Japanese Journal of Clinical Oncology 30:472-477 (2000)
© 2000 Foundation for Promotion of Cancer Research
Clinical Significance of Major and Minor bcr/abl Chimeric Transcripts in Essential Thrombocythemia
1Department of Hematology and 2Department of Clinical Research, Nagoya National Hospital, Nagoya, 3Department of Hematology, Nagoya First Red Cross Hospital, Nagoya, 4First Department of Internal Medicine, Nagoya University, Nagoya and 5Department of Hematology, Kohnan Showa Hospital, Kohnan, Aichi, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Contradictory results have been reported in terms of detecting bcr/abl transcripts in patients with essential thrombocythemia. The aim of this study was to investigate whether the bcr/abl transcript could be found in patients with essential thrombocythemia (ET).
Methods: The bcr/abl transcript was amplified by the RT-nested PCR method using RNA extract from leukocytes taken from 14 essential thrombocythemia patients. The amplified DNAs were electrophoresed in 1% agarose and visualized with ethidium bromide. The DNA bands associated with the bcr/abl transcript were then extracted and followed by DNA sequence analysis.
Results: Major bcr/abl transcripts of the b3a2 type and minor ones of the e1a2 type were found in one and two ET patients, respectively. The incidence of bcr/abl transcripts was 21.4% (three of 14 patients).
Conclusion: Our experiments confirmed that bcr/abl transcripts are present in some patients with essential thrombocythemia.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Essential thrombocythemia (ET) is a myeloproliferative disorder (MPD) that accompanies an increase in megakaryocytes and platelets and is thought to result from abnormal hematopoiesis of stem cells. Cloning ET is controversial because some subjects exhibit monoclonal hematopoiesis whereas others exhibit polyclonal hematopoiesis (1,2). To distinguish ET from other MPD, especially chronic myelogenous leukemia (CML), the absence of the Philadelphia (Ph1) chromosome is required. However, it has recently been reported that the Ph1 chromosome was found in a small population of ET cases (3,4). Stoll et al. (5) reported that about 10% of ET patients possessed the Ph1 chromosome and their prognosis was poor. Blickstein et al. (6) found a chimeric major bcr/abl transcript in 12 (48%) of 25 ET patients. Marasca et al. (7) also reported finding the chimeric major bcr/abl transcript; however, the incidence was only one of 20 ET patients. They mentioned that the Ph1 negative ET with chimeric bcr/abl should be a clinical variant of CML. Actually, 3–15% of CML were reported as Ph1-negative. Biernaux et al. (8) and Bose et al. (9) recently demonstrated the bcr/abl transcript in 22 of 73 healthy adults. Consequently, the clinical significance of the bcr/abl fusion transcript in hematological disorders should be carefully considered. In this study, we investigated whether the bcr/abl transcript could be detected in 14 ET patients by RT-nested PCR methods followed by DNA base sequence analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Patients
ET was diagnosed according to the criteria developed by the Polycythemia Vera Study Group (PVSG) (10). The criteria used in this study were a platelet count >600 000/cmm, the absence of the Ph1 chromosome and the absence of collagen fibrosis. Patients who demonstrated a secondary increase in the platelet count due to inflammation, acute bleeding, iron deficiency, a malignant state or a drug reaction were excluded. The laboratory data and the karyotype analysis of the 14 ET patients are listed in Table 1. Eight were male and six were female and the ages ranged between 24 and 85 years, median 61 years. The platelet counts at the first visit ranged between 60.6 x 104 and 181.1 x 104/cmm (median 113 x 104/cmm). The white blood cell count ranged between 6000 and 10 000/cmm except in four patients who showed counts >10 000/cmm and one with 3700/cmm. The hemoglobin values were between 12 and 16 g/dl in 12 patients. One showed <12 g/dl and another had >16 g/dl. All neutrophil alkaline phosphatase (NAP) scores were within the normal range except patient number (PN) 14 at 56%. PN4 had slight hepatosplenomegaly and PN12 had slight splenomegaly. All 14 patients were determined to be Ph1 chromosome negative by karyotype analyses of their bone marrow samples. All showed normal karyotypes except PN8. All patients are still alive and their conditions have stabilized. The durations after the diagnosis were between 11 months and 10 years 8 months.
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Reagents
Taq DNA polymerase was purchased from Boehringer Mannheim (Mannheim, Germany). dATP, dTTP, dGTP, dCTP, TaKaRa LA Taq and EASYTRAP were purchased from TaKaRa Shuzo (Shiga, Japan). RNase inhibitor, M-MuLV reverse transcriptase and TRIZOL LS were purchased from Gibco BRL (Paisley, UK). An RNA purification kit (EX-R&D) was purchased from Sumitomo Chemicals (Tokyo, Japan). A BigDye terminator kit was purchased from PE Biosystems Japan (Chiba, Japan). Other reagents were purchased from Wako Chemicals (Osaka, Japan) and Katayama Chemicals (Osaka, Japan).
Isolation of Leukocytes and RNA Extraction
Peripheral blood (PB) and/or bone marrow (BM) from patients and PB from 15 age-matched normal volunteers were collected after obtaining informed consent. The leukocytes were prepared from 15 ml of heparinized peripheral blood and/or bone marrow after hypotonic treatment. The cells were washed twice with phosphate-buffered saline (PBS), then lysed with 0.75 ml of TRIZOL LS reagent by pipeting several times. The total cellular RNA was purified according to the instruction manual. The RNA obtained was dried and dissolved in DEPC-treated water. The total RNA extracted from K562 and MR87 (11) was used as a positive control for major and minor bcr/abl transcripts, respectively. HL60 cells were used as an RNA source of the negative control.
Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
The total amount of RNA was reverse transcribed to complementary DNA (cDNA) with 20 µl of taq buffer containing 10 nM 6-mer random primer, 20 U of M-MuLV reverse transcriptase, 700 µM 4dNTP, 5 U of RNase inhibitor and 5 mM dithiothreitol at 37°C for 1 h. cDNA (10 ng) was then transferred into a PCR tube and added to the first PCR mixture. After incubating at 94°C for 5 min, 55°C for 2 min and 72°C for 3 min, the DNA samples were amplified with 30 cycles in a thermal cycler. Each cycle consisted of incubations at 94°C for 30 s, 55°C for 1 min and 72°C for 2 min. The first PCR products were subsequently amplified by the second PCR. The primers were made as in a previous study (12,13). The primers used for major bcr/abl amplification were as follows: first PCR, M-bcr-1 (5'-GAAGTGTTTCAGAAGCTTCTCC-3') and abl-1 (5'-TGATTATAGCCTAAGACCCGGA-3'); second PCR, M-bcr-2 (5'-TGGAGCTGCAGATGCT-3') and abl-2 (5'-ATCTCCACTGGCCACAA-3'). The primers used for minor bcr/abl amplification were: first PCR, m-bcr-1 (5'-ACCATCGTGGGCGTCCGCAAGA-3') and abl-1; second PCR, m-bcr-2 (5'-AGATCTGGCCCAACGAT-3') and abl-2. A ß-actin gene was amplified by using two primers (5'-CGACAACGGCTCCGGCATGTGC-3' and 5'-CGTCACCGGAGTCCATCACGATGC-3') to confirm the quality of the RNA preparation. The sensitivity of our RT-PCR system was estimated as 1:108 cells agreed well with that reported by Biernaux et al. (8). It was determined by RT-PCR using RNA prepared from major bcr/abl-positive K562 cells which were serially diluted with major bcr/abl-negative HL60 cells (Fig. 1).
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Agarose Gel-running and DNA Base Sequence Analysis
The PCR products were applied to a 1% agarose gel and electrophoresed. DNA bands corresponding to major and minor bcr/abl were extracted and purified with EASYTRAP. The DNAs obtained were labeled with the BigDye terminator kit and the sequence of each DNA was determined with an ABI prism 310 genetic analyzer. All experiments were repeated at least twice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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DNA Bands Associated with Major or Minor BCR/ABL Transcripts Found by 1% Agarose Gel Electrophoresis
Fig. 2 shows the DNA bands associated with major bcr/abl transcripts on 1% agarose gel. The samples were amplified by the RT-nested PCR method by using the major bcr/abl specific primers. Lane a shows the molecular weight marker. The negative control sample prepared from HL60 cells and the blank control are shown in lanes c and d, respectively. Lane b shows the positive control of major bcr/abl prepared from K562 cells. The size of the DNA bases was 305 bp. DNA bands with similar size were observed in seven samples from five patients (lanes 1, 2, 3, 4, 5, 2BM and 4BM). Fig. 3 shows the DNA bands associated with a minor bcr/abl transcript. The DNA band of the positive control was prepared from MR87 cells and is shown in lane b. The DNA bands associated with minor bcr/abl transcripts were found in lanes 1, 2 and 2BM. These results are summarized in Table 2. No major or minor bcr/abl transcripts were detected in mononuclear cells from 15 normal volunteers (data not shown).
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DNA Base Sequence Analysis of DNA Bands Associated with BCR/ABL Transcripts
To confirm whether the DNA bands associated with major or minor bcr/abl transcripts found in agarose gel possess true bcr/abl DNA sequences, we analyzed the DNA base sequence after extracting and purifying each positive band of agarose gel. Concerning the major bcr/abl transcripts, the DNA base sequences of PCR products from PN3 were the same as those of b3a2 major bcr/abl chimeric DNA. The remaining six bands were determined to be just ‘ghosts’ by the analysis (Table 2). The PCR products of PN1 and PN2 in Fig. 3 were related to the minor bcr/abl transcript. The fusion type of both products was determined to be e1a2. The incidence of major bcr/abl transcript was 7.1% or higher, considering the possible PCR competition between the real and false target sequences. The incidence of minor transcripts was 14.3%, resulting in a total incidence of at least 21.4% (three of 14 ET patients).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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ET originates in abnormal hematopoietic stem cells and is classified as an MPD accompanied with an increase in the platelet count. This disorder is heterogeneous in terms of the clonality of the hematopoiesis. Fourteen ET patients in this study have been clinically stable without a blastic crisis during the long term follow-up. Harrison et al. (2) studied the clonality of ET in 46 female ET patients by observing X-chromosome inactivation patterns (XCIPs). They found monoclonal hematopoiesis in 10 subjects and polyclonal hematopoiesis in 13. Furthermore, they did not find a clonal progenitor in purified CD34+CD33– or CD34+CD33+ subpopulations from bone marrow samples in two of 13 patients with polyclonal hematopoiesis. They also reported that there was no difference between patients with monoclonal and polyclonal myelopoiesis with respect to age or platelet count at diagnosis, in the duration of follow-up, incidence of hepatosplenomegaly or hemorrhagic complications. However, polyclonal patients were less likely to have experienced thrombotic events than monoclonal patients (10% versus 60%).
The bcr/abl transcript has been evaluated as a specific marker of CML. However, several arguments have been made concerning whether the existence of the transcript can be evaluated as a minimal residual disease (MRD) of chronic myelogenous leukemic cells after treatment (14). Fialkow et al. (1) first demonstrated that some hematopoietic cells of the neoplastic clone were Ph1-negative by using glucose-6-phosphate dehydrogenase as a clonality marker. Faderl et al. (15) evaluated the occurrence of bcr/abl gene fusion as a passage through which antecedent clonal cells must progress to result in CML, rather than the first molecular event concerning the pathogenesis of CML. The usefulness of a bcr/abl transcript detected by PCR to find MRD was reported in a T cell-depleted allogeneic bone marrow transplantation by Mackinnon et al. (16). However, bcr/abl transcripts were found in some patients with long-term remission after an allogeneic bone marrow transplantation (17,18). To predict a relapse by detecting the bcr/abl transcript, some researchers claimed the use of real-time quantitative RT-PCR assays rather than conventional qualitative PCR methods (19,20).
As for ET, Blickstein et al. (6) detected the bcr/abl transcript in 12 (48%) among 25 ET patients without the Ph1 chromosome and also reported that the bcr/abl positive group was clinically indistinguishable from the bcr/abl negative group. Marasca et al. (7) also reported the b3a2 type of bcr/abl transcript. However, the incidence (1/20, 5%) was very low compared with Blickstein’s report. This bcr/abl-positive ET patient showed a transformation to idiopathic myelofibrosis 6 years after the diagnosis, which progressed to a blastic crisis 12 years after the diagnosis. In contrast, one of 19 bcr/abl-negative ET subjects showed an idiopathic myelofibrosis 6 years after the diagnosis. However, Hackwell et al. (21) could not find the bcr/abl transcript in 18 Ph1-negative ET patients. The reason for the discrepant results in different groups is unclear. Najfeld et al. (22) reported they found the Ph1 chromosome after the onset of chemotherapy in a Ph1-negative acute myelogenous leukemia (AML)-M2 patient and this event was evaluated as a secondary effect of the therapy or a late event in the multi-step pathogenesis of AML. Thus, the occurrence of the bcr/abl transcript should also be evaluated from the viewpoint of a therapy modification. Next, we should mention the technical problems underlying the PCR method even though the RT-PCR assay is a useful technique for detecting specific mRNA expressions. The contamination of a bcr/abl-positive sample will cause a false-positive result. An inadequate sample volume and degradation of mRNA cause false-negative results. The lack of sensitivity of junctional probes will cause both false-positive and false-negative results. We found a PCR product whose molecular weight was determined to be from major bcr/abl mRNA in four patients. However, only one showed genuine major bcr/abl fusion DNA judging from the DNA base sequence analysis. This suggests that the presence of a bcr/abl transcript should be judged only by detecting related DNA bands on the agarose gel. The bcr/abl-related DNA band should be confirmed by DNA base sequence analysis or a Southern blot analysis. With regard to the sensitivity of the RT-PCR assay, when the sensitivity was raised to 1.5log-fold from conventional RT-PCR assays, bcr/abl transcripts could be found in normal individuals, as reported by Biernaux et al. (8) and Bose et al. (9). Biernaux et al. (8) found bcr/abl transcripts in 22 adults (30.1%) among 73 healthy donors. In contrast, they found the transcript in only one of 22 children. In addition, bcr/abl transcripts were not observed in 22 patients with umbilical cord blood. They speculated that bcr/abl transcript detection is age-dependent. Bose et al. (9) confirmed the results of Biernaux et al. by finding p210 bcr/abl (b2a2 and/or b3a2) and p190 bcr/abl (e1a2) in four and 11 of 16 normal individuals, respectively. They also found p210 and p190 bcr/abl transcripts in seven cell lines of hematopoietic origin. Their results confirmed the detection of leukemia-associated genes in normal leukocytes and suggested that bcr/abl fusion was generated relatively frequently in hematopoietic cells. However, the frequency of such ‘innocent bcr/abl-carrying leukocytes’ with additional changes necessary to produce leukemia in humans might be low. The possibility of the occurrence of ‘innocent bcr/abl-carrying leukocytes’ in leukemia patients in long-term remission is not negligible. Therefore, such cells might show a bcr/abl transcript in PCR assays.
In this study, we found one major (b3a2) and two minor (e1a2) bcr/abl transcripts in 14 ET patients. The results were verified not only by PCR assay but also by the DNA base sequence analysis. The incidence of bcr/abl transcript detection was 21.4%, three of 14 patients. Our results gave informative suggestions to the clinical significance of bcr/abl transcripts in ET. We are now engaged in expanding the number of patients and also in performing a serial examination during a long-term clinical course.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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This study was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan 10-1 to H.Y. and Y.K. and 11S-4 to M.S.
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FOOTNOTES |
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+ For reprints and all correspondence: Hironori Yamada, Department of Hematology, Nagoya National Hospital, 4–1–1 Sannomaru, Nakaku, Nagoya 460-0001, Japan. E-mail: hayabusa@kctv.ne.jp
Abbreviations: ET, essential thrombocythemia; CML, chronic myelogenous leukemia; MPD, myeloproliferative disorders; Ph1, Philadelphia; PVSG, Polycythemia Vera Study Group; NAP, neutrophil alkaline phosphatase; PN, patient number; PB, peripheral blood; BM, bone marrow; PBS, phosphate-buffered saline; cDNA, complementary DNA; XCIPs, X-chromosome inactivation patterns; MRD, minimal residual disease; AML, acute myelogenous leukemia.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Received February 8, 2000; accepted August 21, 2000.
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