Immunophenotyping of acute leukaemias

Immunology Letters 98 (2005) 9–21

Review

Immunophenotyping of acute leukaemias Marie C. B´en´e∗,1 Laboratoire d’Immunologie du CHU, Facult´e de M´edecine, Universit´e Henri Poincar´e Nancy I, BP 184, 54500 Vandoeuvre les Nancy, France Received 18 September 2004; received in revised form 16 October 2004; accepted 17 October 2004 Available online 11 November 2004

Abstract Progress in the management and understanding of acute leukaemia can only be obtained if these diseases are thoroughly investigated, both clinically and with a series of biological tools. This alone has made and still will make possible the identification of prognostic factors and of useful markers for the follow-up of patients in remission. Among the variety of approaches of acute leukaemia definition, immunophenotyping has taken over the past 25 years a predominant and now well-defined place, although room is left for further improvement. In this review, the current state-of-the-art of immunophenotyping of acute leukaemias will be replaced in the context of physiological leukocyte maturation. The recognized classifications and recommended immunophenotyping panels will then be discussed, and the clinical relevance of several key features will be presented. Finally, more recent openings for the use of immunophenotyping will be evoked. © 2004 Elsevier B.V. All rights reserved. Keywords: Leukaemia; Immunophenotyping panels; Prognostic factors

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10

2.

Immunophenotype of lymphoid cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. B-cell maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. B-lineage differentiation antigens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. B-cell maturation sequence [14,15] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. T-cell maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. T-lineage differentiation antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. T-cell maturation sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 11 11 11 13 13 13

3.

Immunophenotypic classification of ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14

4.

Immunophenotype of myeloid cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Myeloid-lineage differentiation antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Myeloid-lineage maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 15 16

5.

Immunophenotypic classification of AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗

Tel.: +33 383 683 660; fax: +33 383 446 022. E-mail address: [email protected]. 1 On behalf of the GEIL (Groupe d’Etude Immunologique des Leuc´ emies) and EGIL (European Group for the Immunological Classification of Leukemias). 0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.10.008

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M.C. B´en´e / Immunology Letters 98 (2005) 9–21

6.

Immunophenotyping leukaemias: practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Choice of panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Choice of techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 17 17

7.

Immunophenotyping provides unique information independent of other diagnostic tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Immunophenotyping and FAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Immunophenotyping and cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Immunophenotype and prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18 18 18 19

8.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The diagnosis of acute leukaemia (AL) relies on a series of biological investigations. The first step remains the cytological examination of peripheral blood and/or bone marrow samples smears, stained with the classical May Gr¨unwald Giemsa that will allow to objectivate the presence of abnormal immature cells in varying numbers, up to full bone marrow invasion. The morphological characteristics of the malignant clone allow for a first approach of the lineage involved, and makes possible application of the French–American–British classification of AL, first proposed in 1976 [1]. Fresh cells have then to be collected again, preferably from the bone marrow, for further investigations. The latter include immunophenotyping, karyotyping and molecular gene analyses bizarrely truncated. To establish the karyotype of blast cells and identify the frequent but numerous possible chromosomal anomalies of AL (translocations, deletions, aneuploidy, etc.), fresh cells obtained in a sterile fashion must be cultured in order to obtain cell growth and mitoses that are stained or chemically treated to obtain G (Giemsa) or Q (quinacrine)-banding [2]. Images of mitotic cells are then analyzed to identify each chromosome pair and their potential morphological anomalies. New techniques such as “chromosome painting” or fluorescent in situ hybridization (FISH) are still developed to improve the sensitivity and specificity of cytogenetic methods [3,4]. Conventional karyotyping yet remains the choice method to obtain a global genomic analysis in one step. It also detects most anomalies. Molecular gene analyses have been developed more recently, essentially as a result of attempting to better define the genetic anomalies yielded by chromosome translocation. Such translocations may involve crucial regions such as promoters or regulators and influence the transcription of genes not normally submitted to these signals. Identification of the specific structure of a fusion gene is also potentially useful to precisely characterise a given patient’s blasts and engineer highly sensitive tools for the detection of minimal residual disease [5]. In order to perform such investigations, DNA and possibly RNA must be made available, ideally by low-

temperature storage of freshly isolated blast cells, in sterile conditions. Immunophenotyping also requires fresh cells, but, conversely to the two techniques just mentioned, will yield results within a few hours after bone marrow sampling. The fresh marrow must be carefully mixed with anticoagulant, and rapidly transported, at room temperature, to the flow cytometry laboratory. There, several monoclonal antibodies, directed to leukocyte differentiation antigens, will be applied to whole marrow samples or isolated blasts, in one or multicolour staining methods. The pattern of expression of the differentiation antigens investigated will then be useful in four ways: for lineage assignment, to detect biphenotypic acute leukaemia, to apply current immunophenotypic classifications and to devise abnormal sets of markers or patterns useful for the patient’s follow-up. Lineage assignment is the first goal of immunophenotyping, as morphological examination of the blasts only provides a partial approach of the lineage involved. It must also be kept in mind that all hematopoietic lineages may be involved in an acute leukaemic process, i.e. myeloid, lymphoid, erythroblastic and megakaryoblastic, although acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) are the most frequent. Since the development of monoclonal antibodies after the princeps discovery of Kohler and Milstein [6], immunophenotyping has been extensively applied to AL. Interestingly, these large studies allowed to gain new knowledge about the maturation stages of hematopoietic cells, as each AL clone is representative of one of those stages through maturation blockade, and makes available to study an amount of cells never obtainable in normal bone marrow.

2. Immunophenotype of lymphoid cells Through hundreds of studies, a well-established sequence of lymphocyte maturation steps has been now established, first on leukaemic cells or cell lines, then validated on normal bone marrow samples. Although work is still in progress in this field, a reasonable and simple summary can be de-

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Fig. 1. Molecular structure of some B-lineage differentiation antigens.

vised for both B- and T-cell maturation. Interestingly, there are many similarities in these two pathways leading to the development of the cells of cognitive immunity: early lineage commitment by transcription of the molecular “vehicles” of antigen receptors, maturation steps linked to the sequence of antigen receptor genes rearrangement, and drastic selection. 2.1. B-cell maturation 2.1.1. B-lineage differentiation antigens The sequence of B-cell maturation that will be described further down in this manuscript involves the progressive expression of a number of well-identified differentiation antigens. These are represented schematically in Fig. 1. The most B-cell specific of these molecules is the B-cell receptor or BCR [7]. This complex structure of specific antigen recognition is composed of a surface immunoglobulin, with a very short intracytoplasmic tail, associated to two heterodimers of CD79a and CD79b [8]. The latter are characterized by one extracellular domain of the immunoglobulin superfamily (IgSF) and long intracytoplasmic tails carrying ITAM (immunoreceptor tyrosine-based activation motif). Transduction signals will thus be transmitted, upon engagement of the BCR, via these activation motifs on CD79 molecules. CD19 is considered a pan-B antigen, although it may display some lineage infidelity (cf. infra). The CD19 molecule comprises two extracellular IgSF domains separated by a smaller domain and a long intracytoplasmic tail, nearly as big as the extracellular portion of the molecule, carrying one ITAM. CD19 is a key molecule in the development and activation of B-cells, both in the absence and presence of antigen engaging the BCR [9,10]. CD20 is a small molecule with four transmembrane passages and thus both COOH- and NH2-termini in the cytoplasm and two extracellular loops. CD22 however does not belong to the tetraspan family. CD20 appears to act as a cal-

cium ion channel, and to regulate B-cell activation. CD20 is the target of a widely used therapeutic monoclonal antibody of human non-Hodgkin lymphomas, Rituximab [11,12]. CD21 does not belong to the IgSF, but is instead composed of a long series of short consensus repeats, tightlyfolded units of about 60 residues, belonging to the family of regulators of complement activation gene. CD21 is a receptor for degradation fragments of the complement fraction C3 and is supposedly involved in B-cell activation upon recognition of immune complexes or opsonized antigens. CD21 is also used by the Epstein Barr virus as a cell-receptor, explaining the B-cell tropism of this virus, able to immortalize B-cells and induce B-cell lymphomas [10]. CD22 is a molecule of the IgSF which may exist in two isoforms, one with seven domains and an intracytoplasmic tail carrying three ITIMs (immunoreceptor tyrosine-based inhibitory motif) and an ITAM, and a splicing variant with five extracellular domains and an intracytoplasmic tail carrying one ITIM. These inhibitory signals are thought to be involved in the control of B-cell responses to antigens [10]. CD10 was initially thought to be a common antigen of acute lymphoblastic leukaemia and was thus dubbed cALLA. CD10 is an enzyme also known as the neutral endopeptidase or enkephalinase [13]. It cleaves small (3 aa) peptides at the NH2 end of hydrophobic amino acids (val, ile, phe, leu, ala). It is widely expressed by enterocytes in the gut, epithelial cells of renal tubules, but also polymorphonuclears and brain tissue. Among its numerous substrates are the angiotensins and bradykinin or fMLP. Its role in B-cell maturation is still obscure, but it might be involved in regulating the growth and differentiation of B-cell progenitors by interfering with soluble active or pro-active factors. 2.1.2. B-cell maturation sequence [14,15] A schematic representation of B-cell maturation in the bone marrow is presented in Fig. 2.

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M.C. B´en´e / Immunology Letters 98 (2005) 9–21

Fig. 2. B-cell maturation pathway.

As for all hematopoietic cells, a stem cell resulting from the division of a totipotent cell, is at the origin of this sequence. This stem cell is characterized immunophenotypically by the surface expression of the earliest identified differentiation antigen, CD34, and of the MHC class II DR molecule. Its “mother-cell”, which will retain totipotency, is believed to be CD34+/DR− [16]. DR expression could be related with interactions of the developing cells with stromal cells in the bone marrow. In this stem cell, both heavy (H) and light (L) immunoglobulin chain genes are unrearranged, in somatic configuration (H0L0). The first indication of commitment towards the B-lineage is the intracytoplasmic expression of CD79a. This signs the Pro-B cell stage. Interestingly, even before any sign of gene rearrangement is detectable, the cell in some way makes sure that the accessory molecules necessary for BCR membrane expression can be transcribed properly. In such cells, CD22 can also be observed in the cytoplasm but not on the cell surface, while membrane CD19 is already detectable. In the next stage, or “early B-cell” immunoglobulin heavy chain genes will begin to rearrange [17]. This will occur first on one of the two chromosomes 22, then, if the rearrangement is unproductive, on the other one (HxL0). There are thus two opportunities for the cell to manage a successful IgH rearrangement. If a stop codon occurs in the sequence, then the cell will die. This stage is characterized by the expression of surface CD10, perhaps as mentioned above, to stop cell proliferation and activate differentiation factors. CD22 and CD21 can also be detected on the cells surface. The early Bcell stage can probably be divided in two sub-stages, as some ALL display the immunophenotype just described, while oth-

ers are characterized by the additional expression of surface CD20. CD10 expression is transient during B-cell maturation, and disappears during the next stage of “pre-B-cell”. This differentiation step has long been known as it is characterized by the presence of intracytoplasmic mu (␮) chains, readily detectable by polyclonal antibodies in permeabilized cells. This stage seems to translate the fact that, having successfully rearranged an immunoglobulin heavy chain gene (HRL0), the cell is testing that transcription indeed can proceed successfully. It is also known that a small amount of these new mu chains will reach the cells surface, together with pseudo light chains, probably in a first step of selection and possibly also to initiate the rearrangement of an immunoglobulin light chain [18]. This also proceeds sequentially, always on one of the chromosomes 2, carrying the kappa genes, then in a stoichiometric but progressive fashion, until a successful rearrangement is achieved. Thus, one, two, three or four chromosomes may show signs of rearrangement, depending how fast the cell managed to obtain a satisfactory IgL rearrangement. Once this is achieved, the transcripts of the rearranged genes assemble in the cytoplasm to form a full H2L2 immunoglobulin, which is carried to the surface by the heterodimers of CD79. This “na¨ıve B-cell” thus express CD79, CD19, CD22, CD21, CD20, and surface immunoglobulin. However, due to the alternative splicing of a long transcript of the adjacent genes coding for mu and delta constant domains, this na¨ıve B-cell escapes the rule of isotypic exclusion and coexpresses IgM and IgD of the same specificity (associated to the products of the same VDJ and VJ IgH and IgL rearrangements). It is thus also called a “␮␦ B-cell”.

M.C. B´en´e / Immunology Letters 98 (2005) 9–21

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Fig. 3. Molecular structure of some T-lineage differentiation antigens.

The na¨ıve B-cell is submitted to the medullary selection process, aiming at removing self-reacting antibodyproducing cells [19], and, if successful, reaches the peripheral secondary lymphoid organs, in the mantle zone of germinal centres. 2.2. T-cell maturation 2.2.1. T-lineage differentiation antigens The sequence of T-cell maturation similarly involves the progressive expression of well-described differentiation antigens that are represented schematically in Fig. 3. As for B-cells, the most specific of these molecules is the T-cell receptor or TCR [20]. This multimolecular structure of specific antigen recognition is composed of the heterodimeric TCR molecule itself, with a very short intracytoplasmic tail, associated to six CD3 components. The TCR heterodimer is constituted by the association of two molecules of the IgSF with a constant domain near the membrane and a variable domain at the N-terminus, constituted by the rearrangement of TCR-specific VDJ and VJ genes. There are two types of TCR, an ␣␤ heterodimer on more than 95% of mature T-cells and a ␥␦ TCR on about 5%. The CD3 complex comprises four chains with one extracellular domain of the IgSF each and long intracytoplasmic tails with ITAM, respectively dubbed ␥, ␦ and ε which is present twice, likely on each side of the TCR. Two other chains complete the CD3 complex, and can be a ␨␨ (zeta–zeta) homodimer or a ␨␩ (zeta–eta) heterodimer. In both cases, there is virtually on extracellular domain for these mostly intracytoplasmic molecules with several ITAM and a major involvement in T-cell activation upon antigen recognition. The recognition, by mature T-cells, of peptidic epitopes presented by MHC class II or class I molecules involves the presence of co-stimulatory molecules that will specifically bind the constant domain of the proper MHC molecule [21]. CD4 is a molecule of the IgSF with four Ig-like domains that will recognize MHC class II and is present on the surface of T-helper cells. It also is the receptor of the human immunodeficiency virus. CD8 is in most cases an ␣␤ heterodimer comprising two long chains with an N-terminal domain of the IgSF, that will bind to the ␣1 domain of MHC class I

molecules. CD8 cells will be able to recognize abnormal self peptides, derived from tumoral or virus-infected cells, and kill them. They also have regulatory properties and are thus called cytotoxic/suppressor cells [22]. Four other differentiation antigens are important in the definition of T-cell maturation stages. CD2, CD5 and CD7 are all considered pan-T antigens, although all may display some lineage infidelity and be expressed on B-cells for the first two, or on myeloid cells for the last. CD1 is a molecule of restricted expression on thymocytes. CD2 belongs to the IgSF and has two extracellular domains. Its ligand is CD58, expressed on antigen-presenting cells, and curiously also on sheep red blood cell. This characteristic, although the molecular mechanism was unknown, was used at the time when E-rosetting was used to identify T-cells [23]. CD5 is a member of the scavenger receptor superfamily, composed of three cysteine-rich domains of about 100 amino acids each. There is an ITAM on its cytoplasmic tail, yet CD5 is a dual receptor, giving either stimulatory or inhibitory signals depending on the cell type (T or B) and development stage (thymocyte or mature T) on which it is expressed [24]. CD7 is a member of the IgSF, with one N-terminal domain separated from the transmembrane domain by four repeat units rich in Proline, Serine and Threonine. Its functions have long remained obscure, although the presence of an IgSF domain suggests adhesion properties. The study of CD7 deficient mice has shown CD7 to be involved in the regulation of cytokine production by T-cells [25]. CD1 is a peculiar molecule sharing the structure of MHC class I antigens, with a peptide groove between domains ␣1 and ␣2 of its alpha-chain stabilized by the beta-2 microglobulin [26]. Five isoforms of this molecule have been described and although CD1a seems to be restricted to T-cells and Langerhans cutaneous dendritic cells, the other isoforms may be seen on normal or malignant B-cells. 2.2.2. T-cell maturation sequence A schematic representation of T-cell maturation, which takes place first in the bone marrow, then in the thymus, is presented in Fig. 4.

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M.C. B´en´e / Immunology Letters 98 (2005) 9–21

Fig. 4. T-cell maturation pathway.

The same stem cell as described in the B-cell maturation sequence is at the origin of T-cell differentiation. In this stem cell, all TCR genes are in stromal configuration (␥0␦0, ␣0␤0). Again as for B-cells, the first indication of commitment towards the T-lineage is the intracytoplasmic expression of the CD3 complex that will later be used to bring the rearranged TCR to the cells’ surface. This signs the Pro-T cell stage, where the only other marker detectable is membrane CD7. There is evidence in the literature that this stage does not definitely sign commitment to the T-lineage and that such cells may revert towards myeloid differentiation, losing CD3 cytoplasmic transcription. There is another stage or Pro-T cell, characterized by a more complete surface pattern of T-lineage markers expression, where CD7 can be detected together with CD2 and CD5. This stage is the last where maturing T-cells can be found in the bone marrow. It is suspected that the adhesion properties of CD7 and CD2 are used by these cells to reach the peripheral blood and relocalize in the thymic epithelium, although the mechanisms involved are still unclear. As pro-T cells reach the epithelial cells of the thymic cortex, they acquire the transient expression of CD1, characteristic of these “corticothymocytes”. At that stage, TCR genes will begin to rearrange. This will occur first for the ␥␦ TCR although unproductive rearrangements of these genes will be found in most of peripheral T-cells. The VJ and VDJ genes of the ␣␤ TCR will then attempt to rearrange, and only cells with a successful rearrangement will proceed towards full maturation. During these thymic steps, there is first a mas-

sive proliferation, in the cortex, then drastic selection in the medulla [27]. Immunophenotypically, the maturing cells will first co-express CD4 and CD8, which allows, through the recognition of MHC class II or class I molecules on thymic epithelial cells, to decide of the fate of the future mature Tcell that will be either a helper or a cytotoxic/suppressor cell. At the next stage, medullary thymocytes are mature na¨ıve T-cells that have lost surface CD1, and express surface TCR associated to on the cell’s membrane to the CD3 complex. Na¨ıve T-cells that have successfully passed both negative and positive thymic selection will then reach the peripheral secondary lymphoid organs, in the T-cell areas surrounding germinal centres.

3. Immunophenotypic classification of ALL Based on the maturation steps and differentiation antigens patterns described above, the EGIL proposed in 1995 a classification of B and T-ALL [28], shown in Tables 1 and 2. Table 1 Immunological classification of B-lineage ALL according to the EGIL proposal [28]

B-I B-II B-III B-IV

cCD79, CD19, c or s CD22

CD10

c␮

sIg

+ + + +

− + + +

− − + +

− − − +

M.C. B´en´e / Immunology Letters 98 (2005) 9–21 Table 2 Immunological classification of T-lineage ALL according to the EGIL proposal [28]

T-I T-II T-III T-IV

cCD3

CD7

CD2, CD5, CD8

CD1a

CD3+/CD1a−

+ + + +

+ + + +

− + + +

− − + −

− − − +

In both cases, four stages are identified, respectively B-I to B-IV and T-I to T-IV, from the most immature immunophenotype to the most mature one. To apply this classification, it is necessary to test the blast cells of every given patient with MoAbs directed to all the differentiation antigens used for stage definition. It will indeed be as important to detect lineage associated markers allowing proper lineage assignment, that to observe the absence of expression of markers present at later differentiation stages, allowing to properly classify the patient’s leukaemia. Using this classification retrospectively in series of ALL from several countries, EGIL was able to demonstrate that the distribution of the various subsets defined was almost similar in these countries. As shown in Fig. 5, the most frequent subtype, both in children, and adults is B-I, characterized by a proliferation of early-B cells, also called “common B-ALL” in other series. T-lineage ALL are much less frequent than B-lineage ALL, and among them, T-III ALL are the more frequent. These proliferations of blasts with a corticothymocyte immunophenotype may suggest that there diseases, also frequently characterized by mediastinal mass, might originate in the thymus before disseminating back to the bone marrow. The rarest forms of both B- and T-lineage ALL are, at one end of the spectrum, B-I and T-I, i.e. those with the most immature immunophenotype, and at the other end, B-IV and T-IV, i.e. the most mature ones.

Fig. 5. Incidence of ALL sub-types according to EGIL classification, as determined among EGIL members cohorts. Data are expressed as percentages.

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4. Immunophenotype of myeloid cells Hematopoietic cells of the myeloid lineage give rise to cells involved in innate or non-cognitive immunity. These cells do not need to rearrange sophisticated specific receptors, but have to develop abilities to deal promptly with pathogens, and, for some of them, be able to present degraded foreign material in the form of peptides ensconced in the groove of MHC class II antigens. The maturation sequence of these cells is thus more straightforward, and apparently less associated with transitional stages. Another reason to explain that no well-defined immunophenotypic maturation pathway has been described for these cells is that, unlike lymphocytes, they display highly discriminative morphologic features. At the blastic stage of acute myeloid leukaemia (AML), however, it may be difficult to recognize on morphologic grounds the maturation stage, and even sometimes the lineage commitment of the leukaemic cells. This is why immunophenotypic identification of myeloid maturation patterns has begun to develop in the recent years, both in the field of AML and in the neighbour area of myelodysplasia [29–31]. 4.1. Myeloid-lineage differentiation antigens Myeloperoxidase (MPO) is an enzyme expressed at various levels in nearly all myeloid cells [32]. It has an intracytoplasmic localization, within the cell’s phagosomes, and is involved in the generation of hydrogen peroxide. It is composed of two long and two short chains, linked by a globulous domain, as shown in Fig. 6. CD13 is an ectopeptidase sharing many features with CD10, although it is a homodimer with two enzymatic domains [33]. It is also strongly expressed on the brush border of enterocytes and renal tubular epithelial cells. CD33 is a homodimeric member of the IgSF, with two extracellular domains for each monomer [34]. This likely makes it an adhesion molecule involved in cell–cell interactions. CD117 is the receptor of the stem cell factor (SCF) [35]. Is encoded by the c-kit oncogene. It also belongs to the IgSF, and has five extracellular domains. Its cytoplasmic tail harbours a tyrosin kinase domain. CD14 is part of the lipopolysaccharide (LPS)-receptor complex [36]. It is composed of a series of extracellular leucin-rich short repeats. It is linked to the cell’s surface by a glycerophosphatidyl inositol (GPI) anchor. It is able to bind free LPS, or LPS linked to the LPS-binding protein (LBP). CD14 will then interact with toll-like receptors (TLR), mostly TLR-4, which has a long intracytoplasmic tail and will be able to induce cell activation, phagocytosis and the secretion of pro-inflammatory cytokines. CD15 and CD65 are two carbohydrate motifs, respectively the 3-fucosyl-N-acetyl-lactosamine and the ceramide dodecasaccharide [37], that can be carried by various proteins. CD15 is also known as the Lexis × antigen. CD15, bound to CD11b, was recently shown to be the lig-

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Fig. 6. Molecular structure of some myeloid-lineage differentiation antigens.

and of the dendritic cell differentiation antigen DC-SIGN [38]. In order to facilitate phagocytosis, myeloid cells also have to express opsonization receptors, i.e. immunoglobulin and complement fractions receptors. The family of Fc receptors for IgG [39] is composed of three molecules of the IgSF, CD16, CD32and CD64, which may exist in various isoforms. They will allow the recognition and binding of IgG already engaged in an antigen/antibody immune complex, either soluble or on the surface of target cells. A series of complement receptors are also used by myeloid cells to bind opsonized particles or immune complexes having begun to activate the complement cascade. CD35 is also called CR1 (complement receptor 1) [40]. It is a molecule of the family of regulators of complement activation genes, made up of a series of 30 consensus sequences of about 60 aminoacids. It binds C3b, C4b and iC3b. The integrins CD11b/CD18 and CD11c/CD18 are also complement receptors, respectively CR3 and CR4 [41]. They also exert a function of adhesion molecules, able to bind extracellularly to extracellular matrix components, and intracellularly to molecules of the cytoskeleton. They thus contribute to the mobility of myeloid cells, which indeed have to move quickly to inflammation sites. CD36, the thrombospondin receptor, is constituted of a single peptide chain, heavily glycosylated in its extracellular portion [42]. It also binds collagen and oxidized low density lipoproteins. Differentiation antigens on myeloid cells also display a lot of lineage infidelity, and such molecules already described such as CD19, CD2, CD4 or CD7 can be seen on normal or leukaemic cells of myeloid lineages.

4.2. Myeloid-lineage maturation The earliest molecule attesting of a CD34+DR+ stem cell commitment towards myeloid lineage, is the intracytoplasmic enzyme MPO. Several other enzymes, such as lactoferrin or lyzozyme, will also attest of myeloid differentiation, but they will appear later along maturation stages [43]. Three of the myeloid differentiation antigens described above can also be considered as early markers. They are CD13, CD33 and CD117, frequently observed together on the surface of myeloid blasts, or in any type of combination (see below). However, the expression of CD117 seems to disappear at earlier stages of differentiation than the two other, perhaps owing to its function as SCF receptor. Cells engaged in myeloid differentiation, depending on the signals provided by hemopoietic growth factors in their microenvironment, will then proceed to become monocytes or granulocytes, and among the latter, neutrophil, basophil or eosinophil polymorphonuclears. These cells, well differentiated morphologically, may share the expression of most of the molecules described above. Yet CD15 and CD65 can be considered more characteristic of neutrophils, while CD14 would be more specifically expressed by monocytes. During the cells’ maturation however, progressive patterns can be observed, that have not yet been all described [44]. Two other cell types must also be mentioned, although they give rise to leukaemia very rarely, yet have been placed in the myeloid category in the FAB proposal. They are respectively erythroblasts and megakaryoblasts. These cells will not express MPO, but very early acquire specific differentiation antigens. The most characteristic of erythroblasts are CD36 and glycophorins. Megakaryocytes express, first intracytoplasmically then on the cell’s surface, the integrin chains

M.C. B´en´e / Immunology Letters 98 (2005) 9–21 Table 3 Immunological classification of AML (after Casasnovas et al. [46])

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6. Immunophenotyping leukaemias: practical considerations

The first aim is lineage assignment, and it is noteworthy to consider that the earliest markers allowing to determine lineage commitment, for B-ALL, T-ALL and AML, are expressed intracytoplasmically, i.e. cCD79, cCD3 and MPO. This implies that immunophenotyping laboratories must have developed robust techniques of intracytoplasmic labelling, in order to properly detect these important markers, which is trickier than dealing with surface labelling. The recommendations of several groups however engage to identify at last two lineage-specific markers on the blasts’ surface, which implies testing at least three differentiation antigens of each lineage, more of the positivity of two cannot be demonstrated [47]. The second aim is to detect the eventuality that the AL tested is biphenotypic (BAL), i.e. expressing significantly markers from more than one lineage. This is important at the individual patient’s level more than in terms of statistics or frequency, owing to the dramatic issue of these diseases, requiring bone marrow transplantation as soon as possible [48]. As the aberrant expression of markers from different lineages is not exceptional in AL, due to the physiological lineage promiscuity of several antigens, and because of the not infrequent presence of one or two myeloid antigens in ALL [49], the EGIL has proposed a scoring system to diagnose BAL [28,50]. Depending on their lineage specificity, B-, T- and myeloid markers have been attributed a “weight” between 0.5 and 2 points. BAL is thus defined as a leukaemia where blast cells have a score higher than 2 in more than one lineage. In order to apply this scoring system properly, a larger number of markers than three per lineage has to be tested. The third aim of immunophenotyping is to apply the classifications described above, and this again extends the panel of MoAbs to test, as is the fourth aim of determining aberrant patterns useful for the follow-up of patients and detection of minimal residual disease [51]. All in all, and after much debate, a consensus panel was proposed in 2000 by a working group of the International Clinical Cytometry Society [47]. As presented in Table 4, this panel has been completed by the addition of CD35 and CD36, for the sake of AML classification, and CD56 on behalf of the recently described CD4+/CD56+: lineage-entity of precursor dendritic cell leukaemia/lymphoma [52]. Additional to the definition markers described above are CD34 and DR, for two reasons. The first one is that exceptional cases of “undifferentiated” stem-cell AL, only expressing these two markers may exist. The second one is that CD34 is a good marker of immaturity but may also remain on blasts at later stages of maturation providing an abnormal pattern of interest for follow-up as mentioned above.

6.1. Choice of panels

6.2. Choice of techniques

As indicated in Section 1, immunophenotyping has several aims that will guide the choice of markers and the panel of MoAbs to test for a given unknown patient.

The techniques of immunophenotyping have evolved with time, paradoxically gaining in diversity which makes standardization an aim not yet attainable. Initially, the reference

MA MB MC MD ME

MPO and/or CD13 and/or CD33 and/or CD117

CD7

CD35 and/or CD36

CD15

+ + + + −

− + ± ± ±

− − + ± ±

− − − + ±

CD41 and CD61 as well as members of the CD42 family composed of one or several leucin-rich protein domains [45].

5. Immunophenotypic classification of AML As mentioned above, because of the rich aspects of myeloid morphology, much less work has been published on the immunophenotypic features of AML and a fortiori on classifications. However, in 2003, the GEIL published a classification proposal based on the analysis of more than 2000 cases of AML [46]. The strategy used to identify relevant differentiation antigens, was to apply computerized clustering models to the expression patterns of the patients’ blast cells. In a first approach, this allowed to identify four clusters, respectively containing (i) CD13, CD33, CD117; (ii) CD15, CD65; (iii) CD14, CD35, CD36; (iv) CD7. After further analysis of the data, the classification shown in Table 3 was proposed, based on analysis of the expression of (a) CD13, CD33 and/or CD117; (b) CD7; (c) CD35 and/or CD36; (d) CD15. For each additional marker expressed in this progression, the persistence or absence of the previous markers, except group (a), is indifferent, and four stages can thus be defined. They were dubbed MA to MD in order to avoid confusion with the FAB proposal, widely used to describe AML and which is based on a numeric classification, from M0 to M7. A fifth group, ME, was also described, lacking the expression of group (a) antigens. Because of the large series of cases available to develop this classification, it was also possible to determine the frequency of each of the five MA to ME subsets, which was found to be respectively 20%, 6%, 20%, 50% and 5%. Prospective work is currently in progress within the GEIL to see whether these features remain valid on new series of patients more systematically tested for the discriminant markers.

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Table 4 Recommended panel for AL immunophenotyping (modified from Braylan et al. [47]) B-lineage

T-lineage

Myeloid lineage

Other

CD10 CD19 CD20 CD22 cCD79 IgM (cytoplasmic and surface)

CD1a CD2 CD3 (cytoplasmic and surface) CD4 CD5 CD7 CD8

MPO CD11b CD13 CD14 CD15 CD16 CD33 CD35 CD36 CD64 CD65 CD117

CD34 DR CD56 CD41 or CD61 (megakaryocytes) GPA or CD36 or CD71 (red blood cells)

Markers are arranged by CD numbers, not priority choice. This list is by no means exhaustive.

method was applied everywhere. Blast cells were separated by density gradient centrifugation, and enriched cell suspensions were used for indirect labelling first with unconjugated MoAbs (often still in ascites), then with a fluoresceinconjugated anti-mouse Ig polyclonal. After a short period when the labelled cells were examined in UV light under a microscope, flow cytometry was progressively yet quite rapidly available in immunophenotyping centres. The development of technology has now led to a situation where direct labelling associated with multi-colour flow cytometry is more and more currently used, on whole bone marrow samples where erythrocytes are merely lysed and no separation of blast cells is achieved. However, owing to hemodilution of the samples or to the low blast load in some cases, it has become necessary to devise a way to identify blast cells on cytograms. This was overcome by the demonstration of a specific position of blast cells, in most cases, on a biparametric scattergram of side scatter versus CD45 labelling where residual lymphocytes, monocytes and polymorphonuclears display stable patterns, allowing for an accurate gating of blast cells for further analysis with other markers [53,54]. This useful tool however not only uses up one of the flow cytometer photomultiplicator, but imposes the use of a costly reagent in all of the combinations of MoAbs tested. Other technical issues are the choice of lysis reagent, as not all provide the same treatment of the cells, some even erasing the reactivity of some markers [55]. Finally, and consensus is far from reached on this last point, the combinations of clones and labelling fluorochromes remain idiosyncratic to each laboratory. This issue might however be soon overcome by the impossibility for the industry to propose every clone in every possible conjugation form, especially as the choice of fluorochromes keeps growing. Nevertheless, and providing robust and well monitored techniques have been developed by the immunophenotyping laboratory, this diagnostic tool provides extensive data within a few hours after sampling, which probably makes it the second major diagnosis tool of AL after morphology. Moreover, as briefly alluded to below, immunophenotyping provides distinctive information.

7. Immunophenotyping provides unique information independent of other diagnostic tools 7.1. Immunophenotyping and FAB Although immunophenotyping is more and more becoming a support of lineage assignment, sometimes difficult to assess on morphologic grounds, extensive immunophenotyping would be of little interest if it were strongly correlated to the FAB proposal. This is however not the case, neither for ALL nor for AML. For instance, although most L3 cases will indeed be proliferations of mature sIg+ B-IV cells, this immunophenotype can also be observed in L2 or even L1 cases. Another example is provided in Fig. 7, where the expression of CD15 and CD11c is clearly almost equally distributed in the four types of AML exemplified. 7.2. Immunophenotyping and cytogenetics Karyotypic and molecular anomalies are not always observed, and immunophenotyping clearly provides invaluable information in cases with normal karyotypes. One example is that of the t(4;11) translocation, of bad prognosis in ALL. A GEIL study [56] showed that all t(4;11) cases had a B-I immunophenotype, yet not all B-I ALL carried the translocation, although they had the same pejorative prognosis. The

Fig. 7. Expression of CD15 and CD11c among FAB sub-types of AML.

M.C. B´en´e / Immunology Letters 98 (2005) 9–21

immunophenotpype may also orient the costly investigations of cytogeneticists and molecularists. 7.3. Immunophenotype and prognosis Numerous studies in the literature have reported the independent or multivariate value of the expression of given markers or patterns in AL [57,58]. Among recent contributions from our groups, one may mention the description of age as a prognostic factor in M0 AML [59], the various prognoses associated to the AML classification proposal [46], or the dramatic evolution of patients with pDC AL [60]. Another aspect of the utility of immunophenotyping to predict the evolution of treated patients, lies in the increasing capabilities of multi-colour labelling to identify blast cells as rare events. This will most likely find in the near future large practical applications in the detection of minimal residual disease [51]. Several studies have already shown that this tool could work, mostly when aberrant patterns of differentiation antigens have been identified at diagnosis. Of course, this implies that a large enough panel of MoAbs is used at this early stage, essentially to assess the coexistence on the blasts’ surface or cytoplasm of markers from different lineages, which is one of the most common characteristics used to identify these malignant cells when they become very scarce. This strategy has been used for some time for ALL [51,61], but has more recently begun to develop for AML as well [62].

8. Conclusion Immunophenotyping, enhanced by the massive development of monoclonal antibodies and by the progress of flow cytometry techniques has allowed over the past 25 years to progress greatly in the understanding of hematopoiesis physiology and in the definition of hematopoietic disorders, of which acute leukaemia representing a large part. These severe diseases of heavy consequences for the patients and of high cost for the health system, deserve to be as well-defined as possible. This only will help to keep improving an adapted management of each AL, and develop sensitive tools for the early detection and thus faster eradication of relapses. Much hope is also placed in the study of AL with the new technology of microarrays, as such wide screening might help to identify new relevant molecules that could usefully complement the current panels of immunophenotypic definition of leukaemia.

Acknowledgements Many thanks to Gilbert Faure and Marc Maynadi´e for their help with the preparation of this manuscript. Thanks also to the Bulgarian Society of Immunology for the invitation to give this EFIS lecture in Sofia.

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