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Characterization of Immunophenotypic Aberrancies in 200 Cases of B Acute Lymphoblastic Leukemia

Adam C. Seegmiller MD, PhD, Steven H. Kroft MD, Nitin J. Karandikar MD, PhD, Robert W. McKenna MD
DOI: http://dx.doi.org/10.1309/AJCP8G5RMTWUEMUU 940-949 First published online: 1 December 2009

Abstract

Morphologic distinction of leukemic lymphoblasts in B acute lymphoblastic leukemia (B-ALL) from their nonneoplastic counterparts in bone marrow (hematogones) can be difficult. Thus, the presence of aberrant antigen expression detectable by flow cytometry may be critical for diagnosis of B-ALL and detection of minimal residual disease. The current study examined the immunophenotype of B-lineage leukemic lymphoblasts in 200 consecutive, unique, pretreatment patient specimens. We found that all cases of B-ALL exhibited multiple immunophenotypic aberrancies by which they can be distinguished from hematogones. The most frequent aberrancies were uniform or a spectrum of expression of terminal deoxynucleotidyl transferase and CD34, underexpression of CD45, overexpression of CD10, underexpression of CD38, and underexpression of CD20. Asynchronous coexpression of CD34 and CD20 was also frequently observed. Of the 200 cases, 86.5% expressed myeloid-associated antigens, and 19.0% expressed 3 or more. Of 200 cases, 9.0% aberrantly expressed T cell–associated antigens. There were significant differences in antigen-expression patterns between adult and pediatric B-ALL. Specific aberrancies correlate with recurrent cytogenetic abnormalities in B-ALL.

Key Words:
  • Lymphoblastic leukemia
  • Flow cytometry
  • Immunophenotypic aberrancy

B acute lymphoblastic leukemia (B-ALL) is a neoplasm of immature B cells (lymphoblasts) that are committed to the B-cell developmental lineage.1 The nonneoplastic counterparts of leukemic B lymphoblasts, normal bone marrow B-cell precursors, are commonly referred to as hematogones. Hematogones may be abundant in healthy infants and children and are frequently increased in patients with cytopenias of various etiologies.212 They may even be detectable in minute numbers in the blood by flow cytometry.13 Furthermore, hematogones are often increased in the bone marrow of patients who have received chemotherapy or stem cell transplant.2,6,1417 Hematogones, especially if present in large numbers, may confound the diagnosis of B-ALL in 1 of 2 ways: (1) Hematogone hyperplasia in a background of cytopenias may be mistaken for B-ALL at initial diagnosis. (2) Increased hematogones in a patient treated for B-ALL may be mistaken for residual or recurrent leukemia. Consequently, the ability to distinguish hematogones from leukemic lymphoblasts on immunophenotypic or morphologic grounds is critical for accurate diagnosis.

The immunophenotype of hematogones has been well characterized in several large studies.8,13,18,19 Other studies have characterized immunophenotypic aberrancies found in leukemic lymphoblasts that help to distinguish them from developing hematogones.1924 The current study extends these findings by reporting the expression patterns of a more complete set (31) of cell-surface antigens, including not only B-lymphoid antigens but also myeloid, T-lymphoid, and NK-cell markers in one of the largest cohorts (200 patients) of newly diagnosed pretreatment B-ALL examined to date. We demonstrate that there are discernible immunophenotypic aberrancies in every case of B-ALL. Furthermore, for the first time, we show that there are differences in the immunophenotypic patterns of children and adults with this disease, some of which can be explained by differences in the distribution of recurrent cytogenetic abnormalities in these age groups. We also describe several novel immunophenotype-karyotype correlations. All of these findings are considered in light of the most recent classification scheme for acute leukemia.

Materials and Methods

Cases

The University of Texas Southwestern Medical Center at Dallas flow cytometry database was used to identify cases of pretreatment B-ALL. Two sets of 100 consecutive cases each (one set from January 1998 to November 1999 and a second set from November 2005 to August 2007) were collected. These consisted of 136 bone marrow and 64 peripheral blood specimens.

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Table 1

Flow Cytometry

Specimen processing, antibody staining, flow cytometry, and data analysis were performed as described previously.8 Briefly, blood or bone marrow samples were subjected to RBC lysis in 5 volumes of ammonium chloride lysis solution for 10 minutes. The samples were then washed with PAB solution (phosphate-buffered saline, 0.0455% sodium azide, and 0.1% bovine serum albumin) and resuspended in RPMI 1640 culture medium supplemented with 5% newborn calf serum. Cell counts were performed, and 500,000 cells were washed in PAB solution and stained with 4-color antibody cocktails.

Flow cytometry panels included CD3, CD4, CD5, CD8, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD34, CD45, HLA-DR, and κ and λ light chains in all 200 specimens. Other antibodies included CD11b and CD38 (199 cases), CD16, CD36, CD56, and CD64 (197 cases), CD7 (192 cases), intracellular terminal deoxynucleotidyl transferase (TdT; 191 cases), intracellular CD79a and intracellular myeloperoxidase (180 cases), CD1a and CD45RO (179 cases), and CD2 and CD117 (100 cases). Antibody clones, fluorochromes, and manufacturers are listed in Table 1 . Four-color flow cytometry was performed using FACSCalibur flow cytometers, and data were collected using CellQuest software (BD Biosciences, San Jose, CA). Daily fluorescent control experiments were run to ensure consistency of fluorescence intensity. There were no significant changes in equipment, reagents, or methods during the study period.

Data analysis was performed by using multiparameter cluster analysis using Paint-a-Gate software (BD Biosciences), as previously described.8,24,25 In short, leukemic lymphoblast populations were identified as distinct clusters of events delineated in multidimensional space based on forward and orthogonal light scatter properties and the staining intensity of specific lymphoblast-associated antigens, including, but not limited to CD10, CD19, CD22, CD34, and CD45.

Cell populations were designated as positive for a particular antigen if more than 20% of the leukemic lymphoblast events stained beyond an appropriate isotypic cutoff (set at 98% of isotypic control staining); otherwise they were designated as negative. Overexpression or underexpression of an antigen was defined as an increase or decrease, respectively, in fluorescence intensity of greater than one-half log compared with that seen for the same antigen on a normal hematogone population. Whenever possible, hematogones in the B-ALL sample were used as internal controls (as in Image 2). However, in most cases, internal hematogone populations were not present. In these cases, we used as controls hematogone populations in non-ALL bone marrows assayed no more than 1 week before or after the B-ALL specimen in question. Expression of CD34 or TdT in a unimodal manner spanning both negative and positive regions, rather than the typical bimodal expression of these antigens in hematogones,8,19 was designated “abnormal spectrum.” Similarly, abnormal continuous expression of CD10 or CD22 from negative to positive events was designated “variable.”

Cytogenetic Analysis

Conventional karyotypic and fluorescence in situ hybridization studies were performed as previously described.26 Fluorescence in situ hybridization studies used commercially available dual-color/dual-fusion or dual-color/single-fusion probe sets for t(9;22)(q34;q11.2) [BCR-ABL1] and t(12;21) (p13;q22) [TEL-AML1], respectively (Vysis, Downers Grove, IL). Cytogenetic abnormalities were classified according to the International System for Human Cytogenetic Nomenclature and grouped by recurrent cytogenetic abnormalities as defined in the most recent World Health Organization (WHO) classification of B-ALL.27 In total, 162 (44 adult and 118 pediatric) of the 200 cases had available cytogenetic data.

Data Analysis

Immunophenotypic aberrancies in leukemic lymphoblast populations were determined by deviation from normal patterns of B lymphocyte development defined by published immunophenotypes of hematogones from our laboratory.8,19 Categorical variables were analyzed by using the χ2 test. A P value of less than .05 was considered statistically significant.

Results

Patient Characteristics

A total of 200 unique pretreatment patient samples were analyzed. The patients had a very wide age range (range, <1–85 years; median, 7 years). The majority (69.5%) were children (younger than 18 years), most (49.0%) between the ages of 2 and 10 years with a median age of 4 years. Four patients (2.0%) were younger than 1 year. The median age of adults was 46 years. The overall male/female ratio was 1.4:1. This ratio was higher among children (1.5:1) than among adults (1.1:1).

Aberrancies in Hematogone-Associated Antigens

Immunophenotypic aberrancies are defined as patterns of antigen expression on neoplastic cells that are different from those seen on hematogones. Hematogones exhibit a well-defined spectrum of antigen expression as they mature8,19 Image 1 . The most immature hematogones express CD34, TdT, and slightly bright CD10 but are entirely negative for CD20 (stage 1). As they mature, they lose CD34 and TdT expression and become slightly less bright for CD10 (stage 2). They then gradually express CD20 and dim surface immunoglobulin (stage 3). Hematogones express moderate CD22 and bright CD38 throughout. The transition to mature B lymphocytes occurs with the loss of CD10, decreased CD38, increased CD22, and the mature polytypic expression of surface immunoglobulin. CD45 expression is moderate at the earliest stages and increases throughout maturation. CD19, CD79a, and HLA-DR are positive through all stages (not shown).

Image 1

Normal hematogone maturation. Hematogones are represented in yellow; mature, polytypic B-lymphocytes are represented in blue. The arrows designate the changes in antigen expression as the hematogones mature into mature B cells. TdT, terminal deoxynucleotidyl transferase.

In B-ALL, we observed aberrant expression of at least 1 of these 11 hematogone-associated antigens in every case examined Table 2 . These aberrancies included antigen overexpression, antigen underexpression (including lack of expression), asynchronous expression, and an abnormal spectrum of expression. Image 2 is an example of a case showing multiple immunophenotypic aberrancies (uniform positive expression of CD34 and TdT, asynchronous coexpression of CD34 and CD20, increased expression of CD10 and CD22, and decreased expression of CD45) compared with normal hematogones in the same specimen. Abnormal spectrums of expression are illustrated in Image 3 . These include abnormal variable expression of CD10 and CD22 (Image 3A). They also include continuous, unimodal distribution of CD34 (Image 3B) and TdT (Image 3C) expression, as opposed to the normal bimodal pattern seen in hematogones (Image 1).

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Table 2

The number of these aberrancies ranged from 1 to 10 per case. The most common aberrancy was uniform or a continuous spectrum of TdT or CD34 expression, seen in 95.8% and 73.0% of all cases, respectively (Table 2). Including specimens with uniformly negative expression of these antigens, 99.5% and 87.5% of cases showed aberrant expression of TdT and CD34. Asynchronous dual expression of CD34 and CD20 was observed in 37.5% of cases. Other frequent aberrancies included negative or underexpression of CD45 (66.0% of cases), overexpression of CD10 (60.5%), negative or under-expression of CD38 (58.0%), and negative or underexpression of CD20 (46.5%). These and other less frequent aberrancies are cataloged in Table 2.

In most cases, the frequencies of these immunophenotypic aberrancies were similar in adults and children. Notable exceptions included underexpression of HLA-DR, absence of CD10, and overexpression of CD45, which were seen more frequently in adults than in children. Underexpression of CD45 was more common in children than in adults (Table 2).

Image 2

Aberrant expression of hematogone-associated antigens in B acute lymphoblastic leukemia. Leukemic B-lineage lymphoblasts (red events) exhibit patterns of antigen expression distinct from hematogones (yellow events), their nonneoplastic counterparts, and mature B lymphocytes (blue events). Compared with hematogones, the leukemic lymphoblasts show uniform positive expression of CD34 and terminal deoxynucleotidyl transferase (TdT), asynchronous coexpression of CD34 and CD20, increased expression of CD10 and CD22, and decreased expression of CD45.

Image 3

Abnormal spectrums of antigen expression in B acute lymphoblastic leukemia. Leukemic lymphoblasts (red events) exhibiting abnormal expression patterns of 4 hematogone-associated antigens are illustrated in 3 separate cases, including abnormal variable expression of CD10 and CD22 (A) and continuous unimodal expression of CD34 (B) and terminal deoxynucleotidyl transferase (TdT) (C). The blue events represent mature B lymphocytes.

Image 4

Aberrant myeloid antigen expression in B acute lymphoblastic leukemia. Leukemic B-lineage lymphoblasts (red events) often aberrantly express myeloid antigens, but generally express them at levels less than seen in normal myeloid cells, such as granulocytes (green events) and monocytes (blue events). These lymphoblasts exhibit at least dim expression of CD11b, CD13, CD14, CD15, and CD33. Vertical and horizontal lines indicate the cutoff fluorescence intensity values for positive and negative events as determined by analysis of negative isotypic antibody controls (see the “Materials and Methods” section). APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

Aberrant Expression of Myeloid Antigens in B-ALL

Expression of myeloid antigens was a common aberrancy in B-ALL. The leukemic lymphoblasts illustrated in Image 4 exhibit some expression of 5 myeloid antigens, CD11b, CD13, CD14, CD15, and CD33. As seen in this case, the expression of these antigens was generally partial or dim. A total of 86.5% of cases examined showed expression of at least 1 myeloid antigen (range, 1–5) Table 3 . These aberrancies and their frequencies are listed in Table 4 . The most frequently expressed antigen was CD13 (54.5% of cases), followed by CD33 (43.0%), CD15 (36.0%), and CD11b (20.0%). The other myeloid antigens were expressed in fewer than 10% of cases. The majority of cases (67.5%) expressed only 1 or 2 myeloid antigens (Table 3). However, 19.0% of cases showed expression of 3 or more myeloid antigens. Of these 38 cases, 25 (12.5% of total cases) met European Group for the Immunologic Classification of the Leukemias (EGIL)28 criteria for biphenotypic leukemia. However, only 1 case (0.5% of total) met the more stringent WHO criteria for acute leukemias of ambiguous lineage.29

There was little difference in the frequency of myeloid antigen expression in leukemic lymphoblasts in children and adults. The only significant difference was in the expression of CD36, observed in 4.4% of children and 17% of adults (10/60; Table 4). The frequency of EGIL-defined biphenotypic leukemia was nearly equal in the 2 populations (not shown).

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Table 3
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Table 4

Aberrant Expression of T- and NK-Cell Antigens in B-ALL

Expression of T-cell antigens was observed in 18 cases (9.0%) Table 5 . The most common of these was CD4, expressed in 9 cases, although this is not T lineage–specific, as CD4 may also be expressed on myelomonocytic cells. Expressed at lower frequency were CD2, CD5, and CD7. No more than a single T-cell antigen was expressed in any 1 case, and expression of CD3 was not observed. Thus, T-cell/B-cell biphenotypic leukemias were not observed in this patient cohort. Overall, T-cell antigen expression was more than twice as common in adults as in children. Aberrant expression of the T- and NK-cell antigen CD56 was seen in 10 cases (5.1%), nearly all of them children.

Correlation With Cytogenetic Findings

Cytogenetic studies were performed in 162 of the 200 cases and grouped according to known recurrent cytogenetic abnormalities Table 6 . The age distribution of cases in these groups is similar to that seen in previously published studies, in that B-ALL with t(9;22) (Ph+ B-ALL) is more common in adults, whereas the other recurrent changes are more common in children.27 Some immunophenotypic aberrancies were unevenly distributed among these cytogenetic groups. Overexpression of CD22 was increased in cases with hyperdiploidy (36%) relative to all other cases (7%; P < .001). Decreased or absent CD10 expression was observed almost exclusively in 3 groups, patients with MLL (11q23) rearrangements, patients with non-recurrent cytogenetic abnormalities, and patients with normal karyotypes (100.0%, 17%, and 20%, respectively, vs 3% in all other cases; P = .001). Total absence of CD34 expression was most common in cases with t(1;19) (70%) when compared with all other cases (13%; P < .001), as previously described.30 Finally, loss or underexpression of CD45 was more common in cases with hyperdiploidy (92%) than in other cases (54%; P = .01), as also shown previously.31

Myeloid antigen expression was distributed more evenly among the various cytogenetic groups. As has been reported,32,33 most cases of Ph+ B-ALL and B-ALL with t(12;21) exhibited myeloid antigen expression (95% and 91% of cases, respectively), although this was not significantly higher than in other cases. However, expression of CD36 was found more frequently in Ph+ B-ALL than others (26% vs 6%; P = .005), and expression of CD11b was more common in Ph+ B-ALL and hyperdiploidy (37% and 41%, respectively) than in others (10%; P = .004). Of the T- and NK-cell markers analyzed, only CD56 showed an abnormal distribution, with increased prevalence in B-ALL with t(12;21) (27% vs 4% in all other cases; P = .002).

Other reported patterns of antigen expression in different cytogenetic groups27 were confirmed. These include CD10 and TdT expression in Ph+ B-ALL (89% and 100% of cases, respectively), CD15 expression in B-ALL with MLL rearrangements (100%), CD34 and bright CD10 expression in B-ALL with t(12;21) (100% and 91%, respectively), and CD34 expression in B-ALL with hyperdiploidy (90%).

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Table 5
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Table 6

Discussion

In this study of 200 patients with pretreatment precursor B-ALL, we showed that leukemic lymphoblasts exhibited multiple immunophenotypic aberrancies in all cases (mean, 7.6 aberrancies per case; range, 2–14). These aberrancies include abnormal or asynchronous expression of normal hematogone-associated antigens and inappropriate expression of myeloid, T-lymphoid, and NK cell–associated antigens. These findings have important implications for the diagnosis and monitoring of B-ALL by flow cytometry. Multiple reports highlight the challenge of distinguishing B-ALL from hematogone hyperplasia, especially in patients with cytopenias due to another medical condition.7,10,11 The presence of immunophenotypic aberrancies in this setting allows one to effectively distinguish between these 2 entities.34 Hematogone hyperplasia is also commonly seen in patients with recovering bone marrow after chemotherapy or stem cell transplantation.2,6,1417 Therefore, it may be challenging to distinguish residual leukemic lymphoblasts, especially in small numbers, from hematogones in the monitoring of ALL therapy. The presence of immunophenotypic aberrancies in leukemic B-lineage lymphoblasts, however, permits the differentiation of these cell types, even when they are present together35,36 (Image 2). The demonstration by the current study that such aberrancies are essentially universal in B-ALL strengthens the usefulness of flow cytometry as a sensitive diagnostic tool in these settings.

The current study identified several differences in ALL immunophenotypes between children and adults. The primary differences were in the expression of CD10, which is more commonly negative in adults, and CD45, which is more often overexpressed in adults and underexpressed in children. The absence of CD10 expression has been correlated with rearrangements of the MLL gene at 11q23,37 which is commonly seen in infants and older adults.27 Indeed, the 3 cases of B-ALL with MLL rearrangements seen in this study all exhibited decreased or absent expression of CD10. All of these were children, 2 of them younger than 1 year. Decreased CD10 expression was also overrepresented in patients with nonrecurrent or normal cytogenetics. Although these karyotypes were slightly more common in adults (Table 6), this is not sufficient to explain the age distribution of CD10 expression.

The differences in CD45 expression are largely accounted for by the excess of hyperdiploidy in children, which is known to exhibit decreased CD45 expression.31 In fact, if these cases are excluded from the analysis, decreased CD45 expression is no longer overrepresented in children. Given the excellent prognosis of this karyotypic group, this suggests that decreased CD45 expression might portend a good prognosis, although studies examining this question have given conflicting results.31,38

The subject of myeloid antigen expression in B-ALL is complicated (reviewed by Pui et al39). Reports vary widely on the degree of myeloid antigen expression, from 4.3% to 64%.3942 Consequently, the clinical significance of myeloid antigen expression is unclear.39 One potential reason for this variation is differences in the binding characteristics of different monoclonal antibody clones.43 Different thresholds for antigen positivity and different flow cytometry methods, instruments, and reagents may also have a role. The current study reports the highest fraction (86.5%) of myeloid antigen expression in leukemic lymphoblasts reported to date. This is due, at least in part, to the fact that there are more antigens analyzed than in previous studies, which focus primarily on only CD13, CD15, and CD33. In the case of 1 antigen (CD13), the antibody clone used (L138) is one described as staining a higher proportion of ALL cases in a previous study.43 A final consideration is that the current study used cluster analysis of events based on multiple parameters to identify leukemic lymphoblast populations. This is in contrast with most studies that rely on a 2-parameter gating strategy (typically CD45 and orthogonal scatter) to identify the same populations. The advantages of the cluster analysis strategy are that it generates a more homogeneous population of leukemic lymphoblasts and better eliminates contaminating events.

Of the 200 cases, 25 (12.5%) had sufficient myeloid antigen expression to qualify as biphenotypic leukemia by EGIL criteria. However, the B-lineage antigens are clearly dominant in each case, and the myeloid antigens are generally dim or partial in expression. It has been suggested that the diagnosis of biphenotypic leukemia should be reserved for the cases in which there is true lineage ambiguity.44 Indeed, the most recent WHO criteria for acute leukemias of ambiguous lineage are much more stringent, requiring expression of myeloperoxidase or 2 separate monocytic markers to diagnose mixed phenotype acute leukemia, B/myeloid.29 Only 1 (0.5%) of 200 cases in the current study meets these criteria. Although biphenotypic leukemias have been described as having a poor prognosis, the immunophenotype alone does not appear to correlate with prognosis independent of other prognostic factors, including high-risk cytogenetic changes and age.42,45,46 Thus, outside of minimal residual disease detection, there may be little clinical significance to the expression of myeloid antigens in B-ALL.

Correlation of immunophenotype with karyotype largely confirmed previous observations.27 However, we noted several novel correlations. These include increased expression of CD22 in B-ALL with hyperdiploidy and more frequent expression of CD36 in Ph+ B-ALL, CD11b in Ph+ B-ALL and B-ALL with hyperdiploidy, and CD56 in B-ALL with t(12;21).

We presented a large catalog of B-ALL immunopheno-types and demonstrated that there are immunophenotypic differences between pediatric and adult ALL. Furthermore, we demonstrated significant immunophenotype-karyotype correlations. The presence of aberrancies in every case examined strongly supports the continued use of flow cytometry in the diagnosis and monitoring of B-ALL.

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