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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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2. Sample collection and preanalytic procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2.1. Processing of samples for different methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.1.1. Cytomorphology and cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.1.2. Multiparameter flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.1.3. Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2.1.4. Fluorescence in situ hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2.1.5. Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3. Classification in acute leukemias with respect to diagnostic procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.1. Cytomorphology and cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.2. Multiparameter flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
3.3. Cytogenetics and FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
3.4. Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
4. New definition of remission and relapse in acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
5. Important aspects at diagnosis of specific subtypes in acute leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
5.1. Acute myeloid leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
5.2. Acute lymphoblastic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
6. Problems and pitfalls in the diagnosis and at follow-up of acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
7. Conclusions and future developments in the diagnosis of acute leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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: torsten.haferlach@mll-online.com (T. Haferlach).

1040-8428/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2004.04.008
224 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

1. Introduction is increasingly appreciated in the clinical context and was


translated also into the new WHO classification that uses
Acute myeloid and acute lymphoblastic leukemia describe cytogenetic abnormalities as a major criterion in AML and
a heterogeneous group of clonal hematopoietic progenitor in combination with MFC also in ALL [2].
cell disorders. During the last 20 years, the diagnosis of After introducing the different techniques in the diagnosis
acute leukemias emerged from cytomorphology alone to a of acute leukemia and their respective results this paper will
comprehensive bundle of different methods that are neces- suggest an algorithm for laboratory work-up with respect to a
sary not only for the diagnosis and classification but also for comprehensive diagnosis that can be the basis for classifica-
individual treatment decisions. All these therapeutic aspects tion, therapeutic decisions, MRD studies, leads to prognostic
will be further outlined in other articles published in this markers and is cost effective if applied in a step-wise work-
issue. For diagnosis, however, an algorithm that combines flow.
cytomorphology and cytochemistry with immunophenotyp-
ing accompanied by cytogenetics and molecular genetic
methods has to be established in a laboratory setting [1]. 2. Sample collection and preanalytic procedures
As methods not only add important information at diag-
nosis but more and more also define markers for mini- As methods at the diagnosis of acute leukemia and during
mal residual disease (MRD) studies, this has to be con- follow-up studies have to be applied in parallel it is mandatory
sidered at the first time point of any analysis in every to receive optimally prepared samples into the laboratory at
single patient. Furthermore, diagnostic results are equiva- each time point. Therefore, several prerequisites have to be
lent to the most important prognostic parameters in acute fulfilled.
leukemias. If possible, in all cases with suspected acute leukemia the
As new techniques have been established within the last investigation should include blood as well as bone marrow
decade and were brought to routine use it seems necessary to samples in parallel [3]. A trephine biopsy in acute leukemia
define a comprehensive global approach in the diagnosis of is not necessary and should be performed only if the aspirate
acute leukemias. This has to include a step-wise procedure in is dilute, very hypocellular or inaspirable (punctio sicca) [4].
the lab flow in order to save time and money without loosing In these circumstances, peripheral blood should be analysed
important and detailed information. and for cytomorphology smears from trephine cylinders can
Starting from peripheral blood smears and bone marrow be produced. It is also possible to investigate morphology and
cytomorphology, it is mandatory to further perform multipa- immuno-histopathology on paraffin embedded bone marrow
rameter flow cytometry (MFC), and metaphase cytogenetics histologies as well as to perform a cytogenetic analysis after
in every case, in which acute leukemia is suspected. The lat- the trephine biopsy was cultivated in cytogenetic medium and
ter has to be accompanied by FISH and also by PCR analysis processed afterwards to metaphase analysis.
or even screening for specific molecular markers. Although Different methods rely on different sources. It is necessary
MFC is informative in AML only for the former FAB sub- to realize that cytomorphology is hampered by heparin and
types AML M0 and AML M7, it leads to very important that metaphases can not be cultivated after EDTA was added
information in ALL for classification and stratification to to a sample. All further details are listed in Table 1.
adapted therapy. Overall, a comprehensive investigation at diagnosis needs
In 50–70% of patients with acute leukemias acquired at best 3–5 ml EDTA anticoagulated bone marrow and 10 ml
clonal chromosome aberrations can be observed after EDTA peripheral blood in parallel as well as additional
metaphase analyses. The cytogenetic results at diagnosis pro- 5–10 ml heparinized bone marrow and 10–20 ml heparinized
vide the most important single parameter for prognostication peripheral blood. We are aware that investigation can be
so far. Numerous recurrent karyotype abnormalities have performed also with much less cells but with respect to
been described. These findings on the chromosomal level the definition of markers for further MRD studies the first
are followed and complemented in some parts by molecular approach should not be hampered due to insufficient num-
studies that have identified genes involved in leukemogen- bers of leukemia cells in a sample. The material should reach
esis. Even more, molecular markers, such as MLL partial the laboratory at latest within 24 h after biopsy and should
tandem duplications (MLL-PTD) or FLT3 length mutations be shipped at room temperature without adding cool packs
(FLT3-LM) in AML, or BCR-ABL in ALL were found to or dry ice. If this is guaranteed a successful investigation is
characterize specific subtypes and complete the panel of possible in over 98% of cases including also metaphase cyto-
genetic markers. The identification of specific chromosomal genetics, which is indeed the most susceptible technique [5].
abnormalities or molecular markers and their correlation to But also for measurements of protein expression by MFC
cytomorphologic features as well as to clinical outcome led or gene expression by PCR of microarrays these precondi-
to a new understanding of acute leukemias as a heteroge- tions should be considered [6–9]. This also means that for
neous group of different biological entities. The importance the diagnosis of acute leukemias a central reference lab-
of cytogenetic and molecular genetic findings for the classifi- oratory has to be available every day for optimal service
cation and for the understanding of pathogenetic mechanisms [1].
T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234 225

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) Yes* No Yes
Cytochemistry (MPO, NSE) Yes Yes Yes
Cytogenetics No Yes* If needed
FISH (metaphase/interphase) Yes (IP) Yes Difficult
Immunophenotyping Yes Yes No
PCR, real-time PCR Yes Yes Yes
Other molecular techniques Yes 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 fur-
ther fixation, Pappenheim or May Grünwald Giemsa (MGG)
staining should be performed and accompanied by myeloper-
oxidase (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 diag-
nostic set-up of acute leukemias but in myelodysplastic syn-
dromes (MDS). Stainings, such as perjod acid Schiff’s reac-
tion (PAS), acid phosphatase or chloro-acetate esterase (CE)
are not further needed as they did not add important informa-
Fig. 2. Myeloperoxidase reaction in an AML M2 with Auer rods (according
tion if MFC is performed [3,10]. Exceptions may be special to FAB classification) with t(8;21) aberration (according to WHO classifi-
cases, for instance for the demonstration of glycogen in the cation, step 1) (630×).
erythroid lineage (PAS), or CE in histological sections where
it is the best method for the demonstration of neutrophilic work-flow the same syringe (EDTA- or if send over night
granulocytic lineage. better heparinized bone marrow or blood) is processed also
for molecular techniques, many laboratories use ficoll gradi-
2.1.2. Multiparameter flow cytometry ation before MFC. We would also recommend to prefer bone
For multiparameter flow cytometry cells can be processed marrow if available.
after lysis or after ficoll hypaque gradiation [7,12]. If in a

Fig. 1. Pappenheim staining of an AML M5a (according to FAB classi- Fig. 3. Non-specific esterase staining of an AML M5a (according to FAB
fication) with 11q23 aberration (according to WHO classification, step 1) classification) with 11q23 aberration (according to WHO classification, step
(630×). 1). Blasts are strongly positive (630×).
226 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

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. There-
fore, 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 cul-
tivation 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 cap-
ture systems (Fig. 4) (i.e., MetaSystems, Altlussheim, Ger-
many). 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, 24-
color FISH or comparative genomic hybridization (CGH)
are usually hybridized in an overnight procedure and are Fig. 5. (a and b) Metaphase before and after analysis by 24-color FISH
available for analysis 24 h after the sample had reached the in a case with AML and complex aberrant karyotype (software Isis® and
laboratory [16]. hybridization probes by MetaSystems, Altlussheim, Germany).
T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234 227

Table 3
The first hierarchical step in the WHO classification for AML refers to AML
with recurrent balanced chromosomal aberrations
AMLM2(1) t(8;21)
AML M3(v) t(15;17)
AML M4eo inv(16), t(16;16)
AML M4/5 11q23
Most of them correlate clearly with morphological subtypes as formerly
defined by the FAB classification.

3.1. Cytomorphology and cytochemistry

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.
Fig. 6. Interphase FISH with PML/RARA probe showing one PML/RARA Thus, the investigation of MGG, MPO and NSE at least
positive cell with co-localisation of a red and a green signal and two inde- should define if a respective blast population has myeloid or
pendent signals, and one negative cell with two independent red and green
monocytic lineage commitment or fails to show these char-
signals each, respectively.
acteristics 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
2.1.5. Molecular methods
classification based on cytomorphology is already sufficient
For molecular methods, such as PCR, real-time PCR (see
to start treatment. However, the WHO classification for AML
Kern et al. in this issue), sequencing or even gene expres-
sion profiling using microarrays samples should be processed
after Ficoll Hypaque density gradient centrifugation for DNA Table 4
or RNA preparation. This seems to be required for diag- The WHO classification for AML introduces secondary AML after pre-
nostic and for follow-up samples with respect to MRD also ceding MDS and secondary AML after pretreatment with chemotherapy or
[17–19]. radiation, so called therapy-related AML
• 10% of AML and MDS are t-AML
• Two groups can be distinguished
After
3. Classification in acute leukemias with respect to Alkylating agents Topoisomerase
diagnostic procedures II-inhibitors
Often MDS prephase No MDS prephase,
Today the classification of acute myeloid leukemias often M4/M5
Long latency period (4–6 years) Short latency
should follow the WHO proposal published in 2001 (see
period (0.5–3 years)
Tables 2–5) and will only in small parts rely further on −5/5q−, −7/7q−, complex aber- Balanced
the FAB classification [2,20–25]. Furthermore, some specific rant karyotype aberrations, often
classification systems (see Table 6 for EGIL classification) 11q23, 21q22
[26] and specific terminological aspects for cytogenetic and Poor response to therapy Better response to
therapy
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.
Table 5
The WHO classification for AML
• AML minimally differentiated
Table 2
• AML without maturation
The hierarchy of the WHO classification for AML with five steps of sub-
• AML with maturation
classifications
• Acute myelo-monocytic leukemia
1. AML with recurrent genetic abnormalities • Acute monocytic and monoblastic leukemia
(diagnosis of AML is independent of blast • Acute erythroid leukemia
count) • Acute megakaryoblastic leukemia
2. AML with multilineage dysplasia • Acute basophilic leukemia
3. Therapy-related AML and MDS • Acute panmyelosis with myelofibrosis
4. AML not otherwise categorized • Myeloid sarcoma
5. Acute leukemia of ambiguous lineage
Most terms correlate to morphological subtypes as formerly defined by the
New definition: blasts > 20% = AML, no RAEB-T. FAB classification.
228 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

Table 6 translocations t(15;17), inv(16), t(8;21) and 11q23 aberra-


The EGIL classification is used to define acute leukemias of ambiguous tions (see Table 3). This is a very important step for a
lineage and is also included in the WHO classification
more biologically defined approach in classification. There-
Score B-lymphoid T-lymphoid Myeloid fore, also in ALL cytogenetic markers are considered: ALL
2 CD79a cy/sCD3 Anti-MPO with t(9;22), 11q23 aberrations and t(8;14) are considered
2 cyIgM Anti-TZR␣␤ also as unique, biologically defined entities which need spe-
2 cyCD22 Anti-TZR␥␦
cific treatment approaches. Several additional cytogenetic
1 CD19 CD2 CD13 abnormalities with reproducible prognostic impact have been
1 CD10 CD5 CD33
defined within large clinical trials in AML as well as in ALL
1 CD20 CD8 CD65
1 CD10 CD117 in childhood and in adults [37–43].
Subentities like the APL in AML or the BCR/ABL pos-
0.5 TdT TdT CD14
0.5 CD24 CD7 CD15
itive ALL were the first ones to demonstrate clearly that
0.5 CD1a CD64 their recognition at diagnosis not only leads to specific treat-
Leukemia of ambiguous lineage: if score >2 for myeloid and also for B- or
ment circumstances including drugs like ATRA or imatinib,
T-lymphoid. respectively, but also point to the mission of future diagnos-
tic and classification approaches in acute leukemia: define
and ALL pays less attention to these morphological aspects. unique entities with specific biology, prognosis and hope-
Only for the second step of the hierarchical approach in the fully individual treatment.
WHO classification of AML the cytomorphologic perspec-
tive with respect to dysplastic features is needed (Table 2) 3.4. Molecular methods
[2,28]. However, we do not think that this parameter should be
included in future classification systems as outlined in much Reciprocal transloctions that lead to fusion genes can
more detail before by our group [5,29,30]. In conclusion, in also be detected by PCR. This is needed for validation of
AML and ALL cytomorphology classified according to FAB cytogenetic results but even more, serves as a base line for
criteria or analogeously in the WHO (see Table 5) is war- MRD studies in the respective patient. Furthermore, the most
ranted at diagnosis in acute leukemias. But more importantly, often detectable molecular defect in AML, FLT3-LM or
morphology is mandatory to direct all other techniques and point mutations [17,44–49] and MLL-PTD [50–52] are only
to process samples in shortest time. In addition, this approach detectable on the molecular level. Mutations of other genes,
saves money and avoids unnecessary investigations. such as NRAS [46,53–56], CKIT [57,58] or CEBP/A [59]
demonstrate increasing importance in AML and will have to
3.2. Multiparameter flow cytometry
be screened for at diagnosis in every single case in the near
MFC using three, four or nowadays even five color stain- future.
ing should be applied to all cases with the suspected diagnosis In ALL the most important molecular markers are
of acute leukemia. This is absolutely warranted at diagnosis BCR/ABL, all MLL translocations with their respective part-
and achieves increasing importance for MRD also (see Kern ner genes, and c-MYC aberrations that have to be detected at
in this issue) [31–35]. It is quicker and in some cases even diagnosis and for MRD studies [19,36,60,61]. Furthermore,
cheaper in comparison to other MRD techniques, such as real for more than 90% of ALL patients specific markers can be
time PCR for fusion genes or patient specific markers, such detected by IgH mutations and serve as the most important
as FLT3-LM, or IgVH-mutations (see the respective paper MRD marker making even treatment decisions possible in
in this issue by Kern et al. for AML and by Hoelzer et al. most patients in ongoing studies (see Hoelzer et al. in this
for ALL) [18,36]. These two aspects of MFC lead to a broad volume).
spectrum of antibodies to be evaluated at diagnosis in all acute The importance of a global so called multiplex-PCR
leukemias. MFC is not only mandatory for the FAB subtypes screening or gene expression profiling in acute leukemias
AML M0 and AML M7 as defined by FAB or WHO (very for diagnosis, for delineation of specific MRD markers or for
immature, or megakaryoblastic AML) [2,24,25], but leads to prognostic impact needs further investigation and can not be
the most important subclasses in ALL: B-lineage versus T- recommended at this time in a standard diagnostic setting.
lineage 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 4. New definition of remission and relapse in acute
but also add important prognostic parameters in ALL (see leukemias
the paper by Hoelzer et al. in this issue).
The diagnosis of acute leukemias depends on classifica-
3.3. Cytogenetics and FISH tion systems, such as the WHO classification, the FAB clas-
sification and the EGIL classification [2,20,22,23,26]. For
The first hierarchical step in the WHO classification of optimal diagnosis and therapeutic strategies parameters used
AML is based on cytogenetics and includes the balanced in these classifications need to be combined. Furthermore,
T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234 229

due to the broad spectrum of methods at diagnosis and for should call it “morphologic complete remission”. However,
follow-up studies including MRD parameters new definitions in many treatment protocols of AML a second course of
of remission as well as for relapse are needed. In a recent therapy follows closely related to the first induction course.
paper [4], several new and important aspects were included Therefore, for morphologic CR a reconstitution of bone mar-
with focus on AML. The revised recommendations at first row and blood cells for >4 weeks is not further needed.
followed the WHO classification for definition of AML at In cases with abnormal cytogenetics at diagnosis, normal
diagnosis: metaphases and FISH studies have to be detected after a
therapy to reach a cytogenetic complete remission (CRc).
(1) De novo AML should only be referred to if no clinical
However, as data are lacking these two techniques should
history of MDS, MPS or exposure to potentially leuke-
be further evaluated within clinical trials for their prognostic
mogenic therapies or agents is known.
impact in the near future in parallel to others [63].
(2) Secondary AML should refer to patients who have such
The term molecular complete remission (CRm) will be
history and should further be subdivided into secondary
used after MRD studies by MFC for the detection of a
AML after MDS or MPS versus secondary AML after
leukemia-associated immunophenotype (LAIP), or by PCR
proven leukemogenic exposure. We would suggest to fol-
techniques. Both approaches are more sensitive than mor-
low the WHO and call these latter leukemias or MDS
phology alone or cytogenetics and FISH in combination [63].
cases therapy-related AML (t-AML) within the group of
Further studies in parallel are needed for clinical decisions.
s-AML.
However, real time PCR approaches in some molecularly
It seems important to follow the new definition by Cheson et defined subgroups of AML and ALL proved already their
al. that also 1 month or greater preleukemic state should not outstanding prognostic significance in large trials [18,36].
by itself allow the designation of a case into s-AML without Cheson et al. also newly defined the term partial remission
confirmation of pre-existing blood smear that clearly demon- that is relevant for phase I and II trials. A blast count in the
strated morphologic dysplasia. bone marrow aspirate between 5% and 25% or at least half of
Although the absence or presence of dysplasia should the percentage that was measured before treatment is required
be recorded according to the WHO classification and was to call the status partial remission.
also included in the paper by Cheson et al. from our point A relapse after CR was newly defined as a reappearance
of view further studies including interlaboratory confirma- of any leukemic blasts in the peripheral blood, or ≥5% blasts
tion and central review processes are needed. Morphologic in the bone marrow not attributable to regeneration after
characteristics often only demonstrated a limited inter- and chemotherapy treatment.
intraobserver reproducibility, even by thresholds for dyspla-
sia of 50% in AML [28] (and 10% in MDS as defined by
the FAB group and also included in WHO definitions) [2,22] 5. Important aspects at diagnosis of specific subtypes
these criteria have to be evaluated further. Also multivariate in acute leukemia
analyses including cytogenetics and age are needed before
dysplasia can lead to treatment decisions or defines poor risk 5.1. Acute myeloid leukemia
patients in de novo AML.
As new techniques emerged, such as FISH, MFC and PCR In all cases of AML cytomorphology, cytochemistry, MFC
for the detection of MRD, it was necessary to define new cat- and cytogenetics should be performed at diagnosis.
egories of remission. Thus, the paper published by Cheson et The cytogenetic result at diagnosis is still the most
al. at first defined a new so called “early treatment assessment important single prognostic parameter and leads to treatment
7–10 days after therapy”. At this time point blasts in the bone stratification not only in APL. It also serves as a basis for
marrow can be assessed morphologically, by FISH, PCR and strategies in CBF leukemias, i.e., AML with t(8;21) or AML
MFC. Recent papers were able to demonstrate the high prog- with inv(16), in combination with real time PCR [18]. In
nostic impact of this evaluation at such an early time-point as complex aberrant cases decisions with respect to early trans-
assessed by cytomorphology using blast percentages alone plantation strategies may be driven by the cytogenetic results
[62]. As this is the first therapy-dependent parameter to be [64].
measurable in AML it should be investigated further. Paral- In certain cases metaphase cytogenetics the performance
lel studies with MFC and interphase-FISH could clarify the of IP-FISH or WCP-FISH in addition for validation is helpful.
role of each method for the evaluation of residual disease and Twenty four-color FISH provides detailed additional insight
their prognostic impact. in cases with complex aberrant karyotypes (three or more
Within the term “complete remission” three different cat- chromosomes involved) [15]. Although the latter technique
egories should be separated in future studies. In all new CR will only add information usually not needed for therapeutic
categories the neutrophils in the pB should exceed >1000/␮l decisions this method is recommend at least in clinical trials
and the platelet count should be >100,000/␮l, the bone mar- for further pathobiological investigations. The value of com-
row blast count should be <5% (at least 200 cells should parative genomic hybridization in AML genetics for routine
be counted). If these three parameters are measurable, one use is still not defined.
230 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

A cytogenetic result is also mandatory for the first hierar- (see Hoelzer et al. in this issue). Further treatment stratifi-
chical step in the WHO classification [2]. It may further delin- cation will be possible in the near future by PCR studies
eate patients with therapy-related AML (t-AML) between the that are performed with respect to patient specific primers at
most important two subtypes as defined in the WHO to (i) t- defined timepoints during the first months of treatment [36].
AML following treatment with alkylating agents or (ii) after The results will stratify the therapy especially in so called
treatment with topoisomerase II inhibitors. Such mechanism standard risk patients in the near future also in adults (see
of leukemia induction in general lead to two different sub- Hoelzer et al. in this issue) as has been proven very success-
types of t-AML: those with cytogenetic aberrations involving fully in childhood ALL.
5q−, −7 or p53 and in most cases with complex aberrant
karyotypes after alkylating agents, in contrast to 11q23 or
6. Problems and pitfalls in the diagnosis and at
other balanced cytogenetic aberrations after topoisomerase
follow-up of acute leukemias
II inhibitors. Thus, metaphase-cytogenetics gives important
information and also describes in patients with t-AML prog-
Only a comprehensive diagnostic approach in patients sus-
nosis best [65].
pected to have an acute leukemia would lead to an adequate
In addition to standard metaphase cytogenetics interphase
diagnosis and treatment. Most of the parameters defined by
FISH is very helpful not only for verification but can give
a combination of different methods add further information
immediate information with respect to the question, if an
also for prognosis. This can only be achieved by investigation
APL with PML/RARA fusion genes is present or not. As
of blood and bone marrow before any treatment was adminis-
this question is of highly important therapeutic impact and
tered (also no corticosteroids in ALL cases!). Samples have
not in all cases morphology is available interphase FISH is
to be supplemented with EDTA or heparin for the respec-
recommend in all cases of suspected APL [16].
tive analyses (see Table 1). The number of necessary cells
For follow-up studies further investigations with cyto-
to be analysed is important not only at diagnosis but espe-
morphology, MFC, hypermetaphase-FISH, IP-FISH and real
cially for follow-up studies. If hampered by too few cells
time PCR are needed. We hopefully will be able to define the
(low sensitivity) MRD results have to be taken with cau-
value of each technique in an ongoing prospective trial and
tiousness. Parallel investigations applying two methods at the
will define an algorithm for methods at follow-up time points
same time, such as cytomorphology combined with FISH, or
[63].
MFC in combination with real time PCR should be performed
for clinical decisions. Algorithms for diagnostic approaches
5.2. Acute lymphoblastic leukemia in acute leukmias are shown in Tables 8 and 9 summarize the
actual procedures.
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 7. Conclusions and future developments in the
informative in cases with Burkitt-type ALL (formerly called diagnosis of acute leukemias
L3 morphology according to FAB classification). However,
if this subtype is suspected morphologically further cyto- Every treatment in an acute leukemia patient should be
genetic and FISH investigations must be performed for based on a diagnostic set-up that is able to combine cyto-
validation. morphology, cytochemistry, multiparameter flow cytometry,
From the cytogenetic point of view, several ALL specific cytogenetics, fluorescence in situ hybridization and molecu-
aberrations can be defined (see Table 7). Out of these the lar genetic methods if needed. If this can not be guaranteed
t(9;22), t(4;11) and the t(8;14) are important due to their one has to be aware that todays standard therapies in AML
specific prognostic impact and specific treatment protocols and ALL may be inadequate and treatment outcome subop-
timal.
Furthermore, during course of treatment many ongoing
Table 7 studies focus on minimal residual disease. These results have
Cytogenetic aberrations that can be found in ALL and their respective fre- increasing importance for further therapy, i.e., they may even
quencies in children and adults
form the basis for the decision to apply allogeneic periph-
Childhood (%) Adults (%) eral stem cell transplantations for patients with poor risk
t(1;19) Pre-B-ALL 5–6 3 leukemias. On the other hand, MRD markers can also lead to
t(4;11) Pro-B-ALL 2 6 de-escalation of treatment or even an early stop of therapy in
t(9;22) c-ALL 2–5 25–30
t(8;14) B-ALL 3 5
cases with a very low relapse risk.
t(10;14) T-ALL 1 3 Other techniques, such as gene expression profiling and
t(12;21) Pre-B-ALL 10–15 <1 proteomics begin to add important information at diagnosis
9p T, pre-B 7–12 15 and for prediction of response to specific treatment protocols
6q c-, pre-B,T 4–13 6 in acute leukemias [6,8,9,66–70]. It will be very interesting to
14q11 T-ALL 1 6
test gene expression profiling these in comparison to today’s
T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234 231

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 Reviewers
add further clarification to known leukemia subtypes but may
also deliniate new entities, for example, in AML with normal Prof. Fausto Grignani, Istituto Clinica Medicina 1, Poli-
karyotypes. It may even be tested to predict response and may clinico Monteluce, Via Brunamonte, I-06122 Perugia, Italy.
therefore help not only for diagnostic but also for therapeutic Prof. Dr. H. Löffler, Seelgutweg 7, D-79271 St. Peter, Ger-
purposes. However, future studies will have to demonstrate many.
the value of such methods in the context of modern leukemia Prof. Laurent Degos, IUH, Hôpital St. Louis, 1 av. Claude
diagnostics and their possible impact on treatment decisions. Vellefaux, F-75010 Paris, France.
232 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

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234 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234

Biographies PD Dr. rer.nat. Susanne Schnittger: Ph.D. in human


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