Immunophenotyping of acute leukemias and myelodysplastic syndromes

Immunophenotyping of acute leukemias (AL) and myelodysplastic syndromes (MDS) was one of the first areas where monoclonal antibodies were applied (1-3). Initially, indirect immunofluorescence techniques evaluated by fluorescence microscopy were used (4); later immunocytochemistry methods on fixed cells were developed (5). During the last 15 years, multiparameter immunophenotypic approaches using direct immunofluorescence stainings analyzed by flow cytometry have become widely used and the preferred method for the immunophenotypic analysis of AL and MDS (6). The extended use of flow cytometry immunophenotyping and its involvement in routine diagnosis were facilitated by the unique characteristics of this technology that allows an objective analysis of high numbers of cells in a relatively short period of time—information which is simultaneously being recorded about two or more monoclonal antibody stainings for single cells (7). Further development of other alternative or complementary immunophenotypic approaches, such as those based on laser scanning cytometry, never reached the same rate of success (8). Initially, the rationale for the clinical use of immunophenotypic techniques was based on the need for more objective criteria to support the morphological diagnosis and classification of AL and MDS. The underlying hypothesis was that neoplastic cells from patients with these hematological malignancies corresponded to the leukemic counterpart of normal hematopoietic cells usually committed into one, or less frequently more than one, cell lineages, blocked at a specific maturation stage (9). Thus, a detailed analysis of the phenotypic characteristics of these cells would provide useful information to classify them according to their lineage and maturation stage. Classification of AL and MDS according to both parameters had already proven to be clinically useful on morphological grounds (10, 11). Since then, immunophenotyping has provided information that contributed to the refinement of already existing morphological classifications of AL and the definition of new prognostic entities among these patients (12-14). More recently, it has also proven to be of great help for the screening of genetic abnormalities (14-22), the follow-up of minimal residual disease (MRD) (23-25), monitoring of patient-specific therapies (26, 27), and the study of MDS (28, 29). These new applications of flow cytometry immunophenotyping mainly rely on the concept that even if neoplastic cells show a great similarity to normal hematopoietic precursors, they frequently display aberrant phenotypes that allow their specific identification and discrimination from normal cells, even when present at very low frequencies (23-25). To a large extent, such aberrant phenotypes would be a consequence of the genetic abnormalities accumulated by the neoplastic cell (14-22). In this paper, we will briefly review the most outstanding contributions of flow cytometry immunophenotyping for the management of patients with AL and MDS and provide a perspective for future developments. Acute lymphoblastic leukemias (ALL) were the first group of hematological malignancies in which immunophenotyping proved to be clinically useful. More than 20 years ago, ALL was already classified as B, T, or null ALL (non-B, non-T) depending on whether leukemic cells expressed surface immunoglobulins (sIg), formed rosettes with sheep erythrocytes, or lacked on both markers (30). Later on, the identification of the CD10 antigen, present in around two-thirds of all ALL patients, provided the basis for the more recent classifications through the definition of a new subgroup of patients that included most non-B, non-T cases (the common ALL phenotype) (31). The phenotypic immaturity of these morphologically-appearing lymphoid-lineage cells was supported on immunophenotypic grounds by their positivity for the terminal deoxynucleotidyl transferase enzyme (nTdt) (32). Thereafter, the availability of an increasingly high number of monoclonal antibody clones that detected antigens present in lymphoid cells and their precursors, together with the parallel development of the multiparameter capabilities of flow cytometry, contributed to definitively prove that most ALL cases showing either a common or a null phenotype derived from a B-cell precursor (33). In this regard, multidimensional analysis of the immunophenotypic profiles of normal bone marrow (BM) B-cell precursors was crucial. These studies provided a detailed definition of the exact sequence of expression of multiple antigens along the normal B-cell maturation pathways in the BM (34-38). Accordingly, at present it is well accepted that the first B-cell associated antigens to be expressed after commitment of an early CD34+ hematopoietic precursor into the B-lymphoid lineage are CD22, CD10, and CD19 (on the cell membrane), nTdt, and cytoplasmic CD79a (cCD79a) (35-38). Immediately after, the B-cell precursors sequentially start losing CD34 and nTdt, decrease CD10 expression, and display reactivity for CD20 (35-37). Later on, the B-cell precursors produce Ig μ heavy chains which accumulate in the cytoplasm until Ig light chains are produced (37, 39). When this occurs, IgM molecules are expressed on the cell surface of a functionally immature B-lymphocyte (37, 39). Based on the maturation sequence of the normal BM B-cells, precursor-B-ALL patients are currently classified into four major groups (40): BI or null ALL (CD19+, cCD79a+), BII or common ALL (CD10+), BIII or pre-B ALL (cIgμ+), and BIV or B ALL (sIg+). Similar to precursor B ALL, T-ALL is currently divided into four groups (40): pro-T (or TI), pre-T (or TII), cortical or (TIII), and mature (or TIV) ALL. Pro-T ALL typically shows coexpression of two early T-cell markers -CD7 and cCD3− in the absence of other T-cell-associated antigens. In addition to CD7 and cCD3, Pre-T ALL cases express surface CD2, CD5, and/or CD8. As cortical thymocytes (41), leukemic cells from cortical T-ALL display reactivity for CD1a. The TIV/mature T-ALL phenotype (sCD3+, CD1a−, CD4+, or CD8+) is more often observed among patients presenting with T-lymphoblastic lymphomas than a pure T-ALL. In both TIII and TIV T-ALL, surface expression of CD3 may be associated with expression of TCR of either the TCRα/β or TCRγ/d type. Despite the clear association initially reported between the phenotypes of leukemic and normal lymphoid precursors, further studies demonstrated that both groups of cells do not display identical and overlapping phenotypes (42). As an example, accumulating evidence supports the notion that during B-cell ontogeny, CD10 is expressed at a very early stage even prior to CD19 (36, 38). In this case, BI or null ALL, which typically display a cCD79a+, CD19+, CD10− immature (CD34+, Ig−) phenotype (43, 44), would not fit into the normal B-cell maturation scheme (36, 38). Also, the absence of reactivity for CD10 would represent an aberrant phenotype. In fact, during the last decade it has been shown (23, 24, 42, 45-47) that both precursor-B and T ALL display aberrant phenotypes in more than 95% of the cases. This allows for an unequivocal discrimination between normal and leukemic lymphoid precursors in the BM (45-47), peripheral blood (PB) (45), and other body fluids (23, 48). The occurence of these aberrant phenotypes can only be explained because of the existence of underlying genetic abnormalities in leukemic blast cells. Accordingly, CD10− blast cells from pro-B ALL frequently are CD15+, 7.1+, and/or CD65+ (43, 44), a phenotype which has been shown to be closely related to the presence of t(4;11) and other cytogenetic abnormalities involving chromosome 11q23 (43, 44). This concept can also contribute to the understanding of the associations observed between a common-ALL phenotype and hyperdiploidy (49), t(9;22) (18, 49), and t(12;21) (17, 20), as well as the additional correlations reported in adult and childhood common-ALL between the latter two translocations and a CD34high, CD38dim (18), and a CD20−/partial+, CD9− /partial+, CD34−/+heterogeneous phenotype (17, 20), respectively. Moreover, in CD34−, CD20+ pre-B ALL patients, t(1;19) is frequently present (21) and slg+ B-ALL with a bcl2− /dim phenotype commonly display t(8;14), t(2;8), or t(8;22) (16, 22) (Table 1). Altogether, these associations between the phenotype and the genotype of blast cells contribute to explain the prognostic impact and clinical relevance of the immunological classification of precursor-B-ALL (50). At the same time, they also contribute to understanding the apparently controversial associations initially reported in precursor-B ALL, between the expression of individual markers and the prognosis of the disease (e.g., the expression of both CD34 and myeloid-associated antigens has been associated with adverse prognostic features in adults whereas in childhood ALL CD10 and CD34 were considered as favorable prognostic features) and why they have lost their prognostic relevance once the genetic subgroups of precursor-B ALL are separately considered (reviewed in14, 50). In contrast to what is described above for precursor-B ALL, no clear association between the immunological classification of T-ALL and specific T-cell genotypes or prognosis, have been clearly established in the past (16, 50). Despite this, it should be noted that recent reports (51) suggest that with current treatment strategies, cortical T-ALL patients could have a better outcome, which is probably due to a higher susceptibility of leukemic cells from these patients to undergo apoptosis. Immunophenotypic studies are apparently less useful in AML than in ALL; this probably has a multifactorial explanation related to the higher complexity of the former group of leukemias. First, the so-called myeloid cells include up to seven different lineages (neutrophilic, basophilic, eosinophilic, monocytic, mast cell, erythroid, and megakaryocytic) plus dendritic cells (52-56). Moreover, from the phenotypic point of view, leukemic cells from AML patients are significantly more heterogeneous both in phenotypic and cytogenetic grounds, the presence of two or more subpopulations of blast cells being found in most cases (57, 58). Apart from this, information about the normal maturation pathways of different myeloid cell lineages, especially about those less represented in BM, is limited (29, 56). Finally, there is no specific and universal single myeloid marker that would identify early commitment of hematopoietic precursors into any of the myeloid lineages (29, 52-56). CD117 together with CD13 and CD33, is considered the earliest antigen to be detected during differentiation of hematopoietic precursors into myeloid cells (29, 52-56). However, when individually considered, none of these markers is specific to myeloid leukemic cells (46, 47, 59, 60), and their combined expression is also found in the more immature, uncommitted CD34+ hematopoietic precursors (38, 61). At present, cytoplasmic expression of myeloperoxidase (MPO), lisozyme, and tryptase (with the B12 clone) are considered as the most characteristic markers of myeloid cells (40, 62, 63). Despite this, the expression of these markers is typically restricted to a few myeloid lineages. Accordingly, in normal myeloid cells, reactivity to MPO and lisozyme is restricted to the granulomonocytic precursors while B12 (tryptase) appears to be highly characteristic of maturation into the mast cell and basophilic lineages (29). CD15 and CD14 are strongly expressed in mature neutrophils and monocytes, respectively (29, 52, 56). However, these two markers are coexpressed during maturation of myeloid cells into both cell lineages (29, 55, 56), which limits their utility in distinguishing between AML containing neutrophil-(M1, M2, and M3 FAB morphological subtypes) and monocytic-lineage (M5 FAB subtype) blast cells (64). Glycophorin A is a highly specific erythroid marker (29, 56, 65); however, it is only expressed at relatively late stages of maturation of erythroid cells (29, 65), which limits its utility in AML. In contrast, CD36 is expressed early during erythroid maturation, but it is not specific to erythroid cells, since it is also positive in precursor cells of the monocytic, dendritic, and megakaryocytic lineages (29). Regarding the magakaryocytic lineage, CD61, CD41, and CD42 (which recognize gpIIIa, IIb/IIIa, and IX/Ib, respectively) are considered as excellent markers for the detection of megakaryocytic leukemias (AML M7 FAB subtype) (12, 66). Altogether, these results indicate that the utility of individual markers in identifying commitment of leukemic cells into the different myeloid lineages is limited. In fact, it is generally accepted that positivity for two or more myeloid-associated antigens is necessary for the diagnosis of AML (14, 40) and that the utility of immunophenotyping for further classification of AML is almost restricted to the identification of megakaryocytic leukemias, poorly differentiated AML, the microgranular variant of acute promyelocytic leukemia (APL) (14, 40), and a rare subtype of dendritic cell neoplasias that is characterized by coexpression of CD123high, HLADRhigh, CD4+, CD56+, and 7.1+ in the absence of other lineage-specific markers (cMPO-, cCD3-, cCD79a-) (67, 68). In other subtypes of AML, it is frequently claimed that immunophenotyping just stands for confirmation of morphological, cytochemical, and genetic diagnoses (69). In line with this and in contrast to what was described above for ALL, there is still no accepted immunological classification for AML (70). In summary, these results point out the relatively limited utility of individual markers in AML as well as the need for more powerful multiparameter immunophenotypic analytical approaches as also discussed below for MDS. In line with what has been described for ALL, most AML patients (>75%) also display aberrant phenotypes (23, 71-76). These aberrant phenotypes are highly suggestive of the presence of underlying specific genetic abnormalities. Accordingly, leukemic cells from APL patients frequently show an immunophenotype similar to that of normal promyelocytes (CD34−/+heterogeneous, CD117−/+dim, HLADR−, CD13+/++, CD11b−) (29). In contrast to normal promyelocytes, however, these leukemic cells display abnormally low expression of CD15 (CD15-/dim versus CD15high) (Fig. 1, Table 1), a phenotype that is characteristically associated with the presence of associations between immunophenotype and genotype in AML are less clearly (Table and include expression in the of either an immature or a granulomonocytic (CD34+, CD15+, aberrant phenotype and 11q23 abnormalities (16, or (16, respectively. have also been more associated with relatively mature immunophenotypic features or APL Immunophenotypic characteristics of promyelocytes as to leukemic promyelocytes from a with a acute promyelocytic leukemia and The to normal and leukemic promyelocytes and the to other bone marrow cells. that expression of CD15 is observed in normal and leukemic promyelocytes, both being it has been that individual antigens such as and CD34 could be associated with an adverse prognosis in AML, their prognostic could not be definitively (reviewed in more than one immunophenotyping of AL has out the existence of a of cases that show coexpression of immunophenotypic characteristics highly specific of two different myeloid and lymphoid (e.g., and (40, coexpression may in a single cell or in two groups of blast cells in the same and acute leukemias should be as different from both ALL with expression of myeloid associated markers and AML showing reactivity for antigens. These latter cases may represent more than of all AL (40, 47, In they should also be separately considered from ALL patients with phenotypes and from AML cases in which blast cells display phenotypic features characteristic of more than one cell Despite the that the most recent classification of AL by the AL as a new the information currently about their clinical and the most treatment for their management is still limited and poorly In the last the of the presence of residual leukemic cells after using immunophenotypic approaches, has proved to be and from the into clinical that it is that leukemic cells display aberrant since with a few the detection of specific antigens be applied (23, phenotypes are present in most ALL (23, 24, and AML cases (>75%) (23, 24, 71-76). are typically antigen expression (e.g., expression of in AML or in antigen expression (e.g., coexpression of CD34 and CD3 or CD34 and and phenotypes (e.g., and/or CD34+ cells found in or T-cell precursors in the (23, studies have contributed to the of new in such as that of immunological (23, At the same time, these studies allow a better prognostic of AL at an early stage after of and they a follow-up of treatment in individual patients (23, In the availability of new treatment strategies, based on the use of monoclonal antibodies specific for expressed by leukemic cells (e.g., has provided a for the use of immunophenotyping in the of the number of molecules expressed by the cells as a highly for to has been for years that MDS patients display BM that are The and of these especially those involving erythroid, and cells, together with the of and blast cells, are of great utility in the diagnosis and classification of the disease As the availability of an increasingly high number of monoclonal antibody clones and the success of their in the of hematopoietic cells have that these morphological abnormalities could also be by immunophenotypic years, the use of single stainings analyzed by fluorescence microscopy or flow cytometry have restricted the routine applications of immunophenotyping in MDS to the of blast cell after into AL These studies that almost AL an MDS corresponded to an AML, and blast being either rare or respectively During this to MDS at diagnosis were limited in number and their results were (28, 29). This was probably a consequence of the great of the cells present in the BM of MDS and the highly numbers and phenotypes of the cell subpopulations detected in different patients, which can not be with single or even stainings (29, However, these studies clearly demonstrated the occurence of in the expression of individual antigens both in and BM of MDS patients (reviewed Accordingly, neutrophils from a of all MDS patients display expression of and In the existence of abnormally high numbers of and cells is also frequently observed in the of these The latter antigens being in MDS. phenotypic abnormalities of have been reported in the less the existence of reactivity for and together with expression of and have been found to in these cells. In a similar a in the reactivity for antigens expressed on normal myeloid precursors (e.g., and immature cells (e.g., CD33, and together with expression of markers that are characteristic of the last stages of the maturation (e.g., and have also been reported in the BM of MDS Later studies have that these in the expression of individual antigens frequently the existence of underlying abnormalities in the of different BM cell In line with this, it has been shown that in the of CD34+ cells are related to the of blast cells by Accordingly, the number of CD34+ cells from and with to with of and in In a similar expression of markers is frequently found in cases showing numbers of mature these abnormalities also in a decrease in the BM from and to and immunophenotypic abnormalities reported in a of all MDS patients to the expression of aberrant These antigen expression in the (e.g., or and cell lineages (e.g., as well as in the blast cell (e.g., and expression of lymphoid associated antigens on myeloid cells, and of individual antigens such as in erythroid cells, in cells, and in CD34+ BM cells (28, Altogether, these results indicate that the phenotypic present in MDS are highly and that they include abnormalities in the between cells from different lineages and between different a lineage, together with the expression of aberrant phenotypes of this, immunophenotypic analysis of MDS at diagnosis more multiparameter analytical In line with this, the most recent studies to the immunophenotypic of MDS have new analytical First, they on the identification of specific cell by light and they for the presence of phenotypic abnormalities the initially through the use of different objective and/or criteria as in for the To in these latter it is that in the analysis of MDS will multiple stainings for four or more antigens. In the analysis of these stainings to be based on the specific identification of the different cell present in the the analysis cell of the of the cells, the objective of the phenotypic of of the maturation stages and the of the abnormalities Table the immunophenotypic abnormalities found to be clinically useful in of these As a consequence of the utility of these latter strategies, in the last few years there has been an increasingly high on the for new phenotypic parameters that could be of clinical relevance in MDS of the maturation in a normal bone marrow as to different MDS patients with In all cells are to CD34+ cells. Despite the that a high number of antigens have been and phenotypic abnormalities the clinical utility of immunophenotyping of MDS and it has still not become routine (28, 29). This is probably the of multiple studies have reported the existence of characteristic immunophenotypic abnormalities in but few have analyzed its In of the reported abnormalities rely on an expression of individual antigens that are not present in MDS at the same they are also found in other (28, 29). the other abnormalities of the cell precursors are more on immunophenotypic grounds than those of the erythroid and megakaryocytic lineages the prognostic point of view, the expression of individual antigens has been associated with the clinical of MDS (reviewed Accordingly, reactivity for and expression of and in the BM have been associated with both a higher of into AL and a In adverse cytogenetic features are also more frequently found among cases an reactivity for on the BM lineage cells, a expression of on and a higher number of on the surface of CD34+ precursors However, few of these individual markers an prognostic Despite these recent studies in which the expression of antigens is simultaneously evaluated in different BM cell lineages and their according to immunophenotypic analytical show that immunophenotyping is of great utility for the diagnosis of MDS patients in morphological and cytogenetic features are found At the same time, it shows from the for Moreover, it is that this new together with the use of new classifications as well as new based on phenotypic information will contribute to the and prognostic of the disease Despite recent there is still of for immunophenotypic studies of both AL and MDS In the these studies should that either or using new Apart from new markers and of these future studies should of recent in stainings and multiparameter In more approaches at the analysis of all cell present in a mature cells and even the will be since they will probably contribute to the diagnosis between and AML and the identification of respectively. Also, a more detailed analysis of the phenotypic of the neoplastic cells is for a identification and of leukemic and cells. In parallel with this, more detailed studies of normal myeloid differentiation are also especially in the of those cell lineages less represented in BM, to better the impact of specific genetic abnormalities in the of expression and cell Finally, clinical studies in which the of immunophenotypic parameters is analyzed in large of patients should be These studies of new approaches for multiparameter of