Cancer Genetics and Cytogenetics 163 (2005) 62–67

Short communication

CD79a expression in acute myeloid leukemia t(8;21) andthe importance of cytogenetics in the diagnosis ofleukemias with immunophenotypic ambiguity

Igor Kozlova, Kevin Beasona, Cheng Yub, Michael Hughsona,*aDepartment of Pathology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505

bDepartment of Preventive Medicine, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216

Received 29 March 2005; received in revised form 1 June 2005; accepted 2 June 2005

Abstract Acute leukemias that express antigens associated with more than one lineage have been classified asacute lymphocytic leukemia with myeloid markers, acute myeloid leukemia with lymphoidmarkers, or biphenotypic acute leukemia (BAL). Antibody to cytoplasmic CD79a has been recentlyintroduced to flow cytometry. CD79a functions in and has a high degree of specificity for B-celldifferentiation. It has only recently begun to be reported in biphenotypic acute leukemias. Casesof acute leukemia submitted to the flow cytometry laboratory were retrospectively reviewed begin-ning from the time analysis for cytoplasmic CD79a was added to leukemia and lymphoma panels.Among 89 cases of AML, 2 showed strong coexpression of CD79a. Both cases were differentiatedFAB AML-M2 and demonstrated the t(8;21) with cytogenetics and the AML1/ETO rearrangementwith fluorescence in situ hybridization (FISH). These are recurring abnormalities in FAB AML-M2.The immunophenotyping met proposed scoring criteria for a diagnosis of BAL. Nevertheless, thecytogenetic and FISH findings indicate that CD79a, despite its specificity for B-cell differentiation,represented the aberrant presence of a B-cell antigen in leukemias of distinct myeloid linage. It isdoubtful that, in this setting, CD79a expression should be considered a manifestation of lineageambiguity. � 2005 Elsevier Inc. All rights reserved.

1. Introduction

Leukemias are classified using a combination of mor-phologic, cytochemical, immunophenotypic, and cytoge-netic studies. Flow cytometric analysis of leukemias withpanels of monoclonal antibodies now provides 98% accu-racy for distinguishing acute leukemias of myeloid andlymphoid origin. In rare cases, both myeloid and lymphoidantigens are expressed, creating ambiguity for lineage as-signment and difficulties in establishing whether a leukemiarepresents a distinct clinical or biological entity. This cate-gory of acute leukemia has been termed biphenotypic leu-kemia and is included in the WHO classification ofhematopoietic malignancies as acute leukemia of ambigu-ous lineage [1].

Biphenotypic acute leukemias (BALs) are reported toaccount for 4 to 8% of acute leukemias [1,2]. Conventionalmorphologic evaluation of BAL is usually of limited value,

* Corresponding author. Tel.: (601) 984-1540; fax: (601) 984-1531.

E-mail address: mhughson@pathology.umsmed.edu (M. Hughson).

0165-4608/05/$ – see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cancergencyto.2005.06.002

because the majority of cases display blasts with little cyto-logic differentiation. Currently, biphenotypic acute leuke-mia is diagnosed with immunophenotyping [3]. To clarifythe definition of BAL, the European Group for the Immu-nological Classification of Acute Leukemia (EGIL) pro-posed a scoring system based on the number and degreeof specificity of the lymphoid and myeloid markers ex-pressed by leukemic cells (Table 1) [3]. According to thisscoring system, a case is considered biphenotypic whenthe score from two separate lineages is O2.

The coexpression of B-lymphoid and myeloid antigensis the most common combination of markers, being foundin approximately 70% of BAL [2,4]. Among the differentB-cell markers, CD79a has the highest linage-specificityfor B-cell differentiation, with a specificity of 88% anda sensitivity of 100% [5]. It is a cell-surface molecule hav-ing a cytoplasmic domain that is associated physically withmembrane immunoglobulins [6]. CD79a is needed forB-cell differentiation and is expressed in the early and latestages of B-cell development. Approximately eight to ninecases of BAL with coexpressed CD79a have been

63I. Kozlov et al. / Cancer Genetics and Cytogenetics 163 (2005) 62–67

reporteddalthough it must be noted that, in the larger se-ries, most of the leukemias were not tested for the antigen,and several of these series report the same group of patients[2,4,7–10].

We report two cases of AML with differentiation (FABAML-M2) that coexpressed CD79a. Both cases cytogenet-ically demonstrated a t(8;21) translocation, and under fluo-rescence in situ hybridization (FISH) both showed theAML1/ETO rearrangement, recurring cytogenetic and mo-lecular genetic abnormalities associated only with AML.(Note that the genes involved, AML1 and ETO, have sincebeen reclassified and renamed as RUNX1 and RUNX1T1,respectively; the older terminology will be used here, forconvenience.) The cases are presented to emphasize the pri-mary role that cytogenetics or molecular genetics shouldplay in the diagnosis of acute leukemia.

2. Materials and methods

2.1. Case selection

Leukemias submitted for flow cytometry at the Univer-sity of Mississippi Medical Center were retrospectively re-viewed for the period since the laboratory began analyzingspecimens for cytoplasmic CD79a. The cases were classi-fied on the basis of conventional morphology, cytochemis-try, and flow cytometric phenotype. During a 34-monthperiod, 89 cases of AML were diagnosed; of these, 2 casesshowed coexpression of CD79a and myeloid antigens.

2.2. Flow cytometry

Immunophenotyping was performed with flow cytomet-ric analysis of peripheral blood and bone marrow aspiratescollected in sodium heparin anticoagulant using an FC500or Epics XL flow cytometer (Beckman Coulter, Miami,FL). Samples were washed with 30% fetal bovine serumRPMI medium. The portions of the sample to be surface-

Table 1

Scoring system for markers proposed by the European Group for

the Immunologic Classification of Leukemia (EGIL)

Score B-lymphoid T-lymphoid Myeloid

2 CytCD79aa CD3(m/cyt) MPOa

2 Cyt IgM anti-TCR

2 CytCD22

1 CD19a CD2 CD117a

1 CD20 CD5 CD13a

1 CD10 CD8 CD33a

1 CD10 CD65

0.5 TdT TdT CD14

0.5 CD24 CD7 CD15

0.5 CD1a CD64

The EGIL proposals [3] are adapted from the WHO classification of

tumors [1].a Antigens expressed in O20% of the blast population from the

reported cases.

labeled were incubated for 30 minutes at room temperaturewith conjugated monoclonal antibodies and then were pro-cessed into a cell button that was resuspended with 0.5 mLof paraformaldehyde. The cytoplasmic labeled portion ofthe washed sample was surface-stained with CD45. Then,the surfaces of the cells were fixed using IntraPrep fixationreagent (Beckman Coulter) 1 for 15 minutes and afterwashing was treated with IntraPrep permeabilization re-agent 2. The cytoplasmic conjugated monoclonal antibod-ies were added, and following a 30-minute incubation,the cytoplasmic stained cells were washed, and the final cellbutton was resuspended in 0.5 mL of paraformaldehyde.

Abnormal cell populations were gated using right-angleside-scatter versus CD45. Five-color analysis was per-formed with the use of monoclonal antibodies labeled withfluorescein isothiocyanate (FITC), phycoerythrin (PE/RD1), phycoerythrin–TexasRed–x (ECD), phycoerythrin–cyanin 5.1 (PC5/PE-Cy5), and phycoerythrin–cyanin 7(PC7). All samples were stained with an acute leukemiapanel of conjugated monoclonal antibodies purchased fromImmunotech (Beckman Coulter) or Oncomark (BD Bio-sciences, San Jose, CA). Myeloid markers consisted ofCD13, D15, CD33, and cytoplasmic myeloperoxidase(Immunotech clone CLB-MPO-1). B-lymphocyte markerswere CD19, CD20, cytoplasmic CD22, surface and cyto-plasmic anti-k and anti-l, and cytoplasmic CD79a (Immu-notech clone HM47). Monocyte markers were CD4, CD14,and CD64. Markers for stem cells consisted of CD34 andCD117. A threshold of 20% of labeled blasts was set asthe positive cutoff for each marker.

2.3. Conventional cytogenetic analysis

Cytogenetic analysis of metaphase cells was performedon bone marrow specimens using standard techniques.Chromosomes of cultured bone marrow cells from 24-hourcultures were analyzed by applying the GTG bandingtechnique to identify the individual chromosomes. At least15–20 metaphase cells were counted and examined foreach case.

2.4. Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was per-formed on cultured bone marrow cells obtained for cytoge-netic analysis. Cell were dropped on slides and fixed witha 3:1 ethanol acetic acid solution. Dual-color DNA probes(Vysis, Downers Grove, IL) for AML1 at 21q22 (Spectrum-Green) and MTG8 (now renamed RUNX1T1), the ETOgene, at 8q22 (SpectrumOrange) were used to detect theAML1/ETO translocation. In normal interphase nuclei ormetaphase chromosomes, there will be two separate redand green signals. In cells containing the t(8;21), there willone red and one green signal and two fusion signals indicat-ing the translocation between AML1 and ETO on the deriv-ative chromosomes der(8) and der(21).

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3. Case reports

3.1. Case 1

A 41-year-old male presented to the hospital with com-plaints of weakness, nausea, syncope, and shortness ofbreath. His past medical history included cigarette smokingand daily alcohol use. Laboratory investigations showeda white blood cell count of 15,000 mL; hemoglobin of!5.0 g/dL; platelet count of 17,000 mL; and lactate dehy-drogenase of 1448 U/L. The peripheral blood smear dem-onstrated 20% blasts (Fig. 1). A bone marrow aspirateand biopsy showed a cellularity of approximately 80%with 50% of blasts (Fig. 2). There were numerous atypicalneutrophils and eosinophils that were characterized by ab-normal cytoplasmic granularity and many hypolobatedpseudo-Pelger–Huet cells. Auer rods were not identified.Bone marrow flow cytometric studies identified a bipheno-typic blast population expressing myeloid and lymphoidantigens (Fig. 3). The expressed myeloid antigens and theproportion of positive blasts were CD13 (33%), CD33(82%), CD117 (39%), and cMPO (85%). The positive lym-phoid antigens were CD19 (41%) and cCD79a (45%)(Fig. 4). The blasts also expressed CD34 (58%), HLA-DR(65%), and CD71 (45%). TdT was positive in 15% of cells.

3.2. Case 2

A 43-year-old male was admitted to the hospital com-plaining of body aches and intermittent confusion that be-gan gradually a few days previously. His past medicalhistory was significant for prior polysubstance abuse, in-cluding cocaine, alcohol, tobacco, and marijuana. Labora-tory investigations showed a white blood cell count of49,100 mL; hemoglobin of 3.2 g/dL; platelet count of10,000 mL; and lactate dehydrogenase of 2933 U/L. The pe-ripheral blood smear demonstrated 45% blasts (Fig. 5). A

Fig. 1. Case 1. Peripheral blood smear shows circulating blasts and neu-

trophils with abnormal nuclear segmentation, including pseudo-Pelger–

Huet cells (Giemsa–Wright stain; original magnification �1000).

bone marrow aspirate and biopsy showed a cellularity of95% with 80% of blasts (Fig. 6). The morphologic featuresincluded dysplastic megakaryocytes and erythroid cells andmyeloid differentiation with pseudo-Pelger–Huet cells.Auer rods were not identified. Bone marrow flow cytomet-ric studies showed a blast population that expressed the my-eloid and lymphoid antigens: CD33 (56%), CD117 (76%),cMPO (31%), CD19 (50%) and cCD79a (56%) (Figs. 7, 8).The blasts were also positive for CD34 (36%), HLA-DR(43%), and CD71 (63%). TdT was negative.

4. Results

4.1. Cytogenetic and FISH results

Metaphase cells from each case demonstrated a 46,XYkaryotype with t(8;21)(q22;q22). A derivative chromosome

Fig. 2. Case 1. Bone marrow biopsy specimen shows myeloid differenti-

ation with numerous atypical neutrophils, eosinophils, and blasts (hema-

toxylin–eosin stain; original magnification �400).

Fig. 3. Case 1. Flow cytometric analysis of the patient’s bone marrow

demonstrates a discrete blast population (red) gated using side scatter ver-

sus CD45. Abbreviations: PC5, phycoerythrin–cyanin 5.1.

65I. Kozlov et al. / Cancer Genetics and Cytogenetics 163 (2005) 62–67

7 resulting from a whole-arm t(1;7) was detected in case 1.In both cases, FISH showed fusion signals for the AML1/ETO translocation in interphase nuclei (Fig. 9). In a meta-phase of case 1, fusion signals were colocalized on der(8)and der(21) chromosomes (Fig. 10). The cytogenetic andimmunophenotypic features of the two cases are summa-rized in Table 2.

4.2. Treatment response

The patients were treated with high-dose cytarabine con-solidation. At writing, patient 1 has been in remission for14 months and patient 2 for 8 months.

Fig. 4. Case 1. Representative cytogram of the bone marrow blasts

showing expression of the lymphoid-associated cCD79a in 45% of

blasts. Abbreviations: PC7, phycoerythrin–cyanin 7; PE, phycoerythrin;

c, cytoplasmic.

Fig. 5. Case 2. Peripheral blood smear reveals circulating blasts and ab-

normal neutrophils including pseudo-Pelger–Huet cells (Giemsa–Wright

stain; original magnification �1000).

5. Discussion

During a 34-month period, 2 out of 89 cases of AML(2.2%) were found that expressed CD79a. Both cases dem-onstrated myeloid differentiation and were classifiedas FAB AML-M2. The bone marrow aspirate of both pa-tients demonstrated a t(8;21)(q22;q22) translocation withcytogenetic analysis and the AML1/ETO rearrangementwith FISH.

The t(8;21) is found in 40% of karyotypically abnormalFAB AML-M2 [1]. The translocation interrupts AML1 onchromosome 21 and ETO on chromosome 8 that are fusedto form a novel AML1/ETO chimeric gene and transcript[11]. Normally the AML1 protein along with core bindingfactor beta (CBFB) forms an AML1–CBFB transcription

Fig. 6. Case 2. Bone marrow biopsy demonstrates hypercellular marrow

with numerous blasts, dysplastic megakaryocytes and erythroid cells,

and myeloid differentiation with abnormal neutrophils (hematoxylin–eosin

stain; original magnification �400).

Fig. 7. Case 2. Flow cytometric analysis of the patient’s bone marrow re-

veals a discrete blast population (red) gated using side scatter versus

CD45. Abbreviations: SSLin, side scatter linear; PC7, phycoerythrin–

cyanin 7.

66 I. Kozlov et al. / Cancer Genetics and Cytogenetics 163 (2005) 62–67

factor that functions as a protooncogene and regulates nor-mal myeloid cell growth and differentiation [11,12]. TheAML1/ETO fusion product is an oncoprotein that seemsto promote growth of dysplastic hematopoietic cells by op-posing the activity of AML1 and by activating the apoptosisinhibitor BCL-2 [12–15].

In addition to the t(8;21) translocation, one of the leuke-mias we report demonstrated t(1;7). This may relate to thealcohol and substance abuse that was a feature of the

Fig. 8. Case 2. Representative cytogram of the bone marrow blasts

demonstrating expression of the lymphoid-associated cCD79a in 56% of

blasts. Abbreviations: PC5, phycoerythrin–cyanin 5.1; PE, phycoerythrin;

c, cytoplasmic.

Fig. 9. Case 2. FISH probes for AML1 (green signal) and ETO (red

signal) demonstrate two normal and two colocalized, fusion signals for

the AML1/ETO translocation are seen in an interphase nucleus (magnifi-

cation �1000).

clinical history of both cases. The t(1;7) occurs in 3–7%of secondary AML and myelodysplastic syndrome (MDS)due to genotoxin exposure [16]. De novo AML and MDSdemonstrate t(1;7) less frequently, in 0.5% and 2% of cases,respectively [17].

Cytogenetics abnormalities have been reported in sever-al BAL [2,4,9,10]. Carbonell et al. [4] obtained cytogenet-ics on 29 acute biphenotypic leukemias. The t(9;22)Philadelphia chromosome was found in eight cases. Thiswas the most common abnormality, followed by 6q aberra-tions in three cases and 11q23 rearrangements in two cases.The morphologic classification was ALL in 15 patients andAML in 11 patients, with 2 of the latter being differen-tiated FAB AML-M2. One of these FAB AML-M2, whichwas also reported by Killick et al. [9], had the t(8;21)translocation. The authors pointed out that the greatmajority of BALs are morphologically immature or un-classifiable [4,9]. They also questioned whether the FABAML-M2, particularly with t(8;21), should be consideredbiphenotypic.

The prognosis of BAL is regarded as being worse thaneither ALL or AML not showing lineage ambiguity. Theunfavorable prognosis has been related to older age (O15years) and the presence of the Philadelphia chromosome[2,9]. The presence of t(8;21) in AML is associated withhigh remission rates and long-term survival [1]. With thefew cases so far described, it is not known whether B-cellantigen expression will change that outlook. Both of our

Fig. 10. Case 1. In metaphase chromosomes, separate AML1 and ETO

signals are seen on normal chromosomes 8 and 21 and two colocalized

signals representing the AML1/ETO translocation are seen in der(8)

and der(21) chromosomes (arrows) (magnification �1000).

67I. Kozlov et al. / Cancer Genetics and Cytogenetics 163 (2005) 62–67

Table 2

Cytogenetic and immunophenotypic features with EGIL [3] B-lymphoid and myeloid scores of the reported cases

Case no. Karyotype AML1/ETO MPO CD117 CD13 CD33 CD79a CD19 MY B-lym

1 46,XY,t(8;21)(q22;q22+1)der(1;7)(q10;q10) Rearr 1 1 1 1 1 1 5 3

2 46,XY,t(8;21)(q22;q22) Rearr 1 1 1 1 1 1 4 3

Abbreviations: B-lym, B-lymphoid score; MPO, myeloperoxidase; My, myeloid score; Rearr, rearranged.

patients achieved a remission, as did the patient reported byKillick et al. [9]. Scolnik et al. [10] reported the case ofa patient with a CD79a-positive biphenotypic acute leuke-mia having t(15;17) and the PML/RARa rearrangementwho stayed in remission for 3 years after being treatedfor acute promyelocytic leukemia.

Based on the morphologic and cytogenetic studies, webelieve that the leukemias reported here do not show line-age ambiguity but rather represent AML having an aberrantexpression of B-lymphoid antigens. It is doubtful that suchcases should be considered BAL. Immunophenotyping hasassumed a dominant role in the diagnoses of acute leuke-mia, but currently subtypes of AML and ALL are classifiedand their prognosis determined on the basis of cytogeneticabnormalities [1,18]. It is likely that the AML1/ETO rear-rangement will give a better indication of prognosis thanimmunophenotypic ambiguity. The findings emphasizethe primary role that cytogenetics should play in the classi-fication of leukemias.

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