Masked t(15;17) APL with the insertion of PML–RARα fusion gene in 4q21

Leukemia Research 33 (2009) 1552–1555

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Masked t(15;17) APL with the insertion of PML–RAR␣ fusion gene in 4q21 Kouichi Haraguchi a , Nobuhito Ohno a,∗ , Masahito Tokunaga a , Mayumi Tokunaga a , Takahiro Itoyama b , Minako Gotoh c , Masafumi Taniwaki c , Hirohito Tubouchi a a

Department of Digestive and Life-style Related Diseases, Human Environmental Sciences, Health Research Studies, Kagoshima University Graduate School of Medical and Dental Sciences, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan b Department of Hematrogy, Imamura Bun-in Hospital, Kamoike Shinmachi 11-23, Kagoshima 890-0064, Japan c Department of Hematrogy/Oncology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan

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Article history: Received 6 November 2008 Received in revised form 30 March 2009 Accepted 23 April 2009 Available online 27 May 2009 Keywords: Acute promyelocytic leukemia Masked PML/RAR␣ 4q21 All-trans retinoic acid

a b s t r a c t Most cases of acute promyelocytic leukemia (APL) are characterized by the reciprocal translocation t(15;17); however, several complex variant translocations have also been reported. Here we report complex cytogenetic abnormalities without t(15;17) assayed by the G-banding method in a 62-year-old woman with the typical morphology and clinical features of APL. Based on spectral karyotyping and FISH analyses, we confirm the insertion of a cryptic chromosomal segment containing the PML/RAR␣ fusion gene. The patient achieved complete remission after treatment with all-trans retinoic acid (ATRA) alone. Although the mechanism of this cryptic variant insertion is not known, we conclude that the insertion of PML–RAR␣ fusion into 4q21 seems not to alter the effectiveness of treatment with ATRA. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Acute promyelocytic leukemia (APL) is characterized by the reciprocal translocation t(15;17)(q22;21) [1]. This translocation results in the fusion of the PML gene on chromosome 15q22 and the retinoic acid receptor alpha (RAR␣) gene on chromosome 17q21, generating two chimeric fusion genes PML/RAR␣ and RAR␣/PML [2]. The PML/RAR␣ fusion protein is essentially described as an inhibitor of differentiation and may also affect apoptosis but not only to lead to leukemic transformation [3]. In addition to the typical t(15;17)(q22;21), variant translocation and an underlying PML/RAR␣ rearrangement due to insertion as well as other more complex mechanisms have been reported [4]. The most common result of insertion of RAR␣ is the formation of the PML/RAR␣ gene on 15q [4]; the insertion of the PML/RAR␣ gene into other chromosomal locations is rare. Interestingly, there is no significant morphological difference between them. Here we report a masked APL case with the insertion of PML–RAR␣ fusion gene into 4q21, in which treatment with all-trans retinoic acid (ATRA) was successful. 2. Case report A 62-year-old woman was admitted to our hospital in September 2007 because of pancytopenia. Peripheral blood tests showed

∗ Corresponding author. Tel.: +81 99 275 5326; fax: +81 99 264 3504. E-mail address: [email protected] (N. Ohno). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.04.033

hemoglobin 11.1 g/dl, white blood cell count 1.3 × 109 /l (16.1% segmental neutrophils, 80.0% lymphocytes, 0.8% monocytes) and platelet count 100 × 109 /l. The bone marrow aspirate showed hypercellular marrow with 74.4% typical promyelocytic cells, many of which contained azurophilic granules and occasional Auer rods (Fig. 1A), and were positive for peroxidase and N-ASD-CA esterase. Immunosurface and cytoplasmic marker analysis showed that leukemic cells were positive for CD13 (56.9%), CD15 (49.8%), CD33 (91.3%) and CD38 (55.3%), but negative for HLA-DR. Coagulation tests revealed a prothrombin time (PT) of 11.0 s (vs. control PT of 10.6 s), fibrinogen 310 mg/dl (170–410 mg/dl), fibrin degradation products (FDP) less than 5 ␮g/ml, and D-dimer greater than 1.0 ␮g/ml. PML/RAR␣ mRNA was detected by RT-PCR at diagnosis; we treated the patient with all-trans retinoic acid (ATRA) at 60 mg/day. After 34 days, 19% of APL cells matured by ATRA treatment, were seen in a bone marrow aspiration smear (Fig. 1). After achieving complete remission, the patient accepted anthracycline-based consolidation therapies. After 64 days, the fusion signal of PML–RAR␣ was not detected by FISH, and cytogenetic remission was achieved. In November, PML/RAR␣ mRNA was not detected by RT-PCR. Chromosomal analysis was performed on 24 or 48 h cultures of bone marrow cells. The karyotype was described at G-banded metaphase according to ISCN 2005 [5]. The karyotype was interpreted as 46,XX,add(4)(q21),add(5)(p13),add(15)(q22), add(18)(q21)[18]/46,XY[2] (Fig. 2). Spectral karyotyping (SKY) was carried out with a SKyPaint kit (Applied Spectral Imaging, Migdal Ha’Emek, Israel). This examination showed 46,XX, ins(4;15)(q21;?),t(5;18)(p13;q21)[4] (Fig. 3).

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Fig. 1. Bone marrow smear. (A) At the initial presentation, typical APL cells with Auer rods were revealed. (B) Day 34 after starting ATRA treatment. APL cells were matured by ATRA.

Fig. 2. G-banded karyotype of bone marrow cell. The analysis shows no classical translocation t(15;17).

Fig. 3. Spectral karyotyping analysis revealed the insertion on the der(4) and translocation between 5 and 18.

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K. Haraguchi et al. / Leukemia Research 33 (2009) 1552–1555

Fig. 4. Fluorescence in situ hybridization analysis. Probes specific for the PML gene (red) on 15q and the RAR␣ gene (green) on 17q are shown. A fusion signal was detected on der(4)21, indicating an insertion of the PML/RAR␣ fusion gene. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Neither chromosomal analysis nor SKY showed the conventional translocation t(15;17)(q22;q21), therefore we performed fluorescence in situ hybridization (FISH) to detect the PML–RAR␣ fusion genes. FISH analysis was performed using an LSI® PML/RAR␣ dual color, dual fusion translocation probe (Vysis, CA, USA). APL with typical t(15;17) shows one red, one green and two fusion signal pattern with this probe. In the current patient, FISH with this probe showed one red PML on chromosome 15, one green RARA signal on chromosome 17, one small part of red signal on chromosome 15, and one fusion signal on chromosome 4, respectively. Ninety out of 100 metaphase cells were fusion-positive for one of chromosome 4q21. As SKY analysis also showed ins(4;15)(q21;?), whose bands were defined according to counterstained-DAPI banding, we concluded that the PML–RAR␣ fusion signal was located on 4q21 (Fig. 4). To study whether there were any genetic changes in chromosome 4, we performed array comparative genomic hybridization (CGH) analysis as previously reported [6]. There was no gain or loss on chromosome 4 (data not shown).

3. Discussion We report here a case in which reciprocal translocation of t(15;17) was not detected by chromosomal analysis or SKY; however, the PML/RAR␣ fusion gene was detected by FISH. Copy number analysis showed no apparent loss or gain in chromosome 4. These results strongly suggest that the PML and RAR␣ genes were simply inserted into 4q21. Cryptic translocation t(15;17) due to insertion commonly occurs in 15q or 17 [7]. As a result, the PML/RAR␣ fusion gene is generally located on chromosome 15 or 17. Göhring et al. reported a case of an APL patient with a PML/RAR␣ insertion in chromosome 5 [8]. They speculated that the PML gene had first inserted into chromosome 17, with a translocation between chromosome 5 and the derivative chromosome 17 occurring as a second event. In the present case, no genetic change in chromosome 4 was detected by array CGH, and no translocation between chromosome 15 and 17 was detected by SKY. However, insertion of the PML/RAR␣ fusion gene into 4q21 was shown by FISH. t(5;18)(p13;q21) was also observed in all cells analyzed by SKY. Since chromosomal analysis after achieving CR showed a normal karyotype, we suggest that t(5;18)(q13;q21) might occur during leukemogenesis.

Several complex translocations involving chromosome 4 have been previously reported [4], and a breakpoint at 4q21 has been reported in at least three cases excluding ours [7,9,10]. Liu et al. [9] reported a case of t(4;15;17) detected by FISH, and interpreted this complex translocation as follows: 15q22∼qter translocated to 4q21;17q21∼q22 to 15q22; and 4q21∼qter connected to 17q22 located in der(15)(q22). Huret et al. [10] reported a case in which t(15;17) was detected at the initial stage of the illness and t(4;der(15))t(15;17) at the relapse stage. Although the mechanism of the complex translocation seen in the present study is difficult to discern, it appears that the location of the breakpoint might be not random. Various clinical outcomes result from the complex aberrant karyotype with a PML/RAR␣ fusion. Zaccaria et al. reported that a case of APL associated with a PML/RAR␣ fusion gene on chromosome 17 responded poorly to ATRA treatment [11]. However, a case associated with insertion of the PML–RAR␣ gene into chromosome 5 responded well to ATRA and anthracycline-based chemotherapy [8]. Eclache et al. reported a case associated with a PML–RAR␣ fusion on der(1) in which complete remission was achieved with ATRA based chemotherapy [12]. Complete remission with ATRA alone was also achieved in the present report. It seems that the additional aberration had no effect on the successful outcome associated with PML–RAR␣ in these cases involving cryptic translocations. Furthermore, the inserted PML–RAR␣ fusion might be sufficient to give a leukemia. In conclusion, we report here the first APL case with a cryptic translocation in which the PML/RAR␣ fusion gene was inserted into chromosome 4. Since ATRA in this case was as effective as in a typical case of APL, we conclude that this atypical translocation has no therapeutic effect.

Conflict of interest The authors indicated no potential conflicts of interest.

Acknowledgement We thank Dr. T. Ikeda (Ikeda Hospital, Japan) for treating the patient.

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