Identification of DNA Rearrangements at the Retinoic Acid Receptor-a (RAR-a) Locus in All Patients With Acute Promyelocytic Leukemia (APL) and Mapping of APL Breakpoints Within the RAR-a Second Intron zyx By Daniela Diverio, Francesco Lo Coco, Francesca D’Adamo, Andrea Biondi, Marta Fagioli, Francesco Grignani, Alessandro Rambaldi, Vincenzo Rossi, Giuseppe Avvisati, Maria C. Petti, Anna M. Testi, Vincenzo Liso, Giorgina Specchia, Giuseppe Fioritoni, Anna Recchia, Francesco Frassoni, Stefania Ciolli, and Pier Giuseppe Pelicci for the Italian Cooperative Study Group ”GIMEMA Seventy patients with acute promyelocytic leukemia (APL) were characterized at the DNA level using genomic retinoic acid receptor- (RAR-a) probes on Southern blot experi- ments. Sixty-two cases were defined as M3 according to the French-American-British (FAB) criteria, and eight had a diag- nosis of microgranular or variant (M3v) APL. The use of two restriction enzymes and three probes exploring the second intron of the zyxwvutsrqp RAR-a. gene allowed us to detect specific abnormal DNA fragments in every case, with clustering of rearrangements within the 20-kb intronic region between RAR-a exons II and 111. A more detailed mapping of APL breakpoints was performed in 52 cases in which three zyxwvut EcoRl subregions of the RAR-a second intron were analyzed with corresponding probes. Comparison of clinical and hematolog- zyxwvuts CUTE PROMYELOCYTIC leukemia (APL; M3 in A the French-American-British [ F B I classification) is a particular subtype of acute myeloid leukemia (AML).1,2 Morphologically, it is characterized by the presence of hypergranular and dysplastic promyelocytes infiltrating the bone marrow. Several biologic and clinical features allow us to consider APL as a distinct and unique type of AML.3,4 In fact, patients frequently present with a severe bleeding disorder, often accompanied by coagulation defects.’r4 More- over, APL blasts can be induced to undergo terminal differentiation, both in vitro and in vivo, following treat- ment with the vitamin A derivative retinoic This latter agent has recently been used successfully to induce complete remission in APL patients6-10 Regarding cytogenetic features, a reciprocal balanced translocation between chromosomes 15 and 17 is consis- tently detected in APL.” The high frequency of the t(15;17) (70% to 80% of cases) and the fact that this is generally the only karyotypic abnormality suggests that the t(15;17) rearrangement plays a crucial role in APL leukemogenesis. We and others have recently identified the APL translo- cation breakpoints and demonstrated that they occur within the genes coding for the retinoic acid receptor-a zyxwvuts (RAR-a) and a new transcription unit originally called MYL (for myeloid) and now renamed PML (for promyel~cytic).~~-~~ As a consequence of the translocation, a new chimeric PML/RARa gene is generated and actively transcribed as a fusion mRNA. This latter agent encodes an abnormal receptor protein, which is presumably involved in the pathogenesis of the disease.16-18 Regardless of their pathogenetic significance, zyxwvuts RAR-a and PML rearrangements can be considered as genetic markers of the t( 15;17) and, consequently, extremely useful for diagnosis and monitoring of the disea~e.~-l~J~-~~ We have previously reported DNA rearrangements in 100% of APL cases using both RAR-a and PML probes in a small series of patients. However, in those reports, only a limited number of RAR-a or PML probes were available and a ical features in the three subgroups of patients with distinct RAR-a breakpoints did not show significant differences re- garding age, peripheral blood (PB) counts, presence of coag- ulopathy, or FAB classification (M3 Y M3v). Interestingly, a significant difference was observed in the M/F ratio of the three subgroups, with a higher incidence of rearrangements at the 5‘ end of the RAR-a second intron in female patients, and more frequent 3’ breakpoints in males. The results of this study indicate that a unique genomic alteration consistently occurs on the 17q- derivative of the APL specific t(15;17) aberration. Moreover, the clinical relevance of RAR-a gene analysis both at diagnosis and in follow-up studies is further emphasized. zyxwvu 0 1992 by The American Society of Hematology. rather complex Southern blot strategy was designed to optimize the detection of rearrangement^.^^,^^ Further stud- ies on mapping of chromosome 15 breakpoints have indi- cated variable distances within the PML 1ocus,21a while the RAR-a involvement on chromosome 17 seems to be re- stricted to the second intron, which is 20 kb long and may therefore be more easily analyzed by Southern blot hybrid- ization. In this study, we explored the RAR-a gene organization in a consistent series (70 cases) of APL. Using three distinct RAR-a probes and two restriction enzymes, we could demonstrate the occurrence of RAR-a rearrangements in all cases. MATERIALS AND METHODS Seventy patients with AF’L were included in this study. Among these, 45 were newly reported cases, whereas 25 had been previously analysed with two RAR-a probes and were described e l s e ~ h e r e . ~ ~ ~ ~ ~ Of the 70 patients, 54 were de novo cases and 16 were relapsed cases. Following a morphocytochemical Patients and samples. From the Hematology, Human Biopathology Department of the University “La Sapienza, ”Rome, Italy; the Clinica Pediam’ca, Osped- ale S. Gerardo, University of Milano-Monza, Italy; the Clinica Medica I, University of Perugia, Italy; the Divisione di Ematologia, Ospedali Riuniti Bergamo, Italy; the Servizio di Ematologia, University of Ban, Italy; the Divisione di Ematologia, Ospedale Civile of Pescara, Italy; the Divisiorie di Ematologia II, Ospedale S. Martino, Genova, Italy; and the Divisione di Ematologia, Policlinico Careggi, Firenze, Italy. Submitted December 2,1991; accepted February 25, 1992. Address reprint requests to Francesco Lo Coco, MD, Ematologia, Dipartimento di Biopatologia Umana, Universita [‘La Sapienza, I’ VIa Benevento 6, 00161, Rome, Italy. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1992 by The American Society of Hematology. 0006-497119217912-0023$3.00/0 Blood, Vol79, zyxwvutsrqpo No 12 (June 15). 1992: pp3331-3336 3331 Downloaded from http://ashpublications.org/blood/article-pdf/79/12/3331/601128/3331.pdf by guest on 26 August 2022