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July 2002, Volume 16, Number 7, Pages 1233-1258
Table of contents    Previous  Article  Next   [PDF]
Review
Antigen expression patterns reflecting genotype of acute leukemias
O Hrus caronák1 and A Porwit-MacDonald2

1Institute of Immunology/CLIP, Charles University, Prague, Czech Republic

2Department of Pathology, Karolinska Hospital and Institutet, Stockholm, Sweden

Correspondence to: O Hruital scaronák, Institute of Immunology, V uvalu 84, 150 06 Praha 5, Czech Republic; Fax: 4202 2443 5962

Abstract

Multi-parameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to new classification of leukemia. In this review we discuss immunophenotypic characteristics of major genotypic leukemia categories. We describe immunophenotype of: B-lineage ALL with MLL rearrangements, TEL/AML1, BCR/ABL, E2A/PBX1 translocations, hyperdiploidy, and myc fusion genes; T-ALL with SCL gene aberrations and t(5;14) translocation; and AML with AML1/ETO, PML/RARalpha, OTT/MAL and CBFbeta/MYH11 translocations, trisomies 8 or 11 and aberrations of chromosomes 7 and 5. Whereas some genotypes associate with certain immunophenotypic features, others can present with variable immunophenotype. Single molecules (as NG2, CBFbeta/SMMHC and PML/RARalpha proteins) associated with or derived from specific translocations have been described. More often, complex immunophenotype patterns have been related to the genotype categories. Most known associations between immunophenotype and genotype have been defined empirically. Therefore, these associations should be validated in independent patient cohorts before they can be widely used for prescreening of leukemia. Progress in our knowledge on leukemia will show how the molecular-genetic changes modulate the immunophenotype as well as how the expressed protein molecules further modulate cell behavior.

Leukemia (2002) 16, 1233-1258. doi:10.1038/sj.leu.2402504

Keywords

leukemia; immunophenotype; cytogenetics; chromosomes; flow cytometry

Introduction

Chromosomal aberrations are of high prognostic significance both in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).1,2,3 Although it is obvious that genotype is reflected in the immunophenotype of leukemic blasts, there is no absolute concordance between the immunophenotype and genotype categories. Several questions of biological and practical nature emerge:

  • Should we modify the immunophenotype classification to reflect genotype changes?
  • Should we perform molecular genetic studies only in pre-screened subsets of patients?
  • Which genotypes correlate with a specific pattern of antigen expression and which show variable immunophenotypic features?
  • What are the regulatory mechanisms that allow expression of aberrant molecules in leukemic cells?

Those who seek answers to such questions might find useful information in this review.

Methodology

Complex or simple correlation patterns?: The expression of individual molecules in eight important genotypic subtypes of AL is shown in Figures 1 and 2. These figures result from a meta-analysis of available data providing a synopsis of the expression of individual molecules. It is impossible to show the methodological details of each cited study here. These details vary among the cited studies and some of them are mentioned in the respective paragraphs below. Readers should also be aware that the sizes of cohorts differed; however, the graphical tags are identical for all studies, making the larger studies under-represented.

As shown in Figures 1 and 2, the expression of a single molecule can rarely predict a molecular genetic subtype. Only exceptional molecules, eg the fusion protein CBFbeta/SMMHC {product of inv.(16)},4 the aberrant molecule of chondroitinsulfate NG2 (correlating with translocations of MLL gene5,6,7,8) or the hybrid PML/RARalpha protein,9 are known as good molecular-genetic predictors, as mentioned in respective articles. Several authors constructed scoring strategies, which simultaneously take into consideration the expression level of several molecules. These scoring systems used the molecules, that were weak predictors individually, but when combined with Boolean logic operators as many as six antigens could be correlated - resulting in useful diagnostic tools. Percentages of positive cells together with intensity and/or homogeneity of antigen expression have been used.10,11

However, it may be argued that the significance of the scoring strategies can be considered hypothetical, until proven using an adequately sized independent cohort of patients.12 The potential drawback in this sort of scoring strategy (as described in detail in the Appendix) is the fact that, often, these scoring systems are applied to the same cohort from which they are derived. An investigator may design a study using what appears to be a well thought-out strategy using an appropriate antigen array and test population. Each of the antigens may have a slightly different level of expression in samples of leukemia with or without the investigated genotype (G). Just by chance, a combination of some antigens could be expressed in the same way by all Gpos cases (eg all antigens will be positive or all will be heterogeneous, etc). These antigens may be selected and applied as a scoring system. Looking at Gneg cases, only few of them will meet all the scoring criteria. Therefore, a high predictive value is declared in the given study but difficult to reproduce in a different cohort, as reasoned in the Appendix.

Parameters to consider: Classical immunophenotyping by flow cytometry generates several standard parameters that describe the expression of a given molecule. The most commonly used parameters are the percentage of positive cells, mean (or median) fluorescence intensity and coefficient of variation (CV). As shown in Figure 3, the percentage of positive cells reflects the cellular composition of a gated cell population while neglecting the intensity of expression. This parameter is recommended by consensus guidelines.13 The usual cut-off value accepted as positivity is 20% of positive cells. The use of strict cut-off values may obscure the biological nature of some cases, eg B-precursor ALL where most cells display pro-B ('null') immunophenotype, but a minor population of CD10pos cells is also present. Such a case may fulfill the arbitrary criteria for the 'common' ALL (cALL) category (Figure 4a).

Mean fluorescence intensity (MFI) is a principal measure of the intensity of antigen expression. MFI depends on the number of antibody molecules bound to each gated cell. In cases with low numbers of clearly positive cells, MFI may not provide unequivocal information whether positive cells are present or not. Median fluorescence intensity is even less informative in this respect. Coefficient of variation CV is a product of a simple formula (CV = (SD/Mean) ´100), describing homogeneity of expression, ie how much the gated cells vary in the expression of the particular molecule. In general, the expression of commonly investigated molecules is usually more homogenous in cases of ALL than in AML. This reflects the widely known phenomenon that in many AML cases, leukemic cells are found at various stages of differentiation.14

All of these parameters are substantially influenced by the selection of investigated cells. This selection is highly dependent on the quality of the specimen and the gating accuracy. Therefore, immunophenotyping results obtained by flow cytometry should always be correlated with cytomorphologic aspects of bone marrow or blood smears.

When searching for a correlation between immunophenotype and molecular genetics, one has to convert the complex information on immunophenotype into a simple nominal variable (such as 'PML/RARalpha likely/unlikely'). Most of the genotype-immunophenotype associations that we describe in this review show a distinct phenotype pattern. Therefore, an experienced flow cytometry specialist can frequently predict the genotype without using any cut-off values. Setting a threshold value for any of the above-mentioned parameters (% positive cells, MFI or CV) that would correctly predict the genotype, even in the borderline cases, may be difficult.

Aberrant and non-aberrant molecules

Except for rare proteins resulting from specific translocations4,9 or from increased gene expression,15 leukemic blasts in AML or ALL express normal myeloid or lymphoid differentiation antigens, respectively. However, leukemia-associated phenotypes can be identified due to co-expression of markers rarely or never appearing simultaneously in normal hematopoietic differentiation, aberrant marker over-expression or lack of differentiation markers.16,17,18,19,20,21 Considerable effort has been made to determine the patterns of antigen expression in stem cells, myeloid progenitors, B-precursors and T cells in normal bone marrow.22,23,24,25,26,27,28,29,30,31 When compared to their normal counterparts, leukemic blasts in over 90% of ALL and over 70% of AML cases have been reported as displaying aberrant phenotypes.19,30,32 In all leukemia cases carrying specific translocations described below, highly anomalous phenotypes have been detected making flow cytometry an interesting alternative to molecular methods in follow-up of minimal residual disease in these patients.33

Whereas some genotype changes are confined to a specific immunophenotypic subset, two important genetic categories (BCR/ABL fusion and rearrangements of MLL gene) are found in a broad spectrum of leukemia including both AML and ALL.

Acute lymphoblastic leukemia

The genotype categories (Table 1, Figures 4 to 8) will be described according to the differentiation status of the prevalent immunophenotype. Prognosis of the genotype categories is not the scope of this review and is mentioned only briefly in Table 1.

ALL with rearrangements of MLL

Fusion transcripts involving the MLL gene (also called ALL1, located on chromosome 11q23) are found both in ALL and AML. In ALL, the most frequent fusion partner is the AF4 gene on chromosome 4q21. MLL/AF4pos ALL accounts for approximately 50% of ALL cases in infants below 6 months of age. The incidence decreases by two- to three-fold in children 6 to 11 months of age.67 In older children and in adults, the incidence appears to be stable accounting for less than 5% of ALL cases.3,67 Females are over-represented among infants but not within other age categories of MLL/AF4pos patients.67 The MLL/AF4pos ALL is frequently associated with CNS involvement, hyperleukocytosis and hepato-splenomegaly.67 Less common fusion partners of the MLL gene are the ENL gene (chromosome 19p13.3) occurring in approximately 1% of childhood ALL44 and the AF9 gene (chromosome 9p21-22). The MLL/AF9pos leukemias are usually AML, whereas MLL/AF9 has only rarely been described in ALL.68 Overall, rearrangements of MLL gene are found in 6-7% of ALL cases in both children and adults.53

MLL gene rearrangements occur frequently in therapy-related acute leukemia.69,70,71 Topoisomerase inhibitors are considered the main leukemogenic factor in cases of secondary AML. Even though the rarity of therapy-related ALL obscures its causes, topoisomerase inhibitors seem to be involved in most, but not all, cases of secondary ALL.67,69,70 The breakpoint position in the MLL gene is the same in most infant cases with MLL/AF4pos ALL as in therapy-related acute leukemia but different from non-infant de novo MLL/AF4pos ALL.72,73

ALL blasts with MLL rearrangements are typically CD9pos, CD34pos, TdTpos and CD10neg pro-B cells (also called 'null' ALL category) (Figure 4) and frequently express myeloid-associated antigens CD15 and/or CD65.74,75. In some cases, CD10 is present on a subset of cells, although at low intensity. The existence of the prominent pro-B population should be considered biologically more important than the small CD10pos subset, which may become evident at relapse (Figure 4). Several of the other myeloid antigens (CD13, CD33 and CD66c) are only occasionally positive. The expression of CD22 is usually low and CD20 is typically negative, corresponding to early stages of B cell differentiation.

A distinct subset of MLL/ENLpos ALL does not fully conform to the general rules regarding the immunophenotype in MLL-rearranged ALL. A recent study on children with MLL/ENLpos ALL (t(11;19)(q23;p13.3) by cytogenetics) showed that T-lineage ALL is common in these children, especially after infancy.44 It should be pointed out that the prognosis varied depending on the immunophenotype and age.44

NG2 homologue: a cartilage trait pointing to MLL rearrangements in different types of leukemia: Most leukemic cells carrying MLL rearrangements in both ALL and AML cases express NG2 homologue, a chondroitin sulfate molecule reacting with a mAb 7.1 that can be detected by flow cytometry. The expression of NG2 has higher sensitivity and specificity for MLL rearrangement than other molecules both in ALL and in AML. The specificity approaches 100% in ALL and in childhood AML, sensitivity ranges between 50 and 80% in AML and exceeds 80% in ALL.5,6,7,8,76 Adult NG2pos AML cases without MLL rearrangement have been described in one study, indicating lower specificity in adult AML7 (Figures 1 and 2). Physiologically, NG2 homologue is expressed primarily in glial, muscle and cartilage progenitor cells but not in normal hematopoietic cells.77 NG2 homologue binds to matrix molecules including type VI collagen. It also modulates the action of platelet-derived growth factor78 and it appears to be involved in remyelination.79,80 Indeed, the NG2pos oligodendrocyte progenitors are activated by demyelination rather than by inflammation.80 This fact may be related to the frequent CNS involvement in ALL with MLL rearrangement. In malignant diseases, NG2 has been shown to promote metastatic potential of melanoma, which has lead to its synonym human melanoma proteoglycan.79 In leukemia with MLL rearrangements, the significance of NG2 for cell biology has not yet been documented.

TEL/AML1pos ALL

This genotype category is characterized by the presence of a reciprocal translocation of the chromosomes 12p13 and 21q22 resulting in a fusion of the TEL and AML1 genes.81,82 Translocation t(12;21) is virtually undetectable by routine karyotyping (see Ref. 83 for review). TEL/AML fusion can be detected by PCR and/or FISH in approximately 25% of childhood ALL cases,83,84,85 whereas only few adults with TEL/AML1pos ALL have been described.86 The TEL/AML1pos ALL typically accumulates in a pre-school childhood age peak84 and a smaller subset of patients is found around 9 years of age.83,87

ALL cases carrying TEL/AML1 transcripts display exclusively B-precursor phenotype. Most cases are CD19pos, CD34pos, TdTpos and CD10pos (Figures 1 and 5) corresponding to cALL or more seldom pre-B ALL.85 In most patients, at least a fraction of blasts overexpresses CD10 (Figure 5). The TEL/AML1pos cases are often CD33pos and/or CD13pos (Figures 1 and 5), as shown by several independent studies.85,88,89,90 The expression of CD13 and/or CD33 may be heterogeneous and in many cases clearly negative and clearly positive subsets are found (Figure 5). No other myeloid antigens are consistently expressed. Borkhardt et al85 included CD65 together with CD13 and CD33 for definition of the 'My-positive' subset but the usefulness of CD65 was not addressed separately. In our data, among 58 TEL/AML1pos newly diagnosed patients all but two had fewer than 10% CD65pos blasts. The regulatory mechanisms causing more frequent CD13 and/or CD33 expression by comparison to other myeloid antigens in TEL/AML1pos ALL are not yet understood. Notably, some other genotypes like BCR/ABL positivity are also associated with frequent expression of CD13 and/or CD33 (see below).

So far, no single surface molecule that would significantly predict TEL/AML1 positivity has been described. However, positivity of CD66c at 3% cut-off value has been found to significantly correlate with the lack of TEL/AML1 translocation.89,91 This association is certainly accentuated by a positive correlation of CD66c with hyperdiploidy and BCR/ABL positivity (see below). Nevertheless, CD66c positivity remains associated with the lack of TEL/AML1 translocation even when hyperdiploid and BCR/ABLpos cases are excluded. Another molecule, Flt-3 receptor (CD135) has been shown positive (based on MFI) in 40% of TEL/AML1neg cases whereas it was missing in all 21 TEL/AML1pos cases tested.10 Considering B-lineage markers, CD20 is usually negative,89,90 and CD24 is positive (60/60 cases in our cohort, unpublished data) (Figure 1).

CD22 is mostly positive, which is not different from other childhood B-precursor ALL cases (Figure 1).

Several attempts to design scoring systems have been made10,90 but so far only one has been confirmed in a separate, second cohort of patients.90 By that scoring system, presence of TEL/AML1 can be predicted by negativity or only partial expression of both CD9 and CD20.90 For both of these markers, the subjective evaluation is necessary in order to distinguish between positivity in the whole blast population or in the subset of blasts.

ALL with numerical chromosomal changes

High hyperdiploid ALL: High hyperdiploidy represents the most common numerical chromosomal abnormality in ALL and can be readily detected using cytometric DNA analysis.92 High hyperdiploid ALL samples are characterized by the presence of 51 to 65 chromosomes per one leukemic cell, which corresponds to a DNA index of 1.16 or higher and lower than 1.6.50

Typically, this subtype of ALL occurs in the pre-school age peak and is slightly more frequent among girls.93 The mechanisms leading to hyperdiploidy are unknown; the consequences of high hyperdiploidy include high propensity to undergo spontaneous apoptosis in vitro, which cannot be fully reverted by culture on stromal cells.94 High hyperdiploid ALL cases comprise 21% of childhood ALL in developed countries.93 A lower incidence (15%) has been reported in an Indian study.95 In adults, the frequency of high-hyperdiploid ALL is only 6 or 7%.3,53 High hyperdiploidy is typically found in cases lacking MLL rearrangements and fusion genes TEL/AML1 and BCR/ABL. A minority of BCR/ABLpos patients11 and rare cases with TEL/AML1 fusion85 also having highhyperdiploid DNA contents have been described.

Almost all high-hyperdiploid cases have B-precursor immunophenotype. As shown in Figures 1 and 6, hyperdiploid lymphoblasts are mostly CD19pos, TdTpos and CD10pos and usually lack expression of CD45. CD10 is frequently overexpressed.96 CD34 positivity has been found to be slightly more frequent among hyperdiploid ALL cases when a 10% cutoff was used.97 CD22 and CD24 are typically positive and CD20 is positive in approximately 40% of cases. Thus, expression of these antigens is different from non-hyperdiploid B-precursor ALL cases (Figure 1). Among myeloid antigens, CD13, CD33 and CD65 are usually negative, whereas CD66c is highly positive in almost all cases89 (Figures 6 and 9). The expression of CD15 did not differ between hyperdiploid and non-hyperdiploid cases in B-precursor ALL cases studied in our laboratories.

Hypodiploid ALL: Hypodiploid DNA index is found in heterogeneous subsets of ALL. The modal chromosome number ranges from near haploidy (<30 chromosomes) to cases with 45 chromosomes. Hypodiploidy may coincide with other chromosomal aberrations. However, these only rarely include BCR/ABL, MLL/AF4, E2A/PBX1 or TEL/AML1 gene fusion genes. In developed countries, hypodiploid ALL with less than 45 chromosomes is rare in childhood (1%) and uncommon among adults (4%).53 A recent Indian study of 114 patients showed a much higher frequency of hypodiploidy in both childhood and adult ALL (38% and 44%, respectively95), whereas the proportion of hyperdiploidy was lower. However, these results need confirmation in an independent (preferentially population-based) study.

So far, no data has associated hypodiploidy with a consistent set of immunophenotype features.

Other numerical chromosomal abnormalities: Low hyperdiploid as well as (near)-tetraploid cases form a heterogeneous group in terms of immunophenotype, prognosis, and co-presentation with other molecular genetic changes.98 T-lineage immunophenotype is common among (near)-tetraploid cases.99 In addition, (near)-tetraploidy is occasionally observed in AML.100,101,102

BCR/ABLpos ALL

BCR/ABL fusion gene, the product of t(9;22)(q34;q11) is found in 25% of adult ALL patients whereas only 5% childhood ALL cases bear this fusion.53 The incidence of all types of leukemia carrying BCR/ABL gradually increases with age, as does the incidence of very rare BCR/ABLpos cells in the blood of healthy subjects.103 The biologic importance of these non-malignant BCR/ABLpos cells is still unsolved. In malignant cells, the exact position of the breakpoint within the BCR gene is different in ALL and CML, resulting in fusion proteins of different lengths. The BCR/ABL fusion protein may have a molecular weight of either 210 kDa (= 'major-BCR', found in CML) or 190 kDa (= 'minor-BCR', found in most BCR/ABLpos ALL). A third fusion product has 230 kDa molecular weight and is called 'micro-BCR'. It is rarely found in CML cases and in chronic neutrophilic leukemia.104,105 Some cases of acute leukemia may represent a blast crisis of clinically silent CML, which complicates the interpretation of analysis of major-BCR/ABLpos acute leukemia cases.104,106

Most BCR/ABLpos ALL cases have CD9pos, TdTpos and CD10pos B-precursor ALL - 'common' ALL immunophenotype.52,107 Rare BCR/ABLpos cases of T-lineage ALL have been described.52,108,109 In a recent large Pediatric Oncology Group study, t(9;22)(q34;q11) was found in three of 343 patients with T-ALL.110,111 The type of BCR/ABL transcript was either major52 or undocumented in these studies; thus distinction between de novo ALL and T-lymphoblastic blast crisis of CML might not be possible. In a recent multi-national study on BCR/ABLpos ALL in childhood and young adulthood, only six of 300 patients had T-lineage ALL.55 Overall, BCR/ABL positivity can be found in several different subsets of ALL, with a strong predominance of B-precursor phenotype.

Most of the BCR/ABLpos cALL cases present distinct aberrant features. Blasts usually display high CD10 and are CD34pos.112 Expression of CD13 and/or CD33 is frequent52,113,114 (Figure 7). However, these features are not pathognomonic for BCR/ABLpos ALL. There appear to be no differences in the expression of CD20, CD22 and CD24 in comparison to the remaining B-precursor ALL cases (Figure 1).

The expression of CD25 (interleukin 2 receptor alpha chain) is higher in BCR/ABLpos than in other ALL.108 BCR/ABLneg cases with expression of CD25 do exist, but it is not known whether these BCR/ABLneg CD25pos patients share other genotypic or immunophenotypic features.108 In a recent study on BCR/ABLpos ALL, CD38 was expressed at lower intensity and with lower homogeneity. Another feature found in this study was high positivity of CD34.11 Since five of 14 children with BCR/ABLpos ALL were CD34neg or CD34low pos in our combined cohorts, some differences between immunophenotype of adult and childhood BCR/ABLpos ALL might exist. A scoring system proposed by Tabernero et al11 included homogeneous expression of CD10 and CD34 but low and relatively heterogeneous CD38 expression, together with an aberrant reactivity for CD13. However, since all 12 BCR/ABLpos cases in this combined study on childhood and adult ALL were adults, this scoring system needs to be tested in a larger independent study.

CEA family member correlating with three genotypes: High expression of the antigen detected by a mAb KOR-SA3544 has been documented in most BCR/ABLpos ALL cases (Ref. 115 and Figure 9). As mentioned previously, TEL/AML1pos cases do not express this antigen, while most cases of high hyperdiploid ALL are positive (Figure 9). This antigen has not yet been studied in the workshops on human leukocyte differentiation antigens (HLDA). However, a study by Sugita et al116 showed that the KOR-SA3544 mAb reacts with CD66c (synonym, NCA50/90), a member of the carcinoembryonic antigen (CEA) family. CD66c is expressed by normal granulocytes and is constantly lacking in normal lymphocytes. CD66c is involved in cell adhesion and activation, but its physiological function is largely unknown.117 It has been shown that both CD66c and CD66e (CEA) can modulate metastatic potential of human carcinoma cells through macrophage and endothelia activation.118 The consequences of CD66c expression in ALL cells, as well as its regulation, are still poorly understood.

E2A/PBX1pos ALL and other t(1;19)pos cases

Recurring translocation t(1;19)(q23;p13) occurs in approximately 5-6% of children and in less than 5% of adults with ALL.53,119,120 In approximately 90-95% of such cases, this translocation leads to the expression of E2A/PBX1 chimeric mRNA. Studies in transfected animals suggest that E2A/PBX1 fusion is important in oncogenesis but an additional event is required for full malignant transformation.121,122 Transgenic mice with E2A/PBX1 develop mostly T-lineage lymphomas or AML.119 This observation contradicts the strong empirical correlation between E2A/PBX1 positivity and B-precursor ALL immunophenotype. Rare cases with mature B or 'FAB-L3-like' phenotype have been described, some of which displayed monoclonal surface Ig expression proven by flow cytometry.123,124,125 Moreover, E2A/PBX1 positivity has been documented in AML M4, in T-lineage ALL124 and even in meningioma.126

Typical immunophenotype findings in E2A/PBX1pos B-precursor ALL are demonstrated in Figure 8. Lymphoblasts carrying E2A/PBX1 are usually at the pre-B stage of differentiation, which is substantiated by cytoplasmic but not surface expression of IgM. Several independent studies have nevertheless showed that cALL phenotype (ie CD10 expression with lack of cytoplasmic IgM) could be demonstrated in 6 to 30% of E2A/PBX1pos cases.124,127 Reproducibility of pre-B/cALL differential diagnosis could be limited by methodological problems, ie difficulties in the interpretation of cytoplasmic IgM staining.128 Other membrane markers of the differentiation towards pre-B stage, such as CD34 negativity and CD20 and/or CD9 positivity, may be more reliable.129 The expression of CD22 and CD45 in E2A/PBX1pos ALL has not been studied separately in the literature. In our experience, these markers are expressed with variable intensity (unpublished data).

Rare cases of t(1;19)pos ALL have hyperdiploid DNA contents.130 Some t(1;19)pos ALL cases are E2A/PBX1neg by PCR and less likely to have the characteristic immunophenotype.57,129

E2A/HLFpos ALL

Presence of t(17;19)(q21;p13), equivalent to E2A/HLF positivity, apparently correlates with B-precursor immunophenotype.131 The low frequency of this abnormality (up to 1% of children with BCP ALL) precludes thorough analysis of typical immunophenotype.

MYC oncogene and mature B ALL

Juxtaposition of MYC gene with the immunoglobulin gene regulatory sequences on chromosomes 14, 2 or 22 is a result of translocations t(8;14)(q24;q32), t(2;8)(p11;q24) or t(8;22)(q24;q11), respectively.132 The involved partner genes determine the strong correlation with mature B immunophenotype. Rare cases of T-lineage leukemia with t(8;14)(q24;q11), in which the T cell receptor (TCR) gene is involved have been described.133,134,135 By definition, mature B ALL expresses one of the immunoglobulin light chains (either kappa or lambda, resulting from the light chain restriction). There is a strong correlation of MYC translocations with the L3 cytomorphological category.136,137 Approximately half the patients express CD10 with intensity comparable to that of nonmalignant B-precursors or germinal center cells. Myeloid antigens, CD34 and TdT, are negative. No aberrant surface molecule has been demonstrated to be consistently positive in mature B ALL.

Chromosomal changes in T lineage ALL

In most adult and pediatric cases with T-lineage ALL, no cytogenetic or molecular-genetic abnormality can be identified.132,138 In 20% to 25% of T-lineage ALL cases, translocations of TCRalphadelta or TCRbeta genes to an oncogene (including MYC, SCL, TAL2 and others) have been found.132 Juxtaposition of an oncogene under the control of TCR genes drives proliferation in TCR-expressing cells.

Deletions in the SCL gene (synonyms: TAL1 or TCL5 gene) occur in 6-26% of T-ALL cases.132,139,140,141,142,143,144 The SCL gene is silent in normal T lymphocytes, but is expressed in various hematopoietic cells including myeloid progenitors, erythroblasts, committed CD34pos CD38pos progenitors and megakaryocytes.132 The aberrant SCL expression may be caused by deletions in the SCL gene. These deletions are also called TALd. The SCL gene is brought to proximity of the SIL gene, which is upstream of SCL and is normally expressed in T lymphocytes. Other causes are fusion to a TCR gene (most commonly in t(1;14)(q33;q11)) or other, not fully understood, mechanisms.132,145,146

Two recent US studies have demonstrated trisomy 8 identically in 11% of patients with T-ALL, either alone or in combination with t(9;11).111,147 These cytogenetic subgroups are more common in AML (see below) and so far have not been associated with a specific immunophenotype or a different prognosis in T-ALL patients.

Cases with SCL rearrangements (including the TALd cases) more frequently express CD2 but lack CD10 expression.145 In addition, TALd ALL correlates with TCRalphabeta lineage, which can be documented either by TCRalphabeta expression or by bi-allelic deletion of the TCR delta gene.148

The prognosis of TALd ALL appeared to be favorable within the subgroup of T-lineage ALL in two studies.144,145 No cytogenetic relapse risk indicator has been found in a recent Children Cancer Group study on childhood T-ALL. Proportionate improvement of prognosis has been observed using contemporary intensive treatment.147 Patients with T-ALL showing an intermediate stage of differentiation by immunophenotype (defined as CD1apos alone or combined with CD4pos CD8pos phenotype) seem to have a more favorable prognosis.110,111,149 Although once considered associated with a more favorable outcome, neither CD2 nor CD10 expression have been associated with a different prognosis in a recent trial.110

A new t(5;14)(q35;q32) translocation has recently been described.150 According to the original series of patients, it affects approximately 22% of children and adolescents with T-lineage ALL.150 This translocation does not lead to a fusion transcript. The genes in the immediate vicinity of the breakpoint are RanBP17 and Hox11-like2 on chromosome 5 and CTIP2 (chicken ovalbumin upstream promoter transcription factor interacting protein) on chromosome 14. The gene Hox11-like2 is ectopically transcribed in t(5;14)(q35;q32)pos ALL.150 All five t(5;14)(q35;q32)pos cases, published by the time of this review, were of intermediate T-ALL phenotype (CD1apos CD7pos CD5pos CD2pos CD8pos).

Acute myeloid leukemia

Several specific translocations have been found in acute myeloid leukemia and will be presented in order of association with various categories of FAB classification (Table 1, Figures 10 to 13 ).

AML1/ETOpos AML

Translocation t(8;21)(q22;q22) is found in approximately 7% of AML and in approximately 20% of AML M2 according to FAB.151,152 The translocation involves the AML1 gene on chromosome 21q22 and ETO gene on chromosome 8q22.153 The AML1/ETO transcripts can be detected by molecular methods (RT-PCR). These transcripts also occur in a small number of AML cases without detectable t(8;21) translocation, but with other aberrations of chromosome 8.154,155,156 Translocation t(8;21) is found both in adult and childhood AML, with a relatively higher incidence in younger patients, and with equal representation of both genders.151,152,157 Bone marrow smears from most AML cases carrying this translocation show characteristic cytological features. These include the presence of large blasts with needle-like Auer rods, abnormal granules (including pseudo-Chediak granules), large nucleoli and prominent Golgi. Bone marrow eosinophilia is also common.154,157,158,159,160,161

The characteristic immunophenotype features of AML with t(8;21) have been described in several studies.20,154,158,162,163,164,165,166 In most cases an immature subset of blasts is present showing a high expression of CD34 and co-expression of 'dim' CD19 (Figure 10). However, in some studies in both childhood and adult AML the expression of CD19 was not as common as quoted above.167,168,169,170 This may depend on the use of different CD19 mAb clones in these studies. Leu 12 and/or B4 were applied in studies with positive findings.161,162,166 By comparison, HD37 mAb168 or various mAbs167 were used in studies with a low incidence of CD19 positivity. Conversely, CD19 expression was reported in cases with detectable AML1/ETO transcripts without overt t(8;21).154 TdT expression in the immature blast population is common.165,168 There are features of maturation asynchrony with co-expression of high-intensity CD34 concomitant with high-intensity CD15 and myeloperoxidase (MPO) (Figure 10). Differentiating populations of myeloid precursors lacking CD34, but expressing CD15 and MPO are also present. The expression of CD13 is usually high and the expression of CD33 relatively low. Cases without surface myeloid antigens but expressing myeloperoxidase were also described.171 CD56 is often expressed and in some studies it was related to worse prognosis.172 A high expression of CD45RA and CD54 was also reported.166 CD7 and CD2 are negative in most cases.161,166,173,174,175,176

PML/RARalphapos AML

Acute promyelocytic leukemia (APL) or AML M3 according to FAB, first described in 1957 by Hillestad,177 was later related to the translocation t(15;17)(q22;q21).178,179 By morphology, APL is characterized by abnormal promyelocytes, often containing multiple Auer rods and coarse azurophilic granulation. In approximately 20% of cases the promyelocytes are small and hypogranular (AML M3 variant - M3v).180,181 The breakpoint on chromosome 15 has been identified within the PML gene, while the breakpoint on chromosome 17 is located within the retinoid acid receptor alpha gene (RARalpha). The fusion gene results in a fusion protein PML/RARalpha and in 80% of cases also reciprocal fusion protein RARalpha/PML. The fusion gene products are readily detectable by molecular methods (reviewed in Ref. 182). PML/RARalpha AML constitutes 5-15% of all AML cases. A higher incidence was found in Mediterranean countries, in Latinos in the US and in South America than in northern Europe, in the white population of the USA, Japan and Australia.169,176,183,184,185,186,187 The incidence of acute leukemia with t(15;17) appears constant over most of the human life span, implying only one, rate limiting, mutation.188 Therefore, the incidence in young and middle-aged adults is relatively higher than for other types of AML.189

The immunophenotype of t(15;17) AML is considered to be highly specific with the characteristic scatter features187 and expression of CD13, CD33, myeloperoxidase in absence of CD34 and HLA-DR, and variable expression of CD15.190,191,192 Results concerning expression of CD9 and CD68 are not consistent.187,192 Orfao et al184 described in more detail the patterns of myeloid-associated marker expression, pointing out the homogenous expression of CD33 (Figure 11), heterogenous expression of CD13 with lack of expression of HLA-DR (Figure 11) and low expression of CD15 (Figure 11). The CD34 expression was reported to be low or negative in most cases (Figure 11).184,191 In some studies higher CD34 expression was related to M3v morphology.181,193,194 CD7 was negative in most cases.18,161,174,195,196 However, a single case of undifferentiated leukemia positive for PML/RARalpha with immature phenotype (CD34pos, HLA-DRpos, TdTpos, CD7pos) has been reported.197 Similarly, CD38 was reported to be lower in PML/RARalpha.pos cases than in other subtypes of AML.198 CD2 has often been associated with an M3v morphological aspect168,194,199,200 (Figure 11). The expression of CD56 has been found only rarely, and similarly to AML with t(8;21), associated with worse prognosis.201,202

There are no reports of flow cytometric detection of PML/RARalpha protein. However, it has been detected with immunocytochemical methods in over 90% of APL cases tested.9,203

CBFB/MYH11pos AML

AML cases with pericentric inversion of chromosome 16(p13;q22) are associated with the AML M4eo FAB category.180,204 The inv(16) aberrations result in the fusion between the CBFB gene on chromosome 16q22 (encoding core binding factor beta-subunit and the MYH11 gene on chromosome 16p13 (encoding a type II smooth muscle myosin heavy chain). Several CBFB/MYH11 transcripts have been described, with type A being prevalent.205 The fusion results in a chimeric CBFbeta/SMMHC protein that is thought to disrupt normal myelo-monopoiesis.205

Inv(16) and related t(16;16)(p13;q22) are observed in approximately 6-10% of all AML. It is found predominantly in patients of young median age, and often with high white blood cell count or organomegaly.206,207,208,209

The inv(16) AML cases are characterized by a complex immunophenotype with the presence of a more immature CD34pos TdTpos and/or CD117pos blast population together with multiple subsets of blasts differentiating towards granulocytic (CD65pos CD15pos) and monocytic (CD14pos CD11bpos CD4pos) lineage (Figure 12). Maturation asynchrony is a constant feature, with subsets of blasts co-expressing markers characteristic for immature myeloid cells such as CD34 and/or CD117 and markers of granulocyte (CD15, CD65) or monocyte differentiation (CD14, CD4).210 Most cases are CD11bpos and CD13pos.167,170,211 CD7 has been rarely observed,174 while CD2 expression is found in approximately 40% of cases.168,170,211 It should be pointed out that CD2 is expressed on both immature and differentiating blast populations.211

An antibody detecting CBFbeta/SMMHC protein has been developed and applied to detect the chimeric protein in permeabilized cells with the highest fluorescence intensity in the CD34pos blasts.4

Abnormalities of MLL gene

Alterations in chromosome band 11q23 are predominantly found in the AML FAB M4/M5 category. Expression of monocyte-associated markers CD4, CD14, CD11b, CD64 and CD56 is common.212,213,214 Relatively frequent expression of B cell markers has been reported170 but other studies have not confirmed this finding.213,215 Most cases of AML with 11q23 alterations aberrantly express NG2 homologue, similarly to the ALL cases with MLL rearrangements (see section ALL with rearrangements of MLL for details on NG2).6

AML t(9;11) in children is also associated with M5 FAB category and strong expression of NG2, HLA-DR, CD33, CD65w and CD4, while CD13, CD14 and CD34 are low.168,216

OTT/MAL (RBM15/MKL1) fusion and megakaryocytic leukemia

Translocation t(1;22)(p13;q13) has been associated with AML M7 - acute megakaryocytic leukemia (AMKL).217,218,219 Recently, the partner fusion genes have been identified as the OTT (RBM15) gene (one-twenty-two, or RNA binding motif protein-15 on chromosome 1p13) and the MAL (MKL1) gene (megakaryoblastic leukemia-1, chromosome 22q13). Due to their sequence homology to Drosophila genes, these genes are expected to be involved in intracellular signaling and chromatin remodeling, respectively.220,221 AMKL with t(1;22) is most frequent in infants (20% of all infant AML).66,222 It is less common in older children (4%) or adults (2-8%).223,224 Diagnosis of AMKL is primarily based on ultrastructural demonstration of platelet peroxidase.225 By flow cytometry, blasts are positive for platelet-associated antigens CD41a, CD42b and CD61. However, it is important to differentiate the real positivity for platelet-associated markers from non-specific positivity due to platelet adhesion to leukemic blasts. Immunocytochemical staining of smears to show cytoplasmic positivity for CD61 in leukemic blasts can be of help. Myeloid-associated markers CD33, CD13, HLA-DR and CD34 may be positive in AML M7 including OTT/MALpos cases.168,217,224 A reliable immunophenotypic indicator of OTT/MAL within AML M7 has not yet been documented.

AMKL with t(1;22) confers only a subset of megakaryoblastic leukaemias. AMKL in children with Down's syndrome does not usually carry t(1;22), and is instead, often characterized by the presence of complete or partial trisomies involving chromosomes 8 and 1.226 Other frequently found chromosomal changes involved chromosome 3.227 A history of MDS, often presenting as transient myeloproliferative syndrome (TMS), is typical in the Down children.

TMS, which affects approximately 10% of infants with Down's syndrome and rare infants with normal karyotype, can display the same immunophenotype as AMKL.228,229,230,231,232 Given the lack of reliable cytometric distinction between TMS and AMKL, molecular genetics and cytogenetics can provide useful evidence for malignant features.

Trisomy 8 and 11 in AML

The incidence of trisomy 8 (tri8) is approximately 10% of AML cases.1 It occurs both in children and in adults, with predominant M2 FAB category. AML cases with tri8 rarely express CD34 but often express CD13 and CD33.170 No specific immunophenotype has been described.

The incidence of trisomy 11 (tri11) is about 1% of myeloid disorders, mostly MDS and both de novo and secondary AML.233,234 In AML cases, the most common FAB categories are AML M1 or M2. Trisomy 11 may appear as a sole chromosomal change or in association with other aberrations such as monosomy 5 or 7, del(5q) or del(7q), or tri8. Rearrangements of 11q23 MLL gene were confirmed in cases where tri11 was the sole aberration, but not in cases with multiple chromosomal changes.235 In a series described by Slovak et al235 CD34 and HLA-DR were constantly expressed, while there was a heterogeneous expression of all other myeloid-associated markers. Occasional expression of CD19 and CD56 was found.235

BCR/ABLpos AML

The incidence of AML with BCR/ABL major breakpoint is approximately 1% of AML cases (mostly AML M1/M2) and this chromosomal aberration is considered to be associated with poor prognosis.236,237

Cases with de novo AML carrying BCR/ABL are most often positive for CD13, CD18 and HLA-DR. Expression of B cell-related antigens is frequent.170,236,238 Potentially important information regarding the pathogenesis of BCR/ABLpos leukemia comes from case reports. A patient with Philadelphia chromosome (Ph)-positive acute mixed-lineage leukemia that expressed both major and minor BCR/ABL mRNA transcripts has been described.239 This leukemia presented with both myeloid and B cell lineage phenotype as well as with immunoglobulin heavy chain gene rearrangement.

Moreover, a transformation from chronic myelomonocytic leukemia to AML and subsequently to ALL has been reported in a case with a minor BCR/ABL transcript.240 Micro-BCR/ABL genotype, which is typically found in chronic neutrophilic leukemia, has also been described in a patient with AML phenotype, possibly representing an accelerated phase of silent CML.106 Therefore, as in the case of ALL, acceleration of clinically silent CML may mimic de novo AML in many BCR/ABLpos AML cases.

Monosomies and deletions in chromosomes 5 and 7 in AML

The aberrations of chromosomes 5 and 7, such as -5/del (5q), -7/del (7q), are mostly associated with AML secondary to myelodysplastic syndrome and/or AML of the elderly.241,242,243,244 These AML cases are usually positive for at least one of the myeloid-associated antigens CD13, CD15 or CD33.170 High incidence of CD34 expression in both secondary and de novo AML, with aberrations of chromosome 5 and 7, has been reported.245,246,247 CD14 was observed more often in cases with aberrations of chromosome 5 than in cases with aberrations of chromosome 7. In addition, monosomy of chromosome 7 has been documented in cases of AML M7 with or without Down's syndrome.227,248,249

Conversely, cases with aberrations of chromosome 5 more often expressed T cell-related antigens such as CD2 or CD7.170 An Italian study on adult AML showed that TdTposCD7pos immunophenotype correlated with chromosome 5 and/or 7 aberrations and with the multiple-drug resistance phenotype.175

Practical value of immunophenotype pre-screening

From the clinical point of view, rapid diagnosis and classification of all leukemia cases is desirable. Data from literature show that most genotype categories can be predicted from the immunophenotype with a variable degree of accuracy. The biological importance of these observations is indisputable, but the practical value remains to be defined. The decision whether it is ethically justifiable to replace molecular genetic diagnostics with immunophenotyping has to be made by clinical groups. Funding can be an important factor, since most of the antigens necessary to predict the genotype are already included in the basic immunodiagnostics.250 In the treatment protocols, which use molecular genetic markers for risk stratification, a confirmation by PCR or FISH is already required. Nevertheless, rapid indication that the patient may have an unfavorable genotype can justify some clinical measures, such as considering double-luminar catheter (which is suitable in more intensive chemotherapy) and requesting fast processing of the PCR/FISH studies.

Cytometry can depict both simple and complex immunophenotype features correlating with specific genotypes. Exceptions exist in practically all immunophenotype/genotype correlation patterns described. Therefore, we feel that cytometry should not replace PCR or FISH, but rather give an indication in which direction these studies should proceed. Based on the available data, we currently cannot make a general recommendation to narrow down the molecular/FISH techniques to a subset of patients pre-screened by immunophenotype. It is likely that new and more specific immunophenotype-genotype correlations will be discovered in the future as our knowledge on gene expression is growing.

Conclusions and future perspectives

Typical immunophenotype features associated with the major genotype categories are well described. However, the bulk of this knowledge is only empirical. Mostly, we are only beginning to understand why a certain genotype is associated with a certain lineage, why it correlates with a specific stage of differentiation and what triggers the expression of the aberrant molecules. A new generation of important molecules will probably be discovered by the microarray technology.251 The expression of these new markers should be tested in multi-parameter flow cytometry and characteristic expression patterns will distinguish leukemic blasts from normal cells as well as distinguish between the various genotype categories. The growing knowledge about genotype/immunophenotype correlation should help to simplify the immunologic classification of ALL and to establish a useful immunophenotypic classification of AML so that they both closely correlate with genotypes.

Appendix

Statistically, a retrospective study of immunophenotype-genotype correlation, complex data may appear highly predictive even if only composed of several weak predictors. This example is not to say that all similar studies rely on weak predictors. We only want to emphasize the necessity of prospective evaluation, especially in complex associations.

As mentioned in the Methodology section, in a medium-size cohort it is likely that several parameters will be fulfilled by all samples bearing the molecular genetic change (Gpos) under study. Once these parameters have been selected, it is likely that only few of the Gneg samples (ie not displaying the studied change) will conform to all the predicted characteristics although each of the antigens may have quite a high probability of being positive in a Gneg patient. To support this statement, we can consider a simplified example of 20 antigens that are independent of each other in a study of 10 patients with Gpos leukemia and 40 Gneg patients. Let us suppose that expression of these antigens is slightly more frequent in Gpos samples, true probability of positivity for each antigen being, eg 85% and 50% in Gpos and Gneg cases, respectively. Each of the antigens has 20% (= 0.8510) probability of being positive in all Gpos samples. Therefore, we are likely to find four antigens that are positive in all 10 Gpos samples (= 20 ´ 0.2). However, in the Gneg population (n = 40), there is 54% probability (see formula below) of finding two samples or less that will be scored as fulfilling Gpos criteria based on the positivity of all four antigens.

Formula

Variables

k = number of Gneg patients, here 40

z = number of Gneg patients with all four antigens positive, here ranges from 0 to 40 (exact number of false positives)

y = given probability of antigen positivity in a Gneg case, here 0.5

Derivation

  1. Probability that a given patient fulfills all four criteria = y4
  2. Probability that a given patient does not fulfill all four criteria = 1 - y4
  3. Probability that a given group of z patients fulfills all four criteria = y4z
  4. Probability that a given group of z patients fulfills all four criteria whereas none of the others does = (1 - y4)(k-z) y4z
  5. Number of possible combinations of z patients selected in a group of k patients =(k z)
  6. Probability that there will be a combination of z patients who fulfill the criteria and none of the others will (multiplication of lines 4 and 5): = (k z) ´ (1 - y4)(k-z) ´ y4z

Therefore, testing 20 antigens in such a study is a relatively safe way towards a test with a declared 100% sensitivity and at least 95% specificity. However, a study of a subsequent cohort is likely to conclude that the test has much lower sensitivity (57% probability of correctly predicting only 5 cases among 10 patients positive for a given translocation), whereas the specificity would probably remain the same as in the original study. The true overall predictive value of this scoring system would be around 86%, thus only 6% better than if we erroneously presumed all patients to be Gneg (ie all 40 Gneg patients are truly predicted and all 10 Gpos patients are falsely predicted).

Acknowledgements

Supported by Barncancerfonden (The Swedish Childhood Cancer Society), Cancerfonden (The Swedish Cancer Society), Stockholm County Council, and by projects 6406-3 and 111300001 from Czech Ministries of Health and Education. Part of this manuscript is based on the central diagnostic programs of the Czech Pediatric Hematology Working Group. The help of Radek C caronmejla with preparation of the meta-analysis figures is appreciated. The authors thank Mr Lewis Edgel for linguistic consultation.

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247 Sperling C, Buchner T, Creutzig U, Ritter J, Harbott J, Fonatsch C, Sauerland C, Mielcarek M, Maschmeyer G, Loffler H. Clinical, morphologic, cytogenetic and prognostic implications of CD34 expression in childhood and adult de novo AML. Leuk Lymphoma 1995; 17: 417-426. MEDLINE

248 Shitara T, Suetake N, Yugami S, Sotomatu M, Oshima Y, Ijima H, Kuroume T, Nakazawa S. A case of congenital leukemia with monosomy 7. Ann Hematol 1992; 65: 274-277. MEDLINE

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251 Chen JS, Coustan-Smith E, Suzuki T, Neale GA, Mihara K, Pui CH, Campana D. Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia. Blood 2001; 97: 2115-2120. Article MEDLINE

Figures

Figure 1 Meta-analysis of literature data on expression of various CD antigens in B-precursor ALL. The percentages of ALL cases with the selected genotypes positive for a given antigen (x axis) are plotted against the percentages of cases lacking this particular genotype positive for the same antigen (y axis). Thus, antigens with high predictive values for this genotype are close to the lower right corner. Antigens with high predictive values for lack of this genotype are close to the upper left corner of the plot. Antigens with no predictive value are near the hatched diagonal line. Numbers in italics correspond to reference numbers, Un = unpublished data (n = 220, unselected newly diagnosed patients, methods are in Ref. 89). The position of the reference numbers depends on the percentage of positive cases in the cited study, unless an exact overlap occurred and in such cases the points may have been shifted slightly. If more than one study obtained similar results regarding a CD marker, all references are placed around the label with the CD number. See the original articles for methodological details and the sizes of cohorts.

Figure 2 Meta-analysis of literature data on expression of various CD antigens in AML. The percentages of AML cases with the selected genotypes positive for a given antigen (x axis) are plotted against the percentages of cases lacking this particular genotype positive for the same antigen (y axis). Thus, antigens with high predictive values for this genotype are close to the lower right corner. Antigens with high predictive values for lack of this genotype are close to the upper left corner of the plot. Antigens with no predictive value are near the hatched diagonal line. Numbers in italics correspond to reference numbers. The position of the reference numbers depends on the percentage of positive cases in the cited study, unless an exact overlap occurred and in such cases the points may have been shifted slightly. If more than one study obtained similar results regarding a CD marker, all references are placed around the label with the CD number. See the original articles for methodological details and the sizes of cohorts.

Figure 3 Examples of different cytometric parameters commonly used to describe antigen expression in leukemia. Leukemic cells (heavy line) are overlaid over non-leukemic cells shown as gray-tinted histograms (CD19neg cells in (a) and (b) or CD3pos T lymphocytes in (c)) from the same bone marrow specimen. (a) Expression of an antigen in a subset of leukemic blasts illustrated by CD34 expression in CD19/SSC gated leukemic cells in a TEL/AML1pos B-precursor, common ALL. A subset (41%) of CD19pos cells is CD34pos (CD34pos subset values: MFI 92, CV 75%). (b) Bright homogenous antigen expression in the whole blast population illustrated by CD10 expression in the same ALL case. Most (97%) CD19pos cells are CD10pos (CD10pos subset values: MFI 1319, CV 67%. (c) Dim heterogenous expression of an antigen illustrated by the expression of CD4 in a case of AML with CBFB/MYH11pos AML M4. Leukemic cells are CD4neg to CD4dim, with no clear distinction to subpopulations, CD4 MFI 32 and CV78%. The exact position of region would severely influence the reported percentage of CD4pos cells, ranging from 30% to 65% gated cells.

Figure 4 Typical cytometric findings in B-precursor ALL with MLL rearrangement. Gated blasts (in color) are laid over ungated cells (gray) from the same specimen in plots (a) and (c-f) or over non-malignant bone marrow pattern of CD10/CD20 expression in B cells in the contour plot (b). Plot (a) from diagnosis shows a subset of blasts CD10pos (green). However, no CD10 positivity could be found at relapse and the pattern of expression corresponds to most immature CD10neg CD20neg pro-B cells. Plots (c) to (e) illustrate positivity for typical aberrant molecules (NG2, CD15, CD65) shown in specimens of MLL/AF4pos patients. Plot (f) illustrates TdT and CD34 expression in gated CD19pos blasts.

Figure 5 Typical cytometric findings in TEL/AML1pos ALL. Gated blasts (black) are laid over non-malignant bone marrow pattern of CD10/CD20 expression in B cells in the contour plot (a) or over ungated cells (gray) from the same specimen (plots b to e). Plot (a) illustrates CD10 overexpression. Plot (b) and plot (c) illustrate aberrant expression of CD13 (in the whole blast population) and CD33 (in a subset of blasts), respectively. Plot (d) illustrates lack of positivity for CD66c (KOR-SA3544). Plot (e) shows positivity for TdT and dim expression of CD34 in the gated blast population.

Figure 6 Typical cytometric findings in cases of high hyperdiploid ALL. Gated blasts (in color) are laid over non-malignant bone marrow pattern of CD10/CD20 expression in B cells in the contour plot (a), over normal bone marrow pattern of CD14/CD45 expression (c) or over ungated cells from the same specimen (plots b and d to f). Plot (a) illustrates overexpression of CD10 together with aberrant CD20 expression in CD10/high B cells. Plot (b) illustrates aberrant expression of CD66c (KOR-SA3544) and plot (c) negativity of CD45. In plot (d) a characteristic bright CD34 is shown. CD13 and CD33 are absent or detectable in a minor subset of blasts only (examples shown in green in plots (e) and (f).

Figure 8 Typical cytometric findings in E2A/PBX1pos ALL. Leukemic blasts positive for CD19 and with dim CD45 expression (plot b) are found in the lymphocyte region in the forward scatter/side scatter plot (black, plot a). The expression of CD10 varies in different cases, here bright expression together with dim expression of TdT (plot c). Dim expression of CD13 is found in some cases (illustrated in plot d). The expression of cytoplasmic IgM is illustrated together with isotypic control in plot (e).

Figure 7 Typical cytometric findings in BCR/ABLpos ALL. Gated blasts (in color) are laid over non-malignant bone marrow pattern of CD10/CD20 expression in B cells in the contour plot (a) or over ungated cells (gray) from the same specimen (b to f). In plot (a) the overexpression of CD10 is shown and in plot (b) the aberrant expression of CD66c (KOR-SA3544). A homogenous expression of CD34 and heterogenous expression of CD38 are illustrated in plot (c). CD13 and/or CD33 are frequently dim positive as illustrated in plots (d) and (e) respectively. In some cases CD34 may be negative as demonstrated in plot (f) in combination with TdT positivity.

Figure 9 Expression of CD66c in major molecular-genetic subsets of ALL. Data from a cohort of unselected newly diagnosed patients with B-precursor ALL are shown. Each circle represents one ALL case and the position of the circle corresponds to the percentage of cells with given antigen expression in the blast gate. Cells are gated by forward scatter/side scatter. Detailed methods are in Ref. 89.

Figure 10 Typical cytometric findings in AML1/ETOpos AML. The antigen expression in bone marrow cells gated to remove dead cells and debris is presented. Plot (a) and (b) Forward scatter/side scatter plot (a) shows the immature (CD34pos - red and CD34pos CD15pos - purple) population of blasts and differentiating CD15pos (green) population of myeloid precursors (b). Plot (c) illustrates bright expression of CD34 together (red) with dim CD19 expression in a subset of blasts. Plot (d) illustrates dim CD33 expression (blue) present only in a subset of blasts. Plot (e) shows bright CD13 expression (red) together with positivity for myeloperoxidase. Plot (f) illustrates aberrant expression of CD56 (red) that may be found in a fraction of cases.

Figure 11 Typical cytometric findings in PML/RARalphapos acute promyelocytic leukemia. The leukemic cells are shown in color (blue) laid over ungated cells (gray). Plot (a) illustrates typical high side scatter together with homogenous CD33 positivity. Plot (b) illustrates dim CD13 expression together with positivity for myeloperoxidase. Plot (c) shows lack of CD15 and HLA-DR expression. Plot (d) illustrates positivity for CD2 (most often found in cases with PML/RARalpha bcr 3) and dim expression of CD56 that may be found in a fraction of cases. Plot (e) shows a subset of CD34+ blasts (red). CD34pos blasts are negative for HLA-DR. Plot (f) illustrates positivity for CD117 that may be found in a fraction of cases.

Figure 12 Typical cytometric findings in CBFB/MYH11pos AML. The antigen expression in bone marrow cells gated to remove dead cells and debris is presented. Plots (a and b): Forward scatter/side scatter plot (a) shows the immature (CD34pos - red and CD34pos CD15pos - blue) population of blasts and differentiating CD15pos (green) population of myeloid precursors (b). Plots (c and d) show granulocytic differentiation. The CD33pos CD15neg cells (red, c) correspond to immature blasts, while CD33pos CD15pos (blue, c) and CD33neg CD15pos CD65pos (green, c and d) to differentiating precursors. The aberrant expression of CD2 in leukemic cells is shown in plot d (blue and cyan). T cells present in the sample show stronger CD2 expression (red, d). Plots (e and f) show monocytic differentiation. A subset positive for CD14 (blue, e) separate from the immature CD117pos precursors (red, e) is present. Considerable subsets of cells express CD4 and CD11b (violet and red, f).

Figure 13 Typical cytometric findings in AML with MLL rearrangement. Gated blasts (in color) are laid over ungated cells (gray) from the same specimen. Aberrant expression of NG2, CD19 and CD56 is illustrated in plots (a, d and c), respectively. Plot (b) shows expression of CD4 characteristic for AML with monocytic differentiation.

Tables

Table 1 Immunophenotype of leukemia with most common chromosomal aberrations

Received 22 June 2001; accepted 29 December 2001
July 2002, Volume 16, Number 7, Pages 1233-1258
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