Uncommon karyotypic abnormality, t(11;19)(q23;p13.3), in a patient with blastic phase of chronic myeloid leukemia

Cancer Genetics and Cytogenetics 150 (2004) 159–163

Short communication

Uncommon karyotypic abnormality, t(11;19)(q23;p13.3), in a patient with blastic phase of chronic myeloid leukemia Keijiro Suzuki*, Takeshi Sugawara, Shugo Kowata, Taiju Utsugizawa, Shigeki Ito, Kazunori Murai, Yoji Ishida Department of Hematology/Oncology, Iwate Medical University, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan Received 9 July 2003; received in revised form 25 August 2003; accepted 4 September 2003

Abstract

We describe unusual cytogenetic findings in a 33-year-old male with blastic phase of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia. In addition to the t(9;22)(q34;q11), which was detected in all metaphases, a t(11;19)(q23;p13.3) was also identified as an evolutional change in all 20 metaphases. Fluorescence in situ hybridization (FISH) analysis showed that fusion signals of the ABL/BCR probes were found in 95% of blastic cells. Southern blotting and FISH analysis also revealed involvement of the MLL gene on 11q23. Clinical course was aggressive and the patient responded poorly to therapy. These findings suggest an association between Ph and 11q23 with poor prognosis, and that t(11;19)(q23;p13.3) was the essential pathogenic factor in our case. 쑖 2004 Elsevier Inc. All rights reserved.

1. Introduction Chronic myeloid leukemia (CML) is a pluripoteintial stem cell disease that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 in more than 90% of patients, which leads to an overtly foreshortened long arm of one of the chromosomal pair numbers, 22 (22q⫺), referred to as the Philadelphia chromosome (Ph) [1]. In most cases of CML, the patient’s disease eventually changes to the more aggressive accelerated and blastic phases (AP and BP, respectively), which are poorly responsive to therapy that had formerly controlled the chronic phase. BP remains the most malignant leukemia. Disease progression is characterized by increasing proliferation, maturation arrest, development of features of accelerated disease, and karyotypic clonal evolution [2]. About 60 percent of patients in BP have karyotypic abnormalities in addition to the Ph chromosome. A double Ph, trisomy 8, and isochromosome 17 are the secondary changes commonly seen [2,3]. These additional karyotypic abnormalities are associated with shorter survival and lower remission rate [2,3]. Additional karyotypic abnormalities are important determinants of the characteristics of BP.

* Corresponding author. Tel.: ⫹81-19-651-5111, ext. 3755; fax: ⫹8119-626-8040. E-mail address: [email protected] (K. Suzuki). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2003.09.005

We present an uncommon karyotypic change in a male patient with the BP of CML with a t(11;19)(q23;p13.3). Leukemic cells were seen to have rearrangement of the MLL gene by fluorescence in situ hybridization (FISH) and Southern blot analysis. According to immunophenotypic analyses, blastic cells expressed myeloid antigens such as CD13, CD33, CD34, HLA-DR, and CD10, but not myeloperoxidase demonstrable by cytochemistry. To our knowledge, this is the first report of these findings in the BP of CML.

2. Case report A 32-year-old male was transferred to our hospital because of marked leukocytosis during treatment of a gastric ulcer in March 2001. Physical examination showed moderate hepatosplenomegaly. White blood cell (WBC) count was 274.7 × 109/L, with a differential count of 3.7% myeloblasts, 13.3% promyelocytes, 24.3% myelocytes, 8.3% metamyelocytes, 38.7% neutrophils, 4.0% eosinophils, 5.0% basophils, 0.7% lymphocytes, and 2.0% monocytes. Platelet count was 310 × 109/L, and hemoglobin concentration was 11.5 g/dL. Bone marrow (BM) aspiration and biopsy showed a markedly hypercellular marrow with a predominance of myeloid cells, including 2.0% myeloblasts, 3.8% promyelocytes, and a decrease of erythropoiesis. The neutrophil alkaline phosphatase score was low. Cytogenetic analysis

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revealed the t(9;22)(q34;q11) in all of 20 metaphases analyzed. FISH analysis showed that the fusion signals of the major BCR gene and ABL gene were present in 60.3% of BM cells (Table 1). On the basis of these results, he was diagnosed as having CML in chronic phase, and treatment was started with recombinant interferon-α (IFN-α, 5 million U/day) and hydroxyurea (HU, 1000 mg/day) because the patient had no HLA-identical siblings for stem-cell transplantation. Six months after treatment with IFN-α and HU, cytogenetic analysis showed the t(9;22)(q34;q11) in 12 of 20 cells analyzed. FISH analysis showed that the fusion signal of the BCR/ABL gene was found in 23% of BM cells (Table 1). The patient reached a partial cytogenetic response with IFN-α and HU, and these treatments were continued. One year after starting IFN-α and HU (May 2002), the patient was diagnosed as having changed to the BP. WBC count was 147 × 109/L with 97.5% blasts, and anemia (Hb 6.2 g/dL) and thrombocytopenia (platelet count 3.8 × 109/ L) appeared. A bone marrow aspiration showed 96% medium- to large-sized, ungranulated blastic cells with amounts of small cytoplasm. Flow cytometric analysis revealed that these blastic cells expressed the myeloid lineage antigens, but cytochemical analysis was negative for myeloperoxidase. Cytogenetic analysis showed the t(9;22)(q34;q11) in all 20 metaphases analyzed. In addition to the t(9;22)(q34;q11), a t(11;19)(q23;q13.3) was identified as a secondary change in all of 20 cells analyzed (Fig. 1; Table 1). After diagnosis of BP, chemotherapy was started with mitoxantrone and enocitabine and subsequently STI571 (Gleevec), a specific tyrosine kinase inhibitor, was administrated. Despite the resulting severe pancytopenia, blastic cells persisted in the BM. Several other treatments were tried. Although these treatments induced a decreasing number of blastic cells, they did not result in remission. Five months after diagnosis of BP, the patient died due to resistant disease.

3. Methods and results 3.1. Cytogenetic analysis BM cells were cultured and prepared according to standard techniques. Chromosomes were prepared according to

Table 1 Karyotypic and molecular genetic data during the course of disease Date of analysis

Status of disease

Genetic data

March 2001

Chronic phase

December 2001

Chronic phase

June 2002

Blastic phase

46,XY,t(9;22)(q34;q11) 20/20 cells FISH (BM): BCR/ABL 60.3% 46,XY 8/20 cells 46,XY,t(9;22)(q34;q11) 12/20 cells FISH (BM): BCR/ABL 23.0% 46,XY,t(9;22)(q34;q11), t(11;19)(q23;p13.3) 20/20 cells FISH (BM): BCR/ABL 95.0% FISH (BM): MLL 97.5%

standard procedures. Slides were G-banded using a conventional trypsin-Gimesa technique. Cytogenetic data were described according to the guidelines of International System for Human Cytogenetic Nomenclature (ISCN 1995) [4]. Cytogenetic data are summarized in Table 1 and illustrated in Fig. 1. 3.2. Immunophenotypic analyses Mononuclear cells from BM were obtained by Histopaque (Sigma Diagnostics, St. Louis, MO) density gradient centrifugation, and were incubated with fluorescein isothiocyanateor phycoerhytrin-conjugated monoclonal antibodies (5 µg/ mL; Immunotech, Marseille, France) for 60 minutes on ice and washed twice with phosphate-buffered saline (PBS). At least 10,000 gated cells from each study were analyzed with a flow cytometer (FACScallibur; Becton Dickinson, San Jose, CA). Immunophenotypic analyses during BP are summarized in Table 2. 3.3. Southern blotting for MLL gene MLL gene rearrangement was investigated by Southern blot analysis. High-molecular–genomic DNA obtained from blastic cells was digested with BamHI (Takara, Tokyo, Japan), electrophoretically separated in agarose gels, transferred to nylon membrane, and hybridized with a 0.9-kb DIG-labeled MLL cDNA probe that was amplified with specific primers from exon 5 to exon 11 (DIG DNA labeling and detection kit; Roche diagnostics, Mannheim, Germany). In contrast to the digestion pattern of control sample with a single 8.3-kb BamHI fragment, one additional rearranged band was detected above 8.3-kb (Fig. 2). 3.4. FISH analysis for BCR/ABL and MLL genes FISH analysis was performed according to standard methods using methanol–acetic acid–fixed BM cells. Applying a SpectrumOrange-labeled probe specific for the ABL gene (9q34) and a SpectrumGreen-labeled probe for the BCR gene (22q11) (Vysis, Stuttgart, Germany), BCR-ABL fusion signals were found in 95% of blastic cells during the BP of CML (Table 1). To further define the breakpoint region on chromosome 11, LSI MLL Dual Color Break Apart Rearrangement Probe (Vysis) was applied. A 350-Kb portion centromeric of the MLL gene breakpoint cluster region (bcr) was labeled in SpectrumGreen, and a 190-Kb portion largely telomeric of the bcr was labeled in SpectrumOrange. When blastic cells were hybridized with these MLL probes, one fusion signal and two split signals were found in 97.5% of these cells (Table 1; Fig. 3).

4. Discussion CML is a clonal disease arising from a pluripotent stem cell and characterized by the presence of a t(9;22)(q34;q11)

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Fig. 1. G-banded karyotyping showing 46,XY,t(9;22)(q34;q11.2),t(11;19)(q23;p13.3) (horizontal arrow).

in all hematopoietic cells. CML can be divided into two or three phases, chronic, accelerated, and/or blastic. The chronic phase of CML is unstable, and transformation to the AP or BP can occur at any time. The BP is treatment refractory and is fatal in weeks to months in all but a very few patients who have undergone successful stem cell transplants from an HLA-identified donor. In most of these patients, transformation is associated with the development of additional chromosomal abnormalities. Several studies have

shown that the frequent karyotypic abnormalities in patients’ cells before or during the AP or BP include a double Ph, isochromosome 17, and trisomy 8 [2,3]. These changes are more common in the myeloid BP and are associated with a

Table 2 Immunophenotypic analyses at diagnosis of blastic phase by flow cytometry Surface antigen

%

CD2 CD3 CD4 CD5 CD7 CD8 CD10 CD19 CD20 CD16 CD56 CD13 CD14 CD33 CD34 CD41a HLA-DR

0.1 2.0 19.8 0.5 1.0 0.0 77.8 0.3 0.3 1.7 4.9 99.3 0.9 99.1 98.3 7.8 92.6

Fig. 2. Southern blotting for the MLL gene. Single rearranged band was detected upon 8.3 Kb with BamHI digestion (horizontal arrow). Left lane, control; right lane, blastic cells from the patient’s BM.

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Fig. 3. FISH analysis at blastic phase, using a probe covering the breakpoint cluster region of the MLL gene. (A) One fusion signal and two split signals for the MLL gene were found in the patient’s blastic cells. Split signals showed the presence of the MLL gene rearrangement. (B) Normal control.

short remission [2,3]. Other studies described that poorer survival was observed with abnormalities of chromosome 17, other superimposed translocations, or a high percentage of abnormal metaphases [5]. In addition, a large number of other chromosome abnormalities have been described. The secondary karyotypic change of t(11;19)(q23;p13.3) during the BP, observed in this case, is uncommon. To date, involvement of 11q23 in CML has been reported in seven cases in which patients having the t(11;19)(q23;p13.3) were not reported [6–12]. All of these cases showed a BP, short disease-free survival, and a poor prognosis except for one case [11]. In our case, HU, but not topoisomerase II inhibitor, was administered for about 1 year during the chronic phase. Therefore, it is likely that the t(11;19)(q23;p13.3) evolved naturally. Leukemias with 11q23 translocations are clinically unique in that they are common in infant leukemias (acute lymphoblastic leukemia [ALL] and acute nonlymphoblastic leukemia [ANLL]) and secondary acute leukemias associated with topoisomerase II inhibitors which are frequently characterized by biphenotypic antigen expression on these blasts [13]. In these leukemias, the majority of 11q23 translocations disrupt the MLL gene, which encodes a 431-kD protein with significant homology to Drosophilia trithorax [13–16]. Almost all the breakpoints within this gene are tightly clustered in the breakpoint cluster region (bcr) [17]. Almost all breaks of the MLL gene within the bcr facilitate the rapid and reliable detection of respective rearrangements with Southern blotting and FISH analysis [17–23]. The involvement of the MLL gene in our case was clearly demonstrated by Southern blotting and FISH analysis. Almost 30 partner genes that participate with the MLL gene in reciprocal chromosomal translocations associated with acute leukemia have been reported [24]. One of the more common reciprocal translocations involving 11q23 in infant leukemias is the t(11;19). Based on the cytogenetic findings alone, two different forms of t(11;19) with slightly different breakpoints on chromosome19 can be distinguished: one that leads to t(11;19)(q23;13.3) and another to t(11;19) (q23;13.1) [24]. The t(11;19)(q23;p13.3) subtype is found

in ALL as well as in ANLL. The t(11;19)(q23;p13.3) subtypes with myeloid lineage involvement were frequently observed in infant/congenital cases whose characteristics were short survival due to short duration of complete remission. According to the review by Huret et al. [25], the case with t(11;19)(q23;p13.3) as secondary karyotypic change was only one case that was described as therapyresistant acute undifferentiated leukemia after relapsing AML (M5a). The significance of this secondary karyotypic change, both clinically and cytogenetically, should be further investigated because there are few reports of this translocation. In our case, the presence of t(11;19)(q23;p13.3) was detected only in BP, when undifferentiated myeloid blast cells were predominant, suggesting that this translocation might characterize these blast cells and their clinical features. Recent cytogenetic and molecular data have revealed that t(11;19)(q23;p13.3) can involve the ENL gene on chromosome 19 and produce the ENL-MLL fusion protein [14,26]. Structure and function studies have revealed that ENL-MLL contributes to transformation and immortalization of myeloid progenitors in vitro [24,26]. The formation of this fusion gene seems to have a prime role in the development of leukemia. According to these molecular studies, it has been postulated that ENL-MLL fusion gene might have participated in the progression to BP in our case. Unfortunately further molecular studies to clarify the breakpoint in MLL and its partner gene could not be performed because there were no more materials. The t(11;19)(q23;p13.3) is likely to be a probable pathogenic factor in our case. References [1] Lichtman MA, Liesveld JL. Chronic myelogenous leukemia and related disorders. In: Beutler E, Marshall AL, Coller B, Kipps TJ, Seligsohn U, editors. Williams hematology 6th edition. New York: McGraw-Hill, 2001, pp. 1085–123. [2] Kantarjian HM, Keating MJ, Talpatz M, Walters RS, Smith TL, Cork A, McCredie KB, Freireich EJ. Chronic myelogenous leukemia in blast crisis. Analysis of 242 patients. Am J Med 1987;83:445–54. [3] Griesshammer M, Heinze B, Bangerter M, Heimpel H, Fliedner TM. Karyotype abnormalities and their clinical significance in blast crisis at chronic myeloid leukemia. J Mol Med 1997;75:836–8.

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