Application Of Dual Color-Fluorescence In Situ Hybridization In Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia accounting for 10-15% of de novo cases. About 70-100% of APL is characterizaed by a distinct translocation t(15;17) (q22;q21) which fuses the PML gene on chromosome 15q22 to the retionoic acid receptor a (RAR a ) gene on 17q21 leading to the formation of PML/RAR a and RAR a /PML fusion genes. PML/RAR a fusion gene was believed to be critical for leukemgenesis of APL and nearly expressed in all APL patients. However,the effect of RAR a /PML fusion gene was expressed in approximately 70% to 80% of the reported cases,remains to be detemined. About 20 % of the APL patients in which PML/RAR a rearrangements were demonstrated by molecular methods did not display t(15;17) due to the poor qualitative metaphases or cryptic rearrangements. Fluorescence in situ hybridization (FISH) can screen large number of metaphase or interphase cells on bone marrow slides and smears and can identify both numerical and structural chromosome abnormalities as an aid to cytogenetic diagnosis and for monitoring minimal residual disease (MRD). This study is carried out to detect PML/RAR a rearrangement in APL patients and explore the value of dual color-FISH (D-FISH) in diagnosis of APL and monitoring of MRD.Objective: To investigate the value of D-FISH in the early diagnosis and the monitoring of MRD in APL patients.Methods:Sequence special probes for PML and RAR a genes directly labeled with SpectrumGreen and Spectrum Orange respectively and D-FISH were used to detect PML/RARa rearrangement in 55 APL patients and the results were compared with that by conventional cytogenetics (CC) and reverse transcriptase polymerase chain reaction (RT-PCR).Results:(1) In 20 APL patients at diagnosis,11 patients with t (15;17) by CC were found to be positive for PML/RAR a fusion gene by D-FISH. Among them, 9 were comfirmed having PML/RAR a rearrangement by RT-PCR and 2 didn't be examined. In 7 APL patients without t(15;17) by CC,PML/RAR a fusion transcript was identified by RT-PCR showed that they had cryptic 15;17 translocation. Among them,6 patients were found to be positive for PML/RAR a rearrangement by D-FISH. 2 patients diagnosed as APL by mophology each displayed t(8;21) or t(9;22) by CC. They were found to be negative for PML/RAR a rearrangement by D-FISH and RT-PCR simultaneously and finally were diagnosed as having AML-M2. (2) In 15 APL patients in CR,14 didn't display t(15; 17) and 1 failed by CC, 9 were confirmed having PML/RAR a rearrangement by D-FISH and RT-PCR. (3) D-FISH was carried on bone marrow smears of 20 previously untreated APL cases. 8 displayed t (15;17) by CC were found to be positive for PML/RARa rearrangement by D-FISH and RT-PCR,while 2 out of 10 cases without t (15;17) by CC and 1 out of 2 cases lacking cytogenetic analysis was confirmed having PML/RAR a rearrangement by D-FISH and RT-PCR.Conclusions: D-FISH is a sensitive and realiable method indetecting PML/RAR a rearrangement in APL at diagnosis and in CR. It can be used on the slides or bone marrow smears and fit to those patients presenting crypic rearrangement or failed cytogenetics and lacking suitable material for molecular analysis as well as restropective studies.