Impact of Fluorescence in Situ Hybridization in the Detection of Cryptic Fusion Transcript PML/RARA and A Complex t(5;15;17) in a Case of Acute Promyelocytic Leukemia Gönül OGUR*,****, Turgay FEN**, Gülsan SUCAK***, Pierre HEIMANN*, Gaye CANKUÞ****, Rauf HAZNEDAR**** * Univérsité Libre de Bruxelles, Hôpital Erasme, Service Génétique Medicale, Bruxelles, BELGIUM ** Oncology Hospital, Ankara, TURKEY *** Adult Hematology Department, Faculty of Medicine, Gazi University, Ankara, TURKEY **** GEN-MED Genetic Diseases and Prenatal Diagnosis Center, Ankara, TURKEY ABSTRACT Genetic aspects of a 28 year-old female patient with typical morphological and clinical features of acute promyelocytic leukemia is presented. Pml/rara fusion transcript and a complex translocation involving chromosomes 5, 15 and 17 were detected by fluorescence in situ hybridization (FISH) tech- nique which was applied as in adjunct to conventional cytogenetics. The patient deceased soon in spite of the immediate ATRA and cytostatic therapy. Key Words: FISH, PML/RARA, Acute promyelocytic leukemia. Turk J Haematol 2000;17(4):207-212. INTRODUCTION sociated with t(15;17)(q22;q12-21). The diagnosis of APL and the detection of residual disease are Acute promyelocytic leukemia (APL) is a rara based on the presence of this translocati- distinct subtype of myeloid leukemia characterized on[1,3,4,5,6,7,15]. Rarely alternative balanced trans- by invasion of bone marrow by hypergranular le- locations have been described in subtypes of APL, ukemia cells, by specific translocations almost al- and as more cases are being evaluated, complex, ways involving chromosome 17 and by a high sen- variant translocations are increasingly recogni- sitivity of the promyelocytic blasts to retinoic acid zed[17,19,20,22, 24]. (RA). It has been shown to be the first model of dif- ferentiation therapy[5,6]. The disease is mainly as- Here we report a 28 year-old female patient 207 Impact of Fluorescence in Situ Hybridization in the Detection of Cryptic Fusion Transcript PML/RARA and A Complex t(5;15;17) Ogur G, Fen T, Sucak G, Heimann P, Cankuþ G, Haznedar R. in a with typical morphological and cilinical features of MATERIALS and METHODS acute promyelocytic leukemia in whom a complex Cytogenetic Analysis translocation involving chromosomes 5, 15 and 17 was detected by conventional cytogenetics Cytogenetic analysis of the bone marrow aspi- and flourescence in situ hybridization (FISH). ration was performed from direct and 24 hour Conventional cytogenetics and flourescence in si- nonstimulated culture preparations. Chromoso- tu hybridization (FISH). Conventional cytogene- mes were GTG-banded and the karyotype was tics detected a derivative 5 and a derivative 17 described according to the International System chromosome but failed to detect chromosome 15 for Human Cytogenetic Nomenclature (ISCN involvement. Application of FISH with region spe- 1995)[11]. cific probes probes and paint probes, showed that Fluorescence in Situ Hybridization (FISH) the karyotype involved a complex rearrangement of chromosomes 5, 15 and 17 including PML/RA- FISH studies were performed on the speci- RA fusion transcript. mens prepared both from direct and 24-hour incu- bation cultures. To exclue the possibility of a cryp- CASE REPORT tic t(15;17) translocation or a complex t(15;17) A 28 year-old female presented with massive translocation including chromosome 5, direct la- and prolonged hemorrhagia during her menses. A beled locus specific probe, Vysis pml/rara was complete blood count showed: hemoglobin, 70 used (Vysis Inc., IL, USA) Furthermore commerci- g/L; white blood cell count, 13.2 x 109/L, and pla- al digoxine genated chromosome paint probes telet count 23.5 x 109/L. Peripheral blood smear (Cambio, Cambridge, UK) specific for chromoso- yielded more than 80% atypical promyelocytes mes 5, 15 and 17 were respectively used mono- with numerous azurophilic granules and Auer colour hybridization. rods. Bone marrow was markedly hypercellular Slide pretreatment was based on RNAse and and also showed hypergranular atypical promye- pepsin. For direct labeled probe pml/rara, conditi- locytes with Auer bodies which accounted for mo- ons of hybridization and post-hybridization was- re than 90% of all nucleoted cells. Coagulation hings were performed according to Vysis’ own tests showed a prolonged bleeding time, a normal protocol. In case of patient chromosomes, ten prothrombin time: 12 seconds (control 12.3 se- microliters of each probe were denaturated sepe- conds) and a borderline activated partial throm- rately at 70 °C for 10 minutes, left to preanneal at boplastin time: 35. seconds (control 34.8 se- 37 °C for 1 hour and then applied to 3 different sli- conds). Fibrin degradation products were negati- des. After 18 hours of hybridization at 37 °C slides ve in serum and urine. Massive bleeding was att- were washed in 50 % formamide/2xSSC. The ributed to thromboctopenia rather than dissemina- probes were visualized using mouse anti-digoxi- ted intravascular coagulation. Immunohistochemi- genin (Boehringer Mannheim) antibody. FITC sig- cal stainings were strongly positive for myelopero- nal was ampliied with two additional layers of hidase and sudan black B. Immunophenotyping (rabbit anti-mouse), tetrarhodamine-isothiocyona- showed 30% CD13 positivity and was negative for te (TRITC) and (goat anti-rabbit)-TRITC. This sli- surface marker HLA-DR (10.2%). A clinical diag- des were counterstained with propidium iodide (1 nose of APL (FAB-M3) was made. ug/mL). Analyses were performed under a flu- The patient was treated with all-trans retinoic orescence microscope (Axioplan Zeiss) aquipped acid (ATRA, 45 mg/m2 daily), adriablastina (50 with an Applied Imaging Cytovision image analy- mg/m2 for 3 days) and cytosine arabinoside (200 sis system (version 4.4). Both metaphase and in- mg/m2 for 7 days). Partial remission occured inc- terphase cells were examined. luding an increase in platelet counts and a slight RESULTS decrease in leucemic cells. However at day 65 af- ter treatment her relatives reported than she was Cytogenetic evaluation of the bone marrow yi- deceased at home. elded a clone with a rearranged short arm of chro- 208 Turk J Haematol 2000;17(4):207-212 Impact of Fluorescence in Situ Hybridization in the Detection of Cryptic Fusion Transcript PML/RARA and A Complex t(5;15;17) in a Ogur G, Fen T, Sucak G, Heimann P, Cankuþ G, Haznedar R.. mosome 5 and a large derivative chromosome 17. By conventional GTG-banding the derivative chromosome 17 appeared to be disrupted distal to 17q21, chromosome 15 seemed to be intact, pml/rara and chromosome 5 showed a short arm rearran- 15 gement. As the chromosome condensation was 17 severe it was difficult to make a clear identificati- on of the rearrangements, however the source of the daditional material on the derivative 17q was interpreted as 5p and the karyotype was sugges- ted to present 46, XX, t (5;17)(p13:q21)? (51 me- taphases) (Fig. 1). There were also 46, XX normal metaphases (13 metaphases). Figure 2. Fusion transcript PML/RARA by FISH: in FISH studies with direct labeled locus specific an intephase cell and a metaphase (arrows): probe Vysis pml/rara demonstrated three signals nuc.ish t(15;17)(PMLx1)(RARAx1)(RARA- both on the metaphases and the nuclei: a normal conPMLx1) and ish. t(15;17)(PMLx1)(RA- RAx1)(RARAconPMLx1). green coloured chromosome 17, and a normal red coloured chromosome 15 and one double-co- rearranged chromosomes 15 and 17; for chromo- loured (red-yellow) signal which was suggestive some 15, one derived from the infact chromoso- of the fusion transcript pml/rara: (Fig. 2). FISH me 15 and two from the rearranged chromoso- with paint chromosomes 5, 15, and 17 were also mes 15 and 17; for chromosome 5, one derived performed and in each metaphase analysed, the- from the intact chromosome 5, and two from the re seemed to be the triplication of signals for each rearrangements of chromosome 5 and 17 (Table chromosome respectively (three signals for 5 1). Due to chromosome arrangements size of the three for 15 and three for 17 (Figs. 3,4,5). For signals varied (Figs. 3,4,5). each paint probe, three hybridization signals indi- cating chromosome 5, 15 and 17 were detected in The overall neoplasic karyotype after conven- metaphases. For chromosome 17 one derived tional cytogenetics and FISH studies were interp- from the intact chromosome 17 and two from the reted to be 46, XX, t (5;17) (p13;q22; q21).ish t(15;17) (PMLx1) (RARAx1) (RARA conPMLx1) DISCUSSION The vast majority of cases of acute promye- locytic leukemia are associated with a specific 1 2 3 4 5 translocation, t (15;17) (q22-q12-21). By conven- tional cytogenetics, the percentage of APL cases 6 7 8 9 10 11 12 showing t (15;17) varies form 70% to 90%. At the molecular level this translocation results to the for- mation of some specific products namely as 13 14 15 16 17 18 pml/rar-alpha, rar-alpha/pml and other abberrant pml products. In greater than 95% of APL cases the fusion product expressed is pml/rar alpha in 19 22 X Y 20 21 which rar-alpha gene (retionic acid receptor alpha 46,XX, t(5;17)(p13;q21) (?) gene) on chromosome 17q is translocated to a putative transcription factor gene located on chro- Figure 1. Karyotype from bone marrow: 46, mosome 15q known as pml. The formation of this XX,t(5;17)(p13;q21)?. Derivative chromosome 5 fusion product pml/rara is suggested to be essen- and 17 are shown by arrows. Chromosome 15 tial in the pathogenesis of APL and exists in al- seemed to be normal by standart karyotype. Turk J Haematol 2000;17(4):207-212 209 Impact of Fluorescence in Situ Hybridization in the Detection of Cryptic Fusion Transcript PML/RARA and A Complex t(5;15;17) Ogur G, Fen T, Sucak G, Heimann P, Cankuþ G, Haznedar R.