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Leukemia diagnostics comprises the combined application of different methodologies in a standardized fashion, allowing the precise diagnosis, subclassification, and determination of prognostic parameters.
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 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.
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.
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.
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.
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.
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.
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.
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.
Different methods are available, which allow the screening of defined gene regions for mutations without the necessity of sequencing. Some examples are listed below:
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.
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
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.
|FAB- Subtype||FAB-criteria||Association with|
|Granulopoiesis||Monopoiesis||Erythro- poiesis||Immunologic marker||Cytogenetics||Molecular genetics||Frequency|
|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:
- AML with recurrent cytogenetic aberrations
- AML with myelodysplasia-associated features
- Therapy-related AML and MDS
- 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.
|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)|
|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|
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).
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.