(2002) 16, 1233–1258  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu REVIEW

Antigen expression patterns reflecting genotype of acute O Hrus˘a´k1 and A Porwit-MacDonald2

1Institute of Immunology/CLIP, Charles University, Prague, Czech Republic; and 2Department of Pathology, Karolinska Hospital and Institutet, Stockholm, Sweden

Multi-parameter flow cytometry, molecular genetics, and cyto- Methodology genetic studies have all contributed to new classification of leu- kemia. In this review we discuss immunophenotypic character- istics of major genotypic leukemia categories. We describe Complex or simple correlation patterns?: The expression immunophenotype of: B-lineage ALL with MLL rearrangements, of individual molecules in eight important genotypic subtypes TEL/AML1, BCR/ABL, E2A/PBX1 translocations, hyperdiploidy, of AL is shown in Figures 1 and 2. These figures result from and myc fusion ; T-ALL with SCL aberrations and a meta-analysis of available data providing a synopsis of the t(5;14) translocation; and AML with AML1/ETO, PML/RAR␣, expression of individual molecules. It is impossible to show ␤ OTT/MAL and CBF /MYH11 translocations, 8 or 11 the methodological details of each cited study here. These and aberrations of 7 and 5. Whereas some geno- types associate with certain immunophenotypic features, details vary among the cited studies and some of them are others can present with variable immunophenotype. Single mentioned in the respective paragraphs below. Readers molecules (as NG2, CBF␤/SMMHC and PML/RAR␣ ) should also be aware that the sizes of cohorts differed; how- associated with or derived from specific translocations have ever, the graphical tags are identical for all studies, making been described. More often, complex immunophenotype pat- the larger studies under-represented. terns have been related to the genotype categories. Most As shown in Figures 1 and 2, the expression of a single known associations between immunophenotype and genotype have been defined empirically. Therefore, these associations molecule can rarely predict a molecular genetic subtype. should be validated in independent patient cohorts before they Only exceptional molecules, eg the fusion can be widely used for prescreening of leukemia. Progress in CBF␤/SMMHC {product of inv.(16)},4 the aberrant molecule our knowledge on leukemia will show how the molecular– of chondroitinsulfate NG2 (correlating with translocations of genetic changes modulate the immunophenotype as well as MLL gene5–8) or the hybrid PML/RAR␣ protein,9 are known as how the expressed protein molecules further modulate cell good molecular–genetic predictors, as mentioned in respect- behavior. Leukemia (2002) 16, 1233–1258. doi:10.1038/sj.leu.2402504 ive articles. Several authors constructed scoring strategies, Keywords: leukemia; immunophenotype; cytogenetics; chromo- which simultaneously take into consideration the expression somes; flow cytometry 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 anti- Introduction gens could be correlated – resulting in useful diagnostic tools. Percentages of positive cells together with intensity and/or 10,11 Chromosomal aberrations are of high prognostic significance homogeneity of antigen expression have been used. both in acute myeloid leukemia (AML) and acute lymphoblas- However, it may be argued that the significance of the scor- tic leukemia (ALL).1–3 Although it is obvious that genotype is ing strategies can be considered hypothetical, until proven 12 reflected in the immunophenotype of leukemic blasts, there using an adequately sized independent cohort of patients. is no absolute concordance between the immunophenotype The potential drawback in this sort of scoring strategy (as and genotype categories. Several questions of biological and described in detail in the Appendix) is the fact that, often, practical nature emerge: these scoring systems are applied to the same cohort from which they are derived. An investigator may design a study • Should we modify the immunophenotype classification to using what appears to be a well thought-out strategy using reflect genotype changes? an appropriate antigen array and test population. Each of the • Should we perform molecular genetic studies only in pre- antigens may have a slightly different level of expression in screened subsets of patients? samples of leukemia with or without the investigated genotype • Which genotypes correlate with a specific pattern of antigen (G). Just by chance, a combination of some antigens could be expression and which show variable immunophenotypic expressed in the same way by all Gpos cases (eg all antigens features? will be positive or all will be heterogeneous, etc). These anti- • What are the regulatory mechanisms that allow expression gens may be selected and applied as a scoring system. Look- of aberrant molecules in leukemic cells? ing at Gneg cases, only few of them will meet all the scoring Those who seek answers to such questions might find useful criteria. Therefore, a high predictive value is declared in the information in this review. given study but difficult to reproduce in a different cohort, as reasoned in the Appendix.

Parameters to consider: Classical immunophenotyping by Correspondence: O Hrus˘a´k, Institute of Immunology, V uvalu 84, 150 flow cytometry generates several standard parameters that 06 Praha 5, Czech Republic; Fax: 4202 2443 5962 describe the expression of a given molecule. The most com- Received 22 June 2001; accepted 29 December 2001 monly used parameters are the percentage of positive cells, Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1234

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 overlapoccurred and in such cases the pointsmay 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.

mean (or median) fluorescence intensity and coefficient of All of these parameters are substantially influenced by the variation (CV). As shown in Figure 3, the percentage of posi- selection of investigated cells. This selection is highly depen- tive cells reflects the cellular composition of a gated cell popu- dent on the quality of the specimen and the gating accuracy. lation while neglecting the intensity of expression. This para- Therefore, immunophenotyping results obtained by flow cyto- meter is recommended by consensus guidelines.13 The usual metry should always be correlated with cytomorphologic cut-off value accepted as positivity is 20% of positive cells. aspects of bone marrow or blood smears. The use of strict cut-off values may obscure the biological nat- When searching for a correlation between immunopheno- ure of some cases, eg B-precursor ALL where most cells dis- type and molecular genetics, one has to convert the complex play pro-B (‘null’) immunophenotype, but a minor population information on immunophenotype into a simple nominal vari- of CD10pos cells is also present. Such a case may fulfill the able (such as ‘PML/RAR␣ likely/unlikely’). Most of the geno- arbitrary criteria for the ‘common’ ALL (cALL) category type–immunophenotype associations that we describe in this (Figure 4a). review show a distinct phenotype pattern. Therefore, an Mean fluorescence intensity (MFI) is a principal measure of experienced flow cytometry specialist can frequently predict the intensity of antigen expression. MFI depends on the num- the genotype without using any cut-off values. Setting a thres- ber of antibody molecules bound to each gated cell. In cases hold value for any of the above-mentioned parameters (% with low numbers of clearly positive cells, MFI may not pro- positive cells, MFI or CV) that would correctly predict the vide unequivocal information whether positive cells are genotype, even in the borderline cases, may be difficult. present or not. Median fluorescence intensity is even less informative in this respect. Coefficient of variation CV is a pro- duct of a simple formula (CV = (SD/Mean) ×100), describing Aberrant and non-aberrant molecules homogeneity of expression, ie how much the gated cells vary in the expression of the particular molecule. In general, the Except for rare proteins resulting from specific translo- expression of commonly investigated molecules is usually cations4,9 or from increased ,15 leukemic more homogenous in cases of ALL than in AML. This reflects blasts in AML or ALL express normal myeloid or lymphoid the widely known phenomenon that in many AML cases, leu- differentiation antigens, respectively. However, leukemia- kemic cells are found at various stages of differentiation.14 associated phenotypes can be identified due to co-expression

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1235

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 overlapoccurred and in such cases the pointsmay 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 subpopula- tions, 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. of markers rarely or never appearing simultaneously in normal and T cells in normal bone marrow.22–31 When compared to hematopoietic differentiation, aberrant marker over- their normal counterparts, leukemic blasts in over 90% of ALL expression or lack of differentiation markers.16–21 Consider- and over 70% of AML cases have been reported as displaying able effort has been made to determine the patterns of antigen aberrant phenotypes.19,30,32 In all leukemia cases carrying spe- expression in stem cells, myeloid progenitors, B-precursors cific translocations described below, highly anomalous

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1236

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.

phenotypes have been detected making flow cytometry an Acute lymphoblastic leukemia interesting alternative to molecular methods in follow-upof minimal residual disease in these patients.33 The genotype categories (Table 1, Figures 4 to 8) will be Whereas some genotype changes are confined to a specific described according to the differentiation status of the preva- immunophenotypic subset, two important genetic categories lent immunophenotype. Prognosis of the genotype categories (BCR/ABL fusion and rearrangements of MLL gene) are found is not the scope of this review and is mentioned only briefly in a broad spectrum of leukemia including both AML and ALL. in Table 1.

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1237 Table 1 Immunophenotype of leukemia with most common chromosomal aberrations

Chromosomal change Type of leukemia Characteristic phenotype (for refs see text) Prognosis Refs t(4;11) MLL/AF4 B-precursor CD45dim, CD19pos, CD34pos, CD22neg/dim, poor3,34–39 ALL pro-B (‘null’) CD20neg, CD24neg, TdTpos, CD10neg, cyt IgMneg, often CD15dim and/or CD65dim, mostly CD13neg, CD33neg, CD66cneg, NG2pos, CD9pos t(12;21) TEL/AML1 B-precursor CD45dim, CD19pos, CD34pos, CD22dim/pos, favorable but relapses in subset ALL CD20neg, CD24pos, TdTpos, CD10pos, cyt IgMneg, of patients40–45 CD15neg, CD65neg, CD66cneg, often CD13pos and/or CD33pos, NG2neg, CD135neg, CD9neg hyperdiploidy B-precursor CD45neg, CD19pos, CD34pos, CD22pos, favorable46–51 ALL CD20dim/pos, CD24pos, TdTpos, CD10bright, cyt IgMneg, CD15neg, CD65neg, CD66cpos, CD13neg, CD33neg, NG2neg t(9;22) BCR/ABL B-precursor CD45neg/dim, CD19pos, CD34pos/neg, CD22dim/pos, unfavorable52–56 ALL CD20dim/pos, CD24pos, TdTpos, CD10bright, cyt IgMneg, CD15neg, CD65neg, CD66cpos, CD13pos and/or CD33pos, NG2neg, CD25pos, CD38dim t(1;19) E2A/PBX1 B-precursor CD45dim, CD19pos, CD34neg, CD22pos/dim, unfavorable or average57,58 ALL CD20dim/pos, CD24pos, TdTpos, CD10neg/dim, cyt IgMpos, CD15neg, CD65neg, CD66cneg, CD13neg, CD33neg, NG2neg, CD9pos t(8;21) AML1/ETO AML CD34bright, CD117pos, CD13bright, CD33neg/dim, favorable1,59 (FAB M2) CD15bright, CD14neg, CD11bneg, CD4neg,MPObright, HLA-DRpos, CD19dim, TdTpos, CD56pos/neg, CD2neg, CD7neg t(15;17) PML/RAR␣ APL CD34neg, CD117pos/neg, CD13pos, CD33pos, favorable1,60–62 (FAB M3) CD15neg, CD14neg, CD11bneg, CD4neg,MPObright, HLA-DRneg, CD19neg, TdTneg, CD56pos/neg, CD2neg/pos, CD7neg Inv.16 CBF␤/SMMHC AML CD34pos, CD117pos, CD13pos, CD33pos, CD15pos, favorable1,63 (FAB M4) CD14pos, CD11bpos, CD4pos,MPOpos, HLA-DRpos, CD19neg,TdTneg, CD56neg, CD2neg/pos, CD7neg 11q23 AML CD34neg/pos, CD117pos/neg, CD13neg, CD33pos, intermediate to unfavorable1,64,65 (FAB M4/M5) CD15pos/neg, CD14pos/neg, CD11bpos, CD4pos, MPOneg, HLA-DRpos, CD19neg/pos, TdTneg, CD2neg, CD7neg, NG2pos t(1;22) OTT/MAL AMKL CD34neg/pos, CD117neg, CD13neg/pos, CD33neg/pos, unfavorable66 (FAB M7) CD15neg, CD14neg, CD61pos, CD41pos, MPOneg, HLA-DRpos

ALL with rearrangements of MLL MLL gene rearrangements occur frequently in therapy- related acute leukemia.69–71 Topoisomerase inhibitors are Fusion transcripts involving the MLL gene (also called ALL1, considered the main leukemogenic factor in cases of second- located on 11q23) are found both in ALL and ary AML. Even though the rarity of therapy-related ALL AML. In ALL, the most frequent fusion partner is the AF4 gene obscures its causes, topoisomerase inhibitors seem to be on chromosome 4q21. MLL/AF4pos ALL accounts for approxi- involved in most, but not all, cases of secondary ALL.67,69,70 mately 50% of ALL cases in infants below 6 months of age. The breakpoint position in the MLL gene is the same in most The incidence decreases by two- to three-fold in children 6 infant cases with MLL/AF4pos ALL as in therapy-related acute to 11 months of age.67 In older children and in adults, the leukemia but different from non-infant de novo MLL/AF4pos incidence appears to be stable accounting for less than 5% of ALL.72,73 ALL cases.3,67 Females are over-represented among infants but ALL blasts with MLL rearrangements are typically CD9pos, not within other age categories of MLL/AF4pos patients.67 The CD34pos, TdTpos and CD10neg pro-B cells (also called ‘null’ MLL/AF4pos ALL is frequently associated with CNS involve- ALL category) (Figure 4) and frequently express myeloid-asso- ment, hyperleukocytosis and hepato-splenomegaly.67 Less ciated antigens CD15 and/or CD65.74,75. In some cases, CD10 common fusion partners of the MLL gene are the ENL gene is present on a subset of cells, although at low intensity. The (chromosome 19p13.3) occurring in approximately 1% of existence of the prominent pro-B population should be con- childhood ALL44 and the AF9 gene (chromosome 9p21–22). sidered biologically more important than the small CD10pos The MLL/AF9pos leukemias are usually AML, whereas subset, which may become evident at relapse (Figure 4). Sev- MLL/AF9 has only rarely been described in ALL.68 Overall, eral of the other myeloid antigens (CD13, CD33 and CD66c) rearrangements of MLL gene are found in 6–7% of ALL cases are only occasionally positive. The expression of CD22 is usu- in both children and adults.53 ally low and CD20 is typically negative, corresponding to

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1238

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.

early stages of B cell differentiation. arily in glial, muscle and cartilage progenitor cells but not in A distinct subset of MLL/ENLpos ALL does not fully conform normal hematopoietic cells.77 NG2 homologue binds to to the general rules regarding the immunophenotype in MLL- matrix molecules including type VI collagen. It also modulates rearranged ALL. A recent study on children with MLL/ENLpos the action of platelet-derived growth factor78 and it appears ALL (t(11;19)(q23;p13.3) by cytogenetics) showed that T-lin- to be involved in remyelination.79,80 Indeed, the NG2pos oligo- eage ALL is common in these children, especially after dendrocyte progenitors are activated by demyelination rather infancy.44 It should be pointed out that the prognosis varied than by inflammation.80 This fact may be related to the fre- depending on the immunophenotype and age.44 quent CNS involvement in ALL with MLL rearrangement. In malignant diseases, NG2 has been shown to promote meta- static potential of melanoma, which has lead to its synonym NG2 homologue: a cartilage trait pointing to MLL rearrange- human melanoma proteoglycan.79 In leukemia with MLL ments in different types of leukemia: Most leukemic cells rearrangements, the significance of NG2 for cell biology has carrying MLL rearrangements in both ALL and AML cases not yet been documented. express NG2 homologue, a chondroitin sulfate molecule reacting with a mAb 7.1 that can be detected by flow cytome- try. The expression of NG2 has higher sensitivity and speci- TEL/AML1pos ALL ficity for MLL rearrangement than other molecules both in ALL and in AML. The specificity approaches 100% in ALL and in This genotype category is characterized by the presence of a childhood AML, sensitivity ranges between 50 and 80% in reciprocal translocation of the chromosomes 12p13 and AML and exceeds 80% in ALL.5–8,76 Adult NG2pos AML cases 21q22 resulting in a fusion of the TEL and AML1 genes.81,82 without MLL rearrangement have been described in one Translocation t(12;21) is virtually undetectable by routine kar- study, indicating lower specificity in adult AML7 (Figures 1 yotyping (see Ref. 83 for review). TEL/AML fusion can be and 2). Physiologically, NG2 homologue is expressed prim- detected by PCR and/or FISH in approximately 25% of child-

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1239

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). hood ALL cases,83–85 whereas only few adults with ALL cases carrying TEL/AML1 transcripts display exclusively TEL/AML1pos ALL have been described.86 The TEL/AML1pos B-precursor phenotype. Most cases are CD19pos,CD34pos, ALL typically accumulates in a pre-school childhood age TdTpos and CD10pos (Figures 1 and 5) corresponding to cALL peak84 and a smaller subset of patients is found around 9 years or more seldom pre-B ALL.85 In most patients, at least a frac- of age.83,87 tion of blasts overexpresses CD10 (Figure 5). The TEL/AML1pos

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1240

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.

cases are often CD33pos and/or CD13pos (Figures 1 and 5), as expressed. Borkhardt et al85 included CD65 together with shown by several independent studies.85,88–90 The expression CD13 and CD33 for definition of the ‘My-positive’ subset but of CD13 and/or CD33 may be heterogeneous and in many the usefulness of CD65 was not addressed separately. In our cases clearly negative and clearly positive subsets are found data, among 58 TEL/AML1pos newly diagnosed patients all but (Figure 5). No other myeloid antigens are consistently two had fewer than 10% CD65pos blasts. The regulatory mech-

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1241

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). anisms causing more frequent CD13 and/or CD33 expression CD24 is positive (60/60 cases in our cohort, unpublished data) by comparison to other myeloid antigens in TEL/AML1pos ALL (Figure 1). are not yet understood. Notably, some other genotypes like CD22 is mostly positive, which is not different from other BCR/ABL positivity are also associated with frequent childhood B-precursor ALL cases (Figure 1). expression of CD13 and/or CD33 (see below). Several attempts to design scoring systems have been So far, no single surface molecule that would significantly made10,90 but so far only one has been confirmed in a separ- predict TEL/AML1 positivity has been described. However, ate, second cohort of patients.90 By that scoring system, pres- positivity of CD66c at 3% cut-off value has been found to ence of TEL/AML1 can be predicted by negativity or only par- significantly correlate with the lack of TEL/AML1 translo- tial expression of both CD9 and CD20.90 For both of these cation.89,91 This association is certainly accentuated by a posi- markers, the subjective evaluation is necessary in order to dis- tive correlation of CD66c with hyperdiploidy and BCR/ABL tinguish between positivity in the whole blast population or positivity (see below). Nevertheless, CD66c positivity remains in the subset of blasts. 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 ALL with numerical chromosomal changes positive (based on MFI) in 40% of TEL/AML1neg cases whereas it was missing in all 21 TEL/AML1pos cases tested.10 Consider- High hyperdiploid ALL: High hyperdiploidy represents the ing B-lineage markers, CD20 is usually negative,89,90 and most common numerical chromosomal abnormality in ALL

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1242 and can be readily detected using cytometric DNA analysis.92 with 45 chromosomes. Hypodiploidy may coincide with other High hyperdiploid ALL samples are characterized by the pres- chromosomal aberrations. However, these only rarely include ence of 51 to 65 chromosomes per one leukemic cell, which BCR/ABL, MLL/AF4, E2A/PBX1 or TEL/AML1 gene fusion corresponds to a DNA index of 1.16 or higher and lower genes. In developed countries, hypodiploid ALL with less than than 1.6.50 45 chromosomes is rare in childhood (1%) and uncommon Typically, this subtype of ALL occurs in the pre-school age among adults (4%).53 A recent Indian study of 114 patients peak and is slightly more frequent among girls.93 The mech- showed a much higher frequency of hypodiploidy in both anisms leading to hyperdiploidy are unknown; the conse- childhood and adult ALL (38% and 44%, respectively95), quences of high hyperdiploidy include high propensity to whereas the proportion of hyperdiploidy was lower. However, undergo spontaneous apoptosis in vitro, which cannot be fully these results need confirmation in an independent reverted by culture on stromal cells.94 High hyperdiploid ALL (preferentially population-based) study. cases comprise 21% of childhood ALL in developed coun- So far, no data has associated hypodiploidy with a consist- tries.93 A lower incidence (15%) has been reported in an ent set of immunophenotype features. 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 Other numerical chromosomal abnormalities: Low hyper- TEL/AML1 and BCR/ABL. A minority of BCR/ABLpos patients11 diploid as well as (near)-tetraploid cases form a heterogeneous and rare cases with TEL/AML1 fusion85 also having high- group in terms of immunophenotype, prognosis, and co-pres- hyperdiploid DNA contents have been described. entation with other molecular genetic changes.98 T-lineage Almost all high-hyperdiploid cases have B-precursor immu- immunophenotype is common among (near)-tetraploid nophenotype. As shown in Figures 1 and 6, hyperdiploid lym- cases.99 In addition, (near)-tetraploidy is occasionally phoblasts are mostly CD19pos, TdTpos and CD10pos and usually observed in AML.100–102 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 BCR/ABLpos ALL CD22 and CD24 are typically positive and CD20 is positive in approximately 40% of cases. Thus, expression of these anti- BCR/ABL , the product of t(9;22)(q34;q11) is found gens is different from non-hyperdiploid B-precursor ALL cases in 25% of adult ALL patients whereas only 5% childhood ALL (Figure 1). Among myeloid antigens, CD13, CD33 and CD65 cases bear this fusion.53 The incidence of all types of leukemia are usually negative, whereas CD66c is highly positive in carrying BCR/ABL gradually increases with age, as does the almost all cases89 (Figures 6 and 9). The expression of CD15 incidence of very rare BCR/ABLpos cells in the blood of healthy did not differ between hyperdiploid and non-hyperdiploid subjects.103 The biologic importance of these non-malignant cases in B-precursor ALL cases studied in our laboratories. 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. Hypodiploid ALL: Hypodiploid DNA index is found in The BCR/ABL fusion protein may have a molecular weight of heterogeneous subsets of ALL. The modal chromosome num- either 210 kDa (= ‘major-BCR’, found in CML) or 190 kDa (= ber ranges from near haploidy (Ͻ30 chromosomes) to cases ‘minor-BCR’, found in most BCR/ABLpos ALL). A third fusion

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.

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1243 product has 230 kDa molecular weight and is called ‘micro- E2A/PBX1pos ALL and other t(1;19)pos cases BCR’. It is rarely found in CML cases and in chronic neutro- philic leukemia.104,105 Some cases of acute leukemia may rep- Recurring translocation t(1;19)(q23;p13) occurs in approxi- resent a blast crisis of clinically silent CML, which complicates mately 5–6% of children and in less than 5% of adults with the interpretation of analysis of major-BCR/ABLpos acute ALL.53,119,120 In approximately 90–95% of such cases, this leukemia cases.104,106 translocation leads to the expression of E2A/PBX1 chimeric Most BCR/ABLpos ALL cases have CD9pos, TdTpos and mRNA. Studies in transfected animals suggest that E2A/PBX1 CD10pos B-precursor ALL – ‘common’ ALL immunopheno- fusion is important in oncogenesis but an additional event is type.52,107 Rare BCR/ABLpos cases of T-lineage ALL have been required for full malignant transformation.121,122 Transgenic described.52,108,109 In a recent large Pediatric Oncology Group mice with E2A/PBX1 developmostly T-lineage lymphomasor study, t(9;22)(q34;q11) was found in three of 343 patients with AML.119 This observation contradicts the strong empirical cor- T-ALL.110,111 The type of BCR/ABL transcript was either relation between E2A/PBX1 positivity and B-precursor ALL major52 or undocumented in these studies; thus distinction immunophenotype. Rare cases with mature B or ‘FAB-L3-like’ between de novo ALL and T-lymphoblastic blast crisis of CML phenotype have been described, some of which displayed might not be possible. In a recent multi-national study on monoclonal surface Ig expression proven by flow cyto- BCR/ABLpos ALL in childhood and young adulthood, only six metry.123–125 Moreover, E2A/PBX1 positivity has been of 300 patients had T-lineage ALL.55 Overall, BCR/ABL posi- documented in AML M4, in T-lineage ALL124 and even in tivity can be found in several different subsets of ALL, with a meningioma.126 strong predominance of B-precursor phenotype. Typical immunophenotype findings in E2A/PBX1pos B-pre- Most of the BCR/ABLpos cALL cases present distinct aberrant cursor ALL are demonstrated in Figure 8. Lymphoblasts carry- features. Blasts usually display high CD10 and are CD34pos.112 ing E2A/PBX1 are usually at the pre-B stage of differentiation, Expression of CD13 and/or CD33 is frequent52,113,114 (Figure which is substantiated by cytoplasmic but not surface 7). However, these features are not pathognomonic for expression of IgM. Several independent studies have neverthe- BCR/ABLpos ALL. There appear to be no differences in the less showed that cALL phenotype (ie CD10 expression with expression of CD20, CD22 and CD24 in comparison to the lack of cytoplasmic IgM) could be demonstrated in 6 to 30% remaining B-precursor ALL cases (Figure 1). of E2A/PBX1pos cases.124,127 Reproducibility of pre-B/cALL dif- The expression of CD25 (interleukin 2 receptor alpha chain) ferential diagnosis could be limited by methodological prob- is higher in BCR/ABLpos than in other ALL.108 BCR/ABLneg lems, ie difficulties in the interpretation of cytoplasmic IgM cases with expression of CD25 do exist, but it is not known staining.128 Other membrane markers of the differentiation whether these BCR/ABLneg CD25pos patients share other geno- towards pre-B stage, such as CD34 negativity and CD20 typic or immunophenotypic features.108 In a recent study on and/or CD9 positivity, may be more reliable.129 The BCR/ABLpos ALL, CD38 was expressed at lower intensity and expression of CD22 and CD45 in E2A/PBX1pos ALL has not with lower homogeneity. Another feature found in this study been studied separately in the literature. In our experience, was high positivity of CD34.11 Since five of 14 children with these markers are expressed with variable intensity BCR/ABLpos ALL were CD34neg or CD34low pos in our com- (unpublished data). bined cohorts, some differences between immunophenotype Rare cases of t(1;19)pos ALL have hyperdiploid DNA con- of adult and childhood BCR/ABLpos ALL might exist. A scoring tents.130 Some t(1;19)pos ALL cases are E2A/PBX1neg by PCR system proposed by Tabernero et al11 included homogeneous and less likely to have the characteristic immunopheno- expression of CD10 and CD34 but low and relatively hetero- type.57,129 geneous 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 E2A/HLFpos ALL scoring system needs to be tested in a larger independent study. Presence of t(17;19)(q21;p13), equivalent to E2A/HLF positiv- ity, apparently correlates with B-precursor immunopheno- type.131 The low frequency of this abnormality (upto 1% of CEA family member correlating with three geno- children with BCP ALL) precludes thorough analysis of types: High expression of the antigen detected by a mAb typical immunophenotype. 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 MYC and mature B ALL cases of high hyperdiploid ALL are positive (Figure 9). This antigen has not yet been studied in the workshops on human Juxtaposition of MYC gene with the immunoglobulin gene leukocyte differentiation antigens (HLDA). However, a study regulatory sequences on chromosomes 14, 2 or 22 is a result by Sugita et al116 showed that the KOR-SA3544 mAb reacts of translocations t(8;14)(q24;q32), t(2;8)(p11;q24) or with CD66c (synonym, NCA50/90), a member of the carcino- t(8;22)(q24;q11), respectively.132 The involved partner genes embryonic antigen (CEA) family. CD66c is expressed by nor- determine the strong correlation with mature B immunophen- mal granulocytes and is constantly lacking in normal lympho- otype. Rare cases of T-lineage leukemia with t(8;14)(q24;q11), cytes. CD66c is involved in cell adhesion and activation, but in which the T cell receptor (TCR) gene is involved have been its physiological function is largely unknown.117 It has been described.133–135 By definition, mature B ALL expresses one shown that both CD66c and CD66e (CEA) can modulate of the immunoglobulin light chains (either kappa or lambda, metastatic potential of human carcinoma cells through macro- resulting from the light chain restriction). There is a strong cor- phage and endothelia activation.118 The consequences of relation of MYC translocations with the L3 cytomorphological CD66c expression in ALL cells, as well as its regulation, are category.136,137 Approximately half the patients express CD10 still poorly understood. with intensity comparable to that of nonmalignant B-precur-

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1244 sors or germinal center cells. Myeloid antigens, CD34 and Acute myeloid leukemia TdT, are negative. No aberrant surface molecule has been demonstrated to be consistently positive in mature B ALL. Several specific translocations have been found in acute myeloid leukemia and will be presented in order of associ- ation with various categories of FAB classification (Table 1, Figures 10 to 13).

Chromosomal changes in T lineage ALL AML1/ETOpos AML

In most adult and pediatric cases with T-lineage ALL, no cyto- Translocation t(8;21)(q22;q22) is found in approximately 7% genetic or molecular–genetic abnormality can be ident- of AML and in approximately 20% of AML M2 according to ified.132,138 In 20% to 25% of T-lineage ALL cases, translo- FAB.151,152 The translocation involves the AML1 gene on chro- cations of TCR␣␦ or TCR␤ genes to an oncogene (including mosome 21q22 and ETO gene on chromosome 8q22.153 The MYC, SCL, TAL2 and others) have been found.132 Juxta- AML1/ETO transcripts can be detected by molecular methods position of an oncogene under the control of TCR genes drives (RT-PCR). These transcripts also occur in a small number of proliferation in TCR-expressing cells. AML cases without detectable t(8;21) translocation, but with Deletions in the SCL gene (synonyms: TAL1 or TCL5 gene) other aberrations of chromosome 8.154–156 Translocation occur in 6–26% of T-ALL cases.132,139–144 The SCL gene is t(8;21) is found both in adult and childhood AML, with a rela- silent in normal T lymphocytes, but is expressed in various tively higher incidence in younger patients, and with equal hematopoietic cells including myeloid progenitors, erythro- representation of both genders.151,152,157 Bone marrow smears blasts, committed CD34pos CD38pos progenitors and megakar- from most AML cases carrying this translocation show charac- yocytes.132 The aberrant SCL expression may be caused by teristic cytological features. These include the presence of deletions in the SCL gene. These deletions are also called large blasts with needle-like Auer rods, abnormal granules TALd. The SCL gene is brought to proximity of the SIL gene, (including pseudo-Chediak granules), large nucleoli and which is upstream of SCL and is normally expressed in T lym- prominent Golgi. Bone marrow is also 154,157–161 phocytes. Other causes are fusion to a TCR gene (most com- common. monly in t(1;14)(q33;q11)) or other, not fully understood, The characteristic immunophenotype features of AML with 20,154,158,162–166 mechanisms.132,145,146 t(8;21) have been described in several studies. Two recent US studies have demonstrated 8 ident- In most cases an immature subset of blasts is present showing ically in 11% of patients with T-ALL, either alone or in combi- a high expression of CD34 and co-expression of ‘dim’ CD19 nation with t(9;11).111,147 These cytogenetic subgroups are (Figure 10). However, in some studies in both childhood and more common in AML (see below) and so far have not been adult AML the expression of CD19 was not as common as quoted above.167–170 This may depend on the use of different associated with a specific immunophenotype or a different CD19 mAb clones in these studies. Leu 12 and/or B4 were prognosis in T-ALL patients. applied in studies with positive findings.161,162,166 By compari- Cases with SCL rearrangements (including the TALd cases) son, HD37 mAb168 or various mAbs167 were used in studies more frequently express CD2 but lack CD10 expression.145 In with a low incidence of CD19 positivity. Conversely, CD19 addition, TALd ALL correlates with TCR␣␤ lineage, which can expression was reported in cases with detectable AML1/ETO be documented either by TCR␣␤ expression or by bi-allelic transcripts without overt t(8;21).154 TdT expression in the of the TCR delta gene.148 immature blast population is common.165,168 There are fea- The prognosis of TALd ALL appeared to be favorable within 144,145 tures of maturation asynchrony with co-expression of high- the subgroupof T-lineage ALL in two studies. No cyto- intensity CD34 concomitant with high-intensity CD15 and genetic relapse risk indicator has been found in a recent Chil- myeloperoxidase (MPO) (Figure 10). Differentiating popu- dren Groupstudy on childhood T-ALL. Proportionate lations of myeloid precursors lacking CD34, but expressing improvement of prognosis has been observed using contem- 147 CD15 and MPO are also present. The expression of CD13 is porary intensive treatment. Patients with T-ALL showing an usually high and the expression of CD33 relatively low. Cases intermediate stage of differentiation by immunophenotype without surface myeloid antigens but expressing myeloperoxi- pos pos pos (defined as CD1a alone or combined with CD4 CD8 dase were also described.171 CD56 is often expressed and in 110,111,149 phenotype) seem to have a more favorable prognosis. some studies it was related to worse prognosis.172 A high Although once considered associated with a more favorable expression of CD45RA and CD54 was also reported.166 CD7 outcome, neither CD2 nor CD10 expression have been asso- and CD2 are negative in most cases.161,166,173–176 ciated 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 PML/RAR␣pos AML affects approximately 22% of children and adolescents with T-lineage ALL.150 This translocation does not lead to a fusion Acute promyelocytic leukemia (APL) or AML M3 according to transcript. The genes in the immediate vicinity of the breakpo- FAB, first described in 1957 by Hillestad,177 was later related int are RanBP17 and Hox11-like2 on chromosome 5 and to the translocation t(15;17)(q22;q21).178,179 By morphology, CTIP2 (chicken ovalbumin upstream promoter transcription APL is characterized by abnormal promyelocytes, often con- factor interacting protein) on chromosome 14. The gene taining multiple Auer rods and coarse azurophilic granulation. Hox11-like2 is ectopically transcribed in t(5;14)(q35;q32)pos In approximately 20% of cases the promyelocytes are small ALL.150 All five t(5;14)(q35;q32)pos cases, published by the and hypogranular (AML M3 variant – M3v).180,181 The break- time of this review, were of intermediate T-ALL phenotype point on chromosome 15 has been identified within the PML (CD1apos CD7pos CD5pos CD2pos CD8pos). gene, while the breakpoint on chromosome 17 is located

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1245

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. within the retinoid acid receptor alpha gene (RAR␣). The ranean countries, in Latinos in the US and in South America fusion gene results in a fusion protein PML/RAR␣ and in 80% than in northern Europe, in the white population of the USA, of cases also reciprocal fusion protein RAR␣/PML. The fusion Japan and Australia.169,176,183–187 The incidence of acute leu- gene products are readily detectable by molecular methods kemia with t(15;17) appears constant over most of the human (reviewed in Ref. 182). PML/RAR␣ AML constitutes 5–15% of life span, implying only one, rate limiting, .188 There- all AML cases. A higher incidence was found in Mediter- fore, the incidence in young and middle-aged adults is rela-

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1246

Figure 11 Typical cytometric findings in PML/RAR␣pos 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/RAR␣ 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.

tively higher than for other types of AML.189 sistent.187,192 Orfao et al184 described in more detail the pat- The immunophenotype of t(15;17) AML is considered to be terns of myeloid-associated marker expression, pointing out highly specific with the characteristic scatter features187 and the homogenous expression of CD33 (Figure 11), heterogen- expression of CD13, CD33, myeloperoxidase in absence of ous expression of CD13 with lack of expression of HLA-DR CD34 and HLA-DR, and variable expression of CD15.190–192 (Figure 11) and low expression of CD15 (Figure 11). The Results concerning expression of CD9 and CD68 are not con- CD34 expression was reported to be low or negative in most

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1247

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).

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1248

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.

cases (Figure 11).184,191 In some studies higher CD34 described, with type A being prevalent.205 The fusion results expression was related to M3v morphology.181,193,194 CD7 was in a chimeric CBF␤/SMMHC protein that is thought to disrupt negative in most cases.18,161,174,195,196 However, a single case normal myelo-monopoiesis.205 of undifferentiated leukemia positive for PML/RAR␣ with Inv(16) and related t(16;16)(p13;q22) are observed in immature phenotype (CD34pos, HLA-DRpos, TdTpos,CD7pos) approximately 6–10% of all AML. It is found predominantly has been reported.197 Similarly, CD38 was reported to be in patients of young median age, and often with high white lower in PML/RAR␣.pos cases than in other subtypes of blood cell count or organomegaly.206–209 AML.198 CD2 has often been associated with an M3v morpho- The inv(16) AML cases are characterized by a complex logical aspect168,194,199,200 (Figure 11). The expression of CD56 immunophenotype with the presence of a more immature has been found only rarely, and similarly to AML with t(8;21), CD34pos TdTpos and/or CD117pos blast population together associated with worse prognosis.201,202 with multiple subsets of blasts differentiating towards granulo- There are no reports of flow cytometric detection of cytic (CD65pos CD15pos) and monocytic (CD14pos CD11bpos PML/RAR␣ protein. However, it has been detected with immu- CD4pos) lineage (Figure 12). Maturation asynchrony is a con- nocytochemical methods in over 90% of APL cases tested.9,203 stant 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 mono- CBFB/MYH11pos AML cyte differentiation (CD14, CD4).210 Most cases are CD11bpos and CD13pos.167,170,211 CD7 has been rarely observed,174 AML cases with pericentric inversion of chromosome while CD2 expression is found in approximately 40% of 16(p13;q22) are associated with the AML M4eo FAB cate- cases.168,170,211 It should be pointed out that CD2 is expressed gory.180,204 The inv(16) aberrations result in the fusion on both immature and differentiating blast populations.211 between the CBFB gene on chromosome 16q22 (encoding An antibody detecting CBF␤/SMMHC protein has been core binding factor ␤-subunit and the MYH11 gene on chro- developed and applied to detect the chimeric protein in per- mosome 16p13 (encoding a type II smooth muscle myosin meabilized cells with the highest fluorescence intensity in the heavy chain). Several CBFB/MYH11 transcripts have been CD34pos blasts.4

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1249 Abnormalities of MLL gene The incidence of trisomy 11 (tri11) is about 1% of myeloid disorders, mostly MDS and both de novo and secondary Alterations in chromosome band 11q23 are predominantly AML.233,234 In AML cases, the most common FAB categories found in the AML FAB M4/M5 category. Expression of mono- are AML M1 or M2. Trisomy 11 may appear as a sole chromo- cyte-associated markers CD4, CD14, CD11b, CD64 and somal change or in association with other aberrations such as CD56 is common.212–214 Relatively frequent expression of B 5 or 7, del(5q) or del(7q), or tri8. Rearrangements cell markers has been reported170 but other studies have not of 11q23 MLL gene were confirmed in cases where tri11 was confirmed this finding.213,215 Most cases of AML with 11q23 the sole aberration, but not in cases with multiple chromo- alterations aberrantly express NG2 homologue, similarly to somal changes.235 In a series described by Slovak et al235 the ALL cases with MLL rearrangements (see section ALL with CD34 and HLA-DR were constantly expressed, while there rearrangements of MLL for details on NG2).6 was a heterogeneous expression of all other myeloid-associa- AML t(9;11) in children is also associated with M5 FAB cat- ted markers. Occasional expression of CD19 and CD56 was egory and strong expression of NG2, HLA-DR, CD33, CD65w found.235 and CD4, while CD13, CD14 and CD34 are low.168,216

pos OTT/MAL (RBM15/MKL1) fusion and megakaryocytic BCR/ABL AML leukemia The incidence of AML with BCR/ABL major breakpoint is Translocation t(1;22)(p13;q13) has been associated with AML approximately 1% of AML cases (mostly AML M1/M2) and M7 – acute megakaryocytic leukemia (AMKL).217–219 Recently, this chromosomal aberration is considered to be associated the partner fusion genes have been identified as the OTT with poor prognosis.236,237 (RBM15) gene (one-twenty-two, or RNA binding motif pro- Cases with de novo AML carrying BCR/ABL are most often tein-15 on chromosome 1p13) and the MAL (MKL1) gene positive for CD13, CD18 and HLA-DR. Expression of B cell- (megakaryoblastic leukemia-1, chromosome 22q13). Due to related antigens is frequent.170,236,238 Potentially important their to Drosophila genes, these genes are information regarding the pathogenesis of BCR/ABLpos leuke- expected to be involved in intracellular signaling and chroma- mia comes from case reports. A patient with Philadelphia tin remodeling, respectively.220,221 AMKL with t(1;22) is most chromosome (Ph)-positive acute mixed-lineage leukemia that frequent in infants (20% of all infant AML).66,222 It is less com- expressed both major and minor BCR/ABL mRNA transcripts mon in older children (4%) or adults (2–8%).223,224 Diagnosis has been described.239 This leukemia presented with both of AMKL is primarily based on ultrastructural demonstration myeloid and B cell lineage phenotype as well as with of platelet peroxidase.225 By flow cytometry, blasts are positive immunoglobulin heavy chain gene rearrangement. for platelet-associated antigens CD41a, CD42b and CD61. Moreover, a transformation from chronic myelomonocytic However, it is important to differentiate the real positivity for leukemia to AML and subsequently to ALL has been reported platelet-associated markers from non-specific positivity due to in a case with a minor BCR/ABL transcript.240 Micro-BCR/ABL platelet adhesion to leukemic blasts. Immunocytochemical genotype, which is typically found in chronic neutrophilic staining of smears to show cytoplasmic positivity for CD61 in leukemia, has also been described in a patient with AML leukemic blasts can be of help. Myeloid-associated markers phenotype, possibly representing an accelerated phase of CD33, CD13, HLA-DR and CD34 may be positive in AML silent CML.106 Therefore, as in the case of ALL, acceleration M7 including OTT/MALpos cases.168,217,224 A reliable immuno- of clinically silent CML may mimic de novo AML in many phenotypic indicator of OTT/MAL within AML M7 has not yet BCR/ABLpos AML cases. been documented. AMKL with t(1;22) confers only a subset of megakaryoblas- tic leukaemias. AMKL in children with Down’s syndrome does not usually carry t(1;22), and is instead, often characterized and deletions in chromosomes 5 and 7 by the presence of complete or partial trisomies involving in AML chromosomes 8 and 1.226 Other frequently found chromo- somal changes involved chromosome 3.227 A history of MDS, The aberrations of chromosomes 5 and 7, such as −5/del (5q), often presenting as transient myeloproliferative syndrome −7/del (7q), are mostly associated with AML secondary to (TMS), is typical in the Down children. myelodysplastic syndrome and/or AML of the elderly.241–244 TMS, which affects approximately 10% of infants with These AML cases are usually positive for at least one of the Down’s syndrome and rare infants with normal karyotype, can myeloid-associated antigens CD13, CD15 or CD33.170 High display the same immunophenotype as AMKL.228–232 Given incidence of CD34 expression in both secondary and de novo the lack of reliable cytometric distinction between TMS and AML, with aberrations of chromosome 5 and 7, has been AMKL, molecular genetics and cytogenetics can provide use- reported.245–247 CD14 was observed more often in cases with ful evidence for malignant features. 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 and 11 in AML Down’s syndrome.227,248,249 Conversely, cases with aberrations of chromosome 5 more The incidence of trisomy 8 (tri8) is approximately 10% of AML often expressed T cell-related antigens such as CD2 or cases.1 It occurs both in children and in adults, with predomi- CD7.170 An Italian study on adult AML showed that nant M2 FAB category. AML cases with tri8 rarely express TdTposCD7pos immunophenotype correlated with chromo- CD34 but often express CD13 and CD33.170 No specific some 5 and/or 7 aberrations and with the multiple-drug resist- immunophenotype has been described. ance phenotype.175

Leukemia Immunophenotype and genotype of acute leukemias O Hrus˘a´k and A Porwit-MacDonald 1250 Practical value of immunophenotype pre-screening analysis figures is appreciated. The authors thank Mr Lewis Edgel for linguistic consultation. From the clinical point of view, rapid diagnosis and classi- fication of all leukemia cases is desirable. Data from literature show that most genotype categories can be predicted from the Appendix immunophenotype with a variable degree of accuracy. The biological importance of these observations is indisputable, Statistically, a retrospective study of immunophenotype–geno- but the practical value remains to be defined. The decision type correlation, complex data may appear highly predictive whether it is ethically justifiable to replace molecular genetic even if only composed of several weak predictors. This diagnostics with immunophenotyping has to be made by clini- example is not to say that all similar studies rely on weak cal groups. Funding can be an important factor, since most predictors. We only want to emphasize the necessity of pro- of the antigens necessary to predict the genotype are already spective evaluation, especially in complex associations. included in the basic immunodiagnostics.250 In the treatment As mentioned in the Methodology section, in a medium- protocols, which use molecular genetic markers for risk strati- size cohort it is likely that several parameters will be fulfilled fication, a confirmation by PCR or FISH is already required. by all samples bearing the molecular genetic change (Gpos) Nevertheless, rapid indication that the patient may have an under study. Once these parameters have been selected, it is unfavorable genotype can justify some clinical measures, such likely that only few of the Gneg samples (ie not displaying the as considering double-luminar catheter (which is suitable in studied change) will conform to all the predicted character- more intensive chemotherapy) and requesting fast processing istics although each of the antigens may have quite a high of the PCR/FISH studies. probability of being positive in a Gneg patient. To support this Cytometry can depict both simple and complex immuno- statement, we can consider a simplified example of 20 anti- phenotype features correlating with specific genotypes. Excep- gens that are independent of each other in a study of 10 tions exist in practically all immunophenotype/genotype cor- patients with Gpos leukemia and 40 Gneg patients. Let us sup- relation patterns described. Therefore, we feel that cytometry pose that expression of these antigens is slightly more frequent should not replace PCR or FISH, but rather give an indication in Gpos samples, true probability of positivity for each antigen in which direction these studies should proceed. Based on the being, eg 85% and 50% in Gpos and Gneg cases, respectively. available data, we currently cannot make a general rec- Each of the antigens has 20% (= 0.8510) probability of being ommendation to narrow down the molecular/FISH techniques positive in all Gpos samples. Therefore, we are likely to find to a subset of patients pre-screened by immunophenotype. It four antigens that are positive in all 10 Gpos samples (= 20 × is likely that new and more specific immunophenotype–geno- 0.2). However, in the Gneg population (n = 40), there is 54% type correlations will be discovered in the future as our probability (see formula below) of finding two samples or less knowledge on gene expression is growing. that will be scored as fulfilling Gpos criteria based on the posi- tivity of all four antigens.

Conclusions and future perspectives Formula Typical immunophenotype features associated with the major genotype categories are well described. However, the bulk of Variables this knowledge is only empirical. Mostly, we are only begin- = neg ning to understand why a certain genotype is associated with k number of G patients, here 40 = neg a certain lineage, why it correlates with a specific stage of z number of G patients with all four antigens posi- differentiation and what triggers the expression of the aberrant tive, here ranges from 0 to 40 (exact number of false positives) = neg molecules. A new generation of important molecules will y given probability of antigen positivity in a G probably be discovered by the microarray technology.251 The case, here 0.5 expression of these new markers should be tested in multi- parameter flow cytometry and characteristic expression pat- terns will distinguish leukemic blasts from normal cells as well Derivation as distinguish between the various genotype categories. The 1. Probability that a given patient fulfills all four criteria = y4 growing knowledge about genotype/immunophenotype corre- 2. Probability that a given patient does not fulfill all four cri- lation should helpto simplifythe immunologic classification teria = 1 − y4 of ALL and to establish a useful immunophenotypic classi- 3. Probability that a given groupof z patients fulfills all four fication of AML so that they both closely correlate with geno- criteria = y4z types. 4. Probability that a given groupof 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 Acknowledgements k a groupof k patients = ͩ ͪ z Supported by Barncancerfonden (The Swedish Childhood 6. Probability that there will be a combination of z patients Cancer Society), Cancerfonden (The Swedish Cancer Society), who fulfill the criteria and none of the others will Stockholm County Council, and by projects 6406-3 and k − 111300001 from Czech Ministries of Health and Education. (multiplication of lines 4 and 5): = ͩ ͪ ϫ (1 − y4)(k z) ϫ z Part of this manuscript is based on the central diagnostic pro- y4z grams of the Czech Pediatric Hematology Working Group. The helpof Radek Cˇ mejla with preparation of the meta- Therefore, testing 20 antigens in such a study is a relatively

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