Acute myeloid leukemia (AML-M2) associated with variant t(8;21): report of three cases

Cancer Genetics and Cytogenetics 199 (2010) 31e37

Acute myeloid leukemia (AML-M2) associated with variant t(8;21): report of three cases Sook Young Baea, Jang Su Kima, Bung Jun Ryeua, Kap No Leea, Chang Kyu Leea, Young Kee Kima, Chae Seung Lima, Yunjung Choa, Chul Won Choib, Sook-Won Ryuc, Soo-Young Yoona,* a

Department of Laboratory Medicine, Korea University College of Medicine, Anam-Dong 5-Ga, Seongbuk-Gu, Seoul 136-705, Republic of Korea b Department of Internal Medicine, Korea University Medical Center, Korea University College of Medicine, 80, Guro Dong, Guro Gu, Seoul, 152-703, Republic of Korea c Department of Laboratory Medicine, Kangwon National University Hospital, Kangwon National University College of Medicine, Hyoja 3 Dong, Chuncheon City, Kangwondo, 200-722 Republic of Korea Received 16 July 2009; received in revised form 23 September 2009; accepted 8 October 2009

Abstract

Variants of the t(8;21)(q22;q22) involving chromosome 8, 21, and other chromosomes account for approximately 3% of all t(8;21)(q22;q22) found in patients with acute myeloid leukemia (AML). The clinicopathologic features of AML with the variant t(8;21) have not been well established. We report three cases of AML with variants of t(8;21) characterized, respectively, by derivative 8 with the interstitial inverted insertion of 21q and concurrent monosomy 21, t(8;18;21)(p22;q11.3;q22), and t(2;21;8)(q11.2;q22;q22). Fluorescence in situ hybridization or reverse transcriptaseepolymerase chain reaction assay confirmed the presence of RUNX1eRUNX1T1 gene (previously AML1eETO) rearrangements. Among these cases, three-way breakpoints 18p11.3 and 2q11.2 have not been previously reported. The present report deals with the results of hematologic, immunophenotypic, cytogenetic, fluorescence in situ hybridization, and molecular analyses of these variants. The possible role of the genes in this region in leukemogenesis, response to treatment, and clinical implications are discussed. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Multiple recurrent chromosomal aberrations in acute myeloid leukemia (AML) have been identified by conventional cytogenetic analysis, and these findings have been used as the most important diagnostic and prognostic markers in patients with AML [1]. The t(8;21)(q22;q22) is considered a distinct AML subtype associated with a favorable prognosis and is found in approximately 5% of cases of AML and in 10% of the prior AML with maturation (M2) category of the FrencheAmericaneBritish classification [2]. However, approximately 3e4% of AML associated with t(8;21) have variant translocations [3], and the clinicopathologic features of AML carrying variant t(8;21) are less well characterized [4]. Molecularly, the RUNX1 (AML1) gene located at 21q22 fuses to the RUNX1T1 (ETO) gene located at 8q22, generating a chimeric RUNX1eRUNX1T1 fusion gene on the der(8). Translocation (8;21) usually correlates with specific * Corresponding author. Tel.: 82-31-412-5300; fax: 82-31-412-5314. E-mail address: [email protected] (S.-Y. Yoon). 0165-4608/$ e see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.10.002

morphological features, which include large blast cells with Auer rods, often containing numerous azurophilic granules and very large pseudoeChe´diak-Higashi granules as well as homogeneous pink-colored cytoplasm in neutrophils [2,5]. The leukemic cells generally respond to chemotherapy, even after relapse. Thus, detection of the RUNX1eRUNX1T1 fusion gene allows patients to be assigned to the appropriate risk group for treatment. The major investigators have reported that patients who have AML with variant t(8;21) have a worse prognosis [6e9]. In this study, we report the clinicopathologic features of three cases, including two novel cases, of AML associated with variants of t(8;21).

2. Materials and methods 2.1. Case selection The files of the Clinical Cytogenetics Laboratory, Department of Laboratory Medicine, Korea University

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Guro Hospital, were searched for cases of t(8;21)(q22;q22) between October 2003 and February 2009. Cases of AML with variant t(8;21)(q22;q22) were selected for this study. Peripheral blood smears, bone marrow aspirate, and core biopsy specimens were reviewed.

2.4. Molecular analysis 2.4.1. Reverse transcriptaseepolymerase chain reaction (RTePCR) for detection of RUNX1eRUNX1T1 transcript Total RNA was isolated with the QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany) from bone marrow mononuclear cells. The RNA was reverse transcribed to cDNA by means of random hexamer oligonucleotides and reverse transcriptase (Fermentas, Ontario, Canada). RTePCR analyses were performed according to the standardized RTePCR analysis of fusion gene transcripts for the detection of RUNX1eRUNX1T1 fusion gene expression as described previously [13].

2.2. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) Cytogenetic analysis was performed on unstimulated short-term cultured bone marrow cells, and preparation of metaphase cells was performed as previously described [10], followed by karyotyping according to ISCN 2009 guidelines [11]. FISH studies were performed with the dual-color, dual-fusion AML1/ETO probe (Abbott Molecular, Des Plaines, IL) specific for RUNX1 labeled with SpectrumGreen and for RUNX1T1 labeled with SpectrumOrange, respectively. At least 400 interphase nuclei and 10 metaphase cells were evaluated in each study [12]. Poseidon Whole Chromosome Paint (WCP) probes for chromosomes 8 (Kreatech, Amsterdam, The Netherlands) were applied on metaphase spreads according to the manufacturer’s specifications.

2.4.2. Sequence analysis To confirm that the amplified fragment was indeed derived from the RUNX1eRUNX1T1 fusion transcript, the PCR product was directly sequenced in patient 3 as described previously [14].

3. Results 3.1. Clinical findings We identified 3 cases of variant t(8;21)(q22;q22) from a group of 12 cases of AML with t(8;21), for a frequency of 25%. All patients were men who ranged in age from 19 to 51 years and who came to our institution at the time of initial diagnosis. Two patients received treatment with combination chemotherapy consisting of idarubicin and cytarabine initially, and one patient (patient 2) left to be treated at another institution. In patient 1, complete remission lasted for 15 months after induction chemotherapy with mitoxantrone, high-dose cytarabine, and allogeneic bone marrow transplantation. The disease subsequently reappeared, and further treatment with high-dose cytarabine,

2.3. Flow cytometric immunophenotyping We performed three-color flow cytometry immunophenotypic analysis on bone marrow aspirate specimens with a Coulter Epix XL flow cytometer (Beckman Coulter, Miami, FL). Blasts were gated for analysis with CD45 expression and side scatter. All cases were assessed for CD2, CD3, CD5, CD7, CD10, CD13, CD14, CD19, CD20, CD22, CD33, CD34, CD41, CD64, CD117, human leukocyte antigen (HLA) DR, myeloperoxidase, and terminal deoxynucleotidyl transferase. We also assessed two patients for CD56.

Table 1 Clinical and hematologic features of three AML patients with variant t(8;21) Variable

Patient 1

Patient 2

Patient 3

Sex/age (y) WBC (/mL) Hemoglobin (g/dL) Platelets (/mL) Blasts (%) Blast morphologic features

Male/46 10,500 7.4 12,000 74.0 Basophilic cytoplasm; salmon-pink granules; Auer rods CD13þ, CD19, CD33þ, CD34þ, CD117þ, MPOþ Not done Second allogeneic BMT 30 Complete CR

Male/19 27,900 10.1 41,000 34.8 Basophilic cytoplasm; salmon-pink granules; Auer rods CD13þ, CD19, CD33þ, CD34þ, CD56þ, CD117þ, MPOþ Not done Not done Not available Not available

Male/51 1,380 6.6 42,000 38.4 Basophilic cytoplasm; salmon-pink granules; large granules; Auer rods CD13þ, CD19þ, CD33þ, CD34þ, CD56þ, CD117þ, MPOþ Positive Second consolidation CTx 5 Complete CR

Immunophenotypea RTePCR Therapy Follow-up (mo) Status

Abbreviations: AML, acute myeloid leukemia; BMT, bone marrow transplantation; CR, clinical remission; CTx, chemotherapy; MPO, myeloperoxidase; RTePCR, reverse transcriptaseepolymerase chain reaction assay; WBC, white blood cell count. a CD56 was not assessed in patient 1.

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Fig. 1. Acute myeloid leukemia with t(2;21;8) in patient 3. Bone marrow aspirates show blasts with long, slender Auer rods (left) and dysplastic granulocytes containing large granules resembling Che´diak-Higashi granules (right) (WrighteGiemsa stain, original magnification 1,000).

etoposide, mitoxantrone, cyclosporine, and another allogeneic bone marrow transplantation was provided. The patient was in a second complete remission for 10 months at the time of writing this report. Clinical remission in patient 3 was achieved and maintained up to 5 months until the present. The clinical and hematologic features of the three patients are summarized in Table 1.

cytometry demonstrated that the blasts in all cases expressed CD13, CD33, CD34, CD117, HLA-DR, and myeloperoxidase and were negative for CD2, CD3, CD5, CD7, CD10, CD14, CD20, CD22, CD41, CD64, and terminal deoxynucleotidyl transferase. CD19 was expressed in patient 3. CD56, assessed in patients 2 and 3, was positive in both.

3.2. Morphologic and immunophenotypic findings

3.3. Cytogenetic and molecular diagnostic findings

The morphologic and immunophenotypic findings are summarized in Table 1. The bone marrow biopsy specimens showed a cellularity that ranged from 50% to 95% with markedly increased immature myeloid elements. In all cases, the blasts in bone marrow aspirate smears showed long, slender Auer rods, and myeloid elements had basophilic cytoplasm and salmon-pink granules. Large granules resembling Che´diak-Higashi granules were identified in patient 3 (Fig. 1). Immunophenotypic analysis by flow

The results of conventional cytogenetic, FISH, and RTePCR studies are summarized in Table 1, Table 2, and Figure 2. In patient 1, cytogenetic studies of 20 metaphases revealed 18 cells showing an unbalanced rearrangement between chromosomes 8 and 21 leading to partial loss of chromosome 21. FISH analysis performed on metaphase and interphase cells revealed one RUNX1T1 signal on the normal chromosome 8, one RUNX1 signal on the normal chromosome 21, and a single

Table 2 Summary of sequential bone marrow cytogenetics and FISH results using the AML1/ETO probe of three AML patients Patient

Date

Karyotype

FISH

1

1/26/2007a

45,XY,der(8)ins(8;21)(q22;q22q21),21[18]/46,XY[2]

2/21/2007 4/6/2008b

46,XY[20] 45,XY,der(8)t(8;21)(q22;q22),21[20]

2

7/1/2008 9/11/2008a

3

2/25/2009a

//46,XX[20] 46,XY,del(7)(q32q34),t(8;18;21)(q22;p11.3;q22)[16]/ 46,XY[4] 45,X,Y,t(2;21;8)(q11.2;q22;q22),t(5;9)(q15;q22)c[7]/ 46,XY,t(5;9)(q15;q22)c[13] 46,XY,t(5;9)(q15;q22)c[20]

nuc ish(RUNX1T1,RUNX1)2(RUNX1T1 RUNX11)[328/400] nuc ish(RUNX1T1,RUNX1)2[400] nuc ish(RUNX1T1,RUNX1)2(RUNX1T1 RUNX11)[270/400] nuc ish(RUNX1T1,RUNX1)2[400] nuc ish(RUNX1T1,RUNX1)3(RUNX1T1 RUNX11)[493/500] nuc ish(RUNX1T1,RUNX1)3(RUNX1T1 RUNX11)[383/500] nuc ish(RUNX1T1,RUNX1)3(RUNX1T1 RUNX11)[281/400]

3/19/2009

Abbreviation: FISH, fluorescence in situ hybridization; AML, acute myeloid leukemia. a Date of diagnosis. b Date of relapse.

con

con

con con con

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Fig. 2. G-banded karyogram and fluorescence in situ hybridization analysis results using the AML1/ETO dual-color, dual-fusion probe on bone marrow cells of the patients with variant t(8;21). Structurally abnormal chromosomes are denoted with arrows in karyotypes. (A1) Karyotype of patient 1 showing der(8)ins(8;21) and monosomy 21. (A2) One fusion signal is present on the der(8). One orange signal is present on normal chromosome 8, and one green signal is present on normal chromosome 21. (B1) Karyotype of patient 2 showing a del(7)(q32q34) and a t(8;18;21)(q22;p11.3;q22). (B2) One fusion signal is located on the der(8), one small orange signal is located on the der(18), and one small green signal is located on der(21). (C1) Karyotype of patient 3 showing a t(2;21;8)(q11.2;q22;q22), loss of the Y, and a constitutional t(5;9). (C2) One fusion signal is located on the der(8), one small orange signal is located on the der(2), and one small green signal is located on the der(21).

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RUNX1eRUNX1T1 fusion on the der(8). Thus, the karyotype was as follows: 45,XY,der(8)ins(8;21)(q22;q22q21), 21[18]/46,XY[2] (Fig. 2A). In patient 2, cytogenetic studies performed at the time of admission to our institution demonstrated a three-way translocation involving chromosomes 8, 18, and 21 in 16 of 20 metaphases examined. All 16 metaphases also showed a del(7)(q32q34). The karyotype was as follows: 46,XY,del(7)(q32q34),t(8;18;21)(q22;p11.3;q22)[16]/ 46,XY[4]. FISH analysis performed on metaphase chromosomes demonstrated RUNX1T1 signals on the abnormal chromosome 18 and on the normal chromosome 8, RUNX1 signals on the normal and abnormal chromosomes 21, and a single RUNX1eRUNX1T1 fusion on the abnormal chromosome 8 in all three metaphases examineddthat is, FISH on metaphase chromosomes 46,XY,t(8;18;21)(q22;p11.3; q22)[3].ish t(8;18;21)(RUNX1T1þ,RUNX1þ;RUNX1T1þ; RUNX1þ) (Fig. 2B). In patient 3, cytogenetic studies demonstrated a threeway translocation involving chromosomes 2, 8, and 21 in 7 out of 20 metaphases examined. In addition, loss of Y chromosome was also found in the same clone. All of the 20 metaphases also showed a t(5;9)(q15;q22), most likely a constitutional abnormality unrelated to the patient’s leukemia, because the same abnormality was demonstrated in T lymphocyte culture of peripheral blood. Thus, the karyotype was as follows: 45,X,eY,t(2;21;8)(q13;q22; q22),t(5;9)(q15;q22)c[7]/46,XY,t(5;9)(q15;q22)c[13]. FISH performed on metaphase chromosomes demonstrated RUNX1T1 signals on the abnormal chromosome 2 and the normal chromosome 8, RUNX1 signals on the normal and abnormal chromosomes 21, and a single RUNX1eRUNX1T1 fusion on the abnormal chromosome 8dthat is, FISH on metaphase chromosomes 45,X,eY,t(2;21;8)(q13; q22;q22).ish t(2;21;8)(RUNX1T1þ;RUNX1þ;RUNX1T1þ, RUNX1þ) (Fig. 2C). In all cases, we performed WCP for chromosome 8 and found that the insertion of 21q material into chromosome 8q and segment from 8q was translocated to der(18p) and der(2q), respectively (Fig. 3).

35

RTePCR for the detection of RUNX1eRUNX1T1 fusion gene in patient 3 was performed and demonstrated the expected size bands of 395, 338, and 260 bp with RUNX1-A/RUNX1T1-B, RUNX1-E/RUNX1T1-D, and RUNX1-C/RUNX1T1-D primers, respectively. The DNA sequence consisted of the junction of RUNX1 exon 5 to RUNX1T1 exon 2, identical to that of the RUNX1e RUNX1T1 fusion transcript described previously in a t(8;21)-positive patient [14] (Fig. 4).

4. Discussion AML with t(8;21)(q22;q22) is a distinct AML subtype with characteristic morphology and clinical manifestations. In the clinical course, approximately 87% of AML patients with t(8;21) experience remission; however, relapse is frequent, and these patients have worse prognoses, with an overall survival of 50% at 5 years [15]. Leukemic blasts are frequently positive for CD19 and CD56 surface markers. CD19 positivity is not considered to influence patient prognosis, but cases of CD56-positive AML with t(8;21)(q22;q22) tend to have a shorter remission duration and survival time compared to CD56-negative cases [8,16,17]. Variants of t(8;21) are uncommon, and the clinical implication is not well established. Here we report three cases of AML with variants of the t(8;21), one with an derivative 8 with the interstitial inverted insertion of 21q and concurrent monosomy 21, and two with three-way translocations. FISH and RTePCR analyses confirmed the presence of the RUNX1eRUNX1T1 fusion gene or its transcripts. Direct sequencing revealed the same RUNX1eRUNX1T1 fusion transcript in the single case assessed. Morphologically, all cases variably displayed the morphologic features of AML with the classic t(8;21)(q22;q22), and were classified as AML-M2 by French-American-British criteria. Leukemic blasts in the present cases expressed CD19 in one case (1/3) and CD56 in all tested cases (2/2). One patient was lost to

Fig. 3. In patient 1 (A), whole chromosome paint for chromosome 8 (WCP8; green) shows inserted material into 8q; in patient 2 (B) and patient 3 (C), the translocation with three segments is distributed over three chromosomes.

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RUNX1T1 genes and/or RTePCR for the detection of RUNX1eRUNX1T1 fusion transcripts.

References Fig. 4. In patient 3, sequence analysis confirmed RUNX1eRUNX1T1 fusion involving junction of RUNX1 exon 5 to RUNX1T1 exon 2, identical to that of the standard RUNX1eRUNX1T1 fusion transcript.

follow-up after the initial diagnosis and two patients are in complete remission. The total follow up period for the two patients was 5 to 30 months after initial diagnosis. At the molecular level, the two genes involved in t(8;21) are RUNX1 (also referred to CBFA2 or PEBP2aB) at 21q22 and RUNX1T1 (also known as CDR or MTG8) at 8q22. RUNX1 gene fuses to the RUNX1T1 gene in generating a chimeric RUNX1eRUNX1T1 fusion gene on the der(8) [18]. RUNX1 and RUNX1T1 are both involved in transcriptional regulation of genes in the hematopoietic precursor cells. The hybrid transcript is invariably composed by splicing of RUNX1 exon 5 to RUNX1T1 exon 2 [13,19]. Our first case highlights the role of the rearranged chromosome 8, which encodes the RUNX1eRUNX1T1 fusion product in the pathogenesis of AML-M2. The insertion characterized as ins(8;21), leading to the RUNX1e RUNX1T1 fusion is believed to be a rare event, and descriptions of concurrent monosomy 21 have not been published [20]. The other two patients represent the first reported cases of novel three-way breakpoints 18p11.3 and 2q11.2, respectively. In the t(8;21) group, loss of sex chromosome and del(9q) occurs frequently; these findings have been regarded as prognostic marker for shorter overall survival or intermediate prognosis [15,21]. Our patients showed additional cytogenetic abnormalities such as monosomy 21 (patient 1), loss of sex chromosome (patient 3), and partial deletion of 7q (patient 2). The overall frequency of variant t(8;21) in our study was 25% (3 of 12), which is higher than previous studies from Europe and United States [3,4,22,23]. In previous reports of AML, the frequency of variants was estimated to be 3.4% and 4% [3,24]. The recent report from Huang et al. showed a similar frequency (6%; 4 of 64) [4], and Gallego et al. reported that the frequency of variants was 9.5% (7 of 74) in childhood AML [22]. A high frequency has been reported in Japan (28%; 7 of 25) [23]. These findings suggest that the frequency of variant t(8;21) varies among different populations. In summary, the clinical significance of the variant t(8;21) is not certain because of the small number of cases. Poor response to initial remission induction therapy in some patients may suggest a poor prognosis. Further accumulation of similar cases is needed to determine the clinical significance. In addition, all such cases should have FISH analysis to assess for colocalization of RUNX1T1 and

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