Int J Hematol (2008) 87:132–136 DOI 10.1007/s12185-008-0029-z
CASE REPORT
B-Lymphoid and myeloid lineages biphenotypic acute leukemia with t(8;21)(q22;q22) Guangsheng He Æ Depei Wu Æ Aining Sun Æ Yongquan Xue Æ Zhengming Jin Æ Huiying Qiu Æ Xiaowen Tang Æ Miao Miao Æ Zhengzheng Fu Æ Xiao Ma Æ Xiuli Wang Æ Zixin Chen Æ Changgeng Ruan
Received: 28 December 2006 / Accepted: 18 October 2007 / Published online: 22 February 2008 Ó The Japanese Society of Hematology 2008
Abstract By analyzing the characteristics of morphology, immune phenotype, chromosome karyotype and clinical manifestations of six cases of B-lymphoid and myeloid lineages biphenotypic acute leukemia (BAL) with t(8;21)(q22;q22), a new subgroup of BAL was reported. Bone marrow eosinophilia (more than 5%) and pseudoChediak abnormalities were not found. Auer rods were also not identified in four of six cases. Immunophenotype revealed B-lymphoid and myeloid lineages positive, together with frequent and high expression of CD34 and CD33, and weak expression of HLA-DR. In addition to t(8;21) chromosomal translocation, deletion of Y chromosome and complex chromosome abnormalities were also found. Chemotherapy for myeloid and lymphoid leukemia simultaneously produced good response in the patients. BAL with t(8; 21)(q22; q22) might be a new subgroup of BAL, and it was suggested that the leukemia clone with t(8;21)(q22;q22) might have originated from an early phase of hematopoiesis, and AML1/ETO fusion gene might be related to differentiation of B lymphocyte. Keywords Leukemia Biphenotypic Acute t(8; 21)(q22; q22) AML1/ETO
G. He D. Wu (&) A. Sun Y. Xue Z. Jin H. Qiu X. Tang M. Miao Z. Fu X. Ma X. Wang Z. Chen C. Ruan Jiangsu Insititute of Hematology, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, People’s Republic of China e-mail:
[email protected]
123
1 Introduction The t(8;21)(q22;q22) resulted AML1/ETO fusion oncogene is one of the most common karyotypic abnormalities found in acute myeloid leukemia (AML)-M2 subtype, with a minority of cases presenting M1 or M4 [1]. The immunophenotypical features of AML with t(8;21)(q22;q22) was high expression of CD34 and CD19; in addition to myeloid-lineage antigen, other antigens of T or B lymphocytic lineage such as CD10, CD20, CD22, CD2, CD7 and T lymphocyte receptor (TCR) gene rearrangement were usually negative [1]. We report on six cases of acute leukemia with t(8;21)(q22;q22), which revealed B-lymphoid and myeloid lineages biophenotypic changes.
2 Patients and methods 2.1 Patients There were 200 cases (18.9%) with t(8;21) in our total AML cases (1,058). Six cases of BAL (biphenotypic acute leukemia) with t(8;21)(q22;q22) hospitalized from January 2003 to April 2006, three males and three females, aged between 13 and 56 years with a median age of 17 years, fulfilled the standard diagnostic criteria of EGIL [2].
2.2 Characteristics of morphology, immunophenotype and cytogenetics Morphological classification was based on French–American–British criteria for AML [3], and the morphologic parameters were analyzed by weighted score [4]: 2 points for FAB-M2 subtype, marrow eosinophilia (more than
B-Lymphoid and myeloid lineages BAL
133
Table 1 General clinical features of patients P
Age (Y)
Gender
PB WBC (9109/L)
Hb (g/L)
PLT (9109/L)
Hemorrhage
Soakage
Treatment/ response
1
56
F
4.8
87
11
N
Skin
MAOP/NR
2
17
M
2.1
56
62
N
Lymph node
MAOP/CR
3
13
M
6.8
44
20
N
N
DAOP/CR
4
17
F
4.4
63
15
Petechia, rhinorrhagia
N
MA/NR
5
34
F
8.84
48
7
Petechia, rhinorrhagia
N
MA/NR
6
15
M
1.48
65
51
N
Lymph node
MAOP/CR
P patient, Y years, F female, M male; WBC white blood cell, Hb hemoglobin Plt platelet, N no, NR not in remission, CR complete remission
5%), luminous Auer rods, and pseudo-Chediak abnormalities; 1 point for abnormal cytoplasmic granules, large blasts with prominent Golgi, FAB-M1 subtype, and rare Auer rods. It was considered to be positive at a value of 5 or more. Immunophenotype was analyzed with immunofluorescent labels by flow cytometry. The monoclonal antibodies for T-lymphoid lineage were CD2, CD7, and cytoplasmic CD3(cCD3); for B-lymphoid lineage were CD10, CD19, CD20 and cCD79a or cCD22; for myeloid lineage were CD13, CD14, CD33 and cMPO; and for hematopoietic stem cell/progenitor lineage was CD34. It was positive for CD34 and other markers if the percent of expression exceeded 10 or 20%, respectively. Whole bone marrow was stained with CD45-PC7, and blast cells region was gated on the CD45/SSC display. Chromosomes were checked by R bands technology and classified according to an international system for human cytogenetic nomenclature (ISCN, 1995) [5]. The AML1/ETO fusion gene was detected by fluorescence in situ hybridization (FISH). 2.3 Management The patients were treated with chemotherapy for acute myeloid leukemia and lymphocytic leukemia simultaneously: D/MAOP [daunorubicin (DNR) 45 mg m-2 d-1/ mitoxantrone (MTZ) 10 mg m-2 d-1, d1*d3, d15; cytosine arabinoside (Ara-c) 100 mg m-2 d-1, d1*d7; vincristine (VCR) 1.4 mgm-2 d-1, d1, d8, d15 d22; prednisone (Pred): 1 mg kg-1 d-1, d1*d28], or MA [MTZ 10 mg m-2 d-1, d1*d3, Ara-c 100 mg m-2 d-1, d1*d7], which was used for acute myeloid leukemia only.
3 Results 3.1 General characteristics (Table 1) There were three males and three females, an equal number of each gender. All the patients were young, except for one
Table 2 Morphological features of weighted score Weighted score
P1
P2
P3
P4
P5
P6
FAB M2/M1 subtype
2
1
2
2
2
2
Bone marrow eosinophilia (more than 5%)
0
0
0
0
0
0
Auer rods
2
0
2
0
2
0
Pseudo-Chediak abnormalities
0
0
0
0
0
0
Abnormal cytoplasmic granules
1
0
1
1
1
1
Large blast with prominent Golgi
1
0
1
0
0
1
Score
6
1
6
3
5
4
P patient
who was older than 40 years. Almost all suffered from anemia and thrombocytopenia, while the white cell counts were low or in normal range. Extramedullary infiltration was found in three patients, with lymphadenopathy and skin soakage, respectively.
3.2 Features of morphology, immunophenotype and cytogenetics and molecular biology (Tables 2, 3, 4) Morphologically, patient 2 showed M1 and the other five patients showed M2 according to the FAB criteria. Reviewing the six indexes of weighted score, in all the six patients reported here, except in FAB-M2, the morphologic feature that was assigned 2 points was rare (Table 2). Bone marrow eosinophilia (more than 5%) and pseudo-Chediak abnormalities were not found (Fig. 1). In four cases, who could not attain 5 points, Auer rods were also not identified. B-lymphoid and myeloid lineage antigen was coexpressed in all patients (Fig. 2). Besides expression of CD19, the blast cell expressed the typical marker of B lymphoid: cCD79a or cCD22, while the markers of T cell, such as CD2 and CD7, were weak. The blast cells presented with cMPO, and expression of CD33 simultaneously in four patients. In patient 5, in whom cMPO was not
123
134
G. He et al.
Table 3 Immune phenotype of patients (%) P
CD34
DR
cCD3
CD2
CD7
1
95.9
ND
2
71.5
93.2
1.9
1.8
17.5
ND
1.3
8.1
3
81.3
ND
ND
1.4
5.8
4 5
81.6
ND
ND
4.2
98.4
1.2
ND
0.2
6
96.8
6.4
ND
0.6
cCD79a
cCD22
CD10
CD19
CD20
cMPO
CD13
CD14
CD33
82.5
ND
0.4
88.3
1.1
95.2
12.4
0.5
48.5
ND
36.2
ND
46.6
1.1
94.1
6.5
1.4
65.5
ND
32.1
1.1
25.4
0.8
96.6
8.2
0.9
83.5
4.3
47.4
ND
0.8
41.9
3.1
80.6
6.0
1.0
91.4
0.7
33.3
ND
0.3
94.0
0.8
ND
70.9
ND
63.8
5.3
25.6
ND
0.5
92.3
1.0
72.3
91.2
ND
12.0
P patient, ND not done
Table 4 Changes of chromosome karyotype and molecule of patients P
Chromosome karyotype
Fusion gene
1
46,XX,1p+,t(5;11),t(8;21) (q22;q22),15p+,16p+,18q_[12]/ 46,XX[2]
AML1/ETO
2
45,X,-Y,t(8;21) (q22;q22) [8]/46,XY[2]
AML1/ETO
3
45,X,-Y, t(8;21) (q22;q22) [6]/46,XY, t(8;21) (q22;q22) [4]
AML1/ETO
4
46,XX, t(8;21)(q22;q22)[8]/46, XX[3]
AML1/ETO
5
46,XX, t(8;21)(q22;q22)[4]/46, XX[1]
AML1/ETO
6
46,XY, t(8;21) (q22;q22) [7]/46,XY[9]
AML1/ETO
P patient
Fig. 2 The blast cells of patient 4 co-expressed CD33 and cCD79a
found. One female patient revealed complex karyotypic abnormality. AML1/ETO fusion gene was revealed in each patient by FISH.
3.3 Management (Table 1)
Fig. 1 On Wright-Giemsa staining, the blast cells of patient 4 (arrow) showed no Auer rods, pseudo-Chediak abnormalities
measured, it was positive to CD117 (not showed), CD13 and CD33. CD33 was only negative in patient 6 with 12.0%. HLA-DR was only positive in one patients of the three detected. Besides the t(8;21)(q22;q22), it was associated with loss of Y chromosome (-Y) in two of the three male patients. However, another frequent additional karyotypic aberration, depletion of chromosome 9 [del(9q)], was not
123
Treated with chemotherapy for AML and ALL simultaneously, three of the four patients achieved complete remission (CR), while the other two patients who were treated with chemotherapy with a scheme of MA only for AML was not in remission (NR).
4 Discussion Biphenotypic acute leukemia (BAL) and bilineal acute leukemia are categorized as subtype of acute leukemia of ambiguous lineage, according to the WHO classification [5]. BAL was believed to be derived from multipotent
B-Lymphoid and myeloid lineages BAL
hemapoietic stem cells having the capacity to express antigens of more than a single cell lineage [5], representing a distinct clinical and biological entity [6]. The European Group for the Immunological Characterization of Leukemia (EGIL) designed a scoring system, based on antigens of myeloid or lymphoid origin, which assigned a score of 2, 1, or 0.5 depending on the specificity of the marker [2]. BAL was considered when the score of different lineage was greater than 2. Although the expression of CD79a in acute leukemia with t(8;21) was unusual [7, 8], the abnormality was recurring. Among 158 cases of AML, 6 patients with acute leukemia with t(8;21) expressed cCD79a or cCD22 in our department during 2003 to 2006. It was similar to the rate of 2 of 89 AML reported by Kozlov [8]. Kozlov proposed that acute leukemia with t(8;21), expressing CD79a, should be considered as acute myeloid leukemia, although the immunological score exceeded 5 [8]. However, we found that the acute leukemia with t(8;21), expressing cCD79a or cCD22, possessed different features in contrast to typical AML with t(8;21). Andrieu [4] developed a well-defined morphologic weight score system to the anticipated occurrence of AML1/ ETO: 2 points assigned since the morphological parameters were significantly different in AML1/ETO-positive/negative cases in AML-M1 and M2; 1 point assigned since it was observed that the morphological parameters did not reach statistical significance. Using the weight score system, no false negatives, while false positives, occurred in anticipation of the presence of the AML1/ETO fusion gene [4]. In the total of six patients reported here, however, the typical morphologic features that were assigned 2 points, such as marrow eosinophilia (more than 5%), luminous Auer rods and pseudo-Chediak abnormalities ,were not revealed (Table 2), and three cases could not attain 5 points. Marrow eosinophilia was seen in one of the two cases reported by Kozlov [8], of which the percentage of eosinophils was not shown. Auer rods and pseudo-Chediak abnormalities were also not found in the report of Kozlov. This group of leukemic cells expressing cCD79a or cCD22 did not exhibit typical morphological features characteristic of AML carrying t(8;21). Different from AML with normal or other kinds of karyotypes, the blast cells from AML with t(8;21) showed high levels of CD34, DR, CD19 and CD56; conversely, CD33 expreession was characteristically weak [1]. In this group of patients, not only the the expression of CD34 and CD19 were positive, but also frequent positivities for CD33 were revealed in five of six patients in our report and all two patients reported by Kozlov [8]. The DR was weak and positive in only one of the three patients detected in our group, but both were with positive expression of DR in Kozlov’s report [8].
135
All cases cytogenetically demonstrated t(8;21) translocation, and under FISH all showed AML/ETO rearrangement. Loss of chromosome Y, another common additional karyotypic aberration associated with t(8;21) translocation, was found in two of the four male patients. The patients were responsive to chemotherapy for myeloid and lymphoid leukemia, simultaneously. In the report of Kozlov et al., two patients treated with high-dose Ara-c achieved CR [8]. However, as is known, high-dose Ara-c is also an effective scheme for acute lymphoblastic leukemia. Experimental data have shown that AML1/ETO is not sufficient to induce leukemia by itself; activating mutations such as FLT3 or C-KITs might arise from a ‘‘preleukemic’’ clone that harbors AML1/ETO and act as a second but crucial ‘‘hit’’ to promote the transformation of the cells to leukemia [9]. That AML1/ETO was a very early event in leukemogenesis was coincident with BAL deriving from multipotent hemapoietic stem-cells. Miyamoto [10] found that although primitive CD34+Thy-1+Lin-CD10-CD38-/ low hematopoietic stem-cells (HSC) and CD34+Thy1 Lin-CD10+CD38+ common lymphoid progenitors (CLP) were depleted in leukemic bone marrow of patients with t(8;21), the AML1/ETO fusion gene was detectable in HSC and CLP at the remission phase. However, at both leukemic and remission phases, the AML1/ETO fusion gene was expressed in CD34-CD20+IgM+CD3-CD14- B cells of all t(8,21)/AML1/ETO cases detected, but not in CD34-CD3+TCRab+CD20-CD14- T cells. The AML1/ ETO-positive B cells population did not express MPO mRNA, which indicated that these B cells were not contaminated with myeloid cells. More strikingly, the frequency of AML1/ETO-positive B cells changed with the status of leukemia; the frequency of the AML1/ETO-positive B cells decreased approximately 10-fold in remission bone marrow compared with that of leukemic bone marrow. The authors suggested that the acquisition of the t(8;21) occurs at the level of stem cell, capable of differentiating into B cells as well as all myeloid lineages [10]. So, it was also possible that BAL clone could emerge from HSC bearing the AML1/ETO fusion gene. The AML1 bind in vitro to transcription factor paired box 5 (PAX5) through its RUNT domain and cooperated with PAX5 in the activation of the B-cell-specific blk gene promoter [11]. The PAX5, also named B-cell-specific activator protein (BSAP), played a key role in the commitment of hematopoietic precursors to the B-cell lineage [12], and the B-cell-specific genes: CD19 [13], CD79a [14, 15], Blk [16] and RAG2 [17] were targets of PAX5. As t(8;21) chromosome translocation causes the AML1 gene to fuse with the ETO gene, the Runt domain was retained in the AML1/ETO fusion protein [11]. It could be speculated theoretically that AML1-ETO fusion protein might interact
123
136
with PAX5 in leukemic cells and interfere in the development and differentiation of B-cell and then cause BAL with B-lymphoid and myeloid immune phenotype. By integrating morphologic features, immunophenotypic changes, correlation between AML1/ETO and differentiation of B-cell or pathogenesis of leukemia, and the response to chemotherapy, it is suggested that acute leukemia with t(8;21), coexpressing cCD79a or cCD22, represented an entity of BAL. Acknowledgments This work was supported partly by the Natural Science Fund of Jiangsu Province (2004BK424), the 135 Key Department Fund of Jiangsu Province (135XY0416), and the Outstanding Person Fund of First Affiliated Hospital of Soochow University (2004YQG05).
References 1. Ferrara F, Vecchio LD. Acute Myeloid Leukemia with t(8;21)(q22;q22) /AML1/ETO: A distinct biological and clinical enity. Haematologica. 2002;87:306–19. 2. Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, van’t Veer MB; European Group for the Immunological Characterization of Leukemia (EGIL). Proposals for the immunological classification of acute leukemias. Leukemia 1995;9:1783–6. 3. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia. Ann Intern Med. 1985;103:620–5. 4. Andrieu V, Radford-Weiss I, Troussard X, et al. Molecular detection of t(8,21)/AML1/ETO in AMLM1/M2: correlation with cytogenetics, morphology and immunophenotype. Br J Haematol. 1996;92:855–65. 5. Brunning RD, Matutes E, Borowitz MJ, et al Acute leukemia of ambiguous lineage. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, (eds). Pathology and genetics of tumors of the haematopoietic and lympholoid tissue. Lyon: IARC Press; 2001:106–7.
123
G. He et al. 6. Legrand O, Perrot J-Y, Simonin G, et al. Adult biphenotypic acute leukemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression. Br J Haematol. 1998:100:147–55. 7. Carbonell F, Swansbury J, Min T, et al. Cytogenetic findings in acute biphenotypic leukemia. Leukemia. 1996;10:1283–7. 8. Kozlov I, Beason K, Yu C, Hughson M. CD79a expression in acute myeloid leukemia t(8;21) and the importance of cytogenetics in the diagnosis of leukemias with immunophenotypic ambiguity. Cancer Genet Cytogenet. 2005;163:62–7. 9. Kuchenbauer F, Feuring-Buske M, Buske C. AML1-ETO needs a partner: new insights into the pathogenesis of t(8;21) leukemia. Cell cycle. 2005;4:1716–8. 10. Miyamoto T, Weissman IL, Akashi K. AML/ETO1-expressing non-leukemia stem cells in acute myelogenous leukemia with t(8;21) chromosomal translocation. Proc Natl Acad Sci USA. 2000;97:7521–6. 11. Libermann TA, Pan Z, Akbarali Y, et al. AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter. J Biol Chem. 1999;274:24671–6. 12. Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature. 1999;401:556–62. 13. Kozmik Z, Wang S, Dorfler P, Adams B, Busslinger M. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol Cell Biol. 1992;12:2662–72. 14. Nutt SL, Morrison AM, Dorfler P, Rolink A, Busslinger M. Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. EMBO J. 1998;17:2319–33. 15. Fitzsimmons D, Hodsdon W, Wheat W, Maira SM, Wasylyk B, Hagman J. Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter. Genes Dev. 1996;10:2198–211. 16. Zwollo P, Desiderio S. Specific recognition of the blk promoter by the B-lymphoid transcription factor B-cell-specific activator protein. J Biol Chem. 1994;269:15310–7. 17. Kishi H, Wei XC, Jin ZX, et al. Lineage-specific regulation of the murine RAG-2 promoter: GATA-3 in T cells and Pax-5 in B cells. Blood. 2000;95:3845–52.
