ABL mRNA transcripts in chronic myelogenous leukemia and related disorders

Leukemia Research 23 (1999) 477 – 481

ALL- and CML-type BCR/ABL mRNA transcripts in chronic myelogenous leukemia and related disorders Akihiko Yokohama *, Masamitsu Karasawa, Kiyoshi Okamoto, Hirotaka Sakai, Takuji Naruse Third Department of Internal Medicine, Gunma Uni6ersity School of Medicine, Maebashi, Gunma 371 -8511, Japan Received 10 September 1998; accepted 26 September 1998

Abstract Using the reverse transcription polymerase chain reaction, we investigated acute lymphoid leukemia (ALL)-type, and chronic myelogenous leukemia (CML)-type BCR/ABL mRNA expression in a total of 66 patients with chronic myeloproliferative disorder (CMPD). Thirty-six of 37 patients with CML were positive for CML-type mRNA. Thirteen of the 25 CML had ALL-type mRNA expression. The patients with ET, PV, MF, and CMML did not have any detectable BCR/ABL expression. The most remarkable finding was that two patients, a Ph1-positive CML patient and a patient with a presumptive diagnosis of essential thrombocythemia (ET), showed only ALL-type chimeric mRNA expression. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: BCR; ABL; CML; ET; CMML

1. Introduction The Philadelphia chromosome (Ph1), t(9;22)(q34; q11), is found in more than 90% of patients with chronic myelogenous leukemia (CML). As the result of the reciprocal translocation, a head-to-tail joining of BCR and ABL sequences is formed on chromosome 22. In the majority of CML patients, the breakpoint on chromosome 22 falls in a 5.8 kb region called the major breakpoint cluster region (M-BCR), and the fusion of exon 2 (b2) or exon 3 (b3) of M-BCR to the second exon of ABL (a2) is transcribed from this derivative sequence [1,2]. Approx. 20% of adult acute lymphocytic leukemia (ALL) patients have the Ph1. Approx. one-half of these Ph1-positive ALL patients have a breakpoint within the Abbre6iations: CML, chronic myelogenous leukemia; ALL, acute lymphoid leukemia; PV, polycythemia vera; ET, essential thrombocythemia; MF, myelofibrosis; CMPD, chronic myeloproliferative disorders; CMML, chronic myelomonocytic leukemia; Ph1, Philadelphia chromosome; M-BCR, major breakpoint cluster region; m-BCR, minor breakpoint cluster region; FISH, fluorescence in situ hybridization; NAP, neutrophil alkaline phosphatase. * Corresponding author. Tel.: +81-27-2208166; fax: +81-272208173.

M-BCR. The remaining half have a breakpoint within the first intron of the BCR gene, called the minor breakpoint cluster region (m-BCR). In ALL with mBCR, the first exon of BCR (e1) is fused to a2 [3,4]. The b2/a2 or b3/a2 hybrid gene generates a p210BCRABL protein, while e1/a2 generates a p190BCR-ABL protein. Each has been considered to play an important role in leukemogenesis. Rare cases of CML with only the ALL-type fusion gene, lacking the CML-type, have been reported [5–12]. Moreover, Melo et al. have suggested that CML patients with only ALL-type transcripts have atypical clinical features such as monocytosis [10,13]. These findings prompted us to investigate the presence of ALL-type mRNA in patients with CML, chronic myeloproliferative disorder (CMPD), and CMML.

2. Material and method We investigated CML-type BCR/ABL transcripts (b2/a2, b3/a2) in a total of 66 patients with CML, CMPD, or CMML diagnosed and treated at our institute from 1990 to 1996. We studied 37 cases of CML, 17 of ET, two of PV, three of MF, and seven of

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CMML cytogenetically. Ph1 was positive in 35 of 37 CML patients (the other two were confirmed to have CML-type BCR/ABL mRNA as described below), whereas none of the CMPD or CMML patients were Ph1 positive. Bone marrow biopsies were performed in all three MF patients to confirm the diagnosis. Simultaneous expression of ALL-type BCR/ABL mRNA (e1/ a2) was evaluated in 25 of the 37 patients with CML, and in all the patients with CMPD or CMML.

2.1. Cell preparation, RNA isolation, and first strand cDNA synthesis Mononuclear cells were prepared using lymphocyte separation medium (Organon Teknika, Durham, NC). All nucleated cells were obtained using 6% Dextran/ 0.9% NaCl, or a hypotonic cell lysis method. Total RNA was extracted by the modified acid guanidinium thiocyanate–phenol – chloroform method [14]. First strand cDNA synthesis was performed with 0.5 – 5 mg of total RNA. The reaction was carried out for 1 h at 37 in a 20 ml reaction mixture containing 400 units murine leukemia virus reverse transcriptase (GIBCO BRL, Gaithersburg, MD), 0.5 mM deoxyribonucleotide triphosphates (dNTPs), 50 mM Tris – HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 100 ng of random hexamer (TAKARA, Kyoto, Japan), 0.4 units/ml of RNase inhibitor (TAKARA), and 10 mM DTT.

2.2. Polymerase chain reaction (PCR) The primer sequences for the e1/a2 fusion product were prepared according to van Rhee et al. [15]. Nested PCR was performed for the amplification of the ALL chimeric mRNA. The first PCR was carried out in a 40 ml reaction mixture containing 8 ml cDNA, 0.5 mM dNTPs, 10 mM Tris – HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl2, 1.0 units of Taq polymerase (TOYOBO, Osaka, Japan), and 20 pmol primer pairs (ALL-F and ALL-R). The PCR was performed for 30 cycles using a GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, CT) (denaturation at 96 for 1 min, annealing at 68 for 30 s, and extension at 72 for 2 min). Two microlitres of the first PCR product were transferred into the second PCR mixture, which had the same contents as the first PCR reaction mixtures except for the primers (ALL-Fn and ALL-R). The second PCR was performed (denaturation at 96 for 1 min, annealing at 64 for 30 s, extension at 72 for 2 min) for 30 cycles. Five microlitres of each second PCR product were electrophoresed on a 2% agalose gel, stained with ethidium bromide, and photographed. We used K562 cells as a positive control, and sterile water as a negative control in every experiment. The primer sets for detecting standard CML-type BCR/ABL mRNA were produced according to

Thompson et al. [16], and nested PCR was performed as previously described [12]. All primers sequences are shown in the Table 1.

2.3. Fluorescence in situ hybridization (FISH) The two probes used in this study were obtained from Vysis (Downers Grove, IL.). The ABL probe, which is directly labeled with SpectrumGreen fluorophore, begins between exons 4 and 5, and continues for approx. 200 kb toward the telomere of chromosome 9. The BCR probe, which is directly labeled with SpectrumOrange fluorophore, begins between BCR exons 13 and 14, and extends centromeric on chromosome 22 approx. 300 kb, crossing well beyond the minor BCR region. Interphase nuclei of whole white blood cells were hybridized with these two probes at a commercial laboratory (BML, Saitama, Japan). Cells with ALL-type or CML-type chromosomal translocations result in fused yellow signals or adjacent red and green spots Table 2.

3. Results A presumptive ET case (case 1) and a CML case (case 2) were positive for ALL-type mRNA without CML-type expression. The ET case was a 67-year-old woman, who had a history of myocardial infarction and hypertension, who presented with thrombocytosis and a mild leukocytosis for 4 years. She had no palpable organomegaly at the time of admission. The hemoglobin was 11.9 g/dl, the platelet count 567× 109/l, the leukocyte count 12.3× 109/l (neutrophils 74%, eosionphils 4%, basophils 1%, monocytes 2.5%, lymphocytes 18.5%). Due to a normal karyotype (46 XX) on cytogenetic analysis, a high neutrophil alkaline phosphatase (NAP) score (326), and negativity for CML type BCR/ABL transcripts, she was diagnosed with ET. A hypercellular bone marrow with megakaryocytic hyperplasia and slight fibrosis was observed. On FISH, only 16 cells of 100 counted cells showed a positive BCR/ABL fusion gene (Fig. 1). RT-PCR for ALL-type transcript revealed that she had an ALL-type BCR/ABL fusion gene (Fig. 2b). The Table 1 Sequence of the primers CML-F CML-R CML-Fn CML-Rn ALL-F ALL-R ALL-Fn

5%-AGTTACACGTTCCTGATCTC-3% 5%-TTATCTCCACTGGCCACAAA-3% 5%-TCTGACTATGAGCGTGCAGA-3% 5%-AGTGCAACGAAAAGGTTGGG-3% 5%-CCCCCGGAGTTTTGAGGATTGC-3% 5%-GGCCGCTGAAGGGCTTCTGCG-3% 5%-GAACTCGCAACAGTCCTTCGAC-3%

A. Yokohama et al. / Leukemia Research 23 (1999) 477–481

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Table 2 The expression of CMLa-type and ALLb-type bcr/abl transcripts Diagnosis

Total no.

Evaluated no.c

CML-type

ALL-type Both ALL- and CML-type

ALL-type only

CML ET PV MF CMML

37 17 2 3 7

25 17 2 3 7

24 0 0 0 0

13 0 0 0 0

1 1 0 0 0

Total

66

44

24

13

2

a

CML, chronic myelogenous leukemia. ALL, acute lymphoid leukemia. c Evaluated no A number of the patients who were investigated for the simultaneous expression of ALL-type BCR/ABL. b

RT-PCR analysis was repeated three times, and the presence of the ALL-type transcript was confirmed on every occasion. Based on the molecular findings, we diagnosed her with CML. The patient with CML was a 28-year-old man admitted to our hospital because of leukocytosis. At the time of admission, his spleen was palpable. The hemoglobin was 14.4 g/dl, the platelet count 876× 109/l, and the leukocyte count 25.3×109/l with 5% myelocytes, 4% metamyelocytes, 66% neutrophils, 1% eosinophils, 12% basophils, 1% monocytes, and 11% lymphocytes. His bone marrow was typical of chronic-phase CML, hypercellular with granulocytic hyperplasia. His NAP score was 9 (markedly decreased). All 20 metaphases were Ph1 positive. He had two BCR/ABL fusion transcripts, a fusion of a segment of e2 to a2 with a 21 bp ABL intron 1b insertion between e2 and a2, and a standard ALLtype transcript (e1/a2). The molecular characteristics of his BCR/ABL gene have been previously described [12]. The other 36 patients with CML were positive for b2/a2 or b3/a2 chimeric transcripts. Of these, 13 of the 24 CML patients (54%) investigated for the presence of e1/a2 transcripts also had ALL-type BCR/ ABL mRNA expression (Fig. 1b). Patients in blast crisis or accelerated phase had a higher frequency (7/ 8 cases) of ALL-type expression, compared to those in chronic phase (7/18 cases). Usually, this coexistent ALL-type expression was so weak that we could not recognize it until nested PCR was performed. The hemoglobin concentration, leukocyte count and platelet count, and NAP score were not significantly different between ALL-type-positive and -negative cases. No CML- or ALL- type BCR/ABL transcripts were detected in the CMML and CMPD patients, except for the patient with presumed ET described above.

4. Discussion We investigated the presence of ALL-type BCR/ ABL mRNA expression in patients with CML, CMPD, and CMML. Of these, two CML cases, one diagnosed clinically as ET, were found to have only ALL-type mRNA transcripts. The report by Melo et al. describes that CML patients with only ALL-type transcripts may have a prominent absolute and relative monocytosis [10,13]. Our two cases, however, did not show such a feature, similar to a CML patient reported by Zaccaria et al. [7]. However, two newly diagnosed Ph1 positive, CML-type BCR/ABL negative, ALL-type BCR/ABL-positive CML patients with

Fig. 1. Fluorescence in situ hybridization (FISH) analysis of case 1. A representative interphase nucleus, negative for BCR/ABL fusion, is shown. In this FISH analysis, cells positive for the fused BCR/ABL gene were observed in only 16 of the 100 cells counted (normal control cells had positive signals in four of 100).

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Fig. 2. Ethidium bromide staining of amplified BCR/ABL fusion transcripts after nested PCR. The lanes are as follows: M, molecular marker (fX 174 Hae III digestion); lanes 1–3, CML patients with simultaneous expression of CML/ALL-type mRNA; lane 4, case 1; lane 5, case 2; and lane 6, a negative control (sterile water). (a) PCR-amplified CML-type transcripts. b3a2 CML-type transcripts (362 bp) are seen in lanes 1 and 2. A b2a2 CML-type transcript(287 bp) is seen in lane 3. No amplified band was detected in lanes 4, 5 or 6. (b) PCR-amplified ALL-type transcripts. ALL-type transcripts (179 bp) are seen in every lane except lane 6. Stronger expression is seen in lanes 4 and 5, compared with lanes 1 to 3. As previously reported [12], case 2 (lane 4) had a novel transcript, the fusion of a segment of e2 to a2 with a 21 bp ABL intron 1b insertion between e2 and a2, in addition to beside a e1/a2 ALL-type transcript. However, only standard ALL-type transcripts were detected in this study because the e1/a2 junctional primer (nested e1 reverse) was used. PCR, polymerase chain reaction; CML, chronic myeloid leukemia; ALL, acute lymphocytic leukemia.

marked monocytosis were presented at the 59th Annual Meeting of the Japanese Society of Hematology [17,18]. No CMML patients in this study had ALL-type BCR/ ABL expression, confirming a previous report [19]. There have been some reports of Ph1-positive ET or BCR/ABL transcripts positive ET [20 – 23]. However, we revised the diagnosis of our patient from ET to CML because the platelet count was lower than that of typical ET, and no Ph1 positive ET case with ALL-type BCR/ABL has been reported. Although the precise mechanism for juxtaposing BCR and ABL was unclear in this patient, one possible explanation might be that a minor population of cells had the insertion of the ABL gene in juxtaposition to BCR, because more cells with the BCR/ABL fusion signal were detected in this patient than in the control on FISH analysis. However, as a considerable number of control cells had false-positive signals for BCR/ABL, the difference in the number of positive cells between our patient and the control might not be significant. Another possibility is that the fragment of ABL was inserted next to the BCR gene by a mechanism other than chromosomal translocation,

and was therefore too short for the large probe used in the FISH analysis to identify it. There are two large clinical studies on the coexpression of ALL- and CML-type mRNA in CML. Saglio et al. [24] have reported that virtually all 20 CML patients investigated had ALL-type transcripts in addition to the CML-type, and Rhee et al. [15] have reported that ALL-type transcript was detected in 99 of 133 CML cases (74%). In the present study, 13 of the 25 CML patients (52%) had both CML- and ALL-type BCR/ ABL transcripts. The percentage of positive patients was lower than in the two previous reports, but the frequent expression of both types of mRNA in CML was confirmed. Although the pathophysiologic significance of the coexpressed ALL-type transcripts is unclear, these ALL-type transcripts could be derived from the alternative splicing of CML-type mRNA. In cases with only ALL-type transcripts, the breakpoint on chromosome 22 is expected to be located in BCR intron 1 [8] or in the flanking region of BCR intron 1 [12]. One of 20 CML cases (5%) reported by Selleri [5,25], one of 19 (5.3%) by Sawyers [6], one of 63 (1.6%) by Kunieda [11], one of 81 (1.2%) by Nakamura [8], and two of 37 (5.4%) in the present study expressed only ALL-type mRNA. Therefore, a considerable number of patients may have only ALL-type mRNA. Because PCR for ALL-type mRNA is not routinely used in CML cases, these cases could be overlooked, and could be misdiagnosed as other CMPDs, especially when Ph1 is not detected. Rare types of BCR/ABL transcripts have recently been found in Ph1 positive CML, such as c3/a2 (c3; corresponding to BCR exon 19) [26–28], and e6/a2 (e6; BCR exon 6) in Ph1 negative case [29]. PCR is a widely used important tool, and an exact diagnosis is possible using a standard primer for CML in a majority of CML cases. However, the existence of several reported variants of BCR/ABL, including the ALL-type, indicates that when a standard BCR/ABL amplified product is not detected, it is important to consider the possibility of the presence of an unusual BCR/ABL fusion transcript.

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