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Multifaceted approach to the diagnosis and classification of acute leukemias.

Information obtained from immunophenotyping, cytogenetics, and molecular analysis has substantially advanced our understanding of the biology of hematologic malignancies. The evolution of these techniques from primarily research applications to routine components in the diagnosis of hematopoietic neoplasms has expanded the diagnostic capabilities of hematopathology laboratories. This report will discuss how these techniques contribute to a multifaceted approach to diagnosis and classification of hematopoietic neoplasms and provide important treatment and prognostic information. Although acute leukemia will serve as the model to illustrate the value of immunophenotyping and cytogenetic/molecular analysis, these studies also play an important role in the diagnosis of virtually all other types of hematopoietic malignancies.

Diagnosis of Acute Leukemia

The diagnosis of acute leukemia entails a stepwise approach. First in sequence and importance is the distinction of acute leukemia from other neoplastic diseases and reactive disorders. Second is differentiating acute myeloid (AML) [1] and acute lymphoblastic (ALL) leukemia. The third facet is the classification of AML and ALL into categories that define treatment and prognostic groups.


In most cases, the first two facets of the diagnosis of acute leukemia can be achieved by careful morphological assessment of blood and bone marrow smears and marrow trephine biopsy sections. The usual cytologic features of AML and ALL are listed in Table 1 (1, 2). By assessing these morphologic features together, a majority of cases of AML and ALL can be accurately diagnosed (Figs. 1 and 2). In some cases of poorly differentiated acute leukemia, however, the morphologic features may be equivocal, requiring additional studies. Cytochemical stains often are useful in distinguishing poorly differentiated AML from ALL and in identifying subsets of AML (1, 2). Table 2 shows the cytochemical profiles of acute leukemias for the most commonly applied cytochemical stains. The myeloperoxidase and Sudan black B stains are the most commonly used and the most valuable in distinguishing AML from ALL. In the majority of cases of AML, a variable proportion of the leukemic cells (blasts) are reactive for myeloperoxidase and Sudan black B, whereas the stains are uniformly negative in ALL. With the addition of cytochemistry to the morphologic assessment, most cases of acute leukemia can be appropriately designated as either AML or ALL. However, there remains a significant minority of cases that cannot be definitively diagnosed by these methods.


The lineage of most cases of morphologically and cytochemically poorly differentiated acute leukemia can be accurately characterized by immunophenotyping. Additionally, immunophenotypic subsets of AML and ALL can be determined (3, 4). Multiparametric flow cytometry is the preferred method for immunophenotyping acute leukemias. There is an abundance of monoclonal and polyclonal antibodies available to assess myeloid and lymphoid lineage-associated antigens by cytometry, and blood and bone marrow aspirate specimens lend themselves particularly well to flow cytometric analysis because the cells are naturally in a fluid suspension. Multicolor flow cytometry allows for the characterization of up to four different antigens on a single cell. This permits precise immunophenotypic characterization of leukemic cells even when they are present in low numbers. Immunohistochemical staining can be used to immunophenotype leukemias when a specimen is not submitted for flow cytometry or only bone marrow trephine biopsies are available for examination. An array of antibodies to myeloid- and lymphoid-associated antigens is also available for immunohistochemical stains.

The lineage of hematopoietic cells is defined both by antigens expressed and the absence of expression of antigens associated with a different lineage. Leukemia cells, however, may aberrantly express some antigens of another lineage or lack expression of an expected antigen (5). It is important, therefore, to use panels that include sufficient numbers of antibodies to assess a spectrum of both myeloid and lymphoid antigens. The choice of antibody panels varies among laboratories; some choose to use a large panel routinely, others screen with a small panel and add additional antibodies as necessary.

Table 3 shows profiles of antigen expression for various categories of AML (see Classification of Acute Leukemias). In AMLs, immunophenotyping is most important in distinguishing poorly differentiated cases from ALL and in characterizing a few AML subsets (Fig. 1). For ALLs, the immunophenotypic categories are particularly important because they identify distinctive treatment and prognostic groups (4, 6, 7). The immunophenotypic classification of ALL is shown in Table 4. Approximately 80% of cases of ALL have a B-cell precursor immunophenotype (Fig. 2) (4, 7, 8). Lymphoblasts in B-cell precursor ALL express a variable spectrum of early and pan B-cell antigens but lack surface immunoglobulin, which is found on mature B lymphocytes. They also contain terminal deoxynucleotidyl transferase, a nuclear enzyme present in both B- and T-lymphocyte precursors but not in mature lymphocytes. Approximately 15% of ALLs have an antigen profile of T-cell precursors (thymic T cells) (6). T-cell lymphoblasts variably express early and pan T-cell antigens and terminal deoxynucleotidyl transferase. They often lack surface CD3 but express it in their cytoplasm. A small group of cases (<5%) of ALL have the immunophenotypic profile of more mature B cells, i.e., surface immunoglobulin. The importance of defining the immunophenotype in ALL lies in its correlation with response to treatment and prognosis (4, 7). In childhood ALL, immunophenotype is a major factor in determining the chemotherapy protocol. The immunophenotypic prognostic groups of ALL are shown in Table 5. B-cell precursor ALLs have a more favorable prognosis than the other groups; however, within the B-cell precursor category, there are subsets with a poor prognosis (7). Most of the favorable and unfavorable prognostic groups of B-cell precursor ALL can be identified by their cytogenetic karyotype or molecular features (8).

Immunophenotyping has become a standard diagnostic procedure in evaluation of acute leukemias. Immunophenotype should be assessed for diagnosis in all cases of morphologically poorly differentiated acute leukemia and in every case of ALL because of the important treatment and prognostic information that it provides.



Clonal cytogenetic abnormalities are identified in 60-80% of cases of AML and ~80% of cases of ALL (9-11). Both numerical and structural abnormalities are common. Tables 6 and 7 1ist some of the more frequently encountered cytogenetic abnormalities and their relationships to prognosis (9,10,12-16).

Hyperdiploidy with >50 chromosomes is the most common cytogenetic abnormality ([approximately equal to]25%) in B-cell precursor ALL (9). It generally is found in patients between 2 and 10 years of age and is associated with low or intermediate leukemic cell counts in the blood. There often is an extra copy of chromosomes) 4 and/or 10, which seems to impart a particularly favorable prognosis. ALLs with hyperdiploidy with >50 chromosomes in children are highly sensitive to antimetabolite drugs and have a complete remission rate approaching 100% with an ~80% long-term disease-free survival (9). Structural abnormalities in childhood B-cell precursor ALL are more often associated with an intermediate or poor prognosis. One exception is the 12;21 translocation [t(12;21)(p12; q22)], which is observed in ~20-25% of cases (17). This change usually is not evident by cytogenetic karyotyping and must be identified by molecular cytogenetic studies [fluorescent in situ hybridization (FISH)] or PCR (17,18). Cases of ALL with a t(12;21) are always B-cell precursor type but are distinct from the hyperdiploidy with >50 chromosomes group. They are highly sensitive to antimetabolite drugs and have a high rate of complete remission and presumably a high incidence of long-term disease-free survival (19). Patients with B-cell precursor ALL with a 9;22 translocation [t(9/22)(q24;q11)] or abnormalities involving chromosome 11q23, most often a t(4;11)(q21; q23), have an unfavorable prognosis (Fig. 2) (14, 20-24). In cases with either of these chromosome rearrangements, the complete remission rate is lower and the relapse rate is very high; the long-term prognosis is poor. Allogeneic bone marrow transplantation should be considered as a primary treatment in patients with these cytogenetic findings. In childhood B-cell precursor ALL, the likelihood of long-term disease-free survival or relapse and the decision for low-risk or more aggressive chemotherapy or a bone marrow transplant are commonly dictated by cytogenetic findings.


In AMLs, cytogenetic findings are also clinically important. This is particularly true of cases with the 15;17 translocation [t(15;17)(q22;q21); Fig. 1]. The t(15;17) is always associated with acute promyelocytic leukemia (PML), which usually has distinct clinical and morphologic features (13,15). The translocation involves the PML gene on chromosome 15 and the retinoic acid receptor [alpha] (RAR[alpha]) gene on chromosome 17 (25). The fusion messenger RNA product that results inhibits maturation of the affected cells, leading to a proliferation of large numbers of atypical promyelocytes. Treatment with all-trans-retinoic acid (ATRA) can overcome the maturation blockage in most cases and lead to temporary complete remission of the disease (25-27). Treatment with standard chemotherapy with or after ATRA therapy is required to sustain remission. Other types of AML do not respond to ATRA therapy.

Other important cytogenetic abnormalities in AML include t(8;21)(q22;q22); an inverted chromosome 16 [inv(16)(p13;q22)]; abnormalities involving 11q23, -7 or deletions of 7q, -5 or 5q; and various translocations involving chromosome 17 (10, 24, 28-31). Several of these have been found to have either good or bad prognostic significance as shown in Table 6.

Bone marrow cytogenetic findings are a major independent indicator of prognosis for both AML and ALL, and define treatment groups. They are essential in the assessment of patients with acute leukemia and should be performed in every case.


In the diagnosis of acute leukemias, molecular analysis may be used to establish clonality or to identify molecular translocations producing fusion gene products (8,15, 17, 24, 32, 33). Molecular studies are also powerful tools for the identification of minimal residual disease and early relapse (34, 35). Techniques for molecular analysis of leukemias include Southern blot, PCR, and FISH.

Molecular techniques for identifying T-cell receptor or immunoglobulin gene rearrangements are valuable in diagnosis of some cases of lymphoma and other lympho-proliferative disorders but are less commonly required in the diagnosis of acute leukemia. However, gene rearrangements may serve as a fingerprint for later identification of minimal residual leukemia when there are too few leukemic cells present to be recognized by morphologic examination or immunophenotyping (34).

Identification of the fusion genes that result from cytogenetic translocations such as the PML-RAR[alpha] gene in acute PML and the ABL-BCR (p190) gene in ALL has treatment and prognostic importance as described in Cytogenetics in the Diagnosis of Acute Leukemia above (11,21,25,26). In some cases, molecular translocations are present when karyotypic changes are not evident. An example of this is the TEL-AML1 fusion gene resulting from the t(12;21)(p12;q22) translocation. This chromosomal translocation generally is cryptic and can only be identified by molecular analysis, i.e., PCR or FISH (17,19). This is occasionally the case with other well-established translocations in acute leukemia in which the involved chromosome segments are too small for detection by karyotyping or because the translocation is complex and involves several chromosomes. It is important, therefore, to perform molecular analysis when the presence of a fusion gene that would impact treatment decisions is suspected. Tables 8 and 9 show examples of major molecular genetic abnormalities and their associated cytogenetic translocations in AML and ALL, respectively (8).

In some cases, PCR or FISH studies may be performed in search of a specific fusion gene without first doing cytogenetic karyotyping. The advantages of this approach are a shortened result turnaround time and reduced expense. However, the molecular probes for each of the fusion products are specific and identify only that product. Because routine cytogenetic studies identify a spectrum of chromosome abnormalities that may occur in acute leukemia, they should always be performed at initial diagnosis. Molecular analysis should be used to supplement cytogenetics when a specific question is being addressed. Molecular studies may be performed in the absence of parallel cytogenetic analysis when there is a focused purpose for the study, such as identification of minimal residual disease.

Classification of Acute Leukemias

It is obvious that immunophenotyping, cytogenetics, and molecular analysis are vitally important in the diagnosis of acute leukemia. The information provided by these techniques and its direct impact on treatment decisions and prognosis presents a logical argument for incorporating results from these studies into a new classification of acute leukemias. The following discussion deals with issues and controversies related to classification and how immunophenotyping and cytogenetic/molecular information is changing the classification of acute leukemias.

The modern era for classification of acute leukemias dates to 1976 when two proposals appeared. One was published by a cooperative group of hematologists and hematopathologists from France, America, and Britain and was designated the French-American-British (FAB) classification (36). The other was the WHO proposal (37). Although the WHO classification was never widely used, the FAB proposal was adopted internationally. It provided long needed standard terminology for the acute leukemias and was quickly accepted by most of the multiinstitutional study groups.

The FAB classification of AML divides cases into eight major groups with subtypes for three of them (Table 10) (38). The classification criteria are based on morphologic and cytochemical features; for some of the categories, immunophenotyping is necessary (39, 40). The FAB classification of AML is a lineage-based morphologic classification that categorizes cases according to the degree of maturation of the leukemmic cells and their lineage differentiation. The FAB classification of ALL is simpler than the one for AML, but the criteria that distinguish the categories are less precise (Table 11) (41). The major advantage of the FAB lineage-based classification system is its ease of use. The cytologic criteria are well defined; they do not require high technology and can be applied in most laboratories throughout the world. The classifications are also applicable to the majority of cases of acute leukemia, and they partially define prognostic groups. The major disadvantage is their modest clinical relevance; they do not adequately define biologic and treatment groups.

Several clinically important categories of acute leukemia have been defined by cytogenetic/molecular studies during the past two decades. As a result, there are proponents for abandoning the lineage-based classifications and developing an exclusively cytogenetic/molecular analysis-based classification of acute leukemias. Clearly, cytogenetic/molecular analysis-based groupings better define biologic and prognostic groups. The requirement for technology that is not always available at present is a negative aspect of an exclusively cytogenetic/molecular classification. In addition, currently the majority of cases of AML do not express recurrent cytogenetic changes, and only slightly more than one-half of cases of childhood ALL do.

In the mid 1990s, the Society for Hematopathology in the United States and the European Association for Hematopathology were enlisted by WHO to update the WHO classification of hematopoietic neoplasms. The revised WHO classifications of AML and ALL have recently been completed and will be published in monograph form within the next year. A preview of the WHO classifications was published recently in a journal article (42). The newly revised WHO classifications of acute leukemias are shown in Tables 12 and 13. These classifications address the problems of an exclusively lineage-based or an exclusively cytogenetic/molecular classification by combining the best features of both. The result is a classification that enhances clinical and prognostic utility and retains usability.

In the who classification of AML there are four major categories:

* AML with recurrent cytogenetic translocations

* AML with multilineage dysplasia

* therapy-related (secondary) AML

* AML not otherwise categorized (includes the former FAB categories)

Within the major categories, there are several subtypes (Table 12). This proposal retains and even expands morphologic criteria but includes categories defined by cytogenetic/molecular studies that have important prognostic implications.

There are three major categories of ALL in the revised WHO classification, defined by immunophenotype (Table 13):

* B-cell precursor

* T-cell precursor

* Burkitt cell leukemia

Within the B-cell precursor category there are several subtypes identified by cytogenetic/molecular abnormalities. The major treatment and prognostic groups in childhood ALL are identified in this classification.

With the present explosion of cytogenetic and molecular information, the new WHO classifications of acute leukemia must be considered works in progress. It will be necessary to update them periodically as new discoveries provide a better understanding of the biology of the acute leukemias and as additional distinctive clinical and prognostic subtypes are identified.

The evolution of new technology in hematopathology has added several new tools for diagnosis, classification, patient management, and determining prognosis of leukemia. The refinements in diagnosis that they provide have set the stage for more specifically directed treatment regimens. Defining the appropriate clinical indications for new techniques, understanding their limitations, and integrating them with existing standard diagnostic methods are all vitally important in realizing their full potential.


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[1] Nonstandard abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblasfic leukemia; FISH, fluorescent in situ hybridizafion; PML, promyelocytic leukemia; RAR[alpha], retinoic acid receptor [alpha]; ATRA, all-trans-retinoic acid; and FAB, French-American-British.

Department of Pathology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9072. Fax 214-648-4070; e-mail

Received April 10, 2000; accepted May 22, 2000.
Table 1. Usual cytology of AML and ALL.


Blast size Large and uniform Small to medium; variable
Chromatin Finely dispersed Rather coarse
Nucleoli 1 to 4, often prominent Absent or 1 or 2;
Cytoplasm Moderately abundant Scant to moderate
 Granules often present Granules lacking in
 nearly all cases
Auer rods 60-70% of cases Absent
Myelodysplasia Often present Absent

Table 2. Cytochemical profiles in acute leukemia.

 Myeloperoxidase and Nonspecific Periodic acid
 Sudan Black B esterase Shiff (PAS)

AML + (a) + [+ or -]
 (Monocytic, diffuse)
ALL - [+ or -] +
 (Focal) (75%)

(a) +, positive; -, negative; [+ or -], not defnitive.

Table 3. Antigen expression in AML by FAB classification

Antigen Mo, M1, M2 M3 M4, M5 M6 M7
CD13 and 33 + (a) + + + [+ or -]
HLA-DR + - + [+ or -] [+ or -]
CD64 [+ or -] [+ or -] + - -
CD14 - - [+ or -] - -
CD36 [+ or -] - + + +
CD71 [+ or -] [+ or -] [+ or -] + [+ or -]
Glycophorin A - - - [+ or -] -
CD41 and 61 - - - - +

(a) +, positive; -, negative; [+ or -], not definitive.

Table 4. Immunophenotypic classification of ALL.

 T-cell-- B-cell--
 % of FAB associated associated Surface
 ALL category antigens antigens Ig (a)

B-cell precursor >80 L1, L2 - (b) + -
B cell <5 L3 - + +
T-cell precursor 15 L1, L2 + - -

(a) Ig, immunoglobulin.

(b) -, negative; +, positive.

Table 5. Prognosis of ALL related to immunophenotype.

Favorable B-cell precursor (CD10+)
 (Cytogenetic findings influence prognosis)

Less favorable B-cell precursor (CD10-)
 B cell (Slg+; Burkitt cell leukemia)
 T cell

Table 6. Prognosis of AML related to cytogenetics.

Prognostic group Chromosome finding

Favorable Inv(16) or t(16;16)(p13;g22)
 Single miscellaneous

Intermediate t(15;17)(g22;q21), +8,
 t(6;9), normal

Unfavorable -7 or 5, del 7q
 t(v;11)(v;g23), inv(3q)
 Complex abns (a)

(a) abns, abnormalities.

Table 7. Prognosis of ALL related to cytogenetics.

Favorable Hyperdiploidy >50
 Cryptic t(12;21)(p12;q22)

Intermediate Hyperdiploidy 47-50
 Normal (diploidy)

Unfavorable Hypodiploidy: near haploid
 Near tetraploid
 del 17p
 t(11q23), usually t(4;11)

Table 8. Major molecular genetic abnormalities in AML.

Cytogenetic translocation Molecular genetic

t(8;21)(g22;q22) ETO-AML1
inv(16)(p13;g22) MYH11X-CBF[beta]
t(15;17)(g22;q21) PML-RAR[alpha]
t(6;9)(p23;g34) DEK-CAN
t(9;11)(p22;g23) AF9-MLL

Table 9. Major molecular genetic abnormalities in
precursor B-cell ALL.

Cytogenetic abnormality Molecular genetic abnormality

Hyperdiploidy >50
t(12;21)(p12;q22) TEL/AML-1
t(1;19)(q23;p13) PBX/E2A
t(9;22)(q34;q11) ABL/BCR (p190)
t(v;11)(v;q23) (a) V/MLL (a)

(a) In children, the t(4;11)(q21;q23) AF4/MLL is most common.

Table 10. FAB classification of AML.

Myeloblasuc leukemia minimally differentiated M0
Myeloblastic leukemia without maturation M1
Myeloblastic leukemia with maturation M2
Hypergranular promyelocytic leukemia M3
 Microgranular variant
Myelomonocytic leukemia M4
 With bone marrow eosinophilia (M4E0)
Monocytic leukemia M5
 Poorly differentiated (M5A)
 Differentiated (M5B)
Erythroleukemia M6
Megakaryoblastic leukemia M7

Table 11. FAB classification of ALL.

 L1 L2 L3

Cell size Small Large, often Large, homogeneous
Amount of Scant Moderately Moderately abundant
cytoplasm abundant
Nucleoli Inconspicuous Prominent Present, may be
Cytoplasmic Variable Variable Prominent

Table 12. WHO classification of AML.

AML with recurrent cytogenetic translocations
 AML with t(8;21)(q22;q22)
 Acute PML with t(15;17)(q22;q21)
 Variant acute PML with t(v;17)
 AML with abnormal bone marrow eosinophils inv(16)(p13;q22) or
 AML with 11q23 abnormalities
AML with multilineage dysplasia
 With prior myelodysplastic syndrome
 Without prior myelodysplastic syndrome
AML and myelodysplastic syndrome, therapy related
 Alkylating agent related
 Topoisomerase II inhibitor related
 Other types
AML not otherwise categorized
 AML minimally differentiated
 AML without maturation
 AML with maturation
 Acute myelomonocytic leukemia
 Acute monocytic leukemia
 Acute erythroid leukemia
 Acute megakaryocytic leukemia
 Acute basophilic leukemia
 Acute panmyelosis with myelofibrosis

Table 13. WHO classification of ALL.

Precursor B-cell ALL (cytogenetic subgroups)
Precursor T-cell ALL
Burkitt cell leukemia
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Author:McKenna, Robert W.
Publication:Clinical Chemistry
Date:Aug 1, 2000
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