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. 1992 Apr 1;89(7):2694–2698. doi: 10.1073/pnas.89.7.2694

Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia.

W H Miller Jr 1, A Kakizuka 1, S R Frankel 1, R P Warrell Jr 1, A DeBlasio 1, K Levine 1, R M Evans 1, E Dmitrovsky 1
PMCID: PMC48728  PMID: 1372989

Abstract

The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR-alpha) gene on chromosome 17 to a gene on chromosome 15 called PML, a putative transcription factor. This distinct translocation results in a fusion mRNA detected by Northern analysis. Two cDNAs have been isolated that differ in the extent of 3' PML nucleic acid sequence contained. This study describes a reverse transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha fusion transcript, which amplifies PML/RAR-alpha mRNA from APL cells with either reported breakpoint. DNA sequencing of the predominant RT-PCR products from 6 patients showed identical RAR-alpha exonic breakpoints and two PML breakpoints. This RT-PCR assay was positive in leukemic cells from 30/30 APL patients with the molecular rearrangement confirmed by cytogenetics or Northern analysis. In leukemic cells of patients with a morphologic diagnosis of APL lacking the t(15;17) by routine cytogenetics, a positive RT-PCR assay predicted clinical response to all-trans-retinoic acid (RA) therapy. Dilutional studies with leukemic cells that express (NB4) or do not express (HL-60) a PML/RAR-alpha fusion mRNA reveal that this RT-PCR assay detects the transcript from as little as 50 pg of total RNA. In APL cells from 5/6 patients treated with RA alone, a complete response by clinical and cytogenetic criteria accompanied a persistently positive RT-PCR assay. This preceded relapse by 1-6 months. RT-PCR for PML/RAR-alpha mRNA provides a more-sensitive test for the t(15;17) than routine cytogenetics or Northern analysis. This molecular rearrangement detected by RT-PCR best defines this RA-responsive malignancy. The RT-PCR assay for the PML/RAR-alpha transcript yields important diagnostic and prognostic information in the management of APL patients.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alcalay M., Zangrilli D., Pandolfi P. P., Longo L., Mencarelli A., Giacomucci A., Rocchi M., Biondi A., Rambaldi A., Lo Coco F. Translocation breakpoint of acute promyelocytic leukemia lies within the retinoic acid receptor alpha locus. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1977–1981. doi: 10.1073/pnas.88.5.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A., Gralnick H. R., Sultan C. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med. 1985 Oct;103(4):620–625. doi: 10.7326/0003-4819-103-4-620. [DOI] [PubMed] [Google Scholar]
  3. Biondi A., Rambaldi A., Alcalay M., Pandolfi P. P., Lo Coco F., Diverio D., Rossi V., Mencarelli A., Longo L., Zangrilli D. RAR-alpha gene rearrangements as a genetic marker for diagnosis and monitoring in acute promyelocytic leukemia. Blood. 1991 Apr 1;77(7):1418–1422. [PubMed] [Google Scholar]
  4. Castaigne S., Chomienne C., Daniel M. T., Ballerini P., Berger R., Fenaux P., Degos L. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood. 1990 Nov 1;76(9):1704–1709. [PubMed] [Google Scholar]
  5. Chen Z. X., Xue Y. Q., Zhang R., Tao R. F., Xia X. M., Li C., Wang W., Zu W. Y., Yao X. Z., Ling B. J. A clinical and experimental study on all-trans retinoic acid-treated acute promyelocytic leukemia patients. Blood. 1991 Sep 15;78(6):1413–1419. [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Chomienne C., Ballerini P., Balitrand N., Huang M. E., Krawice I., Castaigne S., Fenaux P., Tiollais P., Dejean A., Degos L. The retinoic acid receptor alpha gene is rearranged in retinoic acid-sensitive promyelocytic leukemias. Leukemia. 1990 Dec;4(12):802–807. [PubMed] [Google Scholar]
  8. Dalton W. T., Jr, Ahearn M. J., McCredie K. B., Freireich E. J., Stass S. A., Trujillo J. M. HL-60 cell line was derived from a patient with FAB-M2 and not FAB-M3. Blood. 1988 Jan;71(1):242–247. [PubMed] [Google Scholar]
  9. Giguere V., Ong E. S., Segui P., Evans R. M. Identification of a receptor for the morphogen retinoic acid. Nature. 1987 Dec 17;330(6149):624–629. doi: 10.1038/330624a0. [DOI] [PubMed] [Google Scholar]
  10. Huang M. E., Ye Y. C., Chen S. R., Chai J. R., Lu J. X., Zhoa L., Gu L. J., Wang Z. Y. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood. 1988 Aug;72(2):567–572. [PubMed] [Google Scholar]
  11. Hughes T. P., Morgan G. J., Martiat P., Goldman J. M. Detection of residual leukemia after bone marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapse. Blood. 1991 Feb 15;77(4):874–878. [PubMed] [Google Scholar]
  12. Kakizuka A., Miller W. H., Jr, Umesono K., Warrell R. P., Jr, Frankel S. R., Murty V. V., Dmitrovsky E., Evans R. M. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell. 1991 Aug 23;66(4):663–674. doi: 10.1016/0092-8674(91)90112-c. [DOI] [PubMed] [Google Scholar]
  13. Kawasaki E. S., Clark S. S., Coyne M. Y., Smith S. D., Champlin R., Witte O. N., McCormick F. P. Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5698–5702. doi: 10.1073/pnas.85.15.5698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lanotte M., Martin-Thouvenin V., Najman S., Balerini P., Valensi F., Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood. 1991 Mar 1;77(5):1080–1086. [PubMed] [Google Scholar]
  15. Larson R. A., Kondo K., Vardiman J. W., Butler A. E., Golomb H. M., Rowley J. D. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med. 1984 May;76(5):827–841. doi: 10.1016/0002-9343(84)90994-x. [DOI] [PubMed] [Google Scholar]
  16. Longo L., Pandolfi P. P., Biondi A., Rambaldi A., Mencarelli A., Lo Coco F., Diverio D., Pegoraro L., Avanzi G., Tabilio A. Rearrangements and aberrant expression of the retinoic acid receptor alpha gene in acute promyelocytic leukemias. J Exp Med. 1990 Dec 1;172(6):1571–1575. doi: 10.1084/jem.172.6.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miller W. H., Jr, Warrell R. P., Jr, Frankel S. R., Jakubowski A., Gabrilove J. L., Muindi J., Dmitrovsky E. Novel retinoic acid receptor-alpha transcripts in acute promyelocytic leukemia responsive to all-trans-retinoic acid. J Natl Cancer Inst. 1990 Dec 19;82(24):1932–1933. doi: 10.1093/jnci/82.24.1932. [DOI] [PubMed] [Google Scholar]
  18. Mohamed A. N., Clarkson B. D., Chaganti R. S. High resolution banding of chronic myeloid leukemia chromosomes. Cancer Genet Cytogenet. 1986 Feb 15;20(3-4):209–222. doi: 10.1016/0165-4608(86)90076-2. [DOI] [PubMed] [Google Scholar]
  19. Noonan K. E., Roninson I. B. mRNA phenotyping by enzymatic amplification of randomly primed cDNA. Nucleic Acids Res. 1988 Nov 11;16(21):10366–10366. doi: 10.1093/nar/16.21.10366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pandolfi P. P., Grignani F., Alcalay M., Mencarelli A., Biondi A., LoCoco F., Grignani F., Pelicci P. G. Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties. Oncogene. 1991 Jul;6(7):1285–1292. [PubMed] [Google Scholar]
  21. Petkovich M., Brand N. J., Krust A., Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 1987 Dec 3;330(6147):444–450. doi: 10.1038/330444a0. [DOI] [PubMed] [Google Scholar]
  22. Rodeghiero F., Avvisati G., Castaman G., Barbui T., Mandelli F. Early deaths and anti-hemorrhagic treatments in acute promyelocytic leukemia. A GIMEMA retrospective study in 268 consecutive patients. Blood. 1990 Jun 1;75(11):2112–2117. [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Tkachuk D. C., Westbrook C. A., Andreeff M., Donlon T. A., Cleary M. L., Suryanarayan K., Homge M., Redner A., Gray J., Pinkel D. Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization. Science. 1990 Oct 26;250(4980):559–562. doi: 10.1126/science.2237408. [DOI] [PubMed] [Google Scholar]
  26. Wang Z. Y., Sun G. L., Lu J. X., Gu L. J., Huang M. E., Chen S. R. Treatment of acute promyelocytic leukemia with all-trans retinoic acid in China. Nouv Rev Fr Hematol. 1990;32(1):34–36. [PubMed] [Google Scholar]
  27. Warrell R. P., Jr, Frankel S. R., Miller W. H., Jr, Scheinberg D. A., Itri L. M., Hittelman W. N., Vyas R., Andreeff M., Tafuri A., Jakubowski A. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med. 1991 May 16;324(20):1385–1393. doi: 10.1056/NEJM199105163242002. [DOI] [PubMed] [Google Scholar]
  28. de Thé H., Chomienne C., Lanotte M., Degos L., Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus. Nature. 1990 Oct 11;347(6293):558–561. doi: 10.1038/347558a0. [DOI] [PubMed] [Google Scholar]
  29. de Thé H., Lavau C., Marchio A., Chomienne C., Degos L., Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991 Aug 23;66(4):675–684. doi: 10.1016/0092-8674(91)90113-d. [DOI] [PubMed] [Google Scholar]

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