Bone Marrow WT1 Levels In Long-Term Survivors Of Core-Binding Factor AML and Acute Promyelocytic Leukemia
Josep NomdedéuMontserrat HoyosMaite CarricondoElena BussagliaMaria-Concepcion Garcia-DabrioIsabel BadellCamino EstivillAna GarridoClara MartínezSalut BrunetAnna Aventı́nJorge Sierra
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Minimal Residual Disease
Minimal Residual Disease
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This study was purposed to comparatively analyse the value of PCR and FCM for dynamic monitoring minimal residual disease (MRD) of acute promyelocytic leukemia. The patients with acute promyelocytic leukemia hospitalized in our hospital from January 2011 to December 2012 were observed and all achieved complete remission after remission induction therapy. Before the chemotherapy, the bone marrow cell morphology examination, polymerase-chain reaction (PCR) and multi-parameter flow cytometry (FCM) were performed for each patient. Then the detection results were statistically analyzed. The 477 specimens were achieved from 159 detections for 48 patients. The results showed that 3 specimens were found to be relapsed by bone marrow cell examination, and other specimens were complete remission;PCR detection confirmed 7 positive, and the FCM confirmed 19 positive. There wasn't significant difference between PCR and FCM by kappa test (P > 0.05). It is concluded that FCM is as sensitive as PCR in evaluating the treatment effect of acute promyelocytic leukemia.
Minimal Residual Disease
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The International Consortium on Acute Promyelocytic Leukaemia (IC-APL) is a collaborative effort led by the International Members Committee of the American Society of Hematology. Its aim is to improve the outcomes of patients with APL in developing countries, by adapting European and American diagnostic, supportive and therapeutic guidelines. As a result of this initiative, differences in outcomes of patients with APL in developed and developing countries have been significantly reduced (Jácomo et al, 2007; Rego et al, 2013). Nevertheless, an important obstacle faced by the IC-APL study was to adapt to logistical conditions for minimal residual disease (MRD) monitoring in these countries. In Brazil, participating centres were located at distances of up to 2500 km from the central national laboratory, and the time for delivery of the samples was up to 2 days. In the original protocol, the detection of promyelocytic leukaemia – retinoic acid receptor alpha (PML-RARA) fusion gene was performed by endpoint reverse transcription polymerase chain reaction (endpoint PCR)(Rego et al, 2013). The results of the Europe Against Cancer (EAC) Program suggest that real-time quantitative polymerase chain reaction (RT-qPCR) enables more reliable detection, and provides information regarding sample quality through parallel amplification of a housekeeping gene (Gabert et al, 2003). However, very few studies have compared these two assays for longitudinal MRD monitoring in APL. To address this issue in the context of developing countries, we compared the results obtained with endpoint PCR and RT-qPCR within an IC-APL study. The baseline characteristics of 103 patients diagnosed, treated and monitored according to the IC-APL protocol are summarised in Table 1; methodology details are provided as online Data S1. The median follow-up among survivors was 49 months (range: 10–99 months). The normalised copy number (NCN) at diagnosis was 0·5391 (n = 57) and 0·4382 (n = 40) copies of PML-RARA/104 copies of ABL1 for patients with breakpoint cluster region protein gene isoforms 1 and 3 (BCR and BCRP3), respectively. Only one patient had PML BCRP2 (NCN: 5·0775). After induction, PML-RARA transcripts were detected in 45/73 (62%) and 40/73 (55%) samples analysed by RT-qPCR and endpoint PCR, respectively. Samples from two patients (3%) had poor sample quality (i.e., absence of ABL1 gene amplification within 40 cycles). The median value of NCN after induction was 0·00043 (n = 47) and 0·00026 (n = 25) for subtypes bcr1 and bcr3, respectively. Of the 45 patients that showed positive results by RT-qPCR in samples after induction, two (Patients 1 and 5) relapsed in the maintenance phase. Of the 41 positive patients for PML-RARA according to endpoint PCR after induction, only one relapsed (Patient 5). It is important to point out that bone marrow (BM) collection was not compulsory at this point according to the protocol. The complete haematological response rate was 91% (94/103 patients) and the mortality rate during induction was 9% (9/103 patients). After the third consolidation cycle, RT-qPCR analysis was carried out in 80/83 (96·3%) samples that had originally been analysed by endpoint PCR. The median value of NCN after the third consolidation was 0·00001, regardless of the PML breakpoint. Two patients died during consolidation therapy due to infectious complications. According to endpoint PCR analysis, 96·3% (80/83) patients achieved complete molecular remission (CRm). Analysis by RT-qPCR showed NCN of 0·0021, 0·0046 and 0·0011 in the three patients who did not achieve CRm. In addition, four patients were considered not to have achieved CRm based on RT-qPCR evaluation (NCN: 0·0002). However, in the confirmatory samples the fusion gene was not detected by either technique. Our findings confirm the absence of primary resistance in APL and support the prognostic importance of molecular analysis after consolidation, as well as the relevance of obtaining confirmatory samples within an interval of at least 15 days (Sanz et al, 2009). During maintenance, 84·4% (430/509) and 94·8% (483/509) of the samples were negative for PML-RARA according to RT-qPCR and endpoint PCR analysis, respectively. Relapse was detected in seven patients in our cohort, including two patients with a concomitant molecular and central nervous system (CNS) relapse (Patients 5, 6) (Fig 1). In addition, there were three cases of molecular relapse and two of haematological relapse. The RT-qPCR technique detected PML-RARA transcripts before molecular relapse in six of seven patients with relapsed disease. Patient 1 had PML-RARA detected by RT-qPCR in samples collected 240, 163 and 86 days prior to the diagnosis of molecular relapse (Fig 1). The respective values of NCN were 0·00032, 0·000532 and 0·00104. Patient 2 had PML-RARA transcripts detected by RT-qPCR (NCN: 0·00046) in a BM sample collected 98 days before diagnosis of molecular relapse. One of the three patients that had a molecular relapse (Patient 3) presented six samples that tested negative by endpoint PCR. However, the retrospective analysis by RT-qPCR of the same samples failed to pass the quality control evaluation. In samples obtained 138 and 34 days before haematological relapse, RT-qPCR, but not endpoint PCR, detected PML-RARA transcripts in Patients 4 and 7. Patient 5 had a molecular relapse diagnosed in the CNS in the 6th month of maintenance, after a cerebrospinal fluid puncture was requested for investigation of headaches. The PML-RARA transcripts were detected by RT-qPCR 84 days before the first positive result obtained by the conventional techniques. Patient 6 was also diagnosed in the CNS, with the RT-qPCR data predictive of relapse 10 months before the first positive result was obtained by endpoint PCR. In summary, our results show that the detection and quantification of PML-RARA transcripts after consolidation therapy is associated with haematological relapse after a short period. It is well established that the molecular relapse indicates the need for immediate pre-emptive treatment and is particularly beneficial for patients at high-risk (Grimwade et al, 2009). The major obstacle for monitoring molecular relapse in APL was appropriate sample collection, reinforcing the importance of continued medical education. Although the difference in cost between the two techniques is relatively high, we recommend RT-qPCR as the standard assay for MRD monitoring in APL, in light of its higher precision and reliability in terms of quality sample assessment. We gratefully acknowledge all members of the International Consortium on Acute Leukemia of the American Society of Hematology. This investigation was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP Grant #2013/08135-2). A.P.L. received a fellowship from FAPESP (Grant#2011/17111-4). A.P.L. performed experiments, analysed and interpreted data, and drafted the article. A.S.L. performed experiments and reviewed the paper. A.R.L.A. reviewed and drafted the manuscript. R.H.J., R.A.M., R.B., R.P., K.P., E.M.F., M.L.C. and C.S.C., provided the samples, updated the clinical data and reviewed the paper. M.A.S., F.L.C., D.G., and E.M.R. designed the treatment protocol, reviewed the paper and gave final approval of the submitted version. The authors have no competing financial interests to declare. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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Minimal Residual Disease
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Promyelocytic leukemia protein
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Acute promyelocytic leukemia (APL) is a disease described as definite morphological and cytogenetical abnormalities and leads to coagulopathy leaving the patient in a life-threatening condition. A specific chromosomal translocation of 15 and 17 chromosomes leads to retinoic acid receptor-α (RARα) and promyelocytic leukemia (PML) genes fusion that produces an abnormal gene mutation forming an oncogenic protein which is (PML-RARα). Those APL patients, who have been treated with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) commonly lead a complicated condition called differentiation syndrome which is rarely severe. This case report explains the 37-years old male diagnosed with acute promyelocytic leukemia and later developed a differentiation syndrome after initiation of all-trans retinoic acid and arsenic trioxide induction therapy.
Arsenic Trioxide
Promyelocytic leukemia protein
Differentiation Therapy
Tretinoin
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To evaluate factors predictive for relapse in a cohort of adult patients with acute promyelocytic leukemia monitored by molecular methods during consolidation and during at least one month of maintenance therapy. The charts and laboratory data of 65 adult patients with acute promyelocytic leukemia treated according to the International Consortium on Acute Promyelocytic Leukemia 2006 protocol were reviewed. The identification of the promyelocytic leukemia-retinoic acid receptor-alpha gene rearrangement at diagnosis, post-induction, post-consolidation and during maintenance treatment was performed by qualitative and quantitative reverse transcription polymerase chain reaction. Eighty-nine patients were diagnosed with acute promyelocytic leukemia over a seven-year period and of these 65 were eligible for treatment with the protocol. Among the 45 patients who received consolidation and maintenance treatment, six (13%) relapsed, three of whom presented hematologic and three presented molecular relapse. The first relapses occurred at a median of 39 months. Relapsed patients were from all risk groups (low, intermediate and high) and both morphological types (M3 and M3variant) were found. Three of these patients are alive and in molecular remission after salvage treatment. There were no statistically significant differences regarding gender, age, risk group, morphology, promyelocytic leukemia breakpoint cluster region, use of all-trans retinoic acid, development of differentiation syndrome and number of days to complete remission between the patients who relapsed and those who did not. Our results reinforce the importance of prolonged monitoring of acute promyelocytic leukemia patients using molecular methods to detect relapse early.
Promyelocytic leukemia protein
Tretinoin
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Minimal Residual Disease
Arsenic Trioxide
Tretinoin
Single Center
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Objective To study the clinical significance of monitoring minimal residual disease(MRD)in acute promyelocytic leukemia(APL,M3)by flow cytometry.Methods Three-color combinations of monoclonal antibodies(CD15,CD13,CD11b,CD33,CD34,CD45)were used to detect 50bone marrow specimens of patients with APL diagnosed by FAB and immunophenotype before therapy and to follow up 20bone marrow specimens after initial induction therapy.Meanwhile,ten bone marrow specimens from normal controls were detected.Results Ninety-Six percents of M3leukemia cells expressed CD15-,CD11b-,CD13+and CD33+before therapy at CD45/SSC gate;after initial induction therapy,the recurrence rate of MRD-positive group was 57.0%,significantly higher than that of the MRD-negative group,there were significant difference between the two groups.Conclusion Flow cytometry is a fast,simple and effective method to detect the minimal residual disease in cute promyelocytic leukemia.
Minimal Residual Disease
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Immunophenotyping
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Objective To investigate the kinetics of PML-RARα fusion gene in acute promyelocytic leukemia(APL)to monitor minimal residual disease(MRD). Methods In induction therapy,consolidation and maintenance therapy courses, PML-RARα fusion gene was performed by RT-PCR. Results The long-term follow-up of 18 cases achieved complete remission (CR),two cases experienced molecular relapse. One case relapsed at 4 months after CR1 and achieved CR2 after induction therapy. However, molecular and hematology relapsed again at 2 months after CR2 and re-achieved CR3. The other case relapsed at 74 months after CR1 and achieved CR2 after induction treatment, who had survived for 106 months until the end of follow-up. Conclusion RT-PCR assay for detection of PML-RARα should be performed regularly during CR period so as to find molecular relapse eady. Hematological relapse could potentially be averted through treatment modification according to molecular monitoring results of PML-RARα.
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Acute promyelocytic leukemia; PML-RARα fusion gene; Minimal residual disease (MRD)
Minimal Residual Disease
Hematology
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Objective:To investigate the PML-RARα fusion gene on monitor minimal residual disease(MRD) in acute promyelocytic leukemia(APL).Method:PML-RARα fusion gene was detected by RT-PCR in induction therapy,consolidation and maintenance therapy.Result:The long-term follow-up of 18 cases who achieved complete remission(CR), two cases relapsed in molecular level. One case relapsed after 4 months of CR1 and achieved CR2 after induction therapy. However, molecular and hematological relapse occured again 2 months later after CR2 and it achieved CR3 after one induction therapy. The other case relapsed after 74 months and achieved CR2 by induction treatment, and survived for 106 months untile the end of follow-up. Conclusion:RT-PCR assay for detection of PML-RARα should be performed regularly during CR period to monitor molecular relapse. Hematological relapse could be potentially averted by modification treatment according to monitoring PML-RARα.
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