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Leukemia Diagnostics

Definition

Leukemia diagnostics comprises the combined application of different methodologies in a standardized fashion, allowing the precise diagnosis, subclassification, and determination of prognostic parameters.

Characteristics

Introduction

The diagnosis of leukemias today is based on a comprehensive approach applying a variety of different methods in combination. While cytomorphology and cytochemistry have been the mainstay of diagnostics for decades and have been used to differentiate between acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and myelodysplastic syndromes (MDS) the current up-to-date approach includes immunophenotyping/Multiparameter flow cytometry (MFC) as well as cytogenetics and molecular genetics to correctly diagnose and subclassify leukemias.

Multiparameter flow cytometry (MFC) is used for immunophenotyping of the leukemic cells which is essential particularly to diagnose and classify lymphatic leukemias. Karyotyping and fluorescence in situ hybridization (FISH) are applied to detect chromosomal alterations which are considered disease-defining lesions in an increasing number of entities. Different molecular genetic techniques (PCR, fragment analysis (Genescan), heteroduplex analysis, melting point analysis, nested PCR, real time PCR, sequence analysis, Single stranded conformation polymorphism analysis (SSCP)) allow the identification of leukemia-specific fusion genes and gene mutations and are also an integral part of the diagnostic work-up of leukemias.

The basis for establishing this diversified diagnostic approach has been the growing body of insight into the genetics of leukemias, which not only allowed the definition of recurring genetic aberrations but also their strong correlation to distinct disease subtypes with specific clinical characteristics, including prognostic impact. As a consequence, today the decision to treat a patient suffering from a particular leukemia and the specific therapeutic option to select for him or her largely depends on the presence or absence of these genetic alterations.

The monitoring of minimal residual disease (MRD) increasingly gains clinical relevance in patients with leukemias. While conventional methods like cytomorphology can be used to assess the prognostically highly relevant achievement of complete remission, newly developed methods like multiparameter flow cytometry and quantitative real time PCR allow the exact quantification of the amount of residual malignant cells in patients with complete remission. The level of this MRD in many cases significantly correlates with the further course of the disease and is incorporated into the guidance of a risk-adapted therapy.

The present essay provides, in the first step, an overview of the methods applied in leukemia diagnostics and then focuses on the respective leukemia subentities and their specific diagnostic findings. The content is then summarized by comprehensive algorithms detailing the modern diagnostic work-up for the different leukemias.

Methods Applied in Leukemia Diagnostics

Cytomorphology and Cytochemistry

Cytomorphologic analysis and cytochemistry are performed on peripheral blood and bone marrow smears which are air-dried without fixation before staining. A panoptic staining (Pappenheim or May Gruenwald Giemsa) is used for the general assessment of cell characteristics. Cytochemistry applies myeloperoxidase staining and non-specific esterase staining for the identification of myeloid and monocytic cells, respectively, while the use of Schiff's reaction, acid phosphatase, and chloroacetate esterase (CE) have been substituted by immunophenotyping. Iron staining is applied in the diagnostic setting of MDS.

Immunophenotyping

Immunophenotyping by multiparameter flow cytometry allows the identification, quantification, and characterization of cell populations in peripheral blood or bone marrow samples. Cells are differentiated from each other, based on their light scatter features during their pass across a laser beam (higher cell sizes result in higher forward scatter signal [FSC]; higher heterogeneity of cell content results in higher side scatter signal [SSC]) as well as on their antigen expression patterns. As 1,000–2,000 cells are analyzed per second, the simultaneous analysis of the expression of five antigens in 10,000 cells within seconds is current standard. Populations can be characterized even if their concentration is only around 1%. During the monitoring of minimal residual disease (MRD), 250,000 cells are analyzed.

While the detection of FSC and SSC signals is possible in unmanipulated cells, the detection of antigens requires the use of monoclonal antibodies against the respective antigens, which are coupled to a fluorescent dye. Five or even more different fluorescent dyes can be routinely used as the light emitted differs in wave length and is specifically recognized by different detectors. Sophisticated compensation programs, however, are necessary to account for overlaps in the spectrum of the emitted light.

Due to its expression on all peripheral blood and bone marrow at different levels, CD45 represents an ideal antigen for performing a differential count. Thus, monocytes feature the strongest expression of CD45 and may be separated from lymphocytes, which also express CD45 quite strongly, based on differences in the SSC signal. Granulocytes express CD45 at a lower level and feature the strongest SSC signal. Importantly, erythrocytes hardly express CD45 and blasts show a CD45 expression level similar to granulocytes; however, both the latter populations are easily separated from each other, based on their SSC signal (low SSC signal in blasts). Therefore, CD45 gating is useful for the analysis of different cell populations, particularly for the analysis of blasts.

The simultaneous detection of the expression of multiple antigens within one tube allows the comprehensive assessment of antigen expression patterns in different cell populations, which, in comparison to single and dual color approaches, leads to quick and valid results in diagnostics, particularly in the quantification of MRD. The panel of antibody combinations is selected, respectively, in consideration of the suspected diagnosis and may be supplemented based on primary results.

Cytogenetics

Importance of Chromosome Analysis, Indication

Chromosome analysis today is an essential part of the diagnosis in hematologic neoplasias. The results help in establishing the diagnosis. Most importantly, however, chromosome analysis provides prognostic information which is derived from the karyotype of the malignant cells. The neoplasia-associated chromosome aberrations are acquired genetic alterations and are limited to the malignant cells. Thus, the non-malignant cells in patients with hematologic neoplasias are cytogenetically normal.

Material

For chromosome analysis, bone marrow is preferred to peripheral blood because malignant cells are present at higher percentages and have a higher proliferative activity. If bone marrow cannot be obtained, the cytogenetic analysis may be done on peripheral blood cells. Since viable cells are needed for metaphase cytogenetics, the bone marrow should be shipped into the cytogenetic laboratory within 24 h. The cells must not be frozen.

Conventional Chromosome Analysis Using Banding Techniques

A sufficient number of good quality metaphases is needed for chromosome analysis. Bone marrow cells are arrested in the metaphase by the addition of colchicine either directly after drawing the sample or after short-term cultivation (24–72 h). To maximize the gain of metaphases the leukemic cells can be stimulated by cytokines during cultivation. The addition of hypotonic potassium chloride solution swells the cells and they are fixed in multiple steps by the use of a methanol acetic acid solution. Then the cells are dropped on to glass slides. To allow the unequivocal identification of each chromosome, a banding technique must be applied. The most frequently applied banding techniques are G- (Giemsa-), Q- (Quinacrin-), and R- (reverse) banding. These techniques lead to the appearance of light and dark bands on the chromosomes which are specific for each chromosome and allow an unequivocal identification of each chromosome. To allow a valid report a complete analysis of 20–25 metaphases is required according to the respective international consensus.

Nomenclature – ISCN Cytogenetics

Chromosomes are classified according to their size, the position of the centromere (which divides both arms of the chromosomes), and the characteristic banding patterns. Each chromosome has a short arm (p) and a long arm (q). Based on the banding pattern, each chromosome is divided into regions and bands, which are numbered from the centromere to the telomere. The internationally accepted cytogenetic system of nomenclature (ISCN: International System of Cytogenetic Nomenclature) allows the exact description of all numeric and structural aberrations in a karyotype formula. The karyotype formula in the first place gives the number of chromosomes followed by the sex chromosomes. Thus, the normal female karyotype is 46,XX and the normal male karyotype is 46,XY.

The numeric chromosome aberrations include monosomies (loss of a chromosome) and trisomies (gain of a chromosome). Furthermore, the complete set of chromosomes may be multiplied. In normal cells a double set of chromosomes (diploid chromosome set) is present. Three- and fourfold sets of chromosomes are designated as triploid and tetraploid.

The most frequently occurring structural chromosome aberrations are deletions (losses of parts of chromosomes), chromosomal translocations (exchange of parts of chromosomes between different chromosomes), inversions (twisting of a part of a chromosome by 180°), and isochromosomes (a chromosome consisting of either two short arms or two long arms while the respective other arms are lost).

In the karyotype formula a gain of a chromosome is indicated by a “+” and a loss of a chromosome is indicated by a “−”; e.g. 47,XX,+8 is a trisomy of chromosome 8 and 45,XY,−7 is a monosomy of chromosome 7. There are abbreviations for structural chromosome aberrationens which are internationally agreed on, e.g. “t” for translocation and “inv” for inversion: t(8;21)(q22;q22) indicates a break in chromosome 8 at band q22 and a break in chromosome 21 at band q22 as well as an exchange of the fragments between both chromosomes. Different chromosomes and breakpoints in different chromosomes are divided by in the karyotype formula, while breaks within a chromosome are listed without a separator, e.g. inv(16)(p13q22): breaks occurred in chromosome bands p13 and q22 of a single chromosome 16 with a fragment being twisted by 180°. Another example is del(5)(q13q31): breaks occured in the bands q13 and q31 of a single chromosome 5 with a loss of the area between q13 and q31.

Chromosome aberrations are designated clonal if an identical structural aberration or a gain of a chromosome is observed in at least two metaphases or if a loss of the same chromosome is observed in at least three metaphases.

Comparative Genomic Hybridization (CGH)

Comparative genomic hybridization allows a comprehensive analysis of the tumor genome with regard to over- and underrepresented DNA sequences (losses and gains of chromosomes, deletions, amplifications). The technique is based on the simultaneous staining of both test DNA of a healthy volunteer and tumor DNA using two different fluorescent dyes. Identical aliquots of both DNA samples are then mixed and hybridized on normal metaphases. Differences in the numbers of copies in different sequences between normal and tumor DNA are detected by the quantification of the ratio of fluorescence intensity (tumor DNA to normal DNA) in each region of the normal metaphase chromosomes. Balanced translocations, inversions, or other aberrations which are not accompanied by a change in the number of copies are thus not detectable by CGH. CGH may play an important role particularly if a chromosome analysis is not possible, e.g. if living tumor cells are not available or if tumor cells do not proliferate in vitro.

Fluorescence In Situ Hybridization (FISH)

The FISH technique relies on the hybridization of DNA probes which identify specific chromosomal structures. Probes can be used which are specific for the centromeric region of particular chromosomes, for genes, or for complete chromosomes. The DNA of both the applied probe and of the patient sample are denaturated, i.e. both DNA strands of the double helix are separated. During the following renaturation, the DNA probes attach to the complementary section of the patient DNA (hybridization). The DNA probes are either directly conjugated to a fluorescent dye or are analyzed using fluorescence conjugated antibodies. The respective chromosome structures therefore are assessable as fluorescence signals.

A significant advantage of the method lies in its applicability not only to metaphases but also to interphase nuclei. A disadvantage is that information is obtained only on chromosomes and genes for which probes are used.

The role of the FISH technique differs between the different leukemia subgroups.

Interphase FISH

Due to the multitude of different chromosome aberrations, which are observed particularly in acute leukemias, a screening based on FISH on interphase nuclei covers only a fraction of potentially present aberrations and therefore cannot substitute the classic chromosome analysis. However, if a specific question should be answered, e.g. the detection of the translocation t(15;17)(q22;q12) when acute promyelocytic leukemia is suspected, the FISH technique represents a fast and reliable method, providing a result within 4 h.

In follow-up assessments during therapy the FISH technique can be used for the detection of residual disease if at diagnosis aberrations have been found by chromosome analysis for which FISH probes are available. The sensitivity for this method is higher than for the chromosome analysis; however, it is lower than for PCR.

Metaphase FISH

In addition to the probes applicable to interphase nuclei, so-called chromosome painting probes can be applied to metaphases which specifically bind to the complete DNA of a chromosome. This technique is used mainly for the confirmation of the conventional chromosome analysis in difficult cases.

The 24-color-FISH method allows the display of all 22 different pairs of chromosomes as well as of the sex chromosomes in one single hybridization. It is applicable to metaphase chromosomes only and helps in identifying complex structural aberrations.

Molecular Genetics

PCR

PCR is the most frequently applied method to detect molecular genetic changes in leukemia. By using oligonucleotides specific for certain sequences, regions of interest can be amplified and further analyzed. All the following methods are based on this standard method.

RT-PCR

In specific cases, especially when fusion genes are analyzed in which breakpoints are distributed over a wide genomic range, it is advantageous to use cDNA as the template for PCR.

Nested PCR

Using this method, both the sensitivity and specificity of a PCR can be increased. In hematology, it is frequently applied to detect small amounts of molecules, i.e. mainly for the detection of minimal residual disease. For a nested PCR a fraction of a finished PCR is used for a new PCR. Oligonucleotides that hybridize within the first amplificate are used as primers. This additional amplification increases the sensitivity significantly. Depending on the type of mutation and the initial material, one malignant cell in 104 to 108 normal cells can be detected. Thus, this is the most sensitive method currently available for most sequences targeted for the detection of minimal residual disease (MRD). However, due to the high sensitivity, this method also carries the highest risk of contamination. In order to minimize this risk, different precautions must be applied and control reactions must be performed in parallel.

Real Time PCR

Another method for the amplification and detection of PCR products is the real time PCR. Different from other methods of detection, this is not an end-point analysis, but the measurement is performed during the phase of PCR when a logarithmic amplification of PCR products occurs. This allows an exact quantification of the target sequences in the material which is assessed. The method is based on the addition of fluorescence-conjugated probes to the specific primers required for the PCR. These probes hybridize during the running PCR with the continuously increasing amplification products and release fluorescence signals, which are detected in an optical device specifically constructed for this approach. Thus, an increase of fluorescence intensity occurs during PCR. The time point (PCR cycle) at which a fluorescence higher than baseline is detected for the first time in a sample correlates with the number of targeted molecules in the sample. The fluorescence intensity of the targeted gene is normalized to a constantly present gene or transcript, based on which the number of malignant cells present in the sample can be calculated. Also, for this method of detection, the application of specific PCR machines equipped with optical devices is needed. Using these machines it is also possible to conventionally detect PCR products; however, the strength of real time PCR is its capacity for an exact quantification for follow-up analyses.

Mutation Screening

Different methods are available, which allow the screening of defined gene regions for mutations without the necessity of sequencing. Some examples are listed below:

Heteroduplex Analysis

As the first step in this analysis, a region with a suspected mutation is amplified by PCR. Since in man two alleles of each locus are normally present, two different PCR products occur. After completion of the PCR, these are denaturated (conversion to single-strand state) and then renaturated. Consequently, four renaturated products are possible: a normal double strand, a mutated double strand, and two heteroduplices, in which one base, respectively, is not paired to the other strand, i.e. a mismatch is present. These heteroduplices are detectable on specific gels or by a dHPLC device (WAVE).

SSCP Analysis (Single Stranded Conformation polymorphism Analysis)

In this analysis denaturated single strands are separated using a non-denaturating surrounding by gel or capillary electrophoresis. It takes advantage of the fact that mutated single strands lead to other secondary structures as compared to unmutated ones and thereby have different features within the electric field.

Melting Point Analysis

In addition to the classic real time PCR, a further step is applied. Fluorescence-marked probes, which lie on a potentially mutated area, are slowly melted off the PCR products. In case of a mutation under the probes, a so-called mismatch occurs. Thus, the probes fit less well in the wildtype and faster melt off the PCR product.

Fragment Analysis (Genescan)

As an alternative to the gel analysis, PCR fragments can be detected by a fragment analysis which is also called Genescan-analysis. This method is performed using sequencing machines equipped with special software. To allow the detection, one of the two PCR primers is conjugated to a fluorescent dye. The advantage of this method is its capacity to determine the length of the amplificates exactly. In addition, with some limitations, it is possible to quantify the amplificates in comparison with another gene, which is generally the unmutated wild type. The method needs more extensive equipment and resources, due to the use of primers conjugated to fluorescent dyes, as compared to the standard gel analysis. It is frequently used in the context of multiplex PCR reactions, i.e. reactions employing multiple primers with the parallel amplification of up to four different loci. An example of this application is the chimerism analysis after allogeneic stem cell transplantation with microsatellite markers.

Sequence Analysis

The exact delineation of the base sequence of a region of a gene is frequently needed. To allow this, sequencing reactions are performed with nucleotides being integrated during PCR based reactions. Each of the four different nucleotides is labeled to a fluorescent dye and a dideoxy group, respectively, and leads to a chain stop in the PCR reaction. Subsequently, the products are separated according to their length on a matrix (e.g. a gel or a polymer) and are detected by an optical device allowing the exact determination of the base sequence.

For some gene regions the sequence analysis is feasible as a primary approach in a limited number of patients. In case of a screening of multiple gene regions in one patient or of one gene in many patients, the direct sequencing requires large resources. In this instance, different screening approaches are available for the assessment of mutations in particular gene regions without the need for a full sequence analysis.

Diagnostic Procedures in Leukemia

Diagnostic Workup of AML

Cytomorphology

The examination of both bone marrow and peripheral blood smears by cytomorphology and cytochemistry is necessary to diagnose AML according to FAB criteria (Table 1). Based on the FAB (French-American-British) classification, which was established in 1976 and revised in 1985, AML is subdivided into 11 morphologically different groups. Some of these groups correlate with distinct genetically defined forms of AML; however, most of them do not. Increasing insights into the biology of AML as well as the identification of specific chromosome aberrations prompted the WHO to suggest a new classification in 2001, based on biology and cytogenetics.

Table 1 Definition of subgroups of AML according to FAB, association with genetic aberrations
FAB- Subtype FAB-criteria Association with
Granulopoiesis Monopoiesis Erythro- poiesis Immunologic marker Cytogenetics Molecular genetics Frequency
M0 <10%
POX <3%
<20% <50% Myeloid positive
Lymphoid negative
M1 <10%
POX >3%
<20% <50% t(8;21) AML1-ETO 1.7%
M2 >10% maturation <20% <50% t(8;21) AML1-ETO 12.5%
M3 Hypergranular
Auer-rods
<20% <50% HLA-DR negative t(15;17) PML-RARA 98%
M3v Microgranular
monocytoid nuclei
<20% <50% HLA-DR negative t(15;17) PML-RARA
M4 >20% >20% <50%
M4Eo >20%
abnormal eosinophils
>20% <50% inv(16)/t(16;16) CBFB-MYH11 100%
M5a <20% >80%
immature
<50% 11q23 Aberr. MLL-Fusion 31%
M5b <20% >80%
mature
<50% 11q23 Aberr. MLL-Fusion 17%
M6 >30% of NEC are blasts variable >50%
M7 >30% megakaryoblasts variable <50% CD41/CD61 positive

Bold = typical finding; NEC = non-erythroid cells.

The FAB classification subdivides AML based on cytomorphology and cytochemistry into different subgroups. With only a few exceptions, the diagnosis of AML requires bone marrow blasts of at least 30% of all nucleated cells; at least 3% of the blasts must react positive for myeloperoxidase. The threshold for blasts is 20% in the WHO classification.

The AML classification suggested by the WHO combines the methods previously used for the FAB classification, i.e. cytomorphology, cytochemistry, and immunophenotyping, with cytogenetics and molecular genetics as well as with clinical parameters (Table 2). The genetic characterization of AML, in particular, not only allows a classification according to the prognosis, but provides the basis for the definition of distinct subgroups with recurrent balanced translocations. Thus, the WHO classification divides AML into four groups:

  1. AML with recurrent cytogenetic aberrations
  2. AML with myelodysplasia-associated features
  3. Therapy-related AML and MDS
  4. AML not otherwise categorized

The first group comprises biologically and clinically largely consistent subgroups of AML. The WHO classification thus represents a significant development of the FAB classification. The independent biologic and prognostic impact of groups 2 to 4, however, has not been demonstrated yet. Importantly, the subdivision of group 4 essentially reflects the FAB classification. A further new definition in the WHO classification is the deletion of MDS-RAEB-t and the reduction of the limit between MDS and AML from 30 to 20% bone marrow blasts.

Table 2 WHO classification of AML
AML with recurrent cytogenetic aberrations AML with t(8;21)(q22;q22), AML1(CBF-alpha)-ETO
Acute promyelocytic leukemia (AML with t(15;17)(q22;q11-12) and variants, PML-RARA)
AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(16;16)(p13;q22), CBFB-MYH11)
AML with 11q23(MLL)-fusions
AML with multilineage dysplasia AML following a myelodysplastic syndrome
AML without antecedent myelodysplastic syndrome
AML and myelodysplastic syndrome, therapy-related Alkylating agent-related
Topoisomerase type II inhibitor-related (some may be lymphoid)
Other types
AML not otherwise categorized Minimally differentiated AML
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic or monocytic leukemia
Acute erythroid leukemia
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
Immunophenotyping

AML is diagnosed on the basis of cytomorphology and cytochemistry. The only exceptions are AML M0 and AML M7 in which immunophenotyping is also necessary. Furthermore, some genetically defined subgroups feature typical although not fully specific immunophenotypes which are therefore used to guide further diagnostics only.

According to the abundance of promyelocytes, the AML M3 shows characteristic findings in the scatter plot and in addition is negative for HLA-DR and has a strong unspecific fluorescence signal. However, some of these findings can be present also in AML M2.

Typically, in AML M2 with t(8;21) there is an aberrant coexpression of CD19 and CD56; this can also be found in some AML without t(8;21).

The same is true for the immunophenotype of AML M4Eo with positivity for CD2 and an asynchronous coexpression of CD15 and CD34 which, however, may be present in other AML subtypes as well.

AML M0 is undifferentiated and lacks myeloperoxidase positivity in cytochemistry. Thus, the diagnosis cannot be made based on morphology alone. Immunophenotyping allows the distinction from ALL based on the expression of CD13, CD33, and CD117 and other myeloid antigens and at the same time a lack of lymphatic antigens or, respectively, a low lymphatic score not sufficient to diagnose a biphenotypic acute leukemia. In half of the cases an expression of MPO can be detected although cytochemistry shows negativity for myeloperoxidase.

AML M7 is negative for myeloperoxidase in cytochemistry. The expression of CD41 or CD61 is detected flow cytometrically. Due to the frequently occurring myelofibrosis a cytologic examination may not be possible in some cases.

Monitoring of Minimal Residual Disease (MRD)

In patients with AML a significant prognostic impact of immunologically determined levels of MRD has been demonstrated. Due to the similarity of immunophenotypes between AML and normal bone marrow in some cases early work focused on highly aberrant leukemia-associated aberrant immunophenotypes (LAIPs) including mainly the aberrant expression of lymphoid markers (CD19, CD7, CD56) and the asynchronous expression of progenitor cell and differentiation markers (e.g. CD34+CD117-CD15+).

For MRD levels determined after the achievement of complete remission and completion of consolidation therapy, a significant impact on both relapse-free and overall survival has been demonstrated, which has been largely independent of other prognostic parameters.

Newer studies have aimed at extending the immunologic MRD analysis to patients with less aberrant LAIPs and thus at the applicability of the method to each patient with AML. By the use of a comprehensive panel of monoclonal antibodies this task has been accomplished with a median sensitivity of 0.05%. Further analyses using this approach have confirmed the prognostic impact of MRD levels. Thus, as early as on day 16 of the induction therapy, patients can be divided into two prognostically differing groups based on the MRD level (2-year event-free survival: 53% vs. 19%, p<0.0001; 2-year overall survival: 58% vs. 43%, p=0.0133). Furthermore, the MRD levels determined after both achievement of complete remission and completion of consolidation therapy, demonstrate a similar prognostic impact, which is independent of other parameters.

In patients with AML, thus, multiparametric flow cytometry allows the quantification of MRD in virtually all cases, while molecular techniques allow this in half of the cases. Current studies will determine the role and place of both methods in AML. Table 3 shows a comprehensive panel of antibodies, which can be used to define a LAIP in AML.

Table 3 AML panel for the detection of leukemia-associated aberrant immunophenotypes (LAIP)
Combination FITC PE ECD PC5 PC7
1 Isotype Isotype Isotype Isotype Isotype
2 CD64 CD87 CD56 CD4 CD45
3 CD65 CD2 CD13 CD34 CD45
4 CD9 HLA-DR CD33 CD34 CD45
5 CD11b CD116 CD117 CD34 CD45
6 CD34 CD56 CD33 CD19 CD45
7 CD15 CD7 CD33 CD34 CD45
8 CD36 CD61 CD235a CD14 CD45
9 CD4 7.1 CD13 CD14 CD45
10 CD38 CD135 CD90 CD34 CD45
11 CD15 CD133 CD117 CD34 CD45
12 Isotype Isotype Isotype Isotype Isotype
13 MPO LF cCD33 cCD34 cCD45
14 TdT cCD22 CCD79a cCD3 cCD45

C=cytoplasmic; combinations 12 to 14: cytoplasmic analysis; 7.1=antibodies for the detection of the NG2 antigen (associated with 11q23 aberrations).

Cytogenetics

More than 50 recurrent structural chromosome aberrations have been described in AML. The karyotype of the leukemic blasts is the most important independent prognostic parameter in AML.

50 to 75% in adult AML and 75 to 85% in childhood AML carry clonal chromosome aberrations. The incidences of the respective chromosome aberrations are age-dependent. However, the prognostic relevance of the karyotype is largely independent of age.

A variety of characteristic chromosome aberrations are known in AML, which define distinct entities with typical morphology and clinical course. The newly defined WHO classification of AML includes cytogenetic aberrations as central classification criteria. Thus, the classification is primarily based on specific cytogenetic rearrangements:

  • AML with t(8;21)(q22;q22), AML1/ETO
  • acute promyelocytic leukemia/AML M3/M3v with t(15;17)(q22;q11–12) and variants, PML/RARA
  • AML with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13;q22); CBFB/MYH11
  • AML with 11q23(MLL)-anomalies

Based on cytogenetics and pathogenesis AML can be separated into three groups according to the karyotype:

  1. AML with normal karyotype (40 to 45%).
  2. AML with balanced chromosome aberrations (20 to 25%), the most frequent ones being t(8;21)(q22;q22), inv(16)(p13q22)/t(16;16)(p13;q22), t(15;17)(q22;q12), and 11q23-rearrangements involving the MLL gene. Less frequent ones include inv(3)(q21q26), t(6;9)(p23;q34), and t(3;21)(q26;q22).
  3. AML with unbalanced karyotype abnormalities (30 to 40%) including trisomies (e.g. +8, +11, +13, +21), monosomies (e.g. −7), and deletions (e.g. 5q-, 9q-) as well as the large group of complex aberrant karyotypes (3 and more chromosome aberrations, but none of the recurrent balanced aberrations).

With regard to prognosis AML are divided into three groups based on the karyotype:

  1. Favorable karyotype: t(15;17)(q22;q12), inv(16)(p13q22)/t(16;16)(p13;q22), t(8;21)(q22;q22)
  2. Intermediate karyotype: normal karyotype, all aberrations not grouped into 1 or 3
  3. Unfavorable karyotype: complex aberrant karyotype, -5/5q-, -7/7q-, 17p aberrations, 11q23/MLL-rearrangements, inv(3)(q21q26), t(6;9)(p23;q34)

For a variety of infrequent karyotype aberrations the prognostic impact has not been clearly defined yet. Significant insights into correlations between genetic alterations and response to therapy led to the approach of increasingly selecting therapy according to the karyotype.

The prognostic relevance of the karyotype is valid within all age groups as well as in both de novo and therapy-associated AML.

Fluorescence In Situ Hybridization

In the diagnostic setting of AML, the FISH analysis is used mainly in addition to the classical chromosome banding analysis. Since the karyotype aberrations occurring in AML are highly heterogeneous, even a large-scale FISH screening on interphase nuclei would cover only a small part of these aberrations. Thus, FISH cannot replace the classic chromosome banding analysis.

However, in targeting a specific alteration, e.g. the translocation t(15;17)(q22;q12) (on the molecular level the rearrangement of the PML and RARA genes) when an acute promyelocytic leukemia is suspected, the FISH technology on interphase nuclei provides a fast and valid result within 4 h. Furthermore, the frequently occurring genetic aberrations, t(8;21)(q22;q22), inv(16)(p13q22) and rearrangements of the MLL gene are used for stratification in the newly defined WHO classification of AML. These alterations are detectable by FISH as are the frequently occurring deletions, e.g. deletions on the long arm of chromosomes 5 or 7, and monosomies (−7) and trisomies (+8, +11, +13, +21). A large portion of AML with complex aberrant karyotype is detectable by FISH with the help of a medium-scale set of probes.

The so-called chromosome painting using 1 to 3 or even 24 colors (24-color-FISH) on metaphase chromosomes is applied in addition to the classic chromosome analysis, if the karyotype cannot be fully resolved on the basis of the chromosome analysis after classic banding, which is not uncommon in complex aberrant karyotypes.

FISH can further be used during the course of therapy for the detection of residual disease. It is more sensitive and specific compared to cytomorphology or chromosome banding analysis; however, it is less sensitive compared to real time PCR and immunophenotyping. FISH has its role in the detection of residual disease, mainly in AML with complex aberrant karyotype, since this subgroup in general lacks genetic alterations detectable by PCR.

Molecular Genetics

Detection of Fusion Genes

The detection of fusion genes using RT-PCR plays an important role in the molecular diagnosis of AML. Reciprocal chromosome rearrangements are found in about 25% of all AML. The molecular correlates and fusion genes, respectively, of most reciprocal cytogenetically detectable rearrangements, are known. The most frequently occurring reciprocal translocations t(15;17), t(8;21), and inv(16)/t(16;16) are represented by the fusion genes PML-RARA, AML1-ETO, and CBFB-MYH11, respectively, on the molecular level. Rearrangements of the MLL gene are present in 5% of all AML with more than 50 different translocation partner genes. Furthermore, some reciprocal rearrangements occur infrequently and are present in less than 1% of AML; however, they may be useful for diagnostic purposes and defining prognosis. The most frequent fusion genes in AML are listed here.

The following fusion genes are detectable by RT-PCR (Table 4):

Table 4 Translocations and respective fusion genes in AML
Frequency
Cytogenetics Fusion gene Subtype Children Adults
t(8;21)(q22;q22) AML1-ETO M2/(M1) 10–15% 8–12%
inv(16)(p13q22) CBFβ-MYH11 M4eo 6–12% 8–12%
t(15;17)(q22;q12) PML-RARA M3/M3v 8–15% 8–10%
t(6;11)(q27;q23) MLL-AF6 M4/M5a 2–5% <1%
t(9;11)(p22;q23) MLL-AF9 M5a 8–10% 1–2%
t(10;11)(p13;q23) MLL-F10 M5a <1% 1–2%
t(11;19)(q23;p13) MLL-ENL M5a <1% <1%
t(11;19)(q23;p13) MLL-ELL M5a <1% <1%
t(3;21)(q26;q22) AML1-EVI1 1% <1%
t(6;9)(p23;q34) DEK-CAN M1/M2/M4 1–2% <1%
t(8;16)(p11;p13) MOZ-CBP M4/M5 <1% <1%
t(1;22)(p13;q13) OTT-MAL M7 2%
t(7;11)(p15;p15) HOXA9-NUP98 M2 <1%
t(10;11)(p13;q14) CALM-AF10 M0/M1/M5 <1%
t(16;21)(p11;q22) FUS-ERG <1%

AML1-ETO, AML1-EVI1, BCR-ABL, ETV6-MDS/EVI1, CBFB-MYH11, PML-RARA, RARA-PML, MLL-ENL, MLL-ELL, MLL-AF6, MLL-AF9, MLL-AF10, MLL-AF1q21, MLL-AF17, MLL-MSF, MLL-p300, CALM-AF10, MLL-PTD, FUS-ERG, EFG-FUS, CHIC2-ETV6, NUP98-HOX9, DEK-CAN, MOZ-CBP.

For the fusion genes PML-RARA, AML1-ETO, and CBFB-MYH11 it has been demonstrated that the quantification of their expression at diagnosis is prognostically relevant. For all other fusion genes, the quantification at diagnosis is also useful since this evaluation may be used as the starting point for assessment during follow-up.

Detection of Molecular Mutations

In recent years a variety of mutations and small gene rearrangements, which are not detectable cytogenetically, have been described. Nonetheless, they play an important role in molecular diagnosis and estimation of prognosis in AML.

Among these molecular mutations are the partial tandem duplications of the MLL gene (MLL-PTD) and the length mutation of the FLT3 gene. These mutations are found mainly in AML with a cytogenetically normal karyotype and are associated with an unfavorable prognosis. The FLT3-LM is found in 10 to 15% of all childhood AML and in 20 to 25% of all adult AML. Furthermore, in another 6 to 7% point mutations are found within the tyrosine kinase domain of the FLT3 gene (TKD mutations). Thus, with a total of about 30% FLT3 is one of the most frequently mutated genes known so far in AML.

In an additional 10% of all AML, mutations are found in the transcription factors CEBPA and AML1, which play an important role in hematopoiesis. Furthermore, 30% of all AML and 55% of AML with normal karyotype, carry NPM1 mutations. Mutations of both CEBPA and NPM1 are considered prognostically favorable.

Mutation Most frequent subtypes Frequency (total) Prognosis
MLL-PTD Normal karyotype (11%)
trisomy 11 (20 to 50%)
6.5% Unfavorable
FLT3-LM Normal karyotype (40%)
t(15;17) (35%)
23% Unfavorable
FLT3-TKD All AML 6.5–7% Dependent on additional defects
KITD816 t(8;21) (12%) 1.5% Unfavorable
KITexon8 inv(16) (10%) <1% Unfavorable
NRAS inv(16) (45%)
inv(3)/t(3;3)
10% Intermediate
KRAS inv(16); t(8;21) (5–20%)
(in childhood AML)
<1%
AML1 M0 (22%), trisomy 21 (30%), trisomy 13 (80%) 5% Unfavorable
CEBPA Normal karyotype (18%) 10% Favorable
NPM1 Normal kayotype (55%) 30% Favorable

Some point mutations like those of KIT and RAS are not specific for AML; however, they contribute significantly to leukemogenesis, in particular in cooperation with AML1-ETO and CBFB-MYH11. Accordingly, a two-hit-hypothesis has been suggested for leukemogenesis. Mutations of tyrosine kinase genes like ABL, FLT3, and KIT as well as RAS mutations are designated type I mutations, which lead to an increased proliferation of hematopoietic cells. Fusion genes and mutations of transcription factors are designated type II mutations, which lead to a stop in differentiation. Only the cooperation of both types of mutations results in the clinically evident acute leukemia.

A variety of methods are available for the detection of these molecular mutations. Among others these comprise conventional RT-PCR (FLT3-LM, MLL-PTD), (FLT3-LM, NPM1), melting curve analysis (FLT3-TKD, NPM1, NRAS, KITD816), dHPLC/WAVE (FLT3, AML1, CEBPA, NPM1, KIT), and consequently sequence analysis (for all mutations).

In addition to the prognostic relevance in AML, these molecular markers may be used as targets for the PCR-based detection of minimal residual disease.

Monitoring of Minimal Residual Disease (MRD)

The detection of minimal residual disease using PCR-based methods is feasible for all markers with a sensitivity between 1:100 and 1:1,000. The methods mainly applied are conventional and nested RT-PCR, fragment analysis, and WAVE analysis.

An even higher sensitivity (1:10,000 to 1:1,000,000) is achieved by “real time PCR.” This method is established for the fusion transcripts: AML1-ETO (applicable in 7–10% of all AML cases), CBFB-MYH11 (7–10%), PML-RARA (7–10%), DEK-CAN (1%), different MLL translocations (5%), MLL-PTD (6%), and NPM1 mutations Typ A, B, and D (25%). Furthermore, it is possible to build patient-specific real-time PCR assays for the FLT3-LM (23%) and rare NPM1 mutation types (5%).

Diagnostic Workup of ALL

Cytomorphology

Cytomorphology and cytochemistry are used to diagnose bone marrow blasts negative for myeloperoxidase and non-specific esterase in ALL; however, the exact diagnosis and further subclassification rely on immunophenotyping. The exception is the typical L3-morphology of blast cells in Burkitt's lymphoma and mature B-ALL.

Immunophenotyping

Acute lymphoblastic leukemias (ALL) are grouped into B-precursor- and T-precursor-leukemias, based on the immunophenotype. They are further subdivided according to the degree of maturation of the leukemic blasts into Pro-B-ALL, common-ALL, Pre-B-ALL, and mature B-ALL, and into Pro-T-ALL, Pre-T-ALL, cortical T-ALL, and mature T-ALL, respectively. In general, in case of the respective morphologic findings (negativity for MPO) a B-precursor-ALL is diagnosed if both cCD22 and CD19 are expressed. A T-precursor-ALL is present if c/sCD3 and CD7 are expressed. The definition of ALL with aberrant expression of myeloid antigens as a separate entity is not preferred since these findings in most cases are associated with genetically defined and clinically relevant aberrations. Table 5 provides the antigen expression patterns of the respective entities.

Table 5 Classification of ALL
Antigen B-precursor-ALL T-precursor-ALL
Pro-B-ALL c-ALL Pre-B-ALL Mature B-ALL Pro-T-ALL Pre-T-ALL Cortical T-ALL Mature T-ALL
cCD22 + + + +
CD79α + + + +
CD19 + + + +
CD24 +/− + + +
CD20 −/(+) +/− +/− +
sIg +
cIgM + +
cCD3 + + +/−
sCD3 −/+ +
CD7 + + + +
CD5 +/− + +
CD2 +/− + +
CD1a +
CD4 −/+ +/− +/−
CD8 −/+ +/− +/−
CD10 + +/− +/− −/+ −/+ −/+
HLA−DR + + + + −/+ −/+
CD34 + + + −/+ −/+ −/+
TdT + + + −/+ + + + +/(−)

c=cytoplasmic; s=membrane.

Monitoring of Minimal Residual Disease (MRD)

In T-precursor-ALL the coexpression of cCD3 and TdT is mainly useful as LAIP. In B-precursor-ALL it is the coexpression of CD19 and CD10. Furthermore, in many cases the aberrant coexpression of myeloid antigens as well as the expression of CD34 can be used.

The first area in which the significant prognostic impact of immunologically determined MRD levels has been demonstrated has been after the achievement of complete remission in childhood ALL: the detection of residual leukemic cells was associated with a significantly increased risk of relapse. This association was independent of other prognostic parameters. Further analyses have demonstrated that as early as on day 19 of the induction therapy MRD levels are equally significant with regard to prognosis.

These results have been reproduced in adult ALL. Thus, patients with low-level MRD after completion of induction therapy have a longer relapse-free survival. Also in this context, the impact of MRD was independent of other prognostic parameters.

Multiparameter flow cytometry is, compared to molecular techniques, feasible for the quantification of MRD in virtually all patients with ALL. Current trials will define the respective roles of both methods, based on direct comparisons.

Cytogenetics

A large number of chromosomal aberrations with prognostic and therapeutic relevance have been described. On the one hand, ALL are subdivided into ploidy groups based on the karyotype, i.e. according to the number of chromosomes (Table 6). On the other hand, they are grouped according to structural aberrations (Table 7). The most frequent aberration in adult ALL is the t(9;22)(q34;q11) which is associated with a very unfavorable prognosis. With the availability of the tyrosine kinase inhibitor, imatinib, the detection of this Philadelphia-translocation and of its molecular genetic correlate, the BCR-ABL rearrangement, is even more important as treatment with this specific drug improves outcome. In addition, the detection of translocations involving the MLL-gene, which is located on the long arm of chromosome 11, is prognostically important and requires specific therapeutic approaches.

Table 6 Frequencies of groups according to ploidy in adult ALL
Group Frequency (%)
Normal karyotype 26–34
Hypodiploidy <46 2–8
Pseudodiploidy 7–59
Hyperdiploidy 47–50 7–17
Hyperdiploidy > 50 4–9
nearly triploid 3
nearly tetraploid 2
Table 7 Frequent chromosomal aberrations in adult ALL
Aberration Gene Frequency
t(1;19)(q23;p13) Pre-B-ALL E2A-PBX1 3%
t(4;11)(q21;q23) Pro-B-ALL MLL-AF4 6%
t(9;22)(q34;q11) c-ALL BCR-ABL 25–30%
t(8;14)(q24;q32) Mature B-ALL IGH-MYC 5%
t(10;14)(q24;q11) T-ALL HOX11-TCR 3%
t(12;21)(p13;q22) Pre-B-ALL ETV6-ALL1 <1%
9p T, Pre-B p16INK4A 15%
6q c, Pre-B,T 6%
14q11 T-ALL TCR 6%

Cases with a t(8;14)(q24;q32), a t(2;8)(p12;q24), or a t(8;22)(q24;q11) – all of which come along with rearrangements of the CMYC gene – have a high chance of cure in case of the application of a specific therapeutic protocol which significantly differs from protocols applied in cases with other ALL subtypes.

Fluorescent In Situ Hybridization

In the diagnostic setting of ALL FISH analysis is used mainly in addition to the classic chromosome analysis. Since multiple karyotype abnormalities occur in ALL, even an extensive FISH “screening” using interphase nuclei would detect only a part of these abnormalities. Therefore, the chromosome analysis cannot be replaced by FISH.

However, in case of focusing on a specific aberration, the FISH technique on interphase nuclei represents a fast and reliable method, giving the result within 24 h. The genetic aberrations occurring frequently in ALL, i.e. the Philadelphia-translocation t(9;22)(q34;q11) (rearrangement of BCR and ABL on the molecular level), an MLL-rearrangement, or a CMYC-rearrangement, all of which have therapeutic implications, can be identified using FISH on interphase nuclei.

A FISH-screening targeting the most frequent aberrations is useful in particular in cases with no valid result of the chromosome analysis. This should be considered also in cases with a normal karyotype with the selection of probes according to the immunophenotype.

The so-called “chromosome painting” with 1 to 3 or 24 colors (24-color-FISH) on metaphase-chromosomes is applied in addition to the classic chromosome analysis if the karyotype cannot be fully described after the classic banding approach, e.g. in complex aberrant karyotypes.

Furthermore, the FISH method can be used for the detection of residual disease during the course of therapy. It is more sensitive and more specific than cytomorphology; however, it is less sensitive than real time PCR and immunophenotyping. The FISH method is used for the detection of residual disease mainly in ALL with unbalanced karyotypes as these cases lack genetic alteration detectable by PCR.

Molecular Genetics

Two types of genetic alterations are present in lymphatic leukemias and lymphomas: Somatic mutations which are involved in the pathogenesis and rearrangements of immunoglobulin genes and T-cell receptor genes as markers of clonality.

The molecular diagnostic approach in ALL primarily focuses on the detection of high-risk cases with t(9;22) and t(4;11) in which an RT-PCR for BCR-ABL and MLL-AF4 is performed as a substitute or supplement to cytogenetics and FISH.

In contrast, the ETV6-AML1-rearrangement, which occurs frequently in childhood ALL, and is associated with a favorable prognosis, can be identified exclusively on the molecular level and/or using FISH.

Activation of the MYC gene due to various Ig rearrangements (Tables 8 and 9) is found in 4% of adult ALL representing the leukemic form of Burkitt's lymphoma. The molecular detection is accomplished by Southern blot. The use of FISH makes this diagnostic step simpler and faster; however, it requires the smear of intact cells.

Table 8 Fusion genes in ALL
Frequency
Cytogenetics Fusion gene Subtype Childhood Adult
t(1;19)(q23;p13) E2A-PBX1 Pre-B-ALL 5–6% 3%
t(4;11)(q21;q23) MLL-AF4 Pro-B-ALL 2% 6%
t(11;19)(q23;p13) MLL-ENL Pro-B-ALL
Pre-T
<1% <1%
t(9;22)(q34;q11) BCR-ABL c-ALL
Pre-B
2–5% 25–30%
t(8;14)(q24;q32) MYC-IGH Mature B-ALL 3% 5%
t(10;14)(q24;q11) HOX11-TCR T-ALL 1% 3%
t(12;21)(p13;q22) ETV6-AML1 c-ALL 10–20% <1%
Table 9 MYC-rearragements in ALL
Frequency
Cytogenetics Fusion gene Subtype Childhood Adult
t(2;8)(p12;q24) IGK-MYC B-ALL <1% <1%
t(8;14)(q24;q23) IGH-MYC B-ALL 3% 2–4%
t(8;22)(q24;q11) IGL-MYC B-ALL <1% <1%

Between 4 and 7% of all T-ALL cases feature rearrangements of the HOX11-gene (Tables 10 and 11). HOX11 codes for a transcription factor. The transforming action of overexpressed HOX11 leads to an immortalization of T-cells.

Table 10 T-cell receptor(TCR)-B-rearragements in ALL
Frequency
Cytogenetics Fusion gene Subtype Childhood Adult
t(1;7)(p32;q35) TAL1-TCRβ T-ALL <1% <1%
t(1;7)(p34;q35) LCK- TCRβ T-ALL <1% <1%
t(7;9)(q35)(q32) TCRβ−TAL2 T-ALL <1% <1%
t(7;9)(q35;q34) TCRβ−TAN1 T-ALL <1% <1%
t(7;10)(q35;q24) TCRβ−HOX11 T-ALL <1% <1%
t(7;11)(q35;p13) TCRβ−RHOM2 T-ALL <1% <1%
Table 11 T-cell receptor(TCR)-α- and -δ-rearragements in ALL
Frequency
Cytogenetics Fusion gene Subtype Childhood Adult
t(1;14)(p23;q11) TAL1-TCRδ T-ALL <1% <1%
t(8;14)(q24;q11) MYC-TCRα T-ALL <1% <1%
t(10;14)(q24;q11) HOX11-TCRδ T-ALL <1% 1–3%
t(11;14)(p13;q11) RHOM2-TCRδ T-ALL <1% 2–3%
t(11;14)(p15;q11) RHOM1-TCRδ T-ALL <1% <1%
inv(14)(q11q32.1) TCRα−TCL1 T-ALL <1% <1%
inv(14)(q11q32.3) TCRα−IGH T-ALL <1% <1%

In cases without fusion genes a characterization of clonal Ig- and TCR-gene-rearrangements should be performed. These can be used as markers for residual disease during the course of therapy. Since the latter alterations are identified in DNA while fusion genes are analyzed in RNA it is important to asservate both DNA and RNA at the time of diagnosis.

Diagnostic Workup of CML

Cytomorphology

Cytomorphology typically reveals hypercellularity in both peripheral blood and bone marrow with an increase of immature myeloid cells as well as of eosinophils and basophils.

Immunophenotyping

MFC is applied in CML only in case of blast crisis in order to delineate the cell lineage, i.e. lymphatic or myeloid.

Cytogenetics

In 90 to 95% of all patients, a Philadelphia translocation is present on the cytogenetic level: t(9;22)(q34;q11). This is a translocation between the long arm of a chromosome 9 and a long arm of a chromosome 22. The remaining patients carry so-called variant Philadelphia translocations or a normal chromosome set. The variant Philadelphia translocations are divided into simple ones, in which the long arm of chromosome 22 is translocated to another chromosome (and not to chromosome 9), and complex ones, in which further chromosome are involved besides chromosoms 9 and 22. Patients with CML and a normal karyotype carry a BCR-ABL-rearrangement detectable by both FISH and RT-PCR. By applying FISH and using metaphases the BCR-ABL-fusion gene is detected either on chromosome 22 or, less frequently, on chromosome 9. Submicroscopic insertions are discussed as underlying mechanisms which are not detectable by classic cytogenetics. This group of CML is designated Philadelphia-negative BCR-ABL-positive CML. The clinical and hematologic course of patients with classic and variant Philadelphia-translocation does not differ from the course in patients with Philadelphia-negative BCR-ABL-positive CML.

During the progression into the accelerated phase or into blast crisis in 75 to 85% of the patients, additional karyotype aberrations occur. The most frequently occurring aberrations (so-called clonal evolution) are trisomy 8 (+8), isochromosome of the long arm of chromosome 17 (i(17)(q10)), an additional Philadelphia-chromosome (+der(22)t(9;22)), as well as trisomy 19 (+19). The occurrence of additional karyotype aberrations is a prognostically unfavorable parameter and precedes the clinical manifestation of a blast crisis by 2–6 months in most cases.

In CML follow-up, assessments during therapy are prognostically most important with consequences for the further management of the disease. The classic chromosome analysis represents the gold standard for the assessment of a cytogenetic remission. At least 20 metaphases should be analyzed to obtain a valid result. A complete cytogenetic remission (no Ph+ metaphases) is differentiated from a “partial” remission (1 to 32% Ph+ metaphases), a “minor” remission (33 to 66% Ph+ metaphases), a “minimal” remission (67–95% Ph+ metaphases), and no remission (≥95% Ph+ metaphases). The complete and the partial remission are grouped together as “major” remission.

During therapy with imatinib, the occurrence of karyotype aberrations has been observed in some patients in clones lacking a Philadelphia translocation. Most frequently this aberration is the trisomy 8. Further aberrations are monosomy 7 as well as different translocations. The clinical impact of this phenomenon is yet unclear and is being evaluated in clinical trials.

Fluorescence In Situ Hybridization

Using FISH on interphase nuclei, the presence of a BCR-ABL rearrangement can be detected or ruled out within 24 h. The FISH analysis has some advantages as compared to the classic chromosome analysis: Using this method a CML can be diagnosed and monitored even in patients with Philadelphia-negative BCR-ABL-positive CML. Furthermore, it is more sensitive compared to the classic chromosome analysis in detecting residual disease. FISH may be applied on interphase nuclei but also on metaphases. Using the so-called “Hypermetaphase-FISH”-technique, up to 500 metaphases may be assessed for the presence of a BCR-ABL rearrangement, while only 20 to 25 metaphases are analyzed by the classic chromosome analysis. However, additional cytogenetic aberrations and Ph-negative clones cannot be detected using FISH alone and require conventional chromosome banding analysis.

Molecular Genetics

The molecular correlate of the Philadelphia translocation is the BCR-ABL fusion gene. The diagnosis of a CML can be made only if the Philadelphia translocation is detected by cytogenetics or by FISH or if the BCR-ABL rearrangement is detected by RT-PCR. Since 5% of all CML cases carry a cryptic BCR-ABL rearrangement that is not detectable cytogenetically, a FISH and/or PCR assessment should be done in parallel to cytogenetics at diagnosis. Furthermore, a PCR-based quantification of the BCR-ABL expression provides a starting value for follow-up assessments.

The PCR-based monitoring of BCR-ABL during therapy is a standard procedure in CML. Independent of the therapy applied, a response to therapy is most effectively assessed by real time PCR with regard to both quantity of response and sensitivity. In contrast to most other leukemias, follow-up assessment in CML is possible using peripheral blood. It is recommended to do a peripheral blood evaluation every 3 months.

In patients under imatinib therapy, an association between an increase of the BCR-ABL expression and the occurrence of a resistance against imatinib has been described. In 2 to 5% of all CML cases, a primary resistance against imatinib is present. Many of these resistances are due to point mutations of the ABL gene within the BCR-ABL fusion gene. Other patients acquire secondary mutations in the tyrosine kinase loop of the BCR-ABL fusion protein during therapy with imatinib, which lead to a resistance against the drug. In some cases this resistance can be superseded by an increase of the dose of imatinib or by the combination with IFN-α. Therefore, the early detection of these resistances increasingly carries clinical importance. Mutations have been described in 23 different codons so far. Some of these lead to changes in conformation while others directly inhibit the binding of imatinib to the activating domain of the tyrosine kinase ABL. The type of mutation therefore provides information on the usefulness of an increase of the imatinib dose. The point mutations can be detected by sequencing the ABL part of the fusion gene.

Diagnostic Workup of CLL

Cytomorphology

The diagnosis of CLL per definition requires a lymphocytosis >5,000/µl, although a chronic elevation of the absolute lymphocyte count in the peripheral blood and the presence of both the typical morphologic findings and the typical immunophenotype are increasingly considered diagnostic. Cytomorphologically, CLL cells are small lymphocytes with a narrow cytoplasm and a dense nucleus without nucleoli. Furthermore, shadow cells of Gumprecht are present.

Immunophenotyping

CLL features a typical immunophenotype with a coexpression of CD5 and the B-cell-associated antigens CD19, CD20, and cCD79a. A weak surface expression of CD22 and Ig is present, with a clonal light chain restriction. The positivity of CD23 which is commonly present in CLL allows the distinction from mantle cell lymphoma. The latter is further characterized by a strong surface expression of Ig as well as by an expression of CD22 and FMC7. Overall, no single marker allows the diagnosis of CLL. Rather, the comprehensive evaluation of all the antigens assessed and the application of the Matutes-score have been proved useful (Table 12).

Table 12 Matutes-score of B-CLL
Characteristics of B-CLL
CD5+
CD23+
FMC7−
sIgM(+)
sCD22(+) or CD79b(+)

The presence of four or five of the findings characteristic for B-CLL was found in a large series in 87% of all B-CLL and in only 0.3% of other B-cell lymphomas. Conversely, only 0.4% of B-CLL and 72% of other B-cell lymphomas had none or only one of these criteria.

The expression of CD38, as well as the cytoplasmic expression of ZAP70, is prognostically important. Both findings are associated with an inferior prognosis.

If CLL/PL is diagnosed by cytomorphology (10 to 55% prolymphocytes in peripheral blood) often accompanied by a trisomy 12, which occurs more frequently in these cases, the immunophenotype may show a stronger expression of CD20 and of sIg as compared to CLL. Furthermore, there is a weaker expression of CD23 as well as a positivity for CD22 and CD79b.

In contrast to B-CLL, the co-expression of CD5 is not, or only weakly, present in B-PLL (>55% prolymphocytes in peripheral blood), the surface expression of Ig and of CD20 is stronger and CD22 and FMC7 are expressed.

Besides diagnosis, immunophenotyping allows the quantification of minimal residual disease (MRD). The CLL cells typically carry the phenotype CD19+CD20+CD79-CD5+ and thereby differ from normal B-lymphocytes. The prognostic relevance of the MRD-level, which can be assessed with high sensitivity, has been demonstrated in multiple studies.

Cytogenetics

The classic chromosome analysis has played only a minor role in the past due to the difficulties in cultivating the cells in vitro. By using FISH analysis, the strong prognostic impact of chromosome aberrations could be demonstrated. Recently, a reliable cultivation of CLL cells has been shown to be feasible and chromosome aberrations have been detected by chromosome analysis at higher frequencies compared to FISH analysis.

Fluorescence in Situ Hybridization

FISH analysis detects the most frequent chromosomal aberrations in CLL in a targeted way. These comprise 13q deletions, trisomy 12, 11q deletion, and 17p deletion. The presence of a 17p deletion or of a 11q deletion indicates a more aggressive course of disease compared to a normal karyotype, while the sole presence of a 13q deletion confers a favorable prognosis. Furthermore, the assessment of t(11;14) is useful for the distinction between CLL and mantle cell lymphoma.

Molecular Genetics

Half of the patients with CLL carry so-called somatic mutations in the variable region of immunoglobulins. The presence of 2% or less mutations in this area of the immunoglobulins as compared to the original DNA sequence is designated “unmutated” while the presence of more than 2% mutations is designated “mutated.” The unmutated status is associated with an unfavorable prognosis even in early stages of the disease.

Diagnostic Workup of MDS

Cytomorphology

During the cytomorphologic examination at least 200 bone marrow cells and 20 megakaryocytes should be evaluated. Dysplastic findings should be present in at least 10% of the cells. A particular diagnostic role is played by the so-called pseudo-Pelger neutrophils, ringed sideroblasts, mikromegakaryocytes, and augmented blasts. These morphologic aberrations correlate in part with clonal markers and show a low inter-observer variability. This is true particularly for the prognostically favorable and therefore clinically relevant 5q-syndrome. The assessment of hypogranulation in neutrophils should not be the only diagnostic criterion for dysplasia. Accordingly, an early stage of refractory anemia (RA) with cytopenia in only one lineage is often difficult to diagnose and requires the assessment of follow-up samples. With regard to the differentiation between hypoplastic MDS and aplastic anemia, it is important to notice that dysplastic findings in erythropoiesis may be present also in the latter. They therefore play no diagnostic role in this instance, unlike dysplastic findings in the other lineages and augmented bone marrow blasts. PNH should be considered as differential diagnosis. With regard to the separation of MDS and AML a cut-off of 20% bone marrow blasts has to be used according to the presently applied WHO classification.

Immunophenotyping

Multiparameter flow cytometry allows the qualitative assessment of dysplasia in the different cell lineages, granulopoiesis, monopoiesis, and erythropoiesis through the detection of aberrant antigen expression patterns. Furthermore, the quantification of blasts can be done with a high correlation to cytomorphologic findings. The flow cytometric findings are of particular diagnostic value in cases difficult to judge by cytomorphology. Further studies will define the role of multiparameter flow cytometry in comparison to cytomorphology and cytogenetics with regard to both diagnostics and prognostication.

Cytogenetics

In the context of diagnostic assessment of MDS the chromosome analysis plays a significant role by detecting karyotype aberrations typical for MDS and particularly so in cases difficult to judge by cytomorphology.

The typical aberrations, which are also considered for the prognostically highly relevant IPSS (Tables 13 and 14), include loss of Y chromosome, del(5q), del(20q), as well as complex aberrations and aberrations of chromosome 7. In cytomorphologically defined border-line cases a distinction between AML and MDS can be accomplished by the detection of t(8;21)(q22;q22), t(15;17)(q22;q11-12), or inv(16)(p13q22)/t(16;16)(p13;q22) which define an AML, respectively.

Table 13 IPSS, basis
Points
0 0.5 1 1.5 2
% bone marrow blasts 5 5–10 11–20 21–30
Karyotype Favorable Intermediate Unfavorable
Cytopenias 0/1 2/3

Karyotype favorable: normal, -Y sole, del(5q) sole, del(20q).

Karyotype unfavorable: complex aberrant (≥3 aberrations), aberrations of chromosome 7.

Table 14 IPSS, prognostic grouping
Points 0 0.5–1.0 1.5–2.0 ≥2.5
Risk group Low Int-1 Int-2 High

Fluorescence In Situ Hybridization

FISH analysis may be used in case of lack of adequate material for cytogenetics, e.g. in case of a dry tap, smears may be obtained from a bone marrow biopsy and analyzed by FISH. The analysis includes probes targeting the aberrations most relevant for determining the prognosis: -Y, del(5q), del(20q), aberrations of chromosome 7, del(17p).

Molecular Genetics

In contrast to AML there are no specific molecular markers for MDS. In case of rare reciprocal translocations fusion gene-specific PCRs can be applied which, however, do not play a major role in routine diagnosis. Some of the AML-specific mutations like the partial tandem duplication of MLL (MLL-PTD), FLT3 length mutations (FLT3-LM), RAS mutations, as well as mutations of AML1 and CEBPA may be present in MDS with high blast counts. They are indicative of a progress of MDS to AML. Similarly, an increasing expression of WT1 represents a marker for the progress of MDS.

Diagnostic Algorithms in Leukemia Diagnostics

Based on the data provided above, algorithms for the diagnostic work-up of leukemias have been proposed. These are outlined below and should be applied and evaluated in the context of large clinical trials. Leukemia diagnosis has undergone steady development and is expected to become even more refined within the next few years, particularly taking into consideration gene expression profiling with microarrays, which may be incorporated.

Diagnostic Algorithm in AML

In AML (Fig. 1) a combination of cytomorphology, cytochemistry, immunophenotyping, and cytogenetics should be applied in the first step. Results of cytomorphology and cytochemistry guide the selection of antibody panels to be used for immunophenotyping. Results of these three methods guide the selection of culture conditions for cytogenetics.

Leukemia Diagnostics. Figure1 Algorithm for diagnosis and follow-up in AML.

In case of specific cytomorphologic findings, the respective genetic alterations should be analyzed by FISH and PCR (AML M3/M3v: t(15;17)/PML-RARA; AML M4eo: inv(16)/CBFB-MYH11; AML M1/2 with characteristic long Auer rods: t(8;21)/AML1-ETO; AML M5a: 11q23/MLL rearrangements). The combined analysis of genetic alterations by both FISH and PCR provides a maximized diagnostic security as well as information on variant translocations or submicroscopic deletions, which are only detectable by interphase FISH.

Based on cytogenetic results, FISH analysis using specific probes is applied for numerical (e.g. +8, −7) and structural aberrations (e.g. 5q-, 7q-). Cases with complex aberrant karyotype can further be investigated by 24-color FISH. FISH may be used in infrequent cases without cytogenetic result to identify the most frequent and prognostically relevant aberrations.

RT-PCR is used, according to cytogenetic results, for the detection of fusion genes as well as for the analysis of FLT3-LM, MLL-PTD, or NPM1.

Follow-up assessment during complete remission should be performed by cytomorphology, FISH, PCR, and MFC, whenever a specific marker has been identified at diagnosis. The latter two highly sensitive methods are particularly useful for the quantification of the prognostically important MRD levels.

Diagnostic Algorithm in ALL

Cytomorphology and immunophenotyping should be applied in the first step in ALL (Fig. 2). Cytomorphology identifies acute leukemia negative for peroxidase and reveals cases suspicious for Burkitt's lymphoma/mature B-ALL. MFC allows the diagnosis of ALL as well as the subclassification into different B-precursor ALL and T-precursor ALL classes.

Leukemia Diagnostics. Figure 2 Algorithm for diagnosis and follow-up in ALL.

Cytogenetics follows the results of MFC with specific culture conditions for B- and T-precursor ALL.

FISH analysis for BCR-ABL and MLL rearrangements follows MFC in case of B-precursor ALL.

FISH analysis for CMYC rearrangements follows cytomorphology in case of findings typical for Burkitt's lymphoma as well as MFC in case of mature B-ALL.

In childhood B-precursor ALL, FISH analysis for ETV6-AML1 is applied.

In case of cytogenetics not yielding a result, FISH and PCR are applied for the detection of BCR-ABL and MLL rearrangements.

Real time PCR is applied for MRD monitoring, targeting either fusion transcripts or IgH receptor-/T-cell receptor rearrangements. MFC is also used for MRD monitoring.

A complete reevaluation should be performed at relapse.

Diagnostic Algorithm in CML

The diagnosis of CML is made by a combined approach of cytomorpholgy, cytogenetics, FISH, and PCR,identifying characteristic peripheral blood and bone marrow features as well as the t(9;22) and the BCR-ABL fusion gene (Fig. 3).

Leukemia Diagnostics. Figure 3 Algorithm for diagnosis and follow-up in CML.

During therapy all methods are applied in combination to quantify the amount of residual disease as well as additional chromosomal abnormalities.

In case of failure or suboptimal response an analysis for BCR-ABL mutations conferring resistance to imatinib should be performed.

Immunophenotyping should be performed in blast crisis to delineate the lineage (lymphatic or myeloid).

Diagnostic Algorithm in CLL

Cytomorphology and immunophenotyping should be applied in the first step in CLL (Fig. 4). Cytomorphology identifies mature lymphocytosis. MFC allows the diagnosis of CLL and its discrimination from other indolent lymphomas according to the Matutes score.

Leukemia Diagnostics. Figure 4 Algorithm for diagnosis and follow-up in CLL.

In addition, MFC is used for the determination of the expression of both CD38 and ZAP-70.

FISH analysis is performed to identify the most common and prognostically important chromosomal abnormalities, i.e. 6q-, 11q-, trisomy 12, 13q-, and 17p- as well as t(11;14). A cytogenetic analysis is capable of identifying additional chromosomal abnormalities which may yield further prognostic information.

Molecular genetics is applied to determine the IgVH mutational status.

Monitoring during therapy is performed by a combination of cytomorpholgy, MFC, and FISH.

Diagnostic Algorithm in MDS

The diagnosis of an MDS is based, besides the presence of cytopenias, on cytomorphology (Fig. 5). Cytogenetics is used to identify chromosomal abnormalities which subclassify MDS and yield prognostic information. In cases of equivocal findings in cytomorphology cytogenetics can assure the diagnosis. Immunophenotyping may be used to identify aberrant antigen expression patterns typical for MDS. PCR may be performed in MDS.

Leukemia Diagnostics. Figure 5 Algorithm for diagnosis and follow-up in MDS.

During the course of therapy, which may be limited to supportive measures, cytomorphology in combination with cytogenetics and FISH should be applied.

In case of progression to AML, cytomorpholgy, cytogenetics, PCR, and MFC should be applied.

References

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