Clinical Implication of PML/RAR breakpoints in Acute Promyelocytic Leukaemia (APML): Experience From a Universiti Hospital in MalaysiaRoslineHassan, Abu Dzar Abdullah, Azlan Hussin, Rapiaah Mustaffa, Wan Zaidah Abdullah, Abdul Aziz Baba Department of Hematology 1, Department of Medicine2, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia Abstract INTRODUCTION : Acute Promyelocytic Leukaemia is the third commonest AML subtypes among Malaysian. Reciprocal translocation between chromosome 17 and 15 is the hallmark for APML. The treatment of APML was with all-transretinoic acid and more recently with arsenic trioxide (As2O3) has increased longlasting complete remissions (CRs). Three different PML/RAR isoforms have been described; S-form, L-form and V-form. Our aim was to determine the clinical implication of these three different isoforms of PML/RAR in APML patients. METHOD: Nineteen patients diagnosed as APML were identified. Clinical and laboratory data was retrieved from record office and haematology laboratory respectively. Total RNA was extracted from nineteen bone marrow samples within 24 hours of collection using standard methods as described in manufacturers¶ protocol for QIAamp® RNA Blood Mini Kits (Qiagen GmbH, Hilden, Germany). Multiplex RT-PCR was performed on all patients diagnosed as APML. RESULTS : PML/RAR fusion transcript was detected in all them. Of these patients, 57.9% (11patients) exhibited V-form, 36.8% (7 patients) S-form, and 5.2% L-form. Total white blood cell count was higher in S-form compared to V-form (P < 0.05). Four years survival was 100% and 42.8% for V-forms and S-forms respectively. (p<0.005). CONCLUSION : Patients with PML/RAR V- forms survived better that S-form. Molecular detection of isoforms is indicated in all patients diagnosed as APML. Further study is in progress to identify the underlying mechanism Subsequently split nested PCR was carried. PCR product from the RT-PCR step above was used as template. The PCR mixture contain 2 l of PCR product and 1U of Qiagen HotStarTaq polymerase (Qiagen GmbH, Hilden, Germany) with final concentration of 1´ Qiagen Buffer, 100 M dNTP and 500nM of each PML-INT and RAR-INT primers. PCR was performed in Mastercycler Gradient at 94oC for 20 seconds, 55oC for 20 seconds and 72oC for 30 seconds followed by a 5 minute final extension at 72oC. PCR amplification was preceded by 1 cycle of enzyme activation or preheating at 95oC for 15 minutes. Positive control was performed for every run and no template control (NTC) for the detection of PCR contamination. Each PCR product was run on 1.7% agarose gel prepared stained with ethidium bromide. Sample amplified with the PML INT and RAR INT primers which show bands sized either 714 base pair (bp), 714-312bp (often several bands due to alternative splicing) or 214bp in the presence of 366bp PCR product of ABL housekeeping gene were reported as positive for L-form, V-form and S-form respectively. Figure 2: Kaplan Meier analysis Results RNA was extracted from nineteen Malay patients who was newly diagnosed as APML. PML/RAR fusion transcript was detected in all them. Of these patients, 63.1% (12 patients) exhibited L/Vform,and 36.8% (7 patients) S-form (Figure 1) Those exhibited S-form were older with male predominant compared to L/V-form. Blood counts were almost similar except for platelet counts which were lower in S-form (Table 1). Coagulation profiles were normal but D-dimer was present in all of them. Discussion Studies have shown that incidence of various fusion transcripts varies significantly among different populations. Incidence of Lform, S-form and V-form were 50-55%, 27-49% and 8-20% respectively as reported in literatures from America [4]. The L/Vform was found to be higher than S-form in population such as the Latinos as reported by Dan Douer et al., 2003 [5] which was similar to our study. We noted that there was no significant difference in Hb, and TWBC count at diagnosis between the isoforms. Although there was also no significant difference in platelet counts between the two forms but S-form presented with a lower count. Fibrinogen, APTT and PT were normal in all cases. We also observed elevated D-Dimer level in both forms denoting occult DIC. We therefore recommend screening of D-Dimer level in all cases of APML as part of the management protocol. Debate exists over the clinical relevance of molecular heterogeneity of APML Studies on the prognostic significance of PML/RAR isoforms have reported contradictory results. In our study, four years survival was 100% and 42.8% for L/V-forms and S-forms respectively. Kaplan Meier analysis was performed and patients with L/V-form have significantly better survival than the S-form. (p<0.005). Median survival for S-form was 19.9 months while all patients with L/V- form has survived to date. A larger cohort of patients is required to consider the presence of the S-form as a high-risk feature and to design the future risk-adapted treatment strategies for APML. Introduction Acute promyelocytic leukemia (APML) is a form of acute leukemia representing 10 ± 15% of all acute myeloid leukemia (AML) cases. [1]. Translocation (15;17)(q22;q11.2) is a balanced reciprocal chromosomal translocation that fuse the PML gene on chromosome 15 with the RAR gene on chromosome 17. The resulting PML/RAR chimeric gene is expressed and subsequently PML/RAR fusion protein inhibits the granulocytic differentiation and promoting survival of hematopoietic progenitor cells. Three types of PML/RAR isoforms have been identified. Type A or the short form (S-form) arises when the breakpoint occurs within intron 3 of PML (breakpoint cluster region 3, Bcr-3) and type B or the long form (L-form) occurs within intron 6 (Bcr-1) whereas the third type B variant or variable form (V-form) occurs within exon 6 (Bcr-2). Breakpoint of RAR is invariably in intron 2. [2] Some literatures have described correlation between PML/RAR isoforms and several patient characteristics and outcomes but with discrepant results. In the study done by Gonzalez et al., 2001, there was no difference was observed between the three isoforms in complete remission (CR) rate. However 3-year disease-free survival was lower for V form than L- and S-form (62% vs. 94% and 89%). Both V-form and S form were associated with some negative prognostic features at time of diagnosis. [3] Thus the aim of this study was to to determine the clinical implication of these three different isoforms of PML/RAR in APML patients. Table 1: Clinical and Laboratory features between PML/RAR alpha isoforms PML/RAR alpha isoforms Short-form n=7 (SD) 38.4 (15.9) 5:2 8.2 (1.8) 2.1 (1.0) 24.7 (22.4) L/V-form n=12 28.6(9.9) 4:8 8.3 (2.9) 2.5 (2.0) 43.6 (52.6) Age yrs Sex (M:F) Hb g/L TWBC x 109/L Platelet x 109/L Conclusion Patients with PML/RAR V- forms survived better that S-form. Identification of molecular isoform is important in the management of patients with APML. Further study is in progress to identify the underlying mechanism Material and Method Nineteen Malay patients diagnosed with APML from September 2004 to October 2008. Informed and written consent were taken from them and the study has been approved by ethical committee. Total RNA was extracted from 1ml peripheral blood (PB) or bone marrow (BM) specimen using standard methods described in manufacturers¶ protocol for QIAamp® RNA Blood Mini Kits (Qiagen GmbH, Hilden, Germany). The extracted RNA was subjected to spectrophotometric quantification (Eppendorf, Germany). Primers were designed similar to the ones used in the BIOMED-1 European group (van Dongen et al., 1999). Complementary DNA (cDNA) was synthesized from 200 ± 500ng of patients total RNA using QIAGEN OneStep RT-PCR Kit (Qiagen GmbH, Hilden, Germany). Total RNA was added to RT-PCR master mix to make a 20 l reaction mixture containing final concentration of 1× Qiagen One Step RT-PCR Buffer, 80 M dNTP, 1.25mM MgCl2, 5 ± 50nM of external primers for ABL transcripts and PML/RAR fusion transcripts, 0.6 l Qiagen OneStep Enzyme Mix, 0.08U RNasin Ribonuclease Inhibitor (Promega, Madison, WI) and water for irrigation. Reverse transcription was carried out at 50oC for 30 minutes followed by 1 cycle preheating for enzyme activation, 35 cycles of PCR and terminated by cooling the reaction mixture to 4oC. References Fig 1: PCR for PML/RAR alpha fusion gene Arrow showing transcript 500bp in size. M: Marker of 100bp in size. Lane 1 & 3: Normal ABL gene (366bp). Lane 2: Patient showed PML/RAR fusion gene exhibit the variant form (312-714bp). Lane 4: Patient with no PML/RAR transcript Lane 5 & 6: Negative control using water. Lane 7 & 8: A positive control with normal ABL gene present and PML/RAR fusion gene observed (312-714bp). Four years survival was 100% and 42.8% for L/V-forms and Sforms respectively. Kaplan Meier analysis was performed and patients with L/V-form have significantly better survival than the Sform. (p<0.005).Median survival for S-form was 19.9 months meanwhile all patients with L/V- form has survived to date (fig:2) 1.Barnard DR, Kalousek DK, Wiersma SR. Morphologic, immunologic and cytogenetic classification of acute myeloid leukemia and myelodysplastic syndrome in childhood: a report from the Children's Cancer Group. Leukemia. 1996;10:5-12. 2.Rousselot P, Hardas B, Patel A, Guidez F, Gaken J, Castaigne S, et al. The PML-RAR alpha gene product of the t(15;17) translocation inhibits retinoic acid-induced granulocytic differentiation and mediated transactivation in human myeloid stem cells. Oncogene. 1994;9:545 3. Gonzalez M, Barragan E, Bolufer P, Chillon C, Colomer D, Borstein R, et al. Pretreatment characteristics and clinical outcome of acute promyelocytic leukaemia patients according to the PML-RAR isoforms: a study of the PETHEMA group. Brit J Haematol. 2001;114:99-103. 4.Chang E, Levine AM, Slovak M, Forman S, Douer D. Acute promyelocytic leukemia in Latino patients is associated with a high frequency of the BCR1 breakpoint site in the rearranged PML gene. Blood. 1996 NOV 15;88(10):2647 5. Dan Douer, Sergio Santillana, Laleh Ramezani, Cesar Samanez, Marilyn L. Slovak, Ming S. Lee, et al. Acute promyelocytic leukaemia in patients originating in Latin America is associated with an increased frequency of the bcr1 subtype of the PML/RARa fusion gene. Brit J Haematol. 2003;122:563±70. This work was supported by Research University Grant (1001/PPSP/8121009), Universiti Sains Malaysia, Kelantan, Malaysia ISLH 2011, the XXIVth International Symposium on Technological Innovations in Laboratory Hematology 2011,