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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. RAPID COMMUNICATION A Possible Correlation Between the Type of bcr-abl Hybrid Messenger RNA and Platelet Count in Philadelphia-Positive Chronic Myelogenous Leukemia By Koiti Inokuchi, Tokiko Inoue, Arinobu Tojo, Makoto Futaki, Koichi Miyake, Takashi Yarnada, Yoshihiro Tanabe, lchiro Ohki, Kazuo Dan, Keiya Ozawa, Shigetaka Asano, and Takeo Nornura The Philadelphia (Phl) chromosome, in which the hybrid bcr-ab/ gene is formed, is thought to be the initial event in chronic myelogenous leukemia (CML). The position of the breakpoint within the breakpoint cluster region (bcr) on Phl chromosome and the splicing pattern determine the species of the fused bcr-ab/ messenger RNA (mRNA). We tried to detect the two types of fused mRNAs in 57 chronic-phase cases of Phl-positive CML using the polymerase chain reaction procedure (RT-PCR). The bcr exon 2/ab/ exon 2 fused mRNA (b2-a2) was detected in 17 patients, the bcr exon Blab/ exon 2 fused mRNA (b3-a2) was detected in 34 patients, and both types of mRNA were detected in six patients. The platelet counts of patients who expressed b3-a2 mRNA or both types were significantly higher than those of patients who expressed only b2-a2 (841.5 v 373.5 x 109/L: P < .015), although there was no significant difference in the white blood cell counts or hemoglobin. This finding suggests a possibility that the type of bcr-ab/ mRNA may affect the thrombopoieticactivity in CML. o 1991 by The American Society of Hematology. A The total RNA RT-PCR for detection of bcr-ab1 fusion “A. of mononuclear cells was extracted by the CsCl method.’’ RT-PCR was performed using the methods described by Roth et all4and by Kawasaki et al” with a slight modification.” Approximately 2 pg of total RNA was used as the template with 10 U of avian myeloblastosis virus reverse-transcriptase (Boehringer-Mannheim, Indianapolis, IN) for 1 hour at 37°C in 50 mmol/L Tris-HCI, pH 8.3, 50 mmol/L KCl, 8 mmol/L MgCl,, 10 mmol/L dithiothreitol, dNTPs at 500 pmol/L each in a total volume of 50 pL with 0.05 OD,, units of oligo-primer (abl-RT 5’-AACGAAAAGGTTGGGGTC-3‘, antisense strand). Ten microliters of the reverse transcriptase reaction mixture was adjusted to PCR conditions (10 mmol/L Tris-HCI pH 8.3,50 mmol/L KCl, 15 mmol/L MgCI,, 2 mmol/L dithiothreitol, 200 pmol/L dNTPs) with 0.0125 OD,, units of one of two sets of primers (the first set, A 5’-AGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAA3‘, coding strand, and B: 5’-TCACTGGGTCCAGCGAGAAGGTTMCCTTGGAGTT-3’,antisense strand; the second set; C 5’-GGAGCTGCAGATGCTGACCAAC3‘, coding strand and D: 5’-TCAGACCCTGAGGCr‘CAAAGTC3’, antisense strand) and 2 U of Taq polymerase (Thermus aquaticus DNA polymerase; Perkin Elmer Cetus, Nonvalk, CT) in a 50 pL reaction volume (Fig 1).PCR of 40 cycles was performed, consisting of 30 seconds at 94°C (denaturation), 30 seconds at 55°C (annealing), and 1 minute at 75°C (extention). Finally, the PCR products (20 pL) were phenol-extracted, ethanol-precipitated, and electrophoresed through a 2% agarose gel. The PCR products were visualized directly in ethidium bromide-stained gels, photographed, and transferred onto Gene Screen Plus membranes (New England Nuclear, Boston, MA). Southern hybridization was performed using a 0.6HB probe corresponding to the bcr exon 2 (provided by Dr S. Hirosawa, Tokyo Medical and Dental University), or two kinds of oligonucleotides (E: 5’-GCTGAAGGGClT”- SHORTENED chromosome 22, designated as the Philadelphia (Phl) chromosome, is observed in the leukemic cells of more than 90% of chronic myelogenous leukemia (CML) patients.’ The Phl chromosome, which can be considered to be the first karyotypic marker consistently present in a human neoplasm, is the result of a reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;qll).*This translocation moves the c-ab1protooncogene from chromosome 9 to chromosome 22.’ The breakpoint of chromosome 22 has been shown to be clustered within a limited region of about 5.8 kb, referred to as the breakpoint cluster region ( b ~ r ) .Almost ~ , ~ all of the patients have genomic breakpoints between exons b2 and b3 or between exons b3 and b4 within bcr.‘ The two chimeric genes may be distinguished by Southern blot analysis. The first indication that the precise location of the breakpoint on chromosome 22 might have some clinical significance came from a study by Schaefer-Rego et al.’ Next, a study by Eisenberg et al’ showed that the mean duration of the chronic phase for patients who had progressed to blast crisis was significantly shorter for patients with a 3’-breakpoint site than for those with a 5’-breakpoint site within the bcr. Many studies have reported on this controversial Even a small difference in the location of the breakpoint within the bcr gene may cause a change in the amino acid sequence of the fused protein, which in turn may affect the clinical features of CML patients. Recently, we reported that CML patients with a 3’ breakpoint within the bcr gene have higher thrombopoietic activity than those with a 5‘ breakpoint.“ Here, we tried to ascertain the relationship between thrombopoiesis and the two potential types of fused bcr-ab1 messenger RNAs (mRNAs) using the polymerase chain reaction (RT-PCR) in 57 CML patients. As expected, we found that patients with fused b3-a2 mRNA had higher platelet counts than those with fused b2-a2 mRNA. MATERIALS AND METHODS Patient population. Blood and bone marrow samples were obtained from 57 Phl-positive CML patients in the chronic phase of the disease. Most of the patients had not received therapy before RNA analysis. The clinical data of each patient are that at diagnosis. Blood, Vo178, No 12 (December 15). 1991: pp3125-3127 From the Third Department of Internal Medicine, Nippon Medical School; and the Department of Hematology-Oncology,the Institute of Medical Science, the University of Tokyo, Tokyo, Japan. Submitted August 26, 1991; accepted September 17, 1991. Address reprint requests to Koiti Inokuchi, MD, PhD, The Third Department of Intemal Medicine, Nippon Medical School, Sendagi 1-1-5,Bunkyo-ku, Tokyo 113, Japan. The publication costs of this article were defrayed in part by page charge payment. This aricle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1991 by The American Society of Hematology. 0006-4971191 / 7812-0029$3.00/0 3125 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 3126 INOKUCHI ET AL I 1 b l b2 b3 a2 111 D B AT Fig 1. Schematic representation of the bcr-ebl mRNA with position of oligomers numbered A, B, C, and D used in the PCR reaction, and ab/-RT oligomer for making cDNA synthesis as described."" (0) bcr-specific sequences; (0) ab/-specific sequences. analysis of 57 patients showed the presence of b3-a2 mRNA in 34 cases and b2-a2 mRNA in 17 cases, and coexpression of both junctions in six cases. The relationship between the type of bcr-ab1 mRNA and platelet count is shown in Fig 3. The platelet counts of patients expressing b3-a2 mRNA were significantly higher than those of patients expressing only b2-a2 mRNA (841.5 v 373.5 x lo9& P < .015). We also examined the relationship with the white blood cell count and hemoglobin, but no significant correlations were found. DISCUSSION TGAACTCKY, specific for bcr exon31abl exon 2; and F 5'GCTGAAGGGCTTCTTCCTTATTGATG-3', specific for bcr exon2Iabl exon 2)'' RESULTS RNA samples from 57 CML patients were analyzed for specific bcr-ab1 mRNA. The bcr-ab1 mRNA junctions were amplified by the RT-PCR method using two pairs of primers. The two junctions, bcr2-ab12 and bcr3-ab12, were identified by their electrophoretic mobility and hybridization to bcr exon2 (0.6HB) probe or junction-specific oligoprobes. An example of the RT-PCR analysis in which a pair of primers (primers A and B) was used is shown in Fig 2. The RT-PCR products of RNA samples from K562 cells and five CML patients were electrophoresed and subsequently visualized by ethidium bromide staining and hybridization to the 0.6HB probe. The 319-bp RT-PCR product is the band specific for b3-a2 mRNA, while the 244-bp RT-PCR product is specific for b2-a2 mRNA. The RT-PCR Phl-positive CML is thought to be a clonal myeloproliferative disorder resulting from oncogenic transformation of a bone marrow stem cell. In the chronic phase, the disease phenotype is characterized by pronounced granulocytosis and thrombocytosis in peripheral blood and increased myeloid and megakaryocytecompartments in the hypercelMar bone marrow. Recently, our understanding of the pathogenesis of CML has been greatly advanced by the molecular studies. Phlpositive CML is characterized by the presence of a bcr rearrangement. Attempts have been made to elucidate the relationship between the breakpoint site and the duration of survival or the other clinical features by Southern analysis at the DNA level. Shtalrid et a1,9Jaubert et a1,Ioand Morris et al" reported that there was a significant correla- p<0.015 n e v) g l 2 3 4 5 6 e bP 872 :&LA - - 603 4 - Y ~ 234 194 - A w +b3-a2 b2-a2 + 0 Fig 2 Examples of Southern blot hybridization of RT-PCR products of the bcr-ebl mRNA in Phl-positive patients. Products from RT-PCR analyses were size-fractionated by 2.0% agarose gel electrophoresis, transferred to a nylon membrane, and hybridizedto a 0.6HB probe. K562 cells and patients 1, 4, and 6 have the b3a2 junction. Patients 2 and 5 have the b2a2 junction only. Patient 3 produces both junctional products. b242 b3-a2 type bcr-ab/ mRNA Fig 3. Correlationof platelet count with the type of bcr-abl mRNA. The platelet counts were obtained at diagnosis before the therapy. The normal range is indicated by the hatched box. The bars indicate the mean values. The significance of platelet counts in bothtypes was tested with the Wlcoxon-2 sample test. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. BCR-ABL 3127 MRNA AND PLATELET COUNT IN CML tion between the site of the bcr breakpoint and the duration of survival. CML patients have one of two bcr-ab1 mRNA types or b ~ t h . ’ ~Application ,’~ of the RT-PCR method to determine the type of bcr-ab1 mRNA in CML patients has made it easier to identify the site of the bcr rearrangement. The studies using the RT-PCR method are now prevailing in this research area.””8 Recently, we found that CML patients with a 3’ breakpoint site within the bcr gene have a tendency to have higher thrombopoietic activity than those with a 5’ breakpoint.’* Therefore, to confirm this preliminary observation, we extensively studied the relationship between the type of bcr-ab1 mRNA and the patient’s platelet count in 57 Phl-positive CML cases using the PCR method. As expected, the platelet counts were higher in the patients expressing b3-a2 mRNA. A recent study reported similar finding in Phl-positive essential thrombocythemia.” The b3-a2 type mRNA was observed in five patients and the b2-a2 type mRNA was observed in only one patient. In our present study, 16 patients who had platelet counts of more than 900 x 109/L expressed only b3-a2 type mRNA, and almost all of the patients with b2-a2 type mRNA had a platelet count of less than 600 x 109/L. There were no apparent differences in hematologic findings between the patients with b3-a2 type mRNA and the CML patients with b2-a2 type mRNA, except for the platelet count. Thus, we speculate that the type of bcr-abl mRNA may affect the thrombopoietic activity in CML, although we are currently unable to explain why the platelet count correlates with the type of bcr-ab1mRNA. ACKNOWLEDGMENT We thank Dr S. Hirosawa for the 0.6HB probe, the bloodresearch group of the Third Department of Internal Medicine of the Nippon Medical School for supplying blood samples, and Dr S. Inokuchi for help with the preparation of the manuscript. REFERENCES 1. Rowley JD: A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and giemsa staining. Nature 243:290,1973 2. De Klein A, van Kessel AG, Gromeld G, Bartram CR, Hagemeijer A, Bootsma D, Spur NK, Neisterkamp N, Groffen J, Stephenson JR: A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelogenous leukemia. Nature 300: 765,1982 3. Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, de Klein A, Bartram CR, Grosveld G: Localization of the c-ab1 oncogene adjacent to a translocation breakpoint in chronic myelocytic leukemia. Nature 306:239,1983 4. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G: Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93, 1984 5 . Heisterkamp N, Stam K, Groffen J, de Klein A, Grosveld G: Structural organization of the bcr gene and its role in the Ph’ translocation. Nature 316:758,1985 6. Shtivelman E, Lifshiftz B, Gale RP, Canaani E: Fused transcripts of ab1 and bcr genes in chronic myelogenous leukemia. Nature 315:550,1985 7. Schaefer-Rego K, Dudek H, Popenoe D, Arlin Z, Mears JG, Bank A, Leibowitz D: CML patients in blast crisis have breakpoints localized to a specific region of the BCR. Blood 70:448,1987 8. Eisenberg A, Silver R, Soper L, Arlin Z, Coleman M, Bernhardt B, Benn P: The location of breakpoints within the breakpoint cluster region (bcr) of chromosome 22 in chronic myeloid leukemia. Leukemia 2542,1988 9. Shtalrid M, Talpaz M, Kurzrock, R, Kantarjian H, Trujillo J, Gutterman J, Galina Y, Mark B: Analysis of breakpoints within the bcr gene and their correlation with clinical course of Philadelphiapositive chronic myelogenous leukemia. Blood 72485,1988 10. Jaubert J, Martiat P, Dowding C, Ifrah N, Goldman JM: The position on the M-BCR breakpoint does not predict the duration of chronic phase or survival in chronic myeloid leukemia. Br J Haematol74:30,1990 11. Morris SW, Daniel L, Ahmed CMI, Elias A, Lebowitz P: Relationship of bcr breakpoint to chronic phase duration, survival, and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase. Blood 75:2035,1990 12. Inokuchi K, Futaki M, Yamada T, Tanabe Y, Dan K, Shinohara T, Kuriya S, Nomura T: The relationship between the site of breakpoints within the bcr gene and thrombopoiesis of Philadelphia-positive chronic myelocytic leukemia. Leuk Res (in press) 13. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294,1979 14. Roth MS, Antin JH, Bingham EL, Ginsburg D: Detection of Philadelphia chromosome-positive cells by the polymerase chain reaction following bone marrow transplant for chronic myelogenous leukemia. Blood 74:882,1989 15. Kawasaki ES, Clark SS, Coyne MY, Smith SD, Champlin R, Witte ON, McCormick FP: Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci USA 855698, 1988 16. Futaki M, Inokuchi K, Dan K, Nomura T: Activation of bcr-ab1 fusion gene and ras oncogenes in chronic myelogenous leukemia. Leuk Lymphoma (in press) 17. Morgan GJ, Hernandez A, Chan LC, Hughes T, Martiat P, Wiedemann LM: The role of alternative splicing patterns of BCR/ABL transcripts in the generation of the blast crisis of chronic myeloid leukemia. Br J Haematol76:33,1990 18. Marcelle C, Gale RP, Prokocimer M, Berrebi A, MerleBeral H, Canaani E: Analysis of BCR-ABL mRNA in chronic myelogenous leukemia patients and indentification of a new BCR-related sequence in human DNA. Gene Chromosomes Cancer 1:172,1989 19. Martiat P, Ifrah N, Rassool F, Morgan G, Giles F, Gow J, Goldman JM: Molecular analysis of Philadelphia positive essential thrombocythemia. Leukemia 3:563,1989 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1991 78: 3125-3127 A possible correlation between the type of bcr-abl hybrid messenger RNA and platelet count in Philadelphia-positive chronic myelogenous leukemia [see comments] K Inokuchi, T Inoue, A Tojo, M Futaki, K Miyake, T Yamada, Y Tanabe, I Ohki, K Dan and K Ozawa Updated information and services can be found at: http://www.bloodjournal.org/content/78/12/3125.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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