MRD marker making even treatment decisions possible in most patients in ongoing studies (see Hoelzer et al. in this volume). The importance of a global so called multiplex-PCR screening or gene expression profiling in acute leukemias for diagnosis, for delineation of specific MRD markers or for prognostic impact needs further investigation and can not be recommended at this time in a standard diagnostic setting.

4. New definition of remission and relapse in acute leukemias The diagnosis of acute leukemias depends on classification systems, such as the WHO classification, the FAB classification and the EGIL classification [2,20,22,23,26]. For optimal diagnosis and therapeutic strategies parameters used in these classifications need to be combined. Furthermore,

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due to the broad spectrum of methods at diagnosis and for follow-up studies including MRD parameters new definitions of remission as well as for relapse are needed. In a recent paper [4], several new and important aspects were included with focus on AML. The revised recommendations at first followed the WHO classification for definition of AML at diagnosis: (1) De novo AML should only be referred to if no clinical history of MDS, MPS or exposure to potentially leukemogenic therapies or agents is known. (2) Secondary AML should refer to patients who have such history and should further be subdivided into secondary AML after MDS or MPS versus secondary AML after proven leukemogenic exposure. We would suggest to follow the WHO and call these latter leukemias or MDS cases therapy-related AML (t-AML) within the group of s-AML. It seems important to follow the new definition by Cheson et al. that also 1 month or greater preleukemic state should not by itself allow the designation of a case into s-AML without confirmation of pre-existing blood smear that clearly demonstrated morphologic dysplasia. Although the absence or presence of dysplasia should be recorded according to the WHO classification and was also included in the paper by Cheson et al. from our point of view further studies including interlaboratory confirmation and central review processes are needed. Morphologic characteristics often only demonstrated a limited inter- and intraobserver reproducibility, even by thresholds for dysplasia of 50% in AML [28] (and 10% in MDS as defined by the FAB group and also included in WHO definitions) [2,22] these criteria have to be evaluated further. Also multivariate analyses including cytogenetics and age are needed before dysplasia can lead to treatment decisions or defines poor risk patients in de novo AML. As new techniques emerged, such as FISH, MFC and PCR for the detection of MRD, it was necessary to define new categories of remission. Thus, the paper published by Cheson et al. at first defined a new so called “early treatment assessment 7–10 days after therapy”. At this time point blasts in the bone marrow can be assessed morphologically, by FISH, PCR and MFC. Recent papers were able to demonstrate the high prognostic impact of this evaluation at such an early time-point as assessed by cytomorphology using blast percentages alone [62]. As this is the first therapy-dependent parameter to be measurable in AML it should be investigated further. Parallel studies with MFC and interphase-FISH could clarify the role of each method for the evaluation of residual disease and their prognostic impact. Within the term “complete remission” three different categories should be separated in future studies. In all new CR categories the neutrophils in the pB should exceed >1000/␮l and the platelet count should be >100,000/␮l, the bone marrow blast count should be <5% (at least 200 cells should be counted). If these three parameters are measurable, one

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should call it “morphologic complete remission”. However, in many treatment protocols of AML a second course of therapy follows closely related to the first induction course. Therefore, for morphologic CR a reconstitution of bone marrow and blood cells for >4 weeks is not further needed. In cases with abnormal cytogenetics at diagnosis, normal metaphases and FISH studies have to be detected after a therapy to reach a cytogenetic complete remission (CRc). However, as data are lacking these two techniques should be further evaluated within clinical trials for their prognostic impact in the near future in parallel to others [63]. The term molecular complete remission (CRm) will be used after MRD studies by MFC for the detection of a leukemia-associated immunophenotype (LAIP), or by PCR techniques. Both approaches are more sensitive than morphology alone or cytogenetics and FISH in combination [63]. Further studies in parallel are needed for clinical decisions. However, real time PCR approaches in some molecularly defined subgroups of AML and ALL proved already their outstanding prognostic significance in large trials [18,36]. Cheson et al. also newly defined the term partial remission that is relevant for phase I and II trials. A blast count in the bone marrow aspirate between 5% and 25% or at least half of the percentage that was measured before treatment is required to call the status partial remission. A relapse after CR was newly defined as a reappearance of any leukemic blasts in the peripheral blood, or ≥5% blasts in the bone marrow not attributable to regeneration after chemotherapy treatment.

5. Important aspects at diagnosis of specific subtypes in acute leukemia 5.1. Acute myeloid leukemia In all cases of AML cytomorphology, cytochemistry, MFC and cytogenetics should be performed at diagnosis. The cytogenetic result at diagnosis is still the most important single prognostic parameter and leads to treatment stratification not only in APL. It also serves as a basis for strategies in CBF leukemias, i.e., AML with t(8;21) or AML with inv(16), in combination with real time PCR [18]. In complex aberrant cases decisions with respect to early transplantation strategies may be driven by the cytogenetic results [64]. In certain cases metaphase cytogenetics the performance of IP-FISH or WCP-FISH in addition for validation is helpful. Twenty four-color FISH provides detailed additional insight in cases with complex aberrant karyotypes (three or more chromosomes involved) [15]. Although the latter technique will only add information usually not needed for therapeutic decisions this method is recommend at least in clinical trials for further pathobiological investigations. The value of comparative genomic hybridization in AML genetics for routine use is still not defined.

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A cytogenetic result is also mandatory for the first hierarchical step in the WHO classification [2]. It may further delineate patients with therapy-related AML (t-AML) between the most important two subtypes as defined in the WHO to (i) tAML following treatment with alkylating agents or (ii) after treatment with topoisomerase II inhibitors. Such mechanism of leukemia induction in general lead to two different subtypes of t-AML: those with cytogenetic aberrations involving 5q−, −7 or p53 and in most cases with complex aberrant karyotypes after alkylating agents, in contrast to 11q23 or other balanced cytogenetic aberrations after topoisomerase II inhibitors. Thus, metaphase-cytogenetics gives important information and also describes in patients with t-AML prognosis best [65]. In addition to standard metaphase cytogenetics interphase FISH is very helpful not only for verification but can give immediate information with respect to the question, if an APL with PML/RARA fusion genes is present or not. As this question is of highly important therapeutic impact and not in all cases morphology is available interphase FISH is recommend in all cases of suspected APL [16]. For follow-up studies further investigations with cytomorphology, MFC, hypermetaphase-FISH, IP-FISH and real time PCR are needed. We hopefully will be able to define the value of each technique in an ongoing prospective trial and will define an algorithm for methods at follow-up time points [63]. 5.2. Acute lymphoblastic leukemia The most important method for the diagnosis of ALL is MFC. In combination with cytogenetic and molecular genetic analyses therapy can be directed. Cytomorphology is only informative in cases with Burkitt-type ALL (formerly called L3 morphology according to FAB classification). However, if this subtype is suspected morphologically further cytogenetic and FISH investigations must be performed for validation. From the cytogenetic point of view, several ALL specific aberrations can be defined (see Table 7). Out of these the t(9;22), t(4;11) and the t(8;14) are important due to their specific prognostic impact and specific treatment protocols

Table 7 Cytogenetic aberrations that can be found in ALL and their respective frequencies in children and adults

t(1;19) t(4;11) t(9;22) t(8;14) t(10;14) t(12;21) 9p 6q 14q11

Pre-B-ALL Pro-B-ALL c-ALL B-ALL T-ALL Pre-B-ALL T, pre-B c-, pre-B,T T-ALL

Childhood (%)

Adults (%)

5–6 2 2–5 3 1 10–15 7–12 4–13 1

3 6 25–30 5 3 <1 15 6 6

(see Hoelzer et al. in this issue). Further treatment stratification will be possible in the near future by PCR studies that are performed with respect to patient specific primers at defined timepoints during the first months of treatment [36]. The results will stratify the therapy especially in so called standard risk patients in the near future also in adults (see Hoelzer et al. in this issue) as has been proven very successfully in childhood ALL. 6. Problems and pitfalls in the diagnosis and at follow-up of acute leukemias Only a comprehensive diagnostic approach in patients suspected to have an acute leukemia would lead to an adequate diagnosis and treatment. Most of the parameters defined by a combination of different methods add further information also for prognosis. This can only be achieved by investigation of blood and bone marrow before any treatment was administered (also no corticosteroids in ALL cases!). Samples have to be supplemented with EDTA or heparin for the respective analyses (see Table 1). The number of necessary cells to be analysed is important not only at diagnosis but especially for follow-up studies. If hampered by too few cells (low sensitivity) MRD results have to be taken with cautiousness. Parallel investigations applying two methods at the same time, such as cytomorphology combined with FISH, or MFC in combination with real time PCR should be performed for clinical decisions. Algorithms for diagnostic approaches in acute leukmias are shown in Tables 8 and 9 summarize the actual procedures. 7. Conclusions and future developments in the diagnosis of acute leukemias Every treatment in an acute leukemia patient should be based on a diagnostic set-up that is able to combine cytomorphology, cytochemistry, multiparameter flow cytometry, cytogenetics, fluorescence in situ hybridization and molecular genetic methods if needed. If this can not be guaranteed one has to be aware that todays standard therapies in AML and ALL may be inadequate and treatment outcome suboptimal. Furthermore, during course of treatment many ongoing studies focus on minimal residual disease. These results have increasing importance for further therapy, i.e., they may even form the basis for the decision to apply allogeneic peripheral stem cell transplantations for patients with poor risk leukemias. On the other hand, MRD markers can also lead to de-escalation of treatment or even an early stop of therapy in cases with a very low relapse risk. Other techniques, such as gene expression profiling and proteomics begin to add important information at diagnosis and for prediction of response to specific treatment protocols in acute leukemias [6,8,9,66–70]. It will be very interesting to test gene expression profiling these in comparison to today’s

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Table 8 Proposal for an algorithm at diagnosis and for follow-up studies in AML

Table 9 Proposal for an algorithm at diagnosis and for follow-up studies in ALL

standard methods as outlined in this paper. This may not even add further clarification to known leukemia subtypes but may also deliniate new entities, for example, in AML with normal karyotypes. It may even be tested to predict response and may therefore help not only for diagnostic but also for therapeutic purposes. However, future studies will have to demonstrate the value of such methods in the context of modern leukemia diagnostics and their possible impact on treatment decisions.

Reviewers Prof. Fausto Grignani, Istituto Clinica Medicina 1, Policlinico Monteluce, Via Brunamonte, I-06122 Perugia, Italy. Prof. Dr. H. L¨offler, Seelgutweg 7, D-79271 St. Peter, Germany. Prof. Laurent Degos, IUH, Hˆopital St. Louis, 1 av. Claude Vellefaux, F-75010 Paris, France.

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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.

PD Dr. rer.nat. 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.

PD Dr. med. 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.

Biographies