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
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 neces-
sary 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 immunophenotyp-
ing accompanied by cytogenetics and molecular genetic
methods has to be established in a laboratory setting [1].
As methods not only add important information at diag-
nosis but more and more also define markers for mini-
mal residual disease (MRD) studies, this has to be con-
sidered at the first time point of any analysis in every
single patient. Furthermore, diagnostic results are equiva-
lent 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 multipa-
rameter flow cytometry (MFC), and metaphase cytogenetics
in every case, in which acute leukemia is suspected. The lat-
ter 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 sub-
types 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 pro-
vide 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 leukemogen-
esis. 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 heteroge-
neous group of different biological entities. The importance
of cytogenetic and molecular genetic findings for the classifi-
cation 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 classifica-
tion, therapeutic decisions, MRD studies, leads to prognostic
markers and is cost effective if applied in a step-wise work-
flow.
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 num-
bers 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 cyto-
genetics, 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 precondi-
tions should be considered [6–9]. This also means that for
the diagnosis of acute leukemias a central reference lab-
oratory has to be available every day for optimal service
[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
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 CD79a cy/sCD3 Anti-MPO
2 cyIgM Anti-TZR␣␤
2 cyCD22 Anti-TZR␥␦
1 CD19 CD2 CD13
1 CD10 CD5 CD33
1 CD20 CD8 CD65
1 CD10 CD117
0.5 TdT TdT CD14
0.5 CD24 CD7 CD15
0.5 CD1a 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 perspec-
tive 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 war-
ranted 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 stain-
ing 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 T-
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
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 aberra-
tions (see Table 3). This is a very important step for a
more biologically defined approach in classification. There-
fore, 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 spe-
cific 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 pos-
itive ALL were the first ones to demonstrate clearly that
their recognition at diagnosis not only leads to specific treat-
ment circumstances including drugs like ATRA or imatinib,
respectively, but also point to the mission of future diagnos-
tic and classification approaches in acute leukemia: define
unique entities with specific biology, prognosis and hope-
fully 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 part-
ner 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 classifica-
tion systems, such as the WHO classification, the FAB clas-
sification 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,
 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234
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 leuke-
mogenic 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 fol-
low 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 demon-
strated 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 confirma-
tion and central review processes are needed. Morphologic
characteristics often only demonstrated a limited inter- and
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]
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 cat-
egories 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 prog-
nostic 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. Paral-
lel 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 cat-
egories 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 mar-
row blast count should be <5% (at least 200 cells should
be counted). If these three parameters are measurable, one
229
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 mar-
row 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 mor-
phology 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 trans-
plantation 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 com-
parative genomic hybridization in AML genetics for routine
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-
chical step in the WHO classification [2]. It may further delin-
eate patients with therapy-related AML (t-AML) between the
most important two subtypes as defined in the WHO to (i) t-
AML following treatment with alkylating agents or (ii) after
treatment with topoisomerase II inhibitors. Such mechanism
of leukemia induction in general lead to two different sub-
types 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 prog-
nosis 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 cyto-
morphology, 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 cyto-
genetic 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 fre-
quencies in children and adults
Childhood (%)
t(1;19) Pre-B-ALL 5–6
t(4;11) Pro-B-ALL 2 6
t(9;22) c-ALL 2–5
t(8;14) B-ALL 3 5
t(10;14) T-ALL 1 3
t(12;21) Pre-B-ALL 10–15
9p T, pre-B 7–12
6q c-, pre-B,T 4–13
14q11 T-ALL 1 6
(see Hoelzer et al. in this issue). Further treatment stratifi-
cation 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 success-
fully in childhood ALL.
6. Problems and pitfalls in the diagnosis and at
follow-up of acute leukemias
Only a comprehensive diagnostic approach in patients sus-
pected 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 adminis-
tered (also no corticosteroids in ALL cases!). Samples have
to be supplemented with EDTA or heparin for the respec-
tive analyses (see Table 1). The number of necessary cells
to be analysed is important not only at diagnosis but espe-
cially for follow-up studies. If hampered by too few cells
(low sensitivity) MRD results have to be taken with cau-
tiousness. 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 cyto-
morphology, cytochemistry, multiparameter flow cytometry,
cytogenetics, fluorescence in situ hybridization and molecu-
lar 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 subop-
timal.
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 periph-
Adults (%) eral stem cell transplantations for patients with poor risk
3 leukemias. On the other hand, MRD markers can also lead to
de-escalation of treatment or even an early stop of therapy in
25–30
cases with a very low relapse risk.
Other techniques, such as gene expression profiling and
<1 proteomics begin to add important information at diagnosis
15 and for prediction of response to specific treatment protocols
6 in acute leukemias [6,8,9,66–70]. It will be very interesting to
test gene expression profiling these in comparison to today’s
 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234
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.
231
Reviewers
Prof. Fausto Grignani, Istituto Clinica Medicina 1, Poli-
clinico Monteluce, Via Brunamonte, I-06122 Perugia, Italy.
Prof. Dr. H. Löffler, Seelgutweg 7, D-79271 St. Peter, Ger-
many.
Prof. Laurent Degos, IUH, Hôpital St. Louis, 1 av. Claude
Vellefaux, F-75010 Paris, France.
232 T. Haferlach et al. / Critical Reviews in Oncology/Hematology 56 (2005) 223–234
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Biographies
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. 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. 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 anal-
ysis, sequencing.
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 multi-
color FISH and gene expression analysis with microarrays.