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The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B‐F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs.

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Acute promyelocytic leukemias (APLs) are characterized by a reciprocal balanced translocation that involves chromosomes 15 and 17 [t(15;17) [t(15;17)] that results in the formation of two marker chromosomes: 15q+ and 17q-. The t(15;17) is present in 70-90% of APLs but is never seen in other types of malignancy (3,4). Although the high frequency and specificity of the t(15;17) and the fact that it is often the only karyotypic aberration present (4) are evidence that it plays a crucial role in the pathogenesis of APL, the gene(s) directly involved in the chromosome 15 and 17 breakpoints have never been identified. We have reported that the APL chromosome 17 breakpoint and the retinoic acid receptor a (RARa) locus map to the same cytogenetic band (5) and that the RARa gene is translocated (5) and rearranged in APLs (6). The RARa gene product is a nuclear receptor that acts as a transcription enhancer in response to the binding of retinoic acid, a physiological metabolite of vitamin A with strong celldifferentiating and morphogenic potential (7-13). We herein report the isolation and sequencing of the recombination site of the RARa genetf and chromosome 15-derived material from one APL patient and provide direct evidence that the RARa gene rearrangements in APLs are the consequence of chromosome 17 breaking within the RARa locus.MATERIALS AND METHODS Pathologic Samples and Cytogenetic Analysis. Involved bone marrow was collected from untreated patients during the course of diagnostic procedures. Diagnosis of APL was established in each patient by standard clinical and cytologic criteria, according to the FAB (French-American-British) recommendations (2). All 10 APL patients included in this study carried the typical t(15;17).Isolation of Genomic A Clones. Clones A8C, A2A, and Aal were isolated from a commercially available genomic library obtained from human embryo lung fibroblasts in the A-FIX-1I vector (Stratagene) by screening with the K/S probe. The AW6A clone was isolated from the WI-38 genomic library by screening with the RH15 DNA probe. Clones AR2 and AR13A were isolated with the HB probe from a genomic library constructed by cloning partially Mbo I-digested DNA from patient 10 into the Xho I site of the A-Fix-II vector (Stratagene). Genomic library screening and plaque purification were performed by standard procedures (14). Hybridization was at 37°C in 50% (vol/vol) formamide/3 x SSC/5 X Denhardt's solution/10% (wt/vol) dextran sulfate containing sonicated and denatured salmon sperm DNA (100 l g/ml). (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0; lx Denhardt's solution = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin.) The stringency of the final wash was 0.2x SSC/0.2% SDS at 60°C. Isolated plaques were grown and phage DNA was analyzed by restriction enzyme mapping (14). Inserts were subcloned in plasmid vector pGEM-3 (Promega Biotec) for further analysis.DNA Sequencing. The 2.7-kilobase (kb) Kpn I-EcoRI fragment from Aal and the 2

Acute promyelocytic leukemia (APL;t M3 of the FAB 1 .1 classification) is a distinct, well-characterized clinical and morphological subtype of acute myeloid leukemia (AML) (1,2). It is cytogenetically distinguished by a reciprocal chromosome 15;17 translocation, which is present in 70-90% of cases and never seen in other AMLs or other types of malignancy (3, 4). Although the high frequency and specificity of the t(15;17), and the fact that it is often the only karyotypic aberration present (4), is strong evidence that it plays a crucial role in the pathogenesis ofAPL, the gene(s) directly involved in the chromosome 15 and 17 breakpoints have never been identified . We have previously shown that the APL chromosome 17 breakpoint lies between the c-erbB-2 and the retinoic acid receptor a (RARci) loci (5) . We report here the findings of RARct gene rearrangement and aberrant expression in APLs. Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor a (RARa) locus. Investigation of20 APLs and a large series ofother neoplastic patients and normal controls revealed RARa gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RARce gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes. Materials and Methods Rearrangements and Aberrant Expression of theMolecular Studies. The K/S, IT, and P/R RARct cDNA probes were obtained by subcloning portions of the p63 plasmid (6) in 'Abbreviations used in this paper: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; BM, bone marrow ; RARa, retinoic acid receptor a.